纳豆激酶原核表达纯化及多克隆抗体的制备

Expression of nattokinase in E.coli.and preparation of its polyclonal antibody

纳豆激酶是一种源于日本传统食品纳豆且具有强烈溶栓作用的丝氨酸蛋白酶.本研究将编码信号肽、前导肽和成熟肽在内的纳豆激酶基因克隆到原核表达载体pET28a+中,获得重组表达质粒pETNK.将此重组表达载体质粒转化到大肠杆菌BL21中,经IPTG诱导表达.SDS-PAGE结果显示纳豆激酶在大肠杆菌中成功表达,表达的纳豆激酶融合蛋白分子量大约为31kD.亲和层析法纯化表达产物,并以此纯化产物作为免疫原免疫新西兰大白兔,制备兔抗纳豆激酶血清,用ELISA和Western blotting对制备的兔抗血清中的多克隆抗体进行了鉴定.纤维蛋白平板实验显示,表达的纳豆激酶融合蛋白具有较好的溶纤活性.

Nattokinase is a strong fibrinolytic serine protease from the Japanese traditional food natto. In this study, the nattokinase gene was cloned into a prokaryon expression vector pET28a^＋ to construct the recombinant plasmid pETNK . The recipient cell of Escherichia coli BL21 was transformed with pETNK. Nattokinase was expressed under the induction of IPTG and identified by SDS-PAGE. The molecular mass of expressed rNK is about 31kD. An emulsion was prepared with the purified Nattokinase and injected into rabbits. The polyclonal antibody in the rabbits serum was identified by ELISA and Western blotting . The fibrin plate assay indicated that the nattokinase fusion protein can cleavage fibrin effectively.