CN107267432B - Recombinant bacterium of Brucella 104M vaccine strain with Per gene knocked out and application - Google Patents

Abstract

The invention discloses a recombinant bacterium of Brucella 104M vaccine strain with a Per gene knocked out and application thereof, and provides the recombinant bacterium, which is a bacterium obtained by knocking out the Per protein activity in Brucella 104M.

Description

Recombinant strain of Brucella 104M vaccine strain knocking out Per gene and its application

technical field

The invention relates to the field of biotechnology, in particular to a recombinant strain of Brucella 104M vaccine strain knocking out Per gene and its application.

Background technique

Brucellosis is a chronic infectious disease caused by Brucella (BruceLLa) infection, and it is also a major zoonotic infectious disease that endangers public health safety worldwide. Among livestock, cattle, sheep, and pigs occur most frequently, and can be transmitted to humans and other livestock, characterized by inflammation of reproductive organs and fetal membranes, causing abortion in females, infertility in males, and localized lesions in various tissues. Cattle, sheep, pigs, dogs, etc. are also the main sources of infection of human brucellosis. Human infection with brucellosis can cause fever, joint pain, fatigue and weakness, and some patients turn into chronic incurable patients. Brucellosis is widespread all over the world. Since 2000, the incidence of brucellosis in humans and animals in my country has increased year by year, which has brought great harm to the development of animal husbandry and brought a huge threat to my country's public health security. , the prevention and control situation is very serious. Vaccine immunization against brucellosis is an effective method to control brucellosis, but there are still many problems in the safety of human brucellosis vaccine 104M currently used in my country.

The global vaccines to prevent Brucella infection in animals mainly include S19, Rev1 and RB51. Human Brucella vaccines are BA-19 (Br. Abortus abbreviation) and 104M (Mockba abbreviation) vaccines. In 1923, American Buck isolated an attenuated strain of bovine species No. 19 from the bovine body and made 19 live veterinary vaccines. In 1946, Soviet researchers selected a pure smooth-type bacterium from the mutant colony of No. 19 strain, called BA-19 bacterin, which was officially used for population inoculation in 1951. The 104M bacterin was a strain of bovine species isolated from the placenta of sick cows in the central Soviet Union in the 1950s. A small number of guinea pig experiments have shown that the bacterial species is more immunogenic than BA-19 and more virulent than BA-19. In the following ten years, Soviet scholars have carried out a lot of research on 104M using experimental animals and livestock. And a small amount of research on humans has proved that it is effective for humans and animals under certain conditions. After citing this strain in my country, a systematic study was carried out from 1959 to 1965, which proved that it is effective in preventing brucellosis infection in the population, which is better than the BA-19 strain. In 1965, my country officially approved the production of live scratch 104M Brucella vaccine for human use. However, during use, it was found that 104M vaccine, like BA-19, can cause allergic reactions in the inoculated body, showing increased sensitivity of local skin test and some clinical symptoms. There are some serious problems and it is gradually not used. . The main problems are: 1. The method of skin scratching is not only painful but also unacceptable; 2. Because it is an attenuated vaccine strain, the side reactions after vaccination are also large, which can cause infection of the vaccinators; 3. After immunization Indistinguishable from natural infection, it seriously affects the diagnosis and quarantine of brucellosis.

The virulence factors of Brucella are closely related to the pathogenesis, immune mechanism, treatment and prevention of brucellosis. Due to the continuous progress in the field of modern molecular biology, the understanding of Brucella virulence factors has gradually deepened. Currently recognized Brucella virulence-related factors include: lipopolysaccharide synthesis gene, BvrR/BvrS two-component regulatory protein gene, type IV secretion system, H ₂ O ₂ enzyme gene, superoxide dismutase gene, outer membrane protein, Heat shock proteins, etc.

Perosamine synthase (per, GDP-4-keto-6-deoxymannose-4 aminotransferase) The Perosamine synthase gene has been cloned and sequenced. In Vibrio cholerae (V. choLerae), GDP-4-keto-6-deoxymannosamine is derived from fructose-6-phosphate through 4 intermediates: mannose-6-phosphate, mannose-1-phosphate, GDP -Mannose, 4-keto-6-deoxymannose, and finally converted to GDP-perosamine by Perosamine synthase. Because the synthetic pathway for the final step is identical in V. cholerae and B. ovine, it is assumed that the same is also true in earlier processes. The formyl group was added to Brucella GDP-perosamine as a substrate, then polymerized into O side chain, transported to the periplasmic space, converted into lipid A nuclear oligosaccharide, and then transported to the cell surface. Complete lysis of Per deprives 16M B. malta of the ability of O chain biosynthesis. Gene substitution mutations demonstrate that transposon insertions are more likely to result in mutant phenotypes than spontaneous mutations. In fact, not only on the cell surface, but within the bacterial cytoplasm, per cleavage products protect any O-chain products, demonstrating that the mutation does not affect O-chain transfer to the outer membrane, but rather early biosynthesis.

