Abstract:CASPASES are a class of highly conserved cysteine-dependent endoproteases, whose member is composed of an N-terminal domain and large and small catalytic subunits. CASPASES can hydrolyze substrates at specific aspartic acid residues, thereby playing a role in programmed cell death induced by pathogenic infections and environmental stress. Studies in mammals have shown that CASPASE-3/6/7/8/9/10 is mainly involved in cell apoptosis, while CASPASE-1/4/5/11/12 is associated with inflammatory responses. The activated CASPASE-1 can cleave GASDERMIN D (GSDMD), thereby promoting the formation of cell membrane pores and cell lysis, and initiating cell pyroptosis. Several reports have reported the role of CASPASE-3 and CASPASE-1 in apoptosis, pyroptosis, and inflammatory response in fish, but it is not possible to conduct research at the protein level to reveal the relevant regulatory mechanisms due to the lack of specific antibodies. Under high-density aquaculture conditions, the accumulated ammonia in water leads to tissue damage and cell death in fish. Largemouth bass is a representative species in intensive aquaculture model. In order to further explore the regulatory mechanism of CASPASES in ammonia-induced programmed cell death in largemouth bass, this study firstly analyzed the antigenic epitopes, hydrophilicity, and structural domains of CASPASE-1 and CASPASE-3 in largemouth bass. Their 0-200aa and 0-150aa regions were selected as the target expression fragments, respectively, and pET-32a-MsCASPASE-1 and pET-32a-MsCASPASE-3 prokaryotic recombinant plasmids were constructed by PCR amplification, double enzyme digestion, and ligation. Sequencing results showed that the constructed recombinant plasmids contained the target gene fragments. Subsequently, the two successfully constructed recombinant plasmids were transformed into BL21 (DE3) competent cells and induced for expression under optimal conditions (1.0 mM and 0.6 mM IPTG induced overnight at 16 ℃). The expressed soluble supernatant proteins were purified by affinity chromatography using Ni-NTA Beams 6FF gravity column, and single bands with protein molecular weights of approximately 46 KDa and 36 KDa were identified by SDS-PAGE, demonstrating the successful acquisition of recombinant MsCASPASE-1 and MsCASPASE-3 proteins. Afterwards, the purified recombinant MsCASPASE-1 and MsCASPASE-3 proteins were immunized with one Japanese large eared rabbit and three Balb/C mice to obtain anti-serum, respectively. The titers and specificity of antibodies were detected by enzyme-linked immunosorbent assay and western blotting. Results showed that the anti-serum obtained after immunization could specifically recognize the recombinant proteins CASPASE-1 and CASPASE-3 and endogenous proteins of largemouth bass, with a single target band that was consistent with the expected molecular weight, with molecular weights of 46 KDa/44Da and 36 KDa/31kDa, respectively. At the same time, the rabbit/mouse serum titers of CASPASE-1 and CASPASE-3 were 1:10240 K/1:1024 K and 1:10240 K/1:1024 K, respectively. All these results suggested that the present study has successfully prepared the antibodies against CASPASE-1 and CASPASE-3 proteins in largemouth bass, which exhibits good specificity and high titers, and is available for subsequent analysis. Thus, an ammonia-stress experiment was subsequently conducted in largemouth bass to detect the regulatory roles of CASPASE-1 and CASPASE-3. Kidney is the important urinary organ in fish including largemouth bass, which is composed of renal corpuscles and tubules. Especially, the renal corpuscle, functioning as the filtering unit of the kidney, is mainly composed of a small group of capillaries that form the glomerulus and is responsible for filtering waste and excess fluid from the blood into the renal small sac cavity to become primary urine. The renal tubules include structures such as distal tubules and proximal tubules. The surface of the proximal tubules is covered with wrinkled microvilli, which can reabsorb nutrients and water from the filtered fluid. The inner wall of the distal tubules has no microvilli, and the lumen inside the tubules is larger, which secretes and reabsorbs different ions to further adjust urine and make it suitable for excretion from the body. In the present study, histopathological results revealed significant pathological changes in the kidney of largemouth bass. After ammonia stress, the renal corpuscle cells proliferated and the renal capsule cavity expanded in the kidney of largemouth bass, which structural changes significantly affected the filtration function of glomerular. On the other hand, under ammonia stress, the epithelial cell structure of the distal tubules of kidney was gradually destroyed, and the intercellular space gradually increased. The epithelial cells of the proximal tubules expanded and dissociated, with the cell structure also disrupted. These changes indicate that ammonia stress changed the kidney structure, thus affecting its secretion function and its ability to reabsorb nutrients and water in the filtrate. Using the polyclonal antibodies against CASPASE-1 and CASPASE-3 prepared in this study, western blotting experiments detected the significantly increased protein levels of CASPASE-1 and CASPASE-3 in the kidneys of largemouth bass after ammonia stress. The increased CASPASE-1 and CASPASE-3 in kidney is responsible for the subsequent programmed cell death, consistent with histopathological results. Therefore, this study successfully prepared rabbit- and mouse-derived polyclonal antibodies that can specifically recognize CASPASE-1 and CASPASE-3 in largemouth bass, and revealed the effect of ammonia stress on their expression, providing an important basis for further research on the programmed cell death mechanism of largemouth bass under environmental stress.
