CN112852698B - Construction method and application of Brucella A19 strain asd gene deletion strain - Google Patents
Construction method and application of Brucella A19 strain asd gene deletion strain Download PDFInfo
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Abstract
The invention discloses a construction method and application of a Brucella A19 asd gene deletion strain, and relates to the technical field of genetic engineering, wherein the construction method of the Brucella A19 asd gene deletion strain comprises the following steps: designing and synthesizing a primer; step two, PCR amplification of the asd-kan fusion gene; step three, constructing a pMD18-asd-kan deletion vector; step four, constructing the A19-delta asd deletion strain. According to the invention, asd genes of the Brucella A19 strain are knocked out at fixed points through a genetic engineering technology, so that auxotroph Brucella is knocked out through the asd genes, and experiments prove that the Brucella A19 strain asd gene deletion strain has good safety and immunological characteristics and can be used as a candidate vaccine strain for preventing Brucella infection.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a construction method and application of an asd gene deletion strain of a Brucella A19 strain.
Background
Brucellosis is a widespread and serious-hazard zoonosis caused by brucella worldwide. In recent years, brucellosis has been on the rise. Not only harms the health of the public, but also greatly restricts the development of animal husbandry.
Immunization with vaccines is an important measure for the control of brucellosis. The brucella vaccines used in the current market are mainly live attenuated vaccines, such as brucella vaccines A19, M5, S2 and the like widely used in China, and have the defects of poor safety, strong side effect and the like, for example, the vaccine of the A19 vaccine strain can cause abortion of pregnant female animals after immunization and can also infect people. The gene deletion vaccine has the characteristics of further weakened toxicity, high safety, clear genetic background, good immune effect, no interference to diagnosis and the like, so that the gene deletion vaccine is widely concerned by scientists.
Aspartate beta-galactose dehydrogenase (ASD) is an essential enzyme in the biosynthetic pathway of Diaminopimelic Acid (DAP), an important chemical component of the wall of synthetic bacteria of gram-negative bacteria. If the coding gene (ASD) encoding ASD is knocked out, gram-negative bacteria cannot form a perfect bacterial wall without exogenous DAP, and hypotonic bacteriolysis dies.
Until now, no report of the asd gene deletion strain of the Brucella A19 strain of the cattle species has been found.
Disclosure of Invention
The invention aims to provide a construction method and application of an asd gene deletion strain of a Brucella A19 strain, wherein the constructed asd gene deletion strain has good safety and immunological characteristics and can be used as a candidate vaccine strain for preventing Brucella infection.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the invention discloses a construction method of an asd gene deletion strain of a Brucella melitensis A19 strain, which comprises the following steps:
designing and synthesizing a primer;
step two, PCR amplification of the asd-kan fusion gene;
step three, constructing a pMD18-asd-kan deletion vector;
step four, constructing the A19-delta asd deletion strain.
