【作者】 秦莲花； 柏文娟； 胡忠义； 刘忠华； 杨华；

【Author】 BAI Wen-juan,HU Zhong-yi,YANG Hua,LIU Zhong-hua,QIN Lian-hua School of Pre-clinical Medicine and Life Science,Soochow University,Suzhou 215123,China

【机构】 同济大学附属上海市肺科医院/上海市结核病(肺)重点实验室；

【摘要】 目的利用SELEX技术筛选获得结核患者血清IgG抗体的特异性适体。方法体外合成长度为78个核苷酸的随机s sDNA文库,利用SELEX技术筛选,以结核患者血清中的IgG抗体为靶标进行筛选;筛选得到的饱和ssDNA适体文库,经克隆测序获得单个适体,生物素-亲和素显色系统检测适体与靶标的亲和性,DNAMAN软件分析适体的二级构象,血清学检测进行适体特异性的鉴定。结果纯化获得的IgG抗体浓度为1.3mg/mL。SELEX筛选10轮后,随机挑选13个适体进行亲和性检测和二级结构分析。结果显示,亲和性吸光度值大于1.0的适体有5个。二级构象分析揭示,适体与靶分子的结合基础为口袋颈环鼓包结构和分枝的连接角度。对亲和性吸光度值大于1.0的5个适体,经29份临床标本检测进行特异性的进一步验证显示,其中有4个适体对结核病组与健康对照组的检测结果具有显著差异。结论初步筛选获得结核患者血清IgG抗体的适体,并有较高的特异性。更多还原

【Abstract】 Objective To obtain DNA oligonucleotide aptamers specifically binding to TB IgG antibodies.Methods An random DNA library was subjected to rounds of selection by SELEX method against IgG antibodies purified from TB patient serum(the concentration of IgG antibodies was 1.3 mg per ml).Results After 10 rounds of selection,high-affinity aptamers to TB IgG antibodies were obtained.4 aptamers from 13 random selected aptamers showed high absorbency to TB IgG antibodies(A>1.0) by biotin-streptavidin-horseradish peroxidase system.Available ampters were cloned and sequenced.Pocket and stem-loops was the basis of aptamers binding to the antibodies by the analysis of sequences and second structures in DNAMAN package.5 aptamers(A>1.0) were selected to build the mixed-sandwich ELISA detection systems,respectively.The serum samples of TB patients(n=13) and healthy individuals(n=16) were tested in these systems.The results showed that the differences of TB group and healthy group were statistically significant(P<0.05) by 4 from 5 mixed-sandwich ELISA detection systems.Conclusion A set of aptamers affinity to TB IgG antibodies were successfully selected from the initial random DNA library and had considerably specifical binding ability.更多还原