CN119242550B - New functional lactic acid bacteria expressing ETEC antigens and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of microorganisms, in particular to a novel functional lactic acid bacterium for expressing ETEC antigens, a preparation method and application thereof. The recombinant lactobacillus plantarum for expressing the ETEC antigen takes lactobacillus plantarum NC8 as a host bacterium and a pPG612.1 expression vector, wherein the pPG612.1 expression vector contains FaeC genes or LngC genes of the ETEC. The invention innovatively integrates FaeC and LngC genes of ETEC into lactobacillus plantarum NC8 for expression, and can activate intestinal mucosa immune response in an oral administration mode, so that not only can intestinal injury caused by ETEC be directly resisted, intestinal villus structure is protected, but also the body can be stimulated to generate systemic immune response by adjusting intestinal microenvironment, the capability of resisting ETEC infection of the body is improved, the morbidity and mortality of ETEC infection are reduced, and the economic benefit of pig industry is improved.

Description

Novel functional lactic acid bacteria expressing ETEC antigen, and preparation method and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to novel functional lactic acid bacteria expressing ETEC antigens, a preparation method 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.

Enterotoxigenic escherichia coli (ETEC) infection is a major challenge facing the current pig industry, especially the most serious hazard to piglets in lactation and weaning. ETEC bind to specific receptors of small intestine epithelial cells through its surface-specific pili, release enterotoxins after intestinal colonisation, destroy intestinal mucosa, cause massive loss of water and electrolytes, and cause severe diarrhea. It was investigated that diarrhea caused by ETEC infection in large-scale farms is about 26.4% of cases of diarrhea in piglets, resulting in a huge economic loss to the breeding industry.

At present, the prevention and treatment of ETEC infection faces a plurality of technical bottlenecks. The traditional antibiotic treatment method has the remarkable limitations that ETEC is easy to generate drug resistance to cause the reduction of treatment effect, and the early use of antibiotics not only can influence the growth and development of piglets, but also can interfere the normal colonization of intestinal symbiotic flora. In the aspect of immune protection, the passive immune protection effect of piglets obtained through breast milk is limited, and the level of maternal antibodies can be rapidly reduced after weaning, so that the piglets are very easy to infect ETEC. The prior vaccine technology has a plurality of defects that effective vaccines which can be widely applied are not available at present, traditional vaccines mainly excite systemic immune response, have unsatisfactory local immune effect on intestinal tracts, and are easily interfered by maternal antibodies to influence the immune effect.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a safe and effective prevention and treatment scheme in the field of ETEC (enterotoxigenic escherichia coli) infection prevention and control, and particularly aims at diarrhea of livestock and poultry such as piglets. Currently, ETEC infection prevention and control mainly depends on antibiotic treatment, which not only easily causes drug resistance problems, but also affects animal growth and development and intestinal flora balance. Although conventional vaccines can prevent ETEC infection, there are still disadvantages in terms of immunoprotection effect, convenience of use, and the like. Therefore, there is a need to develop a new and sustainable ETEC prevention and control technology.

In order to solve the problems, the invention specifically aims at providing an ETEC-resistant functional lactic acid bacterium constructed by genetic engineering means, which can express a specific antigen protein (FaeC or LngC) of ETEC, so that the use of antibiotics is avoided, and the generation risk of drug-resistant strains is reduced. Second, a composition or product is provided that can prevent and/or treat ETEC infections, immunomodulation, anti-inflammatory and achieve protection of the gut. The composition or product can activate intestinal immune response, regulate inflammatory response, protect intestinal structure, etc., enhance intestinal immune defenses of host, and effectively reduce ETEC infection risk. Thirdly, a disease prevention and control means suitable for large-scale cultivation is provided. The lactobacillus has the characteristics of high safety, acid resistance, bile salt resistance and the like, is suitable for long-term use, can form a sustainable disease prevention scheme, and effectively improves the culture benefit. Fourth, a preparation method of the functional lactic acid bacteria and application thereof in the aspects of preventing and/or treating ETEC infection, immunoregulation, anti-inflammatory, intestinal tract protection and the like are provided.

Through realizing the aim, the invention not only provides a new technical scheme for ETEC infection prevention and control, but also can reduce the use of antibiotics, reduce environmental pollution and promote the green sustainable development of the aquaculture.

Specifically, the invention provides the following technical scheme.

In a first aspect of the present invention, there is provided a novel functional lactic acid bacterium expressing ETEC antigen, wherein the novel functional lactic acid bacterium expressing ETEC antigen uses lactobacillus plantarum NC8 as a host bacterium and pg612.1 as an expression vector, and the pg612.1 expression vector comprises a gene FaeC of a unit of ETEC bacterium Mao Ya or a gene LngC of an outer membrane protein subunit (hereinafter referred to as FaeC gene or LngC gene).

In an embodiment of the present invention, the expression of the novel functional lactic acid bacteria expressing ETEC antigen produces ETEC pilus antigen FaeC or outer membrane protein LngC (hereinafter abbreviated as FaeC protein or LngC protein). In some embodiments of the invention, the FaeC gene has a nucleotide sequence shown as SEQ ID NO. 1. In some embodiments of the invention, the LngC gene has a nucleotide sequence shown as SEQ ID NO. 2. In some embodiments of the invention, the FaeC protein has the amino acid sequence shown in SEQ ID NO. 3. In some embodiments of the invention, the LngC protein has the amino acid sequence shown in SEQ ID NO. 4.

In a second aspect of the present invention, there is provided a composition comprising the novel functional lactic acid bacterium expressing an ETEC antigen described in the first aspect above. In some embodiments of the invention, the composition comprises a combination of recombinant lactobacillus plantarum with lactobacillus plantarum NC8 as a host bacterium, with ppg612.1 as an expression vector, and recombinant lactobacillus plantarum comprising FaeC genes of ETEC in the ppg612.1 expression vector, and lactobacillus plantarum NC8 as a host bacterium, with ppg612.1 as an expression vector, and recombinant lactobacillus plantarum comprising LngC genes of ETEC in the ppg612.1 expression vector.

In a third aspect of the present invention there is provided a product comprising a novel functional lactic acid bacterium expressing an ETEC antigen as described in the first aspect above or a composition as described in the second aspect above. In an embodiment of the invention, the product is a probiotic, a pharmaceutical preparation or a feed additive.

In some embodiments of the invention, the pharmaceutical formulation is a biologic formulation. In some embodiments of the invention, the biological agent is a vaccine, in particular an oral live vector vaccine. In some embodiments of the invention, the product or composition comprises at least 1X 10 ⁹ CFU/mL of recombinant Lactobacillus plantarum.

In a fourth aspect of the present invention, there is provided a method of constructing the novel functional lactic acid bacterium expressing the ETEC antigen according to the first aspect described above, comprising obtaining FaeC gene or LngC gene fragment of ETEC, constructing the gene fragment into pPG612.1 expression vector, and introducing the pPG612.1 expression vector containing the gene fragment into Lactobacillus plantarum NC8.

In some embodiments of the invention, step (1) obtains the gene fragment by a PCR method. In some embodiments of the invention, the gene fragment is digested and ligated with ppg612.1 expression vectors to obtain recombinant expression vectors. In some embodiments of the invention, step (3) introduces the expression vector into lactobacillus plantarum NC8 by an electrotransformation method.

In some embodiments of the invention, the construction method further comprises a verification step of detecting integration of the gene of interest and expression of the protein of interest. Specifically, in one embodiment of the invention, the construction method comprises the following steps of (1) obtaining FaeC gene or LngC gene fragment of ETEC by using a PCR method, (2) cloning the gene fragment into a pEasyBlunt vector, (3) carrying out double enzyme digestion on a pEasyBlunt recombinant vector and a pPG612.1 vector, (4) connecting a target gene fragment subjected to double enzyme digestion with the pPG612.1 vector to obtain a recombinant expression vector, (5) transforming the recombinant expression vector into escherichia coli MC1061 for amplification, and (6) electrically transforming the amplified recombinant expression vector into lactobacillus plantarum NC 8.

