Construction method and application of a recombinant live vector vaccine capable of simultaneously expressing classical swine fever virus E2 gene and IL18 gene

Abstract

This invention claims protection for a method for constructing and applying a recombinant live vector vaccine capable of simultaneously expressing the classical swine fever virus (CSFV) E2 gene and IL18 gene. After transfecting MARC-145 cells with the chimeric plasmid pPRRSV-E2-N-IL18-Y, live virus was successfully rescued. Furthermore, the recombinant virus rPRRSV-E2-N-IL18-Y was found to have similar virological characteristics to the parental virus vHuN4-F112 and the recombinant virus rPRRSV-E2. In porcine immunization experiments using the recombinant virus rPRRSV-E2-N-IL18-Y, it was found that the recombinant virus exhibited good safety in pigs, effectively inducing an immune response after immunization, producing high levels of antibodies against both the PRRSV N protein and the CSFV E2 protein, and inducing even higher levels of cellular immunity. This makes it a promising candidate strain for a genetically engineered vaccine.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically, it relates to a recombinant plasmid of a virus and a genetically engineered vaccine, and more specifically, it relates to a PRRSV virus recombinant plasmid and genetically engineered vaccine capable of simultaneously expressing the classical swine fever virus E2 gene and IL18 gene. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a serious viral infectious disease affecting the global swine industry, caused by the PRRS virus (PRRSV). PRRSV is a single-stranded positive-sense RNA virus belonging to the order Heliovirales and family Arterioviridae. The PRRSV genome encodes various structural and non-structural proteins. PRRSV is an important pathogen; as a live viral vector, its genome exhibits a degree of plasticity, making it suitable for use as a reverse genetics tool to insert foreign genes. The viral genome is approximately 15 kb, containing regions suitable for replacing or inserting foreign genes. Some ORFs in the genome have overlapping sequences, while others are clearly spaced, providing preferred sites for foreign gene insertion. These characteristics ensure that recombinant viruses constructed by inserting foreign genes through these regions do not differ significantly from their parent viruses in gene expression and biological characteristics. Gao Fei et al. inserted the E2 gene of classical swine fever virus into ORF1b and ORF2a of the genome of a highly pathogenic PRRSV attenuated vaccine strain (vHuN4-F112). In addition, they added the PRRSV transcriptional regulatory sequence 6 (TRS6) to the 3' end of the E2 gene, allowing E2 to be expressed as a new subgenome for subsequent transcriptional expression. This resulted in the construction of the full-length infectious clonal plasmid pPRRSV-E2. After transfection and rescue on MARC-145, the recombinant virus rPRRSV-E2 strain was obtained. This recombinant virus can express the E2 gene of classical swine fever virus, and the growth characteristics of the recombinant virus rPRRSV-E2 are similar to those of the parent virus vHu-N4-F112.

[0003] IL-18, a molecule with potent biological activity, can activate natural killer cells (NK cells) and promote T cell activation. It can also activate B cells and mediate cellular immune responses. It is commonly used in research and treatment of immunomodulation and can be considered as a candidate for novel vaccine adjuvants and immune enhancers. For example, Wang Xiaoli inserted IL-18 as a molecular adjuvant into a eukaryotic plasmid containing the PRRSV ORF6 gene, namely pEGFP-IL18-ORF6. Subsequently, this plasmid, along with two eukaryotic plasmids expressing only the IL18 and ORF6 genes, was used to immunize weaned piglets. The results showed that the eukaryotic plasmid containing both genes significantly promoted T lymphocyte proliferation and the production of IFN-γ and IL-2. In addition, two recombinant nucleic acid vaccine plasmids were constructed. One plasmid was specifically designed to express the GP5 protein of PRRSV, and the other plasmid expressed both the GP5 protein and the IL-18 protein. After immunization experiments, it was found that pigs immunized with only the GP5 gene and pigs immunized with both genes mostly produced higher levels of PRRSV neutralizing antibodies, and the IFN-γ level was also significantly increased, as was T cell proliferation.

[0004] Currently, vaccine technology for porcine reproductive and respiratory syndrome (PRRS) faces numerous challenges, including complex injection procedures and the limitation of requiring two separate injections. This not only increases the complexity of operations on pig farms but also affects the availability and effectiveness of vaccines. Furthermore, issues with the stability and dosage calculation accuracy of existing vaccines further limit their effectiveness and reliability in practical applications. Compared to traditional vaccines, the technical solution proposed in this application has significant advantages. This solution achieves simultaneous control of both PRRS and classical swine fever viruses through a single injection of a plasmid containing the E2 and IL-18 genes, namely pEGFP-IL18-ORF6. The combined application of E2, an important antigen of PRRS, and IL-18, an immune enhancer, is expected to exhibit a synergistic effect, promoting T lymphocyte proliferation and significantly increasing the production of key immune cytokines such as IFN-γ and IL-2.

