Chimeric recombinant plasmid pPRRSV-VP2-Y, chimeric virus thereof and application of chimeric recombinant plasmid pPRRSV-VP2-Y

Abstract

The invention relates to the technical field of veterinary biological products, in particular to a chimeric recombinant plasmid pPRRSV-VP2-Y, a chimeric virus thereof and application of the chimeric recombinant plasmid pPRRSV-VP2-Y. According to the invention, a VP2-Y gene derived from porcine parvovirus is inserted between ORF1b and ORF2a of a highly pathogenic PRRSV cell attenuated strain HuN4-F112, and a chimeric recombinant plasmid pPRRSV-VP2-Y is constructed; a virus rescued after MARC-145 cells are transfected by using the constructed chimeric recombinant plasmid has virus biological characteristics similar to that of a parent virus vHuN4-F112, and meanwhile, the constructed chimeric recombinant vaccine strain based on the recombinant plasmid has relatively good safety to a pig body, and can effectively induce an organism to generate immune response after immunization, so that the vaccine strain can be used for preparing a recombinant vaccine strain for preventing and treating the MARC-145 cells. A high-level PRRSVN protein antibody similar to a parent vaccine can be generated, and a PPVVP2 antibody can be induced to be generated.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to a chimeric recombinant plasmid pPRRSV-VP2-Y, a chimeric virus thereof and applications thereof. Background Art

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is a viral infectious disease that seriously affects the global pig industry. Its pathogen PRRSV is a single-stranded positive-strand RNA virus belonging to the order Nidovirales, the family Arteriviridae, and the genus Arterivirus. PRRSV is used as a live viral vector by researchers using reverse genetics due to its special genome structure and gene regulation mechanism. The genome of PRRSV encodes multiple structural and non-structural proteins. First of all, in terms of genome structure, the genome of PRRSV has a certain tolerance for exogenous genes. Its genome is about 15kb, part of which can be replaced or inserted with exogenous genes. In addition, some ORFs in the genome of PRRSV have overlapping sequences, while another ORFs inter-region has gene spacing, so these regions become preferred regions for inserting exogenous genes. The recombinant virus obtained by inserting exogenous genes in this interval has no significant difference in the expression of its own genes and the biological characteristics of the virus from the parental virus. Gao Fei et al. inserted the E2 gene of classical swine fever virus into the ORF1b and ORF2a of the genome of the highly pathogenic PRRSV attenuated vaccine strain (vHuN4-F112). In addition, the transcription regulatory sequence 6 (TRS6) of PRRSV was added to the 3' end of the E2 gene, allowing E2 to be subsequently transcribed as a new subgenome for expression. In this way, a full-length infectious clone plasmid pPRRSV-E2 was constructed. After transfection and rescue on MARC-145, the recombinant virus rPRRSV-E2 strain was obtained. The recombinant virus can express the E2 gene of classical swine fever virus. The growth characteristics of the recombinant virus rPRRSV-E2 are also similar to those of the parent virus vHu-N4-F112.

[0003] VP2 protein is the main capsid protein of PPV, accounting for more than 60% of the viral capsid, containing important neutralizing antigenic epitopes of the virus, and is the main immunogenic protein. It can participate in the nuclear entry of the viral genome and the assembly of mature viral particles, and can be assembled into virus-like particles in vitro, with high immunogenicity and stability. The present invention optimizes the VP2 gene to make it more conducive to expression in the vector, and also makes the constructed rescued recombinant virus more stable.

[0004] Once a pig herd is infected with PPV, it is difficult to eliminate it. There is currently no effective treatment method, so the prevention and treatment of porcine parvovirus disease is still mainly based on vaccination. The PPV vaccines currently in use and newly registered are all inactivated vaccines. Although inactivated vaccines are safer, they are difficult to form effective protection because they stimulate weak adaptive immune responses after immunizing animals with inactivated vaccines. The existing prevention and control of PRRSV mainly relies on live attenuated vaccines and inactivated vaccines. Existing evidence shows that although live attenuated vaccines can induce the body to produce immune protection against viral infections with high homology to vaccine strains in production practice, they can cause the immunized animals to excrete toxins (vaccine strains), and the vaccine strains exist in the immunized pig herds for a long time. At the same time, during the long-term existence of vaccine strains in the immunized pig herds, there are disadvantages of virulence reversion and recombination with wild epidemic strains to re-evolve into pathogenic strains, so it is considered to have safety risks. Summary of the invention

