Screening Of Cell Interacting Protein Of NS1 Protein Of Bluetongue Virus By Yeast Two-hybrid Technique

Bluetongue is an acute non-contact infectious disease caused by the bluetongue virus(BTV).It is mainly transmitted by insect vectors such as Culicoides,mainly infecting ruminants such as sheep,cattle,and deer.In recent years,bluetongue has broken out many times in Europe,which has caused severe economic losses to animal husbandry.It has been listed as a statutory notifiable animal infectious disease by the World Organization for Animal Health(OIE).The first case of BTV was found in Yunnan in 1979.Up to now,it has spread all over 29 provinces in China.Because of its rapid spread and severe economic losses caused by the disease,our country lists it as the first category of animal infectious diseases.NS1 protein of BTV is the most highly expressed non-structural protein in infected cells and is conserved between various serotypes and can be used as a potential target for antiviral studies.The interaction of proteins plays an important role in the viral life cycle.Studying the interaction between NS1 protein and host cell protein is of great significance for clarifying the function of NS1 protein and the pathogenesis of BTV.Based on this,a c DNA yeast expression library of sheep kidney cells was constructed in this study,host cell proteins interacting with BTV NS1 were screened by yeast two-hybrid technology,and the mutual interaction protein FLNA was selected to further identify its role during BTV infection.In this study,BTV NS1 protein was first expressed truncated(1 ? 320 aa)using the E.coli expression system,purified and immunized to New Zealand white rabbits to prepare a highly specific polyclonal antibody,which could react not only with the recombinant expressed NS1 protein,but also with the native NS1 protein in BTV-infected cells,which provided an antibody tool for further study of the function of NS1 protein.Total RNA was extracted from sheep kidney cells,and the sheep kidney c DNA library was successfully constructed using Gateway cloning technology,and the library quality met the requirements of subsequent experiments by experimental verification.At the same time,the p GBKT7-NS1 bait plasmid was successfully constructed,which had no self-activation effect and could be used for yeast two-hybrid screening;the bait plasmid and c DNA library plasmid were co-transformed into Y2 H GOLD yeast cells,and finally after rotary verification,a total of 18 host cell proteins interacting with NS1 were selected.Filamin A(FLNA)protein was selected for further study,and the intracellular interaction between FLNA protein and BTV NS1 protein was verified by co-immunoprecipitation and cell co-localization experiments,which were co-localized in the cytoplasm,and FLNA protein was found to positively regulate BTV replication using FLNA gene overexpression and si RNA knockdown experiments.