Construction and rescue of infectious clone of porcine reproductive and respiratory syndrome virus

Abstract

The present application relates to a porcine reproductive and respiratory syndrome virus infectious clone construction and rescue method, and belongs to the technical field of bioengineering. The porcine reproductive and respiratory syndrome virus infectious clone contains a full genome sequence of the porcine reproductive and respiratory syndrome virus, and the nucleotide sequence of the full genome sequence is the 26th to 15384th nucleotide of SEQ ID NO: 1 or a fragment having more than 80% identity with SEQ ID NO: 1 and having the same function. The present application also provides a plasmid pCAGGS-HA-CD163 expressing a porcine CD163 gene. By co-transfecting the aforementioned two plasmids into HEK-293T cells, the virus is rescued in vitro, a first porcine reproductive and respiratory syndrome virus infectious clone rescue platform based on HEK-293T cells is successfully built, and the porcine reproductive and respiratory syndrome virus is successfully rescued.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for constructing and rescuing an infectious clone of porcine reproductive and respiratory syndrome virus. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a major infectious disease in pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is an enveloped, single-stranded, positive-sense RNA virus belonging to the order Heliovirales and family Arterioviridae. It can be divided into two species, PRRSV-1 and PRRSV-2. The PRRSV genome is approximately 15 kb in length and contains at least 10 identified open reading frames (ORFs). ORF1a and ORF1b translate pp1a and pp1ab polyproteins, respectively. After treatment with viral proteases, pp1a and pp1ab release 14 non-structural proteins, including four proteases (NSP1α, NSP1β, NSP2, and NSP4), an RNA-dependent RNA polymerase (NSP9), a helicase (NSP10), and a nuclease (NSP11). ORFs 2-5 encode glycosylated proteins GP2-GP5, ORF6 encodes non-glycosylated membrane proteins (M), ORF7 encodes nucleocapsid proteins (N), ORF2a encodes GP2a protein, ORF2b is located in ORF2a and encodes small, non-glycosylated E proteins, and ORF5a encodes GP5a protein.

[0003] The target cells of PRRSV infection in pigs are porcine alveolar macrophages (PAMs). In vitro, PRRSV can also replicate and proliferate in the African green monkey kidney cell line MARC-145, which is also widely used in scientific research and vaccine production.

[0004] PRRS seriously threatens the healthy development of the global pig industry. Infected pig herds mainly exhibit reproductive disorders in breeding pigs and respiratory diseases in pigs of all ages. The mortality rate of highly pathogenic strains can reach up to 100%. PRRSV is mainly transmitted horizontally through the respiratory tract. Pigs can become infected through intranasal, oral, intramuscular injection, intrauterine, and vaginal contact with PRRSV. Indirect transmission includes transmission through inanimate objects (such as equipment, instruments, and clothing) or substances (such as water and feed), aerosols, and physical carriers such as mosquitoes and flies. In addition, PRRSV in the blood of sows can be transmitted vertically to the fetus through the placenta, resulting in stillbirths, weak piglets, or asymptomatic infected piglets.

[0005] Currently, except for a few original breeding farms and breeding pig farms with good natural barriers that implement "immunization-free" purification, vaccination remains the main means of disease control. Commercially available vaccines are mainly divided into two categories: inactivated vaccines and modified live virus (MLV) vaccines. While inactivated vaccines have good safety, their protective effect is not reliable because they cannot activate specific cellular immunity, and the PRRSV infection process involves a mechanism that allows antibodies to escape neutralization. Since the replication of attenuated MLV strains is similar to that of wild-type strains, the host's immune response to MLV is also similar to that following natural infection with wild-type strains. Previously, the main circulating strains in my country were PRRSV-2 lineages 1, 3, 5, and 8. Currently, commercially available MLV parental strains are mainly lineage 5 VR-2332 and R98 strains, lineage 8 CH-1a classic strain, and lineage 8 highly pathogenic PRRSV (HP-PRRSV) strain. Recently, lineage 1 NADC30-like strains have gradually become the dominant strains in clinical practice in my country, but there are currently no vaccines constructed specifically for lineage 1 strains. Although MLV can successfully induce a protective immune response against homologous viruses, there is still a problem of insufficient cross-protection against heterologous viruses. Furthermore, MLV also poses safety issues such as infection, transmission, immunosuppression, virulence reversion, and gene recombination. Therefore, current products cannot meet clinical needs.

[0006] Reverse genetics is a molecular biology research method that allows scientists to determine the biological function of genes by observing the phenotypic effects of altered genetic material, such as DNA and RNA, after modifying the genetic structure of an organism. This technology can also be used to purposefully alter the genome of viruses; artificially constructing viral strains with different characteristics by rescuing viruses has become an important means of developing novel genetically engineered vaccines.

[0007] Currently, the method for rescuing PRRSV involves transfecting the full-length infectious viral clone plasmid into the MRAC-145 cell line and waiting 3-10 days until cytopathic effect (CPE) appears, or continuing multiple blind passages until CPE occurs. However, due to the low transfection efficiency of MRAC-145 cells, this method suffers from slow production of infectious viral particles and a low success rate in virus rescue. Therefore, this method often requires 3-10 days and multiple repetitions to successfully rescue a single virus. Summary of the Invention

[0008] The technical problem to be solved by this invention is to provide a method for constructing and rescuing an infectious clone of porcine reproductive and respiratory syndrome virus (PRRSV). The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a recombinant vector containing the whole genome sequence of porcine reproductive and respiratory syndrome virus, wherein the whole genome sequence is the genome of strain A or a fragment having more than 80% identity with the genome of strain A and having the same function, and the nucleotide sequence of one strand of the genome of strain A is nucleotides 26 to 15384 of SEQ ID NO:1.