Currently recognized traditional serological detection methods for brucellosis are tiger red plate agglutination reaction and standard test tube agglutination reaction. The detection of these two methods is for the O-chain antibody produced by the O-chain antigen of Brucella lipopolysaccharide, that is, as long as the O-chain antibody exists in the host serum, the reaction is positive. Therefore, the current traditional detection methods for brucellosis cannot distinguish whether humans and animals are vaccinated or infected with wild strains. Rough-type (R-type) Brucella is generally regarded as an attenuated strain. Because the real R-type Brucella lacks O-chain antigen, the animals infected by it also have no anti-O-chain specific antibodies. Based on these characteristics, vaccinated and wild-infected animals can be distinguished by traditional serological methods. The strains of Brucella bovis S19, 45/20 and RB51, which are currently used as vaccines, are obtained by repeated passage in vitro to make them mutated from S to R. However, rough strains obtained with this method have the potential risk of virulence recovery. In order to fundamentally change the S-type Brucella strain into an R-type Brucella strain, researchers currently mainly use molecular biological methods such as gene knockout to destroy or delete the related genes of S-LPS synthase, so that the This strain was ultimately unable to produce O-chain antibodies. Now it has been found that the genes related to the synthesis of S-LPS mainly include: WboA, pgm, gmd, per, wbkA, wbkC, etc. The related recombinant strains have been successfully constructed, and the virulence and immune protection test results still need a lot of animal tests to confirm.

As one of the new genetic engineering vaccines, gene deletion vaccine has broad research prospects for the prevention and control of brucellosis. At present, the Brucella attenuated vaccine strain is the most effective vaccine for controlling Brucella in practical applications, but most vaccines show certain toxicity. When used before pregnancy, it will cause miscarriage, and the antibodies produced by immunization are difficult to interact with natural infection. the difference. Therefore, there is an urgent need to develop a Brucella vaccine that can be used for human safe and effective immunity.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a recombinant strain of Brucella 104M vaccine strain knocking out Per gene and its application.

The recombinant bacteria provided by the present invention are bacteria obtained by reducing and/or inhibiting the activity of Per protein in Brucella 104M.

In the above-mentioned recombinant bacteria, the reduction and/or suppression of the activity of the Per protein in the Brucella 104M is to suppress or silence the expression of the Per protein-encoding gene in the Brucella 104M.

In the above-mentioned recombinant bacteria, the inhibition or silencing of the expression of the Per protein encoding gene in Brucella 104M is to knock out the Per protein encoding gene in Brucella 104M.

In the above-mentioned recombinant bacteria, the knockout of the Per protein-encoding gene in Brucella 104M is to replace the Per protein-encoding gene in Brucella 104M with a resistance gene.

In the above-mentioned recombinant bacteria, in the described Brucella 104M, the Per protein coding gene is replaced by the resistance gene and all adopts the mode of genome-directed editing or homologous recombination;

The homologous recombination is specifically λ-red homologous recombination or sacB gene-mediated homologous recombination or suicide plasmid-mediated homologous recombination.

In the above-mentioned recombinant bacteria, in the described Brucella 104M, the Per protein coding gene is replaced with a resistance gene to import the homologous recombination fragment containing the resistance gene into the Brucella 104M;

The homologous recombination fragment containing the resistance gene includes the upstream homology arm of the Per protein encoding gene, the resistance gene and the downstream homology arm of the Per protein encoding gene.

In the above-mentioned recombinant bacteria, the described homologous recombination fragment containing the resistance gene is imported into Brucella 104M by the recombinant vector;

The recombinant vector is a vector obtained by inserting a homologous recombination fragment containing a resistance gene into an expression vector.

In the above-mentioned recombinant bacteria, the resistance gene is kan;

The nucleotide sequence of the homologous recombination fragment containing the resistance gene is sequence 1.

The application of the above-mentioned recombinant bacteria in the preparation of any product in the following 1)-5) is also the scope of protection of the present invention:

1), Brucella attenuated vaccine;

2), Brucella vaccine;

3) Products that promote the increase of CD3+, CD4+ and/or CD8+ cells;

4), increase the number of CD4+ cells and CD8+ cells than the product;

5) Products that increase the content of cytokine IL-2 and/or reduce the content of cytokine IL-4.

Another object of the present invention is to provide a product of any of the following 1)-5).

The product provided by the present invention, its active ingredient is the above-mentioned recombinant bacteria;

1), Brucella attenuated vaccine;

2), Brucella vaccine;

3) Products that promote the increase of CD3+, CD4+ and/or CD8+ cells;

4), increase the number of CD4+ cells and CD8+ cells than the product;

5) Products that increase the content of cytokine IL-2 and/or reduce the content of cytokine IL-4.

Experiments of the present invention prove that the present invention obtains recombinant bacteria by knocking out the virulence gene Per of Brucella 104M, and through the research on its virulence and immunogenicity, screening Brucella with weakened virulence and maintaining immunogenicity Attenuated vaccine candidate strain △Per.

Description of drawings

Figure 1 shows the PCR product of the kan gene.

Figure 2 shows the PCR amplification of the upper and lower homology arms of the Per gene.

Figure 3 is an electrophoresis diagram of the fusion PCR amplification of the target fragment Per::kan.

Figure 4 shows the identification results of the knockout vector bacterial liquid PCR.

Figure 5 shows the screening results of mutant strains.

Figure 6 shows the results of PCR identification of ΔPer deletion strains by continuous and stable passage.

Figure 7 is PCR identification of Brucella-specific primers.

Figure 8 shows the results of mice infected with different immunization doses.

Figure 9 shows the changes in body temperature and body weight of mice after strain immunization.

Figure 10 shows the changes of spleen weight and spleen index in mice after infection.

Figure 11 shows the average number of bacteria in gram spleen of mice after infection.

Figure 12 shows the results of strain-specific virulence survival analysis.

