Dissertation > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Human Virology ( pathogenic virus)

Construction of Co-expression Plasmids of SARS-CoV’s Structural Protein and Evaluation of Their Immunogenicity

Author BaiRu
Tutor ZhouZuo;WangXiLiang
School Ningxia Medical
Course Pathogen Biology
Keywords SARS co-expression plasmid immune response
CLC R373
Type Master's thesis
Year 2005
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Objective To construct three plasmids co-expression S1 protein and N protein of SARS-CoV、S1 protein and mGM-CSF、N protein and mGM-CSF and assay specific immune responses induced by these co-expression plasmids. This study will further promote DNA vaccine investigations of SARS-CoV in the nation.Methods We selected S1 and N protein of SARS-CoV as target gene, mouse gramulocyte macrophage colony stimulating factor(mGM-CSF) as adjuvant, constructed three co-expression plasmids using technology of molecular biology. S1 gene and N gene were amplified from SARS-CoV by RT-PCR, mGM-CSF gene was amplified by PCR. The three gene fragments were cloned or subcloned into the eukaryotic expression vector and called pVAX1/S1-N, pVAX1/S1-mGM-CSF, and pVAX1/N-mGM-CSF. COS-7 cells were transfected with these three plasmids and the corresponding fuse proteins were det al.cted by western blot and immunocytochemistry. BALB/c mice immunized with pVAX1/S1-N+mGM-CSF, pVAX1/S1-mGM-CSF and pVAX1/N-mGM-CSF. SARS-specfic IgG antibody and content of IgG antibody subclass were det al.cted by ELISA, the quantities of CD4+ and CD8+ T cells and the ratio of CD4+/CD8+ T cells assaied by Flow cytometry (FCM), the level of IFN-γand IL-2 in splenocytes det al.cted by ELISPOT.Results DNA sequences analysis and double restriction enzyme digestion confirmed three co-expression plasmids successfully constructed. The three fusion proteins were det al.cted in COS-7 cells. Anti-SARS antibody in mice immuned with these three plasmids were det al.ctable 15 days after the first injection and enhanced steadily with the increase of

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