In a fifth aspect of the present invention there is provided the use of a novel functional lactic acid bacterium expressing an ETEC antigen as described in the first aspect above or a composition as described in the second aspect above or a product as described in the third aspect above for the preparation of a product having at least one of (1) a product for preventing or treating an ETEC infection, (2) an immunomodulatory product, (3) an anti-inflammatory product, and (4) an enterally protected product.

According to the experiments of the present invention, the novel functional lactic acid bacteria expressing the ETEC antigen or the combination thereof of the present invention can effectively prevent ETEC infection of piglets, including reducing ETEC infection rate, alleviating infection symptoms and improving growth performance.

According to the experiments of the invention, the novel functional lactic acid bacteria expressing ETEC antigens or the combination thereof can realize immunoregulation, particularly enhancement of immunity, which comprises activating T cells in Spleen (SP), mesenteric Lymph Node (MLN) and intestinal lamina propria lymph node (LPL) of piglets, activating T cell immune response, promoting proliferation and differentiation of CD4 ⁺IFN^-γ+、CD4⁺IL-4⁺ and dendritic cells, improving intestinal SIgA level and enhancing mucosal immunity.

According to the experiments of the present invention, the novel functional lactic acid bacteria expressing ETEC antigen or a combination thereof according to the present invention have remarkable anti-inflammatory ability, which is shown to inhibit the expression of inflammatory factors (TNF-alpha, IL-6 and IL-1 beta), reduce the expression of inflammatory factor proteins in serum (reduce the levels of TNF-alpha, IL-6 and IL-1 beta in serum), improve the inflammatory status of the intestinal tract and regulate the immune balance.

According to the experiment of the invention, the novel functional lactic acid bacteria expressing ETEC antigen or the combination thereof has the intestinal tract protecting effect, and can protect intestinal tract villus structure, reduce tissue injury, maintain tissue integrity and improve intestinal tract barrier function according to pathological section results, thereby maintaining intestinal tract health.

In some embodiments of the invention, the product is a probiotic, a pharmaceutical formulation or a feed additive.

Compared with the prior art, the recombinant lactobacillus plantarum NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC constructed by the invention have the advantages of obvious effect in the aspect of preventing ETEC infection. The engineering bacteria not only can activate intestinal immune response, regulate inflammatory response and protect intestinal structures, but also have the characteristics of safety, convenience, economy, feasibility and the like, and an innovative ETEC prevention and control scheme is provided for pig industry.

In the aspect of immune activation, the recombinant lactobacillus plantarum disclosed by the invention can effectively activate T cells in LPL, SP and MLN of piglets, promote proliferation of CD4 ⁺IFN^-γ⁺T Cell、CD4⁺IL^-4⁺ T cells and dendritic cells, improve the level of sIgA in intestinal tracts and strengthen the immune function of local mucous membranes. Especially, the mixed application of the two engineering bacteria can generate a synergistic effect, the effect on immune cells and inflammatory factors is better than that of a single engineering bacteria, and a better immune protection effect is provided.

In the aspects of inflammation regulation and tissue protection, the recombinant lactobacillus plantarum of the invention obviously reduces the expression level of proinflammatory factors such as TNF-alpha, IL-6, IL-8 and the like in intestinal tissues and the protein level of inflammatory factors such as TNF-alpha, IL-6, IL-1 beta and the like in serum. Meanwhile, the engineering bacteria can relieve intestinal injury caused by ETEC infection, protect intestinal villus structural integrity and maintain intestinal barrier function, thereby effectively preventing ETEC infection.

In the aspect of application value, the invention adopts lactobacillus as a carrier, avoids the use risk of antibiotics, does not contain endotoxin, and has good biological safety. The oral administration mode is adopted, the operation is simple and convenient, the injection is not needed, the stress reaction can be reduced, and the preparation method is particularly suitable for large-scale cultivation application. The convenient use mode ensures that the utility model has better practicability and popularization value.

In the aspect of economic benefit, the recombinant lactobacillus plantarum provided by the invention can obviously reduce the incidence rate and death rate of ETEC infection and improve the growth performance of animals. Meanwhile, the use of antibiotics and disease loss are reduced, so that the cultivation cost can be effectively reduced, and the economic benefit is improved. As an antibiotic substitute, the application of the technology has important significance for promoting the green development of the pig industry.

In conclusion, the recombinant lactobacillus plantarum developed by the invention has remarkable advantages in the aspects of immune activation, inflammation regulation, safety, economic benefit and the like. As an innovative ETEC prevention and control means, the novel ETEC prevention and control method not only can effectively prevent diseases and improve the breeding benefit, but also can promote the green sustainable development of the pig industry, and has important practical application value and industrial development significance.

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 shows the results of gel electrophoresis of gene cloning and recombinant plasmid construction. Wherein (a) the result of PCR amplification of the target gene is that M: DNA MARKER DL 2000,2000; 1: lngC;2: faeC. (b) pEasyBlunt-FaeC and pEasyBlunt-LngC PCR, wherein M: DNA MARKER DL 2000;1: faeC;2: lngC. (c) pPG612.1, wherein M is DNA MARKER DL 2000:2000, and 1:pPG612.1. (d) pEasyBlunt-FaeC and pEasyBlunt-LngC, wherein M is DNA MARKER DL 2000, 1:EasyBlunt-FaeC, and 2:pEasyBlunt-LngC. (e) PCR verification of recombinant plasmids pPG-FaeC and pPG-LngC, wherein M is DNA MARKER DL 2000, 1 is negative control, 2 is pPG-LngC, and 3 is pPG-FaeC. (f) Double enzyme digestion verification of recombinant plasmids pPG-FaeC and pPG-LngC, wherein M is DNA MARKER DL 5000:5000, 1:pPG-LngC, and 2:pPG-FaeC.

FIG. 2 shows the results of PCR verification of recombinant plasmids and Western blot verification of recombinant strains. Wherein (a) PCR verification of pPG-FaeC and pPG-LngC plasmids in NC8, wherein M is DNA MARKER DL 2000:2000, 1 is negative control, 2 is pPG-FaeC, and 3 is pPG-LngC. (b) Verification of NC8-pPG612.1-FaeC and NC8-pPG612.1-LNGC WESTEN bloc, wherein M is a protein Marker, 1 is a NC8 control, 2 is a NC8-pPG612.1-FaeC, and 3 is a NC8-pPG612.1-LngC.

Fig. 3 shows the results of CD4 ⁺ T Cell flow cytometry detection in piglet LPL.

Fig. 4 shows the results of CD4 ⁺IL-4⁺ T Cell flow cytometry detection in piglet LPL.

Fig. 5 shows the results of CD8 ⁺ T Cell flow cytometry detection in piglet LPL.

FIG. 6 shows the results of flow cytometry detection of CD21 ⁺ DCs in piglet LPL.

Fig. 7 shows the results of CD4 ⁺IFNγ⁺ T Cell flow cytometry detection in piglets SP.

Fig. 8 shows the results of CD4 ⁺IL4⁺ T Cell flow cytometry detection in piglets SP.

Fig. 9 shows the results of CD4 ⁺IFNγ⁺ T Cell flow cytometry detection in piglet MLN.

FIG. 10 shows the results of detection of the transcript levels of TNF- α, IL-6, IL10 and IL-8 in the small intestine of piglets.

FIG. 11 shows ELISA measurements of TNF-alpha (A), IL-6 (B), IL-1 beta (C) and sIgA (D) in piglet manure after challenge.

Fig. 12 shows piglet jejunal histopathological images of PBS group (a), ETEC group (B), MIX group (C), lngC group (D), faeC group (E), PPG group (F).