[0005] The purpose of the present invention is to provide a chimeric recombinant plasmid pPRRSV-VP2-Y, its chimeric virus and its application. Based on the above chimeric recombinant plasmid, the constructed chimeric recombinant vaccine strain rPRRSV-VP2-Y has good safety for pigs, can effectively induce the body to produce an immune response after immunization, can not only produce a high level of PRRSV N protein antibodies similar to the parent vaccine, but also can induce the production of PPVVP2, so as to achieve the effect of simultaneously preventing and controlling PRRSV and PPV and enhancing the disease resistance of pigs.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a chimeric recombinant plasmid pPRRSV-VP2-Y, the nucleotide sequence of which is shown in SEQ ID NO.1-2.

[0008] The present invention also provides a method for constructing the chimeric recombinant plasmid pPRRSV-VP2-Y, comprising the following steps:

[0009] The VP2-Y gene was inserted between ORF1b and ORF2a of strain HuN4-F112 to obtain the chimeric recombinant plasmid pPRRSV-VP2-Y.

[0010] The present invention also provides a chimeric virus comprising the chimeric recombinant plasmid pPRRSV-VP2-Y.

[0011] The present invention also provides an application of the chimeric recombinant plasmid pPRRSV-VP2-Y or the chimeric virus in preparing a detection reagent for in vitro detection of porcine reproductive and respiratory syndrome.

[0012] The present invention also provides an application of the chimeric recombinant plasmid pPRRSV-VP2-Y or the chimeric virus in preparing a chimeric recombinant vaccine for preventing and treating porcine reproductive and respiratory syndrome.

[0013] Preferably, it further comprises one or more of a carrier, a stabilizer, an excipient or an adjuvant.

[0014] The present invention also provides a method for preparing a chimeric recombinant vaccine for preventing and treating porcine reproductive and respiratory syndrome, comprising the following steps:

[0015] (1) taking a MARC-145 monolayer cell, discarding the cell growth medium, inoculating the chimeric virus into the MARC-145 monolayer cell, adding a cell maintenance medium after adsorption, and continuing to culture, and harvesting the virus culture medium when 70% to 80% of the cells show pathological changes;

[0016] (2) The virus culture medium is mixed with a stabilizer to obtain a chimeric recombinant vaccine.

[0017] Preferably, the volume ratio of the chimeric virus to MARC-145 cells in step (1) is 1:8-12.

[0018] Preferably, in step (2), the volume ratio of the virus culture solution to the stabilizer is 1-2:1-2, and the stabilizer is serum-free DMEM.

[0019] Preferably, the virus content of the chimeric virus in the chimeric recombinant vaccine is ≥10 5.0 TCID 50 / mL.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The PRRSV genetic engineering live vaccine candidate strain of the chimeric VP2-Y gene of the present invention is a chimeric recombinant attenuated vaccine strain of HuN4-F112, a highly pathogenic PRRSV cell-passaged attenuated strain capable of stably expressing VP2-Y protein. The chimeric recombinant plasmid pPRRSV-VP2-Y constructed by the present invention is used to rescue the virus after transfection of MARC-145 cells, which has similar viral biological characteristics to the parental virus vHuN4-F112. And the genetic stability can be maintained during at least 10 consecutive generations of passage. At the same time, based on the above-mentioned recombinant plasmid, the constructed chimeric recombinant vaccine strain rPRRSV-VP2-Y has good safety for pigs, and can effectively induce the body to produce an immune response after immunization, which can not only produce high levels of PRRSVN protein antibodies similar to the parental vaccine, but also induce the production of PPVVP2 antibodies. Because clinically, infection with PRRSV can cause secondary infection with other viruses or bacteria, and the recombinant vaccine obtained in the present invention can enhance the disease resistance of pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic diagram of the construction of the chimeric recombinant plasmid pPRRSV-VP2-Y in Example 1;

[0023] Figure 2 is a structural diagram of plasmid pUC57-PPV-VP2 in Example 1;