Figure 13 shows the effect of gene deletion on the level of antibody level induced by the strain.

Figure 14 shows the results of lymphocyte transformation in mice.

Figure 15 is the result of lymphocyte transformation.

Figure 16 is an antigen-specific spleen lymphocyte proliferation assay.

Figure 17 is the detection of cytokine content in mouse spleen lymphocyte supernatant.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Brucella vaccine strain 104M was provided by Lanzhou Biological Products Co., Ltd.; Escherichia coli DH5α (EscherichiacoLi DH5α), pHSG298 vector plasmid, pMD-19T SimpLe Vector were purchased from TaKaRa Company; tryptic soybean broth medium was purchased from BD Company in the United States . Clean-grade BALB/c mice (female) were purchased from the Animal Center of Beijing Academy of Military Sciences.

The preparation of the main reagents in the following embodiment 1:

(1) Amp stock solution (100 mg/mL): Dissolve 1 g of ampicillin in 5 mL of deionized water, dilute to 10 mL, filter with a 0.22 μm filter membrane, divide into 1 mL tubes, and store in -20°C refrigerator.

(2) Kan stock solution (100 mg/mL): Dissolve 1 g of kanamycin sulfate in 5 mL of deionized water, dilute the volume to 10 mL, filter with a 0.22 μm membrane filter, divide into 1 mL tubes, and store at -20°C refrigerator.

(3) LB liquid medium: weigh 2 g of tryptone, 1 g of yeast powder, and 2 g of sodium chloride, add 200 mL of distilled water, mix well, and sterilize at 121° C. for 20 min.

(4) LB solid medium: add 1.5% agar powder to the LB liquid medium, and sterilize at 121° C. for 20 minutes.

(5) TSB liquid culture medium: Weigh 6 g of TSB powder, add distilled water, mix evenly, dilute to 200 mL, and sterilize at 115°C for 15 min.

(6) TSA solid medium: add 1.5% agar to the TSB liquid medium, and sterilize at 115° C. for 15 minutes.

(7) 75% glycerol: Measure 75 mL of glycerol, add 25 mL of distilled water, mix well, and sterilize at 121° C. for 20 min.

The CCK8 cell proliferation detection kit was purchased from Promega Corporation in the United States; the mouse-derived IFN-γ, IL-2, IL-4 cytokine detection kits, and the mouse lymphocyte separation liquid were purchased from Shenzhen Dakewe Biotechnology Co., Ltd.; Cell culture medium RPMI 1640 and fetal bovine serum were purchased from GIBCO company; double antibody was purchased from HyCLone company; bovine serum albumin was purchased from Beijing Solaibao Technology Co., Ltd.; HRP-labeled goat anti-mouse IgG was purchased from American Earthox company; soluble Type single-component TMB substrate solution was purchased from Tiangen Biochemical Technology Co., Ltd.; anti-mouse CD3e PerCP Cyanine5.5, anti-mouse CD4FITC, and anti-mouse CD8a PE antibodies were purchased from eBioscience, USA; erythrocyte lysate, tryptic soybean The broth culture medium was purchased from BD Company in the United States; the conventional chemical reagents were purchased from the conditions of the Academy of Military Medical Sciences, all of which were of domestic analytical grade.

The preparation of following embodiment 2 main reagent:

(1) Coating solution: Weigh Na2CO3 1.59g, NaHCO3 2.93g, add 900mL distilled water, adjust the pH value to 9.6, add distilled water to dilute to 1000mL, and store at 4°C.

(2) Washing solution: add 1 mL of Tween-20 to 1 L of PBS solution, mix well and store at 4°C for later use.

(3) Blocking solution: Weigh 5 g of BSA, add 500 mL of distilled water, and prepare a BSA solution with a concentration of 1%.

(4) Stop solution: Measure 355.6 mL of distilled water, slowly add 44.4 mL of concentrated sulfuric acid dropwise, and keep stirring to prepare a 2moL/L sulfuric acid solution.

(5) Cell culture medium: 400mL RPMI 1640, 25mL fetal bovine serum, 10mL double antibody, add RPMI 1640 to quantify to 500mL, filter sterilize with 0.22μm filter, and store at 4°C.

The following examples detect the required method

1. Viable count

The 104M strain of Brucella was transferred to the non-resistance TSB liquid medium, 180rpm/min, 37°C air shaker to cultivate until the bacteria became turbid, and the OD ₆₀₀ value was measured with a UV spectrophotometer, and 0.3 were collected. -0.8 logarithmic period of the bacterial liquid, and the bacterial liquid was diluted into a 10 ¹ -10 ⁶ gradient bacterial suspension, 0.1 mL was spread on the non-resistant TSA solid medium, and the total viable count per mL was calculated. (Total viable bacteria per ml = number of colonies at dilution × dilution factor × 10)

2. Statistical analysis

The experimental data were statistically analyzed using Graphpad Prism5 and SPSS 17.0 software. The data were expressed as mean ± standard deviation, and the differences between groups were analyzed by one-way ANOVA. P<0.05 means the difference is significant, P<0.01 means the difference is extremely significant, and P>0.05 means the difference is not significant.

