Porcine reproductive and respiratory syndrome replication-defective virus strain and construction method and application thereof

Abstract

The application discloses a porcine reproductive and respiratory syndrome (PRRS) replication-defective virus strain and a construction method and application thereof, and relates to the technical field of biotechnology.The codons of ORF2a and ORF5 of the PRRS virus TA-12 and NL1207 strain are replaced by synonymous mutations, the knockout of E protein and ORF5a protein expression is realized, E and ORF5a are not expressed, meanwhile, the normal expression of GP2a and GP5 proteins is ensured, the virus strain cannot continuously replicate in normal cells, can only proliferate by using a complementary cell line stably expressing E and ORF5a two proteins, and the replication-defective PRRSV with the expression of E protein and ORF5a protein being deleted is successfully rescued, and the complementary susceptible cell line for the proliferation of the replication-defective virus is established.The replication-defective PRRSV provided by the application is expected to become a new PRRSV live vaccine candidate, effectively solves the problems of poor immunization effect and poor safety of the existing PRRSV commercial vaccine, and has a wide application prospect in the livestock production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a porcine reproductive and respiratory syndrome replication-defective virus strain, its construction method, and its application. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious disease caused by infection with porcine reproductive and respiratory syndrome virus (PRRSV). Its typical clinical symptoms mainly include respiratory diseases in pigs of all ages and reproductive disorders in sows. It has caused huge economic losses to the pig industry in countries around the world.

[0003] PRRSV is a single-stranded positive-sense RNA virus belonging to the genus *β-arteritisvirus* of the family Arteritisviridae. Its primary target cell is porcine alveolar macrophages. The PRRSV genome is approximately 15 kb in length, containing 12 open reading frames (ORFs) encoding 8 structural proteins and 16 non-structural proteins. The structural proteins include 4 envelope glycoproteins (GP2a, GP3, GP4, and GP5), 3 non-glycosylated envelope proteins (E, ORF5a, and M), and a nucleocapsid protein (N). The genes encoding these structural proteins are all essential for PRRSV replication and proliferation; none can be omitted. The heteropolymers formed by the envelope proteins, such as GP2a / GP3 / GP4 and GP5 / M, can mediate viral invasion of susceptible cells by binding to cell receptors. It is worth noting that ORF2b is located within ORF2a and encodes the E protein through a different reading frame than ORF2a. The E protein possesses ion channel activity and participates in the formation of the GP2a / GP3 / GP4 / E heteropolymer, but it is not an essential component for viral particle assembly. However, the absence of the E protein can affect viral uncoating, leading to the failure of recombinant virus rescue. The ORF5a gene is 132–156 bases in length, with approximately 93% of its sequence overlapping with the ORF5 gene. It also encodes the ORF5a protein through a different reading frame than ORF5. Currently, the specific function of the ORF5a protein remains unclear.

[0004] Vaccination is one of the most effective measures for the prevention and control of porcine reproductive and respiratory syndrome (PRRS). While existing live attenuated vaccines provide effective homologous protection, their heterologous protection is limited, and they also suffer from problems such as persistent viral load, poor induction of neutralizing antibodies, and virulence reversion. Therefore, there is an urgent need to develop new vaccines with better immunogenicity and higher safety. Replication-deficient viruses, due to the absence of one or more viral genes, have functional defects, and their replication and proliferation depend on complementary cell lines expressing the missing genes. In normal susceptible cells, these viruses can only cause a single round of infection and cannot package to produce infectious viral particles. This characteristic gives them a natural advantage in safety, making them an important direction for new vaccine development. Compared with inactivated vaccines, replication-deficient virus vaccines, due to their single-round infection characteristic, can more effectively activate the body's cellular immune response.

[0005] Scientifically screening for suitable deleted viral genes is crucial for the rational design of replication-deficient PRRS vaccines. Considering the core requirements of replication-deficient vaccines for single-cycle replication and inducing a comprehensive immune response, the preferentially selected deleted genes must simultaneously meet the following three conditions: 1) The protein encoded by the gene is essential for the virus to complete its life cycle; 2) The protein encoded by the gene is not essential for single-cycle viral replication, and its inactivation will not affect the single-cycle replication process of the defective virus; 3) The protein encoded by the gene is not an important protective antigen (or does not contain neutralizing epitopes), and its inactivation will not impair the ability of the defective virus to induce a protective immune response in the body.

[0006] Previous studies have shown that PRRSV non-structural proteins (nsp1-nsp12) and the N protein together constitute the replication transcriptase complex, regulating viral RNA biosynthesis. The absence of any one of these proteins prevents the virus from completing a single round of replication. PRRSV nsp2TF and nsp2N are non-essential genes for PRRSV to complete its life cycle. Furthermore, it is well known to those skilled in the art that CD163 is a key receptor mediating PRRSV invasion of susceptible cells, and CD163 knockout pigs are completely resistant to PRRSV infection. Further research indicates that the heterotrimer formed by the minor structural proteins GP2a, GP3, and GP4 can interact with CD163 to mediate viral invasion, while the heterodimer formed by GP5 and M also participates in this invasion process. Multiple animal immunization experiments have confirmed that GP4 and GP5 contain neutralizing antigenic epitopes, and subunit vaccines and viral vector vaccines based on GP3, GP4, GP5, and M can induce high levels of neutralizing antibody responses in the body. Therefore, it can be inferred that GP2a, GP3, GP4, GP5, and M envelope proteins are all major protective antigens of PRRSV, and the deletion of any one of these genes will affect the protective immune-inducing ability of replication-defective virus vaccines. Other studies have confirmed that ORF2 or ORF4 knockout replication-defective PRRSV vaccine candidates constructed using gene sequence deletion strategies provide very limited immune protection. In summary, in the design of replication-defective PRRSV vaccines, only the ORF2b and ORF5a structural protein genes can meet the three conditions for preferential selection of deleted genes.

[0007] Currently, deleting the target gene sequence and inactivating the target gene start codon are two common strategies for constructing replication-deficient viruses, which can usually achieve the knockout of specific viral proteins. However, both strategies have unavoidable problems when applied to the construction of replication-deficient PRRSVs. Studies have found that PRRSV is one of the RNA viruses with the highest genome mutation rate, estimated to be 4.71~9.8×10⁻² / base / year. If a strategy of inactivating the start codon through single-point mutation is used to construct replication-deficient PRRSVs, the introduced 1-2 base mutation is highly likely to undergo reversion mutation during viral replication, causing the defective virus to regain pathogenicity.

[0008] Furthermore, a typical feature of the PRRSV genome structure is the varying degrees of overlap and sharing among different structural protein genes, including ORF2a / ORF2b, ORF2a / ORF3, ORF3 / ORF4, ORF5a / ORF5, ORF5 / ORF6, and ORF6 / ORF7. Specifically, ORF2b is the 5' end sequence of ORF2a, and approximately 93% of the sequence of ORF5a overlaps with ORF5. Simultaneously, PRRSV transcribes structural protein genes (ORF2-ORF7) through a discontinuous transcription mechanism unique to nested viruses. The resulting subgenomic mRNAs 2-7 share terminal sequences, encoding structural proteins such as GP2a / E, GP3, GP4, ORF5a / GP5, M, and N. This discontinuous transcription process is strictly regulated by the transcription regulating sequences (TRS) specific to each structural protein gene, and these TRS are all located in the coding regions of upstream genes. Therefore, deleting the ORF2b or ORF5a sequence will not only knock out the expression of the E protein or ORF5a protein, but will also inevitably affect the normal expression of their overlapping genes (GP2a or GP5), thereby destroying the immunogenicity of the virus.

[0009] For the reasons mentioned above, neither of the existing strategies—deleting the target gene sequence and inactivating the target gene start codon—is suitable for constructing replication-deficient PRRSVs. Therefore, providing a strategy suitable for constructing replication-deficient PRRSVs is crucial for the prevention and treatment of porcine reproductive and respiratory syndrome (PRRS). Summary of the Invention

[0010] This invention addresses the technical challenges in the construction of existing PRRS vaccines by providing a porcine reproductive and respiratory syndrome replication-defective virus strain, its construction method, and its application. By employing a synonymous codon substitution strategy, it cleverly overcomes the shortcomings of existing technologies and is expected to provide important strategic support and theoretical basis for the development of novel PRRS vaccines.

