Recombinant foot-and-mouth disease virus strain with high antigen yield as well as construction method and application of recombinant foot-and-mouth disease virus strain

Abstract

The invention provides a recombinant foot-and-mouth disease virus strain with high antigen yield as well as a construction method and application thereof, and belongs to the technical field of biology. The invention relates to a recombinant foot-and-mouth disease virus strain, wherein L protein is mutant L protein with 108th amino acid mutated or deleted. According to the invention, overexpression of host protein RAB20 inhibits replication of FMDV, and L protein is degraded through a proteasome pathway, so that virus replication is inhibited; a recombinant foot-and-mouth disease virus strain which is not inhibited by host protein RAB20 to replicate is constructed by mutating a key amino acid site (108) in FMDV L protein, 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 invention belongs to the technical field of animal husbandry and veterinary medicine, and in particular relates to a recombinant foot-and-mouth disease virus strain with high antigen yield, a construction method and application thereof. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease of even-toed ungulates caused by foot-and-mouth disease virus (FMDV). The disease has a huge economic impact on animal husbandry.

[0003] FMDV belongs to the Picornaviridae family and the genus Foot-and-Mouth Disease Virus. 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 the nonstructural protein L pro , 4 structural proteins VP4, VP2, VP3 and VP1, and 2A, 2B, 2C, 3A, 3B, 3C pro and 3D pol Nonstructural proteins. Among them, the leader protein L pro It is a papain that can cut itself off from polyproteins and cleave the host cell translation initiation factor eIF4G, inhibiting host protein synthesis and promoting viral protein translation; L protein is also a deubiquitinating protease that inhibits the type I interferon signaling pathway by inhibiting the ubiquitination of RIG-I, TBKI, TRAF6 and TRAF3; L protein is also an important virulence factor of FMDV. Its second translation initiation codon has a high utilization frequency. After deletion, the replication ability of the recombinant virus decreases, and the pathogenicity to the host animal is significantly reduced; therefore, the L protein of FMDV plays an important role in the regulation of viral replication, virulence and host cell response. A safer and more effective FMDV vaccine can be developed by deleting or mutating the L protein. In addition, there are also complex interactions between host proteins and FMDV proteins, which regulate viral replication through different mechanisms, thereby affecting the replication performance of FMDV in the host, affecting its stable propagation, and then affecting its application in the preparation of FMDV vaccines. 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 production, by mutating the key amino acid sites of the L protein to eliminate the ability of RAB20 to inhibit FMDV replication, thereby increasing the virus titer and antigen production.

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

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

[0007] Preferably, the amino acid at position 108 of the L protein is a mutant L protein with a mutation;

[0008] The mutant L protein includes a mutation of amino acid 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] 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 a mutant L protein coding sequence; the mutation site of the mutant L protein includes the 108th amino acid of the foot-and-mouth disease virus L protein;

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

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

[0015] 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;

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

[0017] Preferably, the two pairs of homologous recombination mutation 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 KpnI-R is shown in SEQ ID NO:9;

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

[0021] The nucleotide sequence of 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 invention provides a recombinant foot-and-mouth disease vaccine strain, comprising the recombinant foot-and-mouth disease virus strain or the recombinant foot-and-mouth disease virus strain prepared by the construction method.

[0024] The 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] 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 preparing a recombinant foot-and-mouth disease vaccine.

[0026] The present invention provides a method for preparing a recombinant foot-and-mouth disease virus strain that is free from the host protein RAB20 inhibition of replication, comprising the following steps:

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

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

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

[0030] The present invention provides the use of the recombinant foot-and-mouth disease vaccine in the preparation of a drug for preventing and / or controlling animal foot-and-mouth disease;

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

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

[0033] The present invention provides a recombinant foot-and-mouth disease virus strain, whose L protein is a mutant L protein with a mutation or deletion in the 108th amino acid. The present invention first studies and finds that overexpression of RAB20 in host cells inhibits the replication of FMDV, and further determines that the RAB20 protein degrades the L protein through the 108th amino acid site, thereby inhibiting viral replication. By performing site-directed mutagenesis on the 108th amino acid in the L protein, the degradation ability of the RAB20 protein on the L protein is eliminated, and the inhibitory effect of the RAB20 protein on the replication of FMDV is eliminated. The present invention successfully constructs and rescues a recombinant foot-and-mouth disease virus strain by designing and modifying the 108th amino acid of the L protein, which can be stably propagated, does not affect the replication of the foot-and-mouth disease virus, improves the virus titer, has good production performance, and can be used to prepare a recombinant foot-and-mouth disease vaccine.

