CN109999194B

CN109999194B - Rough brucella of recombinant A-type foot-and-mouth disease virus VP1 gene and vaccine production method thereof

Info

Publication number: CN109999194B
Application number: CN201910284347.6A
Authority: CN
Inventors: 孙石静; 王芳; 蒋卉; 朱良全; 秦玉明; 许冠龙; 丁家波; 彭小薇; 冯宇; 范学政; 王楠
Original assignee: China Institute of Veterinary Drug Control
Current assignee: China Institute of Veterinary Drug Control
Priority date: 2019-04-10
Filing date: 2019-04-10
Publication date: 2023-01-31
Anticipated expiration: 2039-04-10
Also published as: CN109999194A

Abstract

The invention relates to a construction of a Rough Brucella of a recombinant A-type foot-and-mouth disease virus VP1 gene and a vaccine production method thereof. The invention successfully constructs Sup>A recombinant BrucellSup>A RA343-VP1-FMDV-A strain capable of efficiently expressing FMDV VP1 genes by taking Sup>A rough BrucellSup>A low virulent strain RA343 strain as Sup>A parent strain and sucrose suicide plasmid as Sup>A vector, and inserting the A-type foot-and-mouth disease virus VP1 genes containing specific promoter sequences into BrucellSup>A genomes after codon optimization without traces. The recombinant vaccine strain not only retains the rough characteristic of the original parent strain RA343 and good immune protection to brucellosis (brucellosis), but also can generate an antibody aiming at FMDV VP1 after the recombinant strain is used for immunizing animals, thereby realizing immune protection to FMD. The recombinant vaccine strain is developed into a novel vaccine with the characteristic of heat-resistant protection according to a specific process, and after an animal is immunized, the dual immune protection of the epidemic disease and the foot-and-mouth disease can be realized at the same time.

Description

Rough brucella of recombinant A-type foot-and-mouth disease virus VP1 gene and vaccine production method thereof

The invention belongs to the technical field of a rough brucella of a recombinant A-type foot-and-mouth disease virus VP1 gene and a vaccine production method thereof, and belongs to the field of biological products for livestock.

Technical Field

Brucellosis (brucellosis) is a human and animal co-morbidity characterized by abortion and fever caused by Brucella, and seriously threatens the life health of people and various animals. The disease not only has serious harm to the reproduction and production performance of animals, but also is difficult to cure after people are infected with brucella, thereby causing serious public health problems. Therefore, in countries where brucella is prevalent, eradication of brucella has always been one of the most important goals in public health programs. The main method for eliminating the disease worldwide is to combine killing and immunization at present, and the vaccine prevention becomes the main control means of the disease in China with relatively serious disease distribution due to high cost of killing.

In 2007-2012, 41 strains of Brucella melitensis, 74 strains of Brucella melitensis and 9 strains of Brucella canis are sequentially isolated from the bodies of animals such as cattle, sheep, dogs and deer in different provinces and regions of China. In the research process, we preliminarily found a bovine brucella isolate with virulence between virulent and vaccine strains, and the patent applicant carried out comprehensive biochemical detection on the bovine brucella isolate and measured the virulence by using mice and guinea pigs respectively. As a result, the virulence of the brucella Abortus is found to be between strong virulence and weak virulence, and the brucella Abortus is determined to be moderate virulence and is named as Abortus 343. The invention induces and domesticates the isolate, screens and obtains a stable rough brucella vaccine strain which is named as RA343, and obtains related patents (ZL 201410240895.6). At present, the vaccine strain has completed all laboratory research and development and clinical tests, and enters a new veterinary drug registration link.

Foot and Mouth Disease (FMD) is an acute, hot, highly contagious disease of cattle, sheep, etc. caused by FMDV (Foot and mouth disease virus). Since 2013 spring, the bovine A-type foot-and-mouth disease situation occurs in China in many places, and compared with the A-type foot-and-mouth disease outbreak in 2009, the bovine A-type foot-and-mouth disease mainly has the following characteristics: one is that the symptoms of the cattle without immunization are typical, the infection transmission speed is high, and the infection rate is as high as 80%. In recent years, the A-type foot-and-mouth disease epidemic situation successively appears in Xinjiang and the like in China, and huge economic loss is caused. The VP1 protein is the main structural protein of FMDV, is also the key of virus infected cell, and has the site combining with cell receptor, so that the VP1 gene is the first choice target antigen for developing gene engineering vaccine.

Disclosure of Invention

The invention aims to successfully construct Sup>A recombinant BrucellSup>A RA343-VP1-FMDV-A strain capable of efficiently expressing an FMDV VP1 gene by taking Sup>A rough BrucellSup>A attenuated strain RA343 as Sup>A parent strain and taking Sup>A sucrose suicide plasmid as Sup>A vector, and inserting the A-type FMDV VP1 gene containing Sup>A specific promoter sequence into Sup>A BrucellSup>A genome in Sup>A seamless manner after codon optimization. The recombinant vaccine strain not only retains the rough characteristic of the original parent strain RA343 and good immune protection to brucellosis (brucellosis), but also can generate an antibody aiming at FMDV VP1 after the recombinant strain is used for immunizing animals, thereby realizing immune protection to FMD. The recombinant vaccine strain is developed into a novel vaccine with the characteristic of heat-resistant protection according to a specific process, and after animals are immunized, the dual immune protection on the disease distribution and the foot-and-mouth disease can be realized at the same time.

The technical scheme of the invention is as follows:

1. a brucellSup>A vaccine of Sup>A recombinant A-type foot-and-mouth disease virus VP1 gene is characterized in that after an animal is immunized by the vaccine, double immune protection of brucellSup>A and A-type foot-and-mouth disease can be realized simultaneously, sup>A production strain is Sup>A recombinant rough brucellSup>A RA343-VP1-FMDV-A strain (hereinafter referred to as brucellSup>A RA343-VP1-FMDV-A strain) containing the VP1 gene capable of efficiently expressing the A-type foot-and-mouth disease virus, codon optimization is carried out on Sup>A P3 promoter and Sup>A VP1 sequence, sup>A gene fragment is artificially synthesized, sup>A sucrose suicide plasmid pUC/P3-VP1+ containing Sup>A P3-VP1 (FMDV-A) sequence and homologous arms of upstream and downstream of brucellSup>A is constructed, sup>A VP1 gene expression frame part of the A-type foot-and-mouth disease virus is inserted into Sup>A brucellSup>A genome without traces, positive clones are selected by ampicillin double screening, subculture is carried out, the stability of the Bovine brucellSup>A (virus RA) recombinant BrucellSup>A 343-A strain is obtained by PCR and identification sequencing. And is delivered to Beijing city, chaoyang district, north Xilu No.1 institute No. 3 China academy of sciences, china general microbiological culture Collection center for microbiological research on microorganisms on 14 days 03 and 14 days 2019, wherein the preservation numbers are as follows: CGMCC No.17332.

