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目的 :用丙型肝炎 (丙肝 )病毒 (hepatitisCvirus,HCV)胞膜蛋白 2 (envelopeprotein 2 ,E2 )胞外区片段 ,构建酵母双杂交体系中“饵”载体 ,明确其在酵母细胞中的表达 ,排除自身激活作用 ,验证其能否用于酵母双杂交体系筛选cDNA文库。方法 :设计引物 ,从本室构建的含中国株Ⅲ型HCVE2片段的载体上 ,扩增出不包含C末端跨膜区的E2片段E2 6 6 1,经EcoRI和PstI双酶切后 ,克隆到酵母双杂交的“饵”载体pAS2 1质粒上 ,构建成载体pAS2 1 E2 6 6 1。阳性克隆经EcoRI和PstI双酶切鉴定后 ,转入酵母中 ,用酵母蛋白抽提物作Western杂交 ,证实其表达 ;滤膜印记杂交验证其有无自身激活作用。结果 :所构建的载体 pAS2 1 E2 6 6 1双酶切后 ,片段大小正确 ;转入酵母后 ,提取酵母质粒 ,PCR扩增出预期大小的片段 ,Western杂交出现阳性条带 ;滤膜杂交 ,转有 pAS2 1 E2 6 6 1和阴性对照pLAM5 ’ 1的酵母菌落没有激活报告基因 ,而GAL4全长的阳性对照菌落变为蓝色。结论 :含有HCVE2 6 6 1的“饵”载体构建正确 ,在酵母细胞中能表达出E2蛋白 ,并且没有自身激活报告基因的功能 ,能够应用于酵母双杂交体系。
OBJECTIVE: To construct the “bait” vector in yeast two-hybrid system by using the extracellular domain of envelope protein 2 (E2) of hepatitis C virus (HCV), and to clarify its expression in yeast cells. Exclude the activation of its own, to verify its ability to yeast two-hybrid screening of cDNA libraries. Methods: Primers were designed to amplify E2 E2 6 6 1 without the C-terminal transmembrane region from the vector containing the Chinese type Ⅲ HCVE2 fragment. After digested with EcoRI and PstI, Yeast two-hybrid “bait” vector pAS2 1 plasmid, construct vector pAS2 1 E2 6 6 1. The positive clones were digested with EcoRI and PstI, and then transformed into yeast. The positive clones were confirmed by Western blotting with yeast protein extract. The positive and negative clones were verified by filter blot hybridization. Results: The constructed vector pAS2 1 E2 6 6 1 double digestion, the fragment size correct; transferred yeast, yeast extract plasmid PCR amplification of the expected size of the fragment, Western blot positive bands; filter hybridization, Yeast colonies that were transfected with pAS2 1 E2 6 6 1 and negative control pLAM 5 ’1 did not activate the reporter, whereas the positive control colony of GAL4 full-length turned blue. CONCLUSION: The “bait” vector containing HCVE2 6 6 1 is constructed correctly and can express E2 protein in yeast cells without the function of activating the reporter gene itself, and can be applied to the yeast two-hybrid system.