CN104316706B - 大泷六线鱼卵黄原蛋白免疫印迹试剂盒及其制备方法、检测方法及应用 - Google Patents
大泷六线鱼卵黄原蛋白免疫印迹试剂盒及其制备方法、检测方法及应用 Download PDFInfo
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Abstract
本发明公开了一种检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒及其制备方法、检测方法及应用。该试剂盒含有大泷六线鱼卵黄原蛋白纯品与兔抗大泷六线鱼卵黄原蛋白多克隆抗体。制备时，首先利用硫酸铵沉淀与Sephadex G‑200过滤层析从17β‑雌二醇诱导的大泷六线鱼血浆中纯化出卵黄原蛋白，然后免疫新西兰大白兔制备兔抗大泷六线鱼卵黄原蛋白多克隆抗血清，并利用Hitrap Protein G层析，纯化获得兔抗大泷六线鱼卵黄原蛋白多克隆抗体。本发明的试剂盒可以用于海洋环境雌激素类物质的筛选，能够灵敏、方便的定性检测大泷六线鱼血液、体表粘液、肝脏组织及肝细胞培养液中的卵黄原蛋白，最低检测限为50 ng mL‑1，为我国近岸海域内分泌干扰物的检测提供了重要工具。
Description
技术领域
本发明属于生态检测领域，具体涉及一种检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒及其制备方法和应用。
背景技术
持久性有机污染物((Persistent Organic Pollutants，POPs)，是指在环境中难以分解，能够在环境中长期存在，可以通过各种传输途径而进行全球尺度的迁移扩散，通过食物链在生物体内累积放大，对人体和环境产生毒性影响的一类有机污染物。这些污染物并不是自然界本身就存在的，而是人类工业革命带来的产物．持久性有机污染物给人类和环境带来的危害已经成为全球性问题。为了解决这一问题，联合国环境规划署(UNEP)和瑞典政府于2001年5月23日在瑞典的斯德哥尔摩联合主持召开全权代表会议，签署了旨在禁止和/或限制使用12类持久性有机污染物的《关于持久性有机污染物的斯德哥尔摩公约》。POPs具有下列四个重要的特性： (1) 能够在环境中持久地存在；(2)能够经过长距离迁移到达偏远的极低地区。(3) 在一定的浓度下会对接触该物质的生物造成有害或有毒影响，POPs大都具有“三致（致癌、致畸、致突变）”效应；(4) 能蓄积在食物链中，对有较高营养等级的生物造成影响。由于POPs具有低水溶性、高脂溶性的特点，导致POPs从周围媒介中富集到生物体内，并通过食物链的生物放大作用达到中毒浓度。
POPs在海洋环境中难降解、分布广、易在生物体内富集，对生态环境的危害性很大，POPs不仅影响到海洋生物的栖息与繁殖，通过食物链的传递，也给人类自身生存和生活造成威胁。各项研究显示POPs浓度水平并不很高，但是由于其生物富集性可通过食物链传递富集，使得处于食物链越高的生物受到的威胁越大。在海洋环境和其他水生生态系统中，POPs的传播链是：空气中的POPs最初是被微生物吸收→较大生物吃微小生物→小鱼食用较大生物→大鱼吃小鱼→有时是鸟类或人类食用大鱼。食肉类物种体内的POPs含量将会达到其捕食对象体内POPs平均含量的10倍之多。这导致了在最高端食肉物种体内极高的POPs含量。根据环境加拿大（Environment Canada）组织的报告，食用鱼类的鸟蛋中POPs污染物达到鱼类本身生活的水中POPs含量的2500万倍。而位于生物链顶端的人类，则又把这些毒性放大了7万倍。
