A chimeric recombinant plasmid pprrsv- vp2-y, chimeric virus thereof and application thereof

Abstract

The present application relates to the technical field of veterinary biological products, and particularly relates to a chimeric recombinant plasmid pPRRSV-VP2-Y, a chimeric virus thereof and application thereof. The present application inserts a VP2-Y gene derived from porcine parvovirus between ORF1b and ORF2a of a high pathogenicity PRRSV cell-attenuated strain HuN4-F112 to construct a chimeric recombinant plasmid pPRRSV-VP2-Y; the virus rescued after transfecting MARC-145 cells with the chimeric recombinant plasmid constructed by the present application has similar biological characteristics to a parent virus vHuN4-F112, and meanwhile, the chimeric recombinant vaccine strain constructed based on the above-mentioned recombinant plasmid has good safety to pigs, can effectively induce an immune response of the body after immunization, can produce a high level of PRRSV N protein antibodies similar to the parent vaccine, and can induce PPV VP2 antibodies.

Description

Technical Field

[0001] The present 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 applications thereof. Background Art

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is a viral infectious disease that severely impacts the global swine industry. Its causative agent, PRRSV, is a single-stranded, positive-sense RNA virus belonging to the order Nidovirales, family Arteriviridae, and genus Arterivirus. Due to its unique genome structure and gene regulation mechanisms, PRRSV has been used as a live viral vector using reverse genetics techniques. The PRRSV genome encodes multiple structural and nonstructural proteins. Firstly, its genome structure is highly tolerant of foreign genes. Its genome is approximately 15 kb, portions of which can be replaced or inserted with foreign genes. Furthermore, some ORFs in the PRRSV genome have overlapping sequences, while others have intergenic regions between ORFs, making these regions ideal for inserting foreign genes. Recombinant viruses obtained by inserting foreign genes into these regions exhibit similar gene expression and biological properties to those of 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 transcriptional 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. In this way, the 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, and the recombinant virus rPRRSV-E2 also has similar growth characteristics to the parent virus vHu-N4-F112.

[0003] The VP2 protein is the primary capsid protein of PPV, accounting for over 60% of the viral capsid. It contains important neutralizing epitopes and is the primary immunogenic protein. It participates in the nuclear import of the viral genome and the assembly of mature virions. It can be assembled into virus-like particles in vitro and is highly immunogenic and stable. The present invention optimizes the VP2 gene to facilitate its expression in a vector and enhance the stability of the recombinant virus that is constructed and rescued.

[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 control of porcine parvovirus disease still relies mainly 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 a high homology to the vaccine strain 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 the vaccine strains in the immunized pig herds, there are disadvantages such as virulence reversion and recombination with wild epidemic strains to re-evolve into pathogenic strains. Therefore, it is considered to have safety risks. Summary of the Invention

[0005] The present invention aims to provide a chimeric recombinant plasmid pPRRSV-VP2-Y, a chimeric virus thereof, and applications thereof. The chimeric recombinant vaccine strain rPRRSV-VP2-Y constructed based on the chimeric recombinant plasmid has good safety for pigs and can effectively induce an immune response after immunization. It can not only produce high levels of PRRSV N protein antibodies similar to those of the parent vaccine, but also induce the production of PPVVP2, thereby achieving the effect of simultaneously 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 the virus 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 a use 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 cell monolayer, discarding the cell growth medium, and inoculating the chimeric virus into the MARC-145 cell monolayer. After adsorption, adding a cell maintenance medium and continuing to culture. When 70% to 80% of the cells show pathological changes, the virus culture medium is harvested;

[0016] (2) The virus culture solution is mixed with a stabilizer to prepare 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, the volume ratio of the virus culture solution to the stabilizer in step (2) is 1-2:1-2, and the type of 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 genetically engineered live vaccine candidate strain containing a chimeric VP2-Y gene of the present invention is a chimeric recombinant attenuated vaccine strain of the highly pathogenic PRRSV cell-passaged attenuated strain HuN4-F112, capable of stably expressing the VP2-Y protein. Viruses rescued from transfection of MARC-145 cells using the chimeric recombinant plasmid pPRRSV-VP2-Y constructed by the present invention exhibit biological properties similar to those of the parental virus, vHuN4-F112. Furthermore, genetic stability is maintained over at least 10 consecutive passages. Furthermore, the chimeric recombinant vaccine strain rPRRSV-VP2-Y constructed based on the aforementioned recombinant plasmid is relatively safe for pigs and effectively induces an immune response after immunization, producing high levels of antibodies to the PRRSV N protein similar to those of the parental vaccine, as well as antibodies to PPV VP2. Clinically, PRRSV infection can lead to secondary infections with other viruses or bacteria, and the recombinant vaccine obtained by the present invention can enhance pigs' resistance to disease. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0025] Figure 4 This is an 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 This is the cytopathic effect after cells were transfected with the chimeric recombinant plasmid pPRRSV-VP2-Y in Example 1;

[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 81 is a graph showing RT-PCR detection of chimeric viruses rPRRSV-VP2-Y of different generations in Example 1;

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

[0031] Figure 10 The Western blotting test of the rPRRSV-VP2-YP5 exogenous gene fragment in Example 2;

[0032] Figure 11 This is a graph showing changes in body temperature of pigs in different groups after vaccination in Example 2;

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

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

[0035] Figure 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 exogenous genes into the infectious clone backbone of the obtained 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 to assemble biologically active virus particles, so as to study the changes in viral biological characteristics between the mutant virus and the parent virus, as well as the possible effects of the exogenous 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 the full-length recombinant plasmid pPRRSV-VP2-Y obtained by gene chimera technology is transfected into MARC-145 cells.

