CN113121657A - 一种人肠道病毒71型和柯萨奇病毒a组2型vp1重组蛋白、多克隆抗体及其应用 - Google Patents
一种人肠道病毒71型和柯萨奇病毒a组2型vp1重组蛋白、多克隆抗体及其应用 Download PDFInfo
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- CN113121657A CN113121657A CN202110357650.1A CN202110357650A CN113121657A CN 113121657 A CN113121657 A CN 113121657A CN 202110357650 A CN202110357650 A CN 202110357650A CN 113121657 A CN113121657 A CN 113121657A
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Abstract
本发明公开了一种人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白、多克隆抗体及其应用。本发明的技术方案要点为：提供了一种EV‑A71和CV‑A2VP1重组蛋白纯化、多克隆抗体制备的方法，并应用制备的小鼠多克隆抗体建立了蛋白质免疫印迹和免疫过氧化物酶单层细胞试验检测EV‑A71和CV‑A2的方法。本发明所述的这种EV‑A71和CV‑A2VP1蛋白纯化方法简便快捷、操作性强、回收率高，多克隆抗体制备方法无需免疫佐剂，节约了成本，制备的小鼠多克隆抗体能检测细胞样品中的EV‑A71和CV‑A2，特异性和重复性好。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种人肠道病毒71型(humanenterovirus A71,EV-A71)和柯萨奇病毒A组2型(coxsackievirus A2,CV-A2)VP1重组蛋白、多克隆抗体及其应用。
背景技术
人肠道病毒71型(human enterovirus A71,EV-A71)和柯萨奇病毒A组2型(coxsackievirus A2,CV-A2)都属于小核糖核酸病毒科(Picornaviridae)肠道病毒属(Enterovirus)A组肠道病毒(Enterovirus A)。EV-A71和CV-A2感染都能引起手足口病(hand,foot and mouth disease,HFMD)。EV-A71感染引起的HFMD大多是自限性的，但有一些感染可以引起严重的神经系统疾病，如无菌性脑膜炎、脑干脑炎，甚至死亡。CV-A2感染主要引起疱疹性咽峡炎，较少引起神经系统疾病，但有报道CV-A2感染会引起小儿麻痹症样麻痹、合并神经表现、心肌炎、严重脑炎甚至死亡。
EV-A71和CV-A2都是单股正链RNA病毒。EV-A71基因组长约7.5kb，只有一个编码了2193个氨基酸的开放阅读框(open reading frame,ORF)，两端分别为5′和3′非翻译区(untranslated regions,UTRs)。在EV71复制过程中，其编码的多聚蛋白被细分为P1、P2和P3区域：P1编码四种结构蛋白(VP1、VP2、VP3和VP4)，而P2和P3编码七种非结构蛋白(2A、2B、2C、3A、3B、3C和3D)。CV-A2基因组长约7.4kb，编码约2190个氨基酸的多聚蛋白，结构与EV-A71类似。
肠道病毒的VP1蛋白是主要的病毒中和决定因素。VP1的核苷酸序列分析可以替代中和试验分型方法，用来区分肠道病毒的血清型。EV-A71的VP1蛋白含有297个氨基酸，显示出与病毒血清型相应的完整的遗传多样性，可作为EV-A71血清型分类的依据。EV-A71的VP1蛋白可诱导对EV71的有效免疫保护，是抗EV71感染的理想候选蛋白，其N末端可能具有重要的中和抗体决定簇，可能作为急性感染诊断和治疗抗体的的靶点。CV-A2全长VP1核苷酸序列有885bp，编码295个氨基酸。目前关于CV-A2 VP1蛋白的研究报道较少。
发明内容
本发明解决的技术问题是提供了一种人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白、多克隆抗体及其应用，本发明分别扩增了EV-A71和CV-A2的全长VP1基因并进行了原核表达，采用超声破碎、差速离心、氯化钾染色切胶回收、液氮研磨等简便易行的方法分别对EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白进行了纯化，通过腹腔注射免疫小鼠成功地制备了小鼠抗EV-A71 VP1多克隆抗体和CV-A2 VP1多克隆抗体，并应用多克隆抗体通过蛋白质免疫印迹(Western blot)和免疫过氧化物酶单层细胞试验(immunoperoxidasemonolayer assay,IPMA)检测EV-A71 VP1和CV-A2 VP1在病毒感染人横纹肌肉瘤细胞RD中的表达。
本发明为解决上述技术问题采用如下技术方案：
一种人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白，其特征在于：该人肠道病毒71型VP1重组蛋白的氨基酸序列如序列表SEQ ID NO.2所示；柯萨奇病毒A组2型VP1重组蛋白的氨基酸序列如序列表SEQ ID NO.4所示。
本发明所述的人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白的制备方法，其特征在于具体步骤为：
步骤S1：构建携带EV-A71 VP1基因的重组质粒pET-28a(+)EV-A71 VP1和携带有CV-A2 VP1基因的重组质粒pET-28a(+)CV-A2 VP1；
步骤S2：将重组质粒分别转化大肠杆菌BL21(DE3)感受态细胞，诱导表达EV-A71VP1重组蛋白和CV-A2 VP1重组蛋白，对EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白分别进行纯化即得人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白。
进一步限定，步骤S2的具体过程为：将含有EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白的细菌裂解液，分别用超声波破碎后进行差速离心，重复四次，最后3000×g离心沉淀包涵体，用3/80初始菌液体积重悬；用12％(m/V)十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离上述重悬液中的EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白，用冰冷的5％(m/V)氯化钾水溶液对分离胶进行漂染，切胶回收乳白色蛋白条带；在回收胶中加入适量磷酸盐缓冲液后用液氮研磨三次，最终获得纯化的EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白。
