| 摘要: |
| 采用层析技术、N 末端氨基酸序列分析及同源基因克隆等技术对中国对虾中国对虾血淋巴中的凝血蛋白(CP)进行了研究。 结果表明, 纯化的CP约为380kDa, 其亚基的分子量约为190kDa, 说明该蛋白是由两个相同的亚基组成的同源二聚体; 中国对虾 CP的 N末端氨基酸序列与凡纳滨对虾、保罗美对虾具有100%的相似性, 与其它对虾的也具有很高的相似性; 获得了CP基因518bp的cDNA片段, 分析表明该基因序列与其它虾类 CP 基因序列的具有很高的同源性; CP 基因的组织表达谱分析发现心脏和表皮是该基因主要的合成表达部位。 |
| 关键词: 中国对虾, 凝血蛋白, 分离纯化, cDNA 克隆, 组织表达谱 |
| DOI：10.11693/hyhz201201025025 |
| 分类号: |
| 基金项目:国家自然科学基金资助项目, 30600458 号 |
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| ISOLATION, CHARACTERIZATION AND EXPRESSION OF THE HEMOLYMPH CLOTTABLE PROTEIN OF CHINESE SHRIMP FENNEROPENAEUS CHINENSIS |
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WANG Bao-Jie1,2, LIU Mei1, JIANG Ke-Yong1, ZHANG Guo-Fan1, WANG Lei1
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1.Institute of Oceanlogy, Chinese Academy of Sciences;2.Graduate School of Chinese Academy of Sciences
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| Abstract: |
| 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. |
| Key words: Fenneropenaeus chinensis, Clottable protein, Purification, cDNA clone, Tissue-specific expression |
