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

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.