Detailed Description

The invention will be further elucidated with reference to specific embodiments. It is to be understood that these examples are provided only for illustrating the present invention and are not to be construed as limiting the scope thereof. Experimental methods not specifically noted in the examples were generally performed according to conventional conditions or conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein shall have the meaning familiar to one of ordinary skill in the art. Unless otherwise indicated, all reagents or materials used in the present invention may be obtained by conventional means and used according to conventional methods or product specifications in the art. In addition, any content similar or equivalent to the described methods or materials may be used in the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

Example 1 construction and verification of novel functional lactic acid bacteria against ETEC

1.1 Materials and methods

1.1.1 Strains and plasmid vectors the strains used in this example include plasmid cloning host E.coli MC1061 and protein expression vector L. plantarumstrain NC8, which were maintained and supplied by the university of Jilin agricultural microecological formulation engineering research center. Plasmids required for the experiment include pPG-612, pEasyBlunt (both saved and supplied by the institute of microecological preparation engineering, jilin university) and pUC-FaeC, pUC-LngC (both purchased from Biotechnology Co., ltd.) each carrying the gene of interest. Wherein the nucleotide sequence of FaeC gene is shown as SEQ ID NO.1, and the nucleotide sequence of LngC gene is shown as SEQ ID NO. 2.

1.1.2 The main reagents required in the embodiment comprise restriction enzymes, DNA MARKER (DL 2000 and DL 5000) and 10× Loading Buffer which are all purchased from TAKARA official network, BCA protein concentration determination kit which is purchased from Biyun biological Co., ltd, ECL developing solution and 80 kDa Prestained Protein Marker which are purchased from ProteinTech company, beef powder, yeast extract and tryptone which are purchased from Beijing Soy Bao technology Co., ltd, SDS-PAGE loading buffer which is purchased from Yam biological Co., SDS-PAGE fast electrophoresis buffer, western Blot ice-bath free fast transfer membrane solution, TBS and PBS buffer which are all purchased from Wohan Sier technology Co., ltd, plasmid miniextraction kit which is purchased from Omega biological company, and T4 DNA ligase which is purchased from full gold biological technology Co.

1.1.3 The apparatus required for this example included a PCR instrument and an inverted fluorescence microscope DMi8 from Leaka, germany, a gel imaging analysis system and an electroporation system from BIO-RAD, a PCR instrument, a centrifuge and a pipettor from Eppendorf, germany, a biochemical incubator HERACELL, 240i from America Thermo Scientific, a full-automatic autoclave from Sigma, germany, a 2mm electric shock cup from BIO-RAD, and a metal bath from TANGEN.

1.1.4 Reagents and configurations required for assays

(1) Configuration of culture Medium

Weighing 10.0 g peptone, 10.0 g yeast extract and 5.0 g sodium chloride, dissolving with double distilled water, fixing volume to 1L, packaging into conical flask and test tube, and sterilizing at 121deg.C for 15min. And (5) preserving at room temperature after sterilizing. When preparing solid LB culture medium, adding 15g bacteriological agar powder per L culture medium, autoclaving, cooling moderately, and pouring into bacterial culture dish.

The MRS liquid culture medium formula comprises weighing 10.0 g beef extract powder, 10.0 g peptone, 20.0 g glucose, 5.0 g sodium acetate trihydrate, 0.05 g yeast extract, 2.0 g ammonium citrate, 2.0 g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 0.05 g manganese sulfate, adding 1 mL Tween-80, dissolving with double distilled water, fixing volume to 1L, packaging into conical flask and test tube, and sterilizing at 121deg.C for 15min. And (5) preserving at room temperature after sterilizing. When preparing solid LB culture medium, adding 15g bacteriological agar powder per L culture medium, autoclaving, cooling moderately, and pouring into bacterial culture dish.

(2) TAE (50×) nucleic acid electrophoresis formulation A242 g Tris base was weighed, added with 57.1 mL acetic acid and 100mL EDTA (0.5 mol/L), dissolved in double distilled water and sized to 1L. The solution needs to be fully dissolved and then stored at room temperature.

(3) Agarose gel (1%) was prepared by weighing 1 g agarose, adding to 100mL 1 XTAE buffer, heating to dissolve completely, cooling to about 60℃and adding 5. Mu.L nucleic acid dye, mixing thoroughly, pouring gel, and solidifying at room temperature.

(4) The cleaning buffer was formulated by adding 10mL potassium acetate (1 mol/L, pH 7.0), weighing 11.8 g calcium chloride, 4.0 g manganese chloride and 2.0 g magnesium chloride, dissolving with double distilled water and metering to 1L. The pH value of the solution is regulated to 6.4, and the solution is split charging after filtration and sterilization by a disposable filter with the aperture of 0.45 mu m and is stored at the temperature of 4 ℃ for standby.

(5) The storage buffer was prepared by adding glycerol to the wash buffer to a final concentration of 25% (V/V), and storing the mixture at 4℃after dispensing using a disposable Nalgene filter (0.45 μm pore size).

(6) NC8 competent preparation of washing buffer preparation 0.5mol/L EDTA was first prepared, filtered and added to solution I, and stored at 4℃for further use. Wherein, the configuration of the solution I and the solution II is as follows:

the preparation of the solution I comprises weighing 34.23 g sucrose, adding 20 mL glycerin, dissolving with double distilled water, fixing volume to 1L, autoclaving at 121deg.C for 15 min, and storing at 4deg.C for use.

Solution II is prepared by taking 1L of solution I, adding filtered 0.5 mol/L EDTA to a final concentration of 0.05 mol/L, mixing well, and preserving at 4 ℃ for later use.

(7) Western Blot separation gel (10%) was prepared by taking 3.3 mL of 30% Acr/Bis (29:1), 2.5 mL 1.5M Tris-HCl (pH 6.8), 100. Mu.L of 10% SDS, 100. Mu.L of 10% PAGE gel coagulant, 10. Mu.L of PAGE gel coagulant, adding 4 mL double distilled water, gently mixing, and pouring the gel immediately.

(8) Western Blot concentrate (5%) was prepared by taking 0.83 mL of 30% Acr/Bis (29:1), 0.625 mL 1M Tris-HCl (pH 6.8), 50. Mu.L of 10% SDS, 75. Mu.L of 10% PAGE gel coagulant, 7.5. Mu.L of PAGE gel coagulant, adding 3.42: 3.42 mL double distilled water, gently mixing, and pouring immediately.

(9) The preparation method of 80% glycerol comprises taking 80 mL glycerol, metering to 100 mL with double distilled water, stirring, sealing the bottle mouth, and autoclaving at 121deg.C for 15 min. After the solution cooled, it was stored at 4 ℃ for further use.

1.2 Experimental method

1.2.1 Construction and identification of engineering bacteria

1.2.1.1 The acquisition and synthesis of the target gene is based on the ETEC FaeC gene sequence (AF 450247.2) and ETEC Lngc gene sequence (LR 883052) reported in GenBanK database. Primer design was performed using PRIMER PREMIER 5.0.0 software. Primers were synthesized by Shanghai (Inc.) of biological engineering. Primer List FaeC-F (SEQ ID NO: 5), faeC-R (SEQ ID NO: 6), lngc-F (SEQ ID NO: 7), lngc-R (SEQ ID NO: 8).

1.2.1.2 Preparation of template DNA ETEC strains were removed from the freezer at-80℃and streaked on LB solid medium and cultured at 37℃for 10h. Single colonies were picked up in LB liquid medium without resistance, after culturing for 12h at 37℃they were boiled in boiling water for 10min, centrifuged at 10,000rpm for 15min at 4℃and the supernatant was used as DNA template for preservation at-20 ℃.

1.2.1.3 Construction of cloning vectors pEasyBlunt-FaeC and pEasyBlunt-LngC, cloning FaeC and LngC fragments obtained by PCR amplification to pEasyBlunt to obtain pEasyBlunt-FaeC and pEasyBlunt-LngC recombinant plasmids respectively, and carrying out DNA sequencing and identification on the recombinant plasmids after PCR amplification.

1.2.1.4 Cloning of vector and target Gene Ppg612.1, pEasyBlunt-FaeC and pEasyBlunt-LngC were digested simultaneously, and the digestion reaction system was prepared by taking 25. Mu.L of plasmid, adding 2. Mu.L of Nco I/Asc I and 2. Mu.L of EcoR V/Xho I restriction enzyme, 5. Mu.L of buffer, 16. Mu.L of double distilled water, mixing, and reacting at 37℃for 6h. 2. Mu.L of the digested product was subjected to agarose gel electrophoresis (150V, 20 min) to verify the digestion effect, and after confirmation of the digestion, the gel was recovered.