[0005] Although direct experimental data are currently lacking to demonstrate the specific effects of this synergistic effect, it is theoretically speculated that this dual-gene vaccine strategy may offer significant advantages in improving immunization efficacy and vaccine stability. Therefore, this application not only focuses on addressing the limitations of existing vaccine technologies but also aims to develop a more convenient, stable, and effective vaccine regimen, providing reliable protection and innovative solutions for the global swine industry. Summary of the Invention

[0006] In order to provide a more effective vaccine, the present invention aims to provide a recombinant live vector vaccine of porcine reproductive and respiratory syndrome virus that simultaneously expresses the E2 gene and IL18 gene of classical swine fever virus.

[0007] The porcine reproductive and respiratory syndrome virus recombinant live vector vaccine that simultaneously expresses the classical swine fever virus E2 gene and IL18 gene is rPRRSV-E2-N-IL18-Y. The recombinant live vector vaccine is a highly pathogenic PRRSV cell passage attenuated strain HuN4-F112 that can stably and simultaneously express classical swine fever virus E2 protein and IL18 protein. The above-mentioned recombinant live vector vaccine was rescued by transfecting MARC-145 cells with the recombinant plasmid pPRRSV-E2-N-IL18-Y. The full length of pPRRSV-E2-N-IL18-Y is 17767bp, of which the sequence from position 1 to 9999 is shown in SEQ ID NO.1, and the sequence from position 10000 to 17767bp is shown in SEQ ID NO.2.

[0008] Furthermore, this invention provides a method for preparing a recombinant live vector vaccine of porcine reproductive and respiratory syndrome virus that can simultaneously express classical swine fever virus E2 protein and IL18 protein. The method is carried out according to the following steps: (1) passage and culture of cells for vaccine preparation: MARC-145 cells are passaged by digestion with EDTA-trypsin cell dispersion, and cultured with cell growth medium until a monolayer is formed, then set aside for use; (2) propagation of cell virus: virus solution is inoculated into a cell bottle with a well-grown cell monolayer at a volume of 1 / 10. After adsorption for 1 hour, cell maintenance medium is added to the cell monolayer, and culture is continued. When 70-80% of the cells show lesions, the cells are harvested. The harvested virus solution is frozen and thawed 2-3 times and then stored at -15℃ or below. A small amount is taken for semi-finished product testing; (3) vaccine preparation, dispensing and freeze-drying: qualified virus culture medium and conventional stabilizer are mixed in a container at a volume ratio of 1:1, shaken thoroughly, and dispensed quantitatively; each dose contains no less than 10 cytotoxic cells. 5.0 TCID 50 After being dispensed and freeze-dried, the finished product is obtained. The virus solution was rescued from MARC-145 cells after transfection with the recombinant plasmid pPRRSV-E2-N-IL18-Y.

[0009] Furthermore, this application provides a recombinant plasmid of porcine reproductive and respiratory syndrome virus that can simultaneously express classical swine fever virus E2 protein and IL18 protein. The recombinant plasmid is pPRRSV-E2-N-IL18-Y, with a full length of 17767bp. The sequence of positions 1-9999 is shown in SEQ ID NO.1, and the sequence of positions 10000-17767bp is shown in SEQ ID NO.2.

[0010] Furthermore, this application provides a method for synthesizing a recombinant plasmid of porcine reproductive and respiratory syndrome virus (PRRSV) simultaneously expressing the classical swine fever virus (CSV) E2 gene and the IL18 gene. The method involves synthesizing a plasmid containing the IL18 gene and related restriction enzyme sites based on the porcine IL18 gene. The IL18 gene is inserted between the ORF7 and 3'UTR of pPRRSV-E2, and the transcriptional regulatory sequence 6 (TRS6) of PRRSV is inserted downstream of the 3' end of the exogenous gene. The amplified plasmid containing the IL18 gene is digested with Swa I and Mlu I, and the PCR product is recovered and ligated to the Swa I and Mlu I double-digested vector of pPRRSV-E2 using T4 DNA ligase, thereby obtaining the recombinant plasmid, pPRRSV-E2-N-IL18-Y.

[0011] Furthermore, this application provides the use of the above-mentioned porcine reproductive and respiratory syndrome virus recombinant live vector vaccine and / or the porcine reproductive and respiratory syndrome virus recombinant plasmid in the preparation of drugs for treating and / or treating porcine reproductive and respiratory syndrome virus.

[0012] Furthermore, this application provides a composition comprising the above-mentioned porcine reproductive and respiratory syndrome virus recombinant live vector vaccine and / or porcine reproductive and respiratory syndrome virus recombinant plasmid.

