β-伴大豆球蛋白及其酶解肽对仔猪空肠上皮细胞机械屏障功能损伤的比较研究
发布时间：2019-06-16 17:31
【摘要】：大豆抗原蛋白能够破坏仔猪等幼龄动物的肠道屏障功能,引起吸收障碍和腹泻,影响动物的生长发育,从而给畜牧业造成了重大损失。因此,本文选取大豆中主要抗原蛋白β-Conglycinin及其酶解肽为致敏原,以仔猪空肠上皮细胞(IPEC-J2)为体外研究模型,通过三个试验,研究了不同浓度的大豆抗原蛋白β-Conglycinin及其酶解肽对IPEC-J2机械屏障功能损伤的影响,为进一步探究大豆对肠道损伤的相关机制提供依据。试验I:大豆抗原蛋白β-Conglycinin酶解肽的分离、纯化及其免疫活性鉴定。本试验通过胃蛋白酶、胰蛋白酶连续水解β-conglycinin,采用凝胶层析技术及超滤法分离纯化酶解肽段,再通过SDS-PAGE电泳试验及Q-E质谱进行鉴定。结果表明:这三条酶解肽段的分子量分别为52kDa、30kDa、25kDa,并且均具有免疫活性,再通过凝胶层析技术分离纯化后,得到分子量为52kDa的酶解肽段,经超滤管超滤得到分子量为30kDa与25kDa酶解肽段的混合产物。试验II:不同浓度的大豆抗原蛋白β-Conglycinin及其酶解肽对IPEC-J2通透性的影响。本试验分别采用不同浓度(0、0.125、0.25、0.5、1或2 mg/mL)β-Conglycinin及其酶解肽处理IPEC-J2不同时间(2 h、4 h、6 h、8 h、12 h或24 h)后,通过测定细胞存活力(MTT值),跨上皮细胞电阻值(TEER)以及细胞培养液中碱性磷酸酶(AP)的活性,检测β-Conglycinin及其酶解肽对细胞通透性的影响。结果发现25+30kDa混合酶解肽对IPEC-J2的破坏程度最强,酶解总产物次之,各处理组之间差异显著(P0.05),β-Conglycinin及其酶解肽对IPEC-J2的MTT值与TEER值呈时间-剂量依赖性下降趋势(P0.05),24h处理后,AP活性呈显升高趋势(P0.05)。即β-Conglycinin及其酶解肽破坏了细胞增殖活力,从而对IPEC-J2屏障造成负面影响。试验III:β-Conglycinin及其酶解肽对IPEC-J2紧密连接蛋白(Occludin、Claudin-3、Claudin-4和ZO-1)的分布与相对表达量的影响。为了进一步验证β-Conglycinin及其酶解肽对IPEC-J2机械屏障功能的损伤,本试验选用浓度为0.5mg/mLβ-Conglycinin及其酶解肽处理IPEC-J2 24h后,采用免疫荧光法和Western Blot法检测紧密连接蛋白(Occludin、Claudin-3、Claudin-4和ZO-1)在细胞中的分布及相对表达含量;结果发现β-Conglycinin及其酶解肽刺激IPEC-J2后细胞间紧密连接处荧光染色程度降低且变得模糊和松散,通过Western Blot方法测得,紧密连接蛋白Occludin、Claudin-3、Claudin-4和ZO-1蛋白的相对表达量与对照组相比均有所下降(P0.05)。可见β-Conglycinin及其酶解肽破坏了紧密连接蛋白完整性,使得上皮屏障及其防护功能受损。综上所述:β-Conglycinin经消化酶水解后,对IPEC-J2械屏障功能的损伤能力加强。即:酶解肽对IPEC-J2的损伤程度高于β-Conglycinin。而在各酶解肽段中,低分子量酶解肽破坏作用最为严重,酶解总产物次之、52kDa酶解肽较轻。以上研究结果为更进一步的揭示大豆的致敏机理提供依据。
[Abstract]:Soybean antigen protein can destroy the intestinal barrier function of piglets and other young animals, cause absorption disorder and diarrhea, and affect the growth and development of animals, thus causing great losses to animal husbandry. Therefore, the main antigen protein 尾-Conglycinin and its hydrolyzed peptide in soybean were selected as allergen, and piglets Jejunoepithelial cells (IPEC-J2) were used as the model in vitro. The effects of different concentrations of soybean antigen protein 尾-Conglycinin and its hydrolyzed peptide on the damage of IPEC-J2 mechanical barrier function were studied through three experiments, which provided the basis for further exploring the related mechanism of soybean injury to intestinal tract. Experiment I: isolation, purification and identification of immune activity of soybean antigen protein 尾-Conglycinin hydrolyzed peptide. In this experiment, 尾-conglycinin, was continuously hydrolyzed by pepsin and trypsin by gel chromatography and ultrafiltration, and then identified by SDS-PAGE electrophoresis and Q / E mass spectrometry. The results showed that the molecular weights of the three hydrolyzed peptides were 52kDa, 30kDaand 25kDa. after being separated and purified by gel chromatography, the hydrolyzed peptides with molecular weight of 52kDa were obtained, and the mixed products with molecular weight of 30kDa and 25kDa hydrolyzed peptides were obtained by ultrafiltration. The effects of different concentrations of soybean antigen protein 尾-Conglycinin and its hydrolyzed peptides on the permeability of IPEC-J2 were studied. The effects of 尾-Conglycinin and its hydrolyzed peptides on cell permeability were detected by measuring cell viability (MTT value), transepithelial cell resistance value (TEER) and alkaline phosphatase (AP) activity in cell culture medium after IPEC-J2 was treated with different concentrations (0, 0.125, 0.25, 0.5, 1 or 2 mg/mL) for different time (2 h, 4 h, 6 h, 8 h, 12 h or 24 h) by measuring cell survival (MTT