Construction Of Cell Lines Transfected With Human CD244Gene And Preparation Of Mouse Anti-human CD244Monoclonal Antibody

Part Ⅰ Construction of cell lines transfectedwith human CD244geneObjective: To construct gene transfected cell lines that stably expressed the humanCD244.Methods: Human CD244full-length cDNA was obtained by RT-PCR andamplified by PCR; then subcloned into retroviral expressing vector pEGZ-Term. Afterproved to be correct by sequencing, the recombinant vector pEGZ-Term/CD244together with its two helper virus vectors were cotransfected into the package cells293Twith Lipofectamine2000. The L929and BaF cells were infected with the culturalsupernatant of the transfected293T cells. The Zeocin resistant cells were obtained withZeocin screening, and then their GFP and CD244expression were detected by FlowCytometry.Results: Human CD244full-length cDNA was cloned successfully, and genetransfected lines L929/CD244and BaF/CD244stably expressed the human CD244were constructed successfully.Conclusion: Gene transfected cell lines stably expressed the human CD244wereconstructed successfully and ready for preparing mouse anti-human CD244monoclonalantibody and studying the biological function of CD244molecule. Part Ⅱ Preparation of mouse anti-human CD244monoclonalantibodyObjective: To prepare mouse anti-human CD244monoclonal antibody.Methods: BALB/c mice were immunized with CD244transfected cell L929/CD244that stably expressed human CD244molecule. The spleen cells of immunizedmouse were fused with myeloma cells SP2/0, and then were cultured with HATselective medium. Hybridoma cells were screened with L929/CD244cells by FlowCytometry. Meanwhile, L929/mock and L929/CD48cells, which were respectivelytransfected with vector pEGZ-Term and the recombinant vector pEGZ-Term/CD48,were used as negative control. The positive clones were repeatedly subcloned, until onehybridoma cell line sustainably and stably secreting specific anti-CD244mAb wasobtained. MAb Ig class was detected with Rapid Qualitative Test Strip; Indirectimmunofluorescence assay was used to identify the specificity of the obtained mAb, andthe recognition of CD244on human T lymphocytes from health donor and differenthuman tumor cell lines by21F8Results: A hybridoma cell line sustainably and stably secreting anti-CD244mAbwas obtained, which named21F8. The mAb21F8was proved to be IgM with κ lightchain. The positive rate of ascites formation was100%, and the average ascitesproductive was8ml each mouse. The mAb was revealed to recognize human leukemiacell line U937and T lymphocytes from health donor.Conclusion: A hybridoma cell line sustainably and stably secreting mouseanti-human CD244mAb was obtained successfully.