Bovine Brucella A19 T4SS-bvfA strain as well as construction method and application thereof

Abstract

The invention discloses a bovine Brucella A19 T4SS-bvfA strain as well as a construction method and application thereof, and relates to the technical field of microorganisms. The bovine Brucella A19 T4SS-bvfA strain is preserved in the China General Microbiological Culture Collection Center on September 27, 2024, and the preservation number of the bovine Brucella A19 T4SS-bvfA strain is CGMCC (China General Microbiological Culture Collection Center) No.46201. The vaccine prepared by the invention can be distinguished from natural infection and vaccine immunity, and has no resistance residue, thalli do not enter host cells, and the safety is higher.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically to a bovine Brucella A19ΔT4SS-bvfA strain, a construction method and an application thereof. Background Art

[0002] Brucellosis, also known as brucellosis, is a worldwide zoonotic infectious disease, and the selection of ideal antigens is a major step in vaccine design. To this end, different research groups have studied different subunit components of Brucella as recombinant protein vaccines in mice and animal models. Although a large number of studies have been conducted on immunogenic and protective Brucella antigens in recent decades, most antigens are not considered to be effective vaccines for brucellosis. In order to design and develop an effective Brucella vaccine, it is necessary to further understand the pathogenesis of Brucella, the bacterial cell wall, the best method for antigen purification, the choice of animal models, and effective adjuvants. In addition, no one has yet used an anti-Brucella vaccine, and the differences in the immune response of humans and animals to the vaccine also need to be considered.

[0003] At present, the smooth vaccines on the market include Brucella vaccine strain S19, B. melitensis Rev. 1 vaccine, B. melitensis H38 vaccine, B. melitensis M5 vaccine and B. suis vaccine S2, and the rough vaccines include B. abortus vaccine RB51, B. abortus 45 / 20 vaccine and B. abortus SR82 vaccine. These vaccines have the disadvantage of being unable to distinguish between natural infection and vaccine immunity. Therefore, it is necessary to develop a new strain to prepare a gene-deficient live vaccine that can distinguish between vaccine immunity and natural infection. Summary of the invention

[0004] To solve the above problems, the present invention provides a bovine Brucella A19△T4SS-bvfA strain, a construction method and an application thereof, so as to solve the shortcoming of the prior art that natural infection and vaccine immunity cannot be distinguished.

[0005] The present invention is achieved through the following technical solutions: A Brucella abortus A19△T4SS-bvfA strain, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 27, 2024, with the deposit number CGMCC No.46201.

[0006] The construction method of the bovine Brucella A19ΔT4SS-bvfA strain is as follows: S1: The upstream homologous recombination sequence and the downstream homologous recombination sequence of the bvfA gene were synthesized to obtain the targeting sequence △bvfA, which was cloned into pCVD442GS to obtain the targeting plasmid pCVD442GS-△bvfA.

[0007] S2: The targeting plasmid pCVD442GS-△bvfA was electroporated into the engineered bacteria to obtain β 2155 / pCVD442GS-△bvfA donor bacteria.

[0008] S3: The β2155 / pCVD442GS-△bvfA donor bacteria were combined with the bovine Brucella A19 recipient bacteria, and gentamicin-resistant clones were selected, which were called A19 / pCVD442GS-△bvfA.

[0009] S4: Take the bacterial liquid of a single A19 / pCVD442GS-△bvfA clone and inoculate it on a TSA plate for screening, and obtain the bovine Brucella bvfA gene deletion strain A19△bvfA by PCR.

[0010] S5: Using the single gene deletion strain A19△bvfA as the initial strain, the genes virB1~virB12 were knocked out simultaneously according to the S1~S4 methods to obtain the bovine Brucella A19△T4SS-bvfA strain.

[0011] The targeting sequence △ Bf The gene sequence is shown in SEQ ID NO.1; virB1~virB12 The gene sequence is shown in SEQ ID NO.6.

[0012] Preferably, the engineered bacteria is Escherichia coli.

[0013] The bovine Brucella A19ΔT4SS-bvfA strain is used in preparing a vaccine for preventing bovine Brucella.

[0014] A vaccine for preventing bovine brucellosis, wherein the active ingredient of the vaccine comprises the bovine brucellosis A19ΔT4SS-bvfA strain or its culture. The vaccine prepared only by the bovine brucellosis A19ΔT4SS-bvfA strain or its culture can be used for preventing bovine brucellosis.

[0015] Preferably, the active ingredient further comprises an adjuvant, and the vaccine prepared by adding the adjuvant to the bovine Brucella A19ΔT4SS-bvfA strain or its culture can be used to prevent bovine Brucella.

