β-伴大豆球蛋白及其酶解肽對仔豬空腸上皮細胞機械屏障功能損傷的比較研究
發(fā)布時間：2019-06-16 17:31
【摘要】：大豆抗原蛋白能夠破壞仔豬等幼齡動物的腸道屏障功能,引起吸收障礙和腹瀉,影響動物的生長發(fā)育,從而給畜牧業(yè)造成了重大損失。因此,本文選取大豆中主要抗原蛋白β-Conglycinin及其酶解肽為致敏原,以仔豬空腸上皮細胞(IPEC-J2)為體外研究模型,通過三個試驗,研究了不同濃度的大豆抗原蛋白β-Conglycinin及其酶解肽對IPEC-J2機械屏障功能損傷的影響,為進一步探究大豆對腸道損傷的相關(guān)機制提供依據(jù)。試驗I:大豆抗原蛋白β-Conglycinin酶解肽的分離、純化及其免疫活性鑒定。本試驗通過胃蛋白酶、胰蛋白酶連續(xù)水解β-conglycinin,采用凝膠層析技術(shù)及超濾法分離純化酶解肽段,再通過SDS-PAGE電泳試驗及Q-E質(zhì)譜進行鑒定。結(jié)果表明:這三條酶解肽段的分子量分別為52kDa、30kDa、25kDa,并且均具有免疫活性,再通過凝膠層析技術(shù)分離純化后,得到分子量為52kDa的酶解肽段,經(jīng)超濾管超濾得到分子量為30kDa與25kDa酶解肽段的混合產(chǎn)物。試驗II:不同濃度的大豆抗原蛋白β-Conglycinin及其酶解肽對IPEC-J2通透性的影響。本試驗分別采用不同濃度(0、0.125、0.25、0.5、1或2 mg/mL)β-Conglycinin及其酶解肽處理IPEC-J2不同時間(2 h、4 h、6 h、8 h、12 h或24 h)后,通過測定細胞存活力(MTT值),跨上皮細胞電阻值(TEER)以及細胞培養(yǎng)液中堿性磷酸酶(AP)的活性,檢測β-Conglycinin及其酶解肽對細胞通透性的影響。結(jié)果發(fā)現(xiàn)25+30kDa混合酶解肽對IPEC-J2的破壞程度最強,酶解總產(chǎn)物次之,各處理組之間差異顯著(P0.05),β-Conglycinin及其酶解肽對IPEC-J2的MTT值與TEER值呈時間-劑量依賴性下降趨勢(P0.05),24h處理后,AP活性呈顯升高趨勢(P0.05)。即β-Conglycinin及其酶解肽破壞了細胞增殖活力,從而對IPEC-J2屏障造成負面影響。試驗III:β-Conglycinin及其酶解肽對IPEC-J2緊密連接蛋白(Occludin、Claudin-3、Claudin-4和ZO-1)的分布與相對表達量的影響。為了進一步驗證β-Conglycinin及其酶解肽對IPEC-J2機械屏障功能的損傷,本試驗選用濃度為0.5mg/mLβ-Conglycinin及其酶解肽處理IPEC-J2 24h后,采用免疫熒光法和Western Blot法檢測緊密連接蛋白(Occludin、Claudin-3、Claudin-4和ZO-1)在細胞中的分布及相對表達含量;結(jié)果發(fā)現(xiàn)β-Conglycinin及其酶解肽刺激IPEC-J2后細胞間緊密連接處熒光染色程度降低且變得模糊和松散,通過Western Blot方法測得,緊密連接蛋白Occludin、Claudin-3、Claudin-4和ZO-1蛋白的相對表達量與對照組相比均有所下降(P0.05)�？梢姦�-Conglycinin及其酶解肽破壞了緊密連接蛋白完整性,使得上皮屏障及其防護功能受損。綜上所述:β-Conglycinin經(jīng)消化酶水解后,對IPEC-J2械屏障功能的損傷能力加強。即:酶解肽對IPEC-J2的損傷程度高于β-Conglycinin。而在各酶解肽段中,低分子量酶解肽破壞作用最為嚴重,酶解總產(chǎn)物次之、52kDa酶解肽較輕。以上研究結(jié)果為更進一步的揭示大豆的致敏機理提供依據(jù)。
[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.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.28
本文編號：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.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.28
【參考文獻】
相關(guān)期刊論文 前1條
1 孫澤威,秦貴信,婁玉杰;大豆抗原及其對仔豬和犢牛的影響[J];動物營養(yǎng)學報;2005年01期
，本文編號：2500698
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