Recombinant PRRSV (porcine reproductive and respiratory syndrome virus) genetic engineering live vector vaccine for expressing interleukin-15 and interleukin-18 and application of recombinant PRRSV genetic engineering live vector vaccine

Abstract

The invention provides a recombinant PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) genetic engineering live vector vaccine for expressing interleukin-15 and interleukin-18 and application of the recombinant PRRSV genetic engineering live vector vaccine. According to the present invention, after the MARC-145 cell is transfected with the two recombinant plasmids such as the pPRRSV-IL15-Y and the pPRRSV-IL18-Y, the live virus can be successfully rescued, and the virological characteristics of the virus vHuN4-F112 are similar to the virological characteristics of the parent virus vHuN4-F112; the two recombinant viruses are subjected to a pig body immune experiment, and the two recombinant viruses are found to have good safety to a pig body, can effectively induce an organism to generate immune response and generate a high-level antibody aiming at PRRSV N protein after immunization, can induce a higher cellular immune level, and are good genetic engineering vaccine candidate strains.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a PRRSV live vector vaccine, and more specifically to a recombinant PRRSV genetic engineering live vector vaccine expressing interleukin-15 and interleukin-18 and an application 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, and another ORFs inter-region has gene intervals, 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 genome ORF1b and ORF2a 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 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 similar to those of the parent virus vHuN4-F112. The success of rPRRSV-E2 shows that the ORF1b and ORF2a of PRRSV are a good insertion site, which is more suitable for the insertion of foreign genes.

[0003] A large number of studies have shown that codon optimization can significantly increase the expression of exogenous proteins. Codon optimization can start from the preference of codons to increase the expression of exogenous proteins. Dong Cong (2019) et al. optimized the codons of FAD-dependent glucose dehydrogenase based on the codon preference of Pichia pastoris, and then obtained a recombinant strain that efficiently expressed FAD-dependent glucose dehydrogenase by constructing an expression vector, screening and optimizing it, solving the problem that FAD-dependent glucose dehydrogenase is difficult to express in a soluble recombinant manner. Stachyra (2016) et al. optimized the hemagglutinin (HA) coding gene of avian influenza virus (H5N1), and the third position of its codons was almost all cytosine (C) or guanine (G), which increased the content of CG in the coding gene. A series of experiments showed that this codon optimization improved the gene expression of DNA vaccines and increased immunogenicity.

[0004] Codons encoding the same amino acid are called synonymous codons. In the process of protein transcription and translation, synonymous codons have different usage frequencies, and codons have preferences, that is, a certain bacterium or a certain virus often uses one or more fixed synonymous codons to synthesize proteins, so a series of commonly used codons that match itself are determined. The use of codons by different viruses is not randomly determined, but has certain preferences. For example, the codon preference of each protein genome of Nipah Virus (NiV) ends with A / U, the B and C types of hepatitis B virus prefer codons ending with U / C, and the L subtype of human immunodeficiency virus type 1 prefers codons ending with A / U. The proportion of codons ending with A / U in each protein codon of the new coronavirus (SARS-CoV-2) is about 84.98%. After the sequence of the exogenous gene inserted into PPRSV is optimized, its stable expression in the viral genome can be ensured. This includes the use of regulatory sequences or promoters suitable for the host virus to promote the transcription and translation of exogenous genes while reducing adverse effects on the biological characteristics of the virus itself, ensuring that the vaccine effectively expresses the target protein in the host and thus stimulates an immune response.

[0005] In the current porcine reproductive and respiratory syndrome virus (PRRSV) prevention and control strategy, although live attenuated vaccines and inactivated vaccines are the main means, they have some technical defects. Although live attenuated vaccines can induce high homologous immune protection, the long-standing vaccine strains may recombine with wild epidemic strains in the immunized pig herd, resulting in the re-evolution of virulence, which poses a safety hazard. On the other hand, although inactivated vaccines are safer, the immune response they stimulate is relatively weak, making it difficult to form effective immune protection, and the market acceptance is not high. In contrast, using PRRSV as a vector to construct a recombinant virus and optimizing the expression of exogenous genes such as IL15 and IL18 through codons can significantly improve the immune effect of the vaccine. Codon optimization can ensure that exogenous proteins are stably and efficiently expressed in the viral genome, thereby avoiding the problems of virulence reversion and recombination evolution that may occur in traditional vaccines. In addition, combined with immune regulatory factors such as IL15 and IL18, it can not only effectively activate the immune response, but also help to enhance the immunogenicity and protective effect of the vaccine. Therefore, this technology not only has important application prospects in vaccine research and development, especially in the treatment of diseases such as immunodeficiency virus infections and tumors, but also demonstrates significant advantages in overcoming the technical defects of traditional vaccines and improving the level of technology. Summary of the invention

