Porcine reproductive and respiratory syndrome mutant virus and construction method and application thereof

Abstract

The application discloses a porcine reproductive and respiratory syndrome mutant virus and a construction method and application thereof. The application accurately analyzes key amino acid sites of PRRSV-1 determining Marc-145 cell tropism, specifically, 88(F), 94(I) and 95(L) amino acids of GP2a, 70(G), 72(N), 80(D), 154(I), 158(H) and 163(L) amino acids of GP3, and 49(D), 53(L), 54(R) and 57(G) amino acids of GP4. Three Marc-145 cell non-adapted strains are precisely point mutated, three PRRSV-1 modified strains which can adapt to Marc 145 cell in vitro passage culture are obtained, the strains can be stably passed in vitro and have good replication efficiency, and Marc-145 cells infected by the strains can produce obvious cytopathic effect, and the strains are expected to be used as a new type of PRRSV-1 vaccine.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a mutant virus of porcine reproductive and respiratory syndrome, its construction method, and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant pathogen that seriously threatens the healthy development of the pig industry. PRRSV infection causes a clinical syndrome including reproductive disorders in sows, respiratory diseases in pigs of all ages, and slowed growth in piglets. It is classified into two distinct species: PRRSV-1 and PRRSV-2. Since the first report of the native wild-type PRRSV-1 strain BJEU06-1 in 2006, reports of PRRSV-1 detection have been increasing. Due to its mild pathogenicity and low clinical detection rate, the presence of PRRSV-1 has not received sufficient attention. However, in recent years, PRRSV-1 has been detected in an increasing number of pig-farming areas, spreading to at least 28 provinces and municipalities, and its virulence is showing a gradual increasing trend, becoming a potential threat to the pig industry.

[0003] PRRSV is an RNA virus of approximately 15 kb in length, belonging to the order Heliovirales, family Arteriviridae, and genus β-Arteriviridae, containing 10 open reading frames (ORFs). Among the eight structural proteins encoded by PRRSV (encoded by ORF2-7), the three small envelope proteins (GP2a, GP3, and GP4) encoded by the ORF2-4 gene form heterotrimers through non-covalent bonds. These proteins are not only crucial for viral infection of cells but also the core protein combination determining PRRSV cell tropism. Our previous study found that mutating amino acids 88, 94, and 95 of PRRSV-1 GP2a and replacing the overlapping regions of GP3 and GP4 with the sequence of the Marc-145 cell-adapted strain Amervac (GenBank accession number: GU067771) can induce Marc-145 cell tropism in the strain (published paper, DOI: 10.1128 / jvi.00048-25).

[0004] The Marc-145 cell line is a continuously passaged cell line obtained through clonal selection of MA-104 embryonic kidney cells from African green monkeys. In the production process of PRRSV live attenuated vaccine, Marc-145 cells serve as a substrate for viral replication, supporting efficient amplification of the vaccine strain and obtaining high-titer viral fluid to meet the stringent requirements of commercial vaccines for antigen yield.

[0005] Currently, PRRS prevention and control mainly relies on vaccination. For the increasingly prevalent and virulent PRRSV-1 strains, a key constraint in the development of specific attenuated live vaccines lies in the fact that wild-type PRRSV-1 strains typically cannot adapt to Marc-145 cells. However, the precise amino acid sites that determine its Marc-145 cell tropism have not yet been fully reported. Summary of the Invention

[0006] Purpose of the Invention: The purpose of this invention is to address the technical bottleneck hindering the development of attenuated live vaccines against the difficulty of wild-type PRRSV-1 strains adapting to Marc-145 cells, and to address the serious situation in my country where the detection rate of PRRSV-1 is increasing and its virulence is becoming more potent, with no effective vaccine available. Specifically, through reverse genetics and multiple sequence alignment analysis and verification, we identified the key sites determining Marc-145 cell tropism for the small membrane proteins GP2a, GP3, and GP4; and through site-directed modification, we successfully obtained three modified PRRSV-1 strains that can stably replicate in Marc-145 cells.

[0007] A second objective of this invention is to provide a method for constructing a mutant virus of porcine reproductive and respiratory syndrome.

[0008] A third objective of this invention is to provide the aforementioned recombinant vector and engineered cell.

[0009] A fourth objective of this invention is to provide a kit for detecting the mutant virus.

[0010] The fifth objective of this invention is to provide a vaccine for the preparation of porcine reproductive and respiratory syndrome mutant virus and recombinant plasmid.

[0011] The sixth objective of this invention is to provide a recombinant vaccine.

[0012] Technical solution: In order to achieve the above objectives, the present invention provides a mutant virus of porcine reproductive and respiratory syndrome, wherein the mutant virus includes a mutation of amino acid D at position 49, amino acid L at position 53, amino acid R at position 54, and amino acid G at position 57 in the small envelope protein GP4 of the mutant strain PRRSV-1.

[0013] And / or; the 70th amino acid of the small membrane protein GP3 of the wild-type PRRSV-1 is mutated to G, the 72nd amino acid to N, the 80th amino acid to D, the 154th amino acid to I, the 158th amino acid to H, and the 163rd amino acid to L.

[0014] In the mutant strain PRRSV-1, the 88th amino acid of the small membrane protein GP2a is mutated to F, the 94th amino acid is mutated to I, and the 95th amino acid is mutated to L.

[0015] Preferably, the mutant strain PRRSV-1 is obtained by performing the following mutations on three Marc-145 cell non-adapted strains HLJB1, SD1291 or AHEU2024-2671: the 88th amino acid of the small membrane protein GP2a is mutated to F, the 94th amino acid is mutated to I and the 95th amino acid is mutated to L.

[0016] The present invention also provides a method for constructing a mutant virus of porcine reproductive and respiratory syndrome, comprising the following steps: mutating the 88th amino acid of the small envelope protein GP2a of strain HLJB1 or strain SD1291 to F, the 94th amino acid to I, and the 95th amino acid to L, and mutating the 49th amino acid of the small envelope protein GP4 to D, the 53rd amino acid to L, the 54th amino acid to R, and the 57th amino acid to G;

[0017] Alternatively, mutate the following amino acids from strain AHEU2024-2671: amino acid 88 of GP2a to F, amino acid 94 to I, and amino acid 95 to L; amino acid 49 of GP4 to D, amino acid 53 to L, amino acid 54 to R, and amino acid 57 to G; and amino acid 70 of GP3 to G, amino acid 72 to N, amino acid 80 to D, amino acid 154 to I, amino acid 158 to H, and amino acid 163 to L.

