链激酶基因的分离和其在大肠杆菌中的表达及表达产物的纯化  

Isolation of Streptokinase Gene and Its Expression in Escherichia coli and Purification

作　　者：周俐梅[1] 王嘉玺[1] 邹民吉[1] 段聚宝[1] 赵春文[1] 

机构地区：[1]军事医学科学院基础医学研究所

出　　处：《生物工程学报》1996年第S1期177-183,共7页Chinese Journal of Biotechnology

摘　　要：从化脓性链球菌的染色体ＤＮＡ分离得到的目的基因片段定向插入载体ｐＢＶ２２０中，获得有目的基因插入的重组质粒ｐＢＶＳＫ。双脱氧链终止法手工测序和Ｂｅｃｋｍａｎ自动测序仪分析ｒＳＫ基因的全长序列，测序结果表明我们所获得的ｒＳＫ基因与文献报道的基本一致。重组质粒ｐＢＶＳＫ转化受体菌Ｅ．ｃｏｌｉＤＨ５α株，温敏诱导后表达一个分子量约４７ｋＤａ的特异蛋白，其表达量为６０％。表达产物以包涵体的形式存在，包涵体经过洗涤、溶解和纯化后，最终纯度达到９５％以上。复性的重组蛋白用体外血块溶解法测定生物活性，结果表明我们所获得的ｒＳＫ蛋白具有较好的溶解血块的活性，其比活性为１３×１０５ｕ／ｍｇ。Streptokinase (SK) is one of the most effective activators of human plasminnogen and effective in dissolving coronary thrombi in Acute Myocardil Infarction. In order to obtain plenty of SK preparation and decrease danger in the process of the production of SK directly from streptococci, we have constructed an engineered strain E.coli DH5α/pBV SK by DNA recombinant technologies, and characterized by restriction analysis, DNA sequencing, SDS PAGE and bioassays. The recombinant protein was over expressed in the form of Inclusion body and constituted 60% of total cell proteins, and the specific activity was 1.3×10 5u/mg. The availability of the recombinant SK in high yield and purity offers a potentially attractive alternative source of this important therapeutic agent.

关 键 词：链激酶 基因表达 大肠杆菌 血纤维蛋白溶酶原 

分 类 号：Q344.13[生物学—遗传学]