中国鲎血蓝蛋白提纯及其纯化物的液相色谱串联质谱分析  

Hemocyanin purification from Chinese horseshoe crab(Tachypleus tridentatus)and analysis of the purified products by liquid chromatography tandem with mass spectrometry

作　　者：赵忠浩 常雪晴 王有基[1,2] 何锦锋 台宗光 朱全刚 胡梦红 ZHAO Zhonghao;CHANG Xueqing;WANG Youji;HE Jinfeng;TAI Zongguang;ZHU Quangang;HU Menghong(International Research Center for Marine Biosciences,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China;Beihai Product Quality Testing Institute,Beihai 536000,China;Shanghai Skin Disease Hospital,Shanghai 200443,China)

机构地区：[1]上海海洋大学,海洋生物科学国际联合研究中心,上海201306 [2]上海海洋大学,水产种质资源发掘与利用教育部重点实验室,上海201306 [3]北海市产品质量检验所,广西北海536000 [4]上海市皮肤病医院,上海200443

出　　处：《水产学报》2022年第8期1499-1508,共10页Journal of Fisheries of China

基　　金：香港海洋公园保育基金(OT031617);2017北海市“十三五”海洋经济创新发展示范项目(Bhsfs006)。

摘　　要：为了获得高纯度的中国鲎血蓝蛋白并探究其潜在的应用价值,实验从中国鲎血淋巴中提取了血蓝蛋白,并用AKTA蛋白纯化系统进行纯化,使用液相色谱串联质谱(LC-MS/MS)技术对纯化后的血蓝蛋白进行定性分析,最后从uniprot蛋白数据库下载了22条分属5个物种的血蓝蛋白的氨基酸序列,并构建了N-J系统进化树。结果显示,制备的血蓝蛋白纯度较高,从纯化后的中国鲎血蓝蛋白中鉴定到了12种血蓝蛋白的亚基,包括Hemocyanin subunitⅠ、Hemocyanin subunitⅡ、Hemocyanin subunitⅢa、Hemocyanin subunitⅢb、Hemocyanin subunitⅣ、Hemocyanin subunitⅤ、Hemocyanin subunitⅥ等。uniprot数据库显示,这些亚基最早在圆尾鲎和美洲鲎体内被发现。N-J系统进化树显示,中国鲎血蓝蛋白与圆尾鲎和美洲鲎血蓝蛋白具有高度的同源性,并且鲎科动物血蓝蛋白与凡纳滨对虾、锯缘青蟹的血蓝蛋白有较近的进化关系,这意味着可根据虾蟹血蓝蛋白的功能推测中国鲎血蓝蛋白的功能。本研究结果有助于中国鲎血蓝蛋白的功能预测和应用研究。Tachypleus tridentatus belongs to the arthropod phylum,and its blood contains a lot of hemocyanin.Hemocyanin plays an important role in the organism,such as transporting oxygen,immune defense,and energy storage.Hemocyanin also has many applications in vitro,such as being made into biosensors and immune products.This study aims to obtain high-purity hemocyanin of T.tridentatus and explore its potential application value.In this study,hemocyanin was extracted from hemolymph of T.tridentatus and purified with AKTA protein purification system.The purified hemocyanin was qualitatively analyzed using liquid chromatography tandem mass spectrometry(LC-MS/MS) technology.Finally,22 amino acid sequences of hemocyanin from 5 species were downloaded from the uniprot protein database,then the N-J phylogenetic tree was constructed.The results showed that the purity of the purified hemocyanin was high.Twelve types of hemocyanin subunits were identified from hemocyanin of T.tridentatus,including Hemocyanin subunit Ⅰ,Hemocyanin subunit Ⅱ,Hemocyanin subunitⅢa,Hemocyanin subunit Ⅲb,Hemocyanin subunit Ⅳ,Hemocyanin subunit Ⅴ,Hemocyanin subunit Ⅵ,etc.According to the uniprot database,these subunits were first found in Carcinoscorpius rotundicauda and Limulus polyphemus.The N-J phylogenetic tree shows that hemocyanin of Tachypleus tridentatus has a high degree of homology with C.rotundicauda and L.polyphemus.It was also found that there was a close evolutionary relationship of hemocyanin between hemocyanin of Limulidae and Litopenaeus vannamei and Scylla serrata,which suggests that the function of hemocyanin in T.tridentatus can be inferred from the function of hemocyanin in other species.This study obtained high-purity hemocyanin from hemolymph of T.tridentatus,and the results of this study are helpful to the function prediction and application research of Chinese horseshoe crab hemocyanin.

关 键 词：中国鲎 血蓝蛋白 纯化 液相色谱串联质谱(LC-MS/MS) 进化树 

分 类 号：S917.4[农业科学—水产科学]