A fusion protein of porcine reproductive and respiratory syndrome virus and application thereof

Abstract

The present application belongs to the field of veterinary biological products, and relates to a fusion protein of porcine reproductive and respiratory syndrome virus and application thereof, the fusion protein comprising a signal peptide of a US6 gene of porcine pseudorabies virus, an extracellular domain of GP2 protein, an extracellular domain of GP3 protein, an extracellular domain of GP4 protein, an extracellular domain of GP5 protein and a transmembrane region of the US6 gene of the porcine pseudorabies virus. The coding sequence of the fusion protein is inserted into a genome of a pseudorabies virus attenuated strain in which US8 gene, US7 gene, US9 gene, US2 gene and UL23 gene are all inactivated by a homologous recombination method to obtain a recombinant virus. The recombinant virus inoculated into pigs can produce neutralizing antibodies against the porcine reproductive and respiratory syndrome virus, can resist an attack of the porcine reproductive and respiratory syndrome virus and has good immunogenicity and challenge protection ability.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products and relates to a fusion protein of porcine reproductive and respiratory syndrome virus and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV).

[0003] PRRSV infection in pigs presents with two distinct clinical manifestations: reproductive disorders in sows and respiratory diseases in piglets and finishing pigs. Reproductive disorders in sows are characterized by abortion, stillbirths or mummified fetuses, premature birth, and weak piglets. In addition to severe respiratory distress, PRRSV-infected piglets and finishing pigs exhibit other clinical manifestations, including depression, loss of appetite, lethargy, and fever.

[0004] PRRSV belongs to the family Arteriviridae within the order Nidovirales. The PRRSV genome is a single-stranded positive-sense RNA, approximately 15 kb in length, with a methylated cap at the 5' end and a poly(A) tail at the 3' end. Its genome contains nine open reading frames (ORFs). ORF1a and ORF1b comprise about three-quarters of the viral genome, encoding the polyproteins pp1a and pp1ab, which are cleaved into at least 14 non-structural proteins that play crucial roles in viral genome replication, transcription, and regulation of the host's innate immune response. The remaining ORFs encode structural proteins. ORF2a, ORF3, ORF4, ORF5, and ORF6 encode the envelope glycoproteins GP2, GP3, GP4, and GP5, and the matrix protein M, respectively. These proteins are essential for viral particle assembly and bubbling. ORF7 encodes the nucleoprotein N. ORF2b encodes the small membrane protein E, which has ion channel activity and participates in viral uncoating. ORF5a encodes a protein crucial for viral survival; its mechanism of action is not fully understood, but it is known to be involved in viral particle assembly and infectivity. Regarding immunogenicity, GP5 is the main target for inducing neutralizing antibodies, but GP2, GP3, and GP4 also contain key neutralizing epitopes. Furthermore, many viral proteins, including GP3, GP4, GP5, M, N, and some non-structural proteins, contain epitopes that can be recognized by T cells.

[0005] PRRS is widespread in major pig-producing countries and regions worldwide. The disease broke out in North America in the late 1980s and Europe in the early 1990s, and subsequently spread rapidly throughout the world. In recent years, highly variable PRRSV strains have continued to evolve, constantly triggering new outbreaks globally.

[0006] Currently, vaccination is the primary means of preventing PRRS. Vaccines used globally include various types, such as live attenuated vaccines (MLV) and inactivated vaccines. However, most existing vaccines are designed for homologous strains, offering limited cross-protection against heterologous viruses. Despite the introduction of numerous PRRS vaccines over the past 30 years, a "perfect" vaccine providing complete protection has yet to emerge. The high genetic diversity of PRRSV and its damaging effects on the pig immune system make developing broad-spectrum, highly effective vaccines particularly challenging. With advancements in new technologies and a deeper understanding of the pathogenic mechanisms and immune responses to PRRSV, the development of various novel vaccines, including MLV, inactivated vaccines, subunit vaccines, live vector vaccines, DNA vaccines, and RNA vaccines, is accelerating.

[0007] Pseudorabies virus (PRV) is the pathogen that causes pseudorabies (PR) in pigs. Belonging to the Herpesviridae family, its natural host is the pig, but it can also infect many other animals. The PRV genome is a double-stranded DNA of approximately 143 kb, including unique long regions, unique short regions, and flanking inverted repeat sequences. The PRV genome encodes approximately 70 ORFs, producing viral proteins including envelope proteins, replicases, and various non-essential proteins. Its large genome has a strong capacity to accommodate foreign genes, allowing the insertion of large DNA fragments without affecting viral replication. Because live vector vaccines using PRV can effectively elicit humoral and cellular immune responses in the host, they are often used as a platform for expressing antigens of various porcine pathogens for the development of multivalent or combined vaccines. Summary of the Invention

[0008] The first aspect of the present invention provides a first biological material, wherein the first biological material is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9, P10 and P11;

[0009] P1: First fusion protein

[0010] The peptide segments in the first fusion protein include a first peptide segment, a second peptide segment, a third peptide segment, and a fourth peptide segment;

[0011] The amino acid sequence of the first peptide is shown in positions 18-187 of SEQ ID NO.21;

[0012] The amino acid sequence of the second peptide is shown in positions 193-253 of SEQ ID NO.21;

[0013] The amino acid sequence of the third peptide is shown in positions 259-392 of SEQ ID NO.21;

[0014] The amino acid sequence of the fourth peptide is shown in positions 398-430 of SEQ ID NO.21;

[0015] P2: Second fusion protein

[0016] The peptides in the second fusion protein include the first fusion protein and the fifth peptide;

[0017] The fifth peptide segment is a transmembrane domain peptide segment;

[0018] P3: Third fusion protein

[0019] The sixth peptide is any one, two, or three of the following: a signal peptide, a fluorescent protein peptide, and a tag peptide used for protein isolation and purification, which can be localized to the pseudorabies virus envelope or the host cell membrane.

[0020] The peptide segment in the third fusion protein includes the first fusion protein and the sixth peptide segment; or

[0021] The peptide segment in the third fusion protein includes the second fusion protein and the sixth peptide segment;

[0022] P4: First protein modifier

[0023] The first protein modifier is a modified protein produced by post-translational modification of the first fusion protein, the second fusion protein, or the third fusion protein in eukaryotic cells;

[0024] P5: First RNA

[0025] The first RNA can be translated to the first fusion protein, the second fusion protein, or the third fusion protein;

[0026] P6: First Gene

[0027] The coding sequence of the first gene can encode the first fusion protein, the second fusion protein, or the third fusion protein;

[0028] P7: First gene expression cassette

[0029] The gene expression product in the first gene expression cassette assembly is the first RNA;

[0030] The promoter of the first gene expression cassette is a constitutive expression promoter or an artificially inducible promoter;

[0031] P8: First Gene Engineering Vector

[0032] The first genetic engineering vector encodes the first expressible RNA;

[0033] P9: First Cell

[0034] The first cell contains the first gene engineering vector;

[0035] P10: First Composition

[0036] The first composition contains the first fusion protein, the second fusion protein, the third fusion protein, the first protein modifier, the first RNA, the first gene engineering vector, or the first cell;

[0037] P11: First Reagent Kit

[0038] The kit contains the first fusion protein, the second fusion protein, the third fusion protein, the first protein modifier, the first RNA, the first genetic engineering vector, or the first cell.

[0039] In some embodiments, the signal peptide is the signal peptide of the porcine pseudorabies virus US6 gene.

[0040] The amino acid sequence of the fifth peptide is shown in positions 431-479 of SEQ ID NO.21;

[0041] The transmembrane domain peptide is the transmembrane region of the porcine pseudorabies virus US6 gene.

[0042] In the first fusion egg, the first peptide, the second peptide, the third peptide, and the fourth peptide are arranged in order from the N-terminus to the C-terminus;

[0043] In the second fusion egg, the first peptide, the second peptide, the third peptide, the fourth peptide, and the fifth peptide are arranged in order from the N-terminus to the C-terminus;

[0044] The first fusion protein, the second fusion protein, or the third fusion protein have a linking peptide or random amino acid sequence of 1-15 amino acids that does not affect the functional independence of the domains between different peptide segments;

[0045] The promoter of the first gene expression cassette is the CMV promoter;

[0046] The backbone of the first gene engineering vector is either the pX330 vector or the pUC19 vector; or

[0047] The host cells of the cells are Escherichia coli cells, 293T cells, or PK-15 cells.

