Construction Of Tumour-targeting And Invasive E.coli Top10

A major obstacle for the development of effective immunotherapy is the ability of tumors to escape the immune system. The possibility to kill tumor cells because they are recognized as infected rather than as malignant could help overcome immune esc- ape mechanisms. Recently, a number of studies have shown that it is possible to incre- ase the visibility of the tumor by inducing an inflammatory state within the tumor mi- croenvironment. This can be achieved by subjecting the tumor by subjecting the tum- or area to irradiation,radiofrequency ablation,or by injecting bacterial products or directly anaerobic bacteria. The original observation of tumor spontaneous recovery and regression of cancer recovery and regression of certain cancer patients from conc- urrent from concurrent bacterial infections was made over 300 years ago. Recently, obligate bacteria such as Bifidobacterium,Clostridia species and Corynebacterium pa- rvum as well as facultative anaerobes such as Salmonella were used to achieve tumor therapy in mice. Facultative anaerobes and non-pathogenic E. coli Top10 is a laboratory strain usually used for cloning purposes that does not produce secreted toxins nor mannose-resistant hemagglutinating adhesins.In order to study the tumour- targeting ability of E. coli Top10 , Five- to six-week-old female C57BL/6 mice were injected mouse melanoma cell. Five hundred microliters of the E.coli Top10 were injected into roliters of the E.coli Top10 were injected into the lateral tail vein of B16 tumorbearing C57BL/6 mice 14 days post-cell implantion.To determine bacterial load in tumor,spleen and liver tissues, mice were sacrificed at 2 day or 6 day, the organs were excised,weighed and homogenized, and serial dilutions plated on LB plates. Resultant colonies were counted, and CFU/g tissue calculated. In this study, in order to enable the E. coli Top10 to invade mammalian cells. We clone the inv gene from the Yersinia pseudotuberculosi genom. To constitutively express invasin, the inv gene was inserted into a plasmid under the control of a tet promoter.Invasiveness towards HeLa cells was assayed by gentamicin protection and was reported as the fraction of added bacteria recovered from lysis. The results showed that the E. coli Top10 strains showed the specificity concerning preferential tumor colonization with a tumor to liver to liver ratio in the order of 137:1 at day 4 and 530:1 at day 6 post-injection in B16 tumor-bearing C57BL/6 mice. Invasion/protection assays were show that 0.4% inv+E. coli Top10 were recovered. Our study show that facultative anaerobic bacteria E. coli Top10 as the tumor-targeting strain that colonizes tumors efficiently, replicates at the tumor. We construct the recombinant E. coli Top10 enable to invade mammalian cells.