人蛔蟲CPI基因的全長克隆與免疫學(xué)功能初步研究
發(fā)布時間：2019-06-26 19:13
【摘要】：蠕蟲類寄生蟲感染可以顯著抑制宿主免疫功能這一現(xiàn)象已經(jīng)被廣泛報道,但對這類寄生蟲中起免疫調(diào)控作用的分子所知甚少。許多研究表明,蠕蟲分泌排泄產(chǎn)物中的半胱氨酸蛋白酶抑制因子(Cysteine Protease Inhibitor, CPI)具有顯著的免疫抑制功能,然而其作用機理和應(yīng)用價值仍需深入研究。 人蛔蟲(Ascaris lumbricoides, Al)是一種人體常見的腸道寄生蠕蟲,其體腔液和提取物具有抗過敏反應(yīng)、抗腫瘤等免疫調(diào)控功能。然而人蛔蟲中具有免疫調(diào)控活性的分子有哪些仍然不甚明確。為此,本文針對人蛔蟲中的半胱氨酸蛋白酶抑制因子進行研究。 本研究首先根據(jù)已知線蟲半胱氨酸蛋白酶抑制因子的保守序列設(shè)計兼并引物,從人蛔蟲cDNA中擴增得到人蛔蟲CPI的保守區(qū)序列片段。再利用RACE技術(shù),成功克隆了人蛔蟲CPI全長cDNA序列。將Al-CPI的編碼區(qū)克隆進原核表達載體pET-32a中,轉(zhuǎn)化大腸桿菌origami之后進行誘導(dǎo)表達和親和層析柱純化,得到重組Al-CPI蛋白。 重組CPI蛋白進行生物活性測定,發(fā)現(xiàn)該蛋白可以抑制Cathepsin B、L、S、C四種組織蛋白酶對其相應(yīng)底物的水解作用,證明該蛋白具有生物活性。 進一步研究Al-CPI的免疫學(xué)功能,發(fā)現(xiàn)Al-CPI可抑制由ConA、PPD等刺激引起的人PBMC的增殖活性,并可以顯著降低PBMC中ConA刺激誘導(dǎo)的IL-4和IFN-γ的分泌。流式細(xì)胞術(shù)檢測PBMC中不同T細(xì)胞亞群的表面分子,發(fā)現(xiàn)ConA刺激引起的CD4~+ T細(xì)胞和CD8~+ T細(xì)胞表面CD25和HLA-DR的表達均被CPI抑制。這說明Al-CPI具有顯著的免疫抑制功能。 在混合淋巴細(xì)胞反應(yīng)(Mixed Lymphocyte Reaction, MLR)中,Al-CPI可以抑制應(yīng)答細(xì)胞中CD4~+ T細(xì)胞的增殖能力。MLR體系中CD14+單核細(xì)胞表面共刺激分子CD40、CD80和CD86的表達在CPI作用下均下調(diào)。同時,在MLR培養(yǎng)上清中IL-12和IFN-γ的分泌均可被CPI抑制。這說明人蛔蟲CPI可以抑制同種異型抗原誘導(dǎo)的淋巴細(xì)胞激活,抑制同種異體移植排斥反應(yīng)。該研究結(jié)果表明Al-CPI具有潛在的臨床藥用價值。
[Abstract]:Worm parasite infection can significantly inhibit host immune function, which has been widely reported, but little is known about the molecules that play an immune regulatory role in these parasites. Many studies have shown that cysteinase inhibitor (Cysteine Protease Inhibitor, CPI), which is secreted by worms, has significant immunosuppressive function. However, its mechanism and application value still need to be further studied. Ascaris lumbricoides (Ascaris lumbricoides, Al) is a common intestinal parasitic worm in human body. Its cavity fluid and extract have anti-allergic reaction, anti-tumor and other immune regulatory functions. However, it is still unclear which molecules have immunomodulatory activity in Ascaris lumbricoides. Therefore, the cysteinase inhibitor in Ascaris lumbricoides was studied in this paper. In this study, according to the conserved sequence of cysteinase inhibitor of nematodes, primers were designed to amplify the conserved region of human Ascaris lumbricoides CPI from human Ascaris lumbricoides cDNA. The full-length cDNA sequence of Ascaris lumbricoides CPI was successfully cloned by RACE technique. The coding region of Al-CPI was cloned into prokaryotic expression vector pET-32a, transformed into E. coli origami and purified by affinity chromatography. The recombinant Al-CPI protein was obtained. The biological activity of recombinant CPI protein was determined. It was found that the protein could inhibit the hydrolysis of Cathepsin B, L, S, C cathepsin on its corresponding substrate, which proved that the protein had biological activity. Further study on the immunological function of Al-CPI showed that Al-CPI could inhibit the proliferation of human PBMC induced by ConA,PPD and other stimuli, and significantly decrease the secretion of IL-4 and IFN- 緯 induced by ConA stimulation in PBMC. The surface molecules of different T cell subsets in PBMC were detected by flow cytometry. It was found that the expression of CD25 and HLA-DR on CD4~ T cells and CD8~ T cells stimulated by ConA was inhibited by CPI. This indicates that Al-CPI has significant immunosuppressive function. In mixed lymphocyte reaction (Mixed Lymphocyte Reaction, MLR), Al-CPI could inhibit the proliferation of CD4~ T cells in response cells. The expression of costimulatory molecules CD40,CD80 and CD86 on the surface of CD14 monocytes in CD14 system was down-regulated by CPI. At the same time, the secretion of IL-12 and IFN- 緯 in MLR culture supernatant could be inhibited by CPI. This suggests that human Ascaris lumbricoides CPI can inhibit the activation of lymphocytes induced by allogenic antigen and inhibit the rejection of allotransplantation. The results of this study show that Al-CPI has potential clinical medicinal value.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1
