HBV PreS1蛋白酵母双杂交诱饵表达载体的构建及其毒性和自激活效应检测

Construction of a bait plasmid containing HBV PreS1 gene in a yeast two-hybrid system and evaluation of its toxicity and self-activation

目的利用Sos招募系统(SRS),构建含HBV PreS1基因的酵母双杂交诱饵质粒,并检测其表达产物对cdc25酵母细胞有无毒性作用及对报告基因有无激活作用。方法聚合酶链反应(PCR)扩增HBV PreS1基因的编码序列,定向克隆到酵母表达载体pSos中,构建诱饵重组质粒pSos-PreS1,经测序正确后其将转化酵母菌cdc25感受态细胞,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自激活作用。结果序列测定证实重组诱饵质粒pSos-PreS1构建成功。重组质粒转化入酵母细胞后,经检测其表达产物对cdc25酵母细胞无毒性作用,对报告基因亦无自激活作用。结论可以利用SRS来研究与HBV PreS1蛋白相互作用的蛋白,为进一步研究乙型肝炎病毒的致病机制奠定了基础。

Objective To construct a yeast expression vector of hepatitis B virus （HBV） PreS1 gene using the Sos-recruitment system （SRS）, and evaluate the effect of the expression product on the growth of the yeast cells and activation of the reporter gene. Methods The coding sequence ofHBV preS1 was amplified by PCR and cloned into the yeast expression plasmid pSos. The recombinant bait plasmid pSos- PreS1 was verified by sequencing before transformation into competent yeast cells. The effects of the expression product on the yeast cell growth and activation of the reporter gene were evaluated. Results The yeast expression vector ofHBV PreS1 gene was constructed sucessfully. The recombinant bait plasmid showed no toxic effect on yeast cdc25H cells without a self-activation of the reporter gene. Conclusion The SRS can be used to study the proteins interacting with HBV PreS1 protein and provides a means for obtaining insight into the pathogenic mechanism of HBV.