Rat Atf3 Gene Cloning And Expression And Of Monoclonal Antibodies

Part I Expression and purification of the recombinant protein of rat ATF3 in Escherichia coliObjection: To construct three truncated prokaryotic plasmids of rat activating transcription factor 3 (ATF3) gene and express the fusion proteins in BL21. Methods: The rat ATF3 gene was amplified, sequenced and subcloned into the expression vector pGEX-4T-1 to generate three recombinant plasmids pGEX-4T-1/ATF3, pGEX-4T-1/ATF3 (129-546bp) and pGEX-4T-1/ATF3 (237-546bp). The three combination plasmids were expressed in BL21 and rosetta by induction with isopropyl-β-D-thiogalactopyranoside (IPTG) and then analyzed by SDS-PAGE and Western blot. The fusion proteins were purified with Glutathione S-transferase (GST) affinity chromatography. Results: The isolated and cloned sequences of pGEX-4T-1/ATF3, pGEX-4T-1/ATF3 (129-546bp) and pGEX-4T-1/ATF3 (237-546bp) were confirmed by restriction enzyme digestion analysis and DNA sequencing. It showed no expected band in the SDS-PAGE, but the fusion protein ATF3-GST was detected by Western blot. The results of SDS-PAGE proved that ATF3-GST fusion protein purified with GST affinity chromatography was of low purity. Conclusion: Three truncated prokaryotic plasmids of rat ATF3 gene were constructed successfully, and pGEX-4T-1/ATF3 was correctly expressed in BL21. PartⅡProduction and characterization of a mouse monoclonal antibody against rat ATF3Objection: To produce mouse monoclonal antibody (mAb) against rat ATF3 protein. Methods: Peptides (168-181 amino acid of rat ATF3 protein) were synthesized using PREDICTED ANTIGENIC PEPTIDES and coupled to keyhole limpet hemocyanin (peptide-KLH). Balb/c mice were intraperitoneally immunized with peptide-KLH. Hybridomas were produced by fusing SP2/0 myeloma cells with spleen cells. The supernatant of the growing hybridoma was used to screen for the presence of anti-peptide-KLH antibody with ELISA. The positive clone was subcloned for three cycles by limiting dilution to select stable secreting hybridoma. A commercially mouse mAb isotyping kit was used to identify the isotype of this mAb. Western blot was used to determine whether the mAb could bind rat ATF3 protein. Results: One hybridoma secreting mAb against ATF3 was produced. Immunoglobulin subclass determination identified that this mAb was the IgG1/κsubtype. The results of Western blot proved that the new produced mAb could specifically recognize native and recombinated rat ATF3 protein. Conclusion: A mouse mAb against rat ATF3 protein was prepared successfully, and which provide a useful tool for studying ATF3 biological function in the future.