CN107056932A - 一种氨基酸序列、含ova表位的融合蛋白及其制备方法和应用 - Google Patents
一种氨基酸序列、含ova表位的融合蛋白及其制备方法和应用 Download PDFInfo
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- CN107056932A CN107056932A CN201611210828.5A CN201611210828A CN107056932A CN 107056932 A CN107056932 A CN 107056932A CN 201611210828 A CN201611210828 A CN 201611210828A CN 107056932 A CN107056932 A CN 107056932A
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Abstract
本发明提供了一种编码OVA表位的氨基酸序列、含OVA表位的融合蛋白及其制备方法和应用，所融合抗体的氨基酸序列包括抗DEC抗体单链可变片段的氨基酸序列和OVA表位。本发明通过鸡蛋过敏患者血清和小鼠OVA IgG1抗体筛选模式化抗原OVA优势T细胞表位，再利用scFv DEC能与树突状细胞受体DEC205特异识别并结合的特性，制备scFv DEC:OVA表位融合蛋白，将T表位肽靶向DC，将为食物过敏及其他过敏性疾病开辟新的治疗药物研发途径。本发明提供含OVA表位的融合蛋白具备良好的结合BMDCs的能力，可抑制OVA诱导的小鼠过敏性炎症的发生，有望应用于防治鸡蛋过敏性炎症、甚至有望应用于防治所有的过敏性疾病。
Description
技术领域
本发明涉及生物医药和诊疗领域，具体涉及一种氨基酸序列、含OVA表位的融合蛋白及其制备方法和应用。
背景技术
食物过敏和相关疾病在全球范围内迅速增加，大约4％-8％的儿童和1％-2％的成年人对食物抗原具有IgE介导的高反应性。食物过敏(food allergy)症状轻则表现为轻度不适，重则表现为威胁生命的过敏性休克，己经成为对社会、经济有巨大影响的疾病。尽管食物过敏的死亡率较为罕见，初步估算每年死亡率约为1.8/1000，000。对食物过敏意外的恐惧及饮食回避导致的社会影响严重影响到过敏性炎症儿童及其照顾者的生活质量。在前瞻性过敏原特异性食物过敏治疗临床研究中，口服耐受治疗已受到诸多关注，然而有研究表明，这种免疫治疗可引起严重不良反应(如胃肠道炎症和过敏)。为确保口服耐受治疗的安全型和有效性，急需发展低风险靶向治疗。
树突状细胞(Dendritic Cells,DCs)是专职抗原提呈细胞，DCs在过敏原特异性免疫治疗中发挥重要作用，可能成为诱导食物过敏原免疫耐受的有力靶标。上皮细胞受体(Dendritic and Epithelium Cells，DEC205)属于I类C型凝集素，几乎专一表达于DC表面，因其分子量约205kDa，(205kD)故被称为DEC205。研究证实免疫系统处于静息状态时，抗原经anti-DEC靶向DC，可诱导T细胞免疫耐受。若同时附加额外刺激，如TLR配体CpG，Poly(I:C)则可有效活化DCs，诱导长时间持续性抗肿瘤应答。
单链抗体(single-chain antibody fragment，scFv)由抗体重链可变区和轻链可变区通过15～20个氨基酸短肽(linker)连接而成，它不仅保留了特异性识别抗原的特性，而且具有自身体积小、穿透力强、免疫原性低等优点。有研究将scFv DEC与髓鞘少突胶质细胞糖蛋白(Myelin Oligodendrocyte Glycoprotein,MOG)基因融合形成scFv DEC:MOG，静脉注射scFv DEC:MOG可诱导脾脏TDC的形成。scFv DEC潜在成为靶向DEC205+DCs诱导免疫耐受的重要工具。
