登革病毒血清型特异单克隆抗体的制备和鉴定  

Development and characterization of serotype-specific monoclonal antibodies against Dengue virus NS1

作　　者：刘杨[1,2] 张元 魏艳秋 贾晓娟[4] 陈启军[1] 刘文军[2,3] 杨利敏[2] Yang Liu;Yuan Zhang;Yanqiu Wei;Xiaojuan Jia;Qijun Chen;Weijun Liu;Limin Yang(Shenyang Agricultural University,Shenyang 110866,Liaoning,China;Key Laboratory of Pathogenic Microbiology and Immunology,Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;University of Chinese Academy of Sciences,Beijing 100049,China;The Biological Safety Level-3(BSL-3)Laboratory of Institute of Microbiology,Chinese Academy of Sciences,Beijing 100101,China;Anhui University,Hefei 230601,Anhui,China)

机构地区：[1]沈阳农业大学,辽宁沈阳110866 [2]中国科学院微生物研究所病原微生物与免疫学重点实验室,北京100101 [3]中国科学院大学,北京100049 [4]中国科学院微生物研究所生物安全三级实验室,北京100101 [5]安徽大学,安徽合肥230601

出　　处：《生物工程学报》2020年第10期2206-2215,共10页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(Nos.2018YFC1200602,2018YFC1200502);国家科技重大专项(Nos.2016ZX10004222-006,2018ZX10734-404)资助。

摘　　要：登革病毒(Dengue virus,DENV)是全球传播最为广泛的虫媒病毒,由于缺乏快速鉴别感染病毒血清型的诊断技术,导致异型交叉感染引起重症登革出血热病例居高不下。为实现免疫学方法快速鉴别诊断不同血清型DENV感染,本研究采用哺乳动物细胞293T表达并纯化了4种DENV血清型NS1蛋白,免疫小鼠后通过杂交瘤技术制备了针对NS1蛋白的单克隆抗体。利用酶联免疫吸附方法(Enzyme-linked immunosorbent assay,ELISA)、间接免疫荧光法(Indirect immunofluorescence assay,IFA)、免疫斑点杂交试验(Dot blotting)以及蛋白质免疫印迹试验(Western blotting)确认所制备的单克隆抗体能够有效识别天然病毒NS1以及重组NS1蛋白。获得的单克隆抗体包含2株可识别1–4型DENV NS1蛋白的通用型抗体及3株分别针对DENV-1、DENV-2和DENV-4的血清型特异抗体。以所制备的DENV NS1抗体为基础,采用双抗体夹心ELISA可快速鉴别不同血清型DENV。DENV血清型特异单克隆抗体的制备和甄别DENV血清型ELISA方法的建立为快速鉴别感染DENV血清型的临床诊断奠定了基础。Dengue virus(DENV)is the most widely transmitted arbovirus in the world.Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients,severe dengue hemorrhagic fever cases caused by repeated infections remain high.To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods,in this study,four DENV serotype NS1 proteins were expressed and purified in mammalian cells.Monoclonal antibodies(MAbs)against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice.Enzyme-linked immunosorbent assay,indirect immunofluorescence assay,dot blotting,and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein.These MAbs include not only the universal antibodies that recognize all DENV 1–4 serotype NS1,but also serotype-specific antibodies against DENV-1,DENV-2 and DENV-4.Double antibody sandwich ELISA was established based on these antibodies,which can be used to achieve rapid differential diagnosis of serotypes of DENV infection.Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.

关 键 词：登革病毒 NS1蛋白 单克隆抗体 血清型特异 

分 类 号：R392[医药卫生—免疫学]