关键词: Cystatin, 三疣梭子蟹, 血细胞, 免疫调节

Abstract: Cystatins， the reversible competitive inhibitors of cysteine proteases， comprise a large superfamily of related proteins and can be categorized into three major families: type Ⅰ （Stefins）， type Ⅱ （cystatins） and type Ⅲ （kininogens）. Cystatins are involved in a number of normal and pathological processes， e.g. immunomodulatory， epidermal homeostasis， apoptosis， tumorigenesis. In this study， a novel member of Cystatin superfamily was identified from swimming crab Portunus trituberculatus （designated PtCystatin） by cDNA library sequencing approaches. The full\|length cDNA of PtCystatin consisted of 960 bp and the 372 bp open reading frame encodeed a polypeptide of 123 amino acids with a putative signal petide in the N\|terminal （M1 to C19）. The putative matured petide of PtCystatin consists of 104 amino acids with the theoretical isoelectric point of 524 and the predicted molecular weight of 11 64030 Da， and had a single Cystatin\|like domain （M26 to H115）. PtCystatin exhibited higher sequence homology to type Ⅰ cystatins than to type Ⅱ cystatins from vertebrate or other invertebrate， and was clustered into type Ⅰ cystatin in the phylogenetic tree. Furthermore， the two conserved binding motifs of human Cystatin A also located in PtCystatin （M26VPGG30， Q71VVAG75）， and the conserved disulphide bond in the C\|terminal of type Ⅱ cystatin was absent in PtCystatin. All of these suggested that PtCystatin belonged to type Ⅰ cystatin， but was secreted to extracellure space. The mRNA transcripts of PtCystatin were highly expressed in stomach， moderately in hepatopancreas， gill， hemocytes， and were marginally detectable in muscle and eyes. After Vibrio parahaemolyticus challenge， the relative expression level of PtCystatin was up\|regulated to 121\|fold （P<005） at 3 h in hemocytes. Subsequently， it was down\|regulated to 047 （P<001） and 057\|fold （P<005） at 6 h and 12 h， respectively. Afterwards， PtCystatin mRNA transcripts recovered to original level at 24 h. These results suggested a potential role for PtCystatin in pathogen host defense mechanisms.

Key words: Cystatin, Portunus trituberculatus, hemocytes, immunomodulatory