特异性免疫疗法是治疗I型超敏反应的唯一可靠方法，借助诱导过敏原特异性T细胞耐受可能达到治疗目的。临床治疗主要是过敏原皮下逐渐加大剂量的脱敏治疗，但伴随的全身性副作用较为常见。大量报道表明T辅助细胞在过敏反应免疫应答的诱导和调节过程中发挥重要作用，然而精确表位鉴定及过敏原特异性CD4+T细胞功能特性在单一表位水平的研究有限，妨碍了新型高效疫苗的研发。T细胞表位不能够与锚定在肥大细胞表面的IgE发生交联，故全身性副作用较少；截止目前，肽免疫治疗已成功应用于昆虫毒液和猫过敏。鸡卵清蛋白(OVA)，是主要的鸡卵过敏原，已作为模式化抗原广泛应用于人类鸡蛋过敏性疾病的研究。
目前，还未有一种既能特异识别并结合树突状细胞受体DEC205，又能高效将T表位肽靶向DC，从而有效抑制食物过敏及其他过敏性疾病的抗体药物。
发明内容
解决的技术问题：本发明提供了一种含OVA表位的融合蛋白及其制备方法和应用。
本发明的技术方案如下：
第一方面，本发明提供了一种氨基酸序列，所述氨基酸序列包括至少一个OVA表位，编码所述OVA表位的氨基酸序列为选自如下序列1)或2)中的任一种：
1)第一抗体的氨基酸序列，所述第一抗体的氨基酸序列包括氨基酸序列IKHIATNAVLFFGRC(SEQ ID NO:1)或由氨基酸序列IKHIATNAVLFFGRC(SEQ ID NO:1)组成；
2)第二抗体的氨基酸序列，所述第二抗体为：包括与第一抗体具有至少60％、70％、80％、90％、95％或99％序列同源性的氨基酸序列或由与第一抗体具有至少60％、70％、80％、90％、95％或99％序列同源性的氨基酸序列组成。
第二方面，本发明提供了一种含OVA表位的融合蛋白，所述含OVA表位的融合蛋白的氨基酸序列包括如本发明第一方面提供的氨基酸序列，还包括抗DEC抗体单链可变片段的氨基酸序列。
在本发明一优选实施例中，所述的本发明第一方面提供的氨基酸序列与所述抗DEC抗体单链可变片段的氨基酸序列之间通过连接肽连接，所述连接肽的氨基酸序列优选为GGGGSAAA(SEQ ID NO:2)。
在本发明一优选实施例中，所述抗DEC抗体单链可变片段的氨基酸序列包括SEQID NO:3所示的氨基酸序列、还包括SEQ ID NO:4所示的氨基酸序列。
具体地，所述SEQ ID NO:3所示的氨基酸序列为重链序列，具体为：
VH:GWSCIILFLVATATGVHSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSS
具体地，所述SEQ ID NO:4所示的氨基酸序列为轻链序列，具体为：
VL:DIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKR
更优选地，所述抗DEC抗体单链可变片段的氨基酸序列包括SEQ ID NO:3、SEQ IDNO:4所示的氨基酸序列，其中，SEQ ID NO:3、SEQ ID NO:4所示的氨基酸序列之间通过连接肽连接，所述连接肽的氨基酸序列优选为GGGGSGGGGSGGGGS(SEQ ID NO:5)。
在本发明一优选实施例中，所述含OVA表位的融合蛋白的氨基酸序列包括SEQ IDNO:6所示的氨基酸序列或由SEQ ID NO:6所示的氨基酸序列组成。
具体地，所述SEQ ID NO:6所示的氨基酸序列为：
在本发明一具体实施例中，在本发明提供的sDI融合基因编码蛋白，由295个氨基酸组成，分子量约为31539.15Da，为DEC205抗体单链C末端通过连接肽连接OVA优势表位(OVA优势表位即IC短肽，氨基酸序列为SEQ ID NO:1)，还包括His标签；可选地，除His标签之外，本发明提供的sDI融合基因编码蛋白还可以采用GST标签等蛋白纯化标签。
在本发明一优选实施例中，编码所述含OVA表位的融合蛋白的核苷酸序列如SEQID NO:7所示。