[0011] Therefore, in a first aspect, the present invention provides a method for constructing an infectious clonal plasmid of a porcine reproductive and respiratory syndrome (PRRS) replication-defective virus, comprising: (1) Synonymous mutations were performed on the codons overlapping with the ORF2b gene in the ORF2a gene of the parent porcine reproductive and respiratory syndrome virus strain and the codons overlapping with the ORF5a gene in the ORF5 gene, respectively, to synthesize template sequence 1 for replacing the ORF2b gene and template sequence 2 for replacing the ORF5a gene. (2) Using the initial vector containing the full-length cDNA of the parent porcine reproductive and respiratory syndrome virus strain as the backbone, gene replacement was performed sequentially through two rounds of reverse genetic operations to obtain an infectious clonal plasmid of porcine reproductive and respiratory syndrome replication defect virus.

[0012] Further, the parent porcine reproductive and respiratory syndrome virus strain mentioned in step (1) is either the TA-12 strain or the NL1207 strain; When the parent porcine reproductive and respiratory syndrome virus strain is TA-12, the nucleotide sequence of template sequence 1 is as shown in SEQ ID NO:1, and the nucleotide sequence of template sequence 2 is as shown in SEQ ID NO:2; When the parent porcine reproductive and respiratory syndrome virus strain is NL1207, the nucleotide sequence of template sequence 1 is shown in SEQ ID NO:3, and the nucleotide sequence of template sequence 2 is shown in SEQ ID NO:4.

[0013] Further, in step (2), when the parent porcine reproductive and respiratory syndrome virus strain is TA-12, the initial vector is pCMV-TA-12M, and the nucleotide sequence of pCMV-TA-12M is shown in SEQ ID NO:5; The two rounds of reverse genetic operations include: (A) PCR amplification was performed using the template sequence 1 and the initial vector pCMV-TA-12M as templates to obtain two viral genome fragments. The pCMV-TA-12M was double-digested to obtain an infectious clone backbone. The fragments were homologously recombinated with the infectious clone backbone to obtain the recombinant plasmid pCMV-TA-dE. The PCR amplification primers for template sequence 1 are TA-BsaBI-F with nucleotide sequences as shown in SEQ ID NO:6 and TA-dE-R with nucleotide sequences as shown in SEQ ID NO:7; the PCR amplification primers for pCMV-TA-12M are TA-dE-F with nucleotide sequences as shown in SEQ ID NO:8 and TA-EcoRV-R with nucleotide sequences as shown in SEQ ID NO:9. (B) Two amplified fragments were obtained by PCR amplification using the template sequence 2 and the recombinant plasmid pCMV-TA-dE as templates, respectively. The pCMV-TA-dE was double-digested to obtain a recombinant infectious clone backbone. The amplified fragments were homologously recombinated with the recombinant infectious clone backbone to obtain the infectious clone plasmid pCMV-TA-d5aE. The PCR amplification primers for the template sequence 2 are TA-BsrGI-F with nucleotide sequences as shown in SEQ ID NO:10 and TA-d5a-R with nucleotide sequences as shown in SEQ ID NO:11; the PCR amplification primers for the recombinant plasmid are TA-d5a-F with nucleotide sequences as shown in SEQ ID NO:12 and TA-NotI-R with nucleotide sequences as shown in SEQ ID NO:13.

[0014] Further, in step (2), when the parent porcine reproductive and respiratory syndrome virus strain is NL1207, the initial vector is pCMV-NL1207, and the nucleotide sequence of pCMV-NL1207 is shown in SEQ ID NO:14; The two rounds of reverse genetic operations include: (A) PCR amplification was performed using the template sequence 1 and the initial vector pCMV-NL1207 as templates to obtain two viral genome fragments. The pCMV-NL1207 was double-digested to obtain an infectious clone backbone. The fragments were homologously recombinated with the infectious clone backbone to obtain the recombinant plasmid pCMV-NL-dE. The PCR amplification primers for template sequence 1 are BciVI-F with nucleotide sequences as shown in SEQ ID NO:15 and NL-ER with nucleotide sequences as shown in SEQ ID NO:16; the PCR amplification primers for pCMV-NL1207 are NL-EF with nucleotide sequences as shown in SEQ ID NO:17 and HpaI-R with nucleotide sequences as shown in SEQ ID NO:18. (B) Using the template sequence 2 as a template, PCR amplification was performed to obtain the amplified fragment. The pCMV-NL-dE was double-digested to obtain the recombinant infectious clone backbone. The amplified fragment and the recombinant infectious clone backbone were homologously recombinated to obtain the infectious clone plasmid pCMV-NL-d5aE. The PCR amplification primers for template sequence 2 are SwaI-F with nucleotide sequences as shown in SEQ ID NO:19 and PsiI-R with nucleotide sequences as shown in SEQ ID NO:20.

[0015] A second aspect of the present invention provides a method for constructing a complementary cell line stably expressing porcine reproductive and respiratory syndrome virus (PRRSV) replication-defective virus (PRS) E protein and ORF5a protein, comprising: S1: Design amplification primers based on the ORF2b and ORF5a gene sequences of the TA-12 strain described above; S2: Using the recombinant plasmid pCMV-TA-dE as a template, the ORF2b fragment was amplified by PCR. The ORF2b fragment was ligated with the double-digested pLVX-CMV-TEVp vector fragment and transformed into E. coli to obtain the recombinant plasmid pLVX-CMV-PRRSV-E. S3: Using the recombinant plasmid pLVX-CMV-PRRSV-E as a template, the PuroR fragment was amplified by PCR, and the ORF5a fragment was amplified by PCR using the initial vector pCMV-TA-12M as a template. The PuroR fragment, the ORF5a fragment, and the double-digested recombinant plasmid pLVX-CMV-PRRSV-E fragment were homologously recombined and transformed into E. coli to obtain the recombinant plasmid pLVX-CMV-PRRSV-5aE. S4: The recombinant plasmid pLVX-CMV-PRRSV-5aE, helper plasmid psPAX2, and helper plasmid pVSV-G were co-transfected into HEK-293T cells to obtain lentivirus. The lentivirus was then transduced into HEK-293T cells and MARC-145 cells, respectively, and screened to obtain complementary cell lines HEK-293T-PRRSV-5aE and MARC-145-PRRSV-5aE, which stably express the E protein and ORF5a protein of porcine reproductive and respiratory syndrome virus replication defective virus.

[0016] Further, the PCR amplification primers for the ORF2b fragment are lvx-ER with nucleotide sequences as shown in SEQ ID NO:21 and lvx-EF with nucleotide sequences as shown in SEQ ID NO:22; the PCR amplification primers for the PuroR fragment are puro-5a-F1 with nucleotide sequences as shown in SEQ ID NO:23 and puro-5a-R1 with nucleotide sequences as shown in SEQ ID NO:24; the PCR amplification primers for the ORF5a fragment are puro-5a-F2 with nucleotide sequences as shown in SEQ ID NO:25 and puro-5a-R2 with nucleotide sequences as shown in SEQ ID NO:26; and the nucleotide sequence of the pLVX-CMV-TEVp vector is shown in SEQ ID NO:27.

[0017] A third aspect of the present invention provides a porcine reproductive and respiratory syndrome (PRRS) replication-defective virus strain, wherein the method for constructing the strain includes: The infectious clonal plasmid of the porcine reproductive and respiratory syndrome replication-defective virus obtained by the construction method was transfected into the complementary cell line HEK-293T-PRRSV-5aE obtained by the construction method to obtain the P0 generation virus. The P0 generation virus is inoculated into the complementary cell line MARC-145-PRRSV-5aE obtained by the construction method of claim 5 or 6 for amplification to obtain the porcine reproductive and respiratory syndrome replication defect virus strain rTA-d5aE or rNL-d5aE.

[0018] Furthermore, the rTA-d5aE is obtained by replacing the codons overlapping with the ORF2b gene in the ORF2a gene of the TA-12 strain with the nucleotide sequence shown in SEQ ID NO:28, and replacing the codons overlapping with the ORF5a gene in the ORF5 gene of the TA-12 strain with the nucleotide sequence shown in SEQ ID NO:29. The rNL-d5aE is obtained by replacing the codons overlapping with the ORF2b gene in the ORF2a gene of the NL1207 strain with the nucleotide sequence shown in SEQ ID NO:30, and replacing the codons overlapping with the ORF5a gene in the ORF5 gene of the NL1207 strain with the nucleotide sequence shown in SEQ ID NO:31.

[0019] A fourth aspect of the invention provides the use of the aforementioned porcine reproductive and respiratory syndrome replication defect virus strain rTA-d5aE or rNL-d5aE in the preparation of products for the diagnosis, prevention or treatment of porcine reproductive and respiratory syndrome.

