Jia Xiujuan, Li Guo, Chen Zhan, Xu Guangwu, Xie Chao, Zhang Di, Zhou Wenzhong, Zheng Sheng, Xie Xiaoyan, Yang Jian, Li Jiping, Luo Min
Shanghai Institute of Endocrinology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Chin Med J (Engl). 2003 Apr;116(4):524-8.
To obtain prokaryotic expressed IA-2 recombinant protein and to identify its immunological activity.
The complimentary DNA (cDNA) coding for the intracytoplasmic part of IA-2 (IA-2ic) was amplified from human fetal brain RNA, and was subcloned into the PinPoint Xa-1 T vector to construct recombinant expression plasmid, and was then expressed in E. coli JM109 cells as a fusion protein with a biotinylated peptide sequence at the aminoterminus. The biotinylated fusion protein was then purified by affinity chromatography and was subsequently dialyzed. Finally, its immunogenicity was evaluated by enzyme linked immunosorbent assay (ELISA).
The purified IA-2ic fusion protein resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single Coomassie brilliant blue stained band with a molecular weight of 59 kDa and its immunogenicity was confirmed by ELISA.
E. coli expressed IA-2ic fusion protein has immunological activity. It can be used for detection of IA-2 autoantibodies (IA-2A) and for further studies on type 1 diabetes in future.
获得原核表达的IA-2重组蛋白并鉴定其免疫活性。
从人胎儿脑RNA中扩增编码IA-2胞浆内部分（IA-2ic）的互补DNA（cDNA），将其亚克隆到PinPoint Xa-1 T载体中构建重组表达质粒，然后在大肠杆菌JM109细胞中表达为在氨基末端带有生物素化肽序列的融合蛋白。然后通过亲和层析纯化生物素化融合蛋白，随后进行透析。最后，通过酶联免疫吸附测定（ELISA）评估其免疫原性。
纯化的IA-2ic融合蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上呈现为一条考马斯亮蓝染色的单带，分子量为59 kDa，其免疫原性通过ELISA得到证实。
大肠杆菌表达的IA-2ic融合蛋白具有免疫活性。它可用于检测IA-2自身抗体（IA-2A），并可用于未来1型糖尿病的进一步研究。