[0011] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0012] In the aforementioned recombinant vector, the nucleotide sequence of the fragment that has more than 80% identity with the genome of strain A and has the same function is nucleotides 26 to 15083 of sequence 2.

[0013] In the above-mentioned recombinant vector, the recombinant vector further includes a backbone, wherein the nucleotide sequence of one strand of the backbone is nucleotides 1-5435 of sequence 3.

[0014] The recombinant vectors mentioned above can be constructed by inserting the genome sequence of the desired strain into the backbone sequence via PCR amplification, enzyme digestion, and homologous recombination.

[0015] Preferably, the specific steps include:

[0016] (1) Double digestion of pcDNA3.1-SP plasmid: The pcDNA3.1-SP plasmid was double digested with restriction endonucleases SbfⅠ and PacⅠ, followed by nucleic acid electrophoresis and fragment recovery and purification to obtain the aforementioned backbone sequence.

[0017] (2) Construction of viral whole genome fragment containing homologous arms: Using primers containing two restriction sites and homologous arms on both sides, viral whole genome DNA fragments with homologous arms are obtained by PCR amplification. The PCR amplification primers are pcDNA3.1-SbfⅠ-PRRSV-F(H) and pcDNA3.1-PacⅠ-PRRSV-R(H).

[0018] (3) Obtaining infectious clonal plasmids: Using the plasmid vector obtained in step (1) and the target fragment obtained in step (2), the desired infectious clonal plasmids are obtained through homologous recombination. A large number of positive plasmids are obtained by transforming competent cells, plating, picking single colonies, sequencing, expanding bacterial culture, and extracting plasmids.

[0019] Secondly, the present invention provides a vector composition comprising a vector expressing a porcine CD163 gene and the above-mentioned recombinant vector, wherein the porcine CD163 gene expression vector contains a gene encoding a CD163 protein, and the CD163 protein is any one of the following:

[0020] A1) The amino acid sequence is sequence 5 of the protein;

[0021] A2) Proteins derived from A1) or with more than 80% identity to the protein shown in A1) by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in Sequence 5.

[0022] A3) A fusion protein with the same function is obtained by fusing a tag protein at the N-terminus and / or C-terminus of A1) or A2).

[0023] In the above vector combination, the nucleotide sequence of the gene encoding the protein is nucleotides 1 to 3333 of sequence 4.

[0024] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0025] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0026] In the above vector composition, the coding sequence of the gene encoding the CD163 protein is nucleotides 1 to 3333 of sequence 4.

[0027] Preferably, the specific steps include:

[0028] (1) Double digestion of pCAGGS-HA plasmid: pCAGGS-HA plasmid was double digested with restriction endonucleases EcoRI and XhoI, followed by nucleic acid electrophoresis and fragment recovery and purification.

[0029] (2) Construction of CD163 protein sequence fragment: RNA was extracted from PAMs cells and reverse transcribed into cDNA. Using the cDNA as a template, the CD163 protein gene was amplified by designing primers to amplify the two segments. The full-length CD163 DNA fragment with homologous arms was obtained by fusion PCR. The PCR amplification primers were EcoRⅠ-CD163-F(H), CD163-SRCR4-R, CD163-SRCR5-F and XhoⅠ-CD163-R(H).

[0030] (3) Obtaining pCAGGS-HA-CD163 plasmid: Using the plasmid vector obtained in step (1) and the target fragment obtained in step (2), the pCAGGS-HA-CD163 plasmid was obtained through homologous recombination. A large number of positive plasmids were obtained by transforming competent cells, plating, picking single colonies, sequencing, expanding bacterial culture, and extracting plasmids.

[0031] Preferably, the CD163 segmental amplification in step (2) is performed using the following method:

[0032] First, primers EcoRⅠ-CD163-F(H) and CD163-SRCR4-R; CD163-SRCR5-F and XhoⅠ-CD163-R(H) were combined to amplify CD163-1 and CD163-2 fragments;

[0033] Next, primers EcoRⅠ-CD163-F(H) and XhoⅠ-CD163-R(H) were combined, and CD163-1 and CD163-2 obtained above were used as templates to amplify the full-length CD163 DNA fragment with homologous arms.

[0034] In the above vector composition, the nucleotide sequence of the vector expressing the porcine CD163 gene is sequence 4.

[0035] Thirdly, the present invention provides a method for rescuing porcine reproductive and respiratory syndrome virus, comprising introducing the aforementioned recombinant vector into cells for virus rescue, and obtaining the virus being porcine reproductive and respiratory syndrome virus.

[0036] In the above method, the cell is a mammalian cell.

[0037] In the above method, the cells are cells that can be infected by porcine reproductive and respiratory syndrome virus (PRRSV), also known as PRRSV-permitted cells.

[0038] In the above method, the PRRSV allows cells including, but not limited to, MARC-145 cells.

