CN114957486B - 一种诺如病毒纳米抗体及其应用 - Google Patents
一种诺如病毒纳米抗体及其应用 Download PDFInfo
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Abstract
本发明提供一种上述特异性结合诺如病毒多基因型VP1蛋白的抗体及其应用，本发明提供的单链纳米抗体Nano‑NoVVP1可以广泛的识别近年来亚洲流行诺如病毒，与其他病毒无交叉反应，特异性高。可用于检测样品中诺如病毒的存在或水平试剂盒的研发，实现样品的快速检测，具有良好的应用前景，可以广泛的应用在食品检测、水样检测、进出口产品安全监测、环境样本检测等方面。
Description
技术领域
本发明涉及生物技术领域，更具体地，本发明涉及一种特异性结合诺如病毒的纳米抗体及其应用。
背景技术
诺如病毒(norovirus，NoV)是引起全球各年龄段人群急性胃肠炎(acutegastroenteritis，AGE)暴发流行及散发的主要病原体，由NoV引起的AGE占全球AGE发病总数的近五分之一。诺如病毒是一种全球范围内重要的食源性病毒，可感染人和动物，引起急性胃肠炎。无论是在发达国家还是发展中国家，诺如病毒流行性感染涉及到各个年龄段人群，并可造成暴发性流行，已成为影响人类日常健康的不可忽视的问题。诺如病毒主要来源于海产品，常见于牡蛎等贝类中。可经食源性途径、水源性途径及人-人直接接触(粪口途径或呕吐物的气溶胶)传播；病毒粒子具有高度传染性，约18到100个病毒粒子就可以让人感染，病毒易在小范围内迅速传播，在半封闭和人口集中场所中常出现聚集性感染及群体暴发。目前市场上没有特异性抗NoV的药物及预防和治疗性疫苗，抗生素对该病毒感染引起的腹泻几乎没有作用。
NoV属杯状病毒科，是单股正链小RNA病毒，直径为27～38nm，基因组全长为7.4～7.7kb，包含3个开放阅读框(Openreading frames，ORFs)。ORF1长约5kb，编码一个多聚蛋白(蛋白前体)，病毒复制过程中经过蛋白酶解后可形成6个病毒复制所必需的非结构蛋白，包括N末端蛋白、NTPase(核苷磷酸酶)、p22(3A样蛋白)、VPg(与病毒基因组共价结合)，Pro(3C样蛋白酶)、RNA依赖的RNA聚合酶等；ORF2和ORF3编码结构蛋白，ORF2长约1.8kb，编码57kD的主要结构蛋白VP1，VP1蛋白可在体外包装成VLPs；ORF3长约0.6kb，编码22kD的次要结构蛋白VP2，VP2的功能可能与基因组包装成病毒体有关。基于VP1氨基酸序列的多样性，可将NoV分为GI～GX共10个基因组，49个基因型。10个基因组中GI、GII和GIV基因组可感染人类，而人类诺如病毒感染中，GI.1和GII.4型占据主导地位，占世界范围内流行NoV的70～80％。
对编码病毒衣壳蛋白的VP1序列的分析研究表明，不同基因组间的差别在50％以上，而同一基因组的不同基因型间差异高达40％。因此现有检测技术，尤其是免疫检测技术中使用的针对VP1的抗体往往只针对一种基因型的诺如病毒，无法实现一种抗体识别多种基因型的缺陷。目前对诺如病毒感染、致病机制、病毒与宿主相互作用、靶细胞受体等机理性研究相对较少，因此针对诺如病毒的疫苗尚不成熟。“诺如病毒”感染性腹泻属于自限性疾病，没有疫苗和特效药物，因此有效的预防食品中诺如病毒污染成为了公共卫生，尤其是针对幼儿园的公共卫生预防的关键。在实际应用中，尤其是涉及到食品检测或者抽样检测领域，检验者往往并不关注食品中受到那种基因型的诺如病毒的污染，而是直接关注只要受到污染就一票否决，因此需要寻找这样一种可以识别多种基因型的诺如病毒抗体，并将其应用于诺如病毒的食品检测的诊断、检测中十分必要。
发明内容
本发明要解决的技术问题是针对现有技术中缺乏更好、更有效的针对多种诺如病毒基因型VP1蛋白的特异性抗体，提供了一种特异性结合诺如病毒多基因型VP1蛋白的抗体及其制备方法和应用。
为了解决上述技术问题，本发明提供的技术方案为：
一种诺如病毒多基因型VP1蛋白的抗原表位融合多肽，其中包含近年来亚洲国家较为流行的GI.3/2019中国台湾株(MN922738)、GII.4/2012Sydney株、GII.P7-GII.17株的VP1主要抗原表位，并对其进行优化，暴露其抗原表位，其氨基酸序列为：DPTTCGSPATAFGSGESVKRLDTCRLVAVAARSSHYTDAYEANADPRNWVRSKLAPGRKTNSPVSDQHRHDVHASGGNGPVVSGVNGMR(SEQ ID NO.1)。
