氧氟沙星噬菌体库的构建、筛选及抗体结构模拟

Construction,Screening and Antibody Structure Homology Modeling of Phage Single Chain Variable Fragment Library Against Ofloxacin

应用噬菌体展示和重组抗体技术制备抗氧氟沙星单链抗体(scFv)库,筛选获得氧氟沙星特异性噬菌体scFv以及同源模拟其三维结构。将从氧氟沙星杂交瘤细胞提取的总RNA,用RT-PCR反转录合成cDNA,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,扩增获得VH和VL可变区基因,通过SOE-PCR法将VH基因和VL基因通过柔性多肽Linker(Gly4Ser)3拼接成全长scFv基因片段,将双酶切后的scFv基因片段插入T7噬菌体,经体外包装后转化宿主菌BLT5403,成功构建库容量为3×105pfu/mL的抗体库,经4轮吸附-洗脱-扩增的富集,采用直接竞争ELISA筛选到4个特异性噬菌体scFv,运用Expasy软件模拟特异性scFv的三维结构。为进一步大量表达氧氟沙星单链抗体奠定了基础。

To construct a library of mouse single chain variable fragment（ scFv） antibody against ofloxacin using phage display and recombinant antibody technique,specific anti-ofloxacin scFv was screened and 3D structure was homology modeling. Total RNA was extracted from hybridoma cell of ofloxacin mAb,and was used to amplify VH and VL gene by RT-PCR using random primer. Then they were linked by a DNA linker encoding（ G1y4Ser）3as VH-linker-VL sequence forming scFv by SOE（ splicing by overlap extension） PCR. These fragments were inserted into phage T7 after double digestion and transformed with host bacteria BLT5403. 3 × 10^5pfu / ml single chain antibody phage libraries were successfully constructed. Four positive phage scFv clones were screened by direct competitive ELISA after four times of enriched procedure in the order of adsorption-elution-amplificatio,3D structure of specific scFv was homology modeling finally. This research lays a foundation for further massive expression of anti-ofloxacin scFv.