纳豆激酶基因的克隆及其在大肠杆菌中的表达  

The Cloning of Nattokinase Gene and Its Expression in E.coli

作　　者：李莹[1] 陈斌[1] 敬俊锋[1] 何正波[1] 

机构地区：[1]重庆师范大学昆虫与分子生物学研究所重庆高校生物活性物质工程研究中心活性物质生物技术教育部工程研究中心,重庆400047

出　　处：《重庆师范大学学报（自然科学版）》2012年第1期77-81,共5页Journal of Chongqing Normal University:Natural Science

基　　金：重庆市自然科学基金重点项目(No.CSTC2008BA5030)

摘　　要：纳豆激酶是一种良好的天然蛋白酶类溶栓物质。国内外许多学者对该酶进行了基因工程研究,但在克隆表达过程中出现了许多长短不同的基因片段。本研究通过原产日本的优质纳豆中分离鉴定出高产纳豆杆菌N07并提取该菌株的全基因组;通过PCR手段扩增出能编码纳豆激酶信号肽,前导肽和成熟肽的前纳豆激酶酶原基因NK1,以及能编码纳豆激酶成熟肽的纳豆激酶基因NK2,构建了纳豆激酶基因的表达载体pET30a-NK1和pET30a-NK2,转化E.coli BL21后在大肠杆菌中表达,并进行了活性分析。结果发现,纳豆激酶酶原基因片段NK1能成功表达出有活性的分泌型纳豆激酶;而纳豆激酶基因片段NK2的表达产物为无活性的包涵体。在对NK1和NK2的比较研究后可知,纳豆激酶酶原基因片段NK1能在大肠杆菌中很好的分泌表达,这将为纳豆激酶基因工程的深入研究奠定基础。Nattokinase is a natural streptokinase. Many scholars at home and abroad for the enzyme of genetic engineering research, but the genetic sequences in cloning expression appeared in much different length. The present study aims to determine which fragment of the nattokinase gene is better for the expression of the gene in E. coli BL21. We first isolated the effective fibrinolytic strain Bacillus subtilis N07 from Japanese natto, and extracted its genomic DNA. The nattokinase gene fragment Pre-Pro-NK （ NK1 ） that encodes signal peptide, propeptide and mature peptide, and the fragment NK（NK2） that only encodes mature peptide were then separately amplified from the genomic DNA using PCR with specific primers. Two expression vectors, pET30a-NK1 for NK1 and pET30a-NK2 for NK2, were constructed and transferred into E. eoli BL21, followed by the multiplying of E. coli BI21 culture induced by IPTG, the isolation and purification of nattokinase and the detection of nattokinase activity on fibrin plates. The results showed that the bacteria from pET30a-NK1 effectively produced the secretive nattokinase with high fibrinolytic activity. However, the bacteria from pET30a-NK2 could not produce active nattokinase. This research has compared the expression of NK1 with NK2 in E. coli BL21, and the NK1 could express well; it would be helpful in producing nattokinase by engineering.

关 键 词：纳豆激酶基因 大肠杆菌 克隆 表达 

分 类 号：Q785[生物学—分子生物学] Q786