抗发热伴血小板减少综合征病毒结构蛋白单克隆抗体的制备和功能分析  

Preparation and Functional Analysis of the Monoclonal Antibodies against Severe Fever with Thrombocytopenia Syndrome Bunyavirus Structural Proteins

作　　者：李阿茜 刘林[1] 张硕[1] 李川[1] 张全福[1] 梁米芳[1] 李德新[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所卫生部医学病毒和病毒病重点实验室,北京102206

出　　处：《病毒学报》2015年第1期18-23,共6页Chinese Journal of Virology

基　　金："重大新药创制"科技重大专项:发热伴血小板减少综合征布尼亚病毒灭活疫苗的研制(2013ZX09102029);国家自然科学基金:SFTS布尼亚病毒转录调控元件的鉴定及功能研究(31270201)

摘　　要：为制备抗发热伴血小板减少综合征病毒（Severe fever with thrombocytopenia syndrome bunyavirus,SFTSV）结构蛋白的单克隆抗体,本研究用灭活纯化的SFTSV病毒颗粒免疫BALB/c小鼠,利用杂交瘤技术获得分别分泌抗糖蛋白单抗和核蛋白单抗的杂交瘤细胞株。用免疫荧光法和免疫沉淀方法对制备的单克隆抗体的抗原特异性进行鉴定,并初步进行单抗效价、中和活性及亲和力等功能分析。结果显示,通过细胞融合和克隆化,共筛选出13株稳定分泌抗糖蛋白（Glycoprotein,GP）单抗和7株稳定分泌抗核蛋白（Nucleoprotein,NP）单抗的杂交瘤细胞株。免疫荧光和免疫沉淀鉴定显示获得的单抗有良好的抗原特异性。抗GP单抗中6株针对Gn,7株针对Gc,大部分的间接免疫荧光（Indirect immunofluorescence assay,IFA）滴度在1 280-20 480之间,其中4株抗Gn单抗具有中和活性。获得的7株抗NP单抗均与NP特异性结合,IFA滴度范围在5 120-20 480,均无中和活性。此外,经非竞争ELISA检测的两株抗GP单抗（1C8和1G8）均有较高亲和力。本研究为SFTS诊断方法的发展及SFTSV致病机制研究奠定了基础。To prepare monoclonal antibodies（mAbs）against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus（SFTSV）,BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens.Subsequently,hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein（NP）and glycoproteins（GP）were obtained using a hybridoma technique.The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays.Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities.After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7hybridoma cell lines secreted mAbs specifically against SFTSV-NP.Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity.Among the 13anti-SFTSV-GP mAbs,6recognized Gn,whereas the others reacted with Gc.IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480,and four anti-SFTSV-Gn mAbs showed neutralizing activity.Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity.Furthermore,two anti-SFTSV-GP mAbs,1C8 and 1G8,showed high levels of affinity via a non-competitive ELISA.Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.

关 键 词：发热伴血小板减少综合征病毒(SFTSV) 核蛋白(NP) 糖蛋白(GP) 单克隆抗体 

分 类 号：R373.32[医药卫生—病原生物学]