Construction Of Cell Lines Transfected With Human CD160Gene And Preparation Of Mise Anti-human CD160Monoclonal Aantibody

Part Ⅰ Construction of cell lines transfected with human CD160geneCD160which is a glycosylphosphatidylinositol (GPI)-anchored protein is newmember of immunoglobulin superfamily.The interaction of CD160with HVEM, thespecific legend for CD160, generate signals in the activation, proliferation and cytokineproduction of T cells during the secondary immune responses. As a negative receptor,CD160molecule is involved in the regulation of immune response. Thus, establishmentof human CD160transfected cell line and monoclonal antibodies against human CD160may provide the valuable tools for the research of complicated relationship ofco-stimulatory signal in immune response. They are also useful for clinical diagnosisand effective treatment.Objective: To construct gene transfected cell lines that stably expressed the humanCD160.Methods: Human CD160full-length cDNA was obtained from human wholeblood cDNA library by RT-PCR and then was inserted into retroviral expressing vectorpEGZ-Term. After proved to be correct by sequencing, the recombinant vectorpEGZ-Term/CD160were cotransfected into the package cells293T with Lipofectamine2000and its two helper virus vectors. The L929cells were infected by the culturalsupernatant of the transfected293T cells. The infected L929cells was further selectedwith Zeocin for a long time and identified by FCM and RT-PCR. Eventually，we obtaingene transfected cell lines that not only have Zeocin resistence but also stably expressthe human CD160. Results: Human CD160full-length cDNA was cloned successfully, and genetransfected lines L929/CD160stably expressed the human CD160were constructedsuccessfully.Conclusion: Gene transfected cell lines stably expressing the human CD160wereconstructed successfully, which lay the foundation for preparing mouse anti-humanCD160monoclonal antibody and studying the biological function of CD160molecule.Part Ⅱ Preparation of mouse anti-humanCD160monoclonal antibodyObjective: To prepare mouse anti-human CD160monoclonal antibody.Methods: BALB/c mouse were immunized with CD160transfected cell L929/CD160that stably expressed human CD160molecule. The spleen cells of immunizedmice were fused with myeloma cells SP2/0, and then cultured in HAT selective medium.Hybridoma cells were selected with L929/CD160cells by Flow Cytometry. Meanwhile,L929/mock cells, which were respectively transfected with vector pEGZ-Term, wereused as negative control. The positive clones were repeatedly subcloned, until onehybridoma cell line sustainably and stably secreting specific anti-CD160mAb wasobtained. By using the methods of Western blot and competitive inhibitionexperiment,we studied biological characterization of the obtained antibody. MAb Igclass was detected with Rapid Qualitative Test Strip. Indirect immunofluorescence assaywas used to detect the expression of CD160on the T cell from health donor.Results: A hybridoma cell line named5A7sustainably and stably secretinganti-CD160mAb was obtained. The mAb5A7was proved to be IgG with κ lightchain. The monoclonal antibody was produced according to ascites-inducing procedure,and the average ascites productive was3ml each mice. The ascites was purified byprotein G affinity chromatography. The result of competitive inhibition experimentrevealed that monoclonal antibody5A7and commercial anti-CD160antibodyrecognized different epitopes. The mAb could recognize human T lymphocytes fromhealth donors. Conclusion: A hybridoma cell line sustainably and stably secreting mouseanti-human CD160mAb was obtained successfully.