As a preferred embodiment, the specific operation process of the step one is as follows:
designing primers according to the asd gene and upstream and downstream sequences thereof in an NCBI database, wherein the names and the sequences of the primers are as follows:
asdkan-N-F：5’-GGAGCCAAACCATGATGCAC-3’；
asdkan-N-R：5’-TTCGCTTGCTGTCCATAAAAATCAGATTGCAAAAGCGC CG-3’；
asdkan-C-F：5’-CTTGACGAGTTCTTCTGACAGGCCAAGGGAATCAGACC-3’；
asdkan-C-R：5’-GCAAAGATGACCTTGCGCTC-3’；
asdkan-F：5’-TTTTATGGACAGCAAGCGAA-3’；
asdkan-R：5’-TCAGAAGAACTCGTCAAG-3’；
asd-I-F：5’-TAGAGCGGATCGACAAGCAG-3’；
asd-I-R：5’-CGCCAGCGTTCCATAGAGAT-3’。
as a preferred embodiment, the specific operation process of the second step is as follows:
(1) Using a cattle Brucella A19 strain subjected to boiling cracking as a template, and amplifying upstream and downstream homologous arms of an asd gene by using high-fidelity enzyme; after the PCR product is successfully verified by electrophoresis, all the residual PCR products are subjected to sample addition electrophoresis and gel recovery;
(2) Using a yTREX-SacB plasmid as a template, and amplifying a kan resistance gene sequence by using high-fidelity enzyme; after the PCR product is successfully verified by electrophoresis, all the residual PCR products are subjected to sample addition electrophoresis and gel recovery;
(3) PCR amplification of asd-kan fusion gene by two-step method
The first step is as follows: using an asd gene N-terminal homologous arm, an asd gene C-terminal homologous arm, a kan resistance gene, taq enzyme and ddH 2 O is a reaction system for amplification;
the second step: amplifying by taking the reaction product of the first step as a template and taking asdkan-N-F and asdkan-C-R as primers; and after the PCR product is successfully verified by electrophoresis, all the residual PCR product is subjected to sample loading electrophoresis and gel recovery.
In a preferred embodiment, in step two (1), the amplification conditions are: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 45s,30 cycles; final extension at 72 deg.C for 10min; 50 mu L of the amplification system of the N-terminal homologous arm of the asd gene: high-fidelity enzyme premix: 25 mu L of the solution; template: 2.5 mu L; asdkan-N-F:2.5 mu L; asdkan-N-R:2.5 mu L; ddH 2 O:17.5 mu L; 50 mu L of asd gene C-terminal homologous arm amplification system: high fidelity enzyme: 25 mu L of the solution; template: 2.5 mu L; asdkan-C-F:2.5 mu L; asdkan-C-R:2.5 mu L; ddH 2 O：17.5μL。
In a preferred embodiment, in step (2) of the second step, the amplification conditions are: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min15s,30 cycles; final extension at 72 ℃ for 10min; the reaction system is 50 μ L and comprises: high fidelity enzyme:25 mu L of the solution; template: 2.5 mu L; asdkan-F:2.5 mu L; asdkan-R:2.5 mu L; ddH 2 O：17.5μL。
As a preferred embodiment, in the (3) of the second step, in the first step, the reaction system is 20. Mu.L, and includes 2. Mu.L of the N-terminal homology arm of the asd gene, 2. Mu.L of the C-terminal homology arm of the asd gene, 2. Mu.L of the kan-resistance gene, 10. Mu.L of taq enzyme, ddH 2 O4 mu L; the amplification conditions were: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 2min30s,15 cycles.
In a preferred embodiment, in step two (3), the reaction system in the second step is 50. Mu.L, and the reaction system comprises 2 × taq enzyme: 25 mu L of the solution; the first step reaction product: 2.5 mu L; asdkan-N-F:2.5 mu L; asdkan-C-R:2.5 mu L; ddH 2 O:17.5 mu L; the amplification conditions were: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min30s, and 30 cycles.
As a preferred embodiment, the specific operation process of step three is as follows:
the connecting system is as follows: pMD 18-T1. Mu.L, asd-kan fusion Gene 3. Mu.L, ddH 2 O1 mu L, solution I5 mu L, transformed into Escherichia coli competent cell DH5 alpha after overnight connection at 16 ℃, then spread on LB plate containing 50 ng/mu L kanamycin, picked up the single clone to LB liquid culture medium containing 50 ng/mu L kanamycin, shake cultured for about 12h at 37 ℃, and taken bacterial liquid is subjected to PCR identification and sequencing.
As a preferred embodiment, the specific operation process of step four is as follows:
the pMD18-asd-kan deletion vector was electroporated into prepared A19 competent cells, applied to TSA plates containing 100 ng/. Mu.L each of diaminopimelic acid and kanamycin after overnight recovery, cultured at 37 ℃ for 4 to 5 days, single colonies were picked up into TSB liquid medium containing 100 ng/. Mu.L each of diaminopimelic acid and kanamycin, shake-cultured at 37 ℃ for 24 hours, and PCR-identified using two pairs of primers asd-I-F, asdkan-R and asdkan-F, asd-I-R.
