Preparation And Characteristic Of Monoclonal Antibodies To Tag Of FLAG And Its Application

With the development of genomics and proteomics, fusion protein purification has become an important task in proteomics. Protein purification accounts for more than 70% of the total later cost .Therefore, it has become a bottleneck to develope a fast, efficient, convenient and economical purification approach for further development. In this study, a strain of anti-FLAG monoclonal antibody which is of valid high titer, high specificity, good reaction, and high affinity was obtained through the application of lymphocyte hybridization technique. Affinity chromatography of the fusion protein with FLAG had good effects. The methods of purification and detection for FLAG fusion protein was successfully established. This will be an important tool for researching structures and functions of the largerly emergence of new moleculars in post-genomic era and have high practical value. At the same time, this method can be, as a technology platform, the technical support for preparation of monoclonal antibodies of a variety of other tagged proteins. This research is aimed to explore the effects of using different methods, various carriers to prepare complete antigen of FLAG and anti-FLAG tag monoclonal antibody and the roles of anti-FLAG tag monoclonal antibody in the purification of the fusion proteins.Using bovine serum albumin (BSA), ovalbumin (OVA) and keyhole hemocyanin (KLH) as a carrier protein by carbodiimide method were synthesized FLAG-BSA, FLAG-OVA and FLAG-KLH antigen, using SDS-PAGE to determine the initial coupling effect, and three kinds of antigens as the immunogen through the abdominal cavity, spleen and subcutaneous immunization in BALB / c mice were obtained after the end of the blood serum was detected by using indirect ELISA titer, thereby finalizing coupling effect, select the best person to use hybrid Preparation of tumor anti-FLAG tag monoclonal antibody hybridoma cell lines, preparation ascites, and its purification, analysis and identification. Preparation of cross-linked anti-FLAGmAb affinity chromatography purified fusion protein with a FLAG tag by SDS-PAGE analysis of purity after TLC, using Western-blot detection of mAb reactive to the fusion protein.By indirect ELISA, mouse tail blood titer, FLAG-KLH coupling effect of the best immune titer of 104 or more, a total of preparing an anti-FLAG tag monoclonal antibody hybridoma cell line, named for the 5F-2. The results of this monoclonal antibody identified as follows: antibodies belong to IgG, the subclasses of IgG1, repeatedly subcultured in vitro secretion of antibodies after the stable. 5F-2 monoclonal antibody ascites titer reached 106. Indirect ELISA, this monoclonal antibody confirmed that no cross-reactivity with other antigens, the specificity high. Prepared by HRP, activity reached 104, by affinity chromatography purified fusion protein purity of more than 85%, and the fusion protein can be used for Western-blot detection, the establishment of labels can be used with a FLAG fusion protein purification methods, and detection methods.Successful coupling of the FLAG complete antigen and prepared anti-FLAG tag monoclonal antibody with a FLAG tag for the purification of fusion proteins provide an important tool.