CN106916215A - 一种用于缓解对虾原肌球蛋白致敏反应的改良蛋白dMete1及其制备方法及应用 - Google Patents
一种用于缓解对虾原肌球蛋白致敏反应的改良蛋白dMete1及其制备方法及应用 Download PDFInfo
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Abstract
本发明公开一种用于缓解对虾原肌球蛋白致敏反应的改良蛋白dMet e 1以及其制备方法和应用，dMet e 1的氨基酸序列如SEQ ID NO：2所示。制备方法如下：(1)切除新对虾属(Metapenaeus)原肌球蛋白Met e 1的九个抗原特异性表位氨基酸残基得到改良蛋白dMet e 1氨基酸序列，并对所得改良蛋白dMet e 1氨基酸序列通过逆向翻译转录得到dMet e 1核酸序列，然后合成对应的cDNA；(2)将所得cDNA与载体质粒通过限制性内切酶处理并结合，获得重组质粒载体并导入宿主菌，经表达纯化得到改良蛋白dMet e 1。本发明具有产量高，生产稳定，成本低的特点。通过小鼠模型以及患者血清ELISA试验证明改良蛋白dMet e 1对对虾原肌球蛋白引起的过敏反应有明显的抑制效果，且不引起相关的交叉反应。
Description
技术领域
本发明涉及水产食品过敏免疫调控领域，介绍了一种以新对虾属(Metapenaeus)原肌球蛋白(Met e 1)为原型，通过针对性的比对，设计制备的一种可用于缓解对虾原肌球蛋白致敏反应的改良蛋白dMet e 1制备方法，旨在为典型水产食品过敏的控制提供支撑。
背景技术
食物过敏是一类典型的过敏反应，是一种由IgE介导的速发性免疫反应，成为近年来关注的热点。对虾及其制品味道鲜美，营养丰富，深受人们的喜爱，但却因具有较高的致敏性，影响了过敏人群的食用。据统计，对虾等甲壳类动物引发的过敏反应广泛存在于全球各个国家当中，最高可占食物过敏总人口的30％以上，食物过敏的儿童中对虾等甲壳类过敏的比例甚至高达60％以上。进一步分析显示，原肌球蛋白是引起人们甲壳类水产食品过敏反应的主要原因之一，能够引起过敏性皮炎、支气管哮喘、过敏性腹泻等症状，严重威胁到人们的健康，且不同对虾品种以及其他甲壳类动物的原肌球蛋白具有一定的同源性，能够引起一定的交叉反应，最终加大了这类人群的过敏反应，因此如何缓解对虾原肌球蛋白致敏反应引起了广泛的关注。
目前缓解和控制对虾等食品过敏普遍采用的方法包括物理法、化学法和生物法，均可以显著降低过敏原的免疫活性。其中物理法包括加热、辐照、微波以及超高压等，化学法包括强酸水解、靶向美拉德反应，生物法多采用酶解法，是目前新兴的一种方法，具有一定的应用前景。
但是以上方法均不能从根本上缓解或者控制对虾等食品过敏，一方面在消除对虾等食品过敏原本身的同时，由于食品不同的基质和自身的结构差异，并不能很好的完全去除过敏原；另一方面改变了食品原有的结构和感官性状，进而影响了作为商品的原有价值。
发明内容
针对现有技术存在问题，本发明提供一种用于缓解对虾原肌球蛋白致敏反应的改良蛋白dMet e 1以及其制备方法。
一种用于缓解对虾原肌球蛋白致敏反应的改良蛋白dMet e 1，其氨基酸序列如SEQ ID NO：2所示。
一种如所述改良蛋白dMet e 1的制备方法，其特征在于，包括如下步骤：
(1)切除新对虾属(Metapenaeus)原肌球蛋白Met e 1序列中的九个抗原特异性表位氨基酸残基序列得到改良蛋白dMet e 1氨基酸序列，并对所得改良蛋白dMet e 1氨基酸序列通过逆向翻译转录得到dMet e 1核酸序列，然后合成对应的cDNA；
(2)将所得cDNA与载体质粒通过限制性内切酶处理并结合，获得重组质粒载体并导入宿主菌，经表达纯化得到改良蛋白dMet e 1。
为了缓解对虾原肌球蛋白致敏反应，本研究结合前期的实验，通过针对性将新对虾属原肌球蛋白Met e 1(SEQ ID NO：1)九个抗原特异性表位氨基酸残基切除，制备了一种可用于缓解对虾原肌球蛋白致敏反应的改良蛋白dMet e 1，实验结果表明，可以通过调节免疫耐受，从而降低过敏反应。为从过敏人群角度从本质上解决对虾等食品过敏问题提供了依据。
本发明采用基因工程的方法，以新对虾属原肌球蛋白Met e 1为基础，删除其蛋白表明九个抗原特异性结合位点(E1:25-30、E2:43-60、E3:87-103、E4:146-154、E5:161-165、E6：191-211、E7:236-241、E8：247-255、E9：269-281)得到仅有171个氨基酸残基的改良蛋白dMet e 1氨基酸序列，并通过逆向转录翻译得到其cDNA并合成重组质粒导入宿主菌进行表达，经纯化得到改良蛋白标准品。本制备方法具有产量高，生产稳定，成本低的特点。通过小鼠模型以及患者血清ELISA试验证明改良蛋白dMet e 1对对虾原肌球蛋白引起的过敏反应有明显的抑制效果，且不引起相关的交叉反应。
