Construction Of MAP30 Gene-containing Anti-tumor Engineered Bacteria And Its Anti-tumor Activity

Although people committed to treat cancer with bacteria have more than 100 years,due to defects and limitations caused by bacterium itself,resulting the treatment of cancer progress very slow.With the development of modern microbial genetic engineering technology,brought a new dawn for us.MAP30,is the 30 kDa Momordica charantia anti-tumor protein.It is isolated from the mature fruit and seed of bitter melon.Such protein belongs to single type I ribosome inactivating protein family and widely existed in higher plants,which named by inactivated the ribosome of eukaryotic cells.It has anti-virus,anti-tumor,anti-bacterial and others biological activity.In our study,mature map30(signal peptide sequence excluded)was amplified from the genome of Momordica charantia and inserted on the expression vector pET28a.The final expression vector pET28a-map30 was transformed into E.coli Rostta(DE3)by addition of IPTG for MAP30 expression.The recombinant MAP30 was identified by SDS-PAGE,Western Blot and LC-MS/MS.After purification by nickel-affinity chromatography,The DNA-cleaving activity was analyzed by electro-phoresis after supercoiled plasmid pUC19 as substrate treated with recombinant MAP30 of various concentrations.Meanwhile recombinant MAP30 was used to treat MCF-7 human breast tumor cells,MTT,DNA Ladder,AO/PI double-stained is used to analysis the anti-tumor activity.Results show that map30 gene was successfully expressed in E.coli Rostta(DE3)and been identified by LC-MS/MS and Western Blot using his-tag mAb.We first discovered that the recombinant MAP30 like natural MAP30 protein also exert the activity conversion supercoiled plasmid pUC19 to relaxed or linear forms.The cytotoxicity assay of recombinant MAP30 showed that it exhibited dose-and time-dependent inhibition to MCF-7,gel electrophoresis detected DNA Ladder,AO/PI double-stained displayed typical of apoptosis,these results suggested that recombinant MAP30 could induce MCF-7 apoptosis in vitro.Meanwhile,MAP30 compared anticancer drugs 5-Fu and ?-Elemene,We through Giemsa staining and Confocal observed their subcellular structure differences after acting on MCF-7 cells.Based on the above research,We further employed Red/ET homologous recombination technology,map30 gene was inserted into a low oxygen promoter to construct the hypoxia-inducible expression vector and bring it transferred into E.coli Nissle 1917.This paper identified anti-tumor activity of MAP30 from Momordica charantia,which provided a theoretical basis for the further research on molecular anti-tumor mechanism of MAP30.At the same time,we constructed the anti-tumor target engineering bacteria E.coli Nissle 1917(pET28a-Pvhb-pelb-map30)which can induce the expression of MAP30 protein in hypoxic environment,it established foundation in remote control of engineering bacteria expressing anti-tumor drugs.We also confirmed the superiority of Red/ET homologous recombination technology in molecular cloning field.