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目的:运用核糖体展示技术制备抗HCV NS5B人源性单链抗体,并进行初步分析。方法:以HCV阳性患者的外周血单个核细胞为材料,扩增全套重链可变区和轻链可变区的基因,构建人单链抗体(scFv)核糖体展示文库;以NS5B蛋白为靶标筛选到scFv基因,并将其连接到pET16b载体并转化至大肠杆菌BL21,诱导表达scFv;对scFv基因测序和特异性分析。结果:成功构建抗HCV scFv库;获得2个具有较强结合活性的scFv;基因分析证实获得预期抗体。结论:成功制备了人源性抗NS5B scFv,为HCV的免疫治疗奠定了基础。
OBJECTIVE: To prepare anti-HCV NS5B human scFv antibody by ribosome display technology and conduct preliminary analysis. METHODS: Peripheral blood mononuclear cells from HCV-positive patients were used as materials to amplify the full set of heavy chain variable region and light chain variable region genes to construct a human scFv ribosome display library. NS5B protein was used as a target The scFv gene was screened and ligated into the pET16b vector and transformed into E. coli BL21 to induce the expression of scFv; the scFv gene was sequenced and analyzed for specificity. Results: The anti-HCV scFv library was successfully constructed; two scFvs with strong binding activity were obtained; and the genetic analysis confirmed that the expected antibodies were obtained. Conclusion: The human anti-NS5B scFv was successfully prepared, which laid the foundation for the immunotherapy of HCV.