CN114272368A

CN114272368A - Swine fever and porcine pseudorabies bivalent vaccine as well as preparation method and application thereof

Info

Publication number: CN114272368A
Application number: CN202111541904.1A
Authority: CN
Inventors: 杨鑫; 刘鑫; 李子鸣; 李伊
Original assignee: Chengdu Bocong Biotechnology Co ltd
Current assignee: Chengdu Bocong Biotechnology Co ltd
Priority date: 2021-12-16
Filing date: 2021-12-16
Publication date: 2022-04-05

Abstract

The invention discloses a swine fever and porcine pseudorabies bivalent vaccine which is prepared by inserting a porcine pseudorabies virus gB antigen and a hog cholera virus E2 antigen into a replication-defective chimpanzee adenovirus shuttle vector, wherein the gene sequence of the porcine pseudorabies virus gB antigen is SEQ ID No.1, and the gene sequence of the hog cholera virus E2 antigen is shown as SEQ ID No. 2. Also discloses a preparation method thereof, which comprises 1) preparing hog cholera virus antigen protein E2 gene and porcine pseudorabies virus antigen protein gB gene; (2) constructing a recombinant replication-defective chimpanzee adenovirus shuttle vector; (3) obtaining replication-defective chimpanzee adenovirus plasmids; (4) and obtaining the recombinant adenovirus with uniform genome structure, and preparing the vaccine with required dosage form according to the requirement. The invention can avoid the immunosuppression phenomenon which occurs when the porcine pseudorabies virus and the classical swine fever virus are simultaneously used as antigens for immunization, and obtains the unexpected effect that an immune receptor can generate the classical swine fever antibody within 7 days of immunization.

Description

Swine fever and porcine pseudorabies bivalent vaccine as well as preparation method and application thereof

Technical Field

The invention relates to the field of swine vaccines, in particular to a swine fever and porcine pseudorabies bivalent vaccine and a preparation method and application thereof.

Background

Classical swine fever (classic swine fever CSFV) is an acute, febrile and lethal disease caused by classical swine fever Virus (classic swine fever CSFV), and causes great loss to the world pig industry. The E2 protein is a main protective antigen of classical swine fever virus, can induce an organism to generate a high-level specific neutralizing antibody, protects pigs against the attack of lethal CSFV, and plays an important role in the development of novel vaccines.

The pseudorabies virus belongs to Herpesviridae (Herpesviridae) and Herpesviridae, the virus particle is circular, the diameter is 150-180 mm, and the diameter of the nucleocapsid is 105-110 mm. The outermost layer of the virion is the viral envelope, which is a lipid bilayer structure derived from the host cell. The surface of the capsular sac is provided with fiber protrusions which are radially arranged and have the length of about 8-10 nm. Vaccination is one of the main measures to prevent, control and even eliminate pseudorabies.

A single vaccine requires multiple immunizations for an organism to prevent multiple diseases, but multiple immunizations can cause multiple stimulations for the animal body, thereby affecting the growth of the animal. Thus, in order to be simple and easy, and to reduce the inconvenience of multiple immunizations of animal organisms, a bivalent or multiple vaccine is put into view. The research on the bivalent vaccine of the swine fever and the porcine pseudorabies is already carried out at present, however, the interference phenomenon of the pseudorabies virus on the swine fever antibody in the immune practice process is ignored, the interference phenomenon causes the whole level of the animal body swine fever antibody of the swine fever vaccine to be low, the swine fever vaccine is difficult to prevent in a period of time, and the infection of the swine fever is caused.

Disclosure of Invention

In order to solve the problems, the invention aims to provide the swine fever and porcine pseudorabies bivalent vaccine, which can avoid the immune interference phenomenon when the porcine pseudorabies virus and the swine fever virus are simultaneously used as antigens, improve the generation speed of the antibody of the swine fever virus, improve the protection efficiency of an injection object after the vaccine is injected, and obtain the unexpected effect that an immune receptor can generate the antibody of the swine fever within 7 days of immunization. Also provides a preparation method and application of the vaccine.

The invention is realized by the following technical scheme:

a bivalent vaccine of swine fever and porcine pseudorabies is prepared by inserting a porcine pseudorabies virus gB antigen and a classical swine fever virus E2 antigen into a shuttle vector, wherein the gene sequence of the porcine pseudorabies virus gB antigen is SEQ ID No.1, and the gene sequence of the classical swine fever virus E2 antigen is shown as SEQ ID No. 2.

As mentioned above, the content of the bivalent vaccine of swine fever and porcine pseudorabies is 10^8.0VP per head.

The preparation method of the swine fever and porcine pseudorabies bivalent vaccine comprises the following steps:

(1) preparing hog cholera virus antigen protein E2 gene and porcine pseudorabies virus antigen protein gB gene: designing a primer, and amplifying antigen protein genes of the classical swine fever virus and the pseudorabies virus of the pig by a PCR method.

(2) Constructing recombinant replication-defective chimpanzee adenovirus plasmid: : inserting the hog cholera virus antigen protein E2 gene and the porcine pseudorabies virus gB gene between a shuttle vector specific promoter and a terminator to construct a recombinant replication-defective chimpanzee adenovirus shuttle vector pShuttle-E2/gB containing fused E2 and gB genes; inserting the recombinant replication-defective chimpanzee adenovirus shuttle vector pShuttle-E2/gB into the replication-defective chimpanzee adenovirus vector by linear enzyme digestion to construct a recombinant replication-defective chimpanzee adenovirus plasmid;

(3) preparing a recombinant replication-defective chimpanzee adenovirus solution: transfecting HEK293 cells with replication-defective chimpanzee adenovirus plasmids to obtain recombinant adenoviruses with uniform genome structures, collecting diseased cells infected by the recombinant adenoviruses, freezing and storing the diseased cells together with supernate, and preparing the required virus liquid according to requirements.

The step (1) is prepared by adopting a primer amplification method, wherein the swine fever virus antigen protein E2 is

The upstream primer is 5'-TAGGCTTCTATCTAGTCTGCGCAATA-3', and the upstream primer is,

the downstream primer is 5'-GAATGAAGATTATGCTATGGTACGTAG-3'.

Preparation of porcine pseudorabies virus protein gB

The upstream primer is 5'-CCAGTCGCAGGCCACCGTGAGTG-3', the upstream primer is,

the downstream primer is 5'-ATCGCGTGCTCTTCATGGAGATC-3'.

The application of the swine fever and pseudorabies bivalent vaccine in preparing the medicines or the medicine boxes for preventing the swine fever and the pseudorabies viruses is disclosed.

In practice, the specific method for obtaining replication-defective chimpanzee adenovirus plasmids is as follows:

the obtained recombinant adenovirus shuttle vector pshuttle-E2/gB was mixed with the pAdC linear vector as the adenovirus backbone, using In-

The HD Cloning Kit performs the recombination reaction. Stbl2 competent cells were transformed, positive clones were identified, and then plasmids, pAdC-E2/gB, were upgraded.

