CN113176411B - 利用唾液检测新型冠状病毒感染的生物标志物及其应用 - Google Patents
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Abstract
本发明涉及利用唾液检测新型冠状病毒感染的生物标志物及其应用,本发明公开了ALB、LCP1、HP、CFB、LRG1和IL1B在新冠病毒感染患者的唾液中高表达,可作为利用唾液检测新冠重症患者的重要生物标志物;SAA1、SERPING1、SERPINA3、C9、C6、LUM、TNF、CRP、SAA2、LBP和CLEC3B也可以在唾液中检测到,可作为利用唾液检测新冠重症患者的参考生物标志物。本发明首次公开以上物质作为生物标志物在制备新型冠状病毒唾液检测试剂盒中的作用,能够快速检测新型冠状病毒感染,样本易获得,检测效率高,速度快,降低操作人员的感染风险,具有很高的敏感性和特异性。
Description
技术领域
本发明涉及分子生物学技术领域,特别涉及利用唾液检测新型冠状病毒感染的生物标志物及其应用。
背景技术
新型冠状病毒感染是由严重急性呼吸综合征冠状病毒2(Severe AcuteRespiratory Syndrome Coronavirus 2,SARS-CoV-2)引起,然而目前,新型冠状病毒感染主要通过临床症状和影像学特征来进行诊断,在微观层面疾病进展机制的改变知之甚少。
核酸检测作为新型冠状病毒检测的金标准,通过获取鼻咽拭子样本、痰液或肺泡灌洗液等样本,经过处理后进行PCR检测样本中是否含有新型冠状病毒的核酸,极易造成暴露感染的风险,同时极易引起不适,并给医护人员带来感染的风险。整个测试需要在PCR实验室完成,耗时2-3小时,且由于样本收集的时间过早或过晚,保存、运输、处理等不当,容易引起结果误判,加之需要设备仪器,反而不利于基层或大规模人群的初筛,极大制约了新型冠状病毒快速检测服务的供给能力。
2020年5月份,西湖大学郭天南团队、温州医科大学台州医院陈海啸团队等合作从微观层面上发现了新冠重症患者重要生物标志物。研究人员对新冠病毒感染患者血液中的蛋白质和代谢物分子进行系统检测,发现重症患者的血清中93种特有的蛋白表达和204个特征性改变的代谢分子。其中50种蛋白,与患者体内的巨噬细胞、补体系统、血小板脱颗粒有关。研究团队在质谱分析数据的基础上,使用机器学习方法进一步筛选出重症患者特征性的22个蛋白质和7个代谢物。若患者血清样本成分符合这一组合,则很可能是重症患者或发展为重症病例。该研究不仅为新冠重症患者血清中发生的分子病理改变提供了全景式的描述,也为预测轻症患者向重症发展提供指导。然而,血清检测会对检测者造成多余的创伤,引起不必要的疼痛。
目前报道使用唾液检测新型冠状病毒感染的方法中,有检测唾液中冠状病毒S蛋白基因的方法,也有通过引入一种试剂,与唾液样本混合,再进行一个短时间加热过程,来释放病毒基因组的方法。这些方法虽然以唾液为样本,但都还是基于对新冠病毒本身的检测,即检测唾液中是否存在新冠病毒,虽然可以解决传统鼻咽拭子取样的缺点,但是依然后续要借助于核酸检测的PCR程序,需要等待很长的时间,不能够提升效率,满足大规模检测的需求。
有鉴于此,为解决目前新型冠状病毒检测中的问题,有必要开发一种可以快速利用唾液检测新型冠状病毒的检测方法,为新型冠状病毒检测提供感染风险低、安全无创、高性价比、快速简便的早期筛查和辅助诊断的手段。
发明内容
针对现有技术中的缺陷,本发明提出了利用唾液检测新型冠状病毒感染的生物标志物及其应用,基于唾液蛋白组和血清蛋白组的重叠性,公开了ALB、LCP1、HP、CFB、LRG1和IL1B在新冠病毒感染患者的唾液中高表达,可作为利用唾液检测新冠重症患者的重要生物标志物;SAA1、SERPING1、SERPINA3、C9、C6、LUM、TNF、CRP、SAA2、LBP和CLEC3B也可以在唾液中检测到,可作为利用唾液检测新冠重症患者的参考生物标志物。
本发明提供一种利用唾液检测新型冠状病毒感染的生物标志物,所述生物标志物包括以下物质中的任意一种或多种:利用唾液检测新冠重症患者的重要生物标志物和利用唾液检测新冠重症患者的参考生物标志物。
进一步的,所述利用唾液检测新冠重症患者的重要生物标志物包括:白蛋白、淋巴细胞胞浆蛋白1、触珠蛋白、补体因子B、富亮氨酸α-2-糖蛋白1、白细胞介素1β中的任意一种。
进一步的,所述利用唾液检测新冠重症患者的参考生物标志物包括:人血清淀粉样蛋白-1、血浆蛋白酶C1抑制剂、丝氨酸蛋白酶抑制剂、补体C6、补体C9、基膜聚糖蛋白、肿瘤坏死因子、C-反应蛋白、人血清淀粉样蛋白-2、内毒素结合蛋白、C-型凝集素域家族3成员B中的任意一种。
本发明还提供一种利用唾液检测新型冠状病毒感染的试剂盒,所述试剂盒为胶体金层析试纸条,所述胶体金层析试纸条包括样品垫、结合垫、硝酸纤维素膜和吸水垫,所述硝酸纤维素膜上有检测线和质控线,所述胶体金层析试纸条上含有胶体金颗粒,所述胶体金颗粒上修饰有白蛋白、淋巴细胞胞浆蛋白1、触珠蛋白、补体因子B、富亮氨酸α-2-糖蛋白1、白细胞介素1β的单/多克隆抗体;所述胶体金颗粒表面标记了辣根过氧化物酶。