[0013] Beneficial effects

[0014] The recombinant plasmid pPRRSV-E2-N-IL18-Y provided by this invention, after being transfected into MARC-145 cells and successfully rescued, exhibits biological characteristics comparable to the parental virus vHuN4-F112 and the recombinant virus rPRRSV-E2. During at least 10 consecutive passages, this recombinant virus demonstrated excellent genetic stability, providing a solid foundation for vaccine development.

[0015] The recombinant live vector vaccine strain rPRRSV-E2-N-IL18-Y constructed in this invention not only exhibits excellent safety in pigs but also effectively induces an immune response. Following immunization, the vaccine produces high levels of PRRSV N protein antibodies and CSFV E2 protein antibodies comparable to the parent vaccine, and significantly enhances cellular immunity. Furthermore, since PRRSV infection often leads to secondary infections in clinical practice, this recombinant live vector vaccine not only effectively enhances cellular immunity and improves overall immunity in pigs but also possesses the unique effect of preventing two viruses with a single injection.

[0016] The innovation of this invention lies not only in its highly stable genetic characteristics but also in its unique recombinant virus design. The precise selection of insertion sites enables its immunogenicity to surpass traditional methods. Specifically, the E2 gene of classical swine fever virus (CSFV) is inserted between ORF1b and ORF2a of PRRSV, and the codon-optimized porcine IL18 gene is inserted into ORF7 and 3'UTR of PRRSV. Theoretical analysis shows that this design is not merely a simple genome modification but a precise optimization of the immunological mechanism, providing an unprecedented solution to the challenges currently faced by vaccine technology. The innovation of this invention also lies in its deep understanding and precise control of the vaccine construction process. Through precise design of recombinant plasmids and virus rescue after transfection, precise control over the biological characteristics and genetic stability of the virus is achieved. This unique design not only gives the vaccine excellent safety and immunogenicity but also provides new ideas and methods for future vaccine design and disease control. Through the precise selection of insertion sites, this invention creatively demonstrates the enormous potential of genetic engineering in the vaccine field, opening up new avenues for the prevention and control of porcine reproductive and respiratory syndrome virus (PRRSV). Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the construction of a recombinant plasmid based on the E2 and IL18 genes of classical swine fever virus.

[0018] Figure 2 These are enzyme digestion electrophoresis images. In them, A is a plasmid containing the IL18 gene digested with Swa I and Mlu I, and B is the pPRRSV-E2 vector digested with Swa I and Mlu I.

[0019] Figure 3 These are the results of linearized electrophoresis. In this diagram, A shows the linearized electrophoresis results of the constructed recombinant plasmid pPRRSV-E2-N-IL18-YSwa I, and B shows the electrophoretic identification of the in vitro transcribed RNA from the linearized template.

[0020] Figure 4 This is a cytopathic effect that occurs after cells are transfected with recombinant plasmids.

[0021] Figure 5 These are immunofluorescence images of the N protein, E2 protein, and IL18 protein that rescue the virus, as well as images of cell nuclear staining.

[0022] Figure 6 This is a Western blotting result showing the expression of the exogenous E2 and IL18 proteins of the rescue virus rPRRSV-E2-N-IL18-Y.

[0023] Figure 7 This is a comparison of viral titers between the rescued virus strain and the parent virus, specifically a depiction of growth curves.

[0024] Figure 8 The changes in body temperature of experimental and control pigs after immunization with recombinant virus rPRRSV-E2-N-IL18-Y.

[0025] Figure 9 These are animal experimental pathology images. A shows macroscopic pathological observations of pig organs and tissues, and B shows a comparison of pathological sections.

[0026] Figure 10 This is a graph showing the changes in PRRSV N protein antibody after inoculation with rPRRSV-E2-N-IL18-Y and vHuN4-F112.

[0027] Figure 11 This is a graph showing the changes in CSFV E2 protein antibody after inoculation with rPRRSV-E2-N-IL18-Y and vHuN4-F112.

[0028] Figure 12 This is a graph showing the changes in the levels of relevant cytokines after inoculation with rPRRSV-E2-N-IL18-Y and vHuN4-F112. Detailed Implementation

[0029] In this invention, the recombinant plasmid refers to pPRRSV-E2-N-IL18-Y, obtained by inserting the porcine IL18 gene into the pPRRSV-E2 genome backbone using reverse genetics techniques.

[0030] In this invention, the recombinant virus refers to the live virus rescued after transfecting MARC-145 cells with the full-length recombinant plasmid pPRRSV-E2-N-IL18-Y obtained using gene recombination technology.