[0018] The method includes the following steps: designing mutant primers to perform site-directed mutagenesis on a vector containing the viral genome to obtain a mutant fragment; ligating the mutant fragment and a vector containing the viral genome to obtain a recombinant plasmid; introducing the recombinant plasmid into a host cell; and rescuing the mutant virus from the cell or culture.

[0019] The mutant primers include sets of mutant primers designed for mutations of amino acid F at position 88, I at position 94, and L at position 95 of small vesicle protein GP2a, and for mutations of amino acid D at position 49, L at position 53, R at position 54, and G at position 57 of small vesicle protein GP4; preferably, the sets of mutant primers also include sets of mutant primers designed for mutations of amino acid G at position 70, N at position 72, D at position 80, I at position 154, H at position 158, and L at position 163 of small vesicle protein GP3.

[0020] The present invention also provides a recombinant plasmid containing the following mutations in the full-length cDNA of wild-type PRRSV-1 virus: the mutations result in the following changes: amino acid 88 of the small envelope protein GP2a of the wild-type virus is changed to F, amino acid 94 is changed to I, and amino acid 95 is changed to L; the mutations result in the following changes: amino acid 49 of the small envelope protein GP4 of the wild-type PRRSV-1 is changed to D, amino acid 53 is changed to L, amino acid 54 is changed to R, and amino acid 57 is changed to G.

[0021] Or, as contained in the full-length cDNA of wild-type PRRSV-1 virus, the following mutations occur: the mutation causes the 88th amino acid of the small envelope protein GP2a of the wild-type virus to change to F, the 94th amino acid to change to I, and the 95th amino acid to change to L; the mutation causes the 49th amino acid of the small envelope protein GP4 of the wild-type PRRSV-1 to change to D, the 53rd amino acid to change to L, the 54th amino acid to change to R, and the 57th amino acid to change to G; the mutation causes the 70th amino acid of the small envelope protein GP3 of the wild-type PRRSV-1 to change to G, the 72nd amino acid to change to N, the 80th amino acid to change to D, the 154th amino acid to change to I, the 158th amino acid to change to H, and the 163rd amino acid to change to L.

[0022] This invention also provides a method for constructing the recombinant plasmid, comprising the following steps: using plasmids rHLJB1-FIL, pACYC177-rSD1291, pACYC177-rAHEU2024, or pACYC177-rAHEU2024-M24 as templates, using mutant primer sets targeting the wild-type small membrane protein GP2a with amino acid mutations of F at position 88, I at position 94, and L at position 95, and targeting the small membrane protein GP4 of the wild-type PRRSV-1 with amino acid mutations of D at position 49, L at position 53, R at position 54, and G at position 57, to obtain a combination of mutant fragments, and then using linear... Homologous recombination was performed between the linearized vector and the mutant fragment to obtain a mutant infectious clonal plasmid; and / or, using plasmids rHLJB1-FIL, pACYC177-rSD1291, pACYC177-rAHEU2024 or pACYC177-rAHEU2024-M24 as templates, PCR amplification was performed using mutant primer sets designed to target amino acid G at position 70, N at position 72, D at position 80, I at position 154, H at position 158 and L at position 163 of the small vesicle protein GP3 to obtain a mutant fragment combination, and homologous recombination was performed between the linearized vector and the mutant fragment combination to obtain a mutant infectious clonal plasmid.

[0023] Preferably, the method for constructing the recombinant plasmid includes the following steps: homologous recombination of a linearized vector and a mutant fragment to obtain a mutant infectious clonal plasmid. The linearized vector and the mutant fragment combination include the combination shown in Table 1. The mutant fragment combination includes different mutant fragment compositions. The different mutant fragments are obtained by PCR amplification of the corresponding plasmid template using the corresponding primer pairs in the mutant primer set in Table 1. Table 1. Methods for constructing mutagenic infectious clonal plasmids

[0024] The present invention also provides an engineered cell or recombinant virus comprising the recombinant plasmid described above.

[0025] The present invention also provides a kit for detecting the mutant virus and the recombinant plasmid, the kit comprising the following mutant primer sets, the mutant primer sets comprising rHLJB1-BGLⅡ-F3 as shown in SEQ ID NO.14 and rHLJB1-DLRG-R as shown in SEQ ID NO.16, rHLJB1-DLRG-F as shown in SEQ ID NO.16 and rHLJB1-XBAⅠ-FU-R as shown in SEQ ID NO.15;

[0026] and / or;

[0027] Examples include rSD1291-NHEⅠ-F3 as shown in SEQ ID NO.18, rSD1291-2-FIL-R as shown in SEQ ID NO.19, rSD1291-2-FIL-F as shown in SEQ ID NO.20, rSD1291-4-DLRG-R as shown in SEQ ID NO.21, rSD1291-4-DLRG-R as shown in SEQ ID NO.21, and rSD1291-NOTⅠ-FU-R as shown in SEQ ID NO.23;

[0028] and / or;

[0029] rAHEU2024-2-ASCⅠ-F3 as shown in SEQ ID NO.24 and rAHEU2024-2-FIL-R as shown in SEQ ID NO.25, rAHEU2024-2-FIL-F as shown in SEQ ID NO.26 and rAHEU2024-4-DLRG-R as shown in SEQ ID NO.27, rAHEU2024-4-DLRG-F as shown in SEQ ID NO.28 and rAHEU2024-NOTⅠ-FU-R as shown in SEQ ID NO.29;

[0030] and / or;

[0031] Examples of rAHEU2024-2-ASCⅠ-F3 shown in SEQ ID NO.24, rAHEU2024-3-70-80-R shown in SEQ ID NO.30, rAHEU2024-3-70-80-F shown in SEQ ID NO.31, rAHEU2024-3-154-163-R shown in SEQ ID NO.32, rAHEU2024-3-154-163-F shown in SEQ ID NO.33, and rAHEU2024-NOTⅠ-FU-R shown in SEQ ID NO.29.

