Fused Expression Of Hantavirus Glycoprotein G1 And Partial NP In Insect Cells And Study On Its Immunological Properties

Hantavirus mainly causes hemorrhagic fever with renal syndrome (HFRS). The immunological research on its structural proteins showed that both nucleocapsid protein(NP) and glycoprotein(GP) involved in the protective immunity. Among them, GP can evoke neutralizing antibody, but has a relative low immunogenicity. NP has a high immunogenicity, and its ability to generate protective immunity is considered relating to cell-mediated one. Our previous research showed that the antigenic sites on NP were mainly on the fragment encoded by the 700bp segment of S gene 5' terminal. The further study on the relationship between the two kinds of structural proteins and colligating their strongpoints is the urgent problem which need to be solved in the fundamental research on the HFRS genetic engineering vaccine.By connecting Gl segment of M gene and 700bp segment of S gene 5' terminal of hantavirus 76-118 strain, the prokaryotic fusion expression vectors PGEX-4T-2-G1S0.7 and pGEX-4T-2-S0.7Gl were constructed firstly. The expression of fusion proteins was induced in E.coli. It was proved that both expression products could bind to hantavirus NP specific mAb, but only the weak binding activity of the product of G1S0.7 with glycoprotein Gl specific mAb could be detected. Western blot showed the degradation of the product of G1S0.7 was much less than the one of S0.7G1.The recombinant baculoviruses carrying SO.7, Gl, chimeric gene G1S0.7 or S0.7G1 were constructed furtherly. By IFA, ELISA and Western blot experiments, it was proved all the recombinant baculoviruses containing SO.7 segment could express biologically active proteins in insect cells. The results showed that fusion protein G1S0.7 could be recognized by both the hantavirus NP and glycoprotein Gl specific mAbs, and its molecular weight was consistent with the predicted full one on Western blot; while the product of S0.7G1 could only be recognized by hantavirus NP specific mAb, and the anticipated integrity one was not obtained. The animal experiment proved that the recombinant baculovirus containing Gl segment of M gene could express active glycoprotein too.By immunizing the mice with the expression product of S0.7, Gl or G1S0.7 in insect cells and detecting the immunization efficacy through IFA, ELISA, microcell-culture neutralizing experiment and T lymphocyte proliferation test, it was proved that the specific humoral and cellular immune responses to hantavirus could be detected in all the experimental groups. Respectively, the antibody litre of G1S0.7 test group was the highest one; mice post-immunizationby G1S0.7 could arouse antibodies specific to both hantavirus NP and GP; the neutralizing activity was detected in the sera of some mice in each group; the lymphocyte proliferation test results showed that, the proliferation indexes of G1S0.7 test group to NP and GP were higher than the negative control groups, and somewhat higher than Gl and SO.7 groups.The research results proved that, the recombinant baculovirus carrying the chimeric gene including Gl segment of M gene and 700bp segment of S gene 5' terminal of hantavirus 76-118 strain could express the full biologically active fusion protein G1S0.7 in insect cells. The expression product possessed the characteristics of the proteins coded lay the two gene segments. It could stimulate not only humoral but also cellular immune response in mice, and the immunity efficacy was better than Gl or SO.7 only. It has provided important theoretical and experimental evidences for choosing the proper immunogen ingredient for research and development of HFRS genetic engineering vaccine.