CN107267431B - Recombinant bacterium of Brucella 104M vaccine strain with VirB2 gene knocked out and application - Google Patents

Abstract

The invention discloses a recombinant bacterium of a Brucella 104M vaccine strain with a VirB2 gene knocked out and application thereof. The invention provides a recombinant bacterium, which is a bacterium obtained by reducing and/or inhibiting the activity of VirB2 protein in Brucella 104M. Experiments prove that recombinant bacteria are obtained by knocking out 104M virulence gene VirB2 of the Brucella, and candidate strain delta VirB2 of the attenuated Brucella vaccine for human use is screened out through researching the virulence and immunogenicity of the recombinant bacteria.

Description

Recombinant bacterium of Brucella 104M vaccine strain with VirB2 gene knocked out and application

Technical Field

The invention relates to the technical field of biology, in particular to a recombinant bacterium of a Brucella 104M vaccine strain with a VirB2 gene knocked out 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.

Brucella forms a brucella Body (BCV) after phagocytosis by phagolysosomes in the early stage of macrophage invasion, survives in the acidic environment in the BCV and establishes extensive contact with Endoplasmic Reticulum (ER) thereafter, thereby obtaining the identification of the ER and escaping the immune recognition of host cells. At present, the research suggests that LPS mediates the invasion of brucella into host cells through the interaction between LPS and lipid rafts on the surfaces of macrophages, and an environment suitable for the proliferation and replication of brucella is established. Brucella LPS is weak in toxicity, and only causes weak inflammatory reaction after entering host cells, so that immune defense reaction of the host can be escaped. The IV secretion system (T4SS) of Brucella is encoded by VirB operon, and is closely related to intracellular transport and intracellular proliferation of Brucella. In the presence of T4SS, fusion of lysosomes with BCV is effectively prevented, so that Brucella can live in phagocytes, and then BCV can continuously interact with reticuloendothelial system (ER) through a T4 SS-dependent pathway, so that ER membrane is accumulated in BCV, BCV is enabled to acquire the characteristic of ER, and the immune mechanism of a host is escaped. However, Brucella with T4SS defect can not regulate interaction between BCV and ER, and BCV is in intermediate and immature state and finally fused with lysosome to cause degradation. It is reported in the literature that T4SS plays a role mainly in regulating the intracellular trafficking of brucella from BCV to ER, and once brucella reaches its site of replication, the secretory system is turned off. The type IV secretion system is a family of polyprotein complexes that secrete bacterial virulence factors, the existence of which has been demonstrated in bordetella pertussis, helicobacter pylori, and legionella, and which has been shown to secrete virulence effectors. Recently, the presence of T4SS encoded by the virB operon was found in the genome of brucella, T4SS being a complex composed of 12 different proteins that span the cell wall of the bacterium. VirB2 is located on the surface of bacteria, is closely related to bacterial invasion and plays a key role.

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 of Brucella 104M vaccine strain with VirB2 gene knocked out.

The recombinant bacterium provided by the invention is a bacterium obtained by reducing and/or inhibiting the activity of VirB2 protein in Brucella 104M.

In the recombinant bacterium, the reduction and/or inhibition of the VirB2 protein activity in the Brucella 104M is to inhibit or silence the expression of VirB2 protein coding genes in the Brucella 104M.

In the recombinant bacterium, the VirB2 protein coding gene in the Brucella 104M is suppressed or silenced, so that the VirB2 protein coding gene in the Brucella 104M is knocked out.

In the recombinant bacterium, the VirB2 protein coding gene in the Brucella 104M is knocked out, so that the VirB2 protein coding gene in the Brucella 104M is replaced by a resistance gene.

In the recombinant bacteria, the VirB2 protein coding gene in the Brucella 104M is replaced by a resistance gene in 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 VirB2 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 VirB2 protein coding gene, the resistance gene and a downstream homology arm of a VirB2 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 6) is also within the protection scope of the invention:

1) brucella attenuated vaccines;

2) brucella vaccines;

3) products for promoting lymphocyte proliferation;

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

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

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

It is another object of the present invention to provide a product as described in any one of 1) to 6) below.