[0006] In order to solve the above technical defects of the vaccines in the prior art, the object of the present invention is, first of all, to provide a porcine reproductive and respiratory syndrome virus recombinant vaccine expressing porcine IL15 gene or IL18 gene.

[0007] The porcine reproductive and respiratory syndrome virus recombinant vaccines expressing the porcine IL15 gene or IL18 gene are rPRRSV-IL15-Y and rPRRSV-IL18-Y respectively. The recombinant vaccines are obtained by transfecting MARC-145 cells with the recombinant plasmids pPRRSV-IL15-Y and pPRRSV-IL18-Y and then rescuing them.

[0008] Among them, the full length of pPRRSV-IL15-Y is 16494bp, wherein the nucleotide sequence at positions 1-9999 is shown in SEQ ID NO.1, and the nucleotide sequence at positions 10000-16494 is shown in SEQ ID NO.2; the full length of pPRRSV-IL18-Y is 16584bp, wherein the nucleotide sequence at positions 1-9999 is shown in SEQ ID NO.3, and the nucleotide sequence at positions 10000-16584 is shown in SEQ ID NO.4.

[0009] Furthermore, the present invention also provides a method for batch preparation of a chimeric vaccine of porcine reproductive and respiratory syndrome virus with IL15 or IL18 gene, the method being carried out according to the following steps: (1) cell passaging and culture for vaccine preparation: MARC-145 cells are digested and passaged with EDTA-trypsin cell dispersion solution, and then cultured with cell growth solution. When a monolayer is formed, the cells are set aside; (2) cell seed virus reproduction: the virus solution is inoculated into a cell bottle with a well-grown cell monolayer at a volume of 1 / 10, and after adsorption for 1 hour, a cell maintenance solution is added to the cell line monolayer, and the culture is continued. When 70-80% of the cells show pathological changes, the cells are harvested, and the harvested venom is frozen and thawed 2 to 3 times and stored below -15°C, and a small amount is taken for semi-finished product inspection; (3) vaccine preparation, packaging and freeze-drying: the virus culture solution that has passed the inspection is mixed with a conventional stabilizer in a container at a volume ratio of 1:1, shaken thoroughly, and quantitatively packaged; each portion contains no less than 10% of the cell venom. 5.0 TCID 50 After subpackaging, freeze-drying is performed to obtain the finished product.

[0010] Furthermore, the present invention provides a method for constructing the porcine reproductive and respiratory syndrome virus recombinant plasmid expressing the porcine IL15 gene or IL18 gene, wherein the method comprises synthesizing relevant plasmids according to the porcine IL15 gene and the porcine IL18 gene, inserting the IL15 gene or the IL18 gene between ORF1b and ORF2a of HuN4-F112, and inserting the transcriptional regulatory sequence 6 (TRS6) of PRRSV downstream of the 3' end of the foreign gene. The two amplified chimeric IL15 fragments and IL18 fragments are double-digested by Asc I and EcoRV, and then the recovered products are connected to the double-digested vector of Asc I and EcoRV of HuN4-F112, thereby obtaining chimeric recombinant plasmids, wherein the chimeric recombinant plasmids are pPRRSV-IL15-Y and pPRRSV-IL18-Y.

[0011] Beneficial Effects

[0012] The genetic engineering live vaccine candidate strain of PRRSV with chimeric IL15 or IL18 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 IL15 or IL18 protein. The viruses rescued after transfection of MARC-145 cells using the recombinant plasmids pPRRSV-IL15-Y and pPRRSV-IL18-Y constructed by the present invention have 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 plasmids, the two chimeric recombinant vaccine strains rPRRSV-IL15-Y and rPRRSV-IL18-Y constructed have 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 parental vaccine, but also can induce a higher level of cellular immunity. Because clinical infection with PRRSV can cause secondary infection with other viruses or bacteria, the recombinant vaccine obtained in this study can enhance the cellular immunity level of pigs and strengthen the body's immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the construction of IL15 or IL18 chimeric recombinant plasmid.

