Recombinant foot-and-mouth disease virus strain with high antigen yield and construction method and application thereof

Abstract

The application provides a recombinant foot-and-mouth disease virus strain with high antigen yield and a construction method and application thereof, and belongs to the technical field of biotechnology. The recombinant foot-and-mouth disease virus strain is characterized in that the L protein is a mutant L protein with a mutation or deletion of the 108th amino acid. The application finds that host protein RAB20 overexpression inhibits the replication of FMDV, and degrades the L protein through a proteasome pathway, thereby inhibiting virus replication; the application constructs a recombinant foot-and-mouth disease virus strain which is not inhibited in replication by host protein RAB20 by mutating a key amino acid site (108) in the L protein of FMDV; the recombinant virus can be stably passaged, has the characteristics of high virus titer on BHK-21 cells, improves virus titer and antigen yield, can be used for FMDV vaccine preparation, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry and veterinary technology, specifically relating to a recombinant foot-and-mouth disease virus strain with high antigen yield, its construction method, and its application. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, and highly contagious disease of cloven-hoofed animals caused by the foot-and-mouth disease virus (FMDV). This disease has a significant economic impact on livestock farming.

[0003] FMDV belongs to the family Picornaviridae, genus Foot-and-Mouth Disease Virus, and is a single-stranded positive-sense RNA virus. Its genome is approximately 8.0 kb long and is translated into a 5' untranslated region, an open reading frame (ORF), and a 3' untranslated region. The ORF encodes the non-structural protein L... pro Four structural proteins: VP4, VP2, VP3, and VP1, as well as 2A, 2B, 2C, 3A, 3B, and 3C. pro and 3D pol Non-structural proteins. Among them, the leader protein L... pro The L protein is a papain that can cleave itself from a polyprotein and cleave the host cell translation initiation factor eIF4G, inhibiting host protein synthesis and promoting viral protein translation. The L protein is also a deubiquitinizing protease that inhibits the type I interferon signaling pathway by suppressing the ubiquitination of RIG-I, TBKI, TRAF6, and TRAF3. The L protein is also an important virulence factor for FMDV, with a high utilization frequency of its second translation initiation codon. Deletion of the L protein reduces the replication capacity of recombinant viruses and significantly reduces their pathogenicity to host animals. Therefore, the FMDV L protein plays a crucial role in regulating viral replication, virulence, and host cell responses. Deletion or mutation of the L protein can lead to the development of safer and more effective FMDV vaccines. Furthermore, there are complex interactions between host proteins and FMDV proteins, which regulate viral replication through different mechanisms, thus affecting FMDV replication performance in the host, its stable passage, and consequently its application in FMDV vaccine preparation. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a recombinant foot-and-mouth disease virus strain with high antigen yield, which eliminates the ability of RAB20 to inhibit FMDV replication by mutating key amino acid sites of the L protein, thereby increasing virus titer and antigen yield.

[0005] This invention provides a recombinant foot-and-mouth disease virus strain, wherein the L protein is a mutant L protein with a mutation or deletion at amino acid position 108.

[0006] Preferably, the parental foot-and-mouth disease virus strain of the recombinant foot-and-mouth disease virus strain includes at least one of the following: wild-type foot-and-mouth disease virus strain, recombinant foot-and-mouth disease virus strain, and recombinant foot-and-mouth disease vaccine strain.

[0007] Preferably, the 108th amino acid of the L protein is a mutant L protein with a mutation.

[0008] The mutant L protein includes a mutation at amino acid position 108 to arginine;

[0009] Preferably, the amino acid sequence of the mutant L protein is as shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0010] This invention provides a method for constructing the recombinant foot-and-mouth disease virus strain, comprising the following steps:

[0011] Prepare a full-length infectious clone of foot-and-mouth disease virus containing a mutant L protein coding sequence; the mutation site of the mutant L protein includes amino acid 108 of the foot-and-mouth disease virus L protein;

[0012] A full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence was transfected into foot-and-mouth disease virus-sensitive cells to obtain a recombinant foot-and-mouth disease virus strain.

[0013] Preferably, the preparation of the full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence includes preparation using gene mutation technology or gene synthesis technology;

[0014] Preferably, the 108th amino acid of the L protein of foot-and-mouth disease virus is mutated to arginine;

[0015] Preferably, the method for preparing a full-length infectious clone of foot-and-mouth disease virus containing a mutant L protein coding sequence using gene mutation technology involves using the full-length infectious clone of foot-and-mouth disease virus as a template and performing PCR amplification using two pairs of homologous recombination mutant primers to obtain two PCR amplification products.

[0016] Two PCR amplification products were homologously recombinated with a linearized vector plasmid to obtain a full-length infectious clone of foot-and-mouth disease virus containing a mutant L protein coding sequence.

[0017] Preferably, the two pairs of homologous recombination mutant primers include KpnI-F / mL108-R and mL108-F / KpnI-R;

[0018] The nucleotide sequence of KpnI-F is shown in SEQ ID NO:8;

[0019] The nucleotide sequence of the KpnI-R is shown in SEQ ID NO:9;

[0020] The nucleotide sequence of the mL108-F is shown in SEQ ID NO:10;

[0021] The nucleotide sequence of the mL108-R is shown in SEQ ID NO:11.