2. The recombinant rough brucellSup>A vaccine is characterized in that the specificity of the recombinant brucellSup>A RA343-VP1-FMDV-A strain for production, which is different from other bacteriSup>A, is that Sup>A specific fragment (sequence 23) with the size of 778bp is amplified by adopting Sup>A pair of specific PCR primers (sequence 21 and sequence 22);

the expression verification of the recombinant BrucellSup>A VP1 (FMDV-A) gene is to verify the expression of the VP1 (FMDV-A) gene transcription level in Sup>A real-time fluorescent quantitative PCR (qPCR) mode;

verifying the expression of the VP1 (FMDV-A) gene at the protein level by Sup>A protein printing method (Western Blot);

the recombinant strain is used for immunizing Sup>A mouse, and the expression condition of the VP1 (FMDV-A) antibody in the mouse is monitored.

3. The invention relates to a recombinant rough brucella vaccine, which is characterized in that the production method of the vaccine uses tryptone soybean broth as a culture medium of brucella CGMCC No.17332; inoculating brucellSup>A CGMCC No17332 strain seed bacterial liquid which accounts for 1-2% of the culture medium after the culture medium is sterilized, fermenting and culturing for 28-38 h at 37 ℃ according to Sup>A conventional method, adding Sup>A veterinary biological product freeze-drying protective agent, fully and uniformly mixing, subpackaging in vaccine bottles, and carrying out freeze-vacuum drying to obtain the rough RA343-VP1-FMDV-A strain brucellosis vaccine.

4. The invention relates to a recombinant live Brucella vaccine, which is characterized in that a freeze-drying protective agent in the vaccine comprises the following components in percentage by weight: 5-15g of PVP, 5-15 g of BSA, 50-100 g of trehalose, 50g of mannitol, 10-15 g of thiourea, 2g of ascorbic acid, 2g of vitamin C, 2g of 199 culture, 1.25g of dipotassium phosphate and 0.52g of potassium dihydrogen phosphate are dissolved in 1000ml of water for injection.

5. The recombinant rough brucella vaccine is characterized in that the vaccine immunization effect is evaluated by respectively adopting brucella and A-type foot-and-mouth disease virus to carry out challenge protection tests after the vaccine is used for immunizing sheep.

The invention has the advantages of

1. Brucellosis and foot and mouth disease are two major epidemic diseases which seriously harm the cattle and sheep breeding industry in China. The invention uses the brucella vaccine RA343 as a carrier, constructs a novel vaccine strain which can successfully express the A type FMDV VP1 gene, skillfully solves the prevention and control problems of two important diseases (brucella disease and A type foot-and-mouth disease) in the current cattle herd in China, realizes the idea of preventing two diseases at one time, and reduces the immunization cost and the labor cost in the production practice; 2. at present, the key technical problem which troubles the immune control of the cloth disease worldwide is that the antibody (smooth antibody) generated by the vaccine immunization interferes the clinical diagnosis of the cloth disease. The invention skillfully adopts rough brucella (RA 343 strain) as a carrier, and rough antibodies are generated after the vaccine is used for immunizing animals, so that the clinical diagnosis of the brucella is not interfered, and the weakness of the existing brucella vaccine is fundamentally improved; 3. the invention makes up the defect that the A-type foot-and-mouth disease vaccine lacks cellular immunity and improves the immune protection effect. Considering the risk of the virulent return of the foot-and-mouth disease attenuated vaccine, the inactivated vaccine is used for preventing the A-type foot-and-mouth disease all over the world at present, and in order to obtain a better immune effect, a high-concentration virus antigen is often required to be used, so that the immune load of an animal body is increased, and the immune potential is excessively consumed. Even so, the inactivated vaccine based on the A-type foot-and-mouth disease mainly based on the humoral immunity often has the phenomenon of immune failure. The invention adopts Brucella RA343 live vaccine strain as carrier, while inducing to produce and directing against A type foot-and-mouth disease virus VP1 antibody, also induce the host to produce the strong cellular immune response effectively, thus has realized the good immune protection to A type foot-and-mouth disease; 4. the invention fundamentally shortens the production period of the conventional foot-and-mouth disease vaccine and greatly reduces the production cost. The classical virus culture is converted into bacterial fermentation, so that the production cost of the A-type foot-and-mouth disease vaccine is substantially reduced. In order to improve the immune protection effect of the A-type foot-and-mouth disease vaccine as much as possible, a large amount of virus is required to be cultured and highly concentrated, so that antigen with sufficient concentration is obtained for producing the vaccine. In the invention, the VP1 gene of the foot-and-mouth disease virus A is stably integrated into the genome of the Brucella roughly-cultured vaccine RA343, so that a large amount of antigen can be produced only by fermentation culture for 36 hours according to a bacterial fermentation mode, the production requirement of the vaccine is met, and the method is time-saving, labor-saving and worry-saving; 5. in order to ensure the stable and efficient expression of the VP1 antigen of the A-type foot-and-mouth disease virus, on one hand, a traceless insertion technology is adopted to replace plasmid mediation, the A-type FMDV VP1 gene is directly inserted into the genome of the Brucella vaccine strain RA343, and the stability of the Brucella vaccine strain RA343 gene is verified through passage, so that the foreign gene is ensured not to be lost in the production passage process. On the other hand, in the implementation process of the invention, the codon which is easy to translate by brucella is used for replacing the rare codon in the VP1 gene, and the method for optimizing the codon promotes the high-efficiency expression of the exogenous gene; 6. the conventional live animal vaccine generally needs to be stored and transported at the temperature of 20 ℃ below zero, 4 groups of different heat-resistant protective agent formulas are designed, and one optimal formula is optimized to serve as a freeze-drying protective agent actually adopted, so that the live animal vaccine can be stored at the temperature of 4 ℃ for a long time, and the storage and transportation cost is reduced.

Information on microbial resources related to the present invention

The microbial resource related to the invention is a Brucella melitensis RA343 strain, which is a coarse attenuated strain obtained by inducing and domesticating a Brucella melitensis (Brucella abortus) isolate A343 strain separated from a cow body aborted in a certain dairy farm in Shandong in 2009 in laboratory, and the strain is delivered to the general microorganism preservation center of China Committee for preservation of microorganisms of national institute of microbiology, no. 3 of national academy of sciences, north Yangzhou, beijing City, no.1 of King, no. 3 of the national academy of sciences, 20 days in 2014, wherein the strain has the following accession number: CGMCC No.8886; the brucella abortus 2308 strain (CVCC 788 strain) of the cattle and the brucella abortus BM28 strain (CVCC 920 strain) of the sheep are preserved and supplied by the China veterinary microbial strain preservation management center, which is the China veterinary medical products institute, the China veterinary microbial strain preservation management center, second edition of China veterinary bacterial catalogs, china agricultural science and technology publishing agency, 2002, p24 and p 29.

