Expression And Immunological Characterization Of The Chimeric G1S0.7 And G2S0.7 Gene Of Hantaan Virus 76-118 Strain

Hemorrhagic Fever with Renal Syndrome (HFRS) is an acute infectious disease characterized by fever, hemorrhage and nephritis which is caused by Hantavirus. In China about 50,000 -100,000 people per year were infected by Hantavirus with the severe symptoms and high mortality. There are still no special therapeutical drugs and prophylactic vaccines directed to HFRS, and the etiologic mechanism of HFRS is not clearly elucidated until now.Hantavirus includes several serologically distinct viruses. Serologic and genetic study suggested that the most cases of HFRS in China were caused by hantaan (HTNV) or seoul (SEOV) serotype of Hantavirus. The Hantavirus is a single-stranded negative-sense RNA virus genus. The genome of the virus is composed of L, M and S segment which encoded RNA dependent RNA polymerase (RDRP), envelope glycoprotein G1 and G2, and nucleoprotein (NP) respectively. It is indicated that the glycoprotein (GP), which was encoded by M segment, couldstimulate organism to produce neutralization antibody and could protect infected animal and human body from HTNV lethal infection. But the immunogenicity of GP is weak. The antibody stimulated by GP is produced later and the liter is low. Whereas the NP, another structural protein, has the strongest immunogenicity and can stimulate an early, high-titer and long-lasting antibody response, and plays an important role in inducing cellular immunity activities against HTNV infection in vivo.Therefore GP and NP may play equal important role in stimulating the humoral or cellular immunity. How to bring each advantages into full play in inducing immune response in vivo is a problem waiting to be solved in HFRS genetic engineering vaccine.In this research, Gl, G2 fragment of M segment and 0.7Kb fragment of S segment 5" terminal of the HTNV 76-118 strain were spliced respectively, and then their fusion expression and immunological characterization were tested using baculovirus and adenovirus expression system respectively.1. The recombinant donor plasmid pFBHTa-G20.7 was constructed firstly. Then the recombinant Bacmid DNA and baculovirus Bac-G2S0.7 were harvested after transforming DHIOBac and transfecting sf9. PCR, IFA, HLISA and western-blot were used to identify the characterization of Bac-G2S0.7 in vitro. The results suggested that the fusion protein was successfully expressed in the sf9 cells.2. The PCR generated HTNV G1S0.7 and G2S0.7 chimeric gene were cloned to pShuttle vector, and connected with Adeno-X DNA after I-CeuI and Pl-Scel digestion, respectively. The recombinant Adeno-G1S0.7 and Adeno-G2S0.7 DNA were harvested by elctrotransformation of E.coli JM109 and identified by PCR. The original recombinant adenoviruses were then harvested by transfection of the package cells HEK. 293 using the Pad digested linear adenovius DNA. EL1SA, IFA and western-blot analysis results suggested that the recombinant adenovirus can infect HEK.293 and Vero-l? cells and express chimeric genes in the cells respectively, and the expressed fusion protein keep its biological activity in vitro.3. The freeze-thawing proteins of sf9 infected by recombinant Bac-GlS0.7 or Bac-G2S0.7 baculovirus, and the recombinant Adeno-GlS0.7 or Adeno-G2S0.7 adenovirus were used to immune the Balb/c mice, respectively. The immune response was tested by IFA, ELISA, microcell-culture neutralizing experiment and T lymphocyte proliferation test (MTT assay). The results showed: (1) the fusion protein of sf9 cells infected by Bac-GlS0.7 or Bac-G2S0.7 can stimulate high-title combining antibodies against NP (>1:3200) and GP (1:200) respectively detected by ELISA, which suggested both GP and NP immune response were activated. The low-titer neutralization antibody reaction (1:20) and clear lymphocyte proliferation were detected simultaneity. (2) the low-title combining antibodies against NP (1:160/1:320) and GP(1:20/1:40) were evoked by administration of recombinant Adeno-GlS0.7 or Adeno-G2S0.7 adenovirus respectively, and similar results of neutralization antibody activity (1:5-1:10) a...