CN118389390B - Lactic acid bacteria expressing African swine fever virus fusion antigen and their applications - Google Patents

Abstract

This invention provides a lactic acid bacterium expressing an African swine fever virus fusion antigen and its application. The lactic acid bacterium integrates a fusion gene, which includes: a gene encoding the OprI protein and at least one gene selected from genes encoding PP62, MGF505-3R, EP364R, K145R, and DP96R proteins. The starting strain of the lactic acid bacterium is *Lactobacillus plantarum*. Immunizing animals with this lactic acid bacterium can induce mucosal immunity, cellular immunity, and humoral immunity, comprehensively enhancing the body's immune capacity. It can be used to produce an oral vaccine for the prevention and control of African swine fever.

Description

Lactic acid bacteria expressing African swine fever virus fusion antigen and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to a lactobacillus for expressing African swine fever virus fusion antigen and application thereof.

Background

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

African Swine Fever (ASF) is a severe infectious, hemorrhagic disease of pigs and is considered to be one of the most severe and damaging viral diseases of domestic pigs. When the epidemic outbreak occurs, it must be reported to the world animal health Organization (OIE). Research has found that outbreaks of african swine fever cause serious economic losses worldwide, and ASF has been seen from the 20 th century to be a serious threat to the development of the global pig industry until now, as no safe and efficient vaccine has been developed yet. The clinical manifestations and pathological features of ASF vary widely, depending on the virulence of the strain and host characteristics. Clinical symptoms of ASFV-infected pigs include high fever, vomiting, diarrhea, loss of appetite, dyspnea, skin bleeding, cyanosis and abortion in pregnant sows. Acute ASF is caused by highly or moderately virulent isolates, with mortality rates as high as 100%. In autopsy, the most typical lesion is severe hemorrhagic splenomegaly observed in the abdominal opening of acute ASF animals, and multiple lymphatic nodes in the whole body are hemorrhagic. Subacute and chronic conditions are generally manifested as respiratory symptoms, intermittent fever, chronic skin ulcers and arthritis, which are all caused by low virulent strains with low mortality. The low-virulent strain has a long incubation period, causes chronic diseases, and causes continuous virus discharge of infected pigs, so that early diagnosis is difficult. Therefore, the development of safe and effective ASF vaccines is very urgent. to date, a range of vaccination strategies have been proposed, including inactivated vaccines, attenuated live vaccines, subunit vaccines, DNA vaccines, live vector vaccines, and the like. Inactivated vaccines are made by killing viruses and aim to inhibit cellular viral infection by inducing antibody production. However, such vaccines often lack sufficient efficacy, mainly because the neutralizing antibodies produced do not completely prevent infection by ASFV and the role of the antibodies in the protection mechanism is ambiguous. In practice, viremia in pigs may persist even when vaccinated with inactivated vaccines. Attenuated live vaccines are prepared by attenuating viral virulence through natural selection or genetic engineering techniques. Although such vaccines show the potential to induce protective immune responses, there is a risk of causing chronic infections such as skin and arthrosis, lymphadenopathy and pneumonia etc. In addition, attenuated strains may recover toxicity after passage in pigs, and the same gene deletion of different strains may produce different results, which increases the uncertainty of their use. Subunit vaccines typically employ recombinant techniques to prepare viral proteins or synthetic peptides that induce protective immune responses. The main challenge of such vaccines is the major reliance on the production of neutralizing antibodies, but these antibodies are often insufficient to provide complete protection, requiring the use of powerful adjuvants to enhance immune responses. In contrast, DNA vaccines, which work by injection of DNA fragments encoding ASFV antigens, induce a strong cytotoxic T cell response, providing greater cytoimmune protection relative to vaccines that rely on antibody responses. However, although specific T cell responses can be induced, the protective effect is still limited. Live vector vaccines are similar to nucleic acid-based vaccines, except that the gene encoding the target antigen is delivered into the host cell using a non-pathogenic attenuated virus or bacteria. Live vector vaccines are viral, bacterial or plasmid-based gene expression vectors as antigen delivery systems to elicit an immune response with the expressed viral antigen. However, the host may have an immune response to the vector itself, which may neutralize the vector, reduce its efficacy, prevent the vaccine from delivering the antigen effectively, and furthermore, challenges the antigen expression efficiency, survival rate, stability, etc.

Disclosure of Invention

Therefore, the invention aims to provide a lactobacillus expressing African swine fever virus fusion antigen and application thereof. The invention constructs a fusion gene which comprises a gene for encoding OprI protein and at least one gene selected from genes for encoding PP62 protein, MGF505-3R protein, EP364R protein, K145R protein and DP96R protein, and constructs lactobacillus with good growth capacity and antigen expression capacity, and can be used for african swine fever vaccine immunization animals, and can induce mucosal immunity, cellular immunity and humoral immunity reaction of animals by oral administration, thereby comprehensively enhancing the immunity of organisms and realizing effective prevention and control of african swine fever.

Specifically, the present invention provides the following technical features, and one or more of the following technical features are combined to form the technical scheme of the present invention.

In a first aspect of the present invention there is provided a lactic acid bacterium expressing an African swine fever virus fusion antigen, the lactic acid bacterium having integrated therein a fusion gene comprising a gene encoding an OprI protein and at least one gene selected from the group consisting of a PP62 protein, an MGF505-3R protein, an EP364R protein, a K145R protein and a DP96R protein, the starting strain of the lactic acid bacterium being Lactobacillus plantarum (Lactobacillus plantarum).

In one embodiment of the invention, the nucleotide sequence of the gene encoding the OprI protein is shown as SEQ ID NO. 1, the nucleotide sequence of the gene encoding the PP62 protein is shown as SEQ ID NO. 2, the nucleotide sequence of the gene encoding the MGF505-3R protein is shown as SEQ ID NO. 3, the nucleotide sequence of the gene encoding the EP364R protein is shown as SEQ ID NO. 4, the nucleotide sequence of the gene encoding the K145R protein is shown as SEQ ID NO. 5, and the nucleotide sequence of the gene encoding the DP96R protein is shown as SEQ ID NO. 6.

In one embodiment of the present invention, the fusion gene is obtained by fusing the gene encoding the OprI protein with the gene encoding the PP62 protein, the gene encoding the MGF505-3R protein, the gene encoding the EP364R protein, the gene encoding the K145R protein and the gene encoding the DP96R protein, respectively. In one embodiment of the present invention, the fusion gene further comprises a gene encoding His-tag. In one embodiment of the invention, the fusion gene further comprises a restriction enzyme recognition site sequence. The restriction enzymes, such as XbaI and/or HindIII, preferably contain both XbaI and HindIII. In one embodiment of the invention, the fusion gene further comprises a stop codon. In one embodiment of the present invention, the gene encoding the OprI protein in the fusion gene is linked to the gene encoding the PP62 protein, the gene encoding the MGF505-3R protein, the gene encoding the EP364R protein, the gene encoding the K145R protein or the gene encoding the DP96R protein via a linker. For example, in one embodiment, the nucleotide sequence of the linker is shown in SEQ ID NO. 7. For example, in some embodiments of the invention, the nucleotide sequence of the fusion gene is selected from the group consisting of the sequence shown in SEQ ID NO. 8, the sequence shown in SEQ ID NO. 9, the sequence shown in SEQ ID NO. 10, the sequence shown in SEQ ID NO. 11, and the sequence shown in SEQ ID NO. 12.

In one embodiment of the invention, the lactic acid bacteria is an alanine racemase gene-deficient lactobacillus plantarum (Lactobacillus plantarum) NC8 Δalr. In one embodiment of the present invention, the lactic acid bacterium uses pSIP-pgsA' vector as an expression vector.

In a second aspect of the invention, a method for constructing the lactic acid bacteria according to the first aspect is provided, which comprises the steps of connecting a fusion gene with an expression vector, transferring the fusion gene into competent cells to obtain a recombinant plasmid, and transferring the recombinant plasmid into an original strain. For example, in one embodiment, lactobacillus plantarum NC 8/delta alr is used as an exogenous antigen delivery vector, asd-alr gene is used for replacing erythromycin gene on an anchored expression plasmid pSIP-pgsA' as a marker gene, positive recombinant bacteria are initially screened by transferring into an intermediate host E.coli chi 6212, and then new functional lactobacillus plantarum strains capable of expressing exogenous proteins are screened by transferring into defective lactobacillus plantarum NC 8/delta alr.

In a third aspect of the present invention, there is provided a fusion gene comprising a gene encoding an OprI protein and at least one gene selected from the group consisting of a gene encoding a PP62 protein, an MGF505-3R protein, an EP364R protein, a K145R protein and a DP96R protein. In one embodiment of the invention, the nucleotide sequence of the gene encoding the OprI protein is shown as SEQ ID NO. 1, the nucleotide sequence of the gene encoding the PP62 protein is shown as SEQ ID NO. 2, the nucleotide sequence of the gene encoding the MGF505-3R protein is shown as SEQ ID NO. 3, the nucleotide sequence of the gene encoding the EP364R protein is shown as SEQ ID NO. 4, the nucleotide sequence of the gene encoding the K145R protein is shown as SEQ ID NO. 5, and the nucleotide sequence of the gene encoding the DP96R protein is shown as SEQ ID NO. 6.

In one embodiment of the present invention, the fusion gene is obtained by fusing the gene encoding the OprI protein with the gene encoding the PP62 protein, the gene encoding the MGF505-3R protein, the gene encoding the EP364R protein, the gene encoding the K145R protein and the gene encoding the DP96R protein, respectively. In one embodiment of the present invention, the fusion gene further comprises a gene encoding His-tag. In one embodiment of the invention, the fusion gene further comprises a restriction enzyme recognition site sequence. For example, in one embodiment, the restriction enzymes are XbaI and/or HindIII, preferably containing both XbaI and HindIII. In one embodiment of the invention, the fusion gene further comprises a stop codon.

In one embodiment of the present invention, the gene encoding the OprI protein in the fusion gene is linked to the gene encoding the PP62 protein, the gene encoding the MGF505-3R protein, the gene encoding the EP364R protein, the gene encoding the K145R protein or the gene encoding the DP96R protein via a linker. For example, in one embodiment, the nucleotide sequence of the linker is shown in SEQ ID NO. 7.

In one embodiment of the invention, the nucleotide sequence of the fusion gene is selected from the group consisting of the sequence shown in SEQ ID NO. 8, the sequence shown in SEQ ID NO. 9, the sequence shown in SEQ ID NO. 10, the sequence shown in SEQ ID NO. 11 and the sequence shown in SEQ ID NO. 12.

In a fourth aspect of the present invention, there is provided a fusion protein produced by expression of the lactic acid bacterium described in the first aspect or encoded by the fusion gene described in the third aspect.

The present invention also provides a method of obtaining the fusion protein described in the fourth aspect above. In one embodiment of the invention, the method of obtaining the fusion protein comprises constructing a fusion gene, inserting the fusion gene into an expression vector to form a recombinant expression vector, transforming the recombinant expression vector into a host cell selected from a lactic acid bacterium, E.coli or other microbial cell suitable for protein expression, and culturing the transformed host cell to promote expression of the fusion gene and to produce the fusion protein. In one embodiment of the invention, the method of obtaining the fusion protein further comprises collecting the fusion protein from the cultured host cells and purifying by affinity chromatography using His-tag to obtain a purified fusion protein.

In a fifth aspect of the invention, there is provided a composition comprising at least one lactic acid bacterium as described in the first aspect above or a fusion protein as described in the fourth aspect above. In some embodiments of the invention, the composition is a microbial agent, a pharmaceutical composition, or a feed.

For example, in one embodiment of the present invention, the composition comprises a lactic acid bacterium according to the first aspect, wherein a fusion gene is integrated in the lactic acid bacterium, wherein the fusion gene is obtained by fusing a gene encoding an OprI protein with a gene encoding a PP62 protein, a gene encoding an MGF505-3R protein, a gene encoding an EP364R protein, a gene encoding a K145R protein and a gene encoding a DP96R protein, respectively. For example, in one embodiment, the nucleotide sequence of the fusion gene is set forth in SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12. For example, in one embodiment, the composition may comprise a lactic acid bacterium having any of the above fusion genes integrated therein or a combination of lactic acid bacteria having each of the above fusion genes integrated therein, such as a combination of lactic acid bacteria having fusion genes as set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, integrated therein.

