| Background Gastric cancer is the No. 1 killer among all malignant tumors in China. Conventional methods work poorly in treating gastric cancer, and with few tumor-specific antigen identified, there is no effective cancer vaccine developed to combat gastric cancer. MG7-Ag, discovered by our institute, is a marker molecule of gastric cancer. The antigenicity of MG7-Ag was found to locate in a suger chain, which suggested that the MG7-Ag was a kind of carbohydrate antigen. By screening the phage display library, we have identified the mimicry peptide of MG7-Ag. competitive antibody binding assay showed that the mimicry peptide could mimic the primary antigen well, which provided the premise for further using it to develop vaccines. After oral immunization of a attenuated Salmonella typhimurium vaccine expressing the gastric cancer MG7-Ag gene, it will elicit a limited special mucosal, humoral and cellular immune. In addition, this kind of vaccines is safe, efficient, convenient and economical. The recombinant plasmid containing MG7-Ag gene is transformed into attenuated Salmonella typhimurium, and immune mice with this vaccine to elicit mucosal, humoral and cellular response. It will be a efficient method for gastric cancer immune therapy. Our test uses a attenuated Salmonella typhimurium containing balanced lethal system as a oral vaccine carrier toestablish a attenuated Salmonella typhimurium vaccine expressing the gastric cancer MG7-Ag gene. It could develop a specific immune response against gastric cancer.Aim To develop an attenuated Salmonella typhimurium vaccine expressing the gastric cancer MG7-Ag gene, observe humoral and cellular response after oral immunization with vaccines. Methods An complementary sequence ofNco I site and a seqence coding for MG7-Ag mimotope were designed at the 5' terminus of forward primer.Using pi.2 II -HBCAg plasmid as template , PCR was performed to get to fusion gene of the mimotope and HbcAg gene. The fusion gene was then subcloned into the plasmid pYA3341 complementary to S. typhimurium X4550, and the recombinant plasmid was then used to transform attenuated S. typhinurium X4550. Balb/c mice were orally immunized with the 5. recombinant typhinurium X4550. The mice were immunized every 2 week's to reinforce the immunity. At5, 6 weeks, serum liter of antibody was detected by EL1SA. and at the 8th week cellular immunity detected by ?1Cr release test. Ehrlich ascites carcinoma cells expressing MG7-Ag were used in tumor challenge assay as a model to evaluate the protective effect of the immunization. Results By sequencing, it was confirmed that the PCR product was a fusion gene of MG7-Ag mimotope gene and HBcAg gene. The recombinant plasmid pYA3341-MG7/HBcAg included genes coding for MG7-Ag mimotope and HBCAg. Western blot showed that expression product of recombinant plasmid pYA3341-MG7/HBcAg in the X4550 displayed a protein band, Mr being about 22 000, binding specifically anti-MG7mAb. Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than control group, while 51Cr release test showed no statistical difference between those three groups. However tumor challenge assay showed that 1 in 5 immunized mice did not form tumor, with none in the control group got protected . Conclusions The attenuated Salmonella typhimurium vaccineexpressing the MG7-Ag mimotope is constructed successfully, which lay the foundation for further investigation of its potentiality in the immunotherapy of gastric cancer. Oral attenuated S. typhinurium vaccine based on the MG7-Ag momitope was immunogenic and could induce notable antigen specific humoral immunity response. Though 51Cr release test showed no statistical difference between immunization group and control, tumor challenge assay suggested the vaccine might have partial protective ability and there might be some cellular immune responses. Taking together, this study indicated the feasibility of including mimotope in a attenuated 5". typhinurium vaccine, supported the use of attenuat... |
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