人生长分化因子15的原核表达、纯化与多克隆抗体制备

Prokaryotic Expression and Purification of Human Growth Differentiation Factor-15 and Preparation of its Polyclonal Antibody

目的:原核表达并纯化、鉴定人生长分化因子15(GDF-15),制备其多克隆抗体。方法:从人结肠癌细胞系HT29的cDNA扩增出GDF-15基因片段并插入pET-32a(+)原核表达载体,转化大肠杆菌BL21,IPTG诱导表达重组GDF-15,用镍亲和柱纯化,SDS-PAGE、Western印迹鉴定重组蛋白。用纯化的重组GDF-15免疫BALB/c小鼠制备多克隆抗体,鉴定并检测其效价。结果:制备了pET-32a(+)-GDF-15表达载体;经IPTG诱导重组蛋白表达后,采用Ni亲和柱纯化蛋白,并经SDS-PAGE和免疫印迹鉴定;免疫BALB/c小鼠后获得了GDF-15多克隆抗体,ELISA检测抗体效价为1∶100000,并应用于肿瘤细胞的GDF-15检测中。结论:用基因工程和免疫学方法制备了重组人GDF-15及其多克隆抗体,为后续的分子机制和靶向治疗研究奠定了基础。

Objective： To express recombinant differentiation factor-15（GDF-15） in Escherichia coli,and to puri-fy and identify the fusion protein,prepare the polyclonal antibody against GDF-15.Methods： The gene of GDF-15 was cloned according to the cDNA from the colon cancer cell HT29.Then the synthesized GDF-15 gene wasinserted into p ET-32 a prokaryotic expression vector and the plasmid was transformed into E.coli BL21.The fusionprotein was expressed with IPTG induction,purified by Ni-affinity chromatography and identified by SDS-PAGEand Western blot.A polyclonal antibody was developed by immunizing BALB/c mice with the purified fusion pro-tein,the titer was determined and the specificity was identified by Western blot.Results： The recombinant plas-mid was verified to be correct by restriction enzyme digestion andDNA sequencing.The fusion protein expressedwith IPTG induction and purified by Ni-affinity chromatography,was proved to be GDF-15 by Western blot.Andget a polyclonal antibody with a titer of 1∶100 000.Conclusion： We have purified GDF-15 fusion protein andprepared the antibody against GDF-15 successfully,which provided the basis for the research of mechanism andthe target of the therapy.