Screening For New Binding Proteins Interacting With The Influenza Virus Nucleoprotein By Yeast Two-Hybird System

Avian Influenza (AI) is one of fatal infectious diseases caused by influenza A virus in avian.The primary function of nucleoprotein (NP) is the major structural protein. It is to encapsidate the virus genome for the purposes of RNA transcription, replication and packaging. The influenza virus NP is much more than a structural RNA-binding protein, but also functions as a key adapter molecule between virus and host cell processes. NP also interacts with cellular polypeptides, including actin, components of the nuclear import and export apparatus and a nuclear RNA helicase. However, the molecular mechanism of transcription, replication and packaging is still unfortunately dimness.We performed yeast two-hybrid technique to screen for proteins interacting with virus nucleoprotein, so as to further elucidate the interaction between virus nucleoprotein and cellular proteins, as well as the interaction between virus and host.The coding region of NP was amplified by PCR using pCDNA3.1-NP fusion plasmids as a template, to produce the pGBKT7-NP bait expression vector which expressed successful and nontoxic.After having examined the library titer, we transformed the pGBKT7-NP bait plasmids into yeast Strains AH109. Clontech's Pretransformed libraries are supplied in strain Y187.After AH109 and Y187 mating, the zygote typically has a 3-lobed structure. The lobes represent the two haploid parental cells and the budding diploid cell. When bait and library fusion, proteins interact in a yeast reporter strain.Rescued the prey plasmids, and then transformed pGBKT7 and the prey plasmid into strains AH 109. There was no clone on the SD/-Trp/-Leu/-His/-Ade plate, but 1～2mm clone on the SD/-Trp/-Leu plate.The pGBKT7-NP bait plasmid for screening proteins in human brain cDNA library. DNA inserts of the positive AD/library plasmids were sequenced. By the BLAST analysis against the genebank databases positive clones resulted in twelve genes. There were GNAS, FERM and PDZ domain containing 4 (FRMPD4), LIM domain only 4(LMO4), AldolasC,fructose-bisphosphate(ALDOC), Homo sapiens protein arginine methyltransferase 1 (PRMT1), Homo sapiens clathrin, ATPase, Homo sapiens peptidylprolyl cis/trans isomerase, Homo sapiens zinc finger protein 668 (ZNF668), hypoxia inducible factor-1, Homo sapiens micro tubule-associated protein1A, Homo sapiens microtubule-associated protein 1B.We transformed pGBKT7-NP bait plasmid and the prey plasmid into strain AH109 to verify protein-protein interactions. The true positive could grow on SD/-Ade/-His/-Leu/-Trp/X-gal plate.