大鼠肾皮质受体相关蛋白克隆基因重组表达质粒的构建及分子生物学鉴定

CONSTRUCTION AND IDETIFICATION OF THE EXPRESSION PLASMIDRE CONBINANT WITH THE RECEPTOR-ASSCIATED PROTEIN GENE

运用ＤＮＡ重组技术，将用ＲＴ－ＰＣＲ方法克隆的大鼠肾皮质受体相关蛋白３１９个氨基酸的核苷酸序列导入载体ｐＢｌｕｅｓｃｒｉｐｔ和表达载体ｐＧＥＸ中，分别构建了重组质粒ｐＢＳＫ９ＲＡＰ和ｐＧＥＸ－９ＲＨ７，转化入大肠杆菌ＸＬ１－Ｂｌｕｅ和ＤＨ５α中，经ＩＰＴＧ、ｘ－ｇａｌ和抗生素筛选阳性克隆，扩增后，重组质粒用限制性内切酶图谱分析及ＤＮＡ测序分析，证明插入子插入方向正确，表达阅读框架正确。

The cDNA encoding receptor-assciated protein (RAP) consisted of 319 anmo acid,we cionned by RT-PCR,which was inserted into pBluescript SK(+) and pGEX vector.After transformation into the XL1-Blue,the pGEX-9RH7 were solected with Blue/while.The recombinated plasmid were constructed and analysised by Restriction Enzymes and DNA sequensing.The result showed that the inserting oriontation of the RAP-cDNA encode are correctted,and acquired correctly reading frame.