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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础。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。它们都具有血凝抑制作用 ,病毒中和实验显示能使病毒滴度下降 30倍和 2 0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。
OBJECTIVE To obtain the genetically engineered anti-influenza virus antibody, which lays the foundation for detecting the anti-viral effect of mucosal drug in animal models. Methods Lymphocytes were isolated from human peripheral blood with enzyme - linked immunosorbent assay (ELISA). Total RNA was extracted and Oligo dT was reverse transcribed into cDNA. Polymerase chain reaction (PCR) was used to amplify human light and heavy chain genes. The phage antibody library was constructed by using pComb3 vector. The Fab fragment of the antibody was screened using the purified influenza A Sydeny 5 97 (H3N2) as a solid phase antigen and secreted in Escherichia coli. Fab antibodies with neutralization were selected by hemagglutination inhibition assay, immunofluorescence assay and virus neutralization assay. Results Two Fab clones were isolated, which were positive by direct ELISA and indirect immunofluorescence assay. The results of indirect immunofluorescence assay showed that two Fab clones, IV 2 and IV 6, were positive for influenza virus antigen and anti-Fab antibody. They all have hemagglutination inhibition and virus neutralization experiments have shown to reduce viral titers 30-fold and 20-fold. Conclusion Two Fab anti-influenza antibodies against human influenza virus Sydney strain were obtained, which could be used as materials for the further purification of expression and mucosal drug delivery.