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 bacterium of Brucella 104M vaccine strain with Per gene knocked out and application

Technical Field

The invention relates to the technical field of biology, in particular to recombinant bacteria for knocking out Per gene of Brucella 104M vaccine strain and application thereof.

Background

Brucellosis is a chronic infectious disease caused by BruceLLa (BruceLLa) infection, and is also a serious zoonosis infectious disease which jeopardizes public health and safety worldwide. Among domestic animals, cattle, sheep and pigs occur most frequently and can be transmitted to people and other domestic animals, and the domestic animals are characterized in that genital organs and fetal membranes are inflamed to cause abortion of female animals, sterility of male animals and local lesions of various tissues, and the affected cattle, sheep, pigs, dogs and the like are also main infection sources of brucellosis of human beings. The brucellosis infection of people can cause fever, arthralgia and fatigue, and part of patients turn into chronic patients which are difficult to cure. The brucellosis is widely prevalent all over the world, and since 2000 years later, the incidence of the brucellosis of people and livestock in China rises year by year, thereby bringing great harm to the development of animal husbandry, simultaneously bringing great threat to public health safety in China, and having very severe prevention and control situation. The vaccine immunization of brucellosis is an effective method for controlling brucellosis, but the safety of the human brucellosis vaccine 104M used in China at present has many problems.

The vaccines for preventing brucella infection of animals in the world are mainly S19, Rev1 and RB 51. Brucella vaccines for human use are BA-19(Br. Abortus abbreviation) and 104M (Mockba abbreviation) bacterins. In 1923, American Buck separated a cattle species No. 19 attenuated strain from cattle to prepare a veterinary 19 viable vaccine. In 1946, Su Union researchers selected a pure smooth type of bacteria from the variant colonies of strain No. 19, called BA-19 bacterin, which was used for population inoculation in 1951. 104M vaccine was a strain of bovine stock, numbered 104M (abbreviation for Mockba), isolated from the placenta of sick cattle in the middle of the Soviet Union in the fifties of the last century. The small amount of guinea pig experiments prove that the immunogenicity of the strain to animals is better than BA-19, the toxicity is stronger than BA-19, and the Soviet Union uses experimental animals and livestock to carry out a large amount of research on 104M in more than ten years later, and a small amount of the strain is used for the research on human bodies, thereby proving that the strain is effective to human and animals when used under certain conditions. After the strain is introduced in China, systematic research is carried out in 1959-1965, and the strain is proved to be effective in preventing brucellosis infection of human groups and is superior to BA-19 strain. China officially approved to produce the human brucella live vaccine with the epithelial scratch 104M in 1965. However, 104M bacterin, like BA-19, has been found to cause allergic reaction in the inoculated organism, and shows high sensitivity of local skin test and some clinical symptoms, and has some serious problems and is not used gradually. The main problems are as follows: firstly, the inoculation adopts a skin scratch method, which is not only painful but also unacceptable; secondly, because the vaccine is a low-toxicity vaccine strain, the side reaction is large after inoculation, and the inoculation personnel can be infected; thirdly, the immunity can not be distinguished from natural infection, and the diagnosis and quarantine work of the epidemic disease are seriously influenced.

The virulence factor of Brucella is closely related to the pathogenesis, immune mechanism, treatment and prevention of Brucella. Due to the continuous progress in the field of modern molecular biology, the knowledge of virulence factors of brucella is also deepened gradually. Currently accepted virulence related factors of brucella are: lipopolysaccharide synthesis gene, BvrR/BvrS dual-component regulatory protein gene, IV-type secretion system and H₂O₂Enzyme gene, superoxide dismutase gene, outer membrane protein, heat shock protein, etc.

Perosamine synthetase (per, GDP-4-keto-6-deoxymannose-4 aminotransferase) the Perosamine synthetase gene has been clonally sequenced. In vibrio cholerae (v. cholerae), GDP-4-keto-6-deoxymannosamine is achieved from fructose-6-phosphate, through 4 intermediates: mannose-6-phosphate, mannose-1-phosphate, GDP-mannose, 4-keto-6-deoxymannose, and finally converted into GDP-Perosamine by Perosamine synthetase. Since the synthetic pathways of the last step are identical in Vibrio cholerae and Brucella melitensis, it is assumed that the processes are also identical in the early stages. Adding formyl radicals into Brucella GDP-perosamine as a substrate, polymerizing the formyl radicals into O side chains, transferring the O side chains to periplasmic space, converting the O side chains into lipid A endooligosaccharides, and further transporting the lipid A endooligosaccharides to the cell surface. Complete lysis of Per abolishes the ability of 16M Brucella melitensis to O chain biosynthesis. Gene substitution mutations demonstrate that transposon insertion is more capable of causing the occurrence of the mutant phenotype than spontaneous mutation. In fact, not only at the cell surface, per lysate protects any O chain product within the bacterial cytoplasm, demonstrating that mutations do not affect O chain transfer to the outer membrane, but rather affect early biosynthesis.

