本文關(guān)鍵詞：抗埃博拉病毒核蛋白NP人-鼠嵌合抗體的研究　出處：《病毒學報》2016年01期 　論文類型：期刊論文

  更多相關(guān)文章： 埃博拉核蛋白 雜交瘤細胞 人鼠嵌合抗體

【摘要】：埃博拉病毒感染致死率最高可達90%,屬烈性傳染病,檢測診斷對于疫情控制就顯得異常重要。目前我國實驗室通過實時定量熒光PCR檢測埃博拉病毒核酸進行確診,但耗時相對較長,對檢測的人員和儀器要求較高。ELISA法檢測血清是病原學診斷的主要指標,該種檢測方法簡單易操作,能夠有效判斷患者是否感染埃博拉以及感染程度,可用于流行病學調(diào)查,血清學指標的檢測可以作為核酸檢測的有力補充。由于埃博拉出血熱在我國境內(nèi)尚無病例出現(xiàn),尤其需要儲備血清學檢測陽性質(zhì)控物。通過基因工程抗體技術(shù)構(gòu)建并表達抗埃博拉病毒核蛋白NP人-鼠嵌合抗體,為埃博拉出血熱血清學檢測做人源陽性對照儲備。采用RT-PCR技術(shù),從分泌抗埃博拉核蛋白單克隆抗體的鼠雜交瘤細胞株中分離克隆抗體輕重鏈可變區(qū)基因,對其進行序列分析。根據(jù)序列分析的結(jié)果,設(shè)計特異性引物將鼠源抗體可變區(qū)基因VH、VL分別克隆至攜帶有人抗體輕重鏈恒定區(qū)基因的真核表達載體HL51-14,瞬時轉(zhuǎn)染293T細胞,表達并純化獲得人鼠嵌合IgG抗體,隨后對其進行了免疫學檢測和功能鑒定。結(jié)果表明正確構(gòu)建抗埃博拉病毒核蛋白人鼠嵌合抗體真核表達載體,成功表達并純化獲得兩株人鼠嵌合單抗。
[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.
【作者單位】： 溫州醫(yī)科大學;中國疾病預(yù)防控制中心病毒病預(yù)防控制所;
【基金】：重大新藥創(chuàng)制(2013ZX09304103001006)
【分類號】：R373.32
【正文快照】： 埃博拉出血熱(Ebola hemorrhagic fever)是一種由埃博拉病毒(Ebola virus,EBOV)引起的急性病毒性傳染病,是一種致死率很高的人獸共患病。最早于1976年在剛果(金)埃博拉河流域的蘇丹南部和扎伊爾地區(qū)首次暴發(fā),導(dǎo)致多人死亡。2014年暴發(fā)了有史以來最為嚴重的埃博拉疫情。此次疫

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