FENG Lei.YE IAn-bai“, RUAN Hua, GAO Jin·rong, XU Jin·ping, LIU Yuan.jie, WU Zhenghui and Study on Expression and Location of Hepatitis C Virus NS5A Protein in Insect Cells[J]. Virologica Sinica, 2002, 17(1): 60-65.
Citation: FENG Lei.YE IAn-bai“, RUAN Hua, GAO Jin·rong, XU Jin·ping, LIU Yuan.jie, WU Zhenghui, . Study on Expression and Location of Hepatitis C Virus NS5A Protein in Insect Cells .VIROLOGICA SINICA, 2002, 17(1) : 60-65.

丙型肝炎病毒NS5A基因在昆虫细胞中的表达及其分布研究

  • 应用PCR方法从古有丙肝病毒全部非结掏蛋白基因的质粒pBAC25中扩增出全长的NSSA基因DNA片段 (约1 34kb).PCR扩增NSSA基因片段克隆到转移载体pBlueBacHisA中。重组转移质粒pBlueBacHL~5ADNA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NSSA基因的重组病AcNSSA。对重 组病毒基因组DNA进行酶切和PCR鉴定,证宴HCV NSSA基因已插入重组病毒基因组中。AcNSSA感染SF.9细 胞后.在细胞中表达出一条64kD的蛋白,用Westert1.hint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NSSA基因已在细胞中得到表选、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,NSSA蛋白在AcNSSA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瞳和细胞核内.在72h则完全布满整个细胞.我f『]认为NSSA蛋白定位 于质膜和细晦核中.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 控。

Study on Expression and Location of Hepatitis C Virus NS5A Protein in Insect Cells

  • Full—length NS5A gene of Hepatitis C Virus was amplified by PCR.using plasmid pBAC25 containing HCV nonstructural protein genes as template.The amplified fragment(about 1.34kb)was cloned into transfer plasmid pBuleBacHisA.The recombinant plasmid DNA was cotransfected with genome DNA of wild type AcNPV to SF-9 il~ect cells.The recombinant baculovirus AcNS5A was ob— rained by plaque selection.Restriction analysis and PCR identification indicated that the NS5A gene was integrated into the genome of AcNSSA.and HCV NSSA gene was expre~sad as a 64kD protein in SF·9 cells infected with AcNS5A.This protein can he recognized by anti.NS5A antib0dy when idend— fied bv Western—blot.The Immune fluorescence assay and immune histo-chemistry assay were used tO study the distribution of NS5A protein inside the cel1.Distribution of NS5A protein are changed with the time in the recombinant virus infected SF一9 cells.Ns5A protein are changed with the time in the recombinant virus infected SF-9 cells.NS5A protein iS 1ocated at the cel1 membrance at 24 hours post infection and located at both the membrance and the nucleus after 48 hours.and ful1 of the cells after 72 hours.The result suggests that NSSA protein Was located on plasma membrance and in nucleus.it may play an important role in RNA replication and in controlling the expression of the cell gene~during the replication of the virus.

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    Study on Expression and Location of Hepatitis C Virus NS5A Protein in Insect Cells

    • 1. Institute of Virology,Wuhan University.Hubei,Wuhan 430072,China

    Abstract: Full—length NS5A gene of Hepatitis C Virus was amplified by PCR.using plasmid pBAC25 containing HCV nonstructural protein genes as template.The amplified fragment(about 1.34kb)was cloned into transfer plasmid pBuleBacHisA.The recombinant plasmid DNA was cotransfected with genome DNA of wild type AcNPV to SF-9 il~ect cells.The recombinant baculovirus AcNS5A was ob— rained by plaque selection.Restriction analysis and PCR identification indicated that the NS5A gene was integrated into the genome of AcNSSA.and HCV NSSA gene was expre~sad as a 64kD protein in SF·9 cells infected with AcNS5A.This protein can he recognized by anti.NS5A antib0dy when idend— fied bv Western—blot.The Immune fluorescence assay and immune histo-chemistry assay were used tO study the distribution of NS5A protein inside the cel1.Distribution of NS5A protein are changed with the time in the recombinant virus infected SF一9 cells.Ns5A protein are changed with the time in the recombinant virus infected SF-9 cells.NS5A protein iS 1ocated at the cel1 membrance at 24 hours post infection and located at both the membrance and the nucleus after 48 hours.and ful1 of the cells after 72 hours.The result suggests that NSSA protein Was located on plasma membrance and in nucleus.it may play an important role in RNA replication and in controlling the expression of the cell gene~during the replication of the virus.

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