日本血吸虫多价膜锚定表达疫pIRES-Sj97-Sj14-Sj26的构建及其免疫原性的研究

发布时间：2018-09-12 08:20

【摘要】： 目的构建日本血吸虫多价膜锚定表达疫苗pIRES-Sj97-Sj14-Sj26,并研究其免疫原性。方法采用分子克隆技术,构建日本血吸虫脂肪酸结合蛋白(Sj14)、GST(Sj26)和副肌球蛋白(Sj97)的跨膜共表达质粒pIRES-Sj97-Sj14-Sj26,转染Hela细胞,通过RT-PCR法检测Sj14、Sj26和Sj97mRNA的表达,间接免疫荧光检测Sj26蛋白的表达。用此疫苗免疫BALB/c小鼠,同时设置生理盐水对照组、空白质粒对照组、pIRES-Sj26组和pIRES-Sj14-Sj26组。用ELISA法检测免疫小鼠血清中的总IgG抗体和小鼠脾细胞分泌IFN-γ的水平。以MTT法检测淋巴细胞的增殖情况。并以FCM分析脾细胞亚群。结果经瞬时转染Hela细胞证明质粒pIRES-Sj97-Sj14-Sj26能在体外进行表达。免疫小鼠后ELISA法检测抗体结果表明pIRES-Sj97-Sj14-Sj26能够较各对照组诱导产生高水平的IgG抗体(P0.01,P0.05).该组的脾淋巴细胞刺激指数较各对照组也有显著增高(P0.01),IFN-γ的水平与空白对照组和空载体组有显著差异(P0.01). CD4+和CD8+T细胞含量百分比有明显增高(P0.05)。结论pIRES-Sj97-Sj14-Sj26疫苗具有较强的免疫原性,能明显增强BALB/c小鼠的免疫应答反应.为该疫苗抗感染效应的研究奠定了基础,并为日本血吸虫病的防治寻求一种新的多基因组合疫苗的免疫策略。
[Abstract]:Objective to construct and study the immunogenicity of the multivalent membrane anchored pIRES-Sj97-Sj14-Sj26, vaccine of Schistosoma japonicum. Methods the transmembrane coexpression plasmid pIRES-Sj97-Sj14-Sj26, of fatty acid binding protein (Sj26) and accessory myosin (Sj97) of Schistosoma japonicum was constructed by molecular cloning technique. The expression of Sj14,Sj26 and Sj97mRNA was detected by RT-PCR method, and the expression of Sj26 protein was detected by indirect immunofluorescence. BALB/c mice were immunized with this vaccine, and the control group of normal saline, the control group of blank plasmid pIRES-Sj26 and the group of pIRES-Sj14-Sj26 were set up at the same time. The total IgG antibody in serum of immunized mice and the level of IFN- 纬 secreted by spleen cells of mice were detected by ELISA method. The proliferation of lymphocytes was detected by MTT method. Spleen cell subsets were analyzed by FCM. Results transient transfection of Hela showed that plasmid pIRES-Sj97-Sj14-Sj26 could be expressed in vitro. After immunizing mice, the results of ELISA assay showed that pIRES-Sj97-Sj14-Sj26 could produce higher level of IgG antibody than that of control group (P0.01P 0.05). The level of IFN- 纬 was significantly higher in this group than that in the control group (P0.01), and there was a significant difference between the control group and the blank carrier group (P0.01). The percentage of CD4 and CD8 T cells was significantly increased (P0.05). Conclusion pIRES-Sj97-Sj14-Sj26 vaccine has strong immunogenicity and can obviously enhance the immune response of BALB/c mice. It lays a foundation for the study of the anti-infective effect of the vaccine and seeks a new immunization strategy for the prevention and control of schistosomiasis japonicum.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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