| The research on Hepatitis C vaccine focuses on two aspects presently. One is about the basic research, such as screening the HCV polypeptide for CTL epitope motif and humoral immune epitope motif, seeking for conservative sequences for protective effects. The other is establishing animal models for protection experiments against HCV infection. Recently, great progresses have been made in these two ways, which makes us more confident that we can develop a HCV vaccine successfully. Nucleic vaccine is a new kind of vaccine, which is constructed by inserting the encoding target gene into a eukaryotic expression vector. When it is injected into the body directly, the muscle cells will intake the recombinant plasmid and express the target antigen, which induces the immune system produce immune responses. The effect of this vaccine is excellent, several models of DNA-based immunization against specific disease have been reported, including influenza virus, HIV and HBV etc. We constructed a recombinant plasmid by inserting the highly conserved HCV-core gene fragment into the eukaryotic expression vector, pEF, under the control of an EF-l a promoter, and then it was injected into the muscles of BALB/c mice with different treatments and by different times. The purpose of this experiment is to seek an optimum method of animal immunization with HCV gene and observe whether the DNA vaccine can induce the HCV specific antibodies and the CTL immune response. Methods: 1. Recombinant pEF-HCV core plasmid was constructed by inserting HCV core gene, amplified by PCR from plasmid PQEHCVc 191, into SaL I and BamH I sites of the eukaryotic expression vector, pEF. Whether the recombinant plasmid was constructed successfully was proved by PCR and enzyme digest methods. 2.Transfecting the recombinant plasmid into SP2/O cells (H~2d type) in vitro with liposome mediated method. The stable and transient expression of HCV C gene in transfected cells were detected by blot hybridization and immuno- histochemical staining from mRNA and protein level respectively. 3. DNA vaccine was inoculated into thigh muscles of BALB/c mice with different treatments and by different times. The optical density (OD) of HCV antibody in sera from immunized mice was assayed by ELISA. 4. For in vitro studies, CTL activity has been measured by LDH substantial assay. Two weeks after the final immunization, the immunized mice were killed and a suspension of spleen cells was prepared from each animal as target cells, while the SP2/O cells expressing HCV core antigen were aimed as effect cells. 5.Four weeks after the final immunization with recombinant plasmid pEF-HCV, SP2/O cells expressing the HCV core protein were inoculated subtaneously into the right and left flank of the mouse. The tumor size and weight as well as the emergency time of tumor, the death time, the animal survival rate, were evaluated as in vivo indexes of CTL activity, generated by immunization with the plasmid DNA constructs. Results: 1 .The recombinant plasmid was proved to construct correctly by PCR and enzyme digest methods, and it was also proved to express stably in SP2/O cells by blot hybridization and immunhistochemical staining. 2.The anti-HCV core humoral immune response to the DNA constructs was assessed by measurement of antibodies in serum of mice. Ab to HCV in the sera of immunized mice can be detected as early as...
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