CN116789776A - 一种芒果果实的抑制蛋白过敏原及其应用 - Google Patents
一种芒果果实的抑制蛋白过敏原及其应用 Download PDFInfo
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Abstract
本发明公开了一种芒果果实的抑制蛋白过敏原及其应用，涉及生物技术相关技术领域。一种芒果果实的抑制蛋白过敏原，为芒果抑制蛋白的亚型，氨基酸序列如SEQ ID No.1或SEQ ID No.2所示。本发明的一种芒果果实的过敏原，为抑制蛋白，该过敏原可被用于制备治疗或预防芒果过敏症的脱敏疫苗，或者用于制备过敏原检测的试剂盒。
Description
技术领域
本发明涉及生物技术相关技术领域，特别是涉及一种芒果果实的抑制蛋白过敏原及其应用。
背景技术
食物过敏性疾病在世界范围内不断增加，影响到8％的成年人和6％的儿童。过敏原特异性免疫治疗是近年来发展起来的一种治疗方法，它通过增加过敏原的剂量来诱导耐受。几项使用过敏原(如花生或榛子)特异性免疫疗法的临床试验报告，超过50％的受试者对该疗法有反应。然而，这些疗法有其疗效局限性，不良反应也与之相关。
人们越来越需要发现新的食物蛋白过敏原分子和更有效的靶向免疫疗法。
芒果是世界上最重要的水果作物之一，在印度、中国、泰国等地产量很高，随着芒果消费量的增加，其引发的过敏问题也愈加严重。芒果过敏是一种罕见的疾病，但可引起过敏性疾病，表现为过敏性松弛、血管性水肿、红斑、荨麻疹、喘息性呼吸困难晚期反应表现为接触性皮炎和眶周水肿。芒果引发的过敏可分为三类，分别是由IgE介导的Ⅰ型速发型过敏反应，由免疫细胞介导的Ⅳ型迟发型过敏反应，以及蛋白质接触过敏，其中由IgE介导的速发型超敏反应近年来的发生率持续增长。而目前临床上用芒果提取物对患者进行体内和体外诊断的效率低下，漏诊率较高，存在安全隐患。
过敏原组分的应用能够大大提高临床诊断的精确度和灵敏度，已经成为过敏性疾病诊断的发展趋势。而芒果果实过敏原分子鉴定的研究远远落后于其它水果，目前为止，仅有两种过敏原分子得到鉴定，分别是14kDa的抑制蛋白以及37kDa的3-磷酸甘油醛脱氢酶，然而这两种过敏原以前并没有被纳入国际过敏原命名委员会(WHO/IUIS AllergenNomenclature Home Page)，也没有应用于芒果过敏的诊断中。抑制蛋白是一种高度保守的小分子蛋白，在植物中普遍存在，能够引起广泛的交叉反应，目前已经鉴定的芒果抑制蛋白过敏原共两种亚型，免疫印迹实验表明该过敏原在芒果过敏患者中的阳性率为55％，且能够引起芒果与桦树之间的交叉反应(Song J,Zhang H,Liu Z,et al.Mango profilin:cloning,expression and cross-reactivity with birch pollen profilin Bet v2.Molecular Biology Reports,2008,35(2):231-237.)。抑制蛋白存在较多亚型，不同亚型之间的IgE结合能力可能存在较大差异，而选择具有较高IgE结合能力的亚型应用于诊断试剂中能够提高临床诊断的灵敏度和精确度，从而降低漏诊率，因此需要对不同亚型的IgE结合能力进行比较，筛选出最适宜应用于诊断制剂开发的过敏原亚型，探索一种IgE结合能力远高于其它芒果抑制蛋白过敏原亚型的蛋白分子，应用于诊断试剂的开发是有必要的。
发明内容
为解决现有技术中的不足，本发明提供了一种芒果果实的过敏原及其应用。
本发明提供了一种芒果果实的抑制蛋白过敏原，为芒果抑制蛋白的亚型，氨基酸序列如SEQ ID No.1或SEQ ID No.2所示。
本发明还提供了编码上述抑制蛋白过敏原的DNA分子，核苷酸序列如SEQ ID No.3或SEQ ID No.4所示。
本发明还提供了包含上述DNA分子的重组表达载体。
本发明还提供了包含上述DNA分子或包含上述重组表达载体的宿主生物体。
