| Baculovirus expression system is one of the widely used eukaryotic expression system for heterologous protein expression in insect cells,in which,the baculovirus were served as a foreign gene carrier and the insect cells were utilized as the virus factory.Baculovirus expression systems suitable for high yields of eukaryotic proteins production,with similar biological activity,post-translational modification,structure and immunological activity to natural proteins.To maximize the expression of foreign proteins in the baculovirus expression system,it is necessary to optimize the parameters such as cell seeding density and the amount of virus seeded,while the multiplicity of infection(MOI)is a key parameter.MOI optimization rely on the accurate virus titers.Therefore,it is urgent to establish a stable method for baculovirus titer determination.GP64 protein is the envelope glycoprotein of baculovirus and plays an important role in the process of cell infection.In this study,the GP64 protein expressed by baculovirus was used as the target protein,and the titer of recombinant baculovirus was determined by immunostaining using GP64 monoclonal antibody(M Ab).Three parts of the study were shown as follows:1.Expression and purification of baculovirus GP64 proteinThe recombinant plasmid of GP64 was transfected into Chinese hamster ovary(CHO)cells.After screenings with two drugs,the highest expression cell pool was identified by western blot.The cell culture supernatant was harvested after the cell pool was fed and cultured,and the protein was purified by His Trap FF affinity chromatography column.Eight cell pools expressing GP64 protein were screened,and the cell pool 1B1-2 with high expression level was selected for production and purification of GP64 protein.2.Preparation and identification of GP64 monoclonal antibodyGP64 protein and wild baculovirus were used as immunogens for mice immunization.After fusion of spleen cells and myeloma cells,three MAbs,5B1,2A12 and 5D1,were screened by indirect enzyme linked immunosorbent assay.The results of Mab isotype assay showed that heavy chain of 5B1,2A12 and 5D1 were IgG2b,IgG1 and IgG1,respectively,and all Mabs showed the light chain type of κ.The purified MAbs were identified by indirect immunofluorescence assay and Western Blot.All three MAbs showed the specificity to recognize the baculovirus and GP64 protein.3.Establishment and application of the method for titer determination of recombinant baculovirusAs a primary antibody,MAb GP64 was used to optimize the working conditions of goat serum,MAb GP64,HRP goat anti-mouse secondary antibody.The results of titers determination of 5 different recombinant baculoviruses tested by established method were basically consistent with the results of the commercial kits,which showed that the assay is fast,convenient,and stable,and can be used for titer determination of recombinant baculovirus.Collectively,eight cell pools of GP64 protein-expressing were obtained by the CHO cell expression system,and the cell pool 1B1-2 with high-expression level was used for GP64 protein production and purfication.GP64 protein and wild-type baculovirus were used to immunize mice,and three MAbs of GP64 were screened by indirect ELISA.GP64 is used as the primary antibody for optimizing of goat serum,GP64,HRP goat anti-mouse secondary antibody and other parameters.The titers of were measured at the same time.The results of titers determination of 5 recombinant viruses were basically consistent with the commercial kits.The results showed that a stable method for titer determination of recombinant baculovirus has been established,which provides a basis to evaluate the parameters of foreign proteins expression by recombinant baculovirus. |
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