Molecular cloning and functional characterization of the pig homologue of integrin-associated protein (IAP/CD47)
Molecular cloning and functional characterization of the pig homologue of integrin-associated protein (IAP/CD47)
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DOI：
10.1046/j.1365-2567.2002.01465.x
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发表时间：
2002-08-01
期刊：
影响因子：
6.4
通讯作者：
de la Lastra, JMP
中科院分区：
文献类型：
--
作者：
Shahein, YEA;de Andrés, DF;de la Lastra, JMP
我们报告的cDNA编码猪同源的人整合素相关蛋白（IAP或CD 47）的克隆。通过聚合酶链反应（PCR）扩增猪白细胞cDNA，使用基于来自不同物种的CD 47的已知序列之间的共有区域的引物，产生猪CD 47特异性探针。筛选猪主动脉平滑肌cDNA文库鉴定出7个克隆，均含有相同的序列。该克隆含有编码18个氨基酸的推定信号肽的开放阅读框（ORF），122个氨基酸的序列由单个细胞外免疫球蛋白可变（IgV）样结构域组成，随后是含有5个跨膜结构域的147个氨基酸的区域和16个氨基酸的胞质尾。克隆的氨基酸序列与人IAP的同源性为73%，因此将其称为猪IAP或CD 47。逆转录-聚合酶链反应（RT-PCR）显示，猪CD 47在广泛的组织中表达，并检测到不同的可变剪接形式。通过流式细胞术显示，单克隆抗体（mAb）BRIC 126（抗人CD 47）可染色猪血小板以及用编码猪CD 47的cDNA转染的中国仓鼠卵巢（CHO）细胞。在非还原条件下，猪血小板和血小板的蛋白质印迹分析显示分子量（MW）分别为43 000-50 000和55 000-65 000。猪CD 47在CHO细胞上稳定表达，并显示与人血小板反应蛋白（TSP）结合。BRIC 126抗体抑制血小板和CD 47转染细胞与人TSP和猪纤维蛋白原的结合，而对对照CHO细胞没有观察到影响。
We report the cloning of cDNA encoding the pig homologue of human integrin-associated protein (IAP or CD47). A pig CD47-specific probe was generated by polymerase chain reaction (PCR) amplification of pig leucocyte cDNA, using primers based on consensus regions among the known sequences of CD47 from different species. Screening of a pig aorta smooth muscle cDNA library identified seven clones, all containing identical sequences. The clones contained an open reading frame (ORF) that encoded an 18 amino acid putative signal peptide, a 122 amino acid sequence consisting of a single extracellular immunoglobulin variable (IgV)-like domain followed by a 147 amino acid region containing five membrane-spanning domains and a 16 amino acid cytoplasmic tail. The amino acid sequence of the clones was 73% homologous to human IAP and therefore it was termed pig IAP or CD47. Reverse transcription-polymerase chain reaction (RT-PCR) showed that pig CD47 was expressed in a wide range of tissues and detected different alternatively spliced forms. The monoclonal antibody (mAb) BRIC 126, anti-human CD47, was shown, by flow cytometry, to stain pig platelets as well as Chinese hamster ovary (CHO) cells transfected with the cDNA encoding pig CD47. Western blot analysis of pig erythocytes and platelets showed a molecular weight (MW) of 43 000-50 000 and of 55 000-65 000, respectively, under non-reducing conditions. Pig CD47 was stably expressed on CHO cells and shown to bind human thrombospondin (TSP). BRIC126 antibody inhibited the binding of platelets and of CD47-transfected cells to human TSP and to pig fibrinogen, whereas no effect was observed on control CHO cells.