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目的:构建对干扰素敏感和不敏感的丙型肝炎患者的干扰素敏感决定区(ISDR)基因;在体外表达含ISDR的丙型肝炎病毒(HCV)NS5A蛋白,探讨表达的NS5A蛋白是否通过ISDR与干扰素诱导的RNA蛋白激酶(PKR)结合来影响干扰素的抗HCV作用.方法:采用合成寡核苷酸,PCR,分子克隆等方法构建ISDR基因“盒”,包括3个对干扰素敏感的丙肝患者ISDR及1个对干扰素耐受的丙肝患者ISDR,将含不同ISDR的NS5A基因克隆进原核表达载体PRSET,转染BL21(DE3)细胞后由IPTG诱导表达,获得蛋白过柱纯化及透析去掉高盐后,与经体外转录和翻译系统获得的PKR进行蛋白结合试验.结果:经测序证实获得4个不同ISDR基因,包括1个对干扰素不敏感基因和3个对干扰素敏感基因;体外表达的含ISDR的NS5A蛋白约58000,体外试验初步证实,无论对干扰素敏感或不敏感的ISDR均可与PKR结合.结论:体外构建的含不同ISDR的NS5A蛋白能在原核细胞系统中获得较好表达;研究显示,ISDR影响干扰素抗病毒作用可能不是由ISDR与PKR直接结合导致的.
OBJECTIVE: To construct an interferon-sensitive determinant (ISDR) gene of interferon sensitive and insensitive Hepatitis C patients and to express ISDR-containing HCV NS5A protein in vitro to investigate whether expressed NS5A protein passes ISDR And interfere with interferon-induced RNA kinase (PKR) to affect the anti-HCV effect of interferon.Methods: ISDR gene “cassette” was constructed by using synthetic oligonucleotides, PCR and molecular cloning methods, including three interferon sensitive Of hepatitis C patients with ISDR and one ISDR resistant to interferon in hepatitis C patients. NS5A gene with different ISDR was cloned into prokaryotic expression vector PRSET and transfected into BL21 (DE3) cells and induced by IPTG. After dialysis to remove the high salt, protein binding assay was performed with PKR obtained from in vitro transcription and translation system.Results: Four different ISDR genes were confirmed by sequencing, including one interferon insensitive gene and three interferon sensitive genes ; ISDR-NS5A protein expressed in vitro was about 58000. Preliminary in vitro tests confirmed that both interferon-sensitive and non-sensitive ISDR could bind to PKR.Conclusion: NS5A protein with different ISDR constructed in vitro can express in prokaryotic cells The results showed that the effect of ISDR on the antiviral effect of interferon might not be caused by the direct binding of ISDR to PKR.