新基因ZNFD的克隆及其多克隆抗体的制备

目的:PCR扩增得到新基因ZNFD(Znf domain containing protein),构建pET32a-ZNFD原核表达载体,诱导蛋白表达及制备多克隆抗体。方法:通过PCR的方法克隆新基因ZNFD,选取部分抗原免疫原性较高的部分ORF序列,克隆到原核表达载体pET32a上,利用大肠杆菌Rosetta-gami(DE3)表达系统表达ZNFD融合蛋白。纯化目的蛋白免疫家兔,制备多克隆抗体。通过琼脂糖双向扩散实验和Western blot鉴定其效价和特异性。结果:经测序鉴定已成功克隆ZNFD基因。通过构建原核表达载体pET32a-ZNFD,在大肠杆菌Rosetta-gami(DE3)中使用IPTG诱导表达,得到相对分子质量约为45 kD的融合蛋白。纯化蛋白免疫家兔,制备的多克隆抗体具有较强的免疫特异性。结论:得到纯化的ZNFD蛋白,制备的多克隆抗体达到了一定的效价,且具有高特异性,为进一步研究ZNFD的功能奠定了实验基础。

Cloning ZNFD gene and preparing polyclonal antibody

Objective:To clone ZNFD gene by PCR,construct expression vector ZNFD-pET32a,prepare and obtain its polyclonal antibody.Methods:The ORF fragment of ZNFD was amplified by PCR and sequenced.Part of ORF was cloned into pET32a prokaryotic expressing vector to form pET32a-ZNFD plasmid.His-ZNFD was expressed in E.coli Rosetta-gami(DE3) induced by IPTG and purified by using affinity chromatography.Purified His-ZNFD was used to immunize rabbits to prepare polyclonal antibody.Rabbit serum specificity and its titer were detected by double diffusion and Western blot.Results: The ZNFD gene was cloned and sequenced,and then pET32a-ZNFD was constructed successfully.His-ZNFD protein,45 kD molecular weight,can be expressed in E.coli Rosetta-gami(DE3) with high efficiency.Its corresponding antibody was obtained by immunizing rabbit with the purified protein.The data of double diffusion and Western blot demonstrated that the purified antigen had high antigenicity.Conclusions:ZNFD gene was cloned and the purified fusion protein ZNFD was obtained,it had high antigenicity.Polyclonal antibody was prepared,which provided reliable tools for the future research.