一种基于人肠道病毒71型VP1重组蛋白的多克隆抗体及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体。
本发明所述的基于人肠道病毒71型VP1重组蛋白的多克隆抗体及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体的制备方法，其特征在于：将纯化后的EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白分别作为抗原通过腹腔注射免疫Balb/c小鼠，制备小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1。
本发明所述的基于人肠道病毒71型VP1重组蛋白的多克隆抗体及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体的制备方法，其特征在于具体过程为：将纯化的EV-A71VP1重组蛋白和CV-A2 VP1重组蛋白分别免疫12周龄Balb/c小鼠，首次免疫用200μL剂量，以后每隔三到四周加强免疫，加强免疫用100～150μL剂量，免疫均采用腹腔注射方法；第四次免疫后三周和第五次免疫后第3-4天分别取少量血清，用蛋白质免疫印迹测定抗体效价，抗体效价在1:5000及以上的小鼠，在第五次免疫后9-10天取血并分离血清，获得小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1。
本发明所述的基于人肠道病毒71型VP1重组蛋白的多克隆抗体及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体在制备人肠道病毒71型和柯萨奇病毒A组2型特异性检测试剂中的应用。
本发明所述的基于人肠道病毒71型VP1重组蛋白的多克隆抗体在制备人肠道病毒71型免疫印迹或免疫过氧化物酶单层细胞试验检测试剂中的应用以及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体在制备柯萨奇病毒A组2型免疫印迹或免疫过氧化物酶单层细胞试验检测试剂中的应用。
本发明与现有技术相比具有以下优点：
1.本发明采用氯化钾染色法切胶回收、液氮研磨回收胶获得纯化的EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白，该方法具有简便快捷、操作性强、回收率高的优点。
2.本发明采用纯化的EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白直接腹腔免疫Balb/c小鼠制备多克隆抗体小鼠anti-EV-A71 VP1和anti-CV-A2 VP1，间隔三到四周加强免疫四次即能获得较高效价的抗体，无需免疫佐剂，节约了成本。
3.本发明制备的小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1分别检测感染细胞样品中的EV-A71和CV-A2 VP1，特异性和重复性好。
4.本发明能为研发EV-A71和CV-A2基于抗原抗体反应的其他检测方法提供参考。
附图说明
图1是琼脂糖凝胶电泳检测PCR扩增EV-A71和CV-A2 VP1基因产物。M：
Plus II DNAMarker；1.EV-A71 VP1基因片段；2.CV-A2 VP1基因片段。
图2是琼脂糖凝胶电泳检测pET-28a(+)-EV-A71 VP1和pET-28a(+)-CV-A2 VP1阳性克隆的BamHI和XhoI双酶切产物。M：
Plus II DNAMarker；1.pET-28a(+)-EV-A71VP1；2.pET-28a(+)-CV-A2 VP1。
图3是SDS-PAGE分析重组EV-A71和CV-A2 VP1蛋白的表达。M：Thermo ScientificTMPageRulerTM Prestained Protein Ladder；1.pET-28a(+)-EV-A71 VP1/BL21(DE3)菌1mMIPTG 37℃诱导5h；2.pET-28a(+)-EV-A71 VP1/BL21(DE3)菌未诱导(对照)；3.pET-28a(+)-CV-A2VP1/BL21(DE3)菌1mM IPTG 37℃诱导5h；4.pET-28a(+)-CV-A2 VP1/BL21(DE3)菌未诱导(对照)。
图4是SDS-PAGE分析重组EV-A71和CV-A2 VP1蛋白的纯化。M：Thermo ScientificTMPageRulerTM Prestained Protein Ladder；1.pET-28a(+)-EV-A71 VP1/BL21(DE3)菌未诱导(对照)；2.pET-28a(+)-EV-A71 VP1/BL21(DE3)菌诱导；3.2经第一次破碎后9384×g离心30min获得的上清；4.2经第一次破碎后9384×g离心30min获得的沉淀；5.2经第四次破碎后3000×g离心10min获得的沉淀；6.5经12％(m/V)SDS-PAGE分离、KCl染色割胶回收、液氮研磨获得的重组EV-A71 VP1蛋白；7.pET-28a(+)-CV-A2 VP1/BL21(DE3)菌未诱导(对照)；8.pET-28a(+)-CV-A2 VP1/BL21(DE3)菌诱导；9.8经第一次破碎后9384×g离心30min获得的上清；10.8经第一次破碎后9384×g离心30min获得的沉淀；11.8经第四次破碎后3000×g离心10min获得的沉淀；12.8经12％(m/V)SDS-PAGE分离、KCl染色割胶回收、液氮研磨获得的重组CV-A2 VP1蛋白。
图5是Western blot测定重组EV-A71和CV-A2 VP1蛋白免疫Balb/c小鼠血清效价。A.重组EV-A71 VP1蛋白免疫Balb/c小鼠血清效价测定。M:Thermo ScientificTMPageRulerTM Prestained Protein Ladder；1～4：一抗分别为免疫小鼠血清1:1000、1:5000、1:10000、1:20000稀释；5.未免疫小鼠血清1:1000稀释。B.重组CV-A2 VP1蛋白免疫Balb/c小鼠血清效价测定。M：Thermo ScientificTM PageRulerTM Prestained ProteinLadder；1～4：一抗分别为免疫小鼠血清1:1000、1:5000、1:10000、1:20000稀释；5.未免疫小鼠血清1:1000稀释。二抗为AP标记羊抗小鼠IgG(1:8000稀释)，BCIP/NBT工作液显色。
图6是Western blot检测病毒感染RD细胞中的VP1。A.小鼠多克隆抗体anti-EV-A71 VP1(1:1000)检测EV-A71感染RD细胞中的EV-A71 VP1。M:Thermo ScientificTMPageRulerTM Prestained Protein Ladder；1.EV71感染RD细胞；2.模拟感染RD细胞。B.小鼠多克隆抗体anti-CV-A2 VP1(1:1000)检测CV-A2感染RD细胞中的CV-A2 VP1。M：ThermoScientificTM PageRulerTM Prestained Protein Ladder；1.CV-A2感染RD细胞；2.模拟感染RD细胞。
