小鼠OAZ2基因的原核表达及抗体制备

Prokaryotic Expression of Mouse OAZ2 and Preparation of Its Antibody

目的:克隆小鼠鸟氨酸脱羧酶抗酶2(OAZ2)功能基因,原核表达、纯化OAZ2蛋白并制备抗OAZ2多克隆抗体。方法:RT-PCR法从鼠黑色素瘤细胞总RNA中克隆OAZ2 cDNA后,通过重叠延伸PCR技术构建无需移码即可全长翻译的功能基因。将OAZ2功能基因克隆入原核表达载体pET15b并原核表达。表达的蛋白经Ni-NTA亲和层析纯化后,用SDS-PAGE和West-ern Blot分析鉴定。用纯化的OAZ2蛋白作为抗原免疫Bab/C小鼠以制备多克隆抗体,制备抗体用ELISA和Western Blot检测抗体滴度和特异性。结果:成功获得小鼠OAZ2 cDNA并构建出无需移码翻译的OAZ2功能基因。OAZ2功能基因在大肠杆菌BL21(DE3)中可诱导性高表达并能用Ni-NTA树脂高效纯化。用纯化蛋白免疫Bab/C小鼠制备的抗血清经ELISA检测有较高的多克隆抗体效价(>1:64 000),经Western blot鉴定可与纯化的OAZ2蛋白质特异性结合。结论:建立了鼠OAZ2蛋白原核表达和纯化技术,制备出高效价和特异性抗OAZ2多克隆抗体,为进一步研究OAZ2基因的功能奠定了基础。

Objective： To clone functional gene of mouse ornithine decarboxylase antizyme -2 （ OAZ2）, to express and purify recombinant OAZ2 in prokaryotic system, and then to generate anti - OAZ2 polyclone antibody. Method： OAZ2 cDNA was amplified by RT - PCR from total RNA of mouse melanoma B16 - F1 cells. The function gene that can translate whole length OAZ2 without frameshifting was obtained by PCR-driven overlap extension. The function gene was then subcloned into the plasmid pET15b and was transformed into BI21 （DE3） host cells. The recombinant OAZ2 was expressed by IPTG induction and purified by Ni - NTA resin. The purified recombinant OAZ2 was used as the antigen tO immunize mice for preparation of polyclonal antibody. The titer and specificity of anti - OAZ2 antibody were detected by ELISA and Western blot. Result： The mouse OAZ2 cDNA and its function gene were obtained successfully. The recombinant OAZ2 can be expressed by IPTG induction and purified by Ni - NTA resin. The results of ELISA and Western blot proved that polyelonal antiserum prepared with purified recombinant OAZ2 as antigen has high titration （ 〉 1：64 000） and specificity. Conclusion： A method for prokaryotic expression and purification of mouse OAZ2 was established and the anti - OAZ2 polyclonal antibody with high titration and specificity were obtained. These results would provide reliable tools for the future study on OAZ2 function.