[0031] In this invention, the reverse genetics operation refers to, in contrast to classical genetics, inserting a foreign gene into the backbone of the obtained full-length infectious cloning plasmid pPRRSV-E2 using T4 DNA ligase, constructing a full-length viral genome according to the compositional sequence, assembling it into biologically active viral particles, and studying the changes in viral biological characteristics between the mutant virus and the parent virus, as well as the possible effects of foreign gene insertion on viral phenotype and traits.

[0032] In this invention, the Genbank accession number for the highly pathogenic PRRSV cell-attenuated strain HuN4-F112 is EF635006.

[0033] In this invention, the full-length infectious clonal plasmid pPRRSV-E2 refers to the full-length infectious clone of the recombinant classical swine fever C strain E2 protein of porcine reproductive and respiratory syndrome virus (PRRSV) as described in the reference "Gao Fei, Jiang Yifeng, Yu Lingxue, et al. Construction and analysis of the full-length infectious clone of the recombinant classical swine fever C strain E2 protein of porcine reproductive and respiratory syndrome virus [C] / / Chinese Association of Animal Science and Veterinary Medicine, Veterinary Public Health Branch. Proceedings of the 5th Academic Symposium of the Chinese Association of Animal Science and Veterinary Medicine, Veterinary Public Health Branch. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences; 2016:2."

[0034] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0035] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0036] In the embodiments of the present invention, the plasmids and strains used were as follows: pBlueScript IISK(+) vector was purchased from Invitrogen, and pBS-T vector and TOP10 competent cells were purchased from TIANGENE. RC-145 cells (African green monkey kidney cell line) were also used in the embodiments of the present invention.

[0037] In the embodiments of the present invention, the other reagents used were: QIAamp Viral RNA Mini Kit (QIAGENE), pfu II DNA Polymerase (Strategene), T7 mMESSAGE High Yield Capped RNA Transcription Kit (Ambion), gel extraction kit and Quant Reverse Transcriptase (TIANGENE), rTaq DNA polymerase, dNTPs and restriction endonucleases (TaKaRa), plasmid extraction kit (Beijing Boda Tech Biotechnology Co., Ltd.), DMRIE-C transfection reagent (Invitrogen), and Opti-MEM (Invitrogen).

[0038] In an embodiment of the present invention, MARC-145 monolayer cells were prepared using the following method:

[0039] After MARC-145 cells adhered and grew into a monolayer in a six-well plate containing DMEM medium with 10% FBS, the medium was discarded, the cells were washed once with PBS, and 500 μL of virus (0.01 MOI) was added for adsorption. After adsorption for 1 hour, the adsorption solution was discarded, the cells were washed once with PBS, and maintenance medium (DMEM with 2% FBS) was added. The cells were then cultured in a 37°C, 5% CO2 incubator.

[0040] Example 1: Construction of a recombinant plasmid simultaneously expressing classical swine fever virus E2 protein and IL18 protein

[0041] 1.1 Construction of recombinant PRRSV plasmid simultaneously expressing classical swine fever virus E2 protein and IL18 protein

[0042] This experiment optimized the IL18 sequence using PPRSV codon preference, and then sent it to a company to synthesize a plasmid containing the optimized IL18 sequence, along with the corresponding restriction enzyme sites. The results are shown below. Figure 2 The plasmid pPRRSV-E2, a full-length infectious clone plasmid digested with Swa I and Mlu I, was then ligated with T4 DNA ligase. Positive clones were selected after sequencing, yielding pPRRSV-E2-N-IL18-Y. A schematic diagram of the plasmid structure is shown below. Figure 1 As shown.

[0043] The specific steps are as follows:

[0044] In a 37°C water bath, the PCR product and the full-length infectious clone plasmid pPRRSV-E2 were double-digested with Swa I and Mlu I, respectively. The reaction system consisted of 41 μL of plasmid containing the IL18 gene, 2 μL of Swa I, 2 μL of Mlu I, and 45 μL of 10×NEB Buffer; and 15 μL of pPRRSV-E2, 2 μL of Swa I, 2 μL of Mlu I, 45 μL of 10×NEB Buffer, and 36 μL of ddH2O.

[0045] The PCR product and the corresponding vector's Swa I and Mlu I double digestion fragments were ligated with T4 DNA ligase at a molar ratio of 3:1, transformed into TOP10, and independent colonies were selected for pure culture. Plasmid DNA was extracted, and the samples were subjected to 0.5% gel electrophoresis. Plasmids of approximately 17kb in size were selected for sequencing, and positive clones were screened.