The asd gene deletion strain constructed by the construction method of the cattle Brucella A19 strain asd gene deletion strain is applied to a candidate vaccine strain for preventing Brucella infection.
The beneficial effects of the invention are:
according to the invention, asd gene of the Brucella A19 strain is knocked out at fixed points by using a genetic engineering technology, so that auxotroph Brucella knocked out by using asd gene is constructed, and experiments prove that the asd gene deletion strain of the Brucella A19 strain has good safety and immunological characteristics, and can be used as a candidate vaccine strain for preventing Brucella infection.
Drawings
FIG. 1 shows the amplification products of the asd homology arm gene and the kan resistance gene. In the figure, 1: an N-terminal homology arm; 2: a C-terminal homology arm; 3: a kan resistance gene; 4: DL2000 marker.
FIG. 2 shows amplification products of the asd-kan fusion gene. In the figure, 1: asd-kan fusion gene; m: DL5000 marker.
FIG. 3 shows the result of PCR identification of the pMD18-asd-kan plasmid. In the figure, 1: DL5000 marker; 2: pMD18-asd-kan plasmid PCR product.
FIG. 4 shows the results of PCR identification of A19-. DELTA.asd-deleted strains. In the figure, 1: amplification and identification of asd-I-F and asdkan-R; 2: amplification and identification of asdkan-F and asd-I-R; m: DL2000 maker.
FIG. 5 is a graph of the change in spleen index of mice infected with the A19 parent strain and the A19-. DELTA.asd-deleted strain.
FIG. 6 shows the change in spleen CFU of mice challenged with the A19 parent strain and the A19-. DELTA.asd-deleted strain on different days.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
1. Primary reagent
The gel recovery kit was purchased from Shanghai bioengineering, inc.
High fidelity enzymes, taq enzymes, etc. were purchased from Novowed.
Other reagents are made in China.
2. Design and Synthesis of primers
Primers used in the experiment are designed according to the asd gene and the upstream and downstream sequences (GenBank accession number CP 044986.1) in the NCBI database, the names and the sequences of the primers are shown in Table 1, and the primers are synthesized by Jilin Cue Mei Biotechnology GmbH.
TABLE 1 primer names and sequences
3. PCR amplification and gel recovery of asd-kan fusion gene
(1) According to the position of the asd gene sequence in the genome, 800bp fasta format sequences are respectively searched at the upstream and the downstream of the gene, and sequence primers with the amplification length of about 500bp-600bp are designed from the fasta format sequences.
(2) The upstream and downstream homologous arm amplification of the asd gene was carried out with high fidelity enzyme (2X PhantaMasterMix) using the boiled and lysed Brucella A19 strain as a template. The amplification conditions were: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 45s,30 cycles; final extension at 72 ℃ for 10min. 50 mu L of the amplification system of the N-terminal homologous arm of the asd gene: high-fidelity enzyme premix: 25 mu L of the solution; template: 2.5 mu L; asdkan-N-F:2.5 mu L; asdkan-N-R:2.5 mu L; ddH 2 O: 17.5. Mu.L. 50 mu L of asd gene C-terminal homologous arm amplification system: high fidelity enzyme: 25 mu L of the solution; template: 2.5 mu L; asdkan-C-F:2.5 mu L; asdkan-C-R:2.5 mu L; ddH 2 O: 17.5. Mu.L. And after the reaction is finished, taking 3 mu L of PCR product for electrophoresis verification, and after the verification is successful, completely adding all the residual PCR product for glue recovery.
