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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: To screen high-specificity and strong-affinity aptamers from gastric cancer serum by using carboxylated agar beads as screening media and reverse transcription-polymerase chain reaction (SELEX) technique. Methods: The serum of gastric cancer treated by centrifugal ultrafiltration (50 000 UF tube) method was used as target. The carboxylated agar beads were used as the carrier. The oligonucleotide library was first bound to reverse-phase magnetic beads (bound to normal serum) Take the supernatant and then with the screening of magnetic beads (combined with gastric cancer serum) to wash away the unbound oligonucleotide molecules, the oligonucleotide molecules bound to the magnetic beads were isolated and amplified, lambda cleavage ss DNA secondary library for 10 rounds of screening. The ds DNA was amplified from the 10th round library, and the ds DNA of the purified target fragment was recovered using a gel recovery kit and ligated with the pTMD18-T vector to transform E. coli DH5α competent cells Positive clones were picked out and sequenced to obtain the aptamer sequence. Meanwhile, the Kd value of gastric cancer aptamers was determined by flow cytometry. Results: After 10 rounds of screening, 20 aptamers that bind to gastric cancer serum were obtained. Conclusion: Specificity tests showed that the binding dissociation constants of serum aptamers and gastric cancer serum were all at nanomolar level. The aptamers 5,7,16,17 and 18 had high specificity and strong affinity Combined with gastric cancer serum, and normal human serum does not. The study provides an experimental basis for the early diagnosis of gastric cancer.