图7是IPMA检测病毒感染RD细胞中的VP1。A.小鼠多克隆抗体anti-EV-A71 VP1检测EV-A71感染RD细胞中的EV-A71 VP1。左上：模拟感染RD细胞不加抗体染色(对照)；右上：模拟感染RD细胞加小鼠anti-EV-A71 VP1(1:1000)染色；左下：EV-A71感染RD细胞不加抗体染色(对照)；右下：EV-A71感染RD细胞加小鼠anti-EV-A71 VP1(1:1000)染色。B.小鼠多克隆抗体anti-CV-A2 VP1(1:1000)检测CV-A2感染RD细胞中的CV-A2 VP1。左上：模拟感染RD细胞，不加抗体染色(对照)；右上：模拟感染RD细胞，加小鼠anti-CV-A2 VP1(1:1000)染色；左下：CV-A2感染RD细胞，不加抗体染色(对照)；右下：CV-A2感染RD细胞，加小鼠anti-CV-A2VP1(1:1000)染色。
具体实施方式
下面通过实施例对本发明的上述内容做进一步阐述，这些实施例只用于说明本发明，并非用于限制本发明的范围。以下实施例中未注明具体条件的实验方法，通常按照常规条件进行。
需特别指出的是，尽管本发明阐述的是一种EV-A71和CV-A2 VP1重组蛋白纯化、多克隆抗体制备的方法及其应用，但应用本发明对感染任何其他细胞的EV-A71和CV-A2的检测也包括在本发明所要求的权利范围之内。
下述实验病毒，本申请人实验室均有保存，在确保生物安全的前提下，可以对外公开发放。
实施例1
EV-A71和CV-A2 VP1基因扩增及目的片段胶回收
实验材料：本实验室保存的携带EV-A71 VP1基因的质粒pMD19-T EV-A71 VP1+和携带CV-A2 VP1基因的质粒pMD19-T CV-A2 VP1+。所用引物由生工生物工程(上海)股份有限公司合成(如表1所示)，单下划线部分表示酶切位点BamHI，双下划线部分表示酶切位点XhoI。高成功率PCR酶KOD FX购自TOYOBO CO.,LTD.。Tris base购自BIOSHIARP。盐酸和冰乙酸购自天津市德恩化学试剂有限公司。乙二胺四乙酸二钠(EDTA-Na2)购自国药集团化学试剂有限公司。琼脂糖(
REGULAR AGAROSEG-10)购自GENE COMPANY LTD。
Plus II DNA Marker购自北京全式金生物技术有限公司。核酸染料GelRedTMNucleic Acid Gel Stain购自Biotium。
Gel Extraction Kit购自Omega Bio-tek,Inc.。
参考《分子克隆实验指南(第三版)》(下册)配制Tris-乙酸缓冲液(TAE)：40mMTris-乙酸，1mM EDTA。
表1 EV-A71和CV-A2 VP1基因扩增用引物
实验方案：
分别以pMD19-T EV-A71 VP1+和pMD19-T CV-A2 VP1+为模板、EV-A71-VP1-F1/EV-A71-VP1-R1和CV-A2-VP1-F1/CV-A2-VP1-R1为引物对，用高成功率PCR酶KOD FX扩增EV-A71和CV-A2的VP1基因。PCR扩增反应体系如表2所示。PCR反应条件为：94℃，2min，(98℃，10sec；56℃，30sec；68℃，1min)35个循环，68℃终延伸10min。取1μL PCR产物用0.8％(m/V)琼脂糖-TAE凝胶电泳检测。剩余PCR产物按照
Gel Extraction Kit进行胶回收。
实验结果：电泳检测PCR产物，发现均在750～1000bp之间有明显的条带(如图1所示)，其大小与预期扩增EV-A71 VP1和CV-A2 VP1基因片段相符，表明成功扩增EV-A71VP1和CV-A2 VP1基因。
表2 扩增EV-A71和CV-A2 VP1基因的PCR体系
实施例2
重组表达载体的构建与筛选
实验材料：pET-28a(+)质粒、CaCl2法制备的大肠杆菌DH5α感受态细胞为本实验室保存。BamHI和XhoI限制性内切酶、T4DNA连接酶购自NEB(北京)有限公司。
Plus IIDNA Marker和
-T DNA Polymerase购自北京全式金生物技术有限公司。核酸染料GelRedTM Nucleic Acid Gel Stain购自Biotium。Tris base和Agar(琼脂，日本进口分装)购自BIOSHIARP。盐酸和冰乙酸购自天津市德恩化学试剂有限公司。乙二胺四乙酸二钠(EDTA-Na2)、氯化钠(NaCl)和氢氧化钠(NaOH)购自国药集团化学试剂有限公司。琼脂糖(
REGULAR AGAROSEG-10)购自GENE COMPANY LTD。
Cycle Pure Kit购自Omega Bio-tek,Inc.。TRYPTONE(蛋白胨)和YEAST EXTRACT(酵母提取物)购自OXOIDLTD.。硫酸卡那霉素购自生工生物工程(上海)股份有限公司。
参考《分子克隆实验指南(第三版)》(下册)准备以下材料：
Tris-乙酸缓冲液(TAE)：40mM Tris-乙酸，1mM EDTA。
LB(Luria-Bertani)液体培养基：10g/L蛋白胨，5g/L酵母提取物，10g/L NaCl，去离子水中加溶质溶解后，用NaOH调节pH至7.0后定容，15psi高压蒸气灭菌20min。
含10μg/mL硫酸卡那霉素的LB固体培养基平板：LB液体培养基灭菌前，加入15g/L琼脂后高压灭菌，降温至50℃左右，加入10μg/mL硫酸卡那霉素，旋动混匀，倒入平板后，完全凝固后，倒置平板并贮存于4℃备用。
实验方案：
按照BamHI和XhoI限制性内切酶说明书，取约1μg pET-28a(+)质粒作为载体和4μg回收片段进行双酶切(体系如表3所示)，37℃保温3h。取3μL酶切产物用0.8％(m/V)琼脂糖-TAE凝胶电泳检测。剩余酶切产物用
Cycle Pure Kit回收，取2μL二次回收产物电泳检测。
用T4DNA连接酶连接回收的载体片段和二次回收的目的片段(连接体系如表4所示)，16℃过夜。全部连接产物与大肠杆菌DH5α感受态细胞混合，冰浴30min，42℃热激90sec后，冰上放置2min，加入1mL 37℃预热的LB液体培养基，37℃180rpm振荡培养1h。2500×g离心3min后，弃去900μL上清，剩余100μL上清重悬沉淀的细菌，涂布含10μg/mL硫酸卡那霉素的LB固体培养基平板。稍微晾干后，将平板倒置于生化培养箱中37℃过夜培养。
表3 EV-A71和CV-A2 VP1基因片段酶切体系
挑取平板上的单菌落，加入10μL无菌超纯水中混匀。分别取1μL菌液为模板、以EV-A71-VP1-F1/EV-A71-VP1-R1和CV-A2-VP1-F1/CV-A2-VP1-R1为引物对，用
-T DNAPolymerase扩增菌落中的EV-A71和CV-A2的VP1基因，即用菌落PCR筛选重组表达载体的阳性克隆。菌落PCR扩增反应体系如表5所示。PCR反应条件为：94℃，10min，(94℃，30sec；56℃，30sec；72℃，1min)30个循环，72℃终延伸10min。取3μL PCR产物用0.8％(m/V)琼脂糖-TAE凝胶电泳检测，扩增出目的片段大小的菌落即为阳性克隆。取阳性克隆菌液4μL接种4mL含有10μg/mL硫酸卡那霉素的LB液体培养基，37℃振荡过夜培养。
