CN105924508A - 烟曲霉Asp f 3的线性抗原表位最小基序肽Asp f 367-72及其扩展短肽 - Google Patents
烟曲霉Asp f 3的线性抗原表位最小基序肽Asp f 367-72及其扩展短肽 Download PDFInfo
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Abstract
本发明属于生物检测技术领域，具体为烟曲霉Asp f 3的线性抗原表位最小基序肽及其扩展短肽。本发明的烟曲霉Asp f 3的线性抗原表位最小基序肽为Asp f 367‑72，最小基序肽的扩展短肽为Asp f 364‑72、Asp f 365‑73、Asp f 366‑74和Asp f 367‑75，其氨基酸序列如SEQ ID No. 1~SEQ ID No.5所示。它们可作为抗原单独或组合用于特异检测烟曲霉感染患者血清。
Description
技术领域
本发明属于生物检测技术领域，具体涉及导致侵袭性曲霉病、变应性支气管炎和曲霉肿发生的烟曲霉主要抗原蛋白Asp f 3的线性B细胞表位（B cell epitope, BCE,或称抗原表位或决定簇）及其最小基序肽，以及这些表位基序肽的应用。
背景技术
烟曲霉（Aspergillus fumigatus）是一种广泛存在于自然界中的条件致病菌，易感人群吸入其孢子，定植于肺部，从而产生三种曲霉病：导致咯血的曲霉肿、导致肺纤维化的变应性支气管炎和具有较高死亡率的侵袭性曲霉病（Warris A,et al. The biology ofpulmonary aspergillus infections. J Infect, 69 Suppl 1:S36-41,2014）。
血清免疫学指标对于曲霉病的诊断非常重要（Chabi ML,et al. Pulmonaryaspergillosis. Diagn Interv Imaging, 96(5):435-42,2015）。GM（Galactomannan，半乳甘露聚糖）和BG（（1,3）-β-D-glucans，（1,3）-β-D -葡聚糖）是烟曲霉细胞壁的多糖成分，随着孢子的萌发和菌丝体的生长而进入宿主的血循环，因此可分别用GM和BG单抗检测血清中相应的循环抗原，为目前临床上诊断烟曲霉感染的常用方法，但在特异性和灵敏度方面存在一定的局限性（廖万清.侵袭性真菌感染的实验室诊断. 检验医学，25(7)：503-506，2010）。已有的研究表明利用烟曲霉免疫原性强的体外重组蛋白，可以检测感染者血清中相应的循环抗体，但单个重组蛋白同样在特异性和灵敏度方面存在一定的局限性（SarfatiJ,et al. Recombinant antigens as diagnostic markers for aspergillosis. DiagnMicrobil infect Dis, 55(4):279-91,2006）。一个蛋白抗原的长表位肽可能与其它抗原蛋白的抗体起交叉反应，因此鉴定蛋白抗原表位的最小基序可以提高检测的特异性；将多个蛋白抗原表位的最小基序组成嵌合肽可以提高检测的灵敏度。
Asp f 3作为变应原之一，功能为过氧化物酶体蛋白，其命名由世界卫生组织和国际免疫学会联合会变应原命名分委员会（ World Health Organization andInternational Union of Immunological Societies (WHO/IUIS) AllergenNomenclature Sub-committee）批准同意（http://www.allergen.org/search.php?TaxSource=Fungi Ascomycota）。人体感染烟曲霉后，免疫系统产生相应的抗Asp f 3的IgG、IgE和IgA抗体（Kurup VP, et al. Specific antibodies to recombinantallergens of Aspergillus fumigatus in cystic fibrosis patients with ABPA.ClinMol Allergy,4:11,2006）。