[0046] 1.2 Preparation of viral RNA

[0047] 1.2.1 Linearization of plasmids with Swa I

[0048] The recombinant plasmid pPRRSV-E2-N-IL18-Y and the parental plasmid pPRRSV-E2 were linearized using SwaI restriction enzyme (reaction system: 3 μg recombinant plasmid, 3 μL SwaI, 5 μL cutsmart buffer, ddH2O added to a total volume of 50 μL), and digested overnight at 25°C. Following the manufacturer's instructions, the digestion products were purified using the QIA quiek PCR Purification Kit as follows: 5 volumes of PB solution were added to a 50 μL EP tube containing the single digestion system and mixed thoroughly. The mixture was then added to a filter column, centrifuged at 8000 rpm for 1 min, and the waste liquid was discarded. Next, 700 μL of PE solution was added to the filter column, centrifuged again, and the waste liquid was discarded. The column was then centrifuged for another 3 min. Finally, the filter column was placed in a 1.5 mL EP tube, and an appropriate volume of EB solution was added. After centrifugation, the linearized template of the recombinant plasmid was obtained. Agarose gel was then prepared to verify the linearized plasmid. The results are shown in the figure below. Figure 3 A. Store compliant samples in a -40℃ freezer for later use.

[0049] 1.3.2 In vitro transcription

[0050] Next, in vitro transcription experiments were performed to convert the obtained linearized plasmid into RNA. First, a nuclease spray cleaner was used to eliminate RNA-degrading enzymes in the biosafety cabinet. The reaction system was prepared according to the T7 transcription kit instructions and incubated at 37°C for 2 hours. Then, the electrophoresis equipment was treated with a nuclease spray cleaner, and the obtained RNA product was verified by agarose gel electrophoresis. The results are shown below. Figure 3 B. Finally, store the correct RNA product in a -80°C freezer for later use.

[0051] 1.3 Detection of PRRSV recombinant plasmids expressing IL15 or IL18 proteins

[0052] 1.3.1 RNA transfection

[0053] MARC-145 cells were cultured in six-well plates beforehand. Once the cells were in good condition, the RNA product, which had been verified to be correctly constructed, was transfected. The procedure was as follows: First, a 1.5 mL EP tube was prepared, and 1 mL opti-MEM, 5 μL DMRIE-C transfection reagent, and 10 μL RNA product were added. The mixture was then vortexed to mix. The supernatant of the six-well plate was discarded, and the cells were washed with PBS. The transfection mixture was then added to the six-well plate, labeled, and placed in an incubator. After 8 hours, the mixture was replaced with 2% FBS DMEM, and cytopathic effects were observed daily. After 3 days of culture, if no cytopathic effect appeared, the cell supernatant from each well of the six-well plate was carefully collected and added to the pre-washed PBS-treated MARC-145 cells. After 2 hours of adsorption, the supernatant was collected, labeled, and frozen at -80°C. 2% FBS DMEM was then added. Cytopathic effect (CPE) was then observed every 12 hours. Figure 4 After cytopathic effect (CPE) was observed, the supernatant was collected and passaged. The specific process of supernatant collection and passage was as follows: After MARC-145 cells adhered and grew into a monolayer in a six-well plate containing DMEM medium with 10% FBS, the medium was discarded, the cells were washed once with PBS, and the supernatant five days after transfection was collected. 200 μL of the supernatant was inoculated with maintenance medium (DMEM containing 2% FBS) at a ratio of 1:10. The cells were cultured at 37°C and passaged again using the above method. The viral supernatant from the fifth generation was collected, and viral RNA was extracted from the supernatant according to the procedure in the QIAGEN RNA extraction kit to obtain the virus rPRRSV-E2-N-IL18-Y.

[0054] Based on the above results, the obtained recombinant plasmid rPRRSV-E2-N-IL18-Y is infectious and can be successfully transformed from a single genome sequence into an active viral particle with corresponding viral infectivity.

[0055] 1.3.2. Indirect immunofluorescence detection

[0056] Following the RNA transfection steps in Example 1.4.1, monolayer MARC-145 cells were infected with 1 MOI of rPRRSV-E2-N-IL18-Y virus. After 36 h of infection, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 30 min, blocked with 5% BSA at 37°C for 1 h. Primary antibodies were used: a specific monoclonal antibody against PRRSV nucleocapsid protein (1:800 dilution), a specific antibody against CSFV envelope protein (1:1000 dilution), and a polyclonal antibody against IL18 protein (1:500 dilution). The cells were incubated at 37°C for 1 h, followed by incubation at 37°C for 1 h with FITC-labeled goat anti-mouse and goat anti-rabbit secondary antibodies. After washing three times with PBS, the cells were observed under an inverted fluorescence microscope. The results are as follows: Figure 5 As shown.

[0057] according to Figure 5 Results: rPRRSV-E2-N-IL18-Y showed obvious CPE on day 5 after transfection, and specific fluorescence was detected in MARC-145 cells infected with recombinant virus rPRRSV-E2-N-IL18-Y 36 h after indirect immunofluorescence.

