【摘要】 目的 :获取人肝再生增强因子 (hALR)阅读框的cDNA及构建其酵母双杂交系统的“诱饵”质粒。方法 :利用RT PCR方法 ,从人胎肝组织中扩增出一约 380bp的DNA片段 ,重组入pGBKT7载体中 ,构建成pGBKT7 hALR ,然后研究此重组质粒在酵母AH10 9中的表达情况并用于筛选人肝cDNA文库。结果 :获得的 378bp的DNA序列与文献报道的人ALR序列一致 ;转化的酵母菌在选择性培养基SD Trp上培养 6 5小时 ,长出约Φ1mm大小的白色菌落 ,而在SD Trp His上不生长 ;酵母提取液的Westernblot分析 ,证实ALR基因在AH10 9以融合蛋白的形式表达 ,并具有免疫活性 ;初步得到hALR相互作用的阳性克隆。结论 :pGBKT7 hALR对宿主菌AH10 9没有毒性作用 ,也没有自身激活报告基因 ,可作为酵母双杂交系统中的“诱饵”质粒。更多还原

【Abstract】 Objective:To obtained the cDNA encoding human augmenter of liver regeneration and construct the bait plasmid of yeast two hybrid system.Methods:About 380 bp DNA fragment was amplified from the human fetal liver by RT PCR.The fragment plasmid was ligated with the vector pGBKT7 to construct pGBKT7 hALR.The recombinant plasmid was used to transform into yeast AH109 to observe BD hALR expression in the yeast and was used to screen the gene encoding hALR associated protein from the human liver cDNA library by using yeast two hybrid system.Results:The nucleotide of RT PCR fragment was the same as reported hALR,and about Φ 1 mm white yeast clone was grown in the SD/ Trp after 65 h,but none can grow in the SD/ Trp/ His.The ALR was expressed as recombinant protein and has immunogenic activity as shown by Western blot.The genes of proteins interacting with hALR were cloned.Conclusion:pGBKT7 hALR is nontoxic to yeast and it cannot activate reporter genes.Therefore,pGBKT7 hALR may serve as a bait plasmid of yeast two hybrid system.更多还原