CN113248578B - 新型冠状病毒(2019-nCoV)重组抗原及多克隆抗体 - Google Patents
新型冠状病毒(2019-nCoV)重组抗原及多克隆抗体 Download PDFInfo
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Abstract
本发明提供一种新型冠状病毒(2019‑nCoV)IgM/IgG抗体用重组抗原，与新型冠状病毒(2019‑nCoV)的抗体IgG/IgM反应，而不与其它肺部感染性冠状病毒等发生反应，也不与正常人血清发生反应，特异性高。本发明新型冠状病毒(2019‑nCoV)抗体诊断抗原可以利用大肠杆菌重组表达制备，具有抗原表位丰富，检测灵敏度更高，成本较低。本发明提供的多克隆抗体，有望作为用于临床治疗的诊断指标，并开发阻断病毒感染的治疗性抗体。
Description
相关申请的交叉引用
本申请要求2020年2月12日提交的中国专利申请202010089098.8的优先权，所述申请的公开内容均援引加入本文。
技术领域
本发明属于生物技术领域，具体涉及一种新型冠状病毒(2019-nCoV)IgM/IgG抗体用重组抗原及其多克隆抗体。
背景技术
冠状病毒(Coronaviruses，CoVs)是一组高度多样化的、包膜的、正向单链RNA病毒，其基因组为26-32 kilobases(kb)，是基因组规模最大的RNA病毒。冠状病毒(coronavirus，CoV)属巢状病毒目，冠状病毒科，分为本α、β、γ三个属，其中α、β属仅对哺乳动物致病，γ属主要引起鸟类感染。CoV主要通过直接接触分泌物或经气溶胶、飞沫传播，也有证据表明可经粪口途径传播。目前已经发现引起人类呼吸道疾病的人冠状病毒(HCoV)已达7种：HCoV－229E、HCoV－OC43、SARS－CoV、HCoV－NL63、HCoV－HKU1、MERS－CoV和新型冠状病毒(2019-nCoV)。基因组序列分析发现，新型冠状病毒(2019-nCoV)与目前发现的感染人肺部的其余六种冠状病毒(HCoV-229E、HCoV-OC43、SARS-CoV、HCoV-NL63、HCoV-HKU1、MERS-CoV)具有一定的序列同源性，其中与SARS的核酸序列同源性最高(79.5％sequenceidentity)，与MERS次之(～50％sequence identity)，而与其余的四种冠状病毒的同源性低于40％。新型冠状病毒(2019-nCoV)编码了ORF1ab polyprotein(GeneID:43560238)、S(surface glycoprotein,GeneID:43560230)、ORF3 protein(GeneID:43560231)、envelopeprotein(GeneID:43560232)、membrane glycoprotein(GeneID:43560233)、ORF6 protein(GeneID:43560234)、ORF7 protein(GeneID:43560235)、ORF8 protein(GeneID:43560236)和N(nucleocapsid phosphoprotein,GeneID:43560237)等。用于CoV物种分类的ORF1ab中7个保守的复制酶区域，nCoV-2019和SARS-CoV之间的氨基酸序列一致率为94.6％，表明两者属于同一物种。
冠状病毒的生命周期(life cycle)分为几个步骤：粘附和入胞、病毒复制酶的翻译、基因组转录和复制、结构蛋白的翻译以及病毒体的组装和分泌。其中S(surfaceglycoprotein,GeneID:43560230)和N(nucleocapsid phosphoprotein,GeneID:43560237)在冠状病毒生命周期中有重要作用，S是病毒与宿主受体结合蛋白质，是病毒粘附入胞实现感染的关键分子；N是病毒基因组RNA的结合蛋白质，参与衣壳组装。同时，参照SARS的有关研究发现，S和N是免疫原性最强的病毒蛋白质，也是病毒免疫检测的重要抗原。
目前，针对新型冠状病毒(2019-nCoV)的检测方法主要是核酸检测，其基本流程是采样→保存送样→病毒灭活→裂解核酸提取→荧光定量PCR检测等。核酸检测需要在有条件和资质的实验室进行，对场地要求比较高；需要专门的核酸提取和定量PCR仪等专业设备和专门的经过培训的检测人员，很多的基层医疗机构难以开展；其检测通量比较有限，出具报告的时间较长。另外，对于阳性病人，核酸检测有一定的“假阴性”检出率，即有些患者在临床上被确认为疑似新冠肺炎，但是其核酸检测结果为阴性。因此迫切需要开发新的新型冠状病毒(2019-nCoV)检测方法。
