L protein mutated foot and mouth disease virus recombinant strain as well as construction method and application thereof

Abstract

The invention provides an L protein mutated foot and mouth disease virus recombinant strain as well as a construction method and application thereof, and belongs to the technical field of biotechnology. The invention relates to an L protein mutated foot and mouth disease virus recombinant strain, wherein L protein is mutated L protein with 18th, 28th and 39th amino acids mutated or deleted. The invention proves that the overexpression of the host protein POLR2H inhibits the replication of the FMDV, and the L protein is degraded through a proteasome pathway so as to inhibit the virus replication; key amino acid sites (18, 28 and 39) in the FMDV L protein are mutated to construct the foot and mouth disease virus recombinant strain which is not inhibited by host protein POLR2H for replication, and the recombinant virus can be stably passaged, has the characteristic of high virus titer on BHK-21 cells, improves the virus titer and antigen yield, can be used for preparing FMDV vaccines, and can be used for preparing FMDV vaccines. Good application prospects are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and particularly relates to a recombinant strain of foot-and-mouth disease virus with an L protein mutation, a construction method thereof, and an application thereof. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease of cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV). The characteristic symptoms of diseased animals are the occurrence of blisters on the mouth, nose, hooves, and teats of female livestock. After the blisters rupture, ulcers or scabs are formed, showing lameness and lying down, and leading to a significant decline in productivity. In the case of young animals being infected or a pandemic occurring in wild animals, FMD can cause a relatively high mortality rate, which has a great impact on the international trade of livestock.

[0003] FMDV belongs to the Picornaviridae family, a single-stranded positive-strand RNA virus of the Foot-and-mouth disease virus genus. Its genome is about 8.0 kb long and is translated into a 5' untranslated region, an open reading frame, and a 3' untranslated region. The open reading frame encodes non-structural protein L pro , four structural proteins VP4, VP2, VP3, and VP1, as well as 2A, 2B, 2C, 3A, 3B, 3C pro and 3D pol non-structural proteins. Among them, the leader protein L pro is an important virulence factor of FMDV and plays an important role in the regulation of virus replication, virulence, and host cell response. By deleting or mutating the L protein, a safer and more effective FMDV vaccine can be developed. At the same time, there are also complex interactions between host proteins and FMDV proteins, which regulate virus replication through different mechanisms. Therefore, studying the mechanism of host protein regulation of FMDV replication has important guiding significance for the design and preparation of highly efficient FMDV recombinant vaccine strains. However, there is currently no relevant report on the regulation of FMDV replication by the host protein POLR2H. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a recombinant strain of foot-and-mouth disease virus with an L protein mutation. By mutating the key amino acid sites in the L protein, the ability of POLR2H to inhibit FMDV replication is eliminated, thereby increasing the virus titer and antigen production.

[0005] The present invention provides a recombinant strain of foot-and-mouth disease virus with an L protein mutation, and the L protein is a mutant L protein in which the 18th, 28th, and 39th amino acids are mutated.

[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: foot-and-mouth disease virus wild strain, foot-and-mouth disease virus recombinant strain, and foot-and-mouth disease recombinant vaccine strain.

[0007] Preferably, the amino acids at positions 18, 28, and 39 of the mutant L protein are the mutant L protein;

[0008] Preferably, the amino acids at positions 18, 28, and 39 in the mutant L protein are mutated 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] The present 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 the coding sequence of the mutant L protein; the mutation sites of the mutant L protein include positions 18, 28, and 39 of the foot-and-mouth disease virus L protein;

[0012] Transfect the full-length infectious clone of foot-and-mouth disease virus containing the coding sequence of the mutant L protein 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 coding sequence of the mutant L protein is prepared by using gene mutation technology or gene synthesis technology;

[0014] Preferably, the amino acids at positions 18, 28, and 39 of the foot-and-mouth disease virus L protein are mutated to arginine;

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

[0016] Using the product after homologous recombination of the two PCR amplification products as a template, perform PCR amplification using another two pairs of homologous recombination mutant primers, and perform homologous recombination on the two obtained PCR amplification products and the linearized vector plasmid to obtain a full-length infectious clone of foot-and-mouth disease virus containing the coding sequence of the mutant L protein;

[0017] Preferably, the four pairs of homologous recombination mutant primers include KpnI-F / mL1828-R, mL1828-F / KpnI-R, KpnI-F / mL39-R, and mL39-F / KpnI-R;

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

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

[0020] The nucleotide sequence of mL1828-F is shown in SEQ ID NO:10;

[0021] The nucleotide sequence of mL1828-R is shown in SEQ ID NO:11;

[0022] The nucleotide sequence of mL39-F is shown in SEQ ID NO:12;

[0023] The nucleotide sequence of mL39-R is shown in SEQ ID NO:13.

[0024] Preferably, the foot-and-mouth disease virus-sensitive cells include BHK-21 cells and / or IBRS-2 cells.

[0025] The present invention provides a foot-and-mouth disease recombinant vaccine, including the foot-and-mouth disease virus recombinant strain or the foot-and-mouth disease virus recombinant strain prepared by the construction method.

[0026] The present invention provides the application of the foot-and-mouth disease virus recombinant strain or the foot-and-mouth disease virus recombinant strain prepared by the construction method in the preparation of a foot-and-mouth disease recombinant vaccine.

