丙型肝炎病毒核心蛋白寡核苷酸适配子的筛选与鉴定  

Screening and characterization of aptamers of hepatitis C virus core protein

作　　者：詹林盛[1] 孙红琰[1] 彭剑淳[1] 邵宁生[2] 王全立[1] 

机构地区：[1]军事医学科学院输血研究所 [2]军事医学科学院基础医学研究所

出　　处：《中华微生物学和免疫学杂志》2002年第5期578-581,共4页Chinese Journal of Microbiology and Immunology

摘　　要：目的　筛选、鉴定抗HCV核心蛋白 (C蛋白 )的寡核苷酸适配子 (aptamers)。方法　利用systematicevolutionofligandsbyexponentialenrichment (SELEX)技术 ,以HCVC蛋白为靶分子 ,从体外合成的 81bp随机单链DNA文库中筛选与HCVC蛋白特异结合的寡核苷酸适配子 ,并进行了解离常数 (Kd)测定和适配子序列测定。再分别利用ClustalW软件包和DNAFoldingSever分析适配子的一级结构和二级结构。结果　经过 9轮循环筛选 ,随机ssDNA库与HCVC蛋白的结合率从 0 .5 %上升到32 .5 %。所有的一级结构没有共同的同源序列 ,但可分 5个家族 ,每个家族具有共同的保守序列。二级结构分析表明 ,适配子形成的茎环、凸环结构可能是与HCVC蛋白结合的结构基础。其中寡核苷酸适配子C4与HCVC蛋白特异结合的亲和力最高 ,Kd值为 6 8nmol L。Objective To screen and characterize aptamers against hepatitis C virus(HCV) core protein. Methods A 81bp single stranded DNA (ssDNA) random library was subjected to 9 rounds of selection against HCV core protein by systematic evolution of ligands by exponential enrichment (SELEX) method. The selected aptamers were cloned and sequenced. The primary sequences and structure of the aptamers were analyzed byClustal W and DNA folding sever and the affinities of aptamers to HCV core protein were determinated. Results After 9 rounds selection, the percentage of the ssDNA pool bound to HCV core protein increased from 0.5% to 32.5%. There were 5 conserved sequences and the structure analysis revealed that the stem loops or bulge was the main motif in the interaction between aptamers and HCV core protein. The affinity of aptamer C4 to HCV core protein was highest with Kd values as low as 68nmol/L. Conclusion Aptamers against HCV core protein has been identified by SELEX methods from a 81bp single stranded DNA random library. [

关 键 词：丙型肝炎 丙型肝炎病毒 核心蛋白 SELEX技术 寡核苷酸适配子 

分 类 号：R346[医药卫生—基础医学]