本发明还提供一种利用唾液检测新型冠状病毒感染的试剂盒,所述试剂盒为胶体金层析试纸条,所述胶体金层析试纸条包括样品垫、结合垫、硝酸纤维素膜和吸水垫,所述硝酸纤维素膜上有检测线和质控线,所述胶体金层析试纸条上含有胶体金颗粒,所述胶体金颗粒上修饰有白蛋白、淋巴细胞胞浆蛋白1、触珠蛋白、补体因子B、富亮氨酸α-2-糖蛋白1、白细胞介素1β、人血清淀粉样蛋白-1、血浆蛋白酶C1抑制剂、丝氨酸蛋白酶抑制剂、补体C6、补体C9、基膜聚糖蛋白、肿瘤坏死因子、C-反应蛋白、人血清淀粉样蛋白-2、内毒素结合蛋白、C-型凝集素域家族3成员B的单/多克隆抗体;所述胶体金颗粒表面标记了辣根过氧化物酶。
本发明还提供使用所述试剂盒的检测方法,包括如下步骤:
(1)唾液样本采集;
(2)将待测唾液样本滴加到样品垫,唾液样本通过毛细作用沿着胶体金试纸条移动,移动到结合垫时被胶体金探针捕获;
(3)步骤(2)下继续层析,直到唾液样本通过硝酸纤维素膜上的检测线和质控线时被捕获,累积形成红色的条带,未反应的物质继续层析最终被吸水垫吸收;
(4)检测完成,观察检测线和质控线的条带颜色。
本发明还提供所述的生物标志物在制备新型冠状病毒唾液检测试剂盒中的应用。
本发明还提供所述的生物标志物在制备新型冠状病毒感染辅助唾液诊断产品中的应用。
综上,与现有技术相比,本发明达到了以下技术效果:
1. 本发明的检测方法成本低,性价比高。
2. 本发明的检测方法具有无创性,不会引起检测者的不适,不会对检测者造成多余的创伤、引起不必要的疼痛。
3. 本发明的检测方法不会对检测者造成暴露感染的风险,也不会对医护人员带来感染的风险。
4. 本发明的检测方法的样本为唾液,样本易于收集,尤其是在难以获取血液样本的情况下。
5. 本发明的检测方法能大大缩短检测时间,用胶体金层析试纸条肉眼就能观察到结果,而核酸检测通过PCR扩增,需要等待较长时间,所以本发明的方法有利于大规模人群的初筛。
6. 血浆和唾液的蛋白质组相似度高,许多在唾液中发现的蛋白质也存在于血液中。近40%的可在唾液中被找到的蛋白质被认为是癌症、心血管疾病和中风等疾病的候选标志物,所以可以使用唾液来发现新标志物和诊断早期症状。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,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相关的附图。
图1为本发明实施例1的唾液样本和血清样本中蛋白标记物的含量图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,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的实施例,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的前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
目前已从新型冠状病毒感染患者的分泌物中检测出病毒,如眼与结膜。2020年3月20日,首都医科大学王松灵院士和华中科技大学陈莉莉、王琳、金阳教授等合作,对COVID-19患者唾液中2019-nCoV的存在情况及口腔相关症状进行全面阐述。该研究证实唾液中可检测到2019-nCoV,提示唾液可能具有2019-nCoV传播的风险。唾液阳性检出率中危重患者比例较高,提示唾液病毒阳性患者可能疾病状况恶化。
已有报道证明血清蛋白质具有描述新型冠状病毒感染分子病理机制与预测轻症患者向重症发展的作用。人唾液是龈沟液与唾液腺分泌物的混合物,其组成与血清相似,本发明公开了唾液蛋白组与血清蛋白组具有很大的重叠性。除润滑口腔、溶解食物等作用外,唾液也构成人体免疫功能的第一道防线,其中多种物质的存在可以表明人体病理生理变化。作为最容易获得和最容易收集的体液之一,唾液收集可以通过无创、无痛和方便的程序来完成。因此,在难以采集全血标本的情况下,唾液具有作为新冠病毒感染危重患者病程发展评估的潜力。
以下是在文献中报道的血清中鉴定到的血清蛋白,这些蛋白大都与免疫调节和疾病相关,它们的表达变化可以在一定程度上反映人体的健康状况,其中有的在唾液中被发现,有的目前还未在唾液中发现:
(1)人血清淀粉样蛋白(Serum Amyloid A,SAA)
郭天南等人在文中列出了人血清淀粉样蛋白-2与人血清淀粉样蛋白-1(SerumAmyloid A-2,SAA2,Serum Amyloid A-1,SAA1)两种蛋白作为新型冠状病毒感染重症患者特征性的特征蛋白。α-淀粉酶(SAA)是唾液中的一种关键酶,它能水解淀粉的α-1,4-糖苷键,使唾液中的α-1,4-糖苷键水解为葡萄糖和麦芽糖,抑制细菌粘附到上皮表面,参与黏膜的免疫反应。唾液SAA在多篇文献中证明有多种作用。唾液SAA作为应激期肾上腺素能活动的标志物,对特别是心理压力的肾上腺素能激活变化敏感性提供了直接证据。Leicht等人表明副交感神经系统刺激SAA水平上升。唾液SAA也可用于口腔鳞状细胞癌的检测和系统性红斑狼疮疾病的预测。
