猪传染性胃肠炎病毒间接免疫荧光检测方法的建立  

Development of an indirect immunofluorescence assay for detection of transmissible gastroenteritis virus of swine

作　　者：朱蕴暖 张鑫[2] 朱向东[2] 陈建飞[2] 时洪艳[2] 石达[2] 王鑫[3] 季朝阳 冯力[2] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]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猪消化道团队,黑龙江哈尔滨150069 [3]东北农业大学生命科技学院,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2017年第1期109-113,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31172350;31572541;31502092);兽医生物技术国家重点实验室资助项目(SKLVBP2015005)

摘　　要：为建立猪传染性胃肠炎病毒(TGEV)直观、特异和快速的检测方法,将TGEV接种PK-15细胞后,以抗TGEV N蛋白的单克隆抗体为一抗,荧光素FITC标记的山羊抗小鼠Ig G为二抗,建立了TGEV的间接免疫荧光(IFA)检测方法。结果显示,TGEV的最佳接种效价为1×102TCID50,培养时间为24 h,细胞最佳固定液为40 m L/L多聚甲醛,最佳封闭条件为10 g/L BSA 37℃封闭30 min,一抗的最佳使用浓度为1 ng/L,二抗的最佳稀释度为1∶100。对常见的猪流行性腹泻病毒、猪轮状病毒和猪圆环病毒2型等猪病病原检测均为阴性。结果表明,建立的检测方法对TGEV具有良好的特异性。本研究建立的方法为TGEV的实验室诊断及TGEV在感染细胞中的定位和动态分布提供了有效的检测手段。In order to develop specific method for detection of transmissible gastroenteritis virus of swine（TGEV） in PK-15 cells,a monoclonal antibody against TGEV N protein was used as the primary antibody,and a FITC labeled goat anti-mouse IgGused as the second antibody. Then,the optimization of the reaction conditions was performed to establish an indirect immunofluorescence assay（IFA） for detec- tion of TGEV. The results showed that the titer of TGEV was 1 × 102TCID50,the incubation time of TGEV was 24 h,the stationary liquid was 40 mL/L paraformaldehyde,the blocking condition was at 37℃ for 30 min with 10 g/L BSA,the concentration of the primary antibody was 1 ng/μ L and the dilution of the second antibody was 1 ： i00. The IFA constructed in this study was specific for detecting TGEV,and had no cross-reactions with other related porcine pathogens,such as porcine epidemic diarrhea virus,porcine rotavirus and porcine circovirus type 2. The results indicated that the constructed IFA was specific and stable for detecting TGEV in PK-15 cells.

关 键 词：猪传染性胃肠炎病毒 间接免疫荧光方法 抗TGEVN蛋白单克隆抗体 

分 类 号：S852.659.6[农业科学—基础兽医学]