Embodiment 1, knock out the Per gene of Brucella vaccine strain 104M

In this study, the cloning vector pMD19-T Vector (hereinafter referred to as T vector) was used as the vector to construct the insertion inactivation mutant of Brucella. On this basis, the pMD19-T plasmid with kana resistance gene was constructed, and the deletion mutant of Brucella was constructed by the method of resistance gene replacement. This method avoids the influence of the vector and only needs one time Mutant strains can be obtained by resistance screening. The fusion PCR method is used to fuse the upstream and downstream homology arms of the gene to be deleted with the kanamycin resistance gene to construct a targeting fragment, wherein the resistance gene is located between the upstream and downstream homology arms of the gene. Then the targeting fragment is connected to the T vector to construct a mutant vector, and then a deletion mutant of Brucella is obtained.

1. Primer design and synthesis

According to the whole genome sequencing results of Brucella vaccine strain 104M, the upper and lower homology arm sequences of Per gene were designed and amplified. The Primer 5.0 software was used to follow the principle of about 50% GC content, and the lengths were all about 40 bases. At the same time, specific primers for identifying Brucella species were cited, and specific primers were analyzed and designed (see Table 1). Primers were synthesized by Beijing Saibaisheng Biotechnology Company.

Table 1 lists the upper and lower homology arm genes and identification primer sequences of the target gene

2. Construction of knockout vector

Using the pHSG298 plasmid with Kan ^R resistance as a template, the 950bp kanamycin resistance gene kan was obtained by amplification with primers K1 and K2 (Figure 1);

Using Brucella 104M bacterial solution as a template, use primers p1 and p2 to amplify the upstream homology arm Per-U of the 1001bp Per gene, and use primers p4 and p5 to amplify the 1124bp Per gene downstream homology arm Per-D (Figure 2 , M: DL2000 DNA Marker; 1: PCR product of upstream homology arm of Per gene; 2: PCR product of downstream homology arm of Per gene);

The pure three fragmented products (50ng/μL) of Per-U, Per-D and kan were mixed at a concentration of 1:1:1 as a fusion amplification template, and the following fusion PCR reaction was performed: The fusion PCR reaction system was: template 3 μL , dNTP 1 μL, Q5 High-FideLity DNA PoLymerase 0.3 μL, 5×Q5Reaction buffer 4 μL, ddH ₂ O was added to the final volume of 20 μL. Reaction conditions: 95°C for 3 min, 95°C for 1 min, 65°C for 1 min, 72°C for 1 min, 10 cycles. The obtained PCR reaction solution was named PCR-A. The PCR-A reaction solution was diluted 10 times as a template for PCR amplification. Reaction system: template 3 μL, dNTP 1 μL, p1 (20 μmoL/L) 1 μL, p 5 (20 μmoL/L) 1 μL, LA Taq DNA PoLymerase 0.5 μL, 10×buffer 2.5 μL, ddH ₂ O was added to the final volume of 25 μL. Reaction conditions: 95°C for 5min, 95°C for 30S, 55°C for 1min, 72°C for 3min, and after 35 cycles, 72°C for 10min extension and storage at 4°C. A 3075bp PCR product was obtained (Figure 3).

The PCR product was subjected to DNA agarose gel electrophoresis and purified by gel cutting, and the obtained target fragment was named: Per::kan.

After sequencing, the nucleotide sequence of the target fragment Per::kan is sequence 1, of which the 1-1001 position of sequence 1 is the upstream homology arm Per-U of the Per gene, and the 1002-1951 position of sequence 1 is kanamycin resistance. The sex gene kan, the 1952-3075th position of sequence 1 is the downstream homology arm Per-U of the Per gene.

The targeting fragment Per::kan was directly connected to the pMD19T vector to obtain a recombinant plasmid pMD19T-Per::kan, which was transferred into E. coli, and identified by colony PCR with p1 and p5, and the identified size was 3075bp (as shown in Figure 4).

After sequencing, the recombinant plasmid pMD19T-Per::kan is a plasmid obtained by TA ligating the targeting fragment shown in Sequence 1 in the sequence table with the pMD19T vector.

3. Acquisition of recombinant bacteria

Mix 3 μL of recombinant plasmid pMD19T-Per::kan (200ng/pL) and 48 μL of 104M strain competent cells into a pre-cooled 0.1 mL electroporation cup, use Bio-Rad GenePuLser electroporator, press 1.8KV, 25μF , 200ohms condition electroporation transformation. Immediately after the shock, 1 mL of non-resistant TSB liquid culture was added. Shake culture at 37°C for 6 hours and spread on kan-resistant TSA solid plates, and positive clones grow.

Genomic DNA was extracted from the positive clones, and the primer 1021 for identification of the target gene Per was used for PCR verification, and the wild-type 104M strain was used as a negative control;

The results are shown in Figure 5, M: DL250 DNA Marker; 1: wild-type colony control; 2-21: screened recombinant colony clones; the positive clone of 1827 bp was obtained as a recombinant bacteria, and the amplified fragments were more amplified than wild-type strains. If the fragment (1021bp) is large, it indicates that the recombinant strain is constructed correctly, and it is named as 104MΔPer (hereinafter referred to as ΔPer, also known as 104M mutant strain).

The recombinant strain 104MΔPer is a recombinant strain obtained by replacing the Per gene in the 104M genome with the kan gene.

4. Detection of recombinant bacteria

1) Genetic stability test

The recombinant bacteria 104M△Per were continuously passaged in non-resistance TSB liquid medium, and each generation of bacteria was recorded and stored, and stored at -80℃. When the Brucella per mutant was passaged to the 12th generation, a large number of bacterial fragments were mixed in the bacterial liquid, and it could not be passed to the 13th generation.