具体地，所述SEQ ID NO:7所示的核苷酸序列为
可选地，所述的含OVA表位的融合蛋白的核苷酸编码序列还可包括任一种His标签、GST标签等蛋白纯化标签的核苷酸编码序列，优选为His标签。
第三方面，本发明提供了一种制备如第二方面所述的含OVA表位的融合蛋白的方法，包括：
1)采用软件预测获得OVA T细胞表位，然后采用临床鸡蛋过敏患者血清和小鼠抗OVA IgG1，并借助Western Blot法和ELISA法验证，筛选获得优势OVA表位；
2)获取抗小鼠DEC基因序列，并将所得抗小鼠DEC基因序列与编码步骤1)所得优势OVA表位的基因序列融合，获得编码如权利要求1所述的含OVA表位的融合蛋白的核苷酸序列；
3)将编码如权利要求1所述的含OVA表位的融合蛋白的核苷酸序列用于构建表达所述的含OVA表位的融合蛋白的重组表达载体；导入宿主细胞；在表达条件下，培养宿主细胞，表达，分离纯化，获得所述含OVA表位的融合蛋白。
第四方面，本发明提供了一种药物组合物，包括至少一种如第一方面所述的氨基酸序列或如第二方面所述的含OVA表位的融合蛋白，及诊断剂、可药用的载剂、赋形剂或稀释剂。
第五方面，本发明提供了一种核酸分子，编码如第一方面所述的氨基酸序列或如第二方面所述的含OVA表位的融合蛋白的氨基酸序列。
第六方面，本发明提供了一种重组载体，具有如第五方面所述的任一核酸分子序列。
第七方面，本发明提供了一种宿主细胞，包含如第六方面所述的重组载体。
第八方面，本发明提供了一种检测试剂盒，固体检测支持物，所述的固体检测支持物包含至少一种如第一方面所述的氨基酸序列或如第二方面所述的含OVA表位的融合蛋白；所述固体支持物为抗体、蛋白质、多肽、肽、多核苷酸的固体表面的任一种。
第八方面，本发明提供了一种含如第一方面所述的氨基酸序列或如第二方面所述的含OVA表位的融合蛋白在制备诊断、预防、治疗、抑制过敏性疾病的试剂或药物中的应用。
优选地，本发明提供了一种含如第一方面所述的氨基酸序列或如第二方面所述的含OVA表位的融合蛋白在制备诊断、预防、治疗、抑制鸡蛋过敏引起的消化道炎症的试剂或药物中的应用。
优选地，本发明提供了一种含如第一方面所述的氨基酸序列或如第二方面所述的含OVA表位的融合蛋白在制备诊断、预防、治疗、抑制小鼠过敏性炎症的试剂或药物中的应用。
本发明的效益在于：
(1)本发明通过筛选获得模式抗原OVA优势T细胞表位，与scFv DEC形成融合基因，制备获取重组蛋白，重组蛋白纯度高、特异性较好、产量较高；
(2)本发明提供的所述含OVA表位的融合蛋白可以靶向结合DC；
(3)本发明提供的所述含OVA表位的融合蛋白可以抑制OVA诱导的小鼠过敏性炎症；抑制OVA特异性T细胞增殖；
(4)本发明提供的所述含OVA表位的融合蛋白可潜在应用于对鸡蛋过敏患者消化道炎症的预防和治疗；
(5)本发明提供的所述含OVA表位的融合蛋白可潜在应用于除食物过敏以外的其他过敏性疾病的预防和治疗。
附图说明：
图1为Western Blot法和ELISA法检测结果；
其中，图1-a软件预测OVA T细胞表位偶联BSA的SDS-Page电泳结果；图1-b是BSA偶联的不同表位与小鼠OVA IgG1抗体反应免疫印迹结果；图1-c是BSA偶联的不同表位与鸡蛋过敏患者血清的免疫印迹结果；图1-d是BSA偶联的不同表位与小鼠OVA IgG1抗体反应的ELISA检测结果；图1-e显示BSA偶联的不同表位与鸡蛋过敏患者血清反应的ELISA检测结果；
图2为本发明实施例提供的融合蛋白sDI的电泳、免疫结果；
其中，图2-a为融合蛋白sDI基因结构设计图；图-2b左为scFv DEC，右为融合蛋白sDI纯化后SDS-Page电泳结果；图2-c为sDI与鸡蛋过敏患者血清反应的免疫印迹结果；图2-d为sDI与小鼠OVA IgG1反应的免疫印迹结果；图2-e为sDI蛋白的测序结果；
图3为本发明实施例提供的融合蛋白sDI结合DC能力的流式细胞检测结果；
