Eukaryotic Expression And Monoclonal Antibodies Preparations Of GdX And P15RS Protein

Abnormal expression of Gd X and p15 RS gene in several diseases, especially autoimmune disease occurrence and development plays an importent role, based on the current domestic and foreign research and application of monoclonal antibodies against the genes have not been very common. Monoclonal antibodies have become necessary research tools and treatment methods in many aspect, how to prepare a high performance Gd X and p15 RS monoclonal antibody and its application has become an hot topic and main trend on Gd X and p15 RS gene researching.In this text two typical gene Gd X and p15 RS has been selected to study eukaryotic expression and preparation of monoclonal antibodies, and then identifing the Gd X and p15 RS monoclonal antibodies’ properties in several ways. In the process, The Gd X and p15 RS gene were inserted into p EF-neo-FLAG and pc DNA3.1-Myc plasmid vector to establish the eukaryotic expressing system to transfected HEK293 T cells. Gd X and p15 RS were purified to immune BALB/C mice and titer by ELISA. With the hybridoma technology and limited dilution, Gd X and p15 RS monoclonal antibodies were obtained,which have high specificity and affinity. The detail results are showed below:(1)Ca Cl2 method had been applied to amplify plasmids using E.coli DH5αwith. The A260/280 were up to 1.8 and then got high purity level plasmid for protein expression; Cell growth conditions and transfec environment were induced according to HEK293 T high-secreting expression cell lines Lipofecter2000 liposomal transfection reagent prepared.The cell lines had high stability and added vitality. The concentrations of recombinant Gd X and p15 RS proteins were 2.28mg/ml and 1.71 mg/m by non-denaturing SDS-PAGE analysis and western blot.(2)Serum antibody was raised with mice via immunizing purified plasmid DNA and recombinant proteins, which titred up to 105. Mice spleen cell which P/N vaule much than 2 were choosed to fuse. After that hybridoma cell were produced by polyethyleneglycol(PEG)by means of fusioning lymphocytes and expanding culture. Two Gd X hybridoma cell lines that can be produced monoclonal antibodies were obtained, which named 1-B2 and 4-D3. Two p15 RS hybridoma cell lines were named 2-C8 and 5-H11. The purity of the four m Abs were higher 95% and the relative affinity constants have reached much than 1.0mol/L. Immunoglobulin subtype results demonstrated that the antibodies were all Ig A. The ELISA titers of ascites produced by Mc Abs were up to 100×25. Antibody ELISAs which detected antigens demonstrated that all four Mc Abs were strongly positive with Gd X or p15 RS.In this study, a preliminary study of the Gd X and p15 RS gene had been done. Meanwhile anti-Gd X and anti-p15 RS protein monoclonal antibody had been successful prepared, whic have stable biological characteristics. It provides a theoretical basis and lay foundation for researching and application of Gd X and p15 RS antibodies in the future.