表4 连接体系
实验结果：菌落PCR分别筛选出数个携带EV-A71和CV-A2 VP1基因的重组pET-28a(+)载体阳性克隆(数据未显示)，分别挑选一个阳性克隆用于下一步实验。
表5 菌落PCR筛选阳性克隆体系
实施例3
重组表达载体阳性克隆的鉴定与测序
实验材料：葡萄糖、乙二胺四乙酸二钠(EDTA-Na2)、氯化钠(NaCl)、氢氧化钠(NaOH)、乙酸钾和丙三醇(甘油)购自国药集团化学试剂有限公司。RNA酶A(RNase A,bovinepancreas)购自北京索莱宝科技有限公司。BamHI和XhoI限制性内切酶购自NEB(北京)有限公司。
Plus II DNA Marker购自北京全式金生物技术有限公司。核酸染料GelRedTM NucleicAcid Gel Stain购自Biotium。TRYPTONE(蛋白胨)和YEAST EXTRACT(酵母提取物)购自OXOID LTD.。硫酸卡那霉素购自生工生物工程(上海)股份有限公司。Trisbase、十二烷基硫酸钠(SDS)和Agar(琼脂，日本进口分装)购自BIOSHIARP。盐酸和冰乙酸购自天津市德恩化学试剂有限公司。琼脂糖(
REGULAR AGAROSEG-10)购自GENECOMPANY LTD。
参考《分子克隆实验指南(第三版)》(下册)配制以下试剂：
Tris-乙酸缓冲液(TAE)：40mM Tris-乙酸，1mM EDTA。
LB(Luria-Bertani)液体培养基配制：10g/L蛋白胨，5g/L酵母提取物，10g/LNaCl，去离子水中加溶质溶解后，用NaOH调节pH至7.0后定容，15psi高压蒸气灭菌20min。
含10μg/mL硫酸卡那霉素的LB固体培养基平板制备：LB液体培养基灭菌前，加入15g/L琼脂后高压灭菌，降温至50℃左右，加入10μg/mL硫酸卡那霉素，旋动混匀，倒入平板后，完全凝固后，倒置平板并贮存于4℃备用。
碱裂解溶液I(制备质粒)：50mM葡萄糖，25mM Tris-Cl(pH8.0)，10mM EDTA(pH8.0)，在15psi压力下蒸气灭菌15min，保存于4℃。
碱裂解溶液II(制备质粒)：0.2M NaOH(从10M贮存液中现用现稀释)，1％(m/V)SDS，现配现用，室温下使用。
碱裂解液溶液III：5M乙酸钾60.0mL，冰乙酸11.5mL，超纯水28.5mL，保存于4℃，用时置于冰浴中。
实验方案：
取实施例3中振荡培养后的菌液1.5mL，12,000×g离心30sec沉淀菌体，弃上清，参考《分子克隆实验指南(第三版)》(上册)采用SDS碱裂解法制备质粒DNA(小量制备)，用20μL含有20μg/mL RNA酶A的无菌超纯水溶解提取的质粒，取1μL质粒DNA溶液用0.8％(m/V)琼脂糖-TAE凝胶电泳检测。按照BamHI和XhoI限制性内切酶说明书，取有目的质粒大小条带的DNA溶液进行双酶切(体系如表6所示)，37℃保温2h。取10μL酶切产物用0.8％(m/V)琼脂糖-TAE凝胶电泳检测。
取相应的菌液在含10μg/mL硫酸卡那霉素的LB固体培养基平板上划线。平板在生化培养箱中37℃过夜培养后，挑取线上的单菌落接种4mL含有10μg/mL硫酸卡那霉素的LB液体培养基，37℃振荡过夜培养。取300μL菌液送到生工生物工程(上海)股份有限公司测序，700μL菌液加入300μL 50％(V/V)无菌甘油水溶液冻存于-80℃备用，其余菌液12,000×g离心30sec，收集菌体沉淀冻存于-20℃备用。
表6 重组表达载体阳性克隆质粒DNA酶切体系
实验结果：电泳检测阳性克隆质粒经BamHI和XhoI双酶切产物，发现大片段均位于5000～8000bp之间，小片段均位于1000bp左右(如图2所示)，其大小分别与pET-28a(+)载体、EV-A71和CV-A2 VP1基因片段相符，表明两种阳性克隆质粒酶切鉴定正确。对阳性克隆分别测序，所得序列与预期序列比对，发现对应序列完全一致。EV-A71 VP1基因核苷酸序列见序列表SEQ ID NO.1，重组EV-A71 VP1氨基酸序列见SEQ ID NO.2，CV-A2 VP1基因核苷酸序列见SEQ ID NO.3，重组CV-A2 VP1氨基酸序列见SEQ ID NO.4。结果表明，构建的携带EV-A71 VP1和CV-A2 VP1基因的重组pET-28a(+)载体pET-28a(+)-EV-A71 VP1和pET-28a(+)-CV-A2 VP1核苷酸、阅读框和方向均正确。
实施例4
EV-A71和CV-A2 VP1重组蛋白表达
实验材料：
Plasmid DNA Mini Kit Ⅰ购自Omega Bio-tek,Inc.。CaCl2法制备的大肠杆菌BL21(DE3)感受态细胞为本实验室保存。氯化钠(NaCl)和氢氧化钠(NaOH)购自国药集团化学试剂有限公司。TRYPTONE(蛋白胨)和YEAST EXTRACT(酵母提取物)购自OXOID LTD.。硫酸卡那霉素、异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-Thiogalactoside,IPTG)、丙烯酰胺/甲叉双丙烯酰胺30％(m/V)溶液(29:1)、过硫酸铵(Ammoniumpersulfate,APS)、N,N,N',N'-四甲基乙二胺(TEMED)、考马斯亮蓝R-250、溴酚蓝和β-巯基乙醇购自生工生物工程(上海)股份有限公司。Tris base、甘氨酸、十二烷基硫酸钠(SDS)和Agar(琼脂，日本进口分装)购自BIOSHIARP。盐酸、冰乙酸、甲醇和无水乙醇购自天津市德恩化学试剂有限公司。10×磷酸盐缓冲液(Phosphate Buffered Saline,PBS)购自博迈德生物。预染蛋白分子量(PageRulerTM Prestained Protein Ladder,10 to 180kDa)购自赛默飞世尔科技(中国)有限公司。
5×SDS-PAGE上样缓冲液配制：参考《分子克隆实验指南(第三版)》(下册)中2×SDS凝胶加样缓冲液配方计算，250mM Tris-Cl(pH6.8)，10％(m/V)SDS，0.5％(m/V)溴酚蓝，50％(V/V)甘油，500mMβ-巯基乙醇，分装成小份-20℃冻存备用。
蛋白胶脱色液配制：参考《分子克隆实验指南(第三版)》(下册)中甲醇：乙酸溶液配方，用乙醇替代甲醇，将900mL乙醇：水(500mL乙醇和400mL水)与100mL冰乙酸混合。
参考《分子克隆实验指南(第三版)》(下册)准备以下材料：
LB(Luria-Bertani)液体培养基配制：10g/L蛋白胨，5g/L酵母提取物，10g/LNaCl，去离子水中加溶质溶解后，用NaOH调节pH至7.0后定容，15psi高压蒸气灭菌20min。
含10μg/mL硫酸卡那霉素的LB固体培养基平板制备：LB液体培养基灭菌前，加入15g/L琼脂后高压灭菌，降温至50℃左右，加入10μg/mL硫酸卡那霉素，旋动混匀，倒入平板后，完全凝固后，倒置平板并贮存于4℃备用。
Tris-甘氨酸：25mM Tris-Cl，250mM甘氨酸，0.1％(m/V)SDS。
考马斯亮蓝R-250染色液：0.05％(m/V)考马斯亮蓝R-250溶解于甲醇：冰乙酸：水(50:10:40，V/V)溶液中，滤纸过滤除去杂质后使用。
