Construction and targeting effect observation of fusion protein anti-CD19 (Fab)-CH3

ZHA Jianying; ZHANG Yizhi; LU Yang; YANG Yuanyuan; KONG Fanni; ZHANG Yanjun

doi:10.3969/j.issn.1002-266X.2017.23.011

Abstract：Objective To construct the fusion protein anti-CD19(Fab)-CH3 targeting CD19 and to observe its targeting effect.Methods Gene cloning technique was used to construct the recombinant plasmid pAZY-anti-CD19(Fab)-CH3.The recombinant plasmids were identified by DNA sequencing, and then were transfected into Escherichia coli 16C9.The expression product was purified by Protein G and the purified protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.The formation of homodimer was detected by high performance liquid-size exclusion chromatography (HPLC-SEC).The antigen binding activity of the fusion protein was observed by fluorescence activated cell sorting (FACS).The binding capacity of HIT19a and Raji cells under the presence of fusion protein anti-CD19 (Fab)-CH3 or anti-CD19 (Fab) was detected by competitive immunofluorescence assay.Results SDS-PAGE and Western blotting indicated that we observed the fusion protein bands with the relative molecular mass of 65kDa, which were consistent with the molecular weight of the fusion protein anti-CD19 (Fab)-CH3.HPLC-SEC of purified antibody revealed that the proportion of homodimer was about 89%.FACS results showed that the recombinant fusion protein anti-CD19 (Fab)-CH3 was more efficient than anti-CD19 (Fab) in combination with Raji cells, and the binding fluorescence intensity of HIT19a-FITC with Raji cells under the action of anti-CD19 (Fab)-CH3 was lower than that under the action of of anti-CD19 (Fab).Conclusions The fusion protein anti-CD19(Fab)-CH3 is successfully constructed and expressed.Compared with the monomer anti-CD19(Fab), the fusion protein anti-CD19(Fab)-CH3 exhibits an better targeting effect with Raji in CD19 positive B-cell lymphoma.