琼脂磁珠消减SELEX技术筛选HIV gp41抗原适配体

摘要

Objective To obtain DNA aptamers with a highly specific affinity to HIV gp41 antigen using SELEX screening for detection of HIV.Methods The specific DNA aptamers of HIV gp41 antigen were screened from the double-stranded DNA derived from the single-stranded DNA (ssDNA) library with agarose beads as the supportive medium and HIV gp41 antigen as the target molecule using SELEX technique and real-time quantitative PCR.Results The secondary ssDNA library obtained after 6 rounds of screening was amplified by PCR to obtain dsDNA.The dsDNA was linked with pMDTM 18-T vector,cloned and sequenced to obtain 4 aptamers of HV gp41 antigen.The affinities of the 4 aptamers (Kd) all reached the nanomolar level.Among the 4 aptamers,the No.15 aptamer showed the strongest affinity.Specificity analysis of the aptamers revealed that all these 4 aptamers had specific affinity to HIV gp41 antigen with no affinity to other non-specific proteins.Conclusion We successfully obtained DNA aptamers with highly specific affinity to the HIV gp41 antigen from random single-stranded oligonucleotide library,and the obtained aptamers have the ability to antagonize HIV gp41 antigen.%目的 通过消减SELEX技术筛选得到高特异性、强亲和力的HIV gp41抗原适配体,为HIV的早期诊断提供了新的检测技术.方法 以琼脂磁珠为载体,HIV gp41抗原为靶标分子,利用消减SELEX技术和实时定量-PCR技术,筛选得到HIV gp41抗原适配体.结果 通过6轮筛选,筛选得到的次级ssDNA库通过PCR扩增得到dsDNA,dsDNA与pMDTM 18载体链接,进行克隆、测序,获得4条HIV gp41抗原适配体.获得的4条适配体亲和力(Kd)均在纳摩尔水平,其中15号适配体的亲和力最强,特异性检测表明筛选得到的适配体几乎只与HIV gp41抗原结合,不与其他非特异性蛋白结合.结论 利用随机单链寡核苷酸文库成功获得与HIV gp41抗原特异性结合的适配体,所获得的适配体具有拮抗HIV gp41抗原的能力.