Drawings

FIG. 1 Green fluorescent plasmid GFP-pZL1790 plasmid map.

FIG. 2 shows PCR specificity results of recombinant BrucellSup>A RA343-VP1-FMDV-A strain, wherein 1 is marker,2 is recombinant BrucellSup>A RA343-VP1-FMDV-A strain, 3 is EscherichiSup>A coli, 4 is staphylococcus, and 5 is salmonellSup>A.

FIG. 3 qPCR verifies The relative expression level of VP1 gene (The relative expression of VP1-FMDV-A gene), wherein 1 is RA343 strain, and 2 is recombinant BrucellSup>A RA343-VP1-FMDV-A strain.

FIG. 4 shows Western Blot identification of recombinant BrucellSup>A strain RA343-VP1-FMDV-A with an absorbance at 450nm (adsorbance at 450 nm) on the ordinate; the abscissa represents the time after infection (day post infection). 1 is RA343 strain, 2 is recombinant BrucellSup>A RA343-VP1-FMDV-A strain, and 3 is marker.

FIG. 5 the result of specific antibody response induced by VP1 gene.

FIG. 6 is a schematic graph of the lyophilization curve with temperature (. Degree. C.) plotted on the ordinate; the abscissa represents the lyophilization time.

Detailed description of the invention

1. Construction of recombinant BrucellSup>A RA343-VP1-FMDV-A strain

(1) Selection of promoter for foreign Gene expression by Brucella RA343 (for details, see example 1)

According to the analysis of brucella RA343 (CGMCC No.8886, ZL 201410240895.6) whole genome and gene function prediction, three molecular chaperone promoters P1, P2 and P3 which can be expressed in brucella at a high level continuously are screened out, and brucella promoter P is inserted into the upstream of a green fluorescent group of a green fluorescent plasmid GFP-pZL1790 (constructed in the laboratory) respectively ₁ 、P ₂ 、P ₃ And common promoters tac, trc and T7, and six green fluorescent plasmids P carrying different promoters ₁ -GFP-pZL1790，P ₂ -GFP-pZL1790，P ₃ GFP-pZL1790, tac-GFP-pZL1790, trc-GFP-pZL1790, and T7-GFP-pZL1790. Respectively transferring the gene into brucella RA343 strains, and screening out proper strong promoter P for exogenous gene expression according to the relative expression quantity of green fluorescent protein ₃ 。

(2) Is prepared by mixing P ₃ Carrying out codon optimization on the promoter and the VP1 gene of the foot-and-mouth disease virus type A (FMDV), artificially synthesizing a gene segment, and constructing a gene containing P ₃ -VP1-FMDV-A sequence and sucrose suicide plasmid vector pUC/P of upstream and downstream homology arms of BrucellSup>A ₃ -VP1 ⁺ And inserting the foot-and-mouth disease gene expression frame part into the brucellSup>A genome in Sup>A traceless manner, screening out positive clones by ampicillin and sucrose double screening, carrying out subculture, and identifying the stability of the clones by PCR and sequencing to obtain the recombinant brucellSup>A RA343-VP1-FMDV-A strain.

2. General biological characteristics of recombinant BrucellSup>A RA343-VP1-FMDV-A strain

(1) Morphological and biochemical Properties

The colony edge of the recombinant BrucellSup>A RA343-VP1-FMDV-A strain is neat, round and smooth, and is in Sup>A drop shape, and the colony is irradiated by oblique light and observed by back light to form slight blue opalescence. The staining morphology is coccobacillus, single scattered and does not form spores and capsules. The size is between 0.3 and 0.6 mu m. Gram staining was negative. The growth of the bacterium is independent of CO ₂ Capable of growing on medium containing thionine and basic rubine, H ₂ The S test is strong positive.

(2) The results of Sup>A thermal agglutination test, an acridine yellow test and Sup>A colony crystal violet test show that the recombinant brucellSup>A RA343-VP1-FMDV-A strain keeps the rough characteristic of the brucellSup>A RA343 strain.

(3) Specificity of the drug

1) The serological specificity is that the recombinant brucellSup>A RA343-VP1-FMDV-A strain culture is used for preparing antigen, and the antigen and the smooth brucellSup>A positive serum do not agglutinate, and the rough serum agglutinate.

2) The PCR result shows that 1 specific PCR band (figure 2) with 778bp size should appear when the amplified product is identified by electrophoresis with 1.5% agarose gel.

(4) Virulence

The culture cultured for 48-72 h on a solid medium is washed down by normal saline and diluted into 10 hundred million suspension containing viable bacteria per milliliter, and 5 guinea pigs (Hartley strain) weighing 350-400 g are injected subcutaneously in the groin, 1ml per pig. Killing after 14-15 days, taking spleens, mixing and weighing, preparing into emulsion, inoculating a TSA plate, and calculating the bacteria content of the phagocytosed spleens according to the number of growing colonies, wherein the bacteria content of each 1g of the spleens is not more than 20 ten thousand.

(5) Safety of mice

Balb/C mice weighing 20-20g are divided into 5 mice/group, and the recombinant strain RA343-VP1-FMDV-A is diluted into 1 multiplied by 10 ¹² CFU/ml to 1X 10 ⁸ CFU/ml bacterial suspension, inguinal injection of 0.1 ml/one, and the control group. The health of the mice was observed and recorded within 1 week after inoculation, and as a result, inoculation was 10 ¹¹ CFU/mice in the immunized group of only the following recombinant bacteria were all alive for 6 days. The result shows that the recombinant vaccine has good safety when used for immunizing non-target animals at different doses.

3. Preparation of rough brucella vaccine of recombinant A-type foot-and-mouth disease virus VP1 gene

The vaccine uses Sup>A recombinant brucellSup>A RA343-VP1-FMDV-A strain expressing the VP1 gene of the A-type foot-and-mouth disease virus as Sup>A production strain, and the production method comprises the following steps: tryptone soybean broth is used as a culture medium of Brucella CGMCC No.17332; inoculating brucellSup>A CGMCC No.17332 strain seed bacteriSup>A liquid with the amount of 1-2% of the culture medium after sterilizing the culture medium, fermenting and culturing for 28-38 h at 37 ℃ according to Sup>A conventional method, adding Sup>A freeze-drying protective agent which is commonly used by veterinary biological products, preferably Sup>A heat-resistant protective agent, fully mixing uniformly, subpackaging in Sup>A vaccine bottle, and then freezing and drying in vacuum to obtain Sup>A rough RA343-VP1-FMDV-A strain brucellosis vaccine; after the vaccine is used for immunizing animals, the dual immune protection to the cloth disease and the type A foot-and-mouth disease can be realized at the same time.

