摘要
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13nm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。
Objective To examine an analytical method for rapid detection of cervical tumor biomarker P16INK4 A.Methods Recombinant plasmid P16INK4A-pET28 awas constructed.P16INK4 A 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 P16INK4 A.An aptamer with high affinity and specificity was finally selected through analysis of its affinity to P16INK4 A.Thirteen nm gold nanoparticles were prepared by means of sodium citrate reduction method and characterized.An analysis method was established based on label-free gold nanoparticles and the aptamer and its methodology was investigated.Results The P16INK4 Aprotein was successfully expressed and purified.P16INK4A-targeting aptamer with high affinity and specificity was successfully isolated.An analytical method for detecting the cervical cancer marker P16INK4 A 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.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2015年第6期667-673,677,共8页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
湖北省科技厅资助项目(No.2011GIA051)
