关键词: 家蚕, 丝氨酸蛋白酶抑制剂, 基因克隆, 原核表达, 蛋白纯化, 多克隆抗体

Abstract: Serine protease inhibitor 4 (serpin-4) is a member of serine protease inhibitor family. This investigation aims to research and produce high-titer polyclonal antibody against serpin-4 in Bombyx mori, which will lay a material foundation to further inquiry the physiological function of serpin-4 gene. The serpin-4 gene was first cloned from fat body of B. mori and then was cloned into pET28a prokaryotic expression vector by using genetic recombinant technology. The recombinant vector was transferred into Escherichia coli and the transferred E. coli was induced by IPTG in order to acquire prokaryotic expression recombinant fusion protein which was subsequently purified by Ni column. The molecular weight of the purified protein identified by SDS-PAGE and polyclonal antibody against His-tag was consistent with prediction, indicating that the interest protein with high purity was obtained. The acquired protein was employed as antigen to immunize KM mouse by four separate immunizations and ultimately the polyclonal antibody serum against serpin-4 was achieved. The serum titer validated by ELISA and Western blot was 1∶20 000. The successful preparation of polyclonal antibody against serpin-4 in B. mori demonstrated that the method applied in other organisms of preparing polyclonal antibody was also applicable to study serpin-4 gene of B. mori, and this study laid a material foundation to further investigate the physiological function of serpin-4 gene. 

Key words: Bombyx mori, serine protease inhibitor, gene cloning, prokaryotic expression, protein purification, polyclonal antibody