纳豆激酶基因在毕赤酵母中的表达纯化及抗体制备  

Expression,purification of nattokinase in Pichia pastoris and preparation of its polyclonal antibody

作　　者：蔡立涛[1] 徐祥[1] 王婷婷[1] 喻梅星[1] 杨艳燕[1] 

机构地区：[1]湖北大学生命科学院,湖北武汉430062

出　　处：《中国生化药物杂志》2010年第1期10-13,共4页Chinese Journal of Biochemical Pharmaceutics

基　　金：湖北省自然科学基金(2003ABA116);湖北省中药生物技术重点实验室基金资助项目

摘　　要：目的实现纳豆激酶(NK)在巴斯德毕赤酵母中的分泌表达,以纯化产物为抗原制备兔抗NK血清。为建立双抗夹心酶联免疫吸附反应法测定生物体内NK含量,进一步研究NK在体内代谢与功能奠定基础。方法将NK基因克隆到毕赤酵母表达载体pHBM905A中,鉴定后的重组质粒用SalⅠ酶切线性化后电转化到毕赤酵母宿主菌GS115中,甲醇诱导表达,经盐析和Millipore超滤膜过滤分离纯化表达产物。纤维蛋白法检测纯化产物的活性,并以纯化产物为抗原免疫新西兰大白兔制备兔抗NK血清。结果SDS-PAGE结果显示NK成功表达,其相对分子质量(Mr)约为27 000,纤维蛋白平板实验显示表达产物具有较好的纤溶活性。酶联免疫法测得多克隆抗体效价约为1∶8 000,Western blot结果显示在Mr27 000附近有1条特异带出现。结论NK在巴斯德毕赤酵母中实现了分泌表达,表达产物具有较好的纤溶活性和免疫原性。Purpose To indicate the expression of nattokinase（NK） in Pichia pastoris, an emulsion was prepared with the purified NK to prepare polyclonal antibody. In order to establish sandwich enzyme-linked im- munosorbent assay（ELISA） to assay NK in organism, furthermore to lay the foundation for researching in vivo metabolism and function of NK. Methods The NK gene was cloned into a Pichia pastoris expression vector pI-IBM905A to construct the recombinant plasmid pPRONK. The recipient cell of Pichia pastoris GS115 was transformed with pPRONK which had been cut by restriction enzyme Sal I , under the induction of methanol. The expressed production is purified by salting out and uhrafihration membrane. An emulsion was prepared with the purified NK and injected into rabbits to prepare polyclonal antibody. Results NK was expressed and identi- fied by SDS-PAGE. The molecular mass of expressed production is about 27 kD. The fibrin plate assay indicat- ed that the NK protein can cleavage fibrin effectively. ELISA analysis indicated that the polyclonal antibody titer is about 1 ： 8 000. Western blot demonstrated that there was a special strap nearby 27 kD. Conclusion NK was successfully expressed in Pichia pastoris, the production can cleavage fibrin effectively and it had great im- munogenicity.

关 键 词：纳豆激酶 巴斯德毕赤酵母 纤溶活性 多克隆抗体 

分 类 号：Q78[生物学—分子生物学]