[0039] Fourthly, the present invention provides a method for rescuing porcine reproductive and respiratory syndrome virus, comprising introducing the aforementioned recombinant vector into cells for virus rescue, and obtaining the virus as porcine reproductive and respiratory syndrome virus.

[0040] In the above method, the cell is a mammalian cell.

[0041] In the above method, the cells include cells that can be infected by porcine reproductive and respiratory syndrome virus (PRRSV) and cells that cannot be infected by PRRSV. The cells that can be infected by PRRSV are also referred to as PRRSV-permitted cells, and the cells that cannot be infected by PRRSV are also referred to as non-PRRSV-permitted cells.

[0042] In the above method, the non-PRRSV permitted cells include, but are not limited to, HEK-293T cells.

[0043] In the above method, the PRRSV allows cells including, but not limited to, MARC-145 cells.

[0044] The above method includes the following steps: the plasmid expressing the porcine CD163 gene (pCAGGS-HA-CD163) and the aforementioned recombinant plasmid (i.e. the aforementioned vector combination) are co-transfected into HEK-293T cells and cultured at 37°C in a 5% CO2 cell culture incubator for 48 hours to obtain the porcine reproductive and respiratory syndrome virus inserted into the infectious clone from the cell culture medium.

[0045] Fifthly, the present invention provides a method for constructing cells that can be infected by porcine reproductive and respiratory syndrome virus (PRRSV), wherein the aforementioned plasmid expressing the porcine CD163 gene is introduced into recipient cells to obtain cells that can be infected by PRSV.

[0046] The cells in question are cells that cannot be infected by porcine reproductive and respiratory syndrome virus.

[0047] The cells that cannot be infected by porcine reproductive and respiratory syndrome virus include, but are not limited to, HEK-293T cells.

[0048] Sixthly, the present invention provides a vector that can be used to construct a recombinant vector for a reverse genetics platform for porcine reproductive and respiratory syndrome virus. The vector is named pcDNA3.1-SP and can be obtained by the following method: the vector uses pcDNA3.1 as a vector and contains SbfⅠ and PacⅠ restriction sites. The fragment used to insert the restriction sites is obtained by primer annealing, and the primers used are pcDNA3.1-SP-F(H) and pcDNA3.1-SP-R(H).

[0049] Preferably, the specific steps include:

[0050] (1) Double digestion of pcDNA3.1 plasmid: The pcDNA3.1 plasmid was double digested with restriction endonucleases BamHI and XhoI, followed by nucleic acid electrophoresis and fragment recovery and purification.

[0051] (2) Construction of fragments containing SbfⅠ and PacⅠ restriction sites: Using primers containing two restriction sites and homologous arms on both sides, double-stranded DNA fragments are generated by annealing.

[0052] (3) Obtaining pcDNA3.1-SP plasmid: Using the plasmid vector obtained in step (1) and the target fragment obtained in step (2), pcDNA3.1-SP plasmid was obtained through homologous recombination. A large number of positive plasmids were obtained by transforming competent cells, plating, picking single colonies, sequencing, expanding bacterial culture, and extracting plasmids.

[0053] The nucleotide sequence of the above vector (pcDNA3.1-SP) is sequence 3.

[0054] Seventhly, the present invention provides an application of a method for rescuing an infectious clone of porcine reproductive and respiratory syndrome virus in the fields of basic research and vaccine development.

[0055] Preferably, the basic research includes, but is not limited to, reverse genetics studies related to viral gene mutations, deletions, recombinations, and chimeras, as well as the construction of infectious clones of newly isolated viral strains. Preferably, the vaccine development field includes, but is not limited to, vaccine candidate strains constructed using viral modification methods such as mutations, deletions, recombinations, and chimeras.

[0056] The beneficial effects of this invention are: (1) This invention provides a recombinant vector and an infectious clonal plasmid construction method for constructing a reverse genetics operation platform for porcine reproductive and respiratory syndrome virus (PRRSV). The method allows insertion of the recombinant vector into the PRRSV genome via double digestion with SbfⅠ and PacⅠ. (2) This invention provides a transient transfection strategy to transform the non-susceptible PRRSV cell line HEK-293T into a susceptible cell line. By transfecting the PRRSV essential receptor, HEK-293T cells become cells capable of being infected by PRRSV, and this strategy can be applied to other cell lines. (3) This invention provides a rapid and stable method for rescuing the virus based on HEK-293T cells, which can reduce the time required for virus rescue and improve the success rate of virus rescue. (4) The infectious clonal rescue method described in this invention can be applied to various situations requiring the rescue of PRRSV infectious clones, including but not limited to basic research in reverse genetics and the construction of vaccine candidate strains, which is of great significance for the prevention and control of PRRS.