The 104M strain and the 1-15th generation of the △Per strain were verified by PCR (target gene Per identification primer 1021), the results showed that the 1st to 12th generation strains could amplify the expected bands, and the 13th generation could amplify the cloth. The size of the Rutella mutant strain was 1827 bp; there was also a wild-type band with a size of 1021 bp (Fig. 6A, M: DL2000 DNA Marker; 1: wild-type colony control; 2-16: △per passages 1 to 12) strains).

At the same time, the 10th-generation bacteria stored in the -80°C refrigerator were revived again, and were also continuously passaged in the non-resistance TSB liquid medium, and they could be further passaged 0 times respectively (see Figure 6B, M: DL2000 DNA marker; 1: wild-type colony control; 2-8: △per strains of the 10th to 16th generations).

At the same time, the strains of Brucella Per gene mutant strains △Per 1st generation and △Per 10th generation strains were sequenced and verified, and DNAMAN software was used to compare the 104M strain with the Per gene sequence, and the Per gene was homologous to the parental strain. is 100%; the kan gene sequence is compared with the sequencing results of the first and tenth generation strains of ΔPer, and the homology is 100%.

It can be seen that the resistance gene successfully replaced the target gene, and it was also stably transmitted to the 10th generation.

2) PCR identification of strains

The serially passaged cultures of the ΔPer strain were identified using Brucella identification primers (primers 699 and 279).

The results are shown in Figure 7, Figure 7A is the amplification product of primer 699, the size is 699bp, Figure 7B is the amplification product of primer 279, the size is 279bp; M: DL2000 DNA Marker; 2-14: △Per 1-12 Continuous and stable passage strain; recombinant strain △Per mutant has the same characteristic bands as 104M, which are 669bp and 279bp respectively. It indicates that the screened mutant strains come from the starting strains, not other contamination.

3) Identification of culture characteristics

The strains of Brucella 104M, the first generation of △Per strains, and the 10th generation of △Per strains were inoculated into non-resistant TSB liquid medium, and cultured at 37°C, 180rpm/min in an air shaker for 2 days, and the bacteria were collected. 2.5×10 ⁹ CFU/mL bacterial suspension was prepared and 100 μL was spread on the non-resistant TSA solid medium containing 1:1000 of fuchsin and thionine. Incubate in a 37°C incubator for 2-3 days to observe the growth of bacteria.

The results are shown in Table 2. There was bacterial growth on the magenta plate, but no bacterial growth on the thionine medium.

Table 2 Identification results of strain culture characteristics

"+": bacterial growth; "-": no bacterial growth

4) Variation inspection of strains

The fresh culture 104M strain, the 1st generation of △Per strain and the 10th generation of △Per strain were inoculated into the non-resistant TSB liquid medium with physiological saline to make 2.5×10 ⁹ -3.0×10 bacteria. ⁹ /mL bacterial suspension was placed in a 90°C water bath for 30 minutes to observe the agglutination phenomenon; at the same time, the bacterial suspension of the same concentration was mixed with an equal amount of 1:1000 Sansheng flavin aqueous solution, and placed at 37°C for 24 hours to observe the agglutination phenomenon.

The results are shown in Table 3, the parental strain 104M and the 1st generation of ΔPer strain, the 10th generation of ΔPer strain and the 20th generation of ΔPer strain did not produce agglutination phenomenon.

Table 3 Results of strain culture characteristics inspection

"+": agglutination; "-": no agglutination

Example 2. Effect of Per gene deletion strain ΔPer on bacterial virulence and immunogenicity

1. Immunization program

1. Determination of immunization dose

In order to select a suitable infection dose, mice were infected with 1×10 ⁵ -1×10 ⁸ CFU/mL of 104M bacterial solution by intraperitoneal inoculation, and the mice were sacrificed at different time points (4-8 days) after infection, and then isolated. The spleen was plated to calculate the average spleen weight and the average number of bacteria per gram of spleen (spleen index = spleen weight (mg)/mice body weight (g); number of bacteria per gram of spleen = total number of bacteria in mouse spleen/spleen weight).

Mice were immunized 4-8 days after infection with the 104M strain, and the mice were observed and sacrificed, and the spleens were isolated and plate counted (see Figure 8). No bacteria were isolated from the spleen of mice in the negative control group. The spleen bacterial count in group 10 ⁸ reached a peak on the 4th day after infection and then began to decline; the spleen bacterial count in group 10 ⁷ reached a peak on day 6 after infection and then began to decline. Although the mice in the 10 ⁸ group did not die, they were obviously lethargic and stood upright. The virulence of Brucella to animals is mainly manifested in its viability in vivo. The evaluation index is mainly to compare the colonization and replication ability of bacteria in vivo. Therefore, the infection dose that is too high or too low cannot correctly reflect the effect of bacteria on mice. virulence. Therefore, the amount of infectious bacteria of 10 ⁷ CFU/mL was selected.

The strains of Brucella 104M and △Per were spread on the non-resistant TSA solid medium, placed at 37 °C for 4 days, and then a single colony was picked and inoculated in the non-resistant TSB liquid medium, and cultivated to the logarithmic phase. (OD ₆₀₀ : 0.435) After centrifuging the bacterial liquid at 5000 r/min for 2 min, discard the supernatant, wash and resuspend the bacterial cells with PBS three times, and finally resuspend the bacterial cells in PBS and dispense 1 mL in 1.5 mL centrifuge tubes. Dilute the bacteria with PBS to 5×10 ⁷ CFU/mL, namely the vaccine for immunization with Brucella 104M and the immunization with ΔPer strain.

