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Fusion protein (fusion protein) of two or more genes are connected through a certain way together express a recombinant protein. Tag fusion protein (tag fusion protein) is a fusion protein, it is by the target protein (target protein) and the fusion tag (fusion tag) together constitute the protein chimera. The common fusion tags include: poly-histidine (poly-His), FLAG peptide, staphylococcal protein A (SPA), the Fc portion of the immunoglobulin and glutathione transferase (GST). The use of fusion tags not only facilitate the expression and purification of the target protein, and at the same time provide a convenient means for the purpose of study of protein structure and function. With the completion of the Human Genome Project (Human Genome Project, HGP), after genomics (post-genomics) and proteomics (proteomics) has become the focus of the development of the life sciences, in this period, the biggest challenge faced by the scientists in the whole level system, a comprehensive study of the structure of proteins, the interaction between the function as well as protein-protein, nucleic acids and other biological macromolecules. The expression and purification of proteins is a key link in the study of the protein. Provides a high-throughput (high-through) platform of expression and screening of functional proteins tagged protein fusion expression thus extensive attention of scholars, has become an important link between a contact of the genome and proteome. In addition, the fusion protein in the separation of protein complexes, drug screening, and clinical treatment also plays important role. The monoclonal antibody technology is used to produce co-founded in 1975 by Khler and Milstein for a single epitope antibody immunological techniques. Monoclonal antibody has the advantages of high purity, specificity and high titer, with little or no cross-reaction, is widely used in various fields of life sciences. In proteomics research, not only for the monoclonal antibody of the fusion tag can be used as affinity chromatography media of the fusion protein were isolated and purified, can also facilitate the study of the distribution and function of the target protein, is the importance of the post-genome studies tools. An experiment, we have prepared seven stable hybridoma cell lines secreting anti-Ig fusion protein the FC paragraph monoclonal antibody FMUI7CI ~ 7, which FMUFCZ, F just FC4, FMUFCS and F deleted FC6 of IgGI (K); FMUFCI F puncturing FC3 and FMwC7, IgGZb (K). 7 InAb titers in the 0.5 ~ IXIO-', microtiter a titer is called in the 10 to 10 Diao. The competitive ELISA u biosensor analysis shows that the obtained monoclonal antibody recognizes seven different epitopes, wherein FMUFCS, FMUFC6, and FMUFC3 epitope relatively close 7 FMUF. 2 and FMU'FC4 epitope difference furthest. On the basis of the analysis of the epitope, we use FMUFC4 as coating antibody H belly F as the enzyme-labeled antibody to construct a dual mAb sandwich ELISA system for detecting Ig fusion protein, and by selecting the package is the antibody and enzyme-labeled antibody the optimal working concentration of its optimization. The sensitivity and specificity of the test showed that the level of detection of the system to achieve Zv A pair of IgG did not cross-react with other species. Expression COS River cells using the system LAIRl workers g fusion protein and anti of Notch ligand Deftal the chimeric antibodies were measured, and an A value of concentration dependent curve. This indicates that the sandwich ELISA system for the detection of the Ig fusion proteins and chimeric antibody levels in the supernatants can be used for cell culture. Interestingly, despite the FC3 F FMUFCS and F just FC6 epitope very close, but in the Western blot only FMUFC6 can be used for both Ig fusion protein and hlgG immunoblot detection, suggesting that in the process of protein denaturation, with the protein space structural damage, many of the antibodies recognize an epitope will disappear or weakened, and further illustrate a specific reaction between the antigen-antibody depends on the identification of the antigen and the antibody molecules in the spatial structure, rather than depend on the amino acids in the protein of the linear order. Utilization the affinity chromatography FMUFCS InAb prepared fusion proteins after transfection, the cell culture supernatant of COS River LAIR1 purified product was purified by SDS. Page displayed after chromatography the molecular weight of the protein obtained with the theoretical value, the purity is greater than 80%. This shows that the immunoaffinity chromatography can be effective purification medium of the Ig fusion protein, for the Ig fusion protein was purified to provide a new effective way. In addition, we have prepared my room five anti-GST, 5 strains of anti-m and an anti-FLAG monoclonal antibody to carry out a more systematic identification and some monoclonal antibodies initial application. The results show that in addition to the lower titers of anti-FLAG monoclonal antibody, can not be used for a corresponding fusion protein immunoblot, the other 10 monoclonal antibody titers were between 10 'to 10', and all can be used detecting in the corresponding fusion protein immunoblotting. The comparison found that the purified fusion protein of the anti-His tag monoclonal antibody affinity chromatography and for m superior N iu ten TA matrix metal affinity chromatography. FasL is important apoptosis-inducing ligand (poptosis inducing ligand, AIL), plays an important role in the immune defense and immune-steady. FasL antigen and anti-FasL ti body great value in basic and clinical research. Using genetic engineering techniques to construct and express people the FasL cytomembrane outside area with GST fusion protein and monoclonal antibody anti-GST and anti hFasL them identified, provided favorable conditions for FasL antigen and antibody preparation.
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