基于Ser-SELEX技术的结肠癌血清适配体的筛选及其应用研究  

Screening and Application of Serum Aptamer for Colon Cancer Based on Ser-SELEX

作　　者：张广鑫 王佳 石明[1,2,3] 刘志伟[1,2,3] 丁婷婷[1,2] 岳金媛 李健[1,2] 栗坤 ZHANG Guang-Xin;WANG Jia;SHI Ming;LIU Zhi-Wei;DING Ting-Ting;YUE Jin-Yuan;LI Jian;LI Kun(School of Environmental and Chemical Engineering,Yanshan University,Qinhuangdao 066000,China;Hebei Key Laboratory of Nano-technology,Yanshan University,Qinhuangdao 066000,China;Metastable Materials Science and Technology State Key Laboratory,Yanshan University,Qinhuangdao 066000,China)

机构地区：[1]燕山大学环境与化学工程学院,秦皇岛066000 [2]燕山大学河北省纳米生物技术重点实验室,秦皇岛066000 [3]燕山大学亚稳材料制备技术与科学国家重点实验室,秦皇岛066000

出　　处：《生物化学与生物物理进展》2023年第9期2220-2229,共10页Progress In Biochemistry and Biophysics

基　　金：河北省自然基金创新群体项目(C2022203003)资助。

摘　　要：目的为了解决癌症早期诊断困难的问题,实现结肠癌的早期检测。方法采用Ser-SELEX技术筛选了结肠癌血清的特异性适配体。通过前期结肠癌血清正筛-正常人血清反筛步骤和后期结肠癌血清正筛-其他癌症血清反筛步骤循环的方式,经过16轮筛选流程,共选取4条候选适配体,并对其进行序列分析、二级结构和三级结构模拟、特异性分析等。结果候选适配体主要形成的结构是茎环和假结两种,q PCR法测试亲和力,Kd约为10 nmol/L,适配体与结肠癌患者血清特异性结合情况分析表明,候选适配体APT-2具有良好特异性,且此种方法检出率约为82.5%。结论适配体(APT-2)应用于结肠癌早期诊断具有良好的发展前景。Objective Colon cancer is a common malignant tumor of the gastrointestinal tract and the second leading cause of cancer-related death.It is a type of cancer which is difficult to detect in early stage and easy to metastasize in late stage operation.The aim of this work is to solve the difficult problem of early diagnosis of cancer and realize the early detection of colon cancer and provide a new idea for early diagnosis of colon cancer.Methods Serum-SELEX technique was used to screen the specific aptamers of colon cancer serum.The aptamer sequence with higher affinity and more stable properties can be obtained through the cyclic process of prophase positive screening for colon cancer and prophase positive screening for colon cancer and prophase positive screening for other cancers,and gradually increasing the screening pressure,such as reducing the incubation time of positive screening targets,increasing the incubation time of reverse screening targets and elution steps.qPCR was used to quantitatively monitor the positive screen template obtained to ensure that the aptamer pool was enriched.In this process,the aptamers of common protein binding of negative and positive targets were continuously reduced,so that the sequences in the pool had high specificity.After 16 rounds of screening,a total of four candidate aptamers were selected.Results The results showed that APT-1,APT-2 and APT-5 had high family homology,and the four candidate aptamers mainly formed stem ring and pseudojunction.Affinity was tested by qPCR.The Kd values of the four candidate aptamers binding to the serum of colon cancer patients were(4.466±0.384),(6.122±0.615),(5.610±0.202)and(6.964±0.181)nmol/L,respectively,which were all smaller than 10 nmol/L.The specificity of the serum of about 60 patients was analyzed.It was found that candidate APT-2 had good specificity,and the detection rate of this method was about 82.5%.Conclusion An excellent functional APT-2 was selected,and the APT-2 could be specifically bound to the serum of colon cancer

关 键 词：结肠癌 核酸适配体 SELEX 磁珠 qPCR 

分 类 号：Q52[生物学—生物化学]