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

本研究分离山羊外周血单核细胞,设计引物,采用RT-PCR方法扩增出Viperin基因完整阅读框。经BLAST分析,该基因的核苷酸序列和推导的氨基酸序列与GenBank中已有序列的最高同源性均为99%。同时将不同物种(山羊、猪、人、小鼠)的氨基酸序列进行比对分析,发现N端结构域在不同物种间是高变区。然后,将目的基因酶切克隆入真核表达载体pc DNA3.1中构建重组表达质粒。经PCR、酶切和测序鉴定正确的阳性质粒分别被命名为pcDNA-g Vi HA和pcDNA-HAg Vi。使用脂质体Lipofectamine 2 000将重组质粒转染BHK21细胞,使用HA抗体进行间接免疫荧光检测,发现重组蛋白均得到表达,荧光分布于细胞质中;Western-blot结果表明,两个质粒转染后均可检测到分子量约为43 ku的条带。使用内质网标志物Calnexin抗体进行共聚焦检测,发现表达的Viperin蛋白与内质网存在共定位。上述研究结果为进一步研究山羊Viperin的抗病毒作用及其机制奠定了基础。

Goat PBMCs was separated from blood sample and the viperin coding region gene was amplified by RT-PCR.BLAST analysis showed the nucleotide and amino acid sequences shared 99% identity with other goat viperin sequences in Gen Bank.Viperin protein sequences of different species（goat,pig,human and mouse） were analyzed and N-terminal region showed high variety.Viperin gene with a HA tag at 5′or 3′end were then amplified and cloned into expression vector pc DNA 3.1.The resulting positive plasmids,named as pc DNA-g Vi HA and pc DNA-HAg Vi,respectively,were determined by PCR,enzyme digestion and sequencing.BHK21 cells were transfected with these plasmids using Lipofectamine 2 000.As detected by indirect immunofluscent assay with anti-HA antibody,the recombinant proteins（HAg Vi and g Vi HA） 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.