Abstract: To reconstruct noncanonical pyroptosis in vitro, the coding sequences of Caspase-4, hGSDMD-FL and hGSDMD-p30 were amplified by PCR, and these PCR products were inserted into corresponding plasmids to get the following recombinant eukaryotic expression vectors:pCMV-MYC-Caspase-4, p3XFLAG-hGSDMD-FL. The expressions of both recombinant vectors in HEK293T cells were confirmed by Western blot. Whether the noncanonical pyroptosis model was successfully constructed was confirmed by testing the production of LDH, active N-terminal GSDMD-p30 and PI staining after transfection into HEK293T cells. The results showed that recombinant vectors including pCMV-MYC-Caspase-4, p3XFLAG-hGSDMD-FL, and pcDNA3.1-hGSDMD-p30-MYC were constructed and each protein was successfully expressed in HEK293T cells. After co-transfection, significant production of LDH was detected, Caspase-4 cleaved the full-length GSDMD into active N-terminal GSDMD-p30 domain and obvious PI staining was observed by fluorescence microscope, which indicated that the noncanonical pyroptosis was successfully reconstructed in vitro. Therefore, the noncanonical pyroptosis is reconstructed in vitro, which establishes foundation for further revealing the mechanism of noncanonical pyroptosis activation.

Key words: noncanonical pyroptosis, gasdermin D, GSDMD, plasmid construction, protein expression