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从ConA刺激的小鼠脾细胞中抽提总RNA,用RT-PCR的方法克隆出小鼠IL-12R β2亚基胞内区基因。再将其插入到原核表达载体pGEX-4T-1中,转化宿主菌BL-21(DE3),用IPTC诱导宿主菌表达蛋白。SDS-PAGE分析结果表明,蛋白主要存在于包涵体中。利用割胶回收的方法纯化目的蛋白,免疫兔子制备多抗。经ELISA检测,证明免疫后兔血清中有高滴度的针对IL-12Rβ2亚基胞内区的抗体。
Total RNA was extracted from ConA-stimulated mouse spleen cells and the intracellular region of murine IL-12R β2 subunit gene was cloned by RT-PCR. Then, it was inserted into the prokaryotic expression vector pGEX-4T-1 and transformed into host strain BL-21 (DE3). IPTC was used to induce the host bacteria to express the protein. SDS-PAGE analysis showed that the protein mainly exists in the inclusion body. The target protein was purified by tapping and immunized rabbit to prepare polyclonal antibody. The results of ELISA showed that there was a high titer antibody against the intracellular region of IL-12Rβ2 subunit in the rabbit serum after immunization.