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

1) brucella attenuated vaccines;

2) brucella vaccines;

3) products for promoting lymphocyte proliferation;

4) promoting the increase of CD3+, CD4+ and CD8+ cells;

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

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

Experiments prove that recombinant bacteria are obtained by knocking out 104M virulence gene VirB2 of the Brucella, and a candidate strain delta VirB2 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 the upstream and downstream homology arms of VirB2 gene.

FIG. 3 shows the electrophoresis of kan for the target fragment VirB2 of fusion 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 the delta VirB2 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 provided by lanzhou biologicals limited; coLi DH5 alpha (Escherichia coLi DH5 alpha), pHSG298 Vector plasmid, pMD-19T Simplevector were purchased from TaKaRa; tryptic soy broth was purchased from BD, usa. Clean-grade BALB/c mice (female) were purchased from the animal center of the Beijing military institute.

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 VirB2CP 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 is smeared on a non-resistant TSA solid culture medium, and the total viable bacteria per milliliter is 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 VirB2 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

According to the brucella vaccine strain 104M whole genome sequencing result, the upstream and downstream homologous arm sequences of the amplified VirB2 gene are designed. 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

With Kan^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;

taking brucella 104M bacterial liquid as a template, amplifying by using primers V1 and V2 to obtain 694bp VirB2 gene upstream homology arm VirB2-U, and amplifying 726bp VirB2 gene downstream homology arm VirB2-D by using primers V4 and V5 (figure 2, M: DL2000 DNAmarker; 1: VirB2 gene upstream homology arm PCR product; 2: VirB2 gene downstream homology arm PCR product);

VirB2-U, VirB2-D and kan 3 fragmentation products (50 ng/. mu.L) were sequenced at 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, Q5High-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 V1 (20. mu. moL/L), 1. mu.L V5 (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. A2370 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: and (5) VirB2: kan.

Through sequencing, the nucleotide sequence of the targeting fragment VirB2 is shown in the specification, the nucleotide sequence of kan is shown in sequence 1, wherein the 1 st-694 th site of the sequence 1 is the VirB2 gene upstream homology arm VirB2-U, the 695 th-1644 th site of the sequence 1 is kanamycin resistance gene kan, and the 1645 th-2370 th site of the sequence 1 is the VirB2 gene downstream homology arm VirB 2-U.

The targeting fragment VirB2:: kan was directly ligated to pMD19T vector to obtain recombinant plasmid pMD19T-VirB2:: kan, which was transferred into E.coli, and then subjected to colony PCR using V1 and V5 to determine the size of 2370bp (see FIG. 4).

After sequencing, the recombinant plasmid pMD19T-VirB2 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 the recombinant plasmid pMD19T-VirB2, kan (200ng/pL) and 48. mu.L of 104M strain competent cells were mixed well and added to a pre-cooled 0.1mL cuvette and transformed by electroporation under the conditions of 1.8KV, 25. mu.F and 200ohms using a Bio-Rad GenePulser electrotransfer. 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 genome DNA from the positive clone, performing PCR verification by using a target gene VirB2 identification primer 279, and taking a wild 104M strain as a negative control;

the results are shown in FIG. 5, where M: DL250DNA Marker; 1-5: cloning the screened recombinant colonies; 6: wild type colony control; the obtained 1180bp positive clone is a recombinant bacterium, and the amplified fragment is larger than that (279bp) of a wild strain, so that the recombinant bacterium is constructed correctly and is named as 104M delta VirB2 (hereinafter referred to as delta VirB2, also referred to as 104M mutant strain).

The recombinant bacterium 104M delta VirB2 is obtained by replacing a gene VirB2 in a 104M genome with a kan gene.