[0022] Preferably, the foot-and-mouth disease virus-sensitive cells include BHK-21 cells and / or IBRS-2 cells.

[0023] The present invention provides a recombinant foot-and-mouth disease vaccine strain, comprising the recombinant foot-and-mouth disease virus strain or a recombinant foot-and-mouth disease virus strain prepared by the construction method.

[0024] The present invention provides a recombinant foot-and-mouth disease vaccine, comprising the recombinant foot-and-mouth disease virus strain or the recombinant foot-and-mouth disease virus strain prepared by the construction method.

[0025] This invention provides the application of the recombinant foot-and-mouth disease virus strain or the recombinant foot-and-mouth disease virus strain prepared by the construction method in the preparation of recombinant foot-and-mouth disease vaccines.

[0026] This invention provides a method for preparing a recombinant foot-and-mouth disease virus strain that is immune to replication inhibition by the host protein RAB20, comprising the following steps:

[0027] The 108th amino acid of the L protein in the foot-and-mouth disease virus strain was mutated or deleted.

[0028] Preferably, the mutation method involves mutating the 108th amino acid of the L protein in the foot-and-mouth disease virus strain to arginine;

[0029] Further preferred, the amino acid sequence of the mutated L protein is shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0030] This invention provides the use of the recombinant foot-and-mouth disease vaccine in the preparation of medicaments for the prevention and / or control of foot-and-mouth disease in animals;

[0031] Preferably, the animal includes an even-toed ungulate;

[0032] More preferably, the even-toed ungulate includes at least one of the following: pigs, cattle, and sheep.

[0033] This invention provides a recombinant foot-and-mouth disease virus (FMDV) strain, in which the L protein is a mutant L protein with a mutation or deletion at amino acid position 108. This invention first discovered that overexpression of RAB20 in host cells inhibits FMDV replication, and further determined that the RAB20 protein degrades the L protein through the 108th amino acid site, thereby inhibiting viral replication. By performing a site-directed mutation on amino acid position 108 of the L protein, the degradation ability of the RAB20 protein on the L protein was eliminated, as was its inhibitory effect on FMDV replication. This invention, through the design and modification of amino acid position 108 of the L protein, successfully constructed and rescued a recombinant FMDV strain that can be stably passaged without affecting FMDV replication, increases viral titer, and exhibits good production performance, making it suitable for preparing recombinant FMDV vaccines.

[0034] This invention also provides a method for constructing a recombinant foot-and-mouth disease virus strain. Utilizing reverse genetics, this invention involves transfecting a full-length infectious clone of foot-and-mouth disease virus containing a mutated or deleted coding sequence at amino acid position 108 of the L protein into foot-and-mouth disease virus-sensitive cells for virus rescue. The resulting recombinant virus exhibits high viral titer and antigen content. Therefore, the construction method and recombinant virus strain provided by this invention significantly improve virus production performance. Obtaining a recombinant foot-and-mouth disease vaccine strain using this method is of great significance for improving vaccine quality and efficacy, and has promising application prospects. Attached Figure Description

[0035] Figure 1 The effect of RAB20 protein overexpression on FMDV replication;

[0036] Figure 2 The effect of different doses of RAB20 protein on FMDV L protein expression levels;

[0037] Figure 3 The results show that RAB20 protein degrades FMDV L protein via the ubiquitin-proteasome pathway;

[0038] Figure 4 A schematic diagram illustrating the construction of recombinant foot-and-mouth disease virus with mutations at key amino acid sites in the L protein;

[0039] Figure 5 The rescue result of recombinant foot-and-mouth disease virus with a mutation at a key amino acid site in the L protein;

[0040] Figure 6 The results show the effect of RAB20 protein on the expression level of L protein due to mutations at key amino acid sites;

[0041] Figure 7 The results show the effect of RAB20 protein on the replication of recombinant foot-and-mouth disease virus with mutations at key amino acid sites in the L protein;

[0042] Figure 8 Results of virulence determination for recombinant foot-and-mouth disease virus with mutations at key amino acid sites in the L protein. Detailed Implementation

[0043] This invention provides a recombinant foot-and-mouth disease virus strain, whose L protein is a mutant L protein with a mutation or deletion at amino acid position 108.

[0044] In this invention, the parental foot-and-mouth disease virus strain of the recombinant foot-and-mouth disease virus strain includes at least one of the following: a wild-type foot-and-mouth disease virus strain, a recombinant foot-and-mouth disease virus strain, and a recombinant foot-and-mouth disease vaccine strain. In embodiments of this invention, the parental foot-and-mouth disease virus strain preferably includes an O-type foot-and-mouth disease virus strain or a recombinant O-type foot-and-mouth disease virus strain. The 108th amino acid of the mutant L protein is preferably mutated. The 108th amino acid of the mutant L protein is preferably mutated to arginine. The amino acid sequence of the mutant L protein is preferably as shown in SEQ ID NO: 6(MNTTDCFIALLYAFRQIKTLLLPRTQGKMELTLHNGEKKTFYSRPN NHDNCWLNTILQLFRYVDEPFFDWVYNSPENLTLDAIEQLEEITGLELREGGP PALVIWNI R HLLNTGIGTASRPSEVCMVDGTDMCLADFHAGIFLKGQEHAVFA CVTSNGWYAIDDEDFYPWTPDPSDVLVFVPYDQEPLNGEWKSKVQKRLR) or SEQ ID NO: 7 (MSTTDCFIALLYAFREIKTLFLSRAQGKMEFTLHNG EKKTFYSRPNNHDNCWLNTILQLFRYVDEPFFDWVYYSPENLTLDAIKQLEEITGLELHEGGPPALVIWNIRHLLNTGIGTASRPSEVCMVDGTDMCLADFHAGIFLKGQEHAVFACVTSNGWYAIDDEEFYPWTPDPSDVLVFVPYDQEPLNGEWKARVQRRLK).