[0027] The present invention provides a preparation method of a foot-and-mouth disease virus recombinant strain that is not inhibited by the host protein POLR2H from replicating, including the following steps:

[0028] Mutate or delete the 18th, 28th, and 39th amino acids of the L protein in the foot-and-mouth disease virus strain.

[0029] Preferably, the mutation method is to mutate the 18th, 28th, and 39th amino acids of the L protein in the foot-and-mouth disease virus strain into arginine;

[0030] More preferably, the amino acid sequence of the mutated L protein is shown in SEQ ID NO:6 or SEQ ID NO:7.

[0031] The present invention provides the application of the foot-and-mouth disease recombinant vaccine in the preparation of a drug for preventing and / or controlling foot-and-mouth disease in animals;

[0032] Preferably, the animals include cloven-hoofed animals;

[0033] More preferably, the cloven-hoofed animals include at least one of the following: pigs, cows, and sheep.

[0034] The present invention provides a recombinant foot-and-mouth disease virus strain with a mutated L protein, wherein the L protein is a mutated L protein with mutations or deletions at amino acid positions 18, 28, and 39. The present invention first discovered that overexpression of POLR2H in host cells inhibits the replication of FMDV, and further determined that the POLR2H protein degrades the L protein through amino acid positions 18, 28, and 39, thereby inhibiting virus replication. By mutating the amino acids at positions 18, 28, and 39 of the L protein, the present invention eliminates the effect of the POLR2H protein on degrading the L protein and simultaneously eliminates the inhibitory effect of the POLR2H protein on FMDV replication; by designing and modifying the amino acids at positions 18, 28, and 39 of the L protein, the present invention successfully constructs and rescues a recombinant foot-and-mouth disease virus strain, which can be stably passaged, does not affect the replication of foot-and-mouth disease virus, improves the virus titer, has good production performance, and can be used for preparing recombinant foot-and-mouth disease vaccines.

[0035] The present invention also provides a method for constructing the recombinant foot-and-mouth disease virus strain. Using reverse genetics technology, the full-length infectious clone of foot-and-mouth disease virus containing the coding sequence with mutations or deletions at amino acid positions 18, 28, and 39 of the L protein is transfected into foot-and-mouth disease virus-sensitive cells for virus rescue. The obtained recombinant virus has the characteristics of high virus titer and antigen content. It can be seen that the construction method and recombinant virus strain provided by the present invention significantly improve the production performance of the virus. Obtaining a recombinant foot-and-mouth disease vaccine strain by this method is of great significance for improving the quality and efficacy of the vaccine and has good application prospects. Description of the Drawings

[0036] Figure 1 Results of the effect of overexpressing POLR2H on FMDV replication;

[0037] Figure 2 Results of the effect of different doses of POLR2H on the expression level of FMDV L protein;

[0038] Figure 3 Results of POLR2H degrading FMDV L protein through the ubiquitin-proteasome pathway;

[0039] Figure 4 Schematic diagram of the construction of a recombinant foot-and-mouth disease virus strain with mutations at key amino acid sites of the L protein;

[0040] Figure 5 Results of the rescue of a recombinant foot-and-mouth disease virus strain with mutations at key amino acid sites of the L protein;

[0041] Figure 6 Results of the effect of POLR2H on the expression level of the L protein with mutations at key amino acid sites;

[0042] Figure 7Results of the effect of POLR2H on the replication of a recombinant foot-and-mouth disease virus strain with key amino acid site mutations in the L protein. Detailed implementation mode

[0043] The present invention provides a recombinant foot-and-mouth disease virus strain with an L protein mutation, wherein the L protein is replaced with a mutant L protein with mutations at amino acid positions 18, 28, and 39.

[0044] In the present 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 foot-and-mouth disease virus strain, a recombinant foot-and-mouth disease virus strain, and a recombinant foot-and-mouth disease vaccine strain. In an embodiment of the present invention, the parental foot-and-mouth disease virus strain of the recombinant 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 amino acid mutations at positions 18, 28, and 39 in the mutant L protein are preferably arginine. The amino acid sequence of the mutant L protein is preferably as shown in SEQ ID NO:6 (MNTTDCFIALLYAFRQI R TLLLPRTQG R MELTLHNGEK R TFYSRPNNHDNCWLNTILQLFRYVDEPFFDWVYNSPENLTLDAIEQLEEITGLELREGGPPALVIW NIKHLLNTGIGTASRPSEVCMVDGTDMCLADFHAGIFLKGQEHAVFACVTSN GWYAIDDEDFYPWTPDPSDVLVFVPYDQEPLNGEWKSKVQKRLR) or SEQ ID NO:7 (MSTTDCFIALLYAFREI R TLFLSRAQG R MEFTLHNGEK R TFYSRPNNH DNCWLNTILQLFRYVDEPFFDWVYYSPENLTLDAIKQLEEITGLELHEGGPPA LVIWNIKHLLNTGIGTASRPSEVCMVDGTDMCLADFHAGIFLKGQEHAVFAC VTSNGWYAIDDEEFYPWTPDPSDVLVFVPYDQEPLNGEWKARVQRRLK).