[0024] Figure 3 1 is a diagram showing the results of double enzyme digestion in Example 1; wherein A is a plasmid containing the VP2-Y gene double-digested by EcoRV and Asc I, and B is a pHuN4-F112 vector double-digested by EcoRV and Asc I;

[0025] Figure 4 is the electrophoresis detection diagram of the linearized recombinant plasmid pPRRSV-VP2-Y Swa I in Example 1;

[0026] Figure 5 This is an electrophoresis detection diagram of the in vitro transcribed RNA of the linearized recombinant plasmid pPRRSV-VP2-Y Swa I in Example 1;

[0027] Figure 6 It is the cytopathic result after the chimeric recombinant plasmid pPRRSV-VP2-Y in Example 1 was transfected into cells;

[0028] Figure 7 is an immunofluorescence photograph of the N protein and VP2-Y protein of the chimeric virus rPRRSV-VP2-Y in Example 1;

[0029] Figure 8It is the RT-PCR detection diagram of the chimeric virus rPRRSV-VP2-Y of different generations in Example 1;

[0030] Fig. 9 is a growth curve graph of the virus titer of the chimeric virus rPRRSV-VP2-Y and the parental virus in Example 1;

[0031] Fig.10 The rPRRSV-VP2-YP5 exogenous gene fragment was tested by Western blotting in Example 2;

[0032] Fig.11 is a graph showing changes in body temperature of different groups of pigs after inoculation in Example 2;

[0033] Fig.12 These are the autopsy observations of different groups of pigs after inoculation in Example 2; A is the macroscopic pathological observation of pig organs and tissues, and B is the comparison of pathological sections;

[0034] Fig.13 This is a graph showing changes in PRRSV N protein antibodies in different groups of pigs after inoculation in Example 2;

[0035] Fig.14 This is a graph showing the changes in PPVVP2 protein antibodies in different groups of pigs after vaccination in Example 2. DETAILED DESCRIPTION

[0036] In the present invention, the chimeric recombinant plasmid refers to pPRRSV-VP2-Y obtained by inserting the VP2-Y gene of porcine parvovirus into the HuN4-F112 genome backbone using reverse genetic manipulation technology, and its nucleotide sequence is shown in SEQ ID NO.1-2. The construction process of the chimeric recombinant plasmid pPRRSV-VP2-Y is as follows: Figure 1 shown.

[0037] In the present invention, the reverse genetic operation refers to: relative to classical genetics, it is to insert foreign genes into the infectious clone skeleton of the highly pathogenic PRRSV cell attenuated strain HuN4-F112 using T4 DNA ligase, and then construct the full-length viral genome in the order of composition, so that it can assemble biologically active virus particles, study the changes in viral biological characteristics between mutant viruses and parental viruses, and the possible effects of foreign gene insertion on the phenotype and traits of the virus.

[0038] In the present invention, the Genbank accession number of the highly pathogenic PRRSV cell attenuated strain HuN4-F112 is EF635006.

[0039] In the present invention, the cell attenuated strain HuN4-F112 refers to an infectious clone constructed by the method of reference Shanrui Zhang, Yanjun Zhou, Yifeng Jiang, Guoxin Li, Liping Yan, Hai Yu, Guangzhi Tong. Generation of an infectious clone of HuN4-F112, an attenuated live vaccine strain of porcine reproductive and respiratory syndrome virus.

[0040] In the present invention, the chimeric virus refers to the live virus rescued after transfecting MARC-145 cells with the full-length recombinant plasmid pPRRSV-VP2-Y obtained by gene chimera technology.

[0041] The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0042] In the examples of the present invention, the plasmids and strains used were: pBlueScript II SK(+) vector purchased from Invitrogen, pBS-T vector and TOP10 competent cells purchased from TIANGENE, and MARC-145 cells (African green monkey kidney cell line) purchased from ATCC, USA.

