羊口疮病毒ORF125基因编码蛋白的结构预测及其在真核表达质粒转染细胞中的定位

Structure Prediction and Localization of ORF125 from Orf Virus on Transfected Cells

旨在预测羊口疮病毒(ORFV)ORF125蛋白结构及其在真核表达质粒转染细胞中的定位。设计引物PCR扩增ORF125基因,并将其定向克隆至pEGFP-N1真核表达载体,经酶切鉴定及序列测定后得到阳性重组表达质粒pEGFP—ORF125。利用DNAStar和MEGA4.0元件分析不同毒株间ORF125基因的遗传进化关系,接着通过在线Expasy工具预测其二级结构并与Bcl-2家族成员进行比较。利用脂质体介导法转染山羊皮肤成纤维细胞(GSFs),经DAPI染色后在激光共聚焦显微镜下观察ORF125的亚细胞定位。结果显示:本次克隆的ORFV ORF125基因与国外公布的其他羊口疮毒株核苷酸水平相似性为97.1%~98.1%,与同属另外2株羊伪牛痘病毒相比,核苷酸相似性分别为84.1%(GQ329669)和85.6%(GQ329670);蛋白二级结构预测分析发现ORF125与Bcl-2家族成员α-螺旋位置相近,预测空间结构有很大的相似性;激光共聚焦显微镜观察发现ORF125在GSFs细胞质中呈弥散性分布。结果提示,羊口疮病毒ORF125基因非常保守,预测与Bcl-2蛋白有着相似的功能,重组质粒pEGFP-ORF125构建成功,ORF125基因在GSFs中得到表达,定位于细胞质中。

The current study was undertaken to forecast the secondary structure and find out the localization of transfected cells of ORF125 from orf virus(ORFV).ORF125 gene was acquired by PCR and cloned into pEGFP-Nl eukaryotic expression vector directionally.The recombinant expression plasmid was identified by restriction enzyme digestion analysis,sequencing and named pEGFP-ORF125.Then the phylogenetic analysis of the gene was made by DNAStar and MEGA4.0.Also the secondary structure of ORF125 was predicted and compared with cellular Bcl-2 proteins by Expert Protein Analysis System(EXPASY).The recombinant plasmid was transfect into goat skin fibroblasts(GSFs).subcellular localization of ORF125 was examined with laser con-focal microscopy after DAPI staining.The results of sequencing alignment showed that the homology of China Luoyang 2013 shared 97.l%-98.1%with other ORFV isolates and 84.1%(GQ329669)and 85.6%(GQ329670) with another two Pseudocowpox isolates from different regions at nucleotides level respectively.The sequence identity of ORF125 and cellular Bcl-2 proteins is low,but the secondary and tertiary structures are conserved.Laser con-focal microscopy results showed that ORF125 distributed dispersively in the cytoplasm of GSFs.This study helped to further understanding the function of ORF125.Results verified the ORFV ORF125 gene is conserved and have similar function with Bcl-2 protein,in addition,the ORF125 gene could be expressed successfully in GSFs,and the ORF125 protein localizes in the cytoplasm.