[0045] In the embodiments of the present invention, the nucleotide sequence of the coding sequence of the wild-type foot-and-mouth disease virus L protein is as shown in SEQ ID NO: 3 (ATGAACACGACTGACTGTTTCATCGCTTTGCTGTACGCCTTCAGACAGATCAAAACACTGCTTTTACCACGAACACAAGGAAAGATGGAACTCACACTTCACAACGGTGAAAAGAAGACATTCTACTCCAGGCCCAACAACCACGACAACTGCTGGCTGAACACCATCCTCCAGTTGTTTAGGTACGTTGACGAACCCTTCTTTGACTGGGTTTACAACTCGCCCGAGAACCTCACACTTGATGCTATTGAGCAATTGGAAGAAATTACAGGTCTTGAACTCCGCGAGGGCGGTCCACCCGCCCTCGTCATCTGGAACATTAAACACCTGCTCAATACCGGAATCGGCACCGCTTCGCGCCCCAGCGAAGTGTGCATGGTAGACGGGACGGACATGTGTTTGGCTGACTTCCACGCTGGCATTTTCCTGAAAGGACAGGAACACGCTGTGTTCGCCTGTGTCACCTCCAACGGGTGGTACGCGATTGATGACGAGGACTTTTACCCCTGGACACCGGACCCGTCCGACGTCCTGGTGTTTGTTCCGTACGATCAGGAGCCACTCAACGGAGAATGGAAATCAAAGGTTCAAAAACGACTCAGG). The amino acid sequence of the wild-type foot-and-mouth disease virus L protein is as shown in SEQ ID NO: 4 (MNTTDCFIALLYAFRQIKTLLLPRTQGKMELTLHNGEKKTFYSRPNNHDNCWLNTILQLFRYVDEPFFDWVYNSPENLTLDAIEQLEEITGLELREGGPPALVIWNIKHLLNTGIGTASRPSEVCMVDGTDMCLADFHAGIFLKGQEHAVFACVTSNGWYAIDDEDFYPWTPDPSDVLVFVPYDQEPLNGEWKSKVQKRLR).

[0046] In the present invention, the recombinant foot-and-mouth disease virus strain is not affected by the degradation of the L protein by the host protein POLR2H and virus replication. In the case of overexpressing the POLR2H protein in the host, its replication ability is not inhibited. It has good replication ability and production performance in cells such as BHK-21, and is a recombinant foot-and-mouth disease virus strain with high virus titer and antigen production capacity, which can be used for the preparation of vaccines. The nucleotide sequence of the coding sequence of the host protein POLR2H is as shown in SEQ ID NO: 1 (ATGGCGGGCATTCTGTTCGAGGATATTTTTGATGTGAAAGACATTGACCCAGAGGGCAAGAAGTTTGACCGAGTGTCTCGACTGCATTGTGAGAGTGAATCTTTCAAGATGGACCTCATCTTAGATGTAAACATTCAGATTTATCCTGTTGACCTGGGTGACAAGTTCCGGTTGGTGATAGCCAGTACTTTGTATGAAGATGGTACTCTGGATGATGGTGAATACAACCCCACAGATGATAGGCCTTCCAGGGCTGACCAATTTGAGTATGTAATGTATGGGAAAGTGTACAGGATTGAGGGAGATGAAACTTCTACTGAGGCAGCAACACGCCTCTCTGCCTATGTGTCCTATGGTGGCCTGCTCATGAGGCTGCAGGGTGATGCCAACAACCTGCATGGATTTGAAGTGGATTCCAGAGTTTATCTGCTGATGAAGAAACTGGCCTTCTGA). The amino acid sequence of the host protein POLR2H is as shown in SEQ ID NO: 2 (MAGILFEDIFDVKDIDPEGKKFDRVSRLHCESESFKMDLILDVNIQIYPVDLGDKFRLVIASTLYEDGTLDDGEYNPTDDRPSRADQFEYVMYGKVYRIEGDETSTEAATRLSAYVSYGGLLMRLQGDANNLHGFEVDSRVYLLMKKLAF).

[0047] In one embodiment of the present invention, overexpression of POLR2H in host cells such as PK-15 cells inhibits the viral protein abundance of FMDV in a dose-dependent manner, that is, overexpression of POLR2H in host cells inhibits the viral replication of FMDV. In another embodiment of the present invention, in order to further explore the mechanism by which the host protein POLR2H inhibits FMDV replication, the present invention finds that POLR2H decreases the expression level of the L protein of FMDV in a dose-dependent manner, which indicates that POLR2H inhibits FMDV replication by inhibiting the expression of the L protein; and the degradation of the L protein caused by POLR2H can be inhibited by MG132, indicating that POLR2H causes the degradation of the L protein through the proteasome pathway. In another embodiment of the present invention, in order to further clarify the key amino acid sites of POLR2H on the foot-and-mouth disease virus L protein, the present invention constructs and screens to identify that the 18th, 28th, and 39th positions are the key amino acid sites for POLR2H to degrade the L protein. At the same time, in another embodiment, it is proved that overexpression of POLR2H cannot affect the replication of the foot-and-mouth disease virus recombinant strain with mutated L protein, indicating that the 18th, 28th, and 39th positions in the L protein are the key amino acid sites for POLR2H to act on the foot-and-mouth disease virus strain.

