[1]陈贤华,潘炜伦,李 博,等.外泌体双膜蛋白共表达检测平台:基于磁性分离和催化发夹组装[J].南方医科大学学报,2019,(12):1453-1460.[doi:10.12122/j.issn.1673-4254.2019.12.09]
 Construction and application of a magnetic and catalytic hairpin assembly-based platform for detecting dual membrane proteins on exosomes[J].Journal of Southern Medical University,2019,(12):1453-1460.[doi:10.12122/j.issn.1673-4254.2019.12.09]
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外泌体双膜蛋白共表达检测平台:基于磁性分离和催化发夹组装()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2019年12期
页码:
1453-1460
栏目:
出版日期:
2020-01-01

文章信息/Info

Title:
Construction and application of a magnetic and catalytic hairpin assembly-based platform for detecting dual membrane proteins on exosomes
作者:
陈贤华潘炜伦李 博郑 磊
关键词:
外泌体膜蛋白磁性分离催化发夹组装
Keywords:
exosome membrane proteins magnetic separation method catalytic hairpin assembly
DOI:
10.12122/j.issn.1673-4254.2019.12.09
文献标志码:
A
摘要:
目的 构建基于磁性分离和催化发夹组装(CHA)信号放大策略的外泌体双膜蛋白共表达检测平台。方法 细胞培养与细胞上清液中外泌体的分离纯化和表征。超分辨成像技术与免疫印迹实验表征CD63在外泌体膜上的表达。外泌体与AptEpCAM-T催发链反应电泳图验证EpCAM适配体与外泌体结合。荧光检测法验证CHA核酸序列的可行性、优化反应条件以及验证该方法特异性。结果 超分辨成像技术与免疫印迹实验显示乳腺癌MDA-MB-231细胞来源外泌体富含CD63膜蛋白。外泌体与AptEpCAM-T催发链反应电泳结果显示EpCAM适配体能够识别并结合外泌体。检测平台特异性试验:人正常乳腺上皮细胞外泌体(MCF-10a exo)荧光检测信噪比:1.10±0.01,乳腺癌细胞外泌体(MDA-MB-231 exo)荧光检测信噪比:2.09±0.08。结论 构建基于磁性分离和CHA信号放大策略的外泌体双膜蛋白共表达检测平台。可同时检测外泌体膜上表达的两种蛋白:CD63和EpCAM。乳腺癌细胞外泌体与人正常乳腺上皮细胞外泌体膜蛋白CD63和蛋白EpCAM表达有区分度。
Abstract:
Objective To construct a magnetic and catalytic hairpin assembly-based platform for detection of dual membrane proteins on exosomes. Methods Exosomes in supernatant of breast cancer MDA-MB-231 cell culture were separated, purified and characterized. Super-resolution imaging and Western blotting were performed to confirm the expression of the membrane protein CD63 on the exosomes. Polyacrylamide gel electrophoresis was used to verify the combination of AptEpCAM-T and exosomes. Fluorescence experiments were carried out to test the feasibility of CHA nucleic acid sequence, optimize the reaction conditions, and determine the specificity of the detection platform. Results Super-resolution imaging and Western blotting showed that breast cancer MDA-MB-231 cell-derived exosomes expressed abundant membrane protein CD63. Polyacrylamide gel electrophoresis indicated that AptEpCAM-T could recognize and bind to exosomes. The results of specificity test showed that the signal-to-noise ratio of the detection platform was 1.10±0.01 for detecting normal human breast epithelial cell-derived exosomes, and was 2.09±0.08 for breast cancer cell-derived exosomes. Conclusion Magnetic and catalytic hairpin assembly-based detection platform allows simultaneous detection of two membrane proteins expressed on exosomes and identification of the expressions of membrane proteins on exosomes from different sources.

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更新日期/Last Update: 2019-12-27