Construction And Application Of Escherichia Coli Targeting For Cancer Therapy

Recently,the bacteria-targeted therapy has become a research hotspot.a variety of natural and genetically modified non-pathogenic bacterial species are being explored as potential antitumor agents.The facultative anaerobic E.coli Nissle1917(O6:K5:H1)is a kind of probiotics with a higher safety for human,which has been proven to be capable of homing to tumors,not only in tumor hypoxic regions and necrotic area,also in oxygen enrichment area,which is not restricted by tumor size and tumor microenvironment,Thus it will be an ideal carrier for the treatment of cancer.Based on these advantages,we selected Nissle1917 as the carrier to deliver azurin to tumors.But Nissle1917 is extracellular bacteria,not invasive to tumor cells,which greatly reduces the efficiency of drug delivery and treatment.So we used Red/ET Recombination technology to construct pR6K-inv-hly translocation vector,which contains the inv gene from Yersinia pseudotuberculosis and the hly locus from Listeria monocytogenes,Then introduction of the inv and hly genes by transposon into the chromosomes of Nissle1917 confers it the characteristics of the intracellular bacteria,lysis cell membrane,begins to grow in the cytoplasm and invade a variety of tumor cells.Secondly,invasion and listeriolysin were indentified by RT-PCR,SDS-PAGE and LC/MS techniques in the level of gene and protein expression.Effects on tumor cells were detected by Laser confocal microscopy,results show that the egineered bacteria EcNVY has good activity for a wide variety of tumor cells such as B16 and HCT-116.Lastly,we transformed the egineered bacteria EcNVY with plasmid pET28a-vhb-azurin expressing of azurin protein,then azurin were indentified by SDS-PAGE and Western Blotting.In cell activity assay experiments,we found that the egineered bacteria significantly enhanced anti-tumor effect on B16 cells,which may relate to engineering bacteria directly delivering drugs to the tumor cells,but the specific mechanism remains to be further researched.This study indicates that egineered bacteria EcNVY could be a potential carrier to deliver antitumor drugs effectively for cancer therapy,which would be able to lay foundation for applications of Nissle1917 as a vector in gene therapy.