当前急需全面提升海洋POPs实时监测、预警能力，对海水养殖的污染源进行研究，探讨鱼类饲料、人类活动及环境因素等对水产品质量的影响，为海水养殖产品的质量监控和海域环境的管理提供科学依据。因此建立起快速、高效、大通量针对POPs特别是新增加种类污染物的有效检测方法是加强和完善对环境中POPs的检测及风险控制管理的前提和基础。研究表明，大多数POPs具有环境雌激素效应，是环境雌激素类似物，因此可以将环境雌激素生物检测方法用于POPs的生物检测。
美国、欧盟和日本相继建立起以鱼类为模式生物的环境雌激素筛选评价体系，其中卵黄原蛋白(Vitellogenin, Vtg)作为重要的生物筛选指标，已经得到广泛应用。鱼类卵黄原蛋白是卵黄蛋白的前体，是一种大分子量的脂磷聚糖蛋白。卵黄形成期，在雌激素的刺激下卵黄原蛋白由肝脏合成，并通过血液运输到发育的卵巢中，被卵巢吸收；通常，卵黄原蛋白只能在卵黄形成期的雌鱼体内检测到，但是，雄鱼和幼鱼体内也含有卵黄原蛋白基因，在环境雌激素的诱导下，也能合成和分泌卵黄原蛋白。因此，卵黄原蛋白是环境雌激素筛选的特异性生物标志物，通过检测雄鱼体内卵黄原蛋白水平可以评价环境化学物的雌激素活性。然而，至今我国还未见海洋环境POPs生物监测技术的研发。
大泷六线鱼(Hexagrammos otakii Jordan et Starks )又名欧式六线鱼，俗称黄鱼，隶属于鲉形目(Scorpaeniformes)、六线鱼科(Hexagrammidae)、六线鱼属(Hexagrammos)。大泷六线鱼属冷温性近海底层鱼类，在我国主要分布于黄海和渤海沿岸多岩礁海区，在青岛等近海水域中容易捕获。以黄渤海常见鱼种大泷六线鱼为实验生物，建立其卵黄原蛋白的检测技术，有助于开展我国近岸海域POPs生物监测工作。
发明内容
本发明所要解决的技术问题是在于提供一种检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒及其制备方法，以满足现有技术的上述需求。
为解决上述技术问题，本发明采用的技术方案如下：
本发明提供的一种检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒，包括一个盒体，该盒体内装有：1)PVDF膜2张；2)辣根过氧化物酶标记的羊抗兔二抗1支；3)封闭液、洗涤液、显色液各1支，所述的洗涤液为TBST；封闭液为含5%脱脂奶粉的TBST；显色液为含0.06%(m/V) 3’-二氨基联苯胺的10mM Tris-HCl；其特征在于该盒体还装有：4)大泷六线鱼卵黄原蛋白纯品1支(10 μg/ml)；5)兔抗大泷六线鱼卵黄原蛋白多克隆抗体1支。
本发明还提供了上述检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒制备方法，包括以下步骤：1)制备大泷六线鱼卵黄原蛋白纯品；2)利用步骤1）得到的大泷六线鱼卵黄原蛋白纯品制备兔抗大泷六线鱼卵黄原蛋白多克隆抗体；3)将步骤1)得到的大泷六线鱼卵黄原蛋白纯品1支、步骤2)得到的兔抗大泷六线鱼卵黄原蛋白多克隆抗体1支、PVDF膜2张，以及封闭液、洗涤液、显色液和辣根过氧化物酶标记的羊抗兔二抗1支共同装入盒体，得到检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒。
所述的大泷六线鱼卵黄原蛋白纯品的制备方法如下：