[0045] In this invention, the recombinant foot-and-mouth disease virus strain is unaffected by the degradation of the L protein and viral replication caused by the host protein RAB20. Its replication ability is not inhibited when the RAB20 protein is overexpressed in the host. It exhibits good replication ability and production performance in cells such as BHK-21, and is a recombinant foot-and-mouth disease virus strain with high viral titer and antigen production capacity, suitable for vaccine preparation. The nucleotide sequence encoding the host protein RAB20 is shown in SEQ ID NO: 1 (ATGAGAAAGCCAGACGGGAAGATCGTGCTTTTGGGGGACATGAACGTG).The amino acid sequence of the host protein RAB20 is shown in SEQ ID NO: 2 (MRKPDGKIVLLGDMNVGKTSLLQRYMERRFPDTVSTVGGAFYLKQWRSY NISIWDTAGREQFHGLGSMYCRGAAAVILTYDVNHAQSLLELEDRFLGLTDTASADCLFAIVGNKVDLSEEGPGDGGQDGGHGPGPAGVGAAPRLPKQVQPEDAVALYKKILKYKMLDEKDVPAAEQMCFETSAKTGHNVDLLFETLFDMVVPVILRQRAQGPPQTVDIAPCGPPARTRSGCCA).

[0046] In the present invention, the amino acid sequence of the wild-type FMDV L protein is shown in SEQ ID NO: 4 (MNTTDCFIALLYAFRQIKTLLLPRTQGKMELTLHNGEKKTFYSRPNNHDNC WLNTILQLFRYVDEPFFDWVYNSPENLTLDAIEQLEEITGLELREGGPPALVIW NIKHLLNTGIGTASRPSEVCMVDGTDMCLADFHAGIFLKGQEHAVFACVTSN GWYAIDDEDFYPWTPDPSDVLVFVPYDQEPLNGEWKSKVQKRLR); the nucleotide sequence of the coding sequence of the wild-type FMDV L protein is shown in SEQ ID NO: 3 (ATGAACACGACTGACTGTTTCATCGCTTTGCTGTACGCCTTCAGACAGAT CAAAACACTGCTTTTACCACGAACACAAGGAAAGATGGAACTCACACTTCACAACGGTGAAAAGAAGACATTCTACTCCAGGCCCAACAACCACGACAACTGCTGGCTGAACACCATCCTCCAGTTGTTTAGGTACGTTGACGAACCCTTCTTTGACTGGGTTTACAACTCGCCCGAGAACCTCACACTTGATGCTATTGAGCAATTGGAAGAAATTACAGGTCTTGAACTCCGCGAGGGCGGTCCACCCGCCCTCGTCATCTGGAACATTAAACACCTGCTCAATACCGGAATCGGCACCGCTTCGCGCCCCAGCGAAGTGTGCATGGTAGACGGGACGGACATGTGTTTGGCTGACTTCCACGCTGGCATTTTCCTGAAAGGACAGGAACACGCTGTGTTCGCCTGTGTCACCTCCAACGGGTGGTACGCGATTGATGACGAGGACTTTTACCCCTGGACACCGGACCCGTCCGACGTCCTGGTGTTTGTTCCGTACGATCAGGAGCCACTCAACGGAGAATGGAAATCAAAGGTTCAAAAACGACTCAGG).

[0047] In one embodiment of the present invention, overexpression of RAB20 in host cells, such as PK-15 cells, inhibits the abundance of FMDV viral proteins in a dose-dependent manner, meaning that overexpression of RAB20 protein in host cells inhibits FMDV viral replication. In another embodiment of the present invention, to further explore the mechanism by which the host protein RAB20 inhibits FMDV replication, the present invention found that RAB20 decreases the expression level of FMDV L protein in a dose-dependent manner, indicating that RAB20 inhibits FMDV replication by inhibiting the expression of L protein; and the degradation of L protein induced by RAB20 can be inhibited by MG132, indicating that RAB20 degrades L protein through the proteasome pathway. In another embodiment of the present invention, in order to further clarify the key amino acid site for RAB20 to degrade the L protein, the present invention screened and identified the 108th amino acid (K108) as the key amino acid site for RAB20 to degrade the L protein. In another embodiment, it was demonstrated that RAB20 overexpression could not affect the replication of recombinant foot-and-mouth disease virus strains with the K108R mutation in the L protein, indicating that the 108th amino acid of the L protein is the key amino acid site for RAB20 to inhibit the replication of foot-and-mouth disease virus.

