Construction Of Helicobacter Pylori UreI DNA Vaccine And Evaluation Of Immunocompetence In C57BL/6 Mice

Objectives:(1) To construct an eukaryotic vector for gene encoding the urea channel protein (ureI) of Hel icobacter pylori , to detect its expression in HeLa cells.(2) To observe the humoral and cellular immune responses in C57BL/6 mice induced by the H.pylori ureI DNA vaccine injected intramuscularly, and to lay a foundation for further development of DNA vaccine against H.pylori infection.Methods:Primers were designed by PRIMER5.0 software according to the ureI gene sequence of H.pylori SS1. The ureI genes were amplified by polymerase chain reaction (PCR). Then the eukaryotic expression vector pcDNA3.1(+)/ureI was constructed and then transfected into HeLa cells using Liposome. After verifying that the ureI antigen gene could be expressed in HeLa cells by Western-blot, 6 weeks old C57BL/6 mice were immunized with pcDNA3.1(+)/ureI, pcDNA3.1(+) or PBS buffer intramuscularly at 1-week interval for four times. Six weeks after the last injection, H.pylori ureI expression in mouse tissue was detected by immunohistochemical method. ELISA were used for the quantitative detection of the specific IgG antibody in the sera of C57BL/6 mice and the cytokine IFN-γand IL-4 in mice spleen lymphocyte culture medium after stimulating by ureI. The proliferation response of spleen cells was detected by MTT assay. The ureI gene in muscle was identified by PCR .Results:(1) The eukaryotic expression recombinant pcDNA3.1(+)/ureI was successfully constructed, ureI gene was expressed in HeLa cells .(2) The significant specific antibody titers were detected by ELISA in pcDNA3.1 (+)/ureI DNA vaccine groups and the highest titer was 1:2048 after ten weeks.(3) The cytokines IFN-γand IL-4 in cultural supernatant of T lymphocytes from immunized mice injected with pcDNA3.1(+)/ureI were increased and reached (275.20±43.21) pg/mL and (436.5±68.97)pg/mL, respectively.(4) After stimulated by corresponding antigen, the stimulation index of pcDNA3.1(+)/ureI group was 1.76±0.16 , which were higher than that of control groups (1.20±0.14 VS1.14±0.12) respectively.(5) UreI genes can exsist constantly in musculature cells of mice by PCR.Conclusions:(1) The eukaryotic expression recombinant pcDNA3.1(+)/ureI was successfully constructed, ureI gene was expressed in HeLa cells.(2) Strong cellular immunity and humoral immunity can be induced by DNA vaccines of pcDNA3.1(+)/ureI in C57BL/6 mice.(3) UreI genes can exsist constantly in musculature cells of mice...