团头鲂NK-lysin基因鉴定和表达分析

Identification, characterization and expression of NK-lysin in Megalobrama amblycephala

为研究抗菌肽NK-LYSIN在团头鲂免疫系统中的可能功能,本实验从团头鲂转录组中钓取到2个NK-lysin基因(nkla和nklb),通过PCR扩增并测序验证其ORF序列。通过荧光定量PCR检测其在健康组织和嗜水气单胞菌感染后免疫组织中的表达,并构建其原核表达体系。结果显示,团头鲂nkla和nklb分别编码122和136个氨基酸,NKLA和NKLB均属于鞘脂激活蛋白样蛋白家族成员,各含有6个高度保守的半胱氨酸及1个Sap B结构域。在健康团头鲂各组织中,nkla在脾脏中表达量极高,在肌肉和血液中表达量极低;而nklb则在肠中表达量较高,在头肾中表达量较低,在其他组织均有表达。nkla和nklb不同的表达模式暗示它们可能存在功能上的分化。经嗜水气单胞菌感染后,团头鲂nkla与nklb表达变化模式相似,头肾中,表达量在4 h时显著下降;肝脏中,表达量在4 h达到最高,而后回落到正常水平;在脾脏和肠中4 h时开始下降,72 h时达到峰值,暗示NK-lysin可能在团头鲂抗嗜水气单胞菌感染过程中发挥重要作用。另外,本实验对nkla和nklb进行了原核表达,为进一步研究NK-lysin的功能奠定了基础。

NK-LYSINs are small cationic antimicrobial peptides produced by cytotoxic T lymphocytes and natural killer cells. Antimicrobial peptides, the important components of innate immune systems, have attracted scientists＇ attention because they can be served as novel antimicrobials. Megalobrama amblycephala is an important freshwater fish cultured in our country. A serious disease, bacterial septicemia caused by Aeromonas hydrophila is a big threat to M. amblycephala, which might lead to a high mortality. In order to understand the possible function of NK-lysin in the M. amblycephala immune system, we amplified and characterized the nkla and nklb genes in M.amblycephala, which encode 122 and 136 amino acids respectively. Sequences analysis showed that NK-lysin belong to the saposin-like protein family, with 6 highly conserved cysteines and a Sap B domain. In different healthy fish tissues, the M. amblycephala nkla was highly expressed in the spleen, lowly in the muscle and blood,While nklb had the highest expression in the intestine and the relative lower level in the head kidney. However,after A. hydrophila infection, the M. amblycephala nkla and nklb shared the same expression trend. In the head kidney, the m RNA level decreased remarkablely at 4 h. In the liver, the expression increased to the peak significantly at 4 h and then fell down to the normal level. However, in the spleen and intestine, the expression fell to the bottom at 12 h and then reached the peak at 72 h. These results suggest that NK-lysin might play a vital role in the M. amblycephala defense against A. hydrophlia infection. What＇s more, we constructed prokaryotic expression system and obtained the recombinant proteins of M. amblycephala nkla and nklb, which may provide information for NK-lysin＇s further functional study.