抗埃博拉病毒核蛋白NP人-鼠嵌合抗体的研究

发布时间：2018-01-10 01:24

本文关键词：抗埃博拉病毒核蛋白NP人-鼠嵌合抗体的研究　出处：《病毒学报》2016年01期 　论文类型：期刊论文

【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。
[Abstract]:The highest mortality caused by Ebola virus infection is 90%, which is a severe infectious disease. Detection and diagnosis is very important for the control of the epidemic situation. At present, the detection of Ebola virus nucleic acid by real-time quantitative fluorescent PCR in our laboratory is confirmed, but it takes a relatively long time. The detection of serum by Elisa is the main indicator of etiological diagnosis. This method is simple and easy to operate and can effectively judge whether patients are infected with Ebola and the degree of infection. It can be used in epidemiological investigation, serological indicators can be used as a powerful complement to nucleic acid detection. There are no cases of Ebola haemorrhagic fever in China. In particular, it is necessary to store serologically positive quality control material and construct and express anti-Ebola virus nucleoprotein NP human-mouse chimeric antibody by genetic engineering antibody technology. RT-PCR technique was used to provide a positive control reserve for the serological detection of Ebola haemorrhagic fever. The variable region gene of light and light chain of antibody was isolated from murine hybridoma cell line secreting monoclonal antibody against Ebola nucleoprotein, and the sequence analysis was carried out according to the result of sequence analysis. Specific primers were designed to clone VHG VL into the eukaryotic expression vector HL51-14, which carried the constant region gene of the heavy and heavy chain of human antibody, and was transiently transfected into 293T cells. The expression and purification of human mouse chimeric IgG antibody was carried out. The results showed that the eukaryotic expression vector of human mouse chimeric antibody against Ebola virus ribosomal protein was constructed correctly. Two human mouse chimeric McAbs were successfully expressed and purified.
【作者单位】：温州医科大学;中国疾病预防控制中心病毒病预防控制所;
【基金】：重大新药创制(2013ZX09304103001006)
【分类号】：R373.32
【正文快照】： 埃博拉出血热(Ebola hemorrhagic fever)是一种由埃博拉病毒(Ebola virus,EBOV)引起的急性病毒性传染病,是一种致死率很高的人兽共患病。最早于1976年在刚果(金)埃博拉河流域的苏丹南部和扎伊尔地区首次暴发,导致多人死亡。2014年暴发了有史以来最为严重的埃博拉疫情。此次疫

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