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Abstract：
CD48 is a glycosylphosphatidylinositol-anchored protein involved in lymphocyte adhesion, activation, and costimulation. In this study, the CD48 gene of tilapia (Oreochromis niloticus, named On-CD48), was cloned from the head kidney of tilapia. The coding sequences is 654 bp and encoding 217 amino acids. The deduced amino acid sequence of On-CD48 with an estimated molecular weight of 24.4 kDa and a theoretical pI of 5.03. Amino acid alignment indicated that it had two immunoglobulin-like domain conserved region. In healthy tilapia, the On-CD48 could be detected in all the examined tissues and the highest expression level in the spleen. The expression of On-CD48 in the spleen and head kidney was decreased after immunized by formalin-inactivated Streptococcus agalactiae, and the peak was observed in the spleen at 24 h and appeared again at 96 h, and in the head kidney gradual decline before 48 h then gradually increased to the original level. qPCR analysis of inactivated S. agalactiae, LPS and Poly I:C stimulated at the whole lymphocyte level showed that the stimulation of the Poly I:C was more sensitive. Prokaryotic expression results showed that efficient expression of On-CD48 protein could be realized after induced with 0.5 mmol L-1 IPTG in E. coli BL21 (DE3) for 10 h at 18 °C. The result of subcellular localization showed that On-CD48 were evenly distributed in the whole cell of HEK-293T. Western Blot confirmed that the molecular weight of the recombinant On-CD48 was about 21 kDa, consistent with the predicted result. The results of this study will lay a strong foundation for the further study of On-CD48 molecular function in tilapia.
摘要：
CD48是一种参与淋巴细胞粘附的糖基磷脂酰肌醇锚定蛋白，激活,和共同刺激。在这项研究中,罗非鱼（Oreochromisniloticus，名为On-CD48），是从罗非鱼的头肾中克隆出来的.编码序列为654bp,编码217个氨基酸。推断的On-CD48的氨基酸序列具有估计的24.4kDa的分子量和5.03的理论pI。氨基酸比对表明它具有两个免疫球蛋白样结构域保守区。在健康的罗非鱼中，可以在所有检查的组织中检测到On-CD48，并且在脾脏中表达水平最高。福尔马林灭活无乳链球菌免疫后，脾脏和头肾中On-CD48的表达降低，在24h时在脾脏中观察到峰值，并在96h时再次出现，并在头肾48h前逐渐下降，然后逐渐增加到原来的水平。灭活无乳链球菌的qPCR分析，LPS和PolyI：C在整个淋巴细胞水平上的刺激表明PolyI：C的刺激更敏感。原核表达结果表明,0.5mmolL-1IPTG在大肠杆菌BL21(DE3)中18℃诱导10h后,可实现On-CD48蛋白的高效表达。亚细胞定位结果显示,On-CD48在HEK-293T全细胞中均匀分布。Western印迹证实重组On-CD48的分子量约为21kDa，与预测结果一致。本研究结果为进一步研究罗非鱼On-CD48分子功能奠定了坚实的基础。
