Expression,purification and identification of fully human monomolecular antibody against Clostridium perfringens type A α-toxin
Objective To express,purify and identify human monomolecular antibody against Clostridium perfringens type A α-toxin,in order to lay a foundation for the prevention and treatment of various diseases caused by this toxin.Methods The fully human single-chain fragment variable(ScFv)gene against Clostridium perfringens type A was linked with the constant region of light chain and heavy chain of human antibody in different combinations to construct multiple monomolecular antibody expression plasmids against Clostridiumperfringens α-toxin,which were expressed in competent E.coLi BL21(DE3).Indirect ELISA was used to detect the immunobinding activity of the monomolecular antibodies,and the monomolecular antibody with the highest immunobinding activity was purified by SepharoSe 4 FF and rProtein-A FF affinity chromatography,The purified products were analyzed by 12％SDS-PAGE.Indirect ELISA was used to detect the immune binding activity of each monomolecular antibody.Results Five recombinant plasmids,PTS-ScFv-CL-CH2-CH3,PTS-ScFv-CH2-CH3,PTS-ScFv-CL-CH2,PTS-ScFv-CH2,and PTS-ScFv-CL,were constructed.After transfection into E.coli BL21(DE3)and purification,the corresponding monomolecular antibodies,ScFv-CL-CH2-CH3,ScFv-CH2-CH3,ScFv-CL-CH2,ScFv-CH2,and ScFv-CL,were obtained,which had the relative molecular mass of about 60 000,and the concentrations of about 1 mg/mL.Among them,ScFv-CH2-CH3 showed the highest immune binding activity,and the A450 value reached 3.9,much higher than the other four monomolecular antibodies,with the concentration of about 1 mg/mL and the purity about 86％.Conclusion A fully human monomolecular antibody ScFv-CH2-CH3 with high affinity,low immunogenicity and internalization activity was obtained,which lays a foun-dation for the further study of therapeutic antibody against CPA.
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