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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。结论 利用随机寡核苷酸文库成功获得抗HCVC蛋白的寡核苷酸适配子
Objective To screen and identify oligonucleotide aptamers against HCV core protein (C protein). Methods Using the systematicevolutionofligandsbyexponentiationrich SELEC (SELEX) technique, we screened the oligonucleotide aptamers that bind specifically to HCVC protein from the 81bp randomized single-stranded DNA library synthesized in vitro using the HCVC protein as target molecule, and determined the dissociation constant (Kd) And aptamer sequence determination. The primary and secondary structures of the aptamers were then analyzed using the ClustalW software package and DNA Folding Sever, respectively. Results After 9 cycles of screening, the binding rate of random ssDNA library to HCVC protein increased from 0.5% to 32.5%. All primary structures have no common homologous sequences, but can be divided into 5 families, each family has a common conserved sequence. Secondary structure analysis showed that the stem and loop structures formed by the aptamers may be the structural basis of binding to the HCVC protein. Among them, oligonucleotide aptamer C4 had the highest affinity to HCVC protein with a Kd value of 68 nmol L. Conclusion The oligonucleotide aptamers of anti-HCVC protein were successfully obtained by random oligonucleotide library