| The innate immune system is an ancient form of host defense that relies upon a wide range of pattern recognition receptors, as Toll like receptors (TLR), G-protein coupled receptors, mannose receptors, Fc receptors, etc., which are expressed on neutrophils, monocytes,macrophages, NK cells and other immune cells.These receptors can recognize and bind pathogens,then mediate effector cells playing an immune function. Besides these receptors, effector immnune cells also express other pattern recognition receptors,these receptors often include stimulate receptor and inhibitory receptor. Triggering receptors expressed by myeloid cells(TREM)are one of this kind of recepyors.Trem-Like Transcript2 (TLT-2) gene is one of the conserved TREM family members,which expressed on B cell ,T cell and macrophage. The mouse TLT-2 is suggested to play an impotant role in the immune system, for it is not only higher expressed in inflammation,but also promote the proliferation of T cell and the produce of cytokines. However, the biological function of TLT-2 remains unclear.. This study aims to clone human TLT-2 gene, establish transgenic cell line L929/TLT-2, and then use this cell line as an immungen to prepare mouse anti-human TLT-2 monoclonal antibody. Finally, L929/TLT-2 transgenic cell line and the monoclonal antibody are used to research the biological characteristics and function of human TLT-2 molecule.Part1: Cloning of human TLT-2 gene, construct PIRES2-EGFP-TLT-2 expression vector and establishment of transgenic cell lineObjective:To clone human complementary TLT-2 gene from the human whole blood cDNA library, establish transgenic cell expressing TLT-2 molecule and afford effective immungen for preparing mouse anti-human TLT-2 monoclonal antibody.Methods:The full-length human TLT-2 gene coding region was cloned from the human whole blood cDNA library by RT-PCR and then inserted into the eukaryotic expression vector PIRES2-EGFP to construct the recombinant PIRES2-EGFP-TLT-2 after double digestion with Xho I and BamH I. The recombinant plasmid was transfected to murine L929 cells after induction with lipfectAMINETM2000 and the cells were further selected with G418. Flow cytometric, RT-PCR and Western-blot were used to identify the expression of transfected L929/TLT-2.Results: Sequencing results showed that the full-length human TLT-2 gene was entirely consistent with the gene sequences in the Genbank. PIRES2-EGFP-TLT-2 was digested to release the target gene fragment of about 956bp; Flow cytometric can idengtify GFP was expression on the transfected cell line; RT-PCR can found the TLT-2 expression in gene level and Western-blot can also found it expression in protein level.Part2: Preparation of mouse anti-human TLT-2 monoclonal antibodies and analysis of their biological characteristicsObjective: To prepare mouse anti-human TLT-2 monoclonal antibodies applied to illuminate the characteristics of TLT-2 molecule and the biological function on T cell. Methods: BALB/c mice were immunized with human TLT-2 transfectant L929/TLT-2 as an immungen. Mouse spleen B cells were fused with mouse plasmocytoma cells SP2/0. L929/TLT-2 was used as positive screening cell line.Hybirdoma cells wew eventually obtained through repeated sub-cloning and screening.Fast-strip analysis was performed to identify Ig subclass of the generated monoclonal antibodies.The methods of immunophenotyping and western blot were designed to identify the specificity of generated mAbs.The epitopes recognized by mAbs were analyzed by competition assay.The expression of TLT-2 on different human tumor cell lines,na?ve immune cells was detected by FCM. CCK8 incorporation assay and ELISA were used to study biological characterization of the obtained antibody.Results: After multiple screening and subcloning, one monoclonal antibodies named 8C10 were obtained. The hybirdoma cells grew well after long-term storage in liquid nitrogen and culture in vitro. Identified by test paper, the isotope of 8C10 heavy chain was mouse IgG1 white the isotype of light chain isκ. 8C10 could bind to the same protein as the commercial TLT-2 polyclonal antibody could, which strongly suggested the 8C10 would specially recognized the hTLT-2 protein. Flow cytometric analysis showed that TLT-2 highly expressed on monocytes and B cells, and weakly expressed on actived T cells. 8C10 could inhibit T cells proliferationConclusion: This study has successfully cloned humanTLT-2 gene and constructed L929/TLT-2,and on this basis the hybridomas specifically secreting mouse anti-human TLT-2 monoclonal antibody were generated. These results above have provided a valuable foundation and experimental parameters of the material.For further study of TLT-2 molecule-mediated signal pathway and its role in disease... |
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