A Brucella abortus A19△T4SS-bvfA strain, its construction method and applications

Abstract

The present invention discloses a Brucella abortus A19△T4SS-bvfA strain, a construction method thereof and an application thereof, relating to the technical field of microorganisms. The Brucella abortus A19△T4SS-bvfA strain was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 27, 2024, and the deposit number is CGMCC No. 46201. The vaccine prepared by the present invention can distinguish natural infection from vaccine immunization, has no resistance residue, the bacteria do not enter host cells, and has higher safety.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically to a Brucella abortus A19△T4SS-bvfA strain, a construction method thereof and an application thereof. Background Art

[0002] Brucellosis, abbreviated as brucellosis, is a worldwide zoonotic infectious disease. Selecting an ideal antigen is the main step in vaccine design. To this end, different research groups have studied different subunit components of Brucella as recombinant protein vaccines in mouse and animal models. Although a large number of studies on Brucella antigens with immunogenicity and protectiveness have been carried out in recent decades, most antigens are not yet considered effective vaccines for brucellosis. To design and develop an effective Brucella vaccine, it is necessary to further understand the pathogenesis of Brucella, the bacterial cell wall, the optimal method for antigen purification, the selection of animal models, and effective adjuvants. In addition, no one has used an anti-Brucella vaccine so far, and the differences in the immune responses of humans and animals to the vaccine also need to be considered.

[0003] Currently, the smooth vaccines on the market are 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 ones are B. abortus vaccine RB51, B. abortus 45 / 20 vaccine and B. abortus SR82 vaccine. Among them, 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 for preparing a gene-deleted live vaccine that can distinguish vaccine immunity and natural infection. Summary of the Invention

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

[0005] The present invention is achieved by the following technical solutions:

[0006] A Brucella abortus A19△T4SS-bvfA strain, which was deposited with the China General Microbiological Culture Collection Center on September 27, 2024, and the deposit number is CGMCC No. 46201.

[0007] The construction method of the Brucella abortus A19△T4SS-bvfA strain is as follows:

[0008] S1: Synthesize the upstream homologous recombination sequence and downstream homologous recombination sequence of the bvfA gene to obtain the targeting sequence △bvfA, and clone it into pCVD442GS to obtain the targeting plasmid pCVD442GS-△bvfA.

[0009] S2: Electrotransform the targeting plasmid pCVD442GS-△bvfA into the engineered bacteria to obtain the donor bacteria β 2155 / pCVD442GS-△bvfA.

[0010] S3: Conjugate the donor bacteria β 2155 / pCVD442GS-△bvfA with the Brucella abortus A19 recipient bacteria, and screen for gentamicin-resistant clones, which are designated as A19 / pCVD442GS-△bvfA.

[0011] S4: Inoculate the bacterial solution of a single A19 / pCVD442GS-△bvfA clone on a TSA plate for screening, and obtain the Brucella abortus bvfA gene deletion strain A19△bvfA by PCR.

[0012] S5: Using the single gene deletion strain A19△bvfA as the initial strain, simultaneously knock out the genes virB1-virB12 according to the methods of S1-S4 to obtain the Brucella abortus A19△T4SS-bvfA strain.

[0013] The gene sequence of the targeting sequence △ bvfA is shown in SEQ ID NO.1; the gene sequence of the virB1 to virB12 is shown in SEQ ID NO.6.

[0014] Preferably, the engineered bacteria are Escherichia coli.

[0015] Use of the Brucella abortus A19△T4SS-bvfA strain in the preparation of a vaccine for preventing Brucella abortus.

[0016] A vaccine for preventing Brucella abortus, the active ingredient of which comprises the Brucella abortus A19△T4SS-bvfA strain or its culture, and the vaccine prepared only by the Brucella abortus A19△T4SS-bvfA strain or its culture can be used to prevent Brucella abortus.

[0017] Preferably, the active ingredient further comprises an adjuvant, and the vaccine prepared by adding an adjuvant to the Brucella abortus A19△T4SS-bvfA strain or its culture can be used to prevent Brucella abortus.

[0018] Preferably, the adjuvant is one or more of aluminum salts, oil emulsions or immunomodulators.

[0019] Preferably, the vaccine further comprises conventional auxiliary components for preparing the vaccine.