[0048] This invention provides a method for constructing the recombinant foot-and-mouth disease virus strain, comprising the following steps:

[0049] Prepare a full-length infectious clone of foot-and-mouth disease virus containing the coding sequence of the mutant L protein; the mutation site of the mutant L protein includes amino acid 108 of the L protein;

[0050] A full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence was transfected into foot-and-mouth disease virus-sensitive cells to obtain a recombinant foot-and-mouth disease virus strain.

[0051] This invention prepares a full-length infectious clone of foot-and-mouth disease virus containing a mutant L protein coding sequence.

[0052] In this invention, the preparation of the full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence preferably includes preparation using gene mutation technology or gene synthesis technology. The 108th amino acid in the mutant L protein is mutated to arginine. The gene synthesis technology refers to obtaining the mutant L protein coding sequence or the full-length infectious clone of foot-and-mouth disease virus through gene synthesis methods. In the embodiments of this invention, the nucleotide sequence of the mutant L protein coding sequence is shown in SEQ ID NO:5 (ATGAACACGACTGACTGTTTCATCGCTTTGCTGTACGCCTTCAGACAGAT).

[0053] In this invention, a method for preparing a full-length infectious clone of foot-and-mouth disease virus (FMDV) containing a mutated L protein coding sequence using gene mutation technology is preferred. Using a full-length infectious clone of FMDV as a template, PCR amplification is performed using two pairs of homologous recombination mutant primers to obtain two PCR amplification products. These products are then homologously recombinated with a linearized vector plasmid to obtain a full-length infectious clone of FMDV containing the mutated L protein coding sequence. The full-length infectious clone of FMDV preferably includes the recombinant plasmid prO-FMDV containing the L gene, P1 gene, and P2 gene of the O / JSCZ / 2013 strain. The recombinant plasmid prO-FMDV is disclosed in patents with publication numbers CN 107029231A, CN106916832A, and CN107041951A. The recombinant plasmid prO-FMDV is a reverse genetics operating system for strains with strong cellular immune responses, and the rescued type O recombinant FMDV vaccine strain rO-FMDV exhibits cross-protective activity against different lineages of type O FMDV. The two pairs of homologous recombination mutant primers preferably include KpnI-F / mL108-R and mL108-F / KpnI-R; the nucleotide sequence of KpnI-F is preferably as shown in SEQ ID NO:8; the nucleotide sequence of KpnI-R is preferably as shown in SEQ ID NO:9; the nucleotide sequence of mL108-F is preferably as shown in SEQ ID NO:10; and the nucleotide sequence of mL108-R is preferably as shown in SEQ ID NO:11.

[0054] After the preparation, the present invention transfects the full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence into foot-and-mouth disease virus sensitive cells to obtain a recombinant foot-and-mouth disease virus strain.

[0055] In this invention, the foot-and-mouth disease virus (FMDV) sensitive cells preferably include BHK-21 cells and / or IBRS-2 cells. This invention does not impose any particular limitation on the transfection method; any transfection method well-known in the art can be used. After transfection, the virus is preferably harvested when approximately 90% of the cells exhibit cytopathic effects. The morphology of the cytopathic effects preferably includes cell rounding, aggregation into a grape-like distribution, and eventual cell disintegration. This invention also preferably includes verifying the gene mutations of the collected recombinant FMDV strain. The gene mutation verification method preferably includes amplifying the L-containing gene fragment, purifying and recovering it, and sequencing it. The sequencing results are compared with the coding sequence of the mutated L protein; if they match, it indicates that a recombinant FMDV strain has been obtained.

[0056] This invention involves determining the viral titer of recombinant foot-and-mouth disease virus strains obtained by the above construction method. The results show that, compared with the control FMDV, the viral titer of recombinant FMDV with mutations at key amino acid sites of the L protein is significantly increased. This indicates that mutations at key amino acid sites of the L protein can eliminate the inhibitory effect of RAB20 on FMDV replication, increase the viral titer and antigen yield of FMDV, and improve viral production performance.

[0057] Given that the recombinant foot-and-mouth disease virus strain has a high viral titer, excellent production performance and high antigen yield, the present invention provides a recombinant foot-and-mouth disease vaccine, comprising the recombinant foot-and-mouth disease virus strain or a recombinant foot-and-mouth disease virus strain prepared by the construction method.

[0058] In this invention, the recombinant foot-and-mouth disease vaccine preferably includes an adjuvant. This invention does not impose any special limitations on the type of adjuvant; adjuvants well-known in the art, such as ISA206 or ISA201, can be used. This invention does not impose any special limitations on the antigen content of the recombinant foot-and-mouth disease vaccine strain in the vaccine; a content well-known in the art can be used. This invention does not impose any special limitations on the preparation method of the vaccine; inactivated vaccine preparation methods well-known in the art can be used.

[0059] This invention provides the application of the recombinant foot-and-mouth disease virus strain or the recombinant foot-and-mouth disease virus strain prepared by the construction method in the preparation of recombinant foot-and-mouth disease vaccines.

