Construction And Validation Of New Type Anti-Human CD45 Antibody In Mammalian Expression System

Objective: CD45, which was originally called leukocyte common antigen(LCA), is widely present on all types of leukocyte and plays a crucial role in cellular signaling transduction. In this article,two kinds of new vectors of anti-human CD45 antibody were constructed and validated.These two vectors were transfected into eukaryotic expressing system, aiming to express target antibodies efficiently and stably.Methed: We constructed the recombinant vectors which carried the target genes.After identification by PCR and gene sequencing,the recombinant DNA was transfected into HEK-293 T cells,and the products were harvested and purified.The cell supernatants were utilized separately to detect the difference of antibody expression between these two vectors by ELISA.The binding activity of these two antibodies with membrane CD45 molecules on the surface of multiple cell lines was measured by Flow Cytometry,while the binding specificity of these antibodies with CD45 molecules was observed by Western Blotting.The vector with higher and more stable expression,better binding activity and higher binding specificity was chosen to be used in further study.DHRF system was used to screen out a DG44-CHO cell line for its steady and efficient expression of antibody.Results: The PCR-amplified anti- human CD45 antibody gene fragments of VH and VL were identified by 1.8% agarose gel electrophoresis.The DNA sequences of the vectors p Budce-CD45[H+L] and Anti-CD45-sc Fv-Fc were verified through gene sequencing. After transfection into HEK-293 T cells, the transient antibody expression in the cell supernatant were measured by enzyme-linked immunosorbent assay,and the concentration of p Budce-CD45[H+L] and Anti-CD45-sc Fv-Fc were respectively0.3mg/L and 2mg/L.The antibody expressed by Anti-CD45-sc Fv-Fc vector showed higher binding activity with multiple CD45 highly expressing cell lines(KG-1a/K562/Jurkat), while no significant binding activity was shown with non- CD45 expressing cell lines(Hep G2/A549/B16/HU).The expression level of p Budce-CD45[H+L] vector was so low that no obvious binding activity with CD45 was shown even using concentrated cellular supernatants.Hence, Anti-CD45-sc Fv-Fc vector was chosen to be used in further exploration.Western blot showed the binding of antibody expressed by Anti-CD45-sc Fv-Fc vector with total protein of CD45 highly expressing cell lines at 250 k D.And the non-binding with total protein of CD45non-expressing cell lines at the same site further confirms the specific binding of the antibody with CD45.Biotin-avidin system showed the binding of rh CD45 with Anti-CD45-sc Fv-Fc and sc Fv-1/2Fc.The DG44-CHO cell line is screened out for its steady expression and high efficiency by electro-transfection method and DHRF system,which is chosen to be utilized to produce antibodies in the further effect experiments.Conclusion: We have successfully transformed monoclonal Anti-CD45 antibody and constructed Anti-CD45-sc Fv-Fc in eukaryotic expressing system.The DG44-CHO cell line which can stably express anti- human CD45 antibody was screened out.The experiments above lay the foundation for research about human CD45 based therapy.