发明内容
针对上述现有技术，本发明的目的是提供一种新型冠状病毒(2019-nCoV)重组抗原及其应用。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面，提供一种新型冠状病毒(2019-nCoV)重组抗原，其具有如表1所示氨基酸序列。
表1重组抗原的氨基酸系列信息
本发明新型冠状病毒(2019-nCoV)编码的六个病毒蛋白质的重组抗原，与新型冠状病毒(2019-nCoV)的抗体IgG/IgM反应，不与正常人血清发生反应，特异性高。
本发明的第二方面，提供编码上述新型冠状病毒(2019-nCoV)重组抗原的基因，其特征在于，是具有如表2所示的核苷酸序列。
表2核苷酸序列系列信息
本发明的第三方面，提供一种重组载体，其含有上述新型冠状病毒(2019-nCoV)重组抗原的基因。进一步的，本发明还提供一种重组菌，其被上述的重组载体转化。更进一步，经重组载体转化的重组菌选自重组大肠杆菌。
本发明的第四方面，提供上述新型冠状病毒(2019-nCoV)的重组抗原或编码上述新型冠状病毒(2019-nCoV)重组抗原的基因在新型冠状病毒免疫检测试剂中的应用。本发明新型冠状病毒(2019-nCoV)六个病毒蛋白质的重组抗原在新型冠状病毒免疫检测试剂中的应用，该应用使得新型冠状病毒抗体诊断抗原可以利用大肠杆菌重组表达制备，具有抗原表位丰富，检测灵敏度更高，成本较低。
本发明的第五方面，提供上述新型冠状病毒(2019-nCoV)的重组抗原或编码上述新型冠状病毒(2019-nCoV)重组抗原的基因在制备表位疫苗中的应用。
本发明的第六方面，提供上述新型冠状病毒(2019-nCoV)的重组抗原或编码上述新型冠状病毒(2019-nCoV)重组抗原的基因在病毒感染中和性抗体检测中的应用。
一种检测新型冠状病毒(2019-nCoV)感染的中和性抗体，包含上述新型冠状病毒(2019-nCoV)的重组抗原或编码上述新型冠状病毒(2019-nCoV)重组抗原的基因。本发明提供的六种新型冠状病毒(2019-nCoV)重组抗原来检测病毒感染中和性抗体，有望作为用于临床治疗的诊断指标，并开发阻断病毒感染的治疗性抗体。
本发明的第七方面，提供上述新型冠状病毒(2019-nCoV)的重组抗原在制备检测抗新型冠状病毒(2019-nCoV)抗体或者抗新型冠状病毒(2019-nCoV)多肽抗体的试剂盒中的应用。
本发明的第八方面，提供上述新型冠状病毒(2019-nCoV)的重组抗原、编码新型冠状病毒(2019-nCoV)重组抗原的基因、含有新型冠状病毒(2019-nCoV)重组抗原的基因的重组载体或重组载体转化成重组菌在制备治疗新型冠状病毒(2019-nCoV)药物中的应用。
一种疫苗组合物，包含上述新型冠状病毒(2019-nCoV)的重组抗原。进一步，上述疫苗组合物还包含可药用佐剂。
有益效果：
本发明提供一种新型冠状病毒(2019-nCoV)IgM/IgG抗体用重组抗原，与新型冠状病毒(2019-nCoV)的抗体IgG/IgM反应，而不与其它肺部感染性冠状病毒等发生反应，也不与正常人血清发生反应，特异性高。本发明新型冠状病毒(2019-nCoV)抗体诊断抗原可以利用大肠杆菌重组表达制备，具有抗原表位丰富，检测灵敏度更高，成本较低。本发明提供的六种新型冠状病毒(2019-nCoV)重组抗原来检测病毒感染中和性抗体，有望作为用于临床治疗的诊断指标，并开发阻断病毒感染的治疗性抗体。
附图说明
图1是Spike蛋白质(nCoV)的三维结构模拟图。
图2是Nucleocapsid Protein蛋白质N端结构域(nCoV)的三维结构模拟图。
图3是Nucleocapsid Protein蛋白质C端结构域(nCoV)的三维结构模拟图。
图4是图4.抗原(NN、NC、S1、S2、S3和S4)基因PCR图。
图5是NN抗原菌落PCR图。
图6是NC抗原菌落PCR图。
图7是S1抗原菌落PCR图。
图8是S2抗原菌落PCR图。
图9是S3抗原菌落PCR图。
图10是S4抗原菌落PCR图。
图11是NN、NC、S1、S2、S3和S4双酶切鉴定图。