Examples

This example is provided to further illustrate the present invention and is not intended to limit the invention thereto.

Example 1 construction of recombinant BrucellSup>A roughii RA343-VP1-FMDV-A Strain

1. Preparation of Brucella RA343 competent cells

Picking out a single colony of the Brucella RA343 strain, inoculating the colony and culturing the colony in 100ml of TSB culture medium until the bacteria grow to logarithmic phase, and cooling the colony in ice water. Centrifuging at 12000r/min for 10min, discarding the liquid culture medium, and repeatedly washing with sterile deionized water of different volumes for several times. Finally, the obtained thalli is resuspended in 1ml of 10% glycerol aqueous solution, and the prepared parent strain RA343 strain infected state bacteria are stored at-80 ℃ for standby.

2. Promoter screening of Brucella RA343 expression exogenous gene

(1) Construction of Green fluorescent plasmid GFP-pZL1790

pZL1790 plasmid was stored in the laboratory, GFP green fluorescent gene was synthesized by Shanghai Biotech, primers (SEQ ID NO: 28 and SEQ ID NO: 29) were designed to amplify the green fluorescent gene and to add restriction sites, and the sequences of the primers were as follows:

sequence 28 GFP-F: tgagtcgaca tggttcgaa gggcgagagagagaa 30

Sequence 29 GFP-R: ataccgcggt tacttataca gttca 25

Adding an upstream primer into a Sal I restriction site, adding a downstream primer into a Sac I restriction site, and carrying out PCR by using the primers and a GFP green fluorescent gene as a template, wherein the reaction system is as follows:

PCR reaction (50. Mu.L):

max 2 XBuffer 25. Mu.L, upstream and downstream primers (10. Mu.M) 1. Mu.L each, ddH ₂ O22. Mu.L, template DNA 1. Mu.L.

The reaction conditions are as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 56 ℃, 30s at 72 ℃ and 31 cycles; 10min at 72 ℃.

After fully mixing 6 XLoading Buffer 100. Mu.L and PCR amplification product 20. Mu.L, adding into 0.8% agarose gel well, carrying out electrophoresis at 150V for 25min, carrying out imaging analysis by using a gel imaging system, cutting an agarose gel block containing a target fragment, and carrying out recovery and purification of PCR products according to the instruction of a Takara gel recovery kit (Code No: 9762). After the green fluorescent gene recovery product and pZL1790 plasmid are quantified, double enzyme digestion is respectively carried out, and the reaction system is as follows:

sal I1. Mu.L, sac I1. Mu.L, 10 XBuffer 5. Mu.L, nucleic acid 1. Mu.g, ddH ₂ The content of O is filled to 50 mu L.

After the enzyme digestion is carried out for 30min at 37 ℃, the enzyme digestion products are connected, and the reaction system is as follows:

1 μ L of T4 ligase, 2 μ L of 10 XBuffer, 2 μ L of pZL1790 plasmid after digestion, and 15 μ L of green fluorescent gene after digestion. The reaction was placed in a 4 ℃ freezer overnight. The recombinant green fluorescent plasmid GFP-pZL1790 after connection is transformed into DH5 alpha competent cells by a heat shock transformation method for molecular cloning, screening is carried out by using a culture medium containing chloramphenicol, and sequencing is carried out to obtain a positive plasmid (figure 1).

(2) Three Brucella promoters and three common promoters

According to the analysis of brucella RA343 whole genome and gene function prediction, three molecular chaperone promoters P1, P2 and P3 capable of continuously and highly expressing in brucella are screened out, and the promoters P are synthesized in Shanghai's company after codon optimization ₁ ，P ₂ ，P ₃ And the common promoters tac, trc and T7, and the promoter P is amplified by using the respective primers of different promoters (Table 1) ₁ ，P ₂ ，P ₃ PCR products of tac, trc and T7 (adding KpnI and EcoRI cleavage sites), the PCR reaction system and conditions were as follows:

PCR reaction bodySeries (50 μ L):

Reaction conditions are as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 56 ℃, 30s at 72 ℃ and 31 cycles; 10min at 72 ℃.

TABLE 1 promoter PCR amplification primer Table

(3) Promoter PCR product electrophoresis and purification

mu.L of 6 XLoading Buffer and 20 mu.L of PCR amplification product are fully mixed, added into a 0.8% agarose gel hole, electrophoresed at 150V for about 25min, analyzed by a gel imaging system, cut into agarose gel blocks containing target fragments, and recovered and purified according to the instructions of a Takara gel recovery kit (Code No: 9762).

(4) Construction of Green fluorescent protein plasmid carrying promoter

After the PCR product is purified, the PCR product is subjected to double enzyme digestion by KpnI and EcoRI, and then the PCR product is respectively cloned to a plasmid GFP-pZL1790 subjected to double enzyme digestion by the same restriction enzyme, and 6 plasmids which can theoretically express green fluorescent protein in Brucella are constructed: p ₁ -GFP-pZL1790，P ₂ -GFP-pZL1790、P ₃ GFP-pZL1790, tac-GFP-pZL1790, trc-GFP-pZL1790 and T7-GFP-pZL1790. After sequencing verification, the constructed plasmid is electrically transformed into Brucella RA343 strain.

(5) Electric conversion

Mu.g of YGT-pZL1790 (as a negative control), P ₁ -YGT-pZL1790、P ₂ -YGT-pBBR1mcs2、P ₃ The plasmids-YGT-pZL 1790, tac-YGT-pZL1790, trc-YGT-pZL1790 and T7-YGT-pBBR1mcs2 were added to 50. Mu.L of RA343 competent bacteria, mixed well and transferred to a cuvette for electric shock. The shock voltage and time are respectively: 1.8kv,3ms. After completion of the electric shock, 1ml of TSB medium was immediately added thereto and cultured by shaking at 37 ℃ toAnd 4h, then spreading the bacterial liquid on 2 TSA culture dishes containing 50 mu g/mL kanamycin, and culturing at 37 ℃ for more than 48h to obtain grown bacterial colonies, namely the positive electrotransformation bacteria. The obtained recombinant gene engineering bacteria are named as RA343-YGT-pZL1790 (as negative control) and RA343-P ₁ -YGT-pZL1790、RA343-P ₂ -YGT-pZL1790、RA343-P ₃ -YGT-pZL1790, RA343-tac-YGT-pZL1790, RA343-trc-YGT-pZL1790 and RA343-T7-YGT-pZL1790.

