摘要
目的制备四种血清型交叉反应性的登革病毒包膜蛋白结构域Ⅲ(DENV-EDⅢ)特异性单克隆抗体(mAb),并鉴定其特异性及体外中和活性。方法通过毕赤酵母表达具有良好抗原性的1~4型DENV-EDⅢ蛋白,并以四种DENV血清型EDⅢ蛋白混合免疫BALB/c小鼠,采用聚乙二醇融合技术制备杂交瘤细胞,通过间接ELISA筛选出稳定分泌抗DENV-EDⅢ抗体的杂交瘤细胞,进一步通过间接免疫荧光法对所获得mAb的特异性进行鉴定,并通过改良的酶联免疫斑点微中和实验对所获得的mAb进行体外中和活性测定。结果获得6株特异性针对1~4型DENV-EDⅢ蛋白的mAb和11株特异性针对某一血清型DENV的EDⅢmAb,其中有1株mAb同时对四种血清型DENV有均衡的体外强中和活性,其对四种血清型DENV的半数抑制剂量(IC)分别为(0.05、1.89、0.02、3.91)μg/mL。结论成功筛选获得了一株可同时中和四种血清型DENV的EDⅢ特异性mAb。
Objective To prepare neutralizing monoclonal antibodies(mAbs)against envelope protein domainⅢof the four dengue virus serotypes(DENV-EDⅢ)and identify its specificity.Methods BALB/c mice were immunized with recombinant EDⅢprotein(rDENV-EDⅢ)of the four DENV serotypes.Hybridoma cells secreting DENV-EDIII antibodies were screened by indirect ELISA.The specificity of positive hybridoma cells were further tested by indirect immunofluorescence assay(IFA).The neutralizing activities of DENV-EDⅢmAbs in vitro were determined by the enzyme-linked immunospot microneutralization test(ELISPOT-MNT).Results 6 mAbs specific for the EDⅢof the four DENV serotypes and 11 mAbs specific for only one serotype of DENV-EDⅢprotein were obtained,of which one monoclonal antibody had a balanced strong neutralizing activity against the four DENV serotypes in vitro,and its 50%inhibitory concentrations(IC)for the four DENV serotypes was 0.05μg/mL,1.89μg/mL,0.02μg/mL,3.91μg/mL,respectively.Conclusion A specific monoclonal antibody against DENV-EDⅢis successfully screened and obtained to neutralize the four DENV serotypes.
作者
丁细霞
丘立文
陈月
温坤
DING Xixia;QIU Liwen;CHEN Yue;WEN Kun(Division of Laboratory Medicine,Zhujang Hospital,Southerm Medical University,Guangzhou 510282,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2022年第8期748-753,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
广东省自然科学基金(2020A1515011171)。
