ABSTRACT
Objective To construct bispecific antibody BsAb1/17 against both IL-1β and IL-17A,express and purify the biologically active BsAbl/17 protein in prokaryotic system for further studies and applications. Methods VH1VL17-CL and VL1VH17-CH1 gene segments were constructed by overlap-PCR.Restriction enzyme sites Nco Ⅰ and BamH Ⅰ were designed at the 5'and 3' end primers respectively. The products of overlap-PCR were ligated to the Nco Ⅰ/BamH Ⅰ -prepared pET-27b vector. The recombinant plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 ( petB ) were transformed into E. coliRosetta separately. The expressing products were analyzed by SDS-PAGE and Western blot. Neutralization activity of the bispecific antibody for blocking the induction of IL-18 expression by IL-1β in human T cells was determined by real-time PCR. Neutralization activity of the bispecific antibody for blocking the induction of IL-6 expression by IL-17A in HeLa cells was determined by ELISA assay. Results The structure of the plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 (petB)was confirmed by DNA sequencing. After induction, the fusion proteins were expressed mainly as inclusion bodies. The purity of the both proteins exceeded 90%. SDS-PAGE analysis suggests the relative molecular mass of both products expressed by petA and petB were approximately 38× 103, which is in accordance with the theoretical value. The results of Western blot and ELISA test demonstrated that BsAb1/17 molecule had binding ability to both IL-1β and IL-17A. The BsAb1/17 could block IL-1β to stimulate human T cell to express IL-18 and block IL-17A to stimulate HeLa cell to express IL-6. Conclusion We successfully constructed a novel bispecific antibody BsAb1/17 against both IL-1 β and IL-17A, and expressed biologically active BsAb1/17 protein in prokaryotic system.