抗牛传染性鼻气管炎病毒囊膜gD糖蛋白的单链抗体在昆虫细胞中表达及活性检测

发布时间：2018-08-18 12:17

【摘要】：为了获得具有生物学活性的牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)gD蛋白特异性单链抗体,试验采用PCR方法扩增杂交瘤细胞的cDNA单链抗体重链可变区(VH)和轻链可变区(VL)基因,通过重叠延伸PCR及Linker序列获得完整的单链抗体基因,将单链抗体基因克隆至杆状病毒载体pFB-LICBse中,构建重组转移载体pFB-VH-VL,将其转化至大肠杆菌DH10Bac感受态细胞中制备重组杆粒rBacmid-VHVL,将重组杆粒rBacmid-VH-VL转染至Sf9细胞中获得携带单链抗体基因的重组杆状病毒。该重组杆状病毒在Sf9细胞中表达了分子质量约为30ku的单链抗体蛋白。Western blotting结果显示,重组单链抗体蛋白能被His标签抗体特异性结合,也能特异性结合gD蛋白。间接免疫荧光试验结果显示,该单链抗体蛋白能够识别感染牛肾细胞MDBK中的IBRV。本研究在昆虫细胞中成功表达了具有识别IBRV的gD蛋白特异性单链抗体,为牛传染性鼻气管炎的诊断与治疗奠定了基础。
[Abstract]:In order to obtain the specific single-chain antibody of bovine infectious rhinotracheitis virus (infectious bovine rhinotracheitis virusus IBRV) GD protein, PCR was used to amplify the heavy chain variable region (VH) and light chain variable region (VL) gene of cDNA in hybridoma cells. Complete scFv gene was obtained by overlapping extension of PCR and Linker sequences. The scFv gene was cloned into baculovirus vector pFB-LICBse. The recombinant transfer vector pFB-VH-VLwas constructed and transformed into Escherichia coli DH10Bac competent cells to prepare rBacmid-VHVL. the recombinant rBacmid-VH-VL was transfected into Sf9 cells to obtain recombinant baculovirus carrying single-chain antibody gene. The recombinant baculovirus expressed scFv protein with molecular weight of about 30ku in Sf9 cells. Western blotting showed that the recombinant scFv protein could be specifically bound by His tag antibody and GD protein. The results of indirect immunofluorescence assay showed that the scFv protein could recognize IBRV in MDBK infected bovine renal cells. In this study, the gd protein specific single-chain antibody was successfully expressed in insect cells to recognize IBRV, which laid a foundation for the diagnosis and treatment of bovine infectious rhinotracheitis.
【作者单位】：江西农业大学动物科学技术学院;北京市农林科学院畜牧兽医研究所畜禽疫病防控技术北京市重点实验室;
【基金】：国家重点研发计划-畜禽重大疫病防控与高效安全养殖综合技术研发(2016YFD0500900) 现代农业产业技术体系北京市奶牛创新团队岗位专家资助经费项目(bjcystx-ny-3)
【分类号】：S852.65

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