玉米大斑病菌STK1原核表达载体的构建及其表达

Construction and Expression of Prokaryotic Expression Vector of STK1 from Setosphaeria turcica

【目的】构建玉米大斑病菌STK1原核表达载体并进行表达,以期得到带有His标签的目的蛋白。【方法】根据GenBank中STK1(AY849317)的cDNA序列及原核表达载体pET28a(+)中的多克隆位点设计引物,进行STK1的克隆和原核表达载体的构建。将重组载体在原核表达体系中进行表达。通过SDS-PAGE电泳鉴定蛋白的表达,并利用Western blot技术验证该蛋白是否为目的蛋白。【结果】STK1在大肠杆菌中的表达主要以包涵体形式存在;蛋白的分子量约为40.8kD;经1mmol·L-1 IPTG在37℃下诱导,9h后蛋白产量达到最高;经Western blot检测该表达产物具有His-6抗原性。【结论】玉米大斑病菌MAPK信号转导途径中的STK1在大肠杆菌中获得了表达,为今后STK1蛋白的多克隆抗体制备及功能研究奠定基础。

Objective Prokaryotic expression vector of STK1 from Setosphaeria turcica was constructed and expressed to obtain His-tagged protein.Method A pair of primers were designed according to multiple clone sites in prokaryotic expression vector pET28a（＋） and STK1 which was obtained in authors＇ previous experiment（GenBank accession：AY849317）.The open reading frame of 1 071 bp was amplified by PCR from S.turcica and was linked with pET28a（＋）,and then transformed into the host E.coli strain DH5α.The fragment was conformed to the original sequence.It indicated that the expression vector pET28a（＋）-STK1 was constructed successfully.The pET28a（＋）-STK1 plasmid was taken and transformed into BL21（DE3） for expression.Induced by IPTG at 37℃,the expression product of STK1 was identified by SDS-PAGE and Western blot.Result The expected protein Stk1 had been expressed successfully in the form of inclusion bodies.The molecular weight of protein was 40.8 kD,and the quantity produced reached the highest when it was induced with 1 mmol·L-1 IPTG for 9 h.Meanwhile,the expressed protein showed the antigenic characteristics of His-6 tagged protein.Conclusion STK1 in signal transduction pathway of mitogen activated protein kinase from S.turcica was successfully expressed in E.coli,thus laying a foundation for the preparation of STK1 polyclonal antibody and for the study of its function.