中国当代儿科杂志2008，Vol.10Issue(6)：681-685,5.
抗呼吸道合胞病毒Fab噬菌体抗体库的构建及初步筛选
Construction and preliminary panning of Fab phage display antibody library against respiratory syncytial virus
汪治华 1张国成 1李安茂 1周南 1陈一 1李小青 1苏字飞 1邓阳彬 1王治静1
作者信息
摘要
Abstract
Objective To construct a human phage display antibody library,which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic.This can provide a platform for human antibody preparation and diagnosis,prophylaxis and therapy of RSV infection in children.Methods Peripheral blood lymphocytes were isolated from 52 children with RSV infection,cDNA was synthesized from the total RNA of lymphocytes.The light and heavy chain Fd (VH-CH1)fragments of immunoglobulin gene were amplified by RT-PCR.The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue.The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs.The plasmids extracted from amplified E.coil were digested with restriction endonucleases Sac 1,Xba I,Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes.RSV virions were utilized as antigens to screen Fab antibodies.Results By recombination of light and heavy chain genes,an immune Fab phage display antibody library against RSV containing 2.08×107 different clones was constructed,in which 70% clones had light chains and heavy chain Fd genes.The capacity of Fab phage antibody gene library was 1.46×107 and the titre of the original Fab antibody library was about 1.06 x 1012 pfu/mL The antibody library gained an enrichment in different degrees after the preliminary panning.Condusions Utilizing the technology of phage display,an immune Fab phage display antibody library against RSV was successfully constructed in this study,which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis,therapy and prophylaxis of RSV infection in children.关键词
抗体库/噬菌体展示技术/Fab抗体/呼吸道合胞病毒/儿童Key words
Antibody library/Phage display technology/Fab antibody/Respiratory syncytial virus/Child分类
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汪治华,张国成,李安茂,周南,陈一,李小青,苏字飞,邓阳彬,王治静..抗呼吸道合胞病毒Fab噬菌体抗体库的构建及初步筛选[J].中国当代儿科杂志,2008,10(6):681-685,5.