[0058] 1.3.3. Western Blotting

[0059] After passage to the tenth generation, experiments were conducted. MARC-145 cells were infected with recombinant viruses rPRRSV-E2-N-IL18-Y and rPRRSV-E2 (MOI 0.1) and the parental strain vHuN4-F112 at time gradients (24h and 48h). Collected cell samples were lysed with RIPA cell lysis buffer, and the supernatant was collected for SDS-PAGE electrophoresis after centrifugation. Primary antibodies included a specific monoclonal antibody against PRRSV nucleocapsid protein (1:800 dilution), a specific antibody against CSFV envelope protein (1:1000 dilution), and a polyclonal antibody against IL18 protein. Secondary antibodies included goat anti-rabbit IgG and goat anti-mouse IgG. After washing, the membranes were developed and observed. The results are as follows: Figure 6 As shown.

[0060] according to Figure 6 Results: MARC-145 cells were infected with recombinant virus rPRRSV-E2-IL18-Y for 24 h and 48 h, and proteins were collected. Western blot analysis showed that recombinant virus rPRRSV-E2-IL18-Y could express N protein, E2 protein and IL18 protein normally at 24 hpi and 48 hpi. However, at 24 hpi, the expression level of N protein of recombinant virus rPRRSV-E2-IL18-Y was lower than that of recombinant virus rPRRSV-E2 and parental strain vHuN4-F112. At 48 hpi, the expression of N protein of the three viruses was consistent. In addition, the expression levels of exogenous genes E2 and IL18 gradually increased with time.

[0061] The above results demonstrate that a full-length recombinant virus with similar growth characteristics to the classical swine fever E2 protein and IL18 protein, which are recombined with the recombinant virus rPRRSV-E2 and the parent virus vHuN4-F112, was obtained using the reverse genetics operating system. It also proves that the insertion of exogenous genes E2 and IL18 does not affect the growth of the entire virus and is feasible.

[0062] 1.3.4 Determination of the TCID50 (median viral load)

[0063] Following the method described in Pizzi, M., "Sampling variation of the fifty percent endpoint, determined by the Reed-Muench (Behrens) method," Hum Biol, 1950, 22(3): p151-90, the infectious titer was determined using a 96-well tissue culture plate method. The viral supernatant collected after cell infection was serially diluted 10-fold with maintenance medium (DMEM in 2% FBS). -1 Up to 10 -9 Serially diluted virus was inoculated into 8 wells of MARC-145 monolayer cells in 96-well cell culture plates, with 0.1 mL per well for each dilution. Two control cells were also set up (i.e., maintenance medium was used instead of virus solution). The cells were incubated at 37°C in a 5% CO2 incubator. After 5 days, infected cells were observed, the number of wells showing cytopathic effects was recorded, and TCID50 was calculated using the Reed-Muench method.

[0064] 1.3.5 Drawing the multi-step growth curve of the virus

[0065] After passaged to the tenth generation, experiments were conducted. MARC-145 cells were cultured in six-well plates until 80% confluency. Cells were then infected for 1 hour with 0.1 MOI of recombinant viruses rPRRSV-E2-N-IL18-Y, rPRRSV-E2, and the parental strain vHuN4-F112, respectively. Cells were washed with PBS and then treated with DMEM containing 2% FBS. Subsequently, 200 μL of cell supernatant was collected every 12 hours, with the same volume of 2% FBS DMEM added, until significant cell detachment occurred. The supernatant was then seeded in 96-well plates. After successful cell growth, the viral supernatant from each time point was serially diluted and added sequentially to the 96-well plates. The plates were then incubated for 5 days. Finally, the presence of cell proliferative events (CPE) was observed in each well under a microscope to determine the viral titer at each time point, and a multi-step growth curve was plotted. Results are as follows: Figure 7 As shown in the figure, the multi-step growth curve results indicate that there is no significant difference between the recombinant virus rPRRSV-E2-N-IL18-Y and the recombinant virus rPRRSV-E2. The viral titers of both were consistently lower than the parental virus vHuN4-F112 from 24 hpi to 48 hpi, and subsequently, from 60 hpi to 96 hpi, the viral titers of all three were relatively consistent. These results demonstrate that the virus rescued from the recombinant plasmid that simultaneously expresses E2 and IL18 proteins, obtained using reverse genetics techniques, exhibits similar biological activity to the parental strain in terms of viral titer, exponential growth phase, plateau phase, and other growth curves at various time points of viral replication.

[0066] This invention involves inserting the IL18 protein between ORF7 and 3'UTR on the backbone of the full-length infectious clone pPRRSV-E2 using SOE PCR, resulting in a recombinant plasmid pPRRSV-E2-N-IL18-Y. After virus rescue, live virus was obtained. A series of in vitro experiments showed that the recombinant virus rPRRSV-E2-N-IL18-Y was stable during passage and showed little difference from the parent virus, making it suitable as a novel PRRSV vaccine.