本文編號：2506422
[Abstract]:Worm parasite infection can significantly inhibit host immune function, which has been widely reported, but little is known about the molecules that play an immune regulatory role in these parasites. Many studies have shown that cysteinase inhibitor (Cysteine Protease Inhibitor, CPI), which is secreted by worms, has significant immunosuppressive function. However, its mechanism and application value still need to be further studied. Ascaris lumbricoides (Ascaris lumbricoides, Al) is a common intestinal parasitic worm in human body. Its cavity fluid and extract have anti-allergic reaction, anti-tumor and other immune regulatory functions. However, it is still unclear which molecules have immunomodulatory activity in Ascaris lumbricoides. Therefore, the cysteinase inhibitor in Ascaris lumbricoides was studied in this paper. In this study, according to the conserved sequence of cysteinase inhibitor of nematodes, primers were designed to amplify the conserved region of human Ascaris lumbricoides CPI from human Ascaris lumbricoides cDNA. The full-length cDNA sequence of Ascaris lumbricoides CPI was successfully cloned by RACE technique. The coding region of Al-CPI was cloned into prokaryotic expression vector pET-32a, transformed into E. coli origami and purified by affinity chromatography. The recombinant Al-CPI protein was obtained. The biological activity of recombinant CPI protein was determined. It was found that the protein could inhibit the hydrolysis of Cathepsin B, L, S, C cathepsin on its corresponding substrate, which proved that the protein had biological activity. Further study on the immunological function of Al-CPI showed that Al-CPI could inhibit the proliferation of human PBMC induced by ConA,PPD and other stimuli, and significantly decrease the secretion of IL-4 and IFN- 緯 induced by ConA stimulation in PBMC. The surface molecules of different T cell subsets in PBMC were detected by flow cytometry. It was found that the expression of CD25 and HLA-DR on CD4~ T cells and CD8~ T cells stimulated by ConA was inhibited by CPI. This indicates that Al-CPI has significant immunosuppressive function. In mixed lymphocyte reaction (Mixed Lymphocyte Reaction, MLR), Al-CPI could inhibit the proliferation of CD4~ T cells in response cells. The expression of costimulatory molecules CD40,CD80 and CD86 on the surface of CD14 monocytes in CD14 system was down-regulated by CPI. At the same time, the secretion of IL-12 and IFN- 緯 in MLR culture supernatant could be inhibited by CPI. This suggests that human Ascaris lumbricoides CPI can inhibit the activation of lymphocytes induced by allogenic antigen and inhibit the rejection of allotransplantation. The results of this study show that Al-CPI has potential clinical medicinal value.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1
【共引文獻】
相關(guān)期刊論文 前2條
1 張為宇;黃維義;盧秀紅;吳文德;嚴(yán)紅;鄭啟超;;綿羊和水牛實驗感染大片吸蟲后外周血白細(xì)胞計數(shù)的分析[J];廣西農(nóng)業(yè)生物科學(xué);2006年03期
2 李肖梁;劉剛;張彥明;帥江冰;方維煥;;中華鱉脾淋巴細(xì)胞體外轉(zhuǎn)化的實驗條件研究[J];浙江大學(xué)學(xué)報(農(nóng)業(yè)與生命科學(xué)版);2008年04期
，本文編號：2506422
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