[0041] The experimental methods in the following examples where specific conditions are not specified were generally performed 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 HighYield 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 with reference to the embodiments, but they should not be construed as limiting the scope of protection 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 (detailed structure see Figure 2 ) and the parental virus full-length plasmid pHuN4-F112 were double-digested with Asc I and EcoRV, respectively. 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 successfully construct the 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-enzyme digested with Asc I and EcoRV, respectively, and recovered and purified. The reaction system was: pUC57-PPV-VP2 plasmid 41 μL, Asc I 2 μL, EcoRV 2 μL, 10×NEB Buffer 45 μL; pHuN4-F112 plasmid 15 μL, Asc I 2 μL, EcoRV 2 μL, 10×NEB Buffer 45 μL, ddH2O 36 μL.

[0051] The pUC57-PPV-VP2 plasmid and the Asc I and EcoRV double-digested fragment of the parental virus full-length plasmid pHuN4-F112 were ligated using T4 DNA ligase at a molar ratio of 3:1 and transformed into TOP 10 competent cells. Independent colonies were selected for pure culture, and plasmid DNA was extracted and subjected to 1% gel electrophoresis. A plasmid of approximately 17 kb was selected for sequencing to screen for positive clones. The nucleotide sequence of the chimeric recombinant plasmid pPRRSV-VP2-Y is shown in SEQ ID NOs. 1-2.

[0052] 2. Preparation of viral RNA

[0053] 2.1 Swa I linearization of plasmid

[0054] In a 37°C water bath, linearize the chimeric recombinant plasmid pPRRSV-VP2-Y with Swa I (reaction system: chimeric recombinant plasmid pPRRSV-VP2-Y 3μg, Swa I 3μL, cutsmart buffer 5μL, add ddH2O to a total volume of 50μL) and digest overnight. Purify the digestion product with the QIAquiek PCR Purification Kit according to the instructions to obtain the purified linearized plasmid. The target band size is about 16723bp, see Figure 4 .

[0055] 2.2 In vitro transcription

[0056] The linearized plasmid was purified by in vitro transcription using T7 mMESSAGE High Yield Capped RNA Transeription Kit (purchased from Ambion) according to the instructions 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. The mixture was shaken and mixed in 1 mL of Opti-MEM reduced serum medium. MARC-145 cells were then transfected according to the instructions of the transfection reagent, and the cytopathic effects were 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., cell rounding, detachment, aggregation, etc., the supernatant was collected and passaged. The supernatant collection and passage process was as follows: after the transfected MARC-145 cells were attached to the wall and grown into a full monolayer in a six-well plate containing DMEM medium with 10% FBS, the medium was discarded, and the cells were washed twice with PBS. 200 μL of the culture supernatant of the MARC-145 cells five days after transfection was aspirated and inoculated with the maintenance medium (DMEM containing 2% FBS) at a ratio of 1:10 into 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. 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 a specific monoclonal antibody against PRRSV nucleocapsid protein (1:800 dilution) and a polyclonal antibody against VP2 protein (1:500 dilution) prepared in the laboratory for 2 hours at room temperature. 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 Results: 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 passage, and 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 show that the reverse genetic operating system was used to obtain a full-length recombinant plasmid containing VP2 protein with similar growth characteristics to the parent virus vHuN4-F112, and it was also proved that the insertion of the exogenous gene VP2 does not affect the growth of the entire virus and is feasible.

[0068] 3.4 Virus cell half infectious dose (TCID 50 ) determination

[0069] The infectious titer was determined using a 96-well tissue culture plate method, referring to the method described in 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 medium (2% FBS in DMEM) and 10 -1 ~10 -9 Serially diluted viruses were inoculated into MARC-145 monolayer cells in 96-well cell culture plates. 8 wells were inoculated for each dilution, with 0.1 mL per well. Two columns of controls were set up (i.e., maintenance medium (2% FBS in DMEM) was used instead of virus solution). The cells were cultured in a 37°C 5% CO2 incubator. After 5 days, the infected cells were observed, the number of wells showing cytopathic effects was recorded, and the TCID was calculated according to the Reed-Muench method. 50 .