1.2.1.5 Ligation of expression vector and target gene sequence the recovered linear vector was ligated to the target sequence by T4 DNA ligase. The reaction system was ligated by taking 2. Mu.L of the linear vector recovered product, 5. Mu.L of the target sequence recovered product, 1. Mu.L of 10 XT 4 DNA ligase buffer and 1. Mu. L T4 DNA ligase, adding 1. Mu.L of double distilled water, mixing, and reacting at 16℃overnight. The next day the transformation experiment was performed.

Preparation of competent of 1.2.1.6E.coli M1061 (1) MC1061 was streaked on LB solid medium surface using an inoculating loop, and cultured in a37℃bacterial incubator for 14-16h. (2) MC1061 single colonies were picked the next day in 5mL LB medium and shake-cultured at 37℃for 14-16h. (3) The mixture was inoculated into 100mL of LB at a ratio of 1.5%, and shake-cultured at 37℃until the OD600 became about 0.5 (2.5-3 h). (4) The bacterial liquid was packed into sterile 50mL centrifuge tubes, placed on ice for 10min, centrifuged at 5000 rpm for 10min at 4℃and the supernatant discarded. (5) The cells were resuspended in 10mL of pre-chilled wash buffer, allowed to stand on ice for 10min, centrifuged at 5,000 rpm for 10min at 4℃and the supernatant discarded. (6) The cells were resuspended in 10mL of pre-chilled wash buffer, placed on ice for 20min, centrifuged at 4℃and 5,000 rpm for 10min, and the supernatant discarded. (7) The cells were resuspended in pre-chilled storage buffer and placed on ice for 30min. The competent cells were aliquoted into pre-chilled 1.5mL EP tubes at 50. Mu.L/tube, snap frozen with liquid nitrogen and stored at-80 ℃. All the above operations are performed in an ultra clean bench to prevent contamination.

1.2.1.7 Ligation product transformation ligation products were transformed into MC1061 competent cells by heat shock transformation. The method comprises the steps of (1) taking 5 mu L of connection products and 50 mu L of competent cells, gently mixing, incubating on ice for 30min hours, (2) preheating a water bath to 42 ℃, carrying out heat shock for 90 seconds, carrying out cooling on ice for 2 minutes, (3) adding 400 mu L of LB liquid medium, carrying out shaking table culture at 37 ℃ for 2 hours at 200rpm, (4) centrifuging at 5 000rpm by a centrifuge for 5 minutes, taking 100 mu L of coated plates, (5) carrying out normal culture for 30 minutes in a 37 ℃ bacterial incubator, carrying out inverted culture (12-14 hours), and after single colony appears, picking up a single colony to carry out shaking table culture at 37 ℃ in a 5mL of LB liquid medium test tube, and storing the strain for subsequent experiments.

1.2.1.8 The extraction and primer synthesis of the recombinant expression plasmid in MC1061 were carried out by extracting the plasmid using the plasmid miniprep kit as follows. (1) E.coli with plasmids was inoculated into 5mL LB (containing chloramphenicol) and cultured at 37℃for 12-16 h on a shaker. 5.0mL of the bacterial liquid was collected by centrifugation at 10,000Xg for 1min at room temperature. (2) discarding the medium. Adding 250 mu L SolutionI/RNaseA mixture, blowing to suspend the cells completely, adding 250 mu L Solution II, mixing for 4-6 times with gentle inversion, and performing lysis reaction for no more than 5min. (3) Add 350. Mu.L Solution III, gently invert 7-9 times to white floc, centrifuge 10min at 10 000 Xg. (4) The supernatant was transferred to HiBind DNA conjugate column fitted with a 2mL collection tube, centrifuged at 10,000Xg for 1min at room temperature, and the filtrate in the collection tube was discarded. (5) The combined column was collected into a collection tube, 500. Mu.L of HB Buffer was added, and the mixture was centrifuged under the above-mentioned conditions, and the filtrate was discarded. (6) The column was reloaded into the collection tube, 700. Mu. L DNA Wash Buffer was added, centrifuged as above, and the filtrate was discarded. Note that the concentrated DNA Wash Buffer was added with absolute ethanol at the label tip prior to use. (7) discarding the filtrate, and repeating the step 6 once. (8) The filtrate was discarded, the column was fitted with a recovery header, centrifuged at 10,000Xg, and the column was emptied for 2min to spin-dry the column matrix. (9) The column was placed in a 1.5mL centrifuge tube, 30-50. Mu.L of the solution Buffer was added to the column matrix, and the column was allowed to stand for 2min and centrifuged at 10,000Xg for 1min to elute the DNA. The extracted plasmid is preserved at-20 ℃ for PCR and enzyme digestion identification. The primer sequence and the restriction enzyme site are shown in SEQ ID NO. 9, the restriction enzyme site Nco I (primer F) of the pPG612 plasmid and the restriction enzyme site Asc I (primer R) of the pPG612 plasmid are shown in SEQ ID NO. 10.

1.2.1.9 Recombinant expression plasmid PCR identification

The PCR reaction system is that 1 mu L of template plasmid, 1 mu L of forward primer (F) and 1 mu L of reverse primer (R) are taken, 25 mu L of 2× PRIMESTAR Mix and 22 mu L of double distilled water are added, and the mixture is rapidly centrifuged and then placed on a PCR instrument for amplification reaction.

The PCR reaction conditions were 94℃for 5min, 94℃for 40 s min, 60℃for 40 s and 72℃for 4 min cycles, 72℃for 10 min and 4℃for final extension. 10. Mu.L of the PCR product was subjected to agarose gel electrophoresis.

1.2.1.10 Double digestion identification of pPG612.1-FaeC and pPG612.1-LngC recombinant expression plasmids, wherein the double digestion reaction system is the same as that in 1.2.1.4. And (3) performing enzyme digestion for 6 hours at 37 ℃, taking 2 mu L of enzyme digestion products, performing agarose gel electrophoresis under 170V for 20min, and comparing the sizes of the strips, wherein the method is the same as that of the instruction book.

1.2.1.11 Sequencing and identifying the correct plasmid sample by PCR and enzyme digestion, sequencing and identifying by the biological engineering Co., ltd, and drawing a plasmid map by using SnapGen software.

1.2.1.12 L, plantarum strain NC < 8 > competent preparation, (1) inoculating NC8 strain into 5mL MRS culture medium, and anaerobic culturing in 37 ℃ incubator for 14-16 h. (2) Subculture-1% was inoculated in 100mL of MRS medium containing 2% GLy. OD values were measured at 2h intervals (visual inspection was possible, frequent testing was not required, and the final OD value was 0.3-0.5). (3) Centrifugation, in which the cells were collected by packing into 50mL sterile tubes, centrifuging at 4℃and 5,000rpm for 10min, and discarding the supernatant. (4) The cells were suspended in 20mL of the solution I at 4℃and 5,000rpm for 10min, and collected. (5) The cells were collected by washing with 20mL of solution II 1 time at 4℃and 5,000rpm for 10 min. (6) The cells were suspended in 20mL of the solution I, allowed to stand on ice for 15min at 4℃and 20min at 5,000rpm, and collected. (7) repeating the step 6, and sufficiently washing the EDTA. (8) The cells were suspended in 1mL of solution I, dispensed at 100. Mu.L/tube, snap frozen in liquid nitrogen, and stored at-80 ℃.

1.2.1.13 Electrotransformation of recombinant expression plasmids the correctly sequenced recombinant plasmids were transformed into NC8 competent cells by means of electrotransformation. (1) NC8 competent cells were thawed on ice, 10. Mu.L of plasmid was added to the competence, gently mixed, and incubated on ice for 5min. (2) The cuvette was pre-chilled on ice, and the plasmid competent mixture was transferred to the cuvette (to avoid air bubbles as much as possible) and ice-bathed for 10min. (3) electric shock conditions of 2kV,200Ω and 25. Mu.F. (4) After completion of the electric shock, 900. Mu.L of MRS medium (medium preheated 37℃in advance) was rapidly added to the shaking table at 37℃and 200rpm, and cultured for 60 minutes. (5) 100 mu L of bacterial liquid is evenly coated on an MRS resistant agar plate, and is cultivated for 1h in an upright way and is cultivated for 12-16h in an inverted way to see single colony. (6) Single colonies were picked in 10mL MRS resistant medium, shake incubated at 37℃for 12-16h at 200 rpm. The strain is then preserved for subsequent testing.

