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

ABSTRACT

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 (G1y4 Ser) 3 as 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 ×105 pfu / 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.