小鼠抗人Nanog多克隆抗体的制备及其鉴定

Preparation and identification of mouse polyclonal antibody against human Nanog

目的用基因免疫方法制备小鼠抗人Nanog多克隆抗体并进行鉴定。方法应用Accelrys软件分析Nanog抗原表位，选择其中免疫原性较强的抗原表位（A16-V101），从Nanog cDNA全长质粒中扩增其cDNA（258bp）序列，克隆到pBQAP-TT构建基因免疫载体，鉴定正确后与辅助载体pCMVi-GMCSF和pCMVi-Fh31。同时免疫小鼠，12周后用ELISA方法检测血清抗体滴度，Western blot方法鉴定抗体对人肾组织的特异性。同时将Nanog-AAV2病毒转染到HKC细胞，免疫荧光定位Nanog在细胞的表达。结果成功构建了Nanog基因免疫载体，经酶切及序列分析鉴定完全正确。ELISA结果显示抗体滴度为1：32000。Western blot结果显示小鼠抗Nanog多克隆抗体识别人肾组织34kD的Nanog蛋白。免疫荧光结果表明转染的Nanog基因主要表达在HKC细胞细胞核。结论用基因免疫的方法可以成功制备高效价和高特异性抗Nanog多克隆抗体。

Objective To prepare mouse polyclonal antibody against human Nanog by genetic immunization and to identify this antibody by Western blot and immunofluorescence. Method The antigenicity fragment （A16-V101） of human Nanog （hNanog） was chosen by analysis of Accelrys software, and its cDNA （258bp） was amplified from plasmid containing full-length cDNA of hNanog, then it was cloned into pBQAP-TT to construct recombinant plasmid pBQAP-TT-hNanog for genetic immunization. Mice were immunized with this recombinant plasmid and two other adjuvant plasmids-pCMVi-GMCSF and pCMVi-FIT3L, which help to enhance the antibody＇s generation. After 12 weeks, we obtained mouse anti-hNanog antibody from mice blood serum.The antibody titer was determined by enzymelinked immunoadsordent assay （ELISA）, and its specificity was identified by Western blot in human renal protein. Using this antibody, we detected hNanog expression in HKC cells of hNanog-AAV2 transfection. Results Recombinant plasmid pBQAP-TT-hNanog for genetic immunization was confirmed to be correct by restriction digestion and sequencing. The result of ELISA showed that the antibody titer was 1：3200. This antibody recognized a band of 34kD hNanog protein in human renal protein by Western blot. Immunofluorescence showed that Nanog protein was mainly located in the nuclei in hNanog transgene HKC cells. Conclusion Genetic immunization can offer mouse anti-hNanog polyclonal antibody of high titer and high specificity.