Recombinant vector and recombinant virus for preparing swine triple vaccine and application of recombinant vector and recombinant virus

Abstract

The invention belongs to the field of biological agriculture, and discloses a recombinant vector for preparing a swine triple vaccine, a recombinant virus and application of the recombinant vector and the recombinant virus. The technical problem to be solved by the invention is how to prepare the triple vaccine of porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and hog cholera virus. The recombinant vector for preparing the swine triple vaccine disclosed by the invention is a vector which is obtained by inserting an encoding gene of E2-Cap2 fusion protein into a recombinant vector capable of rescuing porcine reproductive and respiratory syndrome virus and enabling the encoding gene of the E2-Cap2 fusion protein to be expressed, wherein the E2-Cap2 fusion protein is a protein with an amino acid sequence of SEQ ID No.3. Experiments prove that the porcine triple vaccine obtained by using the recombinant vector has good immunogenicity, and can be used for simultaneously preventing diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and hog cholera virus.

Description

Technical Field

[0001] The invention belongs to the field of biological agriculture, and specifically relates to a recombinant vector and a recombinant virus for preparing a swine triple vaccine and applications thereof. Background Art

[0002] Porcine circovirus type 2 (PCV2) belongs to the genus Circovirus of the family Circoviridae and is the most important pathogen of porcine circovirus-related diseases. PCV2 infection is distributed globally. PCV2 invades the lymphatic system of pigs, leading to immunosuppression, which in turn leads to viral infectious diseases such as blue ear disease and swine fever, and even death, aggravating the epidemic and causing huge economic losses to the pig industry.

[0003] Porcine reproductive and respiratory syndrome (also known as blue ear disease) is mainly caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV can cause reproductive disorders in pregnant sows and respiratory diseases in pigs at all stages. It is a highly contact-transmitted pathogen with huge economic harm. Infection with its highly pathogenic strain PRRSV can induce acute lung injury, manifested as pulmonary hemorrhage, pulmonary edema and a large number of inflammatory cell infiltrations, which is one of the important reasons for high mortality. Blue ear disease can cause sow abortion, premature birth, stillbirth, mummified fetuses, and an increase in the proportion of weak piglets, delayed estrus in sows, increased return of estrus rate, and poor semen quality in boars; pigs at all stages have varying degrees of fever, loss of appetite, and respiratory problems; the proportion of secondary infections with bacterial diseases such as Haemophilus parasuis, Streptococcus suis, and porcine contagious pleuropneumonia increases. Blue ear disease has caused significant economic losses to the pig industry.

[0004] Classical swine fever (CSF) is a major infectious disease caused by classical swine fever virus (CSFV) that seriously harms the pig industry. It is a notifiable disease specified by the World Organization for Animal Health (OIE). The disease is characterized by rapid onset, persistent high fever, systemic hemorrhage and leukopenia, with high morbidity and mortality. Pigs (including domestic pigs and wild boars) are the only susceptible hosts of CSFV, and classical swine fever is also a major obstacle to the development of the global pig industry.

[0005] Before the outbreak of ASF, blue ear disease was the number one killer in the pig farming industry. However, in the post-AFS era, the status of blue ear disease has not changed. Blue ear attenuated vaccine is very effective in reducing the severity of clinical symptoms, the duration of viremia, and the frequency of virus spread and wild virus infection. Therefore, it is very necessary to choose a blue ear attenuated vaccine that is safe and has good immunogenicity during the immunization process of other viruses (such as PCV2 and CSFV) in pig farms. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to prepare a triple vaccine of porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and classical swine fever virus.

[0007] In order to solve the above technical problems, the present invention first provides a recombinant vector, wherein the recombinant vector is a vector for inserting a gene encoding an E2-Cap2 fusion protein into a recombinant vector capable of rescuing porcine reproductive and respiratory syndrome virus and expressing the gene encoding the E2-Cap2 fusion protein;

[0008] The E2-Cap2 fusion protein is as follows A1) or A2):

[0009] A1) a protein whose amino acid sequence is SEQ ID No. 3;

[0010] A2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1).

[0011] In the above recombinant vector, the sequence of the gene encoding the E2-Cap2 fusion protein may be positions 1055-2767 of SEQ ID No.2.