[0060] This invention provides a method for preparing a recombinant foot-and-mouth disease virus strain that is resistant to replication inhibition by the host protein RAB20, comprising the following steps:

[0061] The 108th amino acid of the L protein in foot-and-mouth disease virus was mutated or deleted.

[0062] In this invention, the preferred method of mutation is to mutate the 108th amino acid of the L protein in the foot-and-mouth disease virus strain to arginine.

[0063] In this invention, the parental foot-and-mouth disease virus strain of the recombinant foot-and-mouth disease virus strain includes at least one of the following: a wild-type foot-and-mouth disease virus strain, a recombinant foot-and-mouth disease virus strain, and a recombinant foot-and-mouth disease vaccine strain. In embodiments of this invention, the parental foot-and-mouth disease virus strain of the recombinant foot-and-mouth disease virus strain preferably includes a type O foot-and-mouth disease virus strain or a recombinant type O foot-and-mouth disease virus strain. The method for mutating or deleting the 108th amino acid of the L protein in the recombinant foot-and-mouth disease virus strain is preferably prepared using gene mutation technology or gene synthesis technology, and the specific method is the same as described in the above technical solution, and will not be repeated here. In embodiments of this invention, the amino acid sequence of the mutated L protein is preferably as shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0064] This invention provides the use of the recombinant foot-and-mouth disease vaccine in the preparation of medicaments for the prevention and / or control of foot-and-mouth disease in animals.

[0065] In this invention, the animal preferably includes an even-toed ungulate. The even-toed ungulate preferably includes at least one of the following: pigs, cattle, and sheep.

[0066] The following detailed description, in conjunction with embodiments, illustrates a recombinant foot-and-mouth disease virus strain with high antigen yield, its construction method, and its applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0067] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the experimental materials used in the embodiments are all purchased from conventional biochemical reagent companies.

[0068] The relevant experiments described in this embodiment of the invention obtained biosafety permits and foot-and-mouth disease laboratory activity permits: In accordance with the relevant requirements for biosafety level 3 (BSL-3) laboratories and foot-and-mouth disease-related biosafety, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, through a hierarchical reporting process involving the Biosafety Committee of the Lanzhou Veterinary Research Institute, the Biosafety Committee of the Chinese Academy of Agricultural Sciences, and the Experimental Animal Ethics Committee of the Lanzhou Veterinary Research Institute, obtained permission from the Ministry of Agriculture and Rural Affairs to conduct research on highly pathogenic FMDV pathogens and animals, and has been registered with the Ministry of Agriculture and Rural Affairs, meeting the requirements of the national biosafety level.

[0069] The FMDV strains (O / BY / CHA / 2010 and O / GD / CHA / 2015) used in this invention are deposited by the National Foot-and-Mouth Disease Reference Laboratory designated by the Ministry of Agriculture and Rural Affairs, and can be obtained by the public through a letter of authorization approved by the Veterinary Bureau of the Ministry of Agriculture and Rural Affairs. The FMDV strain O / BY / CHA / 2010 is a known virus strain reported in the prior art (Genetic Determinants of Altered Virulence of Type O Foot-and-Mouth Disease Virus. Fan Yang, Zixiang Zhu, Weijun Cao et al. Journal of Virology. Pub Date: 2020-03-17, DOI: 10.1128 / JVI.01657-19). The FMDV O / GD / CHA / 2015 strain is a known viral strain reported in the prior art (The Pseudoknot Region of the 5' Untranslated Region Is a Determinant of Viral Tropism and Virulence of Foot-and-MouthDisease Virus. Zixiang Zhu, Fan Yang, Weijun Cao et al. Journal of Virology. PubDate:2019-04-03, DOI:10.1128 / jvi.02039-18). The recombinant plasmid prO-FMDV has been disclosed in Chinese patents ZL201710256450.0, ZL201710256371.X, and ZL201710256542.9.

[0070] Example 1

[0071] Effect of RAB20 overexpression on FMDV replication

[0072] 1. Construction of RAB20 eukaryotic expression plasmid

[0073] Query the RAB20 gene sequence from the NCBI database, design primers, RAB20-NheI-F: CGTCTA G CTAGC ATGAGAAAGCCAGACGGGAAGATCG (SEQ ID NO: 12) (underlined is the NheI restriction site); RAB20-BamHI-R: CGC GGATCCGGCGCAGCACCCAGAT CTGGTCCG (SEQ ID NO: 13) (underlined indicates BamHI restriction site). RNA was extracted from PK-15 cells, reverse transcribed into cDNA, and used as a template to amplify the RAB20 gene (nucleotide sequence shown in SEQ ID NO: 1). The amplified fragment was recovered by nucleic acid electrophoresis and double-digested with NheI and BamHI restriction endonucleases. At the same time, the pcDNA3.1 / myc vector plasmid was double-digested with the same restriction endonucleases. The RAB20 gene and the linearized vector fragment were purified and recovered, ligated overnight at 4°C with T4 ligase, transformed into DH5α competent cells, and the plasmid was extracted and sequenced. The successfully constructed plasmid was named RAB20-Myc.