[0043] In the embodiments of the present invention, other reagents used: QIAamp Viral RNA Mini Kit was purchased from QIAGENE, pfu II DNA Polymerase was purchased from Strategene, T7mMESSAGE High Yield Capped RNA Transcription Kit was purchased from Ambion, gel recovery kit and Quant Reverse Transcriptase were purchased from TIANGENE, rTaq DNA polymerase, dNTP and restriction endonuclease were purchased from TaKaRa, plasmid extraction kit was purchased from Beijing Broadtech Biogene Technology Co., Ltd., DMRIE-C transfection reagent was purchased from Invitrogen, and Opti-MEM was purchased from Invitrogen.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] Construction of recombinant plasmid expressing VP2-Y protein and its virus rescue

[0047] 1. Construction of recombinant plasmid expressing VP2-Y protein

[0048] The synthesized plasmid pUC57-PPV-VP2 (for details, see Figure 2 ) and the parent virus full-length plasmid pHuN4-F112 were double-digested with Asc I and EcoRV, respectively. The results are shown in Figure 3 After double enzyme digestion, a larger fragment of the parental virus full-length plasmid pHuN4-F112 of about 15163bp and a smaller VP2 target gene fragment of about 1551bp were produced. The two were connected by T4 DNA ligase, and positive clones were screened after sequencing identification to obtain the successfully constructed chimeric recombinant plasmid pPRRSV-VP2-Y.

[0049] The specific steps are as follows:

[0050] In a 37°C water bath, the plasmid containing the optimized VP2 gene and the parental virus full-length plasmid pHuN4-F112 were double-digested with Asc I and EcoRV, respectively, and recovered and purified. The reaction system was: pUC57-PPV-VP2 plasmid 41μL, Asc I 2μL, EcoRV 2μL, 10×NEB Buffer 45μL; pHuN4-F112 plasmid 15μL, Asc I 2μL, EcoRV 2μL, 10×NEB Buffer 45μL, ddH 2 O 36 μL.

[0051] The pUC57-PPV-VP2 plasmid and the Asc I and EcoRV double-digested fragments of the parent virus full-length plasmid pHuN4-F112 were connected using T4 DNA ligase at a molar ratio of 3:1, and transformed into TOP 10 competent cells, and independent colonies were picked for pure culture, plasmid DNA was extracted, 1% gel electrophoresis was performed, and a plasmid of about 17 kb was picked for sequencing to screen and obtain positive clones. The nucleotide sequence of the chimeric recombinant plasmid pPRRSV-VP2-Y is shown in SEQ ID NO.1-2.

[0052] 2. Preparation of viral RNA

[0053] 2.1 Swa I linearization of plasmid

[0054] In a 37°C water bath, the chimeric recombinant plasmid pPRRSV-VP2-Y was linearized with Swa I (the reaction system was: chimeric recombinant plasmid pPRRSV-VP2-Y 3 μg, Swa I 3 μL, cutsmart buffer 5 μL, ddH 2 O was added to a total volume of 50 μL) and digested overnight. Referring to the instructions, the digested product was purified using QIAquiek PCR Purification Kit to obtain a purified linearized plasmid. The target band size was about 16723 bp. Figure 4 .

[0055] 2.2 In vitro transcription

[0056] Referring to the method in the manual, the linearized plasmid was purified by in vitro transcription using T7 mMESSAGE High Yield Capped RNA Transeription Kit (purchased from Ambion) and identified by RNA electrophoresis. Figure 5 , and obtained the in vitro transcribed RNA of pPRRSV-VP2-Y.

[0057] 3. Harvest, amplification and detection of chimeric virus rPRRSV-VP2-Y

[0058] 3.1 RNA transfection

[0059] MARC-145 cells were inoculated and cultured in six-well plates. When the cell density reached 80-90%, in vitro transcribed RNA of pPRRSV-VP2-Y and 5 μL of DMRIF-C transfection reagent were added to each well, and the mixture was shaken and mixed in 1 mL of Opti-MEM reduced serum medium. Then, MARC-145 cells were transfected according to the instructions of the transfection reagent, and the cytopathic effect was observed every day.

