Preparation and activity analysis of hu3D3/core-streptavidin fusion protein as a universal carrier targeting to lung cancer / 中华微生物学和免疫学杂志

Objective To prepare a novel fusion protein of hu3D3 and core-streptavidin(hu3D3/cSA) as a universal carrier targeting to lung cancer,and analyze its activities. MethodscSA gene was acquired by PCR,and inserted into plasmid hu3D3/pET-22b(+)to construct a recombinant plasmid hu3D3/cSA/pET-22b(+)．The fusion gene was expressed in E. Coli BL21(DE3).The fusion protein was purified through nickel-affinity chromatography column and renatured using TEA buffer. The tetrameric hu3D3/cSA complex was analyzed by SDS-PAGE and Western blot.The hu3D3/cSA protein was labeled with FITC,then its antigen-binding activity was analyzed using fluorescence microscopy,and the functional affinity of hu3D3/cSA and hu3D3 were analyzed and compared by flow cytometry. The biotin-binding activity of hu3D3/cSA was tested bv EUSA and Western blot. Results The recombinant plasmid hu3D3/cSA/pET-22b(+)with correct sequence was obtained. The fusion protein was found after expression in E. Coli BL21 (DE3)mainly in the form of inclusion body. After being purified and refolded,tetrameric complex was formed. The purified tetrameric hu3D3/cSA complex retained both antigen-binding activity of hu3D3 moiety and biotin-binding activity of cSA moiety:furthermore,the avidity of the hu3D3/cSA to its target antigen increased by about 14 times as compared with that of monomeric hu3D3. Conclusion The fusion protein hu3D3/cSA with both antigen- and biotin-binding activity is SHCCessfully prepared,and the avidity of hu3D3 moiety to 3D3 antigen is enhanced. Consequently,hu3D3/cSA could be a universal carrier targeting to lung cancer, and could be an alternative,convenient method to realize target therapy to lung cancer by the combination of multiple biotinylated anti-tumor drugs or agents.

Expression and Identification of Single Chain Antibody of Prokaryotic Expression Vector pOPE101-8E5 / 中国生物工程杂志

In the present study, we inserted the core-streptavidin cDNA into downstream of multi-cloning site of plasmid pOPE101-8E5 by DNA gene recombination technology. And then, the variable fragments of heavy and light chain of the scFv-8E5 were replaced by the scFv-C4 variable fragments to construct the expression vector pOPE101-C4∷core-streptavidin. After transformed the vector pOPE101-C4∷core streptavidin into E.coil, the fusion protein C4∷core streptavidin-His-tag can be expressed by inducing with IPTG, and the expression level and activity of the expressed fusion protein analyzed by SDS-PAGE and Western blot. The results show that a scFv-C4∷core-streptavidin fusion protein of 45kDa was obtained, which can bind proteins of 60kDa & 45kDa from the KG1a cells lysate simultaneously. The binding function can be detected by the binding of core-streptavidin and biotin directly.