The currently accepted traditional serological methods for brucellosis detection are the tiger red plate agglutination and the standard test tube agglutination. Both methods detect O-chain antibodies against the O-chain antigen of lipopolysaccharide of Brucella, i.e., the reaction is positive as long as O-chain antibodies are present in the host serum. Therefore, the traditional detection means for brucellosis cannot distinguish whether people and animals are vaccinated or infected by wild strains at present. The rough type (R type) Brucella is generally regarded as an attenuated strain, the real R type Brucella lacks O-chain antigen, and the infected animals do not have specific antibodies against O-chain, so that the traditional serological method can be used for distinguishing the animals which are vaccinated and infected by wild virus according to the characteristics. The bovine Brucella rouxii species S19, 45/20 and RB51, which are currently used as vaccines, are obtained by repeated passage in vitro to cause mutation from S to R type. However, the rough strains obtained in this way have the potential risk of virulence recovery. In order to change S-type Brucella strain into R-type Brucella strain fundamentally, researchers mainly use molecular biology means such as gene knockout to destroy or delete related genes of S-LPS synthetase, so that the strain can not generate O-chain antibody finally. It has now been found that the genes involved in S-LPS synthesis mainly include: WboA, pgm, gmd, per, wbkA, wbkC and the like, and related recombinant strains are successfully constructed, and the toxicity and immune protection test results of the recombinant strains are proved by a large number of animal tests.

The gene deletion vaccine is one of novel genetic engineering vaccines, and has wide research prospects for preventing and controlling the disease distribution. Currently, the Brucella attenuated vaccine strain is the most effective vaccine for controlling Brucella in practical application, but most vaccines show certain toxicity, can cause abortion when used before pregnancy, and antibodies generated by immunization are difficult to distinguish from natural infection. Therefore, there is an urgent need to develop a brucella vaccine that can be used for safe and effective immunization of humans.

Disclosure of Invention

The invention aims to provide a recombinant bacterium for knocking out Per gene of Brucella 104M vaccine strain and application thereof.

The recombinant bacterium provided by the invention is a bacterium obtained by reducing and/or inhibiting the activity of Per protein in Brucella 104M.

In the recombinant bacterium, the reduction and/or inhibition of the activity of the Per protein in the Brucella 104M is to inhibit or silence the expression of the Per protein coding gene in the Brucella 104M.

In the recombinant bacterium, the expression of the Per protein coding gene in the Brucella 104M is suppressed or silenced, so that the Per protein coding gene in the Brucella 104M is knocked out.

In the recombinant bacterium, the Per protein coding gene in the Brucella 104M is knocked out because the Per protein coding gene in the Brucella 104M is replaced by a resistance gene.

In the recombinant bacteria, the substitution of Per protein coding genes in the Brucella 104M into resistance genes is carried out by adopting a mode of genome site-specific editing or homologous recombination;

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

In the recombinant bacterium, the Per protein coding gene in the Brucella 104M is replaced by a resistance gene, so that a homologous recombination fragment 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.

In the recombinant strain, the homologous recombinant fragment containing the resistance gene is introduced into the brucella 104M through a recombinant vector;

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

In the recombinant bacterium, the resistant gene is kan;

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

The application of the recombinant bacterium in preparing any one of the following products 1) to 5) is also within the protection scope of the invention:

1) brucella attenuated vaccines;

2) brucella vaccines;

3) promoting CD3+, CD4+, and/or CD8+ cell-increasing product;

4) the product improves the quantity ratio of CD4+ cells to CD8+ cells;

5) products for increasing the content of cytokine IL-2 and/or reducing the content of cytokine IL-4.

It is another object of the present invention to provide a product as any one of the following 1) to 5).

The active component of the product provided by the invention is the recombinant bacterium;

1) brucella attenuated vaccines;

2) brucella vaccines;

3) promoting CD3+, CD4+, and/or CD8+ cell-increasing product;

4) the product improves the quantity ratio of CD4+ cells to CD8+ cells;

5) products for increasing the content of cytokine IL-2 and/or reducing the content of cytokine IL-4.

Experiments prove that recombinant bacteria are obtained by knocking out virulence gene Per of brucella 104M, and a candidate strain △ Per of the attenuated brucella vaccine with attenuated virulence and maintained immunogenicity is screened out through researching the virulence and the immunogenicity of the recombinant bacteria.

Drawings

FIG. 1 shows the PCR product of kan gene.

FIG. 2 shows PCR amplification of upstream and downstream homology arms of Per gene.

FIG. 3 shows the map of kan electrophoresis of the target fragment Per:byfusion PCR amplification.

FIG. 4 shows the result of PCR identification of the knock-out vector bacterial liquid.

FIG. 5 shows the results of screening for mutants.

FIG. 6 shows the result of continuous stable passage PCR identification of △ Per deletion strain.

FIG. 7 shows PCR identification of Brucella specific primers.

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

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

FIG. 10 shows the change in spleen weight and spleen index after infection of mice.

FIG. 11 shows the average gram spleen bacteria count after infection of mice.