2. Immunization and grouping of animals

(1) Grouping scheme: 6-8 week old BALB/c female mice were randomly divided into 3 groups in each experiment (including safety experiment, humoral immunity experiment, and cellular immunity experiment), with 5 mice in each group.

(2) Immunization program:

104M group: Brucella 104M immunization vaccine, the immunization dose is 1×10 ⁷ CFU/vaccine;

△Per group: vaccine for △Per strain immunization, the immunization dose was 1×10 ⁷ CFU/vaccine;

Blank control group: PBS was immunized with 200 μL per mouse.

(3) Immunization method: intraperitoneal injection of mice.

2. Toxicity testing

1. Fur, weight, body temperature detection

The 104M group, the ΔPer group and the PBS group were immunized with mice respectively, and the fur posture and body temperature of the mice were observed, and the average body weight and body temperature for 2 weeks were recorded.

The results are shown in Figure 9, A is the body weight of the mice after immunization; B is the body temperature of the mice after immunization; the results showed that the mice in the 104M group, the △per group and the PBS group behaved normally, with no obvious abnormality, and the weight of the mice in each group was There was no significant difference; however, the mice in the 104M group showed lack of energy, shrugging, and sluggishness in the first week of immunization, and their average body weight decreased significantly. After the first week, the mice in the 104M group recovered and their body weight gradually increased. There was no significant difference in body weight between the mice in the per group and the mice in the 104M group; there was no significant change in body temperature. The average body weight of the mice in each group was detected (see Figure 9A), and the changes in the average body temperature of the mice in each group (see Figure 9B).

2. Calculation of spleen index and gram spleen bacteria

From the 1st to the 10th week, the mice in each group were sacrificed by cervical dislocation, soaked in 75% alcohol for 5 minutes, the spleen was aseptically taken and weighed, and the average spleen index was calculated, spleen index = spleen weight (mg)/mouse body weight (g ).

Within 1 week after infection, the spleen weight of mice infected with strain 104 changed greatly, reaching the highest value in the 2nd week, showing a downward trend after 5 weeks, and the spleen weight trend gradually stabilized after the 7th week. The spleen of mice infected with the mutant strain reached the highest weight at the fifth week, which was significantly lower than that of the parent strain 104M. After the sixth week, the spleen weight of the Δper mutant was stable and had no significant difference with the blank control PBS group (see Figure 10A). Combined with the results of the spleen index, the spleen index of the parental strain 104M and the Δper mutant strain were 0.0134 and 0.0051 respectively at the 8th week, and the mutant strain was significantly different from the parental strain (see Figure 10B).

Mice were taken from 1-10 weeks after infection, the neck was severed, sterilized, spleen taken, ground, the spleen was appropriately diluted with PBS, and the tissue was graded with non-resistant TSA solid medium, cultured for 4-6 days, and the growth rate was calculated. colonies. Finally, the average number of bacteria per gram of spleen was calculated, the number of bacteria per gram of spleen = the total number of bacteria in mouse spleen/spleen weight.

It was found by enumeration that after 10 weeks of infection, the mutant strain was significantly different from the parental control group (Fig. 11). There was no bacterial growth in the PBS blank control group. The number of spleen bacteria in the mutant strain was significantly lower than that in the positive mutant strain. The number of colonies of the △per mutant strain reached the highest value in the fourth week after infection. After the fourth week, the control parental strain showed no downward trend, while the number of spleen bacteria infected by the mutant strain showed a downward trend. By the tenth week, the parental strains 104M and △ The average spleen counts of the per mutant were significantly different, indicating that the virulence of the mutant △per was significantly lower than that of the parent 104M.

3. Specific toxicity test

According to the regulations on specific toxicity test in the identification of Brucella new vaccine strains in the Regulations of Live Brucella Vaccines for Human Use on Skin Scratch - Pharmacopoeia of the People's Republic of China, 2010 Edition, Part III.

104M group: mice were subcutaneously injected with 0.5mL 1.0×10 ⁹ /mL Brucella 104M immunization vaccine;

△per group: mice were subcutaneously injected with 0.5mL 1.0×10 ⁹ /mL △per strain immunization vaccine;

Blank control group: mice were injected subcutaneously with 0.5 mL of PBS.

Each group used 6 mice weighing 18-20 g.

All the mice in the PBS group survived with good body weight and mental state. On the second day of immunization, the mice in the 104M group and the △per group had severe weight loss, erect coat, sluggish mental state, and closed eyes. The mice in the 104M group died on the 2nd and 4th days, and only one mouse survived on the 7th day. All the 6 mice in the Δper group survived on the 7th day with an upward trend in body weight, and their mental state gradually recovered (see Figure 12), indicating that the virulence of the mutant strain Δper was significantly lower than that of the parent 104M.

3. Immunogenicity test

1. Humoral immunoassay

From the 1st week to the 10th week after immunization, the blood of mice in the 104M group, the △Per group and the PBS group was collected by tail docking. After the whole blood was placed at room temperature for 2 hours, centrifuged at 4°C for 10 minutes at 8000 r/min, and the upper serum was collected and detected by indirect ELISA method. Mouse serum specific IgG levels, the steps are as follows:

(1) Antigen treatment: Inoculate Brucella strain 104 in non-resistant TSB liquid medium, cultivate at 37°C for 2 days, collect the bacteria, and plate a part of them appropriately diluted for counting, and the rest are resuspended in PBS solution and incubated at 80°C. Heat inactivation treatment for 2h.

