【摘要】 目的研究BI-1完整编码区能否在酵母细胞中表达,排除自身激活作用,并验证它能否用于酵母双杂交系统。方法以人正常肺组织cDNA为模板,扩增BI-1完整编码区,经EcoRⅠ、SalⅠ双酶切后克隆到酵母双杂交系统诱饵载体pGBKT7上,构建诱饵蛋白载体pGBKT7-BI-1,转化酵母细胞,通过β-半乳糖苷酶活性分析验证有无自激活,Western印迹验证其表达。结果DNA测序显示BI-1编码区内无突变,β-半乳糖苷酶活性分析显示转入pGBKT7-BI-1和阴性对照的酵母菌落没有激活报告基因LacZ,而阳性对照菌落呈现蓝色。结论含有BI-1完整编码区的诱饵载体不存在自身激活作用,并能在酵母细胞中表达BI-1蛋白,能够应用于酵母双杂交系统筛选相互作用蛋白。更多还原

【Abstract】 Objective To study whether the full lengh human BI-1 gene could be used for yeast two-hybrid analysis to look for the proteins which can interact with BI-1.Methods Using PCR to amplify the target gene from normal lung tissue cDNA library.Digest the vector pGBKT7 and the production of PCR with restriction enzymes EcoRI and SalI.Ligate the vector and the PCR production to construct DNA-BD recombination.After verified by sequencing,transform the construction into yeast cell AH109.Colony-lift filter assay was performed to test autoactiviation of the construction.Western blot was used to detect the expression of BI-1 protein.Results The target gene which does not have mutations in its whole ORF was inserted into pGBKT7 to construct pGBKT7-BI-1.Colony-lift filter assay suggested that the construction could not activate the reporter gene alone.Western blot showed that the yeast cell transformed with pGBKT7-BI-1 had positive signal which was not found in the control.Conclusion It could express BI-1 fusion protein in the yeast cell,but not activate transcription of LacZ reporter gene,so the construction can be used for yeast two-hybrid analysis.更多还原