Study On The Interaction Between NS2 Of Influenza A Virus And Host Factors By The Use Of Yeast Two-Hybrid System

H5N1 NS2 protein is conversation among influenza A virus with a nuclear localization signal which is required as a bait in traditional yeast two-hybrid system. According to literatures reported, roles of NS2 whose sequence was rare were established in exporting viral ribonucleoprotein (vRNP) from the nucleus, relatively to other proteins of influenza virus, while other functions for the NS2 were not reported. To discover new NS2 interaction with host factors may not only expand the existing network between influenza virus and host, but also open a new way for preventing and controlling disease.As a high-throughput method, Yeast two-hybrid has been used to study the interactions between influenza virus proteins and host proteins. In this thesis, yeast two-hybrid was used to study the interactions between the influenza virus NS2 protein and host proteins. The principal results were described as the following:1) Screening proteins that interact with from a human fetal brain cDNA library using NS2 of Avian influenza virus A/chicken/Hubei/327/2004 (H5N1) protein as a baitNS2 was cloned into yeast two-hybrid bait vector (pGBKT7 vector), pGBKT7-NS2(+) AH109 yeast showing no self activation. Y187 yeast was transformed in the human fetal brain cDNA library.Let the both of yeast above mate. Screening 3 cycles, pGADT7-preys were extracted and sequenced. At last,10 different clones turned into positive clones for candidates through blast analysing in NCBI.2) GST pull-down testing the interaction in vivo and in vitroIn GST pull-down assay, NS2 was cloned to pGEX-6P-1 vector to express NS2 byBL21.10 preys cloned to pET32a vector to express the HIS tagged fusion prey proteins. It was found NS2 and 6 of 10 Preys could be soluble.The 6 preys were stathmin-like protein 2 (STMN2), DDB1 and CUL4 associated factor 6 (DCAF6), the required copper ion metabolism domain of the protein (COMMD1),a member of kinesin family C2 (KIFC2) respectively,eukaryotic translation elongation factorl alpha 1 (EEF1A) and microtubule associated protein 1B(MAP1B).STMN2 showed interaction with NS2 by GST pull-down test.The infection of host cell with influenza virus had an effect on the cell morphology, which was concerned with NS2.