【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pFastBac^TMDual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。更多还原

【Abstract】 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 pFastBac^TMDual to obtain the recombinant plasmid pFBD-TGR containing TGEV-RBD gene.The pFBD-TGR was transformed into DH10 Bac 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.更多还原