草鱼胶原凝集素基因的克隆及其功能分析.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2010, January 25; 26(1): 22−27 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ ©2010 CJB, All rights reserved. 动物及兽医生物技术 草鱼胶原凝集素基因的克隆及其功能分析 李琴，马梅生，胡成钰 南昌大学生物科学系，南昌 330031 摘 要: 从草鱼 Ctenopharyngodon idella 肝肾 cDNA 文库中克隆得到胶原凝集素基因。草鱼胶原凝集素全长cDNA 为 1128 bp，其中5′非编码区229 bp ，3′非翻译区 104 bp，最大开放阅读框为795 bp ，编码264 个氨基酸。系统进化分析 表明草鱼胶原凝集素与斑马鱼的亲缘关系最近。根据草鱼胶原凝集素序列特征，克隆了包含糖基识别域 (CRD) 的 cDNA ，并进行原核表达、纯化获得其重组蛋白PCRD 。进行PCRD 与6 种细菌的凝集和糖抑制实验，结果表明半乳糖、 葡萄糖、甘露糖和麦芽糖 4 种糖都会使 PCRD 与嗜水气单胞菌的凝集明显下降甚至极大地干扰凝集;麦芽糖使金黄色葡 萄球菌的凝集明显下降，而肽聚糖和甘露糖会使凝集受到抑制;此外，PCRD 的凝集反应不依赖Ca2+ 。 关键词: C- 型凝集素，胶原凝集素，糖基识别域，表达，草鱼 Cloning and functional analysis of the collectin gene from the grass carp Ctenopharyngodon idella Qin Li, Meisheng Ma, and Chengyu Hu Department of Bioscience , Nanchang University, Nanchang 330031, China Abstract: The grass carp (Ctenopharyngodon idella) collectin gene was cloned from mixed liver and kidney cDNA library. The full length sequence of grass carp collectin was 1128 bp, contained a 5′ untranslated region of 229 bp and a 3′ untranslated region of 104 bp. The open reading frame of grass carp collectin was 795 bp which could code a 264 amino acids polypeptide, including a terminal codon. Phylogenetic analyses showed that grass carp collectin shared the highest homology with that of zebrafish (Danio rerio). To understand the function of grass carp collectin, we expressed and purified the recombinant protein (PCRD) that comprised carbohydrate recognition domain (CRD). Agglutination of Aeromonas hydrophila and Staphylococcus aureus etc. and sugars inhibition experiments showed that: galactose, glucose, mannose and maltose could inhibit the agglutination of