Construction method and application of a PEDV Nsp6 attenuated vaccine gene mutant strain

Abstract

This invention belongs to the field of biotechnology, and specifically relates to a method for constructing and applying a PEDV Nsp6 attenuated live vaccine mutant strain. Based on previous research on the interaction mechanism between the non-structural protein Nsp6 of the PEDV GDU strain and the innate immune transcription factor IRF3, we identified amino acid regions 220-280 of the Nsp6 protein as the key structural domain mediating this interaction. This invention successfully rescued the recombinant mutant strain rPEDV-ΔNsp6 by directionally knocking out this key structural domain of the Nsp6 protein and using reverse genetics. Compared with the parental strain GDU, the rPEDV-ΔNsp6 strain significantly upregulated the production of type I interferon in IPEC-J2 cells, effectively activating the host's innate immune response, thereby constructing a novel genetically engineered attenuated live vaccine candidate strain with innate immune enhancement function. This candidate strain provides a new technical means and scientific basis for improving the immunoprotective effect of PEDV vaccines and optimizing clinical prevention and control strategies.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for constructing and applying a PEDV Nsp6 attenuated vaccine gene mutant strain. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is the pathogen that causes porcine epidemic diarrhea (PED), characterized by acute watery diarrhea, vomiting, and severe dehydration. PED is particularly harmful to newborn piglets, with a mortality rate as high as 80%-100%. Even if the piglets survive, their growth performance is significantly affected, leading to prolonged time to market and decreased feed conversion ratio. Although the mortality rate is lower in adult pigs, infection can cause problems such as decreased production performance and reduced feed utilization, resulting in huge economic losses to the global pig industry. PEDV belongs to the genus Alphacoronavirus of the family Coronaviridae. It is an enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 28 kb, consisting of a 5'-UTR, a 3'-UTR, and seven open reading frames (ORF1a, ORF1b, ORF2-6). ORF2-6 encode four structural proteins (spiking protein S, envelope protein E, membrane protein M, and nucleocapsid protein N), and the prosthetic protein ORF3. ORF1a and ORF1b encode two replicase-associated polymers, pp1a and pp1ab, which are cleaved by papain-like protease Nsp3 and 3C-like protease Nsp5 to form 16 mature non-structural proteins (Nsp1-Nsp16). These PEDV non-structural proteins are key elements involved in viral replication and transcription complexes, as well as evading the immune system. These genes work synergistically to ensure viral replication, assembly, and infectivity. Numerous studies have confirmed that PEDV can encode multiple IFN signaling pathway antagonistic proteins. These viral proteins target key molecules in the host's innate immune signaling pathway, interfering with the transcriptional activation or signal transduction of IFN-α / β through multiple mechanisms, thereby inhibiting the host's innate immune response and creating conditions for viral replication and spread within the body to escape immune responses. Currently, there are no effective clinical treatments for PEDV infection; prevention mainly relies on vaccination. Currently, traditional PED vaccines used clinically mainly include two types: attenuated live vaccines and inactivated vaccines. The continuous mutation of wild-type PEDV strains is one of the reasons for the rampant spread of PEDV, rendering existing vaccines insufficient to provide adequate protection. Therefore, rapidly developing new vaccines is crucial to blocking the spread of PEDV. Finding and utilizing a reverse genetics system to modify viral virulence genes is an important approach to obtaining attenuated live vaccines. Therefore, in-depth research into the pathogenesis of PEDV, the interaction between the virus and its host, and the development of highly effective vaccines and antiviral drugs are of great significance for controlling the spread of PED, reducing economic losses, and ensuring the sustainable development of the global swine industry.

[0003] Currently, research reports on the knockout or mutation of key immune domains or sites in PEDV are relatively scarce. Existing studies have shown that some attempts to mutate immune-related sites have failed to successfully rescue recombinant viruses; others have only targeted immune sites in single genes and preliminarily explored their functional changes. Therefore, current research on the mechanisms of key immune domains or sites in PEDV remains in the preliminary exploratory stage, and a stable research platform for systematically elucidating these mechanisms has not yet been established, greatly limiting a deeper understanding of the molecular mechanisms of PEDV immune escape and the development of novel vaccines. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing and applying a PEDV Nsp6 attenuated vaccine gene mutant strain.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A PEDV encodes a non-structural protein Nsp6. The nucleotide sequence of the mutated Nsp6 is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0006] Furthermore, the PEDV-encoded non-structural protein Nsp6 is obtained by site-directed knockout mutation of amino acid 220-280 in the domain of the Nsp6 protein in the PEDV GDU strain that interacts with the transcription factor IRF3 in the RLR signaling pathway.