[0074] 2. Effects of RAB20 overexpression on FMDV replication

[0075] PK-15 cells were seeded into 6-well plates and cultured at 37°C in a 5% CO2 cell culture incubator until the cell density reached approximately 70%. The cells were then transfected with different doses of the RAB20 eukaryotic expression plasmid RAB20-Myc (0 μg, 1 μg, 3 μg). After 24 h, the cells were infected with FMDV (O / BY / CHA / 2010 strain) and Western blot analysis was performed.

[0076] See results Figure 1 The results showed that RAB20 protein overexpressed on PK-15 cells suppressed the abundance of FMDV viral proteins in a dose-dependent manner.

[0077] Example 2

[0078] Effect of RAB20 on FMDV L protein expression

[0079] 1. Construction method of Flag-L plasmid

[0080] The FMDV L gene sequence was retrieved from the NCBI database, and specific primers were designed. The specific sequences are as follows:

[0081] L-EcoRI-F: CGC GAATTC AATGAACACGACTGACTGTTTCATC (underlined is the EcoRI restriction site, SEQ ID NO: 14);

[0082] L-BamHI-R: CGC GGATCC TTACCTGAGTCGTTTTTGAACCTTTG (underlined is the BamHI restriction site, SEQ ID NO: 15).

[0083] RNA was extracted from strain O / GD / CHA / 2015, and cDNA was synthesized by reverse transcription. The L gene was amplified using the cDNA as a template (nucleotide sequence as shown in SEQ ID NO: 3, amino acid sequence as shown in SEQ ID NO: 4). The amplified fragment was recovered by nucleic acid electrophoresis and double-digested with EcoRI and BamHI restriction endonucleases. At the same time, the p3×FLAG-CMV-7.1 vector plasmid was double-digested with the same restriction endonucleases. The L gene and the linearized vector fragment were purified and recovered, ligated overnight at 4°C with T4 ligase, transformed into DH5α competent cells, single clones were picked, cultured, and the plasmid was extracted and sequenced. The successfully constructed plasmid was named L-Flag.

[0084] 2. Effect of RAB20 on FMDV L protein expression

[0085] HEK293T cells were seeded into 6-well plates and cultured at 37°C in a 5% CO2 cell culture incubator until the cell density reached approximately 70%. The cells were then transfected with the FMDV L protein eukaryotic expression plasmid L-Flag and different doses of the RAB20 eukaryotic expression plasmid RAB20-Myc. After collection, Western blot analysis was performed.

[0086] See results Figure 2 The results showed that RAB20 reduced the expression level of FMDV L protein in a dose-dependent manner, indicating that RAB20 inhibits FMDV replication by suppressing the expression of L protein.

[0087] 3. RAB20 protein degrades FMDVL protein via the ubiquitin-proteasome pathway.

[0088] HEK293T cells were co-transfected with the L protein eukaryotic expression plasmid L-Flag and the RAB20 eukaryotic expression plasmid RAB20-Myc. After 16 h of transfection, 20 μM of the proteasome inhibitor MG132, 20 μM of the lysosomal inhibitor NH4Cl, and 20 μM of the apoptosis inhibitor Z-VAD-FMK were added, respectively. After culturing for another 8 h, the cells were harvested and Western blot analysis was performed.

[0089] See results Figure 3 The results showed that the degradation of L protein induced by RAB20 was inhibited by the proteasome inhibitor MG132, indicating that RAB20 induces the degradation of L protein through the proteasome pathway.

[0090] Example 3

[0091] Methods for constructing eukaryotic expression plasmids of L protein key amino acid site mutants

[0092] Based on Example 1, the L protein of FMDV O / GD / CHA / 2015 strain was mutated, and a eukaryotic expression plasmid of the mutated L protein was constructed. Using the method described in this example, eukaryotic expression plasmids of mutated L protein bodies of other foot-and-mouth disease virus strains can also be constructed. The specific process is as follows:

[0093] Based on the wild-type FMDV L protein eukaryotic expression plasmid L-Flag (nucleotide sequence as shown in SEQ ID NO: 3, amino acid sequence as shown in SEQ ID NO: 4), the coding sequence for amino acid 108 (lysine) of L protein to arginine was mutated using gene synthesis technology or point mutation PCR technology (nucleotide sequence as shown in SEQ ID NO: 5). The product was transformed into DH5α competent cells, single clones were picked, cultured, and the plasmid was extracted and sequenced. The mutation at amino acid 108 of L protein was confirmed to be successful. The key amino acid mutant plasmid was named L-Flag-K108R.

[0094] Example 4

[0095] Construction and identification of recombinant type O foot-and-mouth disease virus with L protein mutation

[0096] 1. Construction of an infectious clone of recombinant type O foot-and-mouth disease virus