[0057] This invention optimizes the system and, for the first time, applies the HEK-293T cell line, a cell line with high transfection efficiency, to virus rescue. By transfecting the cells with the CD163 receptor, the cells become PRRSV-permitted cells, combining the characteristics of high transfection efficiency and infectivity, thus solving the problems of slow and difficult PRRSV rescue. This invention can not only provide biological materials for reverse genetics research related to the structure and function of PRRSV, but can also be directly used for the construction and development of vaccine candidate strains. Attached Figure Description

[0058] Figure 1 This is a schematic diagram of the pcDNA3.1-SP recombinant vector construction strategy in Example 1;

[0059] Figure 2 This is a schematic diagram of the construction strategy of infectious cloning plasmids pcDNA3.1-CHsx1401 and pcDNA3.1-JXwn06 in Example 1;

[0060] Figure 3 This is a schematic diagram of the pCAGGS-HA-CD163 plasmid construction strategy in Example 2;

[0061] Figure 4 This is a graph showing the IFA and Western Blot detection results of pCAGGS-HA-CD163 plasmid expression in Example 2;

[0062] Figure 5 This is a graph showing the detection results of HEK-293T cells infected with PRRSV after transient transfection with pCAGGS-HA-CD163 in Example 2;

[0063] Figure 6 This is a graph showing the detection results of HEK-293T cells co-transfected with pCAGGS-HA-CD163 and pcDNA3.1-CHsx1401 to rescue the virus in Example 3;

[0064] Figure 7 This is a graph showing the results of comparing the transfection efficiency of MARC-145 cells and HEK-293T cells in Example 4;

[0065] Figure 8 This is a graph showing the detection results comparing the virus rescue cycles of MARC-145 cells and HEK-293T cells in Example 4;

[0066] Figure 9 This is a graph showing the results of comparing the virus rescue success rates of MARC-145 cells and HEK-293T cells in Example 5. Specifically, pcDNA3.1-CHsx1401 includes two plasmid repeats, pcDNA3.1-CHsx1401-1 and pcDNA3.1-CHsx1401-2, and pcDNA3.1-JXwn06 includes two plasmid repeats, pcDNA3.1-JXwn06 and pcDNA3.1-JXwn06. Each plasmid contains three repeats (image arranged horizontally). Detailed Implementation

[0067] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0068] The conventional experimental methods used in the following examples are based on M.R. Green et al.'s *Molecular Cloning: A Laboratory Manual* (4th edition) (Beijing: Science Press, 2017). Instrument usage is governed by the instrument's instruction manual. Unless otherwise specified, all methods are conventional and performed according to the techniques or conditions described in the literature or the product instructions. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0069] Definitions: "Virus rescue cycle" refers to the time required to produce infectious viral particles after virus rescue through transfection; "Virus rescue success rate" refers to the percentage of cell wells that can produce infectious viral particles after virus rescue through transfection out of the total number of transfected wells.

[0070] The restriction endonucleases used in this invention were purchased from New England Biolabs; the Gel Extraction Kit (D2500) was purchased from Omega; the ClonExpress Ultra One Step Cloning Kit and HiScript III All-in-one RT SuperMix Perfect for qPCR reverse transcription kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; the PureYield™ PlasmidMidprep System was purchased from Promega; the Trelief® 5α competent cells were purchased from Beijing Qingke Biotechnology Co., Ltd.; the KOD One PCR polymerase was purchased from TOYOBO; the MagZol RNA extraction reagent was purchased from Guangzhou Meiji Biotechnology Co., Ltd.; and the Gibco DMEM medium, Gibco PRMI-1640 medium, Opti-MEM medium, fetal bovine serum (FBS), and Lipofectamine® LTX&PLUS™ Reagent transfection reagent were purchased from Thermo. Fisher Scientific; penicillin-streptomycin was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; HRP-labeled goat anti-mouse IgG secondary antibody and FITC-labeled goat anti-mouse IgG secondary antibody were purchased from Zhongshan Jinqiao Co., Ltd.; chloroform, isopropanol, anhydrous ethanol, and other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Unless otherwise specified, all reagents used in this invention were used according to the methods described in the instructions.

[0071] The primers and sequencing services used in this invention were provided by Beijing Qingke Biotechnology Co., Ltd.

[0072] The mouse-derived PRRSV N and CD163 protein monoclonal antibodies were prepared by the inventor's institution. They were obtained by immunizing Balb / c mice with purified prokaryotic PRRSV N protein and porcine CD163 protein, and by intraperitoneally injecting mice with specific hybridoma cells obtained by fusing spleen cells with SP2 / 0 cells, followed by collecting ascites fluid.

[0073] The pcDNA3.1 plasmid is preserved by the inventor's institution and described in non-patent literature (Yin L, Liu X, Yao Y, Yuan M, Luo Y, Zhang G, Pu J, Liu P. Gut microbiota-derived butyratepromotes coronavirus TGEV infection through impairing RIG-I-triggered localtype I interferon responses via class I HDAC inhibition. Journal of Virology. 2024 Feb; 98(2):e0137723). It is available to the public from China Agricultural University. This biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0074] pCAGGS-HA and pEGFP-N2 plasmids are described in non-patent literature (Chen M, Zhang X, Kong F, Gao P, Ge X, Zhou L, Han J, Guo X, Zhang Y, Yang H. Senecavirus A inducesmitophagy to promote self-replication through direct interaction of 2C protein with K27-linked ubiquitinated TUFM catalyzed by RNF185. Autophagy. 2024 Jun; 20(6):1286-1313). HEK293T cells, MARC-145 cells, BHK-21 cells, and primary porcine alveolar macrophages (PAMs) were all preserved by the inventors' institution and are available to the public from China Agricultural University. This biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0075] Porcine reproductive and respiratory syndrome virus (PRRSV) strains JXwn06 (EF641008.1) and CHsx1401 (KP861625.1) were isolated and preserved by the Key Laboratory of Animal Epidemiology, Ministry of Agriculture and Rural Affairs, China Agricultural University, and described in non-patent literature (Zhou L, Zhang J, Zeng J, Yin S, Li Y, Zheng L, Guo X, Ge X, Yang H. The 30-amino-acid deletion in the Nsp2 of highly pathogenic porcinereproductive and respiratory syndrome virus emerging in China is not related to its virulence. Journal of Virology. 2009 May; 83(10):5156-5167). These strains are available to the public from China Agricultural University. This biological material is intended solely for repeating experiments related to this invention and should not be used for any other purpose.