[0020] Preferably, the vaccine is prepared by the following method: inoculate the Brucella abortus A19△T4SS-bvfA strain into a culture medium for culturing, harvest the culture, add conventional auxiliary components, and obtain the vaccine through drying. The conventional auxiliary component is a stabilizer; the viable bacteria count in the vaccine is 6.0×10 10 CFU / g~6.5×10 10 CFU / g.

[0021] Preferably, the culture medium is TSB liquid culture medium.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a Brucella abortus A19△T4SS-bvfA strain. By using genetic engineering technology, 12 genes of virB1~virB12 and the bvfA gene are simultaneously knocked out on the Brucella abortus vaccine strain A19, and a Brucella abortus T4SS-bvfA combined knockout strain A19△T4SS-bvfA strain is constructed. BvfA is a virulence factor unique to the genus Brucella. Its specific protein can distinguish natural infection from vaccine immunity, which is beneficial for distinguishing other genera and has stronger specificity. Also, because it itself is a virulence factor, after being knocked out, it can also reduce the toxicity of the strain, making it the best choice for an excellent vaccine. Simultaneously knocking out 12 genes of virB1~virB12 can not only prevent the strain from entering host cells through immune escape and reduce chronicity caused by persistent infection, but also can be used for subsequent secondary differentiation of 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 conventional serological detection of immunized animals and naturally infected animals. The present invention proves through experiments that the vaccine prepared from this strain can distinguish natural infection from vaccine immunity. Through the invasion cell experiment, it is found that the double-knockout A19△T4SS-bvfA strain constructed by the present invention does not enter host cells, with higher safety. In addition, the present invention directly docks homologous upstream and downstream arms, and the operation of constructing the T4SS-bvfA combined knockout strain is simple. After antibiotic screening, it is automatically removed without resistance residue. In practical applications, if a vaccine cannot distinguish natural infection from vaccine immunity, it will be difficult to judge whether an individual has obtained immunity through natural infection or vaccination in subsequent detections. It is difficult to accurately evaluate the vaccination effect of the vaccine and the immune status of the vaccinated individuals. In addition, for diseases that require distinguishing natural infection from vaccine immunity to formulate subsequent prevention and control strategies, this indistinguishable situation will increase the difficulty and complexity of prevention and control.

[0024] Biological material preservation

[0025] Brucella abortus A19△T4SS-bvfA The strain was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 27, 2024, with the deposit number CGMCC No. 46201, and the strain is classified and named as Brucella bovis. Brucella abortus The deposit address is Beijing, China. Brief Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 This is a PCR product diagram of the targeting sequence of the present invention; M: Standard DNA molecular weight. From top to bottom, the molecular weights are: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp, among which 750bp is highlighted; 1: PCR product of the targeting sequence, 1719bp.

[0028] Figure 2 This is the outer primer bvfA -outF and bvfA -outR PCR identification result diagram; Figure 2 In it, A: 1-24 are the PCR amplification results of the outer primers of 24 monoclonal colonies picked after the conjugation experiment, 2161 / 1825bp, B: 1-19 are the PCR amplification results of the outer primers of 19 monoclonal colonies picked after the conjugation experiment, 2161 / 1825bp, 20 is the negative control, that is, the parental strain A19, 21 is the blank control, that is, no template; M in A and B is the standard DNA molecular weight. From top to bottom, the molecular weights are: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp, among which 750bp is highlighted.

[0029] Figure 3 This is the PCR identification result diagram of the inner primers bvfA-inF and bvfA-inR of the present invention; M1: Standard DNA molecular weight. From top to bottom, the molecular weights are: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; among which 750bp is highlighted. 3, 6, 21 are the PCR amplification results of the knockout strain A19△bvfA without bands, and the rest with bands are the PCR amplification results of the parental strain A19.

[0030] Figure 4This invention is an outer primer virB -outF and virB -outR PCR identification result graph. In A and B, samples with the same number above and below are two parallel samples; Figure 4 In it, A: M is the standard DNA molecular weight. From top to bottom, the molecular weights are: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp. Above and below, 1-12 are the amplification results of 24 monoclonal outer primers picked after the conjugation experiment; B: M is the standard DNA molecular weight. From top to bottom, the molecular weights are: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; Above 1-12 and below 1-10 are the amplification results of 22 monoclonal outer primers picked after the conjugation experiment, a single band of 1864bp. Below B, 11 is the negative control, that is, the parental strain A19, and below B, 12 is the blank control, that is, no template. The amplification product length of the outer primer of the A19△ bvfA strain is 13009bp, and the amplification product length of the A19△T4SS- bvfA knockout strain is 1864bp.