(6) Screening of strong promoters by brucella cell infection experiments

Mouse macrophage RAW264.7 is passaged to six-hole plate, and the cells are respectively divided into RA343 group and RA343-P ₁ -YGT-pZL1790、RA343-P ₂ -YGT-pZL1790 and RA343-P ₃ Groups of-YGT-pZL 1790, RA343-tac-YGT-pZL1790, RA343-trc-YGT-pZL1790 and RA343-T7-YGT-pZL1790 were incubated until the bottom of the 6-well plate was confluent. Inoculating the corresponding strain into 20ml of TSB culture medium, and carrying out shaking culture at constant temperature of 180r/min and 37 ℃ until the logarithmic phase. Collecting the bacteria, and repeatedly resuspending the bacteria by PBS. Infection was performed according to the brucella and RAW364.7 infection ratio (MOI) 50 ₂ A constant temperature incubator at 37 ℃ with the concentration of 5 percent. After 1h of infection, gentamicin with the final concentration of 50 mug/ml is added into each hole of the six-hole plate for 45min of action. Mouse macrophages after 24h, 48h, 72h infection were washed 3 times with PBS to stop the infection. Adding formaldehyde fixing solution for fixing, then using a fluorescence microscope for fluorescence detection under exciting light, and screening out a strong promoter P according to the intensity of green fluorescence under the same exciting light intensity ₃ . Codon optimization and traceless insertion of type A Foot and Mouth Disease Virus (FMDV) VP1 Gene

(1) Construction of shuttle plasmid pUC/P ₃ -VP1 ⁺

Promoter P artificially synthesized and codon-optimized by Shanghai bioengineering GmbH ₃ And a foot-and-mouth disease VP1 gene sequence, wherein the VP1 gene is amplified by taking a cloned plasmid as a template and taking VP1-F and VP1-R as primers. The Brucella RA343 genome DNA is used as a template, and primers up-F, up-R, down-F and down-R are designed to respectively amplify 941bp bases at the upstream of the 814594 site of the chromosome 1 as an upper homologous arm and 781bp bases at the downstream of the 814594 site as a lower homologous arm (the upstream and the downstream are homologous)XbaI and BamHI restriction sites added to the source arm, respectively), and the promoter P ₃ And the foot-and-mouth disease VP1 gene is inserted into the brucella genome without trace, and the primers are shown in Table 2:

TABLE 2 upstream and downstream homology arm primers for wboA gene

The PCR reaction systems are as follows: in a 50. Mu.L reaction system, 25. Mu.L of 2 XBuffer, 2. Mu.L of mixed primer stock solution, 1. Mu.L of Taq enzyme, 1. Mu.L of template DNA, and ddH ₂ O 21μL。

The PCR reaction procedures were as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 56 ℃ and 60s at 72 ℃ for 31 cycles; 10min at 72 ℃.

Purifying and recycling PCR products of PCR amplified Brucella upstream and downstream homology arms and foot-and-mouth disease VP1 gene fragments, and calculating the dosage of each template in fusion PCR according to a formula after determining the concentration:

and the upper homology arm, the VP1 gene and the lower homology arm are fused together by fusion PCR.

And (3) PCR reaction system: in a 50. Mu.L reaction system, 25. Mu.L of 2 XBuffer, 2. Mu.L of mixed primer stock solution, 1. Mu.L of Taq enzyme, 2. Mu.L of template DNA, and ddH ₂ O 20μL。

PCR reaction procedure: 5min at 95 ℃; 30s at 95 ℃, 30s at 56 ℃ and 3min at 72 ℃ for 31 cycles; 10min at 72 ℃.

The product and the pUC19 plasmid were recovered by double digestion of PCR gel with XbaI and BamHI restriction enzymes, and the fusion fragment was ligated to the multiple cloning site of the shuttle plasmid pUC19 to obtain a recombinant shuttle plasmid named pUC/VP1 ⁺ 。

(2) Electrotransfer shuttle plasmid pUC/P _dnak -VP1 ⁺ Into Brucella RA343

RA343 competent bacteria were prepared according to the above method (1.4) by adding 1-10ng of pUC/P _dnak -VP1 ⁺ The plasmid was added to 50ul RA343 strain competent bacteria and mixed well. After ice-cooling for 10min, the sample was taken out into a 2nm electrotransformation cup for clicking. Setting parameters: 2.5kV,5ms. After the electric shock is finished, adding 1ml of TSB culture medium into the electric conversion cup, sucking the bacterial suspension, adding the bacterial suspension into a TSB culture medium triangular flask, placing the TSB culture medium triangular flask in the flask, and shaking and culturing for at least 4 hours at 37 ℃.

(3) Screening of positive recombinant bacterium RA343-VP1-FMDV-A

Ampicillin screening: the bacterial solution cultured with shaking at 37 ℃ for 4h is centrifuged at 6000r/min for 3min, part of the supernatant is gently aspirated, about 200. Mu.l of the supernatant is reserved, the precipitated bacteria are gently blown to be uniform, and then all colonies are spread on 1 TSA medium plate containing 50. Mu.g/ml ampicillin and cultured at 37 ℃ for at least 72h. The recombinants containing ampicillin were screened.

And (3) sucrose negative screening: single colonies were picked on TSA plates containing ampicillin, inoculated into TSB liquid medium containing no ampicillin, cultured overnight with shaking at 37 ℃ for 24 hours, and then cultured with physiological saline at a ratio of 1:10 ² 、1:10 ³ 、1：10 ⁴ Diluting bacterial liquid, taking 100uL bacterial liquid for each dilution, coating 5&Sucrose TSA plates were incubated at 37 ℃ for at least 72h.

Randomly picking a single colony from a 5% sucrose plate, carrying out colony PCR (polymerase chain reaction) screening on positive recombinant bacteria by using primers (sequence 19 and sequence 20), wherein the size of the positive bacteria is 1579bp, and carrying out sequencing verification on a promoter P ₃ And the VP1 gene is inserted into the genome of the Brucella without any trace. The positive recombinant strain is named as RA343-VP1-FMDV-A strain.

The sequence 19F,

sequence 20R

Simultaneous sequencing to verify promoter P ₃ And the gene is inserted into the genome of the Brucella without any trace with the type A foot-and-mouth disease VP1 gene. The positive recombinant bacteriSup>A are Bovine brucellSup>A (Bovine brucellSup>A) capable of efficiently expressing the VP1 gene of the A-type foot-and-mouth disease, and Sup>A BrucellSup>A RA343-VP1-FMDV-A strain (BrucellSup>A RA343-VP1-FMDV-A strain for short) is delivered to Beijing city, no.1 North Chen of the national academy of sciences, chinSup>A Committee for culture Collection and management, ministry of microorganisms, chinSup>A Committee for culture Collection and management, 3 th national institute of microbiology, national institute of sciences, north and Yan, and 3 th, of Beijing City, 03 and 14 days in 2019The microorganism culture is preserved by the microorganism center with the preservation number as follows: CGMCC No.17332;

example 2 general biological Properties of recombinant BrucellSup>A RA343-VP1-FMDV-A

1. Morphological and biochemical Properties

The colony edge of the recombinant bacterium is neat and round, the recombinant bacterium is in a drop shape, oblique light irradiation is carried out, and micro-strip blue opalescence is observed in a backlight mode. The staining morphology is coccobacillus, single scattered and does not form spores and capsules. The size is between 0.3 and 0.6 mu m. Gram staining was negative. The growth of the bacterium is independent of CO ₂ Capable of growing on medium containing thionine and basic rubine, H ₂ The S test is strong positive.

