犬细小病毒层析纯化方法的建立  

Establishment of a method for chromatographic purification of canine parvovirus

作　　者：代昕宇 胡博[1] 邓效禹[1] 张成琪 李昀真 李甜甜 孙亚杰 许丽文 白雪[1] DAI Xinyu;HU Bo;DENG Xiaoyu;ZHANG Chengqi;LI Yunzhen;LI Tiantian;SUN Yajie;XU Liwen;BAI Xue(Institute of Special Animal and Plant Sciences,CAAS,Changchun 130112,China)

机构地区：[1]中国农业科学院特产研究所,吉林长春130112

出　　处：《畜牧与兽医》2024年第4期49-53,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(32100408);吉林省科技发展计划基金(20200201126JC,20200402111NC)。

摘　　要：病毒纯度是抗体制备和疫苗免疫效果提升的关键,本文利用Purose shell V15离子交换层析和Sepharose 4FF分子筛层析分别建立了犬细小病毒(CPV)的纯化方法。将CPV经过离心澄清和膜包浓缩后,用AKTA蛋白纯化仪结合Purose shell V15和Sepharose 4FF分别进行纯化,最后使用超滤管浓缩,通过病毒含量测定、总蛋白回收率测定、SDS-PAGE、Western blot和电镜观察分析病毒的纯化效果。结果:2种方法纯化后病毒形态典型,病毒颗粒大小约20 nm,总蛋白去除率97%以上,回收率71%以上;Purose shell V15纯化后的病毒经SDS-PAGE和Western blot分析显示无细胞成分的杂带,而Sepharose 4FF纯化后的病毒有少量的牛血清白蛋白条带。本试验结果表明这2种方法均可用于CPV的纯化,Purose shell V15纯化后的病毒纯度更高。The purity of the virus is the key basis for the preparation of antibodies and the improvement of vaccine immunity.In this paper,methods for purifying canine parvovirus(CPV)were established with Purose shell V15 ion-exchange chromatography and Sepharose 4FF molecular sieve chromatography.CPV was clarified by centrifugation and was concentrated with tangential flow filtration.Then,virus purifica⁃tion was performed on AKTA purifier 100 combined with Purose shell V15 or Sepharose 4FF,and was next concentrated by ultrafiltration spin columns.Finally,the virus content was analyzed with hemagglutination test,total protein removal rate was determined by BCA assay,and the purified virus was determined by SDS-PAGE,Western blot test and electron microscope observation.The results showed that the parvovirus purified with the two methods was typical in shape,and the diameter of the virus particle was about 20 nm.The total protein removal rate was over 97%.The virus purified by Purose shell V15 showed no disturbing bands of cellular components in the SDS-PAGE and Western blot ex⁃periments,while the virus purified by Sepharose 4FF had a few bovine serum albumin bands.The experiments showed that both the methods can be used for the purification of CPV,and the virus purified by Purose shell V15 was of higher purity.

关 键 词：犬细小病毒 纯化 离子交换 分子筛 

分 类 号：S855.65[农业科学—临床兽医学]