hCu,Zn-SOD-TAT PTD融合基因的构建和表达

Construction and expression of human Cu,Zn-SOD-TAT PTD

目的 构建人hCu ,Zn SOD TATPTD表达载体及其原核表达与表达产物的纯化。方法 从人肺cDNA文库中PCR扩增hCu ,Zn SOD基因 ,插入 pUC19测序 ;以测序质粒为模板 ,再用含TATPTD编码序列的 3′引物PCR扩增得hCu ,Zn SOD TATPTD融合基因 ,并插入pUC19。将SOD TATPTD融合基因克隆至温控表达载体 pJW 2 ,转化大肠杆菌DH 5α。温度诱导表达 ,表达产物用离子交换层析法初步纯化。结果 经限制性内切酶酶切图谱分析和DNA序列测定表明所构建质粒与设计相同 ,hCu ,Zn SOD TATPTD融合蛋白在转化大肠杆菌获得了高效表达并纯化。结论 成功地获得了hCu ,Zn SOD TATPTD融合基因的表达产物 ,为进一步研究及临床应用奠定了基础。

Objective To construct recombinant vector for expression of human Cu,Zn-SOD-TAT PTD in E.coli. Methods Human Cu,Zn-SOD gene was amplified by PCR from human lung cDNA library, inserted into pUC 19 and identified by sequencing.The plasmid was used as the template for PCR amplification of Cu,Zn-SOD-TAT PTD fusion gene with a primer which contained TAT PTD coding frame. The Cu,Zn-SOD-TAT PTD fusion gene was cloned into temperature-inducing expression vector pJW 2 and the recombinant plasmid was transformed and expressed in E. coli DH5α.The Cu,Zn-SOD-TAT PTD fusion protein was purified by chromatography. Results Restriction endonucleases mapping analysis and DNA sequncing demonstrated that the constructed plasmid was the recombinant one.The fusion protein was successfully expressed in E. coli and purified. Conclusion The results may lay a good basis for manufacture and clinical application of the rhCu,Zn-SOD-TAT PTD.