The Expression,Renaturation And Biological Activity Identification Of A Recombinant Antibody For Cancer Specific Antigen Ccsp

Single chain antibody, fragment (single chain Fv, scFv) is a recombinant protein which fuses the antibody heavy chain variable region VH and light chain variable region VL with a flexible connecting linker. The linker is a synthetic peptide containing 15 to 20 amino acids. As the smallest functional antigen-binding structural unit, its relative molecular weight is about 25KD, only one-sixth of a full size of a typical antibody. Due to its simple preparation method, small molecular weight, high penetrating capacity and low immunogenicity, scFv is widely used in cancer diagnosis and treatment.Ccsp specific scFv cDNA was subcloned into E. coli expression vector pET-11a to generate scFv-pET-11a. The recombinant antibody was produced in E. coli and used to detect the cancer specific antigen ccsp.First, scFv-pET-11a was transformed into E.coliBL21 (DE3). The expression of scFv was induced by IPTG and detected using SDS-PAGE and western blot. Our results demonstrated that scFv antibody was expressed in the form of inclusion body in BL21 (DE3). Large quantity of scFv inclusion body was harvest after optimization of the induction time. Then, three renaturation methods were used for scFv re-folding, including dialysis, adding different concentrations of renaturation promoter and discontinuous adding renaturation buffer at a high protein level. We found that the binding and specificity of scFv antibody for ccsp can only be achieved by the third renaturation method. We also constructed a excretion vector scFv-pET-22b for the direct acquisition of the soluble active recombinant antibody without the complicated procedures of protein denaturation and renaturation.