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Updated: May 15, 2026
Laboratory Scale Production and Purification of a Therapeutic Antibody
Published on: January 24, 2017
[Construction and expression of recombinant prokaryotic expression vector for disulfide-stabilized anti-TeNT
Han Wang1, Rui Yu, Xiaopeng Zhang
1Vaccine and Antibody Engineering Research Laboratory, Army Key Laboratory of Biological Weapon Injury Prevention Pharmaceuticals, Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100071, China. wanghan129@sina.com
Objective:
To construct the prokaryotic expression vector for disulfide-stabilized anti-TeNT single-chain Fv antibody (27G-scdsFv), express the 27G-scdsFv and characterize its bioactivity.
Methods:
27G-scdsFv gene was constructed by PCR-based point mutagenesis strategy and cloned into plasmid pET22b(+). The plasmid was then transformed into E.coli BL21(DE3) competent cells. The target protein was expressed under the induction of IPTG. SDS-PAGE and Western blotting were used to identify the expression products. The specific binding activity to TeNT-Hc and relative stability in vitro of 27G-scdsFv were assayed by ELISA, and the affinity of the antibody was measured by non-competitive enzyme-linked immunosorbentassay. The method of immunofluorescence was employed to determine whether or not the 27G-scdsFv kept the neutralizing activity in vitro.
Results:
Sequencing analysis proved that 27G-scdsFv gene was correctly constructed. The target protein was expressed in the form of inclusion body, accounting for about 50% of total bacterial proteins. After renaturated, the 27G-scdsFv protein still maintained specific binding activity to TeNT-Hc, with a higher affinity constant (K(D);) of 0.93×10(-7); mol/L as compared with that of 27G-scFv, and the yield of the protein was about 5 mg/L induced culture. The relative stability of 27G-scdsFv was improved obviously as compared with the scFv form. 27G-scdsFv strongly inhibited the binding of TeNT-Hc to PC-12 in vitro.
Conclusion:
The porkaryotic expression vector of 27G-scdsFv has been successfully constructed and the activated target protein has been obtained, which lays a foundation for the further study on the biological function of 27G-scdsFv.