[0076] The porcine reproductive and respiratory syndrome virus (PRRSV) strain CHsx1401 (KP861625.1) was isolated and preserved by the Key Laboratory of Animal Epidemiology, Ministry of Agriculture and Rural Affairs, China Agricultural University, and described in non-patent literature (Zhou L, Wang Z, Ding Y, Ge X, Guo X, Yang H. NADC30-like Strain of Porcine Reproductive and Respiratory Syndrome Virus, China. Emerging Infectious Diseases. 2015 Dec; 21(12):2256-2257). It is available to the public from China Agricultural University. This biological material is for the purpose of replicating experiments related to this invention and should not be used for other purposes.

[0077] The genome-wide infectious clonal low-copy plasmid pCMV-JXwn06 was constructed and preserved by the inventor's institution and described in non-patent literature (Song J, Gao P, Kong C, Zhou L, Ge X, Guo X, Han J, Yang H. Thensp2 Hypervariable Region of Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06 Is Associated with Viral Cellular Tropism to Primary Porcine Alveolar Macrophages. Journal of Virology. 2019 Nov; 93(24):e01436-19). It is publicly available from China Agricultural University. This biological material is solely for the purpose of replicating experiments related to this invention and should not be used for any other purpose.

[0078] The genome-wide infectious clonal low-copy plasmid pWSK-CHsx1401 is described in non-patent literature (Kong C, LiD, Hu Y, Gao P, Zhang Y, Zhou L, Ge X, Guo X, Han J, Yang H. The Genetic Variation of Porcine Reproductive and Respiratory Syndrome Virus Replicase Protein nsp2 Modulates Viral Virulence and Persistence. Journal of Virology. 2023 Mar; 97(3):e0168922). It is publicly available from China Agricultural University. This biological material is intended solely for replicating experiments related to this invention and should not be used for any other purpose.

[0079] The following examples used GraphPad Prism 10.4.0 statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and a 2-way ANOVA test was used. P <0.05 (*) P <0.01 (**) P < 0.001 (***) P < 0.0001 (****) indicates a significant difference.

[0080] Example 1: Construction of PRRSV infectious cloning plasmid

[0081] 1. Construction of infectious cloning plasmid recombinant vectors

[0082] according to Figure 1 The construction strategy shown involves digesting the pcDNA3.1 plasmid with restriction endonucleases BamHI and XhoI, followed by 1% agarose gel electrophoresis to excise the bands, purify and recover them, and obtain the linearized vector. 20 μL each of the synthesized complementary primers pcDNA3.1-SP-F(H) and pcDNA3.1-SP-R(H) were mixed and incubated at 95°C for 1 minute. After annealing at the primer melting temperature (Tm) of 76°C for 3 minutes, the mixture was allowed to cool naturally to room temperature to obtain the double-stranded DNA fragment SP(H). This DNA fragment SP(H) contains nucleotides of the SbfⅠ recognition site (cctgcagg), nucleotides of the PacⅠ restriction enzyme recognition site (ttaattaa), and homologous arms on both sides (nucleotides 1 to 16 of pcDNA3.1-SP-F(H) are homologous arms with the same nucleotide sequence as one side of the pcDNA3.1 plasmid, and nucleotides 77 to 95 of pcDNA3.1-SP-F(H) are homologous arms with the same nucleotide sequence as the other side of the pcDNA3.1 plasmid). The SbfⅠ and PacⅠ restriction enzyme sites are very important for the construction of the recombinant plasmid pcDNA3.1-SP because these are restriction enzyme sites in the viral sequence that are not prone to conflict. Homologous recombination of the linearized vector and DNA fragment SP(H) was performed using the ClonExpress Ultra One Step Cloning Kit. Trelief® 5α competent cells were transformed and plated on ampicillin-resistant LB agar plates. After 12 hours of culture, single colonies were picked and sequenced. Successfully sequenced colonies were expanded and plasmids were extracted to obtain recombinant plasmids containing SbfⅠ and PacⅠ restriction sites, named pcDNA3.1-SP, and stored at -20°C.

[0083] pcDNA3.1-SP-F(H): 5'-CTTGGTACCGAGCTCGGATGTAAAACGACGGCCAGTCCACTGCTTAcctgcaggGTTTAGTCTACCttaattaaGGCGAGCATGCATCTAGAGGG-3'.

[0084] pcDNA3.1-SP-R(H): 5'-CCCTCTAGATGCATGCTCGCCttaattaaGGTAGACTAAACcctgcaggTAAGCAGTGGACTGGCCGTCGTTTTACATCCGAGCTCGGTACCAAG-3'.

