利用DNA免疫建立分泌登革2型病毒E蛋白单抗的杂交瘤细胞系

Establishment of hybridoma cell lines which secreted monoclonal antibodies against DEN2 envelope glycoprotein by DNA immunization

目的:利用DNA免疫建立分泌登革2型病毒E蛋白特异单抗的杂交瘤细胞系,为研究E蛋白的结构和功能及其抗原表位提供新手段。方法:以构建的病毒全长prM-E基因的真核重组质粒DNA作为免疫原,免疫BALB/c小鼠后将其脾细胞和SP2/0瘤细胞融合,通过IFA、间接ELISA和空斑减少中和试验对杂交瘤细胞系进行筛选和鉴定。结果:获得了4株分泌登革2型病毒E蛋白单抗的杂交瘤细胞系(2B4,6B4,4C10和2D5),它们结合的抗原表位均位于E蛋白Ⅲ区,其中4C10对登革2型病毒具有中和活性。这些单抗与登革1、3和4型病毒有强的交叉反应,但与黄病毒其他成员反应较弱。结论:DNA免疫法可用于分泌登革2型病毒E蛋白特异单抗的杂交瘤细胞系的建立,该结果有利于登革病毒E蛋白特异抗原表位的研究及新的登革病毒诊断试剂的研制。

Objective： To construct hybridoma cell lines which secrete monoclonal antibodies （mAbs） against the DEN2 E glycoprotein by DNA immunization. Methods： Plasmid DNA encoding the premembrane and envelope glycoproteins of DEN2 was used as an antigen to immunize BALB/c mice, then the spleen cells of immunized mice were fused with SP2/0 myeloma cells. Selection and identification of hybridoma cell lines were performed by IFA, ELISA and PRNT. Results： Four hybridoma cell lines （2B4, 2D5, 4C10 and 6B4） which secreted mAbs against the DEN2 E glycoprotein were obtained, and the recognized epitopes were located in domain Ⅲ of E glycoprotein. All the mAbs obtained were crossreactive with dengue virus 1,3 and 4 serotypes and several other flaviviruses, and mAbs 4C10 neutralized DEN2 infection in BHK-21 cells. Conclusion： DNA immunization can be used to establish hybridoma cell lines which secrete mAbs against DEN2 E glycoprotein. These results are helpful for the specific epitopes of envelope glycoprotein and development of a novel diagnosis reagent of dengue virus.