幽门螺杆菌Lpp20-IL2核酸疫苗免疫活性

Construction of Helicobacter pylori Lpp20-IL2 DNA vaccine and evaluation of its immunocompetence in C57BL/6 mice

摘要:

摘要：【目的】观察pcDNA3.1(+)/Lpp20-IL2免疫C57BL/6小鼠后所产生的体液免疫和细胞免疫应答水平，为研制高效、新型的幽门螺杆菌核酸疫苗提供实验依据。【方法】构建pcDNA3.1(+)/Lpp20-IL2重组载体，并转染HeLa细胞，用Western-blot观察鉴定其在真核细胞得到表达后免疫C57BL/6小鼠， ELISA间接法测定小鼠血清中抗Lpp20 IgG抗体水平，ELISA双抗体夹心法检测脾淋巴细胞培养上清中IFN-γ、IL4水平，MTT比色法检测脾淋巴细胞增殖反应，免疫荧光

Abstract:

Abstract：[Objectives] 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-γ 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-γ 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.