[0034] The present invention also provides a method for constructing a recombinant foot-and-mouth disease virus strain. The present invention utilizes reverse genetics technology to transfect foot-and-mouth disease virus sensitive cells with a full-length infectious clone containing a coding sequence in which the amino acid at position 108 of the L protein is mutated or deleted, and 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. The use of this method to obtain a recombinant foot-and-mouth disease vaccine strain is of great significance for improving the quality and efficacy of the vaccine, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 To investigate the effect of overexpression of RAB20 protein on FMDV replication;

[0036] Figure 2 The results show the effects of different doses of RAB20 protein on the expression of FMDV L protein;

[0037] Figure 3 The result is that RAB20 protein degrades FMDV L protein through the ubiquitin proteasome pathway;

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

[0039] Figure 5 The rescue results of recombinant foot-and-mouth disease virus with mutations in key amino acid sites of L protein;

[0040] Figure 6 The results show the effect of RAB20 protein on the expression of L protein with 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 in key amino acid sites of L protein;

[0042] Figure 8 These are the results of the toxicity test of the recombinant foot-and-mouth disease virus with mutations at the key amino acid sites of the L protein. DETAILED DESCRIPTION

[0043] The invention provides a recombinant foot-and-mouth disease virus strain, wherein the L protein of the recombinant foot-and-mouth disease virus strain is a mutant L protein in which the 108th amino acid is mutated or deleted.

[0044] In the present invention, the parent foot-and-mouth disease virus strain of the recombinant foot-and-mouth disease virus strain includes at least one of the following: a wild strain of foot-and-mouth disease virus, 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 parent 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 at position 108 of the mutant L protein is preferably mutated. The amino acid at position 108 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 the present invention, the recombinant foot-and-mouth disease virus strain is not affected by the host protein RAB20 on L protein degradation and virus replication. When the RAB20 protein is overexpressed 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 vaccine preparation. The nucleotide sequence of the coding sequence of 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 (MRKPDGKIVLLGDMNVGKTSLLQRYMERRFPDTVSTVGGAFYLKQWRSYNISIWDTAGREQFHGLGSMYCRGAAAVILTYDVNHAQSLLELEDRFLGLTDTASADCLFAIVGNKVDLSEEGPGDGGQDGGHGPGPAGVGAAPRLPKQVQPEDAVALYKKILKYKMLDEKDVPAAEQMCFETSAKTGHNVDLLFETLFDMVVPVILRQRAQGPPQTVDIAPCGPPARTRSGCCA).

[0046] In the present invention, the amino acid sequence of the wild-type FMDV L protein is as 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 as 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 viral proteins of FMDV in a dose-dependent manner, that is, overexpression of RAB20 protein in host cells inhibits viral replication of FMDV. In another embodiment of the present invention, in order to further explore the mechanism by which the host protein RAB20 inhibits FMDV replication, the present invention found that RAB20 decreased the expression of L protein of FMDV in a dose-dependent manner, indicating that RAB20 inhibits FMDV replication by inhibiting the expression of L protein; and the degradation of L protein caused 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 of RAB20 degradation of L protein, the present invention screened and identified that the 108th amino acid (K108) is the key amino acid site of RAB20 degradation of L protein. At the same time, in another embodiment, it was proved that RAB20 overexpression could not affect the replication of the recombinant foot-and-mouth disease virus strain with the L protein K108R mutation, indicating that the 108th amino acid of the L protein is the key amino acid site of RAB20 inhibiting the replication of foot-and-mouth disease virus.

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

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

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

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

[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 preparation by 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 by gene synthesis. In an embodiment of the present invention, the nucleotide sequence of the mutant L protein coding sequence is shown in SEQ ID NO:5 (ATGAACACGACTGACTGTTTCATCGCTTTGCTGTACGCCTTCAGACAGAT).

