Screening Research On The Interaction Between Chicken Infectious Anemia Virus VP1Protein And Host Protein

Chicken infectious anemia（CIA）,caused by chicken infectious anemia virus(CIAV), is animmunosuppressive diseases, which is distinguished by aplastic anemia,atrophy of the thymus andbone marrow in two weeks chickens. It was first reported in Japan in1979. Now, it can be foundeverywhere worldwide. CIA has caused severe economic damage to poultry husbandry.There are three proteins in CIAV: VP1、VP2and VP3. VP1is structural protein of the virus,located in capsid. As studies shown, VP1is a key factor associated with immunogenicity andpathogenicity of CIAV. The protein-protein interaction between virus and host exerts an importantimpact on replication and pathogenicity of the virus. Therefore carrying out the researches on theinteractions of CIAV VP1and host proteins will benefit to further understand the biological functionsof the protein and the pathogenicity of CIAV. In this study, the technologies of studying theprotein-protein interactions, such as the yeast two-hybrid system (H2Y), coimmunoprecipitation(Co-IP)and confocal assays were applied to investigate the potential interactions between VP1andhost proteins. The results were shown as follows:Bait vector about VP1was constructed for yeast two hybrid (YTH). Transforming the yeast todetect the expression of MEL1and ADE2, the result showed that VP1-pGBKT7is qualified withoutself-activation and toxicity. In this study, we constructed the full-length cDNA library aboutMDCC-MSB1used SMART skill. The ds DNA were cloned into the pGADT7vector, which is usedfor YTH. By detection, we found the titer of cDNA library is1.7×106cfu/ml. The squesence ofinsertion range from0.3~1.5kb, and recombination rate is100%. Random drawing10samples toanalysis the insert sequence, the sequence results showed that all sequences were highly homology toGullus. Using the VP1-pGBKT7bait screened the cDNA library, positive prey plasmids wereobtained and analysised. The results showed that Snapin, which was found associated with manybiochemical processes and had antivirus effects, interacts with VP1protein. To co-transform Y2Hyeast, it was confirmed the interaction between VP1and Snapin by contrast to positive and negativecontrol.To further verify the interaction between VP1and Snapin, immunoprecipitation (Co-IP) and laserscanning confocal assay were carried out.. The genes of Snapin were amplified from the RNA ofMDCC-MSB1and cloned to eukaryotic expression plasmid pCAGGS. The Co-IP results indicatedthat VP1can immunoprecipitated by Snapin, which is the evidence of the interaction about them.Besides, the interaction was demonstrated by laser scanning confocal. Meanwhile, VP1was dividedinto theree sections: V1, V2and V3. The results of Co-IP indicated that the interaction domain locatesin1-230amino acid of N terminal of VP1.In this study, we constructed bait plasmid of CIAV VP1and cDNA library of MDCC-MSB1which is susceptible to CIAV for YTH. Screening the cDNA library with VP1bait, the Snapin proteinwas proved to interact with VP1protein. In the further experiment, the interaction between VP1and Snapin proteins was confirmed by Co-IP and scanning confocal.