Choi I S, Hash S M, Collisson E W
Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467, USA.
Vet Immunol Immunopathol. 2000 Aug 31;76(1-2):45-59. doi: 10.1016/s0165-2427(00)00192-6.
Feline CD28 and CTLA-4 (CD152) cDNA were cloned from Con-A stimulated feline peripheral blood mononuclear cells (PBMC) by rapid amplification of cDNA end-PCR (RACE-PCR). Both CD28 and CTLA-4 proteins belong to the immunoglobulin superfamily (Ig SF) and are composed of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain. The open reading frame (ORF) of CD28 cDNA encoded a predicted protein of 221 amino acids and that of CTLA-4 cDNA encoded a predicted protein of 223 amino acids. The B7 ligands binding motif MYPPPY hexamer was found on the extracellular Ig V-like domains of both receptors and phosphatidylinositol 3-kinase (PI 3-kinase) binding motifs pYMNM for CD28 and pYVKM for CTLA-4 were identified in the cytoplasmic domains. Comparisons of amino acid sequences of feline proteins with known sequences of other species indicated that rabbit CD28 and CTLA-4 were most closely related and mouse molecules were the least conserved with feline molecules. Comparison of each domain of both molecules with that of other animals showed that the cytoplasmic domain of CTLA-4 was 100% conserved and that of CD28 was the most conserved domain. The cloned CD28 and CTLA-4 cDNA could be expressed in transfected mammalian cells. Expression of feline CD28 and CTLA-4 mRNA in freshly isolated feline PBMC was demonstrated by RT-PCR. Stimulation of PBMC with Con-A similarly increased the expression of both CD28 and CTLA-4 mRNA.
通过cDNA末端快速扩增PCR（RACE-PCR）从刀豆蛋白A刺激的猫外周血单个核细胞（PBMC）中克隆出猫CD28和CTLA-4（CD152）cDNA。CD28和CTLA-4蛋白均属于免疫球蛋白超家族（Ig SF），由信号序列、胞外结构域、跨膜结构域和胞质结构域组成。CD28 cDNA的开放阅读框（ORF）编码一个预测的221个氨基酸的蛋白质，CTLA-4 cDNA的开放阅读框编码一个预测的223个氨基酸的蛋白质。在两种受体的胞外Ig V样结构域上发现了B7配体结合基序MYPPPY六聚体，在胞质结构域中鉴定出了CD28的磷脂酰肌醇3激酶（PI 3激酶）结合基序pYMNM和CTLA-4的pYVKM。猫蛋白氨基酸序列与其他物种已知序列的比较表明，兔CD28和CTLA-4与猫的关系最密切，而小鼠分子与猫分子的保守性最低。两种分子各结构域与其他动物的比较表明，CTLA-4的胞质结构域100%保守，CD28的胞质结构域是最保守的结构域。克隆的CD28和CTLA-4 cDNA可在转染的哺乳动物细胞中表达。通过RT-PCR证实了猫CD28和CTLA-4 mRNA在新鲜分离的猫PBMC中的表达。用刀豆蛋白A刺激PBMC同样增加了CD28和CTLA-4 mRNA的表达。