The recombinant adenovirus plasmid is used for generating recombinant adenovirus with uniform genome structure, and the operation comprises the following operations:

1) enzyme cutting, the adenovirus plasmid is cut by Pac I,

2) transfecting, transfecting HEK293 cells,

3) the HEK293 cells were inoculated with the culture medium,

4) obtaining recombinant adenovirus with uniform genome structure, collecting virus infected pathological cells, and freezing and storing the cells together with supernatant.

The CSFV and PRV antigen protein gene amplification primers designed in the step (1) are,

the CSFV upstream primer is 5'-GAATCTGGTGGGTCCCTC-3' (containing XbaI restriction enzyme cutting site)

The downstream primer of CSFV is 5'-GTTACCCTCACCTCCTTGG-3' (containing KpnI restriction site)

The PRV gB upstream primer is 5'-CCAGTCCCAGGCCACCGTGAAGTG-3' (containing KpnI restriction site)

The PRV gB downstream primer is 5'-ATCGCCGTGCTCTTCAAGGAGAAC-3' (XbaI restriction site)

Wherein the CSFV E2 gene amplification length is 294bp, and the PRV gB gene amplification length is 632 bp.

In practice, in the process (2), the adenovirus vector promoter is CMV; the terminator is polyA;

the construction of the recombinant replication-defective chimpanzee adenovirus shuttle vector comprises the following operations:

e2 and gB construction were cloned into the pshuttle vector by molecular cloning methods to obtain plasmid pshuttle-E2/gB.

1) Carrying out double enzyme digestion on the recycled target gene E2 by XbaI and KpnI, and carrying out double enzyme digestion on the target gene gB by KpnI and XbaII to form a target gene with a sticky end;

2) meanwhile, the adenovirus shuttle vector pShuttle is subjected to double enzyme digestion by XbaII and KpnII to obtain a linearized vector;

3) performing sticky end ligation on the product by using T4 DNA ligase;

4) transforming the ligation product into Escherichia coli JM109 by conventional method, screening on agar plate containing kanamycin resistance, selecting clone, and extracting plasmid;

5) the positive recombinant replication-defective chimpanzee adenovirus shuttle vector pShuttle-E2/gB is obtained by adopting enzyme digestion and PCR amplification methods for identification.

Further, in the process (3), the process for obtaining replication-defective chimpanzee adenovirus plasmids comprises the following operations:

1) the recombinant replication-defective chimpanzee adenovirus shuttle vector pshuttle-E2/gB is subjected to single enzyme digestion by PacI enzyme to be linearized;

2) mixing pAdC with linearized recombinant shuttle vector, and using In-

The HD cloning kit is used for carrying out recombination reaction to obtain a recombinant adenovirus plasmid pAdC-E2/gB. Transforming Stbl2 competent cells by high voltage electricity;

3) resuscitating the cells in a culture solution after electrotransformation, selecting positive clones, extracting plasmid DNA in a small amount, performing electrophoretic mobility analysis by using 0.8% agarose gel, and performing restriction enzyme map analysis;

4) the obtained recombinant adenovirus plasmid is electrically transformed into host bacteria under the same conditions, so that a large amount of high-quality plasmids can be obtained in the host bacteria, and the target clone is selected.

Further, in the process (4), the operations of generating the recombinant adenovirus with a homogeneous genome structure using the recombinant adenovirus plasmid are specifically:

1) carrying out enzyme digestion by Pac I, and exposing an inverted terminal repetitive sequence ITR;

2) transfection, liposome-mediated transfection, and transfection by seeding HEK293 cells in tissue culture dishes, the procedure was as follows:

diluting Pac I enzyme digestion linearized recombinant adenovirus plasmid into a DMEM culture solution;

secondly, adding the liposome lipofectAMINE2000 suspension into a DMEM culture solution, fully mixing the liposome lipofectAMINE2000 suspension and the DMEM culture solution, and incubating at room temperature;

absorbing and discarding the cell stock culture solution during the period, and washing the cells once by using the DMEM culture solution;

adding the DNA/liposome compound into each plate, incubating in a 5% CO2 incubator, and then sucking away the culture solution containing the DNA/liposome compound;

fifthly, adding DMEM complete culture solution containing 10% calf serum, continuing to culture, collecting cells after lipofection, and repeating freeze thawing.

3) Inoculating, namely inoculating HEK293 cells to virus supernatant, culturing in a culture box by using complete DMEM culture solution containing 2% calf serum, and observing cytopathic CPE day by day until the HEK293 cells are completely diseased;

4) detecting that the E2 and gB antigen protein genes are integrated into an adenovirus vector, and detecting that the E2 and gB antigen protein genes are expressed, wherein the detection items are as follows:

the detection gene is integrated into an adenovirus vector, and PCR and restriction enzyme identification are adopted to carry out agarose gel electrophoresis analysis and detection.

② detecting, obtaining expression, wherein the method adopts a WB monitoring protein expression method.

The porcine pseudorabies virus antigen protein gB gene comes from a gene bank NO. KP710982, and the classical swine fever virus antigen protein E2 gene comes from a gene bank NO. DQ907718. The replication-deficient chimpanzee adenovirus vector is a modified chimpanzee adenovirus vector (pAdC) which is provided as a gift by Shanghai Pasteur, compared with a wild-type adenovirus AdC genome, deletes the coding region of a part E1 at position 458-3026 and the coding region of all E3 at position 27094-31799 of the wild-type adenovirus genome, and introduces I-CeuI and PI-Sce as insertion sites of a foreign gene at positions 2348 and 2380 of the vector sequence.

The specific construction method of the replication-defective chimpanzee adenovirus vector (pAdC) is as follows:

A. the origin sequence derived from pNEB193, the LITR fragment derived from AdC adenovirus genome and the NdeI-AgeI fragment derived from AdC adenovirus genome are fused into a fragment OLN by fusion PCR, and the fragment OLN is digested by Spe I and Age I; the AdC adenovirus genome is digested by Nde I, Age I and Spe I, and the digested fragments Age (4028) -speI (10610) are ligated with the digested fragment OLN to form plasmid pOIN.

B. The AdC genome was digested with Hind III and Avr II, and the excised target fragments Hind III (7153) -Avr II (23363) were inserted into the Hind III and Avr II sites of plasmid pOIN, and the resulting plasmid was named pOINH.

C. Amplifying a fragment positioned between 31800 and 36535 in an AdC adenovirus gene from an AdC adenovirus genome, introducing an Avr II and Rsr II enzyme cutting site at the 5 'end, introducing a Pac I and Asis I site at the 3' end, inserting the amplified fragment into the Avr II and Asis I site of pOINH, and obtaining a plasmid named as pOINHR;

D. amplifying a fragment positioned between 23165-27093 in an AdC adenovirus gene from an AdC adenovirus genome, introducing an Rsr II site at the 3' end, and inserting the amplified fragment into the Avr II and Rsr II sites of pOINHR to obtain the replication-defective recombinant adenovirus expression vector.