In a sixth aspect of the present invention, there is provided a vaccine comprising at least one lactic acid bacterium as described in the first aspect above, or a vector comprising a lactic acid bacterium and comprising the fusion gene as described in the third aspect above. In an embodiment of the invention, the vaccine is a vector vaccine, in particular a live vector vaccine. In some embodiments of the invention, the live vector vaccine is administered orally. The subject is an animal, in particular a pig.

For example, in one embodiment of the present invention, the live vector vaccine comprises a lactic acid bacterium according to the first aspect, wherein a fusion gene is integrated in the lactic acid bacterium, wherein the fusion gene is obtained by fusing a gene encoding an OprI protein with a gene encoding a PP62 protein, a gene encoding an MGF505-3R protein, a gene encoding an EP364R protein, a gene encoding a K145R protein and a gene encoding a DP96R protein, respectively. For example, in one embodiment, the nucleotide sequence of the fusion gene is set forth in SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12. For example, in one embodiment, the live vector vaccine may comprise a lactic acid bacterium having any one of the above fusion genes integrated therein or a combination of lactic acid bacteria each having one of the above fusion genes integrated therein. In one embodiment of the present invention, the lactic acid bacteria are preferably lactobacillus plantarum, particularly preferably lactobacillus plantarum NC8 Δalr deficient in the alanine racemase gene.

In a seventh aspect of the invention there is provided the use of a lactic acid bacterium as described in the first aspect above or a fusion protein as described in the fourth aspect above or a composition as described in the fifth aspect above or a vaccine as described in the sixth aspect above for the manufacture of a product for controlling african swine fever. In some embodiments of the invention, the product is a microbial agent, a pharmaceutical formulation, or a feed, e.g., the pharmaceutical formulation includes a biologic, such as a vaccine.

In an eighth aspect of the invention there is provided a method of controlling african swine fever comprising administering to an african swine fever susceptible animal (particularly a pig) an effective amount of an engineered bacterium as described in the first aspect above or a composition as described in the fifth aspect above or a vaccine as described in the sixth aspect above. The effective dose is sufficient to induce an immune response in the subject, thereby preventing and/or treating african swine fever. The method may be carried out by injection, orally or by other suitable route of administration. The particular dosage and frequency of administration will be determined by a veterinarian or professional depending on the health, age, weight and severity of the condition of the subject.

Compared with the prior art, the invention has the advantages that the invention respectively connects the gene sequences of the encoding PP62 protein, the MGF505-3R protein, the EP364R protein, the K145R protein and the DP96R protein with the gene sequences of the encoding OprI protein to construct lactobacillus (especially lactobacillus plantarum) integrated with the gene sequences, and the lactobacillus plantarum can express and generate specific ASFV fusion antigens which can be anchored on the surface of the strain. These lactic acid bacteria can be used as African swine fever vaccine in oral form for immunization of animals, inducing dendritic cell (DENDRITIC CELL, DC) activation in Peyer's Patch (PP) and initiating protective immune response. Enhancing cellular and humoral immune responses in spleen and Mesenteric Lymph Nodes (MLN), promoting B cell activation in PP and production of IgA antibodies, enhancing mucosal immune responses. And simultaneously, the level of antigen-specific IgG antibodies in serum is increased, and the humoral immune response is enhanced. Among them, the generation and promotion of mucosal immune responses is particularly important for the prevention of pathogens transmitted through the respiratory and digestive tracts, whereas the bi-directional responses of cellular and humoral immunity and the generation of specific antibodies can further enhance the overall defenses against pathogens. In addition, the combined use of the lactic acid bacteria provided by the invention can generate a combined immune effect, so that the immune protection effect is further improved. And the lactobacillus provided by the invention has safety and environmental friendliness, and the non-antibiotic resistance marker is adopted for selection, such as asd-alr fusion gene, so that the risk of antibiotic resistance gene transmission in the environment is reduced, and meanwhile, the biological safety is enhanced.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 strain growth curves.

FIG. 2 is a left graph showing the result of Western blot detection of the target proteins of five novel functional Lactobacillus plantarum strains, wherein ,M：Protein marker 190KD;1：NC8Δ-pSIP409-pgsA';2：NC8Δ-pSIP409-pgsA'-PP62;3：NC8Δ-pSIP409-pgsA'-MGF505-3R;4：NC8Δ-pSIP409-pgsA'-K145R;5：NC8Δ-pSIP409-pgsA'-EP364R;6：NC8Δ-pSIP409-pgsA'-DP96R. is a right graph showing the result of Western blot detection of the target proteins in recombinant E.coli, wherein ,M：Protein marker 170KD;1：BL21-pET28a-K145R;2：BL21-pET28a-EP364R;3：BL21-pET28a-96R;4：BL21-pET28a-PP62;5：BL21-pET28a-MGF505-3R;6：BL21-pET28a.

FIG. 3 shows the immunofluorescence of the novel functional Lactobacillus plantarum.

FIG. 4 shows the results of dendritic cell CD80 and CD86 expression in PP of each group of mice.

FIG. 5 shows the results of IFN-. Gamma.expression in CD4 ⁺ T lymphocytes in the spleen.

FIG. 6 shows the results of IFN-. Gamma.expression in CD8 ⁺ T lymphocytes in the spleen.

FIG. 7 shows the results of IFN-. Gamma.expression in CD4 ⁺ T lymphocytes in MLN.

FIG. 8 shows the results of IFN-. Gamma.expression in CD8 ⁺ T lymphocytes in MLN.

FIG. 9 shows the results of IL-4 expression in CD4 ⁺ T lymphocytes in the spleen.

FIG. 10 shows the results of IL-4 expression in CD4 ⁺ T lymphocytes in MLN.

FIG. 11 shows the results of the change in the number of B220 ⁺IgA⁺ cells in PP of each group of mice.

FIG. 12B 220 ⁺IgA⁺ cell number changes in the duodenum.

FIG. 13B 220 ⁺IgA⁺ cell number changes in ileum.

FIG. 14 shows the results of detection of specific antibody SIgA in the feces of mice in each group.

FIG. 15 shows the results of detection of specific IgG in serum of mice in each group.

FIG. 16 shows the results of detection of IL-2 in serum of mice in each group.

FIG. 17 shows the results of IFN-. Gamma.detection in serum of each group of mice.

FIG. 18 shows the results of detection of IL-4 in serum of mice in each group.

FIG. 19 qPCR was performed to determine the relative expression level of IL-2 mRNA in spleen.

FIG. 20 qPCR shows the relative expression level of IFN-. Gamma.mRNA in spleen.

FIG. 21 qPCR was performed to determine the relative expression level of IL-4 mRNA in spleen.

Detailed Description

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or materials used in the present invention may be purchased in conventional manners, and unless otherwise indicated, they may be used in conventional manners in the art or according to the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

EXAMPLE 1 construction of novel functional Lactobacillus plantarum expressing African swine fever Virus fusion antigen

In the embodiment, the African swine fever virus PP62 protein, the MGF505-3R protein, the EP364R protein, the K145R protein and the DP96R protein are respectively fused and expressed with a lipoprotein adjuvant OprI protein, and exogenous proteins are expressed by anchoring the surface of the non-anti-lactobacillus plantarum, so that the novel functional lactobacillus plantarum is constructed.

The following plasmids, vectors and strains used in this implementation were derived from the Jilin agricultural university Jilin province animal microecological formulation engineering center.

E.coli DH 5. Alpha. PUC plasmid vector, plasmid cloning host. Coli χ6212. Asd Gene deficiency E.coli χ6212, plasmid cloning host. NC 8/Deltaalr. Alr gene defective Lactobacillus plantarum NC8, protein expression host. NC 8. Delta. -pSIP409,409-pgsA': an empty vector Lactobacillus plantarum strain carrying the pSIP409,409 plasmid. BL21 (DE 3) E.coli strains, prokaryotic expression hosts. pLP-1261-asd-alr, non-antibiotic resistance plasmid.

The enzymes and major reagents were PRIME STAR Max Premix (2×), restriction enzymes (Xba I and HindIII), DL2000 DNA MARKER, DL10000 DNA MARKER, 10xLoading Buffer (Takara Shuzo Co., ltd.). DNA plasmid small extract kit, anti-His tag mouse monoclonal antibody, HRP-marked goat anti-mouse IgG, FITC-marked goat anti-mouse IgG (Beijing kang is century biotechnology Co., ltd.). Clon Express IIOne Step Cloning Kit kit (Vazyme). 5X SDS PAGE Loading Buffer, lysozyme, 50 XTAE electrophoresis buffer, PBS phosphate buffer, anti-fluorescence decay caplet (Beijing Soy Corp technologies Co.). BeyoBlue ^TM Coomassie brilliant blue ultrafast staining (Biyun Tian Biotechnology Co.). SppIP inducing peptide and IPTG inducing peptide (institute of animal microecologics engineering, jilin province, jilin agricultural university). Beef powder, yeast extract, tryptone, agar powder (OXOID company, uk). ECL chemiluminescent solution (34077), protein Marker (Beijing full gold biosystems (TransGen Biotech)). Other main reagents are imported products or domestic analytically pure products.

The instrument and manufacturer is an inverted fluorescence microscope DMi8 and a full-automatic paraffin microtome (RM 2245) (Leaka, germany). Gradient PCR apparatus, micropipette (Eppendorf, germany). Low temperature ultracentrifuge, full automatic autoclave GR85DA (Sigma Germany). Gel imaging analysis system (Universal HoodII), vertical electrophoresis apparatus (04cbr 02682), electroporation apparatus (Gene P μ Lser XcellTM System) (BIO-RAD company, usa). Biochemical incubator (HERATHERM IGS) CO ₂ incubator HERACELL i, real-time fluorescent quantitative PCR instrument (7500 Real Time PCR system) (company Thermo Scientific, usa). AI600 chemiluminescent imaging system (GE company, usa). Microplate spectrophotometer (Epoch 2) (BioTek company, usa). Flow cytometry (BD LSRFortessa ^TM) (BD Biosciences, usa). Analytical balance ME204E (Metrehler, switzerland). Ultra clean bench (Beijing Tong Harr instruments Co., ltd.). Ordinary refrigerator and-80 ℃ ultralow temperature refrigerator (sea company). pH acidometer (Shanghai instruments and electrosurgery instruments Co., ltd.). Magnetic heating stirrer (Shanghai Mei Xiang instruments Co., ltd.). Electric heating constant temperature water tank (Shanghai Jing Hongjingjingjingjingjingjingjingji Co., ltd.). SCIENTZ-IID ultrasonic cell grinder (Ningbo Xinzhi Biotech Co., ltd.). Metal bath (Hangzhou Bori technologies Co., ltd.).

LB liquid medium, sodium chloride 20.0g, tryptone 20.0g, yeast extract 10.0g, deionized water 2L. Adding into beaker, completely dissolving, packaging the liquid into test tube or conical flask (1.5 g/mL bacteriology agar powder if solid culture medium is to be prepared), and sterilizing with full-automatic autoclave at 121deg.C for 20min. MRS liquid culture medium comprises 20g of beef protein powder, 20g of tryptone, 10g of yeast extract, 20g of D-glucose anhydrous, 10g of sodium acetate, 4g of diammonium citrate, 4g of potassium hydrogen phosphate, 2mL of Tween-80, 0.4g of magnesium sulfate heptahydrate, 0.1g of manganese sulfate monohydrate and 2L of deionized water. Adding into beaker, completely dissolving, packaging into test tube or conical flask (1.5 g/mL bacteriology agar powder if solid culture medium is to be prepared), and sterilizing at 115 deg.C for 20min.

1% Agarose Gel 2g agarose and 0.2L 1 xTAE buffer (prepared by diluting 50 xTAE solution with deionized water) were added to a Erlenmeyer flask, and heated in a microwave oven to dissolve, and cooled at room temperature for 5 minutes, and 10. Mu.L of Super Gel Blue dye was added and mixed well.

SppIP powder (20 mg/mL) SppIP powder (0.2 g) is added into deionized water (10.0 mL), mixed and dissolved, filtered by a microporous filter (0.22 mu m) to achieve the aim of sterilization, and split-packed into PCR tubes (10 mu L per tube) and stored at-20 ℃.

5 XSDS-PAGE running buffer, tris 15.1g, glycine 94.0g, SDS 5.0g, deionized water 1L. Stored at room temperature and diluted into 1 XSDS-PAGE buffer before use.

And (3) transferring the film, namely adding 1.45g Glycine,2.9g Tris,0.185g SDS into a beaker filled with 0.3L of deionized water, stirring to dissolve the film, fixing the volume to 0.4L, adding 0.2L of methanol solution, uniformly mixing, and preserving at room temperature.

