肺癌患者血清特异性核酸适配体的筛选及鉴定

Sreening and identification of serum specific aptamers for lung cancer

目的以磁珠为筛选介质,利用实时定量-PCR和消减SELEX技术从肺癌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。方法以磁珠为载体、肺癌病人血清为筛选的靶标分子,通过消减SELEX技术及实时定量PCR技术,从随机ss DNA文库中筛选出与肺癌血清特异性结合的aptamers,将得到的第9轮富集文库扩增为ds DNA,送上海生工直接对筛选得到的核酸适配体库进行高通量测序。结果经过9轮筛选得到4条与肺癌血清高特异性结合的aptamers,测序结果显示均为不同序列。结论验证筛选出的与肺癌血清结合的aptamer,特异性检测表明,筛选得到的肺癌血清核酸适配体与肺癌血清的结合解离常数均在纳摩尔级水平,其中Seq-2、Seq-3、Seq-6、Seq-19号核酸适配体能高特异性结合肺癌血清,与正常人血清不结合,再通过200例肺癌血清和200例正常人血清进一步鉴定4条肺癌血清核酸适配体检出肺癌的阳性率,阳性检出率达91%以上,为肺癌的早期诊断提供了新方法。

Objective To obtain high-specificity and strong-affinity serum aptamer of lung cancer from lung cancer serum by real-time quantitative PCR and subtractive SELEX technology, with magnetic beads as the screening medium. Methods Using magnetic beads as the carrier and the serum of lung cancer patients as the screening target molecule, aptamers that bind to the sera of lung cancer were screened from the random ss DNA library by SELEX and real-time quantitative PCR. Then the obtained 9 th round enriched library was amplified to ds DNA and sent to Sangon Biotech（Shanghai）, where the screened library of aptamers went through high-throughput sequencing.Results After four rounds of screening, four aptamers with high specificity of serum were obtained. The sequencing results showed that they were different sequences.Conclusion The specificity of screened aptamer in combination with serum of lung cancer was tested. The results showed that the binding dissociation constants of serum aptamer and lung cancer serum were all within nanomolar level. Seq-2, Seq-3, Seq-6 and Seq-19 can bind to the serum of lung cancer with high specificity, but not with normal human serum. Using the serum of 200 cases of lung cancer and normal human serum of 200 cases, it was showed that the positive detection rate of lung cancers by these four serum aptamers of lung cancers could reach 91% or even higher, thus providing a new method for the early diagnosis of lung cancer.