图12是NN抗原表达的IPTG诱导图。
图13是NC抗原表达的IPTG诱导图
图14是S1抗原表达的IPTG诱导图。
图15是S2抗原表达的IPTG诱导图。
图16是S3抗原表达的IPTG诱导图。
图17是S4抗原表达的IPTG诱导图。
图18是NN抗原表达纯化图：(1:蛋白质marker；2:表达细菌总蛋白质；3:细菌超声破碎上清；4:细菌超声破碎离心沉淀蛋白质；5:Binding buffer洗脱；6:Washing buffer(60mM imidazole)洗脱；7:Washing buffer(80mM imidazole)洗脱；8:Elution buffer(200mN imidazole)洗脱目的蛋白质；9:8:Elutionbuffer(500mN imidazole)目的蛋白质)。
图19是重组NN抗原的临床样本检测结果，其中浅灰色显示为阳性结果，深灰色显示为灰度区域，白色为阴性结果。
具体实施方式
为了使本发明的目的和技术方案更加清楚，下面对本发明的优选实施例进行详细的描述。要说明的是：以下实施例只用于对本发明进行进一步的说明，而不能理解为对本发明保护范围的限制。本领域的技术人员根据本发明的上述内容做出的一些非本质的改进和调整均属于本发明的保护范围。需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作和/或它们的组合。
实施例1：抗原的设计与密码子优化
A.利用生物信息学，根据新型冠状病毒(2019-nCoV)基因组(NC_045512.1)编码的S(surface glycoprotein,GeneID:43560230)和N(nucleocapsid phosphoprotein,GeneID:43560237)氨基酸序列，利用swiss-model(https://swissmodel.expasy.org/interactive)进行了三维结构重建，基于SARS的S蛋白质结构(PDB code：6ACC)和N蛋白质结构(PDB code：1SSK和2JW8)获得了新型冠状病毒(2019-nCoV)的N和S的三维结构模型(见附图1-3)。
B.基于获得三维结构模型，设计六个重组抗原，其中来源N(nucleocapsidphosphoprotein)有两个抗原(分别为：NN和NC)和来源于S(surface glycoprotein)的四个抗原(分别为：S1，S2，S3和S4)(具体氨基酸序列信息见表1)。
表1重组抗原的氨基酸系列信息
C.为避免稀有密码子导致的表达效率低下等问题，进行密码子优化设计(具体核苷酸序列信息见表2)，并进行全基因合成，并构建到克隆载体pUCm-T载体，然后进行基因序列测定验证。
表2重组抗原的核苷酸系列信息
实施例2：表达质粒的构建与抗原表达纯化
A.利用pET系列载体的通用引物进行基因扩增(图4)，并结果PCR产物纯化，然后利用基因5’和3’端预先设计的NdeI和XhoI进行双酶切，产物经过回收后与经过双酶切(NdeI和XhoI)纯化后的pET28a载体进行T4连接酶连接。
B.将连接产物转入DH5α菌株，挑取单克隆用LB培养基培养(kanamycin抗性)，然后利用菌液PCR验证(图5-10)，对阳性克隆提取质粒，然后利用NdeI和XhoI进行双酶切鉴定(图11)，最后进行基因测序验证，获得了六个抗原表达质粒，即：pET28a-NN，pET28a-NC，pET28a-S1，pET28a-S2，pET28a-S3，pET28a-S4。
C.将测序验证准确的表达质粒分别转入表达菌株(BL21或/Rosetta)感受态菌株中,然后挑取单克隆于BL21于37℃培养。待待OD600值达到0.6-1.0时，加入终浓度为0.1-0.5μmol/L的IPTG于37℃诱导培养3-4小时(或18-25℃低温诱导培养10-20小时)(或者利用自诱导培养基进行自诱导)，离心收菌，然后利用SDS-PAGE电泳鉴定抗抗原表达效果(图12-18)。将收集菌体利用Lysis缓冲液重悬，超声波破碎后高速低温(15000g,30分钟，4℃)离心取上清，采用Ni亲和层析、离子交换层析、分子筛层析等纯化抗原，经过优化后纯化工艺后，SDS-PAGE检测，抗原纯度大于80-95％。将抗原透析或者超滤去掉咪唑，分装后于-80℃保存。
实施例3：多克隆抗体的制备
1.兔多抗的制备
