应用酵母双杂交技术研究人睾丸特异表达基因HSD-3.8的功能

Study on the Function of HSD-3.8 Gene Encoding a Testis-specific Protein with Yeast Two-hybrid system

目的探寻本实验室筛选出的与不孕症相关的人睾丸特异表达基因HSD-3.8(GenBank接收号为AF311312,国际上命名为精子相关抗原1)编码蛋白质的作用配体,揭示其参与受精过程的机制。方法采用酵母双杂交体系,构建含有HSD-3.8基因部分序列(HSD-0.7)的诱饵质粒,筛选人卵巢cDNA文库。在获得阳性克隆后,根据蛋白质结构分析并结合PCR方法重新构建一系列缺失体,在酵母体内验证它们与被捕获的蛋白因子之间的结合。结果酵母双杂交实验获得一阳性克隆,其编码氨基酸与人G蛋白β1亚基羧基端的144个氨基酸同源性为100%。所构建的几种诱饵蛋白缺失体在酵母体内均不能与该捕获的蛋白因子相互作用。结论HSD-3.8编码蛋白质片段HSD-0.7可能通过G蛋白信号转导途径在受精过程中发挥功能,与G蛋白β1亚基的结合有赖于其结构的完整。

Objective To explore the protein factors that could interact with the testis -specific protein encoded by HSD-3.8gene(GenBank Accession Number AF3113 12)related with female fertilization.Methods Yeast two -hybrid system was u sed to screen the human ovary MATCHMAKER cDNA library with constructed'bait pla smid'containing the0.7kb fragment (HSD-0.7)of HSD-3.8.The interacti on with the positive fragments using a series of truncated bait plasmids was inv estigated.Results One positive gene fragment was obtained,which coded for14 4amino acids of the C-terminus of human G protein beta subunit1.Truncated bai t plasmids couldn ' t interact with the fish protein fragment in yeast.Conclu sions The protein encoded by HSD-3.8gene may function through G protein sign al transduction pathway and the interaction depends on the integration of the ba it protein.