[0012] The above recombinant vector is a circular double-stranded DNA consisting of 20224 bp, the nucleotide sequence of positions 1 to 15000 of one chain is SEQ ID No.1, and the nucleotide sequence of positions 15001 to 20224 is SEQ ID No.2.

[0013] The present invention also provides a recombinant virus, wherein the recombinant virus is a virus prepared using the recombinant vector.

[0014] The present invention also provides a swine triple vaccine, which contains the inactivated recombinant virus.

[0015] The above-mentioned swine triple vaccine may also contain an adjuvant.

[0016] In one embodiment of the present invention, the adjuvant is a water adjuvant. 605 water adjuvant: 3.5g disodium hydrogen phosphate, 0.6g potassium dihydrogen phosphate, 7g sodium chloride, 7g trehalose, 7g astragalus polysaccharide, 3g sodium glutamate, 13g gelatin, 7g polyethylene glycol 3000, 3g dextran, 1000ml water for injection, mixed, ultrasonically treated, and sterilized at 121°C for 20min.

[0017] Specifically, the swine triple vaccine can be obtained by mixing the recombinant virus with the adjuvant.

[0018] The use of the recombinant vector in the preparation of a product for preventing diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and / or classical swine fever virus also falls within the scope of protection of the present invention. The product may be a vaccine.

[0019] The use of the recombinant vector in the preparation of porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and / or classical swine fever virus vaccines also falls within the protection scope of the present invention.

[0020] The use of the recombinant virus in the preparation of a product for preventing diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and / or classical swine fever virus also falls within the scope of protection of the present invention. The product may be a vaccine.

[0021] The use of the trivalent swine vaccine in the preparation of products for preventing diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and / or classical swine fever virus also falls within the scope of protection of the present invention.

[0022] Experiments have shown that the recombinant vector and the trivalent vaccine of the present invention have good safety, no allergy, redness, swelling and ulceration after vaccination, and overdose vaccination has no effect on its growth performance; antibody detection shows that the antibody level of the trivalent vaccine is equivalent to that of the commercial porcine blue ear and circovirus vaccines for pigs; monitoring of swine fever antibodies shows that the antibody blocking rate of the recombinant virus after the first immunization is 18.28% on the 14th day, and the commercial swine fever virus vaccine reaches 19.98%, and the second immunization reaches 88.91% on the 21st day, and the commercial swine fever virus vaccine is 85.66%. The results show that the recombinant virus rjxPRRSE2&Cap fused with the genes of swine fever virus E2 and porcine circovirus type 2 ORF2 has good immunogenicity and can be used to prevent diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and swine fever virus at the same time. At the same time, the inactivated vaccine prepared by the trivalent vaccine of the present invention according to the content of the commercial vaccine and 605 adjuvant has the advantages of low viscosity of the water adjuvant, easy injection, good absorption, and one shot for multiple protection. The recombinant vector and trivalent vaccine of the present invention have a good application prospect.

[0023] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 .Schematic diagram of RRSVscfvE2&PCV2 ORF2 plasmid. Among them, RRSVscfvE2&PCV2 ORF2 clone represents RRSVscfvE2&PCV2 ORF2 plasmid.

[0025] Figure 2.rjxPRRSE2&Cap strain cytopathic effect. The left picture shows healthy Marc-145 cells, and the right picture shows rescued virus cytopathic effect.

[0026] Figure 3 .RT-PCR identification and sequencing results of each generation of rescued viruses. Lane MK: Marker DL5000; Lane 1: normal Marc-145 cells; Lane 2: F1 generation virus; Lane 3: F 15 Generation virus.

[0027] Figure 4 . Agar amplification titer determination of E2 protein and Cap protein of different generations. The left picture shows the agar amplification results of CSFV E2 protein of different generations; the right picture shows the agar amplification results of PCV2 Cap protein of different generations. DETAILED DESCRIPTION

[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0029] Example 1. Preparation and efficacy testing of swine triple vaccine rjxPRRSE2&Cap

[0030] 1 Materials and methods

[0031] 1.1 Main Reagents

[0032] The JX vaccine PRRS genome plasmid was prepared by the inventors; GXL Premix Fast, Onestep RT-PCR KitExTaqtm enzyme is a product of Takara; restriction endonuclease is a product of Neb; competent cells DH5a, DNA recombination kit, DNA recovery kit, plasmid extraction kit, Lipofectamine TM 2000 transfection kit is a product of Quanshijin Company.

