CN112094326B - 一种新冠病毒抗原及其应用 - Google Patents
一种新冠病毒抗原及其应用 Download PDFInfo
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- CN112094326B CN112094326B CN202011019147.7A CN202011019147A CN112094326B CN 112094326 B CN112094326 B CN 112094326B CN 202011019147 A CN202011019147 A CN 202011019147A CN 112094326 B CN112094326 B CN 112094326B
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Abstract
本发明属于冠状病毒检测技术领域,具体涉及一种新冠病毒抗原及其应用。该抗原具有选自S蛋白的片段和选自N蛋白的片段,所述选自S蛋白的片段具有如SEQ ID NO.1‑5所示的氨基酸序列,所述选自N蛋白的片段具有如SEQ ID NO.6‑11所示的氨基酸序列,所述抗原可与新冠病毒抗体特异性结合。该抗原用于新冠病毒的检测时,可有效避免假阳性的问题。
Description
技术领域
本发明属于冠状病毒检测技术领域,具体涉及一种新冠病毒抗原及其应用。
背景技术
新型冠状病毒(SARS-CoV-2)为β属的冠状病毒,是目前已知的第7种可感染人类的冠状病毒,其余6种分别是HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV。该疾病以发热、干咳、乏力为主要表现,常伴有气促和呼吸困难等,在严重病例中,新冠肺炎可导致严重急性呼吸窘迫综合征、脓毒症休克,多功能衰竭甚至死亡。
新型冠状病毒(SARS-CoV-2)特异IgM和IgG抗体检测写进了国家卫生健康委于2020年3月3日发布的“新型冠状病毒肺炎诊疗方案(试行第七版)”,作为临床确诊标准,国家药品监督管理局(NMPA)也应急审批了广州万孚(胶体金试纸条)、英诺特(唐山)(胶体金试剂条)等多个抗体检测试剂盒。徐万洲等在19例核酸检测阴性但基于临床症状确诊的COVID-19患者中,有16例患者IgM阳性,其阳性率达到84.21%,有18例患者IgG阳性,其阳性率达到94.74%,说明抗体的检测可以有效地弥补核酸检测漏检的风险,发挥其在新型冠状肺炎的及时诊治及防控中的作用。但临床现也有反映出现了较多的假阳性,困扰临床决策。
特异IgM和IgG免疫测定假阳性通常有以下两个方面的原因,一是试剂盒所用抗原本身的特异性及阳性判断值(Cut-off value)的设定;二是患者标本中存在导致免疫测定假阳性的内源性或外源性干扰物质。而试剂盒所用抗原本身的特异性是检测特异性的基础。这是由抗原决定簇和抗体的抗原结合区的空间构象决定的。抗体抗原结合区的空间构象是由该区的氨基酸序列来决定源。氨基酸的序列则由基因序列决定。抗体的特异性,并不是针对抗原,而是针对某个特定的空间构象。这就是为什么有的抗体可以识别道2个毫不相干的抗原。比如抗梅毒抗体,同时可以识别心磷脂。因此,优选特异性抗原片段对减少特异IgM和IgG免疫测定的假阳性有重要意义。
SARS-CoV-2核衣壳蛋白(Nucleocapsid,N)位于病毒内部,在β属冠状病毒之间相对比较保守,与其它6种冠状冠状病毒(HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV)的N蛋白全长具有一定的同源性,检测时容易发生交叉反应性,因此筛选出同源性低,免疫性强的N蛋白片段进行组合,避免交叉反应性,是十分重要的。
发明内容
本发明提供一种新冠病毒抗原,以解决现有技术中新冠病毒检测特异性差的问题。
本发明的第二目的在于提供了一种抗体。
本发明的第三目的在于提供一种标记复合物。
本发明的第四目的在于提供一种试剂盒
本发明的目的还在于提供了上述中的任意一项或几项的应用。
本发明的新冠病毒抗原采用如下技术方案:一种新冠病毒抗原,所述抗原具有选自S蛋白的片段和选自N蛋白的片段,所述选自S蛋白的片段具有如SEQ ID NO.1-5所示的氨基酸序列,所述选自N蛋白的片段具有如SEQ ID NO.6-11所示的氨基酸序列,所述抗原可与新冠病毒抗体特异性结合。
作为进一步的优选技术方案,所述抗原的不同片段之间通过GGGGS连接肽连接。
作为进一步的优选技术方案,所述抗原的氨基酸序列如SEQ ID NO.12所示。
本发明的抗体采用如下技术方案:一种抗体,所述抗体通过采用如上述任意一项所述的抗原作为免疫原免疫动物制备得到。例如,可通过将所述抗原用于免疫动物,制备得到抗所述抗原的抗体。本发明的抗体可为单克隆抗体或多克隆抗体,其可用于制备新冠病毒检测试剂(例如,用作制备试剂盒的原料)或作为阳性对照等。
