Mycobacterium tuberculosis Ag85B-IL.doc

 PAGE \* MERGEFORMAT 16 Mycobacterium tuberculosis Ag85B/IL [Keywords:] Ag85B; IL Prokaryotic expression, purification and identification of Mycobacterium tuberculosis Ag85B/IL2 fusion protein and detection of its biological activity [Abstract] AIM: To express Mycobacterium tuberculosis Ag85B / IL2 fusion protein in E.coli, to purify and identify it, and to detect its biological activity. METHODS: By using genetic engineering techniques, we constructed a recombinant expression plasmid pGEXAg85B/IL2, and expressed Ag85B/IL2GST fusion protein in E.coli BL21 under the induction of IPTG. Moreover, we also studied the optimal expression conditions concerning IPTG concentration and induction time. After renatured by urea in a concentration gradient, and purified by GSTSepharose affinity chromatography and digested by thrombin, the Ag85B/IL2 fusion protein was further purified by anionexchange chromatography and RPHPLC and identified with Western blot. Its Nterminal amino acid sequence and IL2 bioactivity were measured as well. RESULTS: A novel fusion protein Ag85B/IL2GST was expressed in E . coli in a way of inclusion body, accounting for 30% of total lysate protein of bacteria. After purified by GSTSepharose affinity chromatography, anionexchange chromatography and RPHPLC, the desired Ag85B/IL2 fusion protein with purification degree of 98.32% was acquired and confirmed by Western blot. Its Nterminal amino acid sequence was identical to the anticipation, and its specific IL2 bioactivity was 2500 u / mg. CONCLUSION: Ag85B/IL2 fusion protein was successfully expressed in E.coli and purified. This results established a groundwork for the further researches of Ag85B/IL2 fusion protein in the immunotherapy of bladder tumor. [Keywords] Ag85B; interleukin2; prokaryotic expression; fusion protein; purification [Abstract] Objective: Expression in E. coli Ag85B/IL2 fusion protein of Mycobacterium tuberculosis and its purified, identification and activity of the initial d