Screening for proteins interacting with NS5 of Japanese encephalitis virus by yeast two-hybrid technique

Xi-zhen ZHOU, Hui ZHAO, Xiao-feng LI, Yong-qiang DENG, Cheng-feng QIN, E-de QIN, Lan GAO
2011 Medical Journal of Chinese People's Liberation Army  
Objective To screen possible human proteins that interact with the non-structural protein 5(NS5) of Japanese encephalitis virus(JEV) by yeast two-hybrid system.Methods The bait plasmid pSos-NS5 carrying JEV NS5 gene was first constructed and transformed to yeast cells,and then validated by the self-activation and toxicity assay.Then,a human umbilical vein endothelial cells cDNA library was used for screening with the bait plasmid pSos-NS5,and positive clones were selected based on nutritional
more » ... ed on nutritional requirements(Leucine and Uracil) and temperature change,and verified by one to one back-hybridization.Finally,the plasmids from the positive clones were sequenced and analyzed by Blast with sequences from GenBank.Results The bait plasmid pSos-NS5 was successfully constructed and confirmed to be no self-activation activity and toxicity to the host yeast cells.Three positive clones(10,11,and 4'6) were then selected from the human umbilical vein endothelial cells cDNA library with the bait plasmid pSos-NS5.Then,the plasmid from clone 4'6 was sequenced and BLAST analysis within GenBank showed that the sequences were highly homologous to the TC21 gene.Finally,these data indicated TC21 protein might be the putative candidate protein that interacted with the NS5 protein of JEV.Conclusion A candidate host protein has been identified by the yeast two-hybrid system,which will be helpful for understanding the biological function of NS5 of JEV,and further research should be warranted in the future.
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