百日咳毒素S1亚基片段在大肠杆菌中的表达.doc

【基础研究】中国图书分类号 Q78 Q786 文献标识码 A 文章编号 1004-5503（2009）04 【基础研究】 中国图书分类号 Q78 Q786 文献标识码 A 文章编号 1004-5503（2009）04-0320-03 百日咳毒素 S1 亚基片段在大肠杆菌中的表达 时成波 1 曹玉锋 1 张秀霞 1 吴晓娟 1 李雨桐 1 徐光磊 1 尹晓东 2 【 摘要 】 目的 构建百日咳毒素（PT）S1 亚基片段原核表达质粒，并在大肠杆菌中表达重组蛋白。方法 根据 GenBank 中 登录的百日咳毒素 S1 亚基基因序列，人工合成密码子优化的 S1 基因，利用重叠 PCR 技术将 S1 基因上的两段 DNA 序列拼接在 一起，形成一个新的基因序列 S1′。将该序列与 7ZTS 表达载体连接，构建重组原核表达质粒 7ZTS-S1′，转化大肠杆菌 JM109（DE3）， IPTG 诱导表达。表达产物经 SDS和 Western blot 进行鉴定。结果 S1′基因经 PCR 鉴定及测序证明与预期相符；重组表达 质粒经双酶切鉴定证明构建正确；表达蛋白的相对分子质量约 17 400，表达量约占菌体总蛋白的 8%，且具有良好的反应原性。 结论 已成功构建百日咳毒素 S1 亚基片段重组原核表达质粒，并在大肠杆菌中表达了重组蛋白，为 PT 单抗及其检测试剂盒的 研制奠定了基础。 【关键词】 百日咳毒素；S1 亚基；原核表达；重叠 PCR Expression of S1 Subunit Fragment of Pertussis Toxin in E. coli SHI Cheng-bo△, CAO Yu-feng, ZHANG Xiu-xia, et al （△Changchun Institute of Biological Products, Changchun 130062, China） 【Abstract】 Objective To construct a prokaryotic expression vector for the S1 subunit fragment of pertussis toxin（PT）and ex－ press in E. coli. Methods A S1 gene fragment with E. coli-preferred codon was synthesized according to the S1 gene sequence re－ ported in GenBank, based on which a new gene sequence S1 was obtained through splicing the two DNA sequences of S1 gene by overlap PCR and inserted into expression vector 7ZTS. The constructed recombinant plasmid 7ZTS-S1 was transformed to E. coli JM109 （DE3）for expression under induction of IPTG. The expressed product was identified by SDSand Western blot. Results The PCR and sequencing result of S1 gene was identical to those expected. Restriction map proved that recombinant plasmid 7ZTS-S1 was constructed correctly. The expressed product, with a relative molecular mass of about 17 400, contained about 8% of total somatic protein and showed good reactogenicity. Conclusion The prokaryotic expression vector of S1 subunit fragment of pertussis toxin was successfully constructed and expressed in E. coli, which laid a foundation of preparation of PT McAb as well as detection kit for PT