【作者】 李霞；

【导师】 李晓眠； 李梅；

【作者基本信息】 天津医科大学 ， 病原生物学， 2009， 博士

【摘要】 副粘病毒Tianjin株（Paramyxovirus Tianjin strain）是1999年从患呼吸道疾病死亡的普通棉耳狨猴肺组织分离得到的毒株，曾引起狨猴致死性感染。以灭活的全病毒为抗原采用ELISA方法检测婴幼儿呼吸道感染者血清中IgM，阳性率高达36．9％。考虑到狨猴与人类具有较近的亲缘关系以及呼吸道感染者血清中抗体的高阳性率，提示该病毒株与人类的关系密切。目的：建立敏感、特异的副粘病毒Tianjin株检测方法，为了解该株病毒与人类的关系提供更为准确的数据；获得单克隆抗体、基因工程抗体，为下一步进行诊断、治疗，研究宿主亲嗜性改变、突变蛋白与功能等奠定基础。方法：本实验分别采用杂交瘤单克隆抗体制备技术和噬菌体抗体库技术，获得副粘病毒Tianjin株的单克隆抗体；建立了敏感、特异的检测病毒抗原的方法，并对婴幼儿下呼吸道感染标本进行检测。结果：1．采用杂交瘤技术制备副粘病毒Tianjn株HN蛋白单克隆抗体（mAbs），共获得了23株mAbs。其中较为特异的mAbs有3株，分别命名为G7H4D3、G7D9G3和G7G7E7。3株mAbs与副粘病毒Tianjin株有较高结合活性，与甲、乙型流感病毒、新城疫病毒（NDV）、人副流感病毒（hPIV）1型、3型、肺炎支原体均无交叉反应性。ELISA相加试验和阻断试验表明，3株单抗识别相同或相近的表位区域，识别的表位在抗病毒免疫应答中处于免疫优势地位。2．以兔抗副粘病毒Tianjin株多克隆抗体为捕获抗体，单抗G7G7E7为检测抗体，建立双抗体夹心ELISA法，检测523例下呼吸道感染患儿支气管肺泡灌洗液（BALF）标本。检测阳性率为2．1％（11／523）。与RT-PCR和间接ELISA法相比，双抗体夹心ELISA法具有敏感性高、特异性强的优点，适于临床检测使用。检测结果显示，副粘病毒Tianjin株可能是婴幼儿下呼吸道感染的重要致病因子之一。3．构建了以噬菌粒p3MH为载体，副粘病毒Tianjin株为免疫原，库容为1．18×10⁷的鼠源性免疫噬菌体抗体库。分别以纯化病毒和重组蛋白HN为筛选抗原对构建的噬菌体抗体库进行了三轮吸附-洗脱-扩增的筛选富集，并挑取单菌落以ELISA方法进行初步检测，获得7株与副粘病毒Tianjin株有结合活性的阳性克隆。其中有6株单抗克隆制备了可溶性Fab抗体，可溶性Fab均识别副粘病毒Tianjin株，其中1株结合HN3(HN_{375aa～575aa})片段，2株结合HN2(HN_{253aa～452aa})片段。对单抗克隆20进行测序，结果表明轻链V区基因属V_K9亚群，重链V区基因属V_H7亚群，经NCBI BLAST和IMGT分析均为鼠源性抗体基因，最高同源性分别为94．87％和97．85％。结论：本实验通过杂交瘤技术获得了3株特异性单抗。建立了双抗体夹心ELISA方法，并对呼吸道患儿标本进行了检测。获得了更为准确的副粘病毒Tianjin株在人群中感染状况的资料。构建了副粘病毒Tianjin株的免疫噬菌体抗体库，并获得6株单抗，为进一步进行该病毒的诊断和致病机制奠定了基础。更多还原

【Abstract】 Paramyxovirus Tianjin strain was isolated from common cotton-eared marmosetlung in 1999;it caused lethal infection on the marmoset.IgM against paramyxovirusTianjin strain in sera of infants and young children with acute respiratory tractinfection were detected,the positive rate was 36.9%.Taking into account closegenetic relationship between the marmoset and human,high IgM positive rate in seraof children with respiratory tract infection,we consider that paramyxovirus Tianjinstrain may be a common respiratory virus in human and animal.Objective:To assess the infection status of paramyxovirus Tianjin strain in the crowd,andaccurately understand the relationship between human and paramyxovirus Tianjinstrain;To prepare monoclonal antibodes and genetic engineering antibody,provide abasis for further study on paramyxovirus Tianjin strain,including diagnosis,therapy,host range changes,and so on.Methods:Monoclonal antibodies agaist paramyxovirus Tianjin strain were preparedthrough hybridoma technique and phage antibody library technology.A moresensitive detection methods was established,and paramyxovirus Tianjin strain wasdetected directly from clinical specimens.Results:1.Twenty-three hybridoma cell strains were obtained.Three hybridoma cellstrains G7H4D3,G7D9G3 and G7G7E7 among them,show specificity forparamyxovirus Tianjin strain.The three mAbs secreted by these three hybridoma cellsshowed the ability of specific binding to paramyxovirus Tianjin strain,and nocross-reactions were evidently detected with influenza A and B,New castle diseasevirus （NDV）,Human parainfluenza virus （hPIV） type 1 and 3,Mycoplasmapneumoniae.ELISA additivity tests indicated the three mAbs recognize the same orclosely adjacent epitope domains,and which were also immunodominant in theimmune response against paramyxovirus Tianjin strain by blocking assay.2.A sandwich ELISA assay is developed in which the rabbit polyclonal antibodies against paramyxovirus Tianjin strain are used as the capture antibody andmAb G7G7E7 as detection antibody.Paramyxovirus Tianjin strain in BALF samplesof the newborns and young children with severe lower respiratory tract infectionswere detected.The positive rate is 2.1% （11/523）.The sandwich ELISA assay showsbetter sensibility and specificity than RT-PCR,hemagglutination inhibition assay （HI）and indirected ELISA.It is suitable for clinical diagnosis.The detection results alsosuggest that paramyxovirus Tianjin strain have a close relation to infants and youngchildren bronchitis and pneumonia,and may be one of causative agents in the lowerrespiratory tract infections.3.The Fab phage antibody library against paramyxovirus Tianjin strain wasconstructed,and biopanning and identifying of anti-paramyxovirus Tianjin strainantibodies were accomplished.We obtained seven positive phage antibody clonesagainst paramyxovirus Tianjin strain,and produced six soluble Fab antibodies.Among them,one positive clone binds with recombinant protein HN_{375aa～575aa},andtwo positive clones bind with recombinant protein HN_{253aa～452aa}.Sequence analysis ofpositive clone 20 showed that the light chain variable domain （V_K） gene belong toV_K9 subgroup,the heavy chain variable domain（V_H） gene belong to V_H7 subgroup,most high identity is respectively 94.87% and 97.85%.Conclusion:Three specific mAbs were obtained through hybridoma technique,and asandwich ELISA assay is developed.We detected lower respiratory tract infectionssamples by this method,get accurate data on relationship between the virus andhuman.An immune phage antibody library against paramyxovirus Tianjin strain wasconstructed,six mAbs was obtained,and to lay a foundation of further study ondiagnosis and pathogenic mechanism of paramyxovirus Tianjin strain.更多还原