Recombinant vector, recombinant virus and application thereof for preparing swine triple vaccine
By preparing recombinant vectors and viruses containing E2-Cap2 fusion protein and combining them with water adjuvant 605, a safe and effective swine trivalent vaccine was successfully prepared, which solved the problem in the existing technology that it was difficult to simultaneously prevent porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and classical swine fever virus, and achieved good immune effects.
Patent Information
- Application Number
- CN202510062157.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-09-19
- Estimated Expiration
- 2045-01-15
AI Technical Summary
It is difficult with existing technologies to effectively prepare vaccines that can simultaneously prevent porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, and classical swine fever virus, and existing blue ear attenuated vaccines have deficiencies in the duration of viremia and the frequency of wild virus infection.
A recombinant vector is provided, into which the coding gene of the E2-Cap2 fusion protein is inserted and expressed to prepare a recombinant virus. The inactivated recombinant virus is combined with an adjuvant to prepare a swine triple vaccine, including water adjuvant 605, for preventing diseases caused by the above three viruses.
The recombinant vector and porcine trivalent vaccine showed good safety and immunogenicity, could effectively prevent three viruses, had no allergic reaction after immunization, and the antibody level was comparable to that of commercially available vaccines. It had the advantages of easy injection, good absorption, and multiple protections with one shot.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biological agriculture, and in particular 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), belonging to the genus Circovirus in the family Circoviridae, is the primary pathogen of porcine circovirus-associated diseases. PCV2 infection is globally distributed. PCV2 attacks the pig's lymphatic system, leading to immunosuppression, which can lead to secondary viral infections such as blue ear disease (PRRS) and swine fever, and even death, exacerbating epidemics and causing significant economic losses to the pig industry.
[0003] Porcine reproductive and respiratory syndrome (PRRS) (also known as blue ear disease) is primarily caused by the porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV can cause reproductive failure in pregnant sows and respiratory disease in pigs of all stages. It is a highly contagious pathogen with significant economic consequences. Infection with highly pathogenic PRRSV strains can induce acute lung injury, manifested by pulmonary hemorrhage, pulmonary edema, and massive inflammatory cell infiltration, contributing to high mortality rates. PRRS can lead to increased rates of abortion, premature birth, stillbirth, mummified fetuses, and weak piglets in sows, delayed estrus, increased return to estrus rates in sows, and poor semen quality in boars. Pigs of all stages experience varying degrees of fever, loss of appetite, and respiratory problems. The incidence of secondary infections with bacterial diseases such as Haemophilus parasuis, Streptococcus suis, and porcine contagious pleuropneumonia increases. PRRS causes significant economic losses to the swine industry.
[0004] Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), is a major infectious disease that severely harms the swine industry and is a notifiable disease designated by the World Organization for Animal Health (OIE). Characterized by acute onset, persistent high fever, systemic hemorrhage, and leukopenia, it is associated with high morbidity and mortality. Pigs (both domestic and wild boar) are the only susceptible host species for CSFV, and CSF is a major obstacle to the development of the global swine industry.
[0005] Before the outbreak of African swine fever (AFS), blue ear disease (PRRS) was the leading killer of the pig industry. However, in the post-AFS era, its prevalence remains unchanged. Attenuated PRRS vaccines are highly effective in reducing the severity of clinical symptoms, the duration of viremia, and the frequency of viral spread and field infection. Therefore, it is essential to select a safe, immunogenic, and attenuated PRRS vaccine for immunization against other viruses on pig farms, such as PCV2 and CSFV. 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 having an amino acid sequence of 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 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, which 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 further 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, and 1000ml water for injection are mixed, ultrasonically treated, and then sterilized at 121°C under high pressure for 20 minutes.
[0017] Specifically, the swine triple vaccine can be obtained by mixing the recombinant virus and 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 scope of protection 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 triple pig 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 trivalent swine vaccine of the present invention are safe, with no allergic reactions, redness, swelling, or ulceration after vaccination, and that overdosage has no effect on pig growth performance. Antibody testing shows that the trivalent vaccine produces comparable antibody levels in pigs as commercially available blue ear and circovirus vaccines. Classical swine fever antibody monitoring shows that the antibody blocking rate after the first vaccination with the recombinant virus was 18.28% 14 days after the first vaccination, compared to 19.98% for the commercially available swine fever virus vaccine. Twenty-one days after the second vaccination, the blocking rate reached 88.91%, compared to 85.66% for the commercially available swine fever virus vaccine. The results indicate that the recombinant virus rjxPRRSE2&Cap, a fusion of the swine fever virus E2 and porcine circovirus type 2 ORF2 genes, has good immunogenicity and can be used to simultaneously prevent diseases caused by porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Furthermore, the inactivated trivalent vaccine of the present invention, prepared with the 605 adjuvant and the content of the commercial vaccine, has the advantages of low viscosity of the water adjuvant, easy injection, good absorption, and multiple protection with one injection. The recombinant vector and trivalent vaccine of the present invention have excellent application prospects.
