摘要
目的构建hPer1bHLH PAS结构域的酵母双杂交系统 ,以寻找与hPer1相互作用的新蛋白。方法构建pGBKT7 hPer1bHLH PAS重组诱饵质粒及 pGADT7 Rec 脑cDNA文库质粒 ;将两种质粒顺序转染至酵母细胞AH1 0 9中 ,进行营养缺陷筛选阳性克隆株。结果经酶切和基因测序鉴定 ,证实重组诱饵质粒 pGBKT7 hPer1bHLH PAS构建成功。脑cDNA文库转化效率为 1 .2× 1 0 6/3μgpGADT7 Rec。结论 酵母双杂交文库筛选得到 2 4个阳性克隆。这为寻找脑组织中与hPer1相互作用的未知蛋白奠定了基础。
Objective To construct yeast two-hybrid system of the bHLH-PAS domain of hPer1. Method The bHLH-PAS domain of hPer1 cDNA was amplified by PCR. The amplified fragment of hPer1 cDNA was ligased with the vector pGBKT7 to construct recombinant bait plasmid pGBKT7-hPer 1 bHLH-PAS. The cDNA library of human brain was constructed. The recombinant plasmid and the cDNA library were transfected into yeast strain AH109 by LiAc Method. The yeast two-hybrid system was screened by SD/-Ade/-His/-Leu/-Trp. Result Digested with endonuclease and sequenced, the recombinant plasmid of pGBKT7-hPer 1 bHLH-PAS was constructed correctly. The transformation rate was about 1.2×10 6/3 μg pGADT7-Rec. Conclusion Twenty-four positive colonies were selected by the yeast two-hybrid system. The positive colonies paved the way for further screening cDNA library and finding of unknown proteins interacting with the hPer1 protein.
出处
《航天医学与医学工程》
CAS
CSCD
北大核心
2004年第6期461-463,共3页
Space Medicine & Medical Engineering
基金
国家自然科学基金资助 (3 0 0 70 2 78
3 9970 2 75 )
