Influence Of Translation Extension Factor In DEF On Proliferation Of Goose Parvovirus

Goose parvovirus(GPV) is the pathogen of goslings, which is also named as Derzsy’s disease. It causes acute or subacute septicemia of goslings and Muscovy ducklings aged 3 to 20 days. Sick goslings show systemic septic lesions, focal hepatitis, myocarditis and embolic enteritis. The autopsy changes mainly appear in jejunum and ileum, forming catarrhal, fibrinous, necrotic enteritis or intestinal embolization. It has a series of characteristics, such as widespread prevalence, fast propagation velocity and higher incidence and mortality rate, which causes serious economy losses to waterfowl industry in our country.To further investigate the mechanism of replication and colonization of GPV in host cell, the interaction between GPV and cell protein was studied. Duck Embryo Fibroblasts(DEF) library was constructed in yeast two-hybrid. The structural proteins VP1 of GPV were used as bait, and then 3 positive clone plasmids were screened by yeast two-hybrid method. After sequencing and Blank online comparison revealed that one gene among them, with a length of 593 bp, showed a homology up to 96% with the EEF1A1 protein of Junglefowl. To further testify the interaction between the EEF1A1 protein and VP1, and then study its effect on the proliferation of GPV, the experiments were carried out.Expression of prokaryotic induction was conducted by constructing the prokaryotic expression plasmid of pET28a-EEF1A1(containing His tag), and then purified by using the label of resin and analyzed by SDS-PAGE. The results indicated that the purified protein was about 25 kDa. The expression of prokaryotic pGEX4T-1-VP1 constructed in our laboratory was induced, with the VP1 protein of about 108 kDa. Protein complexes were formed by EEF1A1 and VP1 using the protein interaction technology of GST-Pulldown, which proved that EEF1A1 might combine with VP1 in the extracellular. EEF1A1 protein was induced by prokaryotic expression and purified for the interaction with GPV in the extracellular. For the experimental group, duck embryo fibroblasts(DEF) was infected with EEF1A1, while control group, DEF was infected with GPV. The nucleic acid replication of GPV was detected by quantitative PCR after 24 h. The results showed that the copy numbers of GPV in control group were 2.3 times、5.4 times and 7.7 times of experimental group, respectively. Thus, we proposed that the adsorption to the cell surface of duck embryo fibroblast was affected by EEF1A1. It indicated that EEF1A1 played a negative role in the GPV reproduction.After DEF transfected with pcDNA3.0-EEF1A1 and then infected with GPV in 12 h and 24 h,the viruscontent was detected by quantitative PCR with the control groups of DEF infected with GPV, GPV and plasmid pcDNA3.0, GPV and liposome and GPV and autoclaved water respectively. The results showed that, comparing with the control group, the copy numbers of GPV were not obvious when transfected for 12 h. However, the copy numbers of GPV in infected with DEF alone were 2.7 times of the experiments group after 24 h transfection, which indicated that the increase in the expression of EEF1A1 promoted the GPV reproduction in the cell. The results provide a basis for further research of replication mechanism of GPV.