[0014] Figure 2 A is a plasmid containing IL15 or IL18 gene that has been double-digested with EcoR V and Asc I, wherein M is a marker, 1 and 2 are IL15 fragments, and 3 and 4 are IL18 fragments. Figure 2 B is the pHuN4-F112 vector double-digested with EcoR V and Asc I, wherein M is a marker and 1 is a pHuN4-F112 vector fragment.

[0015] Figure 3 A is the Swa I linear electrophoresis result of the constructed chimeric recombinant plasmids pPRRSV-IL15-Y and pPRRSV-IL18-Y. Figure 3 B is the electrophoretic characterization of in vitro transcribed RNA from the linearized template.

[0016] Figure 4 It is the cytopathic effect that occurs after cells are transfected with recombinant plasmids.

[0017] Figure 5These are immunofluorescence photographs of the N protein and IL15 or IL18 protein of the rescued virus, wherein A is the immunofluorescence photograph of the N protein and IL15 protein of the recombinant virus rPRRSV-IL15-Y and the cell nuclear staining picture; B is the immunofluorescence photograph of the N protein and IL18 protein of the recombinant virus rPRRSV-IL18-Y and the cell nuclear staining picture.

[0018] Figure 6 It is RT-PCR to detect exogenous gene fragments in recombinant viruses of different generations.

[0019] Figure 7 It is a comparison of the viral titer of the rescued virus strain and the parental virus, specifically a depiction of the growth curve.

[0020] Figure 8 The temperature changes of the experimental and control group pigs after immunization with the recombinant viruses rPRRSV-IL15-Y and rPRRSV-IL18-Y.

[0021] Fig. 9 A is the macroscopic pathological observation of pig organs and tissues. Fig. 9 B is the pathological section comparison.

[0022] Fig.10 This is a graph showing the changes in PRRSV N protein antibodies after vaccination with rPRRSV-IL15-Y, rPRRSV-IL18-Y, and vHuN4-F112.

[0023] Fig.11 This is a graph showing changes in levels of related cytokines (IL-4, IFN-γ, IL-15 and IL-18) after vaccination with rPRRSV-IL15-Y, rPRRSV-IL18-Y and vHuN4-F112. DETAILED DESCRIPTION

[0024] In the present invention, the chimeric recombinant plasmid refers to pPRRSV-IL15-Y and pPRRSV-IL18-Y obtained by inserting the porcine IL15 and IL18 genes into the HuN4-F112 genome backbone using reverse genetic manipulation technology.

[0025] In the present invention, the chimeric recombinant virus refers to the live virus rescued after transfecting MARC-145 cells with the full-length recombinant plasmids pPRRSV-IL15-Y and pPRRSV-IL18-Y obtained by gene chimera technology.

[0026] 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.

[0027] In the present invention, the Genbank accession number of the highly pathogenic PRRSV cell attenuated strain HuN4-F112 is EF635006.

[0028] 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.

[0029] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0030] The experimental methods in the following examples where specific conditions are not specified were generally carried out under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0031] In the examples of the present invention, the plasmids and strains used were: pBlueScript IISK (+) vector purchased from

[0032] Invitrogen, pBS-T vector and TOP10 competent cells were purchased from TIANGENE. RC-145 cells (African green monkey kidney cell line)

[0033] In the examples of the present invention, the plasmids and strains used were: pBlueScript II SK(+) vector purchased from Invitrogen, and pBS-T vector and TOP10 competent cells purchased from TIANGENE.

[0034] 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, T7 mMESSAGE 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.

[0035] In an embodiment of the present invention, MARC-145 monolayer cells are prepared by the following method:

[0036] 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, the cells were washed twice with PBS, and 500 μl of 0.01 MOI virus was added. After adsorption for 1 hour, the adsorption solution was discarded, the cells were washed twice with PBS, and maintenance medium (2% FBS in DMEM) was added and cultured in a 37°C 5% CO2 incubator.

