【摘要】 目的构建基因1b型丙型肝炎病毒(HCV)不同准种株截短片段核心蛋白(CP)绿色荧光蛋白(GFP)融合蛋白表达质粒。方法用聚合酶链反应(PCR)扩增获得等长度片段的CP基因:氨基酸(aa)长度为癌中心株(T)∶T∶1-172aa、癌旁株(NT)∶1-172aa及C191(HCV-J6)∶1-172aa,扩增产物用NheI及EcoRI双酶切后将其插入到真核表达质粒pEGFP-N1中,转染不同细胞系,用荧光显微镜及流式细胞仪观察GFP的表达。结果PCR扩增、序列分析验证,T、NT及C191截短片段CP基因均成功克隆到pEGFP-N1中,在转染HepG2、chang-liver48h后,用荧光显微镜及流式细胞仪均观察到GFP的表达。结论构建基因1b型HCV不同准种株截短片段CP的真核表达质粒获得成功,可用于不同准种株CP的功能研究。更多还原

【Abstract】 Objective To construct green flurosecent protein (GFP) and truncated core protein (CP) fusion protein eukaryotic plasmid of different quasi-species of genotype 1b hepatitis C virus (HCV).Methods The gene sequences of different truncated HCV genotype 1b CP were amplified from plasmids containing CP sequences derived from tumor (T) and non-tumor (NT) tissues from one patient infected with HCV. Amino acid (aa) lengths were HCV T:1-172 aa,NT:1-172 aa,and HCV C191(HCV-J6):1-172 aa. PCR products were cleaved with restriction enzymes Nhe I and EcoR I and cloned into pEGFP-N1. Positive clones were transfected into different cell lines. CP-GFP was observed by flurosecent microscope and flow cytometry. Results The evidences of DNA sequencing and PCR analysis confirmed that the truncated CP genes from different HCV quasi-species were correctly inserted into pEGFP-N1 vector respectively. 48 hours after transfection to HepG2,chang-liver, CP-GFP was observed by flurosecent microscope and flow cytometry.Conclusion Successful construction of truncated CP of different quasi-species expression plasmids the function of different domains of CP of different HCV strains.更多还原