[0023] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 .Schematic diagram of the RRSVscfvE2&PCV2 ORF2 plasmid. RRSVscfvE2&PCV2 ORF2 clone represents the RRSVscfvE2&PCV2 ORF2 plasmid.
[0025] Figure 2Cytopathic effects of the .rjxPRRSE2&Cap strain. The left image shows healthy Marc-145 cells, and the right image shows cytopathic effects of rescued virus.
[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 and Cap proteins at different passages. The left figure shows the agar amplification results of CSFV E2 protein at different passages; the right figure shows the agar amplification results of PCV2 Cap protein at different passages. DETAILED DESCRIPTION
[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. 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 the 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 and Onestep RT-PCR KitExTaqtm enzymes are products of Takara; restriction endonucleases are products of Neb; competent cells DH5a, DNA recombination kit, DNA recovery kit, plasmid extraction kit, Lipofectamine TM The 2000 transfection kit was 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, F1 / R1 was used to amplify the JX vaccine PRRSV genome vector, and F2 / R2 was used to amplify the synthetic E2-Cap fusion gene vector. 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 ligated using homologous recombinase. The ligation product was transformed into DH5a competent cells, and the positive single clone was sent to Shanghai Shenggong Bioengineering 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 the figure. Figure 1 As shown, the RRSVscfvE2 & PCV2 ORF2 plasmid is a circular double-stranded DNA consisting of 20,224 bp. The nucleotide sequence of one strand is SEQ ID No. 1 from nucleotides 1 to 15,000, and the nucleotide sequence of nucleotides 15,001 to 20,224 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 fusion 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 were grown to 70% to 90% confluency in a 6-well plate, the cell surface was washed with Opti-MEM I medium (i.e., Opti-MEM Reduced Serum Medium, Thermo Fisher Scientific, Cat. No. 31985070). Marc-145 cells were then transfected with 4 μg of the RRSV scfv E2 & PCV2 ORF2 plasmid obtained in step 1.2.1 using Lipfection 2000:
[0046] First, add 4 μg of plasmid to 250 μl of Opti-MEM medium, then dilute 12 μl of Lipofectamine 2000 with a certain amount of serum-free medium, incubate at room temperature for 10 minutes, gently mix the diluted plasmid and diluted Lipofectamine 2000 (total volume 500 μl), incubate at room temperature for 20 minutes, and then 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 to 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 occurs, the cells become round, and are distributed in grape-like clusters. Finally, the cells disintegrate into fragments. Figure 2 The rescued recombinant virus was named rjxPRRSE2&Cap and propagated on Marc-145 cells for multiple generations. The time it took for cytopathic effects to appear was significantly shortened, and the lesions became more typical, allowing for further testing.
[0047] 1.3 Identification of biological characteristics of rescued viruses
[0048] The stable virus rjxPRRSE2&Cap was serially diluted 10-fold in MEM medium. Each dilution was inoculated into 96-well Marc-145 cells at 0.1 ml / well and cultured at 37°C for 48-72 hours. The TCID was calculated according to the Reed-Muench method based on the number of cytopathic effects. 50 The results showed that the first and second generation toxicity were slightly lower, 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, F1, F 15 RT-PCR was performed using 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. The sequence determination 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 virus was cultured in a bioreactor to prepare virus liquid. The E2 and Cap contents in different batches of virus liquid were detected according to the PRRS virus inactivated antigen method, and the production efficiency and stability of each generation of virus 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 amplification titers of target proteins E2 and Cap became weaker and weaker, see 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 next-generation virus can stably express and assemble Cap's Vlps.
[0055] Table 1. Virus titer determination at different passages (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 passages
[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 solution that passed the inactivation test was diluted (using MEM medium to dilute the virus content before inactivation to 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: 3.5 g disodium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 7 g sodium chloride, 7 g trehalose, 7 g astragalus polysaccharide, 3 g sodium glutamate, 13 g gelatin, 7 g polyethylene glycol 3000, 3 g dextran, and 1000 ml water for injection are mixed, treated with ultrasound, and sterilized under high pressure at 121°C for 20 min.