[0016] Preferably, the adjuvant is one or more of aluminum salt, oil emulsion or immunomodulator.

[0017] Preferably, the vaccine further comprises conventional auxiliary components for preparing vaccines.

[0018] Preferably, the vaccine is prepared according to the following method: the bovine Brucella A19△T4SS-bvfA strain is inoculated into a culture medium for cultivation, the culture is harvested, conventional auxiliary components are added, and the vaccine is prepared by drying. The conventional auxiliary components are stabilizers; the number of viable bacteria in the vaccine is 6.0×10 10 CFU / g~6.5×10 10 CFU / g.

[0019] Preferably, the culture medium is TSB liquid culture medium.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The invention provides a bovine Brucella A19△T4SS-bvfA strain, and uses genetic engineering technology to simultaneously knock out 12 genes of virB1~virB12 and the bvfA gene on the bovine Brucella vaccine strain A19, thereby constructing a bovine Brucella T4SS-bvfA combined knockout strain A19△T4SS-bvfA strain, wherein BvfA is a virulence factor unique to the genus Brucella, and its unique protein can be used to distinguish natural infection from vaccine immunity, which is beneficial to distinguish other genera, and has stronger specificity. Since it is a virulence factor itself, the toxicity of the strain can also be reduced after knocking it out, and it is the best choice for becoming an excellent vaccine. Knocking out 12 genes of virB1~virB12 at the same time can prevent the strain from entering the host cell through immune escape, and reduce chronicity caused by persistent infection; and can also perform subsequent secondary distinction between natural infection and vaccine immunity through any one of the 12 proteins of VirB1~VirB. A19 is a smooth Brucella strain, and its LPS still contains O-polysaccharide, which can continuously stimulate animals to produce anti-LPS antibodies, interfering with the routine serological detection of immune animals and naturally infected animals. The present invention proves through experiments that the vaccine prepared by the strain can be distinguished between natural infection and vaccine immunity. Through the invasion cell test, it was found that the double knockout A19△T4SS-bvfA strain constructed by the present invention does not enter the host cell, and is safer. In addition, the present invention directly docks the homologous upstream and downstream arms, and the construction of the T4SS-bvfA combined knockout strain is simple to operate, and the antibiotics are automatically removed after screening, and there is no resistance residue. In practical applications, if a vaccine cannot distinguish between natural infection and vaccine immunity, it is difficult to judge whether an individual has acquired immunity through natural infection or vaccination in subsequent tests. It is difficult to accurately evaluate the vaccination effect of the vaccine and the immune status of the vaccinated object. In addition, for diseases that need to distinguish between natural infection and vaccine immunity to formulate subsequent prevention and control strategies, this indistinguishable situation will increase the difficulty and complexity of prevention and control.

[0021] Biomaterial Deposit Brucella bovis A19△T4SS- BfThe strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 27, 2024, with the deposit number CGMCC No.46201, and the strain classification was named Brucella bovis Brucella abortus , the storage address is Beijing, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is a diagram of the target sequence PCR product of the present invention; M: standard DNA molecular weight. The molecular weights from top to bottom are: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp, among which 750bp is highlighted; 1: target sequence PCR product 1719bp.

[0024] Figure 2 The outer primers of the present invention Bf -outF and f -outR PCR identification results; Figure 2 In the figure, A: 1-24 are the PCR amplification results of 24 monoclonal outer primers selected after the ligation experiment, 2161 / 1825bp, B: 1-19 are the PCR amplification results of 19 monoclonal outer primers selected after the ligation experiment, 2161 / 1825bp, 20 is the negative control, i.e., the parent strain A19, and 21 is the blank control, i.e., no template; M in A and B is the standard DNA molecular weight, and the molecular weights from top to bottom are: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp, among which 750bp is highlighted.

[0025] Figure 3 The figure shows the results of PCR identification of the inner primers bvfA-inF and bvfA-inR of the present invention; M1: standard DNA molecular weight. The molecular weights from top to bottom are: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 750bp is highlighted. 3, 6, 21 are the results of PCR amplification of knockout strain A19△bvfA without bands, and the rest are the results of PCR amplification of parent strain A19.

[0026] Figure 4 The outer primers of the present invention virB -outF and virB -outR PCR identification results, in A and B, the same numbers above and below are two parallel samples; Figure 4 In the figure, A: M is the standard DNA molecular weight, and the molecular weights from top to bottom are: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 1 to 12 above and below are the amplification results of 24 monoclonal outer primers selected after the ligation experiment; B: M is the standard DNA molecular weight, and the molecular weights from top to bottom are: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1 to 12 above and 1 to 10 below are the amplification results of 22 monoclonal outer primers selected after the ligation experiment, with a single band of 1864bp, 11 below B is the negative control, i.e., the parent strain A19, and 12 below B is the blank control, i.e., no template. A19△ f The length of the amplified product of the outer primers of the strain was 13009 bp, A19△T4SS- f The length of the amplified product of the knockout strain was 1864 bp.