(2) Coating antigen: Centrifuge the treated resuspended bacteria at 8000 r/min for 2 min, discard the supernatant, resuspend fully with coating solution and dilute to 1×10 ⁷ CFU/mL, then coat on 96-well ELISA plate, 100 μL/well, placed at 37°C for 2 hours and then coated overnight at 4°C (the coated ELISA plate can be stored at 4°C for 1 week).

(3) Washing the plate: discard the coating solution, pat dry, add 250 μL of washing solution (PBST) and let stand for 1 min, discard the washing solution, pat dry, repeat 3 times.

(4) Blocking: adding blocking solution, 100 μL/well, and blocking at 37° C. for 1 h.

(5) Washing the plate: Repeat step (3).

(6) Add primary antibody: Dilute the serum to be tested at a ratio of 1:200 to 1:12800, 100 μL/well, and incubate at 37°C for 1 h.

(7) Washing the plate: Repeat step (3).

(8) Add secondary antibody: HRP-labeled goat anti-mouse IgG antibody was diluted with PBS at 1:5000, 100 μL/well, and incubated at 37° C. for 1 h.

(9) Washing the plate: Repeat step (3).

(10) Color development: add soluble one-component TMB substrate solution, 100 μL/well, and develop color at 37°C for 15 minutes in the dark.

(11) Stop: Add stop solution, 100 μL/well, and shake gently for 1 min.

(12) Reading: first preheat the microplate reader for 15 minutes, and read the ELISA plate value (wavelength 450) within 15 minutes.

(13) Analysis: result data processing.

The IgG antibody titer was detected by ELISA, and the titer at the wavelength of 450 nm was taken as the ordinate and the sampling time as the abscissa, and the antibody growth and decline curve was drawn (see Figure 13). After 2 weeks of immunization, the level of IgG in the serum of mice gradually increased, and began to decline at the 8th week. From the changing trend, the antibody titers of parental 104M and mutant strain △per immunized mice were comparable to those of mutant strains in the fourth week; while the antibody titers of mutant strains were significantly lower than those of parental strains at other time points.

2. Cellular immune detection

1), the preparation of mouse spleen lymphocytes

(1) The mice in the 104M group, the △Per group and the PBS group were taken from the eyeballs at the 4th week of immunization, and then sacrificed by cervical dislocation, soaked in 75% alcohol for 5 minutes, and the spleen was aseptically removed.

(2) Add 4 mL of mouse lymphocyte separation solution to a 35 mm petri dish under sterile conditions, cover with a 200-mesh nylon mesh, place the spleen on the mesh, and carefully grind the spleen with a syringe plunger until it is fully dissolved in the liquid.

(3) Transfer the spleen cell suspension to a 15 mL centrifuge tube, slowly add 500 μl of RPMI 1640 culture medium (containing 10% serum), and centrifuge at 1200 r/min for 10 min at room temperature.

(4) Gently aspirate 800 μL of liquid from the top of the liquid surface, transfer it to another 15 mL centrifuge tube, add 10 mL of RPMI1640 culture medium, centrifuge at 1000 r/min for 5 min, and discard the supernatant.

(5) Add 4 mL of erythrocyte lysing solution, mix gently, let stand at room temperature for 15 min to break and lyse the erythrocytes, centrifuge at 1000 r/min for 5 min, and discard the supernatant.

(6) Add 10 mL of RPMI 1640 medium to rinse, centrifuge at 1000 r/min for 5 min, discard the supernatant, and repeat twice.

(7) Finally, the cells were resuspended in 2 mL of RPMI 1640 medium, diluted with a cytometer for counting, and the cell concentration was adjusted to 5×10 ⁷ CFU/mL for use as 104M group spleen lymphocytes and ΔPer group splenic lymphocytes cells and spleen lymphocytes in the PBS group.

2), antigen-specific lymphocyte proliferation test (CCK8 method)

5×10 ⁶ cells/mL 104M group spleen lymphocytes, ΔPer group spleen lymphocytes and PBS group 100 μL of spleen lymphocyte dilution were added to the 96-well culture plate, and 100 μL of 5×10 ⁷ CFU/mL inactivated antigen was added. (104M inactivated), negative control is RPMI 1640 medium, blank is RPMI 1640 medium without cells and antigens. 37°C, 5% _CO2 incubator for 24h. 20 μL of CCK8 reagent was added, and after culturing for 2 h, the wavelength was measured at 450 nm.

IS=(OD－OD ₁₆₄₀ )/(OD _PBS －OD ₁₆₄₀ )

The results are shown in Figure 16. The spleen lymphocytes in the 104M group, the splenic lymphocytes in the ΔPer group and the splenic lymphocytes in the PBS group were de-stimulated with inactivated antigens. After detection by the CCK8 method, the SI was calculated. The analysis showed that both the mutant strain and the parental control group could stimulate the proliferation of mouse lymphocytes, and the ability of the △Per mutant strain to stimulate proliferation was lower than that of the positive control group.

3. The effect of Per gene deletion on strain-induced lymphocyte transformation

(1) 104M group spleen lymphocytes, ΔPer group spleen lymphocytes and PBS group spleen lymphocytes were diluted with RPMI1640 medium to 5×10 ⁶ cells/mL.

(2) Take 1 mL of spleen cell suspension, centrifuge at 1200 r/min for 10 min, aspirate 900 μL of supernatant, and mix the remaining liquid gently with a pipette tip.

