解脂耶氏酵母新型表达载体构建及癌基因rho在其中的表达

摘要

To simplify the construction procedure of Yarrowia lipolytica secretion/expression system, eliminate the antibiotic contamination, and use gene mel (encoding tyrosinase) as a new type reporter for constructing Y. Lipolytica secretion/expression vectors. Gene mel was synthesized by assembly PCR, and fused with homologous promoter pTEF, signal sequence XPR2pre and terminator LIP2t by overlap PCR. The resulted fragment was used to construct new type of secretion/expression plasmids of Yarrowia lipolytica expression system, with which human gene rho was expressed. The synthesized mel and pTEF, XPR2pre, LIP2t were successfully fused into gene fragment TXML, which was substituted for the reporter gene ura3d4 of Y. Hpolytica plasmids pINA1297 and pINA1297-a, resulting in new type plasmids pINA1297-M and pINA1297-a-M. This new system could screen the yeast recombinant transformants by phenotype, with which the soluble protein Rho was successfully expressed. In this research, mel gene was used as a new reporter for the first time in non-conventional yeast expression system, and it also lends a basis for applying mel in other eukaryotic expression systems. Additionally, the resulted soluble Rho can be used for further study of its character, structure, functions and the correlation between other members of Rho oncogene family.%为了简化解脂耶氏酵母表达载体构建过程、消除抗生素污染,将mel基因(编码酪氨酸酶)作为新型报告基因用于构建新型酵母表达载体,利用组装PCR人工合成基因mel,并用重叠PCR将其与同源组成型强启动子p TEF、分泌性信号肽XPR2pre及强终止区LIP2t融合,构建新型胞外及胞内表达载体,并利用其在解脂耶氏酵母野生菌株中表达人源癌基因rho.成功获得mel全基因并将其与启动子、信号肽和终止区融合,得到融合片段TXML,用其替换原有表达载体的筛选标记基因ura3d4,构建得到新型胞外及胞内表达载体pINA1297-M和pINA1297-a-M,转化后的酵母阳性转化子性状明显,随后利用此新型表达系统获得可溶性异源蛋白Rho.首次实现了将mel作为一种便捷、价廉、无污染的新型筛选标记基因运用于非常规酵母表达系统中,更为mel在其它真核表达系统中的运用奠定了技术基础；获得的可溶性Rho蛋白可为研究其性质、结构、功能及与Rho癌基因家族其它成员的相互作用提供条件.