Expression Of SARS-CoV Nucleocapsid Protein And The Research About The Immunogenicity Of SARS-CoV Vaccine

Since there is no obvious effect for clinical therapy of SARS,vaccine as an effective preventive measure has been used. As the development of immunology and molecular biology, especially the development of technology of gene recombination, DNA vaccine had been invented and showed powerful advantage. Furthermore, expression and preparation recombinant antigen protein of SARS-CoV is a safe and quick method for diagnose disease and inducement antibody. The nucleocapsid protein is one of major antigens in the SARS-CoV.Compared with other viral structural such as spike protein and membrane protein, this protein is believed to be the most conservative and abundant protein. This study used DNA vaccine and antigen protein to immunized animal and researched the immune response of the animal. We obtained the gene sequence from GeneBank and synthesized the gene encoding nucleocapsid protein. In this gene(1296bp), some low-uasage codon were replaced by synonymous ones in order to increased the expressive opportunity in E.coli and Pichia pastoris. After recombined with the CTL epitope gene(195bp), the gene was cloned into secreted expression vectors pET-28a(+),pPIC9K,pIRES to pET-28a-N,pPIC9K-N,pIRES-N. CTL gene could stimulate the cellular immune response and promote the generation of antibody. pET-28a-N was transformed into E.coli BL21, and then induced by IPTG to translate into N protein. pPIC9K-N was transformed into the Pichia pastoris GS115, and　then　the　positive　recombinant　GS115/pPIC9K-N　was　screened　by　MM　and　MD　plates　and　identified　by　PCR　further.　Nucleocapsid　protein　was　highly　expressed.　We　optimized　the　expression　condition　in　P.pastoris.　The　recombinant　vector　pIRES-N　as　an　DNA　vaccine　was　transfected　into　BHK-21　cells.　After　identified　by　Indirect　immunofluorescence　assay(IFA),　the　DNA　vaccine　was　translated　into　N　protein.　We　injected　the　DNA　vaccine　into　Balb/c　mice　and　collected　the　mice　antisera,　antibody　was　found　in　sera.　Spleen　lymphocytes　in　mice　were　analyzed　by　flow　cytometry(FCM)　to　assess　the　phenotypes　of　cell　population.　The　CD4+　T　cells　and　CD8+　T　cells　had　greatly　increased.　These　results　showed　that　the　DNA　vaccine　could...