[0031] Figure 5 This invention is an inner primer virB -inF and virB -inR PCR identification result graph; M is the Marker. From top to bottom, the molecular weights are: 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1, 2 are the 284bp fragment of the amplification product of the inner primer of the A19△bvfA strain, and 3-8 are the non-banding of the A19△T4SS-bvfA knockout strain.

[0032] Figure 6This figure shows the PCR verification results 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 A19, with double bands of bvfA 202bp and virBs 284bp; 3 is the A19△virBs strain, with a single band of bvfA 202bp; 4 is the A19△bvfA strain, with a single band of virBs 284bp; 5 is the double knockout A19△T4SS-bvfA strain, without a band; 6 is the negative control, without a band; 7 is Marker; 8 is the original strain A19, with a single band of virBs 284bp; 9 is the A19△virBs strain, without a band; 10 is the A19△bvfA strain, with a single band of virBs 284bp; 11 is the double knockout A19△T4SS-bvfA strain, without a band; 12 is the negative control, without a band; 13 is Marker; 14 is the original strain A19, with a single band of bvfA 202bp; 15 is the A19△virBs strain, with a single band of bvfA 202bp; 16 is the A19△bvfA strain, without a band; 17 is the double knockout A19△T4SS-bvfA strain, without a band; 18 is the negative control, without a band; 1-6 are multiplex PCR with two primers of bvfA and virBs; 7-12 are PCR with the inner primer of virBs; 13-18 are PCR with the inner primer of bvfA.

[0033] Figure 7 This figure shows the verification results of the invasion cell test; * represents P<0.05, ** represents P<0.01, *** represents p<0.001.

[0034] Figure 8 This figure shows the determination results of differentiating natural infection and vaccine immunity by the BvfA protein; the abscissa represents the time of antibody production after vaccination, the ordinate represents the S / N value, * represents P<0.05, ** represents P<0.01, *** represents p<0.001. Detailed implementation manners

[0035] 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 given. 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.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention.

[0037] The inventive concept of the present invention is as follows:

[0038] Currently, the smooth vaccines on the market include Brucella abortus vaccine strain S19, B. melitensis Rev.1 vaccine, B. melitensis H38 vaccine, B. melitensis M5 vaccine, and B. suis vaccine S2, and the rough ones include B. abortus vaccine RB51, B. abortus 45 / 20 vaccine, and B. abortus SR82 vaccine. Among them, these vaccines have the disadvantage of being unable to distinguish natural infection from vaccine immunity. Therefore, it is necessary to develop a new strain for preparing a gene-deleted live vaccine that can distinguish vaccine immunity from natural infection.

[0039] Based on this, the present invention provides a Brucella abortus A19△T4SS-bvfA strain. The Brucella abortus virulence factor A, namely BvfA, is a new virulence factor unique to the genus Brucella. It is a basic protein composed of 111 amino acids. Using its unique protein can distinguish natural infection from vaccine immunity, which is beneficial for differentiating other genera of bacteria and has stronger specificity. Also, because it is itself a virulence factor, knocking it out can also reduce the toxicity of the strain, making it the best choice for an excellent vaccine. The B.suis 1330 bvfA::kan mutant proliferates significantly less than the parental strain in THP1 cells, J774 cells, and HeLa cells. In addition, when the B.suis 1330 bvfA::kan mutant infects mice at the 4th and 8th weeks, the number of Brucella abortus in the spleen is significantly less than that of the parental strain. The B. suis 1330 bvfA::kan mutant has highly attenuated virulence in in vitro and in vivo models, indicating that the bvfA gene plays an important role in the survival of Brucella abortus both in vitro and in vivo. In addition, in Brucella abortus, the type IV secretion system, whose English name is type IV secretion system, abbreviated as T4SS, is composed of 12 proteins encoded by the virB operon, namely VirB1~VirB12. Brucella abortus can use the T4SS to secrete into host cells, evade immune surveillance, and maintain its continuous presence in cells, which is also one of the reasons why brucellosis is difficult to cure. Knocking out the 12 genes of virB1~virB12 can not only prevent the strain from entering host cells through immune escape and reduce chronicity caused by persistent infection, but also make subsequent secondary differentiation between natural infection and vaccine immunity through any one of the 12 proteins of VirB1~VirB. Using genetic engineering technology to simultaneously knock out the 12 genes of virB1~virB12 and the bvfA gene on the Brucella abortus vaccine strain A19, a Brucella abortus T4SS-bvfA combined knockout strain A19△T4SS-bvfA was constructed. Experiments showed that the vaccine prepared from this strain can distinguish natural infection from vaccine immunity. Through the invasion cell test, it was found that the double-knockout A19△T4SS-bvfA strain constructed in the present invention does not enter host cells and has higher safety. 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. After antibiotic screening, it is automatically removed without resistance residue.

