抗呼吸道合胞病毒免疫噬菌体抗体库的构建及初步鉴定

发布时间：2018-07-10 11:42

本文选题：抗体库 + 噬菌体展示技术　；参考：《细胞与分子免疫学杂志》2008年09期

【摘要】：目的:构建小儿呼吸道合胞病毒感染患者人源性噬菌体抗体库,搭建人源性抗体制备的技术平台,为小儿呼吸道合胞病毒感染发病机制的研究、诊断、治疗和预防提供新的有效途径。方法:从52例呼吸道合胞病毒感染患儿外周血淋巴细胞中提取总RNA,并逆转录为cDNA。用PCR扩增轻链和重链Fd段(即重链的可变区和第一恒定区)基因,并将扩增的轻链和重链基因片段克隆于pComb3x噬粒载体,电转化XL1-Blue大肠杆菌,经辅助噬菌体M13K07超感染后构建成Fab段噬菌体抗体库。对此抗体库双酶切进行鉴定,并用呼吸道合胞病毒颗粒作抗原进行初步筛选。结果:经过重轻链基因的重组,成功构建一免疫噬菌体抗体基因库,共有2.6×106个不同的克隆菌,其中70%的克隆均含有轻链和重链Fd基因。因此,所构建的噬菌体抗体库的库容量为1.8×106,经初步筛选,抗体库得到了不同程度的富集。结论:利用基因重组技术和噬菌体展示技术,成功构建小儿呼吸道合胞病毒感染患者人源性免疫噬菌体抗体库,为进一步的研究奠定了基础。
[Abstract]:Objective: to construct the human phage antibody library of children with respiratory syncytial virus infection and to set up a technical platform for the preparation of human phage antibody for the study and diagnosis of the pathogenesis of respiratory syncytial virus infection in children. Treatment and prevention provide a new and effective way. Methods: total RNAs were extracted from peripheral blood lymphocytes of 52 children with respiratory syncytial virus infection. Light chain and heavy chain FD segment (i.e. variable region and first constant region of heavy chain) were amplified by PCR. The amplified light chain and heavy chain gene fragments were cloned into pComb3x phagocyte vector and electrotransformed into XL1-Blue Escherichia coli. Fab fragment phage antibody library was constructed after superinfection of bacteriophage M13K07. The antibody library was digested and screened with respiratory syncytial virus particles as antigen. Results: after recombination of heavy chain and light chain gene, an immune phage antibody gene library was successfully constructed. There were 2.6 脳 10 6 different clones, 70% of which contained light chain and heavy chain FD gene. Therefore, the library capacity of the constructed phage antibody library was 1.8 脳 106, and the antibody library was enriched in different degrees after preliminary screening. Conclusion: the human immune phage antibody library of children with respiratory syncytial virus infection was successfully constructed by gene recombination and phage display, which laid a foundation for further research.
【作者单位】：第四军医大学西京医院儿童医学中心;
【基金】：国家自然科学基金资助项目(30571948)
【分类号】：R392.33

【共引文献】