浙江农业学报

• 动物科学 • 上一篇    下一篇

重组猪干扰素\|α单克隆抗体的制备和鉴定

  

  1. (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)
  • 出版日期:2015-03-25 发布日期:2015-03-31

Preparation and identification of monoclonal antibodies against recombinant porcine interfer\|α

  1. (1 Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; 2 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210000, China; 3 Gansu Agricultural University, Lanzhou 730070, China)
  • Online:2015-03-25 Published:2015-03-31

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(rPoIFN\|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rPoIFN\|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rPoIFN\|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IgG1亚类,能与rPoIFN\|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rPoIFN\|α单克隆抗体在猪免疫学以及猪疫病诊断中的应用。

关键词: 重组猪IFN\, α;单克隆抗体;特异性鉴定

Abstract: By the fermentation reactor and Pichia pastoris expression system, the expression of recombinant porcine interfer\|α (rPoIFN\|α) was induced with methanol. The rPoIFN\|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 rPoIFN\|α, then mouse myeloma cells and the spleen cells of the BALB/c mice were fused. Five hybridoma clones secreting antibody against rPoIFN\|α were obtained by screening with the indirect ELISA and 3-5 times of subcloning. The five McAbs belonged to IgG I isotype, and showed specificity reaction to rPoIFN\|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(McAb) against rPoIFN\|α in immunology and disease diagnosis.

Key words: recombinant porcine interfer\, α, monoclonal antibody, specificity identification