羊传染性脓疱病毒ORF047基因表达及其蛋白在细胞中的定位分析

Orf virus ORF047 gene eukaryotic expression and cell localization

目的克隆羊传染性脓疱病毒(Orf virus,ORFV)ORF047基因,并进行真核表达及亚细胞定位,为筛选与其相互作用的蛋白提供依据。方法提取羊传染性脓疱病毒分离株的DNA,以其为模板,参照OV-SA00毒株(NC-005336.1)基因ORF047序列,采用PCR技术扩增ORFV ORF047基因片段,凝胶回收后将其插入pEGFP-N1载体,构建重组质粒pEGFPORF047,测序后重组质粒共转染293T细胞,Western blotting鉴定融合蛋白的表达。后将重组质粒pEGFP-ORF047与pHcRed1-Nuc、pHCRed1-mito、pHcRed1-ER分别共转染Vero-E6细胞,通过激光共聚焦显微镜对融合蛋白进行亚细胞定位分析。结果 ORFV ORF047基因片段大小为735bp;重组质粒pEGFP-ORF047能够在293T细胞中明显表达融合有目的分子的绿色荧光蛋白,Western blotting检测表达的融合蛋白大小约54kD;亚细胞定位分析表明,ORF047蛋白主要定位于胞浆中,少量在线粒体中。结论成功分析了ORF047基因的表达与其蛋白在亚细胞定位。

The object of study was to clone the gene of ORFV ORF047 and study the eukaryotic expression and cell localization,making the theoretical basis for the subsequest screening of protein that interact with ORF047.ORF047 gene was amplificated by the specifical primer from the DNA of ORFV using PCR,the length was 735 bp,compared with L1 published in NC-005336.1,the homologies of the nucleotide acid sequence and amino acid sequence were 98.8%and 98.8%.In order to defined the expression and location of the ORF047 gene in cell,the recombinant plasmid pEGFP-ORF047 was constructed and transfected into 293T cell,after 36 h,the green fluorescence could be observed under fluorescence microscope,and 54 kD protein was detected by western bloting.The plasmid of pHcRed1-Nuc,pHcRed1-Mito and pHcRed1-ER with the recombinant plasmid of pEGFP-ORF047 was cotransfected to veroE6 cell respectively,that fusion protein of ORF047 was mainly located in the cytoplasm,a small amount in the mitochondriabyconfocal microscopy analysis.