1.2.1.14 And (3) identifying NC8 recombinant expression plasmids, namely extracting plasmids in engineering bacteria, carrying out PCR identification on the recombinant plasmids by the same method, and carrying out the same operation step 1.2.1.9.

1.2.2 Engineering bacteria protein expression verification

1.2.2.1 Determination of engineering bacteria growth curve 100L was inoculated in 5mL MRS broth after engineering strain activation, 37℃and 200rpm shaking overnight. The following day was inoculated into 100mL of MRS liquid medium at a 1% inoculum size, and shaking culture was performed at 37℃and 200 rpm. OD600 values were measured and recorded every 2 hours in 2mL of bacterial liquid and strain growth curves were drawn by Prisme for 24 hours of monitoring.

1.2.2.2 Engineering bacteria protein expression identification 100. Mu.L engineering bacteria were inoculated into MRS broth and shaken overnight at 37℃and 200 rpm. The following day was inoculated at 1% inoculum size into 100mL MRS medium containing 2% lactose, and shaking-cultured overnight at 37% 200 rpm. The supernatant was centrifuged off, washed twice with sterile PBS and resuspended in PBS buffer for use. Protein concentration was determined by BCA method, 400. Mu.L of the resuspended bacteria solution was mixed with 100. Mu.L of 5 XSDS PAGE Buffer and boiled to 10 min.12 Centrifugation at 000 rpm at 1 min and collection of supernatant for storage at-40 ℃.

1.2.2.3 Westen blot detection, namely verifying the expression condition of the engineering bacteria protein by applying Westen blot technology. The method comprises (1) detecting the leakage of the glass plate with ultrapure water after the glue plate is assembled, detecting leakage for 15min, adding 10% of the prepared separating glue (lower glue) into the glass plate, sealing with absolute ethyl alcohol, waiting for 20min for solidification, removing absolute ethyl alcohol, sucking residual absolute ethyl alcohol with filter paper, adding concentrated glue (upper glue) into the electrophoresis comb, and waiting for solidification. Setting gel in electrophoresis tank after solidification, adding SDS-PAGE fast electrophoresis buffer, adding 20. Mu.L treated protein sample and 5. Mu.L Marker into the gel hole, electrophoresis under 200V (30 min) bromophenol blue to the lower edge of glass plate, and transferring to obtain gel, and draining sponge, filter paper, PVDF film, gel, filter paper and sponge in the order of positive electrode to negative electrode. Gently removing bubbles, placing in a transfer nip, placing in ice together with an electrophoresis tank, (6) transferring film under constant flow of 400mA for 30min, (7) sealing, placing in sealing liquid, shaking slowly at room temperature for 2h, and (8) adding anti-His primary antibody (diluted with TBST), and standing overnight at 4deg.C. The PVDF membrane is rinsed three times with TBST for 10min each time, and (9) the secondary antibody is incubated by adding enzyme-labeled secondary antibody (diluted with TBST) and shaking slowly for 1h at room temperature. The PVDF membrane was rinsed three times with TBST for 10min each time (10) the membrane was wetted with ECL luminophor and placed in an AI600 imaging system for exposure to observe Western blotting.

1.3 Results

1.3.1 Construction of engineering bacteria

And 1.3.1.1, obtaining and synthesizing the FaeC and LngC gene sequences through PCR, wherein the sizes of FaeC and LngC gene fragments are 546bp and 414bp respectively and meet the expected sizes as shown in (a) of figure 1.

1.3.1.2 Construction and identification of pEasyBlunt-FaeC and pEasyBlunt-LngC cloning vectors the gene sequences of the two entries finally obtained by fusion PCR were ligated to pEasyBlunt vector, faeC, lngC were identified by PCR, and the results were shown in FIG. 1 (b) with the expected size bands at 546bp, 414 bp.

1.3.1.3 And (3) verifying the expression vector and the target gene, namely respectively carrying out double enzyme digestion detection on the target gene sequence on the pPG612 vector plasmid pEasyBlunt. As a result, as shown in FIGS. 1 (c) and (d), the band of interest was visible at 4233bp (c), 546bp, 414bp (d), and was consistent with the expected result.

1.3.1.4 Identification of recombinant expression plasmid in MC1061

And 1.3.1.4.1, carrying out PCR identification on the recombinant expression plasmids, namely, carrying out PCR amplification identification by taking suspected positive recombinant expression plasmids pPG-FaeC and pPG-LngC as templates, wherein the result is shown in (e) of fig. 1, and the target band is visible to meet the expected size at the positions of 428 bp and 586 bp.

1.3.1.4.2 Double restriction identification of recombinant expression plasmids pPG-LngC and pPG-FaeC, and the result is shown in figure 1 (f), and the target bands are visible at the positions of 428 bp and 586bp respectively, so that the target fragment is proved to be successfully connected with the expression vector.

1.3.1.4.3 Sequencing and identifying recombinant expression plasmid, namely sequencing the recombinant plasmid by a biological engineering (Shanghai) Co., ltd, and ensuring correct sequencing result and no base mutation 2.

1.3.1.5 PCR identification of recombinant expression plasmid in NC8

The target gene of the recombinant plasmid in NC8 is amplified by PCR, so as to detect whether the recombinant plasmid is successfully transferred into NC 8. As shown in FIG. 2 (a), the target band was visible at 428 bp and 586bp, which demonstrated that the recombinant plasmid was successfully transferred into vector NC 8.

1.3.2 Protein expression validation

1.3.2.1 Growth curve determination the growth curve determination and colony counting were performed on NC8, NC8-pPG612.1-FaeC, NC8-pPG612.1-LngC, 24h OD ₆₀₀ values were determined and the growth curve was drawn by GRAPHPAD PRISM software. By monitoring the growth curve, the engineering bacteria and the original bacteria have no obvious difference in the stationary phase.

1.3.2.2 Engineering bacteria Westen blot, specific proteins are observed at 51kDa and 40kDa respectively through Westen blot experiment detection, which proves that NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC can be successfully induced to express corresponding proteins through lactose MRS culture, and specific proteins are observed at 51kDa and 40kDa respectively, as shown in (b) of FIG. 2.

Example 2 evaluation of the immune Effect against novel functional lactic acid bacteria against ETEC.

2.1 Materials and methods

2.1.1 The test strains NC8-pPG612.1, NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC (constructed in example 1) were novel functional lactic acid bacteria, and ETEC was supplied by the institute of microecological preparation engineering, jilin university.

2.1.2 Experimental animals 18 piglets were kept at 3 days of age.

2.1.3 The main reagents were nylon screen (Solarbio Co.), FBS erythrocyte lysate, DAPI dye solution, fetal bovine serum, triton X-100 and BSA all purchased from Biyunshan BioCo., ltd., 4% paraformaldehyde and RPMI-1640 medium from biosharp Co., and Anti-pig CD4, anti-pig CD8 and Anti-pig IgA antibodies all purchased from BD (U.S.).

2.1.4 The main instrument, namely the flow cytometer, is purchased from BD company in the United states, and the full-automatic embedding machine and the full-automatic paraffin microtome are all purchased from Leica company.

2.2 Experimental method

2.2.1 Engineering bacteria plate count (1) strain is taken from-80 ℃, engineering bacteria are inoculated into 5mL MRS culture medium containing chloramphenicol according to the amount of 1%, and cultured overnight at 200 rpm. (2) The resuscitated strain was inoculated in an amount of 1% into 200mL MRS culture containing chloramphenicol, and shake-cultured at 37 ℃ until the OD ₆₀₀ was 1. (3) The cultured bacterial liquid is diluted to 10 ^-5、10^-6、10^-7 in proportion, 100 mu L of diluted bacterial liquid is evenly coated in MRS solid culture medium, three repetitions are carried out on each ladder, anaerobic culture is carried out for 24 hours at 37 ℃, and the average value is taken for colony counting.

