Cloning of structural protein VP1 gene of foot and mouth disease virus and its expressionin Escherichiacoli

In this study, the viral RNA was extracted fromtongue epithelium of cattle infected with FMDV ,and then the fragment of VP1 wasamplified with a primer pair by RT PCR.The interest fragment was inserted into pGEM Teasy vector.The recombinant plasmid was identified by restriction analysis and PCR.It was proved by DNA sequencing that theacquired recombinant containscomplete VP1 gene. The homologies of the nucleotide sequence of VP1 gene were 80.44%and 99.06% respectivelycomparing with that of strain O1K/66 and HB/WH/99.Afterwards the complete VP1 gene from the identified recombinant was amplified with another primer pair containing EcoRⅠand BamHⅠsites by PCR and digested it with EcoRⅠand BamHⅠ.The expression vector pGEX 4T 1 were digested by EcoRⅠand BamHⅠrespectively. The target gene VP1 was subcloned into vector pGEX 4T 1.Positive clones named as pGEX VP1 with interest gene were identificated by restriction analysis , PCR and DNA sequencing.Then the recombinant was transformed into Escherichia coli BL21(DE 3 ) for VP1 expression. The interest gene was inducedto express in E.coli with IPTG.The bacteria containing pGEX VP1 were collected at different time and subsequently were examinedby SDS PAGE and western blotting.Results showed that the structural protein VP1 gene of FMDV can express successfully in E.coli. Molecular weight of the fusion protein was 53 ku, and the amount of target proteinis over 20.00% of the total bacteria protein.The fusion protein can be recognized by the positive serum of cattle that was infected with FMDV.