CN113527445A - 检测猫血清中新型冠状病毒SARS-CoV-2抗体的试纸条及制备方法 - Google Patents
检测猫血清中新型冠状病毒SARS-CoV-2抗体的试纸条及制备方法 Download PDFInfo
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Abstract
本发明公开了新型冠状病毒RBD抗原表位SEQ ID：01和SEQ ID：02，含有该抗原表位的重组蛋白可用于抗新型冠状病毒的IgG抗体检测的制备。本发明进一步公开了新型冠状病毒RBD抗原在制备检测猫血清中新型冠状病毒抗体的免疫层析产品尤其是胶体金免疫层析试纸条中的应用。本发明本与核酸检测技术相比，具有检测快速、简单操作、价格便宜、适应于广泛使用的优点。
Description
技术领域
本发明涉及免疫检测技术领域，尤其涉及一种检测猫血清中新冠病毒SARS-CoV-2抗体的胶体金试纸条及制备方法。
背景技术
新型冠状病毒肺炎(COVID-19)的病原体最近已经确认，是一种正链RNA病毒，基因组为单链RNA，含有Poly(A)，总长接近30kb。病毒表面有约20nm左右的棒状结构，呈皇冠状。新型冠状病毒(SARS-CoV-2)是目前第7种已知的可以感染人类的冠状病毒，其可以引起新型冠状病毒肺炎(COVID-19)。其余6种冠状病毒分别是HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)。人感染了SARS-CoV-2后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中，可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。目前已在全球造成了大量的感染病例以及严重的经济损失。由于还没有针对SARS-CoV-2的特效药物和疫苗产品。因此，检测SARS-CoV-2病例并将其隔离成为了阻断这场疫病大流行的唯一办法。
蝙蝠最先被认为是SARS-CoV-2的自然宿主，但蝙蝠并不是SARS-CoV-2的唯一自然宿主。华南农业大学的研究者率先在穿山甲上鉴定到了同源性极高的毒株。随后，比利时的研究人员和哈尔滨兽医研究所的研究人员也都发现猫可以被SARS-CoV-2感染并且传播该病毒。穿山甲作为国家二级保护动物，其受到国家严格的管控。尽管偶尔还有偷猎的情况存在，但是总体上穿山甲与人类直接接触的几率较小，因此未来成为COVID-19爆发的病毒源头的可能性也较小。然而猫作为人类饲养的主要伴侣动物受到民众的普遍喜爱，普通民众家中的饲养量极大。一旦发生SARS-CoV-2通过猫感染人的事件，极有可能成为下次人群COVID-19爆发的导火索。目前，由于民众害怕被猫感染SARS-CoV-2，全国各地已经出现多起虐杀猫的事件，使得因疫情恐慌的民众与爱猫的民众之间产生严重的对立情绪。因此，快速筛查出感染了SARS-CoV-2的猫，对疫情的全面防控和缓解民众对立情绪至关重要。
核酸检测是目前判定COVID-19的主要技术手段，但是核酸检测本身也存在着检测准确率低的问题。并且核酸检测需要专业技术人员操作、购买昂贵的设备、耗时长等问题，不适合普通民众快速筛查感染了SARS-CoV-2的猫。因此，现在急需一种快速、简单操作、价格便宜、广泛使用的猫用COVID-19检测手段。
发明内容
为了解决现有技术的问题，本发明提供了一种简便、快速的检测猫血清中SARS-CoV-2抗体(IgG)的检测胶体金试纸条的技术。
一方面，本发明公开了一种新型冠状病毒RBD抗原，其基因序列如SEQ ID NO：1：
另一方面，本发明公开了一种新型冠状病毒RBD抗原，其氨基酸序列如SEQ ID NO：2所示：
