HCVcore真核表达质粒pcDNA3.1(-)/core的构建

Construction of eukaryotic plasmid pcDNA3.1(-)/core containing the core gene of HCV

目的构建HCVcore基因真核表达质粒,为进一步研究和解析HCV诱发人不死化肝细胞癌化的机制,HCV感染的检测及疫苗和药物研发做好前期基础。方法将含HCV全长基因的pBRTM/HCV1 -3011质粒于大肠杆菌JM109内扩增;提取pBRTM/HCV1-3011质粒;从pBRTM/HCV1-3011质粒中PCR扩增出HCVcore片段并将其插入pGEM-T克隆载体;再与表达载体pcDNA3. 1( -)重组,以得到重组的真核表达载体pcDNA3. 1 ( -) /core;最后限制性酶切鉴定HCVcore表达载体。结果从pBRTM/HCV1-3011质粒中扩增出的HCVcore片段大小正确,经测序证明其碱基序列为编码目的基因的正确序列;凝胶电泳结果证明已将此片段克隆到pcDNA3. 1 /core内。结论 成功的构建了HCVcore基因的真核表达载体pcDNA3. 1( -) /core。

Objective To construct the eukaryotic plasmid containing the core gene of HCV, preparing for the research in the mechanism of the cancerization induced by HCV. Methods After expanding plasmid pBRTM/HCV1-3011 (containing the whole genome of HCV) through coliform JM109, we PCR the core gene with pBRTM/HCV1-3011 as the template; Insert the core segment into the clone plasmid pGEM T. Digest plasmid pcDNA3.1(-)and pGEM T/core with EcoRI and BamHI, then recycle the digested pcDNA and core segment. The pcDNA and core segment were circularized with T4 DNA ligase. Results The PCR results of HCV core has correct size and sequences. Conclusion The eukaryotic plasmid pcDNA3.1(-)/core has successfully been constructed.