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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。
Objective To clone a new subtype of woodchuck hepatitis virus interferon (IFN-α) and use it in the woodchuck hepatitis B virus (HBV) model to explore the strategy of IFN-α in the treatment of chronic hepatitis B. To investigate the mechanism of chronic hepatitis B virus (WHV) Infected marmoset peripheral blood mononuclear cells (PBMCs) Interferon functional status. Methods Poly (I-C) cells were stimulated with PBMC from normal and chronic WHV-infected woodchucks and their expressed interferon biological activity was analyzed. The molecular cloning technique was used to clone the gene of woodchuck IFN-α family. The cloned gene was sequenced, sequenced and eukaryotic expression was performed to detect the biological activity of the expressed product. RESULTS: After poly (I-C) stimulated the PBMC of marmots cultured in vitro, the activity of interferon secreted by PBMC from chronically infected marmots was significantly lower than that of normal woodchucks (P <0.01). 36 cloned parvovirus IFN-α gene sequences were cloned. After sequencing analysis, 10 clones were found to be novel subtypes, of which 8 were functional gene subtypes and 2 were pseudogenes. The virus protection test proved that only Functional genotypes are biologically active. Conclusion The immune function of woodchuck mice infected with chronic WHV is impaired. The cloning of a novel woodchuck IFN-α subtype gene provided new material for interferon gene therapy and interferon therapy strategies in a woodchuck HBV animal model.