AIM: To develop an oral attenuated Salmonella typhimurium vaccine against gastric cancer and to evaluate its efficacy in mice. METHODS: A complementary sequence of Nco Ⅰ site and a sequence coding for MG7-Ag mimotope...AIM: To develop an oral attenuated Salmonella typhimurium vaccine against gastric cancer and to evaluate its efficacy in mice. METHODS: A complementary sequence of Nco Ⅰ site and a sequence coding for MG7-Ag mimotope were designed at the 5'terminus of forward primer. Using p1.2 Ⅱ-HBCAg plasmid as template, PCR was performed to get a fusion gene of the mimotope and a HBcAg gene. The fusion gene was then subcloned into the plasmid pYA3341 complementary to Salmonella typhimurium X4550, and the recombinant plasmid was then transformed into attenuated Salmonella typhimurium X4550. Balb/c mice were orally immunized with the recombinant Salmonella typhimurium X4550. The mice were immunized every 2 wk to reinforce the immunity. At the 6th wk, serum titer of antibody was detected by ELISA, and at the 8th wk, cellular immunity was detected by 51Cr 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 vaccine. RESULTS: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than in control groups (0.9538±0.043 vs 0.6531±0.018, P<0.01; 0.9538±0.043 vs 0.6915±0.012, P<0.01), while in vitro 51Cr release assay of the splenocytes showed no statistical difference in the three groups. Two weeks after tumor challenge, 1 in 5 immunized mice was tumor free, while all the mice in the control group presented tumor. CONCLUSION: Oral attenuated Salmonella typhimurium vaccine against the MG7-Ag mimotope of gastric cancer is immunogenic. It can induce significant humoral immunity against tumors in mice, and has some protective effects.展开更多
AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was is...AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then doned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coliDH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine^(TM)2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.展开更多
Objective: To determine whether attenuated Salmonella typhimurium producing Helicobacter pylori (Hp) urease subunit B(UreB)can elicit specific immune responses against Hp in mice tested by a lavage technique. Methods:...Objective: To determine whether attenuated Salmonella typhimurium producing Helicobacter pylori (Hp) urease subunit B(UreB)can elicit specific immune responses against Hp in mice tested by a lavage technique. Methods: Attenuated Salmonella typhimurium producing Hp UreB immunized orally Balb/c mice twice at a 3-week interval. After 12 weeks,mice intestinal secretions were obtained without harm by administering a lavage solution intragastrically. The mice intestinal secretions of immune group were also directly washed out after the mice were killed. The antibody responses were evaluated by using serum and intestinal fluid with ELISA assay. Results: The multiple oral immunizations with SL3261/ pTC01-UreB induced significantly Hp-specific mucosal IgA response as well as serum IgG response. The IgA was also consistently higher in the intestinal fluid obtained by the lavage solution than by direct washout. In addition, no obvious side effects and changes in gastric inflammation were observed in mice. Conclusion: The attenuated Salmonella typhimurium expressing Hp UreB may be used as an oral vaccine against Hp infection. And the lavage technique is an ideal method in the study of mucosal immune responses.展开更多
基金Supported by the National Natural Science Foundation of China, No. 39870742
文摘AIM: To develop an oral attenuated Salmonella typhimurium vaccine against gastric cancer and to evaluate its efficacy in mice. METHODS: A complementary sequence of Nco Ⅰ site and a sequence coding for MG7-Ag mimotope were designed at the 5'terminus of forward primer. Using p1.2 Ⅱ-HBCAg plasmid as template, PCR was performed to get a fusion gene of the mimotope and a HBcAg gene. The fusion gene was then subcloned into the plasmid pYA3341 complementary to Salmonella typhimurium X4550, and the recombinant plasmid was then transformed into attenuated Salmonella typhimurium X4550. Balb/c mice were orally immunized with the recombinant Salmonella typhimurium X4550. The mice were immunized every 2 wk to reinforce the immunity. At the 6th wk, serum titer of antibody was detected by ELISA, and at the 8th wk, cellular immunity was detected by 51Cr 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 vaccine. RESULTS: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than in control groups (0.9538±0.043 vs 0.6531±0.018, P<0.01; 0.9538±0.043 vs 0.6915±0.012, P<0.01), while in vitro 51Cr release assay of the splenocytes showed no statistical difference in the three groups. Two weeks after tumor challenge, 1 in 5 immunized mice was tumor free, while all the mice in the control group presented tumor. CONCLUSION: Oral attenuated Salmonella typhimurium vaccine against the MG7-Ag mimotope of gastric cancer is immunogenic. It can induce significant humoral immunity against tumors in mice, and has some protective effects.
基金Supported by the National Natural Science Foundation of China,No. 30170427
文摘AIM: To construct a recombinant attenuated Salmonella typhimurium DNA vaccine carrying Helicobacter pylori hpaA gene and to detect its immunogenicity. METHODS: Genomic DNA of the standard H pylori strain 17 874 was isolated as the template, hpaA gene fragment was amplified by polymerase chain reaction (PCR) and cloned into pUCmT vector. DNA sequence of the amplified hpaA gene was assayed, then doned into the eukaryotic expression vector pIRES through enzyme digestion and ligation reactions. The recombinant plasmid was used to transform competent Escherichia coliDH5α, and the positive clones were screened by PCR and restriction enzyme digestion. Then, the recombinant pIRES-hpaA was used to transform LB5000 and the recombinant plasmid isolated from LB5000 was finally used to transform SL7207. After that, the recombinant strain was grown in vitro repeatedly. In order to identify the immunogenicity of the vaccine in vitro, the recombinant pIRES-hpaA was transfected to COS-7 cells using Lipofectamine^(TM)2000, the immunogenicity of expressed HpaA protein was detected with SDS-PAGE and Western blot. RESULTS: The 750-base pair hpaA gene fragment was amplified from the genomic DNA and was consistent with the sequence of H pylori hpaA by sequence analysis. It was confirmed by PCR and restriction enzyme digestion that H pylori hpaA gene was inserted into the eukaryotic expression vector pIRES and a stable recombinant live attenuated Salmonella typhimurium DNA vaccine carrying H pylori hpaA gene was successfully constructed and the specific strip of HpaA expressed by pIRES-hpaA was detected through Western blot. CONCLUSION: The recombinant attenuated Salmonella typhimurium DNA vaccine strain expressing HpaA protein with immunogenicity can be constructed and it may be helpful for further investigating the immune action of DNA vaccine in vivo.
文摘Objective: To determine whether attenuated Salmonella typhimurium producing Helicobacter pylori (Hp) urease subunit B(UreB)can elicit specific immune responses against Hp in mice tested by a lavage technique. Methods: Attenuated Salmonella typhimurium producing Hp UreB immunized orally Balb/c mice twice at a 3-week interval. After 12 weeks,mice intestinal secretions were obtained without harm by administering a lavage solution intragastrically. The mice intestinal secretions of immune group were also directly washed out after the mice were killed. The antibody responses were evaluated by using serum and intestinal fluid with ELISA assay. Results: The multiple oral immunizations with SL3261/ pTC01-UreB induced significantly Hp-specific mucosal IgA response as well as serum IgG response. The IgA was also consistently higher in the intestinal fluid obtained by the lavage solution than by direct washout. In addition, no obvious side effects and changes in gastric inflammation were observed in mice. Conclusion: The attenuated Salmonella typhimurium expressing Hp UreB may be used as an oral vaccine against Hp infection. And the lavage technique is an ideal method in the study of mucosal immune responses.