value), transepithelial cell resistance value (TEER) and alkaline phosphatase (AP) activity in cell culture medium. The results showed that the damage degree of 25 30kDa mixed hydrolyzed peptide to IPEC-J2 was the strongest, followed by the total product of enzyme hydrolysis, and there was significant difference among the treatment groups (P 0.05). The MTT value of 尾-Conglycinin and its hydrolyzed peptide to IPEC-J2 decreased in a time-dose-dependent manner (P 0.05). After 24 h treatment, the activity of AP increased significantly (P 0.05). That is to say, 尾-Conglycinin and its hydrolyzed peptides destroy the cell proliferation activity, which has a negative effect on IPEC-J2 barrier. The effects of III: 尾-Conglycinin and its hydrolyzed peptides on the distribution and relative expression of IPEC-J2 tight junction proteins (Occludin,Claudin-3,Claudin-4 and ZO-1) were studied. In order to further verify the damage of 尾-Conglycinin and its hydrolyzed peptides to the mechanical barrier function of IPEC-J2, the distribution and relative expression of tight junction proteins (Occludin,Claudin-3,Claudin-4 and ZO-1) in cells were detected by immunofluorescence and Western Blot after IPEC-J2 was treated with 0.5mg/mL 尾-Conglycinin and its hydrolyzed peptides for 24 h. The results showed that the fluorescence staining degree of tight junction between cells decreased and became blurred and loose after stimulation of IPEC-J2 with 尾-Conglycinin and its hydrolyzed peptides. The relative expression of tight junction protein Occludin,Claudin-3,Claudin-4 and ZO-1 protein measured by Western Blot method was lower than that of the control group (P 0.05). It can be seen that 尾-Conglycinin and its hydrolyzed peptides destroy the integrity of tight junction protein and damage the epithelial barrier and its protective function. To sum up: after hydrolysis of 尾-Conglycinin by digestive enzyme, the damage ability of 尾-IPEC-J2 to the function of mechanical barrier was strengthened. That is to say, the damage degree of IPEC-J2 induced by hydrolyzed peptide is higher than that of 尾-Conglycinin.. Among the hydrolyzed peptides, the destruction of low molecular weight hydrolyzed peptides was the most serious, the total product of hydrolyzed peptides was the second, and the hydrolyzed peptides of 52kDa were lighter. The above results provide a basis for further revealing the sensitizing mechanism of soybean.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
本文编号：2500698
[Abstract]:Soybean antigen protein can destroy the intestinal barrier function of piglets and other young animals, cause absorption disorder and diarrhea, and affect the growth and development of animals, thus causing great losses to animal husbandry. Therefore, the main antigen protein 尾-Conglycinin and its hydrolyzed peptide in soybean were selected as allergen, and piglets Jejunoepithelial cells (IPEC-J2) were used as the model in vitro. The effects of different concentrations of soybean antigen protein 尾-Conglycinin and its hydrolyzed peptide on the damage of IPEC-J2 mechanical barrier function were studied through three experiments, which provided the basis for further exploring the related mechanism of soybean injury to intestinal tract. Experiment I: isolation, purification and identification of immune activity of soybean antigen protein 尾-Conglycinin hydrolyzed peptide. In this experiment, 尾-conglycinin, was continuously hydrolyzed by pepsin and trypsin by gel chromatography and ultrafiltration, and then identified by SDS-PAGE electrophoresis and Q / E