胸腺五肽的固相合成及免疫學(xué)初步研究
發(fā)布時間：2019-03-14 07:59
【摘要】： 以H-Arg-Lys-Asp-Val-Tyr-OH為序列的胸腺五肽(TP5)是胸腺生成素II(Thymopoietin II)中第32~36位的氨基酸殘基片段,它保留了胸腺生成素II原有的生物活性,對機體的免疫系統(tǒng)具有雙向調(diào)節(jié)作用,現(xiàn)已作為藥物投放市場。本文研究固相法合成胸腺五肽,并對合成的多肽進行理化檢測,研究其純度及活性。 合成方法采用Fmoc固相肽合成法,首先通過C18反相HPLC分析柱摸索純化條件,然后以C18反相HPLC制備柱純化。最后將制備的TP5樣品旋轉(zhuǎn)蒸發(fā)以除去殘存的乙腈、三氟乙酸等,經(jīng)凍干后制成白色粉末,以質(zhì)譜儀測定其分子量,以反相HPLC分析其純度,以E玫瑰花實驗檢測其生物活性。 活性檢測采用小鼠免疫抑制模型,分別測定了不同劑量TP-5對免疫抑制小鼠的脾指數(shù)、胸腺指數(shù)、巨噬細(xì)胞吞噬功能、體內(nèi)T淋巴細(xì)胞轉(zhuǎn)化功能、IL-2水平、血清溶血素含量等指標(biāo)的影響;同時體外培養(yǎng)正常小鼠脾細(xì)胞,分別以不同濃度的TP-5處理,然后測定大鼠的脾指數(shù)、胸腺指數(shù),觀察脾臟和胸腺的病理組織學(xué)變化及T淋巴細(xì)胞亞群等指標(biāo)的改變。 本研究共合成三批胸腺五肽,純化后制備成凍干粉末。精肽產(chǎn)量分別為27 mg,34 mg,32 mg。質(zhì)譜檢測證明合成TP-5分子量為679.8,與理論分子量相符。反相HPLC測定其純度大于95%,表明該純化方法有效。三批樣品的玫瑰花結(jié)百分率均大于10%,證明合成的TP-5有生物活性。 活性測定表明,濃度為0.2 mg/kg、0.4 mg/kg、0.8 mg/kg的合成TP-5均可顯著降低免疫抑制小鼠脾指數(shù),顯著增強免疫抑制小鼠的特異性和非特異性免疫功能,具有免疫原性。濃度為0.2mg/kg、0.4mg/kg、0.8mg/kg的TP-5對正常大鼠的脾指數(shù)、胸腺指數(shù)、脾臟和胸腺的病理組織學(xué)及T淋巴細(xì)胞亞群(CD4/CD8)無明顯影響,無免疫毒性。 本課題的研究證明,用固相合成法可以成功地合成具有生物活性的TP-5,且有免疫調(diào)節(jié)功能,無免疫毒性,為以后實現(xiàn)產(chǎn)業(yè)化生產(chǎn)奠定了堅實的基礎(chǔ)。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392
本文編號：2439799
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392
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