抗猪流感病毒的单链抗体在真核细胞中的表达

Expression of single-chain variable fragment antibody against swine influenza virus in A549 cells

为了研究抗猪流感病毒的单链抗体(scFv)在真核细胞内的表达情况及其生物学特性,通过PCR从前期试验获得的重组噬粒中扩增出scFv基因,将其克隆到真核表达载体p3×FLAG-CMV-7.1中,测序验证后转染A549细胞进行表达,对表达蛋白进行特性分析。结果显示,scFv基因的核苷酸序列的长度为732bp,序列排列方式为VH-Linker-VL,与数据库比对结果表明,符合小鼠抗体的重链和轻链可变区基因的结构特征。间接免疫荧光试验的结果表明,细胞内表达的绿色荧光蛋白主要分布在细胞质中。Westernblot分析显示,转染细胞的培养液上清和细胞裂解液上清中均可检测到一约32.8ku的融合蛋白,与预测结果一致。ELISA结果表明,表达的scFv具有结合猪流感病毒的活性。结果证实,抗猪流感病毒的scFv能够在细胞内正确表达,为进一步对抗猪流感的免疫治疗及免疫检测的研究提供了依据。

To express single-chain variable fragment(scFv)antibody against swine influenza virus(SIV)in A549 cells and to investigate its biological activities,DNA fragment encoding anti-SIV scFv was amplified by PCR from the recombinant phagemid obtained in our previous study.Sequencing results showed that the nucleotide sequence of scFv gene was 732 bp in length,and the sequence arrangement mode was VH-Linker-VL.Compared with the database,the structure of scFv gene was in accordance with mouse antibody heavy chain and light chain variable region.The scFv gene fragment was inserted into the eukaryotic expression vector p3×FLAG-CMV-7.1and the recombinant plasmid was transfected into A549 cells.Green fluorescent protein was observed mainly in the cytoplasm of A549 cells by indirect immunofluorescence assay.Western-blot analysis showed that about 32.8ku fusion protein in size was detected in the cell supernatant and the cell lysate supernatant.ELISA confirmed the expression of scFv in the cells.All the above results showed that scFv was able to be efficiently expressed in A549 cells,and the recombinant scFv had specific binding activities with H1N1 influenza virus.The findings of this study lay the foundation for advanced studies of immune detection and immune therapy against swine influenza.