(3) The kan resistance gene sequence was amplified using high fidelity enzyme (2X Phanta MasterMix) using the ytREX-SacB plasmid as a template. The amplification conditions were: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min15s,30 cycles; final extension at 72 ℃ for 10min. The reaction system was 50. Mu.L (Hi-Fi enzyme: 25. Mu.L; template: 2.5. Mu.L; asdkan-F: 2.5. Mu.L; asdkan-R: 2.5. Mu.L; ddH) 2 O: 17.5. Mu.L). And (3) taking 3 mu L of PCR product after the reaction is finished, carrying out electrophoresis verification, and after the verification is successful, adding all the residual PCR product samples and carrying out gel recovery.
(4) PCR amplification of asd-kan fusion gene by two-step method
The first step is as follows: the amplification conditions were: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 2min30s,15 cycles. The reaction system is 20 μ L, and comprises: 2. Mu.L of the N-terminal homology arm of the asd gene, 2. Mu.L of the C-terminal homology arm of the asd gene, 2. Mu.L of the kan-resistant gene, 10. Mu.L of the 2 XTaq enzyme, ddH 2 O 4μL。
The second step is that: amplification was carried out using the first-step reaction product as a template and asdkan-N-F and asdkan-C-R shown in Table 1 as primers. The amplification conditions were: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min30s, and 30 cycles. The reaction system was 50. Mu.L (2 XTaq enzyme: 25. Mu.L; first-step reaction product: 2.5. Mu.L; asdkan-N-F: 2.5. Mu.L; asdkan-C-R: 2.5. Mu.L; ddH 2 O: 17.5. Mu.L). And (3) after the reaction is finished, taking the 3 mu LPCR product for electrophoretic verification, and after the verification is successful, completely adding the residual PCR product for gel recovery.
(5) PCR amplification result of asd-kan fusion Gene
after the amplification products of the upstream and downstream homology arms of the asd and the amplification product of the kan resistance gene were subjected to 1.5% agarose gel electrophoresis, the results are shown in FIG. 1, and bands appeared around 500bp and 1000bp, respectively, which are consistent with the sizes of the expected target fragments (508bp, 5003bp), indicating successful amplification.
after electrophoresis of the amplification product of the asd-kan fusion gene on 1.5% agarose gel, the result is shown in FIG. 2, and a band appears around 2000bp, which is consistent with the expected size of the target fragment (1961 bp), indicating that the asd-kan fusion gene is successfully amplified.
4. Construction of pMD18-asd-kan deletion vector
Since the T vector cannot replicate in Brucella and is degraded by replication of the bacterial cells, the T vector is used as a shuttle plasmid in the construction. The connecting system is as follows: pMD18-T:1 mu L of the solution; asd-kan fusion gene: 3 mu L of the solution; ddH 2 O:1 mu L of the solution; solution I:5 μ L. Overnight ligation at 16 ℃Then the cells are transformed into escherichia coli competent cells DH5 alpha, then the cells are coated on an LB plate containing 50 ng/mu L of kanamycin, a single clone is selected to be in an LB liquid culture medium containing 50 ng/mu L of kanamycin, shaking culture is carried out for about 12 hours at 37 ℃, bacterial liquid is taken for PCR identification, and the bacterial liquid is sent to Jilin Kuei scientific and technology limited company for sequencing after the identification is successful.
Identification of pMD18-asd-kan deletion vector: the cloned plasmid was extracted, PCR was performed, and the product was subjected to 1.5% agarose gel electrophoresis, the result is shown in FIG. 3, a band appeared around 2000bp, the product was subjected to sequencing, the result was BLAST-compared with NCBI database, and the result showed that both ends of pMD18-asd-kan deletion vector were upstream and downstream sequences of Brucella asd.
