The expression of human CD55 and CD55-TM molecules in mouse fibroblasts cell line NIH3T3 and their function in complement lysis restriction / 中国免疫学杂志

ABSTRACT

Objective:To express the GPI-anchored CD55 and recombinant transmembrane form CD55 molecules on mouse fibroblasts cell line NIH3T3, and compare their inhibitory function of complement lysis.Methods:In previous study,had constructed the transmembrane form CD55 (CD55-TM) cDNA by linking the extracellular portion of CD55 to the transmembrane and cytoplasmic domains of MCP, and then subcloned into retroviral vector pLXSN. In this experiment,had transfected recombinant CD55-TM and GPI anchored CD55 into PA317 packaging cell to generate stable virus-producing cell lines. And then, mouse fibroblasts cell line NIH3T3 was infected with the virus containing CD55, recombinant CD55-TM or pLXSN alone. The expression of these molecules on NIH3T3 cells was detected by FACS analysis. Complement killing assay was carried out using MTT colorimetric method.Results:FACS analysis showed that both CD55 and its TM version were expressed stably on NIH3T3 cells. Both kinds of CD55 molecules can efficiently protect the NIH3T3 cells from complement mediated lysis and there is no significant difference between them. Conclusion:These data showed that both GPI-anchored CD55 and its TM version can normally expressed on NIH3T3 cells and both can protect the NIH3T3 cells from human complement mediated lysis. These results confirmed the feasibility of TM CD55 based gene therapy could be used for PNH, and also provided an excellent model for the study of signal transduction mechanisms mediated by GPI-anchored protein.