【摘要】 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。更多还原

【Abstract】 AIM: To express Nanog fusion protein in Escherichia coli(E.coli),and to prepare rabbit anti-mouse polyclonal antibodies to the Nanog fusion protein.METHODS: Mouse Nanog gene was amplified from the pNA992 recombinant plasmid and inserted into pET-32a vector to construct a recombinant expression vector pET-32a-Nanog.The recombinant vector was transfected into E.coli BL21 and induced by IPTG to express in them.The acquired Nanog fusion protein was purified with HisTrap affinity column and injected as an antigen into rabbits for preparing polyclonal antibodies.At last,the titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA,Western blotting and immunocytochemical staining,respectively.RESULTS: The recombinant expression vector pET-32a-Nanog was successfully prepared,transfected and induced to obtain the high expression of the Nanog fusion protein in a form of inclusion bodies in E.coli.After purification,its purity was up to 97%.The titer of anti-Nanog antibodies was 1∶ 32 000 in the immunized rabbit serum,and exhibited a high specificity to Nanog protein.CONCLUSION: The rabbit anti-mouse polyclonal antibodies have been prepared successfully with a high titer and specificity to the Nanog fusion protein.更多还原