[0037] Example 1 Construction of chimeric recombinant plasmid expressing IL15 or IL18 protein and virus rescue thereof

[0038] 1.1 Construction of recombinant PRRSV plasmids containing IL15 or IL18 genes

[0039] Preliminary experiments have shown that if the sequence of the exogenous gene is not optimized before being inserted into PRRSV, the expression of the exogenous gene in PRRSV will be unstable and will be gradually lost as the recombinant virus is propagated. Therefore, this experiment optimized the codons of IL15 and IL18 by using the codon preference of PPRSV, and then sent them to the company to synthesize plasmids containing the optimized sequences of IL15 or IL18. The two plasmids were double-digested with Asc I and EcoR V. The results are shown in Figure 2A. Then, the large fragment of the parent virus full-length plasmid pHuN4-F112, which had been double-digested with Asc I and EcoR V, was ligated with T4 DNA ligase, and positive clones were screened after sequencing and identification to obtain pPRRSV-IL15-Y and pPRRSV-IL18-Y. The schematic diagram of the plasmid structure is shown in the figure. Figure 1 shown.

[0040] The specific steps are as follows:

[0041] In a 37°C water bath, the plasmid containing the IL15 or IL18 gene and the parental virus full-length plasmid pHuN4-F112 were double-digested with Asc I and EcoRV, respectively. The reaction system was 41 μL of the plasmid containing the IL15 or IL18 gene, 2 μL of Asc I, 2 μL of EcoRV, 45 μL of 10×NEB Buffer; 15 μL of pHuN4-F112, 2 μL of Asc I, 2 μL of EcoRV, 45 μL of 10×NEBBuffer, and 36 μL of ddH2O.

[0042] The exogenous gene fragment and the corresponding vector Asc I and EcoRV double-digested fragment were ligated with T4 DNA ligase at a molar ratio of 3:1, transformed into TOP 10, and independent colonies were picked for pure culture. Plasmid DNA was extracted and subjected to 0.5% gel electrophoresis. A plasmid of about 17 kb was picked for sequencing and positive clones were screened.

[0043] 1.2 Preparation of viral RNA

[0044] 1.2.1 Swa I linearization of plasmid

[0045] In a 37°C water bath, the chimeric recombinant plasmids pPRRSV-IL15-Y and pPRRSV-IL18-Y and the parental plasmid pHuN4-F112 were linearized with Swa I (reaction system: 3 μg of recombinant plasmid, 3 μL of Swa I, 5 μL of cutsmart buffer, and ddH2O was added to a total volume of 50 μL) and digested overnight. The digestion products were purified using QIAquiek PCR Purification Kit according to the instructions to obtain purified linearized plasmids, see Figure 3 A.

[0046] 1.2.2 In vitro transcription

[0047] 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 3 B, In vitro transcribed RNA of pPRRSV-IL15-Y and pPRRSV-IL18-Y was obtained.

[0048] 1.3 Detection of PRRSV recombinant plasmids expressing IL15 or IL18 protein

[0049] 1.3.1 RNA transfection

[0050] MARC-145 cells (African green monkey kidney cell line, purchased from ATCC, USA) were inoculated and cultured in six-well plates. When the cell density was 80-90%, invitro RNA of pPRRSV-IL15-Y and pPRRSV-IL18-Y and 5 μL DMRIF-C transfection reagent were added to each well, respectively, and the mixture was shaken and mixed in 1 mL of Opti-MEM. Then, MARC-145 cells were transfected according to the method in the transfection reagent manual, and the cytopathic effect was observed every day.

[0051] Waiting to appear Figure 4 After the cytopathic effect (CPE) shown in the figure, the supernatant was collected and passaged. The specific process of supernatant collection and passage was as follows: 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 supernatant five days after transfection was aspirated and inoculated with 200 μL of the supernatant in the maintenance medium (DMEM containing 2% FBS) at a ratio of 1:10. The cells were cultured at 37°C and passaged using the above method. The fifth-generation virus supernatant was collected, and then the supernatant virus RNA was extracted according to the operating procedures in the QIAGEN RNA extraction kit to obtain the viruses rPRRSV-IL15-Y and rPRRSV-IL18-Y.

