SELEX法筛选宫颈癌前病变核酸适配子

Selection of DNA aptamers to cervical intraepithelial neoplasia by SELEX

摘要:

构建随机ssDNA文库，通过SELEX技术，以正常、炎性宫颈脱落细胞为反筛细胞，以上皮内低级别病变 (CIN1)、上皮内高级别病变 (CIN2、CIN3) 和鳞状细胞癌脱落细胞为正筛细胞，经过12轮筛选特异性适配子高度富集得到宫颈癌前病变适配子库，经特异性、亲和力分析和细胞免疫荧光确立高特异性适配子CIN-Ap4可作为诊断宫颈癌前病变生物标志物，为宫颈癌前病变分子诊断奠定理论基础，提供新思路。利用Prime Premier 5.0设计构建了随机ssDNA文库并根据文库两端固定序列设计引物，对对称PCR和间接不对称PCR中的退火温度、循环数以及上、下游引物浓度比等条件进行优化，分析确定50 μL反应体系中对称PCR的最佳反应条件为：最佳退火温度为49.5 ℃，最佳循环数为15个循环；间接不对称PCR的最佳反应条件为：50 μL反应体系中上、下游引物浓度的最佳比例为 80∶1，最佳循环数为35个循环。实验结果表明成功构建了寡核苷酸文库，在最适PCR条件下可获得理想的dsDNA和ssDNA，并具有良好的重复性，为顺利筛选适配子提供保证。

Abstract:

An in vitro synthesized random ssDNA library was subjected to 12 rounds of selection against anti-screening cells and sieving cells by SELEX. Normal and inflammatory cervical exfoliation cells were selected as anti-screening cells, and the cervical exfoliation cells of low-grade squamous intraepithelial lesion (CIN1), high-grade squamous intraepithelial lesion (CIN2, CIN3) and cervical carcinoma were selected as sieving cells during the screening process. Then, the highly specific aptamer CIN-Ap4 was established by the analysis of the specificity, affinity and cell immunofluorescence, which can be used as biomarker for Cervical Intraepithelial Neoplasia. Prime Premier 5.0 was applied to design a random ssDNA library. According to the fixed sequence at both ends of the library, a pair of primers were designed and synthesized. At the same time, the optimal annealing temperature, cycle times and primer concentration ratio of PCR procedure were selected. The results under the optimal condition are shown as follows. In the 50 μL reaction system, the optimum reaction conditions of symmetry PCR are as follows: annealing temperature is 49.5 ℃, number of cycles is 15. The optimal reaction conditions of indirect asymmetric PCR are as follows: the primer concentration ratio is 80:1, and the number of cycles is 35. The experiment proves that the oligonucleotide library is constructed successfully, and the highly specific dsDNA and ssDNA can be obtained under optimal PCR conditions with good repeatability, which establishes the foundation for the further exploration and experimentation.