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

[0049] Prepare a full-length infectious clone of foot-and-mouth disease virus containing a mutated L protein coding sequence; the mutation sites of the mutated L protein include the 18th, 28th, and 39th amino acids of the foot-and-mouth disease virus L protein;

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

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

[0052] In the present invention, the preparation of the full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence preferably includes the use of gene mutation technology or gene synthesis technology. The amino acid mutations at positions 18, 28, and 39 of the foot-and-mouth disease virus L protein are preferably arginine. In the embodiments of the present invention, the nucleotide sequence of the mutant L protein coding sequence is as shown in SEQ ID NO:5 (ATGAACACGACTGACTGTTTCATCGCTTTGCTGTACGCCTTCAGACAGAT CAGAACACTGCTTTTACCACGAACACAAGGAAGGATGGAACTCACACTTCACAACGGTGAAAAGAGGACATTCTACTCCAGGCCCAACAACCACGACAACTGCTGGCTGAACACCATCCTCCAGTTGTTTAGGTACGTTGACGAACCCTTCTTTGACTGGGTTTACAACTCGCCCGAGAACCTCACACTTGATGCTATTGAGCAATTGGAAGAAATTACAGGTCTTGAACTCCGCGAGGGCGGTCCACCCGCCCTCGTCATCTGGAACATTAAACACCTGCTCAATACCGGAATCGGCACCGCTTCGCGCCCCAGCGAAGTGTGCATGGTAGACGGGACGGACATGTGTTTGGCTGACTTCCACGCTGGCATTTTCCTGAAAGGACAGGAACACGCTGTGTTCGCCTGTGTCACCTCCAACGGGTGGTACGCGATTGATGACGAGGACTTTTACCCCTGGACACCGGACCCGTCCGACGTCCTGGTGTTTGTTCCGTACGATCAGGAGCCACTCAACGGAGAATGGAAATCAAAGGTTCAAAAACGACTCAGG). 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 by gene synthesis according to the coding sequence of the mutant L protein.

[0053] In the present invention, a method for preparing a full-length infectious clone of foot-and-mouth disease virus containing a coding sequence of a mutant L protein by using gene mutation technology is provided. Preferably, taking the full-length infectious clone of foot-and-mouth disease virus as a template, two pairs of homologous recombination mutant primers are used for PCR amplification to obtain two PCR amplification products; taking the product after homologous recombination of the two PCR amplification products as a template, another two pairs of homologous recombination mutant primers are used for PCR amplification, and the two obtained PCR amplification products and the linearized vector plasmid are subjected to homologous recombination to obtain a full-length infectious clone of foot-and-mouth disease virus containing the coding sequence of the mutant L protein. The full-length infectious clone of foot-and-mouth disease virus 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 the patents with publication numbers CN 107029231A, CN106916832A, and CN107041951A. The recombinant plasmid prO-FMDV is a reverse genetic operating system of a strain with a strong cellular immune response, and the rescued O-type foot-and-mouth disease recombinant vaccine strain rO-FMDV has cross-protective effects against different lineages of O-type FMDV. The four pairs of homologous recombination mutant primers preferably include KpnI-F / mL1828-R, mL1828-F / KpnI-R, KpnI-F / mL39-R, and mL39-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 mL1828-F is preferably as shown in SEQ ID NO:10; the nucleotide sequence of mL1828-R is preferably as shown in SEQ ID NO:11; the nucleotide sequence of mL39-F is preferably as shown in SEQ IDNO:12; the nucleotide sequence of mL39-R is as shown in SEQ ID NO:13.

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

[0055] In the present invention, the foot-and-mouth disease virus-sensitive cells preferably include BHK-21 cells and / or IBRS-2 cells. The present invention has no particular limitation on the transfection method, and the well-known transfection methods in the art can be used. After transfection, the virus is preferably harvested when about 90% of the cells show cytopathic effects. The morphology of the cytopathic effects preferably includes the cells becoming round, aggregating in a grape-like distribution, and finally the cells disintegrating. The present invention also preferably includes verifying gene mutations in the collected recombinant foot-and-mouth disease virus strains. The method for verifying gene mutations preferably includes amplifying the gene fragment containing L, purifying and recovering it, sending it for sequencing, and comparing the sequencing results with the mutant L protein coding sequence. If they are consistent, it indicates that the recombinant foot-and-mouth disease virus strain is obtained.

[0056] The present invention measures the biological characteristics of the recombinant foot-and-mouth disease virus strain obtained by the above construction method. The results show that the mutation of the key amino acid sites of the L protein eliminates the inhibitory effect of POLR2H on the replication of FMDV, and the mutant sites can stably exist without affecting the replication of the foot-and-mouth disease virus.

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

[0058] In the present invention, the foot-and-mouth disease recombinant vaccine preferably further includes an adjuvant. The present invention has no particular limitation on the type of adjuvant, and the well-known adjuvant types in the art can be used, such as ISA206 and ISA201 adjuvants. The present invention has no particular limitation on the antigen content of the recombinant foot-and-mouth disease virus strain in the vaccine, and the well-known content in the art can be used. The present invention has no particular limitation on the preparation method of the vaccine, and the well-known inactivated vaccine preparation methods in the art can be used.

