当前位置: 首页 > 文章 > 兔出血症病毒特异性核酸适配体的筛选 中国动物传染病学报 2017,25 (2) 9-13
Position: Home > Articles > SCREENING OF SPECIFIC APTAMERS FOR RABBIT HEMORRHAGIC DISEASE VIRUS Chinese Journal of Animal Infectious Diseases 2017,25 (2) 9-13

兔出血症病毒特异性核酸适配体的筛选

作  者:
焦美会;刘家森;姜骞;陆涛峰;胡小亮;蒋艳妹;曲连东
单  位:
中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室
关键词:
兔出血症病毒;适配体;SELEX技术
摘  要:
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。
译  名:
SCREENING OF SPECIFIC APTAMERS FOR RABBIT HEMORRHAGIC DISEASE VIRUS
作  者:
JIAO Mei-hui;LIU Jia-sen;JIANG Qian;LU Tao-feng;HU Xiao-liang;JIANG Yan-mei;QU Lian-dong;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,CAAS;
关键词:
Rabbit hemorrhagic disease virus;;aptamers;;SELEX
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.

相似文章

计量
文章访问数: 4
HTML全文浏览量: 0
PDF下载量: 0

所属期刊

推荐期刊