4. Detection of recombinant bacteria

1) Genetic stability test

The recombinant bacterium 104M delta VirB2 was continuously passaged in a non-resistant TSB liquid medium, and each generation of the bacterium liquid was recorded and stored at-80 ℃.

PCR verification (primer name 279) is carried out on the 104M strain and the delta VirB2 strain at 1 st to 20 th generations, and the result shows that the Brucella VirB2 mutant can be continuously cultured after continuously propagating for 20 generations, and each generation is subjected to PCR amplification identification to accord with an expected band, which indicates that the inserted exogenous Brucella genome can be stably replicated, and the VirB2 gene does not generate a back mutation phenomenon in the process, which indicates that the strain has good genetic stability; the wild-type negative band fragment size was: 279 bp; the sizes of the mutant strain band fragments are: 1180 bp. (FIG. 6, M: DL2000DNA Marker; 1: wild type colony control; 2-24: 1 st-13 th generation strain of. DELTA. VirB 2).

Meanwhile, the Brucella VirB2 gene mutant strain delta VirB2 generation 1, delta VirB2 generation 10 and delta VirB2 generation 20 are subjected to sequencing verification, 104M strain and VirB2 gene sequences are compared by using DNMAN software, and the homology of VirB2 gene and parent strain is 100%; the kan gene sequence is compared with the sequencing results of the strain delta VirB2 generation 1 and the strain delta VirB2 generation 20, and the homology 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 culture of Δ VirB2 strain with serial passages was strain-identified using brucella identification primers (primer names 1101, 626).

As shown in FIG. 7, FIG. 7A shows the amplification product of primer 1101 and the amplification product of primer 626 of 1101bp, and FIG. 7B shows the amplification product of 626 bp; m: DL2000DNA Marker; 1: wild type colony control; 2-24: the delta VirB2 serial stable passage strains from 1 to 23; the recombinant bacterium delta VirB2 mutant strain has the same characteristic bands as 104M, namely 1101bp and 626bp respectively. The selected mutant strain was shown to be derived from the original strain, not from other contaminants.

3) Characterization of culture Properties

Inoculating the strains of Brucella 104M, VirB2, VirB2, and VirB2 into nonresistant TSB liquid culture medium, shake-culturing at 37 deg.C and 180rpm/min for 2d, collecting the strains, 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

By physiologyInoculating the bacterial liquid of fresh culture 104M strain, strain delta VirB2 strain 1 generation, strain delta VirB2 strain 10 generation and strain delta VirB2 strain 20 generation into nonresistant TSB liquid culture medium with 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 strain Δ VirB2 at the 1 st generation, the strain Δ VirB2 at the 10 th generation, and the strain Δ VirB2 at the 20 th generation.

TABLE 3 examination results of culture characteristics of strains

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

Example 2 Effect of VirB2 Gene-deleted Strain Δ VirB2 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 listlessness and reversed hair growth. The virulence of the Brucella to animals is mainly shown as the viability of the Brucella in vivo, and the evaluation indexes are mainly comparisonThe in vivo colonization and replication capacity of the bacteria, therefore, the toxicity of the bacteria to mice cannot be correctly reflected by the overhigh or overlow infection dose. Thus, choose 10⁷CFU/mL of infectious bacteria.

The Brucella 104M and delta VirB2 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 cultured 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 delta VirB2 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;

Δ VirB2 group: vaccine for delta VirB2 strain immunization with immunization dose of 1 × 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 104M group, Δ VirB2 group, and PBS group, respectively, and the skin and hair morphology, body temperature, and 2-week average body weight, body temperature were recorded.

The results are shown in fig. 9, a is the body weight of the mice after immunization; b is the body temperature of the immunized mouse; the results showed that mice in the 104M, Δ VirB2 and PBS groups performed normally with no obvious abnormalities. The body weights of the mice in the delta VirB2 group and the mice in the 104M group are not obviously different and are all obviously lower than those of the blank control PBS group. Without significant variation between the body temperatures of the groups. The average body weight of each group of mice was measured (see FIG. 9A), and the average body temperature of each group of mice was measured (see FIG. 9B).