[0059] The present invention provides the use 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 a foot-and-mouth disease recombinant vaccine.

[0060] The present invention provides a method for preparing a recombinant foot-and-mouth disease virus strain that is not inhibited by the host protein POLR2H during replication, including the following steps: mutating or deleting the 18th, 28th, and 39th amino acids of the L protein in the foot-and-mouth disease virus strain.

[0061] In the present invention, the parental foot-and-mouth disease virus strains of the recombinant foot-and-mouth disease virus strains include at least one of the following: foot-and-mouth disease virus wild strains, foot-and-mouth disease virus recombinant strains, and foot-and-mouth disease recombinant vaccine strains. In the embodiments of the present invention, the parental foot-and-mouth disease virus strains of the recombinant foot-and-mouth disease virus strains preferably include foot-and-mouth disease virus serotype O strains or recombinant foot-and-mouth disease virus serotype O strains. The method of mutation is preferably to mutate the 18th, 28th, and 39th amino acids of the L protein in the foot-and-mouth disease virus strain into arginine. The amino acid sequence of the mutated L protein is further preferably as shown in SEQ ID NO: 6 or SEQ ID NO: 7. The specific method is the same as the above technical solution and will not be elaborated here.

[0062] The present invention provides the application of the foot-and-mouth disease recombinant vaccine in the preparation of a medicament for preventing and / or controlling foot-and-mouth disease in animals;

[0063] In the present invention, the animals preferably include cloven-hoofed animals. The cloven-hoofed animals preferably include at least one of the following: pigs, cattle, and sheep.

[0064] The following examples are used to elaborate in detail on a recombinant foot-and-mouth disease virus strain with mutated L protein provided by the present invention, its construction method, and application, but they should not be construed as limiting the protection scope of the present invention.

[0065] The experimental methods in the embodiments of the present invention are all conventional methods unless otherwise specified; the experimental materials used in the embodiments are all obtained by purchasing from conventional biochemical reagent companies unless otherwise specified.

[0066] In the embodiments of the present invention, the relevant experiments obtained biosafety permits and foot-and-mouth disease laboratory activity permits: According to the relevant requirements of biosafety level 3 laboratories (BSL-3) and foot-and-mouth disease-related biosafety, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences reported step by step to the Biosafety Committee of the Lanzhou Veterinary Research Institute, the Biosafety Committee of the Chinese Academy of Agricultural Sciences, and the Laboratory Animal Ethics Committee of the Lanzhou Veterinary Research Institute, and obtained the permission from the Ministry of Agriculture and Rural Affairs to conduct research on highly pathogenic FMDV pathogens and animals, and has been filed with the Ministry of Agriculture and Rural Affairs, meeting the requirements of the national biosafety level.

[0067] The FMDV (O / BY / CHA / 2010 strain, O / GD / CHA / 2015 strain) used in the present invention is preserved by the National Foot-and-Mouth Disease Reference Laboratory designated by the Ministry of Agriculture and Rural Affairs, and the public can obtain it through the letter of entrustment approved by the Bureau of Animal Husbandry and Veterinary Medicine of the Ministry of Agriculture and Rural Affairs; the FMDV O / BY / CHA / 2010 strain 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 virus 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-Mouth Disease Virus. Zixiang Zhu, Fan Yang, Weijun Cao et al. Journal of Virology. Pub Date: 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.

[0068] Example 1

[0069] Effect of overexpressing POLR2H on FMDV replication

[0070] 1. Construction of POLR2H eukaryotic expression plasmid

[0071] Query the POLR2H gene sequence in the NCBI database and design primers, POLR2H-NheI-F: CGTC TA GCTAGC ATGGCGGGCATTCTGTTCGAGGATAT (SEQ ID NO: 14) (the underlined part is the NheI restriction site); POLR2H-BamHI-R: CGC GGATCCGAAGGCCAGTTTCTTCATCAGCAG(SEQ ID NO: 15) (The underlined part is the BamHI restriction site). Extract the RNA of PK-15 cells, reverse transcribe it into cDNA, and use it as a template to amplify the POLR2H gene (the nucleotide sequence is as shown in SEQ ID NO: 1). Recover the amplified fragment by nucleic acid electrophoresis, perform double digestion with NheI and BamHI restriction endonucleases, and at the same time perform double digestion on the pcDNA3.1 / myc vector plasmid with the same restriction endonucleases. Purify and recover the POLR2H gene and the linearized vector fragment respectively, ligate them overnight at 4°C with T4 ligase, transform DH5α competent cells, extract the plasmid and sequence it. Name the successfully constructed plasmid POLR 2H-Myc.

[0072] 2. Effect of overexpressing POLR2H on FMDV replication

[0073] Seed PK-15 cells into 6-well plates and culture them in a 37°C, 5% CO 2 cell incubator until the cell density reaches about 70%. Transfect different doses (0 μg, 1 μg, 3 μg) of the POLR2H eukaryotic expression plasmid POLR2H-Myc. After 24 hours, infect with FMDV (O / BY / CHA / 2010 strain) and perform Western-blot analysis.

[0074] The results are shown in Figure 1 . The results show that overexpressing POLR2H in PK-15 cells inhibits the viral protein abundance of FMDV in a dose-dependent manner.