[0053] In the present invention, 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 is preferably performed using the full-length infectious clone of foot-and-mouth disease virus as a template, using two pairs of homologous recombination mutation primers for PCR amplification to obtain two PCR amplification products; it is homologously recombined 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. The full-length infectious clone of foot-and-mouth disease virus preferably includes a 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 with a strong cellular immune response strain, and the rescued O-type recombinant foot-and-mouth disease vaccine strain rO-FMDV has a cross-protective effect on different lineages of O-type FMDV. The two pairs of homologous recombination mutation primers preferably include KpnI-F / mL108-R and mL108-F / KpnI-R; the nucleotide sequence of the KpnI-F is preferably as shown in SEQ ID NO:8; the nucleotide sequence of the 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; 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 the 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 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 special restrictions on the transfection method, and a transfection method well known in the art can be used. After transfection, the virus is preferably harvested when the cells show about 90% cytopathic effects. The morphology of the cytopathic effects preferably includes cells becoming rounded, aggregating into a grape-like distribution, and eventually cell disintegration. The present invention also preferably includes verifying the gene mutation of the collected recombinant foot-and-mouth disease virus strain. The gene mutation verification method preferably includes amplifying a gene fragment containing L, purifying and recovering it, and sending it for sequencing, comparing the sequencing results with the mutant L protein coding sequence, and if they are consistent, it means that a recombinant foot-and-mouth disease virus strain has been obtained.

[0056] The present invention performs toxicity titer determination on the recombinant foot-and-mouth disease virus strain obtained by the above-mentioned construction method, and the results show that compared with the control FMDV, the virus titer of the recombinant FMDV with mutations in the key amino acid sites of the L protein is significantly increased, indicating that the mutations in the key amino acid sites of the L protein can eliminate the inhibitory effect of RAB20 on FMDV replication, increase the virus titer and antigen yield of FMDV, and enhance the virus production performance.

[0057] In view of the fact that the recombinant foot-and-mouth disease virus strain has a higher virus titer, excellent production performance and higher antigen yield, the present invention provides a recombinant foot-and-mouth disease 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 recombinant foot-and-mouth disease vaccine preferably further includes an adjuvant. The present invention does not impose any special restrictions on the type of the adjuvant, and any adjuvant known in the art can be used, such as ISA206 and ISA201 adjuvants. The present invention does not impose any special restrictions on the antigen content of the recombinant foot-and-mouth disease vaccine strain in the vaccine, and any content known in the art can be used. The present invention does not impose any special restrictions on the preparation method of the vaccine, and any inactivated vaccine preparation method known 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 preparing a recombinant foot-and-mouth disease vaccine.

[0060] The present invention provides a method for preparing a recombinant foot-and-mouth disease virus strain that is free from host protein RAB20 replication inhibition, comprising the following steps:

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

[0062] In the present invention, the mutation method is preferably to mutate the amino acid at position 108 of the L protein in the foot-and-mouth disease virus strain to arginine;

[0063] In the present invention, the parent foot-and-mouth disease virus strain of the recombinant foot-and-mouth disease virus strain includes at least one of the following: a wild strain of foot-and-mouth disease virus, 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 parent 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 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 by gene mutation technology or gene synthesis technology. The specific method is the same as described in the above technical scheme and will not be repeated here. In an embodiment of the present 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] The invention provides the use of the recombinant foot-and-mouth disease vaccine in preparing medicine for preventing and / or controlling animal foot-and-mouth disease.

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

[0066] The following is a detailed description of a recombinant foot-and-mouth disease virus strain with high antigen production provided by the present invention, its construction method and application, in conjunction with the examples. However, they should not be construed as limiting the scope of protection of the present invention.

[0067] The experimental methods in the examples of the present invention are all conventional methods unless otherwise specified; the experimental materials used in the examples are all purchased from conventional biochemical reagent companies unless otherwise specified.

[0068] The relevant experiments described in the embodiments of the present invention obtained biosafety licenses and foot-and-mouth disease laboratory activity licenses: The Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, in accordance with the relevant requirements of biosafety level 3 laboratories (BSL-3) and foot-and-mouth disease-related biosafety, has been reported step by step by 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, and obtained the Ministry of Agriculture and Rural Affairs’ permission to conduct research on highly pathogenic FMDV pathogens and animals. It has been filed with the Ministry of Agriculture and Rural Affairs and meets the national biosafety level requirements.

[0069] 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 a letter of entrustment approved by the Veterinary Bureau 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.