用生理盐水将抗原稀释到2倍终浓度(每只家兔按照200-250ul计算，抗原为50-60ug)，按照1:1充分混匀佐剂(QuickAntibody-Rabbit8W，Biodragon公司)。选取1.5-2.0kg左右的家兔，通过后退小腿肌肉注射免疫家兔，每只左、右各注射1针(200ul左右/针)。三周和六周后分别加强免疫一次。第七周采取微量血进行Elisa测定，抗体滴度约1:10000-1:1000000，随即采取全血。
2.多克隆抗体的纯化
1.将抗血清放入冰水或4℃冰箱中缓慢解冻以避免蛋白质的聚集，然后利用低温(4℃)高速离心机(15,000xg)离心5-10分钟，弃沉淀取上清再经过滤器过滤除去多余的脂。
2.将血清用缓冲溶液以一定比例进行稀释，再用过滤器进行过滤。以低速度(约0.5ml/min)将抗血清上到柱上(Protein A Sepharose，GE Healtcare)，并进行洗脱杂蛋白质，最后以pH2.7缓冲溶液洗脱目的蛋白质，收集目的蛋白质备进一步纯化。
3.重组抗原的活性初步检测
纯化好的重组抗原用包被液(0.05M碳酸盐缓冲液，pH9.6)稀释(0.5mg/ml)，然后依次按照120、1；40、1；80和1；160进一步稀释，各稀释度按100ul/孔滴加到Elisa板条中，4℃包被过夜。弃液，用洗涤液洗三次(200ul/孔，每次3-5分钟)。用封闭液(洗涤液中加终浓度为5％的脱脂奶粉)封闭1小时(每孔200ul)，弃封闭液后按上述洗涤方法再次洗涤3次。然后利用纯化后的多抗为阳性对照，正常人血清为阴性对照，检测阳性病人血清。结果显示，重组获得的至少有2个抗原可以与新型冠状病毒(2019-nCoV)感染病人的IgG和IgM结合，显示阳性检测结果。
实施例4：临床样本测试
发明人将基于大肠杆菌表达的NN和NC抗原进行了纯化，用5份确诊的COVID-19患者血清和10份正常血清与纯化NN和NC抗原进行检测。发现NN和NC具有良好的抗体检测性。鉴于NN的的表达水平更高，也更容易就进行纯化，发明人后续采用NN进行进一步检测。为检测检测方法的特异性，对180例感染其他呼吸道病原体(A型流感病毒、B型流感病毒、副流感病毒、腺病毒、呼吸道合胞病毒等)的患者血清进行检测，均未发现交叉反应，表明使用大肠杆菌表达纯化的NN抗原检测SARS-CoV-2病毒感染的抗体具有较好的特异性。
对50例SARS-CoV-2感染者进行RT-PCR确认后，采用磁微粒化学发光法检测血清抗体IgG和IgM(6例正常人为阴性对照)。初步结果显示，其中60％(50中30人阳性)的患者对SARS-CoV-2的IgG抗体检测阳性，而56％(50人中28人阳性)的患者对SARS-CoV-2的IgM阳抗体检测为阳性。共有34例患者IgM或IgG检测阳性，总阳性率达68％(图19)。
本发明所述新型冠状病毒(2019-nCoV)的重组抗原或编码上述新型冠状病毒(2019-nCoV)重组抗原的基因可以用于病毒感染中和性抗体的检测，有望作为用于临床治疗的诊断指标，并开发阻断病毒感染的治疗性抗体。本发明所述新型冠状病毒(2019-nCoV)的重组抗原或编码上述新型冠状病毒(2019-nCoV)重组抗原的基因也可以用于制备治疗新型冠状病毒(2019-nCoV)药物，如疫苗；所述疫苗可以包含佐剂。
序列表
<110> 重庆医科大学，博奥赛斯（天津）生物科技有限公司
<120> 新型冠状病毒(2019-nCoV)重组抗原及多克隆抗体
<140> 申请号2021101564035
<141> 申请号2021-02-04
<150> 优先权号CN2020100890988
<151> 2020-02-12
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 204
<212> PRT
<213> 已知序列
<400> 1
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
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Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
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Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
