日本脑炎病毒E蛋白在酵母双杂交系统中的表达及其对报告基因激活作用的检测

Reporter gene assay of apanese encephalitis virus E gene in a yeast two-hybrid system

用日本脑炎病毒(JEV)E蛋白基因片段构建酵母双杂交诱饵载体,并检测其表达产物对酵母细胞有无毒性作用及对报告基因有无激活作用。用RT-PCR从JEV感染的鼠脑中扩增出JEVE蛋白基因片段,克隆入pUC19质粒,经测序正确后,再亚克隆入酵母双杂交诱饵载体pGBKT7中。将重组质粒导入酵母菌AH109,检测其表达产物在酵母细胞中对报告基因有无激活作用。成功获得JEVE蛋白基因片段,表达的E蛋白对酵母菌AH109无毒性,对报告基因亦无激活作用。为利用酵母双杂交GAL4系统3进行JEV细胞受体蛋白的研究奠定了基础。

To construct a bait vector with Japanese encephalitis virus(JEV)E protein gene in yeast two-hybrid sys-tem(GAL4),and assay whether E protein gene expression product affects the growth of host yeast cells,and acti-vates the yeast two-hybrid system reporter genes.JEV E protein gene fragments were amplified by RT-PCR from brain homogenates of suckling mice infected by JEV,and cloned into pUC19for DNA sequencing.After verified by DNA sequencing,they were subcloned into the bait vector pGBKT7of yeast two-hybrid system(GAL4),and the re-combinant plasmids were subsequently transferred into yeast cell AH109,and its expression product were tested whether it could activate the reporter genes in the yeast two-hybrid system.JEV E protein gene fragments were suc-cessfully amplified,and their expression products showed to be nontoxic to AH109cells,and did not activate the re-porter genes of the GAL4system.Yeast two-hybrid GAL4system3can be further used to fish JEV binding protein(putative JEV receptor)from human brain cDNA libraries.