纳豆激酶基因的克隆和高效表达

Cloning and high-efficiency expression of nattokinase gene

为了克隆纳豆激酶(Nattokinase,NK)基因,实现纳豆激酶在大肠杆菌中的表达,并对其表达条件进行研究,首先以从枯草芽孢杆菌中提取的基因组DNA为模板,通过PCR扩增包括信号肽、前导肽、成熟肽序列的前纳豆激酶原基因,将其克隆到载体pET28a中,构建重组质粒pET28a-NK,成功转化大肠杆菌BL21(DE3);然后优化影响该基因表达的温度、时间和IPTG添加量等因素。结果表明,重组菌株BL21(DE3)-NK在20℃下培养、IPTG添加量为0.4 mmol·L-1、培养20 h时表达产物的纤溶活性最大,可达到81.73 U·mL-1,SDS-PAGE检测观察到1条38 kDa的蛋白条带,与预期相符。

In this study, the nattokinase gene with signal peptide, propeptide and mature peptide was cloned and expressed in Escherichia coli, and the induction and expression parameters were optimized. The gene was amplified with genomic DNA extracted from Bacillus subtillis as template by RT-PCR, digested and ligated with expression vector-pET28a to construct recombinant plasmid pET28a-NK which was transformed into E coli strain BL21 （DE3）. Induction conditions such as time, temperature and IPTG concentration were studied, and the high- est fibrinolytic activity was obtained and it could reach 81.73 U.mL-1 when recombinant strain BL21 （DE3）-NK was cultivated at 20℃ for 20 h with IPTG at 0.4 mmol.L-1. An expected protein band about 38 kDa was observed by SDS-PAGE.