[0020] In a fifth aspect, the present invention provides a porcine reproductive and respiratory syndrome (PRRS) vaccine comprising the aforementioned PRRS replication-defective virus strain rTA-d5aE or rNL-d5aE and a pharmaceutically acceptable vector.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects: To address the technical problems in the construction of porcine reproductive and respiratory syndrome (PRRS) vaccines in existing technologies, this invention provides a PRRS replication-defective virus strain, its construction method, and its application. By replacing the codons of ORF2a and ORF5 in PRRS virus strains TA-12 and NL1207 with synonymous mutations, and utilizing numerous introduced point mutations, the expression of E and ORF5a proteins is knocked out, resulting in the absence of expression of both E and ORF5a, while ensuring the normal expression of GP2a and GP5 proteins. Therefore, the virus strain of this invention cannot replicate continuously in normal cells and can only proliferate using complementary cell lines that stably express both E and ORF5a proteins.

[0022] Based on reverse genetics, this invention successfully rescued replication-deficient PRRSV with missing expression of E and ORF5a proteins and established a complementary susceptible cell line for the proliferation of this replication-deficient virus. This research is expected to provide important strategic support and theoretical basis for the development of novel PRRS vaccines.

[0023] Experiments have confirmed that the replication-deficient PRRSV vaccine strain provided by this invention replicates normally and exhibits a high viral titer in MARC-145 cell lines stably expressing E and ORF5a. In MARC-145 cell lines, it can only undergo one infection and replication, and cannot package infectious viral particles. This viral strain shows promise as a candidate for novel PRRSV live vaccines, effectively addressing the problems of poor immunogenicity and safety in existing commercial PRRSV vaccines, and has broad application prospects in livestock production. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0025] Figure 1 This is a single-round infection pattern diagram of a replication-defective porcine reproductive and respiratory syndrome virus provided in an embodiment of the present invention; Figure 2 A schematic diagram of the genome of a replication-defective porcine reproductive and respiratory syndrome virus provided in an embodiment of the present invention; Figure 3 A map of the lentiviral plasmid pLVX-CMV-PRRSV-5aE that simultaneously expresses PRRSV E and ORF5a, provided in Example 2 of this invention; Figure 4 This is an image showing the immunofluorescence detection results of the MARC-5aE monoclonal complementary cell line infected with rTA-d5aE, as provided in Example 2 of this invention. Figure 5 The image shows the results of indirect immunofluorescence detection of nsp2 protein in MARC-145 after two consecutive passages of successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rTA-d5aE provided in Example 3 of the present invention. Figure 6 The image shows the results of indirect immunofluorescence detection of nsp2 protein after two consecutive passages of the successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rNL-d5aE in MARC-145, as provided in Example 3 of the present invention. Figure 7The image shows the results of indirect immunofluorescence detection of nsp2 protein in porcine alveolar macrophages and MARC-145 cells after two consecutive passages of successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rNL-d5aE provided in Example 3 of the present invention, in which the virus was successfully rescued and passaged twice in porcine alveolar macrophages and MARC-145 cells. Figure 8 The image shows the ORF2b sequence alignment results between the successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rTA-d5aE and the wild-type virus provided in Embodiment 4 of the present invention. Figure 9 The image shows the sequence alignment results of the successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rTA-d5aE and the wild-type virus ORF5a, provided for Embodiment 4 of the present invention. Figure 10 The image shows the sequence alignment results of the successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rNL-d5aE and the wild-type virus provided in Embodiment 4 of the present invention, along with the ORF2b sequence. Figure 11 The image shows the sequence alignment results of the successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rNL-d5aE and the wild-type virus provided in Embodiment 4 of the present invention; Figure 12 The multi-step growth curve results of the successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rTA-d5aE and wild-type virus in MARC-5aE cells provided for Embodiment 5 of the present invention; Figure 13 The multi-step growth curve results of the successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rNL-d5aE and wild-type virus in MARC-5aE cells provided for embodiment 5 of the present invention; Figure 14 Figure 1. ORF2b sequence alignment results of successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rTA-d5aE P1 and P20 provided for embodiment 6 of the present invention; Figure 15 The ORF5a sequence alignment results of P1 and P20 of the successfully rescued replication-defective porcine reproductive and respiratory syndrome virus rTA-d5aE provided in Embodiment 6 of the present invention are shown in Figure 6. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] A first aspect of this invention provides a method for constructing an infectious clonal plasmid of a porcine reproductive and respiratory syndrome (PRRS) replication-defective virus, comprising: (1) Synonymous mutations were performed on the codons overlapping with the ORF2b gene in the ORF2a gene of the parent porcine reproductive and respiratory syndrome virus strain and the codons overlapping with the ORF5a gene in the ORF5 gene, respectively, to synthesize template sequence 1 for replacing the ORF2b gene and template sequence 2 for replacing the ORF5a gene. (2) Using the initial vector containing the full-length cDNA of the parent porcine reproductive and respiratory syndrome virus strain as the backbone, the gene was replaced sequentially through two rounds of reverse genetic operations to obtain an infectious clone plasmid of porcine reproductive and respiratory syndrome virus replication defect.

[0032] The infectious clonal plasmid of porcine reproductive and respiratory syndrome virus (PRRSV) replication-defective virus constructed in this invention replaces the codons of the ORF2a and ORF5 genes of PRRSV strains TA-12 and NL1207 with synonymous mutations, precisely knocking out the expression of E protein and ORF5a protein, while ensuring the normal expression of GP2a and GP5 proteins. This prevents the virus strain from replicating continuously in normal cells and allows it to proliferate only using complementary cell lines that stably express E and ORF5a proteins, greatly improving biosafety.

[0033] In some embodiments, the parent porcine reproductive and respiratory syndrome virus strain in step (1) is either the TA-12 strain or the NL1207 strain; When the parent porcine reproductive and respiratory syndrome virus strain is TA-12, the nucleotide sequence of template sequence 1 is shown in SEQ ID NO:1, and the nucleotide sequence of template sequence 2 is shown in SEQ ID NO:2. When the parent porcine reproductive and respiratory syndrome virus strain is NL1207, the nucleotide sequence of template sequence 1 is shown in SEQ ID NO:3, and the nucleotide sequence of template sequence 2 is shown in SEQ ID NO:4.

[0034] Specifically, the most prevalent PRRSV-2 strains in clinical practice currently belong to lineages 8 and 1. The TA-12 strain is an HP-PRRSV strain, belonging to lineage 8; the NL1207 strain is a NADC30-like strain, belonging to lineage 1. Selecting these two strains can cover as many major circulating strains from different lineages as possible.

[0035] In some embodiments, in step (2), when the parent porcine reproductive and respiratory syndrome virus strain is TA-12, the initial vector is pCMV-TA-12M, whose nucleotide sequence is shown in SEQ ID NO:5; The two rounds of reverse genetics operations include: (A) PCR amplification was performed using template sequence 1 and the initial vector pCMV-TA-12M as templates to obtain two viral genome fragments. pCMV-TA-12M was double-digested to obtain an infectious clone backbone. Homologous recombination was performed between the genome fragments and the infectious clone backbone to obtain the recombinant plasmid pCMV-TA-dE. The PCR amplification primers for template sequence 1 are TA-BsaBI-F with nucleotide sequences as shown in SEQ ID NO:6 and TA-dE-R with nucleotide sequences as shown in SEQ ID NO:7; the PCR amplification primers for pCMV-TA-12M are TA-dE-F with nucleotide sequences as shown in SEQ ID NO:8 and TA-EcoRV-R with nucleotide sequences as shown in SEQ ID NO:9. (B) Two amplified fragments were obtained by PCR amplification using template sequence 2 and recombinant plasmid pCMV-TA-dE as templates, respectively. pCMV-TA-dE was double-digested to obtain a recombinant infectious clone backbone. The amplified fragments were homologously recombinated with the recombinant infectious clone backbone to obtain infectious clone plasmid pCMV-TA-d5aE. The PCR amplification primers for template sequence 2 are TA-BsrGI-F with nucleotide sequences as shown in SEQ ID NO:10 and TA-d5a-R with nucleotide sequences as shown in SEQ ID NO:11; the PCR amplification primers for the recombinant plasmid are TA-d5a-F with nucleotide sequences as shown in SEQ ID NO:12 and TA-NotI-R with nucleotide sequences as shown in SEQ ID NO:13.

