枯草杆菌DC-2纳豆激酶基因的克隆及其融合蛋白在E.coli中的表达

Cloning of Nattokinase of Bacillus subtilis DC-2 and Its Fusion Protein Expression in E.coli

纳豆激酶是一种纤维蛋白溶解酶 ,有望开发成为新型的溶栓药物 .从中国豆豉中分离的具有较强纤溶活性的枯草杆菌DC 2中提取总DNA ,根据纳豆激酶 (NK)基因序列设计引物 ,用PCR法扩增NK基因 .序列分析表明 ,NK基因成熟肽编码区含有 82 5bp ,编码 2 75个氨基酸残基 ,与文献报道的序列分别有 93 4 %和 94 5 %同源性 .将NK基因插入载体pGEX 4T1构建表达质粒pGEX NK ,转化大肠杆菌JM10 9后 ,经 1mmol LIPTG诱导 4h ,发现大量NK融合蛋白表达 ,并形成包涵体 .SDS PAGE分析表明 ,NK融合蛋白作为包涵体的分子量为 5 3kD .凝胶自动扫描结果显示 ,NK融合蛋白约占菌体可溶性蛋白的 2 6 % .

Nattokinase (NK) is a kind of fibrinolytic enzyme, which is expected to be a new thrombolytic drug. Bacillus subtilis DC-2 that is capable of producing a strongly fibrinolytic enzyme was isolated from Douchi, a Chinese traditional food. The mature peptide-coding fragment of NK gene was amplified from B. subtilis DC-2 total DNA by PCR. The sequence analysis indicated that the fragment of NK gene consisted of 825 bp, which encoded 275 amino acid residues. The sequence of NK gene and its deduced amino acids have 93.4% and 94.5% homology with those of the reported ones, respectively. The expression plasmid pGEX-NK was constructed by inserting NK gene into vector pGEX-4T-1, and then introduced into E.coli JM109. SDS-PAGE analysis revealed that the NK protein was highly expressed and accumulated up to above 26% of the bacterial soluble protein after induction with IPTG for 4 hours. The NK fusion protein of 53 kD was accumulated as an inclusion body in E.coli.