[0048] In some embodiments, the backbone of the first genetic engineering vector is a pseudorabies virus vector; or

[0049] The host cells of the cells are porcine pseudorabies virus susceptible cells.

[0050] In some embodiments, the amino acid sequence of the signal peptide is shown as positions 1-17 of SEQ ID NO.21;

[0051] The amino acid sequence of the transmembrane domain peptide is shown in positions 431-479 of SEQ ID NO.21;

[0052] The amino acid sequence of the third fusion protein is shown in SEQ ID NO.21; or

[0053] The pseudorabies virus vector is a weakened strain of pseudorabies virus.

[0054] In some embodiments, the US8, UL23, US7, US9, and US2 genes in the genome of the attenuated pseudorabies virus strain are all inactivated genes; or

[0055] The first gene expression cassette is located between the UL24 and UL22 genes of the attenuated pseudorabies virus strain.

[0056] In some embodiments, the porcine pseudorabies virus-JM strain is used as the parent virus strain. The US8, UL23, US7, US9, and US2 genes are knocked out in the genome of the parent virus strain to form the attenuated pseudorabies virus strain. The genome sequence of the porcine pseudorabies virus-JM strain is shown in GenBank No. OK338077.1. The microbial accession number of the porcine pseudorabies virus-JM strain is CCTCC NO: V202615.

[0057] The second aspect of the present invention provides a method for preparing recombinant pseudorabies virus, wherein the method comprises transferring the first gene expression cassette described in the first aspect of the present invention into the genome of a attenuated pseudorabies virus strain to obtain the recombinant pseudorabies virus.

[0058] In some embodiments, the method is characterized by transferring the first gene expression cassette described in the first aspect of the present invention into the genome of the attenuated pseudorabies virus strain described in the first aspect of the present invention through homologous recombination to obtain the recombinant pseudorabies virus.

[0059] In some embodiments, the preparation method includes the following first three or first four steps:

[0060] S1: Prepare the first plasmid, the second plasmid, and the third plasmid in parallel or sequentially;

[0061] The third plasmid contains the first gene expression cassette;

[0062] The first gene expression cassette contains a first homologous arm upstream; the first homologous arm is homologous to the upstream of the genome fragment of the attenuated pseudorabies virus strain into which the first gene expression cassette is transferred.

[0063] The first gene expression cassette contains a second homologous arm downstream; the second homologous arm is homologous to the downstream portion of the pseudorabies virus attenuated strain genome fragment into which the first gene expression cassette is transferred;

[0064] The first plasmid contains a Cas9 gene expression cassette and a first sgRNA expression cassette; the sgRNA expressed by the first sgRNA expression cassette can mediate homologous recombination between the first homologous arm and the genome of the attenuated pseudorabies virus strain with the participation of the Cas9 protein.

[0065] The second plasmid contains a Cas9 gene expression cassette and a second sgRNA expression cassette; the sgRNA expressed by the second sgRNA expression cassette can mediate homologous recombination between the second homologous arm and the genome of the attenuated pseudorabies virus strain with the participation of the Cas9 protein.

[0066] S2: First pseudorabies virus susceptible cells are co-transfected with the genomes of the first plasmid, the second plasmid, the third plasmid and the attenuated pseudorabies virus strain;

[0067] S3: Identify and harvest the recombinant pseudorabies virus;

[0068] S4: The recombinant pseudorabies virus is cultured and purified in second pseudorabies virus susceptible cells to obtain purified recombinant pseudorabies virus.

[0069] In some embodiments, the backbone of the first plasmid is selected from the pUC19 vector;

[0070] The backbone of the second plasmid was selected from the pX330 vector;

[0071] The backbone of the third plasmid is selected from the pX330 vector;

[0072] The first pseudorabies virus susceptible cells were selected from HEK293T cells;

[0073] The second pseudorabies virus susceptible cells were selected from Vero cells or PK-15 cells; or

[0074] The transfection operation was performed using Lipofectamine 3000 transfection reagent.

[0075] The third aspect of the present invention provides a recombinant pseudorabies virus, which is prepared by the preparation method described in the second aspect of the present invention.

[0076] The fourth aspect of the present invention provides the use of the first biological material described in the first aspect of the present invention in the preparation of a formulation for use alone, for use in combination with other immunomodulators and / or drugs, or as a component of a compound formulation composed of other immunomodulators and / or drugs to prevent porcine reproductive and respiratory syndrome, wherein the first biological material is selected from the first fusion protein, the second fusion protein, the third fusion protein, the first protein modifier, and the first cell.

[0077] The fifth aspect of the present invention provides a combined recombinant pseudorabies virus, the combined recombinant pseudorabies virus comprising a first recombinant pseudorabies virus, a second recombinant pseudorabies virus, a third recombinant pseudorabies virus, a fourth recombinant pseudorabies virus and a fifth recombinant pseudorabies virus.

[0078] The first recombinant pseudorabies virus uses a weakened strain of pseudorabies virus as a vector virus, and the first recombinant pseudorabies virus contains a gene expression cassette 1.

[0079] The second recombinant pseudorabies virus uses a weakened strain of pseudorabies virus as a vector virus, and the second recombinant pseudorabies virus contains gene expression cassette 2;

[0080] The third recombinant pseudorabies virus uses a weakened pseudorabies virus strain as a vector virus, and the third recombinant pseudorabies virus contains a gene expression cassette 3.

[0081] The fourth recombinant pseudorabies virus uses a weakened pseudorabies virus strain as a vector virus, and the fourth recombinant pseudorabies virus contains a gene expression cassette 4.

[0082] The fifth recombinant pseudorabies virus uses a weakened pseudorabies virus strain as a vector virus, and the fifth recombinant pseudorabies virus contains a gene expression cassette 5.

[0083] The gene expression cassette 1 is capable of expressing the GP2 protein of porcine reproductive and respiratory syndrome virus;

[0084] The gene expression cassette 2 is capable of expressing the GP3 protein of porcine reproductive and respiratory syndrome virus;

[0085] The gene expression cassette 3 is capable of expressing the GP4 protein of porcine reproductive and respiratory syndrome virus;

[0086] The gene expression cassette 4 is capable of expressing the GP5 protein of porcine reproductive and respiratory syndrome virus;

[0087] The gene expression cassette 5 is capable of expressing the M protein of porcine reproductive and respiratory syndrome virus.

[0088] In some embodiments, the US8, UL23, US7, US9 and US2 genes in the genome of the attenuated pseudorabies virus strain are all inactivated genes.

[0089] The amino acid sequence of the GP2 protein is shown in SEQ ID NO.15;

[0090] The amino acid sequence of the GP3 protein is shown in SEQ ID NO.16;

[0091] The amino acid sequence of the GP4 protein is shown in SEQ ID NO.17;

[0092] The amino acid sequence of the GP5 protein is shown in SEQ ID NO.18;

[0093] The amino acid sequence of the M protein is shown in SEQ ID NO.19;

[0094] The promoter of the gene expression cassette 1 is the CMV promoter;

[0095] The promoter of the gene expression cassette 2 is the CMV promoter;

[0096] The promoter of the gene expression cassette 3 is the CMV promoter;

[0097] The promoter of the gene expression cassette 4 is the CMV promoter;

[0098] The promoter of the gene expression cassette 5 is the CMV promoter;

[0099] The gene expression cassette 1 is located between the UL24 gene and the UL22 gene of the attenuated pseudorabies virus strain.

[0100] The gene expression cassette 2 is located between the UL24 and UL22 genes of the attenuated pseudorabies virus strain.

[0101] The gene expression cassette 3 is located between the UL24 gene and the UL22 gene of the attenuated pseudorabies virus strain.

[0102] The gene expression cassette 4 is located between the UL24 and UL22 genes of the attenuated pseudorabies virus strain; or

[0103] The gene expression cassette 5 is located between the UL24 and UL22 genes of the attenuated pseudorabies virus strain.

[0104] In some embodiments, the porcine pseudorabies virus-JM strain is used as the parent virus strain. The US8, UL23, US7, US9, and US2 genes are knocked out in the genome of the parent virus strain to form the attenuated pseudorabies virus strain. The genome sequence of the porcine pseudorabies virus-JM strain is shown in GenBank No. OK338077.1. The microbial accession number of the porcine pseudorabies virus-JM strain is CCTCC NO: V202615. Attached Figure Description

[0105] Figure 1 A schematic diagram of the strategy for constructing rPRV-EGFP-TK.

