摘要
以牛传染性鼻气管炎病毒BarthaNu/67株的DNA作为模板,用PCR扩增gE基因并克隆至pGEM-TEasy载体,再以此质粒作为模板将gE基因分成6个片段,分别插入原核表达载体pET32a并在大肠杆菌中进行了表达。蛋白电泳结果表明6个片段中有2个片段以可溶形式表达,1个片段以包涵体形式表达,另外3个片段没有表达。采用固定化金属离子亲和层析法在非变性条件下对两个可溶性片段进行了纯化。经免疫印迹试验,间接ELISA和交叉试验证明,两个纯化的重组蛋白均与牛传染性鼻气管炎阳性血清样品发生反应,而与牛传染性鼻气管炎阴性血清无任何反应,显示其具有良好的抗原性和特异性,可用于牛传染性鼻气管炎gE-ELISA诊断方法的建立。
The Bartha Nu/67 strain of infectious bovine rhinotracheitis virus （IBRV） was propagated in Madin-Darby bovine kidney （MDBK） cell cultures and the viral DNA was extracted. The entire glycoprotein gE gene was amplified from the viral DNA by polymerase chain reaction （PCR）. The gE gene was cloned into pGEM-T easy vector. Then the gE gene was divided into 6 fragments, and inserted into prokaryotic expression vector pET32a, and expressed in E.coli. The SDS-PAGE showed that two of the 6 fragments were expressed in soluble form and one fragment was expressed in inclusion bodies, and the other three were not expressed. The two soluble fragments were purified by immobilized metal ion affinity chromatography under native conditions. The purified recombinant proteins showed reactivity to IBRV positive serum samples and no reactivity to normal bovine sera in indirect ELISA, immuoblot assay and cross of gE protein had very good antigenicity and specificity assay. These assays demonstrated that the two recombinant fragments and might be used for research on development of gE-ELISA for infectious bovine rhinotracheitis.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2006年第3期289-293,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
黑龙江省"十五"科技攻关项目(编号GA02B501)
国家"十五"863项目(编号2003AA241110)