[0027] Figure 5 The inner primer of the present invention virB -inF and virB -inR PCR identification results; M is Marker, and the molecular weights from top to bottom are: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1, 2 are 284bp fragments of the amplification products of the inner primers of the A19△bvfA strain, and 3~8 are bands without bands of the A19△T4SS-bvfA knockout strain.

[0028] Figure 6The PCR verification result diagram of the inner primers bvfA and virBs of the double knockout strain of the present invention; Marker: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1 is a marker, 2 is the original strain of A19, with double bands of bvfA202bp and virBs284bp; 3 is the A19△virBs strain, with a single band of bvfA202bp; 4 is the A19△bvfA strain, with a single band of virBs284bp; 5 is the double knockout A19△T4SS-bvfA strain, with no band; 6 is a negative control, with no band; 7 is a marker; 8 is the original strain of A19, with a single band of virBs284bp; 9 is the A19△v irBs strain, no band; 10 is A19△bvfA strain, virBs284bp single band; 11 is double knockout A19△T4SS-bvfA strain, no band; 12 is negative control, no band; 13 is Marker; 14 is A19 original strain, bvfA202bp single band; 15 is A19△virBs strain, bvfA202bp single band; 16 is A19△bvfA strain, no band; 17 is double knockout A19△T4SS-bvfA strain, no band; 18 is negative control, no band; 1~6 are multiplex PCRs using bvfA and virBs primers; 7~12 are PCRs using virBs internal primers; 13~18 are PCRs using bvfA internal primers.

[0029] Figure 7 This is a diagram of the validation results of the invasion cell test of the present invention; * represents P<0.05, ** represents P<0.01, and *** represents p<0.001.

[0030] Figure 8 This is the result of BvfA protein distinguishing natural infection and vaccine immunity; the horizontal axis represents the time of antibody production after vaccine injection, the vertical axis represents the S / N value, * represents P<0.05, ** represents P<0.01, and *** represents p<0.001. DETAILED DESCRIPTION

[0031] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] The inventive concept of the present invention is as follows: At present, the smooth vaccines on the market include Brucella vaccine strain S19, B. melitensis Rev. 1 vaccine, B. melitensis H38 vaccine, B. melitensis M5 vaccine and B. suis vaccine S2, and the rough vaccines include B. abortus vaccine RB51, B. abortus 45 / 20 vaccine and B. abortus SR82 vaccine. These vaccines have the disadvantage of being unable to distinguish between natural infection and vaccine immunity. Therefore, it is necessary to develop a new strain to prepare a gene-deficient live vaccine that can distinguish between vaccine immunity and natural infection.

[0034] Based on this, the present invention provides a bovine Brucella A19△T4SS-bvfA strain, bovine Brucella virulence factor A, that is, BvfA is a new virulence factor unique to the genus Brucella, which is an alkaline protein composed of 111 amino acids. The use of its unique protein can distinguish natural infection and vaccine immunity, which is conducive to distinguishing other genera, and has stronger specificity. Because it is a virulence factor itself, the toxicity of the strain can also be reduced after knocking it out, and it is the best choice for becoming an excellent vaccine. The proliferation of the B.suis1330bvfA::kan mutant in THP1 cells, J774 cells and HeLa cells is significantly lower than that of the parent strain. In addition, when the B.suis1330bvfA::kan mutant infected mice at 4 weeks and 8 weeks, the number of bovine Brucella in the spleen was significantly less than that of the parent strain. The toxicity of the B. suis1330bvfA::kan mutant was highly weakened in both in vivo and in vitro models, indicating that the bvfA gene plays an important role in the survival of bovine Brucella both in vivo and in vitro. In addition, in bovine Brucella, the IV type secretion system, also known as type IV secretion system, or T4SS for short, consists of 12 proteins encoded by the virB operon, namely VirB1~VirB12. Bovine Brucella can use T4SS secretion to enter host cells, evade immune surveillance, and maintain its persistent presence in cells, which is also one of the reasons why bovine brucellosis is difficult to cure. Knocking out the 12 genes virB1~virB12 can not only prevent the strain from entering the host cell through immune escape and reduce persistent infection and chronicity; it can also use any one of the 12 proteins VirB1~VirB to perform subsequent secondary distinction between natural infection and vaccine immunity. Genetic engineering technology was used to simultaneously knock out the 12 genes virB1~virB12 and the bvfA gene in the bovine Brucella vaccine strain A19, and a bovine Brucella T4SS-bvfA combined knockout strain A19△T4SS-bvfA strain was constructed. Experiments showed that the vaccine prepared by this strain can be distinguished between natural infection and vaccine immunity. Through the invasion cell test, it was found that the double knockout A19△T4SS-bvfA strain constructed by the present invention does not enter the host cell, and is safer. In addition, the present invention directly docks the homologous upstream and downstream arms, and the construction of the T4SS-bvfA combined knockout strain is simple to operate, and the antibiotics are automatically removed after screening, and there is no resistance residue.