[0040] The construction method of the Brucella abortus A19△T4SS-bvfA strain is as follows:

[0041] The first step: Construct a Brucella abortus A19△bvfA deletion strain.

[0042] (1)Synthesis of the targeting sequence

[0043] The upstream and downstream homologous recombination sequences of the bvfA gene were synthesized to obtain the complete targeting sequence ΔbvfA, which was then cloned into the suicide plasmid pCVD442GS containing the gentamicin resistance gene to obtain the targeting plasmid pCVD442GS-ΔbvfA.

[0044] (2)Obtain the donor bacterium

[0045] pCVD442GS-ΔbvfA was electrotransformed into Escherichia coli E. coli β2155 to obtain the donor bacterium β2155 / pCVD442GS-ΔbvfA.

[0046] (3)Conjugation experiment

[0047] The donor bacterium β2155 / pCVD442GS-ΔbvfA and the Brucella bovis A19 recipient bacterium were subjected to a conjugation experiment. Gentamicin-resistant Brucella bovis clones were screened on a gentamicin plate. Their genomes were integrated with the targeting plasmid and named A19 / pCVD442GS-ΔbvfA.

[0048] (4)Screen for the Brucella bovis A19ΔbvfA deletion strain

[0049] Take a single A19 / pCVD442GS-ΔbvfA clone bacterial solution and streak it on an LB liquid medium containing 10% (v / v) sucrose overnight, then streak it on a TSA plate and culture it at 37 °C in 5% (v / v) CO 2 until single colonies are formed. The bvfA gene deletion clone was screened by PCR technology and named A19ΔbvfA.

[0050] Second step: Continue to knockout 12 virBs genes of T4SS on the Brucella bovis A19ΔbvfA deletion strain.

[0051] (1)Construct the targeting plasmid pCVD442GS-ΔvirBs

[0052] The upstream and downstream homologous recombination sequences of the virB1-virB12 genes were directly synthesized to obtain the complete targeting fragment ΔvirBs, which consists of an upstream homologous arm and a downstream homologous arm. Then it was cloned into the suicide plasmid pCVD442GS containing the gentamicin resistance gene to obtain the targeting plasmid pCVD442GS-ΔvirBs.

[0053] (2)Obtain the donor bacterium

[0054] pCVD442GS-ΔvirBs was electrotransformed into Escherichia coli E. coli β2155 to obtain the donor bacterium β2155 / pCVD442GS-ΔvirBs.

[0055] (3)Conjugation experiment

[0056] The conjugation experiment was carried out between the donor bacterium β2155 / pCVD442GS-△virBs and the recipient bacterium Brucella abortus A19△bvfA. The Brucella abortus clones resistant to gentamicin were screened on the gentamicin plate, and their genomes were integrated with the targeting plasmid, named A19△bvfA / pCVD442GS-△virBs.

[0057] (4)Screening for the Brucella abortus A19△T4SS-bvfA double deletion strain

[0058] Take a single A19△bvfA / pCVD442GS-△virBs clone bacterial solution and streak it on LB liquid medium containing 10% (v / v) sucrose overnight, then streak it on a TSA plate, and culture it at 37°C in an atmosphere of 5% (v / v) CO 2 until single colonies are formed. The T4SS-bvfA gene deletion clones were screened by PCR technology and named Brucella abortus A19△T4SS-bvfA.

[0059] The technical solution of the present invention will be further elaborated below in conjunction with specific embodiments.

[0060] Example 1

[0061] The first step: Construction of the Brucella abortus A19△bvfA deletion strain

[0062] 1. Synthesis of the targeting sequence

[0063] The upstream and downstream homologous recombination sequences of the bvfA gene were directly synthesized to obtain the complete targeting sequence △bvfA, which consists of an upstream homologous arm and a downstream homologous arm.

[0064] 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. As Figure 1 shown.