[0062] The rjxPRRSE2&Cap triple vaccine, blue ear 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 swine fever virus live vaccine Wenbeikang produced by Beijing Huaxia Xingyang Biotechnology Co., Ltd. (denoted as commercial vaccine C) were subjected to safety testing and efficacy experimental evaluation.
[0063] 1.7 Safety Inspection
[0064] Twenty-five healthy 14-day-old piglets (Duroc × Large White × Landrace) were immunized intramuscularly with the rjxPRRSE2 & Cap triple vaccine, commercial vaccine A, commercial vaccine B, or commercial vaccine C, respectively, at 4 mL per piglet. Five piglets served as blank controls and were housed under the same isolation conditions. The piglets were observed for 14 consecutive days, with their mental state, diet, and activity levels recorded. The injection site was also observed for redness, swelling, and ulceration. Thirty days after immunization, all pigs were weighed, and weight gain differences were analyzed between the rjxPRRSE2 & Cap triple vaccine, commercial vaccine A, commercial vaccine B, and commercial vaccine C groups and the blank control group.
[0065] Clinical Manifestations: After superdosing with the rjxPRRSE2&Cap triple vaccine and various commercial vaccines, piglets showed no vomiting, maintained normal spirits and appetite, and showed no redness, swelling, or ulceration at the injection site. Twenty-four hours after immunization, each vaccine group exhibited significant temperature increases, with the triple vaccine group experiencing a maximum temperature increase of 0.9°C, the commercial vaccine group A experiencing a maximum temperature increase of 0.8°C, the commercial vaccine group B experiencing a maximum temperature increase of 0.6°C, and the commercial vaccine group C experiencing a maximum temperature increase of 0.8°C. All experimental groups returned to normal temperatures within 48 hours.
[0066] 1.8 Efficacy test
[0067] 1.8.1 Immunization: Twenty-five healthy, 14-week-old piglets (Duroc × Large White × Landrace) were randomly divided into five groups: the triple vaccine group, commercial vaccine group A, commercial vaccine group B, commercial vaccine group C, and a control group (n=5 piglets each). Each group received intramuscular injection of the rjxPRRSE2 & Cap triple vaccine, commercial vaccine A, commercial vaccine B, and commercial vaccine C, at 2 mL per piglet. The control group received a 2 mL dose of normal saline. Piglets were housed in isolation under the same conditions. Twenty-one days after the first vaccination, a second vaccination was administered using the same method and dosage.
[0068] 1.8.2 Blood collection:
[0069] 1.8.2.1 Blood collection from the blue ear pig 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 for 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] PRRS Antibody Testing: Serum samples from the rjxPRRSE2&Cap triple vaccine, commercial vaccine A, and a control group were tested using the IDEXX antibody kit (REF99-0016411, LOTSN:GU902). The results are shown in Table 3. The samples from the triple vaccine and commercial vaccine A were all positive, with similar S / P ratios, both significantly higher than those in the control group, which had negative samples. These results demonstrate that the PRRS antigens in the recombinant rjxPRRSE2&Cap virus have good immunogenicity and can be used to prevent porcine reproductive and respiratory syndrome.
[0074] Table 3. Results of blue ear antibody test in pigs
[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 no 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 strain 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 serum samples of the rjxPRRSE2&Cap trivalent vaccine, commercial vaccine C and control group, and the blocking rate was calculated. The results are shown in Table 5. Antibodies began to be produced in the 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 antibody blocking rate of the commercial vaccine C remained at 19.98-71.84%. From 14 to 21 days after the second vaccination, the antibody blocking rate of the trivalent vaccine reached 82.34-88.91%, and the antibody blocking rate of the commercial vaccine C remained at 80.05-88.66%. Moreover, the antibody blocking rates of both 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 Classical Swine Fever Antibody Detection (Blocking Rate) (%)
[0081] Group Before immunization 14 days after the first vaccination 21 days after the first vaccination 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 seedling 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 practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.
Claims
1. A recombinant vector, 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.
2. A recombinant virus, characterized in that: The recombinant virus is a virus prepared using the recombinant vector according to claim 1.
3. Pig triple vaccine, characterized by: The swine triple vaccine contains the inactivated recombinant virus according to claim 2.
4. The swine triple vaccine according to claim 3, characterized in that: The swine triple vaccine further contains an adjuvant.
5. Use of the recombinant vector according to claim 1 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.
6. Use of the recombinant vector according to claim 1 in the preparation of porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and / or classical swine fever virus vaccines.
7. Use of the recombinant virus according to claim 2 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 swine triple vaccine according to claim 3 or 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.