Preliminery Screening And Validation Of Host Proteins That Interact With Avian Reovirus ?A Protein By Yeast Two Hybrid System

Avian reovirus usually infects chickens and turkeys,causing a variety of d iseases including viral arthritis,tenosynovitis and chronic respiratory diseases,s uch as dwarfism.ARV causes many syndromesincluding gastrointestinal disorde rs,impaired feed conversion efficiency,diarrhea,growth retardation and someti mes immune-suppression.S2 gene encodes the major core protein ?A.Studies have shown that ?A protein is associated with the anti-interferon effect of ARV.However,the pathogenesisn of aA has not been thoroughly or extensively stu died.The study of interactions between host proteins and aA protein may elu cidate the mechanism that ?A protein acts as an antagonist of interferon.In this study,yeast two-hybrid technique was used to examine the interactions between host proteins and avian reovirus aA protein.Viral RNA was extracted from the ARVS-1133 strain,and a A gene was amplified by RT-PCR using a sets of virus-specific primers.The amplified ?A gene fragment was cloned into the pGBKT7 vector,generating a recombinant bait plasmid pGBKT7-?A.The recombinant bait plasmid was tested for toxicity and self-activation.The validated recombinant plasmid was used for the subsequent yeast two-hybrid experiments.The CLONTECH SMART technology was used to construct full-length cDNA library from SPF chicken liver tissue.The quality of the constructed cDNA library was tested.Construction capacity of the cDNA library was 2.4×106 cfu,and the titer was 1.30×107 cfu/mL.The recombination rate was approximately 100%respectively.The average size of the insert is around in the constructed library.This library with relatively high quality was used for yeast two-hybrid screening.The test results of the cDNA Library generated from the SPF liver tissue meet the requirements of the experiments.No toxicity or self-activation has been detected from the recombinant plasmid pGBKT7-?A.A total of 11 host proteins that interacted with ?A protein was detected and analyzed further.The initial screening on the host protein that interacted with the ARV ?A protein may serve as a basis for further study on ARV pathogenesis.