猫杯状病毒蚀斑纯化方法的建立  

Establishment of Plaque Purification Method for Feline Calicivirus

作　　者：赵静杰 梁琳[1,2] 刘子宁 肖发沂 贾亚雄 梁瑞英[1,2] 崔尚金 ZHAO Jing-jie;LIANG Lin;LIU Zi-ning;XIAO Fa-yi;JIA Ya-xiong;LIANG Rui-ying;CUI Shang-jin(Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Technology of Ministry of Agriculture,Beijing 100193,China;Shandong Vocational Animal Sciene and Veterinary College,Weifang 261061,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京海淀100193 [2]农业农村部兽用药物与诊断技术北京科学观测实验站,北京海淀100193 [3]山东畜牧兽医职业学院,山东潍坊261061

出　　处：《中国兽医杂志》2021年第10期15-19,共5页Chinese Journal of Veterinary Medicine

基　　金：中央级公益性科研院所基本科研业务费专项(2019-YWF-YB-05,2020-YWF-YTS-10);中国农业科学院创新工程计划(ASTIP-IAS15)。

摘　　要：猫杯状病毒(FCV)是引起猫科动物呼吸道疾病的重要病原之一,常用患病猫的口鼻眼拭子进行病原学诊断或病毒分离,病原微生物的混合感染给病毒的分离纯化带来困难。为了快速获得纯化的FCV,本试验将临床检测FCV和猫疱疹病毒Ⅰ型(FHV-1)混合病料处理后进行蚀斑纯化及条件优化。结果显示:将细胞数约为7×10^(5)个/mL的F81细胞以每孔2 mL铺入6孔板中,待病毒吸附90 min后覆盖浓度为2%的第1层低熔点琼脂糖溶液,于37℃、5%CO_(2)培养箱倒置培养48 h,然后加入含0.01%中性红的第2层低熔点琼脂糖溶液,于37℃、5%CO_(2)培养箱培养,此条件所形成蚀斑的数量及大小均适合挑取。通过3次克隆纯化,挑取蚀斑进行了RT-PCR检测,成功从FCV和FHV-1的混合病毒中分离纯化到1株FCV。本试验首次建立的FCV蚀斑克隆方法,有助于FCV以及其他病毒的分离纯化,并为病毒的生物学特性试验提供科学依据。Feline calicivirus(FCV) is one of the important pathogens that cause respiratory diseases in felines.Oral,nose,and eye swabs are common methods for clinical isolation and detection of virus pathogens.As clinical samples often contain many other kinds of microorganisms,they often pose difficulty for virus separation and purification.In order to facilitate quick purification of FCV,the mixed viruses of clinically tested FCV and feline herpesvirus type-1(FHV-1) were inoculated into monolayer F81 cells for establishment and optimization of the FCV plaque purification method.The results showed that by spreading 7×10^(5)/mL of F81 cells onto a six-well plate with 2 mL per well,adsorbing the virus for 90 min,covering with a first layer of low melting agarose solution at a concentration of 2%,incubating at 37°C in a 5% CO_(2) incubator upside down for 48 h,adding a second layer of covering solution containing 0.01% neutral red,and incubating at 37°C in a 5% CO_(2) incubator upright,the number and size of plaques formed were suitable for picking.After three rounds of cloning and purification,the plaques were picked for RT-PCR detection,and a FCV strain was successfully isolated and purified from the FCV and FHV-1 mix.This plaque purification method provides an approach for separation of FCV from other viruses,and forms a sound basis for biological characterization of FCV.

关 键 词：猫杯状病毒 蚀斑纯化 病毒分离 病毒纯化 

分 类 号：S852.659.1[农业科学—基础兽医学]