[0035] The construction method of bovine Brucella A19△T4SS-bvfA strain is as follows: Step 1: Construct the bovine Brucella A19△bvfA deletion strain.

[0036] (1) Synthesis of targeting sequences The upstream and downstream homologous recombination sequences of the bvfA gene were synthesized to obtain a complete targeting sequence △bvfA, which was then cloned into the suicide plasmid pCVD442GS containing the gentamicin resistance gene to obtain the targeting plasmid pCVD442GS-△bvfA.

[0037] (2) Obtaining donor bacteria pCVD442GS-△bvfA was electro-transformed into E. coli β2155 to obtain β2155 / pCVD442GS-△bvfA donor bacteria.

[0038] (3) Bonding experiment The β2155 / pCVD442GS-△bvfA donor bacteria were used for conjugation experiments with the bovine Brucella A19 recipient bacteria, and gentamicin-resistant bovine Brucella clones were screened on gentamicin plates. The targeting plasmid was integrated into its genome and was called A19 / pCVD442GS-△bvfA.

[0039] (4) Screening of bovine Brucella A19△bvfA deletion strain Take a single A19 / pCVD442GS-ΔbvfA clone and streak it into LB liquid medium containing 10% sucrose overnight, then streak it onto TSA plates and incubate at 37°C with 5% CO 2 The cells were cultured until a single clone was formed. A bvfA gene deletion clone was obtained by PCR screening and named A19△bvfA.

[0040] Step 2: Continue to knock out the 12 virBs genes of T4SS in the bovine Brucella A19△bvfA deletion strain.

[0041] (1) Construction of targeting plasmid pCVD442GS-△virBs The upstream and downstream homologous recombination sequences of the virB1-virB12 genes were directly synthesized to obtain a complete targeting fragment △virBs, which consisted of an upstream homologous arm-a downstream homologous arm, and then cloned into the suicide plasmid pCVD442GS containing the gentamicin resistance gene to obtain the targeting plasmid pCVD442GS-△virBs.

[0042] (2) Obtaining donor bacteria pCVD442GS-△virBs was electro-transformed into Escherichia coli β2155 to obtain donor bacteria β2155 / pCVD442GS-△virBs.

[0043] (3) Bonding experiment The β2155 / pCVD442GS-△virBs donor bacteria were used for conjugation experiments with the bovine Brucella A19△bvfA recipient bacteria, and gentamicin-resistant bovine Brucella clones were screened on gentamicin plates. The targeting plasmid was integrated into its genome and was called A19△bvfA / pCVD442GS-△virBs.

[0044] (4) Screening of bovine Brucella A19△T4SS-bvfA combined deletion strain Take a single A19△bvfA / pCVD442GS-△virBs clone and streak it into LB liquid medium containing 10% sucrose overnight, then streak it onto TSA plates and incubate at 37℃ with 5% CO 2 The cells were cultured until a single colony was formed. A T4SS-bvfA gene deletion clone was obtained by PCR screening and named Brucella bovis A19△T4SS-bvfA.

[0045] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0046] Example 1 Step 1: Construction of bovine Brucella A19△bvfA deletion strain 1. Synthesis of Targeting Sequences The upstream and downstream homologous recombination sequences of the bvfA gene were directly synthesized to obtain the complete targeting sequence △bvfA, which was composed of the upstream homologous arm-downstream homologous arm.

[0047] The gene sequence of the targeting sequence △bvfA is shown in SEQ ID NO.1, which consists of an upstream homologous recombination arm and a downstream homologous recombination arm, with a length of 1719 bp. Figure 1 shown.

[0048] SEQ ID NO.1:

[0049] 2. Construction of targeting plasmid pCVD442GS-△bvfA Enzyme digestion: After the pCVD442GS plasmid was digested with SmaI, the enzyme digestion system was as shown in Table 1.

[0050] Table 1 Enzyme digestion system

[0051] After reacting at 37°C for 2 h, 5 μL of dephosphorylase FastAp was added and the reaction was continued for 30 min. After the reaction, the dephosphorylated vector and the targeting sequence △bvfA were separated by 1% agarose gel electrophoresis, purified by column centrifugation, and eluted in 50 μL of deionized water.

