Construction, Expression And Functional Characterization Of Single Chain Variable Fragments Against Human CD33 Antigen (CD33scFv) And Its Chimera With Extracellular Region Of CD80 (ExCD80-CD33scFv)

Background:CD33,a normal self-antigen that is a Mr 67,000 cell surface glycoprotein largely restricted to the myeloid/monocytic lineage.It contains a conserved immunoreceptor tyrosine based inhibitory motif(ITIM) in the cytoplasmic tails,so it may have roles in modulating cellular functions via recruitment of signaling molecules.CD33 was selected as the target antigen because of its over expression on 90%of acute myeloid leukemia(AML) blasts and its lack of expression on pluripotent stem cells (12-14),and it is not expressed on mature granulocytes and other tissues either.Many reports confirmed that lack or low expression of costimulatory molecule CD80 on AML cells surfaces is one of most important reasons that CTL can not recognize and kill cancer cells efficiently.It is a recognized phenomenon that T cells are rendered anergic due to the lack of costimulatory molecules expression by tumor cells.Based on the above information,we constructed CD80-anti human CD33 single chain fragment variable(scFv) fusion gene and express it in prokaryocytic expression vector.By this mean, we can modulate the AML cells with fusion protein to enhance the tumor-specific immunity.Objectives:To construct and express the single chain variable fragments (scFv) gene against human CD33 antigen,and characterize its bioactivity.Ampligy the extracellular region of CD80.Construct CD80-anti human CD33 single chain fragment variable(scFv) fusion gene and express it in prokaryocytic expression vector,and then detect the biological activity of the fusion protein.Methods:The genes encoding the light and heavy chain variable regions were cloned by RT—PCR from a murine hybridoma cell line HIM3-4,which could produce monoclonal antibody(mAb) against human CD33 antigen and is named by International workshop on human leukocyte differentiation antigens.Then the light and heavy chain variable regions were fused together by a short peptide linker containing 15 amino acid(Gly4Ser)3 using splice-overlap extensive PCR.The recombinant anti-CD33 scFv was subcloned into the expression vector pET28a(+)and expressed in E.coli Rosetta after induction by IPTG.Flow cytometry analysis showed that the scFv could react with human CD33 antigen.CD80 sequence was kindly presented by professor Wang Min.The extracellular region of CD80 was amplified by PCR with primers containing designed restriction enzymes.The hydrophilic fragments of 403-427 from domainⅢof HSA was choosed as an interlinker for ExCD80-CD33ScFv construction.The fusion gene was cloned into PET22b(+),after the fusion protein was expressed with IPTG in E.coli.Rosetta(DE3),its biological activity was detected with indirect immunological fluorescence experiment.Results:1 SDS-PAGE and Western blot analysis showed that the recombinant anti-CD33 scFv gene was expressed in the form of inclusion body in E.coli Rosetta(DE3),and the purified fusion protein was obtained after a series of purification steps including cell lysis,inclusion body solubilization, Ni2+ metal affinity chromatography and protein refolding.Flow cytometry(FCM) analysis showed that the scFv could react with human CD33 antigen,the VL was 321bp,the VH was 366bp.2 The extracellular region of CD80 was cloned by PCR,it is about 633bp.3 structing a ExCD80-CD33ScFv sequence in PET22b(+) vector by genetic engineering,then expressing it in the E.Coli Rosetta(DE3). The fusion protein was 55KSD fragment with human CD33-binding specificity after renaturation.Conclusions:(1) The VL and VH gene of CD33ScFv were cloned from hybridoma cell line HIN3-4.(2) Combinant anti-CD33 scFv gene has been successfully constructed and expressed in E.coli Rosetta(DE3).(3) the ExCD80 gene was successfully amplified.4 PET22b(+)-ExCD80-CD33ScFv was successfully expressed in Rosetta(DE3),its biological activity was detected initialy.