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Asia 1 type foot and mouth disease virus acid resistant mutant strains, capsid protein carried by same and encoding gene thereof and application

A foot-and-mouth disease virus, capsid protein technology, applied in applications, viral peptides, gene therapy and other directions, can solve problems such as decline, rising cost, and reduction of FMDV effective antigenic mass

Inactive Publication Date: 2014-07-09
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, FMDV is extremely sensitive to an acidic environment. When FMDV is propagated in vitro, acid production by cell metabolism can easily reduce the pH of the FMDV culture environment. When the pH value is slightly lower than neutral conditions, the capsid of FMDV will be cleaved, resulting in FMDV If the effective antigenicity of the vaccine is reduced, the immune efficacy of the inactivated vaccine thus prepared will decrease (Doel, T.R., and W.K.Chong.1982.Comparative immunogenicity of 146S, 75S and 12S particles of foot-and-mouth disease virus.Arch Virol 73:185-191.)
In addition, FMDV capsids are also sensitive to temperature, which requires a complete cold chain system during the transportation and storage of FMDV inactivated vaccines, resulting in a substantial increase in costs

Method used

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  • Asia 1 type foot and mouth disease virus acid resistant mutant strains, capsid protein carried by same and encoding gene thereof and application
  • Asia 1 type foot and mouth disease virus acid resistant mutant strains, capsid protein carried by same and encoding gene thereof and application
  • Asia 1 type foot and mouth disease virus acid resistant mutant strains, capsid protein carried by same and encoding gene thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Screening of Acid-resistant Asian Type 1 Foot-and-Mouth Disease Virus Mutants

[0037] Take 10 μL (equivalent to 10 6 pfu) Asia1 type FMDV Asia1 / YS / CHA / 05 strain was added to 300 μL PBS (pH 5.0) for 1 hour at room temperature, then neutralized with 100 μL Tris-HCL (pH 7.6), inoculated into a monolayer of BHK-21 cells, and waited for 80% After CPE appeared in the cells, the virus was collected by freezing and thawing three times and the virus titer was measured. The virus was used as the seed for the next round of pH 5.0 acid pressure screening until the 15th generation. In the control group, PBS with pH 7.4 was used to interact with the virus, and it was screened and passaged to the 15th generation in the same way as acid screening. FMDV was treated with PBS at pH 5.0, neutralized with Tris-HCL buffer (pH 7.6) after 60 min of action, and then inoculated into BHK-21 cells. After collecting the virus, freeze and thaw repeatedly 3 times for the second round of ...

Embodiment 2

[0039] Example 2 Extraction of viral RNA, cDNA synthesis and DNA sequence alignment

[0040] According to the instructions of the Simply P Total RNA Extraction kit, the viral RNAs of the two passages of Asian type 1 foot-and-mouth disease virus P10 and P15 obtained by acid pressure screening in Example 1 were respectively extracted, and the extracted RNA was dissolved with 26 μL of DEPC-treated deionized water. Using the extracted viral RNA as a template and Oligo-dT (15T) as a reverse transcription primer, cDNA was synthesized under the action of reverse transcriptase PrimeScript. The reaction conditions are: 25°C for 10 minutes, 42°C for 60 minutes, and 4°C for 20 minutes. The reaction system is 40 μL, including: viral RNA 24.5 μL; dNTP (25mM) 3 μL; PrimeScript reverse transcriptase 1 μL; 5×PSRT-Buffer 8 μL; Oligo-dT 3 μL; RNA Inhibitor 0.5 μL. Using the reverse transcription product as a template, a pair of primers for the Asia1 type FMDV structural protein region was used...

Embodiment 3

[0045] Example 3 Construction, rescue and acid resistance analysis of mutant strains

[0046] 1. Experimental method

[0047] 1. Construction of recombinant plasmids

[0048] According to the sequence comparison and analysis results between the acid screening strain and the parent strain in Example 2, positive clones of the structural protein coding region were selected to construct recombinant plasmids. In order to construct a full-length recombinant plasmid containing VP2 H145Y, VP2 G192D and VP3 K153E mutations, RNA was extracted from the 10th-generation virus of the acid-screened strain, and after reverse transcription, the target fragment was amplified with primers AR1980U / AR3135L. The PCR reaction system For 100 μL, including: ddH 2 O, 58 μL; 5×PS Buffer, 20 μL; dNTP, 8 μL; AR1980U (10 pmol / μL), 4 μL; AR3135L (10 pmol / μL), 4 μL; template, 5 μL; Prime STAR HS DNA polymerase, 1 μL. PCR reaction conditions: pre-denaturation at 94°C for 4min; denaturation at 94°C for 1min, ...

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Abstract

The invention discloses an Asia 1 type foot and mouth disease virus acid resistant mutant strains, capsid protein carried by same and encoding gene thereof and application. The invention includes obtaining Asia 1type foot and mouth disease virus P10 and P15 which are two generations of virus group with acid-resistant ability through acid stress screening and constructing and saving to obtain three Asia 1 type foot and mouth disease virus acid resistant mutant strains by using reverse genetics on the basis of analyzing the mutation sequence of the two generations of virus group. The amino acid sequence of the capsid protein carried by the Asia 1 type foot and mouth disease virus acid resistant mutant strain is respectively showed in SEQ ID No.2, SEQ ID No.4 or SEQ ID No.6. The problem of acid sensitivity of viral capsid during the process of producing and storing FDMV (foot and mouth virus) inactivated vaccine is solved and high quality seed virus is provided for preparing Asia 1 type FDMV inactivated vaccine.

Description

technical field [0001] The present invention relates to a foot and mouth disease virus (Foot and Mouth Disease Virus, FMDV) mutant strain, in particular to an acid-resistant mutant strain of Asian type 1 foot-and-mouth disease virus. The field of prevention and control of foot-and-mouth disease virus. Background technique [0002] Foot and Mouth Disease Virus (FMDV) is a member of the genus Foot and Mouth Virus in the Picornaviridae family and belongs to single-stranded positive-sense RNA viruses. Acid sensitivity of FMDV is required for viral capsid cleavage. After the virus enters the cell, the acidic environment of the endosome induces the disassembly of the viral capsid, thereby allowing the RNA genome to be released in the infected cell. In developing countries, the prevention and control of FMD mainly relies on immunization with inactivated vaccines. For FMD vaccines, 146S virion was the most effective antigen for inactivated vaccines to induce neutralizing antibodi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01C12N15/63C12N15/42C07K14/09A61K39/135A61K48/00A61P31/14C12R1/93
Inventor 于力
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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