【摘要】 鸭乙型肝炎病毒 (DHBV)感染鸭 ,是研究IFN γ在机体自然感染过程中机体与病毒的相互作用和病毒的清除机制的良好动物模型。从PHA刺激后的鸭外周血单核细胞 (PBMC)中提取RNA ,通过RT PCR获得鸭IFN γ (DuIFN γ)cDNA基因 ,构建DuIFN γ真核表达质粒 ,转染COS 7细胞 ,经细胞病变效应 (CPE)抑制分析和MTT法对重组DuIFN γ滴度进行测定。实验表明 ,重组DuIFN γ能够抑制VSV感染鸭胚成纤维细胞而产生的细胞病变效应。抗 DuIFN γ抗体 ,能中和这种抗病毒活性。并且 ,GST DuIFN γ融合蛋白在大肠杆菌中得到表达和进一步纯化。更多还原

【Abstract】 The efficacy of cytokine therapy has been demonstrated in several viral diseases. Interferon γ is a cytokine that has potent antiviral property and immunomodulatory activity. To investigate the role of IFN γ in viral clearance during natural infection and to define the antiviral mechanism, DHBV infected ducks was used as an animal model. To clone, express, and develop the method of quantifying DuIFN γ gene transcription and expression, DuIFN γ cDNA was amplified by RT PCR from PHA stimulated duck PBMC. Recombinant plasmid expressing DuIFN γ was used to transfect COS 7, and the cell culture supernatant was analyzed by CPE inhibitory assay and MTT methods to determine the antiviral titer of IFN γ. The GST DuIFN γ fusion protein was expressed in E.coli and purified using the GST sepharose 4B. Results indicated that the supernatant collected from COS 7 cells transfected with DuIFN γ cDNA was able to prevent duck fibroblasts from VSV induced CPE in a dose dependent manner. An anti DuIFN γ antibody neutralized this antiviral activity.更多还原