实验方案：
按照
Plasmid DNA Mini Kit Ⅰ说明书，采用离心法提取实施例3中冻存于-20℃备用的菌体沉淀中的质粒DNA。洗脱的质粒DNA测定其浓度和纯度后，取1μL与大肠杆菌BL21(DE3)感受态细胞混合，冰浴30min，42℃热激90sec后，冰上放置2min，加入1mL 37℃预热的LB液体培养基，37℃180rpm振荡培养1h。取10μL转化菌液，用LB液体培养基稀释到100μL后，涂布含10μg/mL硫酸卡那霉素的LB固体培养基平板。稍微晾干后，将平板倒置于生化培养箱中37℃过夜培养。
挑取平板上单菌落，分别接种于4mL含10μg/mL硫酸卡那霉素的LB液体培养基，37℃振荡过夜培养。分别取700μL菌液加入300μL 50％(V/V)无菌甘油水溶液冻存于-80℃备用。再分别取40μL菌液接种于4mL含10μg/mL硫酸卡那霉素的LB液体培养基，待菌液生长至吸光度A600 0.6～0.8时，加入1mM IPTG诱导培养或不加入IPTG诱导培养(未诱导对照)。37℃振荡培养5h后，测定A600，4℃12,000×g离心1min，弃上清，沉淀加入1mL PBS重悬菌体，再加入3mL PBS摇匀，4℃12,000×g离心1min洗涤菌体，弃上清。沉淀重复上述洗涤步骤1次，弃上清，加入合适体积(15×A600/mL)PBS重悬沉淀。菌样加入适量5×SDS-PAGE上样缓冲液后煮沸10min，取12.5μL/孔上样，参考《分子克隆实验指南(第三版)》(下册)12％(m/V)十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gelelectrophoresis,SDS-PAGE)分离蛋白，考马斯亮蓝R-250染色液染色，蛋白胶脱色液脱色后，观察重组EV-A71和CV-A2 VP1蛋白的表达情况，拍照记录。
实验结果：SDS-PAGE检测发现，与未诱导菌相比，pET-28a(+)-EV-A71 VP1/BL21(DE3)菌和pET-28a(+)-CV-A2 VP1/BL21(DE3)菌诱导后均在35～40kDa之间出现了特异性条带(如图3所示)，其大小与预期重组EV-A71 VP1(36.3kDa)和CV-A2 VP1(36.6kDa)蛋白的大小相符，表明诱导菌中有目标重组蛋白表达。
实施例5
重组EV-A71和CV-A2 VP1蛋白纯化
实验材料：氯化钠(NaCl)、氯化钾(KCl)和氢氧化钠(NaOH)购自国药集团化学试剂有限公司。TRYPTONE(蛋白胨)和YEAST EXTRACT(酵母提取物)购自OXOID LTD.。硫酸卡那霉素、异丙基-β-D-硫代半乳糖苷(Isopropylβ-D-Thiogalactoside,IPTG)、丙烯酰胺/甲叉双丙烯酰胺30％(m/V)溶液(29:1)、过硫酸铵(Ammonium persulfate,APS)、N,N,N',N'-四甲基乙二胺(TEMED)、考马斯亮蓝R-250、溴酚蓝和β-巯基乙醇购自生工生物工程(上海)股份有限公司。Tris base、甘氨酸、十二烷基硫酸钠(SDS)和Agar(琼脂，日本进口分装)购自BIOSHIARP。盐酸、冰乙酸、甲醇和无水乙醇购自天津市德恩化学试剂有限公司。10×磷酸盐缓冲液(Phosphate Buffered Saline,PBS)购自博迈德生物。预染蛋白分子量(PageRulerTMPrestained Protein Ladder,10to 180kDa)购自赛默飞世尔科技(中国)有限公司。液氮购自新乡市豫新航空低温容器有限责任公司。
5×SDS-PAGE上样缓冲液配制：参考《分子克隆实验指南(第三版)》(下册)中2×SDS凝胶加样缓冲液配方计算，250mM Tris-Cl(pH6.8)，10％(m/V)SDS，0.5％(m/V)溴酚蓝，50％(V/V)甘油，500mMβ-巯基乙醇，分装成小份-20℃冻存备用。
蛋白胶脱色液配制：参考《分子克隆实验指南(第三版)》(下册)中甲醇：乙酸溶液配方，用乙醇替代甲醇，将900mL乙醇：水(500mL乙醇和400mL水)与100mL冰乙酸混合。
参考《分子克隆实验指南(第三版)》(下册)准备以下材料：
LB(Luria-Bertani)液体培养基配制：10g/L蛋白胨，5g/L酵母提取物，10g/LNaCl，去离子水中加溶质溶解后，用NaOH调节pH至7.0后定容，15psi高压蒸气灭菌20min。
含10μg/mL硫酸卡那霉素的LB固体培养基平板制备：LB液体培养基灭菌前，加入15g/L琼脂后高压灭菌，降温至50℃左右，加入10μg/mL硫酸卡那霉素，旋动混匀，倒入平板后，完全凝固后，倒置平板并贮存于4℃备用。
Tris-甘氨酸：25mM Tris-Cl，250mM甘氨酸，0.1％(m/V)SDS。
考马斯亮蓝R-250染色液：0.05％(m/V)考马斯亮蓝R-250溶解于甲醇：冰乙酸：水(50:10:40，V/V)溶液中，滤纸过滤除去杂质后使用。
实验方案：
取实施例4中含有携带EV-A71和CV-A2 VP1基因的重组质粒的菌液，分别在含10μg/mL硫酸卡那霉素的LB固体培养基平板划线，将平板倒置于生化培养箱中37℃过夜培养。挑取平板线上单菌落，分别接种于5mL含10μg/mL硫酸卡那霉素的LB液体培养基，37℃振荡过夜培养。各取2mL菌液接种于2瓶200mL含10μg/mL硫酸卡那霉素的LB液体培养基，待菌液生长至吸光度A6000.6～0.8时，取1mL菌液保存于4℃作为未诱导菌液(未诱导对照)，其余加入0.5mM IPTG诱导培养。37℃振荡培养6～7h后，测定A600，4℃2599×g离心10min收集菌体，弃上清，沉淀加入3mL/管重悬，再补足200mL PBS摇匀，4℃2599×g离心10min洗涤菌体，弃上清。沉淀重复上述洗涤步骤2次，弃上清，加入合适体积(15×A600/mL，CV-A2 VP1基因重组质粒菌液加63mL，EV-A71 VP1基因重组质粒菌液加66mL)PBS重悬沉淀，冻存于-20℃。
菌样反复冻融三次后，冰上超声破碎30min，4℃9384×g离心30min，收上清，沉淀用60mL 20mM Tris-Cl(pH7.4)重悬后，第二次冰上超声破碎30min。第二次破碎后先4℃100×g离心10min去除沉淀的菌体，再4℃9384×g离心30min，弃上清，沉淀用60mL 20mM Tris-Cl(pH7.4)重悬后，第三次冰上超声破碎30min。第三次破碎后先4℃100×g离心10min去除沉淀的菌体，再4℃3000×g离心10min，弃上清，沉淀用30mL 20mM Tris-Cl(pH7.4)重悬后，第四次冰上超声破碎30min。第四次破碎后先4℃200×g离心10min去除沉淀的菌体，再4℃3000×g离心10min，弃上清，沉淀重悬于15mL(占原约400mL诱导菌液的3/80)20mM Tris-Cl(pH7.4)中。
取最后的重悬液～250μL加入适量5×SDS-PAGE上样缓冲液后煮沸10min，用12％(m/V)SDS-PGAE分离蛋白，用冰冷的超纯水漂洗分离胶一次后，再用冰冷的5％(m/V)氯化钾(KCl)水溶液对分离胶进行漂染，切胶回收最明显的乳白色蛋白条带，获得纯化的重组EV-A71 VP1和CV-A2 VP1蛋白。切下的胶带加入适量PBS后，用液氮研磨三次，至200μL移液器枪头容易吸起，获得纯化的重组EV-A71 VP1和CV-A2 VP1蛋白。
