牛传染性鼻气管炎病毒gE蛋白在杆状病毒中的表达及抗原性鉴定  

Expression and antigenic reactivity of gE glycoprotein of infectious bovine rhinotracheitis virus in baculovirus expression system

作　　者：杨木娇 林俊 张娟 王婉 向文杰 薛飞[1] 朱远茂[1] YANG Mu-jiao;LIN Jun;ZHANG Juan;WANG Wan;XIANG Wen-jie;XUE Fei;ZHU Yuan-mao(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2022年第1期34-40,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划课题“牛羊虫媒病和慢病毒病的诊断与检测新技术研究”(2016YFD0500908);国家自然科学基金(31372452)。

摘　　要：为了获得具有良好抗原性的牛传染性鼻气管炎病毒(IBRV)重组gE蛋白,本研究采用PCR方法扩增全长gE基因,并在其5’端引入蜂素信号肽(HBM)以增加表达蛋白的分泌,将其克隆至pFastBac1供体质粒中,构建重组质粒pFB-gE。将测序正确的p FB-gE转化至DH10Bac感受态细胞中获得重组杆粒(rBacmid-gE),经PCR鉴定后将阳性r Bacmid-gE转染Sf21细胞,获得重组杆状病毒(rBV)。对不同代次的rBV进行PCR鉴定,结果表明获得了整合gE基因的r BV,且能稳定传代。间接免疫荧光试验(IFA)结果显示,接种rBV的Sf21细胞与gE抗体阳性牛血清反应后出现绿色荧光,而与阴性牛血清不反应,表明g E基因在rBV中获得正确表达。大量培养rBV,收获培养上清液,对重组gE蛋白(rgE)纯化,纯化后的rgE浓度为0.78 mg/mL。对纯化后的rgE进行western blot检测,结果显示,rgE能与g E抗体阳性兔血清和羊血清反应,与IBRV-56(g E基因缺失株)免疫兔血清和健康羊血清均不反应,表明rgE具有较强的反应原性。采用rgE对4只小鼠进行两次免疫,通过间接ELISA方法检测小鼠血清抗体,结果显示,rgE可诱导小鼠产生特异性抗体,表明rgE具有良好免疫原性。本研究为IBRV gE蛋白结构和功能的研究奠定了一定基础,为gE基因缺失疫苗免疫牛与自然感染牛的鉴别诊断提供了物质基础。To obtain recombinant g E proteins of Infectious bovine rhinotracheitis virus with complete functions and good antigenicity,the full-length g E gene fused with a honeybee melittin signal peptide(HBM) sequence at its 5’ terminal was cloned into pFastBac1.The recombinant plasmid was designated as pFB-gE,which was transformed into DH10 Bac competent cells to obtain a recombinant bacmid,which was named r Bacmid-gE.Subsequently,rBacmid-gE was transfected into Sf21 cells to obtain recombinant baculovirus(rBV).PCR identification of rBV of different passages showed that rBV can stably express g E gene.Indirect immunofluorescence(IFA) results showed that Sf21 cells inoculated with rBV reacted with gE antibody-positive bovine sera rather than negative sera,further indicating that gE was successfully expressed on rBV.The rBV was cultured in large quantities,and the cell culture supernatant was harvested to purify the rgE proteins.The purified rgE was about 0.78 mg/mL.Western blot analysis showed that rg E reacted with gE antibody-positive rabbit sera and goat sera rather than negative rabbit sera or goat sera,indicating that rgE has strong reactogenicity.Four mice were immunized with rgE,and the sera were detected by an indirect ELISA.The results showed that rg E induced gE-specific antibodies,indicating that rgE has good immunogenicity.Together,this study laid a foundation for the study of the structure and function of gE,which could be used for the differential diagnosis of gE-deleted vaccines immunized bovines.

关 键 词：牛传染性鼻气管炎病毒 GE基因 糖蛋白E 重组杆状病毒表达 

分 类 号：S852.65[农业科学—基础兽医学]