Bombyx mori serpin 3 is involved in innate immunity by interacting with serine protease 7 to regulate prophenoloxidase activation

A subfamily of conserved proteins called serpins plays crucial roles in various physiological functions, particularly in the activation pathway of the serine protease cascade, an essential component of insect innate immunity. Here, we found Bombyx mori serpin 3 (BmSerpin3) was most highly expressed in the fat body, and was up-regulated after exposure to bacteria, fungus and virus. Further, the expression of BmSerpin3 in the hemocytes, fat body, midgut of silkworm larvae, and BmN cells was up-regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Through Bac-to-Bac expression system, we obtained the active protein of BmSerpin3, and the enzyme activity assay showed that BmSerpin3 significantly inhibited the activity of both subtilisin and trypsin. In addition, BmSerpin3 could inhibit the activation of prophenoloxidase (PPO) in larvae. The knockdown of BmSerpin3 showed increased phenoloxidase (PO) activity compared to control after BmNPV infection. Ultimately, we confirmed that BmSerpin3 interacts with B. mori Serine Protease 7 (BmSP7). Hence, we hypothesize that BmSerpin3 is involved in innate immunity by interacting with BmSP7 to regulate the PPO activation cascade. Taken together, these results showed that BmSerpin3 play a role in silkworm innate immunity and lay a foundation for studying its functions.