本发明的标记复合物采用如下技术方案:一种标记复合物,所述标记复合物的组成包括如上述任意一项所述的抗原和标记物,所述标记物包括但不限于下述中的任意一种:胶体金、荧光免疫微球或辣根过氧化物酶。
本发明的试剂盒采用如下技术方案:一种试剂盒,所述试剂盒包括下述中的任意一种或几种的组合:如上所述任意一项的抗原、如上所述的抗体和如上所述的标记复合物和固相载体。
作为进一步优选的技术方案,所述试剂盒为ELISA试剂盒,所述ELISA试剂盒包括包被有如上述任意一项所述的抗原的包被板和辣根过氧化物酶标记的抗原。
本发明的应用采用如下技术方案:如上述任意一项所述的抗原/如上所述的抗体/如上所述的标记复合物/如上所述的试剂盒中的任意一项或几项在制备新冠病毒检测试剂中的应用。
本发明的有益效果是:本发明的新冠病毒抗原可与新冠病毒抗体(对IgG、IgM均可检测)特异性结合,可有效避免新冠病毒检测假阳性的情况。
本发明的新冠病毒抗原与其他冠状病毒不会发生特异性结合,可有效提高冠状病毒检测的特异性,避免将其他冠状病毒检测为新冠病毒的情况。
具体实施方式
下面将结合具体实施例对本发明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。
实施例1
1.1利用生物信息学手段,结合以往经验,筛选出下述优选的SARS-CoV-2的S片段:
123-ATNVVIKVCEFQFCNDPFLGVYYH-146(SEQ ID NO.1)
209-PINLVRDLPQGFSALEPLVDLPIGI-233(SEQ ID NO.2)
262-AAAYYVGYLQPRTFLLK-278(SEQ ID NO.3)
485-GFNCYFPLQSYGF-497(SEQ ID NO.4)
504-GYQPYRVVVLSFELLHAPATVCGPK-528(SEQ ID NO.5)
1.2筛选得到的优选的SARS-CoV-2的N片段如下:
52-WFTALTQHGK EDLKFPRGQG VPINTNSSPD DQIGYYRR-89(SEQ ID NO.6)
106-PRWYFYYLGT GPEAGLPYGA NKDGIIWVAT EGALNTPKDH(SEQ ID NO.7)
IGTRNPANNA AIVLQLPQGTT-166(SEQ ID NO.8)
217-AALALLLLDRL-227(SEQ ID NO.9)
243-GQTVTKK-249(SEQ ID NO.10)
267-AYNVTQA-273(SEQ ID NO.11)
1.3将上述多肽通过GGGGS连接肽将各部分进行连接,具体氨基酸序列如下:
ATNVVIKVCE FQFCNDPFLG VYYHGGGGSP INLVRDLPQG FSALEPLVDL PIGIGGGGSA
AAYYVGYLQP RTFLLKGGGG SGFNCYFPLQ SYGFGGGGSG YQPYRVVVLS
FELLHAPATV CGPKGGGGSW FTALTQHGKE DLKFPRGQGV PINTNSSPDD QIGYYRRGGG
GSPRWYFYYL GTGPEAGLPY GANKDGIIWV ATEGALNTPK DHIGTRNPAN NAAIVLQLPQ
GTTGGGGSAA LALLLLDRLG QTVTKKAYNV TQA(SEQ ID NO.12)
Theoretical pI/Mw:8.60/27474.98
1.4表达载体构建:合成编码如SEQ ID NO.12所示的氨基酸序列的核苷酸序列并由上海生工将合成的核苷酸序列连接在pET28a的BamHI和XhoI之间,得到PET28a-Nx质粒。
1.5表达纯化:按照《分子克隆》第二版的常规试验方法将构建好的PET28a-Nx质粒转入到Bl21(DE)3中,进行常规的表达纯化。取﹣80℃下保存的大肠杆菌BL21(DE3)菌株,活化培养后,采用CaCl2法制备感受态细胞。将含目的基因的重组质粒转化至BL21(DE3)感受态细胞中,获得重组工程菌株。对工程菌株表达条件(诱导温度、诱导时间、IPTG浓度)进行优化,目的蛋白以包涵体形式存在,通过镍柱亲和层析,以及利用梯度透析对包涵体蛋白进行复性,利用SDS-PAGE与Western Blot对复性蛋白进行鉴定,得到了免疫原性良好的复性蛋白(下文简称为N1蛋白)。具体步骤如下:
1.5.1感受态细胞制备
(1)从BL21(DE3)平板中挑取单克隆,置于50mL LB液体培养基中,37℃摇床培养3小时,至OD=0.5时方可进行下一步;
(2)在无菌条件下,将活化后的BL21(DE3)菌液转移至离心管中,冰浴10min;
(3)4000r/min离心5min,弃上清,将离心管倒置尽可能减少上清残留;
(4)加入10mL提前预冷的0.1mol/L的CaCl2溶液,重悬沉淀;
