摘要
建立一种简单、高效的从混合斑中鉴定和分离精子的方法是法医学急需解决的难题之一。针对精细胞膜表面特定蛋白质开发的核酸适配体系统,可用于混合斑中精子的分离纯化。人载脂蛋白6(hLCN6)是一种附睾分泌蛋白质,它能结合到精子的头部和尾部,与精子成熟有关。利用指数富集配基的系统进化技术,以hLCN6蛋白为靶标,从96 nt单链DNA随机文库中筛选出与hLCN6高亲和力和特异性结合的适配体。经15轮正向筛选和3轮反向筛选。通过测序分析,采用酶联寡核苷酸吸附实验、斑点印迹和细胞免疫荧光染色等方法检测,获得的核酸适配体H2可特异性识别并结合hLCN6蛋白。hLCN6解离常数(Kd)为(3.21±0.75)nmol/L,且适配体H2在体外表现出与人精子较高的亲和力与特异性。这为依据核酸-蛋白质相互作用分离混合斑中的精细胞奠定了理论基础,提供了新思路。
It is an urgent and difficult task to establish a simple and efficient method for identifying and isolating sperm cells from mixed stains in forensic science.Nucleic acid aptamers targeting sperm cell-surface proteins can be used for the separation and purification of sperms from mixed stain samples.Human lipocalin 6(hLCN6)is an epididymal secreted protein that binds to the head and tail of sperm cells and is associated with sperm maturation.Using the systematic evolution of ligands by the exponential enrichment(SELEX)technique,magnetic bead-bound hLCN6 was used as the target molecule to screen for aptamers with high affinity and specificity to hLCN6 from a random single-stranded DNA(ssDNA)library.Through 15 rounds of positive selection and 3 rounds of negative selection,24 clones were selected and subjected to sequence analysis.Subsequently,4 candidate aptamers were selected and further examined for their binding affinity and specificity by enzyme-linked oligonucleotide adsorption(ELONA)and cell binding assays.One aptamer(H2)against hLCN6 with a high affinity and specificity was isolated and investigated by dot blotting and immunofluorescence staining.The result revealed that the candidate aptamer H2 with a dissociation constant of(3.21±0.75)nmol/L was able to recognize and specifically bind to hLCN6.The aptamer H2 also showed high affinity and specificity to human sperms in vitro,which establishes the foundation for the separation of sperm cells from mixed stain based on nucleic acid-protein interactions and provides a new scheme.
作者
陈炯
朱伟豪
赵越
CHEN Jiong;ZHU Wei-Hao;ZHAO Yue(Department of Forensic Biology,Henan University of Science and Technology,Luoyang 471023,Henan,China;CITIC Heavy Industries Co,Luoyang 471003,Henan,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2021年第10期1357-1365,共9页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目(No.81501634)资助。
关键词
人载脂蛋白6
适配体
指数富集配基的系统进化
混合斑
精子
human lipocalin 6(hLCN6)
aptamer
systematic evolution of ligands by exponential enrichment(SELEX)
mixed stain
sperm cell