2.2.2 The experimental animals are grouped and immunized according to the scheme, piglets are randomly divided into 6 groups, 3 piglets are respectively PBS group (A group), ETEC group (B group), NC8-pPG612.1 (C group), NC8-pPG612.1-FaeC (D group), NC8-pPG612.1-LngC (E group) and engineering bacteria mixed group (F group).

The immunization schemes of the groups are that 1mL PBS is orally taken as a control in the group A, 1mL ETEC is orally taken in the group B, 1mL NC8-pPG612.1 (1X 10 ⁹ CFU/mL) is orally taken in the group C, 1mL NC8-pPG612.1-FaeC (1X 10 ⁹ CFU/mL) is orally taken in the group D, 1mL NC8-pPG612.1-LngC (1X 10 ⁹ CFU/mL) is orally taken in the group E, the F group is an engineering bacteria mixed immunization group, and 1mL engineering bacteria mixed solution (the total bacterial amount is 1X 10 ⁹ CFU/mL, wherein, three bacterial amounts of NC8-pPG612.1, NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC are mixed).

And according to the immunization and toxicity counteracting scheme, the bacterial content of engineering bacteria OD ₆₀₀ =1 is obtained through plate counting, and the bacterial concentration is adjusted for immunization. Each piglet was lavaged 1X 10 ⁹ CFU/mL daily, immunized 1 time at 1 day intervals, and immunized 7 times in total. The toxin is removed by gastric lavage at 1 day interval after the last immunization, and the toxin removing dosage is 1mL ETEC (5.34×10 ⁷ CFU/mL) per kilogram of body weight. The observation was continued for 7 days, and the body weight was counted daily and the body weight increase rate was calculated. After 7 days, the body weight was weighed, and after blood collection, all piglets were sacrificed and samples were collected.

2.2.3 Serum collection all piglets were sacrificed 7 days after the last immunization. First, the forelimbs and hindlimbs of piglets were fixed, and blood was collected by pricking the skin at an angle of 45 ° using a 5mL syringe at the anterior fossa of the left collarbone (jugular vein). Blood was collected with a 5mL centrifuge tube, left to stand in a 37 ℃ incubator for 1 hour, pre-cooled by centrifuge at 4 ℃, at 4 000rpm for 15min, and the supernatant collected and stored in a-80 ℃ refrigerator for later experiments.

2.2.4 Collecting stool supernatant, namely, bleeding and killing jugular veins of piglets, dissecting abdominal cavity after the sacrifice, collecting stool at jejunum section, placing the stool in a 5mL centrifuge tube, adding PBS vortex with triple volume according to the volume of the stool, adding protease inhibitor, pre-cooling the centrifuge at 4 ℃, centrifuging at 4000rpm for 15min, and taking the stool supernatant to be stored in a refrigerator at-80 ℃ for later experiments.

2.2.5 Flow cytometry detection, taking piglet Spleen (SP), mesenteric Lymph Node (MLN) and intestinal lamina propria lymph node (LPL).

2.2.5.1 Single cell suspensions were prepared (1) 200 mesh copper mesh was placed in 35nm cell culture dishes and 1mL of complete medium was added at 4℃for use. (2) Tissue (SP, MLN, LPL) is placed in a copper mesh, tail part of a 1mL syringe is used for grinding, the tissue is ground into a homogenate state, and tissue stock solution filtered by the copper mesh is sucked into a 1.5mL centrifuge tube. (3) To the SP cell suspension, 1mL of pre-chilled erythrocyte lysate was added, gently mixed, ice-bathed for 5min, 500. Mu.L of PBS was added, 4℃was used, 500 Xg was centrifuged for 5min, the supernatant was discarded, PBS was gently suspended, centrifuged again, and the pellet was gently selected using PBS after centrifugation. (4) SP, MLN, LPL cell pellet was resuspended in 1mL PBS, centrifuged at 500 Xg for 5min at 4℃and the supernatant discarded and 1mL PBS buffer. (5) Each group of cells was diluted with PBS buffer at a multiple ratio, and after dilution, the cells were counted by using a red blood cell counter, and the stock solution concentration was calculated from the number of cells. (6) The desired volume of cell stock was taken, resuspended in 1mL PBS, centrifuged at 500 Xg for 5min at 4℃and the supernatant discarded, resuspended in 100. Mu.L PBS and kept at 4 ℃.

2.2.5.2 DCs detection (1) CD14C-percp-cy5.5 (40-fold dilution) and CD21-percp-cy5.5 (40-fold dilution) were added to 100. Mu.L single cell suspension, and each sample was gently stirred and mixed by 20. Mu.L, and incubated at 4℃for 30min in the absence of light. (2) After the incubation, 1,000. Mu.L of PBS buffer was added, and the mixture was centrifuged at 4℃and 500 Xg for 5min, and the supernatant was discarded. (3) Cells were resuspended in 200. Mu.L PBS buffer, the cells were filtered through a nylon screen onto a flow tube, and after mixing with 100. Mu.L PBS buffer, stored at 4℃until on-machine detection.

2.2.5.3 B cell detection (1) CD11 (35-fold dilution) antibody 10. Mu.L was added to 100. Mu.L of single cell suspension, gently swirled and mixed, and incubated at 4℃for 30 min in the absence of light. (2) After the incubation, 500. Mu.L of PBS was added, centrifuged at 1,000 rpm at 5min at 4℃and the supernatant was discarded. (3) To the pellet, 80. Mu. L Fixation/Permeabilization of light-suspended cells were added and incubated at 4℃for 45min in the absence of light. (4) After incubation 150. Mu. L Permeabilization wash was added, centrifuged at 4℃and 500 Xg for 5min, and the supernatant was discarded. (5) IgA-FITC (40-fold dilution) antibody 10. Mu.L was added, gently swirled and mixed, and incubated at 4℃for 45min in the dark. (6) After the incubation, 200. Mu. L Permeabilization wash solution was added, centrifuged at 4℃and 500 Xg at 5min, the supernatant was discarded, 200. Mu.L PBS (7) was added, the cell fluid was filtered using a nylon screen to the flow tube, 100. Mu.L PBS was added, and 4℃was stored and was waiting for the on-machine detection.

2.2.5.4 T cell detection (1) adding CD3-APC (40-fold dilution), CD4-APC (40-fold dilution) and CD8-PE (35-fold dilution) into 100. Mu.L single cell suspension, mixing the three antibodies uniformly, adding 30. Mu.L of each sample, gently blowing and mixing uniformly, and incubating for 45min at 4 ℃ in dark place. (2) After completion, 150. Mu.L of PBS was added to terminate the reaction, the reaction was centrifuged at 4℃and 500 Xg for 5min, the supernatant was discarded, and 200. Mu.L of PBS was added. (3) The cell fluid was filtered through a nylon screen onto a flow tube, mixed well with 100. Mu.L PBS, and stored at 4℃until on-machine detection.

2.2.6 ELISA detection

And (3) detecting the sIgA level in 2.2.6.1 piglet manure, namely taking piglet manure supernatant frozen at the temperature of-80 ℃ and detecting the sIgA content in piglet manure by ELISA.

2.2.6.2 Piglet serum inflammatory cytokines level detection, namely taking piglet blood supernatant frozen at-80 ℃ and adopting ELISA to detect the contents of TNF-alpha, IL-6 and IL-1 beta in piglet feces.

