纳豆激酶原基因的克隆及表达

Cloning and Expression of Pro-nattokinase Gene in E.coli DH5α

从纳豆菌中克隆具有溶血栓性质的纳豆激酶原的基因 ,与质粒PBV2 2 0连接 ,转化大肠杆菌E .coliDH5α .挑取阳性克隆用EcoRⅠ /BamHⅠ双酶切鉴定得到一条约10 0 0bp的条带 .阳性克隆经温度诱导表达 ,用SDS_PAGE电泳鉴定 ,在 38kD处有一条明显的带 ,占菌体蛋白的 2 0 %左右 ,同时还表明该蛋白以包涵体的形式蛋白分子量为存在 .包涵体经收集。

The gene of pro_nattokinase, which is fibrinolytic enzyme,was amplified by PCR from chromosome DNA of Bacillus subtilis natto. The PBV220_Pro_NK was constructed and was transformed into E.coli .DH5 α . The positive clone was confirmed by EcoRⅠ/BamHⅠsequencing. A Mr.38kD protein was expressed in 20% of gross protein in E.coli DH5 α induced by increasing temperature from 32 ℃ to 42 ℃. The protein was wraped in inclusion body and showed fibrinolytic activity after refolding.