[0085] The difference between recombinant plasmid pcDNA3.1-SP and plasmid pcDNA3.1 is that pcDNA3.1-SP is a recombinant plasmid obtained by replacing a small fragment between the BamHI and XhoⅠ restriction sites of plasmid pcDNA3.1 with a portion of the DNA fragment SP(H) obtained by annealing pcDNA3.1-SP-F(H) and pcDNA3.1-SP-R(H) (i.e., positions 17-81 of the nucleotide sequence shown in pcDNA3.1-SP-F(H)) while keeping the other nucleotide sequences of plasmid pcDNA3.1 unchanged (specifically, sequence 3).

[0086] 2. Construction of infectious clonal plasmids

[0087] according to Figure 2 The construction strategy shown involves digesting the pcDNA3.1-SP plasmid with restriction endonucleases SbfⅠ and PacⅠ, followed by 1% agarose gel electrophoresis to excise the bands, purify and recover them, and obtain the linearized vector. Using low-copy plasmids pCMV-JXwn06 and pWSK-CHsx1401 containing the complete viral genome as templates (in this example, the low-copy plasmids pCMV-JXwn06 and pWSK-CHsx1401 were used as templates to clone the complete viral genome into a high-copy plasmid for propagation; alternatively, the complete viral genome cDNA could be used as a template for PCR amplification), PCR amplification was performed using pcDNA3.1-SbfⅠ-PRRSV-F(H) and pcDNA3.1-PacⅠ-PRRSV-R(H) as primers. Agarose gel electrophoresis and gel purification yielded the complete viral genome fragments SbfⅠ-JXwn06-PacⅠ(H) (sequence SEQ ID NO:1) and SbfⅠ-CHsx1401-PacⅠ(H) (sequence SEQ ID NO:1) containing SbfⅠ, PacⅠ restriction sites and flanking homologous walls. The linearized vector and fragment were subjected to homologous recombination as described in 1.1 to obtain plasmids, named pcDNA3.1-CHsx1401 and pcDNA3.1-JXwn06. The constructed infectious clonal plasmids were used for virus rescue in subsequent embodiments.

[0088] pcDNA3.1-SbfⅠ-PRRSV-F(H): 5'-CCACTGCTTACCTGCAGGgtttagtATGACGTATAGGTGT-3'.

[0089] pcDNA3.1-PacⅠ-PRRSV-R(H): 5'-GCCCTCTAGATGCATGCTCGCCTTAATTAATTTTTTTTTT-3'.

[0090] The recombinant plasmid pcDNA3.1-JXwn06 sequence consists of a total of 20,794 nucleotides. Nucleotides 1 to 15,359 are shown as nucleotides 26 to 15,384 of SEQ ID NO:1 (i.e., the complete genome sequence of porcine reproductive and respiratory syndrome virus (PRRSV) strain JXwn06, abbreviated as JXwn06. JXwn06 is an RNA virus, and its nucleotides correspond to nucleotides 26 to 15,384 of SEQ ID NO:1). Nucleotides 15,360 to 20,794 are shown as nucleotides 1 to 5,435 of sequence 3 (i.e., the pcDNA3.1-SP backbone sequence).

[0091] The recombinant plasmid pcDNA3.1-CHsx1401 has a total sequence of 20,493 nucleotides and consists of two parts. The sequence of nucleotides 1-15058 is shown as nucleotides 26 to 15083 of sequence 2 (i.e., the whole genome sequence of porcine reproductive and respiratory syndrome virus (PRRSV) strain CHsx1401, abbreviated as CHsx1401. CHsx1401 is an RNA virus, and its nucleotides correspond to nucleotides 26 to 15083 of sequence 2). Nucleotides 15059 to 20493 are shown as nucleotides 1-5435 of sequence 3 (i.e., the pcDNA3.1-SP backbone sequence).

[0092] Example 2: Construction and identification of PRRSV-permitted HEK-293T cells

[0093] 1. Construction and expression of porcine CD163 gene plasmid

[0094] (1) Plasmid construction

[0095] according to Figure 3The construction strategy shown involves digesting the pCAGGS-HA plasmid with restriction endonucleases EcoRI and XhoI, followed by 1% agarose gel electrophoresis to excise the bands, purification, and recovery to obtain the linearized vector. PAMs cell samples were collected, and total RNA was extracted and reverse transcribed into cDNA. CD163-1 was amplified using EcoRI-CD163-F(H) and CD163-SRCR4-R primers, and CD163-2 was amplified using CD163-SRCR5-F and XhoRI-CD163-R(H) primers. After agarose gel electrophoresis and gel purification of the PCR products, PCR amplification was performed using CD163-1 and CD163-2 as templates, with EcoRI-CD163-F(H) and XhoRI-CD163-R(H) primers. Agarose gel electrophoresis and gel purification yielded the target fragment CD163(H) containing EcoRI and XhoRI restriction sites and homologous arms at both ends. Homologous recombination of the linearized vector and the target fragment was performed using the ClonExpress Ultra One Step Cloning Kit. Trelief® 5α competent cells were transformed and plated on LB plates with ampicillin resistance. After 12 hours of culture, single clones were picked and sent for sequencing. The bacterial cultures with correct sequencing were expanded and plasmids were extracted to obtain plasmids containing the porcine CD163 gene, which were named pCAGGS-HA-CD163.

[0096] EcoRI-CD163-F(H): 5'-GTTCCAGATTACGCTGAATTCATGGTGCTACTTGAAGACTCTGGA-3'.