[0036] In some embodiments, in step (2), when the parent porcine reproductive and respiratory syndrome virus strain is NL1207, the initial vector is pCMV-NL1207, and its nucleotide sequence is shown in SEQ ID NO:14. The two rounds of reverse genetic operations include: (A) PCR amplification was performed using template sequence 1 and the initial vector pCMV-NL1207 as templates to obtain two viral genome fragments. pCMV-NL1207 was double-digested to obtain an infectious clone backbone. Homologous recombination was performed between the genome fragments and the infectious clone backbone to obtain the recombinant plasmid pCMV-NL-dE. The PCR amplification primers for template sequence 1 are BciVI-F with nucleotide sequence as shown in SEQ ID NO:15 and NL-ER with nucleotide sequence as shown in SEQ ID NO:16; the PCR amplification primers for pCMV-NL1207 are NL-EF with nucleotide sequence as shown in SEQ ID NO:17 and HpaI-R with nucleotide sequence as shown in SEQ ID NO:18. (B) PCR amplification was performed using template sequence 2 as a template to obtain the amplified fragment. pCMV-NL-dE was double-digested to obtain the recombinant infectious clone backbone. The amplified fragment and the recombinant infectious clone backbone were homologously recombinated to obtain the infectious clone plasmid pCMV-NL-d5aE. The PCR amplification primers for template sequence 2 are SwaI-F with nucleotide sequences as shown in SEQ ID NO:19 and PsiI-R with nucleotide sequences as shown in SEQ ID NO:20.

[0037] Specifically, in step (A), the preferred PCR amplification conditions are: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 sec, annealing at 56℃~72℃ for 15 sec, extension at 72℃ for 30 sec / kb, for 35 cycles; and supplementary extension at 72℃ for 5 min. The preferred PCR amplification system is: 50 μL containing 10 ng template DNA, 25 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL 2 × Phanta Max Buffer, 1 μL dNTP Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, and the remainder being water. The enzymes used for double digestion are BsaBI and NotI; the preferred molar ratio of the two viral genome fragments to the infectious clonal backbone is 2:2:1; the preferred temperature for homologous recombination is 45℃-55℃, more preferably 48℃-52℃, and even more preferably 50℃; the preferred time for homologous recombination is 0.8-1.2h, more preferably 1h; after homologous recombination, the cells are preferably transformed into competent E. coli cells, with TOP10 cells being the preferred type.

[0038] In step (B), the preferred molar ratio of the two amplified fragments to the recombinant infectious clone backbone is 2:2:1.

[0039] A second aspect of this invention provides a method for constructing a complementary cell line stably expressing porcine reproductive and respiratory syndrome virus (PRRSV) replication-defective virus (PRS) E protein and ORF5a protein, comprising: S1: Design amplification primers based on the ORF2b and ORF5a gene sequences of the TA-12 strain; S2: Using recombinant plasmid pCMV-TA-dE as a template, the ORF2b fragment was amplified by PCR. The ORF2b fragment was then ligated with the double-digested pLVX-CMV-TEVp vector fragment and transformed into E. coli to obtain the recombinant plasmid pLVX-CMV-PRRSV-E. S3: Using the recombinant plasmid pLVX-CMV-PRRSV-E as a template, the PuroR fragment was amplified by PCR, and the ORF5a fragment was amplified by PCR using the initial vector pCMV-TA-12M as a template. The PuroR fragment, the ORF5a fragment, and the double-digested recombinant plasmid pLVX-CMV-PRRSV-E fragment were homologously recombined and transformed into E. coli to obtain the recombinant plasmid pLVX-CMV-PRRSV-5aE. S4: Recombinant plasmid pLVX-CMV-PRRSV-5aE, helper plasmid psPAX2, and helper plasmid pVSV-G were co-transfected into HEK-293T cells to obtain lentivirus. The lentivirus was then transduced into HEK-293T cells and MARC-145 cells, respectively, and screened to obtain complementary cell lines HEK-293T-PRRSV-5aE and MARC-145-PRRSV-5aE, which stably express porcine reproductive and respiratory syndrome virus replication-defective virus E protein and ORF5a protein, respectively.

[0040] Specifically, in step S2, the preferred PCR amplification reaction conditions are: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 sec, 65℃ annealing for 20 sec, 72℃ extension for 30 sec, for 35 cycles; and 72℃ supplementary extension for 10 min. The preferred PCR amplification reaction system, in 50 μL volumes, contains 10 ng template DNA, 25 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL 2 × Phanta Max Buffer, 1 μL dNTP Mix, 0.5 μL 10 μM upstream primer, 0.5 μL 10 μM downstream primer, and the remainder is water. The enzymes used for double digestion are preferably BlpI and EcoRI; the preferred molar ratio of ORF2b fragment to vector fragment is 2-4:1, more preferably 3:1; the preferred temperature for DNA ligation reaction is 3℃-5℃, more preferably 4℃; the preferred time for DNA ligation reaction is 8h-12h, more preferably 9h-11h, and even more preferably 10h; the preferred Escherichia coli competent cells are TOP10.

[0041] In step S3, the enzymes used for double digestion are preferably XbaI and MluI; the preferred ratio of the PuroR fragment, ORF5a fragment, and recombinant plasmid fragment is 2:2:1; the preferred temperature for homologous recombination is 45℃-55℃, more preferably 48℃-52℃, and even more preferably 50℃; the preferred time for homologous recombination is 10min-20min, more preferably 12min-18min, even more preferably 14min-16min, and even more preferably 15min.

[0042] In step S4, the co-transfection time is preferably 45-50 h, more preferably 46 h-48 h, and even more preferably 47 h; the density of HEK-293T and MARC-145 cells during transduction is preferably 70%-80%, more preferably 74%-78%, and even more preferably 75%; polybrene is preferably added during transduction, with a final concentration preferably 5 μg / m-10 μg / mL, more preferably 6 μg / m-8 μg / mL, and even more preferably 7 μg / mL; the transduction time is preferably 45 h-50 h, more preferably 48 h; the screening method preferably uses puromycin for screening, and the screening time is preferably based on the death of all HEK-293T and MARC-145 cells that have not been transduced with lentivirus.

[0043] In some embodiments, the PCR amplification primers for the ORF2b fragment are lvx-ER with nucleotide sequences as shown in SEQ ID NO:21 and lvx-EF with nucleotide sequences as shown in SEQ ID NO:22; the PCR amplification primers for the PuroR fragment are puro-5a-F1 with nucleotide sequences as shown in SEQ ID NO:23 and puro-5a-R1 with nucleotide sequences as shown in SEQ ID NO:24; the PCR amplification primers for the ORF5a fragment are puro-5a-F2 with nucleotide sequences as shown in SEQ ID NO:25 and puro-5a-R2 with nucleotide sequences as shown in SEQ ID NO:26; and the nucleotide sequence of the pLVX-CMV-TEVp vector is shown in SEQ ID NO:27.

[0044] A third aspect of this invention provides a porcine reproductive and respiratory syndrome (PRRS) replication-defective virus strain, the construction method of which includes: The infectious clone plasmid of porcine reproductive and respiratory syndrome replication-defective virus was transfected into the complementary cell line HEK-293T-PRRSV-5aE to obtain the P0 generation virus. The P0 generation virus was inoculated into the complementary cell line MARC-145-PRRSV-5aE for amplification to obtain the porcine reproductive and respiratory syndrome replication defect virus strains rTA-d5aE or rNL-d5aE.

[0045] Specifically, in the embodiments of the present invention, the transfection time is preferably 45h-50h, more preferably 46h-48h, and even more preferably 47h; the cell density of the complementary cell line HEK-293T-PRRSV-5aE is preferably 75%-85%, more preferably 78%-82%, and even more preferably 80%.

[0046] The porcine reproductive and respiratory syndrome (PRRS) replication-defective virus strain provided in this invention has the potential to serve as a safe and highly effective live virus vaccine, and is expected to overcome the problems of poor immunization efficacy and insufficient safety of existing commercial PRRSV vaccines.

[0047] In some embodiments, rTA-d5aE is obtained by replacing the codons overlapping with the ORF2b gene in the ORF2a gene of the TA-12 strain with the nucleotide sequence shown in SEQ ID NO:28, and replacing the codons overlapping with the ORF5a gene in the ORF5 gene of the TA-12 strain with the nucleotide sequence shown in SEQ ID NO:29. rNL-d5aE was obtained by replacing the codons overlapping with the ORF2b gene in the ORF2a gene of the NL1207 strain with the nucleotide sequence shown in SEQ ID NO:30, and replacing the codons overlapping with the ORF5a gene in the ORF5 gene of the NL1207 strain with the nucleotide sequence shown in SEQ ID NO:31.

