Anti gb mouse monoclonal antibody clone ch28

Example 38

HCMV glycoprotein B (gB) expression level in HEK 293 cells or on the cell surface was tested for codon-optimized gB mRNA variants (Var #1-Var #10, SEQ ID NOs: 94, 157, and 95-102, respectively, see Table 13). HEK293 cells were transiently transfected with mRNA encoding the codon-optimized gB mRNA variants using Trans IT®-mRNA Transfection Kit (Mirus Bio LLC) per the manufacturer's recommendations. At 24 hr post-transfection, cells were lysed in 1% Digitonin buffer supplemented with complete mini-EDTA free protease inhibitor cocktail tablets (ThermoFisher Scientific). Precleared lysates were resolved on Novex 4-12% Bis-Tris gels (Invitrogen) and blotted with anti-gB mouse monoclonal antibody (clone CH28, Santa Cruz Biotechnology) and mouse anti-β actin (Cell Signaling Technology). Alexa Fluor 680 goat anti-mouse IgG (ThermoFisher Scientific) was used as secondary antibody. All images were captured on a ChemiDoc MP Imaging System (Bio-Rad Laboratories).

The result shows that all of the codon-optimized variants were expressed. Compared to the wild type gB mRNA, several of the codon-optimized variants (Var #1-Var #4, Var #9, and Var #10) showed enhanced expression in HEK293 cells, among which Var #4 showed the highest expression level (FIG. 52).

Example 38

HCMV glycoprotein B (gB) expression level in HEK 293 cells or on the cell surface was tested for codon-optimized gB mRNA variants (Var #1-Var #10, SEQ ID NOs: 94, 157, and 95-102, respectively, see Table 13). HEK293 cells were transiently transfected with mRNA encoding the codon-optimized gB mRNA variants using Trans 1T®-mRNA Transfection Kit (Mirus Bio LLC) per the manufacturer's recommendations. At 24 hr post-transfection, cells were lysed in 1% Digitonin buffer supplemented with complete mini-EDTA free protease inhibitor cocktail tablets (ThermoFisher Scientific). Precleared lysates were resolved on Novex 4-12% Bis-Tris gels (Invitrogen) and blotted with anti-gB mouse monoclonal antibody (clone CH28, Santa Cruz Biotechnology) and mouse anti-β actin (Cell Signaling Technology). Alexa Fluor 680 goat anti-mouse IgG (ThermoFisher Scientific) was used as secondary antibody. All images were captured on a ChemiDoc MP Imaging System (Bio-Rad Laboratories).

The result shows that all of the codon-optimized variants were expressed. Compared to the wild type gB mRNA, several of the codon-optimized variants (Var #1-Var #4, Var #9, and Var #10) showed enhanced expression in HEK293 cells, among which Var #4 showed the highest expression level (FIG. 52).

Example 38

HCMV glycoprotein B (gB) expression level in HEK 293 cells or on the cell surface was tested for codon-optimized gB mRNA variants (Var #1-Var #10, SEQ ID NOs: 94, 157, and 95-102, respectively, see Table 13). HEK293 cells were transiently transfected with mRNA encoding the codon-optimized gB mRNA variants using Trans IT®-mRNA Transfection Kit (Mirus Bio LLC) per the manufacturer's recommendations. At 24 hr post-transfection, cells were lysed in 1% Digitonin buffer supplemented with complete mini-EDTA free protease inhibitor cocktail tablets (ThermoFisher Scientific). Precleared lysates were resolved on Novex 4-12% Bis-Tris gels (Invitrogen) and blotted with anti-gB mouse monoclonal antibody (clone CH28, Santa Cruz Biotechnology) and mouse anti-D actin (Cell Signaling Technology). Alexa Fluor 680 goat anti-mouse IgG (ThermoFisher Scientific) was used as secondary antibody. All images were captured on a ChemiDoc MP Imaging System (Bio-Rad Laboratories).

The result shows that all of the codon-optimized variants were expressed. Compared to the wild type gB mRNA, several of the codon-optimized variants (Var #1-Var #4, Var #9, and Var #10) showed enhanced expression in HEK293 cells, among which Var #4 showed the highest expression level (FIG. 52).