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目的:在大肠杆菌中表达Nanog融合蛋白,制备兔抗小鼠Nanog融合蛋白抗体。方法:从含小鼠Nanog基因的pNA992质粒中扩增出小鼠Nanog基因并插入pET-32a中构建pET-32a-Nanog重组表达载体,并转化大肠杆菌BL21,经IPTG诱导表达,得到了Nanog融合蛋白,并经Histrap亲和层析柱纯化后,将其作为抗原免疫家兔制备多克隆抗体,用间接ELISA法检测抗体效价,Western blot和免疫细胞化学染色检测抗体特异性。结果:成功地构建了重组表达载体pET-32a-Nanog,经诱导获得了大量Nanog融合蛋白,其主要以包涵体形式表达,纯化后蛋白纯度达到97%,经免疫的兔抗血清效价可达1∶32 000,并表现出较好的特异性。结论:成功地制备出高滴度、高特异性的兔抗Nanog融合蛋白抗体。
Objective: Nanog fusion protein was expressed in E. coli to prepare rabbit anti-mouse Nanog fusion protein. Methods: Mouse Nanog gene was amplified from pNA992 plasmid containing mouse Nanog gene and inserted into pET-32a to construct pET-32a-Nanog recombinant expression vector. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG to obtain Nanog fusion The protein was purified by Histrap affinity chromatography and then used as antigen to immunize rabbits to prepare polyclonal antibody. The antibody titer was detected by indirect ELISA. The antibody specificity was detected by Western blot and immunocytochemistry. Results: The recombinant plasmid pET-32a-Nanog was constructed successfully. A large number of Nanog fusion proteins were obtained and expressed mainly as inclusion bodies. The purity of the purified protein was 97%. The titer of the purified rabbit antiserum was up to 1:32 000, and showed better specificity. Conclusion: High titer and high specificity rabbit anti-Nanog fusion protein was successfully prepared.