Expression And Function Analysis Of Dengue Virus Type 1 To 4 Envelope Domain Iii Recombinant Fusion Protein

Objective To observe the ability of dengue virus type 1-4 envelope domainâ…¢fusion protein to inhibit virus infection and analyze the neutralizing ability of polyclonal antibodies against rEâ…¢;To observe the expression of fusion protein in cells and supernatant of cells; To discuss the effects that expression systems bring to the rEIII function.Methodsâ‘ After being connected by linker peptide, Eâ…¢protein of Dengue virus serotypes 1-4 were expressed in E coli BL2l(DE3) then purified. Fusion proteins were verified by Western Blot and ELISA. Rabbits were immunized with fusion proteins to produce anti-rEâ…¢serum. The activity of anti-rEâ…¢serum were detected through indirect immunofluorescence assay test. Inhibition of dengue virus type 1 to 4 infection in BHK-21 cel1s by rEâ…¢fusion protein were tested. Neutralizing activity of anti-rEâ…¢serum was analyzed.â‘¡Fusion rEIII gene was cloned into pAcSecG2T and was transfected into sf9 cells,strengthen virulence through subculture. Indirect immunofluorescence assay showed the Fusion rEIII proteins are expressed high intracellular level after 5 generations.Collect the cells and supernatant respectively.To detect the expression of fusion protein in cells and supernatant of cells.Results Dengue virus type 1 to 4 envelope domainâ…¢recombinant fusion protein was expressed in Ecoli BL2l and purified successfully. Then rEâ…¢fusion protein and anti-rEâ…¢serum were analyzed respectively and rEâ…¢fusion protein can effectively inhibit dengue virus type 1 to 4 from infecting BHK cells. The anti-rEâ…¢serums can neutralize dengue virus type 1 to 4 but with different neutralizing titer. At the same time, rEIII fusion protein was also expressed and purified in Baculovirus-insect cell expression system. These laid the foundation for dengue epitope screening and vaccine reseaech. Conclusion Dengue virus type 1-4 envelope domainâ…¢fusion protein can directly inhibit DV infection. Antibodies induced by rEâ…¢fusion proteins can neutralize dengue virus type 1-4. Fusion rEIII proteins are expressed high intracellular level but there is no target protein secreted into the supernatant of cells.