引用本文:	郝贵杰,林锋,母昌考,李荣华,姚嘉赟,潘晓艺,袁雪梅,沈锦玉,王春琳.三疣梭子蟹(Portunus trituberculatus)半乳糖凝集素PtGAL的基因克隆与原核重组表达.海洋与湖沼,2016,47(6):1241-1249.

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*三疣梭子蟹(Portunus trituberculatus)半乳糖凝集素PtGAL的基因克隆与原核重组表达*
郝贵杰^1,2, 林锋², 母昌考¹, 李荣华¹, 姚嘉赟², 潘晓艺², 袁雪梅², 沈锦玉², 王春琳¹
1.宁波大学海洋学院宁波 315211;2.农业部淡水渔业健康养殖重点实验室浙江省鱼类健康与营养重点实验室浙江省淡水水产研究所湖州 313001

摘要:

半乳糖凝集素（Galectin）是动物凝集素家族的一员，在脊椎动物和无脊椎动物中都参与了重要的免疫防御反应，是一种重要的免疫因子。通过RT-PCR和RACE技术克隆获得了三疣梭子蟹（Portunus trituberculatus）半乳糖凝集素基因全长，并命名为PtGal。该基因全长875bp，开放阅读框为669bp，编码222个氨基酸，包含一个糖识别结构域CRD（位于16-142位氨基酸），预测蛋白的分子量（Mw）为23529.4Da，理论等电点（pI）为5.21。同源性比对结果显示，PtGal编码的氨基酸序列与中华绒螯蟹、南美白对虾、日本囊对虾galectin的同源性分别为77%、66%、58%。实时荧光定量PCR（qRT-RCR）结果表明，PtGal在三疣梭子蟹肝胰腺、肌肉、肠、胃、鳃、心脏、性腺中都有表达，其中在性腺中的表达量最高，在肝胰腺中的表达量最低，但人工感染溶藻弧菌（Vibrio alginolyticus）后，肝胰腺的表达呈先升高后降低的趋势。利用原核表达系统对三疣梭子蟹PtGal进行了重组表达，并通过纯化复性获得了重组目的蛋白rPtGal，ELISA检测rPtGal与不同细菌及真菌的结合活性，结果表明，该重组蛋白与革兰氏阳性菌、阴性菌和真菌均有较强的结合。本研究为进一步深入研究三疣梭子蟹半乳糖凝集素的功能奠定了基础。

关键词: 三疣梭子蟹半乳糖凝集素实时荧光定量PCR 溶藻弧菌重组蛋白

DOI：10.11693/hyhz20160700147

分类号:

基金项目:国家自然科学基金项目，41376150号；国家农业科技成果转化资金项目，2014GB2C220151号。

附件

CLONING AND PROKARYOTIC EXPRESSION OF GALECTIN FROM PORTUNUS TRITUBERCULATUS

HAO Gui-Jie^1,2, LIN Feng², MU Chang-Kao¹, LI Rong-Hua¹, YAO Jia-Yun², PAN Xiao-Yi², YUAN Xue-Mei², SHEN Jin-Yu², WANG Chun-Lin¹

1.School of Marine Science, Ningbo University, Ningbo 315211, China;2.Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture, Key Laboratory of Fish Health and Nutrition of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China

Abstract:

Galectins are a family of Ca²⁺-independent soluble lectins characterized by their affinity to β-galactosides,and play important roles in innate immunity of vertebrates and invertebrates.A cDNA of galectin with one carbohydrate recognition domain (CRD) was cloned from Portunus trituberculatus (designated PtGAL) in RT-PCR and RACE technique.The full length of PtGAL was 875bp.The open reading frame encoded a polypeptide of 223 amino acids containing one carbohydrate recognition domain.The predicted molecular weight was 23529.4Da,and the theoretical isoelectric point was 5.21.The results of multiple amino acid sequences alignment show that the identity of PtGAL shared with Eriocheir sinensis,Litopenaeus vannamei,and Marsupenaeus japonicas was 77%,66%,and 58%,respectively.Quantitative real-time PCR (qRT-PCR) was employed to investigate the mRNA of PtGAL distribution in all tested tissues,including hepatopancreas,muscle,intestines,stomach,gill,heart,and gonad.The highest expression level was observed in gonad,while the lowest in hepatopancreas.After being challenged with Vibrio alginolyticus,the relative mRNA expression level of PtGAL in hepatopancreas was up-regulated and reached the maximum level at 12h (3.49 fold) post challenge.The recombined PtGAL (rPtGAL) showed strong ability to combine G⁺ bacteria Staphylococcus aureus and Bacillus subtilis,or G^- bacteria such as Vibrio alginolyticus,V.parahaemolyticus and so on,and Saccharomyces cerevisiae.Therefore,as a new member of the galectin family,PtGAL is involved in the immune responses against bacterial infection.

Key words: Portunus trituberculatus galectin quantitative real-time PCR Vibrio alginolyticus recombinant protein