人胰岛素瘤相关蛋白-2基因的克隆及原核表达研究  

Molecular Cloning and Prokaryotic Expression of Human Insulinnoma Associated Protein - 2 Gene

作　　者：贾秀娟[1] 陈战[1] 许光武[1] 谢超[1] 谢晓雁[1] 罗敏[1] 

机构地区：[1]上海第二医科大学瑞金医院上海市内分泌研究所,上海200025

出　　处：《上海第二医科大学学报》2003年第3期196-199,共4页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：上海市卫生局科技发展基金(00418);上海高校自然科学研究基金(2000B16)资助课题

摘　　要：目的 克隆1型糖尿病自身抗原人胰岛素瘤相关蛋白-2(IA-2)的编码基因,并从大肠杆菌中表达具有免疫原性的人IA-2重组蛋白。 方法 抽提胎脑总RNA,通过RT-PCR获得编码IA-2胞内结构域的cDNA,与Pinpoint Xa-1T质粒相连接,构建表达型重组质粒,经转化、筛选并鉴定后,从大肠杆菌诱导表达生物素酰化融合蛋白,进而用亲和层析纯化之,以dot-blot鉴定其免疫原性。 结果 重组质粒转化的大肠杆菌经IPTG诱导表达及亲和层析后得到了纯度较高、分子量约为59kd的融合蛋白,表达产物用dot-blot证明具有免疫原性。 结论 原核表达的生物素酰化人IA-2胞内段融合蛋白具有正确的免疫学构象。Objective To get cDNA that encodes human insulinnoma associated protein - 2 (IA -2) and to get E. coli derived IA -2 recombinant protein with immunological activity. Methods The cD-NA coding for the intracytoplasmic domain of IA -2 (IA -2ic) was amplified from human fetal brain RNA by RT - PCR, and was cloned into the PinPoint Xa -IT vector to construct recombinant expression plas-mid, and was then expressed in E. coli as a fusion protein with a biotinylated peptide sequence at the amin-oterminus. The biotinylated fusion protein was then purified by affinity chromatography and its immunogenicity was evaluated by dot - blot. Results The purified IA -2ic fusion protein resolved on SDS - PAGE as a single Coomassie brilliant blue - stained band with a molecular weight of 59kd and its immunogenicity was confirmed by dot - blot. Conclusion E. coli derived IA -2ic fusion protein has correct immunogen-ic conformation.

关 键 词：胰岛素瘤相关蛋白-2 克隆 原核表达 1型糖尿病 自身抗原 大肠杆菌 RT-PCR 

分 类 号：R736.7[医药卫生—肿瘤]