[0097] CD163-SRCR4-R: 5'-CTCCAACCAGCCTGGGTTTCCTGTGGGCTG-3'.

[0098] CD163-SRCR5-F: 5'-CCCAGGCTGGTTGGAGGGGACATTCCCTGC-3'.

[0099] XhoⅠ-CD163-R(H): 5'-AAAAAGATCTGCTAGCTCGAGTCATTGTACTTCAGAGTGGTCTCCTG-3'.

[0100] The recombinant plasmid pCAGGS-HA-CD163 (a total of 8095 nucleotides, sequence 4, of which nucleotides 1 to 3333 are CD163 (encoding amino acid sequence 1110aa, encoded amino acid sequence 5), and nucleotides 3334 to 8095 are the pCAGGS-HA vector sequence).

[0101] (2) Cell transfection

[0102] The experiment was repeated 3 times, with each repetition as follows:

[0103] BHK-21 cells were cultured in DMEM medium containing 10% FBS at a density of 1.2 × 10⁶ cells / year. 6 Cells were seeded at a density of approximately 80% per well in 6-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. Following the instructions of the Lipofectamine® LTX&PLUS™ Reagent transfection reagent, 2.5 μg of pCAGGS-HA-CD163 plasmid was transfected into each well (the control group was transfected with pCAGGS-HA plasmid). Each plasmid was transfected into one well. 24 h after transfection, indirect immunofluorescence assay (IFA) and Western blotting were performed using a CD163 protein monoclonal antibody.

[0104] The results are as follows Figure 4 As shown, the constructed CD163 plasmid can be expressed normally.

[0105] 2. Construction and identification of HEK-293T cells capable of being infected with PRRSV

[0106] The experiment was repeated 3 times, with each repetition as follows:

[0107] HEK-293T cells were cultured in DMEM medium containing 10% FBS at a density of 1.2 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of approximately 80% per well in 6-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. Following the Lipofectamine® LTX&PLUS™ Reagent transfection reagent instructions, 2.5 μg of pCAGGS-HA-CD163 plasmid was transfected into each well (the control group was transfected with pCAGGS-HA plasmid). One well was transfected with each plasmid. After 6 hours of culture, PRRSV CHsx1401 virus was introduced at a multiplicity of infection (MOI) of 0.5. Simultaneously, MARC-145 cells at 100% confluence and untransfected HEK-293T cells were used as cell controls. 24 hours after infection, IFA (infectious viral adhesion) was performed using a PRRSV N protein monoclonal antibody.

[0108] The results are as follows Figure 5As shown, HEK-293T cells that were not transfected with the aforementioned recombinant plasmid pCAGGS-HA-CD163 were non-PRRSV-permitted cells (PRRSV CHsx1401 virus did not proliferate in the cells, so there was no fluorescence during IFA identification), while transfecting HEK-293T cells with the aforementioned recombinant plasmid pCAGGS-HA-CD163 enabled HEK-293T cells to become PRRSV-permitted cells (PRRSV CHsx1401 virus proliferated in the cells, so there was fluorescence during IFA identification).

[0109] Example 3: Construction and Identification of a Virus Rescue Method Based on HEK-293T Cells

[0110] The experiment was repeated 3 times, with each repetition as follows:

[0111] HEK-293T cells were cultured in DMEM medium containing 10% FBS at a density of 1.2 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of approximately 80% per well in 6-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. Following the instructions of the Lipofectamine® LTX&PLUS™ Reagent transfection reagent, 1.25 μg of pCAGGS-HA-CD163 plasmid and 1.25 μg of pcDNA3.1-CHsx1401 plasmid were transfected into each well (the control group was co-transfected with pCAGGS-HA plasmid and pcDNA3.1-CHsx1401 plasmid). One well was transfected with each plasmid group. After 12 h of culture, the medium was replaced with DMEM containing 2% FBS, and the culture was continued until 48 h post-transfection. IFA was performed using PRRSV N protein monoclonal antibody.

[0112] The results are as follows Figure 6 As shown, co-transfection with 1.25 μg of pCAGGS-HA-CD163 plasmid and 1.25 μg of pcDNA3.1-CHsx1401 plasmid can enable PRRSV CHsx1401 virus to proliferate in HEK-293T cells, thus completing the rescue of PRRSV virus.

[0113] Example 4: Comparison of transfection efficiency and rescue cycle of infectious clones in MARC-145 cells and HEK-293T cells.

[0114] 1. Comparison of transfection efficiency between MARC-145 cells and HEK-293T cells

[0115] The experiment was repeated 3 times, with each repetition as follows:

[0116] HEK-293T cells and MARC-145 cells were cultured in DMEM medium containing 10% FBS at a concentration of 1.2 × 10⁻⁶ cells / mL.6 Cells were seeded at a density of approximately 80% per well in 6-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until the cell density reached approximately 80%. Following the instructions for the Lipofectamine® LTX&PLUS™ Reagent transfection reagent, 2.5 μg of pEGFP-N2 plasmid was transfected into each well. After culturing for 24 h, the cells were observed under a fluorescence microscope.

[0117] The results are as follows Figure 7 As shown, HEK-293T cells showed a large amount of EGFP green fluorescence after transfection, while MARC-145 cells showed only a small amount of EGFP protein expression.