[0007] Another object of the present invention is to provide a PEDV Nsp6 attenuated vaccine gene mutant strain, characterized in that: the Nsp6 in the parent strain is replaced by the above-mentioned mutated non-structural protein Nsp6.

[0008] Another object of the present invention is to provide a recombinant vector carrying the aforementioned mutated non-structural protein Nsp6.

[0009] Another objective of this invention is to provide a method for constructing a PEDV Nsp6 attenuated vaccine gene mutant strain, comprising the following steps: double digestion of the above-mentioned recombinant vector mutant strain with enzymes, and recovery and purification of the double-digested full-length parental vector; homologous recombination of the recovered double-digested product, and screening for positive plasmids; transfection of cells, harvesting positive rescue virus supernatant, and inoculating it into host cells permitted for virus replication to harvest an infectious PEDV attenuated vaccine gene mutant strain.

[0010] Another object of the present invention is to provide the application of one or more of the above-mentioned non-structural protein Nsp6, PEDV attenuated vaccine gene mutant strain, and recombinant vector in the preparation of vaccines for the prevention or treatment of porcine epidemic diarrhea; the PEDV attenuated vaccine gene mutant strain can specifically enhance the activation of the type I interferon innate immune signaling pathway, providing stronger early immune protection for the body.

[0011] The advantages of this invention are as follows: Based on previous research on the interaction mechanism between the non-structural protein Nsp6 of the PEDV GDU strain and the innate immune transcription factor IRF3, this invention identified the 220-280 amino acid region of the Nsp6 protein as the key structural domain mediating this interaction. This invention successfully rescued the recombinant mutant strain rPEDV-ΔNsp6 by directionally knocking out this key structural domain of the Nsp6 protein and using reverse genetics. Compared with the parental strain GDU, the rPEDV-ΔNsp6 strain can significantly upregulate the production of type I interferon in IPEC-J2 cells, effectively activating the host's innate immune response, thereby constructing a novel genetically engineered attenuated vaccine candidate strain with innate immune enhancement function. This candidate strain provides a new technical means and scientific basis for improving the immunoprotective effect of PEDV vaccines and optimizing clinical prevention and control strategies. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the structural domains in which IRF3 interacts with PEDV Nsp6 in this invention.

[0013] Figure 2 This invention relates to the expression and identification of mutant genes. In this diagram, A is a schematic diagram of PCR amplification and identification of the constructed mutant plasmid, and B is a schematic diagram of Western blotting identification using a GFP-tagged antibody.

[0014] Figure 3 This is a diagram showing the effect of the mutant gene afNsp6 on the IFN-β promoter activity in this invention.

[0015] Figure 4 This invention relates to the rescue, verification, and growth kinetics study of rPEDV and rPEDV-ΔNsp6. In this invention, A is a schematic diagram of the proliferation curve, and B is a schematic diagram of the Western Blot identification of PEDV N antibody.

[0016] Figure 5 This is a graph showing the changes in cytokine levels in IPEC-J2 cells infected with rPEDV and rPEDV-ΔNsp6 strains in this invention.

[0017] Figure 6This is a verification diagram of the VSV-GFP bioassay method in IPEC-J2 cells infected with rPEDV and rPEDV-ΔNsp6 strains in this invention. Detailed Implementation

[0018] Example 1 Construction and identification of mutant plasmids Based on our previous research, we have confirmed that the Nsp6 protein in the PEDV GDU (GenBank accession number KU985230) strain interacts with the transcription factor IRF3 in the RLR signaling pathway through domain 220-280. Figure 1 Therefore, we performed a knockout mutation on the key domains 220-280 of the Nsp6 protein. The nucleotide sequence of the mutated Nsp6 is as follows: SEQ ID NO. 1: (SEQ ID NO. 1).

