马γ-干扰素单克隆抗体的制备及其特性分析  

Preparation and Analysis of Monoclonal Antibodies against Equine Interferon-gamma

作　　者：白宇[1] 童铁钢[1] 刘光亮[1] 肖一红[1] 王群[1] 徐树兰[1] 张维军[1] 吴东来[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,哈尔滨150001

出　　处：《农业生物技术学报》2008年第1期10-14,共5页Journal of Agricultural Biotechnology

基　　金：国家高科技发展计划(863)项目(No.2006AA10A203);黑龙江省自然科学基金重点项目(No.ZJN-0602-01)

摘　　要：将原核表达的重组马γ-干扰素成熟蛋白(mEIFN-γ)免疫BALB/c小鼠(Mus musculus),经4次免疫后,取脾细胞与SP2/0骨髓瘤细胞进行融合,以昆虫杆状病毒表达系统获得的重组马γ-干扰素作为检测抗原进行间接ELISA检测,共筛选并最终得到了12株能稳定分泌抗体的单细胞克隆株。利用间接免疫荧光(IFA)试验对12株单克隆抗体进行鉴定,结果证实所获得的12株细胞分泌的抗体均为针对马γ-干扰素的特异性抗体。单克隆抗体亚型鉴定结果显示,4株为IgG1、2株为IgG2a、3株为IgG2b和3株为IgM,而且所有单克隆抗体的轻链均为"链。将此12株单克隆抗体分别与用原核系统分节段表达的重组马γ-干扰素GST-mEIFN-γ(1￣84)和GST-mEIFN-γ(80￣146)蛋白进行特异性ELISA检测,结果表明有7株单克隆抗体识别GST-mEIFN-γ(1￣84),而另5株单克隆抗体与GST-mEIFN-γ(80￣146)发生反应,说明所获得的12株单克隆抗体至少针对2个或2个以上不同的抗原表位。BALB/c mice （Mus musculus） were immunized intraperitoneally with purified recombinant mEIFN-γ protein. Murine myeloma cells were fused with splenocytes of the immunized mice. An indirect ELISA with recombinant mEIFN-γ derived from baculovirus expression system as antigen was used to screen antibody-producing hybridomas. Twelve hybridomas cells could produce McAbs steadily after 3 cycles of subcloning. Additionally, all McAbs showed positive reaction to mEIFN-γ in indirect fluorescence assay （IFA） tests. Four McAbs were IgG1, two McAbs were IgG2a, three McAbs were IgG2b and three McAbs were IgM isotype, respectively. The light chains of all McAbs were K chain. Furthermore, seven McAbs showed positive reaction to GST-mEIFN-γ（1-84） protein, and the others showed positive reaction to GST-mEIFN-γ（80-146） protein in specific ELISA tests. The results indicated that 12 McAbs could recognize two or more mEIFN-γ epitope. Therefore, these McAbs can be useful as a basis of detection methods for equine IFN-γ monitoring immune state and investigating immunologic mechanism.

关 键 词：马γ-干扰素 单克隆抗体 

分 类 号：S188[农业科学—农业基础科学] S185