猴源天然免疫限制性因子SAMHD1单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against innate immune restriction factor SAMHD1

为制备具有广谱反应性的SAMHD1单克隆抗体,通过提取Marc-145细胞的总RNA,采用RTPCR将SAMHD1全长编码基因定向克隆至pCold-TF原核表达载体中,成功构建了重组质粒pColdmSAMHD1。重组质粒转化大肠杆菌Rosseta感受态细胞,经IPTG诱导表达并通过纯化获得了可溶性的重组SAMHD1蛋白。以纯化的重组SAMHD1蛋白为抗原,免疫8周龄BALB/c小鼠,通过间接ELISA和5次杂交瘤细胞亚克隆,筛选出1株针对SAMHD1蛋白的单克隆抗体,遂被命名为M2D9。Western-blot分析显示,M2D9能够与SAMHD1真核表达蛋白以及细胞内源性SAMHD1蛋白发生特异性反应,且具有很好的免疫沉淀效价。交叉反应性鉴定结果显示,M2D9抗体能够与人、鼠源细胞内SAMHD1蛋白发生特异反应。本研究成功获得了具有特异性和广谱反应性的猴源SAMHD1单克隆抗体,可用于ELISA检测、Western-blot分析和免疫沉淀试验,为SAMHD1蛋白功能的深入研究奠定了基础。

To development monoclonal antibodies(MAbs)against SAMHD1 with broad reactivity,the total RNA of Marc-145 cells were extracted and cDNAs were obtained by reverse transcription.The open reading frame of SAMHD1 gene was amplified and cloned into the pCold-TF DNA prokaryotic expression vector for construction of a recombinant plasmid,pCold-mSAMHD1.The recombinant SAMHD1 protein was expressed in Escherichia coli Rosseta under induction with IPTG.The purified recombinant SAMHD1 protein was used as an antigen to immunize eight weeks-old BALB/c mice.A hybridoma cell line secreting MAbs against SAMHD1 was obtained by the indirect ELISA and five sequential subclonings,and named as M2D9.The analyses by immunoprecipitation assay and Western-blot showed that SAMHD1 protein expressed from eukaryotic and the endogenous protein in simian cell lines could be specifically recognized by the MAb produced in this study.Moreover,M2D9 had broad cross-reactivity,and the endogenous SAMHD1 protien from human and mouse cell lines were also specifically recognized by the M2D9.In coclusion,the MAb could be useful for the functional studies of SAMHD1 protein.