氧氟沙星单链抗体库的构建、筛选及蛋白结构模拟

Construction, Screening and Protein Structure Simulation of Single Chain Antiboday Fragment Library against Ofloxacin

利用噬菌体展示技术构建兽药氧氟沙星单链抗体(scFv)库,从中筛选氧氟沙星特异性噬菌体scFv以及模拟其三维结构。将从氧氟沙星杂交瘤细胞提取的总RNA,用RT-PCR反转录合成第一链cDNA,设计以针对鼠源重链可变区(VH)及轻链可变区(VL)基因的兼并引物,通过PCR扩增获得VH和VL可变区基因,采用重叠延伸PCR技术将VH和VL拼接为全长scFv基因片段,将经内切酶EcoRⅠ和HindⅢ双酶切后的scFv基因片段插入到T7噬菌体中,经包装蛋白体外包装后转化宿主菌BLT5403,成功构建库容量为3×105pfu/mL氧氟沙星单链抗体库,经过4轮吸附-洗脱-扩增的富集后,采用直接竞争ELISA筛选得到4个特异性噬菌体scFv,经测序最后运用Expasy软件模拟特异性scFv的三维结构和物理化学性质。为进一步大量表达氧氟沙星单链抗体奠定了坚实的基础。

Single chain antibody fragment （scFv） libraries were conslxucted for Ofioxacin by the phage display technology. Specific anti-Ofloxacin scFv was screened and its three-dimensional structure was simulated. Total RNA was abstracted from hybridoma cell of the Ofloxacin and used to amplify VH and VL gene by RT-PCR by degenerate primers. VH and VL were joined by overlap extension PCR to form the single chain variable fragment （scFv） gene, then scfv was inserted into Phage T7 after incision enzyme EcoR I and Hind III double digestion and was transformed by host bacteria BLT5403. Phage single chain antibody libraries consisting of 3 × 105 pfu/ml were successfully constructed. After four times of enriched procedure in the order of adsorption-elution-amplificatio, four Ofloxacin positive phage scFv clones were screened by direct competitve ELISA. The structure of specific scFv and physicochemical property were also simulated. This research lays a solid foundation for the further expression of anti-Ofloxacin scFv.