[0052] Ligation: Use 5U / μL MBI T4 DNA ligase to ligate the purified pCVD442GS / SmaI and the target sequence △bvfA at 16°C overnight. The ligation system is shown in Table 2.

[0053] Table 2 Connection system

[0054] Transformation: After the ligation product was precipitated with isopropanol, it was washed with 70% ethanol and dissolved in 5 μL deionized water. It was transformed into E. coli DH5αλpir by electroporation and cultured at 37°C on LB plates containing 20 μg / mL gentamicin until a single clone was formed. The clone was picked and transferred to 3 mL LB containing 20 μg / mL gentamicin, cultured at 37°C overnight, and the plasmid DNA was extracted and sequenced. After the correct one was identified, it was named the targeting plasmid pCVD442GS-△bvfA.

[0055] 3. Construction of donor bacteria: The targeting plasmid pCVD442GS-ΔbvfA was electrotransformed into E. coli β 2155 strain, spread on LB plates containing 20 μg / mL gentamicin and 0.5 mM diaminopimelic acid, i.e. DAP, purchased from Aladdin, and cultured at 37°C until a single clone was formed. This clone is the β 2155 / pCVD442GS-ΔbvfA donor strain used in the conjugation experiment.

[0056] 4. Binding Experiment The β 2155 / pCVD442GS-△bvfA donor bacteria and the bovine Brucella A19 recipient bacteria were washed with TSB, 500 μL of each was gently combined, and 5 μL of 0.1M DAP was added and mixed. 100 μL was spread on a 0.5mM DAP plate and incubated at 37°C with 5% CO 2After 4 h of incubation, the cells were washed with TSB and inoculated on TSA plates containing 20 μg / mL gentamicin at 37°C with 5% CO 2 Culture for 72 h until single colony is formed.

[0057] 5.PCR Identification Pick 43 single clones from the above plate and put them into 20 μL TSB respectively, take 1 μL as template, and use outer primers bvfA-outF and bvfA-outR for PCR identification. The bvfA-outF primer sequence is shown in SEQ ID NO.2, the bvfA-outR primer sequence is shown in SEQ ID NO.3, the reaction system is shown in Table 3, and the reaction procedure is shown in Table 4.

[0058] Table 3 Reaction system

[0059] Table 4 Reaction procedure

[0060] Note: “ / ” means no such item.

[0061] SEQ ID NO. 2: 5'-TTGTTGCGGCTTATGTAGATTGCG-3'.

[0062] SEQ ID NO. 3: 5'-ACAATCCGCAGGACTATGTGACG-3'.

[0063] The judgment result is Figure 2 As shown: the length of the amplified product of the A19 outer primer is 2161bp, and the length of the amplified product of A19△bvfA is 1825bp. At this time, the product length of the outer primer may have three situations: a single band of 2161bp, which means no recombination has occurred; a double band of 2161 / 1825bp, which means one and two recombination have occurred; and a single band of 1825bp, which means two recombination has occurred. After this PCR identification, the results showed that there was a single band of 2161bp, which means no recombination has occurred, and a double band of 2161 / 1825bp, which means one and two recombination have occurred.

[0064] 6. Sucrose Screening Select No. 24 because No. 24 is a brighter band of 1825 bp in the lower band. The 1825 bp band is the positive clone for screening. 15 μL of bacterial solution was inoculated into 3 mL of TSB liquid medium and incubated at 37°C with a volume fraction of 5% CO. 2After 48 h of incubation, 1 mL of bacterial culture was taken, centrifuged at 4000 g to precipitate, and then suspended with an equal volume of 10% LB sucrose solution and washed once. The precipitate was centrifuged at 4000 g to add to 3 mL of 10% LB sucrose solution and incubated at 37 °C with 5% CO 2 After 48 h of culture, the culture solution was streaked on TSA plates at the original solution, 1:20, and 1:200 respectively after reverse selection by sucrose, and the culture solution was inoculated at 37°C with a volume fraction of 5% CO 2 Culture for 72 h until single clones are formed.

[0065] 7.PCR and sequencing identification The above monoclonal clone was picked up in 20 μL TSB, 1 μL was taken as a template, and PCR identification was performed using the inner primers bvfA-inF and bvfA-inR. The bvfA-inF primer sequence is shown in SEQ ID NO.4, the bvfA-inR primer sequence is shown in SEQ ID NO.5, the reaction system is shown in Table 5, and the reaction procedure is shown in Table 6.

[0066] Table 5 Reaction system

[0067] Table 6 Reaction procedure

[0068] Note: “ / ” means no such item.

