Expression And Purifcation Of IER5 Fusion Protein

Immediate Early Response gene 5 (IER5) is a novel member of the slow-kinetics immediate early genes. There were very few in both the domestic and international literature with regard to IER5 gene. These researches showed that IER5 gene expression may impact on cell cycle and apoptosis and also play an important role in mediating the cellular response tomitogenic signals. IER5 was previously shown that the mRNA level of IER5 was up-regulated in human lymphoblastoid AHH-1 cells, Hela and HepG2 cells after irradiation. Suppression of IER5 gene resulted in an increased proliferation of Hela and HepG2 cells. Cell growth and survival analyses demonstrated that suppression of IER5 significantly increased the radioresistance of these cells to radiation, but reduced the radiosensitivity of cells. And it found that gene silencing of Hela cells had a larger cell size than normal tumor cells. Vice versa, IER5 gene inhibited cell's growth, increased the radiosensitivity of cells and the proportion of cell apoptosis under certain radiation effect in IER5-overexpression-cells, which had remarkablely smaller type compared with normal tumor cells suggesting that IER5 gene play an important role in inhibiting the occurrence and development of hepatocellular carcinoma and cervical carcinoma. The results indicated that IER5 might be involved in cell cycle and proliferation as well as tumorigenesis. Its function was similar to that of the anti-oncogene. In order to further understand the biological function of IER5, it is necessary to clarify the detailed biochemical pathway and mechanism of IER5 action in tumorigenesis which indicates IER5 might be useful target for the therapeutical treatment of the carcinoma.IER5 gene is probably an intronless gene. It is in a region of high genomic GC content (60.5% for whole cDNA, 71.7% in the opening reading frame) which is probably difficult for protein synthesis and preparation for monoclonal antibodies because of forming into a complex secondary structure for PCR in the experiment. Preparation of monoclonal antibodies have some merits, for example, easy to be produced, homogeneous character in physical and chemical properties , upstair specificity, easily standardized, and so on. Monoclonal antibodies can improve the specificity and accuracy in clinical immunology analysis and clinical management to be of great significance for biology, chemistry, medicine, immunology and other areas.Currently, there is only IER5 polyclonal antibody. It brings some difficulties in protein immunoprecipitation (PIP) and Western-blot, which are needed to detect IER5 protein. Therefore, we decided to manufacture monoclonal antibody of IER5 fusion protein. Preparation for IER5 fusion protein is a key step in the process of manufacturing the monoclonal antibody. In this research, to be prepared for developping monoclonal antibody of IER5, Real-time PCR and SDS-PAGE, Western blot analysis were employed to manufacture considerable recombinant fusion protein, which arranges for further study on IER5 gene in the role of radiation therapy in hepatocellular carcinoma.Objective: The subject of using PCR, SDS-PAGE, Western blot, by inducing expression, ultrasonic crushing, denaturation, renaturation, through the Ni-NTE resin column, dialysis and concentration by polyethylene glycol obtained large quantities of purified recombinant fusion protein in order to prepare for monoclonal antibody.Methods: A 984bp of IER5 gene fragment was synthesized and cloned into vectors pET22b(+) and pGEx-5T E.coli expression vectors. The plasmids were transformed into E.coli DH5αand analysed by BLAST. The pET22b(+)-IER5 plasmid was extractied from DH5α, and then was transformed into E. coli BL21(DE3) and induced to express pET22b(+)-IER5 fusion protein with IPTG. We constructed prokaryon pET22b(+)-IER5, pGEx-5T-IER5 expression vector sacrifice checkout IPTG presence Bacillus coli which stabilized express fusion protein pET22b (+)-IER5. Thein fusion protein was identified by SDS-PAGE and Western blot. The use of imidazole ompete elutted off fusion protein in competition with the combination of nickel ion.Results: We succeed in constructing procaryon pET22b(+)-IER5 expression vector and transforming bacillus coli bolting masculine clone induce express. The critical parameters of IER5 proteinum and brachytmema proteinum prokaryotic expression in E. coli was found, the inducing temperature was 37℃, the inducing time was 4 hours and the dosage of IPTG was 0.01mmol/l. The fusion protein was expressed mostly in the form of inclusion bodies. After inclusion bodies dissolving in 8molPLurea, then flowing through Ni2 + affinity chromatography, dialysis and finally concentration, the purification effects of fusion protein was identified by SDS-PAGE and Western blot. The target proteins expressed by E. coli took account of 20% total cell proteins, the purified recombinant proteins with purity up to 95%.Consultation: Our successful cloning and expression of human IER5 gene and purification of IER5 protein lay a basis for further study on the application of this protein to immuning animals and screening of monoclonal antibodies.