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Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
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Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
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Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
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His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
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Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
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Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
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Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly
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atgagtgata acggtccgca gaaccaacgt aacgcgccac gtataacttt cggcggccct 60
agtgatagca ccggtagtaa ccagaacggc gaacgttcag gcgcgcgtag caagcagcgt 120
cgtccgcagg gcctgccgaa caacacggcg agctggttca ccgcgctgac ccagcacggc 180
aaagaggacc tgaaattccc gcgtggccag ggtgtgccga tcaacaccaa ctctagcccg 240
gatgatcaga tcggctacta ccgtcgtgcg acccgtcgca tccgtggtgg tgatggcaaa 300
atgaaagacc tgagcccgcg ttggtacttc tactacctgg gcaccggccc ggaagcgggc 360
ctgccgtacg gtgcgaacaa agatggcatt atctgggttg cgaccgaagg cgcgctgaac 420
accccgaaag atcacatcgg cacccgtaac ccggcgaaca acgcggcgat cgttctccag 480
ctgccgcagg gcaccaccct gccaaaaggc ttctacgcgg aaggtagccg tggcggtagc 540
caggcgagca gccgtagcag cagccgcagc cgtaactcta gccgtaacag cactccgggt 600
agcagccgtg gc 612
Claims (6)
1.一种新型冠状病毒（2019-nCoV）重组抗原肽，其氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述的新型冠状病毒（2019-nCoV）重组抗原肽的基因，其特征在于，其核苷酸序列如SEQ ID NO.2所示。
3.一种重组载体，其含有权利要求2所述新型冠状病毒（2019-nCoV）重组抗原肽的基因。
4.一种重组菌，其包含权利要求3所述的重组载体。
5.如权利要求4所述重组菌，其特征在于：所述重组菌选自重组大肠杆菌。
6.如权利要求1所述新型冠状病毒（2019-nCoV）的重组抗原肽在制备新型冠状病毒免疫检测试剂盒中的应用。
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