Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation.
Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation.
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开发用于高效癌症免疫化学和基于免疫亲和力的外泌体分离的 CD63 适体
DOI：
10.3390/molecules25235585
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发表时间：
2020-11-27
期刊：
影响因子：
--
通讯作者：
Wang T
中科院分区：
文献类型：
--
作者：
Song Z;Mao J;Barrero RA;Wang P;Zhang F;Wang T
CD 63是四跨膜蛋白超家族的成员，是一种高度N-糖基化的III型溶酶体膜蛋白，已知其调节各种类型癌症如黑色素瘤和乳腺癌的恶性程度，并用作癌症检测的潜在标志物。近年来，它作为一个经典的外泌体标记物的重要作用也受到重视。在这项工作中，通过使用基于磁珠的竞争性SELEX（systematic evolution of ligands by exponential enrichment）程序和引入0.5M NaCl作为洗脱缓冲液，我们鉴定了两种对CD 63蛋白具有高亲和力和特异性的DNA适体（CD 63 -1和CD 63 -2）（Kd分别为38.71 nM和78.43）。此外，发现CD 63 -1有效结合CD 63阳性细胞，包括乳腺癌MDA-MB-231细胞和CD 63过表达的HEK 293 T细胞，如通过流式细胞术评估的，具有中等结合亲和力（Kd ~ 100 nM）。当使用临床乳腺癌活检进行免疫染色测定时，CD 63 -1适体显示出与商业抗体相当的CD 63阳性乳腺癌的诊断功效。在使用CD 63 -1适体开发了基于磁珠的外泌体免疫亲和分离系统后，发现该基于磁珠的系统可以有效地从MDA-MB-231和HT 29细胞培养基中分离外泌体。重要的是，在这项工作中引入NaCl洗脱能够通过简单的0.5M NaCl孵育步骤分离天然外泌体。基于这些结果，我们坚信开发的适体可用于从临床样品中有效分离天然状态外泌体和用于CD 63阳性癌症的各种治疗诊断应用。
CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5 M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71 nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (Kd ~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.
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影响因子：
12.4
作者：
Wang T;Gantier MP;Xiang D;Bean AG;Bruce M;Zhou SF;Khasraw M;Ward A;Wang L;Wei MQ;AlShamaileh H;Chen L;She X;Lin J;Kong L;Shigdar S;Duan W
通讯作者：
Duan W
影响因子：
5.2
作者：
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通讯作者：
Ilié M
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影响因子：
8.5
作者：
Liu M;Ma W;Li Q;Zhao D;Shao X;Huang Q;Hao L;Lin Y
通讯作者：
Lin Y
影响因子：
4.6
作者：
AlShamaileh H;Wang T;Xiang D;Yin W;Tran PH;Barrero RA;Zhang PZ;Li Y;Kong L;Liu K;Zhou SF;Hou Y;Shigdar S;Duan W
通讯作者：
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影响因子：
4.9
作者：
Miao, Peng;Tang, Yuguo
通讯作者：
Tang, Yuguo