[0052] According to the above results, the obtained recombinant plasmids rPRRSV-IL15-Y and rPRRSV-IL18-Y are infectious and can be successfully transformed from a single genome sequence into active virus particles and have corresponding viral infectivity.

[0053] 1.3.2 Indirect immunofluorescence detection

[0054] According to the RNA transfection steps in Example 1.3.1, the purified 1000-fold diluted viruses rPRRSV-IL15-Y and rPRRSV-IL18-Y were used to infect the monolayer of MARC-145 cells, and 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 PRRSV nucleocapsid protein specific monoclonal antibody (1:800 dilution) and IL15 protein polyclonal antibody or IL18 protein polyclonal antibody (1:500 dilution) 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 5 shown.

[0055] according to Figure 5 Results: rPRRSV-IL15-Y and rPRRSV-IL18-Y showed obvious CPE on the 5th day after transfection. The chimeric viruses rPRRSV-IL15-Y and rPRRSV-IL18-Y were passaged and purified by infinite dilution method, and MARC-145 cells infected with chimeric viruses rPRRSV-IL15-Y and rPRRSV-IL18-Y were detected by indirect immunofluorescence 36 hours later, and specific fluorescence appeared.

[0056] At the same time, RT-PCR technology was used to detect primary (P0) and progeny (P2, P4, P6, P8, P10) viruses. Figure 6 It showed that IL15 gene or IL18 gene can stably exist in the PRRSV genome.

[0057] The above results indicate that the reverse genetics operating system was used to obtain a full-length recombinant plasmid containing IL15 or IL18 proteins with similar growth characteristics to the parental virus vHuN4-F112. It also proves that the insertion of exogenous genes IL15 or IL18 does not affect the growth of the entire virus and is feasible.

[0058] 1.3.3 Determination of virus cell half infectious dose (TCID50)

[0059] The infectivity titer was determined by the 96-well tissue culture plate method with reference 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 virus supernatant collected after the above-mentioned infected cells was serially diluted 10 times with the maintenance medium (DMEM with 2% FBS) and 10 -1 -10 -9The 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 the maintenance solution). 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 .

[0060] 1.3.4 Plotting of multi-step virus growth curves

[0061] MARC-145 cells were infected with low dose (0.01 MOI) viruses (rPRRSV-IL15-Y, rPRRSV-IL18-Y and vHuN4-F112), and the cell culture supernatant was collected at different time periods (12h, 24h, 36h, 48h, 72h, 84h, 96h, 108h) after infection, and the virus titer was determined. The virus collected at each time point was expressed as TCID 50 Calculate the titer and draw a multi-step growth curve of the virus based on the titer of the virus at different time points. The results are as follows: Figure 7 As shown in the figure, the virus forms a peak replication period 36 hours after infection; the difference between the parental virus vHuN4-F112 and the chimeric viruses rPRRSV-IL15-Y and rPRRSV-IL18-Y is not significant. The results show that the viruses rescued by the full-length IL15 or IL18 chimeric recombinant plasmids obtained by reverse genetic manipulation technology have similar biological activities as the parental strains in terms of virus titer at each time point of virus proliferation, exponential growth phase, plateau phase and other growth curves, as shown in Figure 7 .

[0062] The present invention inserts IL15 or IL18 protein between ORF1b and ORF2a on the skeleton of the full-length infectious clone pHuN4-F112 of the highly pathogenic PRRSV cell-passaged attenuated strain, obtains two chimeric plasmids pPRRSV-IL15-Y and pPRRSV-IL18-Y, obtains live viruses through virus rescue, and RT-PCR identification shows that the chimeric viruses can be stably passaged 10 times without deletion. Therefore, the above two chimeric viruses can be used as candidate strains for a novel PRRSV vaccine.

[0063] Example 2 Animal Clinical Trial of Chimeric Virus

[0064] 1.3.5 Animal Immunization Test

[0065] First, 20 experimental pigs were randomly divided into 4 groups and fed separately, namely rPRRSV-IL15-Y immunization group, rPRRSV-IL18-Y immunization group, vHuN4-F112 immunization group, and blank group. The recombinant virus with TCID50 determined was diluted to 10 with serum-free DMEM. 5.0 TCID 50 / mL. Each pig in the immunized group was injected with 2 mL of recombinant virus dilution into the neck muscle, and pigs in the blank group were injected with 2 mL of serum-free DMEM. The body temperature and clinical symptoms of all pigs were monitored and blood samples were collected 5, 10, 15, 20, 25, and 30 days after immunization. The IDEXX porcine reproductive and respiratory syndrome ELISA test kit was used to detect the PRRSV N protein antibody levels in the four groups. The enzyme-linked immunosorbent assay kits of porcine interleukins 18, 15, 4 and interferon IFN-γ from Jianglai Biological Company were used to detect the levels of related cytokines in the four groups.