[0070] Example 1

[0071] Effect of overexpression of RAB20 on FMDV replication

[0072] 1. Construction of RAB20 eukaryotic expression plasmid

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

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

[0075] PK-15 cells were seeded into 6-well plates and incubated at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator until the cell density reached about 70%, and transfected with different doses of RAB20 eukaryotic expression plasmid RAB20-Myc (0 μg, 1 μg, 3 μg). After 24 hours, the cells were infected with FMDV (O / BY / CHA / 2010 strain) and analyzed by Western-blot.

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

[0077] Example 2

[0078] Effect of RAB20 on the expression of FMDV L protein

[0079] 1. Construction of Flag-L plasmid

[0080] The FMDV L gene sequence was queried in the NCBI database and specific primers were designed. The specific sequence is 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 (the underline indicates the BamHI restriction site, SEQ ID NO: 15).

[0083] RNA of O / GD / CHA / 2015 strain was extracted and synthesized into cDNA by reverse transcription. L gene was amplified using cDNA as template (nucleotide sequence is shown in SEQ ID NO: 3, and amino acid sequence is shown in SEQ ID NO: 4). Amplified fragment was recovered by nucleic acid electrophoresis and double-digested with EcoRI and BamHI restriction endonucleases. At the same time, p3×FLAG-CMV-7.1 vector plasmid was double-digested with the same restriction endonucleases, and L gene and linearized vector fragment were purified and recovered respectively. The fragments were connected at 4°C overnight with T4 ligase and transformed into DH5α competent cells. Single clones were picked, shaken, plasmids were extracted and sequenced, and 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 incubated at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator until the cell density reached about 70%, and transfected with the FMDV L protein eukaryotic expression plasmid L-Flag and different doses of the RAB20 eukaryotic expression plasmid RAB20-Myc. The samples were collected and analyzed by Western-blot.

[0086] Results Figure 2 The results showed that RAB20 decreased the expression of FMDV L protein in a dose-dependent manner, indicating that RAB20 inhibited the expression of L protein and thus inhibited the replication of FMDV.

[0087] 3. RAB20 protein degrades FMDVL protein through 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 transfection for 16 h, 20 μM proteasome inhibitor MG132 and 20 μM lysosomal inhibitor NH 4 Cl and 20 μM apoptosis inhibitor Z-VAD-FMK, and after culturing for 8 h, the cells were harvested and analyzed by Western-blot.

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

[0090] Example 3

[0091] Method for constructing eukaryotic expression plasmid of mutants at key amino acid sites of L protein

[0092] 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 the mutant L protein. The method described in this example can also be used to construct a eukaryotic expression plasmid of the mutant L protein of other foot-and-mouth disease virus strains. The specific process is as follows:

[0093] On the basis of the wild-type FMDV L protein eukaryotic expression plasmid L-Flag (the nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4), the 108th amino acid (lysine) of the L protein is mutated to the coding sequence of arginine (the nucleotide sequence is shown in SEQ ID NO: 5) by using gene synthesis technology or point mutation PCR technology, and DH5α competent cells are transformed, a single clone is picked, the bacteria are shaken, the plasmid is extracted and sequenced, and it is confirmed that the 108th amino acid site of the L protein is successfully mutated, and the key amino acid mutation plasmid is named L-Flag-K108R.

[0094] Example 4

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

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

[0097] 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 was digested with KpnI to recover the large vector fragment, and the gene fragment containing the mutation of the key site of the L gene was amplified by PCR using the plasmid as a template. 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: 9), respectively recover the PCR amplification products, and perform homologous recombination with the recovered vector fragment to obtain a recombinant plasmid in which the lysine at position 108 of the L protein is mutated to arginine. HSDNA polymerase was used to prepare 50 μL reaction system according to the product manual. The amplification conditions were: 98°C for 10 s, 55°C for 15 s, 72°C for 1 min, 30 cycles, and 4°C for 5 min. Homologous recombination was performed using Novazon Ultra One Step Cloning Kit, 50℃ for 15min, transform DH5α competent cells, pick single clone colonies, shake to extract plasmids, and sequence to identify positive clones. Finally, the recombinant plasmid prO-mL-K108R-FMDV containing L gene mutation was obtained. The construction diagram is shown in the figure. Figure 4 shown.

