摘要
为获得猪伪狂犬病毒（PRV）gE蛋白单克隆抗体,选择原核表达的重组gE蛋白免疫6周龄BALB/c雌性小鼠,将其脾细胞与SP2/0进行融合,经间接ELISA筛选阳性杂交瘤细胞,结果获得了2株能稳定分泌抗PRV gE蛋白的杂交瘤细胞,命名为E3B8和E5C11。间接ELISA检测2株杂交瘤细胞的培养上清液抗体效价为1∶6.4×10~3,腹水的抗体效价分别达到1∶3.28×10~6和1∶6.55×10~6。2株杂交瘤细胞的染色体数分别为105和108。E3B8亚类鉴定重链为IgG1,轻链为κ链;E5C11亚类鉴定重链为IgG2b,轻链为κ链。Western blot检测显示2株单克隆抗体腹水均能与PRV重组gE蛋白发生特异性反应,间接免疫荧光试验（IFA）检测显示2株单克隆抗体均能与PRV分离毒株感染的BHK-21细胞发生特异性反应,交叉反应性检测显示2株单克隆抗体与常见病毒不发生交叉反应。表明制备的2株gE蛋白单克隆抗体效价高、特异性强,为gE蛋白结构与功能分析以及PRV免疫诊断试剂盒的开发奠定了基础。
In order to obtain the pseudorabies virus（PRV） gE protein monoclonal antibody,the recombinant gE protein by prokaryotic expression was immunized into female BALB/c mice aged 6 weeks,the spleen cells and SP2/0 were fused,and the positive hybridoma cells were screened by indirect ELISA.The results showed that two hybridoma cell lines secreting anti-PRV gE protein were obtained,named E3 B8 and E5 C11,respectively.The antibody titer in the supernatants of the two hybridoma cells was 1∶6.4×10~3,while the titers in ascites reached 1∶3.28×10~6 and 1∶6.55×10~6,respectively.The chromosome numbers of the two hybridoma cells were 105 and 108.The heavy chain subtypes of E3 B8 and E5 C11 were IgG1 and IgG2 b,respectively,with a κ light chain.Western blot analysis showed that the two monoclonal antibodies could specifically react with the recombinant gE protein of PRV.Indirect immunofluorescence assay（IFA） detection showed that the two monoclonal antibodies could specifically react with the infected BHK-21 cells with PRV.And the two monoclonal antibodies did not cross-react with other viruses.It suggests that the two monoclonal antibodies established in this study had high titer and specificity,which laid the foundation for the research on the structure and function of gE protein and the development of PRV immunological diagnostic kit.
出处
《畜牧与兽医》
北大核心
2018年第1期109-112,共4页
Animal Husbandry & Veterinary Medicine
基金
青海省农技推广补助项目(青农发[2015]293号)
关键词
伪狂犬病毒
gE蛋白
单克隆抗体
制备
鉴定
pseudorabies virus
gE protein
monoclonal antibodies
preparation
identification
