花生過(guò)敏原Ara h2免疫親和層析分離方法的建立
發(fā)布時(shí)間：2019-05-30 02:04
【摘要】：花生是八大類致敏食物之一,其造成的過(guò)敏癥狀嚴(yán)重,發(fā)生率較高,而且不隨過(guò)敏患者年齡的增長(zhǎng)而消減,引起了社會(huì)的廣泛關(guān)注。在目前已發(fā)現(xiàn)的13種花生過(guò)敏蛋白中,Ara h2蛋白占花生總蛋白含量的10%左右,能夠被90%花生過(guò)敏患者血清識(shí)別,是花生的主要過(guò)敏原之一。過(guò)敏原蛋白的分離提取為其致敏研究所必須,故其純化方法的建立具有重要的研究?jī)r(jià)值。本論文以Ara h2為研究對(duì)象,通過(guò)獲取相應(yīng)抗體,進(jìn)行免疫親和層析柱制備,并將自制柱應(yīng)用于生花生及加工花生產(chǎn)物中Ara h2的純化。論文的研究工作包括利用天然花生Ara h2制備兔抗Ara h2多克隆抗體并分離;Ara h2免疫親和層析柱的制備;熱加工花生蛋白產(chǎn)物中Ara h2純化。研究的主要方法、結(jié)果及結(jié)論如下:1.采用離子交換層析法從天然花生中分離Ara h2,并通過(guò)SDS-PAGE對(duì)分離蛋白純度進(jìn)行鑒定;利用分離得到的高純度的Ara h2作為免疫原,采用多點(diǎn)皮下注射免疫日本大耳白兔,獲得抗Ara h2的兔多克隆抗體,并通過(guò)ELISA和免疫印跡方法檢測(cè)其效價(jià)和特異性;以分離得到的高純度的Ara h2和溴化氫活化的sepharose-4B柱材作為親和介質(zhì)材料,制備了從兔抗Ara h2血清中分離純化抗Ara h2多克隆抗體的親和層析柱,并利用SDS-PAGE和werstern blot進(jìn)行鑒定。結(jié)果顯示:純化得到的天然Ara h2純度高達(dá)90%以上,其多克隆抗體效價(jià)高,特異性強(qiáng)。同時(shí)制備得到抗Ara h2多克隆抗體的親和層析柱偶聯(lián)率95.1%,抗體/柱材結(jié)合比率為17.1mg/g。SDS-PAGE結(jié)果表明通過(guò)該層析柱子分離得到的抗花生過(guò)敏原Ara h2特異性抗體純度高;免疫印跡法鑒定表明該抗體特異性非常高,ELISA檢測(cè)效價(jià)為1:6400,相對(duì)于上樣血清,抗體被富集了100倍以上。2.利用純化得到的抗Ara h2多克隆抗體與溴化氫活化的sepharose-4B柱材相偶聯(lián)制備了用于分離Ara h2蛋白免疫親和層析柱,并對(duì)該自制柱偶聯(lián)率、柱容量、回收率、使用次數(shù)等參數(shù)進(jìn)行了表征。結(jié)果顯示:該Ara h2蛋白免疫親和層析柱偶聯(lián)率為90.4%,抗體的介質(zhì)密度為18.1mg/g,該自制柱柱容量約0.55-0.60mg,Ara h2回收率在92.7%-97.8%之間,在使用11次后其柱容量仍保持原柱容量的87.5%,說(shuō)明自制柱具有優(yōu)良的重復(fù)使用性能。3.以生花生粗蛋白液為基本樣品分別進(jìn)行水煮15、30、45、60、90min加工處理,之后對(duì)兩種樣品進(jìn)行15min、30min、45min、60min、90min體外模擬胃消化和5min、15min、30min、60min、90min體外模擬胃腸消化。以Gly-HCl(p H2.7)作為洗脫液,利用自制Ara h2蛋白免疫親和層析柱對(duì)生花生、水煮花生、胃消化、腸消化產(chǎn)物中Ara h2蛋白進(jìn)行分離,并通過(guò)SDS-PAGE和werstern blot對(duì)分離產(chǎn)物進(jìn)行鑒定,利用ELISA方法分別測(cè)定生花生、水煮花生胃消化、腸消化產(chǎn)物中Ara h2的提取率。結(jié)果顯示:水煮處理造成了花生粗蛋白復(fù)雜聚集,不同的水煮時(shí)間形成的電泳條帶差異不顯著,尤其是Ara h2蛋白帶;體外模擬胃消化使生花生和水煮花生蛋白粗提液發(fā)生不同程度消化,Ara h2蛋白具有較強(qiáng)抗胃液消化;在體外模擬胃腸消化中生花生和水煮花生蛋白粗提液中的蛋白質(zhì)都被消化降解,Ara h2蛋白消化也很明顯。利用自制Ara h2蛋白免疫親和層析柱從生花生、水煮花生胃消化、腸消化產(chǎn)物成功分離出Ara h2蛋白,SDS-PAGE結(jié)果顯示生花生粗提液樣品蛋白純度為87.4%,水煮花生粗提液分離產(chǎn)物中Ara h2純度為79.5%,二者體外模擬消化產(chǎn)物分離產(chǎn)物純度分別為72.7%和70.7%左右,并且層析柱能夠分離獲得水煮加工形成Ara h2蛋白二聚體。ELISA結(jié)果顯示生花生、水煮花生胃消化、腸消化產(chǎn)物提取率分別為94.4%、88.7%、73.4%、79.2%,說(shuō)明自制Ara h2蛋白免疫親和層析柱對(duì)該蛋白有較好分離能力。
[Abstract]:Peanut is one of the eight kinds of sensitizers. The cause of the allergy is serious, the incidence rate is high, and is not reduced with the increase of the age of the allergic patients, which causes the wide attention of the society. Of the 13 peanut allergic proteins that have been found at present, Ara h2 protein accounts for about 10% of the total protein content of the peanut, and can be identified by the serum of 90% of the peanut allergy patients, and is one of the main allergens of the peanut. The separation and extraction of the allergen protein is necessary for its sensitization research institute, so the establishment of its purification method has important research value. In this paper, Ara h2 is used as the research object, and the corresponding antibody is obtained, and the preparation of the immunoaffinity chromatography column is carried out, and the self-made column is applied to the purification of Ara h2 in the peanut and the processed peanut product. The research work of the paper includes the preparation of Ara h2 polyclonal antibody by using the natural peanut Ara h2, the preparation of the Ara h2 immunoaffinity chromatography column, and the purification of Ara h2 in