猫细小病毒适配体的筛选与鉴定  

Screening and identification of aptamers for Feline panleukopenia virus

作　　者：董丽丽 许鑫燕 刘迪 郑亚婷 康洪涛 姜骞[1] 杨鸣发 刘家森[1] 曲连东[1] DONG Li-li;XU Xin-yan;LIU Di;ZHENG Ya-ting;KANG Hong-tao;JIANG Qian;YANG Ming-fa;LIU Jia-sen;QU Lian-dong(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第8期826-831,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0501001)。

摘　　要：为筛选猫细小病毒(FPV)特异性核酸适配体,本研究利用体外指数富集的配体系统进化技术(SELEX),从80 nt的单链DNA随机文库中筛选出与FPV高亲和力、高特异性结合的核酸适配体。对FPV适配体共进行14轮筛选,采用dot blot鉴定,确定其中的Apt27和Apt52为候选适配体。MTT试验表明,Apt27和Apt52对CRFK细胞(猫肾细胞)无毒性作用。利用酶联寡核苷酸法(ELONA)测定Apt27和Apt52的解离常数,结果显示分别为18.3249±7.1723 nmol/L和24.7882±10.0067 nmol/L。同时采用dot blot、间接免疫荧光试验(IFA)方法检测Apt27和Apt52对不同病毒的结合情况,结果显示其对猫杯状病毒、猫疱疹病毒的结合力弱,而对FPV的结合能力较强。预测二者二级结构可见,Apt27和Apt52均具有环状、发卡状结构,其中茎环结构可能为适配体特异性识别FPV的基本结构。本研究首次筛选到特异性识别FPV的适配体序列,为FPV的检测与治疗制剂的开发提供了新思路。To screen Feline panleukopenia virus(FPV) specific nucleic acid aptamers, in this study, aptamer candidates with high affinity and specificity against FPV were screened from a 80 nt single-stranded DNA random library by systematic evolution of ligands exponential enrichment(SELEX). Through fourteen rounds of selection, Apt27 and Apt52 were identified by dot blot and selected as the candidate aptamers. The MTT test results showed that Apt27 and Apt52 have no toxic effect on CRFK cells.The dissociation constant(Kd) of Apt27 and Apt52 were identified by enzyme-linked oligonucleotide assay(ELONA), with the Kd value of 18.3249 ±7.1723 nmol/L and 24.7882 ±10.0067 nmol/L, respectively. The binding activity of aptamers to different viruses were detected by dot blot and indirect immunology fluorescence assay(IFA). The results showed the binding ability of aptamers to feline calicivirus(FCV) and feline herpesvirus(FHV) were weak, but to FPV were strong. This study is the first successfully selected the aptamer sequence that specifically recognizes FPV, which provides a new idea for the development of diagnostic and therapeutic agents against FPV.

关 键 词：猫细小病毒 适配体 SELEX技术 

分 类 号：S852.4[农业科学—基础兽医学]