In practice, after the recombinant adenovirus is inoculated with 1 dose of 1 dose for 28 days, the stimulated antibody level of the anti-swine fever virus and the anti-pseudorabies virus is more obvious than that of the induced antibody level of commercial swine fever vaccines and pseudorabies vaccines of the market beasts; compared with commercial swine fever vaccines and pseudorabies vaccines of market animals, the recombinant adenovirus can generate antibodies 7 days after being inoculated with 1 agent with high dose, the antibody reaction is quicker, and piglets with reduced maternal antibodies in a 'blank window' period can be better protected; compared with the commercial swine fever vaccine of the market animal, which is inoculated at intervals of 21 days by 2 doses, the recombinant adenovirus only needs to be inoculated at a high dose by 1 dose, so that the economic value and the market value are extremely high; the recombinant adenovirus is inoculated with the 1 agent in high dose, the immunosuppressive reaction caused by the simultaneous immunization of the swine fever live virus vaccine and the porcine pseudorabies vaccine does not occur, the problem that the two vaccines cannot be immunized simultaneously in actual production can be solved, and the production value is extremely high.

Compared with the prior art, the invention has the following advantages and beneficial effects:

according to the invention, the specific swine fever virus antigen protein E2 gene and the swine pseudorabies virus antigen protein gB gene are inserted into the adenovirus vector, so that the immune interference phenomenon generated when the swine pseudorabies virus and the swine fever virus are simultaneously used as antigens can be avoided, the speed of producing the swine fever virus antibody is increased, the protection efficiency of an injection object after vaccine injection is improved, and the unexpected effect that an immune receptor can produce the swine fever antibody within 7 days of immunization is obtained.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:

FIG. 1 is a West blot diagram of recombined swine fever and pseudorabies combined vaccine infected cell sap.

FIG. 2 is a diagram of the lesion of HEK293 cells infected by the recombined swine fever and pseudorabies combined vaccine.

FIG. 3 is a body temperature curve diagram of the recombined swine fever and pseudorabies bigeminal vaccine piglet after challenge.

Fig. 4 is a temperature profile of piglets in the blank control group after challenge.

FIG. 5 is a comparison of tissue lesions (post-challenge tissue dissection monitoring of piglets).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.

Example 1

Preparation of vaccines

(one) preparation of the target Gene

Designing a primer, and amplifying antigen protein genes of the classical swine fever virus and the porcine pseudorabies virus by a PCR method; the designed amplification primers of the antigen protein genes in the first process are as follows:

CFSV E2 upstream primer 5'-TAGGCTTCTATCTAGTCTGCGCAATA-3' (containing XbaI cleavage site)

CFSV E2 downstream primer 5'-GAATGAAGATTATGCTATGGTACGTAG-3' (containing KpnI restriction site)

The PRV gB upstream primer is 5'-CCAGTCGCAGGCCACCGTGAGTG-3' (containing KpnI restriction site)

The PRV gB downstream primer is 5'-ATCGCGTGCTCTTCATGGAGATC-3' (XbaI restriction site)

(II) construction of recombinant replication-defective chimpanzee adenovirus shuttle vector

Inserting the antigen protein genes of the hog cholera virus and the porcine pseudorabies virus antigen protein between a specific promoter and a terminator of a replication-defective chimpanzee adenovirus shuttle vector to construct a recombinant replication-defective chimpanzee adenovirus shuttle vector pshuttle-E2/gB containing a target gene;

in the present process, it is preferred that,

the adenovirus vector promoter is CMV; the terminator is polyA;

the construction of the recombinant adenovirus shuttle vector comprises the following operations:

1. carrying out double enzyme digestion on the recovered target gene by I-Ceu I and PI-Sce to form a target gene with a sticky end;

2. meanwhile, carrying out double enzyme digestion on the adenovirus shuttle vector pshuttle through I-Ceu I and PI-Sce to obtain a linearized vector;

3. performing sticky end ligation on the product by using T4 DNA ligase;

4. transforming the ligation product into Escherichia coli by conventional method, screening on agar plate containing kanamycin resistance, selecting clone, and extracting plasmid;

5. the positive recombinant replication-defective chimpanzee adenovirus shuttle vector pshuttle-E2/gB is obtained by adopting enzyme digestion and PCR amplification methods for identification.

(III) obtaining replication-defective chimpanzee adenovirus plasmid

Carrying out homologous recombination between the obtained recombinant replication-defective chimpanzee adenovirus shuttle vector pshuttle-E2/gB and an adenovirus backbone vector pAdC in escherichia coli to obtain a recombinant adenovirus plasmid; in the third process, the process for obtaining the replication-defective chimpanzee adenovirus plasmid comprises the following operations:

further, the method for preparing a recombinant adenovirus (the combined live vaccine of the recombinant classical swine fever virus and the porcine pseudorabies virus) vaccine by expressing the antigenic protein genes of the classical swine fever virus and the porcine pseudorabies virus by using the adenovirus vector according to claim 1, which is characterized in that: in the process (4), the specific procedures of generating the recombinant adenovirus with uniform genome structure by using the recombinant adenovirus plasmid are as follows:

1. the recombinant shuttle vector pshuttle-E2/gB is singly cut by Pme I enzyme respectively for linearization;

2. mixing 0.lmg pAdC with 1.0mg of linearized recombinant shuttle vector, and transforming Escherichia coli through high-voltage electricity;

3. after electrotransformation, recovering the cells in LB culture solution at 37 ℃ for 30min, taking the cells, coating the cells into a culture plate containing 50mg/ml kanamycin, and culturing at 37 ℃ for 16-20 h;

4. selecting kanamycin-resistant clones, extracting plasmid DNA in small quantity, carrying out electrophoretic mobility analysis by using 0.8% agarose gel, and carrying out restriction enzyme map analysis;

5. the obtained recombinant adenovirus plasmid is electrically transformed into host bacteria under the same conditions, a large amount of high-quality plasmid can be obtained in the host bacteria, and the target clone is selected.

(IV) generating recombinant adenovirus with uniform genome structure by using recombinant adenovirus plasmid

1. Enzyme digestion: taking 4ug of each recombinant adenovirus plasmid extracted from host bacteria, carrying out enzyme digestion by Pac I, completely digesting to cut off plasmid components such as ori and kan resistance genes and expose inverted terminal repetitive sequence ITR;

2. transfection: liposome-mediated transfection HEK293 cells were seeded in 35mm tissue culture dishes for transfection. The liposome-mediated transfection comprises the following operations:

(1) diluting Pac I enzyme digestion linearized recombinant adenovirus plasmid into 250ul DMEM culture solution;

(2) then adding the liposome lipofectAMINE2000 suspension into 250ul of DMEM culture solution, fully and uniformly mixing the two, and incubating for 20min at room temperature;

(3) during this period, the cell stock culture solution was aspirated and the cells were washed once with DMEM;

(4) adding 500ul of DNA/liposome complex into each plate, incubating for 3-5 h at 37 ℃ in a 5% C02 incubator, then removing the culture solution containing the DNA/liposome complex,

(5) supplementing 3ml DMEM complete culture solution containing 10% calf serum, continuously culturing for 7 days, after lipofection, collecting cells, and repeatedly freezing and thawing for 3 times.

