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Cystatin is a class of cysteine ??proteases closely, reversible natural inhibitors. Because these cysteine ??proteases are present in all organisms and participate in a variety of biological, pathological process, so cystatin functional regulation of these proteases is especially important. In the immune system, cystatin adjustable cathepsin activity and antigen presentation, induce TNFα and IL-10 synthesis and stimulate IFN-γ activated murine macrophages to produce NO. In our previous studies, the establishment of a poLyI: C-induced large yellow croaker spleen SmartcDNA library, and found the two different coding cysteine ??protease inhibitor EST clones were sequenced than the latter identified as large yellow croaker stefin and cystatin similar things, belong cystatin family. This article first cloned a large yellow croaker cystatin length cDNA. Sequence analysis showed that the full-length cDNA of large yellow croaker cystatin including 688 nucleotides encoding a 118 amino acid protein with molecular weight 13kDa, presumably having a 21 amino acid signal peptide sequence. Respectively, and fish, higher vertebrates and nematodes have 27.1-45.9% ,30.0-31 .7%, and 16.3-30.6% sequence identity, in which the highest sequence identity with the pufferfish, was 45.9%. With human cystatin C with 30.8% sequence identity with the egg white cystatin has 31.7% sequence identity. Large yellow croaker cystatin also contain typical cystatin family conserved domain GLy-GLy (25,26), QLVAG (69-73) and PW (106-107). Four cysteine ??residues, respectively, C 56 , C 87 , C 98 and C 118 . May form two pairs of disulfide bonds to maintain its unique spatial structure. However, large yellow croaker four cysteine ??residues with only three other species is consistent with the position, and another one cysteine ??residue (C 56 ) to the N-terminal side shift, which may affect their spatial structures. In addition, a large fish protein amino acid sequence of cystatin have S 52 N 53 residue, may be inhibition of the active site of Legumain. Genome analysis showed that large yellow croaker cystatin gene from the 3 exons and two introns, and human, mouse and zebrafish genome structure cystatin basically the same. RT-PCR analysis showed that the gene in the intestine, gill, heart, muscle, spleen, liver, blood and kidney is constitutively expressed in the spleen and kidneys in the highest level of gene transcription, the gills, the lowest muscle and blood. Triple inactivated bacterial vaccines were used or poLy (I: C) after stimulation of the gene mRNA levels in the kidney and spleen increases in the blood down, but lowered slightly. With pGEX-4T-2 prokaryotic expression vector was constructed large yellow croaker cystatin (GST) recombinant expression vector, expressed and purified recombinant Lyccys (GST) fusion protein. Casein as a substrate, detection of the fusion protein to a specific activity of papain inhibitory activity unit 40 suppression. Labeled with colloidal gold immune electron microscopy studies of large yellow croaker cystatin proteins in the kidney, spleen intracellular localization. The test showed that the protein was mainly distributed in the cytoplasm and attached to the rough endoplasmic reticulum (rER), intracellular vesicular structures (such as the endoplasmic body or lysosome) there are more, not found within the nucleus. Relative quantitative PCR detection using recombinant cystatin large yellow croaker immune immune-related genes TNF-α2 and IL-10 level of transcription regulation. The result is a large yellow croaker cystatin stimulates the large yellow croaker spleen and kidney TNF-α2 and IL-10 mRNA levels increase. So that may have a large yellow croaker cystatin cystatin similar to mammalian immune function. Meanwhile, we have cloned a large yellow croaker stefin full-length cDNA, containing 556 nucleotides encoding a 99 amino acid protein with molecular weight of about 11kDa. Respectively, and pure, cattle, puffer fish, pigs, rainbow trout and zebrafish stefin protein 47.5%, 47.5%, 46.1%, 42.7%, 38.0%, 38.0% sequence homology. Human and mouse stefin A and B proteins were 43.0% and 38.0%, 38.4% and 32.0% consistency. Stefin family of proteins encoded by the characteristics of a domain, i.e. the N-terminal GLy 6 -GLy 7 binding site, and a conserved QLVAG (48-51) motif. Genome analysis showed that genes from the large yellow croaker stefin three exons and two introns, and human, mouse and zebrafish stefin genome structure basically the same. RT-PCR analysis showed that the gene in the intestine, gill, heart, muscle, spleen, liver, blood and kidney are expressed, in which the blood and kidney highest level of gene transcription, the lowest in the intestine. Respectively triple inactivated bacterial vaccines or poLy (I: C) stimulation, mRNA levels of the gene is upregulated in the kidney and spleen in the blood downward. With pGEX-4T-2 prokaryotic expression vector was constructed large yellow croaker stefin (GST) (Lycstefin (GST)) recombinant expression vector, expressed and purified the active Lycstefin (GST) fusion protein. Casein as substrate, the detection of the fusion protein on papain inhibition of specific activity of 42 inhibition activity unit. Relative quantitative PCR detection using recombinant large yellow croaker stefin immune related genes TNF-α2 and IL-10 level of transcription regulation. The result is a large yellow croaker stefin not cause large yellow croaker spleen and kidney TNF-α2 and IL-10 mRNA level changes. By immune electron microscopy studies of large yellow croaker stefin protein in the kidney, spleen intracellular localization. The test showed that the protein was dispersed in the cytoplasm, not attached to the rough endoplasmic reticulum (rER), in the nucleus and intracellular vesicular structures (such as the endoplasmic body or lysosome) a small amount of the distribution.
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