O-type foot and mouth disease virus acid-resistant mutant strain, capsid protein carried by O-type foot and mouth disease virus acid-resistant mutant strain, coding gene of capsid protein and use of O-type foot and mouth disease virus acid-resistant mutant strain and capsid protein

A technology of foot-and-mouth disease virus and capsid protein, applied in the fields of application, viral peptide, gene therapy, etc., can solve the problems of decreased immune efficacy, reduced effective antigen content of FMDV, and increased cost

Active Publication Date: 2014-06-11
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, because 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 The effective antigenicity of FMDV reduces, and its immune effect of the inactivated v

Method used

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  • O-type foot and mouth disease virus acid-resistant mutant strain, capsid protein carried by O-type foot and mouth disease virus acid-resistant mutant strain, coding gene of capsid protein and use of O-type foot and mouth disease virus acid-resistant mutant strain and capsid protein
  • O-type foot and mouth disease virus acid-resistant mutant strain, capsid protein carried by O-type foot and mouth disease virus acid-resistant mutant strain, coding gene of capsid protein and use of O-type foot and mouth disease virus acid-resistant mutant strain and capsid protein
  • O-type foot and mouth disease virus acid-resistant mutant strain, capsid protein carried by O-type foot and mouth disease virus acid-resistant mutant strain, coding gene of capsid protein and use of O-type foot and mouth disease virus acid-resistant mutant strain and capsid protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Screening and identification of O-type foot-and-mouth disease virus acid-resistant mutant strain

[0029] 1. Screening of O-type foot-and-mouth disease virus acid-resistant mutants

[0030] Take 10 μL (10 6 TCID 50 / 100ul) O-type FMDV O / YS / CHA / 05 strain was added to 300 μL PBS (pH 6.0) for 1 hour at room temperature, neutralized with 100 μL 1M Tris (pH 7.4) and inoculated into BHK-21 cells until 80% of the cells appeared CPE Afterwards, the virus was harvested by repeated freezing and thawing three times and the virus titer was measured. The virus was used as the seed for the next round of acid stress selection until the 15th generation. Parents were treated in the same way with PBS of pH 7.4, and continuously passed for 15 generations. After 15 passages were screened at pH 6.0, the 5th passage (P5), 10th passage (P10) and 15th passage (P15) viruses were selected to test their tolerance to pH 6.0 and pH 7.4 acidity.

[0031] 2. Indirect Immunofluorescence...

Embodiment 2

[0035] Example 2 Construction of infectious cDNA clone plasmid pYS-N17D of O-type FMDV acid-resistant mutant strain, rescue of O-type foot-and-mouth disease virus acid-resistant mutant strain and analysis of acid-resistant characteristics

[0036] 1. Viral RNA extraction, cDNA synthesis and DNA sequence alignment

[0037] In order to analyze the amino acid sites that determine the acid-resistant characteristics of FMDV, the genome sequences of P5, P10, P15 strains and their parent strains were determined and the sequences of the structural protein coding regions were compared.

[0038] According to the instructions of the Simply P Total RNA Extraction kit, respectively extract the virus RNA of three passages of O-type foot-and-mouth disease virus P5, P10 and P15 obtained by acid pressure screening in Example 1, and dissolve the extracted RNA with 26 μL of DEPC water.

[0039] Using the extracted viral RNA as a template and O1igo-dT (15T) as a reverse transcription primer, cDNA...

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Abstract

The invention discloses an O-type foot and mouth disease virus acid-resistant mutant strain, a capsid protein carried by the O-type foot and mouth disease virus acid-resistant mutant strain and a coding gene of the capsid protein and also discloses a key amino acid site for determining O-type foot and mouth disease virus acid-resistant characteristics and a use of the key amino acid site. An O-type foot and mouth disease virus parent strain is subjected to serial passage screening under acid stress so that a mutant strain having strong acid resistance is obtained, and an analysis result shows that a VP1 N17D mutational site is a key amino acid site for determining O-type foot and mouth disease virus acid-resistant characteristics, the O-type foot and mouth disease virus only with the VP1 N17D mutational site has the acid resistance the same as that of the mutant strain and also has a good replication capability and immunogenicity. The key amino acid site for determining O-type foot and mouth disease virus acid-resistant characteristics can be used for reconstruction of acid resistance of a foot and mouth disease virus vaccine and for exploitation of a novel foot and mouth disease gene engineering vaccine. The acid-resistant mutant strain rN17D can be used as a high-quality inactivated vaccine-production parent strain, can improve content of a 146S effective antigen in the inactivated vaccine and can improve vaccine immune efficacy.

Description

technical field [0001] The invention relates to an acid-resistant mutant strain of foot-and-mouth disease virus and the capsid protein and coding gene carried by it, in particular to an infectious cDNA plasmid clone of an acid-resistant mutant strain of an O-type foot-and-mouth disease virus and rescue of the infectious cDNA plasmid clone of an acid-resistant mutant strain of an O-type foot-and-mouth disease virus The acid-resistant mutant strain of type O foot-and-mouth disease virus, the invention also relates to the VP1 N17D mutation site and its application that determine the acid-resistant characteristic of the acid-resistant strain of type O foot-and-mouth disease virus, belonging to the field of foot-and-mouth disease virus prevention and control. Background technique [0002] Foot-and-mouth disease (Foot and Mouth Disease, FMD) is caused by Foot and Mouth Disease Virus (Foot and Mouth Disease Virus, FMDV), an acute, febrile, highly contagious infectious disease that m...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N7/00C12N15/42C07K14/09A61K48/00A61K39/135A61P31/14C12R1/93
Inventor 于力杨德成
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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