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目的获得与丙型肝炎病毒非结构蛋白NS2特异结合的高亲和力的DNA适配子。方法应用配基指数富集的系统进化(SELEX)技术进行HCV-NS2蛋白适配子筛选,将筛选得到的适配子克隆测序,应用DNAMAN软件对适配子一级结构和二级结构进行比对分析。结果重复筛选7轮后,适配子亲和力和特异性都达到最高,克隆测序得到4个适配子序列,命名为A1、A2、A3、A4,其随机序列部分分别为A1:GTGCGTCCCATGCTGCTGACTTAAATGGGTGGAGGGCAG,A2:CGTGAAATTGTTGACCACTCATGGAATCTGATCTCGTTT,A3:GAAAGGGGATAATCACTTAGGCCTCTCGAATAGTTTATC,A4:AGAAAGTTGAGAACTGCTGTTATTTTGTTAACGTACATG。经软件分析,适配子之间没有共同保守序列和同源序列,适配子的二级空间结构以茎环和口袋结构为主。结论获得了能与HCVNS2蛋白高亲和力和特异性结合的DNA适配子。
Objective To obtain a high-affinity DNA aptamer that binds specifically to the nonstructural protein NS2 of hepatitis C virus. Methods Systematic evolution (SELEX) based on ligand index enrichment was used to screen for HCV-NS2 aptamers. The aptamers were cloned and sequenced. DNAMAN software was used to compare the primary structure and secondary structure of aptamers Analysis. Results After repeated 7 rounds of selection, the affinity and specificity of the aptamers reached the highest level. Four aptamers were obtained by cloning and sequencing, named as A1, A2, A3 and A4. The random sequences of the aptamers were A1: GTGCGTCCCATGCTGCTGACTTAAATGGGTGGAGGGCAG, A2: A3: GAAAGGGGATAATCACTTAGGCCTCTCGAATAGTTTATC, A4: AGAAAGTTGAGAACTGCTGTTATTTTGTTAACGTACATG. After software analysis, there is no common conserved sequence and homologous sequence between aptamers. The secondary spatial structure of aptamers is dominated by stem-loop and pocket structure. Conclusion DNA aptamers with high affinity and specificity for HCV NS2 protein were obtained.