Tissue-specific Expression Pattern And Histological Distribution Of NLRP3 Inflammasome Constitute Protein In Goat

NLRP3 inflammasome which is formed by a variety of proteins in the cytoplasm is a protein complexes, mainly including NLRP3, ASC and caspase-1. NLRP3 protein, an important pattern recognition receptors in the cytoplasm, which can perceive metabolic stress and products of intracellular pathogenic microorganisms, is the core part of the NLRP3 inflammasome. NLRP3 inflammation complex which is necessary for caspase-1 activation reaction platform, regulates processing and activation of the interleukin-1?, IL-18 and other proinflammatory cytokines, and activate innate immune system. It can also adjust caspase-1 dependent programmed cell apoptosis, and induce cell death in inflammatory and pathological of stress conditions. Here, NLRP3 and caspase-1 gene of NLRP3 Inflammasome constitute protein in Hainan Black goat was cloned and sequence analyzed. NLRP3 and caspase-1 polyclonal antibodies were produced by means of immune the New Zealand white rabbit. Relative expression levels and tissue distribution of NLRP3?ASC and caspase-1 were investigated by Real-time quantitative PCR and immunohistochemistry analysis. The results will help to elucidate the role of NLRP3 inflammasome in inflammatory diseases of Black goat and the main protein relative distribution in the body, a basis data for further investigations in the function and evolution of NLRP3 inflammasome in different species.The results are as follows:1. Amplification of Black goat NLRP3?ASC and caspase-1 gene and sequence analysisBlack goat NLRP3 and caspase-1 gene was amplified by RT-PCR and the sequence was deposited in Gen Bank(accession no.: KM236558; KP999980), which analyzed with bioinformatics softwares. The results showed that Black goat NLRP3 gene ORF 3130 bp and the deduced protein is consists of 1031 amino acids and its deduced molecular weight is 117.8k Da. Black goat caspase-1 gene ORF 1212 bp and the deduced protein is consists of 403 amino acids and its deduced molecular weight is 45.3k Da.2. Prokaryotic expression of Black goat NLRP3 and caspase-1 protein the preparation of the polyclonal antibodyUsing p MD18-T-NLRP3 and p MD18-T-caspase-1 plamids as template, the gene fragments including enzyme loci is amplified, and is cloned into expression vector p ET32a(+). Double enzyme digestion and nucleotide sequence analysis showed that expression plasmid p ET32a(+)-NLRP3 and p ET32a(+)-caspase-1 was successfully constructed. Transfect correct expression vector into host bacterium BL21(DE3), by IPTG induction, SDS-PAGE analysis show the recombinant His-tagged NLRP3 and caspase-1 was efficiently expressed. SDS-PAGE assay showed that the purified protein band was consistent with the predicted size, which is approximately 130 k Da and 63 k Da. Use purification of proteins into oil emulsion immune rabbit, preparation of rabbit anti goat NLRP3 and caspase-1 polyclonal antibody. Indirect ELISA method detect the two kinds of antibody titer were higher. Western blot, respectively using His-tag antibody and our prepared Ig G, results showed that specifity of the polyclonal anti is very good.3. Tissue-specific expression pattern and histological distribution of NLRP3, ASC and caspase-1 gene and protein in Hainan black goat.NLRP3, ASC and caspase-1 gene fragment of Hainan black goat were amplified by RT-PCR. Melting-curve profile analysis confirmed the specificity of primers for PCR amplification of NLRP3, ASC, caspase-1 and ?-actin fragments. The amplified target gene fragments of 78 bp, 196 bp, 110 bp and 152 bp, respectively, were evaluated by agarose gel electrophoresis and were in accordance with expectations. The sequences of these two target gene fragments showed 100% homology with the original sequences. NLRP3, ASC and caspase-1 gene was expressed in all Black goat tissues examined. The relative expression level of NLRP3 was highest and had significant difference in spleen than that in other tissues examined(P<0.05), but expression level was lowest and had significant difference in brain and lymph nodes than that other tissues examined(P<0.05). The relative expression level of ASC gene was very significantly highest in liver than that in other tissues examined(P<0.05), and the higher in spleen, but the lowest in heart, liver and lymph nodes(P<0.05). The relative expression level of caspase-1 gene was highest and had significant difference in liver and spleen than that in other tissues examined(P<0.05), and the lowest and had significant difference in brain, kidney and heart than that in other tissues examined(P<0.05).Histological distribution of Hainan black goat NLRP3, ASC and caspase-1 proteins were research by immunohistochemistry in various organizations.Immunohistochemistry detection showed that Black goat NLRP3, ASC and caspase-1 was tissue specific distribution and locates in cytoplasm of the positive staining cells. The cytoplasm exhibited weak to medium positive staining in the heart and brain, but strong in the lung, spleen, liver, kidney and lymphaden. In heart, cardiac muscle exhibited weak to moderate staining, while that of the connective tissues was negative. In liver, part of liver cells exhibited medium to strong positive staining. In spleen, the endothelial reticular cells exhibited weak to medium positive staining while lymphocyte showed negative. The alveolar epithelial cells of the pulmonary alveoli were strongly positive for NLRP3, ASC and caspase-1 protein expression in the lung. In kidney, most of the renal tubular epithelial cells exhibited medium to strongly positive staining while the cells in the glomerulus and renal capsules were negative. In the cerebral cortex of the brain, some areas of the matrix exhibited medium to strong positive staining and neurons showed weak positive. In lymphaden, germinal center was strong positive, and diffuse lymphoid tissue was weakly positive.