摘要
目的筛选获得结核患者血清IgG抗体的特异性适体。方法纯化结核患者血清中的IgG抗体,以IgG抗体为靶标,利用SELEX技术进行筛选,筛选得到的ssDNA适体文库,进行克隆测序、亲和性检测、二级构象分析,并用于血清标本检测,进行适体特异性的鉴定。结果纯化获得了IgG抗体,浓度为1.3mg/mL。SELEX筛选10轮后,随机挑选12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4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4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3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。
To obtain DNA oligonucleotide aptamers specifically binding to TB IgG antibodies,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）.After 10 rounds of selection,high-affinity aptamers to TB IgG antibodies were obtained.Of 4 aptamers from 12 random selected aptamers showed high absorbency to TB IgG antibodies（A1.0） by biotin-streptavidin-horseradish peroxidase system.Pocket and stem-loops was the basis of aptamers binding to the antibodies by the analysis of sequences and second structures in DNAMAN package.Of 4 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（P0.05） by 3 from 4 mixed-sandwich ELISA detection systems.In conclusion,a set of aptamers affinity to TB IgG antibodies were successfully selected from the initial random DNA library and had considerably specific binding ability.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2011年第12期1111-1114,1118,共5页
Chinese Journal of Zoonoses
基金
上海市科委自然基金项目(11ZR1430200)和上海市科委医药生物处基金项目(10411955100)联合资助
