乳糖诱导瑞替普酶在大肠杆菌中的可溶性表达  

Soluble expression of Reteplase in E.coli induced by lactose

作　　者：田文静[1] 罗学刚[1] 吕丽慧[1] 倪萌[1] 井晓兰[1] 张同存[2,3] 

机构地区：[1]天津科技大学生物工程学院工业微生物教育部重点实验室,天津300457 [2]武汉科技大学医学院,湖北武汉430065 [3]天津市工业微生物重点实验室,天津300457

出　　处：《中国生化药物杂志》2011年第5期341-344,348,共5页Chinese Journal of Biochemical Pharmaceutics

基　　金：国家高技术研究发展计划(863计划)(NO.2008AA10Z336)

摘　　要：目的构建瑞替普酶(rPA)大肠杆菌基因工程菌,优化乳糖诱导表达条件,并获得活性重组蛋白。方法通过PCR扩增得到rPA编码基因,将该基因片段克隆到载体pET40b中,转化E.coli BL21。优化确定乳糖诱导浓度、时间、温度等参数,将诱导表达后获得的菌体细胞通过超声破碎裂解后利用镍柱亲和色谱进行分离纯化,重组目的蛋白经Xa因子切割去除DsbC融合标签后释放获得rPA产物,最后利用纤维蛋白平板法对其溶栓活性进行检测。结果 rPA重组质粒构建成功,利用终浓度30 mmol/L的乳糖于30℃诱导5 h可以获得很好的表达效果,重组蛋白的表达量、可溶性及酶活与IPTG诱导产物基本接近;经纯化后的rPA目的蛋白可表现出明显的溶栓活性。结论本研究为进一步利用大肠杆菌系统表达生产高活性重组rPA提供了一定的参考依据。Purpose To establish the genetic E.coli for Reteplase,and to optimize the expression conditions induced by lactose.Methods The coding sequence of Reteplase（rPA） was amplified with PCR and then cloned into the pET40b vector.To achieve the high-level expression of soluble active reteplase,the induction expression conditions in E.coli strain BL21,including the concentration of inducer lactose,induction duration and temperature,were optimized.The thrombolytic activity of the recombinant K2S was finally determined using an indirect fibrin plate assay.Results The recombinant plasmid pET40b-rPA was constructed successfully.Lactose could be a satisfying inductor and it obtained nearly the same yield,soluble form and activity with the expression inducted by IPTG.The result of fibrinolysis fibrin plate assay showed that the purified target protein exhibited significant fibrinolysis activity in vitro.Conclusion These researches might establish a significant foundation for the following production of rPA.

关 键 词：瑞替普酶 DSBC 乳糖 大肠杆菌 表达 

分 类 号：Q78[生物学—分子生物学]