草鱼BPI/LBP基因的克隆及特征研究

Cloning and characterization of BPI/LBP gene from grass carp,Ctenopharyngodon idella

【目的】克隆草鱼杀菌通透性增加蛋白/脂多糖结合蛋白(Bactericidal permeability-increasing pro-tein/LPS-binding protein,BPI/LBP)基因的cDNA全长,分析其结构特征,研究其表达规律,为进一步探明其功能奠定基础。【方法】首先用同源克隆及快速扩增cDNA末端技术(RACE),克隆草鱼BPI/LBP基因的cDNA全长,然后用生物信息学方法分析BPI/LBP基因的结构特征,最后用半定量RT-PCR检测BPI/LBP基因在草鱼不同组织(血、脑、眼睛、前肠、中肠、后肠、鳔、鳃、头肾、中肾、心脏、肝胰脏、肌肉、皮肤和脾脏)中的表达模式。【结果】草鱼BPI/LBPcDNA全长1 568 bp,编码473个氨基酸,其中包括18个氨基酸组成的信号肽、BPI1结构域(BPI/LBP/CETP N-ter-minal domain)和BPI2结构域(BPI/LBP/CETP C-terminal domain)。该蛋白的分子质量为51 551 u,等电点为8.69。氨基酸序列同源性分析结果显示,草鱼与鲤鱼BPI/LBP的同源性最高(90%),其次为虹鳟(73%)、斑点叉尾鮰(73%)、香鱼(72%)等。在系统进化树中,草鱼BPI/LBP首先与鲤科的鲤鱼聚为一类。通过半定量RT-PCR分析可知,草鱼BPI/LBP基因在被检测的15个组织中都有表达,其中在鳃中的表达量最高,其次为头肾、中肾等。【结论】成功克隆了草鱼BPI/LBP基因的cDNA全长,该基因在草鱼体内不同组织中广泛表达。

【Objective】 The full-length cDNA sequence of bactericidal permeability-increasing protein/LPS-binding protein（CiBPI/LBP） from grass carp,Ctenopharyngodon idella was cloned to study the characterization and expression profile and to lay a foundation for the functional studies.【Methods】 The CiBPI/LBP cDNA was identified from grass carp gill by homologous cloning and rapid amplification cDNA ends（RACE）.The structural characterization was analyzed by bioinformatics method.The CiBPI/LBP expression pattern was examined in different tissues（blood,brain,eye,foregut,midgut,hindgut,gas blander,gill,head kidney,trunk kidney,heart,liver,muscle,skin and spleen） by semi-quantitative RT-PCR.【Result】 The CiBPI/LBP cDNA was 1 568 bp,encoding 473 amino acid（aa） residues,including signal peptide,BPI1 domain（BPI/LBP/CETP N-terminal domain） and BPI2 domain（BPI/LBP/CETP C-terminal domain）.The molecular weight of the deduced protein was 51 551 u,and the isoelectric point 8.69.The amino acid sequence of CiBPI/LBP possessed 90%,73%,73%,and 72% identities with the BPI/LBPs of Cyprinus carpio,Oncorhynchus mykiss,Ictalurus punctatus and Plecoglossus altivelis altivelis respectively.CiBPI/LBP protein firstly clustered with BPI/LBP in Cyprinidae species,Cyprinus carpio in the phylogenetic analysis.CiBPI/LBP mRNA was detected in all the 15 tested tissues by semi-quantitative real-time RT-PCR,high in gill,head kidney and trunck kidney.【Conclusion】 The full-length CiBPI/LBP cDNA sequence was cloned successfully.CiBPI/LBP expression was ubiquitous.