FIG. 12 shows the survival rate analysis results of strain-specific toxicity.

FIG. 13 is a graph showing the effect of gene deletion on the level of body antibody level elongation induced by the strain.

FIG. 14 shows the results of mouse lymphocyte transformation.

FIG. 15 shows the results of lymphocyte transformation.

FIG. 16 is an antigen-specific splenic lymphocyte proliferation assay.

FIG. 17 shows the measurement of the content of the mouse spleen lymphocyte supernatant cytokine.

Detailed Description

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

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Brucella vaccine strain 104M was obtained from Lanzhou biologicals Inc., E.coli DH5 α (Escherichia coli DH5 α), pHSG298 Vector plasmid, pMD-19T Simplevector were obtained from TaKaRa, tryptic Soy Broth Medium was obtained from BD, clean-grade BALB/c mice (female) were obtained from the animal center of the military Hospital, Beijing.

The following formulation of the main reagents in example 1:

(1) amp stock (100 mg/mL): dissolving 1g of ampicillin in 5mL of deionized water, diluting to 10mL, filtering with 0.22 μm filter membrane, subpackaging into 1mL of each tube, and storing in a refrigerator at-20 deg.C.

(2) Kan stock solution (100 mg/mL): dissolving 1g kanamycin sulfate in 5mL deionized water, diluting to 10mL, filtering with 0.22 μm filter membrane, subpackaging to 1mL per tube, and storing in a refrigerator at-20 deg.C.

(3) LB liquid medium: weighing 2g of tryptone, 1g of yeast powder and 2g of sodium chloride, adding 200mL of distilled water, mixing uniformly, and sterilizing at 121 ℃ for 20 min.

(4) LB solid medium: adding 1.5% agar powder into LB liquid culture medium, and sterilizing at 121 deg.C for 20 min.

(5) TSB liquid medium: weighing 6g of TSB powder, adding distilled water, mixing to a constant volume of 200mL, and sterilizing at 115 ℃ for 15 min.

(6) TSA solid medium: adding 1.5% agar into TSB liquid culture medium, and sterilizing at 115 deg.C for 15 min.

(7) 75% of glycerin: weighing 75mL of glycerol, adding 25mL of distilled water, mixing well, and sterilizing at 121 ℃ for 20 min.

The CCK8 cell proliferation assay kit is purchased from Promega corporation, USA; IFN-gamma, IL-2 and IL-4 cytokine detection kits of mouse sources, and mouse lymphocyte separation liquid are purchased from Shenzhendake biotechnology Limited; cell culture RPMI1640, fetal bovine serum from GIBCO company; diabody was purchased from HyCLone corporation; bovine serum albumin was purchased from beijing solibao technologies ltd; HRP-labeled goat anti-mouse IgG was purchased from earthhox, usa; the soluble single-component TMB substrate solution was purchased from Tiangen Biochemical technology Ltd; anti-mouse CD3e PerCP cyanine5.5, anti-mouse CD4FITC, anti-mouse CD8a PE antibodies were purchased from eBioscience, usa; erythrocyte lysate, trypticase soy broth medium, purchased from BD, usa; the conventional chemical reagents are purchased from the condition of military medical science institute and are all made in domestic analytical purity.

Example 2 formulation of the main reagents:

(1) coating liquid: weighing Na2CO31.59g and NaHCO32.93g, adding 900mL of distilled water, adjusting the pH value to 9.6, adding distilled water to a constant volume of 1000mL, and storing at 4 ℃.

(2) Washing liquid: 1L of PBS is added with 1mL of Tween-20, mixed evenly and stored for standby at 4 ℃.

(3) Sealing liquid: 5g of BSA was weighed, and 500mL of distilled water was added to prepare a 1% BSA solution.

(4) Stopping liquid: 355.6mL of distilled water was weighed, 44.4mL of concentrated sulfuric acid was slowly added dropwise with stirring to prepare a 2moL/L sulfuric acid solution.

(5) Cell culture solution: 400mL of RPMI1640, 25mL of fetal bovine serum and 10mL of diabody, adding the RPMI1640 to the mixture in a fixed amount of 500mL, filtering and sterilizing the mixture through a 0.22-micron filter membrane, and storing the mixture at 4 ℃.

The following examples illustrate the methods required for detection

1. Viable count

Inoculating Brucella 104M strain into nonresistant TSB liquid culture medium, shake culturing at 180rpm/min and 37 deg.C on air shaker until bacteria become turbid, and measuring OD with ultraviolet spectrophotometer₆₀₀Respectively collecting bacterial liquid in 0.3-0.8 log period, and diluting bacterial liquid to 10¹-10⁶0.1mL of the gradient bacterial suspension was smeared on a non-resistant mediumOn TSA solid medium, the total viable count per ml was calculated. (Total viable bacteria count per ml ═ number of dilution colonies × dilution factor × 10)

2. Statistical analysis

Experimental data were statistically analyzed using Graphpad Prism5 and SPSS 17.0 software, data are presented as mean ± standard deviation, and differences between groups were analyzed using one-way anova. The difference is significant when P <0.05, extremely significant when P <0.01 and insignificant when P > 0.05.

