大豆球蛋白、β-伴球蛋白的分离提取及对猪外周血淋巴细胞增殖和免疫功能的影响
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摘要
本研究采用实验研究方法，以分离纯化豆粕中的抗原蛋白作实验材料，研究纯化抗原蛋白的特性和体外对体液免疫及细胞免疫的影响。研究分两个实验：
试验一：大豆球蛋白和β—伴球蛋白分离提取。利用豆粕中7S和11S球蛋白在pH6.1-6.6之间溶解度不同的特点，首先对两种球蛋白进行初步分离，然后利用硫酸铵进行盐析和凝胶过滤，除去非抗原蛋白组分，得到纯度相对较高的大豆球蛋白和β—伴球蛋白。
结果表明：豆粕粉经0.03mMTris-HCl、料液比1：20、室温、PH8.0，提取2h，豆粕总蛋白提取率为32.88％。可分离出蛋白质含量71.53％的11S粗组分9.1％和蛋白质含量60.73％的7S粗组分7.9％；经盐析和凝胶过滤后，可得到蛋白含量84.19％的纯化大豆球蛋白4.3％和蛋白含量79.47％的纯化β—伴球蛋白6.0％；经过SDS-PAGE电泳鉴定，11S粗组分纯度达到87.0％，7S粗组分纯度达到62.5％；纯化后的大豆球蛋白纯度达到93.8％，β-伴大豆球蛋白达到67.2％，单位豆粕含大豆球蛋白182.34g／kg，β-伴大豆球蛋白126.94g／kg。
实验二：利用细胞培养技术，研究了不同水平纯化大豆球蛋白、β-伴球蛋白(0、0.5mg／ml、1.25mg／ml、2.5mg／ml、5mg／ml、10mg／ml)对猪外周血单核淋巴细胞(PBMC)增殖和免疫功能的影响。
结果表明：大豆球蛋白、β—伴球蛋白显著增加体外培养的猪外周单核淋巴细胞数量，极显著影响细胞MTT OD值(P＜0.01)。β—伴球蛋白与培养液IL-2、IL-6、IFN-γ、IgA含量呈显著正相关(P＜0.05)，大豆球蛋白对培养液中IL-2、IL-6、IFN-γ、IgG、IgM、IgA含量影响不显著(P＞0.05)。
本实验研究表明，豆粕可分离出纯度达到93.8％的大豆球蛋白182.34g／kg和纯度达到67.2％的β-伴大豆球蛋白126.94g／kg。大豆球蛋白、β—伴球蛋白促进体外培养断奶仔猪外周淋巴血细胞转化和过度增殖，β—伴球蛋白可促使体外培养断奶仔猪外周血淋巴细胞产生炎性细胞因子IL-2、IFN-γ、IL-6和提高IgA水平。由结果可知：β—伴球蛋白抗原性强于大豆球蛋白。
The research to adopt empirical study, using the isolated and purified soybean as subjects, studying characteristics of purified antigen protein and the influence of humoral immunity and cell immunity in vitro. Two experiment was adopted.
Experiment 1: Glycinin andβ—conglycinin was isolated and purified. by means of the characteristics 7s globulin and 11s globulin of different dissolubility at PH6.1 to PH 6.6. first of all, prefractionating two kinds of globulin, then, salting out with sulfate ammonium and gel filtration, as far as possible to remove constituents of non- antigen protein to obtain glycinin andβ—conglycinin with relative higher purity coefficient.
The result indicated: Soybean powder was extracted by 0.03mMTris-HC1, ratio of dosage liquor: 1 to 20, room temperature, PH8.0, 2 hours, the extraction ratio of total protein was 32.88 percents, Separated 9.1 percents crude 11s with 71.53 percents protein and 7.9 percents crude 7s with 60.73 percents protein. Salting out with sulfate ammonium and gel filtration and obtained 4.3 percents crude 11s with 84.19 percents protein and 6.0 percents crude 7s with 79.47 percents protein. Purity coefficient of 11s and 7s was 87.0 and 62.5 percents, and glycinin andβ—conglycinin was 93.8 and 67.2 percents, the unit of soubean contain glycinin with 182.34g/kg andβ—conglycinin with 126.94 g/kg.
Experiment 2:, Study the effect of different level purified glycinin andβ—conglycinin(0、0.5 mg/ml、1.25mg/ml、2.5mg/ml、5 mg/ml、10 mg/ml) on proliferation and immune function of peripheral blood mononuclear lymphocyte of weaning young pigs by utilizing cell culture technology
The result indicated: The amounts of cells in the group of glycinin andβ—conglycinin increased gradually,. The cell MTT absorption at 570 nm wavelength (MTT OD570) was very significantly affected by glycinin andβ-conglycinin concentration (P＜ 0.01).β—conglycinin was positive correlated significantly with interleukin -2, interleukin-6, interferon-γ, and immune globulinA in medium (P＜0.05), but glycinin was correlated non-significantly with nterleukin -2, interleukin-6, interferon-γ, immune globulin G, immune globulin M and immune globulinA in medium(P＞0.05).
In summary, soybean was separated 182.34g/kg glycinin with 93.8 percents purity coefficient and 126.94 g/kgβ-conglycinin with 67.2 percents purity coefficient, glycinin andβ—conglycinin could promote peripheral blood lymphocyte of weaning pigs to transform and proliferate in vitro culture,β—conglycinin could promote peripheral blood lymphocyte of weaning pigs to produce cytokine with IL-2, INF-γ,、IL-6 and raise the level of immunoglobulin. The results showed that antigen ofβ—conglycinin was stronger than glycinin.
