猪圆环病毒2型ORF2单克隆抗体的制备与鉴定  

Preparation and Identification of Monoclonal Antibodies against ORF2 of Porcine Circovirus Type 2

作　　者：安春敬 李祥敏[2] 张华伟[2] 莫红芳 郭夏阳 赵泽凯 钱平[2] 赵晓玲 AN Chun-iing LI Xiang-min ZHANG Hua-wei MO Hong-fang GUO Xia-yang ZHAO Ze-kai QIAN Ping ZHAO Xiao-ling(Department of Animal Science and Technology ,College of Agriculture and Animal Husbandry,Tibet University,Linzhi,Tibet,860000,China State Key Laboratory of Agriculture Microbiology, Huazhong Agriculture University, Wuhan, Hubei, 430070, China)

机构地区：[1]西藏大学农牧学院动物科学学院,西藏林芝860000 [2]华中农业大学农业微生物重点实验室,湖北武汉430070

出　　处：《动物医学进展》2017年第10期32-37,共6页Progress In Veterinary Medicine

基　　金：广西科学研究与技术开发(桂科合14125008-1-18);华中农业大学自主科技创新基金(2662016PY003;2662016PY004)

摘　　要：以杆状病毒表达系统表达PCV2ORF2重组蛋白为免疫原,旨在获得针对PCV2的特异性单克隆抗体。用Sf9细胞表达PCV2ORF2重组蛋白,通过层析柱和超滤管浓缩法纯化PCV2ORF2重组蛋白。将纯化的PCV2ORF2重组蛋白免疫6周龄的雌性Balb/c小鼠,3次免疫后,取免疫后小鼠脾脏细胞与骨髓瘤细胞SP2/0融合,采用间接ELISA筛选,经过3次亚克隆获得4株能稳定分泌特异性抗体的杂交瘤细胞,命名为1H9、4C11、5B8和4F12株。IFA和Western blot试验说明,4株杂交瘤细胞分泌的抗体能与ORF2重组蛋白作用。特异性试验表明,4F12株单克隆抗体与猪圆环病毒2型(PCV2KQ22株)发生特异性反应,经抗体亚型鉴定1H9、4C11和5B8 3株亚型为IgG1/Kappa,4F12株为IgG2b/Kappa,1H9、4C11、5B8和4F12株小鼠腹水效价分别为105、104、106和105。表明获得了与PCV2特异性作用的单克隆抗体。The cow IgG Fc gene was fused to FnBPB-ClfA gene by SOE-PCR and cloned into pET-32a （＋） vector to express the recombinant protein of rFc-FnBPB-ClfA in E. coli. Then the lactating cows were immunized with PBS or purified recombinant protein （bFc-FnBPB-ClfA） with the adjuvant levamisole hydrochloride （LH） or aluminum hydroxide （ALUM）. Immunizatioin in the ways of muscle-muscle or muscle- muscle-muscle or muscle-teat canal to the nearly dry period of dairy cows. Indirect ELISA method and Western blot were used to monitor the antibody in the sera of cows in each group. The results showed that the antibody level arrived the highest at 21 d using LH as adjuvant,and could reach 1 ： 1 600,and the antibody levels of B group and C group were 1 ： 800 and 1 ： 400. The results of this study indicatd that bFc- FnBPB-ClfA and LH adjuvant can effectively stimulate immune response in dairy cows.

关 键 词：猪圆环病毒2型 核衣壳蛋白 单克隆抗体 

分 类 号：S852.4[农业科学—基础兽医学] S852.659.2[农业科学—兽医学]