图4为本发明实施例提供的融合蛋白sDI在预防、治疗过敏性炎症小鼠的检测结果；
其中，图4-a图为sDI蛋白抑制食物过敏的实验方案(虚线箭头表示OVA暴露24小时前给予腹腔注射50/100μg sDI，或scFv DEC 100μg，或PBS，体系均为100μlPBS；实线为OVA/20μgCT(1-4w))；图4-b,c柱形图显示不同组发生腹泻的小鼠数量差异及体温的变化情况；图4-d显示给予过敏性炎症小鼠sDI融合蛋白预防治疗后各组空肠组织的病理结果(各组小鼠空肠组织嗜酸性粒细胞浸润情况)，1-5对应正常对照组(N)，PBS处理组(S)，scFv DEC100μg组，sDI蛋白50μg组，sDI蛋白100μg组，图6-10分别1-5对应的油镜视野；图4-e显示各组空肠组织甲苯染色肥大细胞的结果，各组对应同病理结果(各组小鼠空肠组织肥大细胞浸润情况)；图4-f,g柱形图显示各组空肠组织中嗜酸性粒细胞及肥大细胞浸润的计数；图4-h为小鼠血清中OVA特异性IgE，IgG1，IgG2a的检测结果；图4-i显示血清中IFN-γ和IL-4水平；图4-j显示各组肠系膜淋巴结单个核细胞中OVA特异性CD4+T细胞增殖情况。
具体实施方式
实施例一、sDI融合蛋白的分子克隆
一、模式抗原OVA主要T细胞表位的预测及筛选
通过软件ProPred.MHC ClassПBinding Peptide Prediction Server软件在线预测模式抗原OVA主要T细胞表位，获得结果如下：
VE-15 VVRFDKLPGFGDSIE；
GI-15 GLFRVASMASEKMKI；
IC-15 IKHIATNAVLFFGRC。
通过临床鸡蛋过敏患者血清和小鼠抗OVA IgG1，并借助Western Blot法和ELISA法验证，获得优势OVA表位。
Western blot实验
以鸡蛋过敏患者血清为一抗进行Western blotting，以带有链酶亲和素-辣根过氧化物酶(HRP)标记的小鼠抗人IgG1为二抗孵育后，加入免疫印迹化学发光试剂(ECL)，放射自显影片曝光和洗片处理。
二、间接ELISA检测实验
具体过程：用1μg/ml OVA及OVA主要表位短肽包被96孔板，过敏性炎症患者血清用2.5％的脱脂奶粉按1:2比例稀释，小鼠IgG1按照1：2000比例稀释；4℃孵育过夜，二抗按1:2000稀释，洗涤后，加入TMB显色完毕后，在450nm处读取吸光度值。
具体实验步骤如下：
1.将抗原按适当浓度溶解于包被液(已配好CBS液)中。1μg/ml，设置空白孔，阳性对照孔OVA；在对应的孔中加入100μl抗原，室温孵育2h或4℃过夜；倒空液体并拍干残留液体，用300μl洗涤液清洗2次。
2.每个孔中加200μl封闭液(5％BSA)，37℃孵育1h；倒空液体并拍干残留液体，用300μl洗涤液清洗两次。
3.每个孔中加100μl一抗(食物过敏患者血清或小鼠IgG1)，37℃孵育1-2h；倒空液体并拍干残留液体，每个孔中加满洗涤液，倒空液体并拍干残留液体，重复3次。
4.每个孔中加100μl二抗，室温孵育1-2h；倒空液体并拍干残留液体，每个孔中加满洗涤液，倒空液体并拍干残留液体，重复3次。
5.用洗涤液浸泡5min，拍干残留液体，这次的洗涤步骤对于减少背景信号是很重要的；每个孔中加满洗涤液，倒空液体并拍干残留液体，重复5次。
6.每个孔中加100μl底物，显色10min，用50μl/孔2mol/L H2SO4终止，立即在450读数。
综上，本发明实施例通过NCBI数据库获取scFv DEC-EDIII(GenBank ID:KC596074)的序列，获得抗小鼠scFv anti DEC基因序列，该基因为NLDC-145杂交瘤细胞系克隆扩增获得。
