The Construction And Detection Of New Tumor Gene Therapy Vector Attenuated Salmonella

Cancer gene therapy is to use gene technology translating target genes into target cells and expression, execution or mediated tumor destruction and inhibition, so as to achieve the goal of treatment. Choose the right carrier has been the focus of the cancer gene therapy research. Salmonella typhimurium is a facultative anaerobic bacteria, it can accumulate in hypoxic tumor tissue and reproduction, and gathered at the tumor site (including metastases)1,000times than the normal tissue. Between it and the host mediated by the type III secretion mechanism, this mechanism of bacterial effector proteins directly transferred to the eukaryotic cells, so that it can dilivery the expression of the effects of genes and expression of a variety of therapeutic proteins. The above two characteristics of Salmonella make it possible to be a targeting vector for cancer gene therapy. Attenuated Salmonella strains can be made by knockout virulence gene with the rapid development of genetic engineering techniques in recent years.In this study, we choose bacteria-directed enzyme prodrug therapy, so the activating prodrug enzyme gene can be transferded to the tumor and be expressed by the selective colonization of Salmonella in tumor tissues. Though the systemic use of non-toxic prodrug, the prodrug can only be activated within the tumor tissue with anti-cancer activity of drugs, so as to achieve the killing of tumor cells and reduce the impact of anti-cancer drugs on normal tissues.The first part focused on the construction of the new vector ST-CD strains by using attenuated Salmonella typhimurium VNP20009. First, we get auxotrophic strains ST/Aasd by the Red homologous recombination technology, then we construct Asd-CD plasmid and introduce it into the auxotrophics strains ST/Aasd, and at last we sucessly screen and identify the new vector of ST-CD strain. We find that the new vector ST-CD can convert exogenous5-fluorocytosine into5-fluorouracil.The second part focused on the hypotoxicity and tumor-targeting ability of the new vector ST-CD. Non-tumor-bearing mice were infected intravenously with ST-CD or wild-type. All mice infected with wild-type S.typhymurium expired by4days. In contrast, all mice infected with ST-CD survived. Then tumor-bearing mice were infected intravenously with ST-CD, the tumor/liver bacteria ratios were1020:1by day6after infection. It seems the new vector ST-CD has the very ability of tumor-targeting and hypotoxicity.The third part focused on the observation of tumor bearing mice by infection intravenously with ST-CD and intraperitoneal injection of5-fluorocytosine. The results showed that compared to the control group, ST-CD/5-Fc system has good anti-tumor effects which may be affected by the inhibition of ERK expression.