论文部分内容阅读
目的:表达和纯化带多聚组氨酸(6×His)标签的Autotaxin(58-157位氨基酸)融合蛋白,制备抗Autotaxin的多克隆抗体。方法:构建pET-21 c-Autotaxin(58-157)重组表达质粒,转入BL21大肠杆菌,IPTG诱导蛋白表达,经镍离子金属螯合树脂纯化后,用纯化的蛋白免疫家兔,制备抗Autotax-in的多克隆抗体。ELISA检测抗体效价,Western blot检测抗体特异性,并用于免疫组化实验。结果:在大肠杆菌中高表达Autotaxin(58-157)-His6融合蛋白,经亲和纯化后免疫家兔,获得了特异性的抗Autotaxin抗血清。结论:成功构建pET-21 c-Autotaxin(58-157)表达质粒,原核表达获得高纯度的目的蛋白,制备出高效价的特异性抗Autotaxin多克隆抗体。
Objective: To express and purify Autotaxin (58-157 amino acid) fusion protein with polyhistidine (6 × His) tag and prepare polyclonal antibody against Autotaxin. Methods: The recombinant plasmid pET-21 c-Autotaxin (58-157) was constructed and transformed into BL21 E.coli. IPTG was used to induce the protein expression. After purifying with nickel ion chelate resin, the purified protein was used to immunize rabbits to prepare anti-Autotax -in polyclonal antibody. Antibody titers were detected by ELISA, antibody specificity by Western blot and immunohistochemistry. Results: Autotaxin (58-157) -His6 fusion protein was overexpressed in Escherichia coli and the rabbit was immunized after affinity purification. Anti-Autotaxin antiserum was obtained. CONCLUSION: The recombinant plasmid pET-21 c-Autotaxin (58-157) has been successfully constructed and expressed in prokaryotic cells with high purity. The polyclonal anti-Autotaxin antibody with high titer was prepared.