兔出血症病毒特异性核酸适配体的筛选

摘要

In the present study, a random oligonucleotide library (80 nt) was constructed to select the aptamers against capsid protein VP60 of Rabbit hemorrhagic disease virus (RHDV) and develop a new diagnostic method. Post SDS-PAGE separation, protein VP60 was transferred onto PVDF membranes. After 20 rounds of screening, the secondary library was obtained, whose affinity and specificity were detected by using biotin-labeled aptamers as the primary antibody and HRP-streptavidin as the secondary antibody. The results showed that VP60 was identified by biotin-labeled subordinated library. In conclusion, the recombinant plasmids were cloned, sequenced, detected and eventually three specific nucleotide sequences were obtained.%为筛选出兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)衣壳蛋白VP60特异性核酸适配体,建立新型的兔出血症(rabbit hemorrhagic disease,RHD)检测方法,本研究构建了一个长80 nt的随机寡核苷酸文库.以固定在PVDF膜上的VP60为靶分子,使用消减SELEX技术(systematic evolution of ligands by exponential enrichment)进行20轮的筛选,以生物素标记的次级文库作为"一抗",以辣根过氧化物酶标记的链酶亲和素作为"二抗",对文库进行Western blot鉴定,结果表明第20轮文库存在可特异性的识别VP60的核酸序列.随机选取第20轮文库的46条序列克隆、测序,得到20个高度同源序列,同时对46个适配体再次鉴定,最终得到3个特异性识别VP60的适配体.