副粘病毒Tianjin株在婴幼儿下呼吸道感染情况调查及噬菌体抗体文库的构建筛选

发布时间：2018-08-02 20:26

【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1．18×10~7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。
[Abstract]:Paramyxovirus Tianjin strain (Paramyxovirus Tianjin strain) was isolated from the lung tissues of common cotton marmosets which died of respiratory diseases in 1999. It has caused fatal infection in marmosets. Using inactivated whole virus as antigen, ELISA method was used to detect IgM in the serum of infantile respiratory tract infection. The positive rate was as high as 36.9%. Considering the close relationship between marmosets and humans and the high positive rate of antibodies in the sera of respiratory tract infections, it is suggested that the virus strain is closely related to human beings. Objective: to establish a sensitive and specific method for detection of paramyxovirus Tianjin strain, to provide more accurate data for understanding the relationship between the strain and human beings, to obtain monoclonal antibody and genetic engineering antibody for further diagnosis. Treatment, the study of host affinity changes, mutant protein and function lay the foundation. Methods: the monoclonal antibody of paramyxovirus Tianjin strain was obtained by using hybridoma monoclonal antibody preparation technique and phage antibody library technique, and a sensitive and specific method for detecting virus antigen was established. The samples of infantile lower respiratory tract infection were detected. Results: 1. A total of 23 mAbs of paramyxovirus Tianjn strain HN protein monoclonal antibody (mAbs),) were prepared by hybridoma technique. Three of the specific mAbs strains were named G7H4D3G7D9G3 and G7G7E7.3 strain mAbs with high binding activity to paramyxovirus Tianjin strain, and to A, B influenza virus, Newcastle disease virus (NDV), human parainfluenza virus (hPIV) 1 type 3. Mycoplasma pneumoniae had no cross reactivity. Elisa addition test and blocking test showed that the McAbs recognized the same or similar epitopes. The identified epitopes are dominant in the antiviral immune response. 2. The double antibody sandwich ELISA method was established using rabbit anti paramyxovirus Tianjin strain polyclonal antibody as capture antibody and monoclonal antibody G7G7E7 as detection antibody. Bronchoalveolar lavage fluid (BALF) specimens were detected in 523 children with lower respiratory tract infection. The positive rate was 2.1% (11 / 523). Compared with RT-PCR and indirect ELISA, double antibody sandwich ELISA has high sensitivity and specificity and is suitable for clinical detection. The results showed that paramyxovirus Tianjin strain might be one of the important pathogenic factors of infantile lower respiratory tract infection. 3. The vector of p3MH was constructed and paramyxovirus Tianjin strain was used as immunogen. Mouse immune phage antibody library with capacity of 1.18 脳 10 ~ (7). Using purified virus and recombinant protein HN as screening antigens, the constructed phage antibody library was screened and enriched by three rounds of adsorption-elution amplification, and a single colony was selected for preliminary detection by ELISA method. Seven positive clones with binding activity to paramyxovirus Tianjin strain were obtained. Soluble Fab antibody was cloned from 6 McAbs. Soluble Fab recognized paramyxovirus Tianjin strain, and one strain was bound to HN3 (HN375aa~575aa) fragment and 2 strains were bound to HN2 (HN253aa~452aa) fragment. McAb clone 20 was sequenced. The results showed that the light chain V gene belonged to V_K9 subgroup and the heavy chain V region gene belonged to V_H7 subgroup. The highest homology was 94.87% and 97.85% by NCBI BLAST and IMGT, respectively. Conclusion: three specific McAbs were obtained by hybridoma technique. A double antibody sandwich ELISA method was established, and the respiratory tract samples were detected. More accurate data of paramyxovirus Tianjin infection in population were obtained. The immune phage antibody library of paramyxovirus Tianjin strain was constructed and 6 monoclonal antibodies were obtained, which laid a foundation for further diagnosis and pathogenesis of paramyxovirus.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R725.6;R392

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