水稻密码子优化的cry1Ca基因在大肠杆菌中表达、纯化及抗体制备

Expression and purification of rice codon optimized His-cry1Ca fusion protein in E.coli and preparation of polyclonal antibody against cry1Ca

摘要: 将水稻偏好密码子优化并人工合成抗虫基因cry1Ca通过限制性内切酶酶切的方法构建原核表达载体pET-28bca,成功表达了带6个组氨酸标签的His-cry1Ca融合蛋白,其表达量约占菌体总蛋白的24%,表达的融合蛋白主要以包涵体的形式存在。对包涵体蛋白进行可溶性处理,亲和纯化出带6个组氨酸标签的融合蛋白,将其作为抗原免疫家兔获得了滴度(ELISA)为l:50000的抗血清,并具有较强的特异性。

Abstract: Rice codon optimized cry1Ca gene was cloned into expression vector pET-28b(+) by means of restricted enzymatic digestion.His-cry1Ca fusion protein was highly expressed by SDS-PAGE analysis accumulating more than 24% of the total bacterial proteins.The main expression production was deposited as inclusion body.His-cry1Ca was then purified with Ni-NTA after treatment of solvents.The target protein was used as the antigen to immunize rabbits.ELISA assay showed that the titer of the prepared polyclonal antibody was 1:50000,and had high specificities.