【摘要】 目的克隆土拨鼠α干扰素(IFN-α)新亚型基因,用于土拨鼠HBV模型探索IFN-α治疗慢性乙型肝炎策略;调查慢性土拨鼠肝炎病毒(woodchuck hepatitis virus,WHV)感染的土拨鼠外周血单个核细胞(PBMC)干扰素功能状况。方法poly(I-C)体外刺激正常和慢性WHV感染的土拨鼠PBMC,分析其表达的干扰素生物学活性。利用分子克隆技术对土拨鼠IFN-α家族基因进行克隆,并对所克隆的系列基因进行测序、分型并进行真核表达后检测表达产物生物学活性。结果poly(I-C)刺激体外培养的土拨鼠PBMC后,慢性感染土拨鼠PBMC分泌的干扰素的活性显著差异低于正常土拨鼠(P<0．01)。获得36个土拨鼠IFN-α基因序列克隆,测序分析后,发现有10个克隆是新亚型基因,其中8个为功能基因亚型,2个为假基因亚型,病毒保护试验证明只有功能基因亚型具有生物学活性。结论慢性WHV感染的土拨鼠细胞免疫功能受损。新的土拨鼠IFN-α亚型基因的克隆为在土拨鼠HBV动物模型上进行干扰素基因治疗和研究干扰素治疗策略提供了新的材料。更多还原

【Abstract】 Objective To investigate the function of interferon alpha(IFN-α) in the woodchuck (Mar-mota monax) model of hepatitis B, the woodchuck IFN-αfamily gene(IFNA) was cloned, and analysis cellular immune status of woodchuck with chronic woodchuck hepatitis virus(WHV) infection. Methods Peripheral blood mononuclear cells(PBMC) of naive and chronic WHV infection woodchuck were cultured, then investigate antiviral biological activity of culture supernatants from PBMC stimulated with different concentration Poly I-C. Plasmids harbored woodchuck interferon alpha gene with different genotype were generated using molecular cloning technology. All sequence were sequenced and subtyped. Biologic activity of interferon was detected by viral protection assay. Results The data indicate that we obtain 10 novel IFNA from 36 wIFNA sequences, including 8 functional sequences and 2 pseudogene sequences. There are lower titer of interferon from PBMC of chronic infection woodchuck than that of naive woodchuck(P < 0.01). Conclusion The cellular immune function of woodchuck with chronic WHV infection is impaired. The novel IFN-αgene of woodchuck was cloned and characterized to provide a tool with which to test the antiviral effect of new forms of IFN-αdelivery and to improve the efficacy of current therapies by woodchuck HBV model.更多还原