虎源猫传染性鼻气管炎病毒gD蛋白基因的原核表达及其间接ELISA检测方法的建立  

Cloning and Prokaryotic Expression of gD Gene of Feline Herpesvirus Type 1 from Tiger and Establishment of Indirect ELISA Method

作　　者：张晓明[1,2] 陈武[1] 徐春忠[3] 彭仕明[2] 胡俊[2] 

机构地区：[1]广州动物园广州,510070 [2]华南农业大学兽医学院,广州 510642 [3]上海野生动物园,上海201300

出　　处：《野生动物学报》2014年第3期257-261,共5页CHINESE JOURNAL OF WILDLIFE

基　　金：中国动物园协会2013年科研项目“虎源传染性鼻气管炎的快速诊断和新型疫苗的研制”

摘　　要：为研究虎源猫传染性鼻气管炎的快速诊断试剂盒和基因亚单位疫苗,根据Genbank公布的猫传染性鼻气管炎病毒的核酸序列,设计了关于虎源猫传染性鼻气管炎病毒gD蛋白基因的引物并利用PCR扩增出gD序列,成功插入到pMD18-T Simple载体中,筛选获得重组质粒,命名为pmd-gD。将其利用HindⅢ和XhoⅠ酶切后,产物克隆入原核表达载体pet-32a(+)中,获得重组质粒,命名pet-gD。用IPTG进行诱导表达,收集菌液进行SDS-PAGE和Western-blot检测,结果显示目的基因在pET-32a(+)中获得了高效融合表达,其表达蛋白相对分子质量约为60kDa,与虎源猫传染性鼻气管炎病毒阳性血清发生特异性反应。以纯化的表达产物作为包被抗原,建立了检测虎源猫传染性鼻气管炎抗体的间接ELISA检测方法。本实验为虎源性猫传染性鼻气管炎诊断试剂盒和基因亚单位疫苗的研究打下了重要的基础。To research the vaccine and the diagnostic kit of Feline Herpesvirus type 1 from tiger,we designed the primers of gD genes of the FHV-1 according to sequences of FHV-1 from GenBank.The gD gene was amplified by PCR and cloned successfully into pMD18-T Simple vector.The gD gene was constructed into prokaryotic expression vector pET-32a（＋） and the recombinant plasmid was named pET-gD and induced by IPTG.The results of SDS-PAGE and Westem-blot indicated that the gD gene was expressed at a high level,and the recombinant fusion protein was about 60 kDa and had apeculiar reaction to Feline Herpesvirus type 1 from tiger positive serum,which has immunological activity.We established the indirect ELISA method for detecting Feline Herpesvirus type 1 antibodies with the purified expression product as a coating antigen.This provides an important foundation for further research on FHV-1.

关 键 词：虎源猫传染性鼻气管炎病毒 PCR 原核表达 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]