Cloning HCV Full Genome From The Patient's Serum In Hubei Province

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 also 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. We use the RT-PCR to get HCV full genome in several fragments. By comparing with other HCV genome we find that the cloned genome is belonged to the HCV subtype 1b and with sequencing and analysing the UTR, the HCV genotype 1 is the empidemic in Wuhan. We also ues the Bac-to-Bac system to express the HCV structural protein E1 in insect cells and by the confocal microscope we find that the location of expressed E1. This thesis includes four chapters. In chapter one, a brief introduction of discovery of HCV, the molecular characteristics of this virus, the genotype of the HCV, the diagnose method to test the genotype and the therapy way to patient infected with HCV. We also introduce the application of baculovirus to express the gene. Chapter two reported by RT-PCR, we get the HCV full genome by fragments from the patient who infected with the HCV in Hubei province. After analyzing the sequece, we find the cloned genome belong to the HCV subtype 1b and most related to the HCV-Shanghai. Cmopared with HCV stain, we find that there is a 363 nt deletion in the genome but another 285 nt insertion. This extraneous gene includes the 153 nt of NS5b's the and 132 nt of the 3'UTR. By analyzing, we think that the function replacing sequence is similar with the original HCV sequence, but it needs the exprement to test. In chapter three, we presented using the sequencing the specific region of the HCV genome to analysis the HCV genotype. By sequencing the region in the 5'UTR and the core, the sequence was presented to the genotype software offered by the NCBI. And after testing 9 positive samples, the genotype 1 is the epidemic genotype in the Wuhan, Hubei Province. Chapter four showed that constructing the recombinant baculoviruses containing HCV structural proteins E1 coding sequence (VAcHCVE1). The HCV structural protein E1 was successfully expressed in the insect cells. And using the E1 expressed in the E.coli, the antibody to E1 was obstained. So after neutralizing the antibody, we used the anti-E1 antibody to detect the location of the expressed E1 by confocal microscope.