兔抗鼠IL-12p35多克隆抗体的制备及应用

Preparation and application of mouse IL-12p35 polyclonal antibody

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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。

The pGEX-4T-1-IL-12a was constructed and then was transformed to the expression strain BL21 （DE3） ;The recombinant protein GST-IL-12p35 was induced and expressed by IPTG and purified with Glu- tathione Sepharose 4B;The polyclonal antibody was obtained from the sera of New Zealand white rabbits immuned with recombinant GST-IL-12p35 protein; Antibody titer was detected by ELISA; Antibody speci- ficity was examined by Western blot;After blocking with anti-IL-12, the IFN-γ expression of splenocytes stimulated with LPS was detected by ELISA. Results：The construction of the recombinant pGEX-4T-l-IL- 12a vector and the expression and purification of the recombinant protein GST-IL-12p35 （about 48 kDa） were successful. The anti-IL-12 polyclonal antibody was obtained from the New Zealand white rabbit im- muned with recombinant IL-12p35 protein effectively. The titer of the anti-IL-12 antibody was about 1 ： 256 000. It was showed that the polyclonal antibody could bind to the expressed protein. The IFN-γ expres- sion of splenocytes stimulated with LPS decreased significantly after addition of anti-IL-12 antibody.