草鱼cadm2b基因的克隆及在成体组织中的表达分析

为研究CADMs（Cell adhesion molecules）在草鱼构建抵御病害感染的第一道防线中发挥的作用，用RT-PCR和RACE方法结合测序分析，在草鱼脑组织中检测到了该基因家族成员cadm2b基因的4条不同的cDNA全长序列。序列比对结果表明这4条全长cDNA在5'端的序列完全相同，在3'端的3个局部区域有不同片段的缺失。因此，可以确定这4条不同的mRNA是cadm2b的不同剪接体。这4条不同的剪接体被分别命名为cadm2b、cadm2bX2、cadm2bX3和cadm2bX6。cadm2b的cDNA序列全长1669 bp，开放阅读框（ORF）1203 bp，编码400个氨基酸。cadm2bX2的cDNA序列全长2783 bp，开放阅读框长1323 bp，编码440个氨基酸；cadm2bX3的cDNA序列全长2755 bp，开放阅读框1296 bp，编码431个氨基酸；cadm2bX6基因的cDNA序列全长2649 bp，开放阅读框1161 bp，编码386个氨基酸。根据碱基序列所进行的氨基酸序列和蛋白结构预测显示这4个CADM2b蛋白亚型都具有CADM家族保守的4个功能区，但其C端的蛋白结合位点存在差异。CADM2b具有近膜4.1蛋白结合位点和Ⅱ型PDZ蛋白结合位点，CADM2bX2、X3缺失了PDZ蛋白结合位点，而CADM2bX6则同时缺失4.1蛋白和PDZ蛋白的结合位点。实时定量RT-PCR检测结果显示cadm2b剪接突变体是该基因mRNA的主要形式。半定量RT-PCR和套式PCR实验检测结果表明cadm2b基因在草鱼成体脑中高水平表达，在肝、肾、心脏和肌肉组织中有微量表达。这种表达模式提示草鱼中CADM2b主要是由非免疫细胞，而不是由免疫肥大细胞合成分泌的细胞黏附因子，可能通过介导免疫肥大细胞与病原靶细胞的黏附而起非特异性抵御病害感染的作用。

MOLECULAR CLONING AND EXPRESSING ANALYSIS OF CADM2B IN ADULT TISSUES OF GRASS CARP,CTENOPHARYNGODONIDELLUS

CADMs (cell adhesion molecules) family play important roles in establishing the first line of defense against illnesses and infections by mediating adhesion between immune mast cells and their target cells. Grass carp (Ctenopha-ryngodon idellus) has low livability at the age of 1 and 2 due to its susceptibility to infection by virus or bacterium. To investigate whether CADMs participate in building the first line of defense against infection in grass carp, we cloned the mRNA of grass carp cadm2b, and identify 4 different full length cDNA of cadm2b from grass carp brain tissue. Ac-cording to sequences alignment, sequences of the 5′terminal are identical among all the four cDNAs while various de-leted fragments found in three different position in their 3′terminals. These results indicated that these four mRNAs, named cadm2b, cadm2bX2, cadm2bX3 and cadm2bX6, are splicing variants from cadm2b gene. The full length of cadm2b is 1669 bp with a 1203 bp long open reading frame (ORF) coding 400 amino acids. The full length of cadm2bX2 is 2783 bp with a 1323 bp long ORF coding 440 amino acids. The full length of cadm2bX3 is 2755 bp with a 1296 bp long ORF coding 431 amino acids. The full length of cadm2bX6 cDNA is 2649 bp with a 1161 bp long ORF coding 386 amino acids. Prediction of amino acid sequences base on nucleotide sequenceds showed that all the four CADM2b isoforms contain the four conserve functional domains of CADM family in the N-terminals, but the C-ter-minals are variance. CADM2b has a juxtamembrane 4.1 protein binding domain and PDZ typeⅡprotein binding do-main in the C-terminal. Both CADM2bX2 and CADM2bX3 lack the PDZ type Ⅱ protein binding domain. CADM2bX6 possesses neither the juxtamembrane 4.1 protein binding domain nor the PDZ typeⅡprotein binding do-main. Quantitative RT-PCR results suggested that splicing variant cadm2b is the main form of cadm2b mRNA. A high-level cadm2b was detected in brain tissue and a very low-level cadm2b was detected in liver, kidney, heart and muscle. These findings suggest that CADM2b is a cell adhesion molecule synthesized and secreted by nonimmune cells, and might play a role in against various infections by mediating adhesion between immune mast cells and their target cells in grass carp.