摘要
本研究是利用我国PRRS病毒分离株CH-1a株浓缩的病毒抗原免疫BAL B/c小鼠,经3次免疫后,取脾细胞与SP2/0骨髓瘤细胞进行融合,同时以PRRS病毒作为抗原进行间接ELISA检测,共筛选到22个阳性细胞株。分别经过3轮单克隆后,最终得到了16株能稳定分泌抗体的单细胞克隆株。利用IDEXX-ELISA检测试剂盒和IFA试验对16株单克隆抗体进行鉴定,结果证实所获得的16株细胞分泌的抗体都是针对PRRSV的特异性抗体。将此16株单克隆抗体分别与用原核系统表达的PRRSV N蛋白r、tM和rtGP5蛋白进行特异性ELISA检测试验,结果表明16株单克隆抗体都仅识别表达的N蛋白,而与其它两种蛋白不发生反应,说明所获得的16株单克隆抗体均为抗PRRSV核衣壳蛋白的。单克隆抗体亚型鉴定试剂盒的鉴定结果显示,除01MAb为IgG2b、N1H11为IgG2a外,其余的单克隆抗体均为IgG1,而且所有单克隆抗体的轻链均为κ链。至此证实我们获得了16株PRRV N蛋白单克隆抗体,这将为今后建立更为灵敏的检测PRRS病原的特异性诊断方法、分析PRRSV核衣壳蛋白的功能及鉴定B细胞抗原表位奠定重要基础。
BALB/c mice were immunized intraperitoneally with purified virions of PRRSV strain CH-1 a. Murine myeloma cells were fused with the splenocytes of the immunized mice after the third immunization. An indirect ELISA with PRRS virions as antigen was used to screen antibody-producing hybridomas. 22 MAbs against PRRSV were obtained. Sixteen hybridomas ceils of them can product MAbs steadily after 3 cydes of doning. All the 16 MAbs were reactive in both IDEXX-ELISA and IFA tests, and all of them showed positive reaction to the N protein of PRRSV, but not to the truncated M and GP5 protein （expressed in E. coli ）. Except 01MAb and N1H11 are IgG2b and IgG2a isotype, respectively, the remained MAbs belong to IgG1 isotype, the light chain of all MAbs is Pc chain. The MAbs against N protein of PRRSV devdoped in the study would be useful as a basis of diagnosis and epitope identification.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2005年第6期498-503,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(30470072)
国家重点基础研究发展规划项目(973)(G1999011902)资助
关键词
猪繁殖与呼吸综合征病毒
单克隆抗体
核衣壳蛋白
porcine reproductive and respiratory syndrome virus
monoclonal antibody
nucleocapsid protein
