CN112175912B - 杂交瘤细胞株3g4 1d6、抗gii.4型诺如病毒p蛋白单克隆抗体和应用 - Google Patents
杂交瘤细胞株3g4 1d6、抗gii.4型诺如病毒p蛋白单克隆抗体和应用 Download PDFInfo
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Abstract
本发明公开了杂交瘤细胞株3G41D6、抗GII.4型诺如病毒P蛋白单克隆抗体和应用。所述的杂交瘤细胞株3G41D6的保藏编号为：GDMCC No：61138。该杂交瘤细胞株3G41D6可分泌抗GII.4型诺如病毒P蛋白单克隆抗体。以杂交瘤细胞株3G41D6分泌的单克隆抗体作为捕获抗体，以辣根过氧化物酶标记的由保藏编号为：GDMCC No：61139的杂交瘤细胞株3G71B10分泌的单克隆抗体作为检测抗体，建立了双抗体夹心ELISA检测方法。本发明具有操作简便快捷，结果清晰等优点，为诺如病毒体外诊断方法的研究提供科学依据，具有应用价值。
Description
技术领域
本发明属于生物技术领域，具体涉及一种杂交瘤细胞株3G4 1D6、抗GII.4型诺如病毒 P蛋白单克隆抗体和应用。
背景技术
诺如病毒(Norovirus,NoV)已成为全球急性非细菌性胃肠炎的主要原因，可感染各个 年龄段的人群，尤其是5岁以下的儿童。NoV可以以约每毫升100个病毒颗粒的低剂量感 染，并易于通过食物、水以及被感染者的呕吐物和粪便传播。为满足公众对健康的需求，需要快速，灵敏的生物传感系统来检测并防止病毒的传播。
NoV是非包膜病毒，具有约7.5-7.7kb的单股正链RNA基因组，属于杯状病毒科，诺如病毒属，其基因组具有高度变异性。NoV的基因组由三个开放阅读框(ORF1、ORF2、 ORF3)组成，其中ORF2编码主要结构蛋白VP1。根据VP1的核苷酸序列可以分为GI-GVII 7个基因群(Genogroup)，其中引起人类感染的主要分布在基因群GI、GII和GIV，其中 GII.4型一直是全球优势流行株。主要结构蛋白VP1包括了N端壳结构域S区(Shell domain) 和C端外部突出结构域P区(Protruding domain)，两者通过一段铰链区连接，由于P结构 域位于病毒颗粒的外部，它包含受体结合元件和主要抗原位点。通过大肠杆菌表达系统单 独在体外表达不含铰链区的P区域可形成P颗粒(P particle)，P颗粒的形态结构、抗原性、 免疫原性及受体结合功能均与天然病毒颗粒极为相似，因原核表达P颗粒具备表达周期短， 稳定性高、操作简单等特点，成为研究NoV的替代工具，可以满足多克隆抗体与单克隆抗 体制备的要求。
NoV的检测方法包括电镜法、分子生物学检测方法及免疫检测方法，其中免疫检测方 法在临床诊断，尤其是在POCT领域应用较为广泛，如胶体金免疫层析试纸条、酶联免疫吸附实验(ELISA)等。免疫检测方法具有操作简单、检测时间短等优点特别适合临床大量检测，但由于人源诺如病毒具有丰富的遗传多样性，包括三个基因群以及三十余种基因型，且每隔几年就出现新的变异株并引发大流行，因此针对多种基因型的检测十分必要。诺如病毒免疫检测方法对比分子生物学检测方法RT-PCR等缺乏对于不同基因型的交叉反应性，常有漏检的情况发生，无法满足人源诺如病毒临床多种基因型检测的需求。因此筛选制备出针对多种NoV基因型具有广泛交叉反应性的单克隆抗体对于免疫检测方法的建立尤为重要，对不同基因型的预警防控研究具有重要意义。
发明内容
本发明的第一个目的是提供两种生产抗GII.4型诺如病毒P蛋白单克隆抗体的杂交瘤细 胞株，分别为：杂交瘤细胞株3G4 1D6和杂交瘤细胞株3G7 1B10，其中，杂交瘤细胞株3G4 1D6的保藏编号为：GDMCC No：61138；杂交瘤细胞株3G7 1B10的保藏编号为：GDMCCNo：61139。
本发明的第二个目的是提供两株抗GII.4型诺如病毒P蛋白单克隆抗体，分别由上述的 杂交瘤细胞株3G4 1D6和杂交瘤细胞株3G7 1B10分泌产生，两株单抗的间接ELISA效价 均在10-6以上，其中杂交瘤细胞株3G4 1D6分泌的单抗的亚型为IgG2a，杂交瘤细胞株3G71B10分泌的单抗的亚型为IgG1，在ELISA和Western-blot检测中两株单抗均可特异性结合GII.4型诺如病毒P蛋白。