[0048] In this embodiment of the invention, the porcine reproductive and respiratory syndrome virus replication-defective strain cannot encode the E protein and ORF5a protein. It can only replicate and proliferate in the MARC-145 cell line that stably expresses porcine reproductive and respiratory syndrome virus E and ORF5a and has a high viral titer. In other susceptible cells, it can only complete a single round of infection and cannot package infectious viral particles.

[0049] A third aspect of the present invention provides the use of porcine reproductive and respiratory syndrome (PRRS) replication-defective virus strains rTA-d5aE or rNL-d5aE in the preparation of products for the diagnosis, prevention, or treatment of PRRS.

[0050] A fourth aspect of the present invention provides a porcine reproductive and respiratory syndrome (PRRS) vaccine, comprising a porcine PRRS replication-defective virus strain rTA-d5aE or rNL-d5aE and a pharmaceutically acceptable vector.

[0051] The embodiments of the present invention can effectively solve the safety problems of existing live attenuated vaccines and the problem of weak cellular immunity induced by inactivated vaccines. They can also effectively solve the problems of poor immunization efficacy and poor safety of existing commercial PRRSV vaccines. The present invention has broad application prospects in livestock production and is expected to become a new type of safe porcine reproductive and respiratory syndrome vaccine.

[0052] The plasmid extraction kit and TIANamp Virus RNA Kit used in this invention were purchased from Tiangen Biotech (Beijing) Co., Ltd.; Opti-MEM, DMEM, and MEM maintenance media were purchased from Gibco; Lipofectamine™ 3000 Transfection Reagent was purchased from Thermo Fisher Scientific (China) Co., Ltd.; Phanta Max Super-Fidelity DNA Polymerase and FastPure Gel DNA Extraction MiniKit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; NEBuilder HiFi DNA Assembly kit and restriction endonucleases were purchased from NEB; T4 DNA ligase kit was purchased from Takara; and other chemical reagents were imported or domestically produced analytical grade.

[0053] The infectious clones pCMV-TA-12M and pCMV-NL1207, and the vector pLVX-CMV-TEVp used in this invention were constructed and preserved in our laboratory. The HEK-293T cells and MARC-145 cells used were purchased from ATCC. The PAM cells were primary alveolar macrophages collected from the lungs of pigs (breed: Duroc, Landrace, Large White, and Triple Crossbred) and prepared in the laboratory and frozen in liquid nitrogen. The TOP10 Escherichia coli competent cells and the PRRSV N antibody were purchased from Guangzhou Qianxun Biotechnology Co., Ltd. The antibody with the nsp2-PLP2 domain was a rabbit polyclonal antibody customized by Nanjing Genscript Biotech Co., Ltd. (antigen epitope: AGKRARKTRSGATT; the polyclonal antibody production service project code of Nanjing Genscript Biotech Co., Ltd. is SC2032-PF).

[0054] Example 1: Construction of a replication-defective PRRSV infectious clonal plasmid I. Experimental Materials and Template Sequence Synthesis 1. Experimental Materials Using porcine reproductive and respiratory syndrome virus (PRRSV) strain TA-12 (HP-PRRSV) and strain NL1207 (NADC30-like) as parental strains, a replication-defective PRRSV infectious cloning plasmid was constructed. The TA-12 strain (GenBank accession number: HQ416720.1) and strain NL1207 (GenBank accession number: MZ399800.1) were preserved in our laboratory.

[0055] 2. Template sequence synthesis 2.1 Based on the genome sequence of the TA-12 strain (see schematic diagram) Figure 2As shown in SEQ ID NO:32), the codons in ORF2a that share the same sequence as ORF2b and the codons in ORF5 that share the same sequence as ORF5a were replaced by synonymous mutations (as shown in SEQ ID NO:33). The resulting sequences were then sent to Genscript Biotech Co., Ltd. to synthesize modified sequences and adjacent viral genome sequences, resulting in template sequence 1 as shown in SEQ ID NO:1 and template sequence 2 as shown in SEQ ID NO:2.

[0056] 2.2 Based on the genome sequence of the NL1207 strain (see schematic diagram) Figure 2 As shown in SEQ ID NO:34), the codons in ORF2a that share the same sequence as ORF2b and the codons in ORF5 that share the same sequence as ORF5a were replaced by synonymous mutations. Then, the sequences were sent to Genscript Biotech Co., Ltd. to synthesize and modify the sequences and the adjacent viral genome sequences, resulting in template sequence 1 as shown in SEQ ID NO:3 and template sequence 2 as shown in SEQ ID NO:4.

[0057] II. Construction of pCMV-TA-d5aE and pCMV-NL-d5aE plasmids Through two rounds of reverse genetics operations, the modified sequences were used to replace the ORF2b and ORF5a sequences in pCMV-TA-12M and pCMV-NL1207 (the infectious cDNA clones of pCMV-TA-12M and pCMV-NL1207 were constructed and preserved in our laboratory) to construct the pCMV-TA-d5aE and pCMV-NL-d5aE plasmids.

[0058] 1. Construction of pCMV-TA-d5aE plasmid 1.1 Using pCMV-TA-12M as the backbone, its ORF2b coding sequence was replaced with SEQ ID NO:28: Using SEQ ID NO:1 and pCMV-TA-12M as templates, and TA-BsaBI-F / TA-dE-R and TA-dE-F / TA-EcoRV-R in Table 1 as primer pairs, the two viral genome fragments were amplified by PCR. The reaction system is shown in Table 2.

[0059] PCR reaction conditions were set as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 56℃~72℃ annealing for 15 sec, 72℃ extension for 30 sec / kb, 35 cycles; and a final extension at 72℃ for 5 min. After confirmation of the PCR amplification products by agarose gel electrophoresis, the plasmid template was removed by digestion with DpnI restriction endonuclease. After incubation at 37℃ for 1 h, the PCR products were purified and recovered using the FastPure Gel DNA Extraction Mini Kit according to the manufacturer's instructions. pCMV-TA-12M was linearized by double digestion with BsaBI and EcoRV, and the large infectious clonal backbone was recovered using the FastPure Gel DNA Extraction Mini Kit. Using the NEBuilder HiFi DNA Assembly kit, two purified viral genome fragments were mixed with an infectious clonal backbone at a ratio of 2:2:1 to prepare a DNA homologous recombination reaction system. After incubation at 50°C for 1 hour, the homologous recombination product was transformed into TOP10 competent E. coli cells. Single colonies were picked and cultured, and plasmids were extracted for DNA sequencing identification. The correctly sequenced plasmid was named pCMV-TA-dE.

[0060] 1.2. Using pCMV-TA-dE as the backbone, its ORF5a coding sequence was replaced with SEQ ID NO:29: Using SEQ ID NO:2 and pCMV-TA-dE as templates, and TA-BsrGI-F / TA-d5a-R and TA-d5a-F / TA-NotI-R as primer pairs in Table 1, two viral genome fragments were amplified by PCR. The reaction system is shown in Table 2, and the reaction conditions are the same as above. pCMV-TA-dE was linearized by double digestion with BsrGI and NotI. The PCR amplification product and the large infectious clone backbone were purified and recovered using the FastPure Gel DNA Extraction Mini Kit. Using the NEBuilder HiFi DNA Assembly Kit, the two purified viral genome fragments and the infectious clone backbone were mixed at a ratio of 2:2:1 to prepare a DNA homologous recombination reaction system. After incubation at 50℃ for 1 h, the homologous recombination product was transformed into TOP10 E. coli competent cells, single colonies were picked for culture, and plasmids were extracted for DNA sequencing identification. The plasmid that was correctly sequenced was named pCMV-TA-d5aE.

[0061] 2. Construction of pCMV-NL-d5aE plasmid 2.1 Using pCMV-NL1207 as the backbone, its ORF2b coding sequence was replaced with SEQ ID NO:30: Using SEQ ID NO:3 and pCMV-NL1207 as templates, and using BciVI-F / NL-ER and NL-EF / HpaI-R in Table 1 as primer pairs, the two viral genome fragments were amplified by PCR. The reaction system is shown in Table 2.

[0062] PCR reaction conditions were set as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 56℃~72℃ annealing for 15 sec, 72℃ extension for 30 sec / kb, 35 cycles; and a final extension at 72℃ for 5 min. After confirmation of the PCR amplification products by agarose gel electrophoresis, the plasmid template was removed by digestion with DpnI restriction endonuclease. After incubation at 37℃ for 1 h, the PCR products were purified and recovered using the FastPure Gel DNA Extraction Mini Kit according to the manufacturer's instructions. pCMV-NL1207 was linearized by double digestion with BciVI and HpaI, and the large infectious clonal backbone was recovered using the FastPure Gel DNA Extraction Mini Kit. Using the NEBuilder HiFi DNA Assembly kit, two purified viral genome fragments were mixed with an infectious clone backbone at a ratio of 2:2:1 to prepare a DNA homologous recombination reaction system. After incubation at 50°C for 1 hour, the homologous recombination product was transformed into TOP10 competent E. coli cells. Single colonies were picked and cultured, and plasmids were extracted for DNA sequencing identification. The correctly sequenced plasmid was named pCMV-NL-dE.

