Dab solution

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.

The technique of immunohistochemical staining was used to determine the localization of the StAR3 protein. Intestine and adductor tissues were dissected from golden and brown scallops and washed by PBS buffer. These samples were fixed in 10 % formalin, dehydrated, cleared, embedded in paraffin and cut to sections of 3 μm thickness before staining with hematoxylin (Boster, Wuhan, China). After 10 min treatment with 3 % H₂O₂ solution, tissue sections were washed in PBS and blocked with 5 % BSA for 30 min in a humidified box at room temperature, and then incubated with anti-rStAR3 rabbit antibody (diluted 1: 250) overnight at 4 °C. Then, the sections were washed with PBS and incubated with the horseradish peroxidase-labeled anti-rabbit IgG goat antibody (diluted 1: 500) (Boster, Wuhan, China) at room temperature for 30 min. After washing three times in PBS, the sections were developed with DAB solution (CWBIO, Beijing, China) for 10 min and counterstained in hematoxylin for 3 min. Photomicrographs were taken (EVOS Cell Imaging Systems).