PEG-it^(TM)纯化浓缩J亚群禽白血病病毒效果的鉴定  

Identification of Purified and Concentrated Avian Leukosis Virus Subgroup J by PEG-it^(TM)

作　　者：程晓薇[1,2] 孔正茹 顾晨曦[1,2] 武宗仪 邵红霞[1,2,3] 秦爱建[1,2,3] 钱琨[1,2,3] 

机构地区：[1]扬州大学教育部禽类预防医学重点实验室,江苏扬州225009 [2]扬州大学江苏省动物预防医学重点实验室,江苏扬州225009 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国家禽》2017年第3期21-25,共5页China Poultry

基　　金：国家重点研发计划项目(2016YFD0500803);江苏省高校自然科学研究项目(13KJA230002);江苏省优势学科项目;公益性行业(农业)科研专项(201203055);扬州大学新世纪人才项目

摘　　要：为建立一种不利用超速离心法纯化浓缩J亚群禽白血病病毒(ALV-J)简便、易行的方法,利用PEG-it^(TM)病毒浓缩试剂与病毒培养细胞上清按比例混合,4℃低速离心,最后用无菌的预冷PBS进行重悬,取100μL病毒液进行TCID50的测定,同时利用电镜负染、SDS-PAGE银染及Western blot的方法对浓缩纯化后的病毒进行鉴定。结果表明:经过PEG-it^(TM)浓缩纯化病毒滴度提高10~100倍,滴度可达到1×10~7TCID50/m L;透射电镜负染观察结果表明,病毒粒子呈卵圆形,直径80~120 nm,并清晰可见囊膜上均匀密布的纤突;Western blot和银染结果显示,经过PEG-it^(TM)浓缩纯化的病毒杂蛋白明显减少,且具有良好的抗原性,能够被鸡多抗血清识别。提示在没有超高速离心机的情况下,可以使用PEG-it^(TM)纯化浓缩ALV-J,该方法使用方便,能满足大多数试验的需求。To establish a kind of simple and easy method to purify avian leukosis virus subgroup J（ALV-J）with-out ultracentrifugation, PEG-itTMvirus precipitation solution was mixed with viral supernatant on a proportional basis,and then the mixture was centrifuged at 1 500 g for 30 minutes at 4 ℃. At last viral pellets were resuspended using precold and sterile PBS. The concentrated and purified viruses were identified with TCID50 measurement,electron microscopy observation,silve stain and wstern blot assay. The results showed that ALV-J could be concentrated 10 to 100 times by PEGitTMvirus precipitation solution for 30 minutes at 4 ℃,resulted the viral titer in 1×10^7 TCID50/mL. The purified ALV-J virions were round in shape with a diameter of approximately 80 to 120 nm under transmission electron microscopy.And we could clearly see spikes on the surface of viral envelope. Western blot and silver stain results showed the impure protein was reduced after virus purification. Meanwhile,the purified virus maintained good immunogenicity,which could be recognized by the chicken antiserum against ALV-J. It＇s suggested that PEG-itTMvirus precipitation solution could concentrate and purify ALV-J without ultracentrifugation, and the solution provide a simple and highly effective method to concentrate ALV-J for most of the experimental requirement.

关 键 词：PEG-it^TM病毒浓缩试剂 ALV-J 浓缩纯化 鉴定 

分 类 号：S855.3[农业科学—临床兽医学]