花生过敏原Ara h2免疫亲和层析分离方法的建立

发布时间：2019-05-30 02:04

【摘要】：花生是八大类致敏食物之一,其造成的过敏症状严重,发生率较高,而且不随过敏患者年龄的增长而消减,引起了社会的广泛关注。在目前已发现的13种花生过敏蛋白中,Ara h2蛋白占花生总蛋白含量的10%左右,能够被90%花生过敏患者血清识别,是花生的主要过敏原之一。过敏原蛋白的分离提取为其致敏研究所必须,故其纯化方法的建立具有重要的研究价值。本论文以Ara h2为研究对象,通过获取相应抗体,进行免疫亲和层析柱制备,并将自制柱应用于生花生及加工花生产物中Ara h2的纯化。论文的研究工作包括利用天然花生Ara h2制备兔抗Ara h2多克隆抗体并分离;Ara h2免疫亲和层析柱的制备;热加工花生蛋白产物中Ara h2纯化。研究的主要方法、结果及结论如下:1.采用离子交换层析法从天然花生中分离Ara h2,并通过SDS-PAGE对分离蛋白纯度进行鉴定;利用分离得到的高纯度的Ara h2作为免疫原,采用多点皮下注射免疫日本大耳白兔,获得抗Ara h2的兔多克隆抗体,并通过ELISA和免疫印迹方法检测其效价和特异性;以分离得到的高纯度的Ara h2和溴化氢活化的sepharose-4B柱材作为亲和介质材料,制备了从兔抗Ara h2血清中分离纯化抗Ara h2多克隆抗体的亲和层析柱,并利用SDS-PAGE和werstern blot进行鉴定。结果显示:纯化得到的天然Ara h2纯度高达90%以上,其多克隆抗体效价高,特异性强。同时制备得到抗Ara h2多克隆抗体的亲和层析柱偶联率95.1%,抗体/柱材结合比率为17.1mg/g。SDS-PAGE结果表明通过该层析柱子分离得到的抗花生过敏原Ara h2特异性抗体纯度高;免疫印迹法鉴定表明该抗体特异性非常高,ELISA检测效价为1:6400,相对于上样血清,抗体被富集了100倍以上。2.利用纯化得到的抗Ara h2多克隆抗体与溴化氢活化的sepharose-4B柱材相偶联制备了用于分离Ara h2蛋白免疫亲和层析柱,并对该自制柱偶联率、柱容量、回收率、使用次数等参数进行了表征。结果显示:该Ara h2蛋白免疫亲和层析柱偶联率为90.4%,抗体的介质密度为18.1mg/g,该自制柱柱容量约0.55-0.60mg,Ara h2回收率在92.7%-97.8%之间,在使用11次后其柱容量仍保持原柱容量的87.5%,说明自制柱具有优良的重复使用性能。3.以生花生粗蛋白液为基本样品分别进行水煮15、30、45、60、90min加工处理,之后对两种样品进行15min、30min、45min、60min、90min体外模拟胃消化和5min、15min、30min、60min、90min体外模拟胃肠消化。以Gly-HCl(p H2.7)作为洗脱液,利用自制Ara h2蛋白免疫亲和层析柱对生花生、水煮花生、胃消化、肠消化产物中Ara h2蛋白进行分离,并通过SDS-PAGE和werstern blot对分离产物进行鉴定,利用ELISA方法分别测定生花生、水煮花生胃消化、肠消化产物中Ara h2的提取率。结果显示:水煮处理造成了花生粗蛋白复杂聚集,不同的水煮时间形成的电泳条带差异不显著,尤其是Ara h2蛋白带;体外模拟胃消化使生花生和水煮花生蛋白粗提液发生不同程度消化,Ara h2蛋白具有较强抗胃液消化;在体外模拟胃肠消化中生花生和水煮花生蛋白粗提液中的蛋白质都被消化降解,Ara h2蛋白消化也很明显。利用自制Ara h2蛋白免疫亲和层析柱从生花生、水煮花生胃消化、肠消化产物成功分离出Ara h2蛋白,SDS-PAGE结果显示生花生粗提液样品蛋白纯度为87.4%,水煮花生粗提液分离产物中Ara h2纯度为79.5%,二者体外模拟消化产物分离产物纯度分别为72.7%和70.7%左右,并且层析柱能够分离获得水煮加工形成Ara h2蛋白二聚体。ELISA结果显示生花生、水煮花生胃消化、肠消化产物提取率分别为94.4%、88.7%、73.4%、79.2%,说明自制Ara h2蛋白免疫亲和层析柱对该蛋白有较好分离能力。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R593.1

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