【摘要】： 副粘病毒Tianjin株(Paramyxovirus Tianjin strain)是1999年從患呼吸道疾病死亡的普通棉耳狨猴肺組織分離得到的毒株，曾引起狨猴致死性感染。以滅活的全病毒為抗原采用ELISA方法檢測(cè)嬰幼兒呼吸道感染者血清中IgM，陽性率高達(dá)36．9％�？紤]到狨猴與人類具有較近的親緣關(guān)系以及呼吸道感染者血清中抗體的高陽性率，，提示該病毒株與人類的關(guān)系密切。 目的： 建立敏感、特異的副粘病毒Tianjin株檢測(cè)方法，為了解該株病毒與人類的關(guān)系提供更為準(zhǔn)確的數(shù)據(jù)；獲得單克隆抗體、基因工程抗體，為下一步進(jìn)行診斷、治療，研究宿主親嗜性改變、突變蛋白與功能等奠定基礎(chǔ)。 方法： 本實(shí)驗(yàn)分別采用雜交瘤單克隆抗體制備技術(shù)和噬菌體抗體庫技術(shù)，獲得副粘病毒Tianjin株的單克隆抗體；建立了敏感、特異的檢測(cè)病毒抗原的方法，并對(duì)嬰幼兒下呼吸道感染標(biāo)本進(jìn)行檢測(cè)。 結(jié)果： 1．采用雜交瘤技術(shù)制備副粘病毒Tianjn株HN蛋白單克隆抗體(mAbs)，共獲得了23株mAbs。其中較為特異的mAbs有3株，分別命名為G7H4D3、G7D9G3和G7G7E7。3株mAbs與副粘病毒Tianjin株有較高結(jié)合活性，與甲、乙型流感病毒、新城疫病毒(NDV)、人副流感病毒(hPIV)1型、3型、肺炎支原體均無交叉反應(yīng)性。ELISA相加試驗(yàn)和阻斷試驗(yàn)表明，3株單抗識(shí)別相同或相近的表位區(qū)域，識(shí)別的表位在抗病毒免疫應(yīng)答中處于免疫優(yōu)勢(shì)地位。 2．以兔抗副粘病毒Tianjin株多克隆抗體為捕獲抗體，單抗G7G7E7為檢測(cè)抗體，建立雙抗體夾心ELISA法，檢測(cè)523例下呼吸道感染患兒支氣管肺泡灌洗液(BALF)標(biāo)本。檢測(cè)陽性率為2．1％(11／523)。與RT-PCR和間接ELISA法相比，雙抗體夾心ELISA法具有敏感性高、特異性強(qiáng)的優(yōu)點(diǎn)，適于臨床檢測(cè)使用。檢測(cè)結(jié)果顯示，副粘病毒Tianjin株可能是嬰幼兒下呼吸道感染的重要致病因子之一。 3．構(gòu)建了以噬菌粒p3MH為載體，副粘病毒Tianjin株為免疫原，庫容為1．18×10~7的鼠源性免疫噬菌體抗體庫。分別以純化病毒和重組蛋白HN為篩選抗原對(duì)構(gòu)建的噬菌體抗體庫進(jìn)行了三輪吸附-洗脫-擴(kuò)增的篩選富集，并挑取單菌落以ELISA方法進(jìn)行初步檢測(cè)，獲得7株與副粘病毒Tianjin株有結(jié)合活性的陽性克隆。其中有6株單抗克隆制備了可溶性Fab抗體，可溶性Fab均識(shí)別副粘病毒Tianjin株，其中1株結(jié)合HN3(HN_(375aa～575aa))片段，2株結(jié)合HN2(HN_(253aa～452aa))片段。對(duì)單抗克隆20進(jìn)行測(cè)序，結(jié)果表明輕鏈V區(qū)基因?qū)賄_K9亞群，重鏈V區(qū)基因?qū)賄_H7亞群，經(jīng)NCBI BLAST和IMGT分析均為鼠源性抗體基因，最高同源性分別為94．87％和97．85％。 結(jié)論： 本實(shí)驗(yàn)通過雜交瘤技術(shù)獲得了3株特異性單抗。建立了雙抗體夾心ELISA方法，并對(duì)呼吸道患兒標(biāo)本進(jìn)行了檢測(cè)。獲得了更為準(zhǔn)確的副粘病毒Tianjin株在人群中感染狀況的資料。構(gòu)建了副粘病毒Tianjin株的免疫噬菌體抗體庫，并獲得6株單抗，為進(jìn)一步進(jìn)行該病毒的診斷和致病機(jī)制奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R725.6;R392

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