摘要
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: Using hepatitis C virus (HCV) E2 gene without transmembrane domains to construct the bait vector, which can express E2 protein in yeast cell, and can be used in yeast two hybrid as “bait plasmid” to look for the gene from the cDNA library, which expresses the protein that can interact with the HCV E2 protein. Methods: PCR was performed to amplify the HCV E2 gene fragment E2 661 from the vector pKK223 E2 constructed by our institute. The production of the amplification was inserted into the “bait” plasmid pAS2 1 after the digestion with the restricted endonuclease of EcoR I and Pst I. After verified by restriction endonucleases, the plasmid was transformated into the yeast cell. PCR was used to verify whether the plasmid was transformed into yeast. The E2 protein expressed in the cell was confirmed by Western blot. Using colony lift filter assay to verify the constructed plasmid alone could not activate the reporter gene in the yeast cell. Results: Digested by two endonucleases, the recombined vectors pAS2 1 E2 661 produced anticipated fragment. PCR verified that there was E2 fragment in the yeast. Having assayed by Western blotting, it was showed that the yeast cell transformated with pAS2 1 E2 661 vector had positive signal which could not be seen in the control. Tested by the colony lift filter assay, the plasmid and negative plasmid pLAM5' 1 could not activate LacZ reporter gene in the yeast, while the cell transformed with positive control plasmid pCL1 the carrying whole GAL4 gene turned to blue, which indicated that the reporter gene was activated. Conclusion: Bait plasmid was constructed correctly and could express HCV E2 661 proteins in the yeast cell but could not activate transcription of LacZ reporter gene alone. The plasmid can be used in yeast two hybrid. (J Beijing Med Univ, 2000,32:113 116)
出处
《北京医科大学学报》
CSCD
2000年第2期113-116,共4页
Journal of Peking University(Health Sciences)