[0063] 2.2 Using pCMV-NL-dE as the backbone, its ORF5a coding sequence was replaced with SEQ ID NO:31. Using SEQ ID NO:4 as a template and SwaI-F / PsiI-R from Table 1 as the primer pair, the viral genome fragment was amplified by PCR. The reaction system is shown in Table 2, and the reaction conditions are the same as above. pCMV-NL-dE was linearized by double digestion with SwaI and PsiI. The PCR amplification product and the large infectious clonal backbone were purified and recovered using the FastPure Gel DNAExtraction Mini Kit. Using the NEBuilder HiFi DNA Assembly Kit, the purified viral genome fragment and the infectious clonal backbone were mixed at a ratio of 2:1 to prepare a DNA homologous recombination reaction system. After incubation at 50℃ for 1 h, the homologous recombination product was transformed into TOP10 E. coli competent cells. Single colonies were picked for culture, and plasmids were extracted for DNA sequencing identification. The correctly sequenced plasmid was named pCMV-NL-d5aE.

[0064] Table 1 PCR Primers

[0065] Table 2 PCR reaction preparation

[0066] Example 2: Establishment of a complementary cell line stably expressing PRRSV E and ORF5a proteins via lentiviral transduction 1. Primer design Based on the ORF2b and ORF5a gene sequences of the TA-12 strain, three pairs of PCR amplification primers were designed (as shown in Table 3).

[0067] Table 3 PCR amplification primers

[0068] 2. Construction of lentiviral recombinant plasmid pLVX-CMV-PRRSV-5aE 2.1 Construction of intermediate plasmid pLVX-CMV-PRRSV-E ORF2b was inserted into pLVX-CMV-TEVp using an enzyme digestion-ligation method: Using pCMV-TA-12M as a template, the ORF2b gene fragment was amplified by PCR using the lvx-ER / lvx-EF primers listed in Table 3. The reaction system is shown in Table 2. The PCR reaction conditions were set as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 sec, 65℃ annealing for 20 sec, 72℃ extension for 30 sec, for 35 cycles; and a final extension at 72℃ for 10 min.

[0069] After the PCR amplification products were confirmed to be correct by agarose gel electrophoresis, the plasmid template was removed by digestion with DpnI restriction endonuclease. After incubation at 37°C for 1 hour, the PCR products were purified and recovered using the FastPure Gel DNA Extraction Mini Kit according to the manufacturer's instructions. pLVX-CMV-TEVp was linearized by double digestion with BlpI and EcoRI, and the large vector fragment was recovered using the FastPure Gel DNA Extraction Mini Kit.

[0070] Using the T4 DNA ligase kit, the purified ORF2b fragment was mixed with the plasmid vector fragment at a molar ratio of 3:1 to prepare the DNA ligation reaction system, and the reaction was carried out overnight at 4°C. The ligation product was transformed into TOP10 E. coli competent cells, single colonies were picked and cultured, and plasmids were extracted for DNA sequencing identification. The correctly sequenced plasmid was named pLVX-CMV-PRRSV-E.

[0071] 2.2. Construction of the final plasmid pLVX-CMV-PRRSV-5aE The PRRSV ORF5a gene sequence was inserted into pLVX-CMV-PRRSV-E to form the open reading frame PuroR-T2A-ORF5a. Using pLVX-CMV-PRRSV-E and pCMV-TA-12M as templates, and puro-5a-F1 / puro-5a-R1 and puro-5a-F2 / puro-5a-R2 as primer pairs (Table 3), the PuroR and ORF5a gene fragments were amplified, respectively. The reaction system is shown in Table 2. The PCR reaction conditions were set as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 sec, 65℃ annealing for 20 sec, 72℃ extension for 30 sec, for 35 cycles; and 72℃ supplementary extension for 10 min.

[0072] After the PCR amplification products were confirmed to be correct by agarose gel electrophoresis, the plasmid template was removed by digestion with DpnI restriction endonuclease. After incubation at 37°C for 1 hour, the PCR products were purified and recovered using the FastPure Gel DNA Extraction Mini Kit according to the manufacturer's instructions. pLVX-CMV-PRRSV-E was linearized by double digestion with XbaI and MluI, and the large vector fragment was recovered using the FastPure Gel DNA Extraction Mini Kit.

[0073] Using the NEBuilder HiFi DNA Assembly kit, the purified two PCR fragments and the vector fragment were mixed at a 2:2:1 ratio to prepare a DNA homologous recombination reaction system, which was incubated at 50°C for 15 min. The homologous recombination product was transformed into TOP10 competent *E. coli* cells, and single colonies were picked and cultured. Plasmids were extracted and sequenced for DNA identification. The correctly sequenced plasmid was named pLVX-CMV-PRRSV-5aE, and its chromatogram is shown below. Figure 3 As shown.

[0074] 3. Lentiviral packaging and cell transduction HEK-293T and MARC-145 cells were revived and cultured in DMEM or MEM medium containing 10% FBS at 37°C in a 5% CO2 cell culture incubator.

[0075] When the HEK-293T cell density in the six-well cell culture plate reached 80-90%, pLVX-CMV-PRRSV-5aE was co-transfected into HEK-293T cells with two lentiviral-packaged helper plasmids, psPAX2 (purchased from Addgene, catalog number 12260) and pVSV-G (purchased from Addgene, catalog number 138479), using Lipofectamine™ 3000 Transfection Reagent. After 48 hours, the lentiviral supernatant was collected. HEK-293T and MARC-145 cells were cultured in six-well cell culture plates. When the cells reached 70-80% confluency and were in good condition, lentiviral transduction was performed. 500 μL of lentivirus was transduced into HEK-293T and MARC-145 cells, respectively, and then polybrene was added to a final concentration of 8 μg / mL. The plates were then placed in a cell culture incubator.

[0076] 4. Puromycin screening for stable cell lines Forty-eight hours after transduction, successfully transduced cells were screened by puromycin treatment. Cells in six-well cell culture plates were trypsinized and transferred to T25 culture flasks. Complete cell culture medium (DMEM medium containing 10% FBS) containing 2 μg / mL (HEK-293T) or 10 μg / mL (MARC-145) puromycin was added for resistance selection. The selection medium (DMEM medium containing 10% FBS, with 2 μg / mL (HEK-293T) or 10 μg / mL (MARC-145) puromycin added according to cell type) was changed every 3 days until all untransduced HEK-293T and MARC-145 cells in the control group died. Cells surviving under puromycin selection pressure were polyclonal HEK-293T-PRRSV-5aE and MARC-145-PRRSV-5aE cell lines.

[0077] 5. Screening and validation of monoclonal cell lines Multiple monoclonal MARC-PRRSV-5aE cell lines were screened using a limiting dilution method. The monoclonal cells were named according to the rows and columns of 96-well plates, with the serial number representing a different 96-well plate. The screening results are as follows: Figure 4 As shown.

[0078] Depend on Figure 4 It is known that, under different infection doses (10µL, 5µL, 1µL), the 2-E7 and 1-B3 monoclonal cell lines of MARC-145-PRRSV-5aE can support efficient infection of replication-deficient PRRSV rTA-d5aE.

[0079] Example 3: Rescue of Replication-Defective PRRSV and Identification of its Replication Defect Characteristics 1. Rescue of PRRSV with Replication Defects HEK-293T-PRRSV-5aE cell lines were cultured in 12-well cell culture plates. When the cell density reached approximately 80%, replication-defective PRRSV was rescued via DNA transfection. Following the manufacturer's instructions, 1 μg of pCMV-TA-d5aE or pCMV-NL-d5aE was transfected into HEK-293T-PRRSV-5aE cells using Lipofectamine™ 3000 Transfection Reagent. Forty-eight hours after transfection, the cell culture supernatant was collected and named P0 generation virus. The P0 virus was then seeded into MARC-145-PRRSV-5aE cells in 12-well cell culture plates. Once significant cytopathic effects were observed, the cell culture supernatant was collected and named P1 generation virus. Subsequently, the recombinant virus was passaged three times in MARC-145-PRRSV-5aE cells, and the virus was identified by determining its genome sequence.