需要说明的是，上述新型冠状病毒RBD抗原可以为重组蛋白如重组新型冠状病毒SARS-CoV-2纤突蛋白rRBD。
在其他可实施的方式中，还可以使用其他表达系统制备重组rRBD蛋白。
又一方面，本发明公开了上述新型冠状病毒RBD抗原在制备检测猫血清中新型冠状病毒抗体的免疫层析产品中的应用。
作为本发明实施方式的进一步改进，所述免疫层析产品为胶体金免疫层析试纸条。
再一方面，本发明公开了上述检测新型冠状病毒抗体的胶体金免疫层析试纸条，以SEQ ID NO：1所示基因序列或SEQ ID NO：2所示氨基酸序列的蛋白为检测抗原。
作为本发明实施方式的进一步改进，所述检测新型冠状病毒抗体的胶体金免疫层析试纸条包括背衬以及粘贴在所述背衬上的样品垫、胶体金结合垫、硝酸纤维素膜和吸收垫，所述硝酸纤维素膜上划有包被SEQ ID NO：1所示基因序列或SEQ ID NO：2所示氨基酸序列的蛋白的检测线。
作为本发明实施方式的进一步改进，所述硝酸纤维素膜置于所述背衬的上面，所述样品垫平贴于所述硝酸纤维素膜的一侧，所述吸收垫平贴于所述硝酸纤维素膜的另一侧；
所述胶体金结合垫平贴于所述样品垫与硝酸纤维素膜之间，所述胶体金结合垫的一端压于所述样品垫之下，另一端覆盖于所述硝酸纤维素膜之上，形成试纸条真空封装。
作为本发明实施方式的进一步改进，所述硝酸纤维素膜上划有质控线(C线)，所述质控线为包被羊抗猫lgG抗体的质控线，或包被兔抗猫lgG抗体的质控线，或包被其他来源的抗猫lgG抗体的质控线。
作为本发明实施方式的进一步改进，所述胶体金结合垫喷涂有胶体金颗粒标记的金黄色葡萄球菌蛋白A、链球菌蛋白A或链球菌蛋白G的复合物。
作为本发明实施方式的进一步改进，所述背衬为聚乙烯背衬。
在其他可实施的方式中，也存在使用链球菌蛋白A或者链球菌蛋白G作为T线。
再一方面，本发明还公开了一种体外检测样品中是否存在新型冠状病毒抗体的方法，包括步骤：
(a)将样品与上述的新型冠状病毒RBD抗原接触；
(b)检测是否形成抗原-抗体复合物：若形成复合物就表示样品中存在新型冠状病毒抗体。
其中，步骤(b)的检测方法是使用上述的检测新型冠状病毒抗体的胶体金免疫层析试纸条。
本发明进一步公开了检测新型冠状病毒抗体的胶体金免疫层析试纸条在猫血清中新型冠状病毒早期诊断中的应用。
本发明具有如下有益效果：
1、本发明通过对新冠病毒潜在抗原蛋白的序列分析，找到了具有很强抗原性的氨基酸序列，通过合成多肽合成这些潜在抗原靶位，再由这些多肽免疫获得的抗体，研制出一种通过检测猫血清新冠病毒，进行猫临床早期新冠病毒诊断的试剂条；
2、本发明可显著提高诊断效率，并且简便、准确率高，克服了现有技术中假阳性率高的问题；
3、因为本发明的新冠病毒特异性多肽、多肽偶联物与抗新冠病毒抗体的亲和力高，本发明抗体与新冠病毒的亲和力高，因此，本发明的检测方法灵敏、简便、准确率高；
4、与现有技术中的核酸检测技术相比，具有检测快速、简单操作、价格便宜、适应于广泛使用的优点。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的检测新型冠状病毒抗体的胶体金免疫层析试纸条的结构示例图；
图2是本发明实施例提供的检测新型冠状病毒抗体的胶体金免疫层析试纸条的检测结果对比示意图；
图中示例表示为：
1-背衬；2-样品垫；3-胶体金结合垫；4-硝酸纤维素膜；5-吸收垫。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
本发明实施例公开了一种新型冠状病毒RBD抗原，其基因序列如SEQ ID NO：1：
本发明实施例还公开了一种新型冠状病毒RBD抗原，其氨基酸序列如SEQ ID NO：2所示：
本发明公开了新型冠状病毒RBD抗原表位SEQ ID：01和SEQ ID：02，含有该抗原表位的重组蛋白可用于抗新型冠状病毒的IgG抗体检测的制备。