[0065] SEQ ID NO.1:

[0066]

[0067] 2. Construction of targeting plasmid pCVD442GS-△bvfA

[0068] Enzyme digestion: After the pCVD442GS plasmid was digested with SmaI, the enzyme digestion system was as shown in Table 1.

[0069] Table 1 Enzyme digestion system

[0070]

[0071] 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.

[0072] 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.

[0073] Table 2 Connection system

[0074]

[0075] 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.

[0076] 3. Construction of donor bacteria:

[0077] 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.

[0078] 4. Binding Experiment

[0079] The above-mentioned donor strain β 2155 / pCVD442GS-△bvfA and recipient strain Brucella abortus A19 were each washed with TSB, and then 500 μL of each was gently combined and mixed with 5 μL of 0.1 M DAP. 100 μL was taken and spread on a 0.5 mM DAP plate, and cultured at 37°C in 5% CO 2 After culturing for 4 h, it was washed with TSB and inoculated on a TSA plate containing 20 μg / mL gentamicin, and cultured at 37°C in 5% CO 2 Cultured for 72 h until monoclonal formation.

[0080] 5. PCR Identification

[0081] Forty-three monoclonal colonies were picked from the above plate and placed in 20 μL of TSB respectively. 1 μL was taken as a template, and PCR identification was carried out using the outer primers bvfA-outF and bvfA-outR. The primer sequence of bvfA-outF is shown in SEQ ID NO.2, and the primer sequence of bvfA-outR is shown in SEQ ID NO.3. The reaction system is shown in Table 3, and the reaction program is shown in Table 4.

[0082] Table 3 Reaction System

[0083]

[0084] Table 4 Reaction Program

[0085]

[0086] Note: " / " means there is no such item.

[0087] SEQ ID NO.2: 5’-TTGTTGCGGCTTATGTAGATTGCG-3’.

[0088] SEQ ID NO.3: 5’-ACAATCCGCAGGACTATGTGACG-3’.

[0089] The judgment results are as Figure 2 shown: The amplification product length of the A19 outer primer is 2161 bp, and the amplification product length of A19△bvfA is 1825 bp. At this time, the product length of the outer primer may show three situations: a single band of 2161 bp, that is, no recombination occurred; a double band of 2161 / 1825 bp, that is, one recombination and two recombinations occurred; a single band of 1825 bp, that is, two recombinations occurred. The results of this PCR identification showed that there was a single band of 2161 bp, no recombination occurred, and a double band of 2161 / 1825 bp appeared, that is, one recombination and two recombinations occurred.

[0090] 6. Sucrose Screening

[0091] Select No. 24. Since No. 24 has a relatively bright band of 1825 bp in the lower band, the clone containing the 1825 bp band is the selected positive clone. Inoculate 15 μL of bacterial liquid into 3 mL of TSB liquid medium, at 37 °C, with a volume fraction of 5% CO 2 After culturing for 48 h, take 1 mL of bacterial liquid, centrifuge at 4000 g to precipitate, suspend and wash once with an equal volume of 10% LB sucrose solution by volume fraction, centrifuge at 4000 g, take the precipitate and add it to 3 mL of 10% LB sucrose solution by volume fraction, at 37 °C, with a volume fraction of 5% CO 2 After culturing for 48 h. After reverse screening with sucrose, the culture solution is streaked and inoculated on TSA plates at the original solution, 1:20, and 1:200 respectively, at 37 °C, with a volume fraction of 5% CO 2 After culturing for 72 h until monoclonal formation.

[0092] 7. PCR and sequencing identification

[0093] Pick the above monoclonal into 20 μL of TSB, take 1 μL as a template, and perform PCR identification with the inner primers bvfA-inF and bvfA-inR. The primer sequence of bvfA-inF is as shown in SEQ ID NO.4, and the primer sequence of bvfA-inR is as shown in SEQ ID NO.5. The reaction system is shown in Table 5, and the reaction program is shown in Table 6.

[0094] Table 5 Reaction system

[0095]

[0096] Table 6 Reaction program

[0097]

[0098] Note: " / " means there is no such item.

[0099] SEQ ID NO.4: 5’-GCTCTGCATCGTATAGAGCCAGC-3’.

[0100] SEQ ID NO.5: 5’-GCAGGTCCGCAAGGATGTGAG-3’.

