草魚膠原凝集素基因的克隆及其功能分析

李琴，馬梅生，胡成鈺

南昌大學生物科學系，南昌 330031

草魚膠原凝集素基因的克隆及其功能分析

李琴，馬梅生，胡成鈺

南昌大學生物科學系，南昌 330031

從草魚Ctenopharyngodon idella肝腎cDNA文庫中克隆得到膠原凝集素基因。草魚膠原凝集素全長cDNA為1128 bp，其中5′非編碼區229 bp，3′非翻譯區104 bp，最大開放閱讀框為795 bp，編碼264個氨基酸。系統進化分析表明草魚膠原凝集素與斑馬魚的親緣關系最近。根據草魚膠原凝集素序列特征，克隆了包含糖基識別域(CRD)的cDNA，并進行原核表達、純化獲得其重組蛋白PCRD。進行PCRD與6種細菌的凝集和糖抑制實驗，結果表明半乳糖、葡萄糖、甘露糖和麥芽糖 4種糖都會使 PCRD與嗜水氣單胞菌的凝集明顯下降甚至極大地干擾凝集;麥芽糖使金黃色葡萄球菌的凝集明顯下降，而肽聚糖和甘露糖會使凝集受到抑制;此外，PCRD的凝集反應不依賴Ca2+。

C-型凝集素，膠原凝集素，糖基識別域，表達，草魚

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 Aeromonas hydrophila.Maltose could lower the agglutination of Staphylococcus aureus, whereas peptidoglycan and glucose inhibited it well.In addition, the activity of grass carp collectin could not dependent on Ca2+.

Keywords:C-type lectin, collectin, carbohydrate recognition domain(CRD), expression, grass carp

C-型凝集素(C-type lectins)是凝集素的重要一員，它是一種Ca2+依賴的糖結合蛋白，在機體的先天免疫和獲得性免疫、控制糖蛋白的生物合成等方面發揮著重要功能[1-3]。雖然在不同的物種中，C-型凝集素差異很大，但一般至少含有1個由約130個氨基酸殘基組成的糖基識別域(Carbohydrate recognition domain，CRD)[4-5]。根據CRD的一級結構的差異可以將C-型凝集素分為蛋白聚糖、Ⅱ型跨膜受體、膠原凝集素、選凝素、白細胞受體、巨噬細胞甘露糖受體、單一或原始凝集素7個家族[6-7]。而且，目前仍有一些新的C-型凝集素成員陸續報道。

膠原凝集素(Collectin)是一種可溶性的模式識別受體，包括甘露……