鉴定Asp f 3的抗原表位最小基序，将其和烟曲霉其它抗原表位肽的最小表位基序构建检测血清嵌合肽，有助于提高诊断烟曲霉感染的特异性和灵敏度。因此，我们用大肠杆菌表达Asp f 3，经镍柱纯化后免疫新西兰白兔，得到Asp f 3抗血清。用改良的生物合成肽策略表达相互重叠9个氨基酸残基、覆盖Asp f 3全长的18肽融合蛋白，继而用Asp f 3抗血清进行免疫印迹，鉴定阳性18肽；对阳性18肽进一步构建相互重叠8个氨基酸残基的9肽融合蛋白，鉴定最小表位基序（Xu, et al. Minimal motif mappingof a known epitope on human zona pellucida protein-4 using peptidebiosynthesis strategy. J Reprod Immunol, 81: 9-16, 2009）。
发明内容
本发明的目的在于提供烟曲霉Asp f 3蛋白的抗原表位最小基序肽，并提供含有所述最小基序肽的扩展短肽，同时提供含所述基序肽的短肽的应用。
本发明提供的烟曲霉Asp f 3蛋白的线性抗原表位最小基序肽，其氨基酸序列为SEQ ID No.1所示，记为Asp f 367-72 ，一字简写的氨基酸序列为：PEYIEK。
本发明还提供烟曲霉Asp f 3蛋白的线性抗原表位最小基序肽Asp f 367-72的扩展短肽（9肽），其序列如SEQ. ID No.2-- SEQ. ID No.5所示，其中：
SEQ. ID No.2：X1X2X3PEYIEK，记为Asp f 364-72。
其中X1是R，X2是H，X3是V，为最小基序Asp f 367-72扩展残基，在化学合成或融合表达9肽融合蛋白的场合，扩展的残基部分可增删或用其它残基替代。
SEQ. ID No.3：X1X2PEYIEKX3，记为Asp f 365-73。其中X1是H，X2是V，X3是L，为最小基序Asp f 367-72扩展残基，在化学合成或融合表达9肽融合蛋白的场合，扩展的残基部分可增删或用其它残基替代。
SEQ. ID No.4：X1PEYIEKX2X3，记为Asp f 366-74。其中X1是V，X2是L，X3是P，为最小基序Asp f 367-72扩展残基，在化学合成或融合表达9肽融合蛋白的场合，扩展的残基部分可增删或用其它残基替代。
SEQ. ID No.5：PEYIEKX1X2X3，记为Asp f 367-75。其中X1是L，X2是P，X3是E，为最小基序Asp f 367-72扩展残基，在化学合成或融合表达9肽融合蛋白的场合，扩展的残基部分可增删或用其它残基替代。
本发明的内容进一步具体描述如下：
1. 根据烟曲霉Af293菌株的Asp f 3（NCBI accession No：XP_747849）的蛋白序列，依据大肠杆菌密码子偏好性，全合成Asp f 3编码基因，从EcoR I和Hind Ⅲ酶切位点克隆于pET-21(b+)质粒，构建pET-21(b)-Asp f 3重组质粒；将C端带有6×His标签的重组质粒pET-21(b)-Asp f 3转入大肠杆菌BL21(DE3)，诱导表达得到重组Asp f 3蛋白；通过镍柱亲和层析后获得纯化Asp f 3蛋白；纯化Asp f 3蛋白免疫新西兰白兔，得到高特异性兔抗Aspf 3抗血清（图1）；
2. 用pXXGST-1融合表达质粒，构建相互重叠9个氨基酸残基、覆盖Asp f 3全长的GST-18肽融合蛋白（Xu, et al. Minimal motif mapping of a known epitope on humanzona pellucida protein-4 using peptide biosynthesis strategy. J ReprodImmunol, 81: 9-16, 2009）。继而用兔Asp f 3抗血清进行免疫印迹，鉴定得到其中的一个阳性18肽：Asp f 364-81Rhvpeyi eklpeirakgv（图2）。构建相互重叠8个氨基酸残基、覆盖Aspf 364-81以及与Asp f 364-72相邻的Asp f 363-71GST-9肽融合蛋白，免疫印迹鉴定得到4个阳性9肽：Asp f 364-72 RHVPEYIEK、Asp f 365-73HVPEYIEKL、Asp f 366-74VPEYIEKLP和Asp f367-75PEYIEKLPE，其共有序列Asp f 367-72PEYIEK为Asp f 3抗原最小表位基序肽（图3）。
