CN117538543A - 一种定量蒿属植物花粉中Art an 1类防御蛋白过敏原的方法 - Google Patents
一种定量蒿属植物花粉中Art an 1类防御蛋白过敏原的方法 Download PDFInfo
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Abstract
本发明公开了一种定量蒿属植物花粉中Art an 1类防御蛋白过敏原的方法,使用单抗‑多抗夹心酶联免疫吸附实验进行定量,A7G4E6单抗,来自杭州艾乐吉生物科技有限公司,货号MA‑A7G4E6;二抗为789多抗,来自杭州艾乐吉生物科技有限公司,货号PA‑789。本发明一种定量蒿属植物花粉中类防御蛋白过敏原的方法具有较高的灵敏性和特异性,能够定量常见蒿属花粉中Art an 1类型过敏原。
Description
技术领域
本发明涉及生物技术检测技术领域,特别是涉及一种定量蒿属植物花粉中Art an1类防御蛋白过敏原的方法。
背景技术
蒿属植物花粉是造成夏秋季节过敏性疾病的主要致敏原之一,据统计,在北方地区尤为严重(超过50%)。我国蒿属植物分布广泛,种类繁多,其中黄花蒿(Artemisiaannua)、艾蒿(A.argyi)、大籽蒿(A.sieversiana)、茵陈蒿(A.capillaris)、野艾蒿(A.lavandulifolia)等最为常见,目前用于体外诊断和免疫治疗的花粉提取物主要来自大籽蒿和黄花蒿。
国际免疫学会联合会(IUIS)下属的过敏原命名专门委员会(AllergenNomenclature Sub-committee)(http://www.allergen.org)数据库中已经收录的蒿花粉过敏原共12种,按照过敏原命名规则,依次命名为Art v 1-6(来源于北艾A.vulgaris)以及来源于黄花蒿(A.annua)的Art an 7、Art an 14以及来自大籽蒿的Art si 8和Art sil2。其中第一组分(Art v 1),第三组分(Art v 3)和第七组分(Art an 7)是主要过敏原。Art v1属于类防御蛋白,被视为蒿花粉过敏的标志过敏原,超过80%的蒿过敏患者对其敏感,成熟Art v 1蛋白包含108个氨基酸,理论等电点8.17,由N端的富含半胱氨酸的防御素结构域和C端富含脯氨酸的区域组成,C端包含羟基化和O-糖基化修饰,因此天然过敏原分子量约为24-28kDa。该过敏原在不同蒿属植物中高度保守,仅在C-端存在部分变异,并且不同蒿属植物中Art v 1同源过敏原的IgE结合活性没有显著差异。在欧洲,约有70%的蒿花粉过敏患者对其敏感,而我国约有25%-66%的患者对其敏感,这种蛋白作为泛过敏原存在于很多种植物中,能够引起广泛的交叉过敏反应,尤其是与蔷薇科水果之间,诱发呼吸道、胃肠道、口腔等的过敏症状,严重影响患者的生活质量。
目前用于临床诊断和脱敏治疗的制剂主要是花粉提取物,而这些提取物由于种类和批次不同,其中所包含的过敏原有效成分差异很大,有些重要的过敏原在提取物中含量很低,用于诊疗中常出现漏检或脱敏治疗效果不显著等问题,因此,为了提高临床诊疗效率,对提取物中的主要过敏原进行定量是非常重要的。用于过敏原定量的方法包括酶联免疫吸附法(ELISA),质谱以及其他的分子生物学手段如PCR等,其中双夹心酶联免疫吸附实验是最常用的过敏原定量方法,尤其在检测微量过敏原方面有很高的应用价值。
发明内容
本发明提供一种用于蒿花粉中Art an 1类型过敏原定量的方法,该方法具有较高的灵敏性和特异性,能够定量常见蒿属花粉中Art an 1类型过敏原。
利用单抗-多抗夹心酶联免疫吸附实验对我国主要的蒿属花粉提取物(黄花蒿、艾蒿、茵陈蒿、大籽蒿、野艾蒿、细裂叶莲蒿(A.gmelinii))中的Art an 1类型过敏原进行定量。