3. Inoculating, namely inoculating HEK293 cells to virus supernatant, culturing in complete DMEM culture solution containing 2% calf serum at 37 ℃ in a 5% C02 culture box, and observing cytopathic CPE day by day until the HEK293 cells are completely diseased;

4. freezing and storing, namely collecting virus-infected diseased cells, and freezing and storing the virus-infected diseased cells together with supernatant in a detection process five at-20 ℃: comprises PCR detection that E2 and gB antigen protein genes are integrated into an adenovirus vector and WB detection that E2 and gB antigen protein genes are expressed.

5. The detection E2 and gB antigen protein genes are integrated into an adenovirus vector, and are identified by PCR and restriction enzyme for agarose gel electrophoresis analysis. The obtained recombinant shuttle vector pshuttle-E2/gB containing the target gene is subjected to double enzyme digestion identification by I-Ceu I and PI-Sce, so that target fragments of about E2294 bp and g B632bp can be cut out. Meanwhile, the target fragments of the E2 and gB antigen protein genes with the same size can be amplified through PCR identification and can be judged through comparison with standard molecular weight.

The detection of E2 and gB antigen protein gene obtains expression, and adopts a protein expression method: the method comprises the step of observing cytopathic effect by using an inverted microscope, wherein the normal growth of control cells can be seen, and obvious cytopathic effect appears in transfected cells. Protein expression can be directly observed using WB assay. And (3) inoculating the qualified recombinant adenovirus (expressing hog cholera E2 protein and porcine pseudorabies g B protein) of the cell virus liquid to an injection animal for immunization.

Immunity efficacy experimental animal test of recombinant classical swine fever virus and rabies virus vaccine

1. Test animal screening

Selecting piglets of 3-4 weeks old.

2. Immunization

The test groups were immunized 2 times at 21 days intervals.

Firstly, the method avoids: 0d, 1ml of total injection;

and (2) avoiding: 21d, 1ml total injection.

3. Serum collection

7, 14, 21 after immunization; blood was collected 21 days after challenge.

4. Counteracting toxic substances

Immunization for 21 days with rabies virus challenge (PRV 2018 strain, 10)^5.5TCID₅₀Ml, proffered by professor of Sichuan Dayangxin), observed for 21 days.

5. Observation after attacking toxin

Body temperature: temperature measurement after toxic material attack

Organizing: after the challenge, all the survival test pigs are examined by a dissecting way, and the pathological changes and the antigen content of each organ are observed.

1.1 assay of recombinant rabies Virus (chimpanzee adenovirus vector) Virus content

By TCID₅₀The method determines the virus titer in each sample. The specific method comprises the following steps:

TCID₅₀(Tissue Culture infection Dose), typical cytopathic effect (CPE) due to replication infection of adenovirus in HEK293 cells, the extent of CPE reflects the virulence of the virus as observed by microscopy.

(1) HEK293 cells cultured in 96-well plates were plated 1 day in advance and incubated overnight in an incubator until the cells attached to form a monolayer.

(2) Continuously diluting the next day virus sample by 10 times gradient, adding 100ul of the diluted sample into each well, culturing in an incubator for 10 days, serially diluting the antigen solution by 10 times, and taking 10 times of the diluted antigen solution^-4、10^-5、10^-6Inoculating HEK293 cell monolayer 96-well cell culture plate with 3 dilutions, inoculating 100 μ l of HEK293 cell monolayer in each well, inoculating 6-well HEK293 cell monolayer in each dilution, simultaneously establishing normal cell and positive control, adsorbing for 1 hr, supplementing 100 μ l of cell-containing maintenance solution in each well, and standing at 37 deg.C with 5% CO₂The cultivation is continued in the incubator.

(3) The pathological changes of the cells were observed day by day, and according to the observation results, the infectious titer was calculated by the karber method, and the formula was simplified to T7 × 10^(S+0.5). The infection titer of the stock solution is not less than 5.0 multiplied by 10⁷PFU/ml。

1.2 analysis of immunogenicity and challenge protection of recombinant adenovirus (expressing classical Swine fever and pseudorabies proteins)

In the experiment, the number of healthy piglets with 21-day-old CSFV and PRV antigen and antibody is 8, and the piglets are randomly divided into 2 groups, 5 vaccine immunization groups and 3 control groups. Test group, inoculation is carried out by intramuscular injection at neck, the inoculation dose is 1.0 ml/head (containing 1 head), and the virus content is 10^6.0VP/ml, while group 1 was not inoculated as a blank. The immunization was repeated 21 days after the first immunization, and 1.0 ml/head (1 head) was injected intramuscularly in the neck. 14 days after the secondary immunization, 2.0 ml/head of PRV 2018 strain is inoculated to each piglet by nasal drip (10)^6.0TCID₅₀). Continuously observing for 21 days after toxin attackThe body temperature and clinical symptoms are monitored each time, the death number of piglets is recorded, all live pigs are killed by dissecting after 21 days, and the pathological changes of tissues are observed.

And (3) clinical observation: the body temperature is measured 1 time every day at regular time, the body temperature is measured 1 time every day after toxin counteracting, 21 days are continuously observed, and the whole body reactions of the test pigs, such as mental and appetite states, are observed at the same time. 21 days after challenge, all surviving pigs were examined by dissection to observe histopathology.

1.3 analysis of immunogenicity of recombinant adenoviruses (expressing classical Swine fever and pseudorabies proteins) at different doses

In the experiment, healthy piglets with 21-day-old CSFV and PRV negative antigens and antibodies are randomly divided into 4 groups, 3 groups (4 heads in each group) of vaccine immunization and 1 group (3 heads) of a control group. The experimental group is inoculated by adopting a neck intramuscular injection mode, and three different doses (the virus content is 10 from low to high in sequence)^6.0VP/ml、10^7.0VP/ml and 10^8.0VP/ml) 1.0 ml/head, immunization program 2 doses 21 days apart.

Observation of immunogenicity: before immunization and after immunization, blood was collected every 7 days for the detection of swine fever and pseudorabies antibodies.

And (3) clinical observation: the body temperature is measured for 1 time every day continuously for 14 days after immunization; after immunization, the general reactions of the pigs, such as mental and appetite states, are continuously observed for 21 days.

1.4 recombinant adenovirus (expressing classical Swine fever and Pseudorabies) immunosuppressive assay

In the experiment, 20 healthy piglets with 21-day-old CSFV and PRV antigens and antibodies which are negative are randomly divided into 4 groups, and each group has 5 piglets. The experimental group was inoculated by intramuscular injection into the neck at a dose of 1.0 ml/head (1 head part). Experimental group piglet immune recombinant adenovirus (expressing hog cholera and pseudorabies protein) according to the highest immune dose of 10^8.0VP/head immunized piglet; the control group comprises three groups, wherein the piglet of experiment 1 group is simultaneously immunized with the commercial swine fever live vaccine (batch No. 2019064) and the porcine pseudorabies live vaccine (batch No. 201901), the piglet of experiment 2 group is immunized with the commercial swine fever live vaccine, the piglet of experiment 3 group is immunized with the commercial porcine pseudorabies live vaccine, and the control group is immunized according to the commercial vaccine specification.

