小麦细胞分裂素氧化酶基因TaCKX1原核表达载体的构建及表达

Construction of Prokaryotic Expression Vector of Wheat TaCKX1 Gene and Its Expression

目的:构建小麦细胞分裂素氧化酶基因TaCKX1原核表达载体并进行表达,以期得到大量的His标签融合蛋白。方法:根据GenBank中的TaCKX1序列以及pET-24a载体中的多克隆位点设计引物,以含有TaCKX1编码基因的批pMD-QRCKX1重组质粒为模板,经PCR扩增得到TaCKX1基因的DNA片段。将所得的片段与pET-24a载体连接,转化DH5α大肠杆菌,筛选阳性克隆,其测序结果与原序列一致,表明原核表达载体pET-TaCKX1已构建成功。提取pET-TaCKX1质粒转化到BL21(DE3)plysS表达菌株中,经IPTG诱导后收集菌体进行SDS-PAGE电泳鉴定,并优化其表达条件。结果:在大肠杆菌中获得TaCKX1基因融合表达,主要以包涵体形式存在;融合蛋白的分子量为58.9kD;IPTG终浓度为0.5、1.0、1.5、2.0mmol/L时,诱导融合蛋白产量相差不大。选用0.5mmol/L诱导15h获得大量的融合蛋白。经用原核表达蛋白纯化试剂盒纯化,得到了单一的融合蛋白。结论:小麦TaCKX1基因在大肠杆菌中获得了高效表达,为今后TaCKX1蛋白多克隆抗体的制备奠定了基础。

Objective： Prokaryotie expression vector of wheat TaCKX1 gene was constructed and was expressed to obtain 6 x His＇Tag fusion protein. Method： A pair of primers was designed according to digestion sites in plasmid pET - 24a and the TaCKX1 gene sequence published by GenBank. The DNA fragment of 1 575bp was amplified by PCR from the pMD- QRCKX1 recombinant plasmid with TaCKX1 gene, then cloned into pET- 24a and transformed into the host E. coli strain DH5α. The fragment was conformed to the original sequence. It indicated that fusion expression vector pET - TaCKX1 was constructed. The pET- TaCKX1 plasmid was transformed into BL21 （DE3） plysS for expression. Induced by IPTG at 37℃, the expression product of TaCKX1 gene was identified by SDS- PAGE, and the expression condition was optimized. Result： TaCKX1 fusion protein had been expressed successfully in the form of inclusion bodies. The molecular weight is 58.9kD. Meanwhile, the condition of TaCKX1 fusion protein expression was induced with 0.5mmol/L IPTG for 15 hours. Wheat TaCKX1 gene was successfully expressed in E. coli, which was prepared for TaCKX1 polyclone antibody. Conclusion： Wheat TaCKX1 gene was successfully expressed in E. coli, which was prepared for TaCKX1 polyclone antibody.