禽白血病病毒群特异性抗原单克隆抗体的制备与鉴定  

Preparation and identification of monoclonal antibodies against the group specific antigen of avian leukosis virus

作　　者：贠炳岭[1] 李德龙[1] 胡晓亮 刘在斯[1] 秦立廷[1] 吴关[1] 刘文[1] 祁小乐[1] 王永强[1] 高宏雷[1] 高玉龙[1] 王笑梅[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所、兽医生物技术国家重点实验室/禽传染病研究室,黑龙江哈尔滨150001 [2]黑龙江出入境检验检疫局,黑龙江哈尔滨150090

出　　处：《中国预防兽医学报》2012年第10期782-785,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：现代农业肉鸡产业技术体系建设(nycytx-42-G3-01);公益性行业(农业)科研专项经费(201203056);哈尔滨市科技攻关计划项目(2010AA6AN034)

摘　　要：为制备禽白血病病毒(ALV)群特异性抗原的单克隆抗体(MAb),本研究从ALV-J病毒株感染DF-1细胞的冻融裂解液中提取DNA,通过PCR方法扩增出ALV群特异性抗原p27基因,并将其克隆至pMD18-T载体中,酶切鉴定后进行序列测定和分析。最后将p27基因亚克隆至载体pET-30a(+)中,转化受体菌BL21,IPTG诱导表达,经SDS-PAGE检测获得融合蛋白。经过western blot检测,表达的蛋白具有良好的反应原性。将纯化的p27蛋白作为抗原,免疫7周龄BALB/c小鼠,利用淋巴细胞杂交瘤技术,获得5株能稳定分泌特异性MAb的杂交瘤细胞株。5株MAb与纯化的p27蛋白以及不同亚群ALV可以发生特异性反应。所制备的MAb为禽白血病的诊断方法研究奠定了基础。To prepare monoclonal antibodies （MAb） against the group specific antigen of avian leukosis virus （ALV）, the p27 gene was amplified by PCR from DNA extracted fi＇om DF-1 cell infected with ALV and cloned into plasmid pET-30a for expressing in E. coli. The recombinant P27 protein was expressed with IPTG induction detected by SDS-PAGE and identified by western blot with ALV positive serum. Subsequently, five hybridoma cell lines secreting monoclonal antibodies were established by fusion of the SP2/0 cells with spleen cells immunized BALB/c mice by the purified p27 protein, which reacted specifically both to recombinant P27 protein and ALV antigen. The preparation of the MAbs would be useful to establish diagnosis method for detection of ALV infection.

关 键 词：禽白血病病毒 P27蛋白 原核表达 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]