重组猪干扰素-α单克隆抗体的制备和鉴定  

Preparation and identification of monoclonal antibodies against recombinant porcine interfer-α

作　　者：周晓丽[1,2] 袁秀芳[1] 杜晓莉[1,3] 李军星[1] 徐丽华[1] 王一成[1] 

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021 [2]南京农业大学动物医学院,江苏南京210095 [3]甘肃农业大学动物医学院,甘肃兰州730070

出　　处：《浙江农业学报》2015年第3期332-337,共6页Acta Agriculturae Zhejiangensis

基　　金：浙江省重大科技专项重点农业项目(2012C12009-1);浙江省"三农六方"科技协作项目(2012R22A60C01)

摘　　要：利用自动发酵罐培养毕赤酵母,甲醇诱导其分泌表达重组猪IFN-α（r Po IFN-α）;经硫酸铵沉淀、G-25琼脂糖柱脱盐处理及阴离子交换柱和分子筛层析纯化。以纯化r Po IFN-α蛋白作为抗原免疫6周龄BALB/c鼠3次后,取小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经间接ELISA筛选和3~5次亚克隆,共获得5株均能稳定分泌抗r Po IFN-α单克隆抗体的杂交瘤细胞株。抗体亚类鉴定、特异性和叠加试验结果表明,这5株抗体均属于Ig G1亚类,能与r Po IFN-α产生特异性反应,其抗原结合位点相同或相近。该研究有助于进一步推动r Po IFN-α单克隆抗体在猪免疫学以及猪疫病诊断中的应用。By the fermentation reactor and Pichia pastoris expression system,the expression of recombinant porcine interfer-α（r Po IFN-α） was induced with methanol. The r Po IFN-α was purified by ammonium sulfate precipitation,Q-Sepharose fast flow chromatography and gel filtration in turn. 6-week-old BALB / c mice were immunized for three times with purified r Po IFN-α,then mouse myeloma cells and the spleen cells of the BALB / c mice were fused. Five hybridoma clones secreting antibody against r Po IFN-α were obtained by screening with the indirect ELISA and 3- 5times of subcloning. The five Mc Abs belonged to Ig G I isotype,and showed specificity reaction to r Po IFN-α by Western-blot. ELISA showed that their antigen binding sites were the same. This paper is beneficial to study the applications of monoclonal antibody（Mc Ab） against r Po IFN-α in immunology and disease diagnosis.

关 键 词：重组猪IFN-α 单克隆抗体 特异性鉴定 

分 类 号：Q78[生物学—分子生物学] S859.7[农业科学—临床兽医学]