[0106] Figure 2 These are photographs of viral fluorescence observation and bright-field observation. The scale bar in the image represents 275 μm.

[0107] Figure 3 The image shows the results of Western blot characterization of EGFP insertion at the UL23 site.

[0108] Figure 4 A schematic diagram of the recombinant PRV construction strategy for expressing the PRRSV protein.

[0109] Figure 5 Photographs showing the results of Western blot identification of recombinant PRV exogenous protein expression.

[0110] Figure 6 The results of IFA identification of recombinant PRV exogenous protein expression were obtained.

[0111] Figure 7 To show the growth characteristics of recombinant PRV, (A) one-step growth curve; (B) photograph of the structure obtained from the etch staining experiment.

[0112] Figure 8 Photograph of the results of a study on the genetic stability of recombinant PRV.

[0113] Figure 9 Images of Western blot results for several mice immunized with recombinant PRV and exogenous gene antibodies.

[0114] Figure 10 The results show the detection of PRRSV neutralizing antibodies in mouse serum.

[0115] Figure 11 These are the results of an immunization test on piglets.

[0116] Figure 12Shows the detection results of neutralizing antibodies against PRV and PRRSV.

[0117] Figure 13 Shows the results of the piglet challenge test.

[0118] Figure 14 Shows the photographs of pathological sections (4×10) of piglet tissues and organs. Detailed Description of the Embodiments

[0119] To make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0120] Materials and instruments not specified in the present invention are conventional materials and instruments in the art, operation details not specified in the present invention are conventional operations in the art, the software used in the present invention is operated by conventional methods with reference to the instructions of the software provider, and the kit used in the present invention is operated by conventional methods with reference to the kit instruction manual. The reagents and consumables involved in the present invention are all derived from formal commercial channels unless otherwise specified. Single-stranded DNA sequences with the same name in the present invention are identical, plasmid DNA sequences with the same name in the present invention are identical, and the genome sequences of virus strains with the same name in the present invention are identical.

[0121] Example 1: Source of Biological Materials and Synthesis of Nucleic Acid Materials

[0122] 1. Source of Virus Strains

[0123] Porcine pseudorabies virus-JM strain: abbreviated as PRV JM strain, PRV JM, PRV-JM strain or JM strain, the genome sequence of this strain can be found under GenBank accession number OK338077.1.

[0124] The Porcine pseudorabies virus-JM strain was deposited in a depository institution recognized by the patent procedure. The depository unit is the China Center for Type Culture Collection (CCTCC); the address is Wuhan University, Wuhan, China; the microbial deposit number is CCTCC NO:V202615; the Chinese taxonomic designation is Porcine pseudorabies virus; the English taxonomic designation is Porcine pseudorabies virus; the culture name is Porcine pseudorabies virus-JM; the deposit date is May 9, 2026; the viability certification date is May 15, 2026.

[0125] Document 1: Construction of Two Recombinant Pseudorabies Viruses withDeletion of Virulence Genes and Evaluation of Their Immune Protection in Miceand Piglets, Shanghui Wang et al., Vaccines 2025, 13, 359. https: / / doi.org / 10.3390 / vaccines13040359.

[0126] The preparation process and main biological characteristics of the porcine pseudorabies virus strain PRV-JM-ΔEI92K are described in reference 1.

[0127] Porcine pseudorabies virus PRV-JM-ΔEI92K strain: abbreviated as PRV-JM-ΔEI92K strain, PRV-JM-ΔEI92K, or PRV-JM-DF. This strain is a genetically engineered virus strain with attenuated virulence obtained by knocking out the US8 gene (encoding gE protein), US7 gene (encoding gI protein), US9 gene (encoding US9 protein), US2 gene (encoding US2 protein), and UL23 gene (encoding TK protein) in the genome of porcine pseudorabies virus-JM strain.

[0128] In Reference 1, the PRV-JM-ΔEK strain was also prepared, and this invention uses the PRV-JM-ΔEI92K strain therein. The PRV-JM-ΔEK strain is an attenuated viral strain obtained by knocking out the coding genes (US8 gene and UL23 gene) of the gE and TK proteins from the PRV-JM strain. The preparation principle of this strain is described in Reference 1. Figure 1 Inoculation with both PRV-JM-ΔEK and PRV-JM-ΔEI92K strains did not cause animal death, and the challenge protection rate was 100%, indicating that PRV-JM-ΔEK strain is also an attenuated strain and is equally suitable as a vector strain for PRRSV gene insertion.

[0129] The genomic sequence of the porcine reproductive and respiratory syndrome virus strain HuN4, also known as HP-PRRSV-HuN4, HuN4 strain, or HuN4, can be found in GenBank (EF635006.1).

[0130] II. Construction of sgDNA plasmid

[0131] (a) Construction of pX330-TK plasmid

[0132] Primer pairs TK sgRNA1-F (SEQ ID NO.1) and TK sgRNA1-R (SEQ ID NO.2) were designed to target one side of the homologous sequence of the UL23 gene (encoding TK protein) of PRV-JM strain. The primers TK sgRNA1-F and TK sgRNA1-R were mixed and annealed to form double-stranded DNA.

[0133] The pX330 vector was linearized by digesting it with the restriction enzyme BbsI.

[0134] The aforementioned double-stranded DNA and linearized vector were ligated using T4 DNA ligase to obtain the ligation product. The ligation product was transformed into competent *E. coli* DH5α strain. Plasmids were extracted using a plasmid mini-prep kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP103-03) according to the manufacturer's instructions. The inserted sequence was then sequenced using the Sanger assay with the specific sequencing primers on the pX330 vector. After verifying the correctness of the inserted sgRNA sequence, the recombinant plasmid was designated pX330-TK1 (the sgRNA carried by this plasmid was designated sgRNA-TK1).

[0135] (ii) Construction of other pX330 recombinant plasmids

[0136] Primer pairs UL23sgRNA2-F (SEQ ID NO.3) and UL23sgRNA2-R (SEQ ID NO.4) for synthesizing sgRNA were designed targeting the homologous sequence on the other side of the UL23 gene of the PRV-JM strain. Using the same method as the pX330-TK1 plasmid, the pX330-TK2 plasmid was obtained (the sgRNA expressed by this plasmid is called sgRNA-TK2).

[0137] To ensure the recombination replacement of the EGFP gene expression cassette in the rPRV-EGFP-TK strain prepared in Example 2, primer pairs EGFP sgRNA2-F (SEQ ID NO. 5) and EGFP sgRNA2-R (SEQ ID NO. 6) were designed downstream of the EGFP gene expression cassette for sgRNA synthesis. Using the same method as constructing the pX330-TK1 plasmid, the pX330-EGFP sgRNA2 plasmid was obtained (the sgRNA expressed by this plasmid is referred to as EGFP sgRNA2). To utilize the CMV promoter in the homologous fragment, primer pairs CMV sgRNA-F (SEQ ID NO. 7) and CMV sgRNA-R (SEQ ID NO. 8) were designed upstream of the EGFP gene expression cassette for sgRNA synthesis. Using the same method as constructing the pX330-TK1 plasmid, the pX330-CMV sgRNA plasmid was obtained (the sgRNA expressed by this plasmid is referred to as CMV sgRNA1).

[0138] III. Construction of pUC19 plasmid

[0139] (a) Construction of pUC19-TK-EGFP plasmid

[0140] Primer pairs TK-LA-F (SEQ ID NO. 9) and TK-LA-R (SEQ ID NO. 10) for amplifying the upstream homologous arm of the UL23 gene of the PRV-JM strain, and primer pairs TK-RA-F (SEQ ID NO. 11) and TK-RA-R (SEQ ID NO. 12) for amplifying the downstream homologous arm of the UL23 gene of the PRV-JM strain, were designed.

[0141] PRV-JM cells were cultured using PK-15 cells in DMEM containing 10% fetal bovine serum. Genomic DNA was extracted from PRV-JM cells using a blood / cell / tissue genomic DNA extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP304-02) following the instructions.

[0142] Using primer pairs TK-LA-F / TK-LA-R and TK-RA-F / TK-RA-R as primers, PCR amplification was performed on PRV-JM genomic DNA to obtain amplification products a and c.

[0143] Primer EGFP-F (SEQ ID NO. 13) was designed targeting the upstream sequence of the CMV promoter of the EGFP expression cassette in the pEGFP-N1 vector, and primer EGFP-R (SEQ ID NO. 14) was designed targeting the downstream sequence of the SV40 PolyA of the EGFP expression cassette. PCR amplification of the pEGFP-N1 vector was performed using primers EGFP-F / EGFP-R to obtain amplification product b.