Observation of immunogenicity: before immunization and after immunization, blood was collected every 7 days for the detection of swine fever and pseudorabies antibodies. And (3) observing the difference between the swine fever antibody and the pseudorabies antibody of the piglet immunized by the recombinant adenovirus (expressing the swine fever and the pseudorabies protein) and the single swine fever vaccine or the pseudorabies vaccine, and analyzing whether the recombinant adenovirus (expressing the swine fever and the pseudorabies protein) can cause the reduction of the swine fever antibody or the pseudorabies antibody.

2. Results

2.1 construction of recombinant Swine fever and Pseudorabies Combined vaccine (chimpanzee adenovirus vector)

Inserting the hog cholera virus E2 sequence and the pseudorabies virus g B sequence into a non-replicative chimpanzee adenovirus vector to construct a recombinant adenovirus plasmid, transfecting HEK293 cells with the plasmid, and collecting virus-infected diseased cell culture solution to obtain the recombinant hog cholera and pseudorabies combined virus (chimpanzee adenovirus vector).

2. Results

2.1 construction of recombinant adenovirus (expressing classical Swine fever and Pseudorabies protein)

Inserting a hog cholera virus E2 sequence and a pseudorabies virus g B sequence into a non-replicative chimpanzee adenovirus vector to construct a recombinant adenovirus plasmid, transfecting HEK293 cells with the plasmid, and collecting virus-infected diseased cell culture solution to obtain the recombinant adenovirus (expressing hog cholera and pseudorabies proteins).

2.1.1 West blot analysis

The recombinant classical swine fever and pseudorabies combined virus (chimpanzee adenovirus vector) infected cell culture solution can be seen as the proteins of classical swine fever virus E2 and pseudorabies virus g B through West blot analysis. The results are shown in detail in FIG. 1.

2.1.2 analysis of Virus content

After the HEK293 cell is infected by the recombinant adenovirus (expressing the classical swine fever and the pseudorabies protein), the cell has obvious pathological changes

(CPE), infection by half Tissue (TCID)₅₀) Detecting that the content of the recombinant adenovirus is not less than 5.0 multiplied by 10^7.0PFU/ml. The result is shown in detail in fig. 2. FIG. 2 is a diagram of the lesion of HEK293 cells infected by the recombined swine fever and pseudorabies combined vaccine. In FIG. 2, A: plasmid-transfected HEK293 cells; b: HEK293 cell

2.2 analysis of immunogenicity and challenge protection of recombinant adenovirus (expressing classical Swine fever and pseudorabies proteins)

2.2.1 immunogenicity: antibody monitoring

Recombinant adenovirus (expressing classical swine fever and pseudorabies protein) at a minimum immune dose of 10^6.0After the VP/head part is used for immunizing piglets, blood is collected according to a plan, and the blood is analyzed by an IDEXX swine fever and pseudorabies antibody detection kit, so that the recombined bivalent vaccine can induce the piglets to generate the swine fever virus antibody and the pseudorabies virus antibody. The positive conversion rate of the pseudorabies virus antibody is 100% 21 days after the first immunization of the bivalent vaccine, and the positive conversion rate of the classical swine fever antibody and the pseudorabies virus antibody is 100% 7 days after the second immunization. The results are shown in Table 2.2.1-1.

TABLE 2.2.1-1 statistical Table of swine fever antibody and pseudorabies antibody after immunization of piglets

2.2.2 challenge protection: clinical symptom monitoring

Recombinant adenovirus (expressing classical swine fever and pseudorabies protein) at a minimum immune dose of 10^6.0The VP/head part is used for immunizing piglets, the toxin is attacked 35 days after the head-on immunization, the immunized group piglets do not have any adverse symptoms, and the control group piglets are high in body temperature and die. The results are shown in Table 2.2.2-1, FIG. 3 (body temperature curve after challenge of the recombinant swine fever and pseudorabies bigeminal vaccine piglet) and FIG. 4 (body temperature curve after challenge of the blank control group piglet).

TABLE 2.2.2-1 statistical Table of clinical symptoms, autopsy changes and protection rates of piglets after challenge

Note: "+" indicates a body temperature of 40.5 ℃ or higher for 3 days; or a decline in mental or appetite; or ataxia, water sampling, circling and other clinical symptoms; or thickening and engorgement of meninges; increased cerebrospinal fluid; or die. "-" means no such symptoms.

2.2.3 counteracting toxic protection: tissue lesions

Recombinant adenovirus (expressing classical swine fever and pseudorabies protein) at a minimum immune dose of 10^6.0VP/head immunization piglets, and the toxin is attacked 49 days after the first immunization. After the toxin is attacked, the autopsy is carried out, the immunized piglet does not have any lesion in the brain tissue, and the control piglet has congestion and bleeding in the brain tissue. The results are shown in detail in FIG. 5. The left is the immunization group and the right is the control group.

2.3 Observation of immunogenicity of recombinant adenovirus (expressing classical Swine fever and Pseudorabies proteins) at different doses

The recombinant adenovirus (expressing classical swine fever and pseudorabies protein) is prepared according to three different dosages of high, medium and low (the virus content is 10 in turn from low to high)^6.0VP/ml、10^7.0VP/ml and 10^8.0VP/ml) immunized piglets were collected as planned and analyzed by IDEXX swine fever and pseudorabies antibody detection kit.

The recombinant bivalent vaccine induces the piglets to generate the classical swine fever virus antibody and the pseudorabies virus antibody which have positive correlation with the immune dose. The antibody of the swine fever and the pseudorabies virus can be generated 7 days after the first dose of the high-dose group immunization, the antibody positive conversion rate of the swine fever and the pseudorabies virus is close to 100% 14 days after the immunization, and is obviously better than that of the low-dose and medium-dose groups after the immunization for 21 days, and the result is detailed in a table 2.3-1.

TABLE 2.3-1 statistical Table of hog cholera antibody and pseudorabies antibody after immunization of piglets

In conclusion, the immunization program of the recombinant adenovirus (expressing the classical swine fever and the pseudorabies protein) can be changed from 2 doses every 21 days into high-dose 1 dose immunization for preventing the classical swine fever virus and the pseudorabies virus.

2.4 Observation of immunosuppression by recombinant adenovirus (expressing classical Swine fever and Pseudorabies proteins)

Immune recombinant adenovirus (expressing classical swine fever and pseudorabies protein) according to the highest immune dose of 10^8.0VP/1 dose of immunized piglet; three groups of control groups are used, and 1 group of piglets is used for simultaneously immunizing commercial swine fever live vaccine and porcine pseudorabies live vaccine, and the swine fever live vaccine refers to commercial instructions for 2 timesThe immunization is carried out every 21 days. Experiment 2 groups of piglets immunized with commercial swine fever live vaccine, and 2 times of immunization with 21 days interval are carried out according to commercial instructions. Experiment 3 groups of piglet immunization commercial porcine pseudorabies live vaccines refer to commercial instructions, 1 dose immunization.

