猪繁殖与呼吸综合征病毒纯化方法的建立  

Development of a purification method for porcine reproductive and respiratory syndrome virus

作　　者：田书苗 郅玉宝[2] 王林建[2] 乔松林[2] 郭军庆[2] 陈静[2] 柴书军[2] 张改平[1,2] 杨艳艳[2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省农业科学院动物免疫学重点实验室,河南郑州450002

出　　处：《中国兽医科学》2016年第5期568-572,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31272546);河南省农科院科研发展专项资金项目(20148405)

摘　　要：采用差速离心、膜包超滤浓缩和Sepharose 4 Fast Flow(4FF)凝胶层析相结合的策略纯化猪繁殖与呼吸综合征病毒(PRRSV),建立了一种温和纯化PRRSV的方法。通过SDS-PAGE、Western-blot和检测病毒含量、蛋白含量等方法分析纯化效果。用纯化后的病毒免疫BALB/c雌性小鼠,通过免疫过氧化物酶单层细胞试验(IPMA)检测其免疫原性,并与超速离心沉淀病毒的纯化方法相比较,评估纯化病毒的免疫效果。结果显示,4FF纯化后的病毒TCID_(50)提高了50倍,与超离方法相比,4FF纯化法将病毒原液杂蛋白的去除比率由97.51%提高到99.68%,SDS-PAGE显示未见明显杂蛋白条带,IPMA结果显示4FF纯化病毒的免疫原性较好,特异性较强,消除了免疫后小鼠血清与Marc-145细胞蛋白的非特异性反应。结果表明,4FF纯化方法优于超离纯化方法,前者的建立为制备单克隆抗体和规模化纯化PRRSV疫苗的工艺开发奠定基础。A method with the differential centrifugation,tangential flow filtration and 4 Fast Flow Sepharose gel chromatography was established to purify the porcine reproductive and respiratory syndrome virus （PRRSV）.Then the purification efficiency was analyzed by detecting virus particles and protein contents,and further analyzed by SDS-PAGE and western-blot. The BALB/c female mice were immu- nized with the purified virus and then immunogenicity was detected by IPMA. Finally,the reliability of 4FF method was evaluated by comparing with the results of sedimentation ultracentrifugation. In re- sults,the TCIDm of virus purified by 4FF was increased by 50 times. Compared with the sedimentation ul- tracentrifugation method,4FF would increase the removal efficiency of impurity protein from 97.51%to 99.68%.SDS-PAGE showed that virus purified by 4FF showed no obvious impurity protein bands. The re- sults of IPMAindicated that virus purified by 4FF had better immunogenicity,high specificity,which e- liminated the nonspecific reaction between the serum of the immunized mice and the protein of Marc-145 cell. These results demonstrate that the efficiency of the purification of PRRSV by using 4FF is better than that of sedimentation ultracentrifugation. Therefore our research would lay the foundation forpreparing the monoclonal antibody and large-scale purification of PRRSV vaccines.

关 键 词：猪繁殖与呼吸综合征病毒 纯化 SEPHAROSE 4FF层析 

分 类 号：S852.659.2[农业科学—基础兽医学]