文章摘要
曾家豫,万 波,马 瑾,袁红霞,李 星,赵运旺,廖世奇.癌胚抗原特异性单克隆抗体(Anti-CEA)核酸适配体的筛选[J].南京医科大学学报,2015,(6):782~786
癌胚抗原特异性单克隆抗体(Anti-CEA)核酸适配体的筛选
Selection of aptamers to Anti-CEA
投稿时间:2015-01-18  
DOI:10.7655/NYDXBNS20150605
中文关键词: 消减SELEX技术  羧基琼脂磁珠  CEA特异性单克隆抗体  核酸适配体  肺癌
英文关键词: subtractive SELEX  carboxyl magnetic microspheres  Anti-CEA  aptamer  lung cancer
基金项目:国家自然科学基金(81360333)
作者单位
曾家豫 西北师范大学生命科学学院,甘肃 兰州 730070 
万 波 西北师范大学生命科学学院,甘肃 兰州 730070 
马 瑾 甘肃省医学科学研究院分子生物中心,甘肃 兰州 730050 
袁红霞 甘肃省医学科学研究院分子生物中心,甘肃 兰州 730050 
李 星 西北师范大学生命科学学院,甘肃 兰州 730070 
赵运旺 西北师范大学生命科学学院,甘肃 兰州 730070 
廖世奇 甘肃省医学科学研究院分子生物中心,甘肃 兰州 730050 
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中文摘要:
      目的:筛选癌胚抗原(carcinoembryonic antigen,CEA)特异性单克隆抗体(Anti-CEA)的核酸适配体(aptamers),为肺癌血清肿瘤标志物核酸适配体的筛选奠定实验基础。方法:利用羧基化琼脂磁珠作为筛选介质,以Anti-CEA为筛选的目标靶分子,通过消减SELEX技术及实时定量PCR技术,从随机ssDNA文库中筛选出与Anti-CEA特异性结合的aptamers,并通过凝胶阻滞实验(EMSA)鉴定筛选到的Anti-CEA-aptamer复合物,然后将得到的第10轮富集文库扩增为双链DNA,通过切胶纯化后,连接PMD18-T载体,转化大肠杆菌DH5α感受态细胞,同时利用交错PCR技术鉴定阳性克隆,经测序,获得aptamers的序列。结果:经过10轮筛选得到了4条与Anti-CEA结合的aptamers,测序结果显示均为不同序列。结论:验证筛选出的与Anti-CEA结合的aptamer,特异性检测结果表明2号aptamer与靶分子结合的特异性很高,且与非特异蛋白无明显吸附,筛选出的aptamers用于识别Anti-CEA,将为肺癌的早期诊断和早期治疗提供新的突破口。
英文摘要:
      Objective: To screen aptamers to carcinoembryonic antigen (Anti-CEA), which will lay the experimental foundation for obtaining aptamers to serum tumor markers of lung cancer. Methods: We used carboxyl magnetic microspheres (CMM) as carrier, anti-CEA for the screening target, and subtractive SELEX as well as real-time quantitative PCR techniques to screen aptamers to anti-CEA from random single-stranded oligonucleotide libraries. We identified the anti-CEA-aptamers via electrophoretic mobility shift assay (EMSA). And then, the 10th round of screening was amplified for double-stranded DNA, purified by gel after cutting, connected with PMDTM18-T vector and transformed in the competent cell of Ecoli.DH5α. Meanwhile, we identified positive clones through interlaced PCR technique. Finally, we obtained sequences of aptamers. Results: The results showed that four aptamers connected with Anti-CEA were obtained with different sequences after 10 rounds of screening. Conclusion: The specificity test of screened binding apatamers demonstrated that with No.2 aptamer has a high specificity to Anti-CEA, and it could not be bound with non-specific proteins. These aptamers could be used to recognize the Anti-CEA, ultimately, to offer a new breakthrough for the early diagnosis and early treatment of lung cancer.
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