(2)白蛋白(Albumin,ALB)
白蛋白是脊椎动物血浆中含量最丰富的蛋白质,合成于肝脏。白蛋白参与多种生理作用过程,渗透压维持,转运多种药物及内源性物质,调控组织液分布,参与血浆与组织液间的进行物质交换。唾液中的白蛋白被视为超滤液,可由血液扩散成为黏膜分泌物,且体内试验表明唾液白蛋白含量与血清中白蛋白水平存在显著相关性。炎症下刺激的ALB血清白蛋白含量升高,血液中高峰度的ALB进入唾液的可能性上升,故而唾液中的ALB水平可用于多种疾病的检测与诊断,如慢性牙周炎、口腔扁平苔癣、儿童反复呼吸道感染等。
(3)C-反应蛋白(C-reactive Protein,CRP)
C反应蛋白作为一种急性期反应物,是公认的炎症指标,被广泛应用于临床。CRP功能之为激活补体,调理作用,清除微生物与坏死细胞等。CRP可同时在血清和唾液中被检测出来。唾液中的CRP被用于多种疾病的检测,如牙周病,牛皮癣,桥本甲状腺炎慢性甲状腺炎。唾液CRP在亚急性甲状腺炎的检测中具有特异性,且相对于血清CRP具有更高的敏感性,可以做为病情严重程度、病情变化、发展及好转的观察指标。有研究表明,唾液与血清中的CRP成正相关。血清中的CRP被定义为冠心病的独立危险因素,唾液CRP检测可能是诊断和监测慢性炎症性疾病(包括冠心病和牙周病)的一种新方法。
(4)透明质酸结合蛋白2(Hyaluronan Binding Protein 2,HABP2)
透明质酸结合蛋白2,也称为FⅦ活化蛋白酶(FactorⅦ-activating Protease,FSAP)是在血浆与组织中发现的一种丝氨酸蛋白酶,通过因子VII和单链尿激酶原型纤溶酶原激活物(pro-uPA)的激活而参与纤维蛋白溶解,从而参与外源性凝血途径。HABP2与多种疾病的进展相关,如动脉粥样硬化,急性肺损伤,深静脉血栓和癌症。但是,由于HABP2参与过程复杂,其致病机制仍旧难以明确。目前未见HABP2在唾液中检测的相关研究。
(5)触珠蛋白(Haptoglobin,HP)
触珠蛋白,又称为结合珠蛋白,是一种α-2-糖蛋白,主要产生于肝脏。作为急性期蛋白,HP的合成受到IL-1和IL-6等炎性细胞因子影响,同时HP与游离血红蛋白结合预防毒性作用。在炎症刺激下,血液中HP可增加数倍,可作为炎症和肿瘤诊断的标志物。当血红蛋白从受损的红细胞释放到循环中(如挤压伤、溶血病)后,HP降低。故而HP可用于不同疾病的生物标志物,如肺癌、肺结核、牙周炎等。随着近年来学者们对唾液检测的研究兴趣加深,HP也作为唾液生物标志物用于不同疾病的检测,如口腔扁平苔癣。目前尚不清楚唾液中HP存在的确切机制,可能有:1.从血液中直接渗出;2.从肝脏外的其他组织生成,如肺、皮肤。
(6)蛋白Z依赖性蛋白酶抑制剂(Protein Z-Dependent Protease Inhibitor,ZPI,SERPINA10)
蛋白Z-ZPI抗凝系统是20世纪90年代被发现的新型抗凝系统。蛋白Z,一种维生素K依赖性血浆蛋白,具有促凝和抗凝双向调节作用。SERPINA10的主要抗凝活性为抑制FⅨa,FⅩa,FⅪa系统。蛋白Z可显着增强SERPINA10对FⅩa的抑制作用。研究表明,鼠类模型显示SERPINA10而非PZ是典型的急性期反应物。血浆蛋白Z在急性缺血性脑卒中患者急性期显著降低。目前未见SERPINA10在唾液中检测的相关研究。
(7)羧肽酶N催化链(Carboxypeptidase N Catalytic Chain,CarboxypeptidaseN1,CPN1)
羧肽酶N是一种血浆锌金属蛋白酶,由两个具有酶活性的小亚基(CPN1)和两个大亚基(CPN2)组成,可保护蛋白质免于降解。小亚基包含酶促活性位点,大亚基可保护蛋白质免受降解或从血液中过滤。血清中的CPN1被证明在大肠癌与肝癌术前术后有统计学差异。目前未见唾液中发现CPN1的文献。
(8)血浆蛋白酶C1抑制剂(Serpin Peptidase Inhibitor,Clade G Member 1,Plasma Protease C1 Inhibitor,SERPING1)
蛋白酶C1抑制剂,为一种丝氨酸蛋白酶抑制剂,在人体中承担多种生理作用。SERPING1可抑制糜蛋白酶和激肽释放酶;作为抗炎蛋白,激活补体系统和人体免疫反应;参与血液循环、血液凝固的内源性途径、纤维蛋白溶解、血小板的激活和脱颗粒。目前未见唾液中发现SERPING1的文献。
(9)丝氨酸蛋白酶抑制剂(Serpin Peptidase Inhibitor Clade A Member 3,α1-Antichymotrypsin,SERPINA3)
丝氨酸蛋白酶抑制剂,又称α1-抗糜蛋白酶,是一种丝氨酸蛋白酶抑制剂,参与多种人体生理过程。SERPINA3可进入细胞核与DNA结合,以抑制细胞分裂和增殖。多篇文献表明,SERPINA3与炎性反应、阿尔茨海默氏病、恶性黑色素瘤、胃癌以及结肠癌等疾病致病机制相关。近年来,唾液中的SERPINA3被用于多种疾病的检测,如慢性移植物抗宿主病、干燥综合征等。
(10)基膜聚糖蛋白(Lumican,LUM)