the hot-working peanut protein product. The main methods, results and conclusions of the study are as follows:1. Ara h2 is separated from the natural peanut by ion exchange chromatography, and the purity of the separated protein is identified by SDS-PAGE; the high-purity Ara h2 obtained by separation is used as an immunogen, and a multi-point subcutaneous injection is adopted to immunize the Japanese large-ear white rabbit to obtain a rabbit polyclonal antibody against Ara h2, and the titer and specificity of the purified anti-Ara h2 polyclonal antibody are separated and purified from the rabbit anti-Ara h2 serum by using an ELISA and an immunoblotting method to detect the potency and specificity of the purified anti-Ara h2 polyclonal antibody, The identification was carried out by SDS-PAGE and werstrn-blot. The results showed that the purity of the purified natural Ara h2 was as high as 90%, and its polyclonal antibody titer was high and the specificity was strong. At the same time, the coupling ratio of the affinity chromatography column of the anti-Ara h2 polyclonal antibody is 95.1%, the binding ratio of the antibody/ column material is 17.1 mg/ g, and the SDS-PAGE results show that the anti-peanut allergen Ara h2 specific antibody obtained by the separation of the chromatographic column is high in purity; the identification of the immunoblotting method indicates that the antibody specificity is very high, The titer of ELISA was 1:6400, and the antibody was enriched by more than 100 times with respect to the loaded serum. The purified anti-Ara h2 polyclonal antibody was coupled with a hydrogen bromide-activated sephose-4B column to prepare the immunoaffinity chromatography column for the separation of Ara h2 protein, and the parameters such as the column coupling rate, the column capacity, the recovery rate, the number of use and the like of the self-made column were characterized. The results showed that the coupling ratio of the Ara h2 protein immunoaffinity chromatography column was 90.4%, the medium density of the antibody was 18.1 mg/ g, the volume of the self-made column column was about 0.55-0.60 mg, the recovery of Ara h2 was 92.7%-97.8%, and the column capacity remained 87.5% of the original column capacity after 11 times. It is indicated that the self-made column has excellent reusability. The main samples of crude peanut protein were treated with water in 15,30,45,60 and 90 min respectively, and then the two samples were subjected to 15 min,30 min,45 min,60 min and 90 min to simulate gastric digestion and 5min,15 min,30 min,60 min and 90 min to simulate gastrointestinal digestion in vitro. Ara h2 protein was isolated from Ara h2 protein in the product of peanut, boiled peanut, stomach, and intestine by using the self-made Ara h2 protein immunoaffinity chromatography column as the eluent, and the isolated product was identified by SDS-PAGE and werstrn blot. The peanut was measured by ELISA. The extraction rate of Ara h2 in the digestion of the boiled peanut and the digestion of the intestine. The results showed that the water-boiling treatment resulted in the complex aggregation of the peanut crude protein, the difference of the electrophoretic bands formed by different water-boiling times was not significant, especially the Ara h2 protein band; in vitro, the digestion of the crude extract of the peanut protein and the water-boiled peanut protein