Blood is collected according to a plan after immunization, and is analyzed by an IDEXX swine fever and pseudorabies antibody detection kit, and the level of the swine fever virus antibody and the pseudorabies virus antibody generated by the piglets can be induced by the recombinant bivalent vaccine only by 1 dose of immunization. Compared with a control group 2 (immunization commercial swine fever live virus vaccine, 2 doses at an interval of 21 days) and a control group 3 (immunization commercial porcine pseudorabies live virus vaccine, 1 dose), the antibody generation level is higher, and the obvious difference is achieved; compared with the control group 1 (which is used for simultaneously immunizing commercial swine fever live vaccine and porcine pseudorabies live vaccine), the serum neutralizing antibody of both swine fever and pseudorabies is obviously higher than that of the control group 1. The results are shown in tables 2.2.4-1 and 2.2.4-2.

TABLE 2.2.4-1 statistical table of immunosuppression of recombinant adenovirus bivalent vaccine (hog cholera antibody blocking rate)

TABLE 2.2.4-2 statistical table of immunosuppression of recombinant adenovirus bivalent vaccine (pseudorabies antibody blocking rate)

As can be seen from the above table, the stimulated anti-swine fever virus antibodies and anti-pseudorabies virus antibodies were more significant than the induced antibody levels of the commercial swine fever vaccine and pseudorabies vaccine of the market animals after the high dose of 1 dose of recombinant adenovirus was inoculated for 28 days; compared with commercial swine fever vaccines and pseudorabies vaccines of market animals, the recombinant adenovirus high-dose 1-dose vaccine can generate antibodies 7 days after inoculation, the antibody reaction is quicker, and the piglets with reduced maternal antibodies in a 'blank window' period can be better protected; compared with the commercial swine fever vaccine of the market animal, which is inoculated at intervals of 21 days by 2 doses, the recombinant adenovirus only needs to be inoculated at a high dose by 1 dose, so that the economic value and the market value are extremely high; the recombinant adenovirus is inoculated with the 1 agent in high dose, the immunosuppressive reaction caused by the simultaneous immunization of the swine fever live virus vaccine and the porcine pseudorabies vaccine does not occur, the problem that the two vaccines cannot be immunized simultaneously in actual production can be solved, and the production value is extremely high.

In the present invention, the prior art is not described in detail.

The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

A sequence table:

DEFINITION：Classical swine fever virus strain hclv_1125envelope glycoprotein E2 gene,partial cds.

ACCESSION：DQ907718

VERSION：DQ907718.1

JOURNAL：Submitted(17-AUG-2006)Department of Inspection Technology Research,China Institute of Veterinary Drug Control,No.8Zhongguancun South Street,Beijing,Beijing 100081,People's Republic of China

Sequence 1：

1 cggctagcct gcaaggaaga ttacaggtac gcaatatcgt caaccgatga gatagggcta

61 cttggggccg gaggtctcac caccacctgg aaggaataca accacgattt gcaactgaat

121 gacgggaccg tcaaggccag ttgcgtggca ggttccttta aagtcacagc acttaatgtg

181 gtcagtagga ggtatttggc gtcattgcat aagaaggctt tacccacttc cgtgacattc

241 gagctcctgt tcgacgggac caacccatca actgaggaaa tgggagatga cttcaggtcc

301 gggctgtgcc cgtttgatac gagtcctgtt gttaagggaa agtacaatac gaccttgttg

361 aacggtagtg ctttctatct tgtctgccca atagggtgga cgggtgtcat agagtgcaca

421 gcagtgagcc caacaactct gaggacagaa gtggtaaaga ccttcaggag agacaagccc

481 tttccgcaca gaatggattg tgtgaccacc atagtggaaa atgaagattt attctattgt

541 aagttggggg gcaactggac atgtgtgaaa ggcgagccag tggtctacac agggggggta

601 gtaaaacaat gtagatggtg tggcttcgac ttcgatgggc ctgacggact cccgcattac

661 cccataggta agtgcatttt ggcaaatgag acaggttaca gaatagtaga ttcaacggac

721 tgtaacagag atggcgttgt aatcagcaca gaggggagtc atgagtgctt gatcggtaac

781 acgactgtca aggtgcatgc atcagatgaa agactgggcc ctatgccatg cagacctaaa

841 gagattgtct ctagtgctgg tcctgtaatg aaaacctcct gtacattcaa ctacacaaaa

901 actttgaaga acaggtacta tgagcccagg gacagctact tccagcaata tatgcttaag

961 ggtgagtatc agtactggtt tgacctggat gcgactgacc gccactcaga ttacttcgca

1021 gaatttgttg tcttggtggt ggtagcactg ttaggaggaa gatatgtcct gtggctgata

1081 gtgacctacg tagttctaac agaacaactc gccgctggtc tacca

Type：DNA

Length：1125

SequenceName：CFSV E2

DEFINITION：Suid herpesvirus 1 strain YY glycoprotein B gene,complete cds.

ACCESSION：KP710982

VERSION：KP710982.1

JOURNAL：Submitted(26-JAN-2015)Hunan Agricultural University,College of Veterinary Medicine,Renmin Road,Changsha,Hunan 410128,P.R.China Sequence 2：

