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目的:比较SELEX筛选中不同筛选介质的富集效果,为高通量筛选奠定基础。方法:以乙肝表面抗原(HBsAg)为靶蛋白,采用两种不同的筛选介质:硝酸纤维素膜和环氧树脂,分别将HBsAg包被其上,利用SELEX技术从随机单链DNA文库中筛选得到富集的亲和配基库,最后通过聚丙烯酰胺凝胶电泳和实时荧光定量PCR检测各自的富集效果。结果:经过16轮筛选,发现实时荧光定量PCR时,硝酸纤维素膜空白管与阳性管的循环阈值均在14循环,无明显区别;而环氧树脂空白管与阳性管的循环阈值区别明显,前者是25循环,后者是18循环。结论:在SELEX筛选中,以环氧树脂为筛选介质更易富集到与靶蛋白特异性结合的核酸适配体。
Objective: To compare the enrichment effect of different screening media in SELEX screening and lay the foundation for high-throughput screening. Methods: Hepatitis B surface antigen (HBsAg) was used as target protein. Two different screening media, nitrocellulose membrane and epoxy resin, were used to coat HBsAg respectively. SELEX technique was used to screen out the single-stranded DNA library Enriched affinity pool and finally detected the enrichment effect by polyacrylamide gel electrophoresis and real-time fluorescence quantitative PCR. Results: After 16 rounds of screening, the circulating thresholds of blank tubes and positive tubes of nitrocellulose membrane were all in 14 cycles when using real-time fluorescence quantitative PCR. There was no significant difference between the two groups. However, The former is 25 cycles, the latter is 18 cycles. CONCLUSION: In SELEX screening, the selection of epoxy resin is more likely to enrich for the aptamers that specifically bind to the target protein.