具体例1重组新型冠状病毒SARS-CoV-2纤突蛋白rRBD的制备。
1)重组纤突蛋白rRBD表达载体的构建
重组蛋白RBD的基因序列如SEQ ID NO.1所示。其对应的氨基酸序列如SEQ IDNO.2所示。按照SEQ ID NO.1中的序列，直接人工合成并克隆到pET-28a的BamHI和EcoRI酶切位点之间，得到的pET-28a-RBD基因重组表达载体。
2)重组纤突蛋白rRBD的诱导表达
将上述得到的重组表达载体转化大肠杆菌工程菌BL21。所得到的菌株命名为RBD。挑取RBD单菌落接种于含有卡那霉素的LB液体培养基中，37℃过夜振摇培养。取过夜培养的菌液，按照1∶1000的比列接种于1L含有卡那霉素(25μg/mL)的LB液体培养基中，37℃200rpm振荡培养3h左右，使OD600达到0.6-0.8。向上述菌液中加入终浓度为0.1mmol/L的IPTG进行重组蛋白的诱导表达，并在加入IPTG诱导3-4h。4℃12000rpm离心30min收集诱导表达的细菌，100ml PBS(pH7.2)重悬。
3)重组纤突蛋白rRBD的纯化
将上述重悬的菌液进行超声波破碎。功率选用200W，在冰盒上操作，超声裂解10s，停顿10s，直至菌液变得清澈。将破碎完成的菌液离心，4℃、12000rpm、15min，收集上清。Ni+填料装柱后，用结合缓冲液(50mM Tris，0.2M氯化钠，pH7.4，20mM咪唑)平衡纯化柱子。将收集的上清缓慢的通过处理后的含Ni+填料的柱子。结合缓冲液洗去杂蛋白。最后洗脱液(50mM Tris，0.2M氯化钠，pH7.4，400mM咪唑)洗脱目的蛋白。即初步纯化目的蛋白rRBD。
4)重组纤突蛋白rRBD的透析
取上述初步纯化的rRBD蛋白，使用购自北京索莱宝科技有限公司的普通干型透析袋进行透析；透析袋使用前用无菌水煮沸10min，冷却至常温；将需透析的蛋白溶液装入透析袋，用透析夹夹紧透析袋；将透析袋完全浸泡在100倍体积的透析液(20mM，Tris-HCl，0.5mMNaCl，pH7.0)中4℃透析24h，每3h更换一次透析液；透析所得蛋白溶液即为可用于胶体金标记的重组新型冠状病毒SARS-CoV-2纤突蛋白rRBD，-40℃保存；
在其他可实施的方式中，还可以使用其他表达系统制备重组rRBD蛋白。
实施例2
本发明公开了上述新型冠状病毒RBD抗原在制备检测猫血清中新型冠状病毒抗体的免疫层析产品中的应用。
具体地，免疫层析产品为胶体金免疫层析试纸条。
本发明所公开的上述检测新型冠状病毒抗体的胶体金免疫层析试纸条，以SEQ IDNO：1所示基因序列或SEQ ID NO：2所示氨基酸序列的蛋白为检测抗原。
在本发明实施例中，检测新型冠状病毒抗体的胶体金免疫层析试纸条的组成结构如图1所示，包括背衬1以及粘贴在背衬1上的样品垫2、胶体金结合垫3、硝酸纤维素膜4和吸收垫5，硝酸纤维素膜4上划有包被SEQ ID NO：1所示基因序列或SEQ ID NO：2所示氨基酸序列的蛋白的检测线(T线)。
具体的结构为：硝酸纤维素膜4置于背衬1的上面，样品垫2平贴于硝酸纤维素膜4的一侧，吸收垫5平贴于硝酸纤维素膜4的另一侧；胶体金结合垫3平贴于样品垫2与硝酸纤维素膜4之间，胶体金结合垫3的一端压于样品垫2之下，另一端覆盖于硝酸纤维素膜4之上，形成试纸条真空封装。
作为本发明实施方式的进一步改进，硝酸纤维素膜上划有质控线(C线)，为包被羊抗猫lgG抗体的质控线，在其他可选的实施例中，质控线也可以为包被兔抗猫lgG抗体的质控线，或包被其他来源的抗猫lgG抗体的质控线。
在本发明实施例中，胶体金结合垫3喷涂有胶体金颗粒标记的金黄色葡萄球菌蛋白A的复合物，也可以选择链球菌蛋白A或链球菌蛋白G的复合物。
在本发明实施例中，背衬1为聚乙烯背衬。
具体例2：制备检测猫血清中新型冠状病毒抗体的胶体金免疫层析试纸条
1)制备标记有金颗粒的SPA的金标结合物