本发明所述的表位最小基序肽Asp f 367-72可用于制备作为临床诊断由烟曲霉感染导致侵袭性曲霉病、变应性支气管炎和曲霉肿血清识别表位标志物。
本发明所述的表位最小基序肽的扩展短肽Asp f 364-72、Asp f 365-73、Asp f 366-74和Asp f 367-75单独或组合用于制备作为临床诊断由烟曲霉感染导致侵袭性曲霉病、变应性支气管炎和曲霉肿血清识别表位标志物。
附图说明
图1 兔抗Asp f 3抗血清ELISA结果。1号、2号、3号和4号为Asp f 3免疫兔血清，对照为免疫前兔血清。
图2 Asp f 3 18肽融合蛋白电泳和免疫印迹图。上方蓝色条带为电泳图谱，下方黑色条带为免疫印迹图谱。0号泳道为pXXGST-2表达的GST188，1至18号泳道为跨域Asp f 3全长、相互重叠9个氨基酸残基的18肽与GST188融合蛋白；由于Asp f 3全长168aa，故18号泳道为Asp f 3154-168。其中8号泳道免疫印迹阳性条带为Asp f 364-81。
图3 Asp f 367-72最小表位肽基序免疫印迹图。7-9表示Asp f 363-71，8-1表示Aspf 364-72，8-2表示Asp f 365-73，8-3表示Asp f 366-74，8-4表示Asp f 367-75，8-5表示Asp f368-76，箭头表示免疫印迹反应阳性条带。最小表位基序肽为Asp f 367-72PEYIEK。
具体实施方式
本发明可进一步通过下列实例描述。这些例子并不意味限制本发明所涉及的范围，该范围在之前的描述中已被充分陈述阐明。下列实施例中未注明具体条件的实验方法，均按照常规条件以及[美]J. 萨姆布鲁克和D.W. 拉塞尔编著黄培堂等翻译的第三版“分子克隆：实验室手册”（科学出版社，2002）和[美]E. 哈洛和D. 莱恩编著沈关心等翻译的“抗体技术实验指南” （科学出版社，2002）中所述的步骤，或按照生产销售商建议的条件进行。
材料和方法：
1. pET-21(b+) 质粒和E.coli BL21（DE3）菌株购自德国Novagen公司，热诱导表达质粒pXXGST-1和对照质粒pXXGST-2由本专利发明人构建(专利号：200710173305.2)；
2. 限制性内切酶EcoR I、BamH I、Sal I购自美国New England Biolabs公司， DL2000DNA分子量标准购于北京康为世纪生物科技有限公司，T4 DNA连接酶和预染蛋白分子量标准购自美国Thermo-Fermentas公司，小鼠6 × His单克隆抗体和弗氏完全佐剂以及弗氏不完全佐剂购于美国Sigma公司，辣根过氧化物酶（HRP）标记的羊抗鼠IgG和辣根过氧化物酶（HRP）标记的羊抗兔IgG购自美国Santa Cruz Biotechnology公司，0.2umPVDF膜购自美国Millipore公司，ECL化学发光显色液购自美国GE公司；
3. Gel Extraction凝胶回收试剂盒和Plasmid Mini质粒小量抽提试剂盒购于美国Omega公司；
4. 新西兰白兔购自上海西普尔-必凯实验动物有限公司；
5. Asp f 3基因全由上海旭冠生物科技发展有限公司合成。两端分别为BamH I和SalI粘性末端，中间为覆盖Asp f 3全长且相互重叠9个氨基酸残基的18肽编码DNA序列加TAA终止密码子的正负链DNA片段以及覆盖免疫印迹反应18肽且相互重叠8个氨基酸残基的9肽编码DNA序列加TAA终止密码子的正负链DNA片段由上海杰李生物技术有限公司合成。DNA重组子测序由上海睿迪生物科技有限公司完成。