Western Blot法和ELISA法检测结果见图1：其中，图1-a为所得优势OVA表位(本发明中亦称“T表位”)偶联BSA的SDS-Page结果；图1-b,1-c为WB结果,在图1-a、1-b、1-c中,泳道M是marker，泳道1、2、3分别是GI-15、VE-15、IC-15的结果，OVA和BSA是对照泳道；图1-d,1-e为ELSIA结果；具体地，图-1a为软件预测OVA T细胞表位偶联BSA的SDS-Page电泳结果；图-1b是BSA偶联的不同表位与小鼠OVA IgG1抗体反应免疫印迹结果；图-1c是BSA偶联的不同表位与鸡蛋过敏患者血清的免疫印迹结果；图-1d是BSA偶联的不同表位与小鼠OVA IgG1抗体反应的ELISA检测结果；图-1e显示BSA偶联的不同表位与鸡蛋过敏患者血清反应的ELISA检测结果。
检测结果显示，IC--15效果较佳，故本发明实施例采用IC-15进行后续验证实验，IC-15包含15个氨基酸的短肽，序列为：IKH IAT NAV LFF GRC(SEQ ID NO:1)。
二、sDI融合基因的合成及其表达载体的构建与酶切鉴定
2.1合成融合基因
通过NCBI数据库获取scFv DEC-EDIII(GenBank ID:KC596074)的序列，获得抗小鼠scFv anti DEC基因序列，将步骤(一)筛选获得的OVA优势T细胞表位编码基因序列IC-15与获得的抗小鼠scFv DEC基因序列通过连接序列进行连接，合成sDI融合基因，所得sDI融合基因的核苷酸序列如SEQ ID NO:8所示(融合蛋白sDI基因结构设计图如图2-a所示)
序列合成为南京金斯瑞生物技术有限公司完成。
2.2构建sDI融合蛋白重组表达载体
将sDI融合基因插入至pET-32a的Bam HⅠ与EcoRI酶切位点之间位置，为明确表达载体融合基因的序列是否符合预期，我们送检小量表达sDI融合蛋白筛选的菌种送至华大基因进行测序，经NCBI Blast序列比对发现，表达载体序列与预期100％匹配。
三、sDI蛋白的诱导表达及纯化
将鉴定好的pET32a-sDI转化至感受态大肠杆菌Rosetta(DE3)，待细菌生长处于对数生长期(A600nm＝0.6～0.9)时，加入20μl 1mol/L的IPTG，诱导蛋白表达。诱导4h后取1ml菌液，离心弃上清液，加100μl去离子水后重悬菌体，加20～25μl 10×SDS-PAGE上样缓冲液混合，沸水浴10min，按照5μl，10μl，20μl上样，进行SDS-PAGE电泳分析，以检测重组蛋白表达情况。诱导表达的重组蛋白，经裂解、溶菌、超声，将上清液以2ml/min的速度上样于已平衡好的Ni-NTA柱。然后用含4mol/L尿素的平衡液充分洗柱，再分别用含2M尿素的40mmol/L、300mmol/L咪唑平衡缓冲液进行洗脱，收集各次洗脱液进行SDS-PAGE分析。
BCA试剂盒检测获得的纯化蛋白样品浓度，对蛋白样品适当稀释，配制复性液，梯度脱尿素，依次4M，3M，2M，1M，0.5M，0M，每个梯度三次换液，每3小时换液一次。复性完毕，收集蛋白样品进行二次纯化，二次纯化按照可溶性蛋白的纯化步骤进行。纯化后PBS 4℃脱盐过夜，换液3次，收集蛋白样品，测定不同接样峰处的蛋白浓度，0.22μm滤器过滤除菌，分装冻存于-20℃备用。纯化后蛋白SDS-Page检测结果见图2-b。
以鸡蛋过敏患者血清和小鼠OVA特异性IgG1为一抗进行Western blotting检测。结果如图2所示。具体地，图-2a为融合蛋白sDI基因结构设计图，其中，scFv mDEC-205为抗小鼠scFv DEC基因序列，IC-15为筛选获得的OVA优势T细胞表位编码基因序列；图-2b左为scFv DEC，右为融合蛋白sDI纯化后SDS-Page 结果；图-2c为sDI与鸡蛋过敏患者血清反应的免疫印迹结果；图-2d为sDI与小鼠OVA IgG1反应的免疫印迹结果；以上实验图片均为三次以上独立重复实验的代表性实验结果。
