Yeh Hung-Yueh, Klesius Phillip H
United States Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit, 990 Wire Road, Auburn, AL 36832-4352, United States.
Vet Immunol Immunopathol. 2007 Dec 15;120(3-4):246-53. doi: 10.1016/j.vetimm.2007.07.016. Epub 2007 Aug 2.
The channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59 gene was cloned and analyzed. Total RNA from tissues was isolated and cDNA libraries were constructed by the rapid amplification cDNA end (RACE) method. The gene-specific primers in conjunction with the RACE primers were used to PCR amplify 5'- and 3'-ends of the CD59 transcript. The complete channel catfish CD59 cDNA comprised 1109 bp including a 132-bp 5'-untranslated, a 360-bp open reading frame, and a 617-bp 3'-untranslated region. The open reading frame encodes a putative protein of 119-amino acid residues with calculated molecular mass (without potential glycosylation) of 13.2 kDa. However, the CD59 protein has a potential N-glycosylation site at the Asn35 residue. The degree of conservation of the channel catfish amino acid sequence to mammalian counterparts is 24-32%, while to those of other fish species is 44-54%. One remarkable feature is that the number and position of cysteine residues were conserved in the mature protein among species examined, suggesting that although the primary amino acid sequences are divergent, the three-dimensional structure of CD59 via disulfide linkages may be conserved through the evolutionary process. The putative protein could be further divided into three domains: a 21-amino acid signal peptide at the N-terminus, a 72-amino acid mature protein, and a 26-amino acid glycosylphosphatidylinositol (GPI) anchoring signal peptide at the carboxyl terminus. CD59 was expressed in all channel catfish tissues studied, suggesting that like mammals, channel catfish CD59 is constitutively expressed.
对斑点叉尾鮰（Ictalurus punctatus）补体膜攻击复合物抑制剂CD59基因进行了克隆和分析。从组织中分离出总RNA，并通过快速扩增cDNA末端（RACE）方法构建cDNA文库。使用基因特异性引物与RACE引物结合，通过PCR扩增CD59转录本的5'端和3'端。斑点叉尾鮰完整的CD59 cDNA包含1109 bp，包括一个132 bp的5'非翻译区、一个360 bp的开放阅读框和一个617 bp的3'非翻译区。该开放阅读框编码一个推定的蛋白质，由119个氨基酸残基组成，计算分子量（无潜在糖基化）为13.2 kDa。然而，CD59蛋白在Asn35残基处有一个潜在的N-糖基化位点。斑点叉尾鮰氨基酸序列与哺乳动物对应序列的保守程度为24%-32%，与其他鱼类的保守程度为44%-54%。一个显著特征是在所研究的物种中，成熟蛋白中半胱氨酸残基的数量和位置是保守的，这表明尽管一级氨基酸序列不同，但通过二硫键连接的CD59三维结构可能在进化过程中得以保留。推定的蛋白质可进一步分为三个结构域：N端的一个21个氨基酸的信号肽、一个72个氨基酸的成熟蛋白和C端的一个26个氨基酸的糖基磷脂酰肌醇（GPI）锚定信号肽。CD59在所有研究的斑点叉尾鮰组织中均有表达，这表明与哺乳动物一样，斑点叉尾鮰CD59是组成性表达的。