1 atgcccgctg gtggcggtct ttggcgcggg ccccgcgggc atcggcccgg gcaccacggc

61 ggtgctggcc tcggacgtct ttggcctgct ccacaccacg ctgcagctgc gcggggcgcc

121 gtcgcgctag cgctgctgct gctggcgctc gccgcgaccc cgacgtgcgg cgcggcggcc

181 gtgacgcggg ccgcctcggc ctcgcccgcg cccgggacgg gcgccacccc agacggcttc

241 tccgcggagg agtccctcga ggagatcgac ggggccgtct cccccggccc ctcggacgcc

301 cccgacggcg agtacggcga cctggacgcg cgcacggccg tgcgcgcggc cgcgaccgag

361 cgggaccgct tctacgtctg cccgccgccg tccggctcca cggtggtgcg cctggagccc

421 gagcaggcct gccccgagta ctcgcagggg cgcaacttca cggaggggat cgccgtgctc

481 ttcaaggaga acatcgcccc gcacaagttc aaggcccaca tctactacaa gaacgtcatc

541 gtcacgaccg tgtggtccgg gagcacgtac gcggccatca cgaaccgctt cacggaccgc

601 gtgcccgtcc ccgtgcagga gatcacggac gtgatcgacc gccgcggcaa gtgcgtctcc

661 aaggccgagt acgtgcgcaa caaccacaag gtgaccgcct tcgaccgcga cgagaacccc

721 gtcgaggtgg acctgcgccc ctcgcgcctg aacgcgctcg gcacccgcgg ctggcacacc

781 accaacgaca cctacaccaa gatcggcgcc gcgggcttct accacacggg cacctccgtc

841 aactgcatcg tcgaggaggt ggaggcgcgc tccgtgtacc cctacgactc cttcgccctg

901 tccacggggg acatcgtgta catgtccccc ttctacggcc tgcgcgaggg ggcccacggg

961 gagcacatcg gctacgcgcc cgggcgcttc cagcaggtgg agcactacta ccccatcgac

1021 ctggactcgc gcctccgcgc ctccgagagc gtgacgcgca actttctgcg cacgccgcac

1081 ttcacggtgg cctgggactg ggcccccaag acgcggcgcg tgtgcagcct ggccaagtgg

1141 cgcgaggccg aggagatgat ccgcgacgag acgcgcgacg ggtccttccg cttcacgtcg

1201 cgggccctgg gcgcctcctt cgtcagcgac gtcacgcagc tcgacctgca gcgcgtgcac

1261 ctgggcgact gcgtcctccg cgaggcctcg gaggccatcg acgccatcta ccggcggcgc

1321 tacaacaaca cgcacgtgct ggccggcgac aagcccgagg tgtacctcgc ccgcgggggc

1381 ttcgtggtgg ccttccgccc gctgatctcg aacgagctgg cgcagctgta cgcgcgcgag

1441 ctcgagcgcc tcggcctcgc cggcgtcgtg ggccccgcgt cccccgcggc cgcccgtcgg

1501 gcccggcgct cccccggccc ggcggggacg cccgagccgc cggccgtcaa cggcacgggg

1561 cacctgcgca tcaccacggg ctcggccgag tttgcgcgcc tgcagttcac ctacgaccac

1621 atccaggcgc acgtgaacga catgctgagc cgcatcgcgg ccgcctggtg cgagctgcag

1681 aacaaggacc gcaccctgtg gggcgagatg tcgcgcctga accccagcgc cgtggccacg

1741 gccgcgctgg gccagcgcgt ctcggcgcgc atgctcggcg acgtgatggc catctcgcgg

1801 tgcgtggagg tgcgcggcgg cgtgtacgtg cagaactcca tgcgcgtgcc cggcgagcgc

1861 ggcacgtgct acagccgccc gctggtgacc ttcgagcaca acggcacggg cgtgatcgag

1921 ggccagctcg gcgacgacaa cgagctcctc atctcgcgcg acctcatcga gccctgcacc

1981 ggcaaccacc ggcgctactt taagctgggc ggtgggtacg tgtactacga ggactacagc

2041 tacgtgcgca tggtggaggt gcccgagacg atcagcacgc gggtgaccct gaacctgacg

2101 ctgctcgagg accgcgagtt cctgcccctc gaggtgtaca cgcgcgagga gctcgccgac

2161 acgggcctcc tggactacag cgagatccag cgccgcaacc agctgcacgc gctcaagttc

2221 tacgacattg accgcgtggt caaggtggac cacaacgtgg tgctgctgcg cggcatcgcc

2281 aacttcttcc agggcctcgg cgacgtgggc gccgccgtcg gcaaggtggt cctgggcgcc

2341 acgggggccg tgatctcggc cgtcggcggc atggtgtcct tcctgtccaa ccccttcggg

2401 gcgctcgcca tcgggctgct ggtgctggcc ggcctggtcg cggccttcct ggcctaccgg

2461 cacatctcgc gcctgcgcca caaccccatg aaggccctgt accccgtcac gacgaaggcg

2521 ctcaaggagg acggcgtcga agaggacgac gtggacgagg ccaagctgga ccaggcccgg

2581 gacatgatcc ggtacatgtc catcgtgtcg gccctcgagc agcaggagca caaggcgcgc

2641 aagaagaaca gcgggcccgc gctgctggcc agccgcgtcg gggcgatggc cacgcgccgc

2701 cggcactacc agcgcctcga gaacgaggac cccgacgccc tgtag

sequence listing

<110> adult-living pet Biotechnology Ltd

<120> swine fever and porcine pseudorabies bivalent vaccine, and preparation method and application thereof

<130> FA1531

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1125

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cggctagcct gcaaggaaga ttacaggtac gcaatatcgt caaccgatga gatagggcta 60

cttggggccg gaggtctcac caccacctgg aaggaataca accacgattt gcaactgaat 120

gacgggaccg tcaaggccag ttgcgtggca ggttccttta aagtcacagc acttaatgtg 180

gtcagtagga ggtatttggc gtcattgcat aagaaggctt tacccacttc cgtgacattc 240

gagctcctgt tcgacgggac caacccatca actgaggaaa tgggagatga cttcaggtcc 300

gggctgtgcc cgtttgatac gagtcctgtt gttaagggaa agtacaatac gaccttgttg 360

aacggtagtg ctttctatct tgtctgccca atagggtgga cgggtgtcat agagtgcaca 420

gcagtgagcc caacaactct gaggacagaa gtggtaaaga ccttcaggag agacaagccc 480

tttccgcaca gaatggattg tgtgaccacc atagtggaaa atgaagattt attctattgt 540

aagttggggg gcaactggac atgtgtgaaa ggcgagccag tggtctacac agggggggta 600

gtaaaacaat gtagatggtg tggcttcgac ttcgatgggc ctgacggact cccgcattac 660

cccataggta agtgcatttt ggcaaatgag acaggttaca gaatagtaga ttcaacggac 720

tgtaacagag atggcgttgt aatcagcaca gaggggagtc atgagtgctt gatcggtaac 780

acgactgtca aggtgcatgc atcagatgaa agactgggcc ctatgccatg cagacctaaa 840

gagattgtct ctagtgctgg tcctgtaatg aaaacctcct gtacattcaa ctacacaaaa 900

actttgaaga acaggtacta tgagcccagg gacagctact tccagcaata tatgcttaag 960

ggtgagtatc agtactggtt tgacctggat gcgactgacc gccactcaga ttacttcgca 1020

gaatttgttg tcttggtggt ggtagcactg ttaggaggaa gatatgtcct gtggctgata 1080

gtgacctacg tagttctaac agaacaactc gccgctggtc tacca 1125

<210> 2

<211> 2745

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgcccgctg gtggcggtct ttggcgcggg ccccgcgggc atcggcccgg gcaccacggc 60