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.

Mean spleen weight in the 104M group was significantly different compared to the blank control group (P < 0.01). The spleen weight of the mice is positively correlated with the spleen index, the spleen weight of the mice in the mutant strain and the parent strain group is greatly changed within 1 week after the mice are infected, the spleen weight reaches the highest value in 2 weeks, and the spleen weight is in a descending trend after 5 weeks. Spleen weights of mice infected with the mutant strain were not significantly different compared to the blank control group (P >0.05) and were very significantly different compared to the parental strain 104M (P <0.01) (see FIG. 10A). Further, in combination with the spleen index results, the Δ VirB2 mutant showed a significant decrease in spleen index (P <0.01) compared to the parent strain (see fig. 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.

By counting, the mutants differed significantly from the parental control group 10 weeks after infection (P <0.01) (fig. 11). The PBS blank control group had no bacterial growth. The colony number of the parent strain 104M and the delta VirB2 mutant strain are obviously reduced in the first 3 weeks after infection, the parent strain has no descending trend and the splenomegaly of mutant strain infection shows a descending trend after 4 weeks, and the difference of the average splenomegaly number of the parent strain 104M and the delta VirB2 mutant strain is obvious (P <0.015) in the tenth week, which indicates that the toxicity of the mutant strain delta VirB2 is obviously reduced compared with that of 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 10⁹Brucella 104M immunization vaccine/mL Brucella 104M immunization vaccineSeedling;

Δ VirB2 group: mice were injected subcutaneously with 0.5ml 1.0X 10⁹A vaccine for immunization of the delta VirB2 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 were all alive and were in good condition for body weight mental status. Mice in both the 104M and Δ VirB2 groups immunized the following day were severely lost, with erect fur, depressed mental status and tight eyes. The 104M group of mice died on both days 2 and 4, and only 1 mouse survived on day 7. In contrast, all of the 6 mice in the Δ VirB2 group survived on day 7 and tended to increase in body weight, and mental status was gradually restored. The parent and mutant strains showed significant differences (p >0.05) by analysis of the survival of both groups (see fig. 12), indicating that the mutant strain Δ VirB2 is significantly less virulent than the parent 104M.

Third, detection of immunogenicity

1. Humoral immunity detection

Collecting blood of mice of 104M group, delta VirB2 group and PBS group at the tail breaking period from 1 week to 10 weeks after immunization, standing whole blood at room temperature for 2h, centrifuging at 4 ℃ and 8000r/min for 10min, collecting upper layer serum, and detecting the level of specific IgG of the mouse serum by using an indirect ELISA method, wherein the method 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 measured wavelength of 450nm is taken as the ordinate, the sampling time is taken as the abscissa, and an antibody extinction curve is drawn (see figure 13). After 2 weeks of immunization of the parent strain and the mutant strain on the mice, the IgG level in the serum of the mice gradually rises, and the antibody level is not obviously reduced by 10 weeks. From the trend of change, the antibody titer of the parent 104M and mutant delta VirB2 immunized mice was not significantly different from that of the mutant at week 8 of immunization (P < 0.05).

2. Cellular immunoassay

1) Preparation of mouse spleen lymphocytes

(1) After the eyeballs of mice in the group 104M, the group delta VirB2 and the group PBS which are immunized in the 4 th week 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, Δ VirB2 and PBS splenic lymphocytes.

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

Respectively mixing 5 × 10⁶100. mu.L of each/mL 104M spleen lymphocyte, Δ VirB2 spleen lymphocyte and PBS spleen lymphocyte dilution was added to a 96-well plate, and 100. mu.L of 5X 10 spleen lymphocyte dilution 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)

As shown in FIG. 16, 104M group spleen lymphocytes, Δ VirB2 group spleen lymphocytes and PBS group spleen lymphocytes were unstimulated with inactivated antigen, and SI was calculated after detection by CCK8 method; the mutant strain and the parent control group are proved to be capable of stimulating the mouse lymphocyte proliferation through analysis, and the delta VirB2 mutant strain has higher capacity of stimulating the mouse lymphocyte proliferation than the parent 104M.