[0069] SEQ ID NO. 4: 5'-GCTCTGCATCGTATAGAGCCAGC-3'.

[0070] SEQ ID NO. 5: 5'-GCAGGTCCGCAAGGATGTGAG-3'.

[0071] The judgment result is as follows Figure 3 As shown: After successful sucrose reverse screening, A19△bvfA was negative and a 202bp fragment was amplified in A19.

[0072] The knockout strain was selected and sequenced with the outer primers, and the results showed that it was correct, and the strain completely lacked bvfA to obtain the A19△bvfA strain.

[0073] Step 2: In bovine Brucella A19△ Bf Continue to knock out 12 T4SS in the deletion strain virB Gene virB1~ virB12 .

[0074] virB1~virB12 The gene sequence 1718bp is shown in SEQ ID NO.6.

[0075] SEQ ID NO.6:

[0076] The methods of steps 1, 2, and 3 are exactly the same as the first step, and the β2155 / pCVD442GS-△T4SS donor bacteria used for conjugation experiments are obtained.

[0077] 4. Binding Experiment Wash the donor bacteria and the recipient bacteria of Brucella bovis A19△bvfA with TSB, take 500 μL of each and gently combine, add 5 μL of 0.1M DAP and mix well. Pipette 100 μL and spread on a 0.5mM DAP plate, incubate at 37°C with 5% CO 2 After 4 h of incubation, the cells were washed with TSB and inoculated on TSA plates containing 20 μg / mL gentamicin at 37°C with 5% CO 2 Culture for 72 h until single colony is formed.

[0078] 5.PCR Identification The above monoclonal clone was picked up in 20 μL TSB, 1 μL was taken as a template, and PCR identification was performed using the outer primers virB-outF and virB-outR. The virB-outF primer sequence is shown in SEQ ID NO.7, the virB-outR primer sequence is shown in SEQ ID NO.8, the reaction system is shown in Table 7, and the reaction procedure is shown in Table 8.

[0079] Table 7 Reaction system

[0080] Table 8 Reaction procedure

[0081] Note: “ / ” means no such item.

[0082] SEQ ID NO. 7: 5'-GGTTCGCTCACCATCGCCGAATG-3'.

[0083] SEQ ID NO. 8: 5'-GGCATTGCGATAGATTTCATTGAGTGCCAG-3'.

[0084] The judgment result is Figure 4 As shown: the length of the amplification product of the outer primers of the original strain is 13009bp, and the length of the amplification product of the knockout strain is 1864bp. At this time, the length of the product of the outer primers of the clone picked on the gentamicin plate may have three situations: a single band of 13009bp, indicating no recombination; a double band of 13009 / 1864bp, indicating primary and secondary recombination; and a single band of 1864bp, indicating secondary recombination.

[0085] After this PCR identification, a single band of 1864bp appeared, indicating that secondary recombination had occurred, indicating that the results showed that secondary recombination had occurred in all clones.

[0086] 6. Sucrose Screening The brightest band was selected and 15 μL of bacterial solution was inoculated into 3 mL of TSB liquid medium at 37°C with 5% CO 2 After 48 h of incubation, 1 mL of bacterial solution was taken, centrifuged at 4000 g to precipitate, suspended with an equal volume of 10% LB sucrose solution, washed once, centrifuged at 4000 g to take the precipitate and added to 3 mL of 10% LB sucrose solution, incubated at 37 °C and 5% CO 2 After 48 h of culture, the culture solution was streaked on TSA plates at the original solution, 1:20, and 1:200 respectively after reverse selection by sucrose, and the culture solution was inoculated at 37°C with a volume fraction of 5% CO 2 Culture for 72 h until single clones are formed.

[0087] 7.PCR and sequencing identification The above monoclonal clone was picked up in 20 μL TSB, 1 μL was taken as a template, and PCR identification was performed using the inner primers virB-inF and virB-inR. The virB-inF primer sequence is shown in SEQ ID NO.9, the virB-inR primer sequence is shown in SEQ ID NO.10, the reaction system is shown in Table 9, and the reaction procedure is shown in Table 10.

[0088] Table 9 Reaction system

[0089] Table 10 Reaction procedure

[0090] Note: “ / ” means no such item.

[0091] SEQ ID NO. 9: 5'-CATGCGCTGTCTCTCTGGCCGC-3'.

[0092] SEQ ID NO. 10: 5'-CCTGACGTTGATATGCTTGGGCACGG-3'.

[0093] The judgment result is as follows Figure 5 As shown: the amplification of this pair of primers should be negative, while the positive control of the original bacteria has a 284bp fragment amplified.