[0033] 1.2 Rescue and identification of recombinant viruses

[0034] 1.2.1 Preparation of recombinant plasmid

[0035] Using primer sequences, the JX vaccine PRRSV genome vector was amplified with F1 / R1, and the synthetic E2-Cap fusion gene vector was amplified with F2 / R2. The primers are as follows:

[0036] JX vaccine PRRSV plasmid fragment amplification primers (18255bp):

[0037] F1: 5′-GGGGCCGGTGGTGACTAATGGGCTGGCATTCTTTGGCACC-3′ (SEQ ID No. 4);

[0038] R1: 5′-TGTTCCGCTGAAACTCTGGTTAAAGGGGTTGCCGCGGAACTCATGCTGAGGGTGATGCT-3′ (SEQ ID No. 5);

[0039] E2-Cap2 gene amplification primers (1755 bp):

[0040] F2: 5′-AGTTTCAGCGGAACAATGGCATTCATGGGCAAGGTGCTG-3′ (SEQ ID No. 6);

[0041] R2: 5′-GTCACCACCGGCCCCTTACTTAGGGTTAAGTGGGGGGTC-3′ (SEQ ID No. 7).

[0042] After the PCR product was purified and recovered, the E2-Cap2 gene PCR fragment and the vector (JX vaccine PRRSV plasmid fragment) PCR fragment were connected with homologous recombinase, and the connection product was transformed into DH5a competent cells, and the positive single clone was sent to Shanghai Shenggong Biotechnology Technology Service Co., Ltd. for sequencing. The recombinant plasmid with the correct sequence was screened and named RRSVscfvE2&PCV2ORF2 plasmid. The schematic diagram of RRSVscfvE2&PCV2 ORF2 plasmid is shown in Figure 1 As shown, the RRSVscfvE2&PCV2 ORF2 plasmid is a circular double-stranded DNA consisting of 20224 bp, the nucleotide sequence of positions 1 to 15000 of one strand is SEQ ID No. 1, and the nucleotide sequence of positions 15001 to 20224 is SEQ ID No. 2). The RRSVscfvE2&PCV2 ORF2 plasmid can also be directly synthesized based on its sequence.

[0043] In SEQ ID No. 2, positions 1055-2767 represent the E2-Cap2 gene (i.e., the E2 fused to the Cap2 gene), encoding the E2-Cap2 protein shown in SEQ ID No. 3. In SEQ ID No. 3, positions 1-361 represent the E2 protein, positions 362-373 represent the GS connecting peptide, and positions 374-571 represent the Cap2 protein.

[0044] 1.2.2 Rescue and identification of recombinant viruses

[0045] When Marc-145 cells grow to 70% to 90% in a 6-well plate, wash the cell surface with OPTI-MEMI medium (i.e., Opti-MEM reduced serum medium, Thermo Fisher Scientific, catalog number 31985070), and transfect Marc-145 cells with 4 μg of the RRSVscfvE2 & PCV2 ORF2 plasmid obtained in step 1.2.1 under the mediation of liposome Lipfection2000:

[0046] First, add 4 μg of plasmid to 250 μl Opti-MEM medium, then dilute 12 μl of liposome 2000 with a certain amount of serum-free medium, incubate at room temperature for 10 minutes, gently mix the diluted plasmid and diluted liposome 2000 (total volume 500 μl), incubate at room temperature for 20 minutes, and add to Marc-145 cells that have been washed with OPTI-MEM medium. At the same time, set up liposome transfection control and normal cell control, place in a 5% CO2 incubator, incubate at 37°C for 4 hours, and then replace the medium with DMEM medium containing 10% serum. Continue to culture for about 48 hours and harvest the virus. After repeated freezing and thawing of the culture for 3 times, inoculate new Marc-145 cells until the virus can stably produce cytopathic effect, CPE phenomenon appears, the cells become round, and are distributed in grape clusters. Finally, the cells disintegrate into fragments, as seen. Figure 2 The rescued recombinant virus was named rjxPRRSE2&Cap and propagated on Marc-145 cells for multiple generations. The time for the onset of cytopathic effects was significantly shortened, and the lesions were more typical, so the next step of the experiment was carried out.