本发明还提供了上述抑制蛋白过敏原或如上述DNA分子在制备治疗或预防芒果过敏症的药物中的应用。
本发明还提供了一种用于治疗或预防芒果过敏症的药物，包含上述抑制蛋白过敏原或上述DNA分子中的至少一种。
优选的，所述药物为脱敏免疫疫苗。
本发明还提供了上述过敏原或上述DNA分子在制备过敏原检测的试剂盒中的应用。
本发明还提供了一种用于过敏原检测的试剂盒，包含上述抑制蛋白过敏原或上述DNA分子中的至少一种。
对于过敏症患者，一个重要的手段是检查出引起过敏的过敏原，通过有针对性的减少与过敏原的接触可有效预防过敏的发生。本发明所述过敏原或所述DNA分子可以用于单独或者与其他过敏原一起使用，制备成用于筛查过敏症患者的过敏原的试剂盒。
本发明一种芒果果实的过敏原，为抑制蛋白，为两个亚型蛋白，该过敏原可被用于制备治疗或预防芒果过敏症的脱敏疫苗，或者用于制备过敏原检测的试剂盒。
附图说明
图1为目前已发现的4种芒果抑制蛋白过敏原亚型的序列比对结果图。
图2为芒果果实中两种抑制蛋白亚型在大肠杆菌中表达纯化后的电泳检测图；泳道1为Man i 4.0101，2为Man i 4.0102。
图3为纯化后的过敏原二级质谱检测结果图，高亮部分代表质谱匹配肽段。
图4为Man i 4.0101和Man i 4.0102与12位芒果过敏患者血清IgE的结合能力对比图。
具体实施方式
芒果果实“凯特”：采集自湛江。
芒果过敏阳性血清：来源于浙江大学医学院附属第二医院以及科研合作的山西省大同市第三人民医院临床病例，山西医科大学附属第一医院，云南曲靖中医院，伦理批号No.2020-050，2015-001，2019K-K0007。所有患者都同意并签有知情同意书。
实施例1
分别提取“凯特”芒果果皮和果肉的mRNA，使用Illumina HiSeqTM2000进行转录组测序。通过SOAPnuke和trimmomatic对测序数据进行过滤，然后使用Trinity对序列进行组装和注释。利用Triple TOF 5600平台对芒果果实总蛋白进行测序，用Mascot将所得谱图与转录组数据库中的序列搜索匹配，最终共鉴定到5354个蛋白，19559个肽段。
搜索果实转录组中的抑制蛋白序列，共找到3个在果实中高表达的抑制蛋白，通过与已鉴定出来的蒿花粉、桃、香蕉等果实中抑制蛋白过敏原的序列比对，有2个过敏原与其同源过敏原的序列高度相似，根据IUIS命名规则，将两个亚型分别命名为Man i 4.0101和Man i 4.0102。在“凯特”芒果果实转录组中没有比对到与已鉴定的两个芒果抑制蛋白过敏原(NCBI序列号分别为DQ270547和DQ400579)完全相同的亚型，Man i4.0101与已鉴定的两个亚型之间的差异较大，而Man i 4.0102与已鉴定的芒果抑制蛋白亚型2(NCBI序列号：DQ400579)仅有一个氨基酸差异(33W/R)，如图1所示。
实施例2
提取“凯特”芒果果实mRNA，逆转录为cDNA，在转录组中找到芒果果实中编码两个抑制蛋白亚型的序列全长，并设计相应的特异性引物，Man i 4.0101的上游引物为cagcaa atgggtcgcggatccATGTCGTGGCAGACCTATGTAGATG，下游引物为gtggtggtggtggtgctcgagTTACAGACCTTGGTCAATTAGATAATCA，Man i4.0102的上游引物为cagcaaatgggtcgcggatccATGTCCTGGCAAGCTTACGT，下游引物为gtggtggtggtggtgctcgagCTAAAGACCTTGTTCCACAAGATA(下划线为引物接头，分别包含BamHⅠ和XhoⅠ酶切位点)用2×I-5高保真酶对该序列进行克隆，反应体系为12.5μL酶，9.5μL水，1μL模板cDNA，上下游引物各1μL，PCR程序为98℃2min，98℃10s；57℃10s，72℃15s，72℃5min，共35个循环；4℃保存。克隆得到的目的片段用胶回收试剂盒回收后，用同源重组酶将将其连接到用BamHⅠ和XhoⅠ酶切后的pET28a载体上，转化到大肠杆菌中，挑取单菌落测序。抑制蛋白Man i4.0101和Man i 4.0102的编码序列全长分别为396bp(核苷酸序列如SEQ ID No.3和SEQ ID No.4所示)，分别了编码131个氨基酸，两种亚型的理论分子量分别为14017.12Da和14016.01Da，理论等电点分别为4.42和4.46，氨基酸序列如SEQ ID No.1和SEQ ID No.2所示。
