摘要
利用已构建的基因工程重组菌BL21(DE3)表达猪圆环病毒2型(PCV2)ORF2编码的结构蛋白,经切胶纯化后作为免疫原。采取抗原量依次递增的免疫方式免疫8周龄BALB/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合。间接酶联免疫吸附试验(ELISA)筛选,有限稀释法进行亚克隆3次,最终获得4株稳定分泌抗PCV2 ORF2重组蛋白单克隆抗体的杂交瘤细胞,分别命名为D2A1、H4G2、B10H10和G1G2。经ELISA检测,其腹水效价分别达到1∶2.048×107、1∶2.56×106、1∶1.28×106、1∶2.56×106。亚型鉴定4株单克隆抗体均属IgG2a亚型,其轻链均为κ链。Western blot鉴定表明获得的4株单克隆抗体均能特异性的识别43 kD的重组PCV2 ORF2蛋白。在间接免疫荧光试验中,单抗D2A1株的反应为阴性,H4G2、B10H10和G1G2反应为阳性,表明后3株单抗能够识别天然的PCV2 ORF2蛋白表位。本试验为进一步研究PCV2ORF2基因的功能及建立快速准确的诊断PCV2的方法奠定了基础。
The structural protein encoded by ORF2 of PCV2 was expressed in recombinant E. coli BL21（DE3） and used as immunogen after purification. Four hybridoma cell lines against PCV2 ORF2 fusion protein named D2A1, H4G2, B10H10 and G1G2 respectively, were developed after fusion between Sp2/0 myeloma cells and spleen cells of BALB/c mice immunized with purified recombinant protein. The ELISA titer of ascites were 1 ： 2. 048×10^7,1 ： 2.56×10^6,1 ： 1.28×10^6,1 1： 2.56×10^6. In a western-blot assay, all the mAbs could specifically recognize a 43 kD recombinant PCV2 ORF2 protein. H4G2,B10H10 and G1G2 showed strong specific fluorescence in PCV2（HBO3） infected cells in IFA, but speeifie fluorescenee was not observed in D2Al. This research provided a powerful tool for the researeh Of PCV20RF2 gene and established the method for diagnosing PCV2 rapidly and exactly.
出处
《中国兽医杂志》
CAS
北大核心
2007年第11期9-11,共3页
Chinese Journal of Veterinary Medicine
基金
国家自然科学基金项目"PCV2突变株影响猪肺泡巨噬细胞免疫功能的分子基础"(30400321)
关键词
猪圆环病毒2型
融合蛋白
单克隆抗体
PCV2
recombinant protein
monoclonal antibodies Corresponding authors
YANG Han-chun ,GUO Xin
