Screening of the proteins interacting with NS1 of TMUV by yeast two-hybrid system and the identification of the function of the interacted protein

Abstract

The NS1protein, a nonstructural protein of Tembusu virus, plays a key role in the pathogenesis of TMUV. To research host proteins that interact with NS1 protein, the cDNA library of duck embryo fibroblasts (DEF) was successfully constructed. The recombinant plasmid, pGBKT7-NS1, was transformed into the yeast Y2H to be cloned and tested for autoactivation and toxicity. The autoactivation and toxicity test of bait showed that the yeast two hybrid test could be carried out normally. A total of 7 clones from the library were got by Yeast Two-Hybrid System, and 4 proteins, including RPS7, MORC3, GABARAPL1 and MTSS1, may be interacted with DTMUV NS1 after sequencing and blast. Then we chose the host protein of RPS7 for GST pull down assay and the recombinant plasmid of pGEX-6p-1-NS1and pEGFP-RPS7 were constructed. Then the proteins of GST-NS1 and GFP-RPS7 were successfully expressed in vitro for GST pull down assay. The results showed that there was a real interaction between the two proteins when the protein of GST-NS1-GFP-RPS7 was obtained eventually. The Real-time RT-PCR was used to detect the expression level of RPS7, MDM2 and P53 mRNA after the recombinant plasmid of pEGFP-NS1 was expressed in 293 T cells. It is showed that the expression of NS1 protein causes the low expression of RPS7 and MDM2 mRNA and eventually causes the high expression of P53 mRNA. This research lays the foundation for clarifying the pathogenic mechanism of Tembusu virus and the function of NS1 protein in virus propagation process.

Keywords: GST pull down; NS1 gene; Recombinant plasmid; Tembusu virus; Yeast two-hybrid.