Effect Of Attenuated Salmonella Carrying With4-1BB Ligand And CEACAM6in Rat Colorectal Cancer Models

Part one: The construction and functional identification of therecombinant eukaryotic expression vector carrying rat4-1BBL andCEACAM6geneObjective: to construct eukaryotic expression vector with4-1BBL and CEACAM6and identify the function. Methods: After constructed a recombinant eukaryotic expressingplasmid with CEACAM6and4-1BBL. We transformed it into COS7cells. Fluorescencemicroscope observation and RT-PCR were used to detect the expression of CEACAM6and4-1BBL. Results: We construct three recombinant eukaryotic expressing plasmid:pIRES-CEACAM6, pIRES-4-1BBL, pIRES-CEACAM6-4-1BBL, successfully. COS7cells could steadily express4-1BBL and CEACAM6. Conclusions: We constructrecombinant eukaryotic expressing vector carrying rat4-1BBL and CEACAM6genesuccessfully.Part two: Construction and indentification of recombination AttenuationSalmonella typhimurium vaccine containing pIRES-CEACAM6-4-1BBLvectorObjective: To develop strain SL3261(Attenuation Salmonella typhimurium) vaccinecontaining rat4-1BBL and CEACAM6gene eukaryotic expression vector. Methods:Vector was converted to LB5000(Salmonella typhimurium) for methylation then modifiedvector was electrotransfered to final host SL3261.screened single colony by antibiotics wastested by plasmid extract and PCR. Results: Similar plasmid to origin was extracted fromStrain SL3261imported which containing the interesting gene sequence identified by PCR.Conclusions: The plasmid was successfully transferred into Attenuated Salmonellatyphimunium. Part three: Effects of recombinant attenuated Salmonella harboringCEACAM6and4-1BBL gene on rat experimental colorectal cancerObjective: To investigate the immune change of tumor-bearing rats and the feasibilityof prevention and treatment of colorectal cancer induced by attenuated Salmonellatyphimurium vaccine strain expressing rat4-1BBL and CEACAM6. Methods: Rats weregiven weekly subcutaneous injections of DMH (20mg/kg) for18weeks. Eight weeks afterfirst injection，rats were averagely and randomly divide into pIRES/SL3261group,pIRES-CEACAM6/SL3261group, pIRES-4-1BBL/SL3261group,pIRES-CEACAM6-4-1BBL/SL3261group, all were fed with2ml respectively4times at2weeks intervals. In18weeks tumor-bearing rats were sacrificed and the number and sizeof colon tumors and metastasis were recorded for tumor incidence. The level of phenotypechange and IFN-γstimulated by PHA in peripheral blood cells and spleen cells wasdetermined by flow cytometry. The transcriptions of CMV in spleen cells were detected byRT-PCR. Results: Tumors were found in pIRES/SL3261group，which was significantlyhigher than the other group(P<0.05),（5.0±1.4in pIRES-CEACAM6/SL3261group，5.7±1.2in pIRES-4-1BBL/SL3261group，4.3±1.4in pIRES-CEACAM6-4-1BBL/SL3261group），While in pIRES-CEACAM6-4-1BBL/SL3261、pIRES-CEACAM6/SL3261andpIRES-4-1BBL/SL3261group, there were similar(P>0.05). The results of peripheralblood and splenocytes showed that CD3+CD8+T cells in thepIRES-CEACAM6-4-1BBL/SL3261group of were obviously higher than that of othergroups (P <0.05). There were significantly more IFN-γ produced in the group withpIRES-CEACAM6-4-1BBL/SL3261group than that of other groups (P<0.05).Conclusions: Attenuated Salmonella carrying with pIRES-CEACAM6-4-1BBL plasmidcan achieve APCs transfered in vivo, which is a promising adjuvant for the body toenhance anti-tumor immune and inhibit the growth of experimental colorectal cancer inrats.