[0118] 2. Comparison of virus rescue cycles between MARC-145 cells and HEK-293T cells

[0119] The experiment was repeated 3 times, with each repetition as follows:

[0120] HEK-293T cells and MARC-145 cells were cultured in DMEM medium containing 10% FBS at a concentration of 1.2 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of approximately 80% in 6-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. Following the Lipofectamine® LTX&PLUS™ Reagent transfection reagent instructions, 1.25 μg of pCAGGS-HA-CD163 plasmid and 1.25 μg of pcDNA3.1-CHsx1401 plasmid were transfected into each well. Each cell type was transfected twice. After 12 hours of culture, the medium was replaced with DMEM containing 2% FBS, and cultured for 24, 48, 72, and 96 hours post-transfection. Samples at different post-transfection time points were freeze-thawed at -80°C and then subjected to 50% tissue culture infection dose (TCID) assays using MARC-145 cells. 50 ) detection.

[0121] The results are as follows Figure 8 As shown, compared with the traditional method of rescuing viruses using MARC-145, the virus rescue method constructed in this application can generate infectious virus particles 72 hours earlier, that is, 24 hours after transfection.

[0122] Example 5: Comparison of rescue success rates of infectious clones in MARC-145 cells and HEK-293T cells.

[0123] The experiment was repeated 3 times, with each repetition as follows:

[0124] HEK-293T cells and MARC-145 cells were cultured in DMEM medium containing 10% FBS at a concentration of 1.2 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of approximately 80% per well in 6-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. Following the Lipofectamine® LTX&PLUS™ Reagent transfection reagent instructions, 1.25 μg of pCAGGS-HA-CD163 plasmid and 1.25 μg of either pcDNA3.1-CHsx1401 or pcDNA3.1-JXwn06 plasmids of different clones were transfected into each well. The different clones were named pcDNA3.1-CHsx1401-1, pcDNA3.1-CHsx1401-2, pcDNA3.1-JXwn06-1, and pcDNA3.1-JXwn06-2, respectively. Each cell type was transfected in triplicate. After 12 hours of incubation, the plates were replaced with a solution containing 2% CO2. FBS was cultured in DMEM medium for 48 hours after transfection. The samples were then frozen and thawed at -80°C and inoculated into MARC-145 cells. After 48 hours of inoculation, IFA was performed using PRRSV N protein monoclonal antibody.

[0125] The results are as follows Figure 9 As shown, compared with the traditional method of rescuing viruses using MARC-145, the virus rescue method constructed in this application (co-transfection of pcDNA3.1-CHsx1401 and pCAGGS-HA-CD163 plasmids, or co-transfection of pcDNA3.1-JXwn06 plasmid and pCAGGS-HA-CD163 plasmid) can increase the rescue success rate by 75%, and the rescue success rate of infectious clone plasmids of different clones reaches 100%.

[0126] In summary, this invention provides a method for constructing and rescuing an infectious clone of porcine reproductive and respiratory syndrome virus (PRRSV). The rescue method involves co-transfecting HEK-293T cells with CD163 and an infectious clone plasmid. Compared to traditional methods, the method used in this invention reduces the required rescue cycle and increases the success rate of virus rescue. This invention also provides a recombinant vector and a method for constructing an infectious clone plasmid for building a reverse genetics platform for PRRSV. This method allows for insertion into the PRRSV genome via double enzyme digestion of the recombinant vector and homologous recombination. The infectious clone construction and rescue method described in this invention can be applied to basic reverse genetics research and the construction of vaccine candidate strains, which is of great significance for PRRS prevention and control.

[0127] SbfⅠ-CHsx1401-PacⅠ(H)(Sequence 2)(15113 bp)

[0128]

[0129] pcDNA3.1-SP vector sequence (Sequence 3) (5440bp)

[0130]

[0131] The recombinant plasmid pCAGGS-HA-CD163 sequence (Sequence 4) (8095 bp)

[0132]

[0133] Sequence 5 (Sus scrofa CD163) (1110aa, GenBank: AFR60310.1, update: MAM 21-APR-2015, URL: https: / / www.ncbi.nlm.nih.gov / protein / AFR60310.1 / )

[0134] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for rescuing porcine reproductive and respiratory syndrome virus, characterized in that, The vector expressing the porcine CD163 gene and the recombinant vector containing the complete genome sequence of porcine reproductive and respiratory syndrome virus were co-transfected into HEK-293T cells to rescue the virus, which is porcine reproductive and respiratory syndrome virus. The nucleotide sequence of the vector expressing the porcine CD163 gene is SEQ ID NO:4; The recombinant vector containing the complete genome sequence of porcine reproductive and respiratory syndrome virus is pcDNA3.1-JXwn06 or pcDNA3.1-CHsx1401; The sequence of pcDNA3.1-JXwn06 contains a total of 20,794 nucleotides, of which nucleotides 1 to 15,359 are as shown in nucleotides 26 to 15,384 of SEQ ID NO:1, and nucleotides 15,360 to 20,794 are as shown in nucleotides 1 to 5,435 of SEQ ID NO:

3. The sequence of pcDNA3.1-CHsx1401 consists of 20493 nucleotides, of which the sequence of nucleotides 1 to 15058 is shown as nucleotides 26 to 15083 of SEQ ID NO:2, and nucleotides 15059 to 20493 is shown as nucleotides 1 to 5435 of SEQ ID NO:3.

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