【作者】 邵红霞；

【导师】 程训佳；

【作者基本信息】 复旦大学 ， 病原生物学， 2007， 博士

【摘要】 变态反应性疾病特别是哮喘是危害严重的全球性公共卫生问题,在大多数国家,哮喘的发病率及死亡率呈持续上升趋势。尘螨变应原是引起变态反应性疾病的重要病因之一,特别是引起儿童变应性哮喘的重要危险因子。变应性疾病的病因复杂,除了复合多基因式遗传外,出生前后与环境变应原的接触也至关重要。鉴于此,WHO提出儿童变应性疾病的三级预防理论并呼吁一级预防应成为今后研究的重点。多项研究表明孕期母体高水平的变应原特异IgG抗体对胎儿今后发生变应性疾病具有抑制作用。因此,本课题拟制备尘螨变应原特异的人源抗体IgG Fab片段用于螨性变应性疾病的防治研究。首先分离接受尘螨注射液脱敏治疗并证实具有高水平抗尘螨IgG的变应性哮喘患者的外周血淋巴细胞,提取总RNA,构建非依赖性克隆滴度为8×108的抗尘螨变应原的IgG抗体库；同时分离近两个月内未曾患感冒等感染性疾病的300名健康志愿者的外周血淋巴细胞,同法构建非依赖性克隆滴度为9×108的人IgG抗体库。以实验室培养的粉尘螨制备粉尘螨螨体浸出液、代谢培养基浸出液并利用基因工程手段制备重组2类变应原rDer f 2。分别以上述天然粗抗原及重组变应原作为抗原,采用克隆印迹法和ELISA法对抗体库进行多轮筛选,共筛选克隆数约为1.8×106个,经反复验证,最终获得两个可与尘螨变应原特异性结合的阳性克隆,命名为AM1和AM2,序列比较的结果显示两个克隆的重链是完全同源的。阳性克隆AM1的轻链与抗尘螨变应原的重链库重组,构成重链更替的抗体库,以重组的Der f 2作为抗原,经克隆印迹法筛选,获得1个亲和力提高的特异性阳性克隆AM1L-Hsh。AM1的轻链可变区近似系为K2-30,基因同源性为87%；重链可变区近似系为VH3-30,基因同源性为96%；AM2的轻链可变区近似系为K3-27,基因同源性为95%；AM1L-Hsh的重链可变区近似系为VH5-1。对上述3个Fab片段进行大量表达,并以金属螯合亲和层析法进行纯化,表面等离子共振BIAcore检测显示均有较高的亲和力,解离常数在1.27×10-9-6.3×10-8之间,链替换改造后亲和力有8倍提高。ELSIA、斑点印迹等检测证实纯化的抗体可特异性识别重组2类变应原及天然粗抗原。肥大细胞脱颗粒抑制试验显示尘螨变应原特异的IgG Fab片段可显著抑制肥大细胞的脱颗粒反应,提示Fab片段具有竞争性抑制抗原结合的作用。尘螨变应原特异的重组人单抗IgG Fab片段的制备和研究工作,为进一步应用变应原特异的IgG抗体预防和治疗螨性变应性哮喘提供了理论和实验依据,至今尚未见国内外相关研究报道。更多还原

【Abstract】 Allergic diseases especially asthma are public health problems worldwide. In most countries, the mobility and mortality of asthma are increasing continuously. House dust mites (HDMs) including Dermatophagoides farinae and Dermatophagoid.es pteronyssinus are among the most important allergen sources. They are the most potent causes of allergic diseases especially for allergic asthma in children. Genetic predisposition and environmental factors influence the development of allergic diseases together. A program entitled Prevention of Allergy and Asthma was launched by WHO under this situation, at the same time, primary prevention was stressed. Several studies showed that the increase of maternal IgG antibody would reduce subsequent sensitization rate and atopic symptoms in the offsprings. Therefore, specific human Fab antibodies are prepared for further study on prevention of mite allergic diseases.Human peripheral blood lymphocytes were isolated from the allergic asthma patients after a long period of immunotherapy with house dust mites extracts. Human IgG genes library with a titer of 8×108 was then constructed using total RNA of the lymphocytes. Another human IgG genes library was prepared from 300 healthy volunteers without any infectious diseases even common cold in the past 2 months. These two libraries were screened for the production of human monoclonal antibody Fab fragments to Der f 2 by cloning blotting assay and ELISA method. About 1.8×106 clones were screened and only 2 positive Fab fragments specific to rDer f 2 (named AM1 and AM2) were obtained from the IgG library of healthy volunteers. The heavy chains of AM1 and AM2 yield completely homologous. A new clone with higher affinity was got from a reshuffling library which was constructed by the light chain of AM1 and the heavy chain library from asthmatic patients. Sequence analysis of both AM1 and AM2 heavy chain genes revealed the nearest V-segment germline were VH3-30 with 96%homology. The closest V-segment germline of the light chain gene were K2-30 and K3-27 with 87%and 95%homology respectively. And the nearest V-segment germline of AM1L-Hsh was VH5-1 with 93%homology. Affinity of these 3 Fab fragments to recombinant Der f 2 and Der p 2 was 1.27×10-9 to 6.3×10-8. The affinity of AM1L-Hsh was eight times higher than AM1. The immunoassay data indicated that these Fab fragments had high specificity to recombinant and native protein of dust mite. In vitro mastocyte degranulation inhibition test showed that mite allergen specific IgG Fab could significantly inhibit mast cell degranulation. As a result, mite allergen specific Fab fragments may inhibit the specific binding of IgE to rDer f 2 competitively.This is the first report of gene analysis and bacterial expression of human monoclonal antibody Fab fragments to mite group 2 antigens. The combinatorial immunoglobulin gene library derived from human seems to be a potential tool for clinical immunoprophylaxis and treatment of mite allergic diseases.更多还原