日本血吸虫SjTGR和SjTSP2 DNA疫苗初步研究

发布时间：2018-03-24 12:21

本文选题：日本血吸虫　切入点：DNA疫苗　出处：《中国农业科学院》2012年硕士论文

【摘要】：【目的】日本血吸虫病是危害严重的热带亚热带消耗性疾病，分布广泛。研制安全、有效的疫苗是重要的研究方向之一。本文以血吸虫重要的体被蛋白—日本血吸虫四跨膜蛋白2（SjTSP2）和硫氧还蛋白谷胱甘肽还原酶（SjTGR）为抗原候选分子，制备DNA疫苗，比较不同免疫途径诱导的保护效果，研究其免疫保护机制。【方法】利用分子克隆方法构建DNA疫苗，应用脂质体转染293T细胞，间接免疫荧光法检测目的蛋白在细胞中的表达，分别采取基因枪轰击法和肌肉注射法免疫接种BABL/c小鼠，进行动物免疫保护试验，计算减虫、减卵率。用ELISA法检测抗体水平，用流式细胞术检测细胞因子，用硫氧还蛋白还原酶试剂盒检测虫体SjTGR的酶活力。【结果】成功构建了DNA疫苗（pVAX1/SjTSP2和pVAX1/SjTGR），，间接免疫荧光法检测表明两种蛋白均在293T细胞中表达良好。采用基因枪轰击法免疫pVAX1/SjTSP2，ELISA检测结果表明免疫组小鼠产生了较高水平的特异性抗体，用流式细胞术检测发现免疫组小鼠产生了较高水平的IL-4、IFN-γ，然而，免疫组小鼠平均成虫数较对照组增加12.04%，每克肝脏虫卵数增加10.01%，差异不显著。大量纯化pVAX1/SjTGR，分别采用小鼠腿部肌肉注射法和腹部皮肤基因枪轰击法免疫小鼠，进行小鼠日本血吸虫病免疫保护效果评估。结果基因枪免疫小鼠获得了27.83%（P0.05）的减虫率和40.38%（P0.05）的减卵率，而肌肉注射组没有显著的保护效果。ELISA检测结果显示免疫组小鼠均产生了较高水平的特异性抗体，且基因枪免疫组产生的抗体水平高于肌肉注射法免疫。用基因枪轰击免疫法进行免疫保护重复试验，结果诱导了38.83%（P0.05）的减虫率和44.51%（P0.05）的减卵率。流式细胞术结果显示基因枪免疫pVAX1/SjTGR，致使小鼠产生了较高水平的IL-4、IFN-γ，体外TGR酶活力测定表明，免疫组虫体的酶活力显著低于对照组，而且高浓度特异性抗SjTGR抗体可抑制SjTGR酶活力。【结论】用基因枪轰击法免疫小鼠pVAX1/SjTSP2DNA疫苗，未诱导理想的免疫保护效果。而基因枪法免疫pVAX1/SjTGR诱导了显著的保护效果，诱导的免疫保护作用可能和高水平的特异性抗体和较强的细胞免疫反应有关。SjTGR具有作为抗日本血吸虫病疫苗候选抗原分子的潜力。本文为血吸虫疫苗研究提供了有益的数据资料。
[Abstract]:[objective] Schistosomiasis japonicum is a serious tropical, subtropical and expendable disease, which is widely distributed. The effective vaccine is one of the important research directions. In this paper, DNA vaccine was prepared by using Schistosoma japonicum four-transmembrane protein (SjTSP2) and thioredoxin glutathione reductase (SjTGR) as antigen candidate molecules. The protective effects of different immune pathways were compared and the mechanism of immune protection was studied. [methods] DNA vaccine was constructed by molecular cloning, and 293T cells were transfected with liposome. Indirect immunofluorescence assay was used to detect the expression of target protein in the cells. BABL/c mice were immunized with gene gun bombardment and intramuscular injection respectively. ELISA method was used to detect antibody level, flow cytometry was used to detect cytokines, and thioredoxin reductase kit was used to detect the enzyme activity of SjTGR. [results] DNA vaccine pVAX1 / SjTSP2 and pVAX1 / SjTGR2 were successfully constructed. Indirect immunofluorescence assay showed that both proteins expressed well in 293T cells. Flow cytometry showed that the immunized mice produced a high level of IL-4 and IFN- 纬, however, Compared with the control group, the average adult number of mice in the immunized group increased 12.04 and 10.01 per gram of liver eggs, but the difference was not significant. A large number of purified pVAX1 / SjTGRwere used to immunize mice with leg intramuscular injection and abdominal skin gene gun bombardment, respectively. Results the rate of worm reduction and egg reduction of mice immunized with gene gun were 27.83% (P0.05) and 40.38% (P0.05), respectively. The results of Elisa showed that the immunized mice produced higher levels of specific antibodies. The antibody level of the gene gun immunized group was higher than that of the intramuscular injection method. Results the worm reduction rate and egg reduction rate of 44.51% and 44.51% were induced. The results of flow cytometry showed that the gene gun immunized pVAX1 / SjTGRR resulted in a higher level of IL-4 and IFN- 纬 in mice. The activity of TGR in vitro was significantly lower than that of the control group, the results of flow cytometry showed that the enzyme activity of the immunized group was significantly lower than that of the control group, and the results of flow cytometry showed that the expression of IL-4 and IFN- 纬 in the immunized group was significantly lower than that in the control group. And high concentration of specific anti SjTGR antibody can inhibit the activity of SjTGR enzyme. [conclusion] immunizing mice with pVAX1/SjTSP2DNA vaccine with gene gun bombardment did not induce the ideal immune protective effect, but pVAX1/SjTGR immunized with gene gun had a significant protective effect. The immune protective effect induced by SjTGR may be related to the high level of specific antibodies and the strong cellular immune response. SjTGR has the potential as a candidate antigen molecule for the vaccine against schistosomiasis japonicum. Useful data.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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