本发明的第三个目的是提供上述的杂交瘤细胞株3G4 1D6和杂交瘤细胞株3G71B10 在生产抗GII.4型诺如病毒P蛋白单克隆抗体中的应用。
本发明的第四个目的是提供上述的由杂交瘤细胞株3G4 1D6和杂交瘤细胞株3G71B10 分泌的抗GII.4型诺如病毒P蛋白单克隆抗体在制备诺如病毒不同基因型P蛋白检测试剂中 的应用。
所述的抗GII.4型诺如病毒P蛋白单克隆抗体可与诺如病毒常见基因型GII.2、GII.3、 GII.4、GII.6、GII.17样本发生免疫反应，与轮状病毒、星状病毒、肠道病毒、札幌病毒均 无交叉反应。
本发明的第五个目的是提供一种试剂盒，该试剂盒包含由杂交瘤细胞株3G4 1D6分泌 的抗GII.4型诺如病毒P蛋白单克隆抗体，以及辣根过氧化物酶标记的由杂交瘤细胞株3G7 1B10分泌的单克隆抗体。
本发明的第六个目的是提供一种诺如病毒不同基因型P蛋白的双抗夹心ELISA检测方 法，该检测方法是以由杂交瘤细胞株3G4 1D6分泌的抗GII.4型诺如病毒P蛋白单克隆抗 体作为捕获抗体，以辣根过氧化物酶标记的由杂交瘤细胞株3G7 1B10分泌的单克隆抗体作 为检测抗体。
优选，上述的双抗夹心ELISA检测方法中，所述的捕获抗体是包被于酶标板上，所述 的捕获抗体的最佳包被浓度为2μg/mL，所述的检测抗体的工作浓度为1:5000。
与现有技术相比，本发明具有以下有益效果：
1、本发明的两株杂交瘤细胞株可稳定分泌抗体，所分泌的两株单克隆抗体配对效果好， 适用于各种双抗体夹心检测方法。
2、针对GII型诺如病毒临床粪便样本检测，其特点是可针对当前所有诺如病毒流行株， 交叉反应性好、特异性强、敏感性高、稳定性好。解决了当前因诺如病毒分型复杂造成的 检测方法普遍覆盖面窄的缺点。
3、操作简单，结果清晰，适合于临床现场的诊断和大规模流行病学调查。也可以用于 医疗卫生、检验检疫等具有诺如病毒检测需求的机构以及相应的科研领域。
本发明的Mus musculus hybridoma 3G4 1D6于2020年8月12日保藏于广东省微生物菌 种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，保藏编号为：GDMCC NO：61138。
本发明的Mus musculus hybridoma 3G7 1B10于2020年8月12日保藏于广东省微生物 菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，保藏编号为：GDMCC NO：61139。
附图说明
图1为GII.4型P片段的PCR扩增(A)及重组质粒pGEX-4T-1-GII.4-L307-P菌落PCR鉴定结果(B)。
图2为纯化后GST-P结合蛋白的SDS-PAGE(A)及切除GST标签后P颗粒的SDS-PAGE(B)。
图3为两株单克隆抗体(3G7 1B10和3G4 1D6)纯化的SDS-PAGE。
图4为Western-Blot检测两株单克隆抗体(3G7 1B10和3G4 1D6)特异性。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。下列实施例中未注明 具体实验条件和方法，所采用的技术手段通常为本领域技术人员所熟知的常规手段。
实施例1杂交瘤细胞株的制备及单克隆抗体的获得
1.GII.4型诺如病毒P蛋白免疫原的制备
根据GII.4-2012型GZ2014-L307毒株(GenBank KT202798)P区域设计扩增引物，上游引物P1、下游引物P2别引入BamHI、EcoRI酶切位点(下划线部分)，并在下游引物5' 端加入CDCRGDCFC氨基酸序列(斜体部分)可促进诺如病毒P颗粒的形成如表1所示。 以GII.4-2012型GZ2014-L307毒株cDNA为模板进行PCR扩增，对扩增产物及pGEX-4T-1 载体(GE通用医疗，美国)分别进行BamHI、EcoRI双酶切。将扩增产物连接到pGEX-4T-1 载体上，转化至大肠杆菌BL21感受态细胞，涂板后进行菌落PCR及测序验证。将重组成 功的质粒(pGEX-4T-1-GII.4-L307-P)进行原核表达，通过SDS-PAGE电泳鉴定重组蛋白表 达情况。GII.4型P片段的PCR扩增及重组质粒pGEX-4T-1-GII.4-L307-P菌落PCR鉴定结 果见图1。利用GST亲和纯化柱对重组融合蛋白进行纯化，纯化后利用凝血酶(Thrombin) 切掉GST标签。超滤离心3次换盐，将缓冲液换成0.01M PBS，再次利用GST亲和纯化柱 纯化并收集流穿液。通过SDS-PAGE电泳分析，重组蛋白为大小为62kDa。经Thrombin 切去GST标签蛋白后，获得约36kDa的目的蛋白(P蛋白抗原)，与预期大小一致(图2)。