ggtgctggcc tcggacgtct ttggcctgct ccacaccacg ctgcagctgc gcggggcgcc 120

gtcgcgctag cgctgctgct gctggcgctc gccgcgaccc cgacgtgcgg cgcggcggcc 180

gtgacgcggg ccgcctcggc ctcgcccgcg cccgggacgg gcgccacccc agacggcttc 240

tccgcggagg agtccctcga ggagatcgac ggggccgtct cccccggccc ctcggacgcc 300

cccgacggcg agtacggcga cctggacgcg cgcacggccg tgcgcgcggc cgcgaccgag 360

cgggaccgct tctacgtctg cccgccgccg tccggctcca cggtggtgcg cctggagccc 420

gagcaggcct gccccgagta ctcgcagggg cgcaacttca cggaggggat cgccgtgctc 480

ttcaaggaga acatcgcccc gcacaagttc aaggcccaca tctactacaa gaacgtcatc 540

gtcacgaccg tgtggtccgg gagcacgtac gcggccatca cgaaccgctt cacggaccgc 600

gtgcccgtcc ccgtgcagga gatcacggac gtgatcgacc gccgcggcaa gtgcgtctcc 660

aaggccgagt acgtgcgcaa caaccacaag gtgaccgcct tcgaccgcga cgagaacccc 720

gtcgaggtgg acctgcgccc ctcgcgcctg aacgcgctcg gcacccgcgg ctggcacacc 780

accaacgaca cctacaccaa gatcggcgcc gcgggcttct accacacggg cacctccgtc 840

aactgcatcg tcgaggaggt ggaggcgcgc tccgtgtacc cctacgactc cttcgccctg 900

tccacggggg acatcgtgta catgtccccc ttctacggcc tgcgcgaggg ggcccacggg 960

gagcacatcg gctacgcgcc cgggcgcttc cagcaggtgg agcactacta ccccatcgac 1020

ctggactcgc gcctccgcgc ctccgagagc gtgacgcgca actttctgcg cacgccgcac 1080

ttcacggtgg cctgggactg ggcccccaag acgcggcgcg tgtgcagcct ggccaagtgg 1140

cgcgaggccg aggagatgat ccgcgacgag acgcgcgacg ggtccttccg cttcacgtcg 1200

cgggccctgg gcgcctcctt cgtcagcgac gtcacgcagc tcgacctgca gcgcgtgcac 1260

ctgggcgact gcgtcctccg cgaggcctcg gaggccatcg acgccatcta ccggcggcgc 1320

tacaacaaca cgcacgtgct ggccggcgac aagcccgagg tgtacctcgc ccgcgggggc 1380

ttcgtggtgg ccttccgccc gctgatctcg aacgagctgg cgcagctgta cgcgcgcgag 1440

ctcgagcgcc tcggcctcgc cggcgtcgtg ggccccgcgt cccccgcggc cgcccgtcgg 1500

gcccggcgct cccccggccc ggcggggacg cccgagccgc cggccgtcaa cggcacgggg 1560

cacctgcgca tcaccacggg ctcggccgag tttgcgcgcc tgcagttcac ctacgaccac 1620

atccaggcgc acgtgaacga catgctgagc cgcatcgcgg ccgcctggtg cgagctgcag 1680

aacaaggacc gcaccctgtg gggcgagatg tcgcgcctga accccagcgc cgtggccacg 1740

gccgcgctgg gccagcgcgt ctcggcgcgc atgctcggcg acgtgatggc catctcgcgg 1800

tgcgtggagg tgcgcggcgg cgtgtacgtg cagaactcca tgcgcgtgcc cggcgagcgc 1860

ggcacgtgct acagccgccc gctggtgacc ttcgagcaca acggcacggg cgtgatcgag 1920

ggccagctcg gcgacgacaa cgagctcctc atctcgcgcg acctcatcga gccctgcacc 1980

ggcaaccacc ggcgctactt taagctgggc ggtgggtacg tgtactacga ggactacagc 2040

tacgtgcgca tggtggaggt gcccgagacg atcagcacgc gggtgaccct gaacctgacg 2100

ctgctcgagg accgcgagtt cctgcccctc gaggtgtaca cgcgcgagga gctcgccgac 2160

acgggcctcc tggactacag cgagatccag cgccgcaacc agctgcacgc gctcaagttc 2220

tacgacattg accgcgtggt caaggtggac cacaacgtgg tgctgctgcg cggcatcgcc 2280

aacttcttcc agggcctcgg cgacgtgggc gccgccgtcg gcaaggtggt cctgggcgcc 2340

acgggggccg tgatctcggc cgtcggcggc atggtgtcct tcctgtccaa ccccttcggg 2400

gcgctcgcca tcgggctgct ggtgctggcc ggcctggtcg cggccttcct ggcctaccgg 2460

cacatctcgc gcctgcgcca caaccccatg aaggccctgt accccgtcac gacgaaggcg 2520

ctcaaggagg acggcgtcga agaggacgac gtggacgagg ccaagctgga ccaggcccgg 2580

gacatgatcc ggtacatgtc catcgtgtcg gccctcgagc agcaggagca caaggcgcgc 2640

aagaagaaca gcgggcccgc gctgctggcc agccgcgtcg gggcgatggc cacgcgccgc 2700

cggcactacc agcgcctcga gaacgaggac cccgacgccc tgtag 2745

Claims

1. The swine fever and porcine pseudorabies bivalent vaccine is characterized by being prepared by inserting porcine pseudorabies virus gB antigen and hog cholera virus E2 antigen into a replication-defective chimpanzee adenovirus vector, wherein the gene sequence of the porcine pseudorabies virus gB antigen is SEQ ID No.1, and the gene sequence of the hog cholera virus E2 antigen is shown as SEQ ID No. 2.

2. The bivalent vaccine of swine fever and porcine pseudorabies according to claim 1, characterized by the content of 10^8.0VP per head.

3. The method for preparing the bivalent vaccine of swine fever and porcine pseudorabies according to claim 1, comprising the following steps:

(1) and preparing a target gene: designing a primer, and amplifying a classical swine fever virus antigen protein E2 gene and a porcine pseudorabies virus antigen protein gB gene by a PCR method;

(2) constructing recombinant replication-defective chimpanzee adenovirus plasmid: inserting the hog cholera virus antigen protein E2 gene and the porcine pseudorabies virus gB gene between a shuttle vector specific promoter and a terminator to construct a recombinant replication-defective chimpanzee adenovirus shuttle vector pShuttle-E2/gB containing fused E2 and gB genes; inserting the recombinant replication-defective chimpanzee adenovirus shuttle vector pShuttle-E2/gB into the replication-defective chimpanzee adenovirus vector by linear enzyme digestion to construct replication-defective chimpanzee adenovirus plasmids;

4. The method for preparing the bivalent vaccine of classical swine fever and porcine pseudorabies according to claim 1, wherein the step (1) is performed by primer amplification, wherein the primer for the swine fever virus antigen protein E2 is 5'-TAGGCTTCTATCTAGTCTGCGCAATA-3',

the downstream primer is 5'-GAATGAAGATTATGCTATGGTACGTAG-3'.

5. The method for preparing the bivalent vaccine of swine fever and porcine pseudorabies according to claim 3, wherein the upstream primer of the porcine pseudorabies virus gB is 5'-CCAGTCGCAGGCCACCGTGAGTG-3',

the downstream primer is 5'-ATCGCGTGCTCTTCATGGAGATC-3'.

6. The bivalent vaccine of swine fever and porcine pseudorabies according to claim 4, wherein the promoter of the shuttle vector in step (2) is CMV and the terminator is polyA.

7. Use of the bivalent vaccine of swine fever and pseudorabies according to claim 1 for the preparation of a medicament or kit for the prevention of swine fever and pseudorabies.