测序正确的重组质粒转化到Rosetta菌株中，在200mL的含卡那霉素的LB培养基中扩增，37℃培养至OD600达到0.6-0.8，加入终浓度为0.5mM的IPTG，16℃诱导12h，离心后将菌体重悬至PBS缓冲液中，超声破碎机破碎20min，离心取上清，加入200μL的Ni-NTA，4℃孵育1h，用20-250mM的咪唑缓冲液梯度洗脱，SDS-PAGE检测(如图2所示)，该蛋白在大肠杆菌中以可溶性蛋白表达，带有6×His标签，纯化后蛋白大小约为17kDa，收集目标蛋白透析至PBS缓冲液，将两种亚型的胶条上的样品进行LC-MS/MS质谱验证，与理论蛋白序列比对，Man i4.0101的覆盖度达到45.7％，Man i 4.0102的覆盖度达到45％(如图3所示)。
实施例3
取实施例2中纯化的抑制蛋白两种亚型各0.5μg，在4℃下过夜包板，5％脱脂奶粉37℃封闭2h后，加入100μL芒果过敏患者血清37℃孵育2h，然后加入100μL HRP标记的羊抗人IgE二抗(1∶3000，Thermo Fisher，货号A18793)37℃孵育2h，TMB避光显色15min后，用2MHCl终止反应，测定450nm处的吸光值，三个健康人的血清作为阴性对照，测试值大于阴性对照平均值加上标准差的三倍(阴性对照平均值+3SD，其中SD表示标准差)即视为阳性。在12位受测的芒果过敏患者中，两种亚型的过敏原的阳性率一致，但Man i 4.0101的IgE结合能力显著高于Man i4.0102(图4)，因此用Man i 4.0101对患者进行了组分诊断更加准确。在61位芒果过敏患者中，24位(39.3％)对该过敏原阳性(表1)。
表1
注：症状缩写：U-荨麻疹；C-结膜炎；OAS-口腔综合症；G-胃肠道症状；D-气紧；FA-面部水肿；S-打喷嚏。N1～N3为阴性对照血清。ImmunoCAP是测试过敏原常用方法，一般是大于0.35为阳性。ELISA方所测定的sIgE值中，加粗的数值表示阳性值。
序列表
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<120> 一种芒果果实的抑制蛋白过敏原及其应用
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Claims (9)
1.一种芒果果实的抑制蛋白过敏原，其特征在于，为芒果抑制蛋白的亚型，氨基酸序列如SEQ ID No.1或SEQ ID No.2所示。
2.编码如权利要求1所述抑制蛋白过敏原的DNA分子，其特征在于，核苷酸序列如SEQID No.3或SEQ ID No.4所示。
3.包含如权利要求2所述DNA分子的重组表达载体。
4.包含如权利要求2所述DNA分子或包含如权利要求3所述重组表达载体的宿主生物体。
5.如权利要求1所述抑制蛋白过敏原或如权利要求2所述DNA分子在制备治疗或预防芒果过敏症的药物中的应用。
6.一种用于治疗或预防芒果过敏症的药物，其特征在于，包含如权利要求1所述抑制蛋白过敏原或如权利要求2所述DNA分子中的至少一种。
7.如权利要求6所述的药物，其特征在于，所述药物为脱敏免疫疫苗。
8.如权利要求1所述过敏原或如权利要求2所述DNA分子在制备过敏原检测的试剂盒中的应用。
9.一种用于过敏原检测的试剂盒，其特征在于，包含如权利要求1所述抑制蛋白过敏原或如权利要求2所述DNA分子中的至少一种。
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