Expression Of HCV Structural Proteins In Insect Cells And Mammalian Cells And Establishment Of SMMC-7721 Cell Clones Supporting Stable Expression Of HCV Proteins

Hepatitis C virus (HCV) is a major etiologic agent of posttransfusion and sporadic community-acquired non-A, non-B hepatitis. No efficient and reliable culture system is available to amplify the virus, preventing the research development of the virus and therapy of diseases caused by the virus. Until now, not only the research on the culture system to amplify the virus is in progress, but molecular techniques have been used to make basic research of the virus. In the condition of rapid development of molecular techniques study, the study level of HCV has also been improved greatly. The study on the structural proteins is a very useful tool to discover not only the structure of the viral particles and the assembly process of the virus but also the mechanisms of early stages of virus infection. In addition, it provides novel opportunities for the development of new protective and therapeutical vaccines. In this work, we successfully expressed HCV structural proteins in insect cells by using recombinant baculovirus expression system and detected the transcription of E1 coding sequence in HeLa cells transduced by recombinant baculoviruses. EM results showed that in insect cells HCV structural proteins assembled into spherical particles 50nm in diameter in large cytoplasmic vacuoles. In addition, by transient transfection, we obtained SMMC-7721 cell clones supporting stably expression of HCV proteins.This thesis includes three chapters.In chapter one, a brief introduction of discovery of HCV, the molecular characteristics of this virus and the study progress of structural proteins of HCV and HCV-like particles were presented. The extensive application of recombinant baculoviruses in expressing interested proteins was also summarized. In addition, we described the study progress of establishing efficient HCV culture system.Chapter two reported the constructing of two recombinant baculoviruses containing HCV structural proteins coding sequence (vAcHCVsp1 and vAcHCVsp2). By using vAcHCVsp1, HCV structural proteins were successfully expressed in insect cells and the transcription of E1 coding sequence was also detected in HeLa cells transduced by the recombinant baculovirus vAcHCVsp2. In addition, RT-PCR results showed that the transcription of E1 coding sequence began at 16 h.p.i and western-blot results showed that protein expression began at 48 h.p.i and reached a maximal level at 72 h.p.i.. EM results showed that in insect cells HCV structural proteins assembled into spherical particles 50nm in diameter in large cytoplasmic vacuoles.In chapter three, we presented the process of obtaining SMMC-7721 cell clones which support stable HCV protein expression. RT-PCR results using reverse transcription products of total mRNA extracted from transfected cells as templates showed that HCV RNA can be transcribed in the cell clones; Western-blot assay using anti-Core antibody as primary antibody indicated that HCV proteins can be expressed in the cell clones. In addition, Immuno-fluorescence lable of the cell clones using anti-Core antibody also showed the structural proteins can be expressed in these cells.