CN112881688A - 一种快速检测鹅星状病毒的免疫荧光方法 - Google Patents
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Abstract
本发明涉及一种快速检测鹅星状病毒的免疫荧光方法，利用鸡肝细胞对鹅星状病毒感染的组织样品或者已经分离到的病毒进行培养，培养基为含1μg/mL TPCK‑胰蛋白酶的DMEM‑F12培养基，用针对鹅星状病毒P2蛋白的6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测；或者直接取新鲜的组织进行冰冻切片，用针对鹅星状病毒P2蛋白的6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测。所述一抗是杂交瘤细胞株6C6分泌的抗星状病毒P2蛋白单克隆抗体6C6制备而成。通过本发明，本发明的快速检测鹅星状病毒的免疫荧光技术可以特异性的检测鹅星状病毒，且阴性背景低，效果显著。
Description
技术领域
本发明涉及一种快速检测鹅星状病毒的免疫荧光方法，将建立检测鹅星状病毒的间接免疫荧光技术，实现临床上快速检测鹅星状病毒。这一检测可以被应用于鹅星状病毒流行病学调查，具有良好的应用价值。
背景技术
鹅星状病毒(Goose astrovirus, GAstV)是一种能引起雏鹅痛风的无囊膜单股正链RNA病毒。自2015年始,我国不同地区雏鹅陆续发生一种以内脏尿酸盐沉积为主要症状,并伴有关节尿酸盐沉积的传染性疾病,该病主要发生于3周以内的雏鹅，死亡率在20%-70%之间。该病的发生与发展已对我国养鹅业造成了极大危害,并已成为危害养鹅业健康发展的一种重要传染病，严重制约了国内家禽业的健康发展。目前对于检测鹅星状病毒仅可以通过PCR鉴定，尚无其他快速检测鹅星状病毒的方法。本发明运用针对鹅星状病毒P2蛋白的单抗6C6通过间接免疫荧光的方法来检测鹅星状病毒。检测结果发现，6C6单抗可以特异性快速的检测鹅星状病毒，在鹅星状病毒流行病学调查中具有良好的应用价值。
发明内容
本发明的目的是在于提供了一种快速检测鹅星状病毒的免疫荧光方法，是利用针对鹅星状病毒P2蛋白的6C6单抗通过间接免疫荧光的方法快速有效检测鹅星状病毒。
本发明的目的是这样实现的，一种快速检测鹅星状病毒的免疫荧光方法，其特征在于，利用鸡肝细胞对鹅星状病毒感染的组织样品或者已经分离到的病毒进行培养，培养基为含1μg/mL TPCK-胰蛋白酶的DMEM-F12培养基，用针对鹅星状病毒P2蛋白的6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测；
或者直接取新鲜的组织进行冰冻切片，用针对鹅星状病毒P2蛋白的6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测。
所述一抗是杂交瘤细胞株6C6分泌的抗星状病毒P2蛋白单克隆抗体6C6制备而成。
鸡肝细胞培养鹅星状病毒时，鸡肝细胞采用含10%胎牛血清的DMEM-F12培养基进行培养，待细胞长满单层后，用PBS洗涤一遍后，以0.25%的胰酶进行消化、传代至所需的细胞培养板中；将鸡肝细胞种至96孔板内培养，在汇合度为约70%时，取100μl鹅星状病毒上清加入900μl含1μg/mL TPCK-胰蛋白酶的DMEM-F12培养基，混匀后每孔加入200微升进行培养；培养3天后，用固定液将96孔板固定干燥，以6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测；
或者直接取新鲜的组织进行冰冻切片，取新鲜的发病鹅的肾脏组织修至合适大小，用包埋剂将组织块包裹后用冰冻切片机进行切片，随后使用冷丙酮固定后室温风干30分钟至干燥，再以6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测。
本发明方法先进科学，通过本发明，本发明所述的一种快速检测鹅星状病毒的免疫荧光方法，利用鸡肝细胞对鹅星状病毒感染的组织样品或者已经分离到的病毒进行培养，培养基为含1μg/mL TPCK-胰蛋白酶的DMEM-F12培养基；或者直接取新鲜的组织进行冰冻切片。用针对鹅星状病毒P2蛋白的6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测；所述的一抗是杂交瘤细胞株6C6分泌的抗星状病毒P2蛋白单克隆抗体6C6制备而成。
