胰岛素瘤相关蛋白-2在HEK293中的重组生产及抗原性分析  

Production and antigenicity analysis of a recombinant insulinoma associated protein-2 in HEK293 cells

作　　者：钱静雯 王蓬勃 张元兴 刘琴 QIAN Jingwen;WANG Pengbo;ZHANG Yuanxing;LIU Qin(State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China;Shanghai Engineering Research Center of Maricultured Animal Vaccines,Shanghai 200237,China)

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]上海海洋动物疫苗工程技术研究中心,上海200237

出　　处：《生物工程学报》2023年第10期4246-4257,共12页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2022YFC3400103);上海市细胞代谢光遗传学技术前沿科学研究基地(2021 Sci&Tech 03-28)。

摘　　要：胰岛素瘤相关蛋白-2(insulinoma-associated protein-2,IA-2),是属于酪氨酸磷酸酶样蛋白家族的跨膜糖蛋白,也是诊断1型糖尿病的重要自身抗原,相关产品已在欧美国家上市。目前,商业化的IA-2抗原主要为重组IA-2ic结构域,或从牛胰岛中天然提取的IA-2,其中重组IA-2抗原在临床上存在弱阳性漏检的问题,无法完全替代天然提取IA-2抗原。本研究利用HEK293表达系统探究IA-2的重组生产。通过瞬时表达IA-2的第449-979位氨基酸跨膜片段(IA-2 transmembrane fragment,IA-2 TMF)这一天然形式的膜蛋白,并优化表达条件和膜蛋白溶解条件,纯化后膜蛋白得率为0.78 mg/L细胞发酵液。随后,通过酶联免疫吸附(enzyme linked immunosorbent assay,ELISA),对比IA-2 TMF与RSR rhIA-2的抗原活性,检测了77位1型糖尿病患者血清和32位健康志愿者血清,测试的结果通过接受者操作特性曲线(receiver operating characteristic curve,ROC)表征敏感性和特异性。结果表明:IA-2 TMF的敏感性为71.4%(55/77),而RSR rhIA-2的敏感性为63.6%(49/77),2种抗原特异性均为100%。2种抗原在特异性上无明显差异,而IA-2 TMF的敏感性略优于进口金标RSR rhIA-2抗原。综上所述,本研究基于HEK293重组表达的IA-2 TMF能够作为1型糖尿病体外诊断试剂开发的原料。Insulinoma-associated protein-2(IA-2)is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes.IA-2 products have been marketed in Europe and the United States.At present,commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets.However,the recombinant IA-2 antigen displays weak positive in clinic practice,which often results in occasional detection failures,thus cannot completely replace the naturally extracted IA-2 antigen.In this study,an HEK293 expression system was used to explore the production of recombinant IA-2.An IA-2 transmembrane fragment(IA-2 TMF)located at amino acid position 449–979,also known as the natural membrane protein form of IA-2,was produced in HEK293 through transfection,and both the expression conditions and dissolution conditions of the membrane protein were also optimized.The purified membrane protein yield was 0.78 mg/L cell culture.Subsequently,the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay.The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected.Receiver operating characteristic curve(ROC)curve was used to characterize the sensitivity and specificity of the test results.The results showed that the sensitivity of IA-2 TMF was 71.4%(55/77),while the sensitivity of RSR rhIA-2 was 63.6%(49/77),and the specificity of both antigens were all 100%.There was no significant difference in specificity between the two antigens,but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen.In conclusion,the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.

关 键 词：1型糖尿病 自身免疫 胰岛素瘤相关蛋白-2 膜蛋白纯化 

分 类 号：R587.1[医药卫生—内分泌]