[0101] The judgment result is as Figure 3 shown: After successful reverse screening with sucrose, A19△bvfA is negative, and A19 has an amplification of a 202 bp fragment.

[0102] Pick the knockout strain and sequence it with the outer primer. The result shows that it is correct, and the strain completely lacks bvfA to obtain the A19△bvfA strain.

[0103] Step 2: In Brucella abortus A19△bvfA Continue to knock out 12 virBs genes virB1~ virB12 。

[0104] virB1 to virB12 The gene sequence of 1718 bp is shown in SEQ ID NO.6.

[0105] SEQ ID NO.6:

[0106]

[0107] Steps 1, 2, and 3 are exactly the same as the first step, obtaining the donor strain β 2155 / pCVD442GS-△T4SS for conjugation experiments.

[0108] 4. Conjugation experiment

[0109] Wash the above donor strain and Brucella bovis A19△bvfA recipient strain with TSB, gently combine 500 μL of each, and add 5 μL of 0.1 M DAP and mix well. Pipette 100 μL and spread it on a 0.5 mM DAP plate, at 37 °C, 5% CO 2 After culturing for 4 h, wash with TSB and inoculate on a TSA plate containing 20 μg / mL gentamicin, at 37 °C, 5% CO 2 Cultivate for 72 h until monoclonal formation.

[0110] 5. PCR identification

[0111] Pick the above monoclonal into 20 μL of TSB, take 1 μL as a template, and perform PCR identification with the outer primers virB-outF and virB-outR. The primer sequence of virB-outF is as shown in SEQ ID NO.7, and the primer sequence of virB-outR is as shown in SEQ ID NO.8. The reaction system is shown in Table 7, and the reaction program is shown in Table 8.

[0112] Table 7 Reaction system

[0113]

[0114] Table 8 Reaction program

[0115]

[0116] Note: " / " means there is no such item.

[0117] SEQ ID NO.7: 5’-GGTTCGCTCACCATCGCCGAATG-3’.

[0118] SEQ ID NO.8: 5’-GGCATTGCGATAGATTTCATTGAGTGCCAG-3’.

[0119] The judgment result is as Figure 4Shown as follows: The amplification product length of the outer primer of the original strain is 13009 bp, and the amplification product length of the knockout strain is 1864 bp. At this time, the product lengths of the outer primers of the clones picked on the gentamicin plate may have three situations: a single band of 13009 bp, indicating no recombination; a double band of 13009 / 1864 bp, indicating one recombination and two recombinations; a single band of 1864 bp, indicating two recombinations.

[0120] After this PCR identification, a single band of 1864 bp appeared, indicating two recombinations, which means that all clones showed two recombinations in this result.

[0121] 6. Sucrose screening

[0122] Select the brightest band, inoculate 15 μL of bacterial liquid into 3 mL of TSB liquid medium, at 37 °C, with a volume fraction of 5% CO 2 After culturing for 48 h, take 1 mL of bacterial liquid, centrifuge at 4000 g to precipitate, suspend and wash once with an equal volume of 10% LB sucrose solution by volume, centrifuge at 4000 g to take the precipitate and add it to 3 mL of 10% LB sucrose solution by volume, at 37 °C, with a volume fraction of 5% CO 2 After culturing for 48 h. After reverse screening with sucrose, the culture solution was streaked and inoculated on TSA plates at the original solution, 1:20, and 1:200 respectively, at 37 °C, with a volume fraction of 5% CO 2 After culturing for 72 h until monoclonal formation.

[0123] 7. PCR and sequencing identification

[0124] Pick the above-mentioned monoclonal into 20 μL of TSB, take 1 μL as a template, and perform PCR identification with the inner primers virB-inF and virB-inR. The primer sequence of virB-inF is shown in SEQ ID NO.9, and the primer sequence of virB-inR is shown in SEQ ID NO.10. The reaction system is shown in Table 9, and the reaction program is shown in Table 10.

[0125] Table 9 Reaction system

[0126]

[0127] Table 10 Reaction program

[0128]

[0129] Note: " / " means there is no such item.

[0130] SEQ ID NO.9: 5’-CATGCGCTGTCTCTCTGGCCGC-3’.

[0131] SEQ ID NO.10: 5'-CCTGACGTTGATATGCTTGGGCACGG-3'.

[0132] The judgment result is as Figure 5 shown: The amplification of this pair of primers should be negative, while the positive control of the original bacteria has an amplified fragment of 284 bp.