[0019] Its amino acid sequence is: SEQ ID NO. 2: MSGYVSRACRNVLLVGSFLTFFWSELVSYTKFFWVNPGYVTPMFACLSLLSSLLMFTLKHKTLFFQVFLIPALIVTSCINLAFDVEVYNYLAEHFDYHVSLMGFNAQGLVNI FVCFVVTILHGTYTWRFFNTPVSSVTYVVALLTAAYNYFYASDILSCAMTLFASVTGNWFVGAVCYKAAVYMALRFPTFVAIFGDIKSVMFCYLVLGYFTCCFYGILY(SEQ ID NO. 2).

[0020] The mutated gene sequence was synthesized by Genscript Biotech Inc. and named a102(afNsp6). Then, using the synthesized a102 gene as a template, PCR amplification was performed using primers afNsp6-F and afNsp6-R, respectively. After gel electrophoresis, the target gene fragments were recovered using a gel extraction kit.

[0021] afNsp6-F: GAGCTCAAGCTTGAATTCATGAGCGGATACGTGTCTAGA (SEQ ID NO.3); afNsp6-R:ATCCCGGGCCCGCGGTACCGTTCAGTACAGGATGCCGTAGAA (SEQ ID NO. 4).

[0022] The eukaryotic expression vector pcDNA3.1(+)-N-eGFP was double-digested with restriction endonucleases BamHI and XhoI. The total volume of the digestion reaction system was 50 μL, and the specific preparation was as follows: 1 μL each of EcoRI and KpnI, 5 μL of 10× buffer, 3 μg of vector template, and the remaining volume was made up with enzyme-free double-distilled water (ddH2O). The prepared reaction system was incubated at 37℃ for 3 h. After digestion, the fragments were separated by agarose gel electrophoresis, and then purified using a gel extraction kit. The target gene fragment obtained by PCR amplification and the purified vector double-digestion product were used for ligation using homologous recombinase. The total volume of the ligation system was 10 μL, containing 5 μL of homologous recombinase, 4 μL of the target fragment recovery product, and 1 μL of the vector digestion recovery product. The ligation was completed at 50℃ for 15 min. After the ligation reaction, 10 μL of the ligation product was transformed into DH5α competent cells, and 800 μL of antibiotic-free LB medium was added. The cells were then incubated at 37°C in a shaker for 45 min. After incubation, the cells were centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of antibiotic-free LB medium. 100 μL of the resuspended bacterial solution was evenly spread onto LB agar plates containing ampicillin resistance and incubated at 37°C for 12-16 h. Single colonies were picked from the cultured plates and inoculated into LB liquid medium. After incubation at 37°C in a shaker for 4 h, bacterial PCR identification was performed. The results showed that the amplified fragment of the Nsp6 mutant gene cloned into the pcDNA3.1(+)-N-eGFP vector was approximately 660 bp, completely consistent with the expected fragment size. Figure 2 A); Positive clones were sequenced for verification, and the correctly sequenced eukaryotic expression plasmid was named afNsp6. The afNsp6 positive plasmid was transfected into HEK293T cells using Lipo2000. Cell samples were collected 48 h after transfection, and the protein expression of the target gene was detected by Western blot using an HA tag-specific antibody. The results showed that afNsp6 protein was successfully expressed in the transfected cells, with a molecular weight of approximately 59 kDa. Figure 2 B), indicating that the Nsp6 mutant gene afNsp6 can be effectively expressed in HEK293T cells.

[0023] To investigate the effect of mutated genes on IFN-β promoter activity, HEK-293T cells were used as a model, and transfection experiments were performed using Lipofectamine 2000 reagent. In the experimental group, each well was transfected with 0.8 μg of the test plasmid, 0.1 μg of IFN-β-Luc reporter plasmid (for detecting IFN-β promoter activity), and 0.1 μg of β-gal internal control plasmid (for correcting transfection efficiency). Simultaneously, the Nsp6 encoding plasmid of the parental strain GDU and the pcDNA3.1 (+)-N-eGFP empty vector were set as control groups. Twenty-four hours after transfection, cells were stimulated with Sendai virus (SeV) to induce IFN-β expression. Cell samples were collected six hours after stimulation, and luciferase activity was detected according to the instructions of the dual-luciferase assay kit. The results showed that… Figure 3 Compared with Nsp6 of the parent strain GDU, afNsp6 significantly enhanced the inhibitory effect on SeV-induced IFN-β promoter activity. These results indicate that the difference in amino acid positions 220-280 of the PEDV Nsp6 protein is a key factor in altering its inhibitory function on type I IFN expression.

