斑马鱼ftr56基因克隆表达及功能研究

MOLECULAR CLONING, EUKARYOTIC EXPRESSION AND FUNCTION STUDY OF FTR56 FROM DANIO RERIO

为进一步了解硬骨鱼类特有的finTRIM(Fish novel tripartite motif,ftr)在斑马鱼(Danio rerio)抗病毒天然免疫中的作用,研究克隆了斑马鱼ftr56基因并分析了其对鲤春病毒血症病毒(Spring viremia of carp virus,SVCV)增殖的抑制作用。根据NCBI中斑马鱼ftr56序列设计引物,采用PCR方法,扩增ftr56 CDS区,连接至真核表达载体pcDNA4.0-His,构建真核表达质粒pcDNA4.0-ftr56-His,进行生物信息学分析。采用实时荧光定量PCR(qRT-PCR)技术检测SVCV感染斑马鱼胚胎成纤维细胞(ZF4)后ftr56 mRNA的变化。系统进化树分析显示,斑马鱼FTR56单独聚为一支。氨基酸多序列比对结果显示,其与黑猩猩、牛、鼠的TRIM56相似度为22%—23%。FTR56二级结构具有1个RING指结构域,1个B-box结构域,1个卷曲螺旋结构域和1个B30.0结构域。qRT-PCR检测结果显示,ftr56在SVCV感染后24h表达量显著上升。在过表达ftr56后,SVCV的G蛋白mRNA水平和蛋白水平在12h和24h相比对照组明显减少,培养基上清中SVCV滴度也明显降低,表明FTR56抑制SVCV的增殖。实验为进一步揭示finTRIM在鱼类病毒性疾病中的免疫调节机制提供参考。

To understand the role of zebrafish finTRIM in antiviral innate immunity,the zebrafish ftr56 gene was cloned and analyzed for its effect on the proliferation of spring viremia of carp virus(SVCV).Primers were designed according to the zebrafish FTR56 sequence.The FTR56 CDS region was amplified by PCR and ligated into the eukaryotic expression vector pcDNA4.0-His to construct the eukaryotic expression plasmid pcDNA4.0-FTR56-His and conducted bioinformatics analysis.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of FTR56 mRNA in SVCV-infected zebrafish embryo fibroblasts(ZF4).Phylogenetic tree analysis showed that the zebrafish FTR56 was individually clustered.The amino acid sequence alignment showed that the similarites with TRIM56 of chimpanzees,cattle and mice were 22%—23%.The FTR56 secondary structure has one RING finger domain,one Bbox domain,one coiled-coil region and one B30.0 domain.The FTR56 mRNA level increased significantly at 24h after SVCV infection.After overexpression of FTR56,the mRNA and protein levels of G protein of SVCV reduced significantly at 12h and 24h compared with the control group associated with significantly decreased SVCV titers in the culture supernatant,indicating that FTR56 inhibited SVCV proliferation.This study provide a reference for further revealing the immunoregulatory mechanism of finTRIM in fish viral diseases.