The Construction Of Anti-Trop-2Human IgG Eukaryotic Expression System And Characteristic Analysis

Human trophoblast cell surface antigen-2(trophoblast cell-the surface antigens, Trop-2), also known as TACSTD2, GA733-1, EGP-1, located in the1p32region, intron gene encoded by a product containing323amino acids,35.7kDa protein. It is a single transmembrane glycoprotein, found to be expressed at high levels by various types of human carcinomas. In contrast, normal epithelial tissues show little or no Trop-2expression. Trop-2expression has been associated with aggressive tumors and poor prognosis in variety of tumors. So Trop-2has become a potential target for tumor targeted therapy, and turned into a new prognostic marker for cancers.with the developmentof modern medicine and the molecular technology,people got to understand more about the tumor’s pathogenesis on the molecular and genetic level. So the tumor targeted therapy has become very popular in the field of cancer treatment in recent years. With the constant improvement of cancer therapy, a variety of treatments have been applied in clinical practice and made great achievements. As for monoclonal antibody treatment, it showed good anti-tumor effects. The monoclonal antibody can specificially target and bind the tumor cell’s surface antigens、complements、vascular endothelial cell growth factors or epidermal growth factors, which could induce the tumor cells’ apoptosis and inhibit tumor blood vessel’s formation by complement-dependent and antibody-dependent cytotoxicity.Compared to small molecule antibodies, the Fc part of whole molecule IgG can play an ADCC function. At present, the small molecule antibody scFv and Fab were often expressed in the prokaryotic cell, but not the whole molecule antibody.In order to transform the human Fab derived from phage display antibody library into full molecule antibody, the eukaryotic cells express system was used in this study. On the basis of anti-Trop-2Fab gene, the eukaryotic expression vector of the anti-Trop-2 fully human antibodies was constructed, and was transferred into CHOdhfr-cells to produce fully functional molecular antibody.Aims1. Construction of the human anti-Trop-2IgG eukaryotic expression system by using anti-Trop-2antibody Fab gene as the template.2. Expression and purification of IgG antibody to analyze the antibody’s activity and its inhibition to the pancreatic cancer cell’s growth, observe the anti-Trop-2monoclonal antibody inhibition of pancreatic cancer cell proliferation.Methods1. Constrution of the anti-Trop-2antibody IgG recombinant expression vector: amplify the heavy chain and light chain gene of anti-Trop-2antibody by PCR respectively,double digested pWS-2vectors and light chain separately, and cloned light chaingene into the vector pWS-2. After determing DNA sequence correctly, extracted the plasmid named pWS-L. Double digested pWS-L vectors and heavy Chain separately, and cloned heavy chain gene into the carrier pWS-L, and when DNA sequence was confirmed right, extracted the plasmid.2. Anti-Trop-2antibody IgG’s expression and purification:Recombinant plasmid transfected into CHOdhfr-cell, screened by dosing to subclone. The positive monoclonal was selected for further culture, and the culture supernatant was collected. Using Protein G affinity purification to obtain anti-Trop-2IgG antibody molecules.3. Identification of Anti-Trop-2antibody:Several immunological methods such as ELISA、SDS-PAGE、FACS and IHC were used to analyze the immunological characteriistics of the anti-Trop-2antibody.4. The proliferation of BxPC3cells was inhibited by anti-Trop-2antibody on significantly by MTT.Results1. After amplification by PCR, cloning and DNA’s sequencing analysis, this study has successfully constructed anti-Trop-2antibody eukaryotic expression system; the monoclonal cell lines which stable expressed anti-Trop-2IgG antibody molecules was selected by vector’s transfected into CHOdhfr-cells and dosing screening,and high purity of the anti-Trop-2humanized IgG antibody molecules was obtained finally.2. Both SDS-PAGE and Western blot analysis all showed two bands:28kD and55kD, respectively, which are consistented with the protein’s supposed molecular weight. Immunofluorescence detection showed anti-Trop-2antibodies canrecognize pancreatic cancer cell surface’s Trop-2membrane protein.3.MTT assay results showed that the as the time prolonged and the increased of the antibody dose,cell growth inhibition is gradually increased with antibody concentration increasing, The highest inhibition rates (84.15%) was induced by the highest concentration of antibody incubating72hours; compared with the control group,there are obvious differences of the cell proliferation inhibition among the different concentrations of antibody (p<0.05).Conclusions1. The anti-Trop-2antibody IgG eukaryotic expression system was constructed, and anti-Trop-2-human IgG antibody molecules were expressed and purified which could recognize native Trop-2protein specificially.2. Anti-Trop-2human IgG antibody could inhibit the proliferation of pancreatic cancer cells.