(5)4000r/min离心5min,弃上清,将离心管倒置尽可能减少上清残留;
(6)加入2mL提前预冷的0.1mol/L的CaCl2溶液,重悬沉淀;
(7)在无菌条件将感受态细胞分装至无菌EP管中,若制备后无需立刻使用时可加入20%甘油,置于-70℃保存。
1.5.2重组质粒转化
采用制备的感受态细胞将确认序列正确的重组质粒进行转化。
(1)采用无菌枪头吸取200μL BL21(DE3)感受态细胞,随后加入2μL确定序列正确的重组质粒,混匀后,冰浴30min;
(2)将离心管立刻转入提前预热至42℃的水浴锅中,静置90s;
(3)随后将离心管立即转入冰中,冷却5min;
(4)取1.3mL LB液体培养基加入至转化后菌液中,200rpm,37℃摇床过夜培养;
(5)无菌条件下,蘸取少量菌液在氨苄卡那霉素平板划线,37℃恒温培养12-16h。
1.5.3蛋白表达
转化后菌种在一定的培养条件下,通过加入诱导剂能够促使蛋白表达。蛋白表达主要包括:目的蛋白初步诱导表达;目的蛋白表达与否;诱导条件的探究;大量诱导表达。
(1)无菌条件下,取1支无菌试管,加入5mL含氨苄LB培养基,从平板中挑取3-5个单克隆至培养基中;
(2)37℃过夜培养,作为诱导表达时初始菌液;
(3)取少量初始菌液接种至250mL液体培养基中继续培养2-3h;
(4)按1:1比例进行扩瓶培养;
待菌液培养至OD=2.0,加入IPTG使其终浓度为0.5mmol/L,37℃诱导6h。
1.6按照上述1.4-1.5的方法分别制备得到SARS-CoV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV的全长N蛋白(下述按照次序依次简称为N2蛋白-N8蛋白)和SARS-CoV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV和MERS-CoV的全长S蛋白(下述按照次序依次简称为S2蛋白-S8蛋白)。
实施例2将实施例1制备得到的N1蛋白用于免疫动物
2.1将纯化的N1蛋白分别免疫兔子,取纯化的N1蛋白约400μg与等体积弗氏完全佐剂完全混匀,免疫长耳白兔。采用皮下注射的免疫方式,4周后以相同剂量加弗氏不完全佐剂加强免疫1次,加强免疫后分别隔2周、4周、6周各采血1次,得到兔多抗血清R1。
2.2将纯化的N2和S2蛋白各200μg免疫兔子,等体积弗氏完全佐剂完全混匀,免疫长耳白兔。采用皮下注射的免疫方式,4周后以相同剂量加弗氏不完全佐剂加强免疫1次,加强免疫后分别隔2周、4周、6周各采血1次,得到兔多抗血清R2。
2.3按照2.2的方法,分别制备得到兔多抗血清R3-R8。
以N3和R3蛋白共同免疫兔子制备得到兔多抗血清R3;以N4和R4蛋白共同免疫兔子制备得到兔多抗血清R4;以N5和R5蛋白共同免疫兔子制备得到兔多抗血清R5;以N6和R6蛋白共同免疫兔子制备得到兔多抗血清R6;以N7和R7蛋白共同免疫兔子制备得到兔多抗血清R7;以N8和R8蛋白共同免疫兔子制备得到兔多抗血清R8。
实施例3制备试剂盒
3.1以稀释后的N1蛋白作为包被抗原,以HRP标记的N1蛋白作为标记抗原,进行总抗体的检测,试剂盒命名为K1。
3.2按照上述3.1的方法,分别以N2-N8蛋白作为包被抗原,以HRP标记的N2-N8蛋白作为标记抗原,制备得到相应的分别检测N2-N8蛋白的试剂盒K2-K8。
3.3按照上述3.1的方法,分别以S2-S8蛋白作为包被抗原,以HRP标记的S2-S8蛋白作为标记抗原,制备得到相应的分别检测S2-S8蛋白的试剂盒K9-K15。
3.4上述试剂盒的检测步骤如下(以试剂盒K1为例):以N1纯化蛋白制备包被板,血清样品用样品稀释液(含有BSA,Tris-Cl以及吐温-20)按照1:100进行稀释,每孔加100μL,37℃孵育0.5h,每孔用PBST洗液洗涤5次(96孔板注满洗涤液)。加入辣根过氧化物酶标记的N1纯化蛋白,37℃孵育0.5h。弃掉孔中液体,用PBST洗液洗涤5次。每孔加50μL TMB,室温避光孵育20min,最后用终止液终止反应,用酶标仪于主波长450nm及参考波长630nm进行检测,读取结果。
实施例4采用实施例3制备得到的试剂盒对实施例2制备得到的兔多抗血清R1-R8进行检测
检测结果如下表1所示
表1
在450nm以及630nm波长检测下,得到OD值在0.1以下判定为阴性,OD值在0.1以上判定为阳性。由表1可知,只有N1蛋白不与其他冠状病毒的N蛋白和S蛋白抗体反应,表现出良好的特异性。
实施例5实施例3制备得到的试剂盒K1、K2和K9用于新冠病毒(SARS-CoV-2)的检测
检测结果如下表2所示
表2
2018年临床血清100份阳性率 | 临床确认总抗体阳性标本10本 | |
K1 | 0/100 | 10/10 |
K2 | 13/100 | 10/10 |
K9 | 2/100 | 9/10 |
