【摘要】 利用PCR方法从分泌纳豆激酶的纳豆杆菌基因组DNA中扩增纳豆激酶基因 ,将该基因克隆到表达载体PBV2 2 0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 ,用凝块溶解时间法 (CLT)测出表达产物具有溶解血栓活性 ,证明该基因可在大肠杆菌中表达 .对重组菌中重组质粒的稳定性进行研究 ,结果表明质粒的插入对宿主菌的生长没有太大影响 ,该质粒在宿主菌中具有良好的分离稳定性 ,但结构稳定性较差 .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 .更多还原

【Abstract】 In this study, Nattokinase(NK) gene was amplified by PCR using Bacillus subtilis chromosomal DNA as template and cloned into expressed vector, PBV220. After being analyzed by restriction enzyme and PCR, the NK gene was expressed in E.coli and expression products were proved to have fibrinolytic activity by CLT. Transformation of recombinant plasmid has no effect on the growth of host cell. The results showed that recombinant plasmid had excellent segregational stability but the structural stability was not so good in the host cell. The expression protein amounted to 12% of the total cell protein by SDS_PAGE.更多还原