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HCVNS3基因片段酵母展示文库的构建和鉴定
引用本文:贾帅争,孙红琰,刘晓达,杜芝燕,杜勇,王全立,章扬培.HCVNS3基因片段酵母展示文库的构建和鉴定[J].中国生物化学与分子生物学报,2001,17(1):56-60.
作者姓名:贾帅争  孙红琰  刘晓达  杜芝燕  杜勇  王全立  章扬培
作者单位:1. 军事医学科学院输血研究所,
2. 军事医学科学院微生物流行病研究所,
基金项目:国家自然科学基金资助课题(No.39800133)
摘    要: HCV NS3特异的 CD4+T细胞反应与 HCV感染的良性转归相关 .为了筛选其 CD4+T细胞表位 ,构建了 HCV NS3基因片段酵母展示文库 .首先 DNase 不完全酶切 HCV NS3基因产生长度为 1 0 0~ 30 0 bp的随机片段 ,然后在它们两端加上含限制性内切酶 Bam H 作用位点的接头 ,再以接头序列作引物进行 PCR扩增 .最后扩增产物用 Bam H 酶切后与 Bam H 线性化的穿梭载体 p YD1连接 ,转化大肠杆菌 (E.coli) DH5α,共得到 2× 1 0 6个转化子 .转化菌落的 PCR扩增结果表明 ,约 50 %转化子含插入片段 .随机选择 5个插入片段测序 ,然后与 DNA序列数据库中的序列比较 .结果显示它们分别与 HCV NS3序列有 96%~ 99%的同源性 .用从转化菌落中提取的质粒转化酵母 (S.cerevisiae)菌株 EBY1 0 0 ,得到含 2× 1 0 5个插入片段的 HCV NS3基因片段酵母展示文库 .半乳糖诱导的酵母细胞通过和 FITC标记的抗体结合 ,用 FACS可以在 2 0 %的细胞表面检测到融合蛋白的表达 .

关 键 词:丙型肝炎病毒  非结构蛋白3  酵母展示  基因片段随机文库
收稿时间:2001-02-20
修稿时间:2000年4月17日

Construction and Identification of HCV NS3 Gene-Fragment Yeast Display Libraries
JIA Shuai\|zheng ,SUN Hong\|yan ,LIU Xiao\|da ,DU Zhi\|yan ,DU Yong ,WANG Quan\|li ,ZHANG Yang\|pei.Construction and Identification of HCV NS3 Gene-Fragment Yeast Display Libraries[J].Chinese Journal of Biochemistry and Molecular Biology,2001,17(1):56-60.
Authors:JIA Shuai\|zheng  SUN Hong\|yan  LIU Xiao\|da  DU Zhi\|yan  DU Yong  WANG Quan\|li   ZHANG Yang\|pei
Abstract:HCV NS3\|specific CD4\++ T cell response from patients with self\|limited infection is relevant to a benign course of HCV infection.In order to screening the CD4\++ T cell epitopes on HCV NS3,its gene\|fragment yeast display libraries were constructed.DNA encoding protein of HCV NS3 was partially digested with RQ1 DNaseⅠ to generate random fragments of 100~300 bp in length.These fragments were bluntly ended by T4 DNA polymerase,ligated with 5′ end phosphated 12\|mer linker which contained Bam HⅠ restriction site.The resulting fragments were ligated with Bam HⅠ digested yeast display vector pYD1.\%E.coli\% strain DH5α was transformed with the ligation mixture and approximately 2×10 6 transformants were obtained.That about 50% transformants contained inserts was confirmed by polymerase chain reaction.Five inserts were sequenced.Compared with those in DNA sequence databases,these sequences showed a high homology with those of HCV NS3.Shuttle plasmid pYD1 and recombinant plasmids were transformed into S.cerevisiae strain EBY 100 and yeast display libraries containing 2×10 5 independent inserts were obtained.After induction with galactose at 20℃ and stained with FITC labelled antibody,fusion protein could be detected on about 20% yeast cells surfaces.
Keywords:HCV  NS3  yeast display  gene\|fragment random libraries
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