[0144] The pUC19 plasmid was double-digested using the restriction endonucleases Hind III and EcoRI to obtain digestion product a.

[0145] Amplification product a, amplification product b, amplification product c, and enzyme digestion product a were mixed, and multi-fragment recombination ligation was performed on the mixed system using Exnase MultiS recombinase.

[0146] The multi-fragment ligation product was transformed into competent *E. coli* DH5α strain. Plasmids were extracted using a plasmid miniprep kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP103-03) according to the manufacturer's instructions. The inserted sequence was then sequenced using the Sanger assay with the specific sequencing primers from the pUC19 plasmid. After verifying the correctness of the recombinant sequence, the recombinant plasmid was designated pUC19-TK-EGFP plasmid. This plasmid contains an EGFP gene expression cassette with a CMV promoter and an SV40 poly(A) sequence downstream. Upstream of the cassette is an upstream homologous sequence of the UL23 gene, and downstream of the cassette is a downstream homologous sequence of the TK gene.

[0147] (II) Construction of PRRSV antigen plasmid for CMV promoter

[0148] To insert genes of different individual proteins from the HuN4 strain or fusion proteins from different protein fragments of the HuN4 strain into the rPRV-EGFP-TK viral strain prepared in Example 2, and obtain recombinant PRV strains, a series of pUC plasmids were designed. The promoter of the inserted gene is the CMV promoter, with an SV40 poly(A) tail downstream.

[0149] Using the pUC19 vector as a backbone, expression cassettes containing the following coding sequences derived from the porcine reproductive and respiratory syndrome virus strain HuN4 were inserted between EcoRI and HindⅢ.

[0150] To determine the immunogenicity, challenge protection, and other indicators of different proteins, the coding sequences and protein sequences of various proteins or fusion proteins were designed as follows.

[0151] (1) GP2 gene (full-length coding sequence): The coding sequence is shown at positions 11983-12753 of the genome sequence shown in GenBank No. EF635006.1; the amino acid sequence is shown at positions 1-256 of GenBank No. ABR26250.1. The amino acid sequence is shown in SEQ ID NO.15.

[0152] (2) GP3 gene (full-length coding sequence): The coding sequence is shown at positions 12606-13370 of the genome sequence shown in GenBank No. EF635006.1; the amino acid sequence is shown at positions 1-254 of GenBank No. ABR26251.1. The amino acid sequence is shown in SEQ ID NO.16.

[0153] (3) GP4 gene (full-length coding sequence): The coding sequence is shown as positions 13151-13687 of the genome sequence shown in GenBank No. EF635006.1; the amino acid sequence is shown as positions 1-178 of GenBank No. ABR26252.1. The amino acid sequence is shown in SEQ ID NO.17.

[0154] (4) GP5 gene (full-length coding sequence): The coding sequence is shown at positions 13698-14300 of the genome sequence shown in GenBank No. EF635006.1; the amino acid sequence is shown at positions 1-200 of GenBank No. ABR26253.1. The amino acid sequence is shown in SEQ ID NO.18.

[0155] (5) M gene (full-length coding sequence): The coding sequence is shown at positions 14285-14809 of the genome sequence shown in GenBank No. EF635006.1; the amino acid sequence is shown at positions 1-174 of GenBank No. ABR26254.1. The amino acid sequence is shown in SEQ ID NO.19.

[0156] (6) The recombinant protein gene is tandemly fused with the extracellular domains of the GP2, GP3, GP4 and GP5 genes, and has an added signal peptide sequence (signal peptide of the porcine pseudorabies virus US6 gene) upstream and a added transmembrane region sequence (transmembrane region of the porcine pseudorabies virus US6 gene) downstream.

[0157] The gene coding sequence of the recombinant protein is shown in SEQ ID NO.20.

[0158] In this sequence, positions 1-51 are the signal peptide coding sequence, positions 52-561 are the GP2 gene coding sequence positions 12100-12609 (118-627) (the sequence positions outside the parentheses are the positions of the GP2 gene in the genome shown in GenBank No. EF635006.1, and the positions inside the parentheses are the positions of the gene fragment used in the coding sequence of the GP2 gene, the same below), positions 562-576 are the linker sequence, and positions 577-759 are the GP3 gene coding sequence positions 12723-12905 (1 The coding sequence is as follows: positions 18-330; positions 760-774 are the linker sequence; positions 775-1176 are the coding sequence of the GP4 gene; positions 13217-13618 (67-468) are the coding sequence of the GP4 gene; positions 1177-1191 are the linker sequence; positions 1192-1290 are the coding sequence of the GP5 gene; positions 13794-13892 (97-195) are the coding sequence of the GP5 gene; positions 1291-1437 are the coding sequence of the transmembrane region; and positions 1438-1461 are the coding sequence of the Flag tag gene.

[0159] The amino acid sequence of the recombinant protein is shown in SEQ ID NO.21.

[0160] Among them, positions 1-17 are the signal peptide sequence, positions 18-187 are the amino acid sequence of GP2 protein positions 40-209, positions 188-192 are the linker sequence, positions 193-253 are the amino acid sequence of GP3 protein positions 40-100, positions 254-258 are the linker sequence, positions 259-392 are the amino acid sequence of GP4 protein positions 23-156, positions 393-397 are the linker sequence, positions 398-430 are the amino acid sequence of GP5 protein positions 33-65, positions 431-479 are the transmembrane region sequence, and positions 480-487 are the Flag tag sequence.

[0161] The aforementioned six gene sequences were chemically synthesized and inserted between the Nhe I and Xba I restriction sites of the pcDNA3.1 vector, and named pcDNA3.1-GP2, pcDNA3.1-GP3, pcDNA3.1-GP4, pcDNA3.1-GP5, pcDNA3.1-M, ​​and pcDNA3.1-4T, respectively. Using the same method employed to construct the pUC19-TK-EGFP plasmid as for the primer pairs TK-LA-F / TK-LA-R and TK-RA-F / TK-RA-R, the aforementioned six protein genes were recombined into the pUC19 plasmid and named pUC19-GP2, pUC19-GP3, pUC19-GP4, pUC19-GP5, pUC19-M, and pUC19-C-4T, respectively. Each plasmid contains an expression cassette for each protein gene. The promoter is the CMV promoter, and the downstream of the cassette has an SV40 poly(A) sequence. The upstream of the expression cassette has an upstream homologous sequence of the UL23 gene, and the downstream of the expression cassette has a downstream homologous sequence of the UL23 gene.

[0162] Example 2: Construction of a recombinant virus with exogenous gene inserted at the UL23 gene site

[0163] I. Construction of rPRV-EGFP-TK

[0164] PRV-JM-DF strain was cultured using PK-15 cells in DMEM containing 10% fetal bovine serum. Genomic DNA was extracted from PRV-JM-DF cells using a blood / cell / tissue genomic DNA extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP304-02) following the instructions.

[0165] Recombinant viruses were constructed using a combination of CRISPR / Cas9 gene editing technology and homologous recombination. The construction strategy is detailed below. Figure 1Using 1 μg of PRV-JM-DF genomic DNA, 500 ng of pX330 plasmid (i.e., a mixture of equal masses of pX330-TK1 and pX330-TK2 plasmids), and 2 μg of pUC19-TK-EGFP plasmid, 293T cells (culture medium was DMEM containing 10% fetal bovine serum) were transfected according to the Lipofectamine 3000 transfection kit (purchased from Thermo Fisher, catalog number L3000-015). After cytopathic effects appeared, the cell culture supernatant was harvested and used to infect Vero cells for plaque purification. Green fluorescent plaques were selected by fluorescence microscopy. After multiple rounds of plaque purification and sequencing to verify sequence accuracy, the recombinant virus was obtained and named rPRV-EGFP-TK. This strain is equivalent to inserting an EGFP gene expression cassette into the original UL23 gene (located downstream of the UL24 gene and upstream of the UL22 gene) of the PRV-JM-DF strain genome.

[0166] The recombinant viral genome was amplified using upstream and downstream primers at the recombination site, and the successful recombination was confirmed by gel electrophoresis.