2. Thermal coagulation test

Inoculating the recombinant strain into a TSA culture medium, culturing at 37 ℃ for 46h, then hooking the culture into a test tube filled with about 100ml of physiological saline, shaking up to enable the culture to be turbidified to bacterial suspension containing 10 hundred million/ml of viable bacteria, dividing the bacterial suspension after being taken out into 2 tubes, heating each tube by 4-5ml in a 90 ℃ water bath for 1h, observing results at 30min and 60min, wherein the recombinant vaccine and the parental strain are both in agglutination positive type and rough type, and the recombinant vaccine keeps the characteristic of RA343 rough type.

3. Acridine yellow test

Preparing a 1. Agglutination appeared within 3min, and the recombinant vaccine was a rough colony.

4. Colony crystal violet test

The recombinant strain is serially diluted by 10 times, inoculated on a TSA plate culture, placed at 37 ℃ for culturing for 72-96 h, the crystal violet stock solution is diluted by 1 to 40 times by distilled water, a proper amount of diluted dye solution is absorbed to cover the TSA plate growing a single bacterial colony for dyeing for 15-20 s, the crystal violet dye solution is discarded in a disinfectant, and the coloration condition of the bacterial colony edge is observed by a magnifying glass or a low power microscope. The recombinant colony has colored edges and unclear boundaries, and is rough.

5. Specificity of

1) Serological specificity is to use culture to prepare antigen, which should not agglutinate with smooth Brucella positive serum, and agglutinate with rough serum.

2) PCR specificity the recombinant BrucellSup>A RA343-VP1-FMDV-A strain can amplify specific RA343-VP1-FMDV-A fragments with the size of 778bp (sequence 23) through Sup>A pair of specific PCR primers (sequence 21 and sequence 22), and the method is used as Sup>A method for identifying the bacterium and other bacteriSup>A.

Sequence 21RA343-FMDV-A-F: gcgaacctcc aactttcacta ctct 24

Sequence 22RA343-FMDV-A-R: catcgtgacg caccaccaatc t 21

Template: RA343 genomic DNA extracted from a colony or kit cultured with RA 343.

And (3) PCR reaction system: mu.L of the reaction mixture containing 5. Mu.L of 10 XBuffer, 8. Mu.L of 2.5mM dNTPs, 2. Mu.L of the mixed primer stock, 2U of Taq enzyme, and 1. Mu.L of the template DNA (or a small amount of picked-up colonies).

PCR reaction procedure: after 5min at 95 deg.C, 28 cycles of 94 deg.C for 1min, 60 deg.C for 1.5min, and 72 deg.C for 1.5min, and finally 72 deg.C for 10min.

The amplified products were identified by electrophoresis on 1.5% agarose gel, and 1 specific PCR band of 778bp in size was observed, while no band was observed when the genome of other strains (E.coli, staphylococcus, salmonella) was used as a template (FIG. 2).

Sequence 23:

6. the culture cultured for 48-72 h on a solid culture medium is washed down by using physiological saline for virulence and diluted into 10 hundred million suspension containing viable bacteria per milliliter, and 5 guinea pigs (Hartley strain) with the weight of 350-400 g are injected subcutaneously in the groin, and the injection amount is 1 ml/mouse. Killing after 14-15 days, taking spleens, mixing and weighing, preparing into emulsion, inoculating a TSA plate, and calculating the bacteria content of the phagocytosed spleens according to the number of growing colonies, wherein the bacteria content of each 1g of the spleens is not more than 20 ten thousand.

7. Safety of mice

Balb/C mice weighing 20-20g were divided into 5 mice/groupThe recombinant strain RA343-VP1-FMDV-A is diluted to 1X 10 ¹² CFU/ml to 1X 10 ⁸ CFU/ml bacterial suspension, inguinal injection of 0.1 ml/one, and the control group. The health of the mice was observed and recorded within 1 week after vaccination and the results showed 10 of the recombinant vaccine ¹¹ CFU/mice in the following immunization groups were fully viable for 6 days. The results show that the recombinant vaccine has good safety when used for immunizing non-target animals at different doses.

Example 3 stability of recombinant RA343-VP1-FMDV-A Strain in target animals

Using the recombinant vaccine RA343-VP1-FMDV-A at 1X 10 ¹⁰ CFU/head immunized adult sheep experimental group, inoculating blood collected at 3d after immunization into a TSA culture medium for bacteria isolation, carrying out PCR identification on isolated bacteria, and continuously passaging non-immunized adult sheep for 5 generations according to the method. The phenotype of the recombinant vaccine strain after continuous passage is not changed, and the VP1 gene inserted into the chromosome 1 stably exists.

Example 4 expression verification of VP1 Gene of recombinant BrucellSup>A Strain RA343-VP1-FMDV-A Strain

qPCR verification of VP1 Gene expression at the transcriptional level

Absorbing a proper amount of recombinant bacteria and RA343 parent strain liquid, killing the viable bacteria liquid in a water bath at a constant temperature of 80 ℃ for more than 2h, extracting bacterial RNA by using a TAKARA bacterial genome extraction kit, carrying out reverse transcription, then taking 16s gene as internal reference, and carrying out relative quantification on mRNA transcribed by VP1 gene by qPCR; the primers used are shown in table 3:

TABLE 3 qPCR primer List

The reaction system is as follows: 2 × Premix Ex Taq 10 μ L, upstream and downstream primers 0.5 μ L each (10 μmol/L), rox 0.4 μ L, template 2 μ L, deionized water 6.6 μ L.

The reaction conditions were as follows: 30s at 95 deg.C, 5s at 95 deg.C, and 34s at 60 deg.C for 40 cycles.

The result shows that the relative expression quantity of mRNA of the recombinant strain VP1 gene is obviously higher than that of the parent strain RA343, and the VP1 gene has higher expression quantity at the transcription level (figure 3).

Western blot identification of recombinant vaccine VP1 gene expression

The BrucellSup>A recombinant strain RA343-VP1-FMDV-A and the parent strain RA343 are inoculated into Sup>A TSB culture medium for shaking culture for 24-48h, and subjected to ultrasonic lysis, sample buffer solution is added, SDS-PAGE and Wsetron Blot analysis after boiling water bath for 10min, and the result shows that the recombinant strain RA343-VP1-FMDV-A has an obvious band at about 35kD relative to the parent strain RA343, and the expression of VP1 protein is verified by Sup>A protein imprinting method (figure 4).