试验一：大豆球蛋白和β—伴球蛋白分离提取。利用豆粕中7S和11S球蛋白在pH6.1-6.6之间溶解度不同的特点，首先对两种球蛋白进行初步分离，然后利用硫酸铵进行盐析和凝胶过滤，除去非抗原蛋白组分，得到纯度相对较高的大豆球蛋白和β—伴球蛋白。
结果表明：豆粕粉经0.03mMTris-HCl、料液比1：20、室温、PH8.0，提取2h，豆粕总蛋白提取率为32.88％。可分离出蛋白质含量71.53％的11S粗组分9.1％和蛋白质含量60.73％的7S粗组分7.9％；经盐析和凝胶过滤后，可得到蛋白含量84.19％的纯化大豆球蛋白4.3％和蛋白含量79.47％的纯化β—伴球蛋白6.0％；经过SDS-PAGE电泳鉴定，11S粗组分纯度达到87.0％，7S粗组分纯度达到62.5％；纯化后的大豆球蛋白纯度达到93.8％，β-伴大豆球蛋白达到67.2％，单位豆粕含大豆球蛋白182.34g／kg，β-伴大豆球蛋白126.94g／kg。
实验二：利用细胞培养技术，研究了不同水平纯化大豆球蛋白、β-伴球蛋白(0、0.5mg／ml、1.25mg／ml、2.5mg／ml、5mg／ml、10mg／ml)对猪外周血单核淋巴细胞(PBMC)增殖和免疫功能的影响。
结果表明：大豆球蛋白、β—伴球蛋白显著增加体外培养的猪外周单核淋巴细胞数量，极显著影响细胞MTT OD值(P＜0.01)。β—伴球蛋白与培养液IL-2、IL-6、IFN-γ、IgA含量呈显著正相关(P＜0.05)，大豆球蛋白对培养液中IL-2、IL-6、IFN-γ、IgG、IgM、IgA含量影响不显著(P＞0.05)。
本实验研究表明，豆粕可分离出纯度达到93.8％的大豆球蛋白182.34g／kg和纯度达到67.2％的β-伴大豆球蛋白126.94g／kg。大豆球蛋白、β—伴球蛋白促进体外培养断奶仔猪外周淋巴血细胞转化和过度增殖，β—伴球蛋白可促使体外培养断奶仔猪外周血淋巴细胞产生炎性细胞因子IL-2、IFN-γ、IL-6和提高IgA水平。由结果可知：β—伴球蛋白抗原性强于大豆球蛋白。
The research to adopt empirical study, using the isolated and purified soybean as subjects, studying characteristics of purified antigen protein and the influence of humoral immunity and cell immunity in vitro. Two experiment was adopted.
Experiment 1: Glycinin andβ—conglycinin was isolated and purified. by means of the characteristics 7s globulin and 11s globulin of different dissolubility at PH6.1 to PH 6.6. first of all, prefractionating two kinds of globulin, then, salting out with sulfate ammonium and gel filtration, as far as possible to remove constituents of non- antigen protein to obtain glycinin andβ—conglycinin with relative higher purity coefficient.
The result indicated: Soybean powder was extracted by 0.03mMTris-HC1, ratio of dosage liquor: 1 to 20, room temperature, PH8.0, 2 hours, the extraction ratio of total protein was 32.88 percents, Separated 9.1 percents crude 11s with 71.53 percents protein and 7.9 percents crude 7s with 60.73 percents protein. Salting out with sulfate ammonium and gel filtration and obtained 4.3 percents crude 11s with 84.19 percents protein and 6.0 percents crude 7s with 79.47 percents protein. Purity coefficient of 11s and 7s was 87.0 and 62.5 percents, and glycinin andβ—conglycinin was 93.8 and 67.2 percents, the unit of soubean contain glycinin with 182.34g/kg andβ—conglycinin with 126.94 g/kg.
Experiment 2:, Study the effect of different level purified glycinin andβ—conglycinin(0、0.5 mg/ml、1.25mg/ml、2.5mg/ml、5 mg/ml、10 mg/ml) on proliferation and immune function of peripheral blood mononuclear lymphocyte of weaning young pigs by utilizing cell culture technology
The result indicated: The amounts of cells in the group of glycinin andβ—conglycinin increased gradually,. The cell MTT absorption at 570 nm wavelength (MTT OD570) was very significantly affected by glycinin andβ-conglycinin concentration (P＜ 0.01).β—conglycinin was positive correlated significantly with interleukin -2, interleukin-6, interferon-γ, and immune globulinA in medium (P＜0.05), but glycinin was correlated non-significantly with nterleukin -2, interleukin-6, interferon-γ, immune globulin G, immune globulin M and immune globulinA in medium(P＞0.05).
In summary, soybean was separated 182.34g/kg glycinin with 93.8 percents purity coefficient and 126.94 g/kgβ-conglycinin with 67.2 percents purity coefficient, glycinin andβ—conglycinin could promote peripheral blood lymphocyte of weaning pigs to transform and proliferate in vitro culture,β—conglycinin could promote peripheral blood lymphocyte of weaning pigs to produce cytokine with IL-2, INF-γ,、IL-6 and raise the level of immunoglobulin. The results showed that antigen ofβ—conglycinin was stronger than glycinin.
引文
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