人源抗发热伴血小板减少综合征布尼亚病毒-Gn蛋白重组抗体的研究  

Development of Human Antibodies against the Gn Protein of Severe Fever with Thrombocytopenia Syndrome Virus

作　　者：陈素华[1] 孙丽娜[2] 刘洋[2] 李川[2] 刘林[2] 梁米芳[2] 仇佩虹[1] 

机构地区：[1]温州医科大学,温州325000 [2]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《病毒学报》2015年第1期24-29,共6页Chinese Journal of Virology

基　　金：重大新药创制(2013ZX09304103001006)

摘　　要：为研制人源抗发热伴血小板减少综合征布尼亚病毒(Severe fever with thrombocytopenia syndrome virus,SFTSV)Gn蛋白重组抗体,本研究利用噬菌体表面展示技术,以SFTSV全病毒颗粒和重组表达SFTSV-Gn蛋白为抗原,从人源抗SFTSV Fab噬菌体抗体库中筛选抗SFTSV-Gn蛋白的重组Fab抗体,通过ELISA对Fab抗体的结合特异性进行检测。将Fab抗体基因克隆入哺乳动物细胞表达载体HL51-14,瞬时转染293T细胞获得分泌表达的IgG抗体。通过ELISA、IFA和Western-blotting检测IgG抗体的结合特异性。采用亲和层析纯化IgG抗体,并用微量中和试验检测IgG抗体的中和活性。结果表明经过三轮富集筛选,以SFTSV病毒颗粒为抗原筛选出364株针对SFTS病毒核蛋白Fab抗体,没有筛选出特异性结合Gn蛋白的阳性克隆,而通过Gn蛋白筛选得到8株特异结合Gn蛋白的Fab抗体,其中5株来自Lambda库,3株来自Kappa库。ELISA、IFA和Western-blotting检测证实这8株IgG抗体均能特异性结合Gn蛋白。微量中和试验显示8株新筛抗体没有中和活性,但仍可为后续SFTSV人源单克隆抗体的研究提供借鉴和参考。To obtain human antibodies against the Gn protein of Severe fever with thrombocytopenia syndrome virus（SFTSV）with phage display technology,this study aimed to screen anti-Gn protein antibodies from an anti-SFTSV Fab human phage display library.Antibody genes were identified by sequence analysis and the specificity of antibodies was confirmed by ELISA.The Fab antibody genes were cloned into the HL51-14 vector and expressed in a mammalian cell expression system.IgG antibodies were then purified by protein A affinity chromatography,and the results were further confirmed by ELISA,IFA,western blotting assays and micro-neutralization tests.The results showed that,after three rounds of panning,there were 390 human Fab antibodies against SFTSV particles,of which 364 were specific for nucleoprotein.Coated with the Gn protein,eight different Fab antibodies specific for Gn protein were obtained after the determination of the subtype and subclass of antibodies by gene sequencing;five of these antibodies were from the Lambda library and three were from the Kappa library.The eight IgG antibodies could specifically bind to Gn protein according to the ELISA,IFA and Western blotting assays.The micro-neutralization test showed that these eight antibodies had no neutralizing activity,but they could still provide a reference for research in human monoclonal antibodies against SFTSV.

关 键 词：噬菌体抗体库 重组抗体 发热伴血小板减少综合征布尼亚病毒 

分 类 号：R373.9[医药卫生—病原生物学]