纳豆激酶基因的克隆及其在大肠杆菌中的表达

Cloning of Nattokinase Gene and Its Expression in E. Coli

利用ＰＣＲ方法以分泌纳豆激酶的纳豆杆菌染色体ＤＮＡ为模板扩增纳豆激酶基因，将该基因克隆到质粒载体ＰＵＣ１９上，筛选重组子，通过限制性内切酶和ＰＣＲ技术分析初步确定该重组子所携外源基因为纳豆激酶基因。利用基因重组技术构建了纳豆激酶基因的表达载体，并在大肠杆菌中进行了表达，凝块溶解时间法（ＣＬＴ）测出表达产物具有溶解血栓活性。在确定了最佳培养时间与诱导时间后，ＳＤＳ－ＰＡＧＥ分析结果表明基因表达产物为分泌型，蛋白表达量占菌体蛋白的１２％左右。

In this study, the nattokinase(NK) gene was amplified by PCR with bacillus subtilis genomic DNA as the template and cloned into PUC19 vector. After analyzed by restriction enzyme and PCR , the NK gene was cloned into the expression vector and expressed in E. coli. It was shown that the expression products have the fibrinolytic activity determined by CLT and the expression protein accounted for 12% of the total cell protein by SDS-PAGE. The optimum cultivating and inducing times were determined as 6 h and 5 h respectively.