[0070] 3.5 Drawing of multi-step viral growth curves

[0071] MARC-145 cells were infected with low doses (0.01 MOI) of viruses (rPRRSV-VP2-Y and vHuN4-F112), and cell culture supernatants were collected at different time periods (12h, 24h, 36h, 48h, 60h, 72h, and 84h) after infection, and the virus titers were measured. The titers of the viruses collected at each time point were calculated using TCID50, and the virus multi-step growth curves were drawn based on the titers of the viruses at different time points. The results are shown in Figure 2. Figure 9As shown in the figure, viral replication peaked 48 hours after infection; no significant differences were observed between the parental virus vHuN4-F112 and the chimeric virus rPRRSV-VP2-Y. These results demonstrate that the viruses rescued from the full-length VP2 chimeric recombinant plasmid obtained using reverse genetic manipulation techniques exhibit similar biological activity to the parental strain in terms of viral titer, exponential growth phase, and plateau phase growth curves at various time points during viral proliferation.

[0072] In summary, the present invention inserted the 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 in 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 adhered to the wall and grew into a monolayer in a six-well plate containing DMEM medium containing 10% FBS, the medium was discarded and the cells were washed twice with PBS. The culture supernatant of the cells in step 3.2 of Example 1 with pathological changes was collected and marked as P1. Subsequently, 200 μL of the P1 culture supernatant was taken and mixed with cell maintenance medium (DMEM medium containing 2% FBS) at a ratio of 1:10 to inoculate a monolayer of MARC-145 cells. The cells were cultured at 37°C for two days and the culture supernatant was collected and marked as P2. The above steps were repeated until the fifth generation, which was marked as P5. The harvested P5 culture supernatant was frozen and thawed 2 to 3 times and stored below -15°C. A small amount was taken for Western blotting semi-finished product inspection.

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

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

[0079] (3) Preparation, packaging and freeze-drying: Mix the virus culture medium that has passed the inspection with DMEM without serum in a volume ratio of 1:1 in a container, shake well, and package in a quantitative manner. After packaging, freeze-dry to obtain the finished product; each portion contains no less than 10% cytotoxic liquid. 5.0 TCID50.

[0080] 2. Animal Immunity Experiment

[0081] Fifteen experimental pigs were randomly divided into three groups and fed separately, namely the rPRRSV-VP2-Y immunization group, the vHuN4-F112 immunization group and the 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 pigs in the blank group were injected with 2 mL of serum-free DMEM. All pigs were monitored for body temperature and clinical symptoms on days 0, 7, 14, 21, 28, and 35 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 enzyme-labeled plate coated with PPVVP2 protein in advance.

[0082] Specific temperature changes can be seen in Figure 11 The autopsy results are shown in Figure 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. Figure 13 All pigs immunized with rPRRSV-VP2-Y and vHuN4-F112 were positive for PRRSV antibodies on day 10 of immunization. Within 20 days of immunization, the levels of antibodies produced by pigs immunized with rPRRSV-VP2-Y did not differ significantly from those in the control group, with the same trend of antibody increase and decrease. The blank group remained negative for PRRSV antibodies.

[0084] After immunizing pigs with PRRSV recombinant vaccine, Figure 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] Thus, the chimeric recombinant vaccine strain rPRRSV-VP2-Y constructed by the present invention has good safety for pigs and can effectively induce an immune response after immunization. It can not only produce high levels of PRRSV N protein antibodies similar to those of the parent vaccine, but also stimulate the body to produce specific antibodies against PPV VP2. Because PRRSV infection can clinically cause secondary infections with other viruses or bacteria, the recombinant vaccine obtained by the present invention can simultaneously prevent and control PRRSV and PPV, enhancing 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 principles 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: Its nucleotide sequence consists of the nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2; The method for constructing the chimeric recombinant plasmid pPRRSV-VP2-Y comprises the following steps: The VP2-Y gene was inserted between ORF1b and ORF2a of the virus strain HuN4-F112 to obtain the chimeric recombinant plasmid pPRRSV-VP2-Y.

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

3. Use of the chimeric recombinant plasmid pPRRSV-VP2-Y according to claim 1 or the chimeric virus according to claim 2 in the preparation of a chimeric recombinant vaccine for preventing and treating porcine reproductive and respiratory syndrome.

4. The use according to claim 3, characterized in that It also contains one or more of a carrier, a stabilizer, an excipient or an adjuvant.

5. 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 cell monolayer, discarding the cell growth medium, and inoculating the chimeric virus described in claim 2 into the MARC-145 cell monolayer. After adsorption, adding a cell maintenance medium and continuing to culture. When 70% to 80% of the cells show pathological changes, the virus culture medium is harvested; (2) The virus culture solution is mixed with a stabilizer to prepare a chimeric recombinant vaccine.

6. The preparation method according to claim 5, characterized in that The volume ratio of the chimeric virus to MARC-145 cells in step (1) is 1:8-12.

7. The preparation method according to claim 5, characterized in that The volume ratio of the virus culture solution to the stabilizer in step (2) is 1-2:1-2, and the type of the stabilizer is serum-free DMEM.

8. The preparation method according to claim 5, 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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