鸡γ-干扰素基因在大肠杆菌中的高效表达及多克隆抗体的制备

Expression of Recombinant Chicken IFN-γ in E.coli and Preparation of Antibody against Recombinant Chicken IFN-γ Protein

将克隆得到的鸡 IFN- γ基因亚克隆到大肠杆菌原核表达载体 p PROEXTMHT的 Pst 和 Kpn 位点上 ,构建了重组表达质粒 p PROEXTMHT- IFN -γ,经序列分析鉴定 ,确证目的基因克隆入载体的预期位点。将重组质粒转化大肠杆菌 DH5 α,于 37℃不同时间诱导培养 ,SDS- PAGE电泳表明 ,该基因在大肠杆菌中发生高水平表达 ,表达的鸡 IFN- γ融合蛋白相对分子质量约为 2 2 0 0 0。重组蛋白占菌体总蛋白的 33%。经 Western blot鉴定 ,该重组蛋白质具有生物学活性。包涵体被 6 mol/ L 盐酸胍裂解后 ,通过镍离子亲和树脂进行了纯化。用所获得的重组鸡 IFN- γ融合蛋白及其纯化产物经 3次肌肉注射于新西兰白兔 ,制备了兔抗鸡 IFN- γ多克隆抗体。琼脂扩散结果表明 ,多克隆抗体可与纯化的鸡IFN

The cDNA of chicken IFN-γ gene was subcloned into PstⅠ and KpnⅠ sites of pPROEX^(TM)HT vector to construct recombinant plasmid pPROEX^(TM)HT-IFN-γ.The recombinant plasmid was transfected into E.coli DH (5α) and the bacterium was induced with IPTG.It was demonstrated by SDS-PAGE and Western blot that a (22 000) protein which was equal to chicken IFN-γ protein in molecular weight was expressed in E.coli DH (5α).This protein was purified and used to prepare the antibody against chicken IFN-γ protein.The result showed that the antibody could react with purified recombinant chicken IFN-γ protein.