Rapid Detection Of Severe Acute Respiratory Syndrome Virus From Humans And Study On DNA Vaccine And Immune Sera Preprared

The first case of severe acute respiratory syndrome (SARS) was reported in late2002 in the Guangdong Province of China. Since then, SARS has shocked the worldand caused panic as it spread around the globe,resulting in the deaths of nearly 800people. In March 2003, a virus of the Coronaviridae family, named SARS Coronavirus(SARS-CoV) was identified as a possible causative agent. The SARS-CoV genomeconsists of a single stranded, approximately 27 kb positive sense RNA. It is similar toother coronaviruses, contains five major open reading frames encoding the replicasepolyprotein, the spike (S), the envelope (E), membrane (M) glycoproteins andnucleocapsid (N) proteins, respectively.Developing a safe and effective SARS-CoV vaccine is essential to prevent the futureepidemics. Currently,inactivated virus-based vaccine and a DNA vaccine, whichagainst SARS, are being extensively studied. The inactivated vaccine has threedisadvantages: T-cell independence, major histocompatibility complex restriction,and serotype conversion caused by vaccination. These disadvantages may effect theimmunological analysis of epidemic monitoring. The DNA vaccine may be a bettercandidate for SARS-CoV since it may induce both cellular immunity and humoralimmunityIt was usually recommended that the most effective method of preventingviral disease was vaccine inoculation. However, the exploitation of a kind of vaccinegenerally needs several years or more time from the preparation to the clinicalapplication. So it was especially important that get a preventing and cure method in ashort time. Out of that the horse immunoglobulin is the most straight and effective waysince its technology was relative maturity, and plays an energetic effect on preventionand cure of infectious disease in history.In this study, we diagnosed SARS patients in the Jinlin province of China anddeveloped a RT-PCR method to detect the SARS-CoV. And we synthesized the geneencoding viral structure proteins(S, M, N, E, S1, S2) according to the gene sequencefrom GeneBank. In these gene,some low-uasage codon were replaced by synonymousones in order to increased the expression opportunity in E.coli and Pichia pastoris andprimates. After recombined with the ISS epitope gene (195bp), the gene was clonedinto expression vectors pET-28a(+), pET-CKS, pPIC9K, pIRES to produce 24recombinant plasmids. Series of pET-28a(+), pET-CKS recombinant plasmids weretransformed into E.coli BL21, and then induced by IPTG to translate structuralproteins.The results showed that only pETISS-N could beexpressed. pPIC9K seriesrecombinant plasmids were transformed into the Pichia pastoris GS115, and then thepositive recombinant GS115 were screened by MM and MD plates and identified byPCR further. The nucleocapsid proteins and S2 proteins could be highly expressed.Further more,we optimized the expression condition in P.pastoris. The pIRES seriesrecombinant plasmids as a DNA vaccine was transfected into BHK-21 cells. Afteridentified by indirect immunofluorescence assay (IFA), the DNA vaccines weretranslated into structural proteins. We injected the DNA vaccines into Balb/c mice andcollected the mice sera, and the antibodies were found in the sera. Spleen lymphocytesof the mice were analyzed by flow cytometry (FCM) to assess the phenotypes of cellpopulation. The CD4+ T cells and CD8+ T cells had different changes. These resultsshowed that the DNA vaccines could induce immune response in mice. We injected theDNA vaccine into horses and strengthened the immunity by continuing to injectantigen proteins. We could detect the antibody in horse sera. The title was 1:80 afterDNA vaccine immunization and 1:2560 after N protein immunization. We can draw aconclusion: the DNA vaccine we constructed could induce immune response in horsesand the antibody title could be enhanced by protein vaccine. The immune effectinoculated by DNA vaccine and protein vaccine was superior to the one only by DNAvaccine or protein vaccine. Finally, Anti-serum was developed by purifying IgG afterthe antibody titer reached the peak. And the purity of the Fab2 fragment reached toover 90%.