[Preparation and assessment of a monoclonal antibody against mouse NLRP3].

Objective To prepare a monoclonal antibody (mAb) against mouse NOD-like receptor family pyrin domain-containing 3 (NLRP3) and assess its specificity. Methods A gene fragment encoding mouse NLRP3 exon3 (Ms-N3) was inserted into the vector p36-G3-throhFc to construct a recombinant plasmid named Ms-N3-throhFc. This plasmid was then transfected into HEK293F cells for eukaryotic expression. NLRP3-/- mice were immunized with Ms-N3 protein purified using a protein A chromatography column, and splenocytes from the immunized mice were fused with SP2/0 myeloma cells to generate hybridoma cells. Specific mAbs against murine NLRP3 from hybridoma cells were screened using ELISA and immunofluorescence assay(IFA). Results The Ms-N3-throhFc recombinant plasmid was successfully constructed and exhibited stable expression in HEK293F cells. Twelve hybridoma cell lines were initially screened using ELISA. IFA revealed that the mAb secreted by the 9-B8-3-2-C5 cell line specifically recognized the native form of mouse NLRP3 protein. The heavy and light chain subtypes of this mAb were identified as IgM and κ, respectively. Conclusion A monoclonal antibody against mouse NLRP3 has been successfully prepared.

Anti-influenza activity of CPAVM1 protease secreted by Bacillus subtilis LjM2.

Bacillus spp. has been considered a promising source for identifying new antimicrobial substances, including anti-viral candidates. Here, we successfully isolated a number of bacteria strains from aged dry citrus peel (Chenpi). Of note, the culture supernatant of a new isolate named Bacillus subtilis LjM2 demonstrated strong inhibition of influenza A virus (IAV) infection in multiple experimental systems in vitro and in vivo. In addition, the anti-viral effect of LjM2 was attributed to its direct lysis of viral particles. Further analysis showed that a protease which we named CPAVM1 isolated from the culture supernatant of LjM2 was the key component responsible for its anti-viral function. Importantly, the therapeutic effect of CPAVM1 was still significant when applied 12 hours after IAV infection of experimental mice. Moreover, we found that the CPAVM1 protease cleaved multiple IAV proteins via targeting basic amino acid Arg or Lys. Furthermore, this study reveals the molecular structure and catalytic mechanism of CPAVM1 protease. During catalysis, Tyr75, Tyr77, and Tyr102 are important active sites. Therefore, the present work identified a special protease CPAVM1 secreted by a new strain of Bacillus subtilis LjM2 against influenza A virus infection via direct cleavage of critical viral proteins, thus facilitates future biotechnological applications of Bacillus subtilis LjM2 and the protease CPAVM1.

Inflammasome activity is controlled by ZBTB16-dependent SUMOylation of ASC.

Inflammasome activity is important for the immune response and is instrumental in numerous clinical conditions. Here we identify a mechanism that modulates the central Caspase-1 and NLR (Nod-like receptor) adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). We show that the function of ASC in assembling the inflammasome is controlled by its modification with SUMO (small ubiquitin-like modifier) and identify that the nuclear ZBTB16 (zinc-finger and BTB domain-containing protein 16) promotes this SUMOylation. The physiological significance of this activity is demonstrated through the reduction of acute inflammatory pathogenesis caused by a constitutive hyperactive inflammasome by ablating ZBTB16 in a mouse model of Muckle-Wells syndrome. Together our findings identify an further mechanism by which ZBTB16-dependent control of ASC SUMOylation assembles the inflammasome to promote this pro-inflammatory response.