本发明包含以黄花蒿花粉提取物免疫小鼠,生产并筛选蒿花粉Art an1类型过敏原的特异性单克隆抗体A7G4E6,对单抗的亚型和效价进行检测;以重组的Art an 1过敏原免疫兔子生产多克隆抗体789。然后以A7G4E6作为包被抗体,789多抗作为检测抗体,构建双夹心酶联免疫吸附定量检测体系。单抗和多抗都来源于杭州艾乐吉生物科技有限公司,其中A7G4E6单抗的货号为MA-A7G4E6,789多抗的货号为PA-789。
本发明提供了6种我国常见蒿属花粉中Art an 1类型过敏原的氨基酸序列,每种花粉中各鉴定到1-2种不同亚型,依据IUIS的过敏原命名规则进行命名。
本发明包含用单克隆抗体(A7G4E6)纯化黄花蒿花粉提取物中的Art an 1类型过敏原,通过电泳(SDS-PAGE)和质谱(LC-MS/MS)对纯化产物进行鉴定,以BCA法测定纯化出的过敏原浓度,并将其用作标准品对不同蒿花粉提取物中的Art an 1类型过敏原进行定量。
本发明包含在大肠杆菌表达黄花蒿花粉中的类防御蛋白过敏原天然Art an 1并进行纯化,通过电泳(SDS-PAGE)和质谱(LC-MS/MS)对纯化产物进行鉴定,用于多克隆抗体的生产。
一种定量蒿属植物花粉中类防御蛋白过敏原的方法,使用单抗-多抗夹心酶联免疫吸附实验进行定量,其中一抗为A7G4E6单抗,来自杭州艾乐吉生物科技有限公司,货号MA-A7G4E6;二抗为789多抗,来自杭州艾乐吉生物科技有限公司,货号PA-789。
优选的,二抗用生物素标记。进一步优选的,使用HRP结合二抗后再加TMB避光显色后进行吸光值检测。进一步优选的,使用蒿属植物对应的Art an 1过敏原标准品进行检测得到吸光值后绘制标准曲线,将待检测的蒿属植物花粉样品检测得到的吸光值代入标准曲线计算得到相应浓度。进一步优选的,蒿属植物使用Art an 1过敏原标准品。该定量检测方法的最低检测阈值为0.25ng/ml,最佳浓度测定范围为0.5-16ng/ml。
优选的,蒿属植物为黄花蒿、艾蒿、茵陈蒿、细裂叶莲蒿、大籽蒿、野艾蒿或北艾。
本发明一种定量蒿属植物花粉中类防御蛋白过敏原的方法具有较高的灵敏性和特异性,能够定量常见蒿属花粉中Art an 1类型过敏原。
附图说明
图1为用黄花蒿(A.annua)花粉提取物免疫小鼠,经过一系列筛选(用重组的rArtan 1.0102)后所得到的蒿花粉Art an 1同种型过敏原的单克隆抗体A7G4E6。A:黄花蒿花粉蛋白提取物电泳检测图,M,蛋白Maker,AnE,黄花蒿花粉提取物;B:黄花蒿花粉提取物免疫小鼠;C:A7G4E6单抗纯化;
图2为重组Art an 1.0102电泳(A)和质谱(B)检测图。M:蛋白Maker;1:未经IPTG诱导的菌液总蛋白;2:经过IPTG诱导的菌液总蛋白;3:纯化后的rArt an 1.0102;B:rArt an1.0102质谱检测,红色肽段为质谱匹配肽段;
图3为重组Art an 1.0102免疫兔子生产多克隆抗体;
图4为Art an 1多抗ELISA检测结果;
图5为6种蒿的Art an 1类型过敏原的氨基酸序列比较;
图6为黄花蒿天然类防御蛋白过敏原Art an 1的纯化(A)和质谱检测(B);标红肽段为质谱匹配到的肽段;
图7为以A7G4E6作为包被抗体,生物素标记的789多抗作为检测抗体,天然Art an1作为标准品,用双夹心酶联免疫吸附法所建立的蒿属花粉类防御蛋白过敏原定量检测的标准曲线图;
图8为以图7所建立的标准曲线计算所得的7种蒿花粉提取物种Art an 1类型过敏原的含量。
具体实施方式
材料
黄花蒿、艾蒿、茵陈蒿、细裂叶莲蒿、大籽蒿:采集自山西省;
野艾蒿:采集自浙江省;
北艾:购自德国默克公司。
实施例1
提取黄花蒿花粉总蛋白。蛋白提取纯化方法:2g花粉溶在35mL PBS(pH 7.0)中,在4℃摇床过夜提取,10000g离心15分钟后收集上清,再用0.22μm Millipore膜过滤获得花粉蛋白提取物。BCA法测定提取物种的总蛋白浓度。
以100μg的黄花蒿花粉提取物免疫小鼠,每隔7-14日即注射50μg蛋白提取物,共6次,重组的Art an 1和蒿花粉蛋白提取物共同用于ELISA和Western-blot筛选特异性单抗,得到的单克隆细胞株制备腹水,检测后生产单抗用Protein G层析柱纯化,得到Art an 1过敏原的特异性单抗A7G4E6,如图1所示。单抗来源于杭州艾乐吉生物科技有限公司,货号为MA-A7G4E6。