[0047] 1.3 Identification of biological characteristics of rescued viruses

[0048] The stable adapted virus rjxPRRSE2&Cap was diluted 10-fold in MEM medium, and each dilution was inoculated into 96-well Marc-145 cells, 0.1 ml / well, and cultured at 37°C for 48-72 hours. TCID was calculated according to the Reed-Muench method based on the number of cytopathic wells. 50 The results showed that the first and second generation toxins had slightly lower titers, 10 3.0TCID 50 / ml, 10 6.0 TCID 50 / ml, and the titer of the recombinant virus reached 10 after the third generation 8.0 TCID 50 / ml.

[0049] 1.4 Genetic stability

[0050] After the virus adapted to Marc-145 cells, it was continuously passaged to the 15th generation, and F1 and F 15 RT-PCR was performed with primers F2 / R2 for each generation. The PCR products were recovered from gel and sent for sequencing. The genetic stability was determined based on the homology of the E2-Cap2 gene sequences of each generation.

[0051] The target gene band of about 1.7 kb was successfully amplified from the RNA product digested with DNase using primers F2 / R2 ( Figure 3 ), the sequence determination showed that the E2-Cap gene fragment sequence was contained between the N protein gene and the 3UTR. Sequencing of different generations of viruses showed that the inserted fragment was in F 15 The homology of the spacer region between E2 and Cap is 100%.

[0052] 1.5 Comparison of virus particle yield

[0053] The stable viruses were cultured in bioreactors to prepare virus liquids. The E2 and Cap contents in different batches of virus liquids were detected according to the PRRS virus inactivated antigen method, and the production efficiency and stability of each generation of viruses were compared.

[0054] The 3rd, 5th, 10th and 15th generation viruses (respectively denoted as F3, F5, F 10 and F 15 ) The titer of the recombinant virus was determined on Marc-145 cells, and the results showed that the titer of the recombinant virus at different generations reached 10 8.0 The 15th generation virus titer is 10 8.0 TCID 50 / ml, but the agar titers of target proteins E2 and Cap became weaker and weaker, as shown in Table 2 and Figure 4 This may be related to the instability of the recombinant blue ear virus, which leads to the loss of the target gene. In addition, according to the electron microscopy results of the virus, F 10 The new virus can stably express and assemble Cap's Vlps.

[0055] Table 1. Virus titer determination at different generations (TCID 50 / ml)

[0056] strain <![CDATA[F3]]> <![CDATA[F5]]> <![CDATA[F 10 ]]> <![CDATA[F 15 ]]> rjxPRRSE2&Cap <![CDATA[10 8.0 ]]> <![CDATA[10 8.0 ]]> <![CDATA[10 8.33 ]]> <![CDATA[10 8.0 ]]>

[0057] Table 2. Agar amplification titer determination of E2 protein and Cap protein at different generations

[0058] protein <![CDATA[F3]]> <![CDATA[F5]]> <![CDATA[F 10 ]]> <![CDATA[F 15 ]]> E2 1∶32 1∶32 1∶32 1∶8 Cap 1∶32 1∶32 1∶32 1∶4

[0059] 1.6 Preparation of Trivalent Vaccine

[0060] rjxPRRSE2&Cap was inoculated into dense monolayer Marc-145 cells at a 5% inoculation rate and cultured at 37°C for 48 hours. The virus culture was harvested and inactivated by BEI method. The virus dilution (diluted with MEM medium, the virus content before inactivation was 10 9.0 TCID 50 / ml) was mixed with 605 water adjuvant in a ratio of 1:1 to prepare the rjxPRRSE2&Cap triple vaccine.

[0061] 605 water adjuvant: 3.5g disodium hydrogen phosphate, 0.6g potassium dihydrogen phosphate, 7g sodium chloride, 7g trehalose, 7g astragalus polysaccharide, 3g sodium glutamate, 13g gelatin, 7g polyethylene glycol 3000, 3g dextran, and 1000ml water for injection are mixed, treated with ultrasound, and sterilized at 121°C and high pressure for 20min.