The above results show that Δ VirB2 is more immunogenic than the parental strain 104M.

3) Influence of VirB2 Gene deletion on the Induction of lymphocyte transformation by the Strain

(1) Respectively using 104M group spleen lymphocytes, delta VirB2 group spleen lymphocytes and PBS group spleen lymphocytes in RPMI1640 dilution of the culture to 5X 10⁶one/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 VirB2CP cyanine5.50.25. mu.L, anti-mouse CD4FITC 0.5. mu.L, anti-mouse CD8a PE 0.5. mu.L antibodies were added, and the mixture was incubated at 4 ℃ for 1h in the dark (wherein 3 fluorescent antibodies were added to the negative control group, respectively, for calibration in a 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).

Spleen lymphocytes of 104M group, spleen lymphocytes of delta VirB2 group and spleen lymphocytes of PBS group were labeled with CD3, CD4 and CD8 fluorescent-labeled antibodies, the labeled cells were counted by flow cytometry, the number of immune cells of CD3+, CD4+ and CD8+ was determined, and the value of CD4+/CD8+ was calculated. The results show that the lymphocyte differentiation of mice is changed after the immunization of the mutant strain and the parental control group, and the mice can cause the increase of CD3+, CD4+ and CD8+ (for example, in the figure 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 delta VirB2, and F is CD8+ cells caused by delta VirB 2). From the results of CD4+/CD8+ (fig. 14), the Δ VirB2 mutant strain was significantly higher than the 104M parent strain.

The above results show that Δ VirB2 is more immunogenic than the parental strain 104M.

4) Cytokine detection

(1) Spleen lymphocytes of 104M group, those of Δ VirB2 group and those of PBS group 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, so that the final concentration of antigen in each hole is equal to the immune dose), and simultaneously setting blank pairsAnd (4) performing group control.

(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 parental strain 104M induced significantly less cytokine IL-2 than the Δ VirB2 mutant (fig. 17A); the Δ VirB2 mutant induced a significant reduction in IL-4 compared to parental strain 104M (fig. 17B); the parental strain 104M induced higher cytokine INF- γ than the Δ VirB2 mutant (fig. 17C).

Claims (3)

1. The recombinant bacteria are obtained by reducing and/or inhibiting the activity of VirB2 protein in Brucella 104M;

the reduction and/or inhibition of the VirB2 protein activity in the Brucella 104M is the inhibition or silencing of the expression of VirB2 protein coding gene in the Brucella 104M;

the expression of the VirB2 protein coding gene in the Brucella 104M is knocked out to obtain the VirB2 protein coding gene in the Brucella 104M;

the VirB2 protein coding gene in the Brucella 104M is knocked out, so that the VirB2 protein coding gene in the Brucella 104M is replaced by a resistance gene;

the VirB2 protein coding gene in the Brucella 104M is replaced by a resistance gene by adopting a mode of genome site-directed 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 VirB2 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 VirB2 protein coding gene, the resistance gene and a downstream homology arm of a VirB2 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 recombinant bacterium of claim 1, which is used for preparing any one of the following products 1) to 6):

1) brucella attenuated vaccines;

2) brucella vaccines;

3) products for promoting lymphocyte proliferation;

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

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

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

3. A product of any one of the following 1) to 6), wherein the active ingredient of the product is the recombinant bacterium of claim 1;

1) brucella attenuated vaccines;

2) brucella vaccines;

3) products for promoting lymphocyte proliferation;

4) promote CD3⁺、CD4⁺、CD8⁺(ii) a cell augmentation product;

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

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