肺癌血清肿瘤标志物寡核苷酸适配体的筛选及其应用研究

In the efforts to explore an approach of Lung Cancer early diagnoses, we proposed a new strategy by an oligonucleotide aptamer(ligand) attaching a polymerase chain reaction(PCR) template to detect tumor marker. The oligonucleotide aptamer is selected by systematic evolution of ligands by exponential enrichment(SELEX), so that after aptamer target binding, the template moiety serves as the PCR template in real-time quantitative PCR(RT-PCR), and therefore, the binding event can be reported by the following RT-PCR signals, the oligonucleotide aptamer is for target(tumor marker) sensing and detection with higher sensitivity and specificity. In our researching, we will efforts to establish a detection and assay method of multiple tumor markers from Lung Cancer serum by oligonucleotide aptamers attaching a PCR template, find new tumor markers, and establish libraries of tumor markers from Lung Cancer serum. The contents include that: <1>selecting and obtaining oligonucleotide aptamers of multiple tumor markers from Lung Cancer serum by SELEX. <2>establishing a oligonucleotide-aptamer-based assay method, which is attached a PCR template,for multiple tumor markers from Lung Cancer serum. <3>establishing libraries of tumor markers from Lung Cancer serum, for early, medium-term and terminal Lung Cancer.

寡核苷酸适配体（配基）可与靶分子特异性识别并相互结合。该项目拟利用消减指数级富集的配体进化系统(SELEX)技术筛选肺癌血清标志物（靶分子）的寡核苷酸适配体；利用具有实时定量PCR模板的核酸信标配基传感检测技术，针对多种肺癌血清标志物，建立肺癌血清多标志物分子检测技术；利用特异性配基获得肺癌血清标志物，研究和发现新的肺癌血清标志物，建立肺癌病人早期、中期和晚期血清标志物谱库，为肺癌早期诊断和治疗新方法的建立奠定科学和技术基础。 研究内容包括以下三个方面:<1>利用SELEX技术筛选多种肺癌血清标志物的寡核苷酸适配体；<2>研究和建立基于PCR模板配基的多分子血清标志物分子检测技术；<3>研究寡核苷酸适配体与肺癌血清标志物的分子间相互作用，建立肺癌病人早期、中期和晚期血清标志物谱库。

肺癌是严重威胁人类健康的恶性肿瘤之一。利用肺癌血清标志物发现肺癌，从而进行临床早期干预，对提高肺癌病人的治愈率和生存率具有重要的意义。本项目构建的核酸信标配基分子，可通过核酸适配体特异性识别和结合靶蛋白，将蛋白信号转化为信标配基的核酸信号。而双链信标配基序列可以进行PCR扩增，实现靶蛋白信号的指数级放大。该方法是一种高灵敏度的微量蛋白检测技术，可望成为一种肺癌病人血清早期检测方法。. 本项目利用SELEX技术，从肺癌血清中筛选获得了可与肺癌血清标志物特异性结合的寡核苷酸适配体,并对其中6条适配体序列与肺癌病人血清结合特异性进行了初步验证；以琼脂磁珠为载体，核酸信标配基为探针，建立了一种基于寡核苷酸适配体的肺癌血清标志物标志物RT-qPCR分子检测技术，优化了检测条件，并对其特异性和稳定性进行了验证；利用适配体Apt-1，从肺癌病人血清中调取获得了857种特异性蛋白质，初步开展了相关蛋白质的分类、比对等生物信息学研究工作。