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【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Lpp20IgG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,免疫荧光组化法检测Lpp20蛋白在小鼠肌肉组织中的表达情况。【结果】成功构建了pcDNA3.1(+)/Lpp20-IL2真核表达载体,且重组质粒能在HeLa细胞内有效表达目的蛋白;小鼠接种pcDNA3.1(+)/Lpp20-IL2核酸疫苗后能产生特异性IgG抗体,8w后ELISA测定血清抗体A450值明显升高。核酸疫苗pcDNA3.1(+)/Lpp20-IL2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、IL4含量明显升高。pcDNA3.1(+)/Lpp20-IL2和pcDNA3.1(+)/Lpp20核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数明显高于空质粒组和PBS组。Lpp20蛋白在小鼠肌肉组织中能够有效表达。【结论】幽门螺杆菌Lpp20-IL2融合基因核酸疫苗和Lpp20单基因核酸疫苗均能刺激机体产生较强细胞免疫应答和体液免疫应答,且前者能诱导更强的细胞免疫应答。
【Objective】 To observe the humoral and cellular immune responses of C57BL / 6 mice immunized with pcDNA3.1 (+) / Lpp20-IL2 and to provide an experimental basis for the development of an efficient and novel H. pylori nucleic acid vaccine. 【Method】 The recombinant plasmid pcDNA3.1 (+) / Lpp20-IL2 was constructed and transfected into HeLa cells. The expression of eukaryotic cells was detected by Western-blot and the immunized C57BL / 6 mice were detected by ELISA. Serum anti-Lpp20 IgG antibody levels, ELISA double antibody sandwich method to detect the splenic lymphocyte culture supernatant IFN-γ, IL4 levels, MTT colorimetric assay spleen lymphocyte proliferation reaction, immunofluorescence staining assay Lpp20 protein in mouse muscle The expression in the organization. 【Results】 The eukaryotic expression vector pcDNA3.1 (+) / Lpp20-IL2 was successfully constructed and the recombinant plasmid could effectively express the target protein in HeLa cells. After the mice were inoculated with pcDNA3.1 (+) / Lpp20-IL2 DNA vaccine Can produce specific IgG antibodies, 8w after ELISA serum antibody A450 value was significantly higher. After stimulated with specific antigen, the levels of IFN-γ and IL-4 in the spleen lymphocytes of the immunized mice vaccinated with pcDNA3.1 (+) / Lpp20-IL2 were significantly increased. The stimulation index of splenic lymphocytes stimulated by specific antigen in pcDNA3.1 (+) / Lpp20-IL2 and pcDNA3.1 (+) / Lpp20 DNA vaccine groups was significantly higher than that in empty plasmid group and PBS group. Lpp20 protein in mice muscle tissue can be effectively expressed. 【Conclusion】 Helicobacter pylori Lpp20-IL2 fusion gene DNA vaccine and Lpp20 single-gene nucleic acid vaccine can stimulate the body to produce a strong cellular and humoral immune response, and the former can induce a stronger cellular immune response.