采用肌肉注射17β -雌二醇(17β -estradiol，E2)的方式诱导大泷六线鱼产生卵黄原蛋白；注射一周后取血，4℃，5000 g离心10分钟，收集上清液；向上清中加入等体积的饱和硫酸铵溶液，冰浴条件下摇晃2小时后离心，向沉淀中加入pH 7.5的25mM Tris-HCl，使沉淀重新溶解；进一步的纯化采用1.5´ 20 cm 的Sephadex G-200层析柱，加入1 ml上述溶液，用pH 7.5的25mM Tris-HCl 洗层析柱，收集第一个洗脱峰，即为大泷六线鱼卵黄原蛋白。
所述的大泷六线鱼卵黄原蛋白多克隆抗体的制备方法如下：
取600 μg纯化的大泷六线鱼卵黄原蛋白，加入等体积的弗氏完全佐剂，充分乳化后对新西兰大白兔进行背部皮下多点注射，每点注射0.1 ml，两周后再次加强免疫，免疫剂量为600 μg/只，用弗氏不完全佐剂充分乳化后进行背部皮下注射。此后，每隔7天按以上方法进行加强免疫。于每5次注射5天后从心脏取血，6000 r/min离心20分钟，收集上清，获得了兔抗大泷六线鱼卵黄原蛋白多克隆抗血清。抗体的纯化分为以下几步，上样前向多克隆抗血浆中加入1/3左右的对照阴性样品(雄鱼匀浆液)，低温振荡2h，4℃过夜，次日离心取上清；向上清中加入等体积的0.02 mol/L磷酸缓冲液(pH 7.4)；在冰浴条件下加入硫酸铵，至50%饱和度，0℃震荡2h后，低温离心(8000 rpm, 15min)，弃上清，沉淀用10 ml 0.02 mol/L磷酸缓冲液溶解；以0.02 mol/L磷酸缓冲液透析24 h，其间换液4次；用0.45微米孔径的滤膜过滤后，上Hitrap Protein G柱，随后以0.02mol/L磷酸缓冲液洗脱10个柱体积，然后以0.1M甘氨酸(pH 2.7)洗脱抗体，即获得兔抗大泷六线鱼卵黄原蛋白多克隆抗体。
上述检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒在海洋内分泌扰乱化学物质调查与筛选中的应用。
本发明的有益效果：
本发明的试剂盒利用抗原抗体之间的特异性结合能力，可灵敏、准确、方便地检测大泷六线鱼血浆、体表粘液、肝脏组织及肝细胞培养液中的卵黄原蛋白，最低检出限可达50ng/ml，为内分泌扰乱化学物质筛选、检测和生态环境风险评价提供了一个有效的手段。
目前，已经纯化了多种鱼类的卵黄原蛋白，并且制备成抗体用于环境雌激素的检测。鱼类的卵黄原蛋白的纯化多采用两步层析法，该方法不仅操作复杂，费时费力，还容易造成卵黄原蛋白的降解。本发明首次建立了六线鱼卵黄原蛋白的纯化方案，采用硫酸铵沉淀预处理血浆，可明显提高卵黄原蛋白的浓度与纯度，然后再采用价格低廉的Sephadex G-200层析柱直接纯化获得了大泷六线鱼卵黄原蛋白，较其它鱼类卵黄原蛋白的纯化方案更加省时、操作简便，还极大提高了纯化蛋白的浓度，本发现的试剂盒利用抗原抗体之间的特异性结合能力，能够灵敏、方便地定性检测大泷六线鱼在不同污染物暴露下体内卵黄原蛋白的生成水平，为评价我国近岸海域的环境雌激素效应提供了重要的手段。
附图说明
图1为本发明的大泷六线鱼卵黄原蛋白的纯化图谱；
图2为本发明的大泷六线鱼卵黄原蛋白的鉴定结果；(泳道1为糖蛋白染色结果；泳道2为磷蛋白染色结果；泳道3为脂蛋白染色结果)
图3为本发明大泷六线鱼卵黄原蛋白多克隆抗体对雄性、雌性大泷六线鱼血浆与卵黄原蛋白纯品的检测结果(泳道1为雄性大泷六线鱼血浆；泳道2为雌性大泷六线鱼血浆；泳道3为纯化的卵黄原蛋白)；
具体实施方式：
实施例1