[0097] The L-containing gene fragment in the recombinant plasmid prO-FMDV, containing the L, P1, and P2 genes of the O / JSCZ / 2013 strain, was digested with KpnI, and the large vector fragment was recovered. Using this plasmid as a template, the gene fragment containing the L gene with a key mutation site was amplified by PCR. The homologous recombination mutation primers were: KpnI-F (5'-TAAGGATGCCCTTCAGGTACCCTGAGGTAACACGCGACACTCG-3', SEQ ID NO: 8); mL108-R (5'-GCAGGTGTCTAATGTTCCAAATAACGAGAGCGG-3', SEQ ID NO: 10); mL108-F (5'-GAACATTAGACACCTGCTCAACACCGGAATCG-3', SEQ ID NO: 11); KpnI-R (5'-ATGGAACAAAGTTCAGGTACCATGGCCACCAGTAGGCAGC-3', SEQ ID NO: 8). NO: 9), the PCR amplification products were recovered and homologous recombination was performed with the recovered vector fragment to obtain a recombinant plasmid with a lysine mutation at position 108 of the L protein to arginine. Amplification was performed using a high-fidelity plasmid from TaKaRa. HSDNA polymerase was prepared according to the product instructions, using a 50 μL reaction system. Amplification conditions were: 98℃ for 10 s, 55℃ for 15 s, 72℃ for 1 min, 30 cycles, 4℃ for 5 min. Homologous recombination was performed using Novizan. Using the Ultra One Step Cloning Kit, transform DH5α competent cells at 50℃ for 15 min. Pick single colonies, extract plasmids by shaking, and identify positive clones by sequencing. The final result is the recombinant plasmid prO-mL-K108R-FMDV containing the L gene mutation. A schematic diagram of its construction is shown below. Figure 4 As shown.

[0098] 2. Rescue of Recombinant Viruses

[0099] Prepare the recombinant plasmid prO-mL-K108R-FMDV obtained in step 1, according to Polyplus. The transfection reagent instructions state that when BHK-21 cells reach approximately 70% confluency, the recombinant plasmid is transfected into BHK-21 cells. Normal cell controls and a transfection reagent control are also established. Cells are incubated at 37°C with 5% CO2, and cell status is observed. The virus is harvested when approximately 90% of the cells exhibit cytopathic effects. After three freeze-thaw cycles, the cells are re-inoculated into BHK-21 cells until the virus stably induces cytopathic effects, characterized by rounded cells, grape-like aggregation, and eventual cell disintegration. The resulting type O foot-and-mouth disease recombinant virus is named rO-mL-K108R-FMDV.

[0100] The results are as follows Figure 5 As shown, compared with normal control BHK-21 cells, infection of BHK-21 cells with recombinant virus rO-mL-K108R-FMDV caused typical CPE.

[0101] 3. Identification of recombinant virus by RT-PCR

[0102] BHK-21 cells were infected with the stably passaged recombinant virus rO-mL-K108R-FMDV. Cell supernatant was collected, total RNA was extracted with Trizol, reverse transcribed, and L-containing gene fragments were amplified with KpnI-F (SEQ ID NO: 8) and KpnI-R (SEQ ID NO: 9). After purification and recovery, the fragments were sent for sequencing.

[0103] The results showed that the L gene of the recombinant O-type foot-and-mouth disease virus obtained was consistent with the theoretical sequence, and its 108th amino acid was arginine. The amino acid encoded by the mutated L gene was the amino acid sequence shown in SEQ ID NO:7.

[0104] Example 5

[0105] The effect of RAB20 protein on the replication of recombinant foot-and-mouth disease virus with mutations at key amino acid sites in the L protein.

[0106] 1. Effect of RAB20 on the expression of L protein with key site mutations

[0107] HEK293T cells were seeded into 6-well plates and cultured at 37°C in a 5% CO2 cell culture incubator until the cell density reached approximately 70%. The cells were co-transfected with the eukaryotic expression plasmid L-Flag-K108R, a mutant of the FMDV L protein key site, and different doses of the eukaryotic expression plasmid RAB20-Myc (0 μg, 1 μg, 2 μg, and 3 μg). Western blot analysis was performed after the samples were collected.

[0108] See results Figure 6 The results showed that RAB20 protein overexpression did not affect the expression level of mutant L protein.

[0109] 2. Effects of RAB20 on the replication of recombinant foot-and-mouth disease virus with mutations at key sites of the L protein

[0110] PK-15 cells were seeded into 6-well plates and cultured at 37°C in a 5% CO2 cell culture incubator until the cell density reached approximately 70%. The cells were then transfected with different doses of the RAB20 eukaryotic expression plasmid RAB20-Myc (0 μg, 1 μg, 3 μg). After 24 h, the cells were infected with recombinant foot-and-mouth disease virus rO-mL-K108R-FMDV, which has a mutation at a key amino acid site of the L protein, and Western blot analysis was performed.

[0111] See results Figure 7 The results showed that overexpression of RAB20 in PK-15 cells had no effect on the viral protein abundance of the recombinant virus rO-mL-K108R-FMDV. Combined with the results of Example 1, it is clear that overexpression of RAB20 inhibits the replication of wild-type FMDV in a dose-dependent manner, but cannot inhibit the replication of the L-protein mutated recombinant virus rO-mL-K108R-FMDV. Therefore, this recombinant foot-and-mouth disease virus can be used as a seed virus for foot-and-mouth disease virus vaccines, increasing viral titer and antigen content, and improving production performance.

