兔抗鼠IL-12p35多克隆抗体的制备及应用

发布时间：2018-05-01 22:59

本文选题：IL-a + IL-p多克隆抗体　；参考：《南昌大学学报(理科版)》2017年01期

【摘要】：利用分子克隆方法构建重组载体pGEX-4T-1-IL-12a,转化BL21(DE3),经IPTG诱导表达,谷胱甘肽琼脂糖凝胶4B亲和层析柱纯化后,获得重组融合蛋白GST-IL-12p35。以此蛋白为免疫抗原免疫新西兰大耳白兔制备IL-12p35多克隆抗体。采用ELISA法评估抗体效价,Western blot检测抗体特异性。LPS刺激小鼠脾细胞,ELISA法检测IL-12抗体阻断后细胞培养上清中的IFN-γ的分泌量。结果:成功构建重组载体pGEX-4T-1-IL-12a,并获得重组融合蛋白GST-IL-12p35(约48kDa)。抗原免疫新西兰大耳白兔制备IL-12p35多克隆抗体,ELISA法测得IL-12p35多克隆抗体效价为1鐩256 000,Western blot证实制备的多克隆抗体能够识别结合重组蛋白。IL-12p35多克隆抗体能够有效地抑制LPS诱导的脾细胞IFN-γ的表达。
[Abstract]:The recombinant plasmid pGEX-4T-1-IL-12awas constructed by molecular cloning and transformed into BL21DE-3. The recombinant fusion protein GST-IL-12p35 was obtained by IPTG induced expression and purified by glutathione agarose gel 4B affinity chromatography. IL-12p35 polyclonal antibodies were prepared by immunizing New Zealand rabbits with this protein. ELISA assay was used to evaluate the titer of antibody. Western blot was used to detect the secretion of IFN- 纬 in the culture supernatant of murine spleen cells stimulated by IL-12 antibody. Results: the recombinant vector pGEX-4T-1-IL-12awas successfully constructed and the recombinant fusion protein GST-IL-12p35 was obtained. Antigen-immunized New Zealand white rabbits with IL-12p35 polyclonal antibodies were obtained by Elisa. The titer of IL-12p35 polyclonal antibodies was 1: 256 000. Western blot confirmed that the prepared polyclonal antibodies could recognize the binding protein. IL-12p35 polyclonal antibodies could be effectively inhibited. LPS induced expression of IFN- 纬 in splenocytes.
【作者单位】：郑州大学生命科学学院分子免疫实验室;郑州人民医院;河南省医药科学研究院;
【基金】：重大新药创制科技重大专项(2012ZX09103301-022) 国家自然科学基金资助项目(U1204817、81373119、81571526)
【分类号】：S852.4

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