[0167] II. Detection of EGFP expression level in recombinant strains

[0168] Using PK-15 cells as test cells, the TCID of the recombinant rPRV-EGFP-TK strain constructed in the previous step was detected. 50 Then 1000 TCID 50 The virus was inoculated and spread into 6-well PK-15 plates (culture medium was DMEM containing 10% fetal bovine serum). Fluorescence was observed under a fluorescence microscope after 48 hours; results are shown below. Figure 2 Therefore, the recombinant virus rPRV-EGFP-TK can produce green fluorescence, indicating that the expression level of the foreign gene inserted at the TK position may be relatively ideal.

[0169] 1000 TCID 50 The virus was inoculated into 6-well plates containing PK-15 cells (culture medium was DMEM containing 10% fetal bovine serum). Cells were harvested after 48 h and analyzed by Western blot. Mouse anti-EGFP monoclonal antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.) was used as the primary antibody, and IRDye800-labeled goat anti-mouse monoclonal antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.) was used as the secondary antibody. Western blot results were observed using an Odyssey CLx scanner. (See attached image for details.) Figure 3 The results showed that the recombinant virus could express the EGFP protein. Three repetitions of the Western blot experiment, followed by grayscale analysis, revealed that the EGFP protein inserted at the TK site expressed at an ideal level.

[0170] Figure 3 In the study, (A) Western blot was used to identify the EGFP expression level of the recombinant strain at the UL23 gene locus; (B) Western blot results were analyzed by grayscale values ​​(**, p<0.01; ***, p<0.001; ****, p<0.0001).

[0171] Example 3: Construction of recombinant PRV expressing PRRSV antigen

[0172] I. Viral Recombination Technology Route

[0173] A recombinant PRV expressing the PRRSV antigen was constructed using a combination of CRISPR / Cas9 gene editing technology and homologous recombination. rPRV-EGFP-TK (prepared in Example 2) was used as the parental strain, and construction was achieved by transfecting 293T cells with the viral genome, pUC19 intermediate transfer plasmid, and sgRNA plasmid. The construction strategy is described in [link to construction strategy]. Figure 4 The advantages of using rPRV-EGFP-TK as the parental strain are that reverse selection can be performed using fluorescence, and the highly efficient cleavage-targeting sgRNA for EGFP can be utilized, thus enabling more efficient acquisition of recombinant viruses.

[0174] The principle and operation process of constructing rPRV-EGFP-TK are the same as those in Example 2, except that the parental viral genome, pUC19 intermediate transfer plasmid and sgRNA plasmid are different.

[0175] Using pUC19-GP2, pUC19-GP3, pUC19-GP4, pUC19-GP5, pUC19-M, and pUC19-C-4T as intermediate transfer plasmids for pUC19, and pX330-CMV sgRNA and pX330-EGFP sgRNA2 as sgRNA plasmids, respectively, six recombinant viral strains were obtained and named rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, rPRV-M, and rPRV-PRRSV-4T, respectively.

[0176] PCR amplification and identification using upstream and downstream primers at the recombination site confirmed the successful construction of the aforementioned six recombinant viruses at the DNA level.

[0177] 2. Western blot

[0178] The six recombinant viruses prepared in the previous step of this embodiment were respectively used at 1000 TCID50. 50PK-15 cells were seeded into six-well plates and cultured in DMEM containing 10% fetal bovine serum. After 48 h, cells were harvested, lysed, and subjected to Western blot analysis. The primary antibody was a 1:1000 dilution of mouse anti-Flag monoclonal antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.), and the secondary antibody was a 1:3000 dilution of IRDye800-labeled goat anti-mouse monoclonal antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.). The results were observed using a membrane scanning instrument (Odyssey CLx).

[0179] Among them, cells inoculated with the PRV-JM-DF strain served as a negative control; PK-15 cells transfected with 2 μg of pUC19-GP2, pUC19-GP3, pUC19-GP4, pUC19-GP5, pUC19-M, and pUC19-C-4T served as positive plasmid controls.

[0180] See results Figure 5 .

[0181] Figure 5 In the middle, AF represents the identification results of rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, rPRV-M and rPRV-PRRSV-4T by Western blot, respectively (M: molecular weight standard; lane 1: negative control sample; lane 2: pUC19-X transfected sample; lane 3: rPRV-X inoculated sample).

[0182] Western blot analysis revealed that all six recombinant PRV strains could express exogenous proteins.

[0183] III. Indirect Immunofluorescence Assay (IFA)

[0184] The six recombinant viruses prepared in this embodiment were used at 1000 TCID50. 50 (1) Inoculate PK-15 cells into 12-well plates with DMEM containing 10% fetal bovine serum; (2) After 48 h, discard the supernatant and gradually fix, permeate, and block the cells; (3) Use mouse anti-Flag monoclonal antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.) as the primary antibody and FITC-labeled goat anti-mouse IgG (purchased from Wuhan Sanying Biotechnology Co., Ltd.) as the secondary antibody and observe under a fluorescence microscope.

[0185] Cells inoculated with the PRV-JM-DF strain served as a negative control. See results below. Figure 6 .

[0186] IFA results showed that all six recombinant PRV-inoculated cells exhibited abundant fluorescence, indicating that the inserted recombinant protein was expressed in large quantities.

[0187] IV. Biological characteristics of recombinant PRV expressing PRRSV antigen

[0188] rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, rPRV-M, and rPRV-PRRSV-4T were seeded into Vero cells at a seeding density of 1000 TCID. 50 The culture medium was DMEM containing 2% fetal bovine serum, and its one-step growth curve was measured after 60 hours.

[0189] See results Figure 7 Therefore, it can be seen that the recombinant PRV and PRV-JM-DF have similar replication capabilities, and there is no significant difference in the etch spot diameter between all recombinant PRV and PRV-JM-DF.

[0190] Biological characteristics studies of the recombinant virus showed that inserting a foreign gene at the UL23 gene locus had almost no effect on viral replication and did not change the size of the plaques. Insertion of a foreign gene at the UL23 gene locus did not affect the replication capacity or intercellular infectivity of the recombinant virus.

[0191] V. Study on the genetic stability of recombinant PRV expressing PRRSV antigen

[0192] rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, rPRV-M, and rPRV-PRRSV-4T were passaged continuously in PK-15 cells for 30 generations. PCR identification was performed every 5 generations using primers TKJD-F (SEQ ID NO.22) and TKJD-R (SEQ ID NO.23). It was found that all strains could be stably inherited up to 30 generations.

[0193] Samples of PK-15 cells transfected with the pUC19 recombinant plasmid used to prepare each recombinant strain were used as positive controls. See the results below. Figure 8 .

[0194] Figure 8 In the image, AF represents the PCR results of genetic stability identification of rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, rPRV-M, and rPRV-PRRSV-4T, respectively. (Lane M: molecular weight standard; Lane +: corresponding pUC19 recombinant plasmid; Lanes 1-7: recombinant viruses of generations 1, 5, 10, 15, 20, 25, and 30).

[0195] VI. Safety and Immunoprotective Experiments of Recombinant PRV Expressing PRRSV Antigen in Mice

[0196] To verify the safety of rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, rPRV-M, and rPRV-PRRSV-4T, 4-week-old female BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 9 groups, with 5 mice in each group. Each mouse in groups 1-6 was intramuscularly injected with 10... 6 TCID 50 The seven groups (rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, rPRV-M, and rPRV-PRRSV-4T) underwent mixed immunization. Group 7 received a mixed injection of five viruses: rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, and rPRV-M. Each virus was injected intramuscularly at a dose of 10 mg / L. 6 TCID 50 (Code name rPRV-GP2 / 3 / 4 / 5 / M), mice in group 8 were injected intramuscularly with 10 6 TCID 50 PRV-JM-DF was used as a blank control, and mice in group 9 were injected with 100 μL LDMEM as a negative control. Mice were observed for 28 days post-immunization, and their survival status and clinical symptoms were recorded. Blood samples were collected on day 28 to detect specific antibodies against the exogenous protein, and blood samples were collected on day 35 to detect neutralizing antibodies against HP-PRRSV-HuN4. Mice were injected intramuscularly with 10 μL LDMEM 35 days after immunization. 4 TCID 50 Mice were challenged with the PRV-JM strain. Survival status and clinical symptoms of the mice were recorded within 14 days post-challenge.

[0197] (I) Statistics on symptoms and mortality

[0198] After inoculation with the recombinant virus, the mice did not show any clinical symptoms, and the survival rate of mice in all groups was 100% within 28 days after inoculation, indicating that 10 6 TCID 50 The recombinant PRV dose was safe in mice, even in the mixed immunization group at doses of 5 × 10⁻⁶. 6 TCID 50 The dosage was also safe for mice.