2.2.7 Cytokine mRNA expression level detection

2.2.7.1 Extraction of total RNA of jejunum, namely, taking out frozen jejunum tissues from a refrigerator at-80 ℃ and extracting total RNA of small intestine by adopting a Trizol method. Comprises (1) adding 1mL Trizol into 50mg intestinal tissue, grinding the tissue to homogenate by using an electric grinder, pre-cooling by a centrifuge at 4 ℃, centrifuging at 12000 rpm for 10min, collecting homogenate supernatant, and placing into a 1.5mL centrifuge tube. (2) The sample was allowed to stand at room temperature for 5min, 0.2mL of chloroform was added to each supernatant sample, vortexed for 15s, and allowed to stand at room temperature for 5min. (3) The supernatant samples were centrifuged at 4℃at 12 rpm for 15min, after which the samples were separated and RNA was present in the upper aqueous phase which was then placed in a 1.5mL centrifuge tube, taking care not to suck onto the white film. (4) Adding 2 times of isopropanol, reversing the centrifuge tube, mixing, standing at room temperature for 5min min, standing on ice for 5min, centrifuging at 4 ℃ at 12,000rpm for 10min, and discarding supernatant. (5) 1mL of pre-chilled 75% ethanol (DEPC water formulation) was added to wash, the sample solution was vortexed, centrifuged at 4℃at 12 rpm for 5min, and the supernatant was discarded. (6) The tube lid was opened, left to air dry (without complete drying) in the operating table, 30. Mu.L of DEPC water was added, and the mixture was placed in a 37℃metal bath for 15min to dissolve RNA. -20 ℃ refrigerator preservation for later experiments.

Reverse transcription reaction of 2.2.7.2 Total RNA of intestinal tissue, namely, carrying out reverse transcription on the extracted total RNA, and storing a reverse transcription product at-20 ℃ for detecting the transcription level difference of inflammatory cytokines in the intestinal tract by subsequent qPCR. The reverse transcription reagent was a reverse transcription kit (containing gDNase) from Bio sharp, and the procedure was as described in the specification.

The reverse transcription reaction was performed in a 40. Mu.L system containing 16. Mu.L of 5 XRT Master mix, 4. Mu.L of 20 Xoligo dT & Random Primer, 0.1-4. Mu.g Total RNA, and finally made up to 40. Mu.L with RNASE FREE H ₂ O. The reaction conditions were 37℃for 15-30min,85℃for 5min, and finally 4 ℃.

2.2.7.3 QPCR detection, namely designing qPCR primers according to the gene sequences of pig TNF-alpha, IL-6, IL-8 and IL-10 in NCBI database by taking GapDH as a qPCR reference gene, and referring to the instruction book and the primer sequence of a reaction system ：GapDH-F（SEQ ID NO: 11）,GapDH-R（SEQ ID NO: 12）,TNF-α-F（SEQ ID NO: 13）,TNF-α-R（SEQ ID NO: 14）,IL-6-F（SEQ ID NO: 15）,IL-6-R（SEQ ID NO: 16）,IL-8-F（SEQ ID NO: 17）,IL-8-R（SEQ ID NO: 18）,IL-10-F（SEQ ID NO: 19）,IL-10-R（SEQ ID NO: 20）.

2.2.8 Histopathological detection

2.2.8.1 Embedding and sectioning of tissue (1) fixation with 4% paraformaldehyde, usually for more than 3 days, can start dehydration for a minimum of 2 days. (2) taking materials, namely trimming the two sides of the intestinal canal by using blades. And (3) dehydration, namely gradient dehydration by adopting alcohol. 70% 3h-4h (3.5 h), 80% 3h-4h (3.5 h), 85% 3h-4h (3.5 h), 90% 3h-4h (3.5 h), 95% I (overnight), 95% II (2 h), 100% I (2 h), 100% II (3 h). (4) Transparent, namely setting xylene I and xylene II, wherein the transparent time is 5min each, and the meat skin is transparent to cooked meat skin. (5) And (3) wax dipping and embedding, wherein the wax dipping temperature is 58 ℃, the wax dipping time of the wax 1, the wax 2 and the wax 3 is 30min respectively, and tissues in the wax 3 are put into a wax block tray of an embedding machine to prepare embedding. (6) the thickness of the slices was 3. Mu.m, and the slices were spread in a water bath at 42 ℃. (7) the sections were placed in an 80 ℃ oven for 1h.

2.2.8.2 HE staining, namely, staining the dried slice according to the following procedures of xylene treatment for 2 times (8 min each), gradient alcohol hydration (1 min each for 100% alcohol for 2 times, 95%, 80% alcohol and 70% alcohol for 1min each), hematoxylin staining for 5min and water washing after water washing, 0.5% hydrochloric acid alcohol differentiation for 5s, light ammonia water bluing for 2min and water washing, 0.5% eosin water solution staining for 5min, gradient alcohol dehydration (80% alcohol is rapidly lifted up and down for several times, 95% alcohol for 1min each for 2 times, 100% alcohol for 1min each) and xylene treatment for 2 times (2 min each).

2.2.9 Data statistics and analysis results were tested using One-way ANOVA with significance levels ns (P > 0.05), P <0.05, P <0.01, P <0.001, P <0.0001, using GRAPHICAL PRISM software.

2.3 Results

2.3.1 Body weight change of immunized piglets after ETEC challenge, namely, recording the body weight change on the 1 st day of initial immunization until the piglets are killed after 7 days of challenge. The body weight growth curve shows that the body weight of the ETEC control group shows a significant decrease trend in the ground, the body weight growth trend of the LngC group, the FaeC group and the PBS group after the challenge is significantly higher than that of the ETEC group, and the body weight growth trend is restored to the level before the challenge after 3 days of the challenge.

2.3.2 And (3) analyzing the results of the flow cytometry, wherein in order to detect the immune effect of the engineering bacteria, the experiment is used for immunizing the piglet with the engineering bacteria in a gastric lavage mode. After 7 days of challenge, indices of piglet Spleen (SP), mesenteric Lymph Node (MLN) and intestinal Lamina Propria Lymphocyte (LPL) were examined using flow cytometry, and flow data analysis was performed using FlowJo v10.6.2, data analysis was performed using GRAPHPAD PRISM and mapping was performed.

Influence of 2.3.2.1 engineering bacteria on immune cells in piglet LPL

In order to detect the effect of engineering bacteria on helper T cells in piglet LPL, the experiment performed on CD4 ⁺ T Cell, the results of which are shown in fig. 3. There was no significant difference between groups compared to the ETEC group (P > 0.05). Meanwhile, the test was performed on CD4 ⁺IL-4⁺ T Cell, and the results are shown in FIG. 4. The MIX group showed very significant differences (P < 0.0001) compared to the ETEC group, and both LngC and FaeC groups showed some differences (P < 0.05) compared to the ETEC group. The test was performed on CD8 ⁺ Cell in LPL, and the results are shown in FIG. 5. The MIX group exhibited significant differences (P < 0.05) compared to the ETEC group, while none of the remaining prophylaxis groups had statistical differences (P > 0.05). In the experiment, the expression content of CD21 ⁺ DCs in piglet LPL cells is detected by the flow cell number, as shown in figure 6, compared with ETEC group, MIX group is extremely significant difference (P < 0.001), and LngC is significant difference (P < 0.05).

The research results show that the engineering bacteria administered orally can effectively activate T cells and dendritic cells in the intestinal lamina propria lymphoid tissues of piglets. This process facilitates the recognition and uptake of pathogenic bacteria and their antigens by intestinal antigen presenting cells, as well as the transmission of immune signals.

Influence of 2.3.2.2 engineering bacteria on immune cells in piglet SP in order to detect the influence of engineering bacteria on helper T cells in piglet SP, CD4 ⁺IFNγ⁺ T Cell in this experiment SP was detected. The results are shown in fig. 7, with the ETEC group, MIX group exhibited very significant differences (P < 0.0001), followed by LngC in FaeC groups all exhibited significant differences (P < 0.01). Meanwhile, the experiment examined CD4 ⁺IL4⁺ Cell in SP. The results are shown in fig. 8, comparing ETEC groups, MIX groups are very significantly different (P < 0.001), faeC groups are significantly different (P < 0.01), lngC groups exhibit a certain variability (P < 0.05). The results show that the gastric lavage engineering bacteria can effectively induce the activation of immune cells in the SP of the mice.