一种检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒，包括一个盒体，该盒体内装有：1)PVDF膜2张；2)辣根过氧化物酶标记的羊抗兔二抗1支；3)封闭液、洗涤液、显色液各1支，所述的洗涤液为TBST；封闭液为含5%脱脂奶粉的TBST；显色液为含0.06%(m/V) 3’-二氨基联苯胺的10mM Tris-HCl；其特征在于该盒体还装有：4) 大泷六线鱼卵黄原蛋白纯品1支；5) 大泷六线鱼卵黄原蛋白多克隆抗体1支。
上述试剂盒的制备方法包括以下两部分：
(1)大泷六线鱼卵黄原蛋白纯品的制备方法
采用肌肉注射17β -雌二醇(17β -estradiol，E2)的方式诱导大泷六线鱼产生卵黄原蛋白。注射一周后取血，4℃，5000 g离心10分钟，收集上清液；向上清中加入等体积的饱和硫酸铵溶液，冰浴条件下摇晃2小时后5000 g离心10分钟，向沉淀中加入25mM Tris-HCl (pH 7.5)，使沉淀重新溶解。进一步的纯化采用Sephadex G-200层析柱(1.5´ 20 cm)，加入1 ml上述溶液，用25mM Tris-HCl (pH 7.5)以流速0.3 ml/min冲洗层析柱，收集第一个洗脱峰，即为大泷六线鱼卵黄原蛋白。
(2) 大泷六线鱼卵黄原蛋白多克隆抗体的制备
取600 μg纯化的大泷六线鱼卵黄原蛋白，加入等体积的弗氏完全佐剂，充分乳化后对新西兰大白兔进行背部皮下多点注射，每点注射0.1 ml，两周后再次加强免疫，免疫剂量为600 μg/只，用弗氏不完全佐剂充分乳化后进行背部皮下注射。此后，每隔7天按以上方法进行加强免疫。于每5次注射5天后从心脏取血，6000 r/min离心20分钟，收集上清，获得了兔抗大泷六线鱼卵黄原蛋白多克隆抗血清。抗体的纯化分为以下几步，上样前向多克隆抗血浆中加入1/3左右的对照阴性样品(雄鱼匀浆液)，低温振荡2h，4℃过夜，次日离心取上清；向上清中加入等体积的0.02 mol/L磷酸缓冲液(pH 7.4)；在冰浴条件下加入硫酸铵，至50%饱和度，0℃震荡2h后，低温离心(8000 rpm, 15min)，弃上清，沉淀用10 ml 0.02 mol/L磷酸缓冲液溶解；以0.02 mol/L磷酸缓冲液透析24 h，其间换液4次；用0.45微米孔径的滤膜过滤后，上Hitrap Protein G柱，随后以0.02mol/L磷酸缓冲液洗脱10个柱体积，然后以0.1M甘氨酸(pH 2.7)洗脱抗体，即获得兔抗大泷六线鱼卵黄原蛋白多克隆抗体。
经过以上操作，得到的检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒具体组成如下：
1)PVDF膜2张；
2)大泷六线鱼卵黄原蛋白纯品1支，使用前用PBS稀释至1 μg/ml；
3)兔抗大泷六线鱼卵黄原蛋白多克隆抗体1支，使用前用封闭液按1:1000的体积比稀释；
4)辣根过氧化物酶标记的羊抗兔二抗1支，使用前用封闭液按1:500的体积比稀释；
5)封闭液、洗涤液、显色液各1支，所述的洗涤液为TBST(100mM Tris-HCl、150mMNaCl、0.05% Tween-20，pH7.5)；封闭液为含5%脱脂奶粉的TBST；显色液为含0.06%(m/V)3’-二氨基联苯胺(DAB)的10mM Tris-HCl，使用前向显色液中加入0.05% (v/v)双氧水。
本发明的试剂盒可用于大泷六线鱼卵黄原蛋白的定性检测。
实施例2
利用本发明的试剂盒定性检测大泷六线鱼卵黄原蛋白的方法，具体包括以下步骤：
1) 将试剂盒中的大泷六线鱼卵黄原蛋白纯品和待测的样品稀释后进行变性聚丙烯酰胺凝胶电泳，所述的待测的样品包括大泷六线鱼血浆、体表粘液、肝脏组织及肝细胞培养液；
2)把电泳凝胶上的蛋白转印到PVDF膜；