[0032] The present invention also provides the porcine reproductive and respiratory syndrome mutant virus and the recombinant plasmid for the preparation of a vaccine against the porcine reproductive and respiratory syndrome mutant virus.

[0033] The present invention also provides a recombinant vaccine comprising the porcine reproductive and respiratory syndrome mutant virus, engineered cells, or recombinant virus.

[0034] The vector containing the viral genome described in this invention includes pACYC177-rHLJB1-F1+F2, which is prepared by double digestion of the circular plasmid pACYC177-rHTA-FIL-3-182-270aa shown in SEQ ID NO.1 with restriction endonucleases BGLⅡ and XBAI to obtain the digested linear plasmid.

[0035] This invention verifies the adaptation of Marc-145 cells to toxic GP3 and GP4 by truncating the overlapping region of pACYC177-rHTA-FIL-3-182-270aa. It then precisely identifies the key amino acid sites within this region that determine Marc-145 cell tropism, namely amino acids 49(D), 53(L), ​​54(R), and 57(G) of GP4. Subsequently, reverse genetics is used to compare the three key amino acid sites previously identified on GP2a (patented, patent number ZL202111619805.0) with the four key amino acid sites identified on GP4 in this patent application in two non-M... Precise point mutations were performed on Marc-145 cell-adapted clones (rSD1291 and rAHEU2024) for validation. The rSD1291-M24 mutant strain, obtained by precise point mutation of 7 amino acids in rSD1291, acquired Marc-145 cell tropism, while the rAHEU2024-M24 mutant strain did not adapt to Marc-145 cells. Multiple sequence amino acid alignment analysis and validation revealed that amino acids at positions 70(G), 72(N), 80(D), 154(I), 158(H), and 163(L) in the GP3 unique region are associated with PRRSV-1. Marc-145 cell tropism is also crucial; rAHEU2024-M234, after further mutation of key amino acid sites on GP3, acquired Marc-145 cell tropism; this discovery is the first to accurately identify the key determinants of Marc-145 cell tropism in PRRSV-1 GP2a, GP3 and GP4, solving the technical bottleneck that hinders the development of new vaccines due to the difficulty of PRRSV-1 wild-type strains adapting to Marc-145 cells.

[0036] Beneficial Effects: This invention precisely resolved the key amino acid sites of PRRSV-1 that determine Marc-145 cell tropism, specifically amino acids 88(F), 94(I), and 95(L) of GP2a, amino acids 70(G), 72(N), 80(D), 154(I), 158(H), and 163(L) of GP3, and amino acids 49(D), 53(L), ​​54(R), and 57(G) of GP4, as well as the gene editing regions 50 amino acids before and after these three key amino acid sites. Based on the resolved key amino acid sites, three Marc-145 cell-adapted strains, HLJB1, SD1291, and AHEU2024, were precisely mutated using reverse genetics to obtain three PRRSV-1 modified strains adapted to in vitro passage culture of Marc 145 cells: rHLJB1-M24, rSD1291-M24, and rAHEU2024-M234. The three PRRSV-1 modified strains obtained in this invention, rHLJB1-M24, rSD1291-M24, and rAHEU2024-M234, can be stably passaged in vitro and have good replication efficiency. Infection of Marc-145 cells produces significant cytopathic effects (CPE). This invention also relates to the key amino acid sites identifying the Marc-145 cell tropism and the application of the constructed PRRSV-1 Marc-145 cell-adapted modified strains in the study of PRRSV cell tropism and infection mechanisms. Furthermore, this invention relates to the application of the multiple Marc-145 cell-adapted modified PRRSV-1 strains (rHLJB1-M24, rSD1291-M24, and rAHEU2024-M234) constructed in the development of novel PRRSV-1 vaccines. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the verification strategy for truncating key amino acid fragments that determine tropism in Marc-145 cells in the overlapping region of GP3 and GP4 of the pACYC177-rHTA-FIL-3-182-270aa infectious clone virus in the embodiments. Figure 1 The rHLJB1-FIL-overlap34 in the text refers to pACYC177-rHTA-FIL-3-182-270aa;

[0038] Figure 2 These are indirect immunofluorescence images of PAM cells and Marc-145 cells used to rescue the selected strains constructed in the examples;

[0039] Figure 3This is a sequence alignment diagram of amino acid positions 49-57 of the overlapping region of GP3 and GP4 of the pACYC177-rHTA-FIL-3-182-270aa infectious clone virus in the example.

[0040] Figure 4 According to the embodiments Figure 3 A schematic diagram of the precise point mutations in the overlapping region of rHLJB1-FIL GP3 and GP4, and indirect immunofluorescence images of PAM cells and Marc-145 cells for rescue.

[0041] Figure 5 This is a schematic diagram illustrating the replacement verification of key amino acid sites in the overlapping regions of GP3 and GP4 as analyzed in the examples with two other non-Marc-145 cell-adapted PRRSV-1 infectious clones (rSD1291 and rAHEU2024).

[0042] Figure 6 The images show indirect immunofluorescence images of PAM cells and Marc-145 cells using the rHLJB1-M24-FIL-DLRG (referred to as rHLJB1-M24), rSD1291-M24-FIL-DLRG (referred to as rSD1291-M24), and rAHEU2024-M24-FIL-DLRG (referred to as rAHEU2024-M24) modified strains in the examples.

[0043] Figure 7 This is an amino acid sequence alignment diagram and a schematic diagram of the substitution modification of the unique region of GP3 in rAHEU2024-M24-FIL-DLRG (referred to as rAHEU2024-M24) in the embodiment;

[0044] Figure 8 This is an indirect immunofluorescence image of PAM cells and Marc-145 cells of the rAHEU2024-M24-FIL-DLRG (referred to as rAHEU2024-M24) strain with key amino acid substitution in the unique region of GP3 in the example.

