胸腺五肽的固相合成及免疫学初步研究

发布时间：2019-03-14 07:59

【摘要】： 以H-Arg-Lys-Asp-Val-Tyr-OH为序列的胸腺五肽(TP5)是胸腺生成素II(Thymopoietin II)中第32~36位的氨基酸残基片段,它保留了胸腺生成素II原有的生物活性,对机体的免疫系统具有双向调节作用,现已作为药物投放市场。本文研究固相法合成胸腺五肽,并对合成的多肽进行理化检测,研究其纯度及活性。合成方法采用Fmoc固相肽合成法,首先通过C18反相HPLC分析柱摸索纯化条件,然后以C18反相HPLC制备柱纯化。最后将制备的TP5样品旋转蒸发以除去残存的乙腈、三氟乙酸等,经冻干后制成白色粉末,以质谱仪测定其分子量,以反相HPLC分析其纯度,以E玫瑰花实验检测其生物活性。活性检测采用小鼠免疫抑制模型,分别测定了不同剂量TP-5对免疫抑制小鼠的脾指数、胸腺指数、巨噬细胞吞噬功能、体内T淋巴细胞转化功能、IL-2水平、血清溶血素含量等指标的影响;同时体外培养正常小鼠脾细胞,分别以不同浓度的TP-5处理,然后测定大鼠的脾指数、胸腺指数,观察脾脏和胸腺的病理组织学变化及T淋巴细胞亚群等指标的改变。本研究共合成三批胸腺五肽,纯化后制备成冻干粉末。精肽产量分别为27 mg,34 mg,32 mg。质谱检测证明合成TP-5分子量为679.8,与理论分子量相符。反相HPLC测定其纯度大于95%,表明该纯化方法有效。三批样品的玫瑰花结百分率均大于10%,证明合成的TP-5有生物活性。活性测定表明,浓度为0.2 mg/kg、0.4 mg/kg、0.8 mg/kg的合成TP-5均可显著降低免疫抑制小鼠脾指数,显著增强免疫抑制小鼠的特异性和非特异性免疫功能,具有免疫原性。浓度为0.2mg/kg、0.4mg/kg、0.8mg/kg的TP-5对正常大鼠的脾指数、胸腺指数、脾脏和胸腺的病理组织学及T淋巴细胞亚群(CD4/CD8)无明显影响,无免疫毒性。本课题的研究证明,用固相合成法可以成功地合成具有生物活性的TP-5,且有免疫调节功能,无免疫毒性,为以后实现产业化生产奠定了坚实的基础。
[Abstract]:Thymopentapeptide (TP5), with H-Arg-Lys-Asp-Val-Tyr-OH as its sequence, is a 32-36 amino acid fragment of thymopoietin II (Thymopoietin II). It retains the original bioactivity of thymopoietin II. It has two-way regulation on the immune system of the body and has been put on the market as a drug. In this paper, the solid-phase synthesis of thymopentapeptide was studied, and the purity and activity of the synthesized peptide were studied by physical and chemical analysis. The Fmoc solid-phase peptide synthesis method was used. Firstly, the purification conditions were explored by C18 reversed-phase HPLC column and then purified by C18 reversed-phase HPLC preparation column. Finally, the prepared TP5 samples were rotated and evaporated to remove the remaining acetonitrile and trifluoroacetic acid. After freeze-drying, the white powder was prepared. Its molecular weight was determined by mass spectrometer, its purity was analyzed by reversed-phase HPLC, and its biological activity was tested by E-rose test. The spleen index, thymus index, phagocytic function of macrophage, T lymphocyte transformation function and IL-2 level of different doses of TP-5 on immunosuppressive mice were determined by immunosuppressive model in mice. The influence of serum hemolysin content and other indexes; At the same time, the spleen cells of normal mice were cultured in vitro and treated with different concentrations of TP-5. Then the spleen index and thymus index of rats were measured. The pathological changes of spleen and thymus and the changes of T lymphocyte subsets were observed. In this study, three batches of thymopentapeptide were synthesized and purified to prepare freeze-dried powder. The production of sperm peptide is 27 mg,34 mg,32 mg. respectively. The results of mass spectrometry showed that the molecular weight of TP-5 was 679.8, which was consistent with the theoretical molecular weight. The purity was more than 95% determined by reverse phase HPLC, which indicated that the purification method was effective. The rosette percentage of the three batches of samples was more than 10%, which proved that the synthesized TP-5 was bioactive. The activity assay showed that the synthesis of TP-5 at the concentration of 0. 2 mg/kg,0.4 mg/kg,0.8 mg/kg could significantly decrease the spleen index of immunosuppressive mice and enhance the specific and non-specific immune function of immunosuppressive mice. Have immunogenicity. When the concentration of TP-5 was 0.2 mg / kg, 0.4 mg / kg, 0.8 mg / kg, the spleen index, thymus index, histopathology of spleen and thymus, and T lymphocyte subsets (CD4/CD8) were not significantly affected, and there was no immunotoxic effect on the spleen index, thymus index, spleen and thymus histopathology and T lymphocyte subsets (CD4/CD8) in normal rats. The research in this paper proves that the solid-phase synthesis method can successfully synthesize bioactive TP-5, with immunomodulatory function and no immunotoxicity, which lays a solid foundation for industrial production in the future.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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