HCVNS3基因片段酵母展示文库的构建和鉴定  

Construction and Identification of HCV NS3 Gene-Fragment Yeast Display Libraries

作　　者：贾帅争[1] 孙红琰[1] 刘晓达[1] 杜芝燕[1] 杜勇[2] 王全立[1] 章扬培[1] 

机构地区：[1]军事医学科学院输血研究所,北京100850 [2]军事医学科学院微生物流行病研究所,北京100071

出　　处：《中国生物化学与分子生物学报》2001年第1期56-60,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金!资助课题 (No.3980 0 133)

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 .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.

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 

分 类 号：R373.21[医药卫生—病原生物学] R512.63[医药卫生—基础医学]