[0062] The rjxPRRSE2&Cap triple vaccine, blue ear virus live vaccine (Blue Ear Vaccine produced by Beijing Huaxia Xingyang Biotechnology Co., Ltd., denoted as commercial vaccine A), porcine circovirus type 2 genetically engineered subunit circovirus vaccine produced by Beijing Huaxia Xingyang Biotechnology Co., Ltd. (denoted as commercial vaccine B), and classical swine fever virus live vaccine Webeikang produced by Beijing Huaxia Xingyang Biotechnology Co., Ltd. (denoted as commercial vaccine C) were tested for safety and efficacy.

[0063] 1.7 Safety Inspection

[0064] Twenty-five healthy piglets at 14 days of age were selected, and 5 piglets (Duroc×(Large White×Landrace)) were immunized by intramuscular injection of rjxPRRSE2&Cap triple vaccine, commercial vaccine A, commercial vaccine B, and commercial vaccine C, respectively, 4 mL / head, and 5 piglets were kept as blank controls and isolated under the same conditions. The piglets were observed for 14 consecutive days, and the spirit, diet, and activity performance of the piglets were recorded. The injection site was observed for redness, swelling, and ulceration. Thirty days after immunization, all experimental pigs were weighed, and the weight gain differences between the rjxPRRSE2&Cap triple vaccine group, commercial vaccine group A, commercial vaccine group B, and commercial vaccine group C and the blank control group were analyzed.

[0065] Clinical manifestations: After the rjxPRRSE2&Cap triple vaccine and the commercial vaccines were overdosed, the piglets did not show vomiting symptoms, their spirit and appetite were normal, and there was no redness, swelling or ulceration at the injection site. 24 hours after immunization, each vaccine group showed obvious temperature reaction, the triple vaccine group had a maximum temperature increase of 0.9℃, the commercial vaccine group A had a maximum temperature increase of 0.8℃, the commercial vaccine group B had a maximum temperature increase of 0.6℃, and the commercial vaccine group C had a maximum temperature increase of 0.8℃. All test groups returned to normal temperature range within 48 hours.

[0066] 1.8 Efficacy test

[0067] 1.8.1 Immunization: 25 healthy piglets (Duroc×(Large White×Landrace)) aged 14 weeks were randomly divided into 5 groups, namely, the triple vaccine group, commercial vaccine group A, commercial vaccine group B, commercial vaccine group C, and control group, 5 pigs in each group. Each group was vaccinated with rjxPRRSE2&Cap triple vaccine, commercial vaccine A, commercial vaccine B and commercial vaccine C through neck muscle injection, 2mL / head, and the control group was injected with normal saline, 2mL / head. The piglets were isolated and fed under the same conditions. 21 days after the first vaccination, the second vaccination was carried out according to the same method and dosage.

[0068] 1.8.2 Blood collection:

[0069] 1.8.2.1 Blood collection from the blue ear group: 28 days after the second vaccination, blood was collected from the triple vaccine group, commercial vaccine group A and control group at the same time.

[0070] 1.8.2.2 Blood collection for porcine circovirus type 2 group: 21 days after the second vaccination, blood was collected simultaneously for the triple vaccine group, commercial vaccine group B and control group.

[0071] 1.8.2.3 Blood collection from swine fever group: 14-week-old healthy susceptible piglets were immunized with the triple vaccine and commercial vaccine C, and blood samples were collected from the triple vaccine group, commercial vaccine C group and control group before immunization, 14 days after the first immunization, 21 days after the first immunization, 7 days after the second immunization, and 21 days after the second immunization.

[0072] 1.8.3 Antibody detection: Serum was separated from all blood samples, labeled by group, and tested for antibodies using Elisa kits.

[0073] Detection of porcine blue ear antibody: The rjxPRRSE2&Cap triple vaccine, commercial vaccine A and control serum samples were tested using the IDEXX antibody kit (REF99-0016411, LOTSN:GU902). The results are shown in Table 3. The samples of the triple vaccine and commercial vaccine A were all positive, with similar S / P values, both much higher than the control group where all samples were negative. The results showed that the porcine blue ear antigen in the recombinant virus rjxPRRSE2&Cap has good immunogenicity and can be used to prevent porcine reproductive and respiratory syndrome.