[0199] (ii) Western blot detection of antibody reactivity

[0200] To detect antibodies against exogenous proteins produced by immunized mice, plasmids expressing exogenous proteins pcDNA3.1-GP2, pcDNA3.1-GP3, pcDNA3.1-GP4, pcDNA3.1-GP5, pcDNA3.1-M, ​​and pcDNA3.1-4T were transfected into 293T cells. After 24 h, the cells were lysed and subjected to SDS-PAGE, with cells transfected with the empty pcDNA3.1 vector serving as a negative control. Mouse serum collected on day 28 was used as the primary antibody, and IRDye800-labeled goat anti-mouse monoclonal antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.) was used as the secondary antibody for Western blot identification. The results showed that each group of mice could produce antibodies against the corresponding exogenous protein inserted into their immunized recombinant virus. Figure 9 This indicates that the constructed recombinant PRVs can all express the inserted exogenous protein in mice and produce corresponding antibodies.

[0201] Figure 9 In the table, AG represents the results of Western blot identification of mouse serum from the rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, rPRV-M, rPRV-PRRSV-4T, and rPRV-GP2 / 3 / 4 / 5 / M groups, respectively (lane M: molecular weight standard; lane 1: negative control sample; lane 2: pcDNA3.1-GP2 transfected sample; lane 3: pcDNA3.1-GP3; lane 4: pcDNA3.1-GP4; lane 5: pcDNA3.1-GP5; lane 6: pcDNA3.1-M; lane 7: pcDNA3.1-4T).

[0202] (III) Neutralizing antibody detection

[0203] On day 35, mouse blood was collected for PRRSV neutralizing antibody detection. The method was as follows: (1) Marc-145 cells were cultured in 96-well plates for 24 h in DMEM containing 10% fetal bovine serum. After forming a monolayer of cells, the cells were ready for use. (2) The serum to be tested was placed in a 56℃ water bath for 20 min to inactivate complement. (3) The serum to be tested was diluted with 2% DMEM by a 2-fold serial dilution. (4) 1 mL of diluted serum of different concentrations was mixed with 1 mL of standard virus (1000 TCID50) respectively. 50 Mix 100 μL of porcine reproductive and respiratory syndrome virus (HuN4 strain) and incubate at 37°C for 1 h; (5) Add 200 μL of the mixture to a 96-well plate that has grown into a monolayer of cells, and inoculate 8 wells for each dilution. At the same time, set up 8 wells of negative control (add porcine negative serum of the same dilution) and 8 wells of 100 TCID100. 50 / 200 μL virus control; (6) Incubate the 96-well cell culture plate in a 37°C, 5% CO2 incubator, and observe the cytopathic effect after 3 days; (7) Calculation of the 50% serum neutralization endpoint: the serum dilution at which 50% of cells do not produce cytopathic effect (CPE);

[0204] The 50% serum neutralization endpoint was calculated using the Reed-Muench method, as follows:

[0205] TCID 50 =(10^(A-(B / BA)))×D

[0206] Among them, TCID 50 A represents the viral titer, B represents the dilution of the last infection, and D represents the dilution factor of the first uninfected infection.

[0207] See results Figure 10 The results showed that all groups produced a certain amount of neutralizing antibodies; among them, the rPRV-PRRSV-4T group and the rPRV-GP2 / 3 / 4 / 5 / M mixed immunization group had the highest PRRSV neutralizing titers, both of which could reach more than 10.

[0208] (iv) Results of protection against infection

[0209] Clinical symptoms and mortality were observed in mice after intramuscular injection of the PRV-JM strain. After 14 days, all groups immunized with recombinant PRV showed the same clinical symptoms and mortality as the PRV-JM-DF group, with no mice exhibiting any clinical symptoms. Mice in the DMEM group began showing itching symptoms 2 days after challenge, biting their legs and causing bleeding; mortality began 3 days after challenge, and all mice died by day 4. These results indicate that the recombinant PRV with inserted exogenous protein still retains effective protection against PRV-JM.

[0210] Animal experiments were conducted on piglets using the rPRV-PRRSV-4T group and the rPRV-GP2 / 3 / 4 / 5 / M mixed immunization group to verify the immunoprotective effect of the recombinant virus on the PRRSV HuN4 strain.

[0211] VII. Evaluation of the immunogenicity of recombinant PRV expressing PRRSV antigen in piglets

[0212] Four-week-old piglets without PRV and PRRSV antigens and antibodies were randomly divided into four groups of five piglets each. (1) rPRV-PRRSV-4T group: each piglet was injected intramuscularly with 10 6 TCID 50rPRV-PRRSV-4T; (2) rPRV-GP2 / 3 / 4 / 5 / M group, each pig was intramuscularly injected with rPRV-GP2, rPRV-GP3, rPRV-GP4, rPRV-GP5, and rPRV-M, with 10 doses of each virus injected. 6 TCID 50 (2) PRRSV vaccine group: Purchase commercial PRRS attenuated vaccine and administer 1 dose per pig intramuscularly according to the instructions; (3) DMEM negative control group: Administer 100 μL DMEM intramuscularly per pig.

[0213] Rectal temperature of all pigs was recorded over 14 days, and weekly body weight was also recorded. Nasal and anal swabs were collected weekly to detect recombinant PRV shedding. Blood samples were collected starting on day 21 to detect neutralizing antibodies against PRV-JM and HP-PRRSV-HuN4. Specific antibodies against exogenous proteins were detected in blood samples from the rPRV-PRRSV-4T and rPRV-GP2 / 3 / 4 / 5 / M groups. 10 mg of PRV was administered intranasally 35 days post-immunization. 5 TCID 50 Pigs were challenged with HP-PRRSV-HuN4. Rectal temperature was recorded daily for all pigs, and weekly weight, clinical symptoms, and survival status were also recorded. Blood samples, nasal swabs, and pharyngeal swabs were collected at 0, 3, 5, 7, 11, 14, 21, and 28 days post-challenge to detect PRRSV shedding. Dead pigs were immediately dissected, and tonsils, lungs, and lymph nodes were removed for pathological examination. 28 days post-challenge, remaining pigs were euthanized, and tonsils, lungs, and lymph nodes were removed for further pathological examination.

[0214] The results showed that, after two weeks of continuous monitoring of piglets' body temperature following immunization, piglets in the PRRS vaccine group experienced a transient increase in body temperature, while the body temperature in other groups remained normal. Figure 11 A); No pigs died during the immunity period. Figure 11 (B) Only the PRRS vaccine group experienced lameness and diarrhea. This indicates that the safety of the vaccine of this invention is higher than that of commercial PRRS vaccines.

[0215] Figure 11 In the image, (A) is the body temperature of pigs after immunization; and (B) is the survival curve of pigs after immunization.

[0216] Subsequently, using F1 (SEQ ID NO.24) and R1 (SEQ ID NO.25) as primers and P1 as a probe (with FAM linked upstream and MGB linked downstream; the nucleic acid sequence is shown in SEQ ID NO.26), the PRV viral load in the serum, nasal swabs, and anal swabs of pigs in each group during the immunization period was detected by qPCR. The results showed no difference between the two experimental groups and the control group, indicating that the constructed recombinant PRV did not produce viremia or shed the virus after immunizing piglets, demonstrating high safety.

[0217] Using the same method as in Section 6 of this embodiment, the specific antibodies against exogenous proteins in the rPRV-PRRSV-4T group were detected by Western blot. The results showed that specific bands against each exogenous protein could be detected in the immunized pig serum, indicating that the constructed recombinant PRV can express exogenous proteins and produce specific antibodies in pigs.

[0218] Using the same method as in Section 6 of this embodiment, the neutralizing antibody titers of PRV were determined using PRV-JM strain as the inoculation strain, and the neutralizing antibody titers of PRRSV were determined using PRRSV HuN4 strain as the inoculation strain (see results). Figure 12 Therefore, the neutralizing titer against PRRSV in the serum of pigs in the rPRV-PRRSV-4T group reached 14, while the neutralizing titer in the serum of pigs in the rPRV-GP2 / 3 / 4 / 5 / M group and the PRRS vaccine group was approximately 4. During the immunization phase, piglets in the rPRV-PRRSV-4T group did not exhibit clinical symptoms such as fever and did not die, demonstrating the good safety of the recombinant PRV expressing the PRRSV antigen; furthermore, immunized pigs could produce specific antibodies against the exogenous protein and neutralizing antibodies against PRRSV and PRRSV, demonstrating the good immunization effect of the recombinant virus constructed in this invention.