经蛋白测序，本发明实施例提供的sD I融合蛋白的氨基酸序列为SEQ ID NO:9所示，具体为：
图2结果显示，在理论值大小处有明显条带，表明本实验重组所得sDI融合蛋白能与鸡蛋过敏患者血清中的特异性IgG1结合(图2-c)，同时与小鼠OVA特异性IgG1有良好的反应性(图2-d)。
实施例二：sDI融合蛋白结合BMDCs的能力检测
取SPF级小鼠的胫骨和股骨，分离骨髓，裂解红细胞，充分洗涤2次1400rpm离心后，计数细胞，铺六孔板，大约(2～3)×106细胞，R10液体培养，含谷氨酰胺，20ng/ml rGM-CSF，于37℃细胞培养箱培养，此为第0天，此后于第3天全部换液，第6天半量换液后，第8天收获细胞，便可用于BMDCs后续实验。
收获BMDCs，分别设置Isotype control组，anti mDEC mAb，sDI，scFv DEC蛋白组，冰上孵育30min，充分洗涤两次，5min，1400rpm离心。sDI，scFv DEC需孵育羊抗大鼠二抗1h。
流式细胞检测sDI结合DC的能力,设置分组：Isotype control；anti DEC205-FITC；scFv DEC；sDI融合蛋白组。结果如图3所示，图3所示为三次独立实验中的一次代表性结果。
如图-3所示，我们在成功制备具有很好的OVA免疫原性的融合蛋白后，进一步验证其结合BMDCs的能力，从左到右依次为：Isotype control为同型抗体对照(阴性对照)，antiDEC-205-FITC商业化抗体作为阳性对照，scFv DEC(工具蛋白)，sDI(融合蛋白)；在阴性对照和阳性对照有效的前提下，结果显示本实验制备的sDI具有较好的结合BMDCs的能力。
实施例三：sDI融合蛋白在OVA诱导的小鼠肠道过敏模型中的预防治疗实验
具体操作步骤：
1)随机分组：正常对照组(注射100μl PBS)和模型组(注射100μl PBS),scFv DEC蛋白组(注射纯化scFv DEC蛋白100μg)，sDI融合蛋白50μg组，sDI融合蛋白100μg组；所有实验小鼠接受的注射体积均为100μl；
2)该食物过敏模型在建模过程中分为皮下致敏，灌胃致敏，间歇期，激发；
3)第0周，第0、3天给予皮下致敏，用量为100μg OVA+20μg CT；第1-4周灌胃给予1mg OVA+20μg CT/只加强致敏，
4)第四次灌胃致敏间歇一周，开始给予小鼠高剂量OVA灌胃激发(予50mg OVA/只)，并于第二天处置小鼠，取组织用于后续实验研究。
小鼠体温、空肠组织病理检测结果如图4所示(N为为正常对照组；S为sham组，为模型组)。
图4-a sDI蛋白抑制食物过敏的实验方案；
图4-b柱形图显示各组发生腹泻的小鼠数量的差异；
图4-c柱形图显示各组发生腹泻的小鼠体温的变化情况；
图4-d显示给予过敏性炎症小鼠sDI融合蛋白预防治疗后空肠组织的病理结果；c1-c5对应N组，S组，scFv DEC蛋白100μg组，sDI蛋白50μg组，sDI蛋白100μg组，c6-c10分别对应c1-c5的油镜放大视野；图-4e显示不同分组空肠组织肥大细胞浸润的甲苯氨蓝染色结果(N组，S组，scFv DEC蛋白100μg组，sDI蛋白50μg组，sDI蛋白100μg组)；图4-f,g包括e1-e6,e1-e6分别为小鼠油镜每油镜视野嗜酸性粒细胞数目,小鼠油镜每油镜视野肥大细胞数目,血清中OVA特异性IgE(1:5稀释原液,OD450)、IgG1(1:400稀释原液,OD450)、IgG2a(1:200稀释原液,OD450)的检测结果,血清中IL-4水平变化结果(pg/mL)；
图-4f、4g显示sDI预防治疗实验的各组肠系膜淋巴结单个核细胞OVA特异性CD4+T细胞增殖情况；
图4-h为小鼠血清中OVA特异性IgE，IgG1，IgG2a的检测结果；
图4-i显示血清中IFN-γ和IL-4水平；
图4-j显示各组肠系膜淋巴结单个核细胞中OVA特异性CD4+T细胞增殖情况；