10 XTBS solution, 236g of sodium chloride, 72.6g of Tris, was added to a beaker containing 2L of ddH ₂ O, the pH was measured after stirring until dissolved, and the pH was adjusted to 7.6 by dropping concentrated hydrochloric acid, and the volume was fixed to 3L with ddH ₂ O, and the mixture was placed in a low-temperature storage tank.

TBST solution 1mL Tween-20 was added to 100mL 10 XTBS solution, and 10 XTST was diluted to 1 XTST with deionized water and stored at 4 ℃.

Sealing buffer solution 5g of skimmed milk powder was added to 100mL of TBST solution, mixed well and stored in a 4 ℃ freezer.

80% Glycerol 80mL, deionized water 20mL, and the procedure for setting the autoclave at 121℃for 20min.

TES solution, lysozyme 10.6mg, sucrose 2.7g, RNase A32. Mu.L, 10 XTE Buffer (pH 8.0) 8mL, ddH ₂ O7.2 mL. Stored in a-20 ℃ refrigerator.

Lysozyme solution (20 mg/mL) 1g lysozyme was dissolved in 50mL deionized water and stored at 4℃and ready to use.

PBS phosphate buffer solution 1 bag PBS phosphate buffer solution powder, 1.6L deionized water, stirring to dissolve completely, and setting the volume to 2L, and setting the procedure of autoclave at 121deg.C for 20min. Preserving at room temperature.

1. Construction and identification of novel functional lactobacillus plantarum

1.1 Synthesis of the Gene of interest

The gene sequences of the African swine fever virus protein PP62 protein, the MGF505-3R protein, the EP364R protein, the K145R protein and the DP96R protein and the gene sequences of the adjuvant OprI protein are respectively shown in SEQ ID NO. 1-6, five African swine fever virus proteins and the adjuvant OprI protein are respectively connected by GGGGS-linker, the constructed recombinant fusion gene is optimized according to the codon preference of lactic acid bacteria, and Xba I and Hind III restriction enzyme sites are respectively added at the 5 'end and the 3' end of the sequence, and the recombinant fusion gene is synthesized by Nanjin Biotech Co-Ltd and connected to pUC-GW-Kan vector. The sequences of the recombinant fusion genes are shown in SEQ ID NOs 8 to 12 respectively.

1.1.1 Designing primers the correctness of the recombinant plasmid is determined by designing primers of five target fragments, and the five sets of primers are mainly used for PCR identification and company sequencing (the primer synthesis and sequencing of the test are all handed over to Shanghai Bioengineering Co., ltd.). The primer names and sequences are as follows:

PP62-F:5’-TCTAGAATGCCAAGTAACATGAAGC-3’;PP62-R:5’-AAGCTTTTAATGATGATGATGATGATGTTTACGTGA-3’。

MGF-F:5’-TCTAGAATGAGTTCAAGTTTACAAG-3’;MGF-R:5’-AAGCTTTTAATGATGATGATGATGATGTTTCCGACT-3’。

EP-F:5’-TCTAGAATGTACTTTTTAGTTGCTG-3’;EP-R:5’-AAGCTTTTAATGATGATGATGATGATGCTTCCGTG-3’。

K145-F:5’-TCTAGAATGGATCATTACTT-3’;K145-R:5’-AAGCTTTTAATGATGATGATGATGATGTTTACG-3’。

DP-F:5’-TCTAGAATGAGTACTCATGATTGTTC-3’;DP-R:5’-AAGCTTTTAATGATGATGATGATGATGTTTACGTGATGC-3’。

1.1.2 acquisition of pSIP409-pgsA '(E) vector frame and target fragment seamless cloning primers for five sets of pUC-PP62, pUC-MGF505-3R, pUC-EP364R, pUC-K145R, pUC-DP96R target fragments and pSIP409-pgsA' (E) vector Xba I and Hind III both ends were designed using Primer 5.2 software for construction of recombinant plasmid pSIP409-pgsA'-PP62(E)、pSIP409-pgsA'-MGF505-3R(E)、pSIP409-pgsA'-EP364R(E)、pSIP409-pgsA'-K145R(E)、pSIP409-pgsA'-DP96R(E),. The names and sequences of the seamless cloning primers are shown in Table 1-1.

TABLE 1-1 primer sequences

Note that the homology arm sequences are underlined.

A seamless cloning vector frame PCR amplification System PRIME STAR Max Premix (2X) 20.0. Mu. L, pSIP409-pgsA' - (E) vector 1.0. Mu.L, 9E-F1.0. Mu.L, 9E-R1.0. Mu. L, ddH ₂ O17.0. Mu.L, and a total of 40.0. Mu.L. The amplification conditions of the pSIP-pgsA' (E) vector frame were set on a gradient PCR apparatus at 98℃for 10s (98℃for 10s,56℃for 5s,72℃for 40 s). Times.30 cycles,72℃for 5min.

A PCR amplification system for seamless cloning of the desired fragment was PRIME STAR Max Premix (2X) 20.0. Mu. L, pUC-the desired fragment (5 plasmids )1.0μL、PP6-F/PM5-F/PE3-F/PK1-F/PD9-F 1.0μL、PP6-R/PM5-R/PE3-R/PK1-R/PD9-R 1.0μL、ddH₂O 17.0μL、 together 40.0. Mu.L).

The amplification conditions of the template pUC-PP62 were set on a gradient PCR apparatus at 98℃for 10s (98℃for 10s,60℃for 30s,72℃for 60 s). Times.30 cycles,72℃for 7min.

The PCR amplification conditions of the template pUC-MGF505-3R were the same as those of the template pUC-PP62, but the annealing temperature was changed to 58 ℃. The PCR amplification conditions of the template pUC-EP364R were the same as those of the template pUC-PP62, and the annealing temperature was changed to 61 ℃. The PCR amplification conditions of the template pUC-K145R were the same as those of the template pUC-PP62, and the annealing temperature was changed to 59 ℃. The PCR amplification conditions of the template pUC-DP6R were the same as those of the template pUC-PP62, and the annealing temperature was the same.

Mu.L of the PCR product was mixed with 1. Mu.L of 10X Loading buffer, and the sizes of the bands were identified by electrophoresis (both the PCR result and the double digestion identification result were identified by 1% agarose gel electrophoresis).

1.1.3 Seamless cloning of the pSIP-pgsA '(E) vector frame and the target fragment the concentration of the amplification product was measured by means of an enzyme-labeled instrument, and the vector frame and the target fragment were connected in an optimal system according to the instructions of the Vazyme company seamless cloning kit, the seamless cloning system being pSIP-pgsA' - (E) 3.0. Mu.L, 4.0. Mu.L of the target fragment, 5 XCE II Buffer 2.0. Mu.L, express II 1.0. Mu.L, and a total of 10.0. Mu.L. The mixture was placed in a PCR apparatus, the connection temperature was set at 37℃and the connection time was 30min.

1.1.4 Conversion of the ligation product to E.coli DH 5. Alpha.: 1) taking DH 5. Alpha. Competent cells from a-80℃incubator, placing them on ice for 10min to bring them into an ice-water mixture, 2) mixing 5. Mu.l ligation product with 100. Mu.l DH 5. Alpha. Competent cells in an ultra-clean bench sterilized by ultraviolet irradiation, placing them on ice for 30min, 3) heat-shock 1min in a 42℃constant temperature water tank for 30s, immediately ice-bathing for 5min, 4) addition of 600. Mu.L of LB liquid medium, then placing them on a shaking table at 37℃for 1.5h, 5) centrifugation at 4000rpm/min for 5min, discarding the supernatant, leaving 200. Mu.L of bacterial liquid to mix bacterial pellet, plating them uniformly on LB solid medium to which 10. Mu.g/mL erythromycin had been added, 6) inverted culturing in a 37℃incubator for 18h, and 7) taking single bacterial pellet to take out and place in LB liquid medium to which 10. Mu.g/mL erythromycin had been added for 16h. 8) The strain is preserved in 600. Mu.L strain liquid and 400. Mu.L 80% glycerin in strain preserving tube and preserved in-80 deg.C refrigerator.

1.1.5 Identification of recombinant plasmids in E.coli DH 5. Alpha. Plasmids are extracted from bacterial solutions by using a DNA plasmid miniprep kit, and the details are shown in the specification. The plasmids were then identified by the double cleavage method using the restriction enzymes Xba I and Hind III, the cleavage system being pSIP-pgsA' -fragment of interest (E) (five recombinant plasmids) 5.0. Mu.L, xbaI0.5. Mu.L, hindIII0.5. Mu.L, 10 XM buffer 1.0. Mu. L, ddH ₂ O3.0. Mu.L, a total of 10.0. Mu.L. After digestion for 2h at 37 ℃, the band size was identified by electrophoresis. The correct plasmid was identified by double restriction method and was sent to the Bio Inc. for sequencing, and plasmid map was made using Snap Gene 4.3.6 software.

1.1.6 Acquisition of the non-anti-vector frame and asd-alr fragment A seamless cloning primer for the non-anti-vector frame with the target fragment in pSIP-pgsA' -target fragment (E) (five recombinant plasmids) and a seamless cloning primer for the asd-alr fragment in the pLP-1261-asd-alr vector were designed for construction of the non-anti-recombinant plasmid pSIP409-pgsA'-PP62(A)、pSIP409-pgsA'-MGF505-3R(A)、pSIP409-pgsA'-EP364R(A)、pSIP409-pgsA'-K145R(A)、pSIP409-pgsA'-DP96R(A),. The names and sequences of the seamless cloning primers are shown in tables 1-2.

TABLE 1-2 primer sequences

Note that the homology arm sequences are underlined.

A seamless cloning of the non-resistant vector frame PCR system was carried out with PRIME STAR Max Premix (2X) 20.0. Mu. L, pSIP409-pgsA ' -fragment of interest (E) (five recombinant plasmids) 1.0. Mu.L, 409pgsA ' -F1.0. Mu.L, 409pgsA ' -R1.0. Mu. L, ddH ₂ O17.0. Mu.L and a total of 40.0. Mu.L. The PCR amplification conditions of the template were the same as those of the template pUC-PP62 of 1.1.2.

The PCR amplification system ：Prime STAR Max Premix(2×)20.0μL、pLP-1261-asd-alr 1.0μL、409ata-F 1.0μL、409ata-R 1.0μL、ddH₂O 17.0μL、 for the asd-alr fragment of the seamless clone was 40.0. Mu.L in total. The PCR amplification conditions for the asd-alr fragment were identical to those for the pSIP-pgsA' (E) vector frame of 1.1.2, and the annealing temperature was changed to 55 ℃. mu.L of the PCR product was mixed with 1. Mu.L of 10X Loading buffer and identified by electrophoresis.

1.1.7 Seamless cloning of the non-antibody vector frame and asd-alr fragment, measuring the concentration of the amplified product with an enzyme-labeled instrument, and connecting with Vazyme company seamless cloning kit, wherein the seamless cloning system comprises pSIP409-pgsA' -target fragment 3.0 mu L/2.0 mu L, asd-alr fragment 4.0 mu L/5.0 mu L, 5 XCE II Buffer 2.0 mu L, express II 1.0 mu L and total 10.0 mu L.

The connection system of the non-anti-vector frame carrying the target fragments PP62 and MGF505-3R and the asd-alr fragment in a seamless cloning system is 3 mu L and 4 mu L, and the connection system of the non-anti-vector frame carrying the target fragments EP364R, K145R, DP R and the asd-alr fragment is 2 mu L and 5 mu L. The mixture was placed in a PCR apparatus, the connection temperature was set at 37℃and the connection time was 30min.

1.1.8 Conversion of ligation products to E.coli χ6212 competence. Conversion of non-anti-ligation products to E.coli χ6212 competence by electrotransformation.

A) Taking out the electric shock cup in 70% alcohol in an ultra clean bench, placing on PE glove, and sterilizing and air drying by ultraviolet irradiation. Placing in a refrigerator at-20deg.C for 10min. b) Taking out from the-80 ℃ ultralow temperature preservation box, placing on ice for 10min to make the ice and water mixture state, C) lightly adding 5 μl of the connection product into 100 μl E.coli χ6212 competence in an ultra clean bench, lightly mixing, transferring to a sterilized and precooled electric shock cup, and standing on ice for 20min. d) The cuvette was blotted with kitchen paper towel and then placed in a electroporation cuvette and electrotransformed according to procedure (4.fwdarw.1.fwdarw.2 (2500V, 200Ω, 25. Mu.F)). e) After the electrotransformation is completed, the mixture is placed on ice for 6min, 700 mu L of LB liquid medium without antibiotics is added, the mixture is uniformly mixed, the mixture is transferred into a 2ml EP tube, and a sealing film is sealed. f) Placing in a shaking table at 37 ℃ for 1h at 220rpm/min, g) centrifuging at 5000rpm/min for 3min, discarding the supernatant, reserving 200 mu L of bacterial liquid for uniformly mixing bacterial precipitation, taking 150 mu L of bacterial liquid for uniformly coating on an antibiotic-free LB solid culture medium, h) placing in a 37 ℃ incubator for inverted culture for 16h, i) picking up single bacterial colonies to be cultured in 5mL of liquid culture medium for 16h, j) firstly performing strain preservation on the bacterial liquid, and then extracting plasmids.