[0045] Figure 9 The images show microscopic examinations of lesions caused by the virus strain infecting Marc-145 cells before and after modification of key amino acid sites GP2a, GP3, and GP4, as well as a schematic diagram of the modification. Detailed Implementation

[0046] The routine experimental procedures involved in the embodiments of this invention are all performed in accordance with the standard procedures in the third edition of Molecular Cloning: A Laboratory Manual, edited by Sambrook et al. (Beijing: Science Press, 2002); the specific operation of various instruments used in the experiments strictly follows the requirements of the official instruction manuals of the corresponding instruments.

[0047] The viral materials used in the embodiments of this invention include HLJB1 isolate (isolated and preserved in our laboratory, published in the article Whole genome characterization of a novel porcine reproductive and respiratory syndrome virus 1 isolate: Genetic evidence for recombination between Amervac vaccine and circulating strains in China, DOI:10.1016 / jmeegid.2017.07.024), SD1291 (isolated and preserved in our laboratory, published in the article Metagenomic and Pathogenic Assessments Identify a Pathogenic Porcine Reproductive and Respiratory Syndrome Virus 1 with New Deletions from Adult Slaughter Pig in 2022, DOI: 10.1155 / 2023 / 1975039), and AHEU2024-2671 (isolated and preserved in our laboratory, published in the article Isolation and Genomic Characterization of a Novel Porcine Reproductive and Respiratory Syndrome Virus 1 from Severely Diseased Piglets in The modified strains rHLJB1-M24, rSD1291-M24, and rAHEU2024-M234 (rescued from infectious clone plasmids pACYC177-rHLJB1-M24, pACYC177-rSD1291-M24, and pACYC177-rAHEU2024-M234) were used in the laboratory. Cells included BHK-21 cells, Marc-145 cells, and primary alveolar macrophages (PAMs) (preserved in our laboratory).

[0048] In this embodiment of the invention, plasmid pACYC177-rHTA-FIL-3-182-270aa is obtained by modifying the recombinant plasmid rHLJB1-M-Ame3 sequence, wherein the recombinant plasmid rHLJB1-M-Ame3 has been disclosed in an existing patent (patent name: Cultivation and application of Marc-145 cell-adapted type 1 porcine reproductive and respiratory syndrome virus, patent number: ZL202111619805.0); the specific modification method is to replace the nucleotide fragments 16367-16909 of the recombinant plasmid rHLJB1-M-Ame3 with the nucleotide sequence corresponding to the HLJB1 strain, the specific sequence of which is shown in SEQ ID NO.1). The recombinant plasmid rHLJB1-FIL and the recombinant virus rHLJB1-FIL have been published in the paper The GP2a 91 / 97 / 98 Amino Acid Substitutions Play Critical Roles in Determining PRRSV Tropism and Infectivity but do not Affect Immune Responses. Journal of Virology, 99:e0004825. DOI: 10.1128 / jvi.00048-25 The TOP10 competent cells used for transformation were purchased from Beijing TransGen Biotech Co., Ltd.

[0049] In this embodiment of the invention, the online tool is: https: / / benchling.com .

[0050] Other reagents used in the embodiments of the present invention are shown in Table 2.

[0051] Table 2 Reagent Sources

[0052]

[0053] Example 1: Screening for key amino acid sites that determine PRRSV-1 Marc-145 cell tropism

[0054] 1. Design primers to screen for key amino acid fragments that determine the cell tropism of pACYC177-rHTA-FIL-3-182-270aa Marc-145 cells.

[0055] The nucleic acid sequence of pACYC177-rHTA-FIL-3-182-270aa (as shown in SEQ ID NO.1) and the nucleic acid sequence of the Marc-145 adapted strain Amervac (GenBank accession number: GU067771) were imported into the web tool https: / / benchling.com. Primer pairs for truncation verification were designed by comparing the two sequences to screen the overlapping region of GP3 and GP4 (amino acids 1-89 of GP4). The truncation and substitution verification of amino acids 1-89 of GP4 was verified by designing primer pairs. Initially, the fragment was divided into amino acids 1-62, 63-89, 49-89, and 49-57, and primer pairs were designed for each. The specific information of the primer pairs is shown in Table 3.

[0056] Table 3. Primers used to screen and determine the key amino acid fragments that determine the cell tropism of pACYC177-rHTA-FIL-3-182-270aa Marc-145 cells.

[0057]

[0058] 2. Construction of modified infectious cloning plasmids for screening key amino acid sites

[0059] First, mutant fragments 1, 2, and 3 were obtained by PCR amplification using the mutant primers designed in Table 3 (the PCR amplification system and method for the mutant fragments are shown in 2.2 below). Then, homologous recombination was performed with the double-digested vector (the vector digestion method and system are shown in 2.1 below) (method is shown in 2.3 below) to obtain the mutant truncated plasmids shown in Table 1'. The specific combinations of mutant truncated plasmids are shown in Table 1'.

[0060] Table 1. Combinations for obtaining mutant truncated plasmids

[0061]

[0062] 2.1 Vector enzyme digestion method and system:

[0063] The circular plasmid pACYC177-rHTA-FIL-3-182-270aa (SEQ ID NO.1) was double-digested with restriction endonucleases BGLⅡ and XBAⅠ to prepare the linearized vector pACYC177-rHLJB1-F1+F2. The specific reaction system is shown in Table 4.

[0064] Table 4. Double enzyme digestion system of rHTA-FIL-3-182-270aa plasmid

[0065]

[0066] After enzyme digestion, the enzyme-digested bands were separated by 0.8% agarose gel electrophoresis. The gel block containing the target band was cut off and recycled to obtain the linearized plasmid pACYC177-rHLJB1-F1+F2 (50 ng / μL), which was stored at -20℃ for subsequent construction of mutant truncated plasmids.

[0067] 2.2 PCR amplification system and method for mutant fragments

[0068] The PCR amplification system and procedure are shown in Tables 5 and 6. The amplification enzyme used was 2×PrimeSTARMAX DNA Polymearse, which was included in the materials.

[0069] Table 5. Reaction System

[0070]

[0071] Table 6. Reaction Procedure

[0072]

[0073] Using the above method, combined with the template and primer pair combinations in Table 3, we can obtain Figure 1 The mutant fragments 1-9 required for each of the modified strains are shown. Electrophoresis was performed on a 1% agarose gel for 30 min. The gel block containing the target band was cut off and the mutant fragments 1-9 were recovered. After the concentration was determined, they were stored at -20℃ for later use.

