ABSTRACT
By combining interleukin2 (IL-2) with a tumor specific antibody, immunotherapy of tumors may become more effective in the future. Anti-GD2 single chain antibody directed to the extracellular domain of GD2 disialoganglioside can result in an antitumor response in some pateins with tumors expressing GD2. In this study, the fusion protein consisting of GD2 single chain antibody (ScFv) and IL-2(Ala125) was constructed. Anti-GD2 ScFv and IL-2 genes were obtained by PCR, then the ScFv-IL-2 gene was constructed by over lap PCR. The gene was inserted into the pMD18-T easy vector. Genes from pMD18-T -vector were inserted into expression vector pSE380. Recombinant expression vector was identified by restriction enzyme-cutting and then was transformed into BL21. SDS-PAGE and Western blot analysis confirmed that the transformed E. Coli BL21 could express ScFv-IL-2 fusion-proteins and the molecular weight is 43 kDa. The fusion protein was purified by affinity chromatograph and Sephacryl S-200HR then was identified through ELISA. The results show that the fusion protein retains the activities of both antigen binding and IL-2.
Subject(s)
Humans , Antibodies , Genetics , Metabolism , Antibodies, Monoclonal , Genetics , Base Sequence , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Gangliosides , Allergy and Immunology , Immunoglobulin Fragments , Genetics , Allergy and Immunology , Interleukin-2 , Genetics , Molecular Sequence Data , Recombinant Fusion Proteins , GeneticsABSTRACT
This study sought to construct a recombinant vector that expresses anti-GD2/anti-CD16 bispecific single-chain antibody(sc-BsAb), and to assess its biological activities. The anti-GD2 gene and the anti-CD16 gene (NM3E2) were obtained using PCR amplification technique, and then the fusion gene was constructed by overlapping PCR. The sc-BsAb gene was subcloned into the pET-22b(+) plasmid from the pMD18-T easy vector by digestion with NcoI, Hind III restriction endonucleases, whose sites exist in both the vectors. Then the combinant plasmids were transferred into E. coli BL21 (DE3). The expression product in the periplasmic was analyzed by both SDS-PAGE and Western blot technique, then was purified with Ni2+ -NTA superflow affinity chromatography. It was demonstrated that the linker in the sc-BsAb fusion protein is SerGly4Ser. and the molecular is 53 KD.