Abstract：
CD28 and CD80/86 are crucial co-stimulatory molecules for the T cell activation. Previous study illustrated that CD28 and CD80/86 present on T cells and antigen-presenting cells in flounder (Paralichthys olivaceus), respectively. The co-stimulatory molecules were closely associated with cell immunity. In this paper, recombinant protein of flounder CD80/86 (rCD80/86) and phytohemagglutinin (PHA) were added to peripheral blood leukocytes (PBLs) in vitro. Lymphocytes were significantly proliferated with CFSE staining, and the proportion of CD4+ and CD28+ lymphocytes significantly increased. In the meantime, genes related to the CD28-CD80/86 signaling pathway or T cell markers were significantly upregulated (p < 0.05). For further study, the interaction between CD80/86 and CD28 was confirmed. The plasmid of CD28 (pCD28-FLAG and pVN-CD28) or CD80/86 (pVC-CD80/86) was successfully constructed. In addition, pVN-ΔCD28 without the conserved motif \"TFPPPF\" was constructed. The results showed that bands of pCD28-FLAG bound to rCD80/86 were detected by both anti-FLAG and anti-CD80/86. pVN-CD28 complemented to pVC-CD80/86 showing positive fluorescent signals, and pVN-ΔCD28 failed to combine with pVC-CD80/86. The motif \"TFPPPF\" in CD28 played a crucial role in this linkage. These results indicate that CD28 and CD80/86 molecules interact with each other, and their binding may modulate T lymphocytes immune response in flounder. This study proved the existence of CD28-CD80/86 signaling pathway in flounder.
摘要：
CD28和CD80/86是T细胞活化的关键共刺激分子。先前的研究表明，CD28和CD80/86存在于比目鱼（Paralichthysolivaceus）的T细胞和抗原呈递细胞上，分别。共刺激分子与细胞免疫密切相关。在本文中,将比目鱼CD80/86（rCD80/86）和植物血凝素（PHA）的重组蛋白体外添加到外周血白细胞（PBLs）中。CFSE染色淋巴细胞明显增殖，CD4+和CD28+淋巴细胞比例显著增高。同时,与CD28-CD80/86信号通路或T细胞标志物相关的基因显著上调(p<0.05)。为了进一步研究，CD80/86和CD28之间的相互作用得到证实.成功构建了CD28（pCD28-FLAG和pVN-CD28）或CD80/86（pVC-CD80/86）的质粒。此外,构建了没有保守基序“TFPPPF”的pVN-ΔCD28。结果表明，抗FLAG和抗CD80/86均检测到pCD28-FLAG与rCD80/86结合的条带。pVN-CD28与pVC-CD80/86互补，显示阳性荧光信号，和pVN-ΔCD28未能与pVC-CD80/86组合。CD28中的主题“TFPPPF”在这种联系中起着至关重要的作用。这些结果表明CD28和CD80/86分子相互作用，它们的结合可以调节比目鱼T淋巴细胞的免疫反应。这项研究证明了比目鱼CD28-CD80/86信号通路的存在。
