CD14蛋白表达、纯化及单克隆抗体制备

Expression and Purification of CD14 Protein and Preparation of Anti-CD14 Monoclonal Antibody

目的:制备CD14重组蛋白及抗CD14单克隆抗体。方法:从人外周血淋巴细胞中克隆CD14编码基因,将其连接至质粒pRSETC,构建表达质粒pRSETC/CD14,转染大肠杆菌表达菌株BL21(DE3),筛选阳性克隆、用IPTG诱导表达,SDS-PAGE检测CD14蛋白表达水平,应用镍柱进行亲和纯化,SDS-PAGE及Western blot进行纯化产物鉴定。纯化后的CD14蛋白免疫BALB/c小鼠,利用杂交瘤技术筛选分泌单克隆抗体细胞株,制备单克隆抗体,利用Western blot鉴定抗体活性。结果:获得CD14编码基因,并成功进行了原核表达,SDS-PAGE显示CD14以包涵体形式表达,纯化产物的纯度超过95%。利用杂交瘤技术筛选出稳定分泌抗CD14抗体的细胞株,并制备了高纯度的单克隆抗体,抗体具有CD14蛋白结合活性。结论:成功表达纯化了CD14蛋白,并制备了抗CD14单克隆抗体,为后续开发CD14检测技术提供抗体。

Objective:To express CD14 protein and prepare anti-CD14 monoclonal antibody.Methods:The CD14 encoding sequence was cloned from human peripheral blood lymphocytes,which was connected to the pRSETC vector,the expression plasmid pRSETC/CD14 was transfected into BL21(DE3).Expression of recombinant protein was induced by using IPTG.SDS-PAGE and western-blot were used to detected the purified recombinant protein.Hybridoma cells stably secreting anti-CD14 monoclonal antibodies were prepared by using hybridoma technology,the activity of purified antibody was identified by Western blot.Results:The coding sequence of CD14 was obtained.SDS-PAGE showed that recombinant protein expressed in the forms of inclusion body.And moreover,SDS-PAGE showed the purification rate of the purified product was higher than 95%.The positive hybridoma clone which secreted the mAb against CD14 protein were obtained,high purity monoclonal antibody which had the binding activity to CD14 protein was prepared.Conclusion:CD14 protein and anti-CD14 monoclonal antibody were successfully prepared,which provided the experimental basis for the development of CD14 detection technology.