摘要
应用ＰＣＲ方法从含有丙肝病毒全部非结构蛋白基因的质粒ｐＢＡＣ２５ 中扩增出全长的ＮＳ５Ａ基因ＤＮＡ片段（约１．３４ｋｂ），ＰＣＲ扩增ＮＳ５Ａ基因片段克隆到转移载体ｐＢｌｕｅＢａｃＨｉｓＡ中。重组转移质粒ｐＢｌｕｅＢａｃＨｉｓ５Ａ ＤＮＡ与野生型杆状病毒（ＡｃＮＰＶ）ＤＮＡ共转染ＳＦ－９昆虫细胞，通过空斑纯化获得带有ＮＳ５Ａ基因的重组病ＡｃＮＳ５Ａ。对重组病毒基因组ＤＮＡ进行 酶切和ＰＣＲ鉴定，证实ＨＣＶ ＮＳ５Ａ基因已插入重组病毒基因组中。ＡｃＮＳ５Ａ感染ＳＦ－９细胞后，在 细胞中表达出一条６４ｋＤ的蛋白，用Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析，结果表明这种蛋白与抗ＨＣＶ ＨＳ５Ａ特 异性抗体发生强烈反应，说明ＮＳ５Ａ基因已在细胞中得到表达，应用免疫荧光技术与免疫组化 技术进一步研究ＮＳ５Ａ蛋白在昆虫细胞中不同时间的表达情况及其分布，结果表明，ＮＳ５Ａ蛋白 在ＡｃＮＳ５Ａ重组病毒感染细胞２４ｈ后主要分布在细胞质膜上，而在４８ｈ后则同时分布于细胞质膜 和细胞核内 ，在７２ｈ则完全布满整个细胞，我们认为ＮＳ５Ａ蛋白定位于质膜和细胞核中，暗示着在病毒复 制过程中ＮＳ５Ａ蛋白可能参与病毒ＲＮＡ在质膜上复制和细胞基因表达的调?
Full-length NS5A gene of Hepatitis C Virus was amplified by P CR,using plasmid pBAC25 containing HCV nonstructural protein genes as template.T he amplified fragment (about 1.34kb) was cloned into transfer plasmid pBuleBacHi sA.The recombinant plasmid DNA was cotransfected with genome DNA of wild type Ac NPV to SF-9 insect cells.The recombinant baculovirus AcNS5A was obtained by pla que selection.Restriction analysis and PCR identification indicated that the NS5 A gene was integrated into the genome of AcNS5A,and HCV NS5A gene was expressed as a 64kD protein in SF-9 cells infected with AcNS5A.This protein can be recogn ized by anti-NS5A antibody when identified by Western-blot.The Immune fluoresc ence assay and immune histo-chemistry assay were used to study the distribution of NS5A protein inside the cell. Distribution of NS5A pr otein are changed with the time in the recombinant virus infected SF-9 cells.NS 5A protein are chang ed with the time in the recombinant virus infected SF-9 cells.NS5A protein is l ocated at the cell membrance at 24 hours post infection and located at both the membrance and the nucleus after 48 hours,and full of the cells after 72 hours.Th e result suggests that NS5A protein was located on plasma membrance and in nucle us,it may play an important role in RNA replication and in controlling the expre ssion of the cell genes during the replication of the virus.
出处
《中国病毒学》
CSCD
2002年第1期60-65,共6页
Virologica Sinica
基金
国家自然科学基金资助项目(30070175)