1.1.9 Identification of non-anti-recombinant plasmid in E.coli χ6212 plasmid was extracted from bacterial solution with DNA plasmid miniprep kit, see the specification for details. And then identified by PCR and double digestion techniques.

1.1.10PCR it was identified that the PCR amplification conditions of the template were identical to those of the template pUC-PP62 of 1.1.2, and the annealing temperature was changed to 58 ℃. The PCR products were verified by electrophoresis. The PCR identification reaction was PRIMESTAR MAX PREMIX (2X) 25. Mu.L, no anti-recombinant plasmid 5. Mu. L, PP 62-F/MGF-F/EP-F/K145-F/DP-F2.5. Mu. L, PP 62-R/MGF-R/EP-R/K145-R/DP-R2.5. Mu. L, ddH ₂ O15. Mu.L, and a total of 50. Mu.L. 1.1.11 double enzyme digestion identification, namely, carrying out electrophoresis identification on the size of the band after enzyme digestion for 2 hours at 37 ℃. The correct plasmid was identified by PCR and double digestion, sequenced by the Bio Inc., and a plasmid map was made using Snap Gene 4.3.6 software. The cleavage reaction system was pSIP to pgsA' -desired fragment (A) recombinant plasmid 4.0. Mu.L, xbaI0.5. Mu.L, hindIII0.5. Mu.L, 10 XM buffer 1.0. Mu. L, ddH ₂ O4.0. Mu.L, and a total of 10.0. Mu.L. 1.1.12 electric transformation of non-anti-recombinant plasmid into NC 8/Deltaalr. Electric transformation of correctly sequenced non-anti-recombinant plasmid into NC 8/Deltaalr competence.

A) Taking out the electric shock cup in 70% alcohol in an ultra clean bench, placing on PE glove, and sterilizing and air drying by ultraviolet irradiation. Placing in a refrigerator at-20deg.C for 10min. b) Taking NC 8/Deltaalr competence out of the ultralow temperature preservation box, placing on ice for 10min to make the NC 8/Deltaalr competence be in an ice-water mixture state, c) lightly adding the recombinant plasmid which is verified to be correct into 50 μl NC 8/Deltaalr competence in an ultra clean bench, lightly mixing, then transferring to a electric shock cup, and standing on ice for 5min. d) The periphery of the cuvette was wiped dry with kitchen paper towel and then placed in a electroporation cuvette, and electrotransformation was performed according to the procedure (4.fwdarw.1.fwdarw.9 (2000V, 400. OMEGA., 25. Mu.F)). e) After the electrotransformation is finished, the mixture is placed on ice for 5min, 600 mu L of non-antibiotic MRS liquid culture solution is added for uniform mixing, then the mixture is transferred into a 2.0mL centrifuge tube, then 150 mu L of 5% sucrose solution is added, the mixture is sealed by a sealing film, and the mixture is placed in a water bath kettle at 30 ℃ for 2h. f) Shaking culture is carried out for 1h at 37 ℃ by shaking table at 220 rpm/min. g) Centrifuging at 5000rpm/min for 3 min, discarding supernatant, collecting 200 μl of bacterial liquid, mixing bacterial precipitate, and uniformly plating 150 μl of bacterial liquid on non-antibiotic MRS solid culture medium. h) Placed in an anaerobic workstation at 37℃for 22h of incubation upside down. i) Single colonies were picked up into MRS liquid medium and placed in an anaerobic incubator at 37℃for 18h.

1.1.13 Verification of non-anti-recombinant plasmid in NC 8/Deltaalr the plasmid in NC 8/Deltaalr was treated with lysozyme before extraction, i.e.lactic acid bacteria liquid was poured into 2.0mL EP tube in a clean bench, centrifuged at 12000rpm/min for 1min, the supernatant was discarded and the cells were collected. 200. Mu.L of 50mg/mL lysozyme was added, and the metal bath was incubated at 37℃for 2h. Centrifuging at 12000rpm/min for 1min, discarding supernatant, and collecting thallus. The subsequent procedures were performed according to the plasmid miniprep kit instructions. And carrying out PCR identification on the extracted non-antibody plasmid. The PCR identification reaction system and the amplification conditions are the same as 1.10.

1.2 Optimization of culture conditions for novel functional Lactobacillus plantarum

1.2.1 Measurement of Lactobacillus plantarum growth curves novel functional Lactobacillus plantarum frozen in an ultra-low temperature incubator was placed on ice, 100. Mu.L of bacterial liquid was added to 5mL of MRS liquid medium in an ultra-clean bench, and placed in an anaerobic incubator at 37℃for overnight culture. The following day 100. Mu.L of overnight culture broth was transferred to 40mL MRS broth and incubated in an anaerobic incubator at 37 ℃. After 1h, 2mL of bacterial liquid is sucked in an ultra clean bench, the OD ₆₀₀,OD₆₀₀ value is smaller than 0.2 by an ultraviolet spectrophotometer, the OD ₆₀₀ is measured again after half an hour, the bacterial liquid OD ₆₀₀ values are all about 0.3, 100 mu L of SppIP induction peptide is added, and the bacterial liquid is cultured in a 37 ℃ anaerobic workstation. 2mL of bacterial liquid was taken out at 1h intervals to determine OD ₆₀₀ values, followed by 24h monitoring and growth curves were drawn with GRAPHPAD PRISM software.

1.2.2 Plate colony counting, namely, carrying out gradient dilution on the bacterial liquid in the logarithmic phase after induction in 1.2.1, respectively diluting to 10 ⁶,10⁷,10⁸, taking 100 mu L of bacterial liquid, and uniformly dripping the bacterial liquid onto an MRS solid culture medium for plate counting.

1.2.3 Verifying the expression of the target protein of the novel functional Lactobacillus plantarum by detecting whether the novel functional Lactobacillus plantarum can express the expected protein or not through a Western blot experiment.

1.2.4 Processing a lactobacillus plantarum protein sample, namely a) resuscitating and activating a new functional lactobacillus plantarum strain in an ultralow temperature preservation box, namely taking 100 mu L of bacterial liquid into an MRS liquid culture medium, culturing overnight in a 37 ℃ anaerobic culture box, b) taking 400 mu L of bacterial liquid for the next day, transferring the bacterial liquid into 40mL of MRS liquid culture medium, carrying out anaerobic culture for 1.5h (OD ₆₀₀ =0.2-0.3) at 37 ℃, adding 100 mu L SppIp induction peptide, carrying out induction culture for 7h (bacterial liquid is in logarithmic growth phase), C) taking 4mL of bacterial liquid, centrifuging for 5min at 5000rpm, discarding supernatant, d) adding 1mL of TES solution to suspend bacterial precipitation, centrifuging for 30min at 37 ℃, e) 2500g for 10min, discarding TES solution, adding 1mL of suspension precipitation, f) 2500g for 5min, discarding PBS (5 min), re-suspending and thawing for 5 times (6 min/time) in a-80 ℃ refrigerator, carrying out ultrasonic disruption for 20min under 60W conditions, boiling for 4 mu C for 20h, centrifuging for 1 mu L of PBS (20 ℃ for 1 mu L), and storing the supernatant, centrifuging for 1 mu L of the supernatant, and storing the supernatant in a refrigerator, and carrying out centrifugation for 20 ℃ for 1 mu L.

1.2.5Western blot verifying the target protein expression, namely 1) preparing 12% of lower layer separating gel and 5% of upper layer concentrating gel into a glass plate fixed on a gel preparation frame, immediately inserting a comb (without bubbles) after adding the upper layer concentrating gel, standing for solidification at room temperature, and standing for storage at 4 ℃ for standby (only 3-5 days). 2) The sample was placed in an electrophoresis tank containing an electrophoresis solution, the comb was pulled out, and 8. Mu.L of protein Marker and 12. Mu.L of protein sample were added to the gel well. 3) The upper layer of gel was run at 80V for 50min, the lower layer of gel was run at 120V until the protein Marker was completely dispersed and bromophenol blue was about to approach the bottom of the electrophoresis tank. 4) Placing into a large tray filled with transfer liquid to obtain sandwich sample with black surface under, sequentially adding filter paper, glue, NC film, filter paper, and foam pad, and rolling out air bubbles between the glue and NC film with small roller, wherein hand cannot touch NC film. 5) According to the black surface as the negative electrode and the white surface as the positive electrode, putting the groove into the groove filled with the film transferring liquid, putting the groove into ice, and transferring the film for 1.5 hours at a constant current of 300 mA. 6) NC films were placed in 5% nonfat milk powder and blocked for 2 hours at room temperature on a shaker. 7) Discard 5% nonfat milk powder, add primary antibody (anti-His tag mouse mab, 1000-fold dilution) and incubate overnight at 4 ℃. 8) The next day the primary antibody was recovered and washed three times with 5min each time by adding TBST. 9) Secondary antibodies (HRP-labeled goat anti-mouse IgG, 2000-fold dilution) were added and incubated for 1.5h at room temperature. 10 Recovering secondary antibody, adding TBST, and washing for 5min each time. 11 Dropping color developing liquid onto NC film, and exposing in AI 600 imaging system.

1.2.6 Immunofluorescence verification of fusion protein expression 1) resuscitating and activating a novel functional lactobacillus plantarum strain in an ultralow temperature preservation box, namely, transferring 100 mu L of bacterial liquid into an MRS liquid culture medium, placing the culture medium in a 37 ℃ anaerobic workstation for overnight, 2) transferring 400 mu L of bacterial liquid into 40mL of the MRS liquid culture medium the next day, performing anaerobic culture at 37 ℃ for 1.5 hours (OD ₆₀₀ = 0.2-0.3), adding 100 mu L SppIp induction peptide, performing induction culture for 7 hours (bacterial liquid is in a logarithmic growth phase), 3) taking 500 mu L of bacterial liquid, centrifuging for 1min at 12000rpm/min, discarding supernatant, re-suspending bacterial sediment by 1mL of PBS, centrifuging for 1min again, and discarding the supernatant. 4) 1mL of PBS (1% BSA) was added and the mixture was placed in a 4℃freezer and blocked for 1 hour, followed by 3 washes with PBS. 5) Primary antibody was added and incubated overnight at 4 ℃. 6) The cells were collected after 3 times washing with 1mL of PBS (0.2% Tween-20). 7) Incubation with FITC-labeled secondary antibody (goat anti-mouse IgG) for 2h at room temperature, starting from this procedure, protected from light. 8) The supernatant was centrifuged, washed again with 1mL of PBS (0.2% Tween-20), and the cells were collected after 3 times. 9) The cells were suspended in 100. Mu.L of PBS, and 10. Mu.L of the bacterial liquid was then dropped onto a slide glass, followed by baking with an alcohol burner. 10 Adding an anti-fluorescence quencher, and observing, photographing and preserving under an inverted fluorescence microscope.

1.2.7 Construction and expression verification of the adjuvant bacteria NC8 delta-pSIP 409-pgsA' -OprI

1.2.7.1 Acquisition of the non-resistant vector frame and OprI Gene of pSIP409-pgsA '(A) to construct five novel functional Lactobacillus plantarum genes each carrying the gene of the adjuvant OprI protein, for constructing the non-resistant recombinant plasmid pSIP-pgsA' -OprI (A), a seamless cloning primer was designed for the non-resistant vector frame and OprI gene fragment of pSIP-pgsA '(A) using the non-resistant recombinant plasmid pSIP-pgsA' -PP62 (A) in E.coli χ6212 as template DNA, and was also synthesized by the biological company. The names and sequences of the primers are shown in tables 1-3.

TABLE 1-3 primer sequences

Note that the homology arm sequences are underlined.

