家蚕丝氨酸蛋白酶抑制剂4(serpin-4)的基因克隆、原核表达和多克隆抗体制备

Cloning,prokaryotic expression and preparation of polyclonal antibody of serine protease inhibitor 4 (serpin-4) from Bombyx mori

丝氨酸蛋白酶抑制剂4(serpin-4)为丝氨酸蛋白酶抑制剂家族中的一员。本研究旨在研制高效价的家蚕Bombyxmoriserpin-4多克隆抗体,为深入研究serpin-4基因的生理功能打下物质基础。首先于家蚕脂肪体中克隆了serpin-4基因,利用基因重组技术构建了pET28a-serpin-4原核表达载体,经IPTG诱导,获得原核表达重组融合蛋白,利用镍柱回收纯化技术,获得目的蛋白;经SDS-PAGE和抗His多抗检测,纯化蛋白的分子量大小与预测的一致,即获得了高纯度的目的蛋白,以此蛋白为免疫抗原,采用4次免疫方式对昆明小鼠进行抗原免疫,最终获得抗serpin-4的多克隆抗体血清;该血清经ELISA和Westernblot验证,效价达到1:20000,特异性较好。家蚕serpin-4多克隆抗体的成功制备,一方面表明应用于其他生物的多克隆抗体制备所采用的方法对于家蚕serpin基因的研究同样适用;另一方面,也为深入研究家蚕serpin-4的生理功能打下了物质基础。

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.