摘要
目的 :利用基因工程技术 ,构建表达具溶栓活性的纳豆激酶的大肠杆菌工程菌。方法 :利用PCR方法扩增纳豆激酶酶原 (pro-NK)基因 ,并克隆到表达载体pET3c上 ,构建表达pro -NK和载体上 2 2氨基酸短肽的融合蛋白的表达质粒pENK ;表达质粒pENK分别转化溶源化宿主菌BL2 1(DE3)pLysS-和BL2 1(DE3)pLysS+ ,获得表达菌pENK - (DE3)pLysS-和pENK - (DE3)pLysS+ 。利用SDS -PAGE和纤维蛋白平板法检测目的蛋白的表达及活性。结果 :SDS-PAGE显示两株菌株均表达 4 2kD的目的蛋白。纤维蛋白平板法显示表达产物具溶栓的活性。在pENK -(DE3)pLysS-中融合蛋白不需异丙基硫代 - β -D -半乳糖苷 (IPTG)诱导就有基础表达 ,融合蛋白的表达使表达菌细胞溶解 ,菌落中空 ,表明表达产物具细胞毒作用。结论 :本研究成功实现了在大肠杆菌表达具有溶栓活性的pro -NK融合蛋白 ,为开发纳豆激酶成为新一代溶栓药物的研究奠定基础。
AIM: To construct engineered E.coli strains which can express nattokinase with fibrinolysis activity using gene engineering technology. METHODS: The pro-nattokinase (pro-NK) gene was amplified by PCR and inserted into expression vector pET3c. The recombined plasmid pENK which expressed the fusion protein of pro-NK and 22 amino acid peptide was then transferred into lysogenic host strains BL21(DE3)pLysS - and BL21(DE3)pLysS +. Both SDS-PAGE and the fibrin plate assay were used to examine the expression and the activity of the target protein. RESULTS: SDS-PAGE assay showed the fused gene encoding 42 kD fusion protein was expressed in both expression strains pENK-(DE3)pLysS - and pENK-(DE3)pLysS +, and the fibrin plate assay indicated that the expression product had fibrinolysis activity. pENK-(DE3)pLysS - exhibited the basal expression of the target gene, while fusion protein was only induced by IPTG in pENK-(DE3)pLysS +. Basal expression of the fused toxic gene in pENK-(DE3)pLysS - led to bacteriolysis and hollow lawns. CONCLUSION: A pro-NK fusion protein with fibrinolysis activity is successfully expressed in E.coli , which lay a foundation for the exploitation of nattokinase.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2003年第6期757-761,共5页
Chinese Journal of Pathophysiology
基金
广东省自然科学基金重点项目 (No .0 2 12 0 2 )
