Research On Preparation,Characterization And Application Of Monoclonal Antibodies Against Fusion Tags

Fusion proteins are recombinant proteins which are encoded by two or more combined genes. One kind of fusion protein is tag fusion protein. A tag fusion protein is composed of two different parts: one is target protein and the other is fusion tag. Most frequently used fusion tags are: poly-His tag, FLAG tag, Staphylococcal protein A, and Glutathione-S-transferase. Fusion tags facilitate the expression and purification of the target protein, while it provides powerful tool for researchinh the structure and function of the target protein. With the accomplishment of Human Genome Project(HGP), the key arena of life science come to post-genomics and proteomics. The major challenge for scientists in this period is the systematic and comprehensive analysis of protein structure, function and interaction between proteins or between protein and other biological macromolecules, eg, nucleic acids. Protein expression and purification is the key progress in the study. Therefore researchers have payed a lot attention to the tag fusion system because it provides a sound platform for high-through expression and screening of the functional proteins. Now tag fusion system is becoming a vital bridge joining the genome research and proteome research. In addition tag fusion proteins pay very important role in other scientific field such as protein complex isolation drug screening -, clinical therapy and so on.Monoclonal antibody was firstly described in 1975 by Kohler andMilstein. It is a immunological technique to produce antibodies, against a single epitope. Due to the high purity, stronger specificity and lower cross-reaction, monoclonal antibodies have been applied in each field of life science. In protoemics research, monoclonal antibodies against fusion tags are not only used as matrix in affinity chromatography, but also employed to study the distribution and function of the target proteins. Thus the monoclonal antibodies against fusion tags have been a very important tool in the post-genome period.In this study we established seven hybridoma cell strains which can stably secrete mAbs against Fc fragment of Ig-fusion protein. We detect the isotype of these mAbs and find that FMUFc2, FMUFc4, FMUFc5and FMUFc6 are IgG1 , FMUFcl, FMUFc3 and FMUFc7 are IgG2b. The ascites titer of these mAbs are between 0. 5-1+10-7and the titer of HRP labeled mAbs are betweenlO -4-10-5. Results of competitive ELISA and pairwise testing of epitope mapping analysed by biosensor showed that these mAbs recognize 7 different epitopes respectively. We find that the epitopes recognized by FMUFc3, FMUFc5 and FMUFc6 are contigeous while the largest difference lies in epitopes recognized by FMUFc2 and FMUFc4. A sandwich ELISA for detecting Ig-fusion protein was developed using FMUFc4 as coating mAb and HRP-FMUFc5 as enzyme labeled mAb. The ELISA system was optimized with the detection limitation of 2ug/L when ABTS used as substrate. Cross-reaction test showed the system is hlgG specific. LAIR-1-Ig fusion protein and chimeric antibody agaist hDeltal in condition medium of COS-7 transfected with corresponding vectors were detected and evident dose-dependent A values were obtains. So the system can be easily used to monitor the expression of the recombinant proteins containing the Fc fragment of hlgG. It' s interesting that only FMUFc6 can be used in immunoblot assay although the epitope recognized by FMUFcG is similar to that of FMUFc3 and FMUFc5. This result suggests that many epitopes may be damaged in the progress of degeneration. Condition medium of COS-7 cells transfected with LAIR-1-Ig vector was purified by affinity chromatography column made of Sepharose 4B cross-linked FMUFc3 mAb. SDS-PAGE shows the MW of purified protein is about 60kDa which is consistent with the theoretical MW of LARI-1-Ig fusion protein. This method provides an effective approach to purify Ig fusion proteinbesides protein A or protein G.In addition, we systematically characterize five anti-GST mAbs, five anti-His mAbs and one anti-FLAG mAb that developed...