ABSTRACT
<p><b>OBJECTIVE</b>To establish attenuated Salmonella typhimurium producing Helicobacter pylori (H. pylori) urease subunit B (UreB) and determine whether it could be used as an oral vaccine against H. pylori.</p><p><b>METHODS</b>H. pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector pTC01 after sequencing, and then transformed into attenuated Salmonella typhimurium SL3261 to acquire SL3261/pTC01-UreB. The expression of H. pylori UreB in SL3261 was detected by Western blot. Twelve weeks after oral immunization of mice, antibody responses were evaluated using serum and intestinal fluid by ELISA assay. Interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in the supernatant of spleen cells culture were also assessed by ELISA. In vitro stability of pTC01-UreB plasmid in SL3261 was confirmed by growing in Luria Broth (LB) medium to 80 generations.</p><p><b>RESULTS</b>The UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB as evidenced by sequence analysis. Enzyme digestion revealed that the correct pTC01-UreB was obtained. Western blot showed that a 61kDa protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H. pylori UreB antiserum. After 80 generations of continuous culture, the recombinant plasmid pTC01-UreB was stable in SL3261 and had no obvious toxicity. Multiple oral immunizations with SL3261/pTC01-UreB could significantly induce H. pylori-specific mucosal IgA response as well as serum IgG response. Moreover, there were significant increases of IFN-gamma and IL-10 in the SL3261/pTC01-UreB group. Finally, no obvious side effects for mice and no change in gastric inflammation were observed.</p><p><b>CONCLUSION</b>Attenuated Salmonella typhimurium expressing H. pylori UreB may be used as oral vaccine against H. pylori infection.</p>
Subject(s)
Animals , Female , Mice , Administration, Oral , Antibodies, Bacterial , Blood , Bacterial Vaccines , Blood , Allergy and Immunology , Helicobacter Infections , Helicobacter pylori , Allergy and Immunology , Immunization , Interferon-gamma , Interleukin-10 , Mice, Inbred BALB C , Plasmids , Protein Subunits , Salmonella typhimurium , Genetics , Urease , Allergy and Immunology , Vaccines, Attenuated , Allergy and Immunology , Vaccines, Synthetic , Allergy and ImmunologyABSTRACT
In order to construct a recombinant attenuated Salmonella typhimurium expressing Helicobacter pylori (H. pylori) urease subunit B(UreB), UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector pTC01 after sequencing, then transformed into attenuated Salmonella typhimurium SL3261 to acquire SL3261/pTC01-UreB. The expression of H. pylori UreB in SL3261 was detected by Western blot. Tewelve weeks after oral immunization of mice with this vaccine, anti-UreB IgA antibodies in mouse intestinal fluid and IgG antibodies in serum were determined by ELISA. IFN-? and IL-10 contents in the supernatant of spleen cell culture were also assessed by ELISA. The results showed that 61kD protein was expressed in SL3261/pTC01-UreB that could be recognized by anti-H. pylori UreB antiserum by Western-blot. The multiple oral immunizations with SL3261/pTC01-UreB could induce significantly H. pylori-specific mucosal IgA response as well as serum IgG responses. Moreover, there was significant increase of IFN-? and IL-10 contents in the supernatant of spleen cell culture in SL3261/pTC01-UreB group. These results suggested that the attenuated Salmonella typhimurium expressing H. pylori UreB may be used as oral vaccine against H. pylori infection. Its effect against H. pylori infection also needs to be further evaluated in animal models.
ABSTRACT
Objective To establish attenuated S. typhimurium expressing Helicobacter pylori (H.pylori) urease subunit B (UreB) and determine whether it could be used as oral vaccine against H.pylori Methods H.pylori (SS1 strain) UreB gene fragment amplified by PCR was cloned into the prokaryotic expression vector PTc 01 after sequencing, then transformed into attenuated S.typhimurium SL3261 to acquire SL3261/PTc 01 UreB. The expression of H.pylori UreB in SL3261 was detected by Western blot. Twelve weeks after oral immunization of mice, antibody responses were evaluated using serum and intestinal fluid by ELISA. In vitro stability of PTc 01 UreB plasmid in S. typhimurium SL3261 was confirmed by growing in Luria Bertani medium to 60 generations. Results The UreB gene fragment amplified by PCR was consistent with the sequence of the H.pylori UreB by sequence analysis. Enzyme digestion revealed that the correct PTc 01 UreB was obtained. Western blot showed that 61kD protein was expressed in SL3261/PTc 01 UreB which could be recognized by anti H.pylori UreB antiserum. Anti UreB IgA antibodies in mouse intestinal fluid and IgG antibodies in serum were determined by ELISA. After 60 generations of continuous culture, the recombinant plasmid PTc 01 UreB was stable in SL3261 and had no obvious toxicity. Conclusion The attenuated Salmonella typhimurium expressing H.pylori UreB may be used as oral vaccine against H.pylori infection.
ABSTRACT
Objective To study the relation between p38 MAPK pathway and multidrug resistance of stomach cancer. Methods The activity of p38 MAPK in gastric cancer cell SGC7901 and multidrug resistant cell SGC7901/VCR was examined by immunoprecipitation after treating with vincristine for 0,2,15,30,60 minutes. Results Vincristine could activate the activity of p38 MAPK in gastric cancer cell SGC7901 and multidrug resistant cell SGC7901/VCR. The difference between them was that in cell SGC7901 vincristine activate the p38 MAPK rapidly whereas in cell SGC7901/VCR slowly. Conclusion Vincristine could affect the activity of p38 MAPK in gastric cancer cell SGC7901 and multidrug resistant cell SGC7901/VCR.
ABSTRACT
To investigatethe relationship between p38 MAPK pathway and multidrug resistance of stomach cancer. The activity of p38 MAP kinase in stomach cancer cell SGC7901 and multidrug resistant cell SGC7901/VCR were examined by immunoprecipition after adriamycin treatment SGC7901 for 0, 2, 15, 30 and SGC7901/VCR for 0, 2, 15, 30, 60 minutes,respectively. The results indicated that adriamycin could activate the activity of p38 MAP kinase in gastric cancer cell SGC7901 with time dependency, but could decrease remarkably the activity of p38 MAP kinase in gastric cancer multidrug resistant cell SGC7901/VCR after adriamycin treatment it for 15 minutes. It suggested that adriamycin could affect the activity of p38 MAPK in gastric cancer cell SGC7901 and multidrug resistant cell SGC7901/VCR.
ABSTRACT
A new group of gastric cancer associated antigens in sera of patients with rectal cancer, benign diseases and normal human were detected by MG series monoclonal antibodies against gastric cancer. The average values of normal persons plus 3 standard deviations was set as the highest limit of normal value. The positive rate were 69.7% (23/33) in sera of patients with rectal cancer, and 4%(1/25) in sera of patients with benign diseases. The values of MG-Ags were decreased to normal level 8-10 days after surgical removal in 5 patients of rectal cancer. The results of test suggest that determination of MG-Ags in serum of patients might be helpful in the diagnosis of rectal carcinoma.