基膜聚糖蛋白是硫酸角质素蛋白聚糖,属于富含亮氨酸的小重复蛋白聚糖,与软骨形成有关。基膜聚糖蛋白分别在唾液腺与唾液中被研究者发现。Kusafuka等人的研究表明正常唾液腺中的Lumican,可能与基质的存留相关,而多形腺瘤表达的Lumican mRNA和蛋白质,可能在肿瘤形成的“间质”样区域中发挥重要作用。Ramachandran等人也在唾液中发现Lumican前体,但未在三大唾液腺的分泌物中发现。
(11)犬尿氨酸(Kynurenine,Kyn)
犬尿氨酸是色氨酸中间代谢产物之一,Kyn被代谢为犬尿喹啉酸或喹啉酸。Kyn和喹啉酸具有神经毒性作用;犬尿喹啉酸是避免兴奋性氨基酸损伤脑组织及神经系统保护因子,有抗惊厥、抗痉挛、保护神经系统等作用。Kyn和犬尿喹啉酸参与机体的多种生理过程,与多种疾病密切相关,如神经精神疾病、肾衰竭、白内障、血管硬化及慢性恶性疾病。同时,唾液Kyn也被用于多种疾病的检测,如糖尿病、口腔癌、牙周疾病。
(12)富亮氨酸α-2-糖蛋白1(Leucine-rich-alpha-2-glycoprotein1,LRG1)
LRG1是富亮氨酸重复(leucine-rich repeat,LRR)蛋白家族高度保守的成员之一。它参与蛋白之间的相互作用,在信号转导、细胞黏附以及发育过程中发挥重要作用。LRG1蛋白的异常表达与多种组织恶性肿瘤的发生、异常血管的生长密切有关,故而研究者开始将LRG1作为治疗靶点。Arantes等和Kawahara等的研究将唾液中的LRG1作为检测口腔鳞状细胞癌的生物标志物,同时研究也证明LRG1与口腔鳞状细胞癌的发生风险有关。
(13)中间α(球蛋白)抑制因子H3(Inter-alpha(globulin) inhibitor H3,ITIH3)
ITIH3是属于α-胰蛋白酶抑制剂家族的5条重链之一(ITIH1,ITIH2,ITIH3,ITIH4和ITIH5),包括一系列蛋白酶抑制剂,可在血液循环等各种器官的细胞外基质中找到。它们可与细胞外基质主要成分的透明质酸共价连接,成为稳定细胞外基质的重要因素,ITIH与肿瘤浸润和转移具有拮抗关系。有研究表明,ITIH3可作为胃癌的生物标志物,早期胃癌检测的灵敏度达90-96%,特异性达47-66%。目前未见唾液中检测ITIH3文献。
(14)C-型凝集素域家族3成员B(C-type Lectin Domain Family 3 Member B,CLEC3B)
C型凝集素域家族3成员B是跨膜Ca2+结合蛋白,位于细胞血浆,细胞外基质和外泌体中。目前在多种疾病中发现CLEC3B的血清中水平下调,可能被作为疾病的有效生物标志物或潜在的治疗靶点,如冠心病,帕金森氏病,卵巢癌。唾液中的CLEC3B也被发现有类似的作用,患有原发性和淋巴结转移的口腔鳞状细胞癌患者的血清和唾液中的CLEC3B均有下调。
(15)内毒素结合蛋白(Lipopolysaccharide Binding Protein,LBP)
内毒素结合蛋白,主要在肝脏合成和分泌,属于I型急性期反应蛋白,在正常人体血清中含量极微,而在急性炎症反应期明显升高。革兰氏阴性细菌的脂多糖被血液中LBP和LPS-LBP复合物识别并转运到CD14和Toll样受体激活树突状细胞、巨噬细胞、中性粒细胞。LBP分布于唾液中,以抵抗外来的病原体微生物,如牙龈卟啉单胞菌等。有研究也发现PLUNC蛋白(palate,lung,and nasal epithelium clones),一种新的分泌蛋白家族,LBP类似物,分布在口腔,鼻和呼吸道上皮细胞中,介导宿主激活防御与免疫功能。
(16)肽聚糖识别蛋白2(Peptidoglycan Recognition Protein 2,PGLYRP2)
肽聚糖识别蛋白(PGRP或PGLYRP),一种先天免疫蛋白,可识别细菌肽聚糖,起抗菌和抗炎作用。哺乳动物有四类肽聚糖识别蛋白,分别为PGRP-PGLYRP1,PGLYRP2,PGLYRP3和PGLYRP4。它们是在以下位置中表达的分泌蛋白:多形核白细胞颗粒(PGLYRP1),肝脏(PGLYRP2),体表、粘膜(眼睛,唾液腺,咽喉,舌,食道,胃和肠)和分泌物(唾液,汗液)(PGLYRP3和PGLYRP4)。PGRP主要与炎症性疾病相关,如牛皮癣、关节炎关节。目前未见PGLYRP2在人类唾液中检测的文献。
(17)甲状腺素(Thyroxine)
甲状腺素为甲状腺分泌的激素,可作用与人体广泛的细胞,促进人体生长发育,对人体代谢产生影响。激素通过超滤方式从血液向唾液转运,故而唾液激素能反映血清中激素水平。鉴于唾液检测的优势,学者们逐渐研发出多种测定唾液激素技术。70年代后期,血清激素方面测定技术发展,80年代Putz等人发展出唾液甲状腺素放射免疫试剂测定技术,改良后的此法此方法易于推广。Higashi等人建立并验证了液相色谱-质谱联用仪(LC/ESI-MS/MS)来测定唾液中甲状腺素(T4)的方法。
(18)补体因子B(Complement Factor B,CFB)和补体(Complement C6,C6,Complement C9,C9)