[0098] 2. Rescue of Recombinant Viruses

[0099] Prepare the recombinant plasmid prO-mL-K108R-FMDV obtained in step 1 and press Polyplus Transfection reagent instructions: When BHK-21 cells grow to about 70%, the recombinant plasmid is transfected into BHK-21 cells. At the same time, normal cell control and transfection reagent control are set up and placed at 37°C and 5% CO 2 Incubator, observe the cell state, harvest the virus when the cells show about 90% cytopathic effect, freeze and thaw three times, and inoculate BHK-21 cells again until the virus can stably produce cytopathic effect, that is, the cells become round, aggregate into grape-like distribution, and finally the cells collapse. The obtained O-type foot-and-mouth disease recombinant virus was named rO-mL-K108R-FMDV.

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

[0101] 3. RT-PCR Identification of Recombinant Virus

[0102] The stably propagated recombinant virus rO-mL-K108R-FMDV was used to infect BHK-21 cells, and the cell supernatant was collected. Total RNA was extracted with Trizol and reverse transcribed. The gene fragment containing L was amplified using KpnI-F (SEQ ID NO: 8) and KpnI-R (SEQ ID NO: 9), and then purified and recovered for sequencing.

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

[0104] Example 5

[0105] Effects of RAB20 protein on the replication of recombinant foot-and-mouth disease virus with mutations in key amino acid sites of 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 incubated at 37°C with 5% CO 2 The cells were cultured in a cell culture incubator until the cell density was about 70%, and co-transfected with the FMDV L protein key site mutant eukaryotic expression plasmid L-Flag-K108R and different doses of RAB20 eukaryotic expression plasmid RAB20-Myc (0 μg, 1 μg, 2 μg and 3 μg), and the samples were collected for Western-blot analysis.

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

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

[0110] PK-15 cells were seeded into 6-well plates and incubated at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator until the cell density reached about 70%, and transfected with different doses of RAB20 eukaryotic expression plasmid RAB20-Myc (0 μg, 1 μg, 3 μg). After 24 hours, the cells were infected with recombinant foot-and-mouth disease virus rO-mL-K108R-FMDV with mutations in the key amino acid sites of the L protein, and Western-blot analysis was performed.

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

[0112] Example 6

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

[0114] The control FMDV and the recombinant FMDV with L protein mutation were used to infect BHK-21 cells at the same MOI. The virus solution was collected after 8 hours and repeated freezing and thawing for 3 times. The samples were diluted 10 times in DMEM medium and inoculated into 96-well culture plates with BHK-21 cells growing in monolayers. Each dilution had 8 wells and was placed at 37°C and 5% CO. 2 After culturing in an incubator for 3 days, the cell cytopathic effect was observed and the TCID of the virus was calculated according to the Reed-Muench method (existing literature: "Reed, LJ 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 the recombinant FMDV with mutations in the key amino acid sites of the L protein was significantly increased, indicating that mutations in the key amino acid sites of the L protein can increase the viral titer of FMDV.

[0116] The above results show that the replication level of the recombinant foot-and-mouth disease virus with mutations in the key amino acid sites of the L protein will not be inhibited by the host protein RAB20, and the mutation at this key site significantly increases the viral titer and antigen production of the foot-and-mouth disease virus, improves the virus production performance, and can be used as a candidate strain for the foot-and-mouth disease virus vaccine.

[0117] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as 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 in which the 108th amino acid is mutated or deleted.

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

3. The recombinant foot-and-mouth disease virus strain according to claim 1 or 2, characterized in that: The amino acid 108 of the mutant L protein is the L protein that has undergone a mutation; The mutant L protein includes a mutation of amino acid 108 to arginine; 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 foot-and-mouth disease virus strain 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 108th amino acid of the foot-and-mouth disease virus L protein; The full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence is 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 by using gene mutation technology or gene synthesis technology; Preferably, the 108th amino acid of the foot-and-mouth disease virus L protein is 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 two PCR amplification products were homologously recombined with the linearized vector plasmid to obtain a full-length infectious clone of foot-and-mouth disease virus containing the mutant L protein coding sequence; Preferably, the two pairs of homologous recombination mutation 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 KpnI-R is shown in SEQ ID NO:9; The nucleotide sequence of mL108-F is shown in SEQ ID NO: 10; The nucleotide sequence of mL108-R is shown in SEQ ID NO:

11.

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 foot-and-mouth disease virus strain that is free from replication inhibition by host protein RAB20, characterized in that: The following steps are involved: The 108th amino acid of the L protein in the foot-and-mouth disease virus strain is mutated or deleted; Preferably, the mutation method is to mutate the 108th amino acid 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 recombinant foot-and-mouth disease 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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