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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。
OBJECTIVE: To construct human phage antibody library for children with respiratory syncytial virus infection and to construct a technology platform for the preparation of human antibody to provide new effective ways for the research, diagnosis, treatment and prevention of the pathogenesis of respiratory syncytial virus infection in children. Methods: Total RNA was extracted from peripheral blood lymphocytes of 52 children with respiratory syncytial virus infection and reverse transcribed into cDNA. The light and heavy chain Fd segments (ie, the heavy chain variable region and the first constant region) genes were amplified by PCR, and the amplified light and heavy chain gene segments were cloned into the pComb3x phagemid vector to electrotransform XL1- Blue Escherichia coli, Fab-phage antibody library was constructed after super-infection with helper phage M13K07. The antibody library double enzyme digestion identification, and respiratory syncytial virus particles as antigen for initial screening. Results: After the recombination of heavy and light chain genes, an immunopharmaceutical phage antibody library was successfully constructed. A total of 2.6 × 106 different clones were found, of which 70% contained light and heavy chain Fd genes. Therefore, the constructed phage antibody library has a capacity of 1.8 × 106. After preliminary screening, the antibody library has been enriched to varying degrees. Conclusion: The successful construction of human immune phage antibody library in children with respiratory syncytial virus infection using gene recombination technology and phage display technology laid the foundation for further research.