[0080] 2. Identification of Virus Rescue Effect The results showed that the recombinant virus rescued 3 days after inoculation caused significant cytopathic effects, with cells exhibiting aggregation, shrinkage, and detachment, which were essentially consistent with the cytopathic effects caused by infection with its parent virus. Therefore, the two recombinant viruses, rTA-d5aE and rNL-d5aE, were successfully rescued in complementary cell lines.

[0081] 3. Infectivity analysis of recombinant virus in ordinary MARC-145 cells after passage Two recombinant viruses (0.1 MOI) were used to infect MARC-145 cells. After 1 hour, the viral load was discarded, and the cells were cultured again. After 24 hours, the cell culture supernatant was collected, and the cells were fixed for immunofluorescence assays to detect PRRSV nsp2 protein. The collected cell culture supernatant was then used to infect MARC-145 cells again. After 1 hour, the viral load was discarded, and the cells were cultured again. After 24 hours, the cells were fixed for immunofluorescence assays to detect PRRSV nsp2 protein. The results are as follows: Figure 5 and Figure 6 As shown.

[0082] Depend on Figure 5 It was found that PRRSV nsp2 protein was detected in P1 generation rTA-d5aE-infected MARC-145 cells, but not in P2 generation rTA-d5aE-infected MARC-145 cells. Meanwhile, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. Figure 6The results of rNL-d5aE infection of MARC-145 cells were consistent. These results indicate that rTA-d5aE and rNL-d5aE have the ability to infect MARC-145 cells and replicate intracellularly, but they cannot package infectious viral particles.

[0083] 4. Validation of the infectivity of recombinant virus on porcine natural target cells (PAM) The infection of rNL-d5aE in porcine alveolar macrophages (PAM), the natural target cells of PRRSV, was further tested using the same assay method described above. The results are as follows: Figure 7 As shown.

[0084] Depend on Figure 7 PRRSV nsp2 protein was detected in P1 generation rNL-d5aE-infected PAM cells, but not in P2 generation rNL-d5aE-infected MARC-145 cells. These results indicate that rNL-d5aE has the ability to infect native target cells and replicate intracellularly, but it cannot package infectious viral particles, ruling out the risk of viral shedding when used as a vaccine.

[0085] Example 4: Identification of replication-defective viruses rTA-d5aE and rNL-d5aE To identify the rescued replication-defective virus, RT-PCR amplification and DNA sequencing were performed on the ORF2b and ORF5a regions of the viral genome.

[0086] 1. Viral genomic RNA extraction Viral genomic RNA was extracted from the rescued replication-defective viruses rTA-d5aE and rNL-d5aE using the TIANamp Virus RNA Kit.

[0087] 2. RT-PCR amplification Using viral genomic RNA as a template, one-step reverse transcription RT-PCR amplification was performed using the primers in Table 1 (TA-BsaBI-F and TA-NotI-R for rTA-d5aE amplification, and BciVI-F and PsiI-R for rNL-d5aE amplification).

[0088] 3. PCR product processing and sequencing The PCR products were purified and recovered using the FastPure Gel DNA Extraction Mini Kit according to the instructions, and the purified PCR products were then sequenced. Results are as follows: Figures 8 to 11 As shown.

[0089] 4. Appraisal Results Sequencing results showed that the ORF2b and ORF5a sequences of the rescued replication-defective virus were completely consistent with the experimentally designed sequences.

[0090] Example 5: Comparison of replication capacity of replication-defective virus and its parent virus in complementary cell lines 1. Viral infection and sample collection The MARC-145-PRRSV-5aE cell line was cultured in 24-well cell culture plates. After the cells reached a confluent monolayer, four viruses—TA-12, rTA-d5aE, NL1207, and rNL-d5aE—were inoculated at a multiplicity of infection (MOI) of 0.01. After 2 hours of adsorption, the virus solution was discarded, and the medium was replaced with MEM maintenance medium containing 2% serum for further culture. Viral supernatant was collected at different time points post-infection: for TA-12 and rTA-d5aE, collection times were 12, 24, 36, 48, 60, and 72 hours post-infection; for NL1207 and rNL-d5aE, collection times were increased to 84 and 96 hours post-infection. Two replicate samples were collected each time.

[0091] 2. Virus titer determination and growth curve plotting Viral titers at each time point were determined using a 96-well plate tissue culture method, and the half-maximal infectious dose (TCID50 / mL) was calculated according to the Reed-Muench method. Based on this, multi-step growth curves of the replication-defective virus and its corresponding parent virus were plotted. Results are as follows: Figure 12 and Figure 13 As shown.

[0092] 3. Results Depend on Figure 12 and Figure 13 It was found that in the MARC-145-PRRSV-5aE complementary cell line, both rTA-d5aE and its parent virus TA-12 could replicate normally, but the viral titer of rTA-d5aE was lower than that of TA-12 at all time points, indicating that its replication ability was weaker than that of WT virus. rTA-d5aE reached its replication peak at 60 h post-infection, with an average maximum titer of 10. 6.585 TCID50 / mL. Similarly, the rNL-d5aE-deficient virus and its parent strain NL1207 can replicate normally in this cell line, and the replication capacity of rNL-d5aE is significantly lower than that of WT virus. Both reach peak replication at 72 h post-infection, with the average highest titer of rNL-d5aE being 10. 5.5 TCID50 / mL.

[0093] Example 6: Study on the genetic stability of the replication-defective virus rTA-d5aE 1. Virus continuous passage culture MARC-145-PRRSV-5aE cells were cultured in 12-well cell culture plates. After the cells reached a confluent monolayer, rTA-d5aE virus was inoculated at a multiplicity of infection (MOI). After 2 hours of adsorption, the virus solution was discarded, and the cells were replaced with MEM maintenance medium containing 2% serum for further culture. After 48 hours of infection, the viral supernatant was collected and designated P1. Subsequently, P1 was used to infect MARC-145-PRRSV-5aE cells cultured in 12-well cell culture plates at a MOI of ~0.1. After 48 hours of infection, the viral supernatant was collected and designated P2. Similarly, rTA-d5aE virus was passaged 20 times in MARC-145-PRRSV-5aE cells to obtain viruses P1 to P20.

[0094] 2. Viral genomic RNA extraction and RT-PCR amplification Viral genomic RNA was extracted from P1 and P20 rTA-d5aE using the TIANamp Virus RNA Kit. One-step reverse transcription RT-PCR amplification was performed using the primers listed in Table 1, with the viral genomic RNA as a template.

[0095] 3. PCR product processing and sequencing The PCR products were purified and recovered using the FastPure Gel DNA Extraction Mini Kit according to the instructions, and the purified PCR products were then sequenced. Results are as follows: Figure 14 and Figure 15 As shown.

[0096] 4. Results Depend on Figure 14 and Figure 15 It can be seen that the ORF2b and ORF5a sequences of P1 and P20 viruses are completely consistent with the experimentally designed sequences.

[0097] In summary, this invention provides a porcine reproductive and respiratory syndrome (PRRS) replication-deficient virus strain. This strain was obtained by synonymously replacing codons in the ORF2a / ORF5 coding regions overlapping with ORF2b / ORF5a in the genomes of parental PRRSV strains TA-12 and NL1207, thereby silencing the expression of E and ORF5a proteins. It possesses the potential to serve as a safe and highly effective live virus vaccine, and is expected to overcome the problems of poor immunization efficacy and insufficient safety of existing commercial PRRSV vaccines, thus having significant application value for PRRSV prevention and control. Furthermore, this invention also provides a complementary cell line that stably expresses PRRSV E and ORF5a proteins. Experiments show that the virus strain can replicate continuously and normally in this complementary cell line, and no reversion mutations were found after 20 passages, while in non-complementary cells such as MARC-145 and PAMs, only a single round of infection can be completed.

[0098] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. A method for constructing an infectious clonal plasmid of a porcine reproductive and respiratory syndrome (PRRS) replication-defective virus, characterized in that, include: (1) Synonymous mutations were performed on the codons overlapping with the ORF2b gene in the ORF2a gene of the parent porcine reproductive and respiratory syndrome virus strain and the codons overlapping with the ORF5a gene in the ORF5 gene, respectively, to synthesize template sequence 1 for replacing the ORF2b gene and template sequence 2 for replacing the ORF5a gene. (2) Using the initial vector containing the full-length cDNA of the parent porcine reproductive and respiratory syndrome virus strain as the backbone, gene replacement was performed sequentially through two rounds of reverse genetic operations to obtain an infectious clonal plasmid of porcine reproductive and respiratory syndrome replication defect virus.