取诱导前后菌液、各次破碎后离心的上清和沉淀以及回收研磨后的纯化蛋白加入适量5×SDS-PAGE上样缓冲液后煮沸10min，用12％(m/V)SDS-PGAE分离、考马斯亮蓝染色检测其中重组EV-A71和CV-A2 VP1蛋白的表达情况。
实验结果：SDS-PAGE检测发现，与未诱导菌相比，重组EV-A71和CV-A2 VP1蛋白出现在诱导菌及其超声波破碎后离心的沉淀中，而超声波破碎后离心的上清中则没有发现目标蛋白(如图4所示)，表明这两种重组蛋白主要以包涵体形式表达。通过对比图4中泳道2和5、泳道8和11，发现通过超声波破碎和梯度离心，可以初步纯化包涵体。而进一步采用KCl染色切胶回收、液氮研磨的方法可以获得更纯的重组蛋白(图4中泳道6和12)。
实施例6
小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1的制备和效价测定。
实验材料：
丙烯酰胺/甲叉双丙烯酰胺30％(m/V)溶液(29:1)、过硫酸铵(Ammoniumpersulfate,APS)、N,N,N',N'-四甲基乙二胺(TEMED)、溴酚蓝、β-巯基乙醇、N',N-二甲基甲酰胺(N',N-Dimethylformamide,DMF)、牛血清白蛋白(bovine serum albumin,BSA)、氯化硝基四氮唑蓝(Nitrotetrazolium blue chloride,NBT)和5-溴-4-氯-3-吲哚基磷酸盐钠盐(5-Bromo-4-chloro-3-indolyl phosphate disodium salt,BCIP)购自生工生物工程(上海)股份有限公司。Tris base、甘氨酸和十二烷基硫酸钠(SDS)购自BIOSHIARP。盐酸和甲醇购自天津市德恩化学试剂有限公司。10×磷酸盐缓冲液(Phosphate BufferedSaline,PBS)购自博迈德生物。碱性磷酸酶(alkaline phosphatase,AP)标记羊抗小鼠IgG抗体购自上海碧云天生物技术有限公司。孔径0.45μm的聚偏二氟乙烯(polyvinylidenefluoride，PVDF)膜购自Millipore Corporation。一次性使用无菌注射器带针(1mL)购自河南曙光汇知康生物科技股份有限公司。预染蛋白分子量(PageRulerTM Prestained ProteinLadder,10 to 180kDa)购自赛默飞世尔科技(中国)有限公司。
转膜缓冲液配制：25mM Tris base，192mM甘氨酸，用前加入20％甲醇。
10×Tris缓冲盐溶液(Tris buffered saline,TBS)配制：0.5M Tris base，4MNaCl，用盐酸调节pH至7.5。
TBST配制：先用超纯水将10×TBS稀释成1×TBS，然后加入0.1％吐温-20，混匀。
AP显色缓冲液：0.1M Tris-HCl(pH9.5)，0.1M NaCl，5mM MgCl2(调节好pH后加入)。
BCIP配制：50mg/mL BCIP溶于DMF。
NBT配制：50mg/mL NBT溶于70％(V/V)DMF水溶液。
5×SDS-PAGE上样缓冲液配制：参考《分子克隆实验指南(第三版)》(下册)中2×SDS凝胶加样缓冲液配方计算，250mM Tris-Cl(pH6.8)，10％(m/V)SDS，0.5％(m/V)溴酚蓝，50％(V/V)甘油，500mMβ-巯基乙醇，分装成小份-20℃冻存备用。
参考《分子克隆实验指南(第三版)》(下册)准备以下材料：
Tris-甘氨酸：25mM Tris-Cl，250mM甘氨酸，0.1％(m/V)SDS。
实验方案：
实施例5制备得到的EV-A71 VP1和CV-A2 VP1重组蛋白分别腹腔注射免疫Balb/c小鼠，首次免疫剂量为200μL/只。以后每三到四周加强免疫，加强免疫剂量为100～150μL/只。第四次免疫后三周或第五次免疫后3～4天分别眼眶后静脉丛取血分离血清，用蛋白质免疫印迹(Western blot)测定抗体效价。
实施例5制备得到的纯化重组EV-A71和CV-A2 VP1蛋白加入5×SDS-PAGE上样缓冲液，煮沸10min后，用12％(m/V)SDS-PGAE分离蛋白，100V湿法转膜45min将蛋白质转移到0.45μm的PVDF膜上；加入含有5％(m/V)BSA的TBS在37℃封闭2h；弃封闭液，剪开各泳道膜，分别加入用含有0.5％(m/V)BSA的TBS稀释的免疫EV-A71 VP1或CV-A2 VP1小鼠血清(1:1000、1:5000、1:10000、1:20000)及未免疫小鼠血清(1:1000)，4℃孵育过夜；TBST洗膜3次，加入用含有0.5％(m/V)BSA的TBS稀释的AP标记羊抗小鼠IgG(1:8000)，37℃孵育1h；TBST洗膜4次，加入AP显色缓冲液洗膜1次；将膜放入加有BCIP/NBT底物溶液的AP显色缓冲液中充分显色；用超纯水漂洗终止显色后，将膜保存在超纯水中，尽快拍照后分析实验结果。
实验结果：Western blot分别测定免疫血清效价发现，重组EV71-VP1和CVA2-VP1蛋白分别免疫小鼠获得的血清在1:1000～1:20000稀释时均在杂交膜泳道35～40kDa之间有条带出现，其大小与重组EV71-VP1和CVA2-VP1蛋白相符，而未免疫小鼠血清在杂交膜泳道无对应大小条带(见图5)。这表明免疫小鼠血清中分别有anti-EV-A71 VP1和anti-CV-A2VP1多克隆抗体。免疫血清中的多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1效价都可以达到1:20000。因此，免疫小鼠在第五次免疫后9～10天摘眼球取血分离血清，断颈处死。
实施例7
用小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1进行Westernblot检测感染RD细胞中的EV-A71和CV-A2。
实验材料：
EV-A71(201713#/XX/CHN/2017，VP1基因的GenBank号为MW477479)和CV-A2(201711#/XX/CHN/2017，VP1基因的GenBank号为MT847606)病毒株为本实验室分离，经RD细胞传代纯化，大量扩增并测定半数组织培养感染剂量(median tissue culture infectivedose,TCID50)后，保存于-80℃超低温冰箱。人横纹肌肉瘤细胞RD购自中国典型培养物保藏中心(China Center for Type Culture Collection,CCTCC)。
Dulbecco'sModified Eagle's Medium(DMEM)高糖培养基和胎牛血清(fetal bevine serum,FBS)购自赛默飞世尔科技(中国)有限公司。0.25％(m/V)胰酶+0.02％(m/V)EDTA溶液、青霉素-链霉素溶液(penicillin-streptomycin solution,PS,100×)购自杭州吉诺生物医药技术有限公司。RD细胞培养液为添加10％(V/V)FBS和1％(V/V)PS的DMEM高糖培养基。