[0024] Using the synthesized a102 gene as a template, PCR amplification was performed using primers A102-F and A102-R (A102-F: CTTTTCCTGAXTGGTACCATGAGCGGATACGTGTCTAGA (SEQ ID NO. 5); A102-R: GCCAGAATGCCAAGAATTCTCAGTACAGGATGCCGTAGAAGCA (SEQ ID NO. 6)). The obtained target fragment was purified by agarose gel electrophoresis. Then, the vector pBAC-GDU containing the full-length GDU gene was digested with restriction endonucleases EcoRI and KpnI at 37℃ for 3 h. The digested vector fragment was then recovered using a gel extraction kit. The recovered PCR amplification product and the double-digested vector were added to a 10 μL homologous recombination reaction system, containing 5 μL of homologous recombinase, 4 μL of the target fragment, and 1 μL of the digested vector. Ligation was performed at 50°C for 15 min. The 10 μL ligation product was then transformed into DH5α competent cells. 800 μL of antibiotic-free LB agar was added, and the cells were incubated at 37°C with shaking for 45 min. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and 100 μL of the bacterial culture was plated on an LB agar plate containing ampicillin. The cells were incubated at 37°C for 12–16 h. Single colonies were picked for enrichment culture, and plasmids were extracted. PCR identification of the bacterial culture was performed using identification primers. Plasmids that tested positive by PCR were sequenced for verification. Plasmids were then extracted using a plasmid extraction kit for subsequent virus rescue.

[0025] Example 2 Biological characteristics analysis of mutant strains The qualified PEDV full-length cDNA clone plasmid was linearized using the restriction endonuclease Mlu I. The total volume of the digestion system was 500 μL, with the following components: 20 μg full-length cDNA plasmid, 10 μL restriction endonuclease Mlu I, 50 μL 10×NEB buffer, and the remaining volume was made up with ddH2O. After incubating the digestion system at 25℃ for 12 h, the products were separated by agarose gel electrophoresis and then purified using a gel extraction kit to obtain the linearized plasmid product. Using the purified linearized plasmid as a template, in vitro transcription was performed using the MEGAscript T7 Plus in vitro transcription kit. The reaction system was incubated in a 37℃ water bath for 2 h. After the reaction, the genomic RNA synthesized in vitro was collected for later use. Simultaneously, cell preparation was performed: Vero CCL-81 cells that had grown into a monolayer were gently washed three times with sterile PBS buffer to remove residual culture medium. The transfection procedure was as follows: 12 μL of Lipofectamine 3000 transfection reagent was added to an EP tube containing Opti-MEM medium and incubated at room temperature for 5 min. Then, the prepared in vitro transcription product was added, gently inverted to mix, and incubated at room temperature to form a transfection complex. This complex was uniformly added to washed Vero CCL-81 cells and cultured at 37°C with 5% CO2 for 6 h. After 6 h of transfection culture, the cell culture supernatant was discarded, and the cells were washed once with sterile PBS buffer. The supernatant was then replaced with DMEM cell maintenance medium containing 2% fetal bovine serum, and cultured daily, observing cytopathic effects. When approximately 80% of the cells showed cytopathic effects under a microscope, the cell culture was frozen at -80°C. The frozen cell culture underwent three freeze-thaw cycles to fully release the viral particles; subsequently, cell debris was removed by centrifugation, and the supernatant was collected for subsequent viral passage culture and identification experiments.

[0026] The rescued mutant strain was passaged in Vero CCL-81 cells for three generations. Viral RNA was extracted from 500 μL of the third-generation viral fluid and then synthesized into cDNA via reverse transcription. Using this cDNA as a template, PCR amplification was performed using afNsp6-ZDF / afNsp6-ZDR primers. The amplified target fragment was cloned into the pBAC-GDU vector and sent to GenScript Biotech Inc. for sequencing analysis. Sequencing results showed that the afNsp6 gene had been successfully inserted into the GDU genome without any amino acid mutations. The correctly identified cDNA clone was named GDU-afNsp6, and the rescued mutant strain was named rPEDV-ΔNsp6.