[0074] 2.3 Homologous recombination methods and systems

[0075] Then, the gel-recovered mutant fragments 1-3, 4 and 5, 6 and 7, and 8 and 9 were subjected to homologous recombination with the linearized vector pACYC177-rHLJB1-F1+F2 (One Step Clone Kit). The amount of vector and fragment used was calculated based on the length and concentration of the vector and fragments using a web-based tool (https: / / tool.vazyme.com:18002 / cetool / restructure.html). The homologous recombination systems for mutant fragments 1-3 are shown in Table 7. The homologous recombination systems for mutant fragments 4 and 5, 6 and 7, and 8 and 9 are shown in Table 7'.

[0076] Table 7 Homologous recombination systems and reaction procedures

[0077]

[0078] Table 7. Homologous recombination systems and reaction procedures

[0079]

[0080] After the reaction was completed, all ligation products were transformed into TOP10 competent cells, and single colonies were picked for pure culture. The bacterial culture was then used as the upstream and downstream primers listed in Table 3 for sequencing. Sequencing-positive bacteria were selected and amplified overnight, after which plasmids were extracted to obtain the following plasmids: pACYC177-rHLJB1-FIL-4-1-62aa (215 ng / μL), pACYC177-rHLJB1-FIL-4-63-89aa (243 ng / μL), pACYC177-rHLJB1-FIL-4-49-89aa (239 ng / μL), and pACYC177-rHLJB1-FIL-4-49-57aa (207 ng / μL). These plasmids were stored at -20℃ for later use.

[0081] 3. Rescue of the modified virus in PAM cells and its cell adaptation test in Marc-145.

[0082] 3.1 Transfection of Mutant Truncated Plasmids

[0083] Prepare BHK-21 cells (cell density approximately 80%) for transfection in advance.

[0084] Transfection method: Dilute the target plasmid (use 50 μL OPTI-MEM to dilute 2 μg of target plasmid (pACYC177-rHLJB1-FIL-4-1-62aa, pACYC177-rHLJB1-FIL-4-63-89aa, pACYC177-rHLJB1-FIL-4-49-89aa, pACYC177-rHLJB1-FIL-4-49-57aa), prepare Lip3000 premix (50 μL OPTI-MEM + 3 μL Lip3000), add 4 μL Lip3000 to each diluted target plasmid, followed by 53 μL Lip3000 premix, and incubate at 25℃ for 15 min. Add dropwise and evenly to the supernatant of BHK-21 cells.

[0085] 48 h after transfecting BHK-21 cells, all supernatant was collected.

[0086] 3.2 Rescue of Modified Viruses and IFA Identification

[0087] Primary PAM cells were pre-treated with RPMI (1640) medium containing 2% FBS at a concentration of 2 × 10⁶ cells / year. 5Cells were seeded into 24-well cell culture plates and incubated at 37°C with 5% CO2. After primary PAM cells adhered, the cell supernatant was replaced with the collected transfection supernatant. After incubation for 2 hours, the supernatant was discarded, and 1 mL of cell maintenance medium (1640 medium containing 2% FBS) was added for further culture. Cells were cultured until cytopathic effects appeared, at which point the cell supernatant was collected. Cells at the bottom of the wells were treated with PRRSV-N specific monoclonal antibody 15A1. (PRRSV-N specific monoclonal antibody 15A1 is derived from the article "Amino Acid Substitutions Play Critical Roles in Determining PRRSV Tropism and Infectivity but do not Affect Immune Responses." Journal of Virology, 99:e0004825. DOI: 10.1128 / jvi.00048-25 And refer to the methods in that article to perform IFA identification.

[0088] The rescue results of modified viral PAMs cells are shown in Figure 2 , Figure 2 After IFA identification, Figure 1 All the modified viruses constructed in this way showed a specific red fluorescence, indicating that Figure 1 All the modified viruses constructed in the study were successfully rescued on PAM cells.

[0089] 3.3 Adaptability test and passage of the modified virus Marc-145 in cells

[0090] Marc-145 cells were pre-coated and cultured in DMEM medium containing 10% FBS at a density of 2 × 10⁶ cells / cells. 5 Cells were seeded at a density of approximately 80% per well in 12-well cell culture plates and incubated at 37°C in a 5% CO2 incubator until the cell density reached approximately 80%. Then, the medium was replaced with DMEM containing 2% FBS.

[0091] 500 μL of the successfully rescued modified virus solution in PAM cells (i.e., all supernatant) was inoculated into Marc-145 cells. The strain was continuously passaged blindly for 5 generations. The modified virus from the 5th generation was inoculated into Marc-145 cells. After 4 days post-inoculation (dpi), IFA was performed using PRRSV-2 anti-N protein monoclonal antibody 15A1.

[0092] like Figure 2The IFA identification results showed that the rHLJB1-FIL-4-1-62aa, rHLJB1-FIL-4-49-89aa, and rHLJB1-FIL-4-49-57aa modified strains showed specific red fluorescence by IFA 4 dpi after infecting Marc-145 cells, while rHLJB1-FIL-4-63-89aa was not detected. This indicates that the key amino acid site determining the Marc-145 cell tropism of the pACYC177-rHTA-FIL-3-182-270aa modified strain is located at amino acids 49-57.

[0093] 4. Precise analysis and validation of key amino acids in GP4 that determine Marc-145 cell tropism

[0094] 4.1. Alignment of amino acid sequences from position 49 to 57 of GP4

[0095] The GP4 amino acid sequences of representative PRRSV-1 strains from each subgroup (specific strain information can be found in Table 8) were introduced. https: / / benchling.com The sequence of amino acids 49-57 was compared, and the comparison results are as follows: Figure 3 As shown, the 49th, 53rd, 54th, and 57th hypervariable amino acid sites were selected, and the amino acid combination pattern (D / L / R / G) of the Amervac Marc-145 cell adaptation mode was used to verify the precise replacement of the overlapping regions of rHLJB1-FIL GP3 and GP4.

