胃癌血清适配体的筛选及其鉴定

Screening and identification of gastric cancer serum aptamers

目的 :以羧基化琼脂磁珠为筛选介质,利用RT-PCR和靶标替换消减SELEX技术从胃癌血清中筛选得到高特异性、强亲和力的适配体。方法:以离心超滤(50 000超滤管)法处理后的胃癌血清作为靶分子,羧基化琼脂磁珠为载体,先将寡核苷酸文库与反筛磁珠(结合正常血清)结合,取上清再与筛选磁珠(结合胃癌血清)结合,洗去未结合的寡核苷酸分子,对结合在磁珠上的寡核苷酸分子进行分离和扩增,λ酶切法制备ss DNA次级库,进行10轮筛选,将第10轮的文库扩增得到ds DNA,利用胶回收试剂盒回收纯化目的片段的ds DNA,并与p MDTM18-T载体连接,转化大肠杆菌DH5α感受态细胞,挑取阳性克隆,测序获得适配体序列,同时用流式细胞术测定胃癌血清适配体的Kd值。结果:经过10轮筛选后,得到20条与胃癌血清结合的适配体。结论:特异性检测表明,筛选得到的胃癌血清适配体与胃癌血清的结合解离常数均在纳摩尔级水平,其中5、7、16、17、18号适配体能高特异性、强亲和力结合胃癌血清,与正常人血清不结合。该研究为胃癌的早期诊断提供了实验基础。

Objective：By using carboxylated agar magnetic beads as selection medium,we sought to use RT-PCR and target replacement subtractive SELEX technology from the serum of gastric cancer screening to obtain aptamers with high specificity and affinity. Methods：The target molecules were gastric cancer serum performed with centrifugal ultrafiltration（ultrafiltration 50 000）,and the carriers were the carboxyled agar magnetic beads. Firstly,oligonucleotide library and anti-screening agar magnetic beads（normal serum binding）were bound,then supernatant was combined with screening magnetic beads（gastric binding serum）. We washed away unbound oligonucleotide molecules,and oligonucleotide molecules binding on the magnetic beads were isolated and amplified. λenzyme digestion method was performed to prepare ss DNA second library for 10 rounds of screening. The library of the 10 thround was amplified to obtain ds DNA,then we used gel extraction kit recovering purified fragment of ds DNA and connected it with p MDTM18-T vector and transformed into E. coli DH5α competent cells. we selected positive cloning group and sequenced them to obtain aptamers sequence. Simultaneous determination of Kdvalues of gastric cancer serum aptamer was performed by flow cytometry. Results：After10 rounds of selection,20 gastric cancer serum specific aptamers were screened. Conclusion：The specific detection showed that the Kdvalues of gastric cancer serum binding aptamers were at the nanomolar level,and No.5,7,16,17,18 aptamers can bind to gastric cancer serum with high specificity and affinity,and didn＇t bind to normal human serum. It provides an experimental basis for the early diagnosis of gastric cancer.