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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ss DNA库通过PCR扩增得到ds DNA,ds DNA与p MDTM 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。
OBJECTIVE: To screen high-specificity and strong-affinity HIV gp41 antigen aptamers by SELEX technology and to provide a new detection method for the early diagnosis of HIV. Methods Using agar beads as the carrier and HIV gp41 antigen as the target molecule, HIV gp41 antigen aptamers were screened by using SELEX technique and real-time quantitative-PCR technique. Results The ds DNA was amplified by PCR from the secondary ss DNA library obtained through 6 rounds of screening. The ds DNA was linked with p MDTM 18 vector, cloned and sequenced, and 4 HIV gp41 antigen aptamers were obtained. The affinity of the four aptamers obtained at the nanomolar level was the highest. Among them, aptamer 15 showed the highest affinity. Specificity tests showed that the aptamers were almost bound to the HIV gp41 antigen only, Heterosexual protein binding. Conclusion The aptamers that specifically bind to the HIV gp41 antigen were successfully obtained using a randomized single-stranded oligonucleotide library. The resulting aptamers have the ability to antagonize the HIV gp41 antigen.