人硫氧还蛋白还原酶cDNA的克隆及在大肠杆菌中的表达

Cloning of Human Thioredoxin Reductase cDNA and Its Expression in E. coli

目的:克隆人硫氧还蛋白还原酶(TR)基因并构建原核表达载体,获得TR蛋白。方法:采用反转录聚合酶链式反应(RT-PCR)技术,从人胚脑组织中扩增出TR cDNA片段,克隆至pGEM-T载体并转化大肠杆菌DH5α,获得重组质粒pGEM-TR;经序列分析证实后,提取pGEM-TR重组体,由双酶切得到目的片段,并插入pET9c原核表达质粒,转化大肠杆菌BL21(DE3),获得重组质粒pET-rhTR。异丙基硫代-β-D-半乳糖苷(IPTG)诱导pET-rhTR在BL21中表达,用SDS-聚丙烯酰胺凝胶电泳(PAGE)及免疫蛋白印迹(Western blot)分析重组蛋白。结果:克隆的TR cDNA与人胎盘组织TR cDNA同源性为99％;pET-rhTR在大肠杆菌中高效表达,目的蛋白占菌体总蛋白量的36％,其分子质量为55 ku。TR cDNA序列被GenBank收录,登录号为AF208018。结论:成功克隆了人脑组织来源的TR基因,该基因在大肠杆菌中得到高表达,为进一步研究其功能奠定基础。

Objective: To clone thioredoxin reductase (TR) gene and express in E. coli. Methods: The cDNA of TR from human brain cell was amplified by reverse transcription-PCR. The amplified fragment was cloned into pGEM-T vector and sequenced. The re-combinant plasmid pGEM-TR was digested with Nde I and BamH I, and the target fragment was inserted into pET9c vector. Then, it was used to transform BL21 (DE3) cell and induced expression by IPTG. The expression of TR was analyzed by the methods of SDS-PAGE and western blot. Results: TR cDNA was cloned from human brain cell, and nucleotide and amino acid sequence were 99% homologous with it from placental tissue. The recombinant plasmid pET-rhTR could express richly in E. coli. The content of expressed protein shared the 36% of total bacterial protein, its molecular weight was 55 ku, The TR cDNA was registered in GenBank, and its No. is AF208018. Conclusion: The TR cDNA has been cloned from human brain and expressed richly in E. coli BL21 (DE3), which can pave the way for further studying its function.