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核苷(酸)类似物[neocleot(s)ide analogues,NUCs]在治疗乙型肝炎病毒(hepatitis B virus,HBV)感染的过程中常见的一个问题就是耐药的产生。耐药的产生与HBV逆转录酶(reverse transcriptase,RT)区特定位点突变具有直接关系。现有的体外表型分析方法存在固有的限制。本文提出一种构建HBV聚合酶特定位点多种突变的新方法,即位点特异性随机突变,通过一轮聚合酶链式反应扩增,在RT区特定位点引入多种突变形式,再结合golden-gate克隆法以及片段替换反应(fragment substitution reaction,FSR)将包含不同突变的DNA片段克隆到相应载体中以进行下一步分析。通过这种方法,构建了阿德福韦(adefovir,ADV)耐药位点rt181和rt236位点的多种突变形式。
One of the most common problems in the treatment of hepatitis B virus (HBV) infection is the development of drug resistance in neocleot (s) ide analogues (NUCs). The generation of drug resistance is directly related to the mutation of specific site of HBV reverse transcriptase (RT). Existing methods of in vitro phenotyping have inherent limitations. In this paper, we propose a new method to construct a variety of mutations in specific sites of HBV polymerase, ie, site-specific random mutagenesis, amplification by a round of polymerase chain reaction, introduction of various mutated forms at specific sites in the RT region, golden-gate cloning and fragment substitution reaction (FSR) DNA fragments containing different mutations were cloned into the corresponding vectors for further analysis. In this way, a number of mutated forms of the rt181 and rt236 sites of adefovir (ADV) resistance sites were constructed.