The PCR amplification system ：Prime STAR Max Premix(2×)20.0μL、pSIP409-pgsA'-PP62(A)1.0μL、AA-F/AP-F 1.0μL、AA-R/AP-R 1.0μL、ddH₂O 17.0μL、 for the seamless cloning of the nonreactive vector frame and the OprI gene fragment amounted to 40.0. Mu.L. The PCR amplification conditions of pSIP-pgsA '(A) without the resistant vector frame were the same as those of pSIP-pgsA' (E) in 1.1.2, and the annealing temperature was changed to 60 ℃. The PCR amplification conditions for the OprI gene fragment were identical to those for the template pUC-PP62 of 1.1.2. The PCR products were identified by electrophoresis.

1.2.7.2 Seamless cloning of pSIP-pgsA '(A) vector-free frame and OprI Gene fragment the concentration of the amplification product was determined with a microplate reader, and pSIP-pgsA' (A) vector-free frame and OprI gene fragment were ligated in an optimal system according to the Vazyme company's instructions for seamless cloning, the seamless cloning system being pSIP-pgsA' (A) 2.0. Mu. L, oprI gene fragment 5.0. Mu.L, 5 XCE II Buffer 2.0. Mu.L, express II 1.0. Mu.L, and a total of 10.0. Mu.L. The mixture was placed in a PCR apparatus, the connection temperature was set at 37℃and the connection time was 30min.

1.2.7.3 Electric transformation of the non-anti-recombinant plasmid pSIP-pgsA' -OprI (A) into E.coli χ6212 competence the procedure was the same as 1.1.8.

1.2.7.4PCR identification of plasmid pSIP-pgsA '-OprI (A) in E.coli χ6212 plasmids were extracted with DNA plasmid miniprep kit and then PCR identification was performed with a PCR identification reaction system of PRIME STAR Max Premix (2X) 25. Mu.L, plasmid pSIP-pgsA' -OprI (A) 5. Mu. L, AP-F2.5. Mu. L, AP-R2.5. Mu. L, ddH ₂ O15. Mu.L, total 50. Mu.L. The PCR amplification conditions of the template are the same as those of the template pUC-PP62 in 1.1.2, and the annealing temperature is changed to 62 ℃. The PCR products were identified by electrophoresis. The correct plasmid was identified by PCR and sent to the Biotechnology for sequencing.

1.2.7.5 The plasmid pSIP-pgsA' -OprI (A) was electrotransformed into NC8/Δalr in the same procedure as 1.1.12.

1.2.7.6PCR identification of recombinant plasmid pSIP-pgsA' -OprI (A) of NC8/Δalr the procedure for plasmid extraction was the same as 1.1.13. The reaction system and amplification conditions for PCR identification were the same as 1.2.7.4.

1.2.7.7Western blot detection of the expression of the protein of interest of Lactobacillus plantarum NC8 delta-pSIP 409-pgsA' -OprI the method steps were identical to 1.2.4 and 1.2.5.

Construction of 1.2.8 recombinant E.coli BL21-pET28 a-target fragment

1.2.8.1 Acquisition of pET28a vector frame and target fragment to construct prokaryotic expression plasmids pET28a-PP62, pET28a-MGF505-3R, pET a-EP364R, pET a-K145R, pET a-DP 96R, seamless cloning primers of five groups of target fragments pUC-PP62, pUC-MGF505-3R, pUC-EP364R, pUC-K145R, pUC-DP96R and seamless cloning primers of two end sequences of pET28a expression vector BamH I and Hind III are designed. The names and sequences of the primers are shown in tables 1-4.

TABLE 1-4 primer sequences

Note that the homology arm sequences are underlined.

The amplification system of the seamless cloning vector frame pET28a was PRIME STAR Max Premix (2X) 20.0 μ L, pET28 1a1.0 μ L, PET-F1.0 μ L, PET-R1.0 μ L, ddH ₂ O17.0 μL and a total of 40.0 μL.

The PCR amplification conditions of the template were the same as those of the template pUC-PP62 of 1.1.2. PRIME STAR Max Premix (2×) 20.0 mu L, pUC-target fragment (5 plasmids )1.0μL、ETP6-F/ETM5-F/ETE3-F/ETK1-F/ETD9-F 1.0μL、ETP6-R/ETM5-R/ETE3-R/ETK1-R/ETD9-R 1.0μL、ddH₂O 17.0μL、 together 40.0 mu L) the PCR amplification conditions of the template are the same as those of the template pUC-PP62 in 1.1.2, the annealing temperature is changed to 65 ℃,2 mu L of PCR product is taken and mixed with 1 mu L of 10X Loading buffer uniformly, and then the mixture is identified by electrophoresis.

1.2.8.2 Seamless cloning connecting pET28a carrier frame and target fragment, connecting with seamless cloning kit, wherein the connecting system comprises pET28a 5.0 mu L, target fragment 2.0 mu L, 5 XCE II Buffer 2.0 mu L, express II 1.0 mu L and total 10.0 mu L. The mixture was placed in a PCR apparatus, the connection temperature was set at 37℃and the connection time was 30min.

1.2.8.3 Transformation of the five prokaryotic expression plasmids pET28 a-fragment of interest into DH 5. Alpha. Amplification was performed in the same manner as 1.1.4 (the LB solid medium and liquid medium supplemented with 10. Mu.g/mL erythromycin were replaced by LB solid and liquid medium supplemented with 10. Mu.g/mL kanamycin).

1.2.8.4 Identification of recombinant prokaryotic expression plasmid in E.coli DH5a bacterial solution is extracted by using high-purity plasmid miniprep kit, and then identified by PCR method.

1.2.8.5 Five recombinant prokaryotic expression plasmids were transferred to BL21 competence in the same manner as 1.1.4 (LB solid medium and liquid medium supplemented with 10. Mu.g/mL erythromycin were exchanged for LB solid and liquid medium supplemented with 10. Mu.g/mL kanamycin; DH 5. Alpha. Competence was exchanged for BL21 competence).

1.2.8.6 Purifying protein, namely inducing and culturing five strains according to the optimal induction condition, transferring the bacterial liquid into a 50mL centrifuge tube, and centrifuging 10000g in a 4 ℃ horizontal centrifuge for 15min. The supernatant was discarded, washed once with PBS, and the cells were suspended in PBS. And repeatedly freezing and thawing for three times by using liquid nitrogen, and then performing ultrasonic crushing (150W ultrasonic for 40 min). Centrifuge at 12000rpm for 10 minutes at 4 ℃, place the supernatant at 4 ℃ for storage. The bacterial pellet was resuspended in Equp Buffer and refrigerated at 4 ℃ overnight. The next day was placed in a horizontal centrifuge at 4℃for 25 minutes at 12000rpm, and the supernatant was collected and filtered with a 0.22 μm filter. The supernatant was then column purified. Repeatedly eluting with Wash Buffer for 5 times, mainly washing off impurity protein, and then adding Elutation Buffer for 10 times to obtain target protein. The protein sample treatment and western blot verification steps are the same as 1.2.4 and 1.2.5.

1.3 Results

1.3.1 Construction and identification of novel functional Lactobacillus plantarum

1.3.1.1 PCR amplification results of the pSIP409-pgsA' (E) vector frame and the fragment of interest

The PCR products were identified by agarose gel electrophoresis using five plasmids synthesized and pSIP-pgsA' (E) vector as templates and corresponding seamless cloning primers for amplification. The vector fragment sequence bands were visible at 6161bp and five-item fragment sequence bands at 1881bp, 1131bp, 1398bp, 730bp, 579 bp.

1.3.1.2 Double restriction identification of recombinant plasmid pSIP409-pgsA'-PP62(E)、pSIP409-pgsA'-MGF505-3R(E)、pSIP409-pgsA'-EP364R(E)、pSIP409-pgsA'-K145R(E)、pSIP409-pgsA'-DP96R(E) and plasmid sequencing results

Double restriction identification of the recombinant plasmid joined by the seamless cloning was performed by using Xba I and Hind III restriction enzymes, and the result showed that the target bands were visible at 1881bp, 1131bp, 1398bp, 730bp and 579 bp. And the double enzyme digestion verifies the correct recombinant plasmid, and the sequencing result is also correct.

1.3.1.3 PCR amplification results of the non-resistant vector frame and asd-alr fragment

Five recombinant plasmids and the pLP-1261-asd-alr vector which are successfully constructed are taken as templates, and are amplified by corresponding seamless cloning primers. As a result, the asd-alr fragment bands at 4000bp and five antibiotic-free vector frame bands with the desired fragments at 6774bp, 6024bp, 6291bp, 5619bp and 5472bp were seen.

1.3.1.4E.coli χ6212 verification of non-anti-recombinant plasmid

1.3.1.4.1PCR identification

The nonreactive recombinant plasmid in E.coli chi 6212 is identified by a PCR method, and five clear target fragment bands of 1881bp, 1131bp, 1398bp, 730bp and 579bp can be seen.

1.3.1.4.2 Double enzyme digestion identification and plasmid sequencing

The non-anti-recombinant plasmid, which was correctly verified by PCR, was again verified by double cleavage. The vector-free band is visible at 8127bp, and the target band is visible at 1881bp, 1131bp, 1398bp, 730bp and 579 bp. And the PCR and double enzyme digestion verify that the correct nonreactive recombinant plasmid is also correct in the plasmid sequencing result.

1.3.1.5PCR identification of the non-anti-recombinant plasmid in Lactobacillus plantarum NC 8/Deltaalr

The non-anti-recombinant plasmid in the lactobacillus plantarum NC 8/delta alr is identified by a PCR method, and the successful transfer of the non-anti-recombinant plasmid into the NC 8/delta alr is determined. As a result, five clear target fragment bands at 1881bp, 1131bp, 1398bp, 730bp and 579bp were found.

1.3.2 Optimization of culture conditions for novel functional Lactobacillus plantarum

Five new functional lactobacillus plantarum strains which are successfully constructed are subjected to growth curve measurement and plate colony counting. The results of the growth curve determination of NC8Δ-pSIP409-pgsA'-PP62、NC8Δ-pSIP409-pgsA'-MGF505-3R、NC8Δ-pSIP409-pgsA'-EP364R、NC8Δ-pSIP409-pgsA'-K145R、NC8Δ-pSIP409-pgsA'-DP96R were plotted using GRAPHPAD PRISM software, see figure 1. As shown in FIG. 1, the optimal induction time was 1.5 hours of the culture of the bacterial liquid, and the optimal feeding time was 7 hours after induction (8.5 hours of the culture of the bacterial liquid).

1.3.3 Verification of expression of novel functional Lactobacillus plantarum target proteins

1.3.3.1Western blot verifying the expression of the target protein, namely treating the novel functional lactobacillus plantarum by repeated freeze thawing and ultrasonic crushing, and detecting by using a Western blot technology. Clear protein bands were visible at 68.8kDa, 41.3kDa, 26.5kDa, 51.1kDa, 21.2kDa (left in FIG. 2).

1.3.3.2 Immunofluorescence to verify the target protein expression, namely, five new functional lactobacillus plantarum strains are treated, wherein the primary antibody is anti-His-tag mouse monoclonal antibody, and the secondary antibody combined with the primary antibody is FITC-tag goat anti-mouse IgG. As a result, the results of the comparison with the empty vector strain are shown in FIG. 3, the strain of the control group does not emit immunofluorescence, and five new functional lactobacillus plantarum constructed successfully emit green fluorescence.

1.3.4 Construction of adjuvant bacteria NC8 delta-pSIP 409-pgsA' -OprI and verification of expression of target protein

1.3.4.1 PCR acquisition of pSIP409-pgsA '(A) no-resistance vector frame and OprI Gene PCR amplification was performed with seamless cloning primers using pSIP-pgsA' -PP62 (A) as template. The electrophoresis detection result shows that the carrier fragment band is visible at 8127bp, and the OprI gene fragment band is visible at 312 bp.

1.3.4.2PCR identification of plasmid pSIP-pgsA '-OprI (A) in E.coli χ6212 the plasmid pSIP-pgsA' -OprI (A) in E.coli χ6212 was identified by PCR, and the OprI gene fragment band was seen at 312 bp.

1.3.4.3PCR identification of recombinant plasmid pSIP-pgsA '-OprI (A) of NC8/Δalr PCR method was used to identify recombinant plasmid pSIP-pgsA' -OprI (A) of NC8/Δalr, and the OprI gene fragment band was seen at 312 bp.

1.3.4.4Western blot detection of expression of target protein of Lactobacillus plantarum NC8 delta-pSIP-pgsA '-OprI expression of OprI protein of Lactobacillus plantarum NC8 delta-pSIP-pgsA' -OprI was detected by Western blot, and a clear protein band was seen at 10.5 kDa.

