本文關(guān)鍵詞：PvMSP1-19重組蛋白的制備及其免疫效應(yīng)的實驗研究　出處：《蚌埠醫(yī)學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： PvMSP1-19 原核表達(dá) 蛋白純化 細(xì)胞因子 免疫球蛋白

【摘要】：目的：(1) PvMSP1-19基因的克隆、表達(dá)和純化（2）PvMSP1-19重組蛋白的細(xì)胞免疫效應(yīng)（2）抗PvMSP1-19重組蛋白免疫球蛋白的類型。 方法：（1）將PvMSP1-19基因克隆入原核表達(dá)載體pET28a中，構(gòu)建重組表達(dá)載體。以重組載體轉(zhuǎn)化大腸桿菌BL21，在IPTG誘導(dǎo)下表達(dá)PvMSP1-19重組蛋白，超聲波破菌，分離上清和沉淀，上清經(jīng)His親和層析柱純化，再通過AKTAexplorer100型快速液相蛋白層析分離系統(tǒng)進(jìn)一步純化并檢測其純度，采用SDS-PAGE電泳和Western-blotting對目的蛋白進(jìn)行分析和驗證。（2）密度梯度離心法分離既往感染者外周血單個核細(xì)胞（PBMC），，用PvMSP1-19重組蛋白和rIL-2（刺激組）以及rIL-2（未刺激組）刺激培養(yǎng)，擴(kuò)增效應(yīng)性T淋巴細(xì)胞，7天后，收集細(xì)胞用PvMSP1-19重組蛋白和Mo（刺激組）以及Mo（未刺激組）刺激培養(yǎng)24小時后，用流式細(xì)胞儀檢測分泌IL-4和分泌IFN-γ的T細(xì)胞亞群的比例、淋巴細(xì)胞的增殖水平（CFSE標(biāo)記法）、淋巴細(xì)胞的活化水平（CD69）。（3）PvMSP1-19重組蛋白包被ELISA板，檢測現(xiàn)癥患者、既往感染者和正常人血漿IgG1、IgG4、IgM、IgD、IgA抗PvMSP1-19重組蛋白抗體的水平。 結(jié)果：(1)成功構(gòu)建了質(zhì)粒PvMSP1-19/pET28a，并在大腸埃希菌中誘導(dǎo)可溶性表達(dá)的目的蛋白。在優(yōu)化條件下大量表達(dá)，經(jīng)His柱純化，AKTA explorer100型快速液相蛋白層析分離系統(tǒng)檢測其純度，蛋白純度達(dá)到95%。表達(dá)的PvMSP1-19重組蛋白能與間日瘧患者血清發(fā)生特異性結(jié)合反應(yīng)。（2）分泌IL-4的CD8+T細(xì)胞比例在刺激組與未刺激組存在顯著的差異（P0.05），而分泌IFN-的CD8+T細(xì)胞比例在兩組間無顯著差異（P0.05）。分泌的IL-4和IFN-的CD4+T細(xì)胞比例在刺激組與未刺激組間無顯著差異（P0.05）。CD8+T細(xì)胞的增殖指數(shù)和CD69的表達(dá)在刺激組與未刺激組之間有顯著的差異（P0.05）。而CD4+T細(xì)胞CD69和增殖指數(shù)在刺激組與未刺激組之間無顯著差異（P0.05）。(3)抗體的亞類水平進(jìn)行檢測顯示現(xiàn)癥感染組抗PvMSP1-19重組蛋白IgG1抗體水平與正常對照組和既往感染組有顯著差異（P0.05）。 結(jié)論： （1）成功的克隆表達(dá)了PvMSP1-19重組蛋白，并純化出可溶性的、純度高達(dá)95%的PvMSP1-19重組蛋白且具有備良好的免疫活性。 （2）PvMSP1-19重組蛋白可特異誘導(dǎo)CD8+T細(xì)胞增殖、活化分泌IL-4。 （3）現(xiàn)癥感染者血漿具有高水平抗重組抗原PvMSP1-19的IgG1抗體。
[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- 緯 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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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