Construction, Expression And Biologic Activity Analysis Of Expressional Vectors Of Envelope Glycoprotein Genes Of Hantavirus

Objective To construct the expressional vector of glycoprotein G1 and G2 of Hantavirus,GM04-38 strain and express them in Vero E6 cells,discover the characteristics of its expression and to observe the cell fusion under acidic conditions, to facilitate study on the relationship between gene and protein,and to lay foundation for further study on the development of diagnostic reagent and subunit vaccine. Construct N-linked glycosylation site mutants and it is of great significance for study the role of N-linked glycosylation on GPs of HTNV in cell fusion.Methods Primers were designed according to the conserved sequence of M,S segment cDNA of GenBank SEO HV.The whole sequence of G1 and G2 gene was amplified with PCR,and corresponding restriction enzymes(RE)sites were created in both ends of the sequence.The gene of G1,G2 and pCAGGS/MCS were digested with KpnⅠand XhoⅠrespectively,ligated and transformed into E.coli DH5α.After being screened by ampicillin resistant,Hantavirus GM04-38 strain G1 and G2 gene were cloned into plasmid pCAGGS/MCS,respectively.After being confirmed by enzyme digestion and sequence analysis,the pCAGGS-G1 and pCAGGS-G2 were co-transfected into Vero E6 cells.After treated with acidic MEM,the cells were fixed and stained by Giemsa and observed under microscope.IFA was performed to observe expression efficiencies.Site-directed mutagenesis was used to construct five GP gene mutants which were designed N134A,N235A,N347A,N399A and N928A according to the position of the substitution.Results 1.The eukaryotic expression vectors of glycoprotein G1 and G2 were constructed.They were digested with KpnⅠand XhoⅠ.The target fragments were 2.0 kb and 1.5 kb,the vector fragment was 4.7 kb.The results were confirmed by sequence analysis.2.IIFA showed the G1 and G2 gene were expressed efficiently,and the fluorescent signal was robust and concentrated in the perinuclear region of the transfected cells.3.Giemsa staining showed cell-cell fusion could be induced under the co-expression of G1 and G2 at acidic conditions.The expression of NP gene could not cause cell-cell fusion,The co-expression of NP gene also could not enhance the fusion.4.Five N-linked glycosylation site mutants were constructed and sequencing pictures showed the asparagine(N)residues at the N-linked glycosylation sites on G1 and G2 were replaced with alanine(A).Conclusion The expressional vectors of glycoprotein G1 and G2 were constructed and expressed successfully in Veto E6 cells,the co-expression of G1 and G2 can induce effective biological function,and cell-cell fusion in Vero E6 cells under acidic conditions was obvious.The results above lay foundation for the study on the structure and function of glycoproteins,and for developing the subunit vaccine.N-linked glycosylation site mutants were got successfully and provide requirement for study the role of N-linked glycosylation on GPs of HTNV in cell fusion.