PvMSP1-19重组蛋白的制备及其免疫效应的实验研究

发布时间：2018-01-02 05:19

本文关键词：PvMSP1-19重组蛋白的制备及其免疫效应的实验研究　出处：《蚌埠医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的：(1) PvMSP1-19基因的克隆、表达和纯化（2）PvMSP1-19重组蛋白的细胞免疫效应（2）抗PvMSP1-19重组蛋白免疫球蛋白的类型。方法：（1）将PvMSP1-19基因克隆入原核表达载体pET28a中，构建重组表达载体。以重组载体转化大肠杆菌BL21，在IPTG诱导下表达PvMSP1-19重组蛋白，超声波破菌，分离上清和沉淀，上清经His亲和层析柱纯化，再通过AKTAexplorer100型快速液相蛋白层析分离系统进一步纯化并检测其纯度，采用SDS-PAGE电泳和Western-blotting对目的蛋白进行分析和验证。（2）密度梯度离心法分离既往感染者外周血单个核细胞（PBMC），，用PvMSP1-19重组蛋白和rIL-2（刺激组）以及rIL-2（未刺激组）刺激培养，扩增效应性T淋巴细胞，7天后，收集细胞用PvMSP1-19重组蛋白和Mo（刺激组）以及Mo（未刺激组）刺激培养24小时后，用流式细胞仪检测分泌IL-4和分泌IFN-γ的T细胞亚群的比例、淋巴细胞的增殖水平（CFSE标记法）、淋巴细胞的活化水平（CD69）。（3）PvMSP1-19重组蛋白包被ELISA板，检测现症患者、既往感染者和正常人血浆IgG1、IgG4、IgM、IgD、IgA抗PvMSP1-19重组蛋白抗体的水平。结果：(1)成功构建了质粒PvMSP1-19/pET28a，并在大肠埃希菌中诱导可溶性表达的目的蛋白。在优化条件下大量表达，经His柱纯化，AKTA explorer100型快速液相蛋白层析分离系统检测其纯度，蛋白纯度达到95%。表达的PvMSP1-19重组蛋白能与间日疟患者血清发生特异性结合反应。（2）分泌IL-4的CD8+T细胞比例在刺激组与未刺激组存在显著的差异（P0.05），而分泌IFN-的CD8+T细胞比例在两组间无显著差异（P0.05）。分泌的IL-4和IFN-的CD4+T细胞比例在刺激组与未刺激组间无显著差异（P0.05）。CD8+T细胞的增殖指数和CD69的表达在刺激组与未刺激组之间有显著的差异（P0.05）。而CD4+T细胞CD69和增殖指数在刺激组与未刺激组之间无显著差异（P0.05）。(3)抗体的亚类水平进行检测显示现症感染组抗PvMSP1-19重组蛋白IgG1抗体水平与正常对照组和既往感染组有显著差异（P0.05）。结论：（1）成功的克隆表达了PvMSP1-19重组蛋白，并纯化出可溶性的、纯度高达95%的PvMSP1-19重组蛋白且具有备良好的免疫活性。（2）PvMSP1-19重组蛋白可特异诱导CD8+T细胞增殖、活化分泌IL-4。（3）现症感染者血浆具有高水平抗重组抗原PvMSP1-19的IgG1抗体。
[Abstract]:Objective: to clone PvMSP1-19 gene. Expression and purification of PvMSP1-19 Recombinant protein (PvMSP1-19) the type of immunoglobulin against PvMSP1-19 recombinant protein. Methods PvMSP1-19 gene was cloned into prokaryotic expression vector pET28a, and the recombinant expression vector was constructed. The recombinant vector was transformed into Escherichia coli BL21. The recombinant PvMSP1-19 protein was expressed under the induction of IPTG. The supernatant was isolated and precipitated by ultrasonic wave. The supernatant was purified by His affinity chromatography. The purity was further purified and tested by AKTAexplorer100 rapid liquid protein chromatography system. SDS-PAGE electrophoresis and Western-blotting were used to analyze and verify the target protein. Density gradient centrifugation was used to isolate peripheral blood mononuclear cells (MNCs) from previously infected patients. PBMC). PvMSP1-19 recombinant protein and rIL-2 (stimulation group) and rIL-2 (unstimulated group) were used to stimulate culture and amplify effectual T lymphocytes for 7 days. The cells were cultured for 24 hours with PvMSP1-19 recombinant protein and Mo-stimulated group and Mo-stimulated group. The proportion of T cell subsets secreting IL-4 and IFN- 纬 and the level of lymphocyte proliferation were detected by flow cytometry. The level of lymphocyte activation was determined by ELISA plate of PvMSP1-19 recombinant protein. The level of IgA antibody against PvMSP1-19 recombinant protein. Results the plasmid PvMSP1-19 / pET28awas successfully constructed and the soluble expressed target protein was induced in Escherichia coli. Its purity was determined by His column purification and rapid liquid-phase protein chromatography (RLPC) system. The purity of the protein was 95%. The expressed PvMSP1-19 recombinant protein could react with vivax malaria serum specifically. The proportion of CD8 T cells secreting IL-4 was significantly different between the stimulated group and the unstimulated group (P0.05). There was no significant difference in the proportion of IFN- secreting CD8 T cells between the two groups (P0.05). The percentage of CD4 T cells secreted by IL-4 and IFN- was not significantly different between the stimulated and unstimulated groups (P0.05). The proliferation index and CD69 expression of CD8 T cells were significantly different between the stimulated group and the unstimulated group (P0.05). However, there was no significant difference in CD69 and proliferation index of CD4 T cells between stimulated and unstimulated groups (P 0.05). The level of IgG1 antibody against PvMSP1-19 recombinant protein in the present infection group was significantly different from that in the normal control group and the previous infection group (P 0.05). Conclusion: The recombinant PvMSP1-19 protein was successfully cloned and expressed, and the soluble PvMSP1-19 recombinant protein with purity up to 95% was purified and had good immunological activity. The recombinant protein PvMSP1-19 can specifically induce the proliferation of CD8 T cells and activate the secretion of IL-4. There is a high level of IgG1 antibody against recombinant antigen PvMSP1-19 in the plasma of current infected patients.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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