[0060] 3.2 Rescue and amplification of chimeric virus rPRRSV-VP2-Y

[0061] Waiting to appear Figure 6After the cytopathic effect (CPE) shown in the figure, i.e., the cells become rounded, fall off, aggregate, etc., the supernatant is collected and passaged. The specific process of supernatant collection and passage is as follows: after the transfected MARC-145 cells are attached to the wall and grow into a full monolayer in a six-well plate containing DMEM medium with 10% FBS, the medium is discarded, and the cells are washed twice with PBS. 200 μL of the supernatant of the culture medium after the transfection of MARC-145 cells is taken out, and it is mixed with the maintenance medium (DMEM containing 2% FBS) at a ratio of 1:10 to inoculate the transfected MARC-145 cells. The cells were cultured at 37°C and continued to be passaged using the above method. Finally, the cell supernatant was collected to obtain the chimeric virus rPRRSV-VP2-Y. In order to further verify the sequence authenticity of the rescued chimeric virus rPRRSV-VP2-Y, the genomic RNA of the rescued virus rPRRSV-VP2-Y was extracted, and after RT-PCR amplification, it was compared with the constructed chimeric recombinant plasmid pPRRSV-VP2-Y. The results showed that the sequence of the rescued chimeric virus rPRRSV-VP2-Y was consistent with expectations.

[0062] According to the above results, the obtained recombinant plasmid rPRRSV-VP2-Y is infectious and can be successfully transformed from a single genome sequence into active virus particles with corresponding viral infectivity.

[0063] 3.3 Indirect immunofluorescence detection

[0064] 4 μL of the chimeric virus rPRRSV-VP2-Y supernatant prepared in step 3.2 was taken and inoculated into a monolayer of MARC-145 cells at a ratio of 1:1000 with the maintenance solution (DMEM containing 2% FBS). Then, the culture medium was discarded 36 hours after infection, and the cells were fixed with ice methanol for 10 minutes, blocked with 5% BSA at room temperature for 1 hour, and incubated with the specific monoclonal antibody of PRRSV nucleocapsid protein (1:800 dilution) and VP2 protein polyclonal antibody (1:500 dilution) prepared in the laboratory at room temperature for 2 hours, and then FITC-labeled goat anti-mouse and goat anti-rabbit secondary antibodies were added and incubated at room temperature for 1 hour. After washing with PBS three times, the cells were observed under a fluorescence microscope. The results are as follows: Figure 7 shown.

[0065] according to Figure 7 The results showed that the chimeric virus rPRRSV-VP2-Y showed obvious CPE on the 5th day after transfection. The chimeric virus rPRRSV-VP2-Y was purified by infinite dilution, and the MARC-145 cells infected with the chimeric virus rPRRSV-VP2-Y for 36 hours were detected by indirect immunofluorescence, and specific fluorescence appeared.

[0066] At the same time, RT-PCR technology was used to detect primary (P0) and progeny (P2, P4, P6, P8, P10) viruses. Figure 8 It showed that the VP2 gene could stably exist in the PRRSV genome.

[0067] The above results indicate that the reverse genetics operating system was used to obtain a full-length recombinant plasmid containing a VP2 protein with similar growth characteristics to the parental virus vHuN4-F112, and that the insertion of the exogenous gene VP2 does not affect the growth of the entire virus and is feasible.

[0068] 3.4 TCID 50 ) determination

[0069] The infectivity titer was determined by 96-well tissue culture plate method according to the method of Pizzi, M. Sampling variation of the fifty percent end-point, determined by the Reed-Muench (Behrens) method, Hum Biol, 1950. 22 (3): p151-90. The P5 virus supernatant collected in step 3.2 was serially diluted 10-fold with maintenance solution (DMEM with 2% FBS) and 10 -1 ~10 -9 The virus was serially diluted and inoculated into the MARC-145 monolayer cells on a 96-well cell culture plate. Each dilution was inoculated into 8 wells, with 0.1 mL per well. Two columns of controls were set up (i.e., the virus solution was replaced with maintenance solution (2% FBS in DMEM)). The cells were cultured in a 37°C 5% carbon dioxide incubator. After 5 days, the infected cells were observed, the number of wells with cytopathic effects was recorded, and the TCID was calculated according to the Reed-Muench method. 50 .

[0070] 3.5 Plotting of multi-step virus growth curves

[0071] MARC-145 cells were infected with low dose (0.01MOI) viruses (rPRRSV-VP2-Y and vHuN4-F112), and cell culture supernatants were collected at different time periods (12h, 24h, 36h, 48h, 60h, 72h, 84h) after infection, and the virus titer was measured. The titer of the collected virus at each time point was calculated using TCID50, and the virus multi-step growth curve was drawn according to the titer of the virus at different time points. The results are shown in Figure 2. Fig. 9As shown in the figure, the virus forms a peak of replication 48 hours after infection; the difference between the parental virus vHuN4-F112 and the chimeric virus rPRRSV-VP2-Y is not significant. The results show that the virus rescued by the full-length VP2 chimeric recombinant plasmid obtained by reverse genetic manipulation technology has similar biological activity to the parental strain in terms of virus titer, exponential growth phase, plateau phase and other growth curves at various time points of virus proliferation.