用ELISA法确定单抗的种类和亚型,将0.5μg蒿花粉提取物包被于酶标板上,加入单抗,再分别加入羊抗鼠IgG1,IgG2a,IgG2b,IgG2c,IgG3,IgM,IgE,IgA(1:5000稀释,购自北京博奥龙免疫技术有限公司,货号BF06004)作为二抗,HRP标记的兔抗羊IgG作为三抗(1:5000稀释),TMB显色,测量450/620nm出的吸光值,以此确定各单抗的亚型。效价检测是用0.5μg的蒿花粉提取物包板,分别加入依次稀释了200,1000,5000,10000,20000,60000,240000倍的单抗,HRP标记的羊抗鼠IgG作为二抗,以此来确定各单抗的效价。检测结果如表1所示,抗体为IgG1亚型。
表1Art an 1特异性单抗的亚型和效价
免疫原 | 抗体编号 | 抗体亚型 | 抗体浓度(mg/ml) | 效价 |
黄花蒿 | A7G4E6 | IgG1κchain | 1.59 | 1∶240000 |
实施例2
提取黄花蒿花粉mRNA,用反转录试剂盒One-Step gDNA Removal and cDNASynthesis SuperMix(全式金,北京)将其反转录为cDNA,体系为7μl RNA样品,1μl Oligo(dt),10μl R Mix,1μl RT/RI enzyme,1μl gDNA Removal,42℃30min,85℃5s,4℃10min。根据Art an 1.0102序列(NCBI序列登录号KR996728),设计特异性引物(上游引物序列:ATGGCAAAGTGTTCATATGTTTTTTG;下游引物序列:TTAGTGAGCGGACGGAGGAG),利用I-5高保真酶(擎科,北京)从花粉cDNA中克隆Art an 1.0102序列,体系为12.5μl I-5Mix,上下游引物各1μl,cDNA 1μl,无菌水9.5μl。98℃2min;98℃10s,57℃15s,72℃15s(35个循环);72℃5min,4℃10min。PCR产物加入上样缓冲液后在1%琼脂糖凝胶上电泳检测,将大小正确的条带从胶上割下,用回收试剂盒Easy Gel Extraction&Clean-up Kit(易思得,杭州)进行回收。BamHI和HindIII酶切pET32a载体,体系为两种酶各1μl,载体1μg,CutSmart缓冲液5μl,用无菌水定容至50μl,37℃酶切2h,65℃反应20min使酶失活,4℃保持10min,酶切完成后的载体用1%琼脂糖凝胶电泳检测并割胶回收纯化。然后用同源重组酶ClonExpressII(NEB,美国)将目的片段和双酶切后的载体连接起来,体系为ExnaseII0.5μl,5X CE buffer 1μl,载体1.5μl,目的片段2μl,37℃反应30min,4℃10 min。然后将5μl重组载体转化到50μl大肠杆菌感受态细胞DH5α中,冰浴30min,42℃热激1min,置于冰上2min后在超净工作台中加入1mlLB培养基,在37℃的摇床中培养1h后涂布于有氨苄抗性的固体LB培养基上,然后将培养皿置于37℃的培养箱中过夜培养,挑取单菌落用T7通用引物进行检测进行检测,PCR产物经电泳检测片段大小正确的重组质粒进行测序验证。
将测序正确的重组质粒转化到表达型的大肠杆菌感受态细胞Rosetta(DE3)中,涂板后挑取单菌落转摇到200ml含有氨苄青霉素的LB培养基中,37℃,200rpm培养至OD600达到0.6,加入IPTG诱导剂至终浓度为0.5mM,16℃,160rpm过夜诱导,10000rpm离心取沉淀重悬于20ml PBS中,在超声破碎仪中破碎20min,破碎完成后10000rpm离心10min,加入Ni-NTA,4℃中震荡孵育1h,将样品转移到蛋白纯化柱中,使未结合至镍胶上的样品自然流出,然后用Binding/Wash Buffer(pH 7.9~8.1)for Ni-NTA or Ni-IDA Sefinose(TM)Resin(含有20mM咪唑)清洗6次,每次5ml,洗去某些非特异性结合的蛋白,然后用梯度浓度的(50mM,100mM,200mM,250mM)Elution Buffer(pH 7.9~8.1)for Ni-NTA or Ni-IDASefinose(TM)Resin将目的蛋白洗脱下来。