抗EV71人源抗体的筛选及常见呼吸道病毒QPCR检测方法的建立

发布时间：2018-05-10 12:46

本文选题：肠道病毒EV71 + 单克隆抗体　；参考：《北京工业大学》2016年硕士论文

【摘要】：手足口病是由肠道病毒感染引起的传染病,多发于5岁以下婴幼儿,其中部分由肠道病毒71型(简称EV71)感染的患儿可引起神经系统感染症状并导致心肺功能衰竭,严重者会导致死亡。EV71病毒感染病例数量庞大,但是只有少数患者发展为重症,因此,研制EV71中和抗体,为手足口病重症患者提供特异性治疗药物,是降低手足口病发病率和死亡率的可能解决方案之一。本课题通过磁珠分选EV71感染者外周血中记忆性B细胞,在饲养层细胞中培养,加入细胞因子刺激细胞分泌抗体,用ELISA法检测细胞上清是否含有阳性抗体,提取检测结果为阳性的细胞总RNA,采用逆转录和巢式PCR方法扩增免疫球蛋白重链和轻链;经测序鉴定后,将序列克隆到真核表达载体中;通过瞬时转染293T细胞得到重组抗体,通过以上方法共分离得到59株单克隆抗体;用硫氰酸铵洗脱法检测抗体的亲和力活性,共得到6株亲和力较高的单克隆抗体;中和实验检测抗体的中和活性,成功筛选到1株抗EV71的人源中和抗体,IC50为5.5μg/m L。本课题成功建立了人源化EV71单克隆抗体的制备方法,为特异性治疗由EV71病毒感染引起的手足口病提供了新的方法和思路,同时为筛选其他人源化单克隆抗体奠定了基础。呼吸道疾病高发于春冬季,能引起呼吸道疾病的细菌和病毒多种多样,本课题建立呼吸道合胞病毒、副流感病毒1型、副流感病毒2型、腺病毒、冠状病毒、流感病毒、EB病毒等12种呼吸道病原体实时荧光定量PCR检测方法,对北京市望京医院2011-2012年采集的178份呼吸道感染病例咽拭子样本进行检测,结果显示178份患者咽拭子样本中检测到阳性样本64例,阳性结果占总样本的36.2%。该工作为呼吸道病原体检测提供了可靠的技术手段,为呼吸道疾病预防控制提供了基础数据。
[Abstract]:Hand, foot and mouth disease (HFMD) is an infectious disease caused by enterovirus infection, mostly in infants under 5 years old. Some of the children infected with enterovirus 71 (EV71) can cause symptoms of nervous system infection and cause heart and lung failure. Severe cases can lead to death. EV71 virus infection is a large number, but only a few patients develop severe. Therefore, the development of EV71 neutralizing antibodies, for hand, foot and mouth disease patients with specific treatment drugs, It is one of the possible solutions to reduce the morbidity and mortality of HFMD. In this study, memory B cells in peripheral blood of EV71 infected patients were separated by magnetic beads, cultured in feeder layer cells, and cytokines were added to stimulate cells to secrete antibodies. ELISA method was used to detect whether the supernatants contained positive antibodies. The total RNAs of positive cells were extracted, the heavy and light chains of immunoglobulin were amplified by reverse transcription and nested PCR, the sequence was cloned into eukaryotic expression vector after sequencing, and the recombinant antibody was obtained by transient transfection of 293T cells. A total of 59 monoclonal antibodies were isolated by the above methods, 6 monoclonal antibodies with high affinity were obtained by ammonium thiocyanate elution assay, and neutralization activities of antibodies were detected by neutralization experiments. The IC50 of a human neutralizing antibody against EV71 was 5.5 渭 g / mL. The preparation method of humanized EV71 monoclonal antibody was successfully established, which provided a new method and thought for the specific treatment of hand-foot-mouth disease caused by EV71 virus infection, and laid a foundation for screening other humanized monoclonal antibodies. Respiratory diseases occur frequently in spring and winter. There are a variety of bacteria and viruses that can cause respiratory diseases. In this study, respiratory syncytial virus, parainfluenza virus type 1, parainfluenza virus type 2, adenovirus, coronavirus were established. Twelve respiratory pathogens such as influenza virus Epstein-Barr virus and other respiratory pathogens were detected by real-time fluorescence quantitative PCR. 178 throat swabs of respiratory tract infection cases collected by Beijing Wangjing Hospital in 2011-2012 were detected. The results showed that 64 of 178 pharynx swab samples were positive, and the positive results accounted for 36.2% of the total samples. This work provides reliable technical means for the detection of respiratory pathogens and provides basic data for the prevention and control of respiratory diseases.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R725.1

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