5. Preparation of cattle Brucella A19 strain competence
Taking out a frozen A19 strain (CVCC 70202), streaking and recovering a bacterium liquid by using an inoculating loop, culturing at 37 ℃ for about 4 days, selecting a single bacterium colony, inoculating the single bacterium colony into a 4mL LTSB liquid culture medium by using the inoculating loop, transferring all the bacterium colony into a 200mL TSB liquid culture medium after culturing for 14-18h, centrifuging at 4 ℃ after culturing for 14-18h, collecting the bacterium body, re-suspending and uniformly mixing the collected bacterium body with precooled sterile deionized water, repeatedly washing for 3 times, fully washing the culture medium to reduce the ion content in A19 competence, finally washing once with 15% glycerol deionized water, removing the liquid as clean as possible after the last washing, and adding 15% glycerol deionized water for re-suspending according to the proportion of 100-200 times of concentration. The resuspended and mixed A19 competence is divided into centrifuge tubes according to the amount of 80 microliter per tube for standby.
6. Construction of A19-. DELTA.asd-deleted Strain
The pMD18-asd-kan deletion vector was electroporated into prepared A19 competent cells, applied to TSA plates containing 100 ng/. Mu.L each of diaminopimelic acid and kanamycin after overnight recovery, cultured at 37 ℃ for 4 to 5 days, single colonies were picked up into TSB liquid medium containing 100 ng/. Mu.L each of diaminopimelic acid and kanamycin, cultured with shaking at 37 ℃ for 24 hours, and PCR-identified with two pairs of primers asd-I-F, asdkan-R and asdkan-F, asd-I-R in Table 1.
Identification of A19-. DELTA.asd deletion strains: after the colonies grown on the plate after the electric transfer were inoculated into a liquid medium containing 100 ng/. Mu.L of kanamycin and a liquid medium containing 100 ng/. Mu.L of each of kanamycin and diaminopimelic acid, PCR identification was performed using two pairs of primers, asdkan-I-F, asdkan-R and asdkan-F, asdkan-I-R, in Table 1 after the lysis of the boiled bacterial liquid, and the results are shown in FIG. 4, wherein a band of about 1500bp was amplified and the amplified band was identical to the expected target fragment (asdkan-I-F, asdkan-R expected amplified size of 1461bp, asdkan-F, asdkan-I-R expected amplified size of 1453 bp), indicating that the A19-. DELTA.asd deletion strain was successfully constructed.
7. Safety experiment of A19-delta asd deletion strain on mice
Selecting 6-week-old BALB/c mice, randomly dividing the mice into 3 groups, wherein each group comprises 20 mice, namely an A19 group, an A19-delta asd group and a negative control group. Infecting mice by intraperitoneal injection at a dose of 1 × 10 7 CFU/ml, 5 mice per group were weighed and sacrificed at different time points (7d, 14d,21d, 28d) after infection, spleens were weighed and ground to 1ml, 0.1% Tritiox-100, and ten serial dilutions of the disrupted lysate were plated on TSA plates for enumeration.
The results of the mouse spleen weight index experiment are shown in FIG. 5. The spleen weight index of mice infected by 7d,14d,21d and 28d and A19 groups after infection is obviously higher than that of mice infected by A19-delta asd groups, which shows that the pathogenicity of the A19-delta asd deletion strain to the mice is obviously lower than that of the A19 strain.
8. Immune protection evaluation experiment of A19-delta asd deletion strain
Balb/c mice of 6 weeks old are selected and randomly divided into 6 groups of 10 mice, namely an A19 immune group, an A19-delta asd immune group and a blank control group. Mice were immunized at an immunization dose of 1X 10 7 CFU/ml. After immunization, 5 mice are respectively taken from each group of 14d and 28d to carry out 2308 virulent strain challenge, and the challenge dose is 1 multiplied by 10 7 CFU/ml. Two weeks after challenge, mice were weighed and sacrificed, spleens were weighed and added with 1ml of 0.1% Tritiox-100 for grinding and crushing, and ten-series multiple dilutions of the crushed lysates were spread on TSA plates for counting.
