虎源传染性鼻气管炎病毒gD基因重组真核表达载体的构建

Construction of Eukaryotic Recombinant Expression Plasmid Containing gD Gene of Feline Herpesvirus Type 1 from Tiger

根据从虎气管组织扩增获得的猫传染性鼻气管炎病毒相关核酸序列,设计扩增编码虎源猫传染性鼻气管炎病毒gD蛋白基因片段的引物,通过PCR扩增出gD基因片段,并成功插入到pMD18-T Simple载体中,筛选获得重组质粒,命名为18T-gD。重组质粒通过HindⅢ和XhoⅠ酶切后,插入pcDNA 3.1(+)真核表达载体构建出gD基因的重组真核表达质粒pcDNA-gD。用脂质体将重组真核表达质粒pcDNA-gD转染293T细胞,通过RT-PCR和Western blot检测,结果显示gD基因在真核细胞中得到正确转录和表达,所表达蛋白分子质量约为42.29ku,为虎源猫传染性鼻气管炎基因疫苗的研究奠定了基础。

The primers of gD genes of the FHV-1 were designed according to sequences of the FHV-1 from GenBank,gD gene was amplified by PCR and cloned successfully into pMD18-T Simple vector.gD gene was constructed into eukaryotic expression vector pcDNA 3.1（＋）,the recombinant plasmid ,named as pcDNA-gD,was transfected into 293T cells.The results of RT-PCR and Western blot indicated that the gD gene was transcripted and expressed,and the recombinant fusion protein was about 42.29 ku.The results lay an important foundation for the further research of FHV-1.