摘要
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.
出处
《生物技术》
CAS
CSCD
北大核心
2009年第1期10-13,共4页
Biotechnology
基金
中国博士后科研基金项目("小麦细胞分裂素氧化酶基因的克隆和功能分析"
20070411101)
山东省博士后创新专项资金项目("小麦细胞分裂素氧化酶基因的功能鉴定"
200701001)资助