幽门螺杆菌Lpp20核酸疫苗的构建及其免疫活性的初步研究

目的 构建幽门螺杆菌脂蛋白Lpp20基因的真核表达载体pcDNA3.1(+)/Lpp20,并在HeLa细胞中进行表达.通过肌肉注射免疫C57BL/6小鼠,观察其诱导小鼠产生的体液免疫和细胞免疫应答水平.方法 用PCR法扩增Lpp20全基因,再将Lpp20基因克隆至pcDNA3.1(+)真核细胞表达载体构建pcDNA3.1(+)/Lpp20重组体,观察其在HeLa细胞中的表达.将核酸疫苗PcDNA3.1(+)/Lpp20、对照空质粒pcDNA3.1(+)及PBS分组通过肌肉注射免疫6周龄C57BL/6小鼠.隔2周免疫一次,共免疫4次.间接ELISA法测定小鼠血清中抗Lpp20 IgG抗体水平,双抗体夹心ELISA法检测脾淋巴细胞培养上清中IFN-γ水平,MTT比色法检测脾淋巴细胞增殖反应.通过PCR法检测小鼠肌细胞中Lpp20基因的存在.结果 小鼠接种pcDNA3.1(+)/Lpp20核酸疫苗后能产生特异性IgG抗体,6周后ELISA测定血清抗体A450值为0.74,效价为1:1024.核酸疫苗免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ含量明显升高[(410.36±56.23)ps/ml],与空质粒组[(25.26±10.85)pg/ml]之间差异有统计学意义(P<0.01).脾淋巴细胞增殖反应测定,核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数(2.37±0.22)明显高于空质粒组(1.53±0.47)和PBS组(1.20±0.13),P<0.01.PCR检测Lpp20基因可在小鼠肌细胞中存在.结论 成功构建了pcDNA3.1(+)/Lpp20核酸疫苗,且其在小鼠体内可诱导较强的特异性体液免疫和细胞免疫应答.为进一步研究该疫苗的免疫保护作用提供实验依据.

Construction of Helicobacter pylori Lpp20 DNA vaccine and primary study of its immunocompetence in mice

Objective To construct an eukaryotic expression plasmid PeDNA3.1 (+)/Lpp20 and to detect its expression in HeLa cells, and to observe the humoral and cellular immune responses in C57BL/6 mice induced by the Helicobacter pylori Lpp20 DNA vaccine injected intramuscularly. Methods The Lpp20 gene was amplified by PCR. PCR product was subcloned into the eukaryotic expression vector pcDNA3.1 (+)/ Lpp20, and the recombinant plasmid was transfected into HeLa cells using Liposome. After verifying that the Lpp20 antigen gene could be expressed in HeLa cells. Six 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 was used for the quantitative detection of the specific IgG antibody in the sera of C57BL/6 mice and the cytokine IFN-γ in 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 The significant specific antibody titers were detected by ELISA in DNA vaccine groups and the highest titer was 1:1024 after 6 weeks. The cytnkine IFN-γ in mice inoculated with pcDNA3.1 (+)/Lpp20 was increased and reached (410.36±56.23) pg/ml. A significant difference was tested between the experiment group and the control group[(25.26±10.85)pg/ml] ,P <0.01. The proliferation response of spleen cells of DNA vaccine group(SI: 2.37±0.22) was significantly higher than those of mice injected with pcDNA3.1 (+) (SI:1.53+0.47) ,P<0.01. Lpp20 gene could exist constantly in musculature cells of mice. Conclusion The eukaryotic expression recombinant pcDNA3.1 (+)/Lpp20 was successfully constructed. Strong humoral and cellular im-munity can be induced by DNA vaccine of pcDNA3.1(+)/Lpp20 in C57BL/6 mice, which might be helpful for further investigation concerning the immunoprotection of DNA vaccine.