Anti-human Cd19 Single-chain Antibody-of Cd80 Fusion Proteins And Expression

Acute lymphoid leukemia (ALL) is one of the most important hematological malignancies. About 70%-80% of ALL patients are B line acute lymphoid leukemia, especially in childhood patients. Whereas great progress has been made in children ALL treatment, the outcome of adult ALL patients is still disappointed. It needs to combine chemical therapy with stem cell transplantation, biological therapy and immunotherapy to prolong the patients' survival. Many experiments found that ALL cells lack of costimulatory molecule B7.1 on their surfaces. It is a recognized phenomenon that T cells are rendered anergic due to the lack of costimulatory molecules , such as B7.1, expression by tumor cells.However, major of the B-ALL cells expression CD19 surface antigen. We desire to construction of anti-CD19 single chain fragment variable (scFv) and B7.1 fusion gene and expression the fusion gene in prokaryocytic expression vector. By this mean, we can modulate the B-ALL cell with fusion protein to enhance the tumor-specific immunity.The genes encoding for the light and heavy chain variable regions were cloned by RT-PCR from a murine monoclonal hybridoma cell line, which could produce monoclonal antibody to recognize CD19 antigen on human B lymphocyte and then fused together by a short peptide linker containing 15 amino acid (Gly₄Ser)₃ using splice-overlap extensive PCR. The recombinant anti-CD 19- scFv was subcloned into the expression vector pET28a and induced by IPTG to express in E.coli BL21. SDS-PAGE and Western blot analysis showed that the recombinant anti-CD19-scFv gene was expressed in E.coli BL21. Flow cytometry analysis showed that the scFv could react with human CD19 antigen.After that, we cloned human CD80 cDNA and linked human CD80 molecular extracell domain gene and anti-CD 19 scFv gene with part of human serum albumin gene. The fusion gene was subcloned into the expression vector pET22b and induced by IPTG to express in E.coli BL21 (rossetta). SDS-PAGE and Western blot analysis showed that the fusion gene was expressed in E.coli BL21 (rossetta). Our result could provide a basic study for the future target therapy to the B lymphoid leukemia and B lymphoma.