ISOLATION, CHARACTERIZATION AND EXPRESSION OF THE HEMOLYMPH CLOTTABLE PROTEIN OF CHINESE SHRIMP FENNEROPENAEUS CHINENSIS
中国对虾(Fenneropenaeus chinensis)血淋巴中凝血蛋白的分离纯化、鉴定及cDNA片段克隆、组织表达谱分析

A clottable protein (CP) was purified from the hemolymph of the Chinese shrimp Fenneropenaeus chinensis by anion-exchange chromatography and hydrophobic interaction chromatography. The shrimp CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+, suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase in shrimp hemocytes. The molecular mass of the CP was found to be 380kDa under non-reducing conditions and 190kDa under reducing conditions determined by SDS-PAGE. CPs exist as disulfide-linked homodimers and oligomers. The N-terminus amino acid sequence of this CP was 100% identical to that of the penaeids Litopenaeus vannamei, F. paulensis, and Penaeus monodon; and 66% to 80% identical to the CPs of other decapods. By Homologous Cloning method, the cDNA fragment of CP was cloned and sequenced, and it contained 518bp and with deduced amino acid sequence of 172 residues. The sequence analysis indicated that it shared high identity with CP gene from other previously reported shrimps. RT-PCR analysis revealed that the sub-cuticular and the heart were identified as the tissues that express the most of CP in F. chinensis.