松江鲈肌酸激酶的基因克隆、表达与功能分析

Molecular identification, expression and function analysis on creatine kinase in Trachidermus fasciatus

前期蛋白质质谱分析发现,松江鲈(Trachidermus fasciatus Heckel)经鳗弧菌(Vibrio anguillarum)刺激后,其皮肤黏液中的肌酸激酶(creatine kinase, CK)的表达明显上调。目前,肌酸激酶在鱼类中的功能研究尚未深入开展。本文利用RACE技术克隆获得了松江鲈CK基因的全长cDNA序列(命名为TfM-CK)。TfM-CK基因的cDNA全长为1 474 bp,开放阅读框为1 146 bp,编码381个氨基酸。序列比对分析显示,TfM-CK序列高度保守。实时荧光定量PCR显示,TfM-CK mRNA广泛表达于松江鲈各组织,在肌肉中的相对表达量最高,其次为血液。鳗弧菌刺激后,TfM-CK mRNA在肌肉、皮肤、脾脏和头肾中均上调表达。其中,脾脏中的表达量高达对照组的900多倍。原核重组表达纯化的TfM-CK(rTfM-CK)蛋白酶活测定实验结果显示,重组蛋白的酶活为22.0 U/mg。rTfM-CK对鳗弧菌、大肠杆菌(Escherichia coli)、巨大芽孢杆菌(Bacillus megaterium)和金黄色葡萄球菌(Staphylococcus aureus)有较强的凝集作用。由以上实验结果推测,TfM-CK可能通过细菌凝集作用参与到了松江鲈抵抗病原菌的先天免疫防御过程中。本研究为认识鱼类肌酸激酶的功能及其在病原菌防御过程中的分子免疫机制机制提供了参考。

In previous study,we found that creatine kinase(CK)was one of the proteins which were up-regulated in the skin mucus of roughskin sculpin Trachidermus fasciatus post Vibrio anguillarum injection.To date,the function of this gene has not been well studied in fish.In this study,the cDNA sequence of CK gene was cloned using3′RACE and5′RACE techniques from roughskin sculpin,named as TfM-CK.The full-length of TfM-CK cDNA was1474bp,which contained an open reading frame(ORF)of1146bp encoding a polypeptide of381amino acids.The sequence BLASTP in NCBI indicated that TfM-CK was highly conserved.Quantitative real-time PCR(qPCR)analysis showed that TfM-CK mRNA expressed in all the detected tissues,with the highest expression in the muscle.After Vibrio anguillarum infection,TfM-CK transcripts were up-regulated significantly in the muscle,spleen,skin and head kidney,with the highest increase of900fold in the spleen.The kinase activity of recombinant protein of TfM-CK(rTfM-CK)was relatively high with22.0U/mg protein.It displayed high agglutination activity to all4tested bacterial including2Gram-negative bacteria Escherichia coli,Vibrio Anguillarum and2Gram-positive bacteria Staphylococcus aureus and Bacillus megaterium.These data indicate that TfM-CK participate in the fish immune response against pathogen infection.The present results may provide a theoretical basis for further investigation into the functions of fish TfM-CK and its molecular mechanisms of regulating fish immune response to pathogens.