was caused by the in vitro simulated gastric digestion, The Ara h2 protein has stronger anti-gastric juice digestion, and the protein in the crude extract of the peanut and water boiled peanut protein in the in vitro simulated gastrointestinal digestion is digested and degraded, and the Ara h2 protein digestion is also obvious. The Ara h2 protein was isolated from the raw peanut, the boiled peanut and the stomach by using the self-made Ara h2 protein immunoaffinity chromatography column, and the protein purity of the crude peanut crude extract was 87.4%, and the purity of Ara h2 in the crude extract of the boiled peanut was 79.5%. The purity of the isolated product of the two in-vitro simulated digestion product is 72.7% and 70.7%, respectively, and the chromatographic column can be separated to obtain water-boiling processing to form the Ara h2 protein dimer. The results showed that the extraction rates of peanut, boiled peanut and stomach were 94.4%, 88.7%, 73.4% and 79.2%, respectively.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R593.1
本文編號(hào)：2488421
[Abstract]:Peanut is one of the eight kinds of sensitizers. The cause of the allergy is serious, the incidence rate is high, and is not reduced with the increase of the age of the allergic patients, which causes the wide attention of the society. Of the 13 peanut allergic proteins that have been found at present, Ara h2 protein accounts for about 10% of the total protein content of the peanut, and can be identified by the serum of 90% of the peanut allergy patients, and is one of the main allergens of the peanut. The separation and extraction of the allergen protein is necessary for its sensitization research institute, so the establishment of its purification method has important research value. In this paper, Ara h2 is used as the research object, and the corresponding antibody is obtained, and the preparation of the immunoaffinity chromatography column is carried out, and the self-made column is applied to the purification of Ara h2 in the peanut and the processed peanut product. The research work of the paper includes the preparation of Ara h2 polyclonal antibody by using the natural peanut Ara h2, the preparation of the Ara h2 immunoaffinity chromatography column, and the purification of Ara h2 in the hot-working peanut protein product. The main methods, results and conclusions of the study are as follows:1. Ara h2 is separated from the natural peanut by ion exchange chromatography, and the purity of the separated protein is identified by SDS-PAGE; the high-purity Ara h2 obtained by separation is used as an immunogen, and a multi-point subcutaneous injection is adopted to immunize the Japanese large-ear white rabbit to obtain a rabbit polyclonal antibody against Ara h2, and the titer and specificity of the purified anti-Ara h2 polyclonal antibody are separated and purified from the rabbit anti-Ara h2 serum by using an ELISA and an immunoblotting method to detect the potency and specificity of the purified anti-Ara h2 polyclonal antibody, The identification was carried out by SDS-PAGE and werstrn-blot. The results showed that the purity of the purified natural Ara h2 was as high as 90%, and its polyclonal antibody titer was high and the specificity was strong. At the same time, the coupling ratio of the affinity chromatography column of the anti-Ara h2 polyclonal antibody is 95.1%, the binding