全新靶向小分子核酸适配体特异性检测肺癌患者血清标志物的应用研究

Study on the application of the novel targeted small molecule aptamer in the specific detection of serum markers in lung cancer patients

目的以纳米磁珠为筛选介质,利用富集配体系统进化技术(SELEX)从肺癌患者血清中筛选得到高特异性、强亲和力的肺癌血清核酸适配体,并进一步构建基于核酸适配体的肺癌血清标志物检测方法。方法以羧基官能团修饰的磁性纳米颗粒为载体、肺癌患者血清为筛选正向靶标、正常人血清为负向靶标,利用SELEX技术从含有77个碱基的随机ssDNA文库中筛选出与肺癌血清特异性结合的核酸适配体,流式细胞术监测筛选进程;当筛选达到饱和时对富集文库进行克隆测序并鉴定克隆的靶标结合特异性。选择性能最优的核酸适配体用于肺癌患者血清标志物的检测,通过50例肺癌患者血清和50例正常人血清鉴定该方法的特异性与敏感度。结果SELEX筛选进行到第9轮时,富集达到最大化。对筛选文库克隆测序,发现seq24和seq85的结合特异性更优,与正常人血清和白蛋白几乎不结合。核酸适配体seq24的Kd值为98.56 nmol/L,seq85的Kd值为128.69 nmol/L;将构建的基于核酸适配体seq24的肺癌患者血清检测方法与目前临床应用的方法比较,两者结果一致,阳性检出率为100%;此外,所构建的方法可区分早期和晚期肺癌患者。结论核酸适配体seq24能以高特异性和亲和力识别肺癌患者血清,以其为基础的全新检测体系可为肺癌的早期诊断提供新方法。

Objective To select high-specificity and strong-affinity lung cancer serum aptamer from lung cancer patients'serum by systematic evolution of ligands by exponential enrichment(SELEX)with nano-magnetic beads as the screening medium,and to further construct the detection method of lung cancer serum markers based on aptamers.Methods The nano-magnetic beads modified by carboxyl functional groups were used as the carrier,the serum of lung cancer patients as positive selection target,and normal human serum as negative selection target,aptamers that could specifically bind to lung cancer serum were selected from a random ssDNA library containing 77-base by SELEX technique,and flow cytometry was used to monitor the screening process.When the screening reached saturation,the enriched library was cloned and sequenced,and the target binding specificity of the clone was identified.The most optimal aptamer was selected for the detection of serum markers in patients with lung cancer,and the specificity and sensitivity of the detection method were evaluated by testing 50 lung cancer patient serum and 50 normal human serum.Results The enrichment of SELEX reached the maximum at the 9th round.It was found that the seq24 and seq85 owned better binding specificity than others by cloning and sequencing of screening library,which almost no binding with normal human serum and albumin.The Kd value of aptamer seq24 was 98.56 nmol/L and seq85 was 128.69 nmol/L.When compared the constructed method of lung cancer serum based on aptamer seq24 with the current clinical method,the results were consistent,and the positive detection rate was 100%.Moreover,the constructed method could distinguish patients with early and advanced lung cancer.Conclusion Aptamer seq24 can identify the lung cancer serum with high specificity and afficity,and the new detection system based on it can provide a novel approach for the early diagnosis of lung cancer.