抗新城疫病毒单链抗体(scFv)载体构建及表达研究-动物遗传育种与繁殖专业毕业论文.docxVIP

600TCID50时，DF．1与DF．1．CAG．scFv细胞发生病变的孔数明显多于 600TCID50时，DF．1与DF．1．CAG．scFv细胞发生病变的孔数明显多于 DF—I．EFla．scFv。本实验表明，EFla启动子对单链抗体的表达翻译效果更 好，利用该启动子构建的新城疫病毒单链抗体能够在DF．1细胞中稳定表达， 并分泌到细胞外，具有良好的抗新城疫病毒效果。将以EFl Q为启动子的 抗新城疫病毒单链抗体质粒转染鸡原始生殖细胞(PGCs)，24 h后的转染 效率为2．9％，经嘌呤霉素筛选后得到稳定表达抗新城疫病毒单链抗体的 PGCs细胞系。本研究为抗新城疫病毒单链抗体的转基因鸡的生产奠定了基 础。 关键词：新城疫病毒单链抗体DF-1细胞转座子PGCs 万方数据 PLASMID PLASMID CONTRUCTIoN AND EXPRESSION 0F ANTI．NDV SINGLE．CHAIN FRAGMENT VARIABLE ANTIBODY AB STRACT Newcastle disease(ND)is avian respiratory disease that caused by Newcastle disease virus(NDV)．ND poses a significant economic threat to the poultry industry worldwide because it can result in high mortality in susceptible hosts T Currently no effective treatment available when the chicken are infected by NDV．Single—chain fragment variable(scFv)antibody is the smallest functional unit of immunoglobulin retaining the antigen-binding activities and could be express in vertebrate and invertebrate．It could be used in medical diagnosis and therapy by neutralizing various virus．In this study,we designed and contructed two eukaryotic expression vectors of anti-NDV scFv,named pB3 1 5B·-CAG·-puro-pGFP--scFv and pB3 1 5B-·EF l a--puro—-pGFP--scFv which were driven by CAG and EF l a promoter,respectively,using a PiggyBac transposal backbone．Two cell lines，DF一1 and 293T were transfected using Lipofect锄iIleTM2000 and a total of four stable cell lines stably expressing the above two vectors were established：DF．1．CAG．scFv、293T-CAG-scFv、 III 万方数据 DF．1．EFlot-scFv DF．1．EFlot-scFv and 293T-EFlot—scFv．The transfection efficiency in 293T cell is 25．8％．higher than in DF．1 cell with 19．1％．RT-PCR results indicated that the gene of anti-NDV-scFv antibody was expressed in all of four cell lines-Then， we detected the scFv in protein level by western—blot using antibody against His．tag protein，which was fused with anti—NDV-scFv antibody．Cell lysates from all four cell lines were negative for His—tag in the westernIblot，whereas supematant co