[0074] Table 3. Results of blue ear antibody test

[0075]

[0076] Porcine circovirus antibody detection: The serum samples of rjxPRRSE2&Cap triple vaccine, commercial vaccine B and control group were tested and the titers were calculated using the Ingena antibody kit (Cat. No.: PCV.K002 / 2). The results are shown in Table 4. The serum samples of the triple vaccine and commercial vaccine B were all positive, with titers above 700 and similar average values. The control group was negative, with titers not higher than 130. The results showed that the Cap antigen in the recombinant virus rjxPRRSE2&Cap has good immunogenicity, and the recombinant virus rjxPRRSE2&Cap can be used as a vaccine candidate for the prevention of porcine circovirus disease.

[0077] Table 4. Results of porcine circovirus antibody test

[0078]

[0079] Classical swine fever antibody detection: The IDEXX antibody kit (lot number: REF99-40939, LOTSN: ELISA) was used to detect the rjxPRRSE2&Cap trivalent vaccine, commercial vaccine C and control group serum samples and calculate the blocking rate. The results are shown in Table 5. Antibodies began to be produced in pigs 14 days after the first vaccination of the trivalent vaccine. The antibody blocking rate of the trivalent vaccine reached 18.28-61.45% from 14 to 21 days after the first vaccination, and the commercial vaccine C maintained at 19.98-71.84%. The antibody blocking rate of the trivalent vaccine reached 82.34-88.91% from 14 to 21 days after the second vaccination, and the antibody blocking rate of the commercial vaccine C was maintained at 80.05-88.66%. Moreover, the antibody blocking rates of the two groups exceeded 75% 14 days after the second vaccination, reaching the classical swine fever challenge antibody blocking protection value. The results showed that the E2 antigen in the recombinant virus rjxPRRSE2&Cap has good immunogenicity, and the recombinant virus rjxPRRSE2&Cap can be used as a vaccine candidate strain for the prevention of classical swine fever.

[0080] Table 5. Results of swine fever antibody detection (blocking rate) (%)

[0081] Group Before immunization 14 days after the first free 21 days after the first free 14 days after the second exemption 21 days after the second exemption Triple vaccine group 8.74％ 18.28％ 61.45％ 82.34％ 88.91％ Commercial seedlings group C 2.83％ 19.98％ 71.84％ 80.05％ 88.66％ Control group 10.66％ 8.61％ 9％ 0.17％ 1.96％

[0082] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A recombinant vector, characterized in that: The recombinant vector is a vector in which the coding gene of the E2-Cap2 fusion protein is inserted into the recombinant vector capable of rescuing porcine reproductive and respiratory syndrome virus and the coding gene of the E2-Cap2 fusion protein is expressed; The E2-Cap2 fusion protein is as follows A1) or A2): A1) a protein whose amino acid sequence is SEQ ID No. 3; A2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1).

2. The recombinant vector according to claim 1, characterized in that: The sequence of the gene encoding the E2-Cap2 fusion protein is positions 1055-2767 of SEQ ID No.

2.

3. The recombinant vector according to claim 1 or 2, characterized in that: The recombinant vector is a circular double-stranded DNA consisting of 20224 bp, wherein the nucleotide sequence of positions 1 to 15000 of one chain is SEQ ID No. 1, and the nucleotide sequence of positions 15001 to 20224 is SEQ ID No.

2.

4. A recombinant virus, characterized in that: The recombinant virus is a virus prepared using the recombinant vector described in any one of claims 1-3.

5. A swine triple vaccine, characterized in that: The swine triple vaccine contains the inactivated recombinant virus of claim 4.

6. The swine triple vaccine according to claim 5, characterized in that: The swine triple vaccine also contains an adjuvant.

7. Use of the recombinant vector described in any one of claims 1 to 3 in the preparation of products for preventing diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and / or classical swine fever virus.

8. Use of the recombinant vector according to any one of claims 1 to 3 in the preparation of vaccines against porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and / or classical swine fever virus.

9. Use of the recombinant virus according to claim 4 in the preparation of products for preventing diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and / or classical swine fever virus.

10. Use of the swine triple vaccine according to claim 5 or 6 in the preparation of products for preventing diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and / or classical swine fever virus.

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