1.3.5 Construction of recombinant E.coli BL21-pET28 a-destination fragment

1.3.5.1 PCR amplification of the pET28a vector frame and the target fragment Using pUC-PP62, pUC-MGF505-3R, pUC-EP364R, pUC-K145R, pUC-DP96R plasmid and pET28a expression vector as templates, amplification was performed with seamless cloning primers, and by electrophoresis detection, a 5344bp vector fragment band and five fragment sequence bands of 1596bp, 846bp, 1113bp, 441bp, 294bp were seen.

13.5.2PCR identification of recombinant prokaryotic expression plasmid in E.coli DH5a by PCR method, five clear target fragment bands at 1596bp, 8406bp, 1113bp, 441bp and 294bp can be seen.

1.3.5.3 Expression verification of purified target proteins five groups of recombinant E.coli are induced by IPTG, target proteins are purified and protein samples are processed, and detection is carried out by a western blot technique. Clear protein bands were visible at 18.8kDa, 47.5kDa, 12.5kDa, 68.1kDa, 36.1kDa (right in FIG. 2).

1.4 Summary in this example, five new functional Lactobacillus plantarum (NC8Δ-pSIP409-pgsA'-PP62、NCΔ-pSIP409-pgsA'-MGF505-3R、NC8Δ-pSIP409-pgsA'-EP364R、NC8Δ-pSIP409-pgsA'-K145R、NC8Δ-pSIP409-pgsA'-DP96R), strains expressing recombinant proteins fused with bacterial lipoprotein OprI by ASFV protein PP62, MGF505-3R, EP364R, K145R, DP R were successfully constructed, and the corresponding target proteins were determined to be expressed on the Lactobacillus plantarum surface by Western blot technique and immunofluorescence technique. The adjuvant bacteria NC8 delta-pSIP-pgsA' -OprI were successfully constructed and the expression of the OprI protein was verified. The PP62 protein, MGF505-3R protein, EP364R protein, K145R protein and DP96R protein were successfully purified and prepared for the animal experiment in example 2.

EXAMPLE 2 study of the immune Effect of novel functional Lactobacillus plantarum

In the embodiment, the BLAB/c mice are orally immunized by using the successfully constructed novel functional lactobacillus plantarum, and the immune index change of the mice is detected by the technologies of flow cytometry, ELISA, immunofluorescence and the like, so that the immune effect of the novel functional lactobacillus plantarum is studied.

2.1 Materials and methods

Five new functional lactobacillus plantarum (NC8Δ-pSIP409-pgsA'-PP62、NCΔ-pSIP409-pgsA'-MGF505-3R、NC8Δ-pSIP409-pgsA'-EP364R、NC8Δ-pSIP409-pgsA'-K145R、NC8Δ-pSIP409-pgsA'-DP96R) were constructed by chapter one. The empty vector Lb.plantarum NC 8. Delta. -pSIP409,409-pgsA' was kept by the laboratory.

Experimental animals 81 SPF-class 4 week old female BLAB/c mice were purchased from Fukang Biotechnology Co., ltd. Beijing and immunized after the Jilin agricultural university animal base was acclimatized for one week.

The main reagents were 200 mesh copper mesh and nylon screen were supplied by Solarbio (Beijing), red blood cell lysate, sodium citrate antigen retrieval solution, DAPI dye solution, goat serum, triton X-100, anti-quenching capper, protease inhibitor (PMSF) by Biyun Biotechnology Co., ltd., 4% paraformaldehyde fixative and RPMI-1640 medium were supplied by Biosharp (Beijing), oprI protein polypeptide (SEQ ID NO: ADEAYRKADEALGAAQK) was synthesized by Sangon Biotech (Shanghai), and flow cytometry antibodies were stored in this laboratory, the other reagents were as in example 1.

Experimental apparatus the same as in example 1.

2.2 Method

2.2.1 Experimental protocol

The mice were randomly divided into 9 groups, namely a PBS group, an empty vector group, an adjuvant group, a PP62 group, an MGF505-3R group, an EP364R group, a K145R group, a DP96R group and a mixed bacterium group, and 9 mice were used in each group. See table 2-1 for specific experimental groupings.

TABLE 2-1 animal immunization and grouping protocol

Immunization procedure, primary immunization at 1,2,3 days, seven days apart, booster immunization at 11, 12, 13 days apart, flow cytometry detection at 20 days, and tertiary immunization at 21, 22, 23 days. Blood and feces were collected from mice prior to primary immunization and on the seventh day after 3 consecutive days of immunization each. The collected blood was placed in a 4℃refrigerator and allowed to stand for 2 hours, centrifuged at 4000rpm/min at 4℃for 20 minutes, and the serum was sucked into a PCR tube and stored at-80 ℃. Mouse faeces were weighed, 3mL of PMSF solution (100 fold diluted in PBS) was added to 1g of faeces, incubated for 2h at 4 ℃ refrigerator, centrifuged at 4000rpm/min for 20min at 4 ℃, and the supernatant was taken into PCR tubes and stored at-80 ℃ refrigerator.

2.2 Flow cytometry

Tissue was collected, 5 mice from each group were subjected to eyeball blood collection and cervical removal, and spleens, peer's group lymph nodes (PP), mesenteric Lymph Nodes (MLN) were removed from the mice in an ultra clean bench.

Grinding on ice, namely placing the mixture into a cell culture dish with a 200-mesh copper net, adding 700 mu L of complete culture medium, and lightly grinding spleen, PP and MLN by using the tail of a 1mL syringe. After tissue grinding, 700. Mu.L of complete medium was aspirated to rinse the copper mesh, then the cell suspension in the cell culture dish was transferred to a 2mL centrifuge tube, placed in a 4℃horizontal centrifuge, spun at 2000rpm for 5min, and the supernatant gently aspirated after centrifugation. PP and MLN cells were washed once with PBS and 1mL of complete medium suspension cell stock was added. Spleen cells require removal of erythrocytes.

Adding 500 mu L of erythrocyte lysate into the centrifuged splenocyte solution, uniformly mixing, performing ice lysis for 3min, centrifuging again by a4 ℃ horizontal centrifuge for 5min, if the splenocyte amount is too large, performing secondary lysis, adding 500 mu L of PBS, standing on ice for 2min to terminate the lysis reaction, centrifuging by the 4 ℃ horizontal centrifuge for 5min, washing once by PBS, and adding 1mL of complete culture medium suspension cell stock solution.

Counting, namely diluting the spleen cell stock solution by 100 times with PBS, diluting the MLN cell stock solution by 50 times with PBS, and counting with the stock solution because of the small number of cells in PP. The diluted spleen cell fluid, MLN cell fluid and PP cell stock solution were counted by a cell counting plate, and the stock solution concentration was calculated by the cell number to calculate the volume of the cell stock solution required for 1.0X10 ⁷ cells. The corresponding volumes of cell stock solution were removed into 2mL centrifuge tubes, washed once with 4 ℃ pre-chilled PBS, and 200 μl of the remaining stock solution was mixed to make a cell suspension, which was stored at 4 ℃ for subsequent experiments.

Diluting the antibody, namely diluting the antibody with PBS on ice and under a light-shielding condition of tinfoil. Antibody names and dilution factors are shown in Table 2-2.

TABLE 2-2 dilution of antibodies

Dendritic cells in PP are stained by mixing three diluted antibodies of CD11C, CD80 and CD86 together, adding 30 mu L of the mixed antibody into 200 mu L of prepared cell suspension, uniformly mixing, incubating for 25min in a 4 ℃ refrigerator in dark place, centrifuging, washing once with PBS, uniformly mixing with 250 mu L of PBS, filtering with a nylon screen, transferring into a flow tube, and detecting by using a BD flow cytometer.

Spleen and MLN T cells staining, namely placing the treated spleen and MLN cell suspension in a 48-well cell culture plate, adding 300 mu L of complete culture medium, 2 mu L of stimulator PMA (with a final concentration of 40ng/mL and ready for use), 1 mu L of blocker and 1 mu L of purified corresponding antigen protein of interest (with a final concentration of 4 ug/mL) (each group of antigen proteins are shown in tables 2-3), incubating for 5 hours in a 37 ℃ cell incubator, taking out the cells out of the incubator, placing the incubator on ice, uniformly mixing the cells, sucking the cells out of the incubator into a 1.5EP tube, adding 1mL of 1% BSA PBS for washing once, leaving 100 mu L of diluted mixed CD3 CD4 CD8 antibody, washing once with pre-cooled 1mL of 1% BSA, adding 200 mu L of formaldehyde fixing solution, fixing in a4 ℃ refrigerator for 20min in a dark place, adding 800 mu L of membrane penetrating solution (diluted with Wash 1:9), incubating the incubator for 3min in a dark place, centrifuging the incubator, and leaving 200 mu L of supernatant. IL-4 and IFN-gamma antibodies were mixed after dilution by adding 20. Mu.L and incubated at 4℃for 20min in the absence of light. Washing with PBS once, mixing with 250 μl PBS, filtering, and detecting.

PP surface (germinal center) antibody staining, namely adding 10 mu L B of antibody into 200 mu L of prepared cell suspension, uniformly mixing, incubating for 25min in a low-temperature preservation box in a dark place, washing once with PBS containing 1% BSA, adding 200 mu L of formaldehyde fixing solution, fixing for 25min in a dark place in the low-temperature preservation box again, adding 800 mu L of penetrating fluid (diluted by Waha 1:9), washing once, adding 1mL of penetrating fluid, uniformly mixing, incubating for 3min at room temperature in a dark place, centrifuging, discarding supernatant, adding 150 mu L of IgA antibody, and incubating for 25min at 4 ℃ in a dark place. After centrifugation for 5min, the mixture was washed once with PBS, and finally mixed with 250. Mu.L of PBS, filtered and detected by BD flow cytometry.

Data processing and analysis streaming data were analyzed using flowjo_v10.6.2 software, using GRAPHPAD PRISM 8.0.2 plots and statistics, and group differences were calculated using One-way ANOVA (P <0.05; P <0.01; P <0.001; P < 0.0001).

Antigen proteins of each group of tables 2 to 3

2.3 Immunofluorescence detection of the number of B cells in the duodenum and ileum of mice by immunofluorescence.

2.3.1 Treatment of intestinal tissue after mice were sacrificed, the duodenum and ileum of each group of mice were fixed in 4% paraformaldehyde for 5 days at the experimental bench. Tissue sampling was performed in a fume hood, and the duodenum and ileum were both circular and trimmed. Placing the trimmed tissue into an embedding box for alcohol gradient dehydration, namely 70% absolute ethyl alcohol (2 h), 80% absolute ethyl alcohol (2 h), 85% absolute ethyl alcohol (overnight), 90% absolute ethyl alcohol (2 h), 95% absolute ethyl alcohol I (1.5 h), 95% absolute ethyl alcohol II (1.5 h), 100% absolute ethyl alcohol I (1 h), 100% absolute ethyl alcohol II (1 h), and beating the tissue on newspapers after each dehydration. The transparent coating is prepared by completely soaking in xylene I and xylene II for 1 min, and the eye is transparent jelly. Wax I, wax II, and wax III were put into a 58 ℃ water bath to completely melt them in advance. The clear finished tissue was placed in wax I, wax II, wax III in sequence for 40 minutes. After wax dipping, the intestinal tissues are placed in an embedding machine preheated in advance, so that the intestinal tissues are embedded in a standing mode. Slicing with a full-automatic paraffin slicer to a slice thickness of 2.5 μm, placing the slice into deionized water at a temperature of 42 ℃ to be fully unfolded, then treating and adhering the glass slide with polylysine, taking out, and placing the glass slide on a glass slide staining rack. The pieces were baked in an oven at 60℃for 2h.

