日本脑炎病毒核心蛋白酵母双杂交诱饵载体构建及鉴定

目的构建日本脑炎病毒核心蛋白（JEV C蛋白）基因的酵母双杂交诱饵载体,并检测其蛋白表达产物对酵母细胞的毒性及对酵母细胞内报告基因的自激活效应。方法 PCR扩增JEV C蛋白cDNA全基序并克隆入载体pGBKT7的EcoRI/BamHI酶切位点之间,酶切及测序鉴定;PEG/LiAc法将构建好的诱饵质粒pGBKT7-JC及载体pGBKT7分别转化入酵母菌株Y2H Gold感受态细胞,经酵母氨基酸缺陷培养及表型筛选检测其对酵母细胞毒性及自激活作用;用Western-blotting法分析酵母菌中诱饵蛋白BD-JC的表达。结果重组质粒pGBKT7-JC经酶切鉴定及测序结果与Genebank公布的JEV SA14-14-2株中JEV C蛋白编码基序完全一致;成功转化质粒pGBKT7-JC及空载体pGBKT7的酵母菌株Y2H Gold感受态细胞均只在SD/-Trp、SD/-Trp/X培养基上生长,二者菌落数、菌落大小、分布无明显差异且不变蓝,且SD/-Trp液体培养基30℃过夜培养测OD600值均〉0.8,在SD/-Trp/X/A培养基上均不生长;经Western-blotting法检测到酵母菌能表达分子量约为35KDa的特异性融合蛋白BD-JC。结论成功构建了酵母双杂交诱饵表达质粒pGBKT7-JC;其诱饵蛋白BD-JC对酵母细胞无细胞毒性及自激活效应,为筛选及验证与pGBKT7-JC相互作用蛋白奠定实验基础。

Construction and verification of a bait vector for the gene encoding core protein derived from Japanese encephalitis virus

Objective To construct a bait vector with the gene encoding core protein derived from Japanese encephalitis virus,toxicity and self-activation of the bait protein were tested.Methods Full-length gene of JEV C protein was amplified by PCR method.The identified PCR product was inserted between EcoRI and BamHI sites of pGBKT7 vector,The bait vector pGBKT7-JC was confirmed by restriction endonuclease enzyme analys and sequencing;The pGBKT7-JC and pGBKT7 were transformed into their respective yeast strain Y2H Glod cells by PEG/LiAc method.Toxicity and self-activation of the bait protein was tested by cultured in SD with different amino acids defects and the phenotype assay.The expression of the bait protein BD-JC was detected by western-blotting.Results The gene fragment of JEV C protein was amplified and cloned into pGBKT7 successfully and its sequence was the same as that of the published JEV SA14-14-2 strains in the Genebank.pGBKT7-JC and pGBKT7 were transformed into their respective yeast strain Y2H Glod cells as well,both groups those cells only grew in SD/-Trp、SD/-Trp/X but not in SD/-Trp/X/A indifference and the colours were not turn blue,and the OD600 were more than 0.8 by cultured overnight at 30℃ in SD/-Trp fluid medium in both groups cells.The size of BD-JC protein expressed in Y2H Glod cells were transformed with pGBKT7-JC was 35KDa.Conclusion The bait expression vector of pGBKT7-JC was constructed successfully,neither toxicity nor self-activation of the BD-JC protein expressed in Y2H Glod cells were found,which layed the foundation for screening and proving target proteins interacting with pGBKT7-JC using the yeast two-hybrid technique.