人类的补体系统,由补体成分、血浆补体调节蛋白、膜补体调节蛋白及补体受体等30多种糖蛋白组成的。多种病原微生物及抗原抗体复合物等可通过经典途径、替代途径或称旁路途径和凝集素激活途径等调节免疫应答。唾液中的补体有多种用途,可作为分泌蛋白,参与口腔局部免疫和炎症反应,故而在医学检测与诊断中也有多种作用。CFB与LCP1可共同作为唾液腺肿瘤标志物。复发性口疮患者的C3含量与正常人有明显差别。也有研究表明,人类唾液中发现C9与C6。
(19)淋巴细胞胞浆蛋白1(Lymphocyte Cytosolic Protein 1,LCP1)
淋巴细胞胞浆蛋白1,一种肌动蛋白结合蛋白,主要通过与肌动蛋白互相作用来调控细胞运动。高浓度的LCP1在龈沟液中的可促进了多形核中性粒细胞在炎症部位的聚集,参与免疫突触的形成、T细胞的激活等。有研究表明,2型糖尿病的牙周炎患者唾液中的LCP1水平上升。LCP1作为生物标志物,可用于预测易患牙周炎的2型糖尿病患者。有研究被在正常口腔黏膜组织、癌旁组织和口腔鳞状细胞癌组织中的检测到LCP1的表达,且具有统计学差异,LCP1被证明可用于唾液腺肿瘤标志物。
(20)破骨细胞活化因子(Osteoclast-activating Factor,OAF)
破骨细胞活化因子最早在1972年由Hörton等提出,为活化淋巴细胞所分泌的一种生物活性因子,具有诱导破骨细胞形成、刺激其活性、引起破骨吸收的功能。OAF并非单一的物质,而是一组多源性、异质性生物活性因子,包括IL-1和TNF等。在唾液中的IL-1和TNF等受多种因素的影响,被用于多种疾病的检测,如TNF唾液用于扁平苔癣恶化的监控,IL-1用于检测口腔肿瘤前病变、口腔鳞状细胞癌和心理变化的患者。
以上的蛋白标志物总结在下表中,按在唾液中的含量由高到低排序,其中ALB、LCP1、HP、CFB、LRG1和IL1B在唾液中高表达,SAA1、SERPING1、SERPINA3、C9、C6、LUM、TNF、CRP、SAA2、LBP和CLEC3B也可以在唾液中检测到,表中余下的蛋白目前未在唾液中鉴定到。
蛋白ID | 基因名称 | 蛋白名称 | 唾液中含量 | 组织特异性 | 功能 |
P02768 | ALB | Serum albumin | ++ | 血浆 | 血液胶体渗透压的调节 |
P13796 | LCP1 | Plastin-2 | ++ | 淋巴结、外周血、恶性肿瘤 | T细胞激活 |
P00738 | HP | Haptoglobin | ++ | 肝脏, 血浆 | 杀菌 |
P00751 | CFB | Complement factor B | ++ | 多种组织,比如肝脏肾上腺、肠、肺等 | 补体结合 |
P02750 | LRG1 | Leucine-rich alpha-2-glycoprotein | ++ | 血浆 | TGFβ受体结合 |
P01584 | IL1B | Interleukin-1 beta | ++ | 骨髓、淋巴组织、OSCC | 有效的促炎细胞因子 |
Thyroxine | ++ | 在大部分组织中广泛存在 | 代谢率、生长发育等 | ||
P0DJI8 | SAA1 | Serum amyloid A protein | + | 肝脏、血浆 | 主要急性期蛋白 |
P05155 | SERPING1 | Plasma protease C1 inhibitor | + | 多种组织,比如肝脏、肺、胃等 | 激活C1复合体 |
P01011 | SERPINA3 | Alpha-1-antichymotrypsin | + | 血浆 | 急性时相反应 |
P02748 | C9 | Complement component C9 | + | 血浆 | 补体激活 |
P13671 | C6 | Complement component C6 | + | 多种组织,比如肝脏、心脏、胰腺、脂肪等 | 补体激活 |
P51884 | LUM | Lumican | + | 角膜、外周血 | 细胞外基质结构成分 |
P01375 | TNF | Tumor necrosis factor | + | 骨髓、淋巴组织、OSCC | 结合TNFR1和TNFBR的细胞因子 |
P02741 | CRP | C-reactive protein | + | 血浆 | 宿主防御 |
P0DJI9 | SAA2 | Serum amyloid A protein | + | 肝脏、血浆 | 主要急性相反应物 |
P18428 | LBP | Lipopolysaccharide binding protein | + | 血清 | 参与先天免疫应答 |
P05452 | CLEC3B | Tetranectin | + | 血浆, OSCC | 参与胞外分泌分子的包装 |
Q14520 | HABP2 | Hyaluronan binding protein 2 | 无信息 | 各种组织均表达 | 细胞粘附、蛋白质水解 |
Q9UK55 | SERPINA10 | Protein Z-dependent protease inhibitor | 无信息 | 肝脏、血浆 | 血液凝固 |
