鹅模式受体MDA5基因克隆及功能分析

黑素瘤差异化相关基因5(melanoma differentiation associated gene 5,MDA5)为一类胞内模式识别受体,能够识别入侵病毒的RNA链,在先天性免疫中发挥重要的抗病毒作用.本研究根据已发表的人和鸡MDA5基因序列设计引物,用overlap PCR技术从鹅成纤维细胞(goose embryo fibroblast cells,GEF)中扩增出鹅的MDA5基因,经序列测定发现它与鸡的MDA5序列有很高的同源性.将鹅MDA5真核表达质粒转染HEK-293T细胞,并用Poly(I:C)进行刺激,发现IFN-β的表达出现上调.同时,用新城疫病毒(Newcastle disease virus,NDV)感染GEF,发现MDA5 mRNA表达也出现了上调.本研究获得的gMDA5为国内首次报道证实的鹅MDA5基因,为进一步研究其在抗病毒中的作用奠定了基础.

SEQUENCING AND FUNCTION ANALYSIS OF GOOSE MDA5 GENE

Melanoma differentiation associated gene 5(MDA5),a family member of pattern recognition receptors(PRRs) that acts as a sensor for RNA virus,plays an important role in antiviral innate immunity.In our study,specific primers for goose MDA5(gMDA5)cDNA have been designed and synthesized according to the previously reported conserved nucleic acid sequence of human and chicken MDA5 cDNA.Goose MDA5 gene was amplified by overlap PCR from total RNA of goose embryo fibroblast cells(GEF).The amino acid alignments of goose and chicken MDA5 showed that they were highly homologous to each other.HEK-293T cells were transfected with gMDA5 or empty vector,then challenged with poly(I:C).We found the IFN-β promoter activity was increased compared with unstimulated cells.We also infected the GEF cells with Newcastle disease virus(NDV),and found the endogenous gMDA5 mRNA was up-regulated post infection.It is the first report of goose MDA5.This study has paved a way for future study on the antiviral function of goose MDA5.