2.3.2 Immunofluorescence staining 1) preparation before experiment, namely diluting sodium citrate antigen retrieval liquid (50 x) into sodium citrate antigen retrieval liquid (1 x) by deionized water, storing at 4 ℃, using at present, and not being reusable, antibody (light-proof), namely searching the optimal dilution of the antibody in advance, centrifuging the diluted antibody for 5min at 5000rpm/min before using, preparing sealing liquid, namely diluting 5% goat serum and 0.3% Triton-100 by PBS under the light-proof condition, diluting 1000 times by DAPI by PBS, and storing at 4 ℃ in light-proof condition. 2) Dewaxing, namely putting the baked slices into xylene I and xylene II for 7min, putting the slices into absolute ethyl alcohol I and absolute ethyl alcohol II for 5min, and putting the slices into distilled water for 2 times. 3) Antigen retrieval, namely placing the slice into a staining jar filled with antigen retrieval liquid (1X), heating the slice in boiling water at 100 ℃ for about 20min, then cooling the staining jar in cold water for 10min, and washing the slice with PBS for 2 times for 4min each time. 4) And closing, namely beginning to avoid light until the sealing sheet is finished. The kitchen paper is used for sucking up the water, and then an immunohistochemical pen is used for circling the tissues, so that the tissues cannot be damaged. The tissue is completely covered with the sealing liquid, the membrane is broken for 60min, the sealing liquid is poured off, and the kitchen paper is used for sucking the liquid. 5) B cell staining the tissues were covered with 50 μl of 200-fold dilutions of B220 and IgA, placed in a wet box and incubated overnight at 4 ℃ freezer. Wash with PBS for 5min,2 times (recovery of antibody before washing). 6) Cell nucleus staining, namely adding 50 mu LDAPI and incubating for 10min in dark. Wash 2 times with PBS, 6 min/time. 7) Sealing, namely sealing the tablet by using an anti-fluorescence quenching sealing tablet, wherein the sealing can be observed by using a microscope after finishing, and the tablet is necessarily finished before being dried or stored at 4 ℃.

2.3.3ELISA detection of SIgA the treated mouse faeces supernatant was subjected to specific SIgA detection.

Coating the antigen proteins of interest were diluted to 2. Mu.g/mL with coating solution, spread on 96-well ELISA plates (see Table 2-3 for antigen proteins of each group) and 100. Mu.L of each well was coated overnight in a 4℃freezer. Washing with 200. Mu.L of PBST (PBS with 0.05% Tween-20 added) three times, washing with shaking for 5 min/time. Blocking 100. Mu.L PBS (1% BSA) was added and blocked for 2h at 37℃in an incubator. Washing, namely adding 200 mu L of PBST, and vibrating and washing for 5min for three times.

The samples to be tested were added, and the mouse fecal supernatant was diluted 50-fold, 100. Mu.L/well with PBS, and repeated three times. Incubate for 2h at 37 ℃, wash. Secondary antibody was added, 2000-fold dilution of HRP-labeled goat anti-mouse IgG was added, incubated at 37 ℃ for 60min, and washed 3 times with PBST. Color development, adding 100. Mu.L of TMB color developing solution, standing for 15min at 37 ℃ in dark, and observing the color of the solution. Stop the color reaction by adding 50. Mu.L of 20% H ₂SO₄. And detecting by using an enzyme-labeled instrument, wherein the set conditions are that the wavelength is 450nm and the vibration plate is 30s. The data is saved and analyzed.

Data analysis was performed using GRAPHPAD PRISM 8.0.2 plots and Two-way ANOVA was used to calculate the differences between the data for each experimental and empty group at each time period (P <0.05, P <0.01, P <0.001, P < 0.0001).

2.3.4ELISA detection of the level of specific IgG in mouse serum the content of specific IgG in serum samples was detected. The method was the same as 2.3.3, but the dilution factor of the antibody was different. Serum samples were diluted 100-fold and HPR-labeled goat anti-mouse IgG H & L diluted 1000-fold.

2.3.5ELISA detecting the level of IL-2, IFN-gamma and IL-4 in serum by ELISA kit.

2.3.6 Fluorescent quantitative PCR (qPCR)

2.3.6.1Trizol method for extraction of tissue Total RNA 1) 1mLTrizol reagent and 100mg spleen tissue or mesenteric tissue were added to a 2mL centrifuge tube and ground using a tissue homogenizer. 2) The supernatant was aspirated into a fresh centrifuge tube, added with 200. Mu.L of chloroform, mixed well, allowed to stand for 5 minutes, centrifuged at 13000g for 20 minutes at 4℃and transferred to an enzyme-free tube. 3) 500. Mu.L of precooled isopropanol was added, mixed well and placed in a 4℃refrigerator for 15 minutes. Centrifuge at 10000g for 20min at 4℃and discard supernatant. 4) The supernatant was discarded after being resuspended in 1mL of 75% ethanol, centrifuged at 4℃for 10min at 8500 g. 5) The mixture was left at room temperature for 5 minutes, 30. Mu.L of enzyme-free water was added thereto for dissolution, and the mixture was stored at-80 ℃.

2.3.6.2 Reverse transcription reaction genomic DNA was removed by reverse transcription kit and RNA was reverse transcribed into cDNA. The specific steps are shown in the specification. The cDNA product was stored in a-20℃refrigerator and used for half a year.

2.3.6.3 Primers were designed by NCBI query of the sequences of mouse IL-2, IL-4, IFN-gamma and qPCR primers were designed by Primer 5 software.

IL-2:Forward:GCAGCAGCAGCAGCAGCAG;Reverse:GCCGCAGAGGTCCAAGTTCATC。

IL-4:Forward:GGACGCCATGCACGGAGATG;Reverse:GAAGCACCTTGGAAGCCCTACAG。

IFN-γ:Forward:AAACCTTGTACTTCTGACGGCTGAG;Reverse:CGAGTCCTGGCTGGTCTGTGAG。

ACT-β:Forward:CTACCTCATGAAGATCCTGACC;Reverse:CACAGCTTCTCTTTGATGTCAC。

2.3.6.4 Fluorescent quantitative PCR, namely detecting the transcription levels of IL-2, IL-4 and IFN-gamma in spleen by a fluorescent quantitative PCR method. qPCR reaction system ：2×Universal SYBR qPCR mix 10μL、IL-2-F/IL-4-F/IFN-γ-F/ACT-β-F 0.8μL、IL-2-R/IL-4-R/IFN-γ-R/ACT-β-R 0.8μL、cDNA template 2 μ L, ROX REFERENCE DYE 0.4 μ L, RNase-Free ddH ₂ O6 μL, total 20 μL. The reaction conditions are 95 ℃ and 60s (95 ℃ and 10s;60 ℃ and 34 s) multiplied by 40cycle in a fluorescent quantitative PCR instrument. Waiting for the program to run and storing the data. Using GRAPHPAD PRISM 8.0.2 plots and statistics, the group differences were counted using One-way ANOVA (P <0.05; P <0.01; P <0.001; P < 0.0001).

2.4.1 Flow cytometry detection results

2.4.1.1 Effect of novel functional Lactobacillus plantarum immunization on DC cells in mouse PP

To evaluate the effect of a new functional lactobacillus plantarum constructed successfully on mouse dendritic cells, the expression of CD80 and CD86 on dendritic cell surfaces in PP was examined by flow cytometry. The results are shown in FIG. 4, and the results show that the expression level of CD80 in dendritic cells of the PP62 group, the MHG505-3R group, the EP364R group, the K145R group and the DP96R group is extremely remarkably increased (P < 0.01) compared with the PBS control group, and the expression level of CD80 in dendritic cells of the mixed bacteria group is extremely remarkably increased (P < 0.001) compared with the PBS control group. The expression level of CD86 in dendritic cells was significantly increased in PP62 group, MHG505-3R group, EP364R group and K145R group DP96R group (P < 0.01) compared with PBS group, and the expression level of CD86 in dendritic cells was significantly increased in mixed bacteria group (P < 0.001) compared with PBS group.

Effect of 2.4.1.2 novel functional lactobacillus plantarum immunization on IFN-gamma expression in CD4 ⁺ and CD8 ⁺ T lymphocytes in the spleen of mice

To examine whether the novel functional Lactobacillus plantarum can cause cellular immune response in mice, the expression of IFN-gamma in CD4 ⁺ T lymphocytes in the spleen of mice was examined. The results are shown in FIG. 5, and the results show that the IFN-gamma content expressed in CD4 ⁺ T lymphocytes is significantly different (P < 0.05) in the PP62 group, the MGF505-3R group, the EP364R group and the K145R group compared with the PBS group, the IFN-gamma content expressed in CD4 ⁺ T lymphocytes is significantly different (P < 0.01) in the DP96R group compared with the PBS group, and the IFN-gamma content expressed in CD4 ⁺ T lymphocytes is significantly different (P < 0.001) in the mixed bacterium group compared with the PBS group.

The expression of IFN-gamma in CD8 ⁺ T lymphocytes in the spleen of mice was examined. The results are shown in FIG. 6, which shows that the IFN-gamma content expressed in CD8 ⁺ T lymphocytes is significantly increased (P < 0.01) in the PP62 group, MGF505-3R group, EP364R group, K145R group and DP96R group compared with the PBS group, and the IFN-gamma content expressed in CD8 ⁺ T lymphocytes is significantly different (P < 0.001) in the mixed bacterium group compared with the PBS group.

Effect of 2.4.1.3 novel functional lactobacillus plantarum immunization on IFN-gamma expression in CD4 ⁺ and CD8 ⁺ T lymphocytes in mouse MLN

To examine the effect of novel functional Lactobacillus plantarum on T lymphocytes in mouse MLNs, IFN-gamma expression in CD4 ⁺ T lymphocytes in each group of mouse MLNs was determined by flow cytometry. The results are shown in FIG. 7, which shows that the IFN-gamma content expressed in CD4 ⁺ T lymphocytes is significantly increased (P < 0.05) in the PP62 group and the K145R group compared with the PBS group, the IFN-gamma content expressed in CD4 ⁺ T lymphocytes is significantly increased (P < 0.01) in the MGF505-3R group, the EP364R group and the DP96R group compared with the PBS group, and the IFN-gamma content expressed in CD4 ⁺ T lymphocytes is significantly different (P < 0.001) in the mixed bacterium group compared with the PBS group.

The expression of IFN-gamma in CD8 ⁺ T lymphocytes in the MLN of each group of mice was examined. The results are shown in FIG. 8, which shows that the IFN-gamma content expressed in CD8 ⁺ T lymphocytes is significantly different (P < 0.05) in the PP62 group, the MGF505-3R group, the EP364R group, the K145R group and the DP96R group compared with the PBS group, and the IFN-gamma content expressed in CD8 ⁺ T lymphocytes is significantly increased (P < 0.01) in the mixed bacterium group compared with the PBS group.

Effect of 2.4.1.4 novel functional lactobacillus plantarum immunization on IL-4 expression in CD4 ⁺ T lymphocytes in spleen

To examine whether the novel functional Lactobacillus plantarum is capable of eliciting a cellular immune response in mice, IL-4 expression in spleen CD4 ⁺ T lymphocytes from each group of mice was examined. The results are shown in FIG. 9, in which the PP62 group, MGF505-3R group, EP364R group, K145R group and DP96R group showed very significant differences in the IL-4 content expressed in CD4 ⁺ T lymphocytes (P < 0.05) compared to the PBS group, and in the mixed bacteria group showed very significant differences in the IL-4 content expressed in CD4 ⁺ T lymphocytes (P < 0.01) compared to the PBS group. 2.4.1.5 Effect of novel functional Lactobacillus plantarum immunization on IL-4 expression in CD4 ⁺ T lymphocytes in MLN

The results of measuring IL-4 expression in CD4 ⁺ T lymphocytes in MLN of mice in each group by flow cytometry showed, as shown in FIG. 10, that PP62 group, EP364R group, K145R group and DP96R group showed a significant increase in IL-4 content in CD4 ⁺ T lymphocytes (P < 0.05) as compared to PBS group, and that MGF505-3R group and mixed bacterium group showed a significant increase in IL-4 content as compared to PBS group (P < 0.01) as compared to CD4 ⁺ T lymphocytes.

Effect of 2.4.1.6 novel functional lactobacillus plantarum immunization on B cells in mouse PP

The example is used for researching whether five new functional lactobacillus plantarum which are successfully constructed influence the activation of B cells or not, B220 ⁺IgA⁺ cells in PP of each group of mice are detected through a flow cytometry, the result is shown in figure 11, and the results show that compared with a PBS control group, the B220 ⁺IgA⁺ cells of the PP62 group and the MGF505-3R group are obviously increased (P < 0.05), the B220 ⁺IgA⁺ cells of the EP364R group, the K145R group and the P96R group and the mixed bacteria group are obviously increased (P < 0.01), so that the five new functional lactobacillus plantarum can effectively activate the B cells of the mice.

2.4.2 Immunofluorescence results to evaluate whether the novel functional Lactobacillus plantarum has an effect on B cells in the mouse intestinal tract, the number of B220 ⁺IgA⁺ cells in the mouse duodenum and ileum was examined by immunofluorescence techniques. The results are shown in FIGS. 12 and 13, which show that in the duodenum, B220 ⁺IgA⁺ cells of mice orally immunized with the novel functional Lactobacillus plantarum are significantly increased compared to PBS and empty groups. In the ileum, the B220 ⁺IgA⁺ cells of mice immunized orally with the novel functional lactobacillus plantarum were significantly increased compared to the PBS group and the empty group.