[0075] Example 2

[0076] Effect of POLR2H on FMDV L protein expression

[0077] 1. Construction method of Flag-L plasmid

[0078] The O / GD / CHA / 2015 strain used is preserved by the National Foot-and-Mouth Disease Reference Laboratory designated by the Bureau of Animal Husbandry and Veterinary Medicine of the Ministry of Agriculture and Rural Affairs. The public can obtain it through the commission letter approved by the Bureau of Animal Husbandry and Veterinary Medicine of the Ministry of Agriculture and Rural Affairs.

[0079] Query the FMDV L gene sequence in the NCBI database and design primers. L-EcoRI-F: CGC GAATTC AATGAACACGACTGACTGTTTCATC(SEQ ID NO: 16, the underlined part is the EcoRI restriction site); L-BamHI-R: CGC GG ATCCTTACCTGAGTCGTTTTTGAACCTTTG (SEQ ID NO:17, the underlined part is the BamHI restriction site).

[0080] Extract the RNA of O / GD / CHA / 2015 strain, reverse transcribe to synthesize cDNA, use the cDNA as a template to amplify the L gene (the nucleotide sequence is as shown in SEQ ID NO: 3, and the amino acid sequence is as shown in SEQ ID NO: 4), recover the amplified fragment by nucleic acid electrophoresis, perform double digestion with EcoRI and BamHI restriction endonucleases, and at the same time perform double digestion on the p3×FLAG-CMV-7.1 vector plasmid with the same restriction endonucleases. Purify and recover the L gene and the linearized vector fragment respectively, ligate overnight at 4°C with T4 ligase, transform DH5α competent cells, pick monoclonal colonies, shake the bacteria, extract the plasmid and then sequence. Name the successfully constructed plasmid as L-Flag.

[0081] 2. Effect of POLR2H on the expression of FMDV L protein

[0082] Inoculate HEK293T cells into 6-well plates, and culture them in a cell incubator at 37°C and 5% CO 2 until the cell density reaches about 70%. Transfect the eukaryotic expression plasmid L-Flag of FMDV L protein and different doses of the eukaryotic expression plasmid POLR2H-Myc of POLR2H, and perform Western-blot analysis after collecting the samples.

[0083] The results are shown in Figure 2 . The results show that POLR2H decreases the expression level of FMDV L protein in a dose-dependent manner, indicating that POLR2H inhibits FMDV replication by inhibiting the expression of L protein.

[0084] 3. POLR2H causes the degradation of L protein through the proteasome pathway

[0085] Co-transfect the eukaryotic expression plasmid L-Flag of L protein and the eukaryotic expression plasmid POLR2H-Myc of POLR2H in HEK293T cells. After 16 hours of transfection, add 20 μM of the proteasome inhibitor MG132, 20 μM of the lysosome inhibitor NH 4 Cl and 20 μM of the apoptosis inhibitor Z-VAD-FMK respectively, continue to culture for 8 hours, collect the cells, and perform Western-blot analysis.

[0086] The results are as shown in Figure 3 . The degradation of L protein caused by POLR2H is inhibited by MG132, indicating that POLR2H causes the degradation of L protein through the proteasome pathway.

[0087] Example 3

[0088] Construction method of eukaryotic expression plasmid of key amino acid site mutants of L protein

[0089] On the basis of Example 1, the L protein of FMDV O / GD / CHA / 2015 strain was mutated to construct a eukaryotic expression plasmid of L protein mutants. By using the method described in this example, eukaryotic expression plasmids of L protein mutants of other foot-and-mouth disease virus strains can also be constructed. The specific process is as follows:

[0090] Based on the eukaryotic expression plasmid L-Flag of the wild-type FMDV L protein coding sequence (nucleotide sequence shown in SEQ ID NO: 3), the coding sequences of the 18th, 28th, and 39th amino acids (lysine) of the L protein were all mutated to arginine coding sequences by using gene synthesis technology or site-directed mutagenesis technology. The product containing the mutated L protein coding sequence (nucleotide sequence shown in SEQ ID NO: 5) was transformed into DH5α competent cells, single colonies were picked, cultured in a shaker, and the plasmid was extracted and sequenced. After confirming that the mutations at the 18th, 28th, and 39th amino acid sites of the L protein were successful, the key amino acid mutant plasmid was named L-Flag-(18+28+39)R.

[0091] Example 4

[0092] Construction and identification of recombinant strains of foot-and-mouth disease virus type O with L protein mutations

[0093] 1. Construction of recombinant foot-and-mouth disease virus type O infectious clone