Example 1 knock-out of Per Gene of Brucella vaccine Strain 104M

In this study, an insertionally inactivated mutant strain of Brucella was constructed using the cloning Vector pMD19-T Vector (hereinafter referred to as "T Vector") as a Vector. On the basis, pMD19-T plasmid with kanamycin resistance gene is constructed, and a resistance gene replacement method is adopted to construct a brucella deletion mutant strain, so that the method avoids the influence of a vector, and the mutant strain can be obtained only by one-time resistance screening. And fusing homologous arms at the upstream and downstream of the gene to be deleted and a kanamycin resistance gene by adopting a fusion PCR method to construct a targeting fragment, wherein the resistance gene is positioned between the homologous arms at the upstream and downstream of the gene. And then connecting the targeted fragment to a T vector to construct a mutant vector, thereby obtaining the deletion mutant strain of the Brucella.

1. Design and Synthesis of primers

Designing and amplifying upstream and downstream homologous arm sequences of Per gene according to brucella vaccine strain 104M whole genome sequencing result. The length was about 40 bases using Primer 5.0 software following the GC content rule of about 50%. Specific primers for identifying Brucella species are introduced at the same time, and the specific primers are analyzed and designed (see Table 1). The primers were synthesized by the Biotech company Baisheng, Beijing.

Table 1 shows the upstream and downstream homology arm genes of the target gene and the identification primer sequences

2. Construction of knockout vectors

To be provided withKan^RThe resistant pHSG298 plasmid is used as a template, and 950bp kanamycin resistance gene kan (figure 1) is obtained by amplification by primers K1 and K2;

using brucella 104M bacterial liquid as a template, amplifying by using primers p1 and p2 to obtain 1001bp Per gene upstream homology arm Per-U, and amplifying by using primers p4 and p5 to obtain 1124bp Per gene downstream homology arm Per-D (figure 2, M: DL2000DNA Marker; 1: PCR product of Per gene upstream homology arm; 2: PCR product of Per gene downstream homology arm);

Per-U, Per-D and kan pure 3 fragmentation products (50 ng/. mu.L) were fragmented at a rate of 1: 1:1, mixing the same amount of the mixture as a fusion amplification template to perform the following fusion PCR reaction: the fusion PCR reaction system is as follows: mu.L template, dNTP 1. mu.L, Q5 High-Fidelity DNA PoLymerase 0.3. mu.L, 5 XQ 5Reaction buffer 4. mu.L, ddH₂O was added to a final volume of 20. mu.L. Reaction conditions are as follows: 95 ℃ for 3min, 95 ℃ for 1min, 65 ℃ for 1min, 72 ℃ for 1min, 10 cycles. The resulting PCR reaction solution was named PCR-A. The PCR-A reaction solution was diluted 10 times and used as A template for PCR amplification. Reaction system: mu.L template, 1. mu.L dNTP, 1. mu.L p1 (20. mu. moL/L), 1. mu.L p5 (20. mu. moL/L), 0.5. mu.L LA Taq DNA PoLymerase, 2.5. mu.L 10 XBuffer, ddH₂O was added to a final volume of 25. mu.L. Reaction conditions are as follows: 5min at 95 ℃, 30S at 95 ℃, 1min at 55 ℃, 3min at 72 ℃, and after 35 cycles, 10min at 72 ℃ and 4 ℃ for extension. A3075 bpPCR product was obtained (FIG. 3).

And (3) carrying out DNA agarose gel electrophoresis on the PCR product and cutting and purifying, and naming the obtained targeting fragment as: kan in Per.

After sequencing, the nucleotide sequence of the targeting fragment Per is shown as sequence 1, wherein the 1 st to 1001 st position of the sequence 1 is the upstream homology arm Per-U of the Per gene, the 1002 nd and 1951 st positions of the sequence 1 are kanamycin resistance gene kan, and the 1952 nd and 3075 th positions of the sequence 1 are the downstream homology arm Per-U of the Per gene.

The targeting fragment of Kan was directly ligated with the pMD19T vector to obtain the recombinant plasmid pMD 19T-Per:Kan, which was transformed into E.coli, and then identified as 3075bp by colony PCR using p1 and p5 (see FIG. 4).

After sequencing, the recombinant plasmid pMD19T-Per shows that kan is a plasmid obtained by TA connecting a targeting fragment shown as a sequence 1 in a sequence table with a pMD19T vector.

3. Obtaining of recombinant bacteria

mu.L of recombinant plasmid pMD19T-Per, kan (200ng/pL) and 48. mu.L of competent cells of 104M strain were mixed and added to a pre-cooled 0.1mL cuvette and transformed by electroporation using a Bio-Rad GenePuLser electrotransfer under conditions of 1.8KV, 25. mu.F and 200 ohms. Immediately after the shock, 1mL of non-resistant TSB broth was added. Positive clones were grown by shaking culture at 37 ℃ for 6h and plating them on kan-resistant TSA solid plates.

