百日咳毒素S1亚基片段在大肠杆菌中的表达

Expression of S1 Subunit Fragment of Pertussis Toxin in E. coli

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-PAGE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17400,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。

Objective To construct a prokaryotic expression vector for the S1 subunit fragment of pertussis toxin（PT）and express in E. coli. Methods A S1 gene fragment with E. colipreferred codon was synthesized according to the S1 gene sequence reported 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 SDS-PAGE and 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.