Construction And Preliminary Identification Of Attenuated Salmonella Strains Carrying EPNP/MePdR Suicide Gene System

Research background: As the world enters an aging society,the increasing incidence rate and mortality of tumors not only force countries to invest a lot of human and financial resources,but also seriously reduce people’s quality of life.So far,all kinds of gene therapy,immunotherapy and molecular targeted drugs combined with traditional surgery,radiotherapy or chemotherapy have greatly extended the survival period of patients,but still can not significantly reduce the mortality of patients with solid tumors.The global incidence rate of tumors is still showing a rapid upward trend.With its unique tumor targeting ability,cell invasion ability,tumor tissue permeability and the rise of synthetic biology in recent years,bacteria therapy has become a rising star in the field of tumor treatment.In the field of bacterial anti-tumor therapy,bacterial detoxification and functional modification are the focus of scientists’ attention.Researchers have knocked out bacterial toxic genes through various pathways and carried out functional modification,hoping to construct strains with high tumor targeting ability and good safety,utilizing the strain’s ability to target tumors and its own immunogenicity to kill tumors.However,they have found significant differences between reality and theory,Because it is difficult to find a balance between bacterial toxicity and efficacy.Subsequently,scientists turned to genetic engineering technology to transform bacteria into miniature "robots" that could carry various substances,utilizing the tumor targeting ability of bacteria to bring exogenous substances to the local tumor area for action.Salmonella can transport recombinant plasmids containing external genes into eukaryotic cell and express them through its own unique secretion mode,so Salmonella can be used as a good carrier for bacterial therapy;Gene directed enzyme prodrug therapy(GDEPT)is a prodrug delivery therapy system,which is mainly composed of three parts: a carrier carrying foreign genes,an inactive drug(prodrug)and a gene encoding an enzyme that converts an inactive prodrug into an active drug;In the GDEPT treatment strategy,the ePNP/MePdR prodrug system has attracted attention due to its powerful anti-tumor and bystander effects.The principle is that ePNP can convert non-toxic MePdR into toxic Me Ps,which affects the survival of cells by interfere with the synthesis of DNA,RNA and protein.Research purposes: Previous studies have shown that the ePNP/MePdR prodrug system mediated by viruses and bacteria can show a strong anti-tumor effect in melanoma,pancreatic cancer,colon cancer and other cancer models,but the anti-tumor study of bladder cancer with Salmonella as the carrier has not been reported at home and abroad.This study first constructed a plasmid carrying the ePNP gene to investigate whether it can be stably expressed in Salmonella;Secondly,in vitro experiments were conducted to verify the killing effect of Salmonella carrying the ePNP/MePdR prodrug system on mouse bladder tumor cells.It is hoped that this will lay the foundation for further research on the ePNP/MePdR prodrug system and Salmonella anti-tumor therapy.Research methods: In this experiment,primers were first designed by the nucleotide sequence of ePNP provided in Gen Bank,and the target gene was cloned from E.coli DH5α,and a eukaryotic expression plasmid carrying the target gene was constructed;Transfer the recombinant plasmid carrying the ePNP gene into Salmonella,a strictly anaerobic attenuated Salmonella typhimurium constructed in the laboratory in the early stage,and conduct preliminary identification through gene sequencing;Finally,the direct killing effect of recombinant Salmonella carrying the ePNP target gene on mouse bladder tumor cells was verified through cell and bacterial co culture experiments in vitro.Conclusion: In this experiment,an attenuated Salmonella carrying an ePNP/MePdR suicide gene system recombinant plasmid was successfully constructed.In vitro experiments showed that recombinant attenuated Salmonella carrying ePNP/MePdR suicide gene system had a powerful killing effect on mouse bladder tumor cells,which provided an experimental basis for subsequent in vivo research..