[0133] The knockout strain was picked and sequenced with the outer primers. The result showed correctness, and it was named strain A19△T4SS-bvfA.

[0134] Example 2

[0135] Verify the amplification of bvfA gene and virBs gene in the double knockout strain. The PCR system and procedure are as shown in Tables 5, 6, 9, and 10 above.

[0136] The primers used are the internal primers mentioned above, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.9, and SEQ ID NO.10; for the procedure and system, refer to Tables 5, 6, 9, and 10 above. Electrophoresis was performed using a 1.5% (v / v) agarose gel. The electrophoresis result is as Figure 6As shown in the figure, it is 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 A19 strain, with double bands of bvfA 202bp and virBs 284bp; 3 is the A19△virBs strain, with a single band of bvfA 202bp; 4 is the A19△bvfA strain, with a single band of virBs 284bp; 5 is the double knockout A19△T4SS-bvfA strain, without a band; 6 is the negative control, without a band; 7 is Marker; 8 is the original A19 strain, with a single band of virBs 284bp; 9 is the A19△virBs strain, without a band; 10 is the A19△bvfA strain, with a single band of virBs 284bp; 11 is the double knockout A19△T4SS-bvfA strain, without a band; 12 is the negative control, without a band; 13 is Marker; 14 is the original A19 strain, with a single band of bvfA 202bp; 15 is the A19△virBs strain, with a single band of bvfA 202bp; 16 is the A19△bvfA strain, without a band; 17 is the double knockout A19△T4SS-bvfA strain, without a band; 18 is the negative control, without a band; 1-6 are multiplex PCR with bvfA and virBs two primers; 7-12 are PCR with the inner primer of virBs; 13-18 are PCR with the inner primer of bvfA. After correct electrophoresis, it was sent to Sangon Biotech in Shanghai for sequence determination and comparison, indicating that the double knockout strain of bvfA gene and virB gene was successfully constructed.

[0137] Example 3

[0138] Verify the invasion of cells experiment.

[0139] Method: After counting J774A.1 cells, 1×10 6 cells were seeded in a 6-well plate until a monolayer of adherent cells was formed. Brucella abortus in the logarithmic growth phase was used to infect the cells at a MOI of 100:1. After 15 min, 30 min and 45 min of infection, extracellular bacteria were killed with gentamicin. The cells were washed 3 times with PBS, lysed with 0.01% (v / v) Triton-100, diluted 10-fold and spread on solid medium, and the invasion was judged by CFU counting. The results are as Figure 7 shown, ● represents the invasion of A19ΔT4SS-bvfA strain; ■ represents the invasion of A19Δ bvfA strain; ▲ represents the invasion of A19Δ virBs strain; ◆ represents the invasion of A19 strain. The column height represents the number of invaded cells in each group. The graphs on the histogram indicate that each group has three replicates, and three ● overlap as one. A19ΔT4SS-bvfA Group, A19Δ bvfA Group and A19Δ virBs The invasion numbers of each group were all less than those of A19, indicating that the invasion ability was weakened after gene knockout. And for the A19ΔT4SS- bvfA group, the value was 0, indicating that it could not invade cells, had the weakest invasion ability and the highest safety. Therefore, this result shows that A19△T4SS- bvfA No colonies were collected from the cells infected with the double-knockout strain, indicating that it did not invade cells, suggesting that the double-knockout A19△T4SS- bvfA strain could not invade J774A.1 macrophages. It shows that the double-knockout A19△T4SS- bvfA strain constructed in the present invention does not enter host cells and has higher safety.

[0140] Example 4

[0141] Experiment for differentiating natural infection and vaccine immunity.

[0142] (1) Purified BvfA protein was obtained by prokaryotic expression, and natural infection and vaccine immunity were differentiated by detecting the BvfA protein.

[0143] (2) Indirect ELISA kit

[0144] The antibody reactivity of the purified BvfA protein with the sera of mice injected with normal saline, A19△T4SS- bvfA and A19 was determined by indirect ELISA. 100 μL of coating solution, pH 9.6, 0.05 mol / L carbonate buffer, was used to coat the BvfA protein at a concentration of 0.2 μg / mL, overnight at 4˚C, with 3 replicates in each group.

