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目的该研究旨在利用噬菌体展示技术,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,解决鼠源性单克隆抗体临床应用的不足,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2.08×107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1.46×107,经过滴定,原始抗体库的滴度为1.06×1012pfu/mL。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,成功构建了小儿呼吸道合胞病毒感染患者人源性Fab噬菌体抗体库,为人源性单克隆抗体的制备提供了良好的条件,为进一步的研究奠定了基础,亦将有益于小儿呼吸道合胞病毒感染的诊断、治疗和预防。
OBJECTIVE: The purpose of this study was to construct phage antibody library of human respiratory syncytial virus infected patients with phage display technology and to build a platform for the preparation of human antibodies to overcome the shortcomings of clinical application of murine monoclonal antibody for pediatric respiratory tract Syncytial virus infection pathogenesis research, diagnosis, treatment and prevention provide a new effective way. 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, a total of 2.08 × 107 different clones were successfully constructed, and 70% of the clones contained light and heavy chain Fd genes. Therefore, the constructed phage antibody library had a library capacity of 1.46 × 107, and the titer of the original antibody library was 1.06 × 1012 pfu / mL after titration. After preliminary screening, the antibody library has been enriched to varying degrees. Conclusion The human Fab Fab phage antibody library in patients with respiratory syncytial virus infection was successfully constructed by using gene recombination technology and phage display technology, which provided a good condition for the preparation of human monoclonal antibodies and laid the foundation for further research. It will also benefit the diagnosis, treatment and prevention of respiratory syncytial virus infection in children.