人锰超氧化物歧化酶的扩增和表达研究

Amplification and Expression of Human Manganese Superoxide Dismutase Gene

构建人锰超氧化物歧化酶的原核表达载体,对影响蛋白表达的因素进行探索,并初步检测其活性。以人293T细胞cDNA为模板,利用PCR方法克隆人锰超氧化物歧化酶基因,将其插入pET15b载体中,经测序鉴定正确后,将重组质粒转化大肠杆菌Rosetta-gami,通过IPTG诱导,SDS-PAGE检测菌体总蛋白。利用SOD检测试剂盒检测粗提物的SOD活性。结果:PCR扩增了一个长597bp的基因片断,该片断编码由198个氨基酸组成的成熟的人锰超氧化物歧化酶蛋白。经过IPTG诱导表达出约25k的蛋白。优化的IPTG浓度为0.25mmol/L,在1～6h范围内随着诱导时间的增加表达量逐渐增加,IPTG总量一样的情况下分次加入比单次加入所诱导的目的蛋白产量高。SOD活性检测表明经过诱导表达的hMn-SOD具有SOD活性。结论:成功扩增人锰超氧化物歧化酶基因,并构建了原核表达载体,经过IPTG诱导表达的目的蛋白有SOD活性。

To construct prokaryotic expression vector of the human manganese superoxide dismutase gene, to explore the factors which infect protein expression, and to detect the SOD activity of protein, PCR was employed to clone the human manganese superoxide dismutase gene using cDNA of 293T as template. The DNA fragment was inserted into pET15b vector. After sequencing identification, the recombinant plasmid was transformed into E. coli Rosetta gami as host strain. Expression of the target protein was induced with IPTG and SDS-PAGE was conducted to analyze the total protein. SOD activity assay kit was used to detect the SOD activity of crude extracts. Results showed that a DNA fragment of 597bp was cloned and sequence analysis indicated that it encoded a mature human manganese superoxide dismutase protein of 198 amino acid residues. After induction with IPTG, an expected molecular mass of 25ku was detected by SDS-PAGE. The optimized IPTG concentration is about 0. 25 mmol/L. At range of 1 to 6 hours, the expression level increases with the increase of time and the expression level is high when IPTG was added separately. The human manganese superoxide dismutase gene was cloned and the prokaryotic expression vector was constructed correctly. The human manganese superoxide dismutase was expressed successfully in E. coli and the expressed protein was functionally activated.