Establishment Of Human CD48 Transgenic Cell Line And Preparation Of Anti-human CD48 Monoclonal Antibody

CD48, a glycosylphosphatidylinisotol(GPI)-anchored glycoprotein, has a molecular weight of 40-45kD. It belongs to CD2 family of Ig-like receptors that comprises the member of CD2, CD58, CD244, Ly9 and SLAM. CD48 broadly expressed on a series of immune cells, such as T cells, B cells, NK cells, dendritic cells, monocytes, neutrophile granulocyte, eosinophile granulocyte and mast cells. CD48 also expressed on endothelial cells and some tumor cells. High level of soluble CD48 is found in plasma of cancer patients as well.CD48 is involved in the function of NK cells and takes part in the homeostasis of NK cells. CD48 plays an important role in stimulating IL-2 production and sustaining T cell further activation. It is reported that CD48/CD244 is a novel target that independent of PD-1 pathway to treat the viral infectious disease. Importantly, it provides a useful tool to precisely define the primitiveness of hematopoietic stem cells by analyzing their CD48, CD244 and CD150 expression.Taken together, CD48 participates the development of immune cells and takes part in immune response and immune regulation. It plays important roles in tumor immunology and autoimmune disease. It is a promising target of immunological intervention as well.Part I Establishment of human CD48 gene transfected cell lineAims:To establish the gene transfected cell line that expressed human CD48 stably.Methods: Total RNA of human peripheral blood mononuclear cells that separated from healthy donor was isolated, full length of open reading frame of human CD48 gene was cloned by RT-PCR and subcloned into retrovirus expression vector pEGZ/term and the recombinated vector named pEGZ/CD48. pEGZ/CD48 together with its two helper virus vectors pHIT456 and pHIT60 were cotransfected into package 293T cells. And the supernatant containing recombinated retrovirus was harvested to infect L929 cells. The stable transfected cell lines were selected by zeocine, and the CD48 expression were analyzed by FACS and RT-PCR.Results:1) Obtained human CD48 gene and established its recombinant retrovirus expression vector named pEGZ/CD48. Restriction analysis and DNA sequencing demonstrated that the correct human CD48 gene was obtained and inserted into vector pEGZ-term.2) Established a stable gene transfected cell line that expressed human CD48. 48h after cotransfected with plasmid pEGZ/CD48, pHIT60 and pHIT456, GFP was expressed in of 293T cells. The retroviral supernatant was harvested and added into culture medium of L929 cells. 2 weeks later, zeocine resistant clones formed and expressed GFP. The CD48 expression on zeocine resistant clones was verified by FACS and RT-PCR. A transfected cell line that stably expressed human CD48, named L/CD48, was established.Conclusion: We successfully cloned full length of open reading frame (ORF) of human CD48 gene, constructed recombinated retrovirus expression vector, established a stable human CD48 gene transfected cell line.Part II Preparation of murine anti-human CD48 monoclonal antibody and analysis its characteristicsAims: To provide a useful tool for studying the bio-function of CD48. Methods: Splenocytes of the Balb/c mice that immunized with leukemia cell line THP-1 were fused with SP2/0 cells according to the hybridoma technique. Hybridoma cell lines were screened by flow cytometry. L/CD48 cells were used as an antigen for immunization and as the positive control, and L/mock cells were used as the negative control. The immunoglobulin isotype was analyzed with test paper. Immunostaining and FACS were performed to analyze the binding pattern of mAb 3H6 to the CD48 molecule on different cell lines. And the competitive binding experiment was applied to determine whether the mAb 3H6 and MEM-102 bind the different epitope. MAb 3H6 was prepared and purified from the ascites and added into the culture medium of CD48+ leukemia cell lines, Raji and THP-1. Cell counting was used to evaluate the effect of mAb 3H6 to the growth of leukemia cells.Results:An anti-human CD48 MAb 3H6, with IgG1 heavy chain andκlight chain, was obtained and the specificity of this mAb was verified by flow cytometry. Consistent to commercial mAb MEM-102, MAb 3H6 effectively recognized the CD48 molecule expressed on a series of malignant cell lines. MAb 3H6 recognized different eptitope in compare with mAb MEM-102. Furthermore, we demonstrated that MAb 3H6 could induce aggregation and inhibit the proliferation of Raji cells and THP-1 cells.Conclusion: We successfully prepared a murine anti-human CD48 monoclonal antibody. It provides a useful tool for further studying of mechanism of CD48 biofunction and immunological intervention that targeting CD48 molecule.