3. VP1 specific antibody of recombinant brucellSup>A RA343-VP1-FMDV-A stimulated mouse

The mice were randomly divided into 3 groups of 30 mice each. First group of inguinal subcutaneous immune recombinant vaccines RA343-VP1 _(FMDV Strain A), 10 ⁹ CFU/only; a second group of inguinal subcutaneous immune parent strain RA343, 10 ⁹ CFU/only; the third group was a blank, immune saline, 1 ml/tube.

After the immunized group of mice is immunized, blood is collected and serum is separated 14d later, and the foot-and-mouth disease VP1 antibody is measured by an indirect ELISA method. The result shows that the recombinant vaccine RA343-VP1-FMDV-A group foot-and-mouth disease VP1 antibody level OD ₄₅₀ Positive when kept at 1.0 or more, parent strain RA343 group and blank group OD ₄₅₀ It remained below 0.5, and was negative (FIG. 5).

Example 5 screening of Heat-resistant protective Agents and validation of Effect of recombinant BrucellSup>A Strain RA343-VP1-FMDV-A

Designing 4 groups of heat-resistant protective agents (respectively marked as

formulas

1, 2, 3 and 4) according to the characteristics and the eutectic performance of RA343-VP1-FMDV-A, respectively carrying out freeze-drying according to Sup>A conventional freeze-drying curve (figure 6), and determining that the freeze-drying RA343-VP1-FMDV-A of the heat-resistant protective agent formulSup>A 1 has the best effect, does not crystallize after freeze-drying and has the freeze-drying bacterial storage rate of 87.7 percent through the test results (table 4) of 7-day aging resistance after the freeze-drying before and after the freeze-drying; the product can be stored at 37 deg.C for 7 days without atrophy, and the viable bacteria reduction rate is 13.1%. Is obviously superior to gelatin and sucrose control.

TABLE 4 Freeze drying results for different protectant formulations (viable count: 10) ⁸ CFU/ml)

The protectant formulations and methods of formulation are shown in table 5 below:

TABLE 5 Heat-resistant Freeze-drying protectant formulations

Note: preparation method

(1) Solution 1: adding proper amount of water for injection (100 ℃) into a clean beaker, sequentially weighing PVP, mannitol, trehalose and thiourea according to the formula, adding the PVP, mannitol, trehalose and thiourea into the beaker, stirring with a glass rod while adding to completely dissolve the PVP, then adding the water for injection to a constant volume of 500ml, and sterilizing for 30min at 116 ℃.

(2) Solution 2: weighing BSA, 199 culture medium, ascorbic acid, vitamin C, potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a beaker according to the formula in sequence, adding water for injection to fully dissolve the BSA, 199 culture medium, ascorbic acid, vitamin C, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and filtering and sterilizing by a sterilizing filter with a filter membrane of 0.22 mu m to a constant volume of 500ml.

Example 6 determination of immunoprotective Effect of recombinant BrucellSup>A Strain RA343-VP1-FMDV-A vaccines against BrucellSup>A and foot-and-mouth disease

(1) Immune protection effect of recombinant vaccine on brucella

The 45 clean grade sheep were randomly divided into three groups of 15 sheep each. Sup>A group of RA343 vaccine strains which are orally immunized with 50 hundred million CFU, sup>A group of RA343-VP1-FMDV-A recombinant strains which are orally immunized with 50 hundred million CFU, and Sup>A group of control normal saline which is orally immunized with the same dose. After immunization, blood was collected at 0d,7d,14d,28d,42d, and 60d, and serum was separated, and brucella antibody IgG was detected by a microtubular agglutination method. The minimum infection dose was 5X 10 at week 8 after immunization in 3 groups ⁶ Melitensis M28 strain conjunctival infection was challenged with CFU/dose b. Observe and record ingestionCondition, weight, mental state, etc. And (3) killing after 30d of challenge, taking spleen and inguinal lymph node, mashing and grinding, inoculating a TSA culture medium, culturing at 37 ℃ for at least 3d, separating bacteria and identifying. The result shows that BrucellSup>A antibody appears 7d after the sheep is immunized by RA343 and RA343-VP1-FMDV-A recombinant strain, the antibody level of 7-28 d is continuously increased, the titer of the antibody reaches the peak 28d after immunization, and then the antibody begins to gradually decline. The control group had no antibody production. The separation results of spleen and inguinal lymph node bacteriSup>A of sheep show that the parental strain RA343 of the recombinant vaccine RA343-VP1-FMDV-A strain can provide 87% of immune protection rate for sheep.

(2) Immune protection effect of recombinant vaccine on foot-and-mouth disease

40 clean-grade sheep are randomly divided into two groups, each group comprises 20 sheep, the experimental group is injected with recombinant vaccine subcutaneously according to the dose of 50 hundred million CFU bacteria to immunize adult sheep, and the control group is injected with normal saline with equal dose subcutaneously. 30 days later, foot and mouth disease virus 10 was inoculated ⁵ TCID ₅₀ . And after 14d, observing the foot-and-mouth disease infection rate of the two groups of sheep, and determining the immune protection effect of the immune recombinant vaccine group. The result shows that 20 sheep in the control group all suffer from the disease, and the foot-and-mouth disease infection rate is 100 percent; only 3 sheep in the experimental group are attacked, and the foot-and-mouth disease infection rate is 85%. The result shows that the immune protection rate of the recombinant vaccine to the foot-and-mouth disease virus reaches 85 percent, and the recombinant vaccine shows higher immune protection effect.

Sequence listing

<110> China institute for veterinary drug inspection

<120> construction of rough brucella of recombinant A-type foot-and-mouth disease virus VP1 gene and vaccine production method thereof

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 31

<212> DNA

<213> Artificial Synthesis (P1-F)

<400> 1

cggggtaccc cgtgtcagcc cgccatccac a 31

<210> 2

<211> 29

<212> DNA

<213> Artificial Synthesis (P1-R)

<400> 2

ccggaattcg gtcacgccct cgaacttgc 29

<210> 3

<211> 33

<212> DNA

<213> Artificial Synthesis (P2-F)

<400> 3

cggggtaccc cgatgcggtg gtaagctcct tca 33

<210> 4

<211> 31

<212> DNA

<213> Artificial Synthesis (P2-R)

<400> 4

ccggaattcc ggcggcagcg cctcatacat t 31

<210> 5

<211> 32

<212> DNA

<213> Artificial Synthesis (P3-F)

<400> 5

cggggtaccc cgaaatgtgc cgctcttctc gc 32

<210> 6

<211> 31

<212> DNA

<213> Artificial Synthesis (P3-R)

<400> 6

ccggaattcc caggagttgg tcgttcccag a 31

<210> 7

<211> 28

<212> DNA

<213> Artificial Synthesis (tac-F)