3)用封闭液封闭PVDF膜的非特异性结合位点，4℃孵育过夜，弃去封闭液；
4)加入用封闭液稀释的兔抗大泷六线鱼卵黄原蛋白多克隆抗体(1:1000)，室温下震荡孵育，弃去溶液，用洗涤液洗涤PVDF膜5次；
5)加入用封闭液稀释的辣根过氧化物酶标记的羊抗兔二抗(1:2000)，室温下震荡孵育，弃去溶液，用洗涤液洗涤PVDF膜5次；
6)加入显色液，待蛋白质条带清晰后，弃去显色液，用蒸馏水终止显色反应，PVDF膜拍照后于避光处保存；
7)在雄性大泷六线鱼样品中发现有显色条带，表明该鱼体内产生了卵黄原蛋白，其生活的水环境受到了环境雌激素类物质的污染。
Claims (1)
1.一种检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒，包括一个盒体，该盒体内装有：1)PVDF膜2张；2)辣根过氧化物酶标记的羊抗兔二抗1支；3)封闭液、洗涤液、显色液各1支，所述的洗涤液为TBST；封闭液为含5%脱脂奶粉的TBST；显色液为含0.06%(m/V) 3’-二氨基联苯胺的10mM Tris-HCl；其特征在于该盒体还装有：4) 大泷六线鱼卵黄原蛋白纯品1支，5) 兔抗大泷六线鱼卵黄原蛋白多克隆抗体1支；
制备所述的检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒，包括下列步骤：
1)制备大泷六线鱼卵黄原蛋白纯品；
2)利用步骤1）得到的大泷六线鱼卵黄原蛋白纯品制备兔抗大泷六线鱼卵黄原蛋白多克隆抗体；
3)将步骤1)得到的大泷六线鱼卵黄原蛋白纯品1支、步骤2)得到的兔抗大泷六线鱼卵黄原蛋白多克隆抗体1支、PVDF膜2张，以及封闭液、洗涤液、显色液和辣根过氧化物酶标记的羊抗兔二抗1支共同装入盒体，得到检测大泷六线鱼卵黄原蛋白的免疫印迹试剂盒；
所述的大泷六线鱼卵黄原蛋白纯品的制备方法如下：
采用肌肉注射17β -雌二醇(17β -estradiol，E2)的方式诱导大泷六线鱼产生卵黄原蛋白；注射一周后取血，4℃，5000 g离心10分钟，收集上清液；向上清中加入等体积的饱和硫酸铵溶液，冰浴条件下摇晃2小时后离心，向沉淀中加入pH 7.5的25mM Tris-HCl，使沉淀重新溶解；进一步的纯化采用1.5´ 20 cm 的Sephadex G-200层析柱，加入1 ml上述溶液，用pH 7.5的25mM Tris-HCl 洗层析柱，收集第一个洗脱峰，即为大泷六线鱼卵黄原蛋白；
所述的兔抗大泷六线鱼卵黄原蛋白多克隆抗体的制备方法如下：
取600 μg纯化的大泷六线鱼卵黄原蛋白，加入等体积的弗氏完全佐剂，对大白兔进行背部皮下多点注射，两周后再次加强免疫，免疫剂量为600μg/只，用弗氏不完全佐剂充分乳化后进行背部皮下注射；此后，每隔10天按以上方法进行加强免疫；于每5次注射5天后从心脏取血，6000 g离心20分钟，收集上清，获得了兔抗大泷六线鱼卵黄原蛋白多克隆抗血清；向抗血清中加入等体积的0.02 mol/L pH 7.4的磷酸缓冲液；在冰浴条件下加入硫酸铵，至50%饱和度，0℃震荡2h后，8000 g低温离心 15min，弃上清，沉淀用10ml 0.02mol/L磷酸缓冲液溶解；以0.02mol/L磷酸缓冲液透析24h，其间换液4次；用0.45微米孔径的滤膜过滤后，上Hitrap Protein G柱，随后以0.02 mol/L磷酸缓冲液洗脱10个柱体积，然后以0.1M、pH2.7的甘氨酸洗脱抗体，即获得兔抗大泷六线鱼卵黄原蛋白多克隆抗体。
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