波纹龙虾C-型凝集素PhLecA的基因克隆与表达*

doi:10.11978/2015155

热带海洋学报 ›› 2016, Vol. 35 ›› Issue (5): 29-37.doi: 10.11978/2015155CSTR: 32234.14.2015155

波纹龙虾C-型凝集素PhLecA的基因克隆与表达^*

李斌, 梁华芳, 陈兆明, 黄启幸, 程志芬, 陈仕坤

广东海洋大学水产学院, 广东湛江 524088

收稿日期:2015-12-24 出版日期:2016-08-29 发布日期:2016-09-22
通讯作者: 梁华芳(1965—), 男, 博士, 教授, 从事甲壳动物生物学, 养殖技术研究。E-mail: hfliang@126.com
作者简介:李斌(1990—), 男, 在读硕士研究生, 从事甲壳动物分子免疫方向研究。E-mail: 1649977084@qq.com
基金资助:
基金项目：广东海洋与渔业科技推广专项-科技攻关与研发项目(A201208D04)

Gene cloning and analysis of a novel C-type lectin PhLecA from Panulirus homaus

LI Bin, LIANG Huafang, CHAN Siuming, HUANG Qixing, CHENG Zhifen, CHEN Shikun

Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China

Received:2015-12-24 Online:2016-08-29 Published:2016-09-22
Contact: Corresponding author: LIANG Huafang. E-mail: hfliang@126.com
Supported by:
Foundation item: This study funded by the Special Project of Science and Technology for Marine Fisheries of Guangdong Province (A201208D04)

摘要/Abstract

摘要： C-型凝集素是存在于所有后生动物中的一类Ca²⁺依赖性糖蛋白, 能够识别并结合细菌和真菌表面的糖类物质, 在机体免疫防御中发挥重要作用。采用RT-PCR和末端快速扩增技术(rapid amplification of cDNA ends, RACE)首次克隆了波纹龙虾Panulirus homaus C-型凝集素PhLecA的cDNA全长, 共863bp, 其中开放阅读框729 bp, 编码一个243个氨基酸残基的蛋白, 其中含一个17个氨基酸残基的信号肽和一个CRD(carbohydrate recognition domain)结构域。通过半定量RT-PCR检测发现, PhLecA的mRNA广泛分布于波纹龙虾各个组织, 胚胎期以及幼体期, 其中在肝胰腺组织表达量最高, 胃肠组织及复眼色素形成期次之。采用定量PCR方法对WSSV(white spot syndrome virus)、哈维氏弧菌免疫刺激后波纹龙虾肝胰脏中的PhLecA 转录水平检测, 结果显示, 波纹龙虾注射WSSV、哈维氏弧菌后, PhLecA表达量均发生了变化, 总体趋势为先上调, 然后下调至正常水平。研究显示, PhLecA可能在波纹龙虾对病毒、细菌的免疫应答过程中起重要作用。

关键词: 波纹龙虾, C-型凝集素, 基因克隆, 组织分布, 免疫刺激

Abstract: C-type lectins (CTLs) are a large family of proteins that exist in all deuterostomia. CTLs can bind to carbohydrate moieties normally in a calcium-dependent manner and play important roles in immune defense. The cDNA sequence of Panulirus homaus LecA (PhLecA) was obtained using rapid amplification of cDNA ends method (RACE). The full-length cDNA sequence of PhLecA was 863 bp, which contained a 729 bp complete open reading frame (ORF) encoding 243 amino acids. The deduced amino acid sequence of PhLecA had a signal peptide containing 17 amino acid residues and a carbohydrate recognition domain (CRD). RT-PCR analysis showed that the mRNA of PhLecA was widely distributed in various tissues in the embryonic period and the larval stage, with highest expression in hepatopancreas and then in stomach, intestine and eye pigment formation stage. Real-time PCR analysis indicated that PhLecA transcripts level showed significant change in hepatopancreas after the lobster was artificially challenged with white spot syndrome virus (WSSV) and Vibrio harveyi, the overall change trend of PhLecA transcripts first went up, and then went down to normal level. The results suggested that PhLecA might be involved in the immune response against virus and bacterial.

Key words: Panulirus homaus, C-type lectin, gene cloning, tissue distribution, immune response

李斌, 梁华芳, 陈兆明, 黄启幸, 程志芬, 陈仕坤. 波纹龙虾C-型凝集素PhLecA的基因克隆与表达^*[J]. 热带海洋学报, 2016, 35(5): 29-37.

LI Bin, LIANG Huafang, CHAN Siuming, HUANG Qixing, CHENG Zhifen, CHEN Shikun. Gene cloning and analysis of a novel C-type lectin PhLecA from Panulirus homaus[J]. Journal of Tropical Oceanography, 2016, 35(5): 29-37.

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波纹龙虾C-型凝集素PhLecA的基因克隆与表达^*

Gene cloning and analysis of a novel C-type lectin PhLecA from Panulirus homaus

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