人APOBEC3G基因克隆、表达、纯化及多克隆抗体制备

为了获得人APOBEC3G蛋白及其多克隆抗体,从H9细胞中提取总RNA,采用反转录-聚合酶链式反应(RT-PCR)技术获得人APOBEC3G基因.将测序鉴定过的APOBEC3G基因克隆到原核表达载体pET-32a上,以包涵体的形式在E.coli BL21(DE3)中高效表达,由于APOBEC3G蛋白C端融合了6×His标签,有助于对蛋白的纯化及鉴定.应用酶切技术、SDS-PAGE及Western Blot等方法确保基因片段的正确性和蛋白的特异性.纯化后的APOBEC3G蛋白用来免疫日本大耳白兔,用间接ELISA法测定兔多克隆抗体滴度,获得了纯度超过80%的APOBEC3G融合蛋白,抗APOBEC3G多克隆抗体滴度高达1:102 400.

Cloning,Expression and Purification of APOBEC3G,and Its Polyclonal Antibodies Production

To prepare human APOBEC3G and to produce its antibodies,the total RNA was extracted from H9 cells,and APOBEC3G gene was achieved by RT-PCR.The resulting DNA construct was cloned into a prokaryotic expression vector(pET-32a)and recombinant pET-APOBEC3G was expressed in Eserichia coli BL21(DE3)as an insoluble protein.The vector also contained a six-histidine(His6)tag at the C-terminus for convenient purification and detection.To express and purify the APOBEC3G in E.coli cells,the accuracy of inserted genes and specificity of APOBEC3G proteins were detected by two enzymes digestion technology, SDS-PAGE,and Western Blot(WB).Rabbits were immuzied by purified APOBEC3G proteins.Serum samples were tested by indirect enzyme-linked immunosorbent assays(ELISAs)to determine the level of antibodies.The purity of APOBEC3G was above 80%.The titer of the antibodies was 1:102 400