[0072] In summary, the present invention inserted VP2 protein between ORF1b and ORF2a on the backbone of the full-length infectious clone pHuN4-F112 of the highly pathogenic PRRSV cell-passaged attenuated strain to obtain the recombinant plasmid pPRRSV-VP2-Y. After virus rescue, live virus was obtained. RT-PCR identification showed that the chimeric virus could be stably passaged 10 times without deletion. Therefore, the above-mentioned chimeric virus can be used as a candidate strain for a new PRRSV vaccine.

[0073] Example 2 Animal Clinical Trial of Chimeric Virus rPRRSV-VP2-Y

[0074] 1. Preparation of chimeric recombinant vaccines

[0075] (1) Cell subculture and culture for seedling preparation: MARC-145 cells were digested and subcultured with EDTA-trypsin cell dispersion solution, and 6×10 6 The cells were cultured with cell growth medium (DMEM medium containing 10% FBS) until a monolayer was formed and set aside;

[0076] (2) Propagation of cell toxins: After MARC-145 cells adhere to the wall and grow into a monolayer in a six-well plate containing DMEM medium containing 10% FBS, the medium is discarded, the cells are washed twice with PBS, and the culture medium supernatant of the cells with pathological changes in step 3.2 of Example 1 is collected and marked as P1. Subsequently, 200 μL of the P1 culture medium supernatant is taken and inoculated with a cell maintenance medium (DMEM medium containing 2% FBS) at a ratio of 1:10 to a monolayer of MARC-145 cells. The cells are cultured at 37°C for two days and then the culture medium supernatant is collected and marked as P2. The above steps are repeated until the fifth generation, which is marked as P5. The harvested P5 culture medium supernatant is frozen and thawed 2 to 3 times and stored below -15°C. A small amount is taken for Western blotting semi-finished product inspection;

[0077] The specific test steps are as follows: 4 μL of the supernatant of the P5 culture medium is taken, and it is inoculated with the maintenance solution (DMEM containing 2% FBS) at a ratio of 1:1000 for a monolayer of MARC-145 cells. Then, the culture medium is discarded 36 hours after infection, and the cells are lysed on ice with 200 μl RIPA for 15 minutes. After centrifugation at 4°C, 10 minutes, and 12000 rpm, the supernatant is taken for SDS-PAGE and transferred to the membrane. The membrane is washed 3 times with TBST, blocked with 5% skim milk at room temperature for 2 hours; the membrane is washed 3 times with TBST, and incubated at room temperature for 2 hours with the prepared VP2 polyclonal antibody (1:500) as the primary antibody; the membrane is washed 3 times with TBST, and incubated at room temperature for 1 hour with HRP-labeled goat anti-rabbit IgG (1:6000) as the secondary antibody. The membrane is washed 3 times with TBST, and finally, a color developing solution is added for development and photography. The results are as follows: Fig.10 shown.

[0078] according to Fig.10 Results: 36 hours after the chimeric virus rPRRSV-VP2-Y infected MARC-145 cells, a Western Blotting experiment was performed, and a specific band appeared near 70KDa, indicating that the exogenous gene VP2 inserted by the chimeric virus rPRRSV-VP2-Y was expressed in MARC-145 cells.

[0079] (3) Vaccine preparation, packaging and freeze-drying: Mix the qualified virus culture medium and serum-free DMEM in a container at a volume ratio of 1:1, shake well, and package in quantitative quantities. After packaging, freeze-dry to obtain the finished product; each portion contains no less than 10% cytotoxic fluid. 5.0 TCID50.