进一步的，本发明提供一种特异性结合诺如病毒多基因型VP1蛋白的抗体，所述抗体为单链纳米抗体Nano-NoVVP1，其序列为DVGLVQQLQELGSGGRPASLGASFNYNNWSVYWIKQEWFRRGKEAPGVSFTNGNKYEKFYKGRFTTDSVDTAARAVYDNLYQMNVYPECAGKTQTLVAYPATVSGGAPDWTQVSTQGGVS(SEQ ID NO.2)。
所述一种特异性结合诺如病毒多基因型VP1蛋白的抗体可使用本领域技术人员公知的方法获得，如利用表达载体进行蛋白表达纯化、多肽固相合成方法等。
进一步的，所述表达载体可以为真核表达载体或原核表达载体，优选的，所述表达载体为真核表达载体，所述表达载体可选自：pCRII、pCR3和pcDNA3.1(Invitrogen，SanDiego，CA)、pB SII(Stratagene，La Jolla，CA)、pET 15(Novagen，Madison，WI)、pGEX(Pharmacia Biotech，Piscataway，NJ)、pEGFP-N1(Clontech，Palo Alto，CA)、pETL(BlueBacII，Invitrogen)、pDSR-α(PCT Pub.No.WO90/14363)和pFastBacDual(Gibco-BRL，Grand Island，NY)等。
所述宿主细胞为大肠杆菌、酵母或真核细胞，优选的，所述宿主细胞为真核细胞，可选自例如：中国仓鼠的卵巢细胞CHO、猴的肾脏细胞COS细胞、人的胚肾细胞HEK-293、人的子宫颈癌细胞HELA等。
进一步的，本发明还提供一种上述特异性结合诺如病毒多基因型VP1蛋白的抗体在制备检测诺如病毒试剂中的应用。
进一步的，所述应用为非疾病诊断目的的检测，例如：食品检测、水样检测、进出口产品安全监测、环境样本检测等方面。
有益效果
本发明提供的诺如病毒多基因型VP1蛋白的抗原表位融合多肽，其中包含近年来亚洲国家较为流行的GI.3/2019中国台湾株(MN922738)、GII.4/2012Sydney株、GII.P7-GII.17株的VP1主要抗原表位，而且高级结构分析显示其核心抗原表位都位于螺旋结构中，可以在高级结构中充分暴露，为诺如病毒的免疫监测提供基础。
本发明提供的单链纳米抗体Nano-NoVVP1可以广泛的识别近年来亚洲流行诺如病毒，与其他病毒无交叉反应，特异性高。可用于检测样品中诺如病毒的存在或水平试剂盒的研发，实现样品的快速检测，具有良好的应用前景。
附图说明
图1为本发明诺如病毒多基因型VP1蛋白的抗原表位融合多肽的高级结构预测。
具体实施方式
除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
为了使本领域的技术人员更好地理解本发明的技术方案，下面结合具体实施例对本发明作进一步的详细说明。
实施例1
首先针对近年来亚洲国家较为流行的GI.3/2019中国台湾株(MN922738)、GII.4/2012Sydney株、GII.P7-GII.17株的VP1主要抗原表位进行同源性分析，并利用生物分析手段筛选抗原片段候选区域，并根据生物信息学分析对其中的部分氨基酸残基进行替换或者优化(参见图1)，最终确定其抗原表位融合多肽的氨基酸序列为DPTTCGSPATAFGSGESVKRLDTCRLVAVAARSSHYTDAYEANADPRNWVRSKLAPGRKTNSPVSDQHRHDVHASGGNGPVVSGVNGMR(SEQ IDNO.1)。
利用overlap PCR方法构建抗原表位融合多肽表达盒，利用双酶切将其连接到克隆至PET-28a(购自Invitrogen公司，货号A11499)，挑取单克隆鉴定插入方向，插入方向正确的质粒送Invitrogen公司测序，测序正确的质粒命名为PET-28a-NoVVP1。将PET-28a-NoVVP1重组阳性质粒进行表达，经SDS-PAGE验证后经镍柱层析法纯化获得可溶性融合蛋白。
实施例2针对抗原表位融合多肽NoVVP1的天然单域抗体的免疫淘选过程
(1)将已建立的天然单域抗体噬菌体文库进行扩增：100μL甘油菌文库加入2×YT培养基，OD600＝0.5时，加入20MOI辅助噬菌体，静置30min，离心后将沉淀用2×YT培养基重悬，再培养1h，再加抗生素培养16h后离心，其上清经预冷PEG-NaCl(1/4体积)沉淀，1mL PBS重悬即为扩增的单域抗体文库；
