Serine protease inhibitor 3 (Serpin3) from Penaeus vannamei selectively interacts with Vibrio parahaemolyticus PirA^vp

Acute Hepatopancreatic Necrosis Disease (AHPND) represents a significant challenge in the field of shrimp aquaculture. This disease is primarily caused by Vibrio parahaemolyticus strains harbouring the pVA1 plasmid encoding the PirA(vp )and PirB(vp) toxins. To combat this epidemic and mitigate its devastating consequences, it is crucial to identify and characterize the receptors responsible for the binding of these pathogenic toxins. Our studied discovered that Penaeus vannamei's Serine protease inhibitor 3 (PvSerpin3) derived from shrimp hepatopancreatic tissues could bind to recombinant PirA(vp), confirming its role as a novel PirA(vp)-binding protein (PABP). Through comprehensive computational methods, we revealed two truncated PirAvp-binding proteins derived from PvSerpin3 called Serpin3(13) and Serpin3(22), which had higher affinity to PirA(vp) than the full-length PvSerpin3. The PABP genes were amplified from a cDNA library that was reversed from total RNA extracted from shrimp, cloned and expressed in Escherichia coli. Three PABP inclusion bodies were refolded to obtain the soluble form, and the recovery efficacy was found to be 100% for Serpin3 and Serpin3(13), while Serpin3(22) had a recovery efficacy of roundly 50%. Co-Immunoprecipitation (co-IP) and dot blot assays substantiated the interaction of these recombinant PABPs with both recombinant PirA(vp) and VPAHPND (XN89)-producing natural toxins.