图-4f～4-j中，均包括N组，S组，scFv DEC蛋白100μg组，sDI蛋白50μg组，sDI蛋白100μg组。
以上均为3次独立重复实验的一次代表性实验结果，以上结果均以mean±SE表示。*，表示p＜0.05；**，代表p＜0.01；***，代表p＜0.001，提示有显著统计学差异。
给予sDI蛋白体内进行预防和治疗，可显著抑制OVA诱导的过敏性炎症的发生(图4-a)。空肠组织病理染色显示经与sham组相比较，sDI预防性治疗的小鼠嗜酸性粒细胞及肥大细胞数目显著减少，组织明显少见破坏，较完好(图4-d,e)。血清OVA特异性IgE，IgG1，IgG2a水平显著较S组降低(图4-e)，同时血清IL-4水平亦可见显著降低，但血清IFN-γ水平则显著升高(图4-I)，以上结果均提示预防治疗性给予sDI融合蛋白可显著抑制OVA诱导的小鼠过敏性炎症。
上述实验结果证实，sDI融合蛋白能够靶向DCs，抑制OVA诱导的过敏性炎症的发生，初步探讨可能与其抑制OVA特异性T细胞增殖相关，潜在可应用于防治鸡蛋过敏引起的腹泻。
本发明实施例通过鸡蛋过敏患者血清和小鼠OVA IgG1抗体筛选模式化抗原OVA优势T细胞表位，再利用scFv DEC能与树突状细胞受体DEC205特异识别并结合的特性，制备scFv DEC:OVA表位融合蛋白，将T表位肽靶向DC，将为食物过敏及其他过敏性疾病开辟新的治疗药物研发途径。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 深圳大学
<120> 一种氨基酸序列、含OVA表位的融合蛋白及其制备方法和应用
<130> 2016
<160> 9
<170> PatentIn version 3.3
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catcacggtt ggtcttgcat tatcctgttt ctggttgcaa ccgctacggg cgtgcattct 60
gaagttaaac tgctggaaag cggcggtggt ctggtccagc cgggtggctc gctgcgtctg 120
agctgtgcgg cctctggttt caccttcaac gatttctaca tgaactggat tcgtcagccg 180
ccgggtcaag caccggaatg gctgggcgtt atccgcaaca aaggcaatgg ttacaccacg 240
gaagtcaaca cctccgtgaa aggtcgtttc accatttcac gcgataacac gcagaatatc 300
ctgtatctgc aaatgaatag cctgcgtgcg gaagacaccg ccatttatta ctgcgcacgc 360
ggtggcccgt attactattc cggcgatgac gctccgtatt ggggccaggg tgtgatggtt 420
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acgtgtcatg cgagccagaa tattaaaggt tggctggcct ggtaccagca aaaaagcggc 600
aacgcaccgc agctgctgat ctataaagct agttccctgc aaagtggcgt gccgtcccgt 660
tttagtggct ccggttcagg caccgattac attttcacga tctctaatct gcagccggaa 720
gacattgcca cctactattg ccagcactat caaagttttc cgtggacctt cggtggcggt 780
