一种基于适配体的快速检测宫颈癌标志物P16IN K4A 的方法

摘要

Objective To examine an analytical method for rapid detection of cervical tumor biomarker P16INK4A.Methods Recombinant plasmid P16INK4A‐pET28a was constructed.P16INK4A was purified through affinity chromatography after it successfully expressed in E.coli ,and verified by Western blotting.Systematic evolution of ligands by exponential enrichment (SELEX)was used to identify specific aptamers of P16INK4A.An aptamer with high affinity and specificity was finally selected through analysis of its affinity to P16INK4A.Thirteen nm gold nanoparticles were prepared by means of sodium citrate reduc‐tion method and characterized.An analysis method was established based on label‐free gold nanoparticles and the aptamer and its methodology was investigated.Results The P16INK4A protein was successfully expressed and purified.P16INK4A‐targe‐ting aptamer with high affinity and specificity was successfully isolated.An analytical method for detecting the cervical cancer marker P16INK4A was established by using the aptamer and label‐free gold nanoparticles.Conclusion This method has good accuracy and precision ,and can significantly improve the detection rate of early cervical cancer.%目的：提出一种可快速检测宫颈癌标志物P16INK4A的方法。方法构建重组质粒P16INK4A‐pET28a ，在大肠埃希菌中表达P16INK4A ，利用亲和层析纯化，Western blot法验证。以纯化的重组蛋白作靶标，利用指数级富集配体的系统进化技术（SELEX）筛选其特异性适配体，通过分析其亲和力最终选出一条高亲和力、高特异性的适配体。枸橼酸钠还原法制备13 nm纳米金颗粒并表征。建立基于免标记纳米金和适配体的分析方法，并对其进行方法学的考察。结果成功表达并纯化了P16INK4A蛋白，筛选出一条抗重组P16INK4A的高亲和力、高特异性的适配体，利用该适配体和免标记的纳米金建立了一种可快速检测宫颈癌标志物P16INK4A的分析方法。结论该方法具有较好的准确度和精密度，有望显著提高早期宫颈癌的检出率。