Influence of 2.3.2.3 engineering bacteria on immune cells in piglet MLN in order to detect the influence of engineering bacteria on helper T cells in piglet MLN, the experiment detects CD4 ⁺IFNγ⁺ Cell in MLN. The results are shown in fig. 9, in the comparative ETEC group, the LngC group and the MIX group both differ significantly (P < 0.0001), followed by FaeC group (P < 0.01). The results show that the engineering bacteria can induce the cell immune response of the mesenteric lymphadenitis of the piglets.

2.3.3 Detection of inflammatory cytokine transcription levels the transcription levels of the TNF- α, IL-6, IL8 and IL-10 genes of the small intestine of each group of mice were detected by RT-PCR in this experiment, and the results are shown in FIG. 10.

By examining the pro-inflammatory factor TNF- α (fig. 10 (a)) it was shown that TNF- α levels were significantly down-regulated in LngC (P < 0.05), faeC (P < 0.05), MIX (P < 0.001) groups compared to ETEC groups, while there was no significant difference (P > 0.05) between LngC, faeC, MIX groups. By examining the pro-inflammatory factor IL-6 (fig. 10 (B)), it was shown that the levels of IL-6 were significantly down-regulated in LngC (P < 0.01), faeC (P < 0.001), MIX (P < 0.0001) and, in addition, that the IL-6 levels were significantly lower in MIX than in LngC (P < 0.05), faeC (P < 0.01) groups compared to ETEC groups. By examining the anti-inflammatory factor IL-10 (fig. 10 (C)), it was shown that the levels of IL-10 were significantly up-regulated in LngC (P < 0.05), faeC (P < 0.001), MIX (P < 0.0001) groups, and at the same time, the levels of IL-10 were significantly higher than LngC (P < 0.001), faeC (P < 0.05) groups, as compared to ETEC. The results by examining the pro-inflammatory factor IL-8 (fig. 10 (D)) showed that the FaeC group was significantly different (P < 0.01) compared to the ETEC group, and that there was no statistical difference (P > 0.05) for the other groups.

The experiment analyzes the gene expression of key cytokines in the intestinal tract of the immunized piglet through RT-PCR. The results showed that the MIX group was significantly lower in TNF- α and IL-6 expression than the ETEC group. At the IL-10 expression level, each immune group was significantly higher than the ETEC group, indicating good regulation of immune response. From the above results, it was found that the large amount of the pro-inflammatory factor released from the intestinal tract due to ETEC infection was closely related to the inhibition of the anti-inflammatory factor. The prevention by using engineering bacteria can help to reduce the secretion of the pro-inflammatory factors and promote the level of the anti-inflammatory factors, thereby reducing the intestinal inflammation of piglets.

2.3.4 Detection of piglet inflammatory cytokine protein levels the levels of TNF-alpha, IL-6 and IL-1 beta cytokines in the serum of each group of piglets were detected by ELISA method, and the detection results are shown in FIG. 11. ELISA detection of TNF- α (FIG. 11 (A)) showed that by comparison with the ETEC group, lngC (P < 0.0001) and FaeC (P < 0.0001) groups exhibited significant differences in TNF- α expression levels from the ETEC group, followed by PPG (P < 0.01) and MIX (P < 0.01) groups. ELISA detection of IL-6 (FIG. 11 (B)) showed that by comparison with the ETEC group, the LngC group showed very significant differences (P < 0.001), followed by FaeC group showed significant differences (P < 0.01), and that there was no statistical difference between the PPG group and the MIX group. ELISA detection of IL-1β (FIG. 11 (C)) showed no significant differences between groups compared to the ETEC group, and no statistical differences (P > 0.05). These experimental results show that inflammatory factor protein content in piglet sera treated with different engineering bacteria has a statistically significant difference. There was a significant difference in inflammatory factors between the PBS group and ETEC group, which can be judged that the experimental model was established. Whereas the pro-inflammatory cytokines down-regulation trend was more pronounced in LngC and FaeC groups, MIX group did not show significant statistical differences in inflammatory cytokines.

2.3.5 Effect of engineering bacteria on secretory IgA expression of piglets in this experiment, the content of sIgA in challenged piglet intestinal chalk was detected by ELISA method, as shown in FIG. 11 (D). In fecal sIgA detection, the MIX group was found to be very significantly different (p < 0.0001) compared to the ETEC group, while the FaeC group was found to be significantly different (p < 0.05) compared to the ETEC group. Whereas MIX group had significantly up-regulated sIgA compared to FaeC group (p < 0.05).

2.3.6 The pathological histology observation result of the piglets is shown in fig. 12. After ETEC challenge, each group of small intestinal villi was damaged to varying degrees. Of these, lngC (fig. 12 (D)), faeC (fig. 12 (E)) had relatively light injury to small intestinal villi, followed by PPG (fig. 12 (F)), MIX (fig. 12 (C)), ETEC (fig. 12 (B)) exhibited relatively heavy injury, exhibited sparse and varying length of intestinal villi, and had structural changes, which were significantly different from PBS (fig. 12 (a)). The research results show that the intestinal villus injury of piglets caused by ETEC infection can be effectively reduced by immunizing lactobacillus plantarum containing LngC and FaeC engineering strains, so that the structure of intestinal mucosa barriers is improved.

The experimental results show that NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC can activate intestinal mucosa immune response, relieve intestinal injury caused by ETEC, protect intestinal villus structure, improve the capability of organism against ETEC infection, and the effect of the mixed engineering bacteria on immune cells and inflammatory factors of piglets is superior to that of single engineering bacteria. These results show that the recombinant lactobacillus plantarum is used as a vaccine carrier, not only can directly resist intestinal damage caused by ETEC, but also can stimulate the organism to generate systemic immune response by adjusting intestinal microenvironment, and reduce the incidence and death rate of ETEC infection, thereby improving the economic benefit of pig industry.

The foregoing description is only of the preferred embodiments of the invention and is not intended to limit the scope of the invention. Although the invention has been described in detail with reference to the above embodiments, various modifications and equivalent substitutions of parts of technical features can be made by those skilled in the art after reading the present specification. Any modification, equivalent replacement or improvement made within the spirit and principle of the present invention should be considered as falling within the protection scope of the present invention.

Claims (8)

1. The novel functional lactic acid bacteria for expressing the ETEC antigen are characterized in that the novel functional lactic acid bacteria for expressing the ETEC antigen take lactobacillus plantarum NC8 as a host bacteria and pPG612.1 as an expression vector, wherein the pPG612.1 expression vector contains LngC genes of ETEC, and the novel functional lactic acid bacteria for expressing the ETEC antigen express LngC proteins;

Wherein the nucleotide sequence of LngC gene is shown as SEQ ID NO. 2.

2. A composition comprising the novel functional lactic acid bacterium expressing ETEC antigen according to claim 1.

3. The composition according to claim 2, wherein the composition further comprises recombinant Lactobacillus plantarum which uses Lactobacillus plantarum NC8 as a host bacterium and pPG612.1 as an expression vector, and wherein the pPG612.1 expression vector contains FaeC genes of ETEC;

wherein, the recombinant lactobacillus plantarum expresses FaeC protein, and the nucleotide sequence of the FaeC gene is shown as SEQ ID NO. 1.

4. A product comprising the novel functional lactic acid bacterium expressing ETEC antigen of claim 1 or the composition of claim 2 or 3.

5. The product of claim 4, wherein the product is a microecological formulation, a pharmaceutical formulation or a feed additive.

6. The product of claim 5, wherein the pharmaceutical formulation is an oral live carrier vaccine.

7. A method of constructing a novel functional lactic acid bacterium expressing an ETEC antigen according to claim 1, comprising:

obtaining LngC gene fragments of ETEC;

constructing the gene fragment into a pPG612.1 expression vector;

the pPG612.1 expression vector containing the gene fragment was introduced into Lactobacillus plantarum NC 8.

8. Use of a novel functional lactic acid bacterium expressing an ETEC antigen according to claim 1 or a composition according to claim 2 or 3 for the preparation of a product having at least one of the following uses:

(1) A product for preventing or treating ETEC infection;

(2) An immunomodulatory product;

(3) An anti-inflammatory product;

(4) An intestinal tract protection product;

wherein the product is a microecological preparation, a pharmaceutical preparation or a feed additive.