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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。
Objective To construct a recombinant plasmid of HCV NS5A from patients with chronic hepatitis C (CHC) in South China and analyze the biological characteristics of HCV NS5A in antiviral response. Methods Plasmid type 1b HCV replicon with efficient replication was used as the backbone to construct the switch plasmid with double restriction enzyme sites of MIu Ⅰ and Bcl Ⅰ. HCV 1b subtype was amplified from the serum of different CHC patients by reverse transcription - polymerase chain reaction NS5A full length fragment was cloned into pMD-18 vector and sequenced to analyze the variation of PKRBD, ISDR, V3 and IRRDR regions. Amplification products without MIuI and BclI digestion sequence, introduced in the primer sequence after amplification and amplification, double digestion after NS5A segment into the HCV 1b replicon skeleton. Results The full-length NS5A fragment was successfully amplified and sequenced. The ISDR-V3 region of NS5A region was replaced by the replicon, and the sequencing result was correct. Sequence alignments showed that the number of amino acid variations of ISDR and PKRBD in response strains was higher than that in non-responders; the mutation rates of V3 and IRRDR were higher. Conclusion The successful construction of a recombinant plasmid containing the core region of HCV 1b subtype NS5A in South China laid the foundation for the study of the biological characteristics of HCV NS5A, the mechanism of interferon resistance and the analysis of individual antiviral therapy in refractory CHC patients.