[0094] The knockout strain was selected and sequenced with outer primers. The results showed that it was correct and was named A19△T4SS-bvfA strain.

[0095] Example 2 The double knockout strain was verified to amplify the bvfA gene and the virBs gene. The PCR system and procedure are shown in Tables 5, 6, 9, and 10 above.

[0096] The primers used were the internal primers mentioned above, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.9 and SEQ ID NO.10; the procedures and systems are shown in Tables 5, 6, 9 and 10 above. Electrophoresis was performed using a 1.5% agarose gel. The electrophoresis results are shown in Figure 6 As shown, the PCR verification result diagram of the inner primers bvfA and virBs of the double knockout strain of the present invention, Marker: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1 is Marker; 2 is the original strain of A19, with double bands of bvfA202bp and virBs284bp; 3 is the A19△virBs strain, with a single band of bvfA202bp; 4 is the A19△bvfA strain, with a single band of virBs284bp; 5 is the double knockout A19△T4SS-bvfA strain, without bands; 6 is the negative control, without bands; 7 is Marker; 8 is the original strain of A19, with a single band of virBs284bp; 9 is the A19△ virBs strain, no band; 10 is A19△bvfA strain, with a single band of virBs284bp; 11 is double knockout A19△T4SS-bvfA strain, no band; 12 is negative control, no band; 13 is Marker; 14 is A19 original strain, with a single band of bvfA202bp; 15 is A19△virBs strain, with a single band of bvfA202bp; 16 is A19△bvfA strain, no band; 17 is double knockout A19△T4SS-bvfA strain, no band; 18 is negative control, no band; 1~6 are multiplex PCRs using bvfA and virBs primers; 7~12 are PCRs using virBs internal primers; 13~18 are PCRs using bvfA internal primers. After the electrophoresis was correct, it was sent to Shanghai Biotechnology for sequence determination and alignment, indicating that the bvfA gene and virB gene double knockout strain was successfully constructed.

[0097] Example 3 Validation of the invasion cell assay.

[0098] Method: After counting J774A.1 cells, 1×10 6Cells were plated in a 6-well plate until they attached to the wall to form a monolayer of cells. Brucella bovis in the logarithmic growth phase was infected with an MOI of 100:1, and extracellular bacteria were killed with gentamicin after 15 min, 30 min, and 45 min of infiltration. The cells were washed with PBS three times, lysed with 0.01% Triton-100, and coated with solid culture medium at a 10-fold dilution. The invasion was determined by CFU count. The results are shown in Figure 7 As shown, ● represents the invasion of A19ΔT4SS-bvfA strain; ■ represents A19Δ f Strain invasion; ▲ represents A19Δ virBs Strain invasion; ◆ represents A19 strain invasion, the column height represents the number of invading cells in each group, and the graph on the bar graph indicates that each group has three replicates, of which three ● overlap into one. A19ΔT4SS- Bf Group, A19Δ f Group and A19Δ virB The number of invasion groups in the A19 group was less than that in the A19 group, indicating that the invasion ability was weakened after the gene was knocked out, while the A19ΔT4SS- Bf The group value is 0, which means that it cannot invade cells, has the weakest invasion ability, and has the highest safety. Therefore, this result shows that A19△T4SS- Bf No colonies were collected from cells infected with the double knockout strain, indicating that it did not invade the cells, indicating that the double knockout A19△T4SS- Bf The strain cannot invade J774A.1 macrophages. This indicates that the double knockout A19ΔT4SS- Bf The strain does not enter the host cells and is safer.

[0099] Example 4 Experiments to differentiate between natural infection and vaccine immunity.

[0100] (1) Purify BvfA protein by prokaryotic expression and distinguish natural infection from vaccine immunity by BvfA protein detection.

[0101] (2) Indirect ELISA kit The indirect ELISA method was used to determine the difference between the purified BvfA protein and normal saline, A19△T4SS- f The cells were coated with 100 μL of coating solution, pH 9.6, 0.05 mol / L carbonate buffer, at a concentration of 0.2 μg / mL, at 4 ˚C overnight, with 3 replicates per group.