P15169 | CPN1 | Carboxypeptidase N catalytic chain | 无信息 | 血浆 | 防止蛋白降解 |
Q06033 | ITIH3 | Inter-alpha-trypsin inhibitor heavychain H3 | 无信息 | 多种组织,比如肝脏、血管、胃等 | 血清中透明质酸的载体 |
Q96PD5 | PGLYRP2 | N-acetylmuramoyl-L-alanine amidase | 无信息 | 肝脏、血清 | 在抗菌防御和一些炎症疾病中发挥作用 |
ar | AADAT | Kynurenine | 无信息 | 多种组织,比如肾脏、心脏、脑、肝脏、胰腺、前列腺等 | 免疫应答调节 |
以下实施例将描述新型冠状病毒感染重症患者特征性的蛋白质与唾液蛋白组的重叠性,证实唾液蛋白组也同样可以作为新冠病毒检测生物标志物。
实施例1验证唾液蛋白组与血清蛋白组具有相似的蛋白组成
一、唾液样本收集
1. 正常健康人清水漱口后静坐10 min;
2. 使用50 mL无菌离心管收集非刺激性唾液,唾液含于口中至少1 min,然后吐到无菌离心管中,此过程可重复多次,最后收集2-5 mL样本;
3. 采集完成后将样本置于冰上,2 h内转运至-80℃冻存。
二、唾液蛋白提取
1. 1,500g 4℃离心唾液5 min,以去除完整细胞和细胞碎片;
2. 每100 μL唾液加入100 μL尿素缓冲液(100 mM Tris-HCl pH 7.5,8 M尿素,2M硫脲),超声10 min,然后10,000 g离心5 min;
3. 用Bradford assay kit试剂盒测定总蛋白浓度。
三、血清样本收集
1. 抽取空腹正常健康人静脉血5-10 mL,室温静置30 min;
2. 4℃,3000 rpm离心10 min,收集上层血清,-80℃冰箱保存。
四、样本处理
1. 从-80℃冰箱取出唾液蛋白与血清,置于冰上溶解。将血清10,000 rpm离心20min,取上清10 μL与20 μL裂解液(9 M尿素,20 g /L CHAPS,10 g /L DTT,50 mM Tris-HCl,pH 9.0)混合,或取10 μg唾液蛋白与20 μL裂解液混合,4℃震荡孵育30 min;
2. 1 μg胰蛋白酶消化样品,37℃过夜。
五、LC-MS/MS分析和非标记定量(lable-free quantification,LFQ)
1. 用0.1%甲酸重新溶解样品多肽混合物,使用Easy-nLC液相色谱仪,将样品上样到预柱C18-A1 Easy-Column(2 cm,ID 100 µm,5 µm particle size);
2. 分析柱使用Biosphere C18 column(15 cm,ID 75 µm,3 µm particle size),所用梯度为100 min内流动相0.1%甲酸缓冲液中乙腈浓度由2%升高至35%,流速为200 nL/min;
3. 使用LTQ-Orbitrap Velos双分压线性阱和静电场轨道阱组合式高分辨质谱扫描并片段化肽段,一级质谱与二级质谱之间采用data-dependent mode模式自动转换;
4. 一级质谱的m/z范围为4000-1,200,分辨率为60,000,AGC target为106;全谱扫描后,选择10个最强烈的离子进行碰撞诱导解离(CID),动态排除设置为30 s,targetvalue设置为10,000,碰撞能量为35%。实验重复两次;
5. 利用MaxQuantity LFQ软件进行非标记定量分析和数据库搜索。选择Uniprot人类数据库用于蛋白的识别。N末端肽段的乙酰化和甲硫氨酸的氧化设置为可变修饰,半胱氨酸的脲甲基化修饰设置为固定修饰。PSM和FDR设置为1%。LFQ为Lable FreeQuantitative Method的缩写,意为非标记定量方法。是常用的质谱定量方法,通过检测LFQ强度可知样品中蛋白的丰度。表1和表2中的数字分别为唾液和血清样品中的LFQ强度数值。
表1 唾液样品的LFQ强度
表2 血清样品的LFQ强度
以上结果表明,在唾液和血清中均能够检测到大量的ALB、LCP1、HP、CFB和LRG1蛋白,通过统计以上蛋白的LFQ强度数值,根据表1和表2的数据做成柱状图,如图1所示,可以直观的看到唾液样品和血清样品中均有高表达的ALB、LCP1、HP、CFB和LRG1蛋白,且上述几种蛋白在唾液和血清中的丰度并没有显著的区别,具有很大程度的重叠性和一致性,以上结果表明本发明的理论依据是正确的,唾液蛋白组与血清蛋白组具有相似的蛋白组成,由此推断由唾液蛋白组检测新型冠状病毒感染是可行的。
实施例2利用新冠病毒感染患者的唾液筛选用于检测新型冠状感染的蛋白标志物
第一步,患者唾液样本采集:
(1)清水漱口后静坐10 min;
(2)使用50 mL无菌离心管收集非刺激性唾液,唾液含于口中至少1 min,然后吐到无菌离心管中,此过程可重复多次,最后收集2-5 mL样本;
(3)采集完成后将样本置于冰上,2 h内转运至-80℃冻存。
第二步,样本处理:
(1)以56℃ 30 min失活并灭菌唾液样本;
(2)每10 μg唾液样本蛋白中加入50 μL 100 mM TEAB(triethylammoriumbicarbonate)缓冲液(其中尿素浓度为8 M),32℃ 30 min,使蛋白变性;
(3)加入10 mM TCEP(tris (2-carboxyethyl) phosphine)32℃ 30 min,使蛋白还原;
(4)然后利用40 mM碘乙酰胺将蛋白烷基化,黑暗条件下室温(25℃)45 min;
(5)用200 μL 100 mM TEAB稀释蛋白提取物,用双步蛋白酶化方法消化蛋白,其中每一步酶与底物的比例为1:20,32℃孵育60 min,随后加入30 μL 10% TFA(trifluoroacetic acid)终止反应;
第三步,液相色谱串联质谱检测
(1)将被消化的肽段在SOLAμ固相萃取孔板中清洗,并用TMTpro 16plex labelreagents进行标记;
(2)使用XBridge peptide BEH C18 column (300 Å, 5 μm×4.6 mm×250 mm),通过纳流DIONEX UltiMate 3000 RSLCnano System对TMT标记的样本进行分馏;
(3)利用乙腈-10 mM氨水溶液,以1 mL/min流速对样本进行液相色谱分离,其中乙腈浓度梯度为5%-35%;
(4)肽段共被分离为120份,将其合并为40份,然后干燥样品,并用2%乙腈/0.1%甲酸将样品溶解;
(5)用相同的液相色谱分离系统串联使用Q Exactive HF-X复合四极杆-轨道阱质谱仪对重新溶解的肽段进行分析,采用DDA(data dependent acquisition)模式;
(6)以6 μL /min的流速将样品上样到预柱(3 μm, 100 Å, 20 mm × 75 mm)上,冲洗4 min。分析柱(1.9 μm, 120 Å, 150 mm × 75 mm)时所用梯度为35 min内流动相B由5%升高至28%,流速为300 nL /min。其中缓冲液B为包含有0.1%甲酸的98%乙腈水溶液。所有试剂均为质谱级。一级质谱的m/z范围为350-1,800,分辨率为60,000,AGC target为3e6,max IT为50 ms。
(7)选择前15个产物进行二级质谱分析,分辨率为45,000,AGC target为2e5,maxIT为120 ms。
(8)利用Ptoteome Discoverer对质谱数据进行分析,数据库选择UniProtKB提供的20412条已经审核过的人类蛋白序列fasta格式数据库,SARS-CoV-2病毒fasta格式序列下载自NCBI。参数设置上,酶设置为胰蛋白酶允许有两个错误剪切;蛋白修饰设置上,半胱氨酸为脲甲基化修饰(+57.021464),赖氨酸残基和N末端肽段为TMTpro修饰(+304.207145),可变修饰设置为甲硫氨酸的氧化(+15.994915)和N末端肽段的乙酰化(+42.010565)。前体离子质量容忍为10 ppm,产物离子质量容忍设置为0.02 Da。肽段光谱匹配的FDR值设置为1%(严格)和5%(宽松)。对总肽段数量进行标准化。其他参数为默认设置。
第四步,结果分析:
通过已有研究报道通过对新冠病毒感染患者血液中蛋白质进行检测,筛选出重症患者的22个特征性蛋白质,分别为albumin(ALB)、plastin-2(LCP1)、haptoglobin(HP)、complement factor B(CFB)、Complement component C9(C9)、Complement component C6(C6)、Leucine-rich alpha-2-glycoprotein(LRG1)、osteoclast-activating factor(OAF)、thyroxine、serum amyloid A protein(SAA1和SAA2)、plasma Protease C1inhibitor(SERPING1)、Alpha-1-antichymotrypsin(SERPINA3)、lumican(LUM)、C-reactive protein(CRP)、lipopolysaccharide binding protein(LBP)、tetranectin(CLEC3B)、Hyaluronan binding protein 2(HABP2)、Protein Z-dependent proteaseinhibitor(SERPINA10)、Carboxypeptidase N catalytic chain(CPN1)、Inter-alpha-trypsin inhibitor heavy chain H3(ITIH3)、N-acetylmuramoyl-L-alanine amidase(PGLYRP2)和Kynurenine(AADAT)。本实施例的质谱检测结果表明ALB、LCP1、HP、CFB、LRG1和IL1B在唾液中高表达,可作为利用唾液检测新冠重症患者的重要生物标志物;SAA1、SERPING1、SERPINA3、C9、C6、LUM、TNF、CRP、SAA2、LBP和CLEC3B也可以在唾液中检测到,可作为利用唾液检测新冠重症患者的参考生物标志物。