[0096] Table 8. Specific information on each subgroup of PRRSV-1 strains

[0097]

[0098] 4.2 Verification of precise replacement of overlapping areas between GP3 and GP4

[0099] Designed mutant primer pairs, the primer pair sequences are shown in Table 9, and the substitution diagram is shown in... Figure 4 As shown.

[0100] Table 9. Primers for precise replacement of GP3 and GP4 overlapping regions.

[0101]

[0102] Using the PCR method described in section 2.2 with the mutant primer pairs rHLJB1-BGLⅡ-F3 and rHLJB1-DLRG-R as a template, mutant fragment 10 was obtained. Similarly, mutant fragment 11 was obtained using primer pairs rHLJB1-DLRG-F and rHLJB1-XBAⅠ-FU-R. Then, the method described in section 2.3 was used to PCR the vector pACYC177-rHLJB1-F1+F2 with mutant fragments 10 and 11. Homologous recombination of fragment 11 yielded the mutant infectious clonal plasmid pACYC177-rHLJB1-M24-FIL-DLRG, abbreviated as pACYC177-rHLJB1-M24; the infectious clonal plasmid was rescued using the method in section 3 to obtain the modified virus rHLJB1-M24-FIL-DLRG, abbreviated as rHLJB1-M24; PAMs cell rescue of the modified virus and Marc-145 cell adaptability test were performed.

[0103] The rescue results and Marc-145 cell adaptation results are as follows: Figure 4 As shown, red specific fluorescence could be detected in both PAMs and Marc-145 cells, indicating that replacing only amino acids 49, 53, 54, and 57 of GP4 can induce Marc-145 cell tropism in rHLJB1-FIL, and that amino acids 49, 53, 54, and 57 of GP4 are key amino acid sites that determine the tropism of PRRSV-1 in Marc-145 cells.

[0104] 5. Verification of non-adapted strains modified based on key amino acid sites of GP2a and GP4.

[0105] Mutation primers were designed to modify two non-Marc-145 cell-adapted strains, SD1291 and AHEU2024-2671. A schematic diagram of the modification is shown below. Figure 5 As shown in Table 10, the specific primer information is as follows.

[0106] Table 10. Primers for modifying key amino acid sites of GP2a and GP4 in SD1291 and AHEU2024-2671.

[0107]

[0108] Using the method described in section 2.2, mutant fragments 12-17 were obtained using the modified primer pairs (see Table 3 for specific correspondences). Then, the vector digestion method described in section 2.1 was used to digest the vectors pACYC177-rSD1291 (SEQ ID NO.2) and pACYC177-rAH2024 (SEQ ID NO.2). NO.3) Infectious cloning plasmids were digested with enzymes. The enzyme digestion systems are shown in Tables 11 and 12 below. Linearized vectors pACYC177-rSD1291-F1+F2 (50 ng / μL) and pACYC177-rAH2024-F1+F2+F3 (50 ng / μL) were obtained after digestion. Using the homologous recombination method described in section 2.3, mutant fragments 12-14 were homologously recombinated with the linearized vector pACYC177-rSD1291-F1+F2 to obtain the modified plasmid pACYC177-rSD1291, containing amino acids 88, 94, and 95 of GP2a and amino acids 49, 53, 54, and 57 of the key amino acid site GP4. -M24-FIL-DLRG, abbreviated as pACYC177-rSD1291-M24, was obtained by homologous recombination of mutant fragments 15-17 with the linearized vector pACYC177-rAH2024-F1+F2+F3 to obtain amino acid positions 88, 94, and 95 of GP2a and amino acid positions 49, 53, 54, and 57 of the key amino acid site of GP4 in rAH2024, respectively. The modified plasmid pACYC177-rAHEU2024-M24-FIL-DLRG, abbreviated as pACYC177-rAHEU2024-M24, was then transfected, rescued, and tested for adaptability to Marc-145 cells using the same method described in section 3.

[0109] Table 11. Double enzyme digestion system of pACYC177-rSD1291 plasmid

[0110]

[0111] Table 12. Double enzyme digestion system of pACYC177-rAHEU2024 plasmid

[0112]

[0113] The results of PAMs cell rescue of IFA are as follows: Figure 6As shown in Figure A, pACYC177-rHLJB1-M24, pACYC177-rSD1291-M24, and pACYC177-rAHEU2024-M24 all showed specific red fluorescence detected using monoclonal antibody 15A1, demonstrating the successful rescue of infectious clonal modified strains of rHLJB1-M24, rSD1291-M24, and rAHEU2024-M24; while the Marc-145 cell adaptive IFA results are as follows... Figure 6 As shown in B, only rHLJB1-M24 and rSD1291-M24 showed specific red fluorescence, while rAHEU2024-M24 did not show specific red fluorescence. This indicates that simply changing the 88th, 94th, and 95th amino acids of GP2a and the 49th, 53rd, 54th, and 57th amino acids of GP4 (FIL-DLRG pattern) is insufficient to make the rAHEU2024 strain acquire Marc-145 cell tropism. Therefore, we infer that there are still key amino acid sites in the unique amino acid region of GP3 that determine the PRRSV-1 Marc-145 cell tropism.

[0114] 6. Analysis and verification of key amino acid sites in the unique GP3 region that determine the cell tropism of PRRSV-1 Marc-145.

[0115] 6.1 Amino acid sequence alignment of the unique region of GP3

[0116] The rHLJB1 and rSD1291 strains achieved Marc-145 cell tropism simply by precisely replacing the key amino acid sites at GP2a and GP4. This indicates that the rAHEU2024 strain contains amino acid sites in the unique amino acid region of GP3 that differ from those of the Marc-145 cell-adapted strains Amervac, rHLJB1, and rSD1291. These sites may be related to the PRRSV-1 Marc-145 cell tropism. Therefore, this invention performs amino acid sequence alignment on the unique region of GP3, focusing on amino acid sites that are the same as those in Amervac, rHLJB1, and rSD1291 but different from those in rAHEU2024.

