| Hepatitis C virus has infected people worldwide. 3% people of our country also afflicted this disease. About fifty to eighty percent people progress to chronic carrying state, which is closely related to cirrhosis and hepatocellular carcinoma (HCC). Today, HCV infection has been one of the most important damaging infectious diseases. There is no distinctive or highly effective prevention and therapeutic procedure. Therefore, it is important to find way to prevent infection. However, HCV genotypes variability is occur frequently. Accordingly, it is significant to finda way to prevent HCV infection by producing effective vaccine.DNA vaccine is a new method for target antigen to induce immunization. The plasmid DNA, which includes encoding external protein gene, can be transmitted directly into man or animal by DNA vaccine. The external gene can be expressed and carried, then stimulate body to produce according humoral and a cellular-mediated response. DNA vaccine also named nucleus vaccine or nude DNA vaccine. DNA vaccine can prevent all kinds of virus infection, including those blood-transmission viruses. DNA vaccine can physic chronic infected individuals and carcinoma. DNA vaccine of HCV includes encoding viral protein (core or envelope protein) gene. Host cell can ingest external DNA, then express viral gene and produce corresponding viral protein. The later can be carried to cell surface by MHOI of host cell. By stimulating CD8+ CTL of cell surface, cellular-mediated immune response can be start-up to exert antiviral effect.Hepatitis C virus is a positive-strand RNA virus.The whole length of HCV RNA is 9,600 bp, which includes a single open reading frame encoding about 3,010 aa viral pre-protein.Among HCV encoding gene, core region shows greater sequence conservation. Core gene expression has favorable antigen stability. The core protein has been shown to induce both a humoral and a cellular-mediated response in the natural infection.The past study has showed that HCV core gene could be cloned into an eukaryotic expression vector and express in SP2/0. After immunization with the HCV core DNA expression constructs-pcDNAHCV-C, the specific antibody production was induced. CTL activity primed by a HCV DNA vaccine in vivo is mediated by cells expressing CD8+ surface phenotype. Liposome technique may promoter the efficiency of immune responses of DNA vaccine in Balb/c mice.Based on this study, we can observe HCV-C antigen expression by using immunohistochemistry method and with laser confocal microscopy. Then we can investigate the effect of DNA immunization ofcore gene on infection of hepatitis C virus. The studies consisted of:1.The recombinant plasmid pcDNAHCV-C was constructed and expressed by using Lipofectamine in SP2/0 cells of mouse. The product was detected by immunofluorescent-stainning and by using HCV C monoclonal antibody. The result indicated that SP2/0 cells were shown to express specific antigen.2.SP2/0-HCV-C was transfected by the plasmid, selected by G418 and could stable express HCV-C antigen. After injection into epidermis of Balb/c mice, the subcutaneous transplanting tumor of HCV-C was formed. Therefore, we can use it as animal-model of HCV infection.3. Observing effect of plasmid DNA, including tumor forming-time, growth speed and size, life of mice. Compare with the control group, we found mice, which have received the HCV DNA vaccine, showed an inhibition of tumor growth.4. Observing HCV-C antigen expression by using immunohistochemistry method. Primary antibody isHCV-C monoclonal antibody of mice. Secondaryantibody is goat-against-mice IgG marked by horseradish peroxidase. At last, DAB display the color. Additional, we distinctively marked with T lymphocyte to see how T and B lymphocyte infiltrate. We observed that HCV-C antigen was mainly expressed in cytoplasm and membrane of Sp2/0 cell, few expressed in nucleolus. Compared with the experimental group, HCV-C antigen was expressed obviously in contr...
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