摘要
为研究羊口疮病毒(ORFV)VIR蛋白的生物活性,扩增了绿色荧光蛋白基因EGFP和ORFV的VIR基因,构建了真核表达载体pVAX1-EGFP和pVAX1-VIR-EGFP,转染至BHK-21细胞,在荧光显微镜下进行了EGFP和VIR与EGFP融合蛋白的绿色荧光检测,用ORFV的全病毒血清作一抗,进行了Western blot检测,应用生物信息学方法,构建了VIR蛋白和VIR与EGFP融合蛋白的三级结构并进行了比较,进行了细胞表位预测。结果显示,pVAX1-EGFP和pVAX1-VIR-EGFP载体在BHK-21细胞中均表达出了绿色荧光,但VIR与EGFP融合蛋白表达的荧光弱于EGFP表达的荧光,Western blot检测出了53 ku左右的融合蛋白条带,说明VIR与EGFP融合蛋白具有较高的特异性。VIR蛋白和VIR与EGFP融合蛋白的三级结构直观地反映了VIR蛋白和VIR与EGFP融合蛋白的差异,预测出VIR蛋白的优势B细胞抗原表位4个,HLA-A*0201限制性CTL优势表位3个,为VIR蛋白的生物学功能研究奠定了基础。
To study the biological characteristics of VIR protein of Orf virus(ORFV),in this study,the EGFP gene and the VIR gene of ORFV were amplified,The eukaryotic expression vector pVAX1-EGFP containing EGFP gene and the eukaryotic expression vector pVAX1-VIR-EGFP containing VIR gene were constructed and transfected into BHK-21 cells using liposomes,the green fluorescence of EGFP protein and VIR fusion protein with EGFP was detected by fluorescence microscope.The Wetern blot analysis was performed by extracted total protein from the serum of orf virus as substrate.The tertiary structure of VIR protein and the fusion protein of VIR and EGFP were constructed and compared,and cell epitope was predicted by bioinformatics method.The results showed that both pVAX1-EGFP and pVAX1-VIR-EGFP vectors expressed green fluorescence in BHK-21 cells,but the fluorescence of VIR-EGFP fusion protein was weaker than that of the EGFP protein.The molecular weight of the VIR fusion protein is about 53 ku by Western blot test,the VIR fusion protein has a good antibody spcificity.The tertiary structures of VIR protein and VIR-EGFP fusion protein intutively reflect the differences between them.Four dominant B cell epitopes and three restricted CTL dominance epitopes of VIR protein were predicted by bioinformatics software.This study laid a foundation for the study of biological function of VIR protein.
作者
成伟伟
杨军祥
常攀峰
李元新
陈伯祥
赵子惠
杨明
常亮
CHENG Wei-wei;YANG Jun-xiang;CHANG Pan-feng;LI Yuan-xin;CHEN Bo-xiang;ZHAO Zi-hui;YANG Ming;CHANG Liang(Gansu Institute of Animal and Veterinary Science,Pingliang,Gansu,744000,China)
出处
《动物医学进展》
北大核心
2022年第5期18-22,共5页
Progress In Veterinary Medicine
基金
甘肃省畜牧兽医研究所创新项目(GMCX-2019-3)。
作者简介
成伟伟(1988-),男,甘肃秦安人,助理研究员,硕士,主要从事动物病毒分子生物学研究。