[0102] The coating solution was discarded, and the cells were washed three times with 280 μL PBST buffer, pH 7.4, 0.01 mol / L PBS: Tween-20, 1:1000, and blocked with 100 μL 1% BSA, pH 7.4, at 37°C for 1 h, then rinsed three times with PBST, and 50 μL of mouse serum immunized with saline, A19△T4SS-bvfA strain, and A19 strain for 2 weeks and 3 weeks was added, respectively, and incubated for 1 h. 100 μL 1:5000 horseradish peroxidase-labeled goat anti-mouse IgG was added, incubated at 37°C for 1 h, and then washed three times with PBST, and 100 μL TMB substrate was added, and the cells were placed in the dark at room temperature for 10 min, and 50 μL 2 mol / L sulfuric acid solution was added to terminate the reaction, and the OD absorbance was measured at 450 nm. Three replicates were performed in each group. The antibody titer is reflected by the S / N value. If S / N ≥ 2.1, it is positive. S / N = sample OD450 -blank OD450 / Negative control OD 450 – Blank OD 450 The negative control is the serum of uninjected mice. S is the abbreviation of Sample; N is the abbreviation of Negative. The negative control is the serum of uninjected mice; the saline group is the control group.

[0103] The judgment result is as follows Figure 8 As shown: The graph on the bar graph indicates that each group has three replicates, the S / N value of the control is equal to the average of the three origin column heights, the S / N value of A19ΔT4SS-bvfA is equal to the average of the three square column heights, and the S / N value of A19 is equal to the average of the three triangle column heights. S / N ≥ 2.1 is natural infection; 2.1 is the cut-off, i.e., the critical value; S / N < 2.1 is injected with A19ΔT4SS- f Vaccine. Verify that BvfA protein is different from saline (control), A19△T4SS- Bf Serum antibody reactivity of mice injected with A19 strain, saline (control) and A19△T4SS- f There is no BvfA protein in the A19△T4SS- Bf None of the groups reacted with BvfA protein, and there was no statistical difference between the two groups. The reaction S / N values ​​were all less than 2.1; while the A19 strain produced anti-BvfA protein antibodies after injection, and the reaction S / N values ​​were all greater than 2.1; natural infection and vaccine immunity can be distinguished based on the S / N value.

[0104] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0105] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Brucella abortus A19ΔT4SS-bvfA strain, characterized in that: The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 27, 2024, with the deposit number CGMCC No.46201.

2. The method for constructing the bovine Brucella A19ΔT4SS-bvfA strain as claimed in claim 1, characterized in that: The specific construction method is as follows: S1: The upstream homologous recombination sequence and the downstream homologous recombination sequence of the bvfA gene were synthesized to obtain the targeting sequence △bvfA, which was cloned into pCVD442GS to obtain the targeting plasmid pCVD442GS-△bvfA; S2: The targeting plasmid pCVD442GS-△bvfA was electroporated into the engineered bacteria to obtain β 2155 / pCVD442GS-△bvfA donor bacteria; S3: The β2155 / pCVD442GS-△bvfA donor bacterium was conjugated with the bovine Brucella A19 recipient bacterium, and gentamicin-resistant clones were selected, which were named A19 / pCVD442GS-△bvfA; S4: Take a single A19 / pCVD442GS-△bvfA clone and inoculate it on a TSA plate for screening, and obtain the bovine Brucella bvfA gene deletion strain A19△bvfA by PCR and sequencing technology; S5: Single gene deletion strain A19△ f The initial strain was used to knock out genes simultaneously according to S1~S4 methods. virB1~ virB12 , and obtained Brucella bovis A19△T4SS- Bf strain; The targeting sequence △ f The gene sequence is shown in SEQ ID NO.1; virB1~virB12 The gene sequence is shown in SEQ ID NO.

6.

3. The method for constructing the bovine Brucella A19ΔT4SS-bvfA strain as claimed in claim 2, characterized in that: The engineered bacteria is Escherichia coli.

4. Use of the bovine Brucella A19ΔT4SS-bvfA strain as claimed in claim 1 in the preparation of a vaccine for preventing bovine Brucella.

5. A vaccine for preventing bovine Brucella, characterized in that: The active ingredient comprises the bovine Brucella A19ΔT4SS-bvfA strain or its culture as described in claim 1.

6. A vaccine for preventing bovine Brucella as claimed in claim 5, characterized in that The active ingredient may also contain an adjuvant.

7. A vaccine for preventing bovine Brucella as claimed in claim 6, characterized in that The adjuvant is one or more of aluminum salt, oil emulsion or immunomodulator.

8. The vaccine according to any one of claims 5 to 7, characterized in that Conventional auxiliary ingredients used in the preparation of vaccines are also included.

9. The vaccine according to claim 8, characterized in that The vaccine is prepared according to the following method: the bovine Brucella A19△T4SS-bvfA strain is inoculated into a culture medium for cultivation, the culture is harvested, conventional auxiliary components are added, and the vaccine is prepared by drying; the conventional auxiliary components are stabilizers; The number of live bacteria in the vaccine is 6.0×10 10 CFU / g~6.5×10 10 CFU / g.

10. The vaccine according to claim 9, characterized in that The culture medium is TSB liquid culture medium.

Images