华南HCV 1b亚型NS5A插入式复制子重组质粒的构建和序列分析

目的构建来自华南不同慢性丙型肝炎(CHC)患者的HCV NS5A的复制子重组质粒并测序分析,探索HCV NS5A在抗病毒应答中生物学特性。方法以能够高效复制的1b型HCV复制子质粒为骨架,构成带有MIuⅠ和BclⅠ双酶切位点的开关质粒,采用逆转录-聚合酶链反应从不同CHC患者血清中扩增获得HCV 1b亚型NS5A全长片段,克隆到pMD-18载体中,测序分析其中的PKRBD、ISDR、V3和IRRDR区域的变异情况。扩增产物中无MIuⅠ和BclⅠ酶切序列的,在引物中引入相关酶切序列后再扩增,双酶切后将NS5A区段置换入HCV 1b复制子骨架中。结果成功扩增出NS5A全长片段并克隆测序,将NS5A区ISDR-V3区域置换入复制子中,测序结果正确。序列比对显示应答株的ISDR和PKRBD氨基酸变异数目比无应答株高;V3和IRRDR区域存在较高氨基酸突变率。结论成功构建包含华南HCV 1b亚型NS5A核心区域的复制子插入式重组质粒,为研究HCV NS5A生物学特性、干扰素抵抗的机制以及难治性CHC患者的个体化抗病毒治疗分析奠定了基础。

Construction and sequence analysis of recombinant HCV-1b replicon by replacing NS5A region

Objective To construct the recombinant HCV-1b replicon by replacing NS5A region using serum samples from patients with chronic hepatitis C(CHC) in South China and explore the biological characteristics of NS5A protein in response to antiviral therapy.Methods The on-off plasmid containing the cutting sites of the restriction endonucleases MIu I and Bcl I was designed based on the backbone of robust HCV 1b replicon.The full-length fragments of HCV NS5A were amplified from different CHC patients by RT-PCR and cloned into pMD-18 vector,followed by sequence analysis of amino acid mutation of ISDR,PKRBD,V3 and IRRDR within the NS5A region.If the amplicon obtained contained no MIu I or Bcl I cutting sites,the NS5A fragment was re-amplified using primers containing the cutting sites and inserted into the replicon for replacement.Results The full-length fragments of NS5A were obtained successfully from CHC patients.The core region of ISDR-V3 of NS5A was replaced in the HCV replicon plasmid and showed correct sequences.The amino acid mutations of ISDR and PKRBD within NS5A were more frequent in patients with sustained viral response(SVR) than those without SVR.A high variability in the amino acid sequence was observed in both IRRDR and V3 regions.Conclusion The plug-in type recombinant HCV replicon for replacement of NS5A region in the virus from CHC patients has been successfully constructed,which provides a basis for further investigation of the biological characteristics of NS5A protein,the mechanisms of interferon-resistance,and antiviral therapy of difficult-to-treat CHC.