[0066] The body temperature and mental status of the three immunization groups were normal, and no obvious pathological changes were found in the autopsy. Figure 8 The autopsy results are shown in Fig. 9 , PRRSV antibody levels Fig.10 , cytokine levels are shown in Fig.11 .

[0067] The PRRSV-specific antibody levels of pigs in the immunization group and the mock group were detected. Fig.10 , pigs immunized with rPRRSV-IL15Y, rPRRSV-IL18-Y and vHuN4-F112 were all positive for PRRSV antibodies on the tenth day of immunization. The level of antibodies produced by pigs immunized with rPRRSV-IL15Y was always higher than that of the control group, while the level of antibodies produced by pigs immunized with rPRRSV-IL18Y was not significantly different from that of the control group within 20 days of immunization, and gradually increased from the control group until 30 days after immunization. The PRRSV antibody level in the blank group was always negative.

[0068] After immunizing pigs with PRRSV recombinant vaccine, Fig.11, the results of detecting IL-4 and IFN-γ in serum showed that the IL-4 cytokine level of the vHuN4-F112 immunization group was higher than that of other immunization groups on the tenth day of immunization, while on the 20th and 35th days of immunization, the IL-4 cytokine levels of the vHuN4-F112 immunization group, the rPRRSV-IL15-Y immunization group, and the rPRRSV-IL18-Y immunization group were not much different, but all higher than the control group. Similarly, in the detection of IFN-γ cytokine levels, it was found that the three immunization groups were able to stimulate higher levels of IFN-γ after the 20th day of immunization, and all were higher than the control group. This shows that the recombinant vaccines rPRRSV-IL15-Y and rPRRSV-IL18-Y can stimulate humoral and cellular immune responses in pigs.

[0069] The results of detecting serum cytokine expression levels before immunization and 10 days, 20 days and 35 days after immunization showed that the IL-15 cytokine level in the rPRRSV-IL15 immunization group gradually increased and reached the highest level on the 35th day after immunization. There was no obvious regular change in the IL-15 cytokine levels in other immunization groups and the control group.

[0070] The two chimeric recombinant vaccine strains rPRRSV-IL15-Y and rPRRSV-IL18-Y constructed in this application have shown that while improving the safety of pigs, they can effectively induce immune responses, produce high levels of PRRSV N protein antibodies similar to those of the parent vaccine, and significantly enhance the level of cellular immunity, which is equivalent to an "upgraded version" of the parent vaccine. These vaccines are not only expected to provide long-term and comprehensive protection, but also show significant advantages in overcoming the limitations of traditional vaccine technology. By introducing strategies such as IL15 and IL18, the immune effect and stability of the vaccine are further improved, providing new solutions for the health and production of the pig industry. In the future, the vaccine of this application is expected to play an important role in PRRSV prevention and control and other disease treatment fields, especially in improving immunity and reducing the risk of secondary infections, showing broad application prospects and economic benefits.

[0071] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A porcine reproductive and respiratory syndrome virus recombinant vaccine, characterized in that The porcine reproductive and respiratory syndrome virus recombinant vaccine chimerically expresses the porcine IL15 gene or IL18 gene.

2. The porcine reproductive and respiratory syndrome virus recombinant vaccine as described in claim 1, wherein the porcine reproductive and respiratory syndrome virus recombinant vaccines expressing the porcine IL15 gene or IL18 gene are rPRRSV-IL15-Y and rPRRSV-IL18-Y, respectively, and the recombinant vaccine is obtained by transfecting MARC-145 cells with the recombinant plasmids pPRRSV-IL15-Y and pPRRSV-IL18-Y and then rescuing them.

