人蛔虫CPI基因的全长克隆与免疫学功能初步研究
发布时间：2019-06-26 19:13
【摘要】：蠕虫类寄生虫感染可以显著抑制宿主免疫功能这一现象已经被广泛报道,但对这类寄生虫中起免疫调控作用的分子所知甚少。许多研究表明,蠕虫分泌排泄产物中的半胱氨酸蛋白酶抑制因子(Cysteine Protease Inhibitor, CPI)具有显著的免疫抑制功能,然而其作用机理和应用价值仍需深入研究。 人蛔虫(Ascaris lumbricoides, Al)是一种人体常见的肠道寄生蠕虫,其体腔液和提取物具有抗过敏反应、抗肿瘤等免疫调控功能。然而人蛔虫中具有免疫调控活性的分子有哪些仍然不甚明确。为此,本文针对人蛔虫中的半胱氨酸蛋白酶抑制因子进行研究。 本研究首先根据已知线虫半胱氨酸蛋白酶抑制因子的保守序列设计兼并引物,从人蛔虫cDNA中扩增得到人蛔虫CPI的保守区序列片段。再利用RACE技术,成功克隆了人蛔虫CPI全长cDNA序列。将Al-CPI的编码区克隆进原核表达载体pET-32a中,转化大肠杆菌origami之后进行诱导表达和亲和层析柱纯化,得到重组Al-CPI蛋白。 重组CPI蛋白进行生物活性测定,发现该蛋白可以抑制Cathepsin B、L、S、C四种组织蛋白酶对其相应底物的水解作用,证明该蛋白具有生物活性。 进一步研究Al-CPI的免疫学功能,发现Al-CPI可抑制由ConA、PPD等刺激引起的人PBMC的增殖活性,并可以显著降低PBMC中ConA刺激诱导的IL-4和IFN-γ的分泌。流式细胞术检测PBMC中不同T细胞亚群的表面分子,发现ConA刺激引起的CD4~+ T细胞和CD8~+ T细胞表面CD25和HLA-DR的表达均被CPI抑制。这说明Al-CPI具有显著的免疫抑制功能。 在混合淋巴细胞反应(Mixed Lymphocyte Reaction, MLR)中,Al-CPI可以抑制应答细胞中CD4~+ T细胞的增殖能力。MLR体系中CD14+单核细胞表面共刺激分子CD40、CD80和CD86的表达在CPI作用下均下调。同时,在MLR培养上清中IL-12和IFN-γ的分泌均可被CPI抑制。这说明人蛔虫CPI可以抑制同种异型抗原诱导的淋巴细胞激活,抑制同种异体移植排斥反应。该研究结果表明Al-CPI具有潜在的临床药用价值。
[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- 纬 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- 纬 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.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
本文编号：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- 纬 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- 纬 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.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
【共引文献】
相关期刊论文 前2条
1 张为宇;黄维义;卢秀红;吴文德;严红;郑启超;;绵羊和水牛实验感染大片吸虫后外周血白细胞计数的分析[J];广西农业生物科学;2006年03期
2 李肖梁;刘刚;张彦明;帅江冰;方维焕;;中华鳖脾淋巴细胞体外转化的实验条件研究[J];浙江大学学报(农业与生命科学版);2008年04期
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