Asp f 367-72最小表位肽基序鉴定的具体步骤如下：
1. 烟曲霉Asp f 3的表达与纯化
1.1 表达载体pET-21(b)-Asp f 3的构建
根据烟曲霉Af293菌株的Asp f 3（NCBI accession No：XP_747849）的蛋白序列，依据大肠杆菌密码子偏好性，由上海旭冠生物科技发展有限公司全合成基因，分别从EcoR I和Hind Ⅲ酶切位点克隆于pET-21(b+)，构建pET-21(b)-Asp f 3重组质粒，Asp f 3蛋白的全长为168个氨基酸。
1.2 重组Asp f 3表达
将C端带有6×His标签的重组质粒pET-21(b)-Asp f 3取1µL（约100ng/µL）转化到10µL大肠杆菌BL21(DE3)感受态细胞中，冰浴30min后42℃热激90s，冰上冷却5min，加入900µLLB液体培养基37℃复苏40min～60min，最后10000r/min离心1min弃去约800 µL培养基，其余混匀后涂氨苄青霉素抗性的LB平板。待菌液被培养基吸收后37℃倒置培养16～24h。
挑取单克隆菌落于含50µg/mL氨苄青霉素的3mL LB液体培养基中，37℃培养过夜。翌日，按照1:50的比例将过夜菌转接至新的3mL LB液体培养基中，氨苄青霉素的工作浓度不变，37℃培养约2.5～3h（OD600为0.6~0.8）后，取出1mL菌液样本作为诱导前对照，其余菌液加入终浓度为1mM的异丙基硫代半乳糖苷（IPTG）诱导重组蛋白表达，表达条件为37℃培养4h。取500 µL诱导后菌液样本，10000r/min离心5min，收集菌体，用150µL 1×PBS缓冲液溶菌，并加入50 µL 4×SDS-PAGE Loading Buffer混匀，沸水煮样7～10min保存待用。
配制2块相同的12%的SDS-PAGE胶，按照同样的顺序（蛋白分子量marker→诱导前全菌蛋白→诱导后全菌蛋白）和同样的上样量（5 µL）在同一电泳系统内进行SDS-PAGE凝胶电泳。电泳完成后，其中一块胶用于考马斯亮蓝R250染色分析诱导表达条带，另一块用小鼠6 × His单克隆抗体为一抗、HRP标记的羊抗鼠IgG为二抗进行Western-blot验证。实验结果表明成功表达了重组Asp f 3蛋白。
1.3 重组Asp f 3蛋白的纯化
选取表达量高的单克隆菌株作为保种菌。取保种菌10µL接入50mL含50ug/mL氨苄青霉素的LB液体培养基中37℃培养过夜。次日，按照1:50的比例将菌液转入500mL含50µg/mL氨苄青霉素的LB液体培养基中，用1mM IPTG诱导4h，5000r/min 4℃离心5min，弃去培养基收集菌体。菌体称重（事先称好500mL塑料瓶的重量）后，加入菌体重量10陪的1×startbuffer（1g菌体加10mL 1×start buffer ；start buffer: 50mM Na2HPO4/NaH2PO4，pH8.0，300mM NaCl，10mM咪唑，1mM β-巯基乙醇），重悬菌体。用高压细胞破碎机在合适的压力下破菌，重复破菌3次后，4℃、15000rpm离心30分钟，收集上清。5mL His Trap HP预装柱事先用5倍柱体积的纯水冲净，并以5倍柱体积以上的Start Buffer平衡。上样完毕后，以StartBuffer冲洗柱子，去除未吸附的蛋白。以含40 mM 咪唑浓度的Start Buffer冲洗柱子，尽量去除杂蛋白，随后设置15mL 长度，12%-100% Elution Buffer （50mM Na2HPO4/NaH2PO4，300mM NaCl，250mM咪唑，pH 8.0）洗脱，收集目的蛋白。与此同时将pET-21(b)空质粒作为阴性对照与实验重组质粒pET-21(b)-Asp f 3做平行实验，并收集两个空质粒的诱导前菌体及IPTG诱导4h后菌体，用于SDS-PAGE电泳。