<400> 7

cggggtgcct cgaatagctg acgtgcca 28

<210> 8

<211> 30

<212> DNA

<213> Artificial Synthesis (tac-R)

<400> 8

ccggaaaaag gacacacttt aacaataggc 30

<210> 9

<211> 25

<212> DNA

<213> Artificial Synthesis (trc-F)

<400> 9

cggggtttga caattaatca tccgg 25

<210> 10

<211> 27

<212> DNA

<213> Artificial Synthesis (trc-R)

<400> 10

ccggaacatt atacgagccg gatgatt 27

<210> 11

<211> 27

<212> DNA

<213> Artificial Synthesis (T7-F)

<400> 11

cggggttaat acgactcact ataggga 27

<210> 12

<211> 24

<212> DNA

<213> Artificial Synthesis (T7-R)

<400> 12

ccggaatctc cctatagtga gtcg 24

<210> 13

<211> 32

<212> DNA

<213> Artificial Synthesis (VP 1-F)

<400> 13

gatactaagc ataatcttta gaggtgagta tg 32

<210> 14

<211> 26

<212> DNA

<213> Artificial Synthesis (VP 1-R)

<400> 14

ctgatagcgg gacaaaaagt taggat 26

<210> 15

<211> 31

<212> DNA

<213> Artificial Synthesis (up-F)

<400> 15

ggctctagaa tacgcttatt gagaatgttc g 31

<210> 16

<211> 32

<212> DNA

<213> Artificial Synthesis (up-R)

<400> 16

agcggcacat tttcttaatg cgcgggaata gc 32

<210> 17

<211> 32

<212> DNA

<213> Artificial Synthesis (down-F)

<400> 17

tcaagcagtc gctgttcttg cttcagcttg tt 32

<210> 18

<211> 31

<212> DNA

<213> Artificial Synthesis (down-R)

<400> 18

ccggatccta agccgacgag caaatagaag g 31

<210> 19

<211> 20

<212> DNA

<213> Artificial Synthesis (F)

<400> 19

atcagttcta gagacaagga 20

<210> 20

<211> 21

<212> DNA

<213> Artificial Synthesis (R)

<400> 20

attgttacac cgctaccctg g 21

<210> 21

<211> 24

<212> DNA

<213> Artificial Synthesis (RA 343-FMDVA-F)

<400> 21

gcgaacctcc aacttcataa ctct 24

<210> 28

<211> 21

<212> DNA

<213> Artificial Synthesis (RA 343-FMDVA-R)

<400> 28

catcgtgacg caccacaatc t 21

<210> 30

<211> 778

<212> DNA

<213> Artificial Synthesis ()

<400> 30

gcgaacctcc aacttcataa ctctagcacc ggggggaaaa cttttgtggc caatgtcacc 60

acaaatgggt ctgcataacg gtccttgcca ttttaactat aaatgagcta ttcccgcgca 120

ttaagagtag acacgggaaa tcagtcgcgg atccgcgaaa tgtgccgctc ttctcgctgg 180

aaaattccag ggcgaaccag aaattcatcg atgcgattta ttccgcgggc agaagcgggg 240

tgagcgagac tgtgtgacgc ctgtaaagag tgcggggcaa ggaatctgtt tttgtctggc 300

tcttgtgact tgcagtgaaa aactgccttt cttatatacg cctcgcacag ggctttgatg 360

acaggccgga aaccggtccg ctttctggaa cccaaccttg aagcaggcat tgtgaaaacc 420

gataggaact gcccatggaa cgctcggtcg aggtcttggc agtttgctga atggagagaa 480

atatggctaa ggttattggt atcgatcttg gtacgaccaa ctcctggccg acgtcagngg 540

ggnaacgntg cagaccctgt caccaccacc gttgagaact acggtggtga gacacaggtt 600

cagcgacgtt accacactga cgtcggcttc attatggaca ggtttgtgaa aatcagccct 660

gtgagcccca cgcacgtcat cgacctcatg caaacacacc aacacgcgtt ggtgggtgcc 720

ttcttgcgtg cagccacgta ctacttctcc gacctggaga ttgtggtgcg tcacgatg 778

<210> 24

<211> 17

<212> DNA

<213> Artificial Synthesis (16S-F)

<400> 24

cacaagcggt ggagcat 17

<210> 25

<211> 17

<212> DNA

<213> Artificial Synthesis (16S-R)

<400> 25

gcaactaagg gcgaggg 17

<210> 26

<211> 21

<212> DNA

<213> Artificial Synthesis (VP 1-F)

<400> 26

ggcaacttga cgtgggtacc c 21

<210> 27

<211> 21

<212> DNA

<213> Artificial Synthesis (VP 1-R)

<400> 27

caccaccagt tgcagagtac t 21

<210> 28

<211> 30

<212> DNA

<213> Artificial Synthesis (GFP-F)

<400> 28

tgagtcgaca tggtctcgaa gggcgaagaa 30

<210> 29

<211> 25

<212> DNA

<213> Artificial Synthesis (GFP-R)

<400> 29

ataccgcggt tacttataca gttca 25

Claims

1. A rough brucellSup>A vaccine for recombining A type foot-and-mouth disease virus VP1 gene is characterized by comprising Sup>A recombinant rough brucellSup>A RA343-VP1-FMDV-A strain, which is delivered to Beijing city, chaoyang district, north ChinSup>A academy of sciences, no.1 Homeh, no. 3 ChinSup>A Committee for culture Collection of microorganisms, wherein the strain is preserved by the general microorganism center of ChinSup>A academy of sciences, with the preservation number of: CGMCC No.17332.

2. The rough brucella vaccine of claim 1, wherein the recombinant VP1 gene of foot-and-mouth disease virus type A is produced by using tryptone soy broth as the culture medium of Brucella CGMCC No.17332; inoculating brucella CGMCC No.17332 strain seed bacterial liquid with the amount of 1-2% of the culture medium after sterilizing the culture medium, fermenting and culturing for 28-38 h at 37 ℃ according to a conventional method, adding a veterinary biological product freeze-drying protective agent, fully and uniformly mixing, subpackaging in vaccine bottles, and then freezing and drying in vacuum to obtain the rough brucella vaccine of the recombinant A-type foot and mouth disease virus VP1 gene.

3. The rough brucella vaccine of the recombinant VP1 gene of a-type foot-and-mouth disease virus of claim 2, wherein the lyophilized protectant of said vaccine comprises the following formulation: 5-15g of PVP, 5-15 g of BSA, 50-100 g of trehalose, 50g of mannitol, 10-15 g of thiourea, 2g of ascorbic acid, 2g of 199 culture medium, 1.25g of dipotassium hydrogen phosphate and 0.52g of potassium dihydrogen phosphate, and the components are dissolved in 1000mL of water for injection.