抗埃博拉病毒核蛋白NP人-鼠嵌合抗体的研究  

Research of Human-mouse Chimeric Antibodies Against Ebola Virus Nucleoprotein

作　　者：周荣苹 孙丽娜[2] 刘洋[2] 芜为[2] 李川[2] 梁米芳[2] 仇佩虹[1] 

机构地区：[1]温州医科大学,温州325035 [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》2016年第1期14-18,共5页Chinese Journal of Virology

基　　金：重大新药创制(2013ZX09304103001006)

摘　　要：埃博拉病毒感染致死率最高可达90%,属烈性传染病,检测诊断对于疫情控制就显得异常重要。目前我国实验室通过实时定量荧光PCR检测埃博拉病毒核酸进行确诊,但耗时相对较长,对检测的人员和仪器要求较高。ELISA法检测血清是病原学诊断的主要指标,该种检测方法简单易操作,能够有效判断患者是否感染埃博拉以及感染程度,可用于流行病学调查,血清学指标的检测可以作为核酸检测的有力补充。由于埃博拉出血热在我国境内尚无病例出现,尤其需要储备血清学检测阳性质控物。通过基因工程抗体技术构建并表达抗埃博拉病毒核蛋白NP人-鼠嵌合抗体,为埃博拉出血热血清学检测做人源阳性对照储备。采用RT-PCR技术,从分泌抗埃博拉核蛋白单克隆抗体的鼠杂交瘤细胞株中分离克隆抗体轻重链可变区基因,对其进行序列分析。根据序列分析的结果,设计特异性引物将鼠源抗体可变区基因VH、VL分别克隆至携带有人抗体轻重链恒定区基因的真核表达载体HL51-14,瞬时转染293T细胞,表达并纯化获得人鼠嵌合IgG抗体,随后对其进行了免疫学检测和功能鉴定。结果表明正确构建抗埃博拉病毒核蛋白人鼠嵌合抗体真核表达载体,成功表达并纯化获得两株人鼠嵌合单抗。The Ebola virus is highly infectious and can result in death in≤90% of infected subjects.Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control.Presently,Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction（RT-PCR）.However,such detection takes a relatively long time and necessitates skilled personnel and expensive equipment.Enzyme-linked immunosorbent assay（ELISA）of serum is simple,easy to operate,and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection.Hence,ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids.Cases of Ebola hemorrhagic fever have not been documented in China,so quality-control material for positive serology is needed.Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out.Genes encoding variable heavy（VH）and variable light（VL）chains were extracted and amplified from murine hybridoma cells.Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR.According to sequence analyses,aprimer was designed to amplify functional sequences relative to VH and VL chain.The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain-and light chain-constant regions was used.IgG antibodies were obtained by transient transfection of 293 Tcells.Subsequently,immunological detection and immunological identification were identified by ELISA,immunofluorescence assay,and western blotting.These results showed that we constructed and purified two humanmouse chimeric antibodies.

关 键 词：埃博拉核蛋白 杂交瘤细胞 人鼠嵌合抗体 

分 类 号：R373.32[医药卫生—病原生物学]