分别收集全菌液、上清和纯化产物20ul，加入工作浓度为1×SDS Loading BufferLoading混匀，煮沸5min，冷却后取10 μL上样，用于变性SDS-PAGE电泳检测。将蛋白条带和预计分子量测吻合，纯度达到80%以上的样品合并，并进行超滤、浓缩处理，用Bradford法估测纯化后蛋白浓度。用像素计算软件估测详细的纯化蛋白的纯度。
2. 兔抗重组蛋白Asp f 3抗血清的制备
取1mg Asp f 3纯化蛋白与等体积弗氏完全佐剂乳化后皮下注射新西兰白兔，进行初次免疫；3周后取0.5mg Asp f 3纯化蛋白与等体积弗氏不完全佐剂乳化后皮下注射新西兰白兔，进行第一次加强免疫；2周后取0.5mg Asp f 3纯化蛋白与等体积弗氏不完全佐剂乳化后皮下注射新西兰白兔，进行第二次加强免疫；第二次加强免疫一周后，在实验兔子的耳缘静脉取血约3～5mL，离心得到抗血清，ELISA法检测抗体效价后备用。
3. Asp f 367-72最小表位肽基序鉴定
3.1 Asp f 364-81阳性表位肽鉴定
依据Asp f 3基因序列公开信息，设计跨越Asp f 3全序列的系列相互重叠9个氨基酸残基的18肽编码DNA正负链片段（正链5’-端加5’-gatcc,3’-端加taag-3’；负链5’-端加5’-tcgactta，3’-端加g-3’），外送DNA合成。将1或2个OD的互补正负链片段用ddH2O溶解成20 µmol/µL储存液（根据DNA合成报告单数据），各取10 µL储存液和20 ddH2O于1.5 mLEppendorf管中，94℃水浴加热5 min,自然降至室温后，在15 µL反应体积中吸入2 µL退火片段、1 µL 约200 ng/µL经BamH I和Sal I双酶切的pXXGST-1质粒、1 µL T4 DNA连接酶和其1.5 µL缓冲液，连接过夜；连接液转化感受态BL21（DE3）宿主菌，涂布含Amp的LB平板上，于37℃培养过夜；第二天挑取氨苄LB平板上长出的单克隆转接3 mL LB培养液诱导表达，以由pXXGST-2表达的GST188蛋白为对照，各表达的GST188-18肽融合蛋白经15％SDS-PAGE分析确认（与对照蛋白电泳迁移率相差约2 kDa），挑取各重组克隆外送进行DNA测序。将插入片段测序结果正确的克隆接种加入Amp的3 mL LB 培养液中，于30℃振荡培养过夜，翌日晨按1/50比例转接新鲜含Amp的LB培养液中，30℃振荡培养2 ～ 3 h 至菌体浓度达到0.6 ～0.8 OD后，调高温度至42℃热诱导4 h，离心收集菌体，加上样裂解液煮沸5 min，进行15％SDS-PAGE。用兔抗Asp f 3抗血清为一抗，羊抗兔IgG /HRP为二抗，进行免疫印迹。Asp f364-81为阳性表位肽之一。
3.2 Asp f 367-72最小表位基序肽鉴定
以pXXGST-1为表达载体，构建表达跨越Asp f 364-81、相互重叠8个氨基酸残基以及与Asp f 364-72相邻的Asp f 363-71系列9肽GST188融合蛋白，用兔抗Asp f 3抗血清为一抗，羊抗兔IgG /HRP为二抗，进行免疫印迹，Asp f 364-72 RHVPEYIEK、Asp f 365-73HVPEYIEKL、Aspf 366-74VPEYIEKLP和Asp f 367-75PEYIEKLPE为阳性表位肽，其共有序列Asp f 367-72PEYIEK为Asp f 3抗原最小表位基序肽。
应用例
应用Asp f 364-72、Asp f 365-73、Asp f 366-74和Asp f 367-75检测血清中Asp f 3抗体
1. 用Asp f 3蛋白免疫新西兰白兔后取血清，以免疫前血清为对照，4只实验兔的Aspf 3抗体滴度均达到1.28×105以上（图1），用于后续免疫印迹反应。
2. 以pXXGST-1为载体，表达Asp f 363-71、Asp f 364-72、Asp f 365-73、Asp f 366-74、Asp f 367-75Asp 和f 368-76融合蛋白。
3. 表达的Asp f 363-71、Asp f 364-72、Asp f 365-73、Asp f 366-74、Asp f 367-75和Asp f 368-76融合蛋白进行15% SDS-PAGE电泳。
