马γ-干扰素单克隆抗体的制备及其特性的鉴定

摘要：将原核表达的重组马-干扰素成熟蛋白（mEIFN-）免疫BALB/c小鼠，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-干扰素作为检测抗原进行间接ELISA检测，共筛选到15个阳性细胞株。分别经过3轮亚克隆后，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）实验对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-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、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-干扰素GST-mEIFN-(1-84)、GST-mEIFN-(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-(1-84)，而另5株单克隆抗体与GST-mEIFN-(80-146)发生反应，说明所获得的12株单克隆抗体至少针对两个或两个以上不同的抗原表位。这将为今后建立马-干扰素检测方法、监测机体免疫状态和研究机体免疫机制奠定了基础。

Preparation and Analysis of Monoclonal Antibodies against Equine Interferon-gamma

Abstract BALB/c mice were immunized intraperitoneally with purified recombinant mEIFN-protein. Murine myeloma cells were fused with the splenocytes of the immunized mice after the fourth immunization. An indirect ELISA with recombinant mEIFN-derived from the baculovirus expression system as antigen was used to screen antibody-producing hybridomas and 15 McAbs against mEIFN-were obtained. Twelve hybridomas cells of them can produce McAbs steadily after 3 cycles of subcloning. All the 12 McAbs were reactive in (indirect fluorescence assay) IFA tests, and all of them showed positive reaction to mEIFN-. Four McAbs are IgG1, two McAbs are IgG2a，three McAbs are IgG2b and three McAbs are IgM isotype, respectively. The light chains of all McAbs are chain. The seven McAbs of them showed positive reaction to GST-mEIFN-(1-84) protein, and the others show positive reaction to GST-mEIFN-(80-146) protein in specific ELISA tests, respectively. The McAbs against mEIFN- protein developed in this study would be useful as a basis of detection methods for equine IFN-, investigating the structure and biological effects of equine IFN-.