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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。
In this study, goat peripheral blood mononuclear cells were isolated and primers were designed. The complete reading frame of Viperin gene was amplified by RT-PCR. According to BLAST analysis, the nucleotide and deduced amino acid sequences of this gene shared 99% identity with the existing sequences in GenBank. At the same time, amino acid sequences of different species (goat, pig, human and mouse) were compared and analyzed. It was found that N-terminal domain was hypervariable between different species. Then, the target gene was digested and cloned into the eukaryotic expression vector pcDNA3.1 to construct a recombinant expression plasmid. The correct positive plasmids were identified by PCR, restriction enzyme digestion and sequencing. They were named pcDNA-g Vi HA and pcDNA-HAg Vi. The recombinant plasmids were transfected into BHK21 cells using Lipofectamine 2000, and indirect immunofluorescence assay with HA antibody showed that all the recombinant proteins were expressed in the cytoplasm. The results of Western-blot showed that after the two plasmids were transfected Bands with a molecular weight of about 43 ku can be detected. The endoplasmic reticulum Calnexin antibody was used for confocal detection, and it was found that the expressed Viperin protein co-localized with the endoplasmic reticulum. These results provide a basis for further study on the anti-viral effect of goat Viperin and its mechanism.