| Hantavirus(HV) is the pathogen of two dangerous viral infectious diseases: Hemorrhagic Fever with Renal Syndrome(HFRS) and Hantavirus Pulmonary Syndrome(HPS). HFRS is a group of clinically similar illnesses which characterized by pyrexia, haemorrhage, and renal lesion. It is found throughout the world, but ninety percent of the total cases worldwide happened in China. HFRS has a broad scope of prevalence with high fatality rate, thus it is a significant public health problem in China. Even now, we are still unable to control HFRS and we have no effective therapeutical drugs to cure it.Hantaviruses belong to the Bunyaviridae family of viruses. They are enveloped viruses with a tripartite, single-stranded negative-sense RNA genome designated S(small), M(medium), and L(large) segments. The S, M, and L genomic segments encode the nucleoprotein(NP), a polyprotein that is cotranslationally cleaved to yield the envelope glycoproteins G1 and G2, and the RNA dependent RNA polymerase(RDRP), respectively. Many researches indicated that both the GP and NP are related to stimulating host body producing immunity activities. The GP protein plays an important role in HV pathogenicity, because the determinants of HV for neutralization, receptor-binding, membrane fusion are all located in it, So the GP is considered as a protective antigen of HV. It could stimulate neutralization antibodies and protect infected animal and human bodies when they are infected by HV. However the GP has some disadvantages, such as its immunogenicity is weak, the anti-GP specific antibodies appear late and the titer is low. What's more, the expression level of the GP in cells is also very low. NP is the other significant structural protein of HV. It was testified that the NP is the most abundant viral component in both virions and infected cells, and it has the strongest immunogenicity among the structural proteins of HV. The anti-NP specific antibodies come forth early and could last for a long time with high titers. According to many researches recently, NP had determinants of B cells and T cells, it mainly induced the cellular immune response against HV infection. A series of research findings in our laboratory showed that a minority of neutralized-antigenic determinants of HV did exist in the NP, in addition, the main antigenic sites located in the N-terminal part of NP which was encoded by 0.7Kb fragment of S gene.In this research, we used HTNV 76-118 strain. We constructed an eukaryotic expression vector containing chimeric gene G1S0.7 of Hantavirus, in order that the fused protein expressed could make up for each other's deficiencies. Fragment G1S0.7 was obtained by cutting from recombinant plasmid pShuttle-G1S0.7 which was constructed formerly in the prophase work in our laboratory and confirmed by sequencing. Then insert the fragment into the expression vector pVAX to construct the recombinant plasmid pVAX-G1S0.7. After transfecting HEK293 cells by lipofectine-mediated method, ELISA and Western-blot were used to identify the expressed product. The eukaryotic expression vector was proved to be successfully constructed by restriction enzyme analysis. According to Western-blot results and ELISA results, the recombinant plasmid expressed in HEK 293 cells transiently and produced protein about 90KD, corresponding to the estimated molecular size, the protein could bind specifically to the hantavirus nucleoprotein specific mAb.On the basis of the prophase work, the recombinant eukaryotic expression vector was used to vaccinate the abdomens of C57 mice mediated by gene gun. ELISA and ELISpot methods were used to determine the humoral immune response and the cellular immune response of immuned mice respectively. ELISA results showed that the recombinant plasmid could induce specific antibodies against the GP of HV, but the anti-GP specific antibody titer was low. The quantity of IFN-γ-producing cells in spleencytes of immuned mice was detected by enzyme-linked immunosorbent spot (ELISpot) assay. It was demonstrated that the spot forming cells (SFC) per 106 cells of mice immunized with pVAX-G1S0.7 were significantly higher than those of control group (P<0.05). This result showed the recombinant plasmid could induce the specific cellular immune response.It is suggested that the successfully constructed recombinant plasmid pVAX-G1S0.7 could induce immuned mice producing specific humoral immunity and cellular immunity with gene gun inoculation, but the specific antibody titer is low. On the other hand, the gene gun technology hasn't showed its superiority in this experiment. So next, we have to optimize the using conditions of genegun, explore various strategies to enhance the immune effect of genetic immunization, including the selection of different plasmid vectors, using various adjuvants and different immune dosage and so on, in order to lay the basis for producing efficient and practical genetic vaccine for Hantavirus in the near future. |
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