鸭肠炎病毒囊膜糖蛋白gE基因序列分析及真核表达载体的构建

摘要

为探索鸭肠炎病毒（ DEV）囊膜糖蛋白gE 基因的相关特性和功能，应用PCR扩增了DEV的gE基因，全长1473 bp，编码490个氨基酸，其中1～22位氨基酸为信号肽。 gE基因与国内分离强毒CHv株、国内疫苗株同源性均为100％，与德国分离强毒2085株同源性达到99％，显示其高度保守。将gE基因完整开放阅读框（ gE）和去信号肽的gE基因（ gE’）分别克隆至真核表达载体pCDNA3．1，获得重组质粒 pCDNA3．1-gE、pCDNA3．1-gE，然后应用脂质体法转染 vero 细胞。RT-PCR扩增证实gE、gE’均在细胞内进行了转录，而间接免疫荧光试验证实只有gE’基因在vero细胞中获得了表达，为研究gE蛋白的功能及研制鸭瘟抗体检测试剂盒奠定了基础。%To explore the correlated properties and functions of glycoprotein gE in duck enteritis virus( DEV) ,the sequence of gE was amplified by PCR in this study. The whole length of gE open reading frame( ORF) is 1473 bp and encodes 490 amino acids, 1to 22 amino acids of which were defined as signal peptide. Gene gE of DEV strain AV1221 share 100% homology with gE of both virulent strain and vaccine strain in China,also 99%with virulent strain 2085 in German, which shows gE of DEV strain AV1221 is highly homological. The complete ORF ( gE) and truncated glycoprotein gE without signal peptide ( gE’ ) were respectively cloned to the eukaryotic expression vetor pCDNA3.1. Recombinant pCDNA3.1-gE and pCDNA3.1-gE’ plasmids were transfected to vero cells with liposome mediated transfection method. Successful transcription of gE and gE ’ in vero cells were confirmed by RT-PCR, while successful expression of gE’ alone in vero cells was verified by indirect immunofluorescence assay. The study provides a fundation for function of DEV gE and development of gE antibody test kit.