取40nm胶体金颗粒2ml用0.1mol/L碳酸钾调节溶液调节pH至6.8，加入10ug金黄色葡萄球菌蛋白A(SPA)，快速混匀并在3D旋转仪上室温混匀30min，然后加入终浓度1％BSA并在3D旋转混合仪上混匀30min，将金标液于4℃12000rpm离心10min，小心弃去上清液，沉淀物用0.01M PBS缓冲液洗涤2次，再离心所得沉淀即为纯化的SPA金标复合物，制备的胶体金标记SPA用0.01MPBS重悬，4℃保存备用。
2)硝酸纤维素膜检测线和质控线喷洒及胶体金结合垫制备
将重组表达的新型冠状病毒SARS-CoV-2纤突蛋白rRBD用0.01M PBS稀释成3mg/ml，以1ul/cm均匀划在硝酸纤维素膜的T线位置。将羊抗猫IgG抗体用0.01M PBS稀释成2mg/ml，以1ul/cm均匀划在硝酸纤维素膜的C线位置，然后放于37℃过夜烘干备用。将SPA-胶体金复合物以8uL/cm均匀喷涂在标记物垫上，37℃过夜烘干备用。
3)试纸条的组装
将样品垫、喷涂有SPA金标结合物的标记垫、喷涂有新型冠状病毒SARS-CoV-2纤突蛋白rRBD作为检测线和喷涂有羊抗猫IgG抗体作为质控线的硝酸纤维素膜、吸收垫按顺序依次粘附在聚乙烯背衬上。
在其他可实施的方式中，也使用链球菌蛋白A或者链球菌蛋白G作为T线。
实施例3
本发明在一种体外检测样品中是否存在新型冠状病毒抗体的方法，具体包括以下步骤：
(a)将样品与上述的新型冠状病毒RBD抗原接触；
(b)检测是否形成抗原-抗体复合物：若形成复合物就表示样品中存在新型冠状病毒抗体。
其中，步骤(b)的检测方法是使用上述的检测新型冠状病毒抗体的胶体金免疫层析试纸条。
本发明进一步公开了检测新型冠状病毒抗体的胶体金免疫层析试纸条在猫血清中新型冠状病毒早期诊断中的应用。
具体例3
S1、应用上述胶体金免疫层析试纸条检测猫血清中新型冠状病毒SARS-CoV-2抗体；
取100ul待测样品液滴在本发明试纸样品垫上，10min后观察结果，同时分别取已知阳性血清样品和已知阴性血清分别作为阳性和阴性对照。结果判定：在样品垫上加入检测样品时，若检测线和质控线均显红色，为新型冠状病毒抗体阳性；若检测线不显色而质控线显红色，为新型冠状病毒抗体阴性；若质控线不显色，则试纸无效。
S2、进行胶体金免疫层析试纸条特异性试验；
采集免疫过重组rRBD蛋白的猫血清，并将此血清作为阳性血清。同时采集未免疫过重组rRBD蛋白的健康猫血清和感染了猫传染性腹膜炎(另一种猫的冠状病毒引起的猫传染病)的猫的血清，并将这些血清作为阴性血清。将这些血清进行交叉实验，结果见图2，其中血清1为rRBD免疫猫血清；血清2为阴性猫血清；血清3为猫传染性腹膜炎阳性猫血清。结果显示，只有免疫过重组rRBD蛋白的猫血清呈阳性反应，而与其他血清反应时，检测线均不显色，呈阴性反应，表明所述试纸条具有很高的特异性。
S3、进行胶体金免疫层析试纸条重复性和准确性试验；
将不同批次的所述免疫层析胶体金试纸分别检测5份来源不同的免疫血清和5份来源不同的阴性血清，每份血清样品平行做5个重复，结果显示批内重复性和批间重复性试验的检测结果均一致且与预设试验结果保持一致，表明所述胶体金免疫层析试纸条条具有良好的重复性和准确性。
S4、进行胶体金免疫层析试纸条的符合性试验；
取同一批号胶体金免疫层析试纸条对60份已知背景的血清样本分别进行检测，观察结果。
本发明的试纸条阳性样品18份，阴性样品42份，阳性符合率100％(18/18)，阴性符合率100％，总符合率100％，表明所述胶体金免疫层析试纸条用于检测新型冠状病毒S蛋白时具有良好的准确性。
