人Epsilon干扰素在CHO细胞中的表达及生物学活性的研究  

Expression of human interferon-ε in Chinese hamster ovary cell and its biological activity

作　　者：马丽丽[1] 盛伟华[1] 谢宇锋[1] 缪竞诚[1] 王金志[1] 庄文卓[1] 李丽娥[1] 杨吉成[1] 

机构地区：[1]苏州大学医学院细胞与分子生物学教研室,215007

出　　处：《中华微生物学和免疫学杂志》2006年第3期279-284,共6页Chinese Journal of Microbiology and Immunology

摘　　要：目的为了研究新发现的人epsilon干扰素(hIFNε)的生物学活性,我们通过RTPCR克隆了hIFNε基因,并构建pcDNA3.1mychis()AhIFNε(以下简称pcDNA3.1AhIFNε)的真核表达载体,在CHO细胞中表达,并研究真核细胞重组表达的rhIFNε的生物学活性。方法用TNFα刺激人宫颈癌HeLa细胞,抽提总RNA,经RTPCR获得全长cDNA,与pcDNA3.1mychis()A真核表达载体连接,构建重组体pcDNA3.1AhIFNε,并在CHO细胞中进行表达,采用微量细胞病变抑制试验研究表达产物中的rhIFNε对多种病毒的抗病毒活性;MTT法检测其对A375、HeLa和A549细胞的生长影响,并通过在Wish、HeLa、A375细胞中诱导MxA抗病毒蛋白的产生研究hIFNε的抗病毒机制。结果经PCR和限制性酶切鉴定以及DNA测序,结果表明已经成功构建了重组体pcDNA3.1AhIFNε,并在CHO细胞中稳定表达了rhIFNε蛋白,该蛋白具有抗HSVⅠ、PolioV、Ad3和VSV病毒的作用,能够抑制HeLa、A375、A549细胞的生长,刺激Wish、HeLa、A375细胞产生MxA蛋白。结论本研究成功地构建了pcDNA3.1AhIFNε真核表达载体并在CHO细胞中获得稳定表达,证明了rhIFNε蛋白具有抗病毒和抗增殖活性,其抗病毒效应的分子机理可能与诱导细胞产生MxA抗病毒蛋白有关,为今后研究rhIFNε的生物学功能和基因重组药物研制及其开展基因治疗奠定了基础。Objective To express human interferon epsilon（hIFN-ε） in CHO cells and evaluate its antiviral activities. Methods Full-length human interferon-ε cDNA was obtained using RT-PCR from HeLa cells stimulated by tumor necrosis factor-α（TNF-α） and then subcloned into pcDNA3.1/myc-his（-）A vector. The construct was transfected into CHO cells. Cytopathic effect reduction assay was used to measure the antiviral activities d the protein against several kinds of virus. MTT assay was performed to analyze the influence of the target protein on the viability of A375, HeLa and A549 cells. Meanwhile, whether recombinant human interferon-ε protein induces these cells to produce MxA protein was also explored. Result The recombinant vector , pcDNA3.1/myc-his （-）A-IFN-ε, was constructed and verified by PCR amplification, restriction endonucleases digestion and following DNA sequencing. The hIFN-ε protein was stably and consistently expressed in CHO cells. The protein showed intrinstic cellular antiviral activity in CHO cells infected by virus, such as HSV-I , PolioV, Ad3 and VSV. The protein also inhibited the proliferation of HeLa, A375, A549 ceils and induced the expression of MxA in these cells. cells Conclusion hIFN-ε is able to induce the production of MxA in cells, which will provide a clue for study of recombination gene drugs related to corresponding intefferons.

关 键 词：pcDNA3.1A-hIFN-ε CHO细胞 抗病毒 抗增殖 MXA 

分 类 号：R346[医药卫生—基础医学]