[0117] The nucleic acid sequences of Amervac, HLJB1, SD1291, and AHEU2024-2671 were simultaneously introduced. https: / / benchling.com The comparison results are as follows Figure 7As shown in Figure A, comparison revealed that amino acid residues at positions 70, 72, 80, 154, 158, and 163 of rAHEU2024 differ from those of Amervac, rHLJB1, and rSD1291. Amervac, rHLJB1, and rSD1291 belong to the G / N / D / I / H / L pattern, while rAHEU2024 belongs to the S / T / T / V / Y / S pattern. Therefore, this invention proposes the hypothesis that amino acids at positions 70, 72, 80, 154, 158, and 163 of GP3 may be related to PRRSV-1 Marc-145 cell tropism.

[0118] 6.2 Validation of the key role of six amino acid sites in the unique GP3 region for PRRSV-1 Marc-145 cell tropism

[0119] Mutation primer pairs were designed targeting six amino acid sites in the unique region of rAHEU2024-M24 GP3. A schematic diagram of the modification is shown below. Figure 7 As shown in B, the modified virus for six amino acid sites of GP3 at amino acid sites 70, 72, 80, 154, 158, and 163 is rAHEU2024-M24-3U-M6 (i.e., rAHEU2024-M234). The modified virus that modifies only the first three amino acid sites of GP3 at amino acid sites 70, 72, and 80 is rAHEU2024-M24-3U-3M. The modified virus that modifies only the last three amino acid sites of GP3 at amino acid sites 154, 158, and 163 is rAHEU2024-M24-3U-M3. The specific primer information is shown in Table 13.

[0120] Table 13. Primers for modifying six amino acid sites in the unique region of GP3

[0121]

[0122] Using the same method as in section 2, the modified primer pairs in Table 10 were combined with the plasmids in Table 3 as templates to obtain mutant fragments 18-20. Then, the homologous recombination method in section 2.3 was used to obtain the modified plasmid pACYC177-rAHEU2024-M234, which modifies six amino acid sites in the unique GP3 region; the modified plasmid pACYC177-rAHEU2024-M24-3U-3M, which modifies only the first three amino acid sites (70, 72, and 80) in the unique GP3 region; and the modified plasmid pACYC177-rAHEU2024-M24-3U-M3, which modifies only the last three amino acid sites (154, 158, and 163) in the unique GP3 region. The modified plasmids were then transfected, rescued, and subjected to Marc-145 cell adaptability testing using the same method as in section 3.

[0123] The results of PAMs cell rescue of IFA are as follows: Figure 8 As shown in Figure A, specific red fluorescence was detected in all three modified plasmids after transfection, indicating that the infectious clones of the three GP3 unique regions were successfully rescued; while the Marc-145 cell adaptive IFA results are as follows. Figure 8 As shown in B, only the rAHEU2024-M24-3U-M6 (i.e., rAHEU2024-M234) infectious clone modified with six amino acid sites modified in the unique region of GP3 can adapt to Marc-145 cells. The results indicate that amino acids 70, 72, 80, 154, 158, and 163 in the unique region of GP3 are also crucial for PRRSV-1 Marc-145 cell tropism.

[0124] In summary, this invention has resolved the key amino acid sites of PRRSV-1 that determine Marc-145 cell tropism, specifically amino acids 88(F), 94(I), and 95(L) of GP2a, amino acids 70(G), 72(N), 80(D), 154(I), 158(H), and 163(L) of GP3, and amino acids 49(D), 53(L), ​​54(R), and 57(G) of GP4. Furthermore, this invention, through precise modification of these key amino acid sites, yielded three PRRSV-1 modified strains adapted to Marc-145 cells: rHLJB1-M24, rSD1291-M24, and rAHEU2024-M234. Microscopic images of these strains before and after modification infecting Marc-145 cells are shown below. Figure 9 As shown, the GP3 key amino acid sites of rHLJB1 and rSD1291 are already in the adaptation mode, so only the GP2a and GP4 key amino acid sites need to be changed to obtain Marc-145 cell tropism. However, rAHEU2024 requires the replacement of the GP2a, GP3 and GP4 key amino acid sites at the same time to obtain Marc-145 cell tropism.

Claims

1. A mutant virus of porcine reproductive and respiratory syndrome, characterized in that, The mutant virus includes mutations in the small envelope protein GP4 of the mutant strain PRRSV-1, with amino acid 49 changed to D, amino acid 53 changed to L, amino acid 54 changed to R, and amino acid 57 changed to G. And / or; the 70th amino acid of the small membrane protein GP3 of the wild-type PRRSV-1 is mutated to G, the 72nd amino acid to N, the 80th amino acid to D, the 154th amino acid to I, the 158th amino acid to H, and the 163rd amino acid to L. In the mutant strain PRRSV-1, the 88th amino acid of the small membrane protein GP2a is mutated to F, the 94th amino acid is mutated to I, and the 95th amino acid is mutated to L. Preferably, the mutant strain PRRSV-1 is obtained by performing the following mutations on three Marc-145 cell non-adapted strains HLJB1, SD1291 or AHEU2024-2671: the 88th amino acid of the small membrane protein GP2a is mutated to F, the 94th amino acid is mutated to I and the 95th amino acid is mutated to L.

2. The method for constructing the porcine reproductive and respiratory syndrome mutant virus according to claim 1, characterized in that, The steps include: mutating the 88th amino acid of the small membrane protein GP2a of strain HLJB1 or strain SD1291 to F, the 94th amino acid to I, and the 95th amino acid to L, and mutating the 49th amino acid of the small membrane protein GP4 to D, the 53rd amino acid to L, the 54th amino acid to R, and the 57th amino acid to G; Alternatively, mutate the following amino acids from strain AHEU2024-2671: amino acid 88 of GP2a to F, amino acid 94 to I, and amino acid 95 to L; amino acid 49 of GP4 to D, amino acid 53 to L, amino acid 54 to R, and amino acid 57 to G; and amino acid 70 of GP3 to G, amino acid 72 to N, amino acid 80 to D, amino acid 154 to I, amino acid 158 to H, and amino acid 163 to L.

3. The method for constructing a porcine reproductive and respiratory syndrome mutant virus according to claim 2, characterized in that, The method includes the following steps: designing mutant primers to perform site-directed mutagenesis on a vector containing the viral genome to obtain a mutant fragment; ligating the mutant fragment and a vector containing the viral genome to obtain a recombinant plasmid; introducing the recombinant plasmid into a host cell; and rescuing the mutant virus from the cell or culture.

