剑尾鱼趋化因子CCL4和CCL19基因的克隆及嗜水气单胞菌感染对其组织表达的影响

Identification and expression analysis of two CC chemokines in swordtail fish(Xiphophorus helleri) responses to Aeromonas hydrophila challenges

趋化因子是一类能够吸引白细胞移行到感染部位的低分子量(8~10 ku)蛋白质,在炎症反应、非特异性免疫反应中具有重要作用。实验克隆了剑尾鱼2个趋化因子基因(CCL4、CCL19)c DNA序列,运用荧光定量PCR技术检测其在健康组织和嗜水气单胞菌感染后肝、脾中的表达情况。结果显示,剑尾鱼CCL4和CCL19开放阅读框长度分别为294和333 bp,分别编码97和110个氨基酸,推导氨基酸序列均含有趋化因子CC家族标志性的4个半胱氨酸残基,与其他物种尼罗罗非鱼、鼠、狗、人、鸡的氨基酸序列相似性比较,CCL4相似度分别为71.1%、33%、33%、31%、23.5%;CCL19相似度分别为62%、26.9%、24.5%、26.6%、27.8%。在检测的健康剑尾鱼8个组织中,CCL4和CCL19均有表达,其中CCL4和CCL19均在脾脏中表达量最高;CCL4在肌肉和肠中表达量较低,而CCL19则在心脏中表达量较低。经嗜水气单胞菌感染后,CCL4在剑尾鱼头肾中12 h表达量达到最高,在肝脏中24 h表达量最高;CCL19在头肾和肝脏中均在24 h时表达量最高。研究表明,趋化因子CCL4和CCL19可能参与剑尾鱼机体免疫调节反应,在抗嗜水气单胞菌感染过程中发挥了重要作用。

Chemokines are a large group of small proteins involved in migration, activation, and differentiation of leukocytes and are key regulators of the immune response and inflammatory response. In this study, we cloned and sequenced the c DNA of two CC chemokines（CCL4 and CCL19） from swordtail fish（X. helleri）. The ORF-length of CCL4 is 294 nucleotides（nt） encoding a protein of 97 amino acids（aa）, and the ORF-length of CCL19 is 333 nucleotides（nt） encoding a protein of 110 amino acids（aa）. Quantitative real-time RT-PCR（q RT-PCR） analysis revealed both two CC chemokines were constitutively expressed in all tissues examined. The CCL4 was highly expressed in the spleen, lowly in the muscle and intestine; the CCL19 had the highest expression in the spleen and the relatively lower level in the heart. After challenge with bacteria, the CCL4 and CCL19 shared the same expression trend. In the spleen and liver, the m RNA level increased remarkably at 12 h, which suggested that the two CC chemokines may be involved in the X. helleri defense against A. hydrophlia infection. In summary, this study demonstrates the basic profiles of two CC chemokines in X. helleri and chemokines take an important part in inflammatory and immune responses to bacteria. Together, our results also indicate that the X. helleri could be utilized as a useful fish model for the research of fish diseases.