摘要
目的 研究高效表达葡激酶载体的构建及其体外表达与产物的纯化。方法 用PCR方法扩增葡激酶cDNA ,插入质粒pET 2 2b中构建成表达载体 ,转化入大肠杆菌BL2 1(DE3) ,经IPTG诱导表达 ,表达产物用Q Poros和S Sepharose纯化。 结果 经限制性内切酶酶切图谱分析和DNA序列测定证明所构建质粒为葡激酶重组质粒 ,体外表达产物经离子交换纯化后HPLC纯度达 98%以上 ,SDS PAGE和WesternBlot实验证实该产物为葡激酶。结论 成功构建了重组葡激酶表达载体 ,并经表达纯化获得高纯度葡激酶 ,为产业化和临床应用奠定了良好基础。
Objective To study construction of vector to overexpress staphylokinase, expression in vitro and purification of the product. Methods PCR was used to amplify cDNA of staphlokinase, which was inserted into pET-22b to construct expression vector. The vector was transformed into BL21(DE3) and IPTG used to induce expression. Q-Poros and S-Sepharose were used to purify expressed products. Results Restriction endonucleases mapping analysis and DNA sequencing demonstrated that the constructed plasmid was recombinant one. The expressed protein was purified by ion exchange, and the purity is over 98% by HPLC analysis. Moreover, the expressed protein was proved to be staphylokinase by SDS-PAGE and Western blotting. Conclusion The expressed vector is constructed and expressed successfully, and the high purity staphylokinase is obtained, which lay a good basis for manufacture and clinical application of the enzyme.
出处
《安徽医科大学学报》
CAS
2001年第1期18-21,共4页
Acta Universitatis Medicinalis Anhui
