【摘要】 目的构建用于筛选人CD4结合蛋白的酵母双杂交饵载体,并对其功能进行鉴定。方法 RT-PCR扩增编码CD4的cDNA。构建双杂交饵载体pAS2-1-CD4,并转化入宿主酵母菌Y190中。通过测定报告基因——β半乳糖苷酶的活性,判断饵载体自激活报告基因能力。将CD4的已知HIV配体gp120 构建人猎物载体pACT2,转入重组菌Y190／pAS2-1-CD4。检测其报告基因活性,鉴定pAS2-1-CD4是否具有筛选CD4结合蛋白的能力。结果DNA序列测定证实,获得了编码CD4蛋白的cDNA序列。β半乳糖苷酶活性测定表明,pAS2-1-CD4单独无自激活能力,与猎物载体pACT2-gp120能够发生相互作用,激活报告基因表达。结论构建了酵母双杂交饵载体pAS2-1-CD4,为筛选CD4结合蛋白(尤其是筛选病毒编码的 CD4配体),提供了技术平台。更多还原

【Abstract】 To construct and identify a yeast two-hybrid system bait vector for screening of Homo sapiens CD4-binding proteins. Methods CD4 cDNA has been amplified by reverse transcription-PCR(RT-PCR), cloned into pAS2-1 vector and then transformed into Saccharamyces oerevisiae strain Y190. To find β-galactosidase, the self-activating effect was tested by filter assay. The known ligand of CD4 encoded by HIV, gp120 was cloned into the prey vector pACT2 and then transformed into Y190/pAS2-l-CD4. Its β-galactosidase activity of the recombinant cells were assayed. Results CD4 cDNA has been cloned and used to construct recombinant bait vector hpAS2-1-CD4. The pAS2-1-CD4 transformed into Y190 didn’t show its self-activation effect. The Y190 transfonned with pAS2-1-CD4 and pACT2-gp120 showed β-galactosidase activity. Expressed proteins of these two vectors can interact each other. Con-clusiom The yeast two-hybrid bait vector offers an applicable platform for screening the proteins, especially the viral ligands that combined with CD4.更多还原