| GL50 is a new member of B7 family. The interaction of GL50 with ICOS, the specific receptor for GL50, is critically involved in the activation, proliferation, differentiation and cytokine production of T cells as well as in the antibody secretion of B cells during the secondary immune responses. It is well-accepted that the ICOS/GL50 signal pathway plays a pivotal role in the regulation of inflammatory responses, in the prevention of transplantation rejection and tumor development, as well as in the modulation of autoimmune diseases and allergic response. As an important regulatory factor in the signaling pathways, mouse anti-human GL50 monoclonal antibodies provide powerful tools for us to further study the co-stimulatory signal pathway and cross-talking in immune response. So we can investigate the essence of immune response. Thus, establishment of human GL50 transfected cell line and monoclonal antibodies against human GL50 may provide a valuable and potential way for the clinical diagnosis, treatment and prevention of diseases.This article consists of two parts:Part I Establishment of cell line transfected with human GL50 gene and preliminary study on its co-stimulatory effect on T cell activation and proliferation in vitroTotal RNA was extracted from B lymphoma cell line Daudi, which was reverse transcripted into cDNA . The human GL50 gene was then amplified by RT-PCR. The target gene was inserted into vector pIRES2-EGFP and confirmed by sequencing. The recombinant pIRES2-EGFP-GL50 vector was transfected into L929 cells with LipofectamineTM 2000, and the cells was further selected with G418 for a long time. GL50 expressed on L929/GL50 cells was analysed by FCM and RT-PCR. Effect of L929/GL50 cells on T cells proliferation and cytokine production in vitro was studied by methods of CCK-8 and ELISA. In vitro, L929/GL50 cells could obviously promote the proliferation of T cells, while up-regulating the production of IL-4,IL-10 and IL-17.Part II Generation and characteration of mouse anti-human GL50 monoclonal antibody with biological functionThe L929/GL50 transgenic cells were used to immunize Balb/C mice. The immunized splenocytes were fused with murine myeloma cells (SP2/0) by the cell fuse technique. By means of HAT selective culture and repeated screening with L929/GL50 as antibody-screening positive cell and L929/mock as negative control, two hybridoma cell lines (2B4 and 4D11) continuously and steadily secreting specific anti-human GL50 were obtained. The two hybridomas grew well after long-term culture in vitro and storage in liquid nitrogen.The monoclonal antibody was produced according to ascites-inducing procedure in mouse abdominal cavity. The average yield of ascites was about 3.5 milliliter (ml) per mouse. After the ascites was purified by protein G affinity chromatography, the concentration of the protein was above 4 milligram (mg)/ml and the ascitic titer was over 1:1000 dilution by immunofluorescence analysis. Every 1×106 cells required 0.2～2 microgram (μg) purified monoclonal antibody in indirect immunofluorescence. Karyotype analysis result showed that the number of the chromosomes of the hybridoma was more than these of mouse somatic chromosomes, which suggested that they are fused cells.Fast-strip method analysis displayed that monoclonal antibodies 2B4 and 4D11 were mouse IgG1 and IgG2a, and the light chain wereκ. Dot-blot and flow cytometry analysis showed that monoclonal antibodies 2B4 and 4D11 exclusively binded GL50 molecules. The mAbs were also identified by ELISA.The competitive inhibition results showed that monoclonal antibodies (2B4,4D11) and MIH12, a commercial anti-GL50 antibody, recognized different epitopes.Flow cytometry analysis showed that GL50 molecule was highly expressed on cell lines such as Daudi, Raji, HO8910, THP-1 and ECV. B lymphocytes, isolated from peripheral blood hardly express GL50 molecules. L929/GL50 cells could obviously promote the proliferation of T cells. Specific mAbs 2B4 and 4D11 could act as blockers in the proliferation of T cells. |
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