收集洗脱液,用12%的SDS-PAGE凝胶检测,将含有目的蛋白的样品洗脱液收集起来,透析至PBS缓冲液中,电泳检测后将目的条带割下,进行质谱鉴定。BCA法测定样品浓度。蛋白纯化结果如图2A所示,获得了纯度较高的重组蛋白,并且质谱验证序列正确无误(图2B)。
实施例3
用实施例2中所得的重组Art an 1.0102作为抗原免疫兔子生产多克隆抗体。抽取抗原与佐剂1:1混匀,免疫2只新西兰兔子,首免抗原浓度为1mg/ml,二免-四免抗原量减半。首免后第14天进行二免,二免到三免间隔时间为7天。兔子三免后第7天耳中动脉采小样血清检测,检测合格,7天后加免,加免完7天后采集全血离心收血清,用黄花蒿花粉提取物作为抗原,免疫印迹法验证血清结合活性:抗原电泳后原位转至PVDF膜上,用5%脱脂奶粉室温封闭1h后,加入稀释过的兔子血清孵育1h,用TBST缓冲液清洗PVDF膜,然后加入HRP标记的羊抗兔IgG抗体孵育1h,再次洗膜,避光加入ECL发光液,在ChemiDoc成像系统(Bio-Rad,美国)中拍照,结果如图3所示,免疫两只兔子分别获得789和790两种血清,其中789与Artan 1结合力更强,因此选择该血清进一步用Protein G层析柱纯化,得到多克隆抗体789,直接ELISA法检测多抗与Art an 1和黄花蒿提取物的结合活性,结果如图4所示,多克隆抗体789可以与黄花蒿提取物,天然Art an 1以及重组Art an 1.0102结合,并且与阴性对照蛋白BSA不结合,可用于蒿属花粉中Art an 1类型过敏原的检测。
实施例4
提取6种我国常见蒿花粉(黄花蒿,艾蒿,茵陈蒿,细裂叶莲蒿,野艾蒿,大籽蒿)的mRNA,逆转录为cDNA,方法同实施例2,根据IUIS数据库中已有的Art an 1序列,设计Art an1类型过敏原的特异性引物(上游引物:ATGGCAAAGTGTTCATATGT;下游引物:TTAGTGASYGGACGGAGGAG),从而获得6种蒿的Art an 1类型过敏原的氨基酸序列,如图5所示。按照IUIS过敏原命名规则对各亚型进行命名,如表2和图5所示,每种蒿中均克隆发现了1-2种Art an 1类型过敏原的亚型,各个亚型序列较为保守,序列相似度超过95%。
表2六种蒿花粉中Art an 1类型过敏原的GeneBank号
实施例5
提取黄花蒿花粉中的蛋白,提取方法同实施例1,提取液放入冷冻干燥机中过夜干燥,然后用0.1M Tris-HCl溶解。将实施例1中所得到的单抗A7G4E6(10mg)透析至偶联缓冲液中(0.1M NaHCO3,0.5M NaCl,pH=8.3),取1ml CNBr-activated Sepharose 4FastFlow,加入4倍体积的1mM预冷的HCl,搅拌并除去液体,加入透析至PBS中的抗体,4℃过夜偶联,用至少5倍体积的偶联缓冲液清洗2-3次,加入10ml的0.1M Tris-HCl缓冲液室温封闭2h,用3倍体积的A液(0.1M NaAC,0.5M NaCl,pH=4.0)和3倍体积的B液(0.1M Tris-HCl,0.5M NaCl,pH=8.0)交替清洗介质3-4次,再用0.1M Tris-HCl清洗两次后,加入黄花蒿花粉提取物,室温孵育30min,然后用0.1M的Tris-HCl清洗4次,再用0.1M甘氨酸溶液洗脱6次,每次5ml,洗脱液干燥后,用PBS缓冲液溶解,电泳检测,并将目的条带割下,进行质谱鉴定。纯化所得到的天然Art an 1如图6A所示,纯度在90%以上,目的条带经质谱后证实为天然Art an 1过敏原,将肽段匹配到实施例4中所得到的氨基酸序列上,质谱匹配序列如图6B所示。
实施例6
