【摘要】 目的制备兔抗小鼠白细胞介素23p19(IL-23p19)多克隆抗体。方法利用分子克隆技术构建重组表达质粒p ET-16b-IL-23p19,转化大肠杆菌BL21(DE3),异丙基-β-D-硫代半乳糖吡喃糖苷(IPTG)诱导蛋白表达,SDS-PAGE及Western blot法分析鉴定蛋白,镍柱亲和层析纯化制备蛋白;以此蛋白为免疫抗原免疫新西兰大白兔,获得抗血清,并利用亲和层析法纯化抗血清,制备兔抗小鼠IL-23p19多克隆抗体;采用ELISA检测抗体效价;Western blot法检测抗体特异性。结果重组表达载体p ET-16b-IL-23p19构建正确且IL-23p19蛋白能够在大肠杆菌BL21(DE3)中有效表达。通过多次IL-23p19蛋白免疫新西兰大白兔,成功制备兔抗小鼠IL-23p19多克隆抗体。ELISA检测确定新西兰大白兔血清效价达1∶256 000,Western blot法证实兔抗小鼠IL-23p19多克隆抗体能特异性识别小鼠IL-23p19蛋白。结论成功制备了兔抗小鼠IL-23p19多克隆抗体,抗体具有高的效价和较好的特异性。更多还原

【Abstract】 Objective To prepare the polyclonal antibody against mouse interleukine-23 p19( IL-23p19). Methods The murine full-length IL-23p19 gene was subcloned into p ET16 expression vector to construct the recombinant plasmid p ET-16bIL-23p19. The plasmid was transformed into E. coli BL 21( DE3) and IL-23p19 protein expression was induced by IPTG and identified with SDS-PAGE analysis and Western blotting. After purified by Ni+affinity column chromatography,the IL-23p19 protein was used as the antigen to immunize New Zealand rabbit to prepare the antiserum. The polyclonal antibody against the mouse IL-23p19 was isolated from antiserum by affinity chromatography. Antibody titer was detected by ELISA. Antibody specificity was evaluated by Western blotting. Results The p ET-16b-IL-23p19 recombinant plasmid was successfully constructed and the IL-23p19 protein was effectively expressed in E. coli BL 21( DE3). The antibody was successfully prepared by immunizing New Zealand rabbit with the IL-23p19 protein four times. ELISA showed that the titer of the anti-mouse IL-23p19 polyclonal antibody was about 1∶256 000. Western blotting confirmed that anti-mouse IL-23p19 polyclonal antibody could specifically recognize the IL-23p19 protein. Conclusion We have successfully prepared the anti-IL-23p19 polyclonal antibody with the high titer and specificity.更多还原