[Construction of Helicobacter pylori Lpp20-IL2 DNA vaccine and evaluation of its immunocompetence in C57BL/6 mice].
OBJECTIVE
To evaluate the humoral and cellular immune responses in C57BL/6 mice induced by pcDNA3.1(+)/Lpp20-IL2 for further development of DNA vaccine against H. pylori infection.


METHODS
The eukaryotic expression vector pcDNA3.1(+)/Lpp20-IL2 was constructed and then transfected into HeLa cells using liposome. Western blot was used to verify the expression of Lpp20 antigen gene in HeLa cells. Then five groups of 6-week-old C57BL/6 mice were immunized intramuscularly with either of the following vaccines: pcDNA3.1(+)/Lpp20-IL2, pcDNA3.1(+)/Lpp20, pcDNA3.1(+)/IL2, mock plasmid pcDNA3.1(+), or PBS four times at 1-week interval. Six weeks after the last injection,the specific IgG in the sera of C57BL/6 mice and the cytokine content of IFN-gamma and IL4 in mice spleen lymphocyte culture medium after stimulation by recombinant Lpp20 (rLpp20) was tested quantitatively by ELISA, respectively. The proliferation of spleen cells was measured by MTT assay. Lpp20-IL2 fusion protein in mouse muscular tissue was detected by immunofluorescence histochemistry.


RESULTS
The eukaryotic expression recombinant pcDNA3.1(+)/Lpp20-IL2 was successfully constructed and could be expressed in HeLa cells . The significant specific antibody titers were detected by ELISA in pcDNA3.1(+)/Lpp20-IL2 DNA vaccine groups and the highest titer was 1: 4096 after eight weeks. The cytokines content of IFN-gamma and IL4 in cultural supernatant of spleen lymphocytes from mice immunized with pcDNA3.1(+)/Lpp20-IL2 increased significantly. After stimulated by corresponding antigen, the stimulation index of pcDNA3.1(+)/Lpp20-IL2 and pcDNA3.1(+)/Lpp20 group were higher than that of control groups (P < 0.01). Lpp20-IL2 fusion protein could be expressed in mouse muscular tissue.


CONCLUSIONS
We have successfully constructed the recombinant eukaryotic expression plasmid pcDNA3.1(+)/Lpp20-IL2 and the Lpp20-IL2 fusion protein could be effectively expressed in HeLa cells. Both pcDNA3.1(+)/Lpp20-IL2 and pcDNA3.1(+)/ Lpp20 DNA vaccines could induce strong cellular immunity and humoral immunity in C57BL/6 mice, and the former could induce more powerful cellular immunologic response.
Yu
Wen
W
Institute of Pathogenic Biology, University of South China, Hengyang 421001, China. yuwen800823@tom.com
Zhang
Yan
Y
Jing
Jifang
J
Liu
Zhijie
Z
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells; D006801:Humans; D056724:Immunity, Humoral / Q000502:physiology; D007121:Immunocompetence / Q000276:immunology*; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D015847:Interleukin-4 / Q000235:genetics / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000378:metabolism; D019444:Vaccines, DNA
D000942:Antigens, Bacterial; D007376:Interleukin-2; D008074:Lipoproteins; C089630:Lpp20 protein, Helicobacter pylori; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; C104514:interferon-gamma (95-133); D015847:Interleukin-4; D007371:Interferon-gamma