HCV核蛋白羧基端信号肽基因缺失突变体的构建、鉴定和真核表达  

Expression of a C-terminal deleted mutant of hepatitis C virus core protein in P815 cell line

作　　者：洪沙[1] 黄长形[1] 杨为松[1] 陈红梅[1] 李光玉[1] 王平忠[1] 陈伟红[1] 张岩[1] 李羽[1] 

机构地区：[1]中国人民解放军第四军医大学唐都医院全军感染病诊疗中心,陕西省西安市710038

出　　处：《世界华人消化杂志》2005年第7期852-855,共4页World Chinese Journal of Digestology

基　　金：国家教育部留学回国人员科研启动基金资助项目;No.2002HG003~~

摘　　要：目的:构建羧基末端缺失核蛋白的真核表达载体并在小鼠肥大细胞瘤细胞株P815细胞中表达.方法:用RT-PCR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,以此为模板扩增羧基末端缺失突变核蛋白基因片段(C507),将其定向克隆入真核表达载体pCI-neo中并转染P815细胞,经间接免疫荧光染色、激光共聚焦显微镜检测细胞中突变核蛋白(P169)的表达.结果:从患者血清中成功克隆出HCVCcDNA,构建了编码羧基末端缺失核蛋白的重组质粒pCI-507,并经酶切鉴定和测序证实;间接免疫荧光染色表明P169主要在P815细胞胞质中表达,少数可见于细胞核内.结论:重组体pCI-507构建正确,其表达产物P169在P815中得到有效表达,为在此基础上的DNA免疫研究提供了实验依据.AIM: To construct a recombinant eukaryotic expression vector encoding C-terminal deleted mutant of hepatitis C virus (HCV) core protein and express it in P815 cell line. METHODS: cDNA for HCV core protein was obtained from patients with chronic HCV infection by RT-PCR and employed as template to amplify the gene fragment of truncated core protein (C507). The truncated core cDNA was modified by two restriction enzymes and cloned into pCI-neo, which was named as pCI-C507. By using lipofectamine 2000, the recombinant was transferred into P815 cells. 24 hours after transfection, the cells were collected and examined by confocal microscopy. RESULTS: The cDNA of HCV core protein was analyzed with phylogenetic analysis and confirmed to be genotype Ib. pCI-C507 was identified by digestion with restriction enzymes and confirmed by DNA sequencing. The expression of pCI-C507 (P169) was observed in the cell plasma under confocal microscopy while only a little was in the nucleus. CONCLUSION: The recombinant pCI-C507 was correctly constructed and effectively expressed in P815 cells, which can be used for DNA vaccination study.

关 键 词：核蛋白 缺失突变体 信号肽基因 间接免疫荧光染色 羧基端 P815细胞 RT-PCR方法 真核表达载体 pCI-neo 丙型肝炎患者 末端缺失 E1区基因 显微镜检测 激光共聚焦 瘤细胞株 肥大细胞 西安地区 基因片段 羧基末端 定向克隆 

分 类 号：R373.21[医药卫生—病原生物学]