流感病毒中和性基因工程Fab抗体的研制

目的 获得基因工程抗流感病毒抗体，为检测其粘膜用药在动物模型中的抗病毒效果奠定基因。方法 从酶联免疫吸附试验阳性的人外周血中分离淋巴细胞，提取总RNA，Oligo-dT逆转录cDNA,聚合酶链反应扩增人IgG轻、重链基因，利用pComb3载体构建噬菌体抗体库。用纯化的流感病毒A／Sydeny/5/97(H3N2）为固相抗原筛选抗体Fab段，并在大肠埃希菌中进行分泌性表达。通过血凝抑制实验、免疫荧光实验和病毒中和实验选择具有中和作用的Fab抗体。结果 分离到两株Fab克隆，IV－2和IV－6，流感病毒抗原和抗Fab抗体直接ELISA检测阳性，间接免疫荧光实验呈阳性，它们都具有血浆抑制作用，病毒中和实验显示能使病毒滴度下降30倍和20倍。结论 获得了两株具有病毒中和活性的人源抗流感病毒悉尼株的Fab段抗体，为进一步的表达纯化及粘膜给药研究其抗病毒效果提供材料。

Generation of human recombinant antibody Fab fragment to influenza virus from combined antibody library.

Objective To acquire the recombinant human monoclonal antibodies to influenza virus which can be used in mucous membrane treatment to prevent the infection of virus in animal model in the future.Methods Construct and pan human Fab antibody library to influenza virus Sydney strain (H3N2) from vaccinated donors by using phage display technology and secreted expression in \%E.Coli\% system.The positive Fab clones with neutralization activity to influenza virus were selected and characterized through HI test,IFA and VN test.Results Separate two Fab clones,IV\|2 and IV\|6,appear positive responses in ELISA and IFA.These two clones can inhibit hemagglutination in HI test and can decrease viral titer by 30 and 20 times in viral neutralization assay,respectively.Conclusion we have got two clones of Fab fragments with neutralization activity to influenza virus Sydney strain (H3N2).