丙型肝炎病毒C,E2,NS3区基因嵌合重组载体的构建

【目的】构建包含丙型肝炎病毒（HCV）C区、E2区及NS3区的嵌合重组载体。【方法】设计合成3对寡核苷酸引物，用PCR法从HCV重组质粒pHCVc、pBE2和pGMXNS3-5中特异扩增出编码HCVC区、E2区和NS3区抗原的部分基因片段（501、603和900bp），分别用HindⅢ、BamHⅠ、EcoRⅠ和XhoⅠ酶切后，逐个定向连接到质粒pcDNA3中，转化宿主菌XL1-Blue，分别用上述内切酶酶切及DNA测序鉴定重组质粒。【结果】酶切鉴定示所切下的片段大小均与预计相符，测序结果与文献报道序列及预计结果一致,证实符合表达框架。【结论】成功构建了HCV嵌合重组质粒pcDNA3-C-E2-NS3。

Construction of the Chimeric Recombinant Plasmid Vector Encoding C,E2,NS3 Antigens of Hepatitis C Virus

【 Objective】 To construct the chimeric recombinant plasmid vectorencoding C、E2 and NS3 antigens of HCV. 【Methods】 The specific primers were designed and synthesized to amplify the DNA fragments(PCR method) of C、E2 and NS3 antigen genes from the recombinant plasmids pHCVc, pBE2 and pGMXNS3-5 of HCV respectively. The length of the acquired fragments were 501 bp, 603 bp and 900 bp.After being digested by appropriate restriction endoenzymes(HindⅢ, BamHⅠ, EcoRⅠ and XhoⅠ), the generated fragments of C、 E2 and NS3 genes were cloned into pcDNA3 one by one according to their specific orientations.Then the recombinant plasmid was identified by DNA sequencing and digesting of endoenzymes.【Results】 The fragments digested by endoenzymes were as large as the predicted results.DNA sequencing was the same with the sequence reported on the literatures. 【Conclusion】 The chimeric recombinant plasmid pcDNA3-C-E2-NS3 was successfully constructed.