4. The method for constructing a porcine reproductive and respiratory syndrome mutant virus according to claim 3, characterized in that, The mutant primers include sets of mutant primers designed for mutations of amino acid F at position 88, I at position 94, and L at position 95 of small vesicle protein GP2a, and for mutations of amino acid D at position 49, L at position 53, R at position 54, and G at position 57 of small vesicle protein GP4; preferably, sets of mutant primers also include sets of mutant primers designed for mutations of amino acid G at position 70, N at position 72, D at position 80, I at position 154, H at position 158, and L at position 163 of small vesicle protein GP3.

5. A recombinant plasmid, characterized in that, It contains gene segments in the full-length cDNA of the wild-type PRRSV-1 virus with the following mutations: the mutations result in amino acid F at position 88, I at position 94, and L at position 95 of the small envelope protein GP2a of the wild-type virus; the mutations result in amino acid D at position 49, L at position 53, R at position 54, and G at position 57 of the small envelope protein GP4 of the wild-type PRRSV-1. Or; it includes gene segments in the full-length cDNA of wild-type PRRSV-1 virus that have undergone the following mutations: the mutations result in the following changes: amino acid 88 of the small envelope protein GP2a of the wild-type virus is changed to F, amino acid 94 is changed to I, and amino acid 95 is changed to L; the mutations result in the following changes: amino acid 49 of the small envelope protein GP4 of the wild-type PRRSV-1 is changed to D, amino acid 53 is changed to L, amino acid 54 is changed to R, and amino acid 57 is changed to G; the mutations result in the following changes: amino acid 70 of the small envelope protein GP3 of the wild-type PRRSV-1 is changed to G, amino acid 72 is changed to N, amino acid 80 is changed to D, amino acid 154 is changed to I, amino acid 158 is changed to H, and amino acid 163 is changed to L.

6. The method for constructing the recombinant plasmid according to claim 5, characterized in that, The steps include: using plasmids rHLJB1-FIL, pACYC177-rSD1291, pACYC177-rAHEU2024, or pACYC177-rAHEU2024-M24 as templates, respectively, and using mutations to the 88th amino acid of the wild-type small membrane protein GP2a (mutated to F), the 94th amino acid of GP2a (mutated to I), and the 95th amino acid of GP4a (mutated to L), as well as mutations to the GP4a of the wild-type PRRSV-1 small membrane protein. PCR amplification was performed using a set of mutant primers containing mutations at amino acid positions 49 (D), 53 (L), 54 (R), and 57 (G) to obtain a mutant fragment combination. Homologous recombination was then performed between the linearized vector and the mutant fragment combination to obtain a mutant infectious clonal plasmid. This plasmid was then used in combination with plasmids rHLJB1-FIL, pACYC177-rSD1291, pACYC177-rAHEU2024, or p... Using ACYC177-rAHEU2024-M24 as a template, PCR amplification was performed using a mutant primer set designed to target amino acid G at position 70, N at position 72, D at position 80, I at position 154, H at position 158, and L at position 163 of the small membrane protein GP3. The resulting mutant fragment combination was then subjected to homologous recombination with the linearized vector to obtain a mutant-modified infectious clonal plasmid. Preferably, the construction method specifically includes the following steps: homologous recombination of the linearized vector and the mutant fragment combination to obtain a mutant-modified infectious clonal plasmid. The linearized vector and the mutant fragment combination include combinations as shown in Table 1. The mutant fragment combination includes different mutant fragment compositions, and the different mutant fragments are obtained by PCR amplification of the corresponding plasmid template using the corresponding primer pairs in the mutant primer set in Table 1. Table 1: Construction Method of Mutant-Modified Infectious Clonal Plasmid 。 7. An engineered cell or recombinant virus, characterized in that, It contains the recombinant plasmid as described in claim 5.

8. A kit for detecting the mutant virus of claim 1 and the recombinant plasmid of claim 5, characterized in that, The kit includes the following mutant primer sets, including rHLJB1-BGLⅡ-F3 as shown in SEQ ID NO.14, rHLJB1-DLRG-R as shown in SEQ ID NO.16, rHLJB1-DLRG-F as shown in SEQ ID NO.16, and rHLJB1-XBAⅠ-FU-R as shown in SEQ ID NO.15; and / or; Examples include rSD1291-NHEⅠ-F3 as shown in SEQ ID NO.18, rSD1291-2-FIL-R as shown in SEQ ID NO.19, rSD1291-2-FIL-F as shown in SEQ ID NO.20, rSD1291-4-DLRG-R as shown in SEQ ID NO.21, rSD1291-4-DLRG-R as shown in SEQ ID NO.21, and rSD1291-NOTⅠ-FU-R as shown in SEQ ID NO.23; and / or; rAHEU2024-2-ASCⅠ-F3 as shown in SEQ ID NO.24 and rAHEU2024-2-FIL-R as shown in SEQ ID NO.25, rAHEU2024-2-FIL-F as shown in SEQ ID NO.26 and rAHEU2024-4-DLRG-R as shown in SEQ ID NO.27, rAHEU2024-4-DLRG-F as shown in SEQ ID NO.28 and rAHEU2024-NOTⅠ-FU-R as shown in SEQ ID NO.29; and / or; Examples include rAHEU2024-2-ASCⅠ-F3 as shown in SEQ ID NO.24, rAHEU2024-3-70-80-R as shown in SEQ ID NO.30, rAHEU2024-3-70-80-F as shown in SEQ ID NO.31, rAHEU2024-3-154-163-R as shown in SEQ ID NO.32, rAHEU2024-3-154-163-F as shown in SEQ ID NO.33, and rAHEU2024-NOTⅠ-FU-R as shown in SEQ ID NO.

29.

9. The porcine reproductive and respiratory syndrome mutant virus of claim 1, the recombinant plasmid of claim 5, or the engineered cells or recombinant virus of claim 7, used to prepare a vaccine for porcine reproductive and respiratory syndrome mutant virus.

10. A recombinant vaccine, characterized in that, It comprises the porcine reproductive and respiratory syndrome mutant virus of claim 1 or the engineered cell or recombinant virus of claim 7.

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