异育银鲫c型溶菌酶全长cDNA序列的生物信息学分析及其组织表达分析

利用RACE及克隆等方法获得了异育银鲫（Carassius auratus gibelio）c型溶菌酶基因全长cDNA序列．序列分析表明，所克隆的异育银鲫溶菌酶的cDNA全长751 bp，包括溶菌酶基因开放阅读框（ORF）438 bp,5′ 非编码区（UTR）为109 bp和3′ UTR为204 bp．438 bp ORF共编码146个氨基酸，其成熟肽的分子量预测值为14　543.6,理论等电点为8.86．通过ClustalW软件,将异育银鲫和其它多个物种c型溶菌酶的氨基酸序列进行多序列比对发现，所克隆的异育银鲫溶菌酶编码的氨基酸序列中存在c型溶菌酶的活性中心（Glu53和Asp69），且与活性位点相邻的氨基酸序列高度保守．同时,8个保守的半胱氨酸残基也与其它物种的c型溶菌酶相一致．结合BLASTN分析的结果，可以确认所获得的异育银鲫溶菌酶cDNA序列属于c型溶菌酶．异育银鲫c型溶菌酶和人c型溶菌酶（pdb 1at6_）在蛋白质序列上有50%相似性，其三维（3-D）结构非常类似．通过氨基酸空间位置比较发现，两者具有类似的酶活中心，异育银鲫c型溶菌酶只能形成3个二硫键，比人少1个．荧光定量RT-PCR检测和溶菌酶活性测定显示，异育银鲫头肾和脾脏c型溶菌酶mRNA的表达量约为肝胰脏的2.9 倍和1.7 倍，异育银鲫头肾和脾脏的溶菌酶活性约为肝胰脏的6.2 倍和4倍．

Molecular Cloning of Full-length c-Type Lysozyme from Carassius auratus gibelio and Its Tissue-specific Expression and Bioinformatic Analysis

We have successfully cloned a 751 bp full-length lysozyme cDNA by rapd amplification of cDNA ends (RACE) methods from Carassius auratus gibelio,which consists of a 438 bp open reading frame (ORF) encoding 146 amino acids, a 109 bp 5 -untranslated region and a 204 bp 3 -untranslated region. The fluorescent quantitative RT-PCR analysis indicated that its mRNA levels in the cephalo-kidney and the spleen were 2.9 and 1.7 folds higher than that of the liver in Carassius auratus gibelio, and the lysozyme catalytic activities were determined to be 6.2 and 4 folds as compared to liver respectively. The molecular weight of this mature peptide was estimated to be 14　543.6 and the pI was 8.86. The BlastN search revealed the cloned lysozyme was homologous to c-type lysozymes from several other species with the conserved Glu53 and Asp69 at the catalytic sites, as well as other eight structural cysteine residues. Multiple alignment analyses with ClustalW have shown the sequences flanking the catalytic sites were also highly conserved. By three-dimensional (3-D) structure prediction suggested this c-type lysozyme of Carassius auratus gibelio was sharing 50% similarity to its human homolog in structural alignment, which possess two enzymatic sites, except the predicted disulfide bonds was 3 instead of 4in human c-type lysozymes.