摘要
目的:构建丙型肝炎病毒(hepatitisCvirus,HCV)第6080位氨基酸多肽缺失核蛋白的真核表达载体,并在真核细胞中表达.方法:用RTPCR方法从西安地区丙型肝炎患者血清中扩增HCVC区及部分E1区基因并进行克隆、测序,以此为模板用重叠延伸拼接方法扩增第6080位氨基酸多肽基因缺失的核蛋白基因片段(C510),将其定向克隆入哺乳动物高效表达载体pCIneo中,通过Lipofectamine2000转染入p815细胞,经间接免疫荧光染色、激光共聚焦显微镜和WesternBlotting方法检测细胞中目的蛋白的表达.结果:从患者血清中成功克隆出HCVC区基因,序列及系统发生树分析结果显示为1b型;测序结果显示构建的第6080位氨基酸多肽缺失核蛋白基因的重组真核表达质粒pCI510基因序列准确;免疫荧光和免疫印迹方法均证实目的蛋白p170可在p815细胞中表达.结论:重组体pCI510构建正确,其目的基因在p815细胞中可有效表达,为进一步的研究奠定了基础.
AIM: To construct an eukaryotic cell expression vector encoding a mutation hepatitis C virus(HCV) core protein deleted amino acids 60-80 and express it in p815 cell. METHODS: HCV core protein cDNA was amplified by RT-PCR from blood serum of chronic hepatitis C patients. The gene fragment of mutation core(C510) was amplified by splicing by overlap extension(SOE) from the core protein cDNA and cloned into pCI-neo, and named as pCI-C510. By aid of lipofectamine2000,the expression vector was transfected into p815 cell. The expression protein was detected by immunofluorescence under confocal microscopy and western-blot. RESULTS : Phylogenetic analysis shows that the HCV core protein cDNA amplified from Shaanxi chronic hepatitis C patients were genotype 1 b.The pCI-C510 was identified by sequencing. The signals of C510(p170) were diffusive in the cytoplasm under confocal microscopy.p170 was proved to be expressed in p815 cells by Western Blotting.CONCLUSION: The recombinant pCI-C510 is correctly constructed and proved to be expressed effectively in p815 cells.
出处
《第四军医大学学报》
北大核心
2005年第12期1101-1104,共4页
Journal of the Fourth Military Medical University
基金
国家教育部归国人员启动基金项目(2002HG003)
关键词
肝炎病毒
病毒核心蛋白质类
突变
真核表达
重叠延伸拼接法
hepacivirus,viral core proteins,mutation,eukaryotic expression, splicing by overlap extension(SOE)
