家蚕GAPDH内参蛋白多克隆抗体的制备及其检测

Preparation and Detection of Polyclonal Antibody of GAPDH Protein from Bombyx mori

制备家蚕GAPDH内参蛋白多克隆抗体,并对该抗体进行检测。利用PCR技术从家蚕中克隆GAPDH基因,构建其原核表达载体,转化大肠杆菌,诱导表达重组蛋白并纯化。纯化后的蛋白作为抗原免疫新西兰大白兔制备GAPDH多克隆抗体。用酶联免疫吸附法和Western blot检测抗体的效价和特异性。结果显示,成功构建GAPDH/p ET-28a原核表达载体,获得高纯度的GAPDH重组融合蛋白;经SDS-PAGE和抗His单抗检测,纯化后蛋白的分子量大小与预测的一致;以该蛋白为免疫抗原,采用4次免疫方式对新西兰大白兔进行免疫,获得GAPDH多克隆抗体血清。酶联免疫吸附检测结果表明,GAPDH抗体的效价为1:8000,并能与天然家蚕蛋白特异性结合。成功制备了家蚕内参蛋白GAPDH多克隆抗体,为深入研究家蚕中不同蛋白的生理功能和作用奠定了坚实的基础。

This work is to prepare and detect the polyclonal antibody of GAPDH protein in Bombyx mori. GAPDH gene was cloned fromsilkworm by PCR,then prokaryotic expression vector was constructed and transformed into Escherichia coli competent cell. The recombinantprotein was induced and purified. The purified protein was used as antigen to prepare the polyclonal antibody by immunizing New Zealandpurebred rabbit. The titer and specificity of the polyclonal antibody was detected by enzyme-linked immune sorbent assay（ELISA）andWestern blot. As results,GAPDH/p ET-28 a vector was constructed successfully,and recombinant GAPDH fusion protein in high purity wasobtained. SDS-PAGE and anti-His monoclonal antibody detection showed that the molecular weight of the purified protein was consistent withthat predicted. GAPDH polyclonal antiserum was obtained by immunized New Zealand purebred rabbit for four times using the purified proteinas an antigen. The titer of GAPDH polyclonal antibody was 1 ：8 000 by ELISA,and the antiserum was able to bind specifically with the totalprotein of B mori. Conclusively,the polyclonal antibody of GAPDH is successfully prepared,which lays a solid foundation for further studyingthe physiological function of different proteins in B. mori.