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IER5 Immediate Early Response gene 5 gene referred to, is one of the early response gene family. Retrieve the literature that people IER5 genetic research are not many, existing research shows IER5 changes in gene expression of the cell cycle and apoptosis has a regulatory role, participated in the cells to promote mitotic signal response regulator. Our previous study results show that: after radiation the lymphoblasts (AHH-1), cervical carcinoma (HeLa) and hepatoma (HepG2) cells IER5 gene expression; inhibit IER5 gene expression becomes faster Hela and HepG2 cells, the cells on the the antagonist enhanced radiation radiation sensitivity weakened, and IER5 gene silencing Hela cell volume than normal tumor cells; contrast, increase IER5 expression, Hela and HepG2 cell proliferation slows down, and IER5 genes highly expressed in Hela cells was much smaller size increased the proportion of apoptosis in normal tumor cells after radiation, IER5 high expression of cell radiation resistance weakened radiation increased sensitivity. These studies suggest that we IER5 gene with liver cancer, cervical cancer occur related to the development, involved in the regulation of the cell cycle, may be similar to the function of the tumor suppressor gene. These findings suggest that it is necessary for us to expand more in-depth study of the IER5 gene further elucidate the biological function of the gene, it is possible for the the tumor radiotherapy looking to new radiation-sensitive genes, to enhance the effect of clinical tumor radiotherapy. IER5 genes without introns, the entire cDNAGC content of 60.5% (open reading frame of 71.7%), in the PCR process into a complex secondary structure, bring difficulty to synthetic IER5 fusion proteins and monoclonal antibodies. The easy preparation of monoclonal antibodies, uniform physical and chemical properties and biological activity of a single, specificity, ease of standardization, etc.. Using monoclonal antibodies, can improve the specificity, and accuracy of the immunological testing and clinical fields such as biology, chemistry, medicine, immunology has important significance [13]. Currently market only IER5 is polyclonal antibodies for protein immunoprecipitation experiments IER5 interacting protein and Western-blot experiments IER5 the protein detection ineffective, difficult biological function of this gene to further study IER5, so our group decided to prepare a IER5 monoclonal antibody synthesis IE5 fusion protein is a key step prepare IER5 monoclonal antibody research time is limited, this study intends to Real-time PCR, SDS-PAGE and Western blot method of preparation of a large number of restructuring IER5 fusion protein is prepared IER5 monoclonal antibody to prepare the foundation for further depth research IER5 genes in the radiation treatment of liver cancer and other tumors. Objective: This topic using PCR, SDS-PAGE and Western blot, and other methods, by inducing the expression, ultrasonication, denaturation, renaturation, over NI-NTE nickel column, dialysis, polyethylene glycol concentrated purified protein of experimental techniques to obtain The large number IER5 recombinant fusion protein, monoclonal antibody preparation to prepare for IER5. Method: synthetic IER5 gene, the gene into the prokaryotic expression vector pET22b () and pGEx-5T, transforming competent cells DH5α, sequenced and analyzed. After sequencing, the plasmid was extracted, and then the plasmid was transformed into competent E. coli BL21 (DE3). I.e. to construct prokaryotic pET22b ()-IER5 expression vector, pGEX-5T-IER5 by isopropyl half 2B2D2 thio-galactoside (IPTG) in the E. coli system stably expressed the fusion protein pET22b ()-IER5, by Western blot and SDS -PAGE analysis of pET22b ()-IER5. Application imidazole fusion protein competition with the nickel ion binding to the fusion protein was eluted. Results: The building was cloned into vector pET22 b ()-IER5 expression vector pGEX-5T-IER5 transformed into E. coli, and positive clones were screened pET22b ()-IER5 induced expression. The key expression parameters determine the prokaryotic expression: induction temperature of 37 degrees, the induction time for 4h and IPTG concentration of 0.01mmol / l, stable expression in E. coli. Through 8M urea degeneration, a HisTrap HP affinity chromatography column, dialysis, concentrated technology to obtain expression product was cloned into vector pET22 b ()-IER5 fusion protein, mainly in the form of inclusion bodies, 20% of the total expression of total bacterial protein purified prove expression products with higher purity of 95%. The prokaryotic expression CONCLUSION: This study successfully constructed human IER5 gene prokaryotic expression vector pET22b ()-IER5 pGEX-5T-IER5 and purification of the gene product pET22b ()-IER5 fusion protein. Obtain a purity of 95% IER5 fusion protein, as the immunized animal and screening of monoclonal antibodies to prepare.
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