[0094] The large vector fragment was recovered after digesting the gene fragment containing L in the recombinant plasmid prO-FMDV containing the L gene, P1 gene and P2 gene of the O / JSCZ / 2013 strain with KpnI. Using this plasmid as a template, a gene fragment with key site mutations in the L gene was amplified by PCR. The homologous recombination mutant primers were: KpnI-F (5'-TAAGGATGCCCTTCAGGTACCCTGAGGTAACACGCGACACTCG-3', SEQ ID NO:8); mL1828-R (5'-GTGCTCGTGATAAGAACAGTGTTCTAATCTCTCTGAAAGCGTACAACAAAG-3', SEQ ID NO:11); mL1828-F (5'-GTTCTTATCACGAG CACAAGGAAGGATGGAGTTCACACTTCACAACGG-3', SEQ ID NO:10); KpnI-R (5'-ATGGAACAAAGTTCAGGTACCATGGCCACCAGTAGGCAGC-3', SEQ ID NO:9). The PCR amplification products were recovered respectively, subjected to homologous recombination, and amplified using them as templates. The primers were KpnI-F and mL39-R (5'-ATGTTCTCTTCTCACCGTTGTGAAGTGTGAAC-3', SEQ ID NO:13); mL39-F (5'-CGGTGAGAAGAGAACATTCTACTCCAGGC CCAACA-3', SEQ ID NO:12) and KpnI-R. The PCR amplification products were recovered respectively, and subjected to homologous recombination with the recovered vector fragment to obtain a recombinant plasmid in which lysine at positions 18, 28 and 39 of the L gene was mutated to arginine. The amplification was carried out using the high-fidelity HSDNA polymerase of TaKaRa Company. A 50 μL reaction system was prepared according to the product instruction manual. The amplification conditions were: 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, 30 cycles, 4°C for 5 min. For homologous recombination, Novoprotein Ultra One Step Cloning Kit was used at 50°C for 15 min. DH5α competent cells were transformed, single colony clones were picked, cultured in liquid medium to extract plasmids, and positive clones were identified by sequencing. Finally, a recombinant plasmid prO-mL(18 + 28 + 39)-FMDV containing the L gene mutation was obtained. The construction schematic diagram is as Figure 4 shown.

[0095] 2. Rescue of recombinant virus

[0096] The recombinant plasmid prO-mL(18 + 28 + 39)-FMDV obtained in step 1 was prepared. According to Polyplus Instructions for transfection reagent: When BHK-21 cells grow to about 70%, transfect the recombinant plasmid into BHK-21 cells. At the same time, set up normal cell control and transfection reagent control, and place them in an incubator at 37°C and 5% CO 2 2. Incubate in an incubator, observe the cell state, harvest the virus when about 90% of the cells show cytopathic effect, freeze-thaw 3 times repeatedly, and then inoculate BHK-21 cells again until the virus can stably produce cytopathic effect, that is, the cells become round, aggregate in a grape-like distribution, and finally the cells disintegrate. Name the obtained recombinant foot-and-mouth disease virus type O as rO-mL(18+28+39)-FMDV.

[0097] The results are as Figure 5 shown. Compared with normal control BHK-21 cells, the recombinant virus strain rO-mL(18+28+39)-FMDV infects BHK-21 cells and causes cytopathic effect.

[0098] 3. Identification of recombinant virus by RT-PCR

[0099] Extract total RNA from the supernatant of BHK-21 cells infected with the stably passaged recombinant virus rO-mL(18+28+39)-FMDV using Trizol. After reverse transcription, amplify the gene fragment containing L using KpnI-F (SEQ ID NO: 8) and KpnI-R (SEQ ID NO: 9), and send it for sequencing after purification and recovery.

[0100] The results show that the L gene of the obtained recombinant foot-and-mouth disease virus type O is consistent with the theoretical sequence, and its amino acids at positions 18, 28, and 39 are arginine. The amino acids encoded by the mutated L gene are the amino acid sequence shown in SEQ ID NO: 7.

[0101] Example 5

[0102] Effect of POLR2H protein on the replication of foot-and-mouth disease virus recombinant strains with key amino acid site mutations in L protein

[0103] 1. Effect of POLR2H on the expression of L protein with key site mutations

[0104] Seed HEK293T cells into 6-well plates and culture them in a cell incubator at 37°C and 5% CO 2 until the cell density is about 70%. Co-transfect the eukaryotic expression plasmid L-Flag-(18+28+39)R of the FMDV L protein key site mutant and the eukaryotic expression plasmid POLR2H-Myc of POLR2H at different doses (0, 0.5 μg, 1 μg, 2 μg). After sampling, perform Western-blot analysis. The results show that overexpression of POLR2H does not affect the expression level of the mutant L protein ( Figure 6 ).

[0105] 2. Influence of POLR2H on the Replication of Recombinant Foot-and-Mouth Disease Virus Strains with Key Site Mutations in the L Protein

[0106] Inoculate PK-15 cells into 6-well plates and culture them in a 37°C, 5% CO 2 cell incubator until the cell density reaches about 70%. Transfect different doses of POLR2H eukaryotic expression plasmids (0 μg, 1 μg, 3 μg). After 24 h, infect with wild-type and recombinant foot-and-mouth disease virus rO-mL(18 + 28 + 39)-FMDV with key amino acid site mutations in the L protein, and perform Western-blot analysis.

[0107] The results showed that overexpression of POLR2H in PK-15 cells had no effect on the viral protein abundance of recombinant virus rO-mL(18 + 28 + 39)-FMDV, but inhibited the viral protein abundance of wild-type FMDV( Figure 7 ), indicating that overexpression of POLR2H inhibits the replication of wild-type FMDV in a dose-dependent manner, but cannot inhibit the replication of recombinant virus rO-mL(18 + 28 + 39)-FMDV with L protein mutations. Therefore, this recombinant foot-and-mouth disease virus strain can be used as a seed virus for foot-and-mouth disease virus vaccines to increase virus titer and antigen content and improve production performance.

