中国兽医科学2017，Vol.47Issue(11)：1392-1398,7.DOI:10.16656/j.issn.1673-4696.2017.11.008
山羊Viperin基因的克隆与真核表达载体的构建
Cloning of caprine viperin gene and construction of eukaryotic expression vector
摘要
Abstract
Goat PBMCs was separated from blood sample and the viperin coding region gene was amplif ied by RT-PCR.BLAST analysis showed the nucleotide and amino acid sequences shared 99％ identity with other goat viperin sequences in GenBank.Viperin protein sequences of different species (goat,pig,human and mouse) were analyzed and N-terminal region showed high variety.Viperin gene with a HAtag at 5'or 3'end were then amplified and cloned into expression vector pcDNA3.1.The resulting positive plasmids,named as pcDNA-gViHAand pcDNA-HAgVi,respectively,were determined by PCR,enzyme digestion and sequencing.BHK21 cells were transfectedwith these plasmids using Lipofectamine 2 000.As detected by indirect immunofluscent assaywithanti-HAantibody,the recombinant proteins (HAgVi andgViHA) were successfully expressed and localized in the cytoplasm.Western-blot analysis showed that the proteins were expressed with a molecular weight of 43 ku.Confocal laser scaning microscope detection showed that both of the recombinant proteins localized in endoplasmic reticulum.The above-mentioned results should be useful for further studies on the antiviral activities and mechanism of goat viperin.关键词
Viperin基因/序列分析/真核表达/亚细胞定位/山羊Key words
viperin gene/sequence alignment/eukaryotic expression/subcellular localization/goat分类
农业科技引用本文复制引用
李照耀,李文良,吴铖楠,李基棕,郝飞,毛立,周斌,江杰元..山羊Viperin基因的克隆与真核表达载体的构建[J].中国兽医科学,2017,47(11):1392-1398,7.基金项目
国家自然科学基金项目(31402180) （31402180）
江苏省自然科学基金项目(BK20130729) （BK20130729）
