Construction Of HV S Gene And Its Segments Eukaryotic Prokaryotic Expression Vectors And Identification Of Expressed Prodocts

Hantavirus mainly causes hamorrhagic fever with renal syndrome (HFRS), which is an acute infectious diseases characterized by fever, hemorrhage, nephritis or thrombocytopenia, and hantavirus pulmonary syndrome(HPS), which main clinical manifestation includes fever, hemorrhage, acute respiratory destress and capillary leakeage syndrome. Four of the hantavirus species, Hantaan virus(HTNV), Seoul virus(SEOV), Dobrava/Belgrade virus(DOBV), and Puumala virus(PUUV), which are all different serotypes, are known to cause HFRS,while Sin Nombre virus(SNV) causes HPS. In China, two serotypes of hantavirus,HTNV and SEOV, are found. About 40,000-100,000 people per year were infected by Hantavirus with the severe symptoms and high mortality. Since the severity of infection depends on the viral serotype, a specific diagnosis of the causative virus is important ,not only for rodent control but also for prevention and therapeutic purposes.Currently, the plaque reduction neutralization test (PRNT) is the most specific serodiagnostic procedure for differentiating between HTN and SEO infections. However, PRNT takes more than 1 week to perform and requires a containment laboratory for virus manipulation , therefore, a simple, safe, and rapid diagnostic method which is able to distinguish HTN from SEO infection serologically is required.Hantavirus nucleocapsid protein(NP) possesses immuno-dominant, linear, cross-reactive epitopes in the first 100 amino acids of the N terminus. The C terminus of NP contains the type-specific B cell epitopes, which occupies C terminus, about half of NP. So in this research, we firstly constructed the eukaryotic expression vectors pcDNA3.1-S and pcDNA3.1-S-C,which contains the 3'-terminal 825bp segment of HTNV S gene. It was proved that the eukaryotic expression vectors pcDNA3.1-S and pcDNA3.1-S-C were successfully constructed by restriction enzyme analysis and sequence analysis. Then the recombinant expression plasmids were transfected into Vero cells in vitro, respectively. The expression products were analyzed by IFA. IFA results showed the expressed proteins both NP and truncated protein of NP could be detected distinctly and peculiarly.In order to obtain sufficient and easily purified NP and its fragment, we successfully constructed the prokaryotic expression vectors pRSET A-S and pRSET A-C, which were identified by PCR and restriction enzyme analysis. Then we transformed the pRSET A-S and pRSET A-C into BL21(DE3)pLySs and the proteins were expressed at high level respectively. According to the SDS-PAGE results, the molecular weight of expressed fusion protein encoded by HTNV S gene 1287bp, 825bp is 55KD and 35KD, respectively. But on Western blot , the expressed proteins could be recognized by HTNV patient sera slightly. The further study is being carried out now.The research results suggested that, the expression product of truncated NP (155 to 429 aa) by the eukaryotic expression vector pcDNA3.1-S-C could be considered as serotyping antigen; whether the prokaryotic expression product of truncated NP (155 to 429 aa) could be biologically active antigen or not, it needs further study.