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

作　　者：曾家豫[1] 万波[1] 马瑾[2] 袁红霞[2] 李星[1] 赵运旺 廖世奇[2] 

机构地区：[1]西北师范大学生命科学学院,甘肃兰州730070 [2]甘肃省医学科学研究院分子生物中心,甘肃兰州730050

出　　处：《南京医科大学学报（自然科学版）》2015年第6期782-786,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81360333)

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ss DNA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。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 mierospheres （CMM） as cartier, 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 eleetrophoretic 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.

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 

分 类 号：R392.33[医药卫生—免疫学]