Extracting genomic DNA from the positive clone, performing PCR verification by using a target gene Per identification primer 1021, and taking a wild 104M strain as a negative control;

the result is shown in FIG. 5, M: DL250DNA Marker, 1: wild colony control, and 2-21: screened recombinant colony clone, wherein the obtained 1827bp positive clone is a recombinant bacterium, and the amplified fragment is larger than the amplified fragment (1021bp) of a wild strain, so that the recombinant bacterium is correctly constructed and is named as 104M △ Per (hereinafter referred to as △ Per, also referred to as 104M mutant strain).

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

4. Detection of recombinant bacteria

1) Genetic stability test

Continuously passaging the recombinant bacterium 104M △ Per in a non-resistant TSB liquid culture medium, recording and storing bacterial liquid of each generation, and storing the bacterial liquid to-80 ℃.

PCR verification is carried out on 104M strain and △ Per strain at 1-15 generations (target gene Per identification primer 1021), and the result shows that the strains at 1-12 generations can amplify expected bands, while the strains at 13 generations can amplify Brucella mutant bands with the size of 1827bp, and wild bands with the size of 1021bp (FIG. 6A, M: DL2000DNA Marker; 1: wild colony control; 2-16: △ Per strains at 1-12 generations).

Meanwhile, the 10 th generation of bacteria stored in a refrigerator at the temperature of-80 ℃ are recovered again, and can be continuously passaged for 0 time respectively through a nonresistant TSB liquid culture medium (see figure 6B, M: DL2000DNA marker; 1: wild type colony control; 2-8: △ per generation 10-16).

Meanwhile, brucella Per gene mutant △ Per generation 1 and △ Per generation 10 strains are subjected to sequencing verification, 104M strains and Per gene sequences are compared by DNAMAN software, the homology of the Per gene and a parent strain is 100%, and the homology of the kan gene sequences and △ Per generation 1 and 10 strain sequencing results is 100%.

It can be seen that the resistance gene successfully replaces the target gene, and the gene stably exists continuously until the 10 th generation.

2) PCR identification of strains

The △ Per strain was serially passaged and the species identified using Brucella identification primers (primers 699 and 279).

As shown in FIG. 7, FIG. 7A shows the amplification product of primer 699 with a size of 699bp, FIG. 7B shows the amplification product of primer 279 with a size of 279bp, M: DL2000DNA Marker, 2-14: △ Per No. 1-12 serial stable passage strains, and the recombinant strain △ Per mutant has the same characteristic bands as 104M, 669bp and 279 bp. respectively, which indicate that the selected mutant strain comes from the original strain and is not contaminated.

3) Characterization of culture Properties

Inoculating the preserved strains of Brucella 104M strain, △ Per strain generation 1 and △ Per strain generation 10 into non-resistant TSB liquid culture medium, shake culturing at 37 deg.C and 180rpm/min for 2d, collecting thallus, and making into 2.5 × 10⁹Each 100. mu.L of CFU/mL suspension was applied to non-resistant TSA solid medium containing 1:1000 fuchsin and thionine. Culturing the strain in a 37 ℃ incubator for 2-3 days, and observing the growth condition of bacteria.

The results are shown in Table 2, with bacteria on the magenta plates and no bacteria on the thionine medium.

TABLE 2 identification of the culture characteristics of the strains

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

4) Examination of mutation of Strain

Inoculating the bacterial liquid of fresh culture 104M strain, △ Per strain 1 generation and △ Per strain 10 generation into non-resistant TSB liquid culture medium with normal saline to obtain 2.5 × 10 strain⁹-3.0×10⁹Placing the suspension in 90 ℃ water bath for 30min, and observing the agglutination phenomenon; meanwhile, the bacterial suspension with the same concentration is mixed with 1:1000 sanshenflavin water solution in equal amount, and the mixture is placed at 37 ℃ for 24h to observe the agglutination phenomenon.

As a result, as shown in Table 3, no agglutination occurred between the parent strain 104M and the △ Per strain at the 1 st generation, the △ Per strain at the 10 th generation, and the △ Per strain at the 20 th generation.

TABLE 3 examination results of culture characteristics of strains

"+": agglutination; "-": does not agglutinate

Example 2 Effect of Per Gene-deleted Strain △ Per on bacterial virulence and immunogenicity

First, immunization protocol

1. Determination of the immunization dose

To select a suitable dose of infection, 1X 10 is added⁵-1×10⁸CFU/mL 104M inoculum infected mice by intraperitoneal inoculation, sacrificed at different time points (4-8 days) after infection, spleens were isolated and plated to calculate the average weight of the spleens and the average number of bacteria in the spleens (spleen index: spleen weight (mg)/mouse weight (g); the number of bacteria in the spleens of mice: total bacteria in the spleens/spleen weight).