2. The construction method according to claim 1, characterized in that, The parent porcine reproductive and respiratory syndrome virus strain mentioned in step (1) is either the TA-12 strain or the NL1207 strain; When the parent porcine reproductive and respiratory syndrome virus strain is TA-12, the nucleotide sequence of template sequence 1 is as shown in SEQ ID NO:1, and the nucleotide sequence of template sequence 2 is as shown in SEQ ID NO:2; When the parent porcine reproductive and respiratory syndrome virus strain is NL1207, the nucleotide sequence of template sequence 1 is shown in SEQ ID NO:3, and the nucleotide sequence of template sequence 2 is shown in SEQ ID NO:

4.

3. The construction method according to claim 1 or 2, characterized in that, In step (2), when the parent porcine reproductive and respiratory syndrome virus strain is TA-12, the initial vector is pCMV-TA-12M, and the nucleotide sequence of pCMV-TA-12M is shown in SEQ ID NO:5; The two rounds of reverse genetic operations include: (A) PCR amplification was performed using the template sequence 1 and the initial vector pCMV-TA-12M as templates to obtain two viral genome fragments. The pCMV-TA-12M was double-digested to obtain an infectious clone backbone. The fragments were homologously recombinated with the infectious clone backbone to obtain the recombinant plasmid pCMV-TA-dE. The PCR amplification primers for template sequence 1 are TA-BsaBI-F with nucleotide sequences as shown in SEQ ID NO:6 and TA-dE-R with nucleotide sequences as shown in SEQ ID NO:7; the PCR amplification primers for pCMV-TA-12M are TA-dE-F with nucleotide sequences as shown in SEQ ID NO:8 and TA-EcoRV-R with nucleotide sequences as shown in SEQ ID NO:

9. (B) Two amplified fragments were obtained by PCR amplification using the template sequence 2 and the recombinant plasmid pCMV-TA-dE as templates, respectively. The pCMV-TA-dE was double-digested to obtain a recombinant infectious clone backbone. The amplified fragments were homologously recombinated with the recombinant infectious clone backbone to obtain the infectious clone plasmid pCMV-TA-d5aE. The PCR amplification primers for the template sequence 2 are TA-BsrGI-F with nucleotide sequences as shown in SEQ ID NO:10 and TA-d5a-R with nucleotide sequences as shown in SEQ ID NO:11; the PCR amplification primers for the recombinant plasmid are TA-d5a-F with nucleotide sequences as shown in SEQ ID NO:12 and TA-NotI-R with nucleotide sequences as shown in SEQ ID NO:

13.

4. The construction method according to claim 1 or 2, characterized in that, In step (2), when the parent porcine reproductive and respiratory syndrome virus strain is NL1207, the initial vector is pCMV-NL1207, and the nucleotide sequence of pCMV-NL1207 is shown in SEQ ID NO:14; The two rounds of reverse genetic operations include: (A) PCR amplification was performed using the template sequence 1 and the initial vector pCMV-NL1207 as templates to obtain two viral genome fragments. The pCMV-NL1207 was double-digested to obtain an infectious clone backbone. The fragments were homologously recombinated with the infectious clone backbone to obtain the recombinant plasmid pCMV-NL-dE. The PCR amplification primers for template sequence 1 are BciVI-F with nucleotide sequence as shown in SEQ ID NO:15 and NL-ER with nucleotide sequence as shown in SEQ ID NO:16; the PCR amplification primers for pCMV-NL1207 are NL-EF with nucleotide sequence as shown in SEQ ID NO:17 and HpaI-R with nucleotide sequence as shown in SEQ ID NO:

18. (B) Using the template sequence 2 as a template, PCR amplification was performed to obtain the amplified fragment. The pCMV-NL-dE was double-digested to obtain the recombinant infectious clone backbone. The amplified fragment and the recombinant infectious clone backbone were homologously recombinated to obtain the infectious clone plasmid pCMV-NL-d5aE. The PCR amplification primers for template sequence 2 are SwaI-F with nucleotide sequences as shown in SEQ ID NO:19 and PsiI-R with nucleotide sequences as shown in SEQ ID NO:

20.

5. A method for constructing a complementary cell line stably expressing porcine reproductive and respiratory syndrome virus (PRRSV) replication-defective virus (PRS) E protein and ORF5a protein, characterized in that, include: S1: Design amplification primers based on the ORF2b and ORF5a gene sequences of the TA-12 strain according to claim 2; S2: Using the recombinant plasmid pCMV-TA-dE described in claim 3 as a template, the ORF2b fragment was obtained by PCR amplification. The ORF2b fragment was ligated with the double-digested pLVX-CMV-TEVp vector fragment and transformed into E. coli to obtain the recombinant plasmid pLVX-CMV-PRRSV-E. S3: Using the recombinant plasmid pLVX-CMV-PRRSV-E as a template, the PuroR fragment is obtained by PCR amplification, and the ORF5a fragment is obtained by PCR amplification using the initial vector pCMV-TA-12M as a template according to claim 3. The PuroR fragment, the ORF5a fragment and the double-digested recombinant plasmid pLVX-CMV-PRRSV-E fragment are homologously recombined and transformed into Escherichia coli to obtain the recombinant plasmid pLVX-CMV-PRRSV-5aE. S4: The recombinant plasmid pLVX-CMV-PRRSV-5aE, helper plasmid psPAX2, and helper plasmid pVSV-G were co-transfected into HEK-293T cells to obtain lentivirus. The lentivirus was then transduced into HEK-293T cells and MARC-145 cells, respectively, and screened to obtain complementary cell lines HEK-293T-PRRSV-5aE and MARC-145-PRRSV-5aE, which stably express the E protein and ORF5a protein of porcine reproductive and respiratory syndrome virus replication defective virus.

6. The construction method according to claim 5, characterized in that, The PCR amplification primers for the ORF2b fragment are lvx-ER (nucleotide sequence as shown in SEQ ID NO:21) and lvx-EF (nucleotide sequence as shown in SEQ ID NO:22); the PCR amplification primers for the PuroR fragment are puro-5a-F1 (nucleotide sequence as shown in SEQ ID NO:23) and puro-5a-R1 (nucleotide sequence as shown in SEQ ID NO:24); the PCR amplification primers for the ORF5a fragment are puro-5a-F2 (nucleotide sequence as shown in SEQ ID NO:25) and puro-5a-R2 (nucleotide sequence as shown in SEQ ID NO:26); and the nucleotide sequence of the pLVX-CMV-TEVp vector is shown in SEQ ID NO:

27.

7. A porcine reproductive and respiratory syndrome replication-defective virus strain, characterized in that, The method for constructing the strain includes: The infectious clonal plasmid of the porcine reproductive and respiratory syndrome replication-defective virus obtained by the construction method according to any one of claims 1-4 is transfected into the complementary cell line HEK-293T-PRRSV-5aE obtained by the construction method according to claim 5 or 6 to obtain P0 generation virus; The P0 generation virus is inoculated into the complementary cell line MARC-145-PRRSV-5aE obtained by the construction method of claim 5 or 6 for amplification to obtain the porcine reproductive and respiratory syndrome replication defect virus strain rTA-d5aE or rNL-d5aE.

8. The porcine reproductive and respiratory syndrome replication-defective virus strain according to claim 7, characterized in that, The rTA-d5aE is obtained by replacing the codons overlapping with the ORF2b gene in the ORF2a gene of the TA-12 strain according to claim 2 with the nucleotide sequence shown in SEQ ID NO:28, and replacing the codons overlapping with the ORF5a gene in the ORF5 gene of the TA-12 strain with the nucleotide sequence shown in SEQ ID NO:

29. The rNL-d5aE is obtained by replacing the codons overlapping with the ORF2b gene in the ORF2a gene of the NL1207 strain according to claim 2 with the nucleotide sequence shown in SEQ ID NO:30, and replacing the codons overlapping with the ORF5a gene in the ORF5 gene of the NL1207 strain with the nucleotide sequence shown in SEQ ID NO:

31.

9. The use of the porcine reproductive and respiratory syndrome replication defective virus strain rTA-d5aE or rNL-d5aE as described in claim 7 in the preparation of products for the diagnosis, prevention or treatment of porcine reproductive and respiratory syndrome.

10. A vaccine for porcine reproductive and respiratory syndrome, characterized in that, Includes the porcine reproductive and respiratory syndrome replication-defective virus strain rTA-d5aE or rNL-d5aE as described in claim 7 and a pharmaceutically acceptable vector.

Images