4. 将电泳凝胶蛋白条带电转移至PVDF膜，印迹膜用封闭液（TBSTM）封闭后，用封闭缓冲液（TBST）洗膜，然后以一定比例稀释的实验兔血清为一抗，4℃振荡孵育过夜。
5. 次日，用TBST洗膜后将印迹膜置于TBSTM中，加入一定比例稀释的HRP标记的羊抗兔IgG为二抗，室温震荡孵育1.5h。用TBST洗膜3遍后将膜置于TBS缓冲液中，加入ECL化学发光剂，约1min后进行曝光，判断检测结果。包含最小表位基序肽Asp f 367-72PEYIEK的Aspf 364-72、Asp f 365-73、Asp f 366-74和Asp f 367-75印迹反应为阳性；而Asp f 363- 71ARHYPEYIE和Asp f 368-76由于缺少包含于最小表位基序肽Asp f 367-72中的第72位残基k和第67位残基P，印迹反应为阴性（图3）。
尽管本发明描述了Asp f 3蛋白抗原表位最小基序Asp f 367-72PEYIEK，以及可单独和/或组合用含最小基序的9肽或9肽融合蛋白研制用作检测人烟曲霉感染的抗原肽（或化学合成或融合表达），但有一点对于相关领域研究技术人员来说是显而易见的，即在不脱离本发明的精神和范围的情况下，可对本发明的表位肽基序和扩展的含最小基序9肽融合蛋白作各种变化改动。因此，所附权利要求覆盖了所有这些在本发明范围内的变动。
SEQUENCE LISTING
<110> 复旦大学
<120> 烟曲霉Asp f 3的线性抗原表位最小基序肽Asp f 367-72及其扩展短肽
<130> 001
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Claims (4)
1. 一种烟曲霉Asp f 3蛋白的最小表位基序肽，其特征在于具有SEQ.ID No.l 所示氨基酸序列，记为Asp f 367-72，其一字简写氨基酸序列为PEYIEK。
2. 一种含如权利要求1所述烟曲霉Asp f 3蛋白的最小表位基序肽的扩展短肽，其特征在于：
具有SEQ. ID No.2所示氨基酸序列，记为Asp f 364-72，扩展9肽一字简写氨基酸序列为RHVPEYIEK；或者，
具有SEQ. ID No.3所示氨基酸序列，记为Asp f 365-73，扩展9肽一字简写氨基酸序列为HVPEYIEKL；或者
具有SEQ. ID No.4所示氨基酸序列，记为Asp f 366-74，扩展9肽一字简写氨基酸序列为VPEYIEKLP；或者，
具有SEQ. ID No.5所示氨基酸序列，记为Asp f 367-75，扩展9肽一字简写氨基酸序列为PEYIEKLPE；
在化学合成或融合表达9肽融合蛋白的场合，扩展的残基部分可增删或用其它残基替代。
3.如权利要求1所述的表位最小基序肽在制备作为临床诊断由烟曲霉感染导致侵袭性曲霉病、变应性支气管炎和曲霉肿血清识别表位标志物中的应用。
4.如权利要求2所述的表位最小基序肽的扩展短肽单独或组合在制备作为临床诊断由烟曲霉感染导致侵袭性曲霉病、变应性支气管炎和曲霉肿血清识别表位标志物中的应用。
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| CN102659923A (zh) * | 2012-05-13 | 2012-09-12 | 上海市计划生育科学研究所 | 人卵透明带蛋白-4的表位最小基序肽及其扩展短肽和应用 |
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| Title |
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| 汪世华主编: "《抗体技术》", 31 March 2009 * |
| 石晶 等: "烟曲霉Asp f3和Asp f4线性B细胞抗原表位最小基序鉴定", 《微生物与感染》 * |
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