Researching Interaction Of GPV Structural Protein VP1 With Host Cell Protein SH3BP4 And Effect On Proliferation Of GPV

Goose parvovirus(GPV)is a member of Parvoviridae depending virus.It is caused by Goose parvovirus(Goose parvovirus).Goslings and Muscovy ducklings are mainly infected by GPV,which are aged 3 to 20 days.Systemic septic lesions,cellulose and exudative enteritis are the main pathological changes,which are finally leaded to enteral embolism.This disease spreads rapidly with a high infectiousness,and the mortality rate of goslings is above 95%.Serious harm and enormous economic losses are caused in waterfowl industry.The susceptible gosling is vertical infected by the egg with GPV.Therefore,it is critical to prevent the vertical transmission of GPV.It is significant to understand the pathogenesis of the GPV and master the interaction of viral structural proteins with host cell proteins for the GPV's effective prevention.In our laboratory,three positive clones were screened by yeast two-hybrid technique.One of the third genes was 479 bp,and the homology with SH3BP4 was up to 99%.The aim of this work was to further verify the interaction between SH3BP4 and VP1 protein and the effect on GPV proliferation.The complete SH3BP4 gene was amplified by PCR,and the prokaryotic expression plasmid of pET-28a-SH3BP4 was constructed,the plasmid was expressed with IPTG and then SH3BP4 was purified,the product was analyzed by SDS-PAGE,and the purified protein was about 106 kDa.At the same time,the pGEX-4T-1-VP1 was expressed,the interacted reaction was proved between VP1 and SH3BP4 in vitro body by the technology of GST-pull down.The eukaryotic expression plasmid of pcDNA-3.0-SH3BP4 was constructed,DEF was transfected by the plasmid,then DEF was infected by GPV.The experimental group was finished,three controlled groups were seted,pcDNA-3.0vector and GPV,lipofectamine and GPV,autoclaved water and GPV respectively.The virus copy numbers were detected by quantitative PCR after 12 h and 24 h.The copy numbers of GPV were not obvious between the experimental group and other three controlled groups after 12 h.After 24 h,the copy numbers of GPV in control groups were 4 times as large as the experimental group,this result indicated that SH3BP4 had an adverse effect on the replication of GPV.The puried SH3BP4 protein was mixed with GPV with different volumes in the virto,and then DEF was infected,the viruscopy numbers was detected by quantitative PCR,and the copy numbers of GPV was decreased gradually with the amount of proteins increasing.It was indicated that the SH3BP4 in the virto had an adverse effect on the replication of GPV.