2308 virulent strain challenge is carried out 14 days and 28 days after mice are immunized by the A19- Δ asd deletion strain and the A19 parent strain, the mice are killed 14 days after challenge, spleen indexes and spleen bacterial loads are calculated, and the results are shown in FIG. 6, wherein the spleen indexes of the mice immunized by the A19- Δ asd deletion strain are obviously lower than those of the A19 parent strain, the spleen bacterial loads of the mice immunized by the A19- Δ asd deletion strain 28 days are obviously lower than those of the A19 parent strain, one of the mice immunized by the A19- Δ asd deletion strain 28 days is killed 2 weeks after challenge, the spleen bacterial loads are 0, and the results obviously show that the protection provided by the A19- Δ asd deletion strain 28 days after challenge is far higher than that of the A19 parent strain.
The invention discloses a construction method of a Brucella A19 strain asd gene deletion strain, and a person skilled in the art can realize the construction method by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.
Sequence listing
<110> military veterinary institute of military medical institute of military sciences
Construction method and application of <120> Brucella A19 strain asd gene deletion strain
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Claims (10)
1. The construction method of the Brucella A19 aspartic acid beta-galactose dehydrogenase asd gene deletion strain is characterized by comprising the following steps:
designing and synthesizing a primer;
step two, PCR amplification of the asd-kan fusion gene;
step three, constructing a pMD18-asd-kan deletion vector;
step four, constructing the A19-delta asd deletion strain.
2. The method for constructing the Brucella melitensis A19 aspartic acid beta-galactose dehydrogenase asd gene deletion strain according to claim 1, wherein the specific operation process of the first step is as follows:
designing primers according to the asd gene and upstream and downstream sequences thereof in the NCBI database, wherein the names and the sequences of the primers are as follows:
asdkan-N-F：5’-GGAGCCAAACCATGATGCAC-3’；
asdkan-N-R：5’-TTCGCTTGCTGTCCATAAAAATCAGATTGCAAAAGCGCCG-3’；
asdkan-C-F：5’-CTTGACGAGTTCTTCTGACAGGCCAAGGGAATCAGACC-3’；
asdkan-C-R：5’-GCAAAGATGACCTTGCGCTC-3’；
asdkan-F：5’-TTTTATGGACAGCAAGCGAA-3’；
asdkan-R：5’-TCAGAAGAACTCGTCAAG-3’；
asd-I-F：5’-TAGAGCGGATCGACAAGCAG-3’；
asd-I-R：5’-CGCCAGCGTTCCATAGAGAT-3’。
3. the method for constructing the Brucella melitensis A19 aspartic acid beta-galactose dehydrogenase asd gene deletion strain according to claim 2, wherein the specific operation process of the second step is as follows:
(1) Amplifying upstream and downstream homologous arms of the asd gene by using high-fidelity enzyme by taking a cattle Brucella A19 strain subjected to boiling cracking as a template; after the PCR product is successfully verified by electrophoresis, all the residual PCR products are subjected to sample addition electrophoresis and gel recovery;
(2) Using a yTREX-SacB plasmid as a template, and amplifying a kan resistance gene sequence by using high-fidelity enzyme; after the PCR product is successfully verified by electrophoresis, all the residual PCR products are subjected to sample addition electrophoresis and gel recovery;
(3) PCR amplification of asd-kan fusion gene by two-step method
The first step is as follows: using an asd gene N-terminal homologous arm, an asd gene C-terminal homologous arm, a kan resistance gene, taq enzyme and ddH 2 O is a reaction system for amplification;
the second step: amplifying by taking the reaction product of the first step as a template and taking asdkan-N-F and asdkan-C-R as primers; and after the PCR product is successfully verified by electrophoresis, all the residual PCR product is subjected to sample loading electrophoresis and gel recovery.