丙烯酰胺/甲叉双丙烯酰胺30％(m/V)溶液(29:1)、过硫酸铵(Ammoniumpersulfate,APS)、N,N,N',N'-四甲基乙二胺(TEMED)、溴酚蓝、β-巯基乙醇、N',N-二甲基甲酰胺(N',N-Dimethylformamide,DMF)、牛血清白蛋白(bovine serum albumin,BSA)、氯化硝基四氮唑蓝(Nitrotetrazolium blue chloride,NBT)和5-溴-4-氯-3-吲哚基磷酸盐钠盐(5-Bromo-4-chloro-3-indolyl phosphate disodium salt,BCIP)购自生工生物工程(上海)股份有限公司。Tris base、甘氨酸和十二烷基硫酸钠(SDS)购自BIOSHIARP。盐酸和甲醇购自天津市德恩化学试剂有限公司。10×磷酸盐缓冲液(Phosphate BufferedSaline,PBS)购自博迈德生物。碱性磷酸酶(alkaline phosphatase,AP)标记羊抗小鼠IgG抗体购自上海碧云天生物技术有限公司。孔径0.45μm的聚偏二氟乙烯(polyvinylidenefluoride，PVDF)膜购自Millipore Corporation。
转膜缓冲液配制：25mM Tris base，192mM甘氨酸，用前加入20％(V/V)甲醇。
10×Tris缓冲盐溶液(Tris buffered saline,TBS)配制：0.5M Tris base，4MNaCl，用盐酸调节pH至7.5。
TBST配制：先用超纯水将10×TBS稀释成1×TBS，然后加入0.1％(V/V)吐温-20，混匀。
AP显色缓冲液：0.1M Tris-HCl(pH9.5)，0.1M NaCl，5mM MgCl2(调节好pH后加入)。
BCIP配制：50mg/mL BCIP溶于DMF。
NBT配制：50mg/mL NBT溶于70％(V/V)DMF水溶液。
5×SDS-PAGE上样缓冲液配制：参考《分子克隆实验指南(第三版)》(下册)中2×SDS凝胶加样缓冲液配方计算，250mM Tris-Cl(pH6.8)，10％(m/V)SDS，0.5％(m/V)溴酚蓝，50％(V/V)甘油，500mMβ-巯基乙醇，分装成小份-20℃冻存备用。
参考《分子克隆实验指南(第三版)》(下册)准备以下材料：
Tris-甘氨酸：25mM Tris-Cl，250mM甘氨酸，0.1％(m/V)SDS。
实验方案：
以常规方法培养RD细胞，生长状态良好时消化成单个细胞，以5×105个细胞/孔接种于直径为35mm的细胞培养皿中。37℃、体积分数5％CO2培养箱中培养至生长成单层后，分别用1mL/孔PBS洗3次，实验组加入800μL EV-A71(5×105TCID50)或800μL CV-A2(5×105TCID50)感染，对照组加入800μL DMEM+2％(V/V)FBS+1％(V/V)PS培养基模拟感染。37℃孵育1h后，分别用1mL/孔PBS洗3次，然后加入2mL DMEM+2％(V/V)FBS+1％(V/V)PS，37℃/5％(V/V)CO2培养箱中培养至感染细胞与模拟感染细胞形态出现明显不同，弃上清液，加入1mL/孔预冷PBS洗2次，再加入1mL/孔PBS吹下细胞，4℃1016×g离心5min，弃上清，沉淀加入含有1mM PMSF的RIPA裂解液(强)冰上裂解30min；4℃15,000×g离心10min，吸取上清，加入SDS-PAGE蛋白上样缓冲液(5×)；煮沸10min，用12％(m/V)SDS-PAGE分离，100V湿法转膜45min将蛋白质转移到0.45μm的PVDF膜上；加入含有5％(m/V)BSA的TBS 37℃孵育2h进行封闭；弃封闭液，加入用含有0.5％(m/V)BSA的TBS稀释的一抗——小鼠anti-EV-A71 VP1、小鼠anti-CV-A2VP1(1:1000)或未免疫小鼠血清(1:1000)，4℃孵育过夜；TBST洗膜3次，加入用含有0.5％(m/V)BSA的TBS稀释的二抗——AP标记羊抗小鼠IgG(1:5000)，37℃孵育1h；TBST洗膜4次，加入AP显色缓冲液洗膜1次；将膜放入加有BCIP/NBT底物溶液的AP显色缓冲液中充分显色；用超纯水漂洗终止显色后，将膜保存在超纯水中，尽快拍照后分析实验结果。
实验结果：Western blot分别检测EV-A71和CV-A2感染RD细胞发现，小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1分别能在EV-A71和CV-A2感染RD细胞样品中杂交出一条约35kDa大小的条带，而模拟感染RD细胞样品中则没有出现相应条带(如图6所示)，并且该条带大小(约35kDa)与EV-A71 VP1(32.7kDa)和CV-A2 VP1(32.9kDa)预期大小相近，说明这两种抗体能分别用于检测EV-A71和CV-A2感染RD细胞样品中的VP1且特异性好，因此这两种抗体能分别用于检测感染RD细胞中的EV-A71和CV-A2。
实施例8
用小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1进行IPMA检测感染RD细胞中的EV-A71和CV-A2
实验材料：
EV-A71(201713#/XX/CHN/2017，VP1基因的GenBank号为MW477479)和CV-A2(201711#/XX/CHN/2017，VP1基因的GenBank号为MT847606)病毒株为本实验室分离，经RD细胞传代纯化，大量扩增并测定半数组织培养感染剂量(median tissue culture infectivedose,TCID50)后，保存于-80℃超低温冰箱。人横纹肌肉瘤细胞RD购自中国典型培养物保藏中心(China Center for Type Culture Collection,CCTCC)。
Dulbecco'sModified Eagle's Medium(DMEM)高糖培养基和胎牛血清(fetal bevine serum,FBS)购自赛默飞世尔科技(中国)有限公司。0.25％(m/V)胰酶+0.02％(m/V)EDTA溶液、青霉素-链霉素溶液(penicillin-streptomycin solution,PS,100×)购自杭州吉诺生物医药技术有限公司。RD细胞培养液为添加10％(V/V)FBS和1％(V/V)PS的DMEM高糖培养基。
牛血清白蛋白(bovine serum albumin,BSA)购自生工生物工程(上海)股份有限公司。10×磷酸盐缓冲液(Phosphate Buffered Saline,PBS)购自博迈德生物。辣根过氧化物酶(horseradish peroxide,HRP)标记羊抗小鼠IgG抗体购自上海碧云天生物技术有限公司。3-氨基-9-乙基咔唑(3-amino-9-ethylcarbazole,AEC)显色试剂盒(20×)购自北京索莱宝科技有限公司。
PBST配制：先用超纯水将10×PBS稀释成1×PBS，然后加入0.05％(V/V)吐温-20，混匀。
实验方案：