[0219] Figure 12 In the study, (A) detection of porcine serum PRV neutralizing antibodies; (B) detection of porcine serum PRRSV neutralizing antibodies (**, p<0.01; ***, p<0.001; ****, p<0.0001).

[0220] 35 days after immunization, administer 10 drops of [amount of medication] intranasally to each pig. 5 TCID 50 All pigs were challenged with HP-PRRSV-HuN4. Fever began on day 2 post-challenge, decreased on day 4, and then rose again; except for the DMEM group, the body temperature in other groups decreased to below 40°C on day 12 post-challenge, while the body temperature in the DMEM group decreased on day 16 post-challenge. Figure 13A). This indicates that rPRV-PRRSV-4T, rPRV-GP2 / 3 / 4 / 5 / M, and PRRS vaccine immunization can all shorten the fever period in piglets infected with PRRSV. Since piglets experience two phases—fever and recovery—the survival curve can be divided into two parts: the fever period (1 to 14 days) and the recovery period (15 to 28 days). During the fever period, pigs in the rPRV-GP2 / 3 / 4 / 5 / M group developed severe clinical symptoms, including respiratory and neurological symptoms, after challenge, and began dying on day 9, with a total of 4 deaths by day 11. In the DMEM group, 2 pigs died on day 11, exhibiting severe respiratory symptoms. No piglets died in the rPRV-PRRSV-4T group or the PRRS vaccine group, but still showed relatively severe respiratory symptoms. During the recovery period, the clinical symptoms of all pigs lessened until they returned to normal. Two piglets in the PRRS vaccine group died on days 16 and 18, respectively, and one piglet in the DMEM group died on day 21. Figure 13 (B) Upon necropsy, all cases were determined to be secondary infections with Haemophilus parasuis. The rPRV-PRRSV-4T group recovered the fastest with no deaths, achieving a 100% protection rate. In the first week after challenge, the rPRV-PRRSV-4T group and the PRRS vaccine group showed a slight increase in body weight, while the other two groups experienced a decrease. In the second week, all piglets experienced a decrease in body weight and began to regain weight in the third week.

[0221] Figure 13 In the text, (A) body temperature of pigs after challenge; (B) survival curve of pigs after challenge.

[0222] Blood, pharyngeal swabs, and nasal swabs were collected from pigs at different time points. Using F2 (SEQ ID NO.27) and R2 (SEQ ID NO.28) as primers and P2 as a probe (the probe is linked to FAM upstream and MGB downstream; the nucleic acid sequence is shown in SEQ ID NO.29), the viral load of PRRSV was detected by qPCR. It was found that the viral load in the rPRV-PRRSV-4T group was lower, especially in serum, which was significantly lower than that in the PRRS vaccine group on days 3 and 7.

[0223] All piglets were euthanized 28 days after challenge. Using F2 and R2 as primers and P2 as a probe, the viral load in the tissues of all pigs was measured by qPCR. The results showed that the viral load in the lungs of pigs in the rPRV-PRRSV-4T group was about 10 times lower than that in other groups. Furthermore, the viral load in other tissues and organs of pigs in the rPRV-PRRSV-4T group was also at a low level, indicating that rPRV-PRRSV-4T can reduce the viral load in the tissues and lymph nodes of immunized pigs after challenge.

[0224] This invention involves necropsy of experimental pigs to observe lesions in their tissues and organs. It was found that the lungs of pigs in the rPRV-PRRSV-4T group and the PRRS vaccine group only had mild nodules and edema, and no apparent lesions in the lymph nodes, brain, and tonsils. However, the lungs of pigs in the DMEM and rPRV-GP2 / 3 / 4 / 5 / M groups were severely damaged, with severe hemorrhage, nodules, consolidation, and edema. There was extensive hemorrhage in the lymph nodes, with the most severe hemorrhage in the mandibular lymph nodes. Hemorrhage was also present in the tonsils of pigs in the rPRV-GP2 / 3 / 4 / 5 / M group.

[0225] Subsequently, HE staining was performed on the experimental pig tissues and organs, and pathological damage was observed. Figure 14 The DMEM group showed severe pathological damage, including severe inflammatory changes in the lungs leading to complete disappearance of alveolar cavities; glial cell proliferation, nerve cell degeneration and necrosis in the brain, and multiple inflammatory cells infiltrating around blood vessels; inflammatory cell infiltration in the tonsillar crypt epithelium and necrosis of surrounding lymphocytes; partial cell necrosis in the parenchyma of the mandibular lymph nodes; and widespread lymphocyte necrosis and reduced number of lymphocytes in the mesenteric and inguinal lymph nodes, along with macrophage proliferation. The rPRV-GP2 / 3 / 4 / 5 / M group also showed severe histopathological damage, with severe alveolar wall capillary congestion and alveolar cavities filled with serous exudate; subpiatric hemorrhage and serous exudation in the brain, inflammatory cell infiltration, and partial cell necrosis; severe lymphocyte necrosis in the tonsils and disappearance of lymphoid follicle structure; and widespread, diffuse, and severe lymphocyte necrosis in the lymph nodes, with hemorrhage in the inguinal and mandibular lymph nodes. The rPRV-PRRSV-4T group of pigs showed mild histopathological damage, with mild perivascular inflammatory cell infiltration in the lungs and a slight decrease in tonsillar lymphocytes. No significant changes were observed in other tissues and organs. The PRRS vaccine group also showed mild pathological damage, with localized alveolar wall thickening in the lungs and a slight decrease in lymphocytes in the mandibular and inguinal lymph nodes. No significant changes were observed in other tissues and organs. These results indicate that rPRV-PRRSV-4T immunization can reduce pathological damage in piglets infected with HuN4. Preliminary evaluation of immunogenicity showed that rPRV-PRRSV-4T immunization can shorten the duration of fever in experimental pigs, reduce viral load in organs, alleviate lung damage, and reduce secondary bacterial infections, indicating that rPRV-PRRSV-4T immunization can effectively resist PRRSV infection.

[0226] Figure 14 Among them, (A) DMEM group; (B) rPRV-GP2 / 3 / 4 / 5 / M group; (C) rPRV-PRRSV-4T group; (D) PRRS vaccine group.

[0227] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A first biological material, wherein the first biological material is any one of the following P3, P4, P5, P6, P7, P8, P9, P10 and P11; P3: Fusion protein The fusion protein is composed of a first peptide segment, a second peptide segment, a third peptide segment, a fourth peptide segment, a fifth peptide segment, and a sixth peptide segment; wherein the sixth peptide segment, the first peptide segment, the second peptide segment, the third peptide segment, the fourth peptide segment, and the fifth peptide segment are arranged in order from the N-terminus to the C-terminus in the fusion protein; The amino acid sequence of the first peptide is shown in positions 18-187 of SEQ ID NO.21; The amino acid sequence of the second peptide is shown in positions 193-253 of SEQ ID NO.21; The amino acid sequence of the third peptide is shown in positions 259-392 of SEQ ID NO.21; The amino acid sequence of the fourth peptide is shown in positions 398-430 of SEQ ID NO.21; The fifth peptide segment is the transmembrane region of the porcine pseudorabies virus US6 gene; The sixth peptide segment is a signal peptide that can be localized to the pseudorabies virus envelope or the host's cell membrane. P4: First protein modifier The first protein modifier is a modified protein produced by post-translational modification of the fusion protein in eukaryotic cells; P5: First RNA The first RNA can be translated to obtain the fusion protein; P6: First Gene The coding sequence of the first gene encodes the fusion protein; P7: First gene expression cassette The gene expression product in the first gene expression cassette assembly is the first RNA; The promoter of the first gene expression cassette is a constitutive expression promoter or an artificially inducible promoter; P8: First Gene Engineering Vector The first genetic engineering vector encodes the first expressible RNA; P9: First Cell The first cell contains the first gene engineering vector; P10: First Composition The first composition contains the fusion protein, the first protein modifier, the first RNA, the first gene engineering vector, or the first cell; P11: First Reagent Kit The kit contains the fusion protein, the first protein modifier, the first RNA, the first gene engineering vector, or the first cell.