[0027] afNsp6-ZDF: AGCGGATACGTGTCTAGA (SEQ ID NO.7) afNsp6-ZDR: TCAGTACAGGATGCCGTAGAA (SEQ ID NO.8) The rescued strain and the parental virus GDU were used to infect Vero CCL-81 cells that had formed a cell monolayer in 6-well plates at a dose of 0.1 MOI. 300 μL of cell supernatant was collected at 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h post-infection for viral TCID50 assay, and multi-step viral growth curves were plotted. The results showed that, compared with the parental virus GDU, the replication efficiency of the mutant strain rPEDV-ΔNsp6 was significantly lower at 6 h post-infection. Figure 4 A). Western blot analysis using PEDV N antibody showed that, starting 24 hours post-infection, the N protein expression level of rPEDV-ΔNsp6 was significantly lower than that of wild-type recombinant porcine epidemic diarrhea virus (rPEDV). Figure 4 B) This indicates that the viral genome replication or subgenomic N gene mRNA transcription of rPEDV-ΔNsp6 is generally weak.

[0028] Example 3 Differential analysis of the regulation of type I interferon innate immune signaling pathway by rPEDV-ΔNsp6 strain Cytokines, as an important component of the inflammatory response, play a crucial role in the host cell's innate immunity. To investigate the innate immune response of the mutant strain rPEDV-ΔNsp6, the levels of cytokines in IPEC-J2 cells infected with rPEDV-ΔNsp6 were detected using relative quantitative PCR. The results showed that, compared with rPEDV, rPEDV-ΔNsp6 infection of IPEC-J2 cells significantly increased the transcription levels of type I interferon IFNβ, interferon-induced genes IFIT1, IFIT2, ISG15, and antiviral proteins Mx and OAS1. Figure 5 The above results indicate that infection with the mutant strain rPEDV-ΔNsp6 can induce a specific innate immune response in IPEC-J2 cells.

[0029] Because vesicular stomatitis virus (VSV) is sensitive to type I interferon treatment, it is often used in interferon biological assays. This study verified that VSV-GFP can inhibit IFNβ-induced expression using a recombinant green fluorescent protein expression VSV-GFP bioassay. Supernatants from IPEC-J2 cell cultures infected with wild-type rPEDV or the mutant rPEDV-ΔNsp6 were collected and co-incubated with Vero cells, followed by VSV-GFP infection. Results showed that VSV-GFP proliferated normally in the supernatants of simulated infected cells or wild-type rPEDV-infected cells; however, the supernatant from rPEDV-ΔNsp6-infected cells, even at the highest dilution of 1:32, still inhibited VSV-GFP proliferation. Figure 6 These results indicate that rPEDV-ΔNsp6 can promote the production of type I interferon.

Claims

1. A PEDV-encoded non-structural protein Nsp6, characterized in that: The nucleotide sequence of the mutated Nsp6 is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The non-structural protein Nsp6 as described in claim 1, characterized in that: It was obtained by site-directed knockout mutation of the 220-280 domain of the Nsp6 protein in the PEDV GDU strain, which interacts with the transcription factor IRF3 in the RLR signaling pathway.

3. A PEDV Nsp6 attenuated live vaccine gene mutant strain, characterized in that: In the parental strain, Nsp6 is replaced by the non-structural protein Nsp6 as described in claim 1.

4. A recombinant vector, characterized in that: It carries the non-structural protein Nsp6 as described in claim 1.

5. A method for constructing a PEDV Nsp6 attenuated live vaccine gene mutant strain, characterized in that, The procedure includes the following steps: double-digesting the recombinant vector mutant strain as described in claim 4, and recovering and purifying the full-length parental vector after double digestion; performing homologous recombination on the recovered double-digested product and screening for positive plasmids; transfecting cells, harvesting the positive rescue virus supernatant, and inoculating it into host cells permitted for virus replication to harvest an infectious PEDV attenuated vaccine gene mutant strain.

6. The use of one or more of the following in the preparation of a vaccine for the prevention or treatment of porcine epidemic diarrhea: the non-structural protein Nsp6 of claim 1, the PEDV Nsp6 attenuated vaccine gene mutant strain of claim 3, and the recombinant vector of claim 4.

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