[0112] Example 6

[0113] Pathogenicity test of recombinant foot-and-mouth disease virus on BHK-21 cells

[0114] BHK-21 cells were infected with control FMDV and recombinant FMDV with L protein mutation using the same MOI. Virus solutions were collected after 8 hours, and the cells were subjected to three freeze-thaw cycles. The samples were serially diluted 10-fold using DMEM medium and seeded into 96-well plates containing a confluent monolayer of BHK-21 cells, with 8 wells per dilution. The plates were incubated at 37°C and 5% CO2 for 3 days. Cytopathic effects were observed, and the TCID of the virus was calculated using the Reed-Muench method (see existing literature "Reed, L.J. and Muench, H. (1938). 'A Simple Method of Estimating Fifty Percent Endpoints'". The American Journal of Hygiene 27:493-497"). 50 .

[0115] The results are as follows Figure 8 The results showed that, compared with the control FMDV, the viral titer of recombinant FMDV with mutations at key amino acid sites of the L protein was significantly increased, indicating that mutations at key amino acid sites of the L protein can increase the viral titer of FMDV.

[0116] In summary, the results indicate that the recombinant foot-and-mouth disease virus with mutations at key amino acid sites in the L protein does not exhibit replication levels inhibited by the host protein RAB20. Furthermore, this key site mutation significantly enhances the viral titer and antigen yield of the foot-and-mouth disease virus, thereby improving viral production performance and making it suitable as a candidate strain for foot-and-mouth disease virus vaccines.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant foot-and-mouth disease virus strain, characterized in that, The L protein is a mutant L protein with only the 108th amino acid mutated to arginine.

2. The recombinant foot-and-mouth disease virus strain according to claim 1, characterized in that, The parental foot-and-mouth disease virus strain of the recombinant foot-and-mouth disease virus strain is a wild-type foot-and-mouth disease virus strain and / or a recombinant foot-and-mouth disease virus strain.

3. The recombinant foot-and-mouth disease virus strain according to claim 1, characterized in that, The amino acid sequence of the mutant L protein is shown in SEQ ID NO: 6 or SEQ ID NO:

7.

4. A method for constructing the recombinant foot-and-mouth disease virus strain according to any one of claims 1 to 3, characterized in that, It consists of the following steps: A full-length infectious clone of foot-and-mouth disease virus containing a mutant L protein coding sequence was prepared; the mutation site of the mutant L protein was amino acid 108 of the foot-and-mouth disease virus L protein; the 108th amino acid of the foot-and-mouth disease virus L protein was mutated to arginine; A full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence was transfected into foot-and-mouth disease virus-sensitive cells to obtain a recombinant foot-and-mouth disease virus strain.

5. The construction method according to claim 4, characterized in that, The preparation of the full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence includes preparation using gene mutation technology or gene synthesis technology.

6. The construction method according to claim 4, characterized in that, A method for preparing a full-length infectious clone of foot-and-mouth disease virus containing a mutant L protein coding sequence using gene mutation technology was described. Using the full-length infectious clone of foot-and-mouth disease virus as a template, PCR amplification was performed using two pairs of homologous recombination mutant primers to obtain two PCR amplification products. Two PCR amplification products were homologously recombinated with a linearized vector plasmid to obtain a full-length infectious clone of foot-and-mouth disease virus containing a mutant L protein coding sequence.

7. The construction method according to claim 6, characterized in that, The two pairs of homologous recombination mutant primers include KpnI-F / mL108-R and mL108-F / KpnI-R; The nucleotide sequence of KpnI-F is shown in SEQ ID NO:8; The nucleotide sequence of the KpnI-R is shown in SEQ ID NO:9; The nucleotide sequence of the mL108-F is shown in SEQ ID NO:10; The nucleotide sequence of the mL108-R is shown in SEQ ID NO:

11.

8. The construction method according to claim 4, characterized in that, The foot-and-mouth disease virus-sensitive cells include BHK-21 cells and / or IBRS-2 cells.

9. A recombinant foot-and-mouth disease inactivated vaccine, characterized in that, Includes the recombinant foot-and-mouth disease virus strain according to any one of claims 1 to 3 or the recombinant foot-and-mouth disease virus strain prepared by the construction method according to any one of claims 4 to 8.

10. The use of the recombinant foot-and-mouth disease virus strain according to any one of claims 1 to 3 or the recombinant foot-and-mouth disease virus strain prepared by the construction method according to any one of claims 4 to 8 in the preparation of a recombinant foot-and-mouth disease inactivated vaccine.

11. A method for preparing a recombinant foot-and-mouth disease virus strain that is immune to replication inhibition by the host protein RAB20, characterized in that, It consists of the following steps: The 108th amino acid of the L protein in the foot-and-mouth disease virus strain was mutated. The mutation method involves mutating the 108th amino acid of the L protein in the foot-and-mouth disease virus strain to arginine.

12. The preparation method according to claim 11, characterized in that, The amino acid sequence of the mutated L protein is shown in SEQ ID NO: 6 or SEQ ID NO:

7.

13. The use of the recombinant foot-and-mouth disease inactivated vaccine of claim 9 in the preparation of a medicament for the prevention and / or control of foot-and-mouth disease in animals.

14. The application according to claim 13, characterized in that, The animals mentioned include even-toed ungulates.

15. The application according to claim 14, characterized in that, The even-toed ungulates include at least one of the following: pigs, cattle, and sheep.

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