中国旱獭干扰素α家族基因在真核细胞和原核细胞中的表达

Expression of interferon alpha family gene of Chinese marmot in eukaryotic and prokaryotic cells

目的将不同基因型中国旱獭干扰素α(IFNα)基因在真核和原核细胞中进行表达,检测其生物学活性,以期利用克隆的中国旱獭IFN在动物模型上探索慢性乙型肝炎有效的IFN治疗方案和策略。方法利用分子克隆技术将14个不同亚型的中国旱獭IFNα家族基因亚克隆到真核表达载体,将中国旱獭IFNα5亚型基因亚克隆到原核表达载体。病毒保护实验检测表达产物的生物学活性,比较不同基因亚型IFNα生物学活性的差异,分析中国旱獭IFNα生物学活性的种属特异性。结果 14个基因亚型中国旱獭IFNα中,8个功能基因亚型真核表达产物有生物学活性,而6个假基因亚型产物没有生物学活性,8个功能基因亚型真核表达产物生物学活性存在差异,且其活性都受种属特异性限制。原核表达纯化产物具有较好的生物学活性。结论中国旱獭IFNα家族基因能在真核和原核细胞中表达,表达产物具有生物学活性。本研究为在中国旱獭乙型肝炎病毒感染模型上探索IFN治疗策略奠定了实验基础。

Objective To investigate the function of interferon alpha （IFNα） in a Chinese marmot model of hepatitis B, we expressed the Chinese marmot （Marmota himalayana） IFNα family gene （IFNA） in eukaryotic cells and prokaryotic cells. Methods Eukaryotic and prokaryotic expression plasmids harboring Chinese marmot interferon alpha gene with different genotypes were generated using molecular cloning technology, We detected the biological activity of all expression products by viral protection assay, and analyzed their differences and species restriction of the biological activity. Results The Chinese marmot functional genotype IFNα was expressed in the baby hamster kidney （BHK） cell line, and these products protected WH12/6 cells challenged by encephalomyocarditis virus （EMCV）, The Chinese marmot IFN-α5 also expressed in E.Coli induced by IPTG, and purified fusion protein had antiviral biological activity. The biologic activity displayed differences among different subtype IFNα, and it had strict species restriction. Conclusion The IFNα family gene of the Chinese marmot can be expressed in both eukaryotic and prokaryotic cells, and the expression products show antiviral activity in a protection assay. This study provides, for the first time, evidence that IFNα from the Chinese marmot has an antiviral function in vitro and can be used to improve the efficacy of current therapies for HBV infection in our Chinese marmot model.