小鼠内源性逆转录病毒Gag抗原在胰岛的表达及单克隆抗体的制备

doi:10.16352/j.issn.1001-6325.2023.01.0059

基础医学与临床 ›› 2023, Vol. 43 ›› Issue (1): 59-67.doi: 10.16352/j.issn.1001-6325.2023.01.0059

小鼠内源性逆转录病毒Gag抗原在胰岛的表达及单克隆抗体的制备

李瑜¹, 张雯^1,2, 袁昱¹, 李姝亭^1,2, 王哲², 贲亚琍^1,2*, 戴飏^1,3*

1.江汉大学免疫疾病研究所, 湖北武汉 430056;
2.江汉大学医学院,湖北武汉 430056;
3.美国南加州生物医学研究所,加利福尼亚州圣地亚哥 92056

收稿日期:2021-11-16 修回日期:2022-06-10 出版日期:2023-01-05 发布日期:2022-12-27
通讯作者: ^*benyali@jhun.edu.cn;yangdai30@hotmail.com
基金资助:
国家自然科学基金(11804121); 省中央引导地方科技发展专项资金([2016]109)

Production of monoclonal antibodies specific for the endogenous retrovirus Gag antigen expressed in pancreatic islets of mice

LI Yu¹, ZHANG Wen^1,2, YUAN Yu¹, LI Shuting^1,2, WANG Zhe²,BEN Yali^1,2*, DAI Yang^1,3*

1. Institute of Immunological Diseases,Jianghan University,Wuhan 430056;
2. School of Medicine, Jianghan University, Wuhan 430056, China;
3. Biomedical Research Institute of Southern California, San Diego, CA 92056,United States/USA

Received:2021-11-16 Revised:2022-06-10 Online:2023-01-05 Published:2022-12-27
Contact: ^*benyali@jhun.edu.cn;yangdai30@hotmail.com

摘要/Abstract

摘要： 目的探究一种新发现的与1型糖尿病(T1D)相关的T细胞抗原[内源性逆转录病毒(ERV)的组特异性抗原(Gag)]与T1D发病的关系。方法首先对一个从非肥胖糖尿病(NOD)模型小鼠胰岛获得的Gag抗原(Gag 194)的氨基酸序列进行分析[多肽位置:193-210(VSRLRGRRDPPAVDSTTS)],确定Gag 194多肽作为靶点,制备单抗;然后,再利用ELISA和Western blot检测单抗的特异性;最后,在免疫组织化学实验中检测不同年龄的T1D敏感的NOD小鼠与抗性的C57BL/6小鼠胰腺Gag抗原表达的差异性。结果成功制备了5株(1F4C8、2D4C6、3C3B9、4D6B3、5F9E4)IgG2b亚型的小鼠ERV Gag单抗;5株单抗的敏感性及特异性具有差异;其中,生物素化后的3株单抗(1F4C8、2D4C6、5F9E4)能特异识别NOD小鼠胰腺中表达的ERV Gag抗原且该抗原的表达可在3周龄的NOD小鼠检测到,Gag抗原多表达在NOD小鼠胰岛β细胞区域内,而非淋巴细胞浸润的区域。此外,C57BL/6小鼠的胰腺Gag抗原的表达为阴性。结论在NOD小鼠幼年时期ERV Gag抗原就已经开始表达,该抗原的表达可能受其特异的基因调控。

关键词: 1型糖尿病, 非肥胖糖尿病(NOD)小鼠, 内源性逆转录病毒, 单克隆抗体, 组特异性抗原Gag

Abstract: Objective To explore a newly discovered T cell antigen [the group-specific antigen(Gag)of endogenous retrovirus (ERV)] associated with type 1 diabetes (T1D) and the pathogenesis of T1D. Methods First, to analyze the amino acid sequence of an islet-expressing Gag antigen, Gag 194 and identified one peptide[P193-P210(VSRLRGRRDPPAVDSTTS)], for production of Gag-specific monoclonal antibodies (mAbs). Second, ELISA and Western blot methods were performed to evaluate antigen specificity of the mAbs. Third, the expression of Gag antigen in pancreatic islets of T1D-susceptible non-obese diabetic (NOD) mice and T1D-resistant C57BL/6 mice was examined by immunohistochemical experiments. Results Five mAbs, namely 1F4C8,2D4C6,3C3B9,4D6B3 and 5F9E4 were obtained, and all of them were IgG2b subtype. Their antigen specificity and titer varied slightly. Three mAbs (1F4C8, 2D4C6 and 5F9E4) specifically recognize the ERV Gag antigen expressed in the pancreatic islets of NOD mice and at the age of 3 weeks, the antigen was detectable. Within the islets, the Gag antigen located near β-cells rather than the area infiltrated by lympho- cytes.In contrast, the T1D-resistant C57BL/6 mice do not express the Gag antigen as detected using the same mAb. Conclusions The expression of ERV Gag antigen in pancreatic islets of NOD mice occurs at very young age, suggesting that genetic factors of this diabetes-prone mouse strain may contribute to the expression.

Key words: type 1 diabetes, non-obese diabetic (NOD) mice, endogenous retrovirus, monoclonal antibody, group-specific antigen Gag

中图分类号:

R392

李瑜, 张雯, 袁昱, 李姝亭, 王哲, 贲亚琍, 戴飏. 小鼠内源性逆转录病毒Gag抗原在胰岛的表达及单克隆抗体的制备[J]. 基础医学与临床, 2023, 43(1): 59-67.

LI Yu, ZHANG Wen, YUAN Yu, LI Shuting, WANG Zhe,BEN Yali, DAI Yang. Production of monoclonal antibodies specific for the endogenous retrovirus Gag antigen expressed in pancreatic islets of mice[J]. Basic & Clinical Medicine, 2023, 43(1): 59-67.

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小鼠内源性逆转录病毒Gag抗原在胰岛的表达及单克隆抗体的制备

Production of monoclonal antibodies specific for the endogenous retrovirus Gag antigen expressed in pancreatic islets of mice

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