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HCV感染相关干扰素L4真核表达载体的构建与表达 预览

Construction and expression of recombinant eukaryotic expression vector of HCV infection related interferonλ4
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摘要 目的构建HCV感染相关干扰素IFNL4蛋白融合His标签的真核表达载体,并将其在293-T细胞中表达后进行初步纯化鉴定。方法采用PCR法从含人IFNL4基因序列的质粒pFC14A-p179中扩增出目的片段,将其定向连接到pcDNA3.1-His真核表达载体中,经酶切和测序鉴定正确后,以脂质体法转染293-T细胞,培养72 h后裂解细胞,采用抗His-Tag抗体行蛋白质印迹法检测目的蛋白的表达。结果成功构建了真核表达载体pcDNA3.1-IFNL4-His,转染后经Western blot法检测显示20 kDa位置有目的蛋白条带,大小与目的蛋白相符。结论我们成功构建了HCV感染相关IFNL4与His标签融合的真核表达载体,体外转染293-T细胞后鉴定其工作良好,为后续对干扰素在HCV感染及抗肝纤维化治疗中的研究奠定了基础。 Objective To construct a eukaryotic vector for recombinant interferon-λ4(IFNL4)protein expression in 293-T cells in vitro.Methods The target fragment of IFNL4 was amplified by PCR from plasmid pFC14A-p179 which contained human IFNL4 gene sequence.The amplified fragment was ligated into pcDNA3.1-His-tag vector and identified by restriction enzyme digestion and sequencing.The vector was then transfected into 293-T cells.After culture for 72 hours,the cells were lysed and the recombinant protein was purified and identified by Western blot.Results The eukaryotic expression vector pcDNA3.1-p179-His was successfully constructed and the recombinant protein was expressed in 293-T cells and successfully purified.Western blot analysis showed that the target protein band was about 20 kDa.Conclusion The eukaryotic expression vector fused with IFNL4 and His-tag is successfully constructed,which might be used as a noval candidate for immune therapy.
作者 徐溪 党引利 吴朔 Xu Xi;Dang Yinli;Wu Shuo(Department of Respiratory Disease,XiJing Hospital,Army Military Medical University,Xi’an 710032,Shaanxi Province,China)
出处 《实用肝脏病杂志》 CAS 2018年第6期833-836,共4页 Journal of Practical Hepatology
关键词 丙型肝炎病毒 干扰素λ4 融合蛋白 真核表达 体外 Hepatitis C virus Interferon-λ4 Fusion protein Eukaryotic expression In vitro
作者简介 第一作者:徐溪,女,31岁,硕士研究生。主要从事分子病毒学研究。E-mail:xuxi198617@163.com;通讯作者:吴朔,E-mail:wushuoywb@163.com
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