表1扩增GII.4-2012型诺如病毒P颗粒的引物序列
2.小鼠免疫
选择8只8周左右雌性Balb/c小鼠，首次免疫将含50μg的P蛋白抗原溶液与等体积弗氏完全佐剂混合乳化至油包水状态，加强免疫用与首次免疫等剂量抗原与弗氏不完全佐剂充分混合乳化，免疫方式为皮下多点注射。加强免疫共三次，每次免疫间隔2周，之后 采血利用间接ELISA方法测定血清效价，选取效价高于1:10000的小鼠在1周内以50μg P 蛋白抗原/只的剂量进行腹腔冲击，本次不使用佐剂。
3.细胞融合与筛选
小鼠腹腔冲击3天后，摘除眼球处死，并收集阳性血，取出脾脏，制备成单细胞悬液， 用50％聚乙二醇(PEG1450)将脾细胞与处于对数期的SP2/0细胞(小鼠骨髓瘤细胞)融合。按常规方法对融合的杂交瘤细胞进行克隆化筛选，最后获得生长良好且能够稳定分泌抗体的杂交瘤细胞株，分别为杂交瘤细胞株3G4 1D6、3G7 1B10。杂交瘤细胞株3G4 1D6、3G7 1B10于2020年8月12日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广 州市先烈中路100号大院59号楼5楼，其中，杂交瘤细胞株3G4 1D6的保藏编号为：GDMCC NO：61138；杂交瘤细胞株3G7 1B10的保藏编号为：GDMCC NO：61139。
4.单克隆抗体的制备与纯化
Balb/c小鼠免疫前一周腹腔注射液体石蜡预处理，细胞定株后选用10％胎牛血清培养 基扩大培养，当细胞密度达到1×106-2×106/mL时，1000rpm离心，收集细胞沉淀，并用PBS 重悬后注入小鼠腹腔。10天后收集腹水，选用Protein G亲和层析柱纯化，分别获得杂交瘤 细胞3G4 1D6和杂交瘤细胞3G7 1B10分泌的两株抗GII.4型诺如病毒P蛋白单克隆抗体， 分别命名为3G4 1D6单抗、3G7 1B10单抗，经亚型鉴定杂交瘤细胞株3G4 1D6分泌的单抗(3G4 1D6单抗)的亚型为IgG2a型，杂交瘤细胞株3G7 1B10分泌的单抗(3G7 1B10 单抗)的亚型为IgG1型。SDS-PAGE分析显示，2株单克隆抗体纯度均在90％以上，在约25 kD和50kD有明显两条带，即为单克隆抗体轻链和重链，WB实验显示均有明显单一目的 条带。两株单克隆抗体(3G4 1D6单抗、3G7 1B10单抗)纯化的SDS-PAGE见图3。通过 Western-Blot验证两株抗体与P蛋白抗原的反应性，结果显示均有明显单一目的条带，如图 4所示。
5.单克隆抗体的评价
经NanoDrop 2000蛋白质定量测定抗体浓度，然后应用间接ELISA方法，以1μg/mL的GII.4P颗粒作为包被抗原，5％脱脂奶粉封闭，PBS溶液做倍比稀释的2株单克隆抗体(3G4 1D6单抗和3G7 1B10单抗)作为一抗，1:3000倍稀释辣根过氧化物酶(HRP)标记的 羊抗鼠IgG作为二抗，TMB显色后测定OD450的值，并计算P/N值(P为检测样品OD450 的值；N为阴性对照OD450的值)，P/N大于2.1的最低稀释度即为抗体效价。应用同样间 接ELISA方法以GI.6、GII.2、GII.3、GII.4-2012、GII.6、GII.17阳性样本作为包被抗原， 确定这2株单抗与NoV不同基因型样本的反应情况。经NanoDrop 2000测定3G4 1D6、3G7 1B10抗体浓度分别为2.2mg/mL、5.5mg/mL。纯化后3G4 1D6、3G7 1B10抗体经间接 ELISA测定，效价均在10-6以上。2株抗体均表现出广谱的结合活性，与GII.2、GII.3、GII.4、 GII.6、GII.17样本反应结果呈阳性，结果见表2。
表2单克隆抗体与诺如病毒样本反应评价
实施例2双抗夹心ELISA方法的建立
1.配对抗体筛选及最佳工作浓度确定