逆向翻译转录得到dMet e 1核酸序列并合成对应的cDNA由生工生物工程(上海)股份有限公司完成。
获得全长cDNA基因序列后，在序列两端引入限制性内切酶EcoRV及HindⅢ酶切位点，与Pet30(a)+原核表达载体酶切、连接，使得插入片段的起始密码子位于翻译的起始密码子位置，构建重组质粒；将构建的重组质粒导入大肠杆菌BL21(DE3)菌株，然后置于大肠杆菌表达培养基MagicMedia上37℃下培养过夜；组氨酸标记大肠杆菌BL21(DE3)菌株表达出的dMet e 1，并通过纯化柱HisPur Cobat Spin Column进行纯化。
纯化后的蛋白通过SDS-PAGE对蛋白进行纯度、分子量进行检测。
发明的改良肌球蛋白dMet e 1用于缓解对虾原肌球蛋白致敏反应，患者血清及小鼠模型试验证明能够有效地缓解对虾原肌球蛋白引起的食品过敏反应。
一种如所述改良蛋白dMet e 1在制备用于缓解对虾原肌球蛋白致敏反应的药物或保健品中的应用。
与现有技术相比，本发明的有益效果：
本发明由获得改良过敏原基因的全长cDNA出发，采用基因工程的方法，合成了经改良的原肌球蛋白dMet e 1，所得改良蛋白纯度高，经患者血清及小鼠模型试验证明能够有效地缓解对虾原肌球蛋白引起的食品过敏反应，本发明所建立的降低对虾原肌球蛋白过敏反应的改良及其制备方法，未见文献报道，为快速、准确的降低对虾原肌球蛋白过敏提供新的手段。
附图说明
图1为改良蛋白dMet e 1的氨基酸序列。
图2为改良dMet e 1对免疫反应的抑制情况。
具体实施方式
以下实施例中所用处理手段均为本领域常规处理方法。
实施例1
1.改良蛋白质序列设计
dMet e 1改良蛋白序列设计：去除新对虾属原肌球蛋白Met e 1(SEQ ID NO：1)的九个抗原特异性表位氨基酸残基(E1:25-30、E2:43-60、E3:87-103、E4:146-154、E5:161-165、E6：191-211、E7:236-241、E8：247-255、E9：269-281)得到改良蛋白dMet e 1氨基酸序列(SEQ ID NO：2)。
2.cDNA序列获取及合成
对所得改良原肌球蛋白序列进行分析，并逆向转录翻译得到相应的cDNA序列，并合成得到相应的cDNA。该步骤送至生工生物工程(上海)股份有限公司完成。
3.载体构建及蛋白质表达
获得全长cDNA基因序列，并在序列两端引入限制性内切酶EcoRV及HindⅢ酶切位点，与Pet30(a)+原核表达载体酶切、连接，使得插入片段的起始密码子位于翻译的起始密码子位置，构建重组质粒，其酶切反应体系与酶联反应体系如表1和表2所示。
表1酶切反应体系
4.转化培养
将构建的重组质粒(5μL)导入大肠杆菌BL21(DE3)菌株(100μL)，然后将所筛选得到的转化菌株接种于大肠杆菌表达培养基MagicMedia上37℃下培养过夜。
5.蛋白纯化
组氨酸标记大肠杆菌BL21(DE3)菌株表达出的dMet e 1，并通过纯化柱HisPurCobat Spin Column进行纯化。纯化后的蛋白通过SDS-PAGE对蛋白进行纯度、分子量进行检测，看到只有一条分子量大约为27kDa的明显单带，经检测纯度达95％以上，将纯化后的蛋白冻干保存。
6.改良蛋白减低致敏效果的鉴定
将3-4周雌性Balb/c小鼠分为Met e 1组与dMet e 1组(每组10只)，对Met e 1组使用原肌球蛋白Met e 1进行口服灌胃处理、dMet e 1组使用原肌球蛋白Met e 1和改良蛋白dMet e 1进行口服灌胃处理，而后将两组小鼠进行组内均分每组5只试验小鼠，对组内不同分组分别腹腔注射原肌球蛋白Met e 1和改良蛋白dMet e 1，30min后取小鼠血清对过敏反应特异性抗体IgE及IgG含量进行检测，以判断小鼠不同组蛋白处理对小鼠过敏反应程度的影响，其结果如表3所示。
表3不同原肌球蛋白蛋白处理后小鼠(n＝5)血清中抗原等级
结果表明，预先经原肌球蛋白Met e 1刺激小鼠在再次摄入原肌球蛋白Met e 1后会发生过敏反应释放过敏反应特异性抗体IgE，而经改良蛋白dMet e 1处理后小鼠再摄入原肌球蛋白Met e 1能有效降低80％的抗原IgE释放，即dMet e 1能显著减缓因原肌球蛋白Met e 1摄入而引起的食品过敏反应。
实施例2