技术方案为，1）鸡肝细胞培养鹅星状病毒；鸡肝细胞采用含10%胎牛血清的DMEM-F12培养基进行培养，待细胞长满单层后，用PBS洗涤一遍后，以0.25%的胰酶进行消化、传代至所需的细胞培养板中。将鸡肝细胞种至96孔板内培养，在汇合度为约70%时，取100μl鹅星状病毒上清加入900μl含1μg/mL TPCK-胰蛋白酶的DMEM-F12培养基，混匀后每孔加入200微升进行培养。培养3天后，用固定液将96孔板固定干燥，以6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测。2）新鲜组织进行冰冻切片；取新鲜的发病鹅的肾脏组织修至合适大小，用包埋剂将组织块包裹后用冰冻切片机进行切片，随后使用冷丙酮固定后室温风干30分钟至干燥，再以6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测。
在本发明实施例中，杂交瘤细胞株6C6是以P2蛋白作为免疫原免疫小鼠后通过杂交瘤技术获得的，本发明对所述获得方法没有特殊的限定。在本发明实施例中，单克隆抗体6C6是杂交瘤细胞株6C6制备小鼠腹水所得。
有益效果：本发明的快速检测鹅星状病毒的免疫荧光技术可以特异性的检测鹅星状病毒，且阴性背景低，效果显著。
附图说明
图1 为鹅星状病毒在鸡肝细胞上感染后用6C6单抗检测；
图2为正常鸡肝细胞培养后用6C6单抗检测；
图3为感染星状病毒的鹅肾组织冰冻切片后用6C6单抗检测；
图4为正常鹅的肾组织冰冻切片后用6C6单抗检测。
具体实施方式
为更好的理解本发明的内容，以下实施方式结合附图给出了快速检测鹅星状病毒的免疫荧光技术的示例。
1）鹅星状病毒感染鸡肝细胞后检测鹅星状病毒；
将鸡肝细胞用含10%胎牛血清的DMEM-F12培养基进行培养，待细胞长满单层后，用PBS洗涤一遍后，以0.25%的胰酶进行消化、传代至6孔或96孔细胞培养板中。将鸡肝细胞接种至6孔板内培养，在汇合度为约70%时，取100μl鹅星状病毒上清加入900μl含1μg/mLTPCK-胰蛋白酶的DMEM-F12培养基，混匀后每孔加入200微升进行培养，并同步设置不加病毒的对照细胞孔。培养3天后将细胞用100μl丙酮乙醇比例为3：2的固定液固定5分钟，干燥；将100μl 1：1000稀释的鹅星状病毒P2蛋白6C6单抗为一抗与固定好的细胞于37℃孵育45分钟，弃去所有液体，以200μl PBS洗3遍；加入100μl 1：150稀释的FITC标记的羊抗鼠IgG为二抗于37℃孵育45分钟，弃去所有液体，以200μl PBS洗3遍，加入50μl PBS，于倒置荧光显微镜下进行观察。结果可见，正常细胞内无特异性荧光，而病毒感染的鸡肝细胞在胞浆部位出现特异性荧光且背景干净。
2）发病鹅新鲜组织冰冻切片检测鹅星状病毒；取新鲜的发病鹅的肾脏组织修至合适大小，并同步设置正常鹅肾脏组织作为对照，用包埋剂将组织块包裹后用冰冻切片机进行切片，随后使用冷丙酮固定十分钟后室温风干30分钟至干燥，将1：1000稀释的6C6单抗为一抗与固定好的组织切片于37℃孵育45分钟，弃去所有液体，以适量的PBS洗3遍；加入1：150稀释的FITC标记的羊抗鼠IgG为二抗于37℃孵育45分钟，弃去所有液体，以PBS洗3遍，滴加适量的甘油后用指甲油进行封片，并于倒置荧光显微镜下进行观察。结果可见，正常的鹅肾脏组织切片无特异性荧光，而病毒感染的鹅肾脏组织切片在胞浆部位出现特异性荧光且背景干净。
Claims (3)
1. 一种快速检测鹅星状病毒的免疫荧光方法，其特征在于，利用鸡肝细胞对鹅星状病毒感染的组织样品或者已经分离到的病毒进行培养，培养基为含1μg/mL TPCK-胰蛋白酶的DMEM-F12培养基，用针对鹅星状病毒P2蛋白的6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测；
或者直接取新鲜的组织进行冰冻切片，用针对鹅星状病毒P2蛋白的6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测。
2.根据权利要求1所述的间接免疫荧光检测方法，其特征在于，所述一抗是杂交瘤细胞株6C6分泌的抗星状病毒P2蛋白单克隆抗体6C6制备而成。
3. 根据权利要求1所述的间接免疫荧光检测方法，其特征在于，鸡肝细胞培养鹅星状病毒时，鸡肝细胞采用含10%胎牛血清的DMEM-F12培养基进行培养，待细胞长满单层后，用PBS洗涤一遍后，以0.25%的胰酶进行消化、传代至所需的细胞培养板中；将鸡肝细胞种至96孔板内培养，在汇合度为约70%时，取100μl鹅星状病毒上清加入900μl含1μg/mL TPCK-胰蛋白酶的DMEM-F12培养基，混匀后每孔加入200微升进行培养；培养3天后，用固定液将96孔板固定干燥，以6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测；
或者直接取新鲜的组织进行冰冻切片，取新鲜的发病鹅的肾脏组织修至合适大小，用包埋剂将组织块包裹后用冰冻切片机进行切片，随后使用冷丙酮固定后室温风干30分钟至干燥，再以6C6单抗作为一抗，FITC标记的羊抗鼠IgG作为二抗进行IFA检测。
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