ratio of the antibody/ column material is 17.1 mg/ g, and the SDS-PAGE results show that the anti-peanut allergen Ara h2 specific antibody obtained by the separation of the chromatographic column is high in purity; the identification of the immunoblotting method indicates that the antibody specificity is very high, The titer of ELISA was 1:6400, and the antibody was enriched by more than 100 times with respect to the loaded serum. The purified anti-Ara h2 polyclonal antibody was coupled with a hydrogen bromide-activated sephose-4B column to prepare the immunoaffinity chromatography column for the separation of Ara h2 protein, and the parameters such as the column coupling rate, the column capacity, the recovery rate, the number of use and the like of the self-made column were characterized. The results showed that the coupling ratio of the Ara h2 protein immunoaffinity chromatography column was 90.4%, the medium density of the antibody was 18.1 mg/ g, the volume of the self-made column column was about 0.55-0.60 mg, the recovery of Ara h2 was 92.7%-97.8%, and the column capacity remained 87.5% of the original column capacity after 11 times. It is indicated that the self-made column has excellent reusability. The main samples of crude peanut protein were treated with water in 15,30,45,60 and 90 min respectively, and then the two samples were subjected to 15 min,30 min,45 min,60 min and 90 min to simulate gastric digestion and 5min,15 min,30 min,60 min and 90 min to simulate gastrointestinal digestion in vitro. Ara h2 protein was isolated from Ara h2 protein in the product of peanut, boiled peanut, stomach, and intestine by using the self-made Ara h2 protein immunoaffinity chromatography column as the eluent, and the isolated product was identified by SDS-PAGE and werstrn blot. The peanut was measured by ELISA. The extraction rate of Ara h2 in the digestion of the boiled peanut and the digestion of the intestine. The results showed that the water-boiling treatment resulted in the complex aggregation of the peanut crude protein, the difference of the electrophoretic bands formed by different water-boiling times was not significant, especially the Ara h2 protein band; in vitro, the digestion of the crude extract of the peanut protein and the water-boiled peanut protein was caused by the in vitro simulated gastric digestion, The Ara h2 protein has stronger anti-gastric juice digestion, and the protein in the crude extract of the peanut and water boiled peanut protein in the in vitro simulated gastrointestinal digestion is digested and degraded, and the Ara h2 protein digestion is also obvious. The Ara h2 protein was isolated from the raw peanut, the boiled peanut and the stomach by using the self-made Ara h2 protein immunoaffinity chromatography column, and the protein purity of the crude peanut crude extract was 87.4%, and the purity of Ara h2 in the crude extract of the boiled peanut was 79.5%. The purity of the isolated product of the two in-vitro simulated digestion product is 72.7% and 70.7%, respectively, and the chromatographic column can be separated to obtain water-boiling processing to form the Ara h2 protein dimer. The results showed that the extraction rates of peanut, boiled peanut and stomach were 94.4%, 88.7%, 73.4% and 79.2%, respectively.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R593.1
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