Zhang Jinzhou, Ao Jingqun, Chen Xinhua
School of Life Sciences, Xiamen University, Xiamen 361005, PR China.
Fish Shellfish Immunol. 2008 Nov;25(5):664-71. doi: 10.1016/j.fsi.2008.09.003. Epub 2008 Sep 16.
A full-length CC chemokine cDNA was isolated from large yellow croaker (Pseudosciaena crocea) by expressed sequence tag (EST) analysis (LycCC2). Its open reading frame (ORF) is 282 nucleotides (nt), encoding a polypeptide of 93 amino acids (aa). The deduced LycCC2 contains a 20-aa signal peptide and a 73-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CC chemokines (C(34), C(35), C(61) and C(75)). It shares 17.4-40.4% and 10.0-24.5% aa sequence identities to other known bony fish and mammalian CC chemokines, respectively. Tissue expression distribution analysis showed that LycCC2 gene was constitutively expressed in eight of all nine tissues examined, except for liver. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, LycCC2 gene expression was obviously up-regulated in blood, kidney, heart, spleen, intestine and gills at 12 h post-induction, and also induced in liver. The time-course analysis using a real-time PCR showed that LycCC2 transcript in spleen and kidney was quickly increased by either poly(I:C) or bacterial vaccine and reached peak levels at 12h, followed by a recovery to normal level after 48h. LycCC2 expression in spleen and kidney was more strongly up-regulated by bacterial vaccine (37.3- and 17.4-fold mRNA increases at 12h post-induction, respectively) than by poly(I:C) (15.5- and 8.5-fold mRNA increases, respectively). Recombinant LycCC2 (rLycCC2) protein produced in Pichia pastoris exhibited marked chemotactic ability for the peripheral blood leucocytes (PBLs) from large yellow croaker. These results suggested that LycCC2 represents a novel member of the fish CC chemokine family, which may be involved in a pro-inflammatory function in the immune response triggered by bacterial vaccine or poly(I:C) in large yellow croaker.
通过表达序列标签（EST）分析，从大黄鱼（Pseudosciaena crocea）中分离出一个全长CC趋化因子cDNA（LycCC2）。其开放阅读框（ORF）为282个核苷酸（nt），编码一个93个氨基酸（aa）的多肽。推导的LycCC2包含一个20个氨基酸的信号肽和一个73个氨基酸的成熟多肽，其具有其他已知CC趋化因子中典型的四个半胱氨酸排列（C(34)、C(35)、C(61)和C(75)）。它与其他已知硬骨鱼和哺乳动物CC趋化因子的氨基酸序列同一性分别为17.4 - 40.4%和10.0 - 24.5%。组织表达分布分析表明，LycCC2基因在所有九个检测组织中的八个中组成性表达，肝脏除外。在用聚肌胞苷酸（poly(I:C)）或灭活三价细菌疫苗刺激后，LycCC2基因表达在诱导后12小时时在血液、肾脏、心脏、脾脏、肠道和鳃中明显上调，在肝脏中也被诱导。使用实时PCR进行的时间进程分析表明，脾脏和肾脏中的LycCC2转录本在受到poly(I:C)或细菌疫苗刺激后迅速增加，并在12小时达到峰值水平，随后在48小时后恢复到正常水平。脾脏和肾脏中LycCC2的表达受到细菌疫苗的上调作用更强（诱导后12小时时mRNA分别增加37.3倍和17.4倍），而受到poly(I:C)的上调作用较弱（分别增加15.5倍和8.5倍）。在毕赤酵母中产生的重组LycCC2（rLycCC2）蛋白对大黄鱼外周血白细胞（PBLs）表现出明显的趋化能力。这些结果表明，LycCC2代表鱼类CC趋化因子家族的一个新成员，它可能在大黄鱼中由细菌疫苗或poly(I:C)触发的免疫反应中参与促炎功能。
