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
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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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