Cloning,Expressing And Fishing Interacting-proteins Of Avian Influenza Viruses Nucleoprotein Gene

Nucleoprotein gene is the fifth segment 9f genome of avian influenza virues.Forepart, when AIV replicate, the RNA segments are contained within the virues envelopein association with the nucleoprotein and three subunits of viral polymerase (PA, PB1, andPB2),which together form the ribonucleoprotein (RNP) complex responsible for RNAreplication and transcription.That is the most important factors for virues infection.Therefore, it makes sense to illuminate the molecule mechanism of virues infection and findthe way of preventive from AI by finding the other proteins associated with NP in hostcells, and studying the relation of each-interacting.This task aims at preparing of NP antibody, then using the antibody to study the NPexpressing protein in cells and relation of mutual-interacting.At the same time, using theCytoTrap yeast two-hybrid system to fish NP-interacting protein. This study aim is to questfor relation of mutual-interacting about AIV-host cells.First, the virues RNA was obtained from the supernatant of MDCK cell infected H5N1Subtype avian influenza virues by RT-PCR, and the complete cDNA strands of NP wascloned.Second, prokaryotic expression vector pTIG/H5NP was constructed, then it wastransformed into E.coli BL21(DE3)plysS for highly expression; NP prokaryotic expressionproduction was purificated.Third, the purification of NP protein was injected into rabbit,and serums was collected toprepare NP antibody; at the same time, NP antibody affinity columniation was prepared topurificate antibody.Fourthly, the bait vector of NP (pSos/NP) was constructed, and fuse proteins hSos-NPexpressing in cdc25H (a) yeast stably was confirmed by Western blotting.At last,the bait vector pSos/NP and lung cell library were comtransformed into cdc25H-(a) yeast,and two putative positive clones combined with NP were fished utilizing CytoTrapyeast two-hybrid system. The two positive clones are respective Homo sapiens poly (ADP-ribose) polymerase family, member 8 (PARPS) and Homo sapiens zinc finger protein 658(ZNF658).