Effect Of CD40 Gene Vaccine Mediated By Attenuated Salmonella On Rat Colorectal Cancer Models

Part one: The construction and functional identification of the recombinant eukaryotic expression vector carrying rat CD40 geneObjective: to construct eukaryotic expression vector with p IRES2-EGFP-CD40. Methods: After cloning rats CD40 gene from rat spleen and constructed a recombinant eukaryotic expressing plasmid with CD40,We transformed it into COS7 cells. RT-PCR and fluorescence microscope observation were used to detect the expression of CD40. Results: We construct successfully the recombinant eukaryotic expressing plasmid: p IRES2-EGFP-CD40. COS7 cells could steadily express CD40. Conclusions: The recombinant eukaryotic expressing vector carrying CD40 gene was constructed successfully.Part two: Construction and indentification of recombination attenuation salmonella typhimurium vaccine carrying p IRES2-EGFP-CD40 vectorObjective: To construct the recombination attenuation salmonella typhimurium vaccine that carries p IRES2-EGFP-CD40 vector. Methods: First the vector was converted into LB5000(salmonella typhimurium) for methylation and then the modified vector was electrotransfered to the final host SL3261.Screened single colony by antibiotics was tested by plasmid extract and PCR. Results: Plasmid similar to the original one was extracted from imported strain SL3261 which contains the same gene sequence identified by PCR. Conclusions: The p IRES2-EGFP-CD40 plasmid was successfully transferred into attenuated salmonella typhimunium.Part three: Effects of recombinant attenuated Salmonella harboring CD40 gene on rat experimental colorectal cancerObjective: To investigate the immune change of tumor-bearing rats and the feasibility of prevention and treatment of colorectal cancer induced by attenuated salmonella typhimurium vaccine strain expressing rat CD40. Methods: 18 six to eight week old SD rats were randomly divided into SL3261 group、empty plasmid(p IRES2-EGFP) group and CD40(p IRES2-EGFP-CD40) group.All rats were given weekly subcutaneous injections of DMH(20mg/kg) for 18 weeks. Eight weeks after first injection,rats were fed with 2 ml different vaccine bacteria respectively 4 times at 2 weeks intervals according to the different group. In 18 weeks tumor-bearing rats were sacrificed and the number and size of colon tumors and metastasis were recorded for tumor incidence. The level of phenotype change and IFN-γ stimulated by PHA in peripheral blood cells and spleen cells was determined by flow cytometry. RT-PCR were exploited to detect the expression of GFP.Results:Tumors were found in CD40 group which was significantly lower than other groups(SL3261 group 12.7±3.1; p IRES2-EGFP group 11.6±2.4; CD40 group 5.8±2.2), P<0.05. However in SL3261 group and p IRES2-EGFP group, they were similar( P>0.05). The results of peripheral blood and splenocytes showed that CD3+CD8+ T cells in the CD40 group were obviously higher than those in other groups(P < 0.05). There were significantly more IFN-γ produced in the group from CD40 group than those from other groups(P<0.05). Conclusions: Attenuated salmonella carried with CD40 plasmid can achieve APCs transfered in vivo which is a promising adjuvant for the body to enhance anti-tumor immune and inhibit the growth of experimental colorectal cancer in rats.