(2)免疫管淘选：将带生物素标签并且纯化后的抗原表位融合多肽NOVVP1按照50μg/管包被免疫管过夜，去除包被液后洗涤3次，用2mL BSA(1％)封闭2h，PBST洗涤3次，加入100μL上述步骤(1)扩增的单域抗体文库作为一抗，37℃作用2h，PBST洗涤3次，用Glycine-HCI(PH2.2)洗脱，洗脱液用Tris-HCI调节至PH 7.4，获得淘选的第一轮天然单域抗体文库；
(3)将(2)步获得的第1轮天然单域抗体文库按照(1)步进行扩增，获得第1轮天然单域抗体重悬文库，然后重复(2)步免疫管淘选步骤，只是一抗加入的是100μL扩增的第1轮天然单域抗体重悬文库，最后获得淘选的第2轮天然单域抗体文库；(4)将(3)步获得的第2轮天然单域抗体文库按照(1)步进行扩增，获得第2轮天然单域抗体重悬文库，然后重复(2)步免疫管淘选步骤，只是一抗加入的是100μL扩增的第2轮天然单域抗体重悬文库，最后获得淘选的第3轮天然单域抗体文库。
实施例3单个克隆的ELISA鉴定
(1)淘选的单个阳性克隆摇菌扩增用于抗体表达：将上述淘选的第3轮天然单域抗体文库接种于2×YT培养基，OD600nm＝0.5时，加入20MOI辅助噬菌体，静置30min，离心后将沉淀用2×YT培养基重悬，再培养1h，然后涂布于含抗生素的2×YT平板，培养过夜，次日挑选40个单菌落接种到2×YT培养基中，OD600＝0.5时，加入20MOI辅助噬菌体，静置30min，离心后将沉淀用2×YT培养基重悬，再培养，并加入IPTG进行诱导表达8h。
(2)ELISA鉴定：将带生物素标签并且纯化后的抗原表位融合多肽NOVVP1(浓度1ng/μL)100μL/孔包被ELISA板过夜，去除包被液后洗涤3次，用200μL/孔BSA(3％)封闭2h，PBST洗涤3次，加入100μL/孔(1)步分别扩增的单域抗体文库作为一抗(文库构建载体带M13)，37℃作用2h，PBST洗涤3次，二抗加入M13-HRP，终止后检测OD450nm值，结果判读：比对照组OD450nm值高出3倍判为阳性。
实施例4阳性克隆的测序鉴定
将实施例3中经ELISA检测为阳性的克隆对应相应菌液提取质粒，利用质粒载体的通用引物进行测序。根据序列比对软件MEGA 6.0分析各个克隆株的基因序列，把CDR1、CDR2、CDR3序列相同的株视为同一克隆株，而其序列不同的株视为不同克隆株，最终获得针对抗原表位融合多肽NOVVP1特异性单域抗体Nano-NoVVP1，其序列为DVGLVQQLQELGSGGRPASLGASFNYNNWSVYWIKQEWFRRGKEAPGVSFTNGNKYEKFYKGRFTTDSVDTAARAVYDNLYQMNVYPECAGKTQTLVAYPATVSGGAPDWTQVSTQGGVS(SEQ ID NO.2)。
实施例5抗体型别特异性的检测
利用间接ELISA法，包被原分别为GI.3/2019中国台湾株(MN922738)、GII.4/2012Sydney株、GII.P7-GII.17株的VP1蛋白，鼠GV型诺如病毒、犬GVI型诺如病毒的VP1蛋白，胎儿弯曲杆菌O蛋白、肠出血性大肠杆菌O157∶H7脂多糖(LPS)蛋白、甲型副伤寒沙门氏菌A群O抗原蛋白、单增李斯特菌表面蛋白LMO1847抗原、副溶血性弧菌FlaE蛋白，100ng/孔。对该纳米抗体进行检测(表1)，结果显示上述抗体对上述感染人的诺如病毒的VP1蛋白反应为阳性，但对其他非人诺如病毒的VP蛋白或者其他常见病毒的检测结果均为阴性，确定该纳米抗体为人感染诺如病毒特异性抗体。
表1抗体型别特异性的检测结果
实施例6特异性单域抗体Nano-NoVVP1的中和活性及稳定性鉴定
在实验室采用微量细胞中和试验进行验证。将单域抗体Nano-NoVVP1用生理盐水进行倍比稀释1:2～1:128，将1份免疫血清作为阳性对照，也进行1:2～1:128的倍比稀释，每个稀释度设置5个重复。在稀释好待检样本的培养板各孔中均加入含100TCID50的病毒液，同时设置阴性血清对照的倍比稀释孔，补加等量的稀释液。将所有培养板放入37℃细胞培养箱中和2h。中和后每孔加Vero细胞悬液，放置37℃培养箱培养5d。同时设正常细胞对照。5d后观察并计数每个培养孔是否发生病变，即能够保护50％细胞不受100TCID50病毒感染的血清最高稀释度为该血清的抗体滴度，全程试验重复3次。单域抗体Nano-NoVVP1最终检测效价为1:64、阳性对照血清的中和效价为1:32(≧1：4判定为阳性)。中和活性试验结果表明，单域抗体Nano-NoVVP1具有较高的中和效价(1:64)，3次重复试验的变异系数<3％，说明该单域抗体具有良好稳定性。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
