猪血凝性脑脊髓炎病毒N蛋白单克隆抗体制备及表位鉴定  

Preparation of monoclonal antibody against N protein of porcine hemagglutinating encephalomyelitis virus and identification of its epitope

作　　者：赵明明 翟晓凤 粟硕[1] ZHAO Mingming;ZHAI Xiaofeng;SU Shuo(Institute of Immunology,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学免疫研究所,江苏南京210095

出　　处：《畜牧与兽医》2023年第3期119-124,共6页Animal Husbandry & Veterinary Medicine

摘　　要：为制备猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus, PHEV)N蛋白的单克隆抗体,本研究构建了pET-32a-N重组质粒,原核表达并纯化重组N蛋白,免疫BALB/c小鼠,细胞融合后利用间接ELISA进行筛选,获得了3株稳定分泌的阳性杂交瘤细胞株,分别命名为2C3、4F3、5C6。Western blot与间接免疫荧光试验表明,3株单抗均能与293T细胞中表达的N蛋白产生特异性反应。经测定,3株单抗的效价均为1×10^(6),重链类型均为IgG1,轻链类型均为κ。利用一系列表达的部分重叠的N截短蛋白片段,经Western blot鉴定单抗所识别的抗原表位,结果显示,^(267)KPRQK^(271)为单抗2C3、4F3、5C6所识别的表位。本研究为PHEV临床检测方法的建立和表位疫苗的研发奠定了基础。To prepare monoclonal antibodies against the nucleocapsid protein of porcine hemagglutinating encephalomyelitis virus(PHEV), pET-32a-N recombinant plasmids were constructed, recombinant N proteins were prokaryotically expressed and purified. BALB/c mice were immunized with the purified N proteins, indirect ELISA was used to screen after cell fusion, and three positive hybridoma cell lines with stable secretion were obtained, named: 2C3, 4F3, 5C6, respectively. Western blot and indirect immunofluorescence assay(IFA) showed that the three monoclonal antibodies specifically recognized the N-flag eukaryotic proteins expressed in 293T cells. The titers of the three monoclonal antibodies were all 1×10^(6), the heavy chains belonged to IgG1, and the light chains were of the κ type. Using a series of expressed partially overlapping N-truncated protein fragments, the epitope recognized by the monoclonal antibodies was identified by Western blot. The results showed that^(267)KPRQK^(271)was the epitope recognized by the monoclonal antibodies 2C3, 4F3 and 5C6. This study laid the foundation for the establishment of clinical detection methods for PHEV and the development of epitope vaccines.

关 键 词：猪血凝性脑脊髓炎病毒 N蛋白 单克隆抗体 表位 

分 类 号：S852.65[农业科学—基础兽医学]