实施例1中所生产的A7G4E6单抗作为包被抗体,实施例3中789多抗作为检测抗体用于构建双夹心ELISA定量检测体系。先用EZ-Link Sulfo-NHS-LC-Biotinylation(ThermoFisher)试剂盒为789多抗标记生物素,取2ml抗体透析至PBS缓冲液中,加入适量现配10mM生物素溶液,混匀后冰浴2h,用脱盐柱去除过量未连接的生物素。将单抗和生物素标记的多抗稀释为不同浓度,实施例5中所得的天然Art an 1稀释为1μg/ml作为抗原,进行双夹心ELISA检测:取100μl A7G4E6单抗包被于酶标板上,4℃过夜,5%脱脂奶粉封闭后加入100μl抗原37℃孵育1h,洗板后加入100μl生物素标记的789多抗37℃孵育1h,HRP标记的链霉亲和素为三抗(1:5000),TMB显色并记录450/620nm值,结果如表3所示,单抗-多抗配对结果较好,随着抗体浓度升高,OD值也随之升高,综合之下,选择单抗和多抗浓度均为3μg/ml。
表3单抗-多抗配对浓度筛选
实施例7
根据实施例5所得结果,将A7G4E6抗体稀释至3μg/ml,取100μl包板,4℃过夜,用5%脱脂奶粉,37℃封闭1h,然后天然Art an 1梯度稀释(32ng/ml,共稀释14个梯度),加入100μl,37℃孵育1h,然后加入100μl的3μg/ml生物素标记的789多抗,37℃孵育1h后,加入100μl HRP(1:10000稀释)37℃孵育1h,然后TMB避光显色15min后,加入2M HCl终止反应,测定450/620nm处的吸光值,用GraphPad绘制标准曲线图(图7),标曲每孔重复两次,绘制标准曲线所用数据如表4所示,该曲线拟合度大于0.99,最低检测阈值(空白对照OD值平均值加上其10倍标准差对应的标准曲线上的最小浓度值)为0.25ng/ml,最佳浓度测定范围(标曲上的线性区域所对应的浓度范围)为0.5-16ng/ml。
表4标准品用于Art an 1类型过敏原定量检测双夹心ELISA标曲的建立
实施例8
按照实施例1中的方法提取6种我国常见蒿属植物及欧洲北艾花粉总蛋白,以实施例7中所建立的标准曲线为基础,测量不同蒿花粉提取物中的Art an 1同种型过敏原含量。将花粉提取物稀释至40ng/ml作为检测样品,按照实施例7中所建立的方法进行检测,每个样品重复测定3次,根据标准曲线计算花粉中Art an 1同种型过敏原的含量如图8所示,Artan 1同种型过敏原在七种蒿花粉中的含量以黄花蒿最高(7.1%),其次是艾蒿(5.6%),细裂叶莲蒿(5.4%),茵陈蒿(4.8%),大籽蒿(4.3%),北艾(4.1%),野艾蒿最低(3.4%)。
Claims (8)
1.一种定量蒿属植物花粉中Art an 1类防御蛋白过敏原的方法,其特征在于,使用单抗-多抗夹心酶联免疫吸附实验进行定量,其中一抗为A7G4E6单抗,来自杭州艾乐吉生物科技有限公司,货号MA-A7G4E6;二抗为789多抗,来自杭州艾乐吉生物科技有限公司,货号PA-789。
2.如权利要求1所述的方法,其特征在于,二抗用生物素标记。
3.如权利要求2所述的方法,其特征在于,使用HRP结合二抗后再加TMB避光显色后进行吸光值检测。
4.如权利要求3所述的方法,其特征在于,使用蒿属植物对应的Art an 1过敏原标准品进行检测得到吸光值后绘制标准曲线,将待检测的蒿属植物花粉样品检测得到的吸光值代入标准曲线计算得到相应浓度。
5.如权利要求4所述的方法,其特征在于,蒿属植物使用Art an 1过敏原标准品。
6.如权利要求5所述的方法,其特征在于,Art an 1过敏原标准品的最低检测阈值为0.25ng/ml,最佳浓度测定范围为0.5-16ng/ml。
7.如权利要求1所述的方法,其特征在于,蒿属植物为黄花蒿、艾蒿、茵陈蒿、细裂叶莲蒿、大籽蒿、野艾蒿或北艾。
8.如权利要求1所述的方法,其特征在于,一抗和二抗的使用浓度均为3μg/ml。
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