Construction Of Helicobacter Pylori Lpp20 DNA Vaccine And Primary Study Of Its Immunocompetence In Mice

Objectives:(1) To construct an eukaryotic expression plasmid containing the lipoprotein Lpp20 gene of Helicobacter pylori and to detect its expression in HeLa cells.(2) To observe the humoral and celluar immune responses in C57BL/6 mice induced by the Helicobacter pylori Lpp20 DNA vaccine injected intramuscularly,and to lay a foundation for further development of DNA vaccine against Helicobacter pylori infection.Methods:Primers were designed by PRIMER5.0 software according to the Lpp20 gene sequence of Helicobacter pylori 26695. The Lpp20 gene was amplified by polymerase chain reaction (PCR). The PCR product was subcloned into the expression vector pGEX-6P-2 to generate recombinant plasmid pGEX-6P-2/Lpp20, and rLpp20 protein was expressed in E.coli BL21, and analyzed by SDS-PAGE and Western-blot. Then construct eukaryotic expression vector pcDNA3.1(+)/Lpp20, the recombinant plasmid was transfected into HeLa cells using Liposome. After verifying that the Lpp20 antigen gene could be expressed in HeLa cells by immunocytochemistry and Western-blot, 6 weeks old C57BL/6 mice were immunized with pcDNA3.1(+)/Lpp20 or pcDNA3.1(+) or PBS buffer intramuscularly at 2-week interval for four times. ELISA were used for the quantitative detection of the specific IgG antibody in the sera of C57BL/6 mice and the cytokine IFN-γin mice spleen lymphocyte culture medium after stimulating by Lpp20. The proliferation response of spleen cells was detected by MTT assay. The Lpp20 gene in muscle was identified by PCR .Results:(1) The Lpp20 gene fragment (528bp) was successfully amplified. The prokaryotic expression recombinant pGEX-6P-2/Lpp20 was successfully constructed. A soluble fusion protein with molecular weight about 44KDa was attained after expression and purification. The result of Western-blot showed that rLpp20 could be recognized by the polyclonal rabbit anti-Helicobacter pylori serum.(2) The eukaryotic expression recombinant pcDNA3.1(+)/Lpp20 was successfully constructed, Lpp20 gene was expressed in HeLa cells and located at cytoplasm.(3) The significant specific antibody titers were detected by ELISA in DNA vaccine groups and the highest titer was 1:1024 after six weeks.(4) The cytokine IFN-γin mice inoculated with pcDNA3.1(+)/Lpp20 was increased and reached 410.36±56.23pg/mL. A significant difference was tested between the experiment group and the control group(25.26±10.85pg/mL) (P<0.01).(5) The proliferation response of spleen cells of DNA vaccine group(SI:2.37±0.22) were significantly higher than those of mice injected with pcDNA3.1(+)(SI:1.53±0.47)(P<0.01).(6) Lpp20 gene can be exsist constantly in musculature cells of mice.Conclusions:(1) The Lpp20 gene fragment was successfully amplified. The prokaryotic expression recombinant pGEX-6P-2/Lpp20 was successfully constructed. And the rLpp20 has good immunoreactivity.(2) The eukaryotic expression recombinant pcDNA3.1(+)/Lpp20 was successfully constructed, Lpp20 gene was expressed in HeLa cells.(3) Strong humoral and cellular imunity can be induced by DNA vaccine of pcDNA3.1(+)/Lpp20 in C57BL/6 mice.(4) Lpp20 gene can be exsist in musculature cells of mice.