3. The porcine reproductive and respiratory syndrome virus recombinant vaccine according to claim 2, wherein The full length of pPRRSV-IL15-Y is 16494 bp, wherein the nucleotide sequence at positions 1-9999 is shown in SEQ ID NO.1, and the nucleotide sequence at positions 10000-16494 is shown in SEQ ID NO.2; the full length of pPRRSV-IL18-Y is 16584 bp, wherein the nucleotide sequence at positions 1-9999 is shown in SEQ ID NO.3, and the nucleotide sequence at positions 10000-16584 is shown in SEQ ID NO.

4.

4. A porcine reproductive and respiratory syndrome virus recombinant plasmid, characterized in that The porcine reproductive and respiratory syndrome virus recombinant plasmid chimerically expresses the porcine IL15 gene or IL18 gene.

5. The porcine reproductive and respiratory syndrome virus recombinant plasmid according to claim 4, characterized in that The porcine reproductive and respiratory syndrome virus recombinant plasmids are pPRRSV-IL15-Y and pPRRSV-IL18-Y, wherein the full length of pPRRSV-IL15-Y is 16494 bp, wherein the nucleotide sequence at positions 1-9999 is shown in SEQ ID NO.1, and the nucleotide sequence at positions 10000-16494 is shown in SEQ ID NO.2; the full length of pPRRSV-IL18-Y is 16584 bp, wherein the nucleotide sequence at positions 1-9999 is shown in SEQ ID NO.3, and the nucleotide sequence at positions 10000-16584 is shown in SEQ ID NO.

4.

6. A method for constructing a porcine reproductive and respiratory syndrome virus recombinant plasmid expressing a porcine IL15 gene or IL18 gene, characterized in that: The method comprises the following steps: synthesizing relevant plasmids based on the porcine IL15 gene and the porcine IL18 gene, inserting the IL15 gene or the IL18 gene between ORF1b and ORF2a of HuN4-F112, and inserting the transcriptional regulatory sequence 6 (TRS6) of PRRSV at the downstream of the 3' end of the foreign gene. The two amplified chimeric IL15 fragments and IL18 fragments are double-digested by Asc I and EcoRV, and then the recovered products are connected to the double-digested vector of Asc I and EcoRV of HuN4-F112, thereby obtaining chimeric recombinant plasmids, which are pPRRSV-IL15-Y and pPRRSV-IL18-Y.

7. A method for preparing a recombinant vaccine of porcine reproductive and respiratory syndrome virus, the method being carried out according to the following steps: (1) cell passage and culture for vaccine preparation: MARC-145 cells are digested and passaged with EDTA-trypsin cell dispersion solution, and then cultured with cell growth solution. When a monolayer is formed, the cells are set aside; (2) cell seed virus reproduction: the virus solution is inoculated into a cell bottle with a well-grown cell monolayer at a volume of 1 / 10, adsorbed for 1 hour, and then a cell maintenance solution is added to the cell monolayer, and the culture is continued. When 70-80% of the cells show pathological changes, the virus solution is harvested, and the harvested virus solution is frozen and thawed 2-3 times and stored at below -15°C, and a small amount is taken for semi-finished product inspection; (3) vaccine preparation, packaging and freeze-drying: the virus culture solution that has passed the inspection is mixed with a conventional stabilizer in a container at a volume ratio of 1:1, shaken thoroughly, and quantitatively packaged; each portion contains no less than 10% of the cell venom solution. 5.0 TCID 50 The finished product is obtained by packaging and freeze-drying, wherein the virus is obtained by rescuing the recombinant plasmids pPRRSV-IL15-Y and pPRRSV-IL18-Y.

8. A porcine reproductive and respiratory syndrome virus recombinant vaccine obtained by the preparation method according to claim 7.

9. Use of the porcine reproductive and respiratory syndrome virus recombinant vaccine according to any one of claims 1 to 3 or 8 and / or the porcine reproductive and respiratory syndrome virus recombinant plasmid according to claim 4 or 5 in the preparation of treatment and / or porcine reproductive and respiratory syndrome virus drugs.

10. A pharmaceutical composition, characterized in that The porcine reproductive and respiratory syndrome virus recombinant vaccine as described in any one of claims 1-3 or 8 and\or the porcine reproductive and respiratory syndrome virus recombinant plasmid as described in claim 4 or 5.

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