草鱼CRP基因全长cDNA的克隆及其表达分析

Molecular cloning and expression of the full length cDNA of C-reaction protein from grass carp

利用草鱼C-反应蛋白(CRP)的EST序列(CK233056)设计引物,采用快速扩增cDNA末端(SMART-RACE)的方法克隆CRP基因的5′末端,获得1个约520bp的cDNA片段.将该片段与EST拼接,得到CRP基因全长cDNA,长度为889bp,含有1个672bp的开放阅读框,两侧分别有74bp和143bp的5′和3′非翻译区域(open reading frame,ORF),CRP基因编码224个氨基酸残基,N端含有15个氨基酸残基的信号肽.利用RT-PCR半定量法对健康及被嗜水产气单胞菌人工感染的草鱼的心脏、脑、前肠、肾脏、肝胰脏、肌肉、脾等组织CRP的mRNA表达水平进行检测,结果表明,CRP基因在7种组织中均有表达,表达水平差异不明显.在人工感染24h后,各组织中CRP的mRNA水平平均升高5倍左右,与哺乳动物和一些鱼类在炎症反应中血清CRP水平升高一致,而人工感染48h后,除心脏和肌肉组织中有显著降低外,其他组织没有明显变化,仍维持较高水平.

A 520 bp of 5＇-end ofCRP cDNA from grass carp, was cloned using the primers based on EST sequence of CRP （CK233056） by SMART-RACE. This 520 bp 5＇-end fragment was processed with the CRP EST and a full-length cDNA of CRP was achieved. CRP cDNA was found to contain an ORF of 672 nucleotides flanked by a 74 nucleotides of 5＇-UTR and a 143 nucleotides of 3＇-UTR. The deduced amino-acid sequence contained 224 amino acid residues with a 15 signal peptide of amino-acid residues in the N-terminal. To detect the mRNA expression of CRP in different tissues and different condition, RT-PCR assay was performed using the tissues of heart, brain, foreintestine, kidneys, hepatopancrease, muscle and spleen obtained from healthy fish and artificially infected grass carp by Aerornonas hydrophila. The results showed that CRP mRNA were expressed in all seven tissues without obvious difference between each other, and expression levels of CRP of the infected fish increased averagely 5 folds after 24 hours of infection which was consistent with the changes of serum CRP level in mammalians and some fishes in response to inflammation, whereas no changes in most tissues after 48 hours of infection except in the heart and muscle.