Construction, Screening and Protein Structure Simulation of Single Chain Antiboday Fragment Library against Ofloxacin
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Abstract:
Single chain antibody fragment (scFv) libraries were constructed for Ofloxacin 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 EcoRⅠand 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.