[0145] The coating solution was discarded, and 280 μL of PBST buffer, pH 7.4, 0.01 mol / L PBS:Tween-20, 1:1000, was used to wash 3 times. 100 μL of 1% BSA by volume, pH 7.4, was used to block for 1 h at 37˚C, and then rinsed 3 times with PBST. 50 μL of the sera of mice immunized with normal saline, A19△T4SS-bvfA strain and A19 strain for 2 weeks and 3 weeks were added accordingly and incubated for 1 h. 100 μL of 1:5000 horseradish peroxidase-labeled goat anti-mouse IgG was added, incubated for 1 h at 37˚C, washed 3 times with PBST, 100 μL of TMB substrate was added, placed in the dark at room temperature for 10 min, 50 μL of 2 mol / L sulfuric acid solution was added to terminate the reaction, and the OD absorbance value was measured at 450 nm. There were 3 replicates in each group. The antibody titer was reflected by the S / N value. If S / N≥2.1, it was positive, S / N = sample OD450 - blank OD450 / negative control OD 450 – blank OD450 The negative control was the serum of non-injected mice. S stands for Sample, which is the abbreviation of the sample; N stands for Negative, which is the abbreviation of the negative control. The negative control was the serum of non-injected mice; the normal saline group was the control group.

[0146] The judgment results are as Figure 8 shown: The graphs on the bar chart indicate that there are three replicates in each group. The S / N value of the control is equal to the average height of the three origin columns, the S / N value of A19ΔT4SS-bvfA is equal to the average height of the three square columns, and the S / N value of A19 is equal to the average height of the three triangular columns. S / N≥2.1 indicates natural infection; 2.1 is the cut-off value, that is, the critical value; S / N<2.1 indicates injection of A19△T4SS- bvfA vaccine. To verify the antibody reactivity of the BvfA protein with normal saline (control), A19△T4SS- bvfA and the sera of mice injected with the A19 strain, there is no BvfA protein in normal saline (control) and A19△T4SS- bvfA and no anti-BvfA protein antibody will be produced after injection. Therefore, the normal saline (control) group and the A19△T4SS- bvfA group do not react with the BvfA protein, and there is no statistical difference between them. The reaction S / N values are both less than 2.1; while anti-BvfA protein antibody will be produced after injection with the A19 strain, and the reaction S / N values are both greater than 2.1; natural infection and vaccine immunity can be identified according to the S / N value.

[0147] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0148] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A bovine Brucella ( Brucella abortus )A19△T4SS- f The strain is 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; The bovine Brucella A19ΔT4SS- f The specific method of constructing the strain is as follows: S1: f The upstream homologous recombination sequence and downstream homologous recombination sequence of the gene are synthesized to obtain the targeting sequence△ f , cloned into pCVD442GS, and obtained the targeting plasmid pCVD442GS-△ f ; S2: The targeting plasmid pCVD442GS-△ f Electroporation into engineered bacteria to obtain β 2155 / pCVD442GS-△ f Donor bacteria; S3: β 2155 / pCVD442GS-△ f The donor bacteria were conjugated with the bovine Brucella A19 recipient bacteria, and gentamicin-resistant clones were selected, which were called A19 / pCVD442GS-△ f ; S4: Take a single A19 / pCVD442GS-△ f The cloned bacterial suspension was inoculated on TSA plates for screening, and bovine Brucella was obtained by PCR and sequencing technology. f Gene deletion strain A19△ f ; S5: Single gene deletion strain A19△ f The initial strain was used to knock out genes simultaneously according to the S1~S4 method. virB1~ virB12 , and obtained Brucella bovis A19△T4SS- f 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.

2. The Brucella bovis A19ΔT4SS- f Application of strains in the preparation of vaccines for preventing bovine Brucella.

3. A vaccine for preventing bovine Brucella, characterized in that: The active ingredient comprises the bovine Brucella A19ΔT4SS- f strains or cultures thereof.

4. A vaccine for preventing bovine Brucella as claimed in claim 3, characterized in that The active ingredient may also contain an adjuvant.

5. A vaccine for preventing bovine Brucella as claimed in claim 4, characterized in that The adjuvant is one or more of aluminum salt, oil emulsion or immunomodulator.

6. The vaccine according to any one of claims 3 to 5, characterized in that Conventional auxiliary ingredients used in the preparation of vaccines are also included.

7. The vaccine according to claim 6, characterized in that Prepared according to the following method: f After the strain is inoculated in 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.

8. The vaccine according to claim 7, characterized in that The culture medium is TSB liquid culture medium.

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