【摘要】 目的 :表达GST p11和 6xHis p11融合蛋白并制备GST p11特异性抗体。方法 :将人p11基因克隆入两种原核表达载体pGEX 4T 2和pQE30 ,分别在大肠杆菌BL2 1和M15中表达 ,用GlutathioneSepharose 4B和Ni NTAagar ose亲和柱分别纯化目的蛋白。利用纯化的GST p11蛋白制备多克隆抗体。结果 :得到高表达量的融合蛋白 ,经亲和层析柱纯化获得较高纯度的GST p11和 6xHis p11蛋白。以GST p11蛋白免疫新西兰兔得到p11多克隆抗体 ,Westernblotting证实该抗体能够识别 6xHis p11蛋白 ,具有较高特异性。结论 :利用原核表达人p11融合蛋白制备的p11多克隆抗体具有较好的特异性 ,为研究p11蛋白在蛋白转运和定位过程中的作用提供重要的技术和材料保障更多还原

【Abstract】 Objective To obtain p11 fusion protein and prepare specific polyclonal antibody against p11. Methods A full length human p11 gene was cloned into expression vectors, pGEX 4T 2 and pQE30, and transformed into E.coli. The expressed proteins were purified from lysates with Glutathione Sepharose 4B and the Ni NTA agarose column, respectively. The purified GST p11 was mixed with Freund’s complete or incomplete adjuvant and immunized rabbits. Results A high level of expression of target proteins was detected after IPTG induction and purified proteins were obtained by affinity chromatography with Glutathione Sepharose 4B and the Ni NTA agarose column, respectively. Western blotting analysis suggested that the polyclonal antibody can recognize 6xHis p11 and GST protein. Conclusion The antiserum against p11 prepared by prokaryotic expression of GST p11 fusion protein has good specificity.更多还原