Expression Of Foot-and-Mouth Disease Virus Structural Protein VP1 Gene In Prokaryotic And Eukaryotic Cells

Foot-and-mouth disease virus (FMDV) causes a highly contagious infectious disease-foot-and-mouth disease (FMD) in cloven-hoofed animals.This disease is widely endemic all over the world and often results in considerable economic losses. FMDV capsid contains 60 copies of each of four structural proteins, VP1-4.VP1 protein named as 1D protein as well, which is composed of 213 aminoacids. Its gene occupy 2977-3615 of FMDV RNA genome. Some researchs about VP1 immune suggest that VP1 take a major importance role in inducing FMDV neutralization antibody.Here,VPl gene was amplified from the recombinant plamids pGEM-VP1 with a pair of primers containing Ncol I and Xhol I sites by PCR and digested with Ncol I and Xhol I .The expression vector pTriEx was digested by Ncol I and Xhol I respectively.The VP1 gene was inserted into the shuttle vector pTriEx.Positive clones plasmid named as pTriEx-VP1 with interest gene were identificated by restriction analysis, PCR and DNA sequencing.Then the recombinant plasmid was transformed into E.coli BL21(DE3) pLySs for VP1 expression.The interest gene was induced to express in E.coli with IPTGThe culture liquid of bacteria containing pTriEx-VPl were collected at different time and were subsequently examined by SDS-PAGE and Western-blotting.Results showed that the structural protein VP1 gene of FMDV could successfully express in E.coli.The amount of target protein in inducing products is over 26% of the total bacteria protein.Molecular weight of the fusion protein was 26 KD.It was proved that the expressed protein was soluble and didn' t existed in the form of inclusion body. The fusion protein purified by ProBond?Column, ELISA assay showed that the reaction of expressed fusion protein with FMDV antibody was positive. When the recombinant plasmid was transfected into BHK21 cells with Lipofectin regent.ELISA and indirect immune-fluorescence tests were positive.However,the expression level was more low than its prokaryotic expression.lt is clear that much efforts and further work would be need to improve VP1 gene expression in eukaryotic BHK21 cells.