大容量人免疫球蛋白G基因文库的构建及初步应用  

Construction and preliminary use of a large human IgG gene library

作　　者：邵红霞[1] 付永锋[1] Hiroshi Tachibana 程训佳[1] 

机构地区：[1]复旦大学上海医学院病原生物学系,上海200032

出　　处：《中国病原生物学杂志》2009年第10期738-741,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30371331);上海市基础研究重点项目(No.08JC1401000)

摘　　要：目的从大容量人免疫球蛋白G基因文库中获得尘螨2类变应原特异的IgG Fab片段。方法通过DNA重组技术,从300名健康志愿者的外周血淋巴细胞中扩增全套人抗体轻链及重链Fd基因,分别插入pFab1-His2相应位置,构建人免疫球蛋白G基因文库;以重组粉尘螨2类变应原(rDer f2)作为抗原,采用克隆印迹法对抗体库进行筛选,ELISA验证以获得阳性克隆;提取阳性克隆质粒,进行核苷酸序列测定并推算氨基酸序列,Kabat数据库分析轻、重链基因的同源性,表面等离子共振BIAcore检测纯化的Fab片段与rDer f2的亲和力。结果从9×108库容的人免疫球蛋白G基因文库中筛选1.28×106个克隆,获得2个可与尘螨变应原特异结合的阳性克隆AM1和AM2。序列比较显示2个克隆的重链完全同源;同源性分析显示,AM1、AM2轻链可变区近似系分别为K2-30和K1-12,重链可变区近似系为VH3-30;BIAcore检测显示,2个克隆的Fab片段与rDer f2的结合常数分别为6.1×107和5.7×107,解离常数分别为3.1×10-8和4.1×10-8。结论从未经免疫的大容量人免疫球蛋白G基因文库获得具有较高亲和力的尘螨变应原特异的IgG Fab片段。Objective To produce human monoclonal antibody Fab fragments specific to group 2 mite allergens from a combinatorial immunoglobulin gene library. Methods Total genes encoding the light chains and Fd region of the heavy chains of human immunoglobulin G were amplified from the peripheral blood lymphocytes of 300 healthy volunteers by recombinant DNA technology. A human IgG gene library was constructed by inserting the light and heavy chain genes into the vector pFabl-His2. The library was screened for the production of human monoclonal antibody Fab fragments with an affinity for Der f2 by cloning blotting assay and results were confirmed by ELISA. Genes of positive clones were identified and the amino acid sequences were deduced from the DNA sequences; the variable regions of the heavy and light chain were compared with a Kabat database. Affinity measurements of purified Fabs by surface plasmon resonance were carried out using the BIAcore 3000. Results After about 1.28 × 10^6 clones were screened, 2 positive Fab fragments specific to rDer f2 named AM1 and AM2 were identified. The heavy chains of both clones were completely homologous. Sequence analysis of both AM1 and AM2 light chain genes revealed that the nearest V-segment germlines were K2-30 and K1-12, respectively. The closest V-segment germline of the heavy chain gene was VH3-30. Plasmon resonance assay revealed that the association and dissociation constants were 6.1 × 10^7 M 1 and 3.1 × 10^-8 M for AM1 and 5.7 × 10^7 M-1 and 4.1 × 10^-8 M for AM2, respectively. Conclusion High-affinity human monoclonal antibody Fab fragments specific to mite allergens were obtained from a large naive combinatorial library of immunoglobulin genes.

关 键 词：尘螨变应原 变应性哮喘 人源抗体Fab片段 变应性哮喘的预防 

分 类 号：R384.4[医药卫生—医学寄生虫学]