Mice were observed and sacrificed 4-8 days after immunization with 104M strain, spleens were isolated and plated for counting (see FIG. 8). Bacteria were not isolated in the spleen of mice in the negative control group. 10⁸Group reached a peak in splenic bacterial load at day 4 post infection and then began to decline; 10⁷Groups peaked at day 6 post infection with splenocytes and then began to decline. 10⁸Although the mice in the group did not die, they showed marked listlessnessThe hair stands up in the opposite direction. The virulence of brucella to animals is mainly shown by the in vivo viability of the brucella, and the evaluation index is mainly used for comparing the in vivo colonization and replication capacities of bacteria, so that the virulence of the bacteria to mice cannot be correctly reflected by excessively high or excessively low infection dose. Thus, choose 10⁷CFU/mL of infectious bacteria.

The Brucella 104M and △ Per strains are spread on a non-resistant TSA solid culture medium, cultured at 37 ℃ for 4 days, then single colonies are picked and inoculated in a non-resistant TSB liquid culture medium, and the culture is carried out to logarithmic phase (OD)₆₀₀: 0.435) centrifuging the bacterial liquid at 5000r/min for 2min, discarding the supernatant, washing the resuspended thallus with PBS for 3 times, finally suspending the thallus in PBS, subpackaging 1mL in a 1.5mL centrifuge tube, diluting the thallus-containing volume with PBS to 5 × 10⁷CFU/mL, namely vaccine for brucella 104M immunization and vaccine for △ Per strain immunization.

2. Immunization and grouping of animals

(1) Grouping scheme: BALB/c female mice of 6-8 weeks old, each experiment (including safety experiment, humoral immunity experiment, cellular immunity experiment) was divided into 3 groups of 5 mice each.

(2) Immunization protocol:

104M groups: vaccine for brucella 104M immunization, and immunization dose is 1 × 10⁷CFU/only;

△ Per group △ Per strain vaccine for immunization, immunization dose 1X 10⁷CFU/only;

blank control group: PBS was immunized at 200. mu.L/mouse.

(3) The immunization mode comprises the following steps: mice were injected intraperitoneally.

Second, toxicity detection

1. Detection of fur, body weight and body temperature

Mice were immunized with the 104M group, the △ Per group, and the PBS group, respectively, and the mouse coat and body temperature were observed and the average body weight and body temperature were recorded for 2 weeks.

The results are shown in FIG. 9, wherein A is the weight of the immunized mice, B is the body temperature of the immunized mice, and the results show that the mice in the 104M group, the △ per group and the PBS group are normal without obvious abnormality and have no obvious difference in the weight of the mice among the groups, while the mice in the 104M group are immunized, the phenomena of lassitude, shrugging and bradykinesia appear at 1 week and the average weight is obviously reduced, the state of the mice in the 104M group is recovered after 1 week, and the weight is gradually increased, the weights of the mice in the per group and the mice in the 104M group are not obviously different, and the body temperature is not obviously changed, the average weight of the mice in each group (see FIG. 9A) and the average body temperature change of the mice in each group (see FIG. 9B) are.

2. Spleen index and number of gram splenocytes

From week 1-10, each group of mice was sacrificed by neck-breaking, immersed in 75% alcohol for 5min, and the spleens were aseptically weighed, and the average spleen index, spleen weight (mg)/mouse weight (g), was calculated.

The spleen weight of mice infected by the mutant strain 104M and 104P is changed greatly within 1 week after the mice are infected, the mice reach the highest value at 2 weeks, show a descending trend after 5 weeks, and gradually stabilize the spleen weight trend after 7 weeks, the spleen weight of mice infected by the mutant strain reaches the highest value at the fifth week and is obviously lower than that of the parent strain 104M, △ P mutant strain has no obvious difference from a blank control PBS group in stable spleen weight trend after 6 weeks (see figure 10A), and further combined with spleen index results, the spleen indexes of the parent strain 104M and △ P mutant strains at 8 weeks are respectively 0.0134 and 0.0051, and the mutant strain has obvious difference compared with the parent strain (see figure 10B).

After infection, respectively taking mice 1-10 weeks, breaking the neck, sterilizing, taking the spleen, grinding, properly diluting the ground spleen tissue with PBS (phosphate buffer solution), coating a non-resistant TSA solid culture medium in a gradient manner, culturing for 4-6 d, and calculating to grow single colonies. Finally, the average number of the gram splenocytes is calculated, and the number of the gram splenocytes is equal to the total number of the splenocytes in the mouse/weight of the spleen.

The mutant strain is found to have a significant difference with the parent control group after 10 weeks of infection by counting (figure 11). the PBS blank control group has no bacteria growth.the number of splenocytes in the mutant strain is reduced significantly compared with the positive mutant strain. △ per mutant strain reaches the highest value in the number of colonies in the fourth week after infection, the number of splenocytes infected by the mutant strain is reduced because the parent strain is not reduced, and the difference between the average number of splenocytes in the parent strain 104M and the mutant strain △ per is significant after the fourth week, which shows that the toxicity of the mutant strain △ per is reduced significantly compared with the parent strain 104M.