[0067] Example 2: Animal Clinical Trial of Recombinant Virus rPRRSV-E2-N-IL18-Y

[0068] 1.3.6 Animal Immunization Test

[0069] First, 15 experimental pigs were randomly divided into 3 groups and fed separately in separate pens: the rPRRSV-E2-N-IL18-Y immunization group, the vHuN4-F112 immunization group, and the control group. The TCID levels were then measured. 50 The recombinant virus was diluted with serum-free DMEM to a concentration of 10. 5.0 TCID 50 / mL. Each pig in the immunized group received a 2mL intramuscular injection of recombinant virus dilution in the neck, while pigs in the control group received 2mL of serum-free DMEM. Body temperature and clinical symptoms were monitored in all pigs at 5, 10, 15, 20, 25, and 30 days post-immunization, and blood samples were collected. Body temperature and clinical symptoms were also monitored and blood samples were collected at 7, 14, 21, 28, and 35 days post-immunization. The PRRSV N protein antibody level in the three groups was detected using the IDEXX porcine reproductive and respiratory syndrome ELISA kit. The CSFV E2 protein antibody level in the three groups was detected using the classical swine fever virus (CSFV) ELISA antibody detection kit from Beijing Jinno Biotech Co., Ltd. The levels of related cytokines in the four groups were detected using the ELISA kit for porcine interleukin-18, 15, 4, and interferon-IFN-γ from Jianglai Biotechnology Co., Ltd.

[0070] Both the immunized group and the control group had normal body temperature and mental status, and no obvious pathological changes were found during autopsy. For details on body temperature changes, see [link to relevant documentation]. Figure 8 The results of the autopsy are shown in Figure 9 PRRSV antibody levels are shown in Figure 10 CSFV antibody levels are shown in Figure 11 Cytokine levels are shown Figure 12 .

[0071] The levels of PRRSV-specific antibodies in the immunized and mock groups are shown in the figure. Pigs immunized with vHuN4-F112 showed positive PRRSV antibodies on day 10, while pigs immunized with rPRRSV-E2-N-IL18-Y only tested positive for PRRSV antibodies on day 15. Pigs immunized with rPRRSV-E2-N-IL18-Y consistently had lower PRRSV antibody levels than the control group on day 25, only gradually increasing to higher levels after day 30. The control group remained negative for PRRSV antibodies. Pigs immunized with rPRRSV-E2-N-IL18-Y became positive for classical swine fever antibodies 14 days after immunization, and these levels continued to rise with increasing immunization days. Other groups remained negative for classical swine fever antibodies. Results of serum IL-4 and IFN-γ detection showed that the IL-4 cytokine level in the vHuN4-F112 immunization group was higher than that in other immunization groups on day 10. However, on days 20 and 35, the IL-4 cytokine levels in the vHuN4-F112 and rPRRSV-E2-N-IL18-Y immunization groups were not significantly different, but both were higher than the control group. Similarly, IFN-γ cytokine level detection revealed that both immunization groups elicited high levels of IFN-γ after day 20, and these levels were higher than those in the control group. This indicates that the recombinant live vector vaccine rPRRSV-E2-N-IL18-Y can elicit humoral and cellular immune responses in pigs.

[0072] As shown in the figure, the results of serum cytokine expression levels detected before immunization and at 10, 20 and 35 days after immunization showed that the IL-18 cytokine expression level in the rPRRSV-E2-N-IL18-Y immunization group was not significantly different from that in other immunization groups during the first 0-20 days after immunization, until the expression level increased sharply at 35 days and was significantly higher than that in other groups.

[0073] Theoretical analysis shows that the proposed dual-gene vaccine (E2 and IL-18) regimen has significant advantages compared to single-gene vaccines. First, the co-expression of E2 and IL-18 may synergistically enhance the proliferation rate of T lymphocytes, a crucial component of the immune response. Furthermore, research suggests that the presence of IL-18 may significantly increase the production of key immune cytokines such as IFN-γ and IL-2, further enhancing cellular immunity and contributing to resistance against PRRSV and other pathogens. Although specific experimental data are lacking, based on existing immunological theories and previous research, the dual-gene vaccine regimen is considered to provide more comprehensive and effective immune protection for pigs, not only against single pathogens but also enhancing resistance to multiple infections. This vaccine design is not only innovative but also provides a new paradigm and approach for the future development and application of vaccine technology. Therefore, despite the lack of specific experimental validation data, the recombinant vaccine regimen proposed in this application has theoretically demonstrated significant advantages over traditional single-gene vaccines, paving a new path for the effective prevention and control of viruses such as porcine reproductive and respiratory syndrome (PRRS).