实施例3本发明的试剂盒及其使用方法
本试剂盒采用胶体金层析试纸条的检测方法。该方法是一种基于比色法的检测方法,利用胶体金的颜色深浅,实现裸眼定性或半定量检测。胶体金层析试纸条具有操作简单、检测快速、应用成本低、能用于即时检测等优点,被广泛用于医学诊断、环境监测和食品安全检测等领域。
本胶体金层析试纸条由样品垫、结合垫、硝酸纤维素膜和吸水垫四部分组成,并有序地粘贴在塑料底板上。样品垫用于滴加样品并确保样品溶液均匀分布到下游组件。结合垫附着在样品垫旁边,用于装载检测分析物的胶体金探针,控制反应物溶液在膜上的释放,并使反应物在整个保质期内保持稳定。硝酸纤维素膜附着在结合垫旁,设有检测线和质控线。吸水垫用于增强毛细作用驱动力并吸收所有未反应的物质。
金纳米颗粒作为示踪剂用于报告检测信号。分别在胶体金上修饰ALB、LCP1、HP、CFB、LRG1、IL1B、SAA1、SERPING1、SERPINA3、C9、C6、LUM、TNF、CRP、SAA2、LBP或CLEC3B单/多克隆抗体,并分别用于制备新冠重症患者胶体金层析试纸条。其中用ALB、LCP1、HP、CFB、LRG1或IL1B单/多克隆抗体修饰的胶体金层析试纸条是必须组分;用SAA1、SERPING1、SERPINA3、C9、C6、LUM、TNF、CRP、SAA2、LBP或CLEC3B单/多克隆抗体修饰的胶体金层析试纸条可有可无,但是如果包含有这些会使检测更加完善,提高准确度。
同时,将辣根过氧化物酶(HRP)标记到胶体金表面,结合酶信号放大技术提高检测的灵敏度。HRP能催化底物3-氨基-9-乙基咔唑(AEC)生成红色不溶物,利用其催化反应能够实现颜色信号的放大。
使用时,收集唾液样本,将待测唾液样本滴加到样品垫,唾液样本通过毛细作用沿着胶体金试纸条移动,移动到结合垫时会被胶体金探针捕获,得到的复合物继续层析,直到通过硝酸纤维素膜上的检测线和质控线时被捕获,累积形成红色的条带,未反应的物质继续层析最终被吸水垫吸收。检测完成后,若检测线和质控线都有红色条带,则为阳性结果;只有质控线有红色条带,则为阴性结果;若质控线无颜色,则此次检测无效。检测过程中,用ALB、LCP1、HP、CFB、LRG1、IL1B、SAA1、SERPING1、SERPINA3、C9、C6、LUM、TNF、CRP、SAA2、LBP或CLEC3B单/多克隆抗体修饰的胶体金层析试纸条中有大于或等于80%的检测结果为阳性,可以表征新冠病毒感染患者有发展为重症的发展趋势。
综上,多种被用于新型冠状病毒感染预测的蛋白可在患者唾液中被检测到,所以唾液蛋白组同血清蛋白组一样,具有作为新冠病毒检测生物标志物的潜力。唾液蛋白组也同样可以作为新冠病毒检测生物标志物。质谱检测结果表明ALB、LCP1、HP、CFB、LRG1和IL1B在唾液中高表达,可作为利用唾液检测新冠重症患者的重要生物标志物;SAA1、SERPING1、SERPINA3、C9、C6、LUM、TNF、CRP、SAA2、LBP和CLEC3B也可以在唾液中检测到,可作为利用唾液检测新冠重症患者的参考生物标志物。本发明的方法成本低,性价比高。本发明的方法具有无创性,不会引起检测者的不适,不会对检测者造成多余的创伤、引起不必要的疼痛,不会对检测者造成暴露感染的风险,也不会对医护人员带来感染的风险。本发明的检测样本为唾液,样本易于收集,尤其是在难以获取血液样本的情况下。本发明的检测方法能大大缩短检测时间,用胶体金层析试纸条肉眼就能观察到结果,而核酸检测通过PCR扩增,需要等待较长时间,所以本发明的方法有利于大规模人群的初筛。血浆和唾液的蛋白质组相似度高,许多在唾液中发现的蛋白质也存在于血液中。近40%的可在唾液中被找到的蛋白质被认为是癌症、心血管疾病和中风等疾病的候选标志物,所以可以使用唾液来发现新标志物和诊断早期症状。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
1.一种利用唾液检测新型冠状病毒感染的试剂盒,其特征在于,所述试剂盒为胶体金层析试纸条,所述胶体金层析试纸条包括样品垫、结合垫、硝酸纤维素膜和吸水垫,所述硝酸纤维素膜上有检测线和质控线,所述胶体金层析试纸条上含有胶体金颗粒,所述胶体金颗粒上修饰有白蛋白、淋巴细胞胞浆蛋白1、触珠蛋白、补体因子B、富亮氨酸α-2-糖蛋白1和白细胞介素1β的单克隆抗体或多克隆抗体;所述胶体金颗粒表面标记了辣根过氧化物酶。
2.生物标志物在制备新型冠状病毒唾液检测试剂盒中的应用,所述生物标志物为以下物质的组合:白蛋白、淋巴细胞胞浆蛋白1、触珠蛋白、补体因子B、富亮氨酸α-2-糖蛋白1和白细胞介素1β。
3.生物标志物在制备新型冠状病毒感染辅助唾液诊断产品中的应用,所述生物标志物为以下物质的组合:白蛋白、淋巴细胞胞浆蛋白1、触珠蛋白、补体因子B、富亮氨酸α-2-糖蛋白1和白细胞介素1β。
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