在本发明实施例中提及的英文术语的翻译为：μg/mL：微克每毫升；rpm：每分钟多少转；h：小时；OD600：吸光度600nm；mmol/L：毫摩尔每升；IPTG：异丙基-β-D-硫代半乳糖苷；min：分钟；ml：毫升；PBS：磷酸盐缓冲液；pH：酸碱度；W：瓦特；s：秒；mM：毫摩尔；Tris：三羟甲基氨基甲烷；M：摩尔；nm：纳摩尔；mol/L：摩尔每升；BSA：牛血清白蛋白；IgG：免疫球蛋白G；mg/ml：毫克每毫升；ul/cm：微升每厘米；ELISA：酶联免疫吸附测定。
本发明具有如下有益效果：
1、本发明通过对新冠病毒潜在抗原蛋白的序列分析，找到了具有很强抗原性的氨基酸序列，通过合成多肽合成这些潜在抗原靶位，再由这些多肽免疫获得的抗体，研制出一种通过检测猫血清新冠病毒，进行猫临床早期新冠病毒诊断的试剂条；
2、本发明可显著提高诊断效率，并且简便、准确率高，克服了现有技术中假阳性率高的问题；
3、因为本发明的新冠病毒特异性多肽、多肽偶联物与抗新冠病毒抗体的亲和力高，本发明抗体与新冠病毒的亲和力高，因此，本发明的检测方法灵敏、简便、准确率高；
4、与现有技术中的核酸检测技术相比，具有检测快速、简单操作、价格便宜、适应于广泛使用的优点。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<120> 检测猫血清中新型冠状病毒SARS-CoV-2抗体的试纸条及制备方法
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Thr Ala Ala Thr Thr Ala Cys Cys Thr Gly Thr Ala Thr Ala Gly Ala
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Claims (10)
1.一种新型冠状病毒RBD抗原，其特征在于，其基因序列如SEQID NO：1：
2.一种新型冠状病毒RBD抗原，其特征在于，其氨基酸序列如SEQ ID NO：2所示：
3.根据权利要求1或2中所述新型冠状病毒RBD抗原在制备检测猫血清中新型冠状病毒抗体的免疫层析产品中的应用。
4.根据权利要求3所述新型冠状病毒RBD抗原在制备检测猫血清中新型冠状病毒抗体的免疫层析产品中的应用，其特征在于，所述免疫层析产品为胶体金免疫层析试纸条。
5.一种检测新型冠状病毒抗体的胶体金免疫层析试纸条，其特征在于，以权利要求1所示基因序列或权利要求2所示氨基酸序列的蛋白为检测抗原。
6.根据权利要求5所述检测新型冠状病毒抗体的胶体金免疫层析试纸条，其特征在于，包括背衬以及粘贴在所述背衬上的样品垫、胶体金结合垫、硝酸纤维素膜和吸收垫，所述硝酸纤维素膜上划有包被权利要求1所示基因序列或权利要求2所示氨基酸序列的蛋白的检测线。
7.根据权利要求6所述检测新型冠状病毒抗体的胶体金免疫层析试纸条，其特征在于，所述硝酸纤维素膜置于所述背衬的上面，所述样品垫平贴于所述硝酸纤维素膜的一侧，所述吸收垫平贴于所述硝酸纤维素膜的另一侧；
所述胶体金结合垫平贴于所述样品垫与硝酸纤维素膜之间，所述胶体金结合垫的一端压于所述样品垫之下，另一端覆盖于所述硝酸纤维素膜之上，形成试纸条真空封装。
8.根据权利要求6所述检测新型冠状病毒抗体的胶体金免疫层析试纸条，其特征在于，所述硝酸纤维素膜上划有质控线，所述质控线为包被羊抗猫lgG抗体的质控线，或包被兔抗猫lgG抗体的质控线，或包被其他来源的抗猫lgG抗体的质控线。
9.根据权利要求6所述检测新型冠状病毒抗体的胶体金免疫层析试纸条，其特征在于，所述胶体金结合垫喷涂有胶体金颗粒标记的金黄色葡萄球菌蛋白A、链球菌蛋白A或链球菌蛋白G的复合物。
10.根据权利要求6所述检测新型冠状病毒抗体的胶体金免疫层析试纸条，其特征在于，所述背衬为聚乙烯背衬。
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Application publication date: 20211022 |