Mouse anti his tag mab

To verify the recombinant gD protein expressed in E. coli Rosetta (DE3) cells, western blot analysis was performed using the following procedures (29 (link)). Three samples of the purified gD protein were subjected to 12% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) using a wet transfer apparatus (Bio-Rad). After blocking with PBS containing 0.05% tween-20 (PBST) and 5% skimmed milk for two hours, one of the PVDF membranes was added to the mouse anti-His-tag mAb (Cwbio, Beijing, China) at a dilution ratio of 1:5000 in 5% skimmed milk solution at 37°C for two hours. After washing with PBST, goat anti-mouse IgG conjugated horseradish peroxidase (Santa Cruz Biotechnology, CA) as secondary antibody, was added at a dilution ratio of 1:3000 and incubated at 37°C for one hour, and then incubated with DAB solution (Cwbio, Beijing, China) for 5 - 10 minutes. The second PVDF membrane was added to monkey B virus serum at a dilution ratio of 1: 500 in 5% skimmed milk solution and the third one was added to monkey negative serum at the same dilution ratio, both of them used the goat anti-rhesus IgG (H + L) HRP (Southern Biotech, USA) as a secondary antibody, this procedure was followed by eECL western blot analysis (Cwbio, Beijing, China). The cell culture of pET-32a-gD-Rosetta (DE3) without IPTG induction was used as the negative control.