SEQUENCE LISTING
〈110〉青岛新万福食品有限公司
〈120〉一种诺如病毒纳米抗体及其应用
〈160〉 2
〈170〉 PatentIn version 3.3
〈210〉 1
〈211〉 89
〈212〉 protein
〈213〉VP1蛋白的抗原表位融合多肽
<400> 1
Asp Pro Thr Thr Cys Gly Ser Pro Ala Thr Ala Phe Gly Ser Gly
1 5 10 15
Glu Ser Val Lys Arg Leu Asp Thr Cys Arg Leu Val Ala Val Ala
20 25 30
Ala Arg Ser Ser His Tyr Thr Asp Ala Tyr Glu Ala Asn Ala Asp
35 40 45
Pro Arg Asn Trp Val Arg Ser Lys Leu Ala Pro Gly Arg Lys Thr
50 55 60
Asn Ser Pro Val Ser Asp Gln His Arg His Asp Val His Ala Ser
65 70 75
Gly Gly Asn Gly Pro Val Val Ser Gly Val Asn Gly Met Arg
80 85
〈210〉 2
〈211〉 120
〈212〉 protein
〈213〉诺如病毒单链纳米抗体Nano-NoVVP1
〈400〉2
Asp Val Gly Leu Val Gln Gln Leu Gln Glu Leu Gly Ser Gly Gly
1 5 10 15
Arg Pro Ala Ser Leu Gly Ala Ser Phe Asn Tyr Asn Asn Trp Ser
20 25 30
Val Tyr Trp Ile Lys Gln Glu Trp Phe Arg Arg Gly Lys Glu Ala
35 40 45
Pro Gly Val Ser Phe Thr Asn Gly Asn Lys Tyr Glu Lys Phe Tyr
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Lys Gly Arg Phe Thr Thr Asp Ser Val Asp Thr Ala Ala Arg Ala
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Val Tyr Asp Asn Leu Tyr Gln Met Asn Val Tyr Pro Glu Cys Ala
80 85 90
Gly Lys Thr Gln Thr Leu Val Ala Tyr Pro Ala Thr Val Ser Gly
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Gly Ala Pro Asp Trp Thr Gln Val Ser Thr Gln Gly Gly Val Ser
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Claims (5)
1.一种诺如病毒多基因型VP1蛋白的抗原表位融合多肽，其特征在于所述抗原表位融合多肽的氨基酸序列为：DPTTCGSPATAFGSGESVKRLDTCRLVAVAARSSHYTDAYEANADPRNWVRSKLAPGRKTNSPVSDQH RHDVHASGGNGPVVSGVNGMR(SEQ ID NO.1)。
2.一种特异性结合诺如病毒多基因型VP1蛋白的抗体，其特征在于，所述抗体为单链纳米抗体Nano-NoVVP1，其序列为DVGLVQQLQELGSGGRPASLGASFNYNNWSVYWIKQEWFRRGKEAPGVSFTNGNKYEKFYKGRFTT DSVDTAARAVYDNLYQMNVYPECAGKTQTLVAYPATVSGGAPDWTQVSTQGGVS(SEQID NO.2)。
3.如权利要求1所述的抗原表位融合多肽或权利要求2所述单链纳米抗体Nano-NoVVP1在制备检测诺如病毒试剂中的应用。
4.如权利要求3所述的应用，其中应用为非疾病诊断目的的检测，所述非疾病诊断目的的检测为食品检测、进出口产品安全监测、环境样本检测。
5.如权利要求4所述的应用，其中的食品检测为白条肉预制菜检测。
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