Screening Of The Avian Influenza Virus Pa Related Protein With Yeast Two-hybrid Techniques

The molecular determinants and related mechanisms that make certain influenza viruses highly pathogenic species,including humans,remain poorly understood.Numerous studies have shown that influenza virus virulence in mammalian species is a polygenic trait.The influenza virus RNA polymerase is a complex composed of three subunits,PA,PB1, and PB2.The polymerase,together with the nucleoprotein(NP) and the viral RNA template, (RNP),which carry out viral transcription and replication.PA not only carry out viral transcription and replication,but also induces a generalized proteolytic process.In this paper, the expression of PA protein and the protein interacting with PA were studied.First,17 deletion mutants of PA protein were constructed and expressed in Ecoli.Rosseta GamiB(DE),we conclusion that the two segments of 61th-120th bp and the 325th-426th bp of PA gene may be the very fragments that affect the expression level of PA protein.At the same time,nine mutants with the deletion between the 1th bp and the 426th bp of the PA gene got higer expression.Second,we use H5N1 PA as bait protein to screen the related protein by yeast Two-hybrid System 3.Eight positive clones were identified,they are GPATCH8,GPR89A,ADH1A,ADH1C,TTR,PMSA,LBP,ETFA.The interaction of these 8 proteins with PA were tested by yeast mating.Third,the full-length of gene GPR89A,ADH1C,TTR,PMSA,LBP and ETFA are cloned and constructed into pCMV-3HA vector and pTIG-Trx-Etag vector.Protein ADH1C,TTR,PMSA,LBP and ETFA can be expressed by A549.In conclusion,based on these results,the function of PA protein and the molecular pathogenic mechanism of AIV could be greatly and deeply enderstood,and the strategy for prevention against and treatment for AIV would be formed.