[0108] Example 6

[0109] Pathogenicity Test of Recombinant Foot-and-Mouth Disease Virus Strains on BHK-21 Cells

[0110] Infect BHK-21 cells with recombinant FMDV with key site mutations in the L protein. Collect the virus solution after 8 h, freeze-thaw it 3 times repeatedly, and serially dilute the sample 10-fold with DMEM medium. Add virus solutions of each dilution (10 -4.0 -10 -9.0 ) to the culture plates, with 4 wells for each dilution, and culture in a 37°C, 5% CO 2 incubator for 3 days. Observe the cytopathic effect and calculate the TCID 50 of the virus according to the Reed-Muench method (refer to the 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").

[0111] Calculate the TCID 50 of the recombinant strain to be 10 -8.5 / mL, indicating that the recombinant FMDV with mutations at key amino acid sites of the L protein has the characteristic of high virus titer.

[0112] In summary, the results show that the replication level of the recombinant FMDV strain with mutations at key amino acid sites of the L protein in cells is not inhibited by the host protein POLR2H, and the mutation at this key site improves the virus titer and antigen production of FMDV, which can be used as a candidate strain for FMDV vaccines.

[0113] The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant strain of foot-and-mouth disease virus with L protein mutation, characterized in that: The L protein is a mutant L protein in which the 18th, 28th and 39th amino acids are mutated or deleted.

2. The recombinant strain of foot-and-mouth disease virus according to claim 1, characterized in that: The parent foot-and-mouth disease virus strain of the foot-and-mouth disease virus recombinant strain includes at least one of the following: a wild strain of foot-and-mouth disease virus, a recombinant strain of foot-and-mouth disease virus and a recombinant vaccine strain of foot-and-mouth disease virus.

3. The recombinant foot-and-mouth disease virus strain according to claim 1 or 2, characterized in that: The amino acids 18, 28 and 39 of the mutant L protein are mutated L proteins; Preferably, the amino acids at positions 18, 28 and 39 in the mutant L protein are mutated to arginine; Further preferably, the amino acid sequence of the mutant L protein is as shown in SEQ ID NO:6 or SEQ ID NO:

7.

4. A method for constructing a recombinant strain of foot-and-mouth disease virus according to any one of claims 1 to 3, characterized in that: The following steps are involved: 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 the 18th, 28th and 39th amino acids of the foot-and-mouth disease virus L protein; The full-length infectious clone of foot-and-mouth disease virus containing the coding sequence of mutant L protein is transfected into foot-and-mouth disease virus sensitive cells to obtain the recombinant strain of foot-and-mouth disease virus.

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 by using gene mutation technology or gene synthesis technology; Preferably, the amino acids at positions 18, 28 and 39 of the foot-and-mouth disease virus L protein are mutated to arginine; Preferably, the method of preparing a full-length infectious clone of foot-and-mouth disease virus containing a mutant L protein coding sequence using gene mutation technology comprises 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 mutation primers to obtain two PCR amplification products; The products of homologous recombination of the two PCR amplification products are used as templates, and PCR amplification is performed using another two pairs of homologous recombination mutation primers. The two PCR amplification products obtained are homologously recombined with the linearized vector plasmid to obtain a full-length infectious clone of foot-and-mouth disease virus containing the coding sequence of the mutant L protein; Preferably, the four pairs of homologous recombination mutation primers include KpnI-F / mL1828-R, mL1828-F / KpnI-R, KpnI-F / mL39-R and mL39-F / KpnI-R; The nucleotide sequence of KpnI-F is shown in SEQ ID NO:8; The nucleotide sequence of KpnI-R is shown in SEQ ID NO:9; The nucleotide sequence of mL1828-F is shown in SEQ ID NO: 10; The nucleotide sequence of mL1828-R is shown in SEQ ID NO: 11; The nucleotide sequence of mL39-F is shown in SEQ ID NO: 12; The nucleotide sequence of mL39-R is shown in SEQ ID NO:

13.

6. The construction method according to claim 5, characterized in that: The foot-and-mouth disease virus-sensitive cells include BHK-21 cells and / or IBRS-2 cells.

7. A recombinant foot-and-mouth disease vaccine, characterized in that: The invention comprises 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 6.

8. 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 6 in the preparation of a recombinant foot-and-mouth disease vaccine.

9. A method for preparing a recombinant strain of foot-and-mouth disease virus that is free from replication inhibition by host protein POLR2H, characterized in that: The following steps are involved: The 18th, 28th and 39th amino acids of the L protein in the foot-and-mouth disease virus strain are mutated or deleted; Preferably, the mutation method is to mutate the amino acids at positions 18, 28 and 39 of the L protein in the foot-and-mouth disease virus strain to arginine; More preferably, the amino acid sequence of the mutated L protein is as shown in SEQ ID NO: 6 or SEQ ID NO:

7.

10. Use of the foot-and-mouth disease recombinant vaccine according to claim 7 in the preparation of a medicament for preventing and / or controlling animal foot-and-mouth disease; Preferably, the animal comprises an even-toed ungulate; Further preferably, the even-toed ungulate comprises at least one of the following: pigs, cattle and sheep.

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