The result of 2.4.3ELISA detection of specific antibody SIgA in the mouse feces, which is shown in FIG. 14 (the marked significant difference in the graph is the result of comparative analysis of the experimental group of oral administration of novel functional lactobacillus plantarum and the empty load group), shows that the SIgA content in the mouse feces of each group has no significant difference in the mouse feces before immunization, the P62 group, the MGF505-3R group, the EP364R group, the K145R group and the DP96R group are significantly increased (P < 0.05) compared with the empty load group in the mouse feces of each group before immunization, after the initial immunization, after the boosting, and after the third immunization, the SIgA content in the mouse feces of the mixed bacteria group is significantly increased (P < 0.01) compared with the empty load group, the SIgA content in the mouse feces of the mixed bacteria group is significantly increased (P < 0.01) compared with the P < 0.01) on the experimental group after the twenty days of oral administration, the MGF505-3R group, the EP R group and the K364R group on the tenth day after immunization (seventh day after the initial immunization), the PP62 group, the P < 62R <0.01 > compared with the P < 0.01) compared with the empty load group, the P <0.001, the mixed bacteria group in the mixed bacteria group is significantly increased (P < 0.01), the mixed bacteria group is significantly increased in the mouse feces of the mixed bacteria group compared with the P < 0.001.

2.4.4ELISA results of detecting specific antibody IgG in mouse serum by ELISA, wherein the results are shown in figure 15 (marked significant differences in the figure are the results of comparative analysis of experimental groups and empty groups of oral administration immune novel functional lactobacillus plantarum) that the specific antibody IgG level in the mouse serum of each group has no obvious difference before immunization; the levels of specific antibody IgG in the serum of mice of PP62, MGF505-3R, EP364R, K145R and DP96R were very significantly increased (P < 0.01) compared to the empty load group on day seven of initial immunization, the levels of specific antibody IgG in the serum of mice of the mixed fungus group were very significantly increased (P < 0.001) compared to the empty load group on day seven after boost immunization, the levels of specific IgG in the serum of mice of PP62 and K145R were very significantly increased (P < 0.01) compared to the empty load group, the levels of specific IgG in the serum of mice of MGF505-3R, EP364R and DP96R were very significantly increased (P < 0.001), the levels of specific antibody IgG in the serum of mice of the mixed fungus group were very significantly increased (P < 0.0001) compared to the empty load group, the levels of specific IgG in the serum of mice of PP62, EP R and K145 group were very significantly increased (P < 0.001) compared to the empty load group on day seven days after the third immunization, and the levels of specific IgG in the serum of mice of MGF505-3R, EP364R and DP96R were very significantly increased (P < 0.0001).

2.4.5ELISA detection of changes in the levels of cytokines IL-2, IFN-gamma, IL-4 in mouse serum

The levels of cytokines IL-2, IFN-gamma, IL-4 in the serum of each group of mice after booster immunization were examined by ELISA. The results are shown in fig. 16, 17 and 18, and the results show that oral administration of the novel functional lactobacillus plantarum can significantly increase the levels of cytokines IL-2, IFN-gamma and IL-4 in the serum of mice. The IL-2 level in the serum of mice of MGF505-03R group, EP364R group and DP96R group was significantly higher than that of PBS group (P < 0.05), the IL-2 level in the serum of mice of PP62 group and K145R group was significantly higher than that of PBS group (P < 0.01), the IL-2 level in the serum of mice of mixed bacteria group was significantly higher than that of PBS group (P < 0.001), the IFN-gamma level in the serum of mice of MGF505-03R group and DP96R group was significantly increased (P < 0.05) compared to PBS control group, the IFN-gamma level in the serum of mice of PP62 group, EP364R group and K145R group was significantly increased (P < 0.01), the IFN-gamma level in the serum of mice of mixed bacteria group was significantly increased (P < 0.001), the IL-4 level in the serum of mice of PP62 group, EP364R group, K145R group and DP96R group was significantly different from that of PBS control group (P < 0.05), and the IL-4 level in the serum of mice of mixed bacteria group was significantly different from that of PBS control group (P < 0.05).

2.4.6 Fluorescent quantitative PCR detection of IL-2, IFN-gamma, IL-4 mRNA transcription levels in mouse spleens the fluorescent quantitative PCR detection of IL-2, IFN-gamma, IL-4 mRNA transcription levels in mouse spleens of each group was performed, and the results are shown in FIG. 19, FIG. 20, and FIG. 21. The expression level of IL-2 cytokine in the spleens of mice of PP62 group, MGF505-3R group, EP364R group, K145R group and DP96R group is significantly higher than that of PBS group (P < 0.01), the expression level of IL-2 cytokine in the spleens of mice of mixed bacteria group is significantly higher than that of PBS group (P < 0.001), the expression level of IL-4 cytokine in the spleens of mice of PP62 group and EP364R group is significantly higher than that of PBS group (P < 0.05), the expression level of IFN-gamma cytokine in the spleens of mice of MGF505-3R group, K145R group and DP96R group is significantly higher than that of P < 0.01), the expression level of IFN-gamma cytokine in the spleens of mice of mixed bacteria group is significantly higher than that of PBS group (P < 0.001), the expression level of IL-4 cytokine in the spleens of mice of PP62 group, MGF505-3R group, EP364R group and DP96R group is significantly higher than that of PBS group (P < 0.01), and the expression level of IFN-gamma cytokine in the spleens of mice of mixed bacteria group is significantly higher than that of P < 0.01.

2.5 Discussion:

Initiation of mucosal immune responses in PP relies on the collection, processing and efficient presentation of foreign antigens by Dendritic Cells (DCs). Among antigen presenting cells, dendritic cells are the cells that are the most capable of initiating antigen-specific responses and are the cells that can induce differentiation of primary CD4 ⁺ and CD8 ⁺ T cells. The surface marker of DCs was CD11c, and DCs induced expression of B7 costimulatory molecules (CD 80 and CD 86) after capture of antigen. The flow results of this example show that oral immunization of novel functional Lactobacillus plantarum can effectively induce activation of DC in mouse PP, thereby initiating antigen specific immune responses. When DCs present the treated antigen, an adaptive immune response is activated. CD4 ⁺ T cells can play a central role in adaptive immunity by recruiting and activating other immune cells by producing a variety of cytokines and chemokines, enhancing their immune activity, and counteracting pathogen infection by coordinating immune responses. After pathogen infection, CD4 ⁺ T cells differentiate into Th1 cells, and the developing Th1 cells release large amounts of IFN- γ, which then induces Th1 cells to differentiate further stably by the positive feedback pathway. Cytotoxic T cells (CD 8 ⁺ T, CTL) are the major cell subset that clear viral infection. IFN-gamma is an effector molecule of activated CTL release. in this example, the flow results indicate that oral immunization with the novel functional lactobacillus plantarum promotes the expression of IFN- γ in mouse CD4 ⁺ T and CD8 ⁺ T cells, thereby promoting a cell-mediated immune response. Th2 is involved in humoral immune response, and interleukin-4 (IL-4) is a key cytokine secreted by Th2 cells, and has important immunoregulatory function on immune cells and non-immune cells. the flow chart of this example shows that oral immunization of novel functional Lactobacillus plantarum promotes IL-4 expression in mouse CD4 ⁺ T cells, thereby mediating the humoral immune response. The ELISA technology detects that the secretion level of cytokines IL-2, IFN-gamma and IL-4 in the serum of mice in the experimental group is obviously increased, and further proves that the novel functional lactobacillus plantarum can enhance the cellular immunity level and the humoral immunity level of organisms. The relative expression level of IL-2, IL-4 and IFN-gamma gene mRNA in the spleen of mice in the experimental group is obviously increased by qPCR, and the analysis is carried out in combination with the flow cytometry result, so that the spleen may have specific immune memory response. PP of the small intestine is the primary induction site of mucosal immune response. Most germinal center cells of PP are B lymphocytes, which are activated by antigens to produce IgA, thereby defending against pathogenic bacterial and viral infections. IgA is a non-inflammatory antibody that is specifically used for intestinal mucosa protection. IgA is the most abundant antibody in mucosal secretions, and monomeric IgA interacts with small plasma cell-derived polypeptides in the gut to form IgA dimers that recognize the polymeric immunoglobulin receptor (pIgR) on the basolateral surface of mucosal Intestinal Epithelial Cells (IEC). The pIgR promotes the release of secretory IgA (SIgA) to the intestinal surface. SIgA can protect mucosal epithelium from pathogen infection. After oral immunization of mice with the novel functional lactobacillus plantarum expressing the african swine fever virus fusion protein constructed in example 1, a significant increase in B220 ⁺IgA⁺ cells in PP germinal center was detected by flow cytometry. Meanwhile, immunofluorescence results also show that B220 ⁺IgA⁺ cells of mice orally immunized with the novel functional lactobacillus plantarum are higher than those of PBS control groups and empty vector control groups in the small intestine. ELISA detection results also show that the content of a specific antibody SIgA in the feces of the mice of the oral administration of the novel functional lactobacillus plantarum is obviously increased, and the results show that the oral administration of the novel functional lactobacillus plantarum can enhance the mucosal immune response of the mice. Th1 and Th2 cells participate in protective immune responses and induce B cells to produce specific IgG antibodies to protect the body. Most of the IgG antibodies produced after B cell activation are present in the blood in a secreted form, and the proportion of the IgG antibodies in the serum is 75%, which plays an important role in humoral immunity. In the detection results of this example, the level of specific IgG antibodies in the serum of mice was significantly increased, which suggests that immunization with the novel functional lactobacillus plantarum may promote humoral immune responses, thereby enhancing the immune ability of the organism.

2.6 Summary the results of the study in this example show that the novel functional Lactobacillus plantarum NC8Δ-pSIP409-pgsA'-PP62、NCΔ-pSIP409-pgsA'-MGF505-3R、NC8Δ-pSIP409-pgsA'-EP364R、NC8Δ-pSIP409-pgsA'-K145R and NC8 delta-pSIP 409-pgsA' -DP96R alone or in combination with oral immunization of mice can induce DC activation in PP and initiate protective immune responses. Enhancing cellular immune response and humoral immune response in spleen and MLN, promoting B cell activation in PP and IgA antibody production, and enhancing mucosal immune response. And simultaneously, the level of antigen-specific IgG antibodies in serum is increased, and the humoral immune response is enhanced.

The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (11)

1. A lactic acid bacterium expressing an African swine fever virus fusion antigen, characterized in that it has a fusion gene, wherein the gene encoding the OprI protein in the fusion gene is linked via a linker to a gene encoding the PP62 protein, a gene encoding the MGF505-3R protein, a gene encoding the EP364R protein, a gene encoding the K145R protein, or a gene encoding the DP96R protein, respectively; the nucleotide sequence of the linker is shown in SEQ ID NO: 7; The nucleotide sequence of the fusion gene is selected from the following nucleotide sequences: the sequence shown in SEQ ID NO: 8, the sequence shown in SEQ ID NO: 9, the sequence shown in SEQ ID NO: 10, the sequence shown in SEQ ID NO: 11, and the sequence shown in SEQ ID NO: 12; The lactic acid bacteria is Lactobacillus plantarum NC8/ΔaLr, which is alanine racemase gene defective. The lactic acid bacteria were expressed using the pSIP409-pgsA’ vector. 2. The method for constructing lactic acid bacteria according to claim 1, comprising: ligating the fusion gene to the expression vector and then transferring it into competent cells to obtain a recombinant plasmid, and transforming the recombinant plasmid into the starting strain. 3. A composition comprising the lactic acid bacteria of claim 1. 4. The composition according to claim 3, wherein the composition is a microbial agent, a pharmaceutical composition, or a feed. 5. A vaccine comprising the lactic acid bacteria of claim 1. 6. The vaccine according to claim 5, wherein the vaccine is a vector vaccine. 7. The vaccine according to claim 5, wherein the vaccine is a live vector vaccine. 8. The use of the lactic acid bacteria of claim 1, the composition of claims 3-4, or the vaccine of claims 5-7 in the preparation of products for the prevention and control of African swine fever. 9. The application according to claim 8, wherein the product is a microbial agent, a pharmaceutical preparation, or feed. 10. The application according to claim 9, wherein the pharmaceutical preparation is a biological product. 11. The application according to claim 9, wherein the pharmaceutical preparation is a live vector vaccine.