mass spectrometry. The results showed that the molecular weights of the three hydrolyzed peptides were 52kDa, 30kDaand 25kDa. after being separated and purified by gel chromatography, the hydrolyzed peptides with molecular weight of 52kDa were obtained, and the mixed products with molecular weight of 30kDa and 25kDa hydrolyzed peptides were obtained by ultrafiltration. The effects of different concentrations of soybean antigen protein 尾-Conglycinin and its hydrolyzed peptides on the permeability of IPEC-J2 were studied. The effects of 尾-Conglycinin and its hydrolyzed peptides on cell permeability were detected by measuring cell viability (MTT value), transepithelial cell resistance value (TEER) and alkaline phosphatase (AP) activity in cell culture medium after IPEC-J2 was treated with different concentrations (0, 0.125, 0.25, 0.5, 1 or 2 mg/mL) for different time (2 h, 4 h, 6 h, 8 h, 12 h or 24 h) by measuring cell survival (MTT value), transepithelial cell resistance value (TEER) and alkaline phosphatase (AP) activity in cell culture medium. The results showed that the damage degree of 25 30kDa mixed hydrolyzed peptide to IPEC-J2 was the strongest, followed by the total product of enzyme hydrolysis, and there was significant difference among the treatment groups (P 0.05). The MTT value of 尾-Conglycinin and its hydrolyzed peptide to IPEC-J2 decreased in a time-dose-dependent manner (P 0.05). After 24 h treatment, the activity of AP increased significantly (P 0.05). That is to say, 尾-Conglycinin and its hydrolyzed peptides destroy the cell proliferation activity, which has a negative effect on IPEC-J2 barrier. The effects of III: 尾-Conglycinin and its hydrolyzed peptides on the distribution and relative expression of IPEC-J2 tight junction proteins (Occludin,Claudin-3,Claudin-4 and ZO-1) were studied. In order to further verify the damage of 尾-Conglycinin and its hydrolyzed peptides to the mechanical barrier function of IPEC-J2, the distribution and relative expression of tight junction proteins (Occludin,Claudin-3,Claudin-4 and ZO-1) in cells were detected by immunofluorescence and Western Blot after IPEC-J2 was treated with 0.5mg/mL 尾-Conglycinin and its hydrolyzed peptides for 24 h. The results showed that the fluorescence staining degree of tight junction between cells decreased and became blurred and loose after stimulation of IPEC-J2 with 尾-Conglycinin and its hydrolyzed peptides. The relative expression of tight junction protein Occludin,Claudin-3,Claudin-4 and ZO-1 protein measured by Western Blot method was lower than that of the control group (P 0.05). It can be seen that 尾-Conglycinin and its hydrolyzed peptides destroy the integrity of tight junction protein and damage the epithelial barrier and its protective function. To sum up: after hydrolysis of 尾-Conglycinin by digestive enzyme, the damage ability of 尾-IPEC-J2 to the function of mechanical barrier was strengthened. That is to say, the damage degree of IPEC-J2 induced by hydrolyzed peptide is higher than that of 尾-Conglycinin.. Among the hydrolyzed peptides, the destruction of low molecular weight hydrolyzed peptides was the most serious, the total product of hydrolyzed peptides was the second, and the hydrolyzed peptides of 52kDa were lighter. The above results provide a basis for further revealing the sensitizing mechanism of soybean.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
【参考文献】
相关期刊论文 前1条
1 孙泽威,秦贵信,娄玉杰;大豆抗原及其对仔猪和犊牛的影响[J];动物营养学报;2005年01期
，本文编号：2500698
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