2. The first biomaterial as described in claim 1, characterized in that, The signal peptide is the signal peptide of the porcine pseudorabies virus US6 gene. The amino acid sequence of the fifth peptide is shown in positions 431-479 of SEQ ID NO.21; The different peptide segments of the fusion protein have a linker sequence with an amino acid sequence as shown in positions 188-192, 254-258 or 393-397 of SEQ ID NO.21; The fusion protein contains a tag peptide for separating and purifying the protein; The promoter of the first gene expression cassette is the CMV promoter; The backbone of the first gene engineering vector is either the pX330 vector or the pUC19 vector; or The host cell of the first cell is an Escherichia coli cell, a 293T cell, or a PK-15 cell.

3. The first biomaterial as described in claim 1, characterized in that, The backbone of the first genetically engineered vector is a pseudorabies virus vector; or The host cell of the first cell is a porcine pseudorabies virus susceptible cell.

4. The first biomaterial as described in claim 2 or 3, characterized in that, The amino acid sequence of the sixth peptide is shown in positions 1-17 of SEQ ID NO. 21; The amino acid sequence of the fifth peptide is shown in positions 431-479 of SEQ ID NO.21; The amino acid sequence of the tag peptide is shown in positions 480-487 of SEQ ID NO.21; or The pseudorabies virus vector is a weakened strain of pseudorabies virus.

5. The first biomaterial as described in claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO.

21.

6. The first biomaterial as described in claim 4, characterized in that, In the genome of the attenuated pseudorabies virus strain, the US8, UL23, US7, US9, and US2 genes are all inactivated genes; or The first gene expression cassette is located between the UL24 and UL22 genes of the attenuated pseudorabies virus strain.

7. The first biomaterial as described in claim 6, characterized in that, Using porcine pseudorabies virus-JM strain as the parent virus strain, the US8, UL23, US7, US9, and US2 genes were knocked out from the genome of the parent virus strain to form the attenuated pseudorabies virus strain. The genome sequence of the porcine pseudorabies virus-JM strain is shown in GenBank No. OK338077.1; the microbial accession number of the porcine pseudorabies virus-JM strain is CCTCC NO: V202615.

8. A method for preparing recombinant pseudorabies virus, wherein the method comprises transferring the first gene expression cassette as described in claim 1, 2, 4 or 5 into the genome of a pseudorabies virus attenuated strain to obtain the recombinant pseudorabies virus.

9. The preparation method according to claim 8, characterized in that, The recombinant pseudorabies virus is obtained by transferring the first gene expression cassette as described in claim 1, 2, 4 or 5 into the genome of the attenuated pseudorabies virus strain as described in claim 6 or 7 through homologous recombination.

10. The preparation method according to claim 9, characterized in that, The preparation method includes the following first three or first four steps: S1: Prepare the first plasmid, the second plasmid, and the third plasmid in parallel or sequentially; The third plasmid contains the first gene expression cassette; The first gene expression cassette contains a first homologous arm upstream; the first homologous arm is homologous to the upstream of the genome fragment of the attenuated pseudorabies virus strain into which the first gene expression cassette is transferred. The first gene expression cassette contains a second homologous arm downstream; the second homologous arm is homologous to the downstream portion of the pseudorabies virus attenuated strain genome fragment into which the first gene expression cassette is transferred; The first plasmid contains a Cas9 gene expression cassette and a first sgRNA expression cassette; the sgRNA expressed by the first sgRNA expression cassette can mediate homologous recombination between the first homologous arm and the genome of the attenuated pseudorabies virus strain with the participation of the Cas9 protein. The second plasmid contains a Cas9 gene expression cassette and a second sgRNA expression cassette; the sgRNA expressed by the second sgRNA expression cassette can mediate homologous recombination between the second homologous arm and the genome of the attenuated pseudorabies virus strain with the participation of the Cas9 protein. S2: First pseudorabies virus susceptible cells are co-transfected with the genomes of the first plasmid, the second plasmid, the third plasmid and the attenuated pseudorabies virus strain; S3: Identify and harvest the recombinant pseudorabies virus; S4: The recombinant pseudorabies virus is cultured and purified in second pseudorabies virus susceptible cells to obtain purified recombinant pseudorabies virus.

11. The preparation method according to claim 10, characterized in that, The backbone of the first plasmid was selected from the pUC19 vector; The backbone of the second plasmid was selected from the pX330 vector; The backbone of the third plasmid is selected from the pX330 vector; The first pseudorabies virus susceptible cells were selected from HEK293T cells; The second pseudorabies virus susceptible cells were selected from Vero cells or PK-15 cells; or The transfection operation was performed using Lipofectamine 3000 transfection reagent.

12. A recombinant pseudorabies virus, wherein the recombinant pseudorabies virus is prepared by the preparation method according to any one of claims 8-11.

13. Use of the first biomaterial of any one of claims 1-7 in the preparation of a formulation for use alone, for use in combination with other immunomodulators and / or drugs, or as a component of a compound formulation composed of other immunomodulators and / or drugs to prevent porcine reproductive and respiratory syndrome, wherein the first biomaterial is selected from the fusion protein, the first protein modifier, and the first cell.

14. A combined recombinant pseudorabies virus, wherein the combined recombinant pseudorabies virus comprises a first recombinant pseudorabies virus, a second recombinant pseudorabies virus, a third recombinant pseudorabies virus, a fourth recombinant pseudorabies virus, and a fifth recombinant pseudorabies virus. The first recombinant pseudorabies virus uses a weakened strain of pseudorabies virus as a vector virus, and the first recombinant pseudorabies virus contains a gene expression cassette 1. The second recombinant pseudorabies virus uses a weakened strain of pseudorabies virus as a vector virus, and the second recombinant pseudorabies virus contains gene expression cassette 2; The third recombinant pseudorabies virus uses a weakened pseudorabies virus strain as a vector virus, and the third recombinant pseudorabies virus contains a gene expression cassette 3. The fourth recombinant pseudorabies virus uses a weakened pseudorabies virus strain as a vector virus, and the fourth recombinant pseudorabies virus contains a gene expression cassette 4. The fifth recombinant pseudorabies virus uses a weakened pseudorabies virus strain as a vector virus, and the fifth recombinant pseudorabies virus contains a gene expression cassette 5. The gene expression cassette 1 is capable of expressing the GP2 protein of porcine reproductive and respiratory syndrome virus; The gene expression cassette 2 is capable of expressing the GP3 protein of porcine reproductive and respiratory syndrome virus; The gene expression cassette 3 is capable of expressing the GP4 protein of porcine reproductive and respiratory syndrome virus; The gene expression cassette 4 is capable of expressing the GP5 protein of porcine reproductive and respiratory syndrome virus; The gene expression cassette 5 is capable of expressing the M protein of porcine reproductive and respiratory syndrome virus; The amino acid sequence of the GP2 protein is shown in SEQ ID NO.15; The amino acid sequence of the GP3 protein is shown in SEQ ID NO.16; The amino acid sequence of the GP4 protein is shown in SEQ ID NO.17; The amino acid sequence of the GP5 protein is shown in SEQ ID NO.18; The amino acid sequence of the M protein is shown in SEQ ID NO.19; Using porcine pseudorabies virus-JM strain as the parent virus strain, the US8, UL23, US7, US9, and US2 genes were knocked out from the genome of the parent virus strain to form the attenuated pseudorabies virus strain. The genome sequence of the porcine pseudorabies virus-JM strain is shown in GenBank No. OK338077.1; the microbial accession number of the porcine pseudorabies virus-JM strain is CCTCC NO: V202615.

15. The combined recombinant pseudorabies virus as described in claim 14, characterized in that, The promoter of the gene expression cassette 1 is the CMV promoter; The promoter of the gene expression cassette 2 is the CMV promoter; The promoter of the gene expression cassette 3 is the CMV promoter; The promoter of the gene expression cassette 4 is the CMV promoter; The promoter of the gene expression cassette 5 is the CMV promoter; The gene expression cassette 1 is located between the UL24 gene and the UL22 gene of the attenuated pseudorabies virus strain. The gene expression cassette 2 is located between the UL24 and UL22 genes of the attenuated pseudorabies virus strain. The gene expression cassette 3 is located between the UL24 gene and the UL22 gene of the attenuated pseudorabies virus strain. The gene expression cassette 4 is located between the UL24 and UL22 genes of the attenuated pseudorabies virus strain; or The gene expression cassette 5 is located between the UL24 and UL22 genes of the attenuated pseudorabies virus strain.

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