Preparation and characterization of monoclonal antibodies against porcine caspase-7.

Abstract

Cysteine-aspartic proteases (Caspases) play a central role in programmed cell death (PCD). Caspase-7 is an effector caspase involved in the execution phase of apoptosis and pyroptosis. Determining the expression and activation of caspase-7 is often an essential experiment in studies on PCD. Currently, commercially available antibodies specific to human and mouse caspase-7 have facilitated research on PCD in these species. A specific antibody against porcine caspase-7 (pcaspase-7) is needed, as PCD has been closely linked to porcine diseases. In this study, a recombinant pcaspase-7 protein (rpcaspase-7) with cysteine replaced by alanine at the 186 position was prokaryotically expressed and purified. This recombinant protein was used to immunize BALB/c mice to generate pcaspase-7-specific monoclonal antibodies (mAbs). The prepared 7-4E1-F4-D2-E1 mAb targets the 90-100 amino acid (aa) oligopeptide (TDKDAEALFKC) of pcaspase-7, while the mAbs 7-3F1-E2-H2-D6, 7-3F1-E2-H2-D1, 7-4E5-C11-C9-B5, and 7-4E5-C11-C9-F11 target the 45-55 aa oligopeptide (GSSVKIPRDRE). All prepared mAbs can discriminate pcaspase-7 from pcaspase-1, -3, -8, and -13. The mAbs targeting the 45-55 aa oligopeptide can also distinguish pcaspase-7 from human, hamster, and bovine caspase-7. The 7-4E1-F4-D2-E1 mAb recognizes pcaspase-7 in Western blot assays but not in immunofluorescence assays. These mAbs can be used to purify prokaryotically expressed pcaspase-7 via immunoprecipitation and to detect pcaspase-7 activation in virus-infected porcine cells. Thus, they represent valuable tools for future structural and functional studies of pcaspase-7. Additionally, this study demonstrated that pcaspase-7 can cleave porcine GSDMD, revealing a previously unrecognized function of pcaspase-7.