上述临床阴性样本来源于河南省中医院,阳性样本来源于河南省疾病预防控制中心。
2018年由于该病毒未传播,所以2018年存的临床发热病人血清样本应为阴性,实际上试剂盒K2(以SARS-CoV-2全长N蛋白作为包被和标记抗原)的检测出现了13份假阳性的结果,试剂盒K9(以SARS-CoV-2全长S蛋白作为包被和标记抗原)的检测出现了2份假阳性的结果,一份假阴性的结果。
以上所述仅为本发明的较佳实施例而已,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。
序列表
<110> 河南省生物工程技术研究中心
郑州倍赛泰生物科技有限公司
<120> 一种新冠病毒抗原及其应用
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<211> 263
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp
1 5 10 15
Pro Phe Leu Gly Val Tyr Tyr His Gly Gly Gly Gly Ser Pro Ile Asn
20 25 30
Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val
35 40 45
Asp Leu Pro Ile Gly Ile Gly Gly Gly Gly Ser Ala Ala Ala Tyr Tyr
50 55 60
Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Gly Gly Gly Gly
65 70 75 80
Ser Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gly Gly
85 90 95
Gly Gly Ser Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu
100 105 110
Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Gly Gly Gly Gly
115 120 125
Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys Phe
130 135 140
Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp Asp
145 150 155 160
Gln Ile Gly Tyr Tyr Arg Arg Gly Gly Gly Gly Ser Pro Arg Trp Tyr
165 170 175
Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala
180 185 190
Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr
195 200 205
Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile
210 215 220
Val Leu Gln Leu Pro Gln Gly Thr Thr Gly Gly Gly Gly Ser Ala Ala
225 230 235 240
Leu Ala Leu Leu Leu Leu Asp Arg Leu Gly Gln Thr Val Thr Lys Lys
245 250 255
Ala Tyr Asn Val Thr Gln Ala
260
Claims (6)
1.一种新冠病毒抗原,其特征在于,所述抗原的氨基酸序列如SEQ ID NO.12所示,所述抗原与新冠病毒抗体特异性结合。
2.一种多克隆抗体,其特征在于,所述多克隆抗体通过采用如权利要求1中所述的抗原作为免疫原免疫动物制备得到。
3.一种标记复合物,其特征在于,所述标记复合物的组成包括如权利要求1-2中任意一项所述的抗原和标记物,所述标记物包括:胶体金、荧光免疫微球或辣根过氧化物酶。
4.一种试剂盒,其特征在于,所述试剂盒包括下述中的任意一种或几种的组合:如权利要求1所述的抗原、如权利要求2所述的多克隆抗体、如权利要求3所述的标记复合物,所述试剂盒还包括固相载体。
5.根据权利要求4所述的试剂盒,其特征在于,所述试剂盒为ELISA试剂盒,所述ELISA试剂盒包括包被有如权利要求1中所述的抗原的包被板和辣根过氧化物酶标记的抗原。
6.如权利要求1中所述的抗原/如权利要求2所述的多克隆抗体/如权利要求3所述的标记复合物中的任意一项或几项在制备新冠病毒检测试剂中的应用。
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