3. Specific toxicity detection

According to the specific toxicity test regulation in the identification of the new Brucella vaccine strain in the < Bluella live vaccine procedure for scarfskin people-pharmacopoeia of the people's republic of China, three parts of 2010 edition >.

104M groups: mice were injected subcutaneously with 0.5ml 1.0X 10⁹Vaccine for 104M immunization with Brucella/mL;

△ per group mice were injected subcutaneously with 0.5ml 1.0X 10⁹Vaccine for immunization with/mL △ per strain;

blank control group: mice were injected subcutaneously with 0.5ml pbs.

6 mice weighing 18-20 g were used per group.

The PBS group mice all survived, the body weight was well-maintained, the 104M and △ per groups immunized the following days, the mice were severely reduced in body weight, erect in hair, depressed in mental state, and tightly locked in eyes, the 104M group mice died on the 2 nd and 4 th days, only 1 mouse survived on the 7 th day, the △ per group 6 mice all survived and gradually recovered in body weight on the 7 th day (see FIG. 12), which indicates that the toxicity of the mutant strain △ per is significantly reduced compared with the parent 104M.

Third, detection of immunogenicity

1. Humoral immunity detection

After the mice in the 104M group, the △ Per group and the PBS group are collected by tail breaking from the 1 st week to the 10 th week after immunization, after the whole blood is placed for 2 hours at room temperature, the whole blood is centrifuged for 10 minutes at the temperature of 4 ℃ and 8000r/min, the upper layer serum is collected, and the level of the specific IgG of the mouse serum is detected by an indirect ELISA method, which comprises the following steps:

(1) antigen treatment: inoculating Brucella 104 strain in nonresistant TSB liquid culture medium, culturing at 37 deg.C for 2d, collecting thallus, diluting part of thallus, coating on plate, counting, suspending the rest with PBS, and heating at 80 deg.C for inactivating for 2 h.

(2) Coating antigen: centrifuging the treated resuspended bacteria at 8000r/min for 2min, discarding supernatant, fully resuspending with coating solution and diluting to 1 × 10⁷CFU/mL, and then coated in 96-well ELISA plates, 100 u L/well, 37 ℃ after 2h 4 ℃ coated overnight (coated ELISA plate can be stored at 4 ℃ for 1 week).

(3) Washing the plate: discarding the coating solution, draining, adding 250 μ L of washing solution (PBST), standing for 1min, discarding the washing solution, draining, and repeating for 3 times.

(4) And (3) sealing: add blocking solution, 100. mu.L/well, block for 1h at 37 ℃.

(5) Washing the plate: and (4) repeating the step (3).

(6) Adding a primary antibody: the serum to be tested is diluted by 1:200 to 1:12800 times, 100 mu L/hole, and incubated for 1h at 37 ℃.

(7) Washing the plate: and (4) repeating the step (3).

(8) Adding a secondary antibody: HRP-labeled goat anti-mouse IgG antibody was diluted 1:5000 with PBS at 100. mu.L/well and incubated at 37 ℃ for 1 h.

(9) Washing the plate: and (4) repeating the step (3).

(10) Color development: adding soluble single-component TMB substrate solution, 100. mu.L/well, and developing at 37 deg.C in dark for 15 min.

(11) And (4) terminating: stop solution was added at 100. mu.L/well and gently shaken for 1 min.

(12) Reading: the microplate reader was preheated for 15min and the ELISA plate number (wavelength 450) was read within 15 min.

(13) And (3) analysis: and (6) processing result data.

The IgG antibody titer is detected by an ELISA method, the titer at the wavelength of 450nm is taken as the ordinate, the sampling time is the abscissa, an antibody extinction curve (shown in figure 13) is drawn, after 2 weeks of immunization, the IgG level in the serum of a mouse gradually rises, and the IgG level begins to fall back to the 8 th week, from the change trend, the antibody titer of the parent 104M and the mutant △ per immunized mouse is equivalent to that of the mutant, and the mutant antibody titer level at the rest time points is obviously lower than that of the parent strain.

2. Cellular immunoassay

1) Preparation of mouse spleen lymphocytes

(1) After the eyeballs of mice in the group 104M, the group △ Per and the group PBS for 4 weeks of immunization are bled, the neck is cut off and killed, the mice are soaked in 75% alcohol for 5min, and the spleen is aseptically taken.

(2) 4mL of mouse lymphocyte isolate was aseptically added to a 35mm petri dish, covered with a 200 mesh nylon screen, the spleen was placed on the screen, and carefully ground with a syringe plunger until sufficiently dissolved in the liquid.

(3) The spleen cell suspension was aspirated and transferred to a 15mL centrifuge tube, and 500. mu.LRPMI 1640 medium (containing 10% serum) was added slowly and centrifuged at 1200r/min for 10min at room temperature.

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

(5) Adding 4mL of erythrocyte lysate, gently mixing, standing at room temperature for 15min to break and lyse erythrocytes, centrifuging at 1000r/min for 5min, and discarding the supernatant.

