A method for constructing extensive mutagenesis of HBV polymerase

Liu Ya; Yang Liuqing; Zhang Wenlu; Huang Ailong; Hu Jieli

doi:10.13406/j.cnki.cyxb.001313

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A method for constructing extensive mutagenesis of HBV polymerase

更新时间：2024-01-17

- A method for constructing extensive mutagenesis of HBV polymerase
- Journal of Chongqing Medical University Vol. 42, Issue 7, Pages: 771-775(2017)
- 作者机构：
  
  重庆医科大学感染性疾病分子生物学教育部重点实验室
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.001313
  CLC： R373.21
- Published：2017，
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Liu Ya, Yang Liuqing, Zhang Wenlu, et al. A method for constructing extensive mutagenesis of HBV polymerase. [J]. Journal of Chongqing Medical University 42(7):771-775(2017)
DOI：

Liu Ya, Yang Liuqing, Zhang Wenlu, et al. A method for constructing extensive mutagenesis of HBV polymerase. [J]. Journal of Chongqing Medical University 42(7):771-775(2017) DOI： 10.13406/j.cnki.cyxb.001313.

摘要

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。

Abstract

A common problem during long term administration of chronic hepatitis B with Neocleot（s）ide analogues（NUCs）is drug resistance. NUCs resistance has been attributed to mutations at specific sites in the reverse transcriptase domain of HBV polymerase.Currently used extensive mutagenesis assay based on plasmid transfection method has some disadvantages. Here

we developed an alternative method for extensive mutagenesis phenotype assay of HBV polymerase. This method combined a randomized mutation method and golden-gate or fragment substitution reaction（FSR） strategy developed previously. Using this way

we successfully constructed multiple mutations at rt181 and rt236

the well-known mutation sites related to ADV resistance.

关键词

克隆生物工程方法核苷类似物耐药HBV聚合酶

Keywords

clonebiotechnologyneocleot（s）ide-analoguesHBV-polymerasedrug resistance

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Related Institution

The Department of Biochemistry,Academy of Basic Medicine,Chongqing Medical University

重庆医科大学基础医学院生物化学教研室

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重庆医科大学附属第一医院眼科

重庆医科大学基础医学研究所

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