Construction Of Eukaryotic Expressing Vector Of Multiple Myeloma MUC1-2VNTR And Its Expression In COS-7 Cells In Vitro

Multiple myeloma,also known as myeloma or plasma cell myeloma,is a progressive hematologic malignacy.It is characterized by excessive number of abnormal plasma cells in the bone marrow,and secretes a monoclonal immunoglobulinprotein or its segment.At the same time,extensive fracture or osteoporosis and anaemia,contract,renal insufficiency constitute a quatemion that is strongly suggestive of multiple myeloma,Without treatment,advanced multiple myeloma patients,the median survival was only 6 months.The efficiency of conventional chemotherapy was only 40%～60%,complete remission rate was less than 5%,with a median survival period of time less than 3 years only.About 25%of patients survive more than 5 years;survival 10 years less than 5%of MM.For this reason,to find a new treatment for this currently incurable disease should be highlighted and will become very important need of current research project. Polymorphic epithelial mucin,encoded by the MUCl gene,is a type I transmembrane protein,which presents at the apical surface of glandular epithelial cells.It is over-expressed and aberrantly glycosylated in many carcinomas particularly in MM resulting in an antigenically distinct molecule and a potential target for active specific immunotherapy.In order to resolve the difficult problem,we construct an eukaryotic expression vector for multiple myeloma mucin MUC1-2VNTR gene and let it to express in COS-7 cells in vitro,so to provide the basic material for further research of multiple myeloma DNA vaccine.MUC1-2VNTR coding gene was used and the KOZAK sequence was inserted before the gene to improve the efficiency of gene expression. HindⅢand XbaⅠrestriction sites were inserted before and after the coding gene. Then the whole sequence was synthesized and inserted into pcDNA3.1/myc-his B vector,and the resulted recombinant vector was transfoemed into E.coil competent cells to get an engineed strain,identified by restriction analysis and DNA sequencing. After that,the recombinant plasmid pcDNA3.1-2VNTR/myc-his B were transfected into COS-7 cells by liposome-mediated gene transfer method.Finally,fluorescent microscopy was used to assess GFP expression and Western blot analysis using MUC1 monoclonal antibody to recognize VNTR,to confirm the expression of VNTR.As expected synthesized MUC1-2VNTR gene was 140 bp.Both restriction analysis and DNA sequencing demonstrated that pcDNA3.1-2VNTR /myc-his B included the whole translation flame region and MUC1-2VNTR gene.Furthermore, by fluorescence microscopy we proved that the recombinant plasmid has been successfully transfected into COS-7 cells.The expression of Mucin-1 protein was observed in the transfected cell and the cell secretion by Western blot.It was concluded that The pcDNA3.1-2VNTR/myc-his B has been successfully constructed and expressed in COS-7 cells in vitro,which could provide the basic material for further researches of MUC1 mucin function and possible multiple myloma DNA vaccine.