摘要
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 medium in systematic evolution of ligands by exponential en- richment （SELEX）, and to lay a foundation for high throughput screening. Method：Hepatictis B surface antigen （HBsAg） was chosen as the target protein and two different screening mediums, nitrocellulose membranes and epoxy resins, were coated with HBsAg and adopted to select the enriched library from random single - stranded DNA library with SELEX technology. At last, polyacrylamide gel electrophoresis and real - time fluorescence quantitative PCR were performed to detect the enrichment effect of two different screening mediums. Result： After 16 rounds of selection,the values of threshold cycle（Ct） of the blank tubes and positive tubes of nitrocellulose membranes were 14 cycles,didn＇t have significant differences. While the Ct value of the blank tubes of epoxy resins were 25 cycles and the positive ones were 18 cycles, showed obvious differences between the two. Conclusion： In SELEX screening, the epoxy resins were easier to enrich the aptam- ers that specifically combine with target proteins.
出处
《生物技术》
CAS
CSCD
北大核心
2012年第6期48-52,共5页
Biotechnology
基金
国家自然科学基金地区基金项目("具有PCR模板的核酸适配体对肝癌肿瘤早期诊断检测技术的研究"
No.81060180)资助~~
