TGEV-RBD基因在昆虫杆状病毒中的表达及鉴定  

Expression and Identification of TGEV-RBD Gene in Baculovirus Expression Vector System

作　　者：伊立超 李乐天 郝嘉翼 时小双 张爽[1] 徐鹏 任世斌 高旭[1] 李昌 YI Li-chao;LI Le-tian;HAO Jia-yi;SHI Xiao-shuang;ZHANG Shuang;XU Peng;REN Shi-bin;GAO Xu;LI Chang(Yanbian University,College of Agricultural,Yanji,Jilin,133000,China;Research Unit of Key Technologies for Prevention and Control of Virus Zoonoses,Chinese Academy of Medical Sciences,Institute of Military Veterinary Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Changchun,Jilin,130122,China)

机构地区：[1]延边大学农学院,吉林延吉133000 [2]军事科学院军事医学研究院军事兽医研究所/中国医学科学院人兽共患病毒病防控关键技术研究创新单元,吉林长春130122

出　　处：《动物医学进展》2021年第9期65-70,共6页Progress In Veterinary Medicine

基　　金：中国医学科学院医学与健康科技创新工程项目(2019RU059)。

摘　　要：旨在获得具有天然结构且有反应原性的猪传染性胃肠炎病毒(TGEV)受体结合域(RBD)蛋白。受体结合域是决定病毒宿主和嗜性的关键因素。根据猪传染性胃肠炎病毒纤突蛋白(S)序列(GenBank:ABC72414.1),设计、优化并合成其受体结合域(RBD)部分,亚克隆至杆状病毒pFastBacTMDual上,获得重组质粒,命名为pFBD-TGR。将该质粒转化DH10Bac感受态细胞中,筛选较大的白色菌落,获得重组杆状质粒Bacmid-TGR,转染至sf9细胞中进行拯救病毒,转染后5 d~7 d,收上清,传至第3代,应用间接免疫荧光法(IFA)和Western blot检测sf9细胞上清,进行目的蛋白表达鉴定。结果显示,重组穿梭质粒Bacmid-TGR构建成功,Western blot结果证明,杆状病毒表达了TGEV-RBD蛋白且能与Strep tag抗体和抗猪传染性胃肠炎病毒多克隆抗体发生特异性反应,间接免疫荧光结果证明,成功地表达了TGEV-RBD蛋白,为进一步开展猪传染性胃肠炎病毒抗体研究以及新型疫苗的研发奠定了基础。The aim of the experiment was to obtain the swine transmissible gastroenteritis virus(TGEV),RBD protein with natural conformation and good reactionogenicity.Receptor binding domain(RBD)is the key factor to determine the host and tropism of virus.According to the sequence of TGEV s protein(GenBank:ABC72414.1),the RBD part was designed,optimized and synthesized,and subcloned into the baculovirus double expression vector pFastBacTMDual to obtain the recombinant plasmid pFBD-TGR containing TGEV-RBD gene.The pFBD-TGR was transformed into DH10Bac competent cells and a blue/white colony screening assay was implemented to obtain the recombinant rod-like plasmid Bacmid-TGR that was transfected into sf9 cells by lipofectamine for viral rescue.5-7 d after transfection,the supernatant of viruses was collected.sf9 cells'supernatant were collected after viruses were cultured for three generations.the target protein was to verify whether it was expressed by using indirect immunofluorescence,and a further verification by using Western blot.The results showed that the recombinant plasmid pFBD-TGR was proved to be correct.The TGEV-RBD protein expressed in baculovirus reacted specifically to strep tag antibody.The specific green fluorescence was detected by IFA showed that the TGEV-RBD protein was expressed successfully.This study laid a foundation for the further preparation and the development of a new vaccine for TGEV antibody.

关 键 词：猪传染性胃肠炎病毒 杆状病毒 受体结合域 蛋白表达 

分 类 号：S852.659.6[农业科学—基础兽医学] S858.28[农业科学—兽医学]