(6) Adding 10mL RPMI1640 culture solution for washing, centrifuging at 1000r/min for 5min, discarding supernatant, and repeating for 2 times.

(7) Finally, the cells were resuspended in 2mL of RPMI1640 medium, diluted with a cell counter for counting, and the cell concentration was adjusted to 5X 10⁷CFU/mL, ready for use, was 104M, △ Per and PBS splenic lymphocytes.

2) Antigen-specific lymphocyte proliferation assay (CCK8 method)

Respectively mixing 5 × 10⁶100. mu.L of a dilution of 104/mL 104M-group spleen lymphocytes, △ Per-group spleen lymphocytes and PBS-group spleen lymphocytes was added to a 96-well plate, and 100. mu.L of a dilution of 5X 10-well plate was added⁷CFU/mL inactivated antigen (inactivated 104M), negative control RPMI1640 medium, blank RPMI1640 medium without cells and antigen. 37 ℃ and 5% CO₂The incubator is used for 24 h. 20 μ L of CCK8 reagent was added, and after further incubation for 2h, the wavelength was measured at 450 nm.

IS＝(OD－OD₁₆₄₀)/(OD_PBS－OD₁₆₄₀)

The results are shown in fig. 16, the inactivated antigen is used for stimulating 104M group spleen lymphocytes, △ Per group spleen lymphocytes and PBS group spleen lymphocytes, after being detected by the CCK8 method, SI. is calculated, and the mutant strain and the parent control group are analyzed to find that the mutant strain can stimulate mouse lymphocyte proliferation, and the △ Per mutant strain has lower proliferation stimulation capability than the positive control group.

3. Effect of Per Gene deletion on Strain-induced lymphocyte transformation

(1) 104M group of spleen lymphocytes and △ Per group of spleen lymphocytesSpleen lymphocytes of the cell and PBS groups were diluted to 5X 10 with RPMI1640 culture medium, respectively⁶one/mL.

(2) 1mL of spleen cell suspension is taken, centrifuged at 1200r/min for 10min, 900 μ L of supernatant is sucked out, and the rest solution is gently mixed by a gun head.

(3) Anti-mouse CD3e PerCP cyanine5.50.25. mu.L, anti-mouse CD4FITC 0.5. mu.L, anti-mouse CD8a PE 0.5. mu.L antibody were added, and incubated at 4 ℃ for 1h in the dark (wherein 3 kinds of fluorescent antibodies were added to the negative control group, respectively, for calibration of the flow cytometer).

(4) Adding 1mL PBS for washing, centrifuging at 1200r/min for 10min, and discarding the supernatant.

(5) Adding 1mL PBS for washing, centrifuging at 1200r/min for 10min, and discarding the supernatant. Resuspend with 300. mu.L PBS.

(6) The detection was carried out by a flow cytometer (model: FACSCALibur, BD Co., USA).

The spleen lymphocytes of 104M group, the spleen lymphocytes of △ Per group and the spleen lymphocytes of PBS group are labeled by using CD3, CD4 and CD8 fluorescent labeled antibodies, the labeled cells are counted by using a flow cytometer, the immune cell numbers of CD3+, CD4+ and CD8+ are measured, and the values of CD4+/CD8+ are calculated, and the result analysis shows that the mouse lymphocytes are changed in differentiation after the immunization of the mutant strain and the parent control group (for example, FIG. 15, A is CD4+ cells caused by PBS, B is CD8+ cells caused by PBS, C is CD4+ cells caused by 104M, D is CD8+ cells caused by 104M, E is CD4+ cells caused by Per573 5, F is CD8+ cells caused by △ Per), the result of CD4+/CD8+ from CD4+/CD 68614 shows that the mutant strain is remarkably higher than the result of Per 104M 104 +/CD △.

The above results show that Δ Omp25 is more immunogenic than the parental strain 104M.

4) Cytokine detection

(1) 104M, △ Per and PBS spleen lymphocytes were diluted to 5X 10 with RPMI1640 medium (5% serum)⁶one/mL.

(2) 1.9mL of cell suspension was added to a 24-well cell culture dish, and 100. mu.L of the corresponding antigen (5X 10 in volume) was added for stimulation⁷CFU/mL, to achieve a final antigen concentration per well with an immunizing agentEqual amount) and a blank control group is set.

(3) The cell culture dish was placed in a 5% CO2 incubator at 37 ℃ for 48 h.

(4) The procedures were performed according to the instructions of the mouse cytokine ELISA kit.

(5) And counting data and drawing a relevant chart.

After mice are immunized, spleen lymphocytes are taken for culture, and a part of important immune related cytokines IFN-gamma, IL-2 and IL-4 are detected in vitro. INF-gamma, IL-2 and IL-4 content was analyzed by ELISA.

The results showed that the 104M parent strain did not significantly differ from the Δ per mutant induced cytokine INF- γ (fig. 17C); the Δ per mutant induced a significant reduction in IL-4 compared to 104M (fig. 17B); the delta per mutant induced cytokine IL-2 significantly higher than 104M (fig. 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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