1.改良蛋白质序列设计
dMet e 1改良蛋白序列设计：切除新对虾属原肌球蛋白Met e 1(SEQ ID NO：1)的九个抗原特异性表位氨基酸残基(E1:25-30、E2:43-60、E3:87-103、E4:146-154、E5:161-165、E6：191-211、E7:236-241、E8：247-255、E9：269-281)得到改良蛋白dMet e 1氨基酸序列(SEQ ID NO：2)。
2.cDNA序列获取及合成
对所得改良原肌球蛋白序列进行分析，并逆向转录翻译得到相应的cDNA序列，并合成得到相应的cDNA。
3.载体构建及蛋白质表达
获得全长cDNA基因序列，并在序列两端引入限制性内切酶EcoRV及HindⅢ酶切位点，与Pet30(a)+原核表达载体酶切、连接，使得插入片段的起始密码子位于翻译的起始密码子位置，构建重组质粒，其酶切反应体系与酶联反应体系如表4和表5所示。
表4酶切反应体系
表5酶联反应体系
4.转化培养
将构建的重组质粒(5μL)导入大肠杆菌BL21(DE3)菌株(100μL)，然后将所筛选得到的转化菌株接种于大肠杆菌表达培养基MagicMedia上37℃下培养过夜。
5.蛋白纯化
组氨酸标记大肠杆菌BL21(DE3)菌株表达出的dMet e 1，并通过纯化柱HisPurCobat Spin Column进行纯化。纯化后的蛋白通过SDS-PAGE对蛋白进行纯度、分子量进行检测，看到只有一条分子量大约为27kDa的明显单带，经检测纯度达95％以上，将纯化后的蛋白冻干保存。、6.改良蛋白减低致敏效果的鉴定
用碳酸盐包被缓冲液将Met e 1与dMet e 1稀释至1-10μg/mL，于96孔板上每孔加200μL抗原，4℃下过夜，次日用洗涤缓冲液洗涤3次，每次5min。每孔中加入200μL封闭液，置37℃恒温箱中保持1h后洗涤。加入稀释的人类患者血清200μL于上述包被抗原相应的孔中，置于37℃下孵化1h后洗涤，同时做空白、阴性、阳性对照孔，对各反应孔加入稀释的酶标二抗(山羊抗人)200μL，37℃下孵育30-60min后洗涤，最后用蒸馏水洗涤2次，置于酶标仪上检测，改良蛋白dMet e 1对IgE反应的抑制情况如图2所示，结果表明，改良原肌球蛋白dMet e1能有效减弱77.4％的IgE释放，证明其对过敏症状具有一定减缓作用。
以上所述仅为本发明专利的具体实施案例，但本发明专利的技术特征并不局限于此，任何相关领域的技术人员在本发明的领域内，所作的变化或修饰皆涵盖在本发明的专利范围之中。
序列表
SEQUENCE LISTING
<110> 浙江工商大学
<120> 一种用于缓解对虾原肌球蛋白致敏反应的改良蛋白 dMet e 1及其制备方法及应用
<130>
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<170> PatentIn version 3.3
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<213> 新对虾属（Metapenaeus）
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Claims (7)
1.一种用于缓解对虾原肌球蛋白致敏反应的改良蛋白dMet e 1，其特征在于，其氨基酸序列如SEQ ID NO：2所示。
2.一种如权利要求1所述改良蛋白dMet e 1的制备方法，其特征在于，包括如下步骤：
(1)切除新对虾属(Metapenaeus)原肌球蛋白Met e 1序列中的九个抗原特异性表位氨基酸残基序列得到改良蛋白dMet e 1氨基酸序列，并对所得改良蛋白dMet e 1氨基酸序列通过逆向翻译转录得到dMet e 1核酸序列，然后合成对应的cDNA；
(2)将所得cDNA与载体质粒通过限制性内切酶处理并结合，获得重组质粒载体并导入宿主菌，经表达纯化得到改良蛋白dMet e 1。
3.根据权利要求2所述制备方法，其特征在于，所述限制性内切酶为EcoRV及HindⅢ。
4.根据权利要求2所述制备方法，其特征在于，所述载体质粒为Pet30(a)+。
5.根据权利要求2所述制备方法，其特征在于，所述宿主菌为大肠杆菌BL21(DE3)。
6.根据权利要求2所述制备方法，其特征在于，纯化采用的纯化柱为HisPur CobatSpin Column。
7.一种如权利要求1所述改良蛋白dMet e 1在制备用于缓解对虾原肌球蛋白致敏反应的药物或保健品中的应用。
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