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.

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

  • Available online: 15 February 2002
  • 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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