首先将2株单抗按照辣根过氧化物酶(HRP)偶联标记试剂盒说明书进行标记，采用直接 ELISA测定标记后抗体灵敏度，并确定检测抗体。利用双抗夹心ELISA的方法，以1μg/mL 的GII.4P颗粒作为检测抗原，利用确定的HRP标记单抗(HRP-3G7 1B10、HRP-3G4 1D6)作为检测抗体，另两株单抗(3G7 1B10、3G4 1D6)分别作为捕获抗体与其配对，确定最佳配对抗体及最适工作浓度。结果表明HRP标记3G7 1B10抗体(HRP-3G7 1B10)灵敏度最佳， 因此选择HRP-3G7 1B10作为检测抗体。配对抗体筛选结果表明HRP-3G7 1B10与3G4 1D6 配对效果更好。棋盘法梯度稀释抗体，确定检测抗体HRP-3G7 1B10工作浓度为1:5000， 捕获抗体3G4 1D6最佳包被浓度为2μg/mL。
2.标准曲线的绘制及最低检出限的确定
应用双抗夹心ELISA方法检测梯度稀释的P颗粒蛋白(0.125、0.25、0.5、1、2μg/mL)， 读取OD450的值。以P颗粒蛋白浓度(μg/mL)为横坐标，OD450为纵坐标，绘制标准曲线，并计算P/N值，P/N大于2.1判定为阳性，根据曲线可知在0.125-2μg/mL范围内具有 良好的线性，标准曲线方程为y＝1.8032x+0.2695，R2＝0.9938，最低检出限为125ng/mL。
实施例3方法的性能测试
对实施例2建立的双抗夹心ELISA方法进行性能测试，具体如下：
1.特异性试验
采用本方法检测对常见的几种腹泻病毒，如轮状病毒、星状病毒、肠道病毒、札幌病 毒的反应性，结果表明本方法与腹泻病毒，如轮状病毒、星状病毒、肠道病毒、札幌病毒均无交叉反应。
2.重复性试验
利用同一批次及不同批次包被好3G4 1D6抗体的酶标板，分别对3份GII型诺如病毒 阳性样本进行批次间和批次内检测，每个重复3次，通过变异系数的计算公式
计算批间、批内变异系数。结果批间和批内变异系数CV均小于7％(见表3)，表明三批次间具有较好的重复性较好。
表3重复性试验结果
3.临床样本检测
同时应用建立的双抗夹心ELISA方法和荧光定量RT-PCR法对24份临床粪便样本进行 检测，结果见表4，其中荧光定量RT-PCR法检出阳性样本13份，13份阳性样本中双抗夹心ELISA方法检出11份为阳性，其余结果均为阴性。两者的阳性符合率约为84.6％，结果 见表4。
表4双抗夹心ELISA与荧光定量RT-PCR检测结果
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发 明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通 技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进 和润饰也应视为本发明的保护范围。
Claims (9)
1.一种杂交瘤细胞株3G4 1D6，其保藏编号为：GDMCC No：61138。
2.一种抗GII.4型诺如病毒P蛋白单克隆抗体，其特征在于，是由权利要求1所述的杂交瘤细胞株3G4 1D6产生。
3.权利要求1所述的杂交瘤细胞株3G4 1D6在生产抗GII.4型诺如病毒P蛋白单克隆抗体中的应用。
4.权利要求2所述的抗GII.4型诺如病毒P蛋白单克隆抗体在制备诺如病毒不同基因型P蛋白检测试剂中的应用；所述的诺如病毒不同基因型包括GII.2、GII.3、GII.4、GII.6、GII.17。
5.根据权利要求4所述的应用，其特征在于，所述的检测试剂通过酶联免疫吸附试验检测样本中诺如病毒不同基因型P蛋白的表达。
6.一种试剂盒，其特征在于，包含权利要求2所述的抗GII.4型诺如病毒P蛋白单克隆抗体。
7.根据权利要求6所述的试剂盒，其特征在于，还含有辣根过氧化物酶标记的由保藏编号为GDMCC No：61139的杂交瘤细胞株3G7 1B10分泌的单克隆抗体。
8.一种非疾病诊断和治疗目的的诺如病毒不同基因型P蛋白的双抗夹心ELISA检测方法，其特征在于，是以权利要求2所述的抗GII.4型诺如病毒P蛋白单克隆抗体作为捕获抗体，以辣根过氧化物酶标记的由保藏编号为：GDMCC No：61139的杂交瘤细胞株3G7 1B10分泌的单克隆抗体作为检测抗体；所述的诺如病毒不同基因型包括GII.2、GII.3、GII.4、GII.6、GII.17。
9.根据权利要求8所述的检测方法，其特征在于，所述的捕获抗体是包被于酶标板上，所述的捕获抗体的最佳包被浓度为2 μg/mL，所述的检测抗体的工作浓度为1:5000。
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