酵母双杂交技术筛选与乙型脑炎病毒NS5相互作用的蛋白

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

目的应用酵母双杂交技术筛选与乙型脑炎病毒NS5蛋白相互作用的宿主蛋白。方法构建表达乙型脑炎病毒NS5蛋白的诱饵质粒pSos-NS5,经自激活和毒性实验后对人脐静脉内皮细胞cDNA文库进行筛选,挑取37℃条件下在缺失亮氨酸和尿嘧啶培养基上生长的克隆,利用一对一酵母回复性杂交实验进行验证,然后测定阳性克隆插入片段序列,通过Blast分析确定可能与NS5相互作用的蛋白。结果构建的诱饵载体pSos-NS5对酵母宿主无毒性,且无自激活活性。经过筛选得到3个阳性克隆(10,11,4′6号克隆),经测序和Blast分析,初步确定4′6号克隆编码的TC21蛋白可能与NS5发生相互作用。结论经酵母双杂交技术筛选到一种可能与NS5蛋白具有相互作用的人类蛋白,为深入探讨乙型脑炎病毒NS5蛋白的生物学功能奠定了基础。

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 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.