以常规方法培养RD细胞，生长状态良好时消化成单个细胞，以2×104个细胞/孔接种于96孔板中。37℃/5％(V/V)CO2培养箱中培养至生长成单层后，分别用200μL/孔PBS洗3次，实验组加入100μL EV-A71(100TCID50)或100μL CV-A2(100TCID50)感染，对照组加入100μL DMEM+2％(V/V)FBS+1％(V/V)PS培养基模拟感染。37℃孵育1h后，分别用200μL/孔PBS洗3次，然后加入200μL/孔DMEM+2％(V/V)FBS+1％(V/V)PS，37℃/5％(V/V)CO2培养箱中培养至感染细胞与模拟感染细胞形态出现明显不同，弃上清液，加入200μL/孔PBS洗3次，加入100μL/孔冰冻的无水乙醇室温固定10min；200μL/孔PBS清洗1次后，加入100μL/孔封闭液【含有5％(m/V)脱脂奶粉的PBST】37℃孵育1h；弃封闭液，用PBST清洗1次；加入用封闭液稀释的一抗——小鼠anti-EV-A71 VP1(1:1000)或小鼠anti-CV-A2 VP1(1:1000)，4℃孵育过夜；PBST清洗3次，加入封闭液稀释的二抗——HRP标记羊抗小鼠IgG(1:1000)，37℃孵育1h；PBST清洗5次，加入AEC工作液充分显色；弃显色液，超纯水洗2次后，加入适量超纯水保存；倒置光学显微镜下观察，拍照后分析实验结果。
实验结果：IPMA分别检测感染RD细胞中的EV-A71和CV-A2发现，经小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1分别染色后，感染EV-A71和CV-A2的细胞样品均出现红棕色圆形细胞样颗粒，而模拟感染细胞样品无此种颜色的细胞样颗粒，同时对照处理的感染病毒细胞样品和模拟感染细胞样品也没有此种颜色的细胞样颗粒出现(如图7所示)，表明这两种抗体能分别用于检测EV-A71和CV-A2感染RD细胞样品中的VP1且特异性好，因此这两种抗体能分别用于检测感染RD细胞中的EV-A71和CV-A2。
以上实施例描述了本发明的基本原理、主要特征及优点，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，在不脱离本发明原理的范围下，本发明还会有各种变化和改进，这些变化和改进均落入本发明保护的范围内。
序列表
<110> 新乡医学院
<120> 一种人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白、多克隆抗体及其应用
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Claims (8)
1.一种人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白，其特征在于：该人肠道病毒71型VP1重组蛋白的氨基酸序列如序列表SEQ ID NO.2所示；柯萨奇病毒A组2型VP1重组蛋白的氨基酸序列如序列表SEQ ID NO.4所示。
2.一种权利要求1所述的人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白的制备方法，其特征在于具体步骤为：
步骤S1：构建携带EV-A71 VP1基因的重组质粒pET-28a(+)EV-A71 VP1和携带有CV-A2VP1基因的重组质粒pET-28a(+)CV-A2 VP1；
步骤S2：将重组质粒分别转化大肠杆菌BL21(DE3)感受态细胞，诱导表达EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白，对EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白分别进行纯化即得人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白。
3.根据权利要求2所述的人肠道病毒71型和柯萨奇病毒A组2型VP1重组蛋白的制备方法，其特征在于步骤S2的具体过程为：将含有EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白的细菌裂解液，分别用超声波破碎后进行差速离心，重复四次，最后3000×g离心沉淀包涵体，用3/80初始菌液体积重悬；用12％(m/V)十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离上述重悬液中的EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白，用冰冷的5％(m/V)氯化钾水溶液对分离胶进行漂染，切胶回收乳白色蛋白条带；在回收胶中加入适量磷酸盐缓冲液后用液氮研磨三次，最终获得纯化的EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白。
4.一种基于人肠道病毒71型VP1重组蛋白的多克隆抗体及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体。
5.一种权利要求4所述的基于人肠道病毒71型VP1重组蛋白的多克隆抗体及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体的制备方法，其特征在于：将纯化后的EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白分别作为抗原通过腹腔注射免疫Balb/c小鼠，制备小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1。
6.一种权利要求4所述的基于人肠道病毒71型VP1重组蛋白的多克隆抗体及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体的制备方法，其特征在于具体过程为：将纯化的EV-A71 VP1重组蛋白和CV-A2 VP1重组蛋白分别免疫12周龄Balb/c小鼠，首次免疫用200μL剂量，以后每隔三到四周加强免疫，加强免疫用100～150μL剂量，免疫均采用腹腔注射方法；第四次免疫后三周和第五次免疫后第3-4天分别取少量血清，用蛋白质免疫印迹测定抗体效价，抗体效价在1:5000及以上的小鼠，在第五次免疫后9-10天取血并分离血清，获得小鼠多克隆抗体anti-EV-A71 VP1和anti-CV-A2 VP1。
7.权利要求4所述的基于人肠道病毒71型VP1重组蛋白的多克隆抗体及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体在制备人肠道病毒71型和柯萨奇病毒A组2型特异性检测试剂中的应用。
8.根据权利要求7所述的应用，其特征在于：所述的基于人肠道病毒71型VP1重组蛋白的多克隆抗体在制备人肠道病毒71型免疫印迹或免疫过氧化物酶单层细胞试验检测试剂中的应用以及基于柯萨奇病毒A组2型VP1重组蛋白的多克隆抗体在制备柯萨奇病毒A组2型免疫印迹或免疫过氧化物酶单层细胞试验检测试剂中的应用。
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