纳豆激酶基因的克隆表达以及活性分析

应用PCR的方法从分泌纳豆激酶的枯草杆菌基因组DNA中扩增得到全长为1056bp的前导肽+成熟肽纳豆激酶原基因（pro-NK）,并构建了纳豆激酶的重组表达载体pET-28a-pro-NK;转化E.coliBL21（DE3）后,在IPTG的诱导下实现了纳豆激酶的高效表达,经SDS-PAGE显示在28ku处有一特异带;表达产物经Ni2+亲和层析纯化后,纤维平板法测得的纳豆激酶的比活力为34245.1IU/mg;纯蛋白经透析和冷冻干燥处理后,纳豆激酶的比活力为16271.5IU/mg;在此基础上,对冻干保护剂的添加进行优化,最佳保护效果为甘露醇与纳豆激酶质量比为1:2,可比不加保护剂时比活力提高了30.9%。这可为基因工程方法生产纳豆激酶纯品,稳定其活性便于贮运,并将其开发成为临床药物提供技术参考。 

Cloning,expression of the nattokinase gene and the activity assay

The pro-nattokinase(pro-NK) gene,1056bp,was amplified from genomic DNA of Bacillus subtilis by PCR.The amplified pro-NK gene was cloned into the expression vector pET-28a to construct the expression vector pET-28a-pro-NK.The recombinant vector pET-28a-pro-NK was transformed into E.coli BL21(DE3),and the protein of mature NK was efficiently expressed with IPTG induction.The SDS-PAGE analysis showed that the expressed NK was about 28ku,which was consistent with the theoretical calculation.The specific activity of mature NK purified by Ni2+ affinity chromatography was 34245.1IU/mg,however,declined to 16271.5IU/mg after the treatments of dialysis and freeze-drying without protectants.The protectant was optimized to keep the activity of mature NK in high level,indicating that the best ratios of the mass of the Mannitol to nattokinase were 1:2,increased by 30.9% compared with the treatments without protectants.The research provided a reference for producing pure nattokinase by genetic engineering,stabilizing the activity,and developing the clinical medicine.