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

摘要

For preparation of bioactive single chain fragment variable(ScFv)which was targeted against envelope glycoprotein D(gD)of infectious bovine rhinotracheitis virus(IBRV),VH and VL genes were amplified from the cDNA of lymphocyte hybridoma,then VH and VL genes were integrated with a Linker by SOE-PCR,and the ScFv gene was cloned into the baculovirus vector pFB-LIC-Bse,which was transformed into Escherichia coli DH10Bac competent cells to construct a recombinant bacmid rBacmid-VH-VL.Finally,ScFv was expressed by transfected rBacmid-VH-VL into insect Sf9 cells,its molecular weight was about 30 ku.The result of Western blotting suggested that ScFv generated in Sf9 cells was specifically binds to His antibody,the protein also showed high affinity to gD of IBRV.The result of indirect immunofluorescence assay showed that ScFv could recognize IBRV which infected bovine kidney cells(MDBK).The study indicated that the recombinant ScFv was expressed in Sf9 cells,and could bind to gD of IBRV specifically.The result could provide the basis for the diagnosis and treatment of infection of IBRV.%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-LIC-Bse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-VH-VL,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30 ku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.