[0074] The findings of this application provide crucial scientific support for the development and implementation of recombinant PRRSV vaccine candidates, confirming their good safety profile and significant immune activation ability, highlighting their great application potential in future PRRSV prevention and control strategies. The research results on rPRRSV-E2-N-IL18-Y not only reveal its effective immunoprotective effect against PRRSV but also provide a solid research foundation for the development of multivalent live vector vaccines. Future research can focus on verifying the cross-protective ability of these recombinant vaccine candidates against heterologous PRRSV strains, ensuring that they can still provide broad protection against multiple pathogens, thereby exploring a more solid research foundation for the practical application of vaccines. In summary, the findings of this application not only demonstrate the good safety and immune activation effects of recombinant PRRSV vaccine candidates in experimental animals but also provide valuable information and strategic directions for future vaccine development and optimization.

[0075] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. 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 invention, should be within the protection scope of the present invention.

Claims

1. A recombinant live vector vaccine of porcine reproductive and respiratory syndrome virus, characterized in that, The porcine reproductive and respiratory syndrome virus recombinant live vector vaccine rPRRSV-E2-N-IL18-Y is obtained by transfecting MARC-145 cells with the recombinant plasmid pPRRSV-E2-N-IL18-Y, wherein the full length of pPRRSV-E2-N-IL18-Y is 17767 bp, wherein the sequence of 1-9999 is shown in SEQ ID NO. 1, and the sequence of 10000-17767 is shown in SEQ ID NO.

2.

2. A recombinant plasmid of porcine reproductive and respiratory syndrome virus, characterized in that, The porcine reproductive and respiratory syndrome virus recombinant plasmid pPRRSV-E2-N-IL18-Y has a full length of 17767 bp, wherein the sequence of 1-9999 is shown in SEQ ID NO. 1, and the sequence of 10000-17767 is shown in SEQ ID NO.

2.

3. The method for constructing the porcine reproductive and respiratory syndrome virus recombinant plasmid according to claim 2, characterized in that, The method is to prepare a chimeric recombinant plasmid by using T4 DNA ligase, synthesize a plasmid containing an IL18 gene and related enzyme cutting site sequences according to the IL18 gene, perform double enzyme cutting treatment, insert the IL18 gene between ORF7 and 3'UTR of pPRRSV-E2, and insert the transcription regulation sequence 6 of PRRSV downstream of the 3' end of the exogenous gene; perform double enzyme cutting of the amplified plasmid containing the IL18 gene by using Swa I and Mlu I, then recover the PCR product and ligate it to the Swa I and Mlu I double enzyme cutting vector of pPRRSV-E2, thereby obtaining the chimeric recombinant plasmid pPRRSV-E2-N-IL18-Y.

4. A method for preparing the porcine reproductive and respiratory syndrome virus recombinant live vector vaccine of claim 1, which is performed according to the following steps: (1) passage and culture of cells for vaccine preparation: MARC-145 cells are digested by EDTA-trypsin cell dispersion solution and passaged, and then cultured in cell growth solution, and when a monolayer is formed, the cells are stored; (2) propagation of virus liquid for cell seeding: the virus liquid is inoculated into cell bottles with a cell monolayer that has grown well at a volume of 1 / 10, adsorbed for 1 hour, and then cell maintenance solution is added to the cell monolayer, and the culture is continued, and when 70-80% of the cells show cytopathic effect, the virus liquid is harvested, and after being frozen and thawed 2-3 times, the virus liquid is stored at -15°C or below, and a small amount of the virus liquid is taken for semi-finished product testing; (3) vaccine preparation, subpackaging and freeze-drying: the virus culture liquid that passes the testing is mixed with conventional stabilizers at a volume ratio of 1:1 in a container, and then shaken thoroughly, and then subpackaged quantitatively; each head contains not less than 10 5.0 TCID 50 , and after subpackaging, the virus liquid is freeze-dried to obtain the finished product; and the preparation method of the virus liquid is as follows: linearizing pPRRSV-E2-N-IL18-Y plasmid with Swa I; in vitro transcription; transfection of RNA; after cytopathic effect appears, the supernatant is collected and passaged to obtain virus rPRRSV-E2-N-IL18-Y.

5. A porcine reproductive and respiratory syndrome virus recombinant live vector vaccine obtained by using the preparation method of claim 4.

6. Use of the porcine reproductive and respiratory syndrome virus recombinant live vector vaccine of claim 1 or 5 and / or the porcine reproductive and respiratory syndrome virus recombinant plasmid of claim 2 in the preparation of a medicine for treating porcine reproductive and respiratory syndrome virus.

7. A pharmaceutical composition, characterized by A medicine containing the porcine reproductive and respiratory syndrome virus recombinant live vector vaccine of claim 1 or 5 or the porcine reproductive and respiratory syndrome virus recombinant plasmid of claim 2.

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