4. The method for constructing the Brucella melitensis A19 aspartic acid beta-galactose dehydrogenase asd gene deletion strain according to claim 3, wherein in the second step (1), the amplification conditions are as follows: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s, extension at 72 ℃ for 45s,30 cycles; final extension at 72 ℃ for 10min; 50 mu L of the amplification system of the N-terminal homology arm of the asd gene: high-fidelity enzyme premix: 25 mu L of the solution; template: 2.5 mu L; asdkan-N-F:2.5 mu L; asdkan-N-R:2.5 mu L; ddH 2 O:17.5 mu L; 50 mu L of asd gene C-terminal homologous arm amplification system: high fidelity enzyme: 25 mu L of the solution; template: 2.5 mu L; asdkan-C-F:2.5 mu L; asdkan-C-R:2.5 mu L; ddH 2 O：17.5μL。
5. The method for constructing the Brucella melitensis A19 aspartic acid beta-galactose dehydrogenase asd gene deletion strain according to claim 3, wherein in the second step (2), the amplification conditions are as follows: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min15s,30 cycles; final extension at 72 deg.C for 10min; the reaction system is 50 μ L and comprises: high fidelity enzyme: 25 mu L of the solution; template: 2.5 mu L; asdkan-F:2.5 mu L; asdkan-R:2.5 mu L; ddH 2 O：17.5μL。
6. The method for constructing the Brucella melitensis A19 aspartic acid beta-galactose dehydrogenase asd gene deletion strain according to claim 3, wherein in the second step (3), the reaction system is 20 μ L in the first step, and the reaction system comprises the N-terminal of the asd gene2. Mu.L of homology arm, 2. Mu.L of C-terminal homology arm of asd gene, 2. Mu.L of kan-resistant gene, 10. Mu.L of taq enzyme, ddH 2 O4 mu L; the amplification conditions were: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, extension at 72 ℃ for 2min30s,15 cycles.
7. The method for constructing the Brucella melitensis A19 strain aspartic acid beta-galactose dehydrogenase asd gene deletion strain according to claim 3, wherein in the second step (3), the reaction system is 50 μ L, and the reaction system comprises 2 × taq enzyme: 25 mu L of the solution; the first step reaction product: 2.5 mu L; asdkan-N-F:2.5 mu L; asdkan-C-R:2.5 mu L; ddH 2 O:17.5 mu L; the amplification conditions were: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min30s, and 30 cycles.
8. The method for constructing the Brucella melitensis A19 aspartic acid beta-galactose dehydrogenase asd gene deletion strain according to claim 3, wherein the specific operation process of the third step is as follows:
the connecting body is: pMD 18-T1. Mu.L, asd-kan fusion gene 3. Mu.L, ddH 2 O1 mu L and solution I5 mu L are transformed into Escherichia coli competent cells DH5 alpha after being connected overnight at 16 ℃, then the Escherichia coli competent cells are coated on an LB plate containing 50 ng/mu L of kanamycin, a single clone is picked up to an LB liquid culture medium containing 50 ng/mu L of kanamycin and is subjected to shake culture at 37 ℃ for about 12 hours, and a bacterial solution is taken for PCR identification and sequencing.
9. The method for constructing the Brucella melitensis A19 aspartic acid beta-galactose dehydrogenase asd gene deletion strain according to claim 8, wherein the specific operation process of the fourth step is as follows:
the pMD18-asd-kan deletion vector was electroporated into prepared A19 competent cells, applied to TSA plates containing 100 ng/. Mu.L each of diaminopimelic acid and kanamycin after overnight recovery, cultured at 37 ℃ for 4 to 5 days, single colonies were picked up into TSB liquid medium containing 100 ng/. Mu.L each of diaminopimelic acid and kanamycin, shake-cultured at 37 ℃ for 24 hours, and PCR-identified using two pairs of primers asd-I-F, asdkan-R and asdkan-F, asd-I-R.
10. The use of the asd gene-deleted strain constructed by the method for constructing the aspartic acid β -galactose dehydrogenase asd gene-deleted strain of the bovine brucella a19 strain according to any of claims 1 to 9 for preparing a candidate vaccine strain for preventing brucella infection.
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