[0080] 2. Animal Immunization Experiment

[0081] Fifteen experimental pigs were randomly divided into three groups and fed separately, namely, rPRRSV-VP2-Y immunization group, vHuN4-F112 immunization group and blank group. Each pig in the immunization group was injected with a volume of 2 mL of the chimeric recombinant vaccine prepared in step 1 of Example 2 into the neck muscle, and the blank group pigs were injected with 2 mL of serum-free DMEM. All pigs were monitored for body temperature and clinical symptoms at 0, 7, 14, 21, 28, and 35 days after immunization, and blood samples were collected. The IDEXX porcine reproductive and respiratory syndrome ELISA test kit was used to detect the PRRSVN protein antibody levels in the three groups, and the PPVVP2 antibody levels in the rPRRSV-VP2-Y immunization group and the vHuN4-F112 immunization group were detected using an ELISA plate coated with PPVVP2 protein in advance.

[0082] Specific temperature changes can be seen in Fig.11 The autopsy results are shown in Fig.12The results showed that the body temperature and mental state of the immunized group were normal, and no obvious pathological changes were found in the autopsy.

[0083] The PRRSV-specific antibody levels of pigs in the immunization group and the mock group were detected. Fig.13 The PRRSV antibodies of pigs immunized with rPRRSV-VP2-Y and vHuN4-F112 were all positive on the tenth day of immunization. The antibody levels of pigs immunized with rPRRSV-VP2-Y and the control group were not significantly different within 20 days of immunization, and the trend of antibody increase and decrease was the same. The PRRSV antibody level of the blank group was always negative.

[0084] After immunizing pigs with PRRSV recombinant vaccine, Fig.14 The results of detecting VP2 in serum showed that the recombinant virus rPRRSV-VP2-Y can also induce pigs to produce specific antibodies against PPV VP2, and the antibody level reached a peak at 21 days.

[0085] It can be seen that the chimeric recombinant vaccine strain rPRRSV-VP2-Y constructed by the present invention has good safety for pigs, and can effectively induce the body to produce an immune response after immunization, which can not only produce high levels of PRRSVN protein antibodies similar to the parent vaccine, but also stimulate the body to produce specific antibodies against PPV VP2. Because clinically, infection with PRRSV can cause secondary infection with other viruses or bacteria, and the recombinant vaccine obtained in the present invention can simultaneously prevent and control PRRSV and PPV, and enhance the disease resistance of pigs.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A chimeric recombinant plasmid pPRRSV-VP2-Y, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO.1-2.

2. A method for constructing the chimeric recombinant plasmid pPRRSV-VP2-Y according to claim 1, characterized in that: The following steps are involved: The VP2-Y gene was inserted between ORF1b and ORF2a of strain HuN4-F112 to obtain the chimeric recombinant plasmid pPRRSV-VP2-Y.

3. A chimeric virus comprising the chimeric recombinant plasmid pPRRSV-VP2-Y according to claim 1.

4. Use of the chimeric recombinant plasmid pPRRSV-VP2-Y according to claim 1 or the chimeric virus according to claim 3 in the preparation of a detection reagent for in vitro detection of porcine reproductive and respiratory syndrome.

5. Use of the chimeric recombinant plasmid pPRRSV-VP2-Y according to claim 1 or the chimeric virus according to claim 3 in the preparation of a chimeric recombinant vaccine for preventing and treating porcine reproductive and respiratory syndrome.

6. The use according to claim 5, characterized in that: It also contains one or more of a carrier, a stabilizer, an excipient or an adjuvant.

7. A method for preparing a chimeric recombinant vaccine for preventing and treating porcine reproductive and respiratory syndrome, characterized in that: The following steps are involved: (1) taking a MARC-145 monolayer cell, discarding the cell growth medium, and inoculating the chimeric virus described in claim 3 into the MARC-145 monolayer cell. After adsorption, adding a cell maintenance medium to continue culturing. When 70% to 80% of the cells show pathological changes, the virus culture medium is harvested; (2) The virus culture medium is mixed with a stabilizer to obtain a chimeric recombinant vaccine.

8. The preparation method according to claim 7, characterized in that: The volume ratio of the chimeric virus to MARC-145 cells in step (1) is 1:8-12.

9. The preparation method according to claim 7, characterized in that: In step (2), the volume ratio of the virus culture solution to the stabilizer is 1-2:1-2, and the type of the stabilizer is serum-free DMEM.

10. The preparation method according to claim 7, characterized in that: The virus content of the chimeric virus in the chimeric recombinant vaccine is ≥10 5.0 TCID 50 / mL.

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