(3) Add 0.25 μL of anti-mouse CD3e PerCP Cyanine5.5, 0.5 μL of anti-mouse CD4FITC, and 0.5 μL of anti-mouse CD8a PE, and incubate at 4°C for 1 h in the dark (the negative control group was added with 3 kinds of fluorescent antibodies, respectively, with calibration on flow cytometry).

(4) Add 1 mL of PBS for washing, centrifuge at 1200 r/min for 10 min, and discard the supernatant.

(5) Add 1 mL of PBS to wash, centrifuge at 1200 r/min for 10 min, and discard the supernatant. Resuspend with 300 μL PBS.

(6) Detect with flow cytometer (model: FACSCaLibur, BD Company, USA).

The spleen lymphocytes in the 104M group, the splenic lymphocytes in the △Per group and the spleen lymphocytes in the PBS group were labeled with CD3, CD4, and CD8 fluorescently labeled antibodies, and the labeled cells were counted by flow cytometry to determine the levels of CD3+, CD4+, and CD8+. The number of immune cells was calculated, and the value of CD4+/CD8+ was calculated. The results of the analysis showed that the differentiation of lymphocytes in mice after immunization of the mutant strain and the parental control group changed, and all of them could cause an increase in CD3+, CD4+, and CD8+ cells (as shown in Figure 15, A is CD4+ cells induced by PBS, and B is CD8+ cells induced by PBS). cells, C is CD4+ cells induced by 104M, D is CD8+ cells induced by 104M, E is CD4+ cells induced by ΔPer, and F is CD8+ cells induced by ΔPer). From the results of CD4+/CD8+ ( FIG. 14 ), the ΔPer mutant strain was significantly higher than the 104M parent strain.

The above results indicated that the immunogenicity of ΔOmp25 was higher than that of the parental strain 104M.

4) Cytokine detection

(1) The 104M group spleen lymphocytes, the ΔPer group spleen lymphocytes and the PBS group spleen lymphocytes were diluted with RPMI 1640 medium (5% serum) to 5×10 ⁶ cells/mL.

(2) Add 1.9 mL of cell suspension to a 24-well cell culture dish, add 100 μL of the corresponding antigen to stimulate (5×10 ⁷ CFU/mL, so that the final concentration of antigen in each well is equal to the immunization dose), and set a blank control at the same time Group.

(3) Place the cell culture dish in a 37°C, 5% CO2 incubator for 48 hours.

(4) Follow the steps in the instructions of the mouse cytokine ELISA detection kit.

(5) Statistical data and draw related charts.

After immunizing mice, spleen lymphocytes were cultured, and some important immune-related cytokines IFN-γ, IL-2 and IL-4 were detected in vitro. INF-γ, IL-2 and IL-4 contents were analyzed by ELISA.

The results showed that the cytokine INF-γ induced by the 104M parental strain was not significantly different from that of the Δper mutant (Figure 17C); the IL-4 induced by the Δper mutant was significantly reduced compared to 104M (Figure 17B); the cells induced by the Δper mutant Factor IL-2 was significantly higher than 104M (Figure 17A).

Claims (3)

1. The recombinant bacteria are obtained by reducing and/or inhibiting the activity of Per protein in Brucella 104M;

the reduction and/or inhibition of the activity of the Per protein in the Brucella 104M is the inhibition or silencing of the expression of a Per protein coding gene in the Brucella 104M;

the expression of the Per protein coding gene in the Brucella 104M is the knockout of the Per protein coding gene in the Brucella 104M;

the Per protein coding gene in the Brucella 104M is knocked out, so that the Per protein coding gene in the Brucella 104M is replaced by a resistance gene;

the substitution of Per protein coding gene in the Brucella 104M into resistance gene is carried out by adopting genome site-specific editing or homologous recombination;

the homologous recombination is lambda-red homologous recombination or homologous recombination mediated by sacB gene mediated screening or homologous recombination mediated by suicide plasmid;

the Per protein coding gene in the Brucella 104M is replaced by a resistance gene, so that a homologous recombination segment containing the resistance gene is introduced into the Brucella 104M;

the homologous recombination fragment containing the resistance gene comprises an upstream homology arm of a Per protein coding gene, the resistance gene and a downstream homology arm of the Per protein coding gene;

the homologous recombination fragment containing the resistance gene is introduced into the brucella 104M through a recombination vector;

the recombinant vector is obtained by inserting homologous recombinant fragments containing resistance genes into an expression vector;

the resistance gene is kan;

the nucleotide sequence of the homologous recombination fragment containing the resistance gene is sequence 1.

2. The use of the recombinant bacterium of claim 1 in the preparation of any one of the following products 1) to 5);

1) brucella attenuated vaccines;

2) brucella vaccines;

3) promote CD3⁺、CD4⁺And/or CD8⁺(ii) a cell augmentation product;

4) improve CD4⁺Cells and CD8⁺Number of cells to product;

5) and the product for increasing the content of the cell factor IL-2.

3. A product of any one of the following 1) to 5), wherein the active ingredient of the product is the recombinant bacterium of claim 1;

1) brucella attenuated vaccines;

2) brucella vaccines;

3) promote CD3⁺、CD4⁺And/or CD8⁺(ii) a cell augmentation product;

4) improve CD4⁺Cells and CD8⁺Number of cells to product;

5) and the product for increasing the content of the cell factor IL-2.

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