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ccgggtggct cgctgcgtct gagctgtgcg gcctctggtt tcaccttcaa cgatttctac 180
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tcgggtggtg gtggcagcga tatccagatg acccaatcac cgtcgttcct gagcacgtct 540
ctgggtaact cgattaccat cacgtgtcat gcgagccaga atattaaagg ttggctggcc 600
tggtaccagc aaaaaagcgg caacgcaccg cagctgctga tctataaagc tagttccctg 660
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atctctaatc tgcagccgga agacattgcc acctactatt gccagcacta tcaaagtttt 780
ccgtggacct tcggtggcgg tacgaaactg gaactgaaac gcggcggtgg cggtagcgcg 840
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Claims (10)
1.一种氨基酸序列，其特征在于，所述氨基酸序列包括至少一个OVA表位，编码所述OVA表位的氨基酸序列为如下序列1)或2)中的任一种：
1)第一抗体的氨基酸序列，所述第一抗体的氨基酸序列包括氨基酸序列IKHIATNAVLFFGRC(SEQ ID NO:1)或由氨基酸序列IKHIATNAVLFFGRC(SEQ ID NO:1)组成；
2)第二抗体的氨基酸序列，所述第二抗体为：包括与第一抗体具有至少60％、70％、80％、90％、95％、99％或99.9％序列同源性的氨基酸序列或由与第一抗体具有至少60％、70％、80％、90％、99％或99.9％序列同源性的氨基酸序列组成。
2.一种含OVA表位的融合蛋白，其特征在于，编码所述含OVA表位的融合蛋白的氨基酸序列包括如权利要求1所述的氨基酸序列，还包括抗DEC抗体单链可变片段的氨基酸序列。
3.如权利要求2所述的含OVA表位的融合蛋白，其特征在于，所述抗DEC抗体单链可变片段的氨基酸序列包括SEQ ID NO:2、SEQ ID NO:3所示的氨基酸序列。
4.一种制备如权利要求2所述的含OVA表位的融合蛋白的方法，其特征在于，包括：
1)采用软件预测获得OVA T细胞表位，然后采用临床鸡蛋过敏患者血清和小鼠抗OVAIgG1，并借助Western Blot法和ELISA法验证，筛选获得优势OVA表位；
2)获取抗小鼠DEC基因序列，并将所得抗小鼠DEC基因序列与编码步骤1)所得优势OVA表位的基因序列融合，获得编码如权利要求1所述的含OVA表位的融合蛋白的核苷酸序列；
3)将编码如权利要求1所述的含OVA表位的融合蛋白的核苷酸序列用于构建表达所述的含OVA表位的融合蛋白的重组表达载体；导入宿主细胞；在表达条件下，培养宿主细胞，表达，分离纯化，获得所述含OVA表位的融合蛋白。
5.一种药物组合物，包括至少一种如权利要求1所述的氨基酸序列或至少一种如权利要求2所述的含OVA表位的融合蛋白；还包括诊断剂、可药用的载剂、赋形剂或稀释剂。
6.一种核酸分子，其特征在于，编码如权利要求1所述的氨基酸序列或如权利要求2所述的含OVA表位的融合蛋白的氨基酸序列。
7.一种重组载体，其特征在于，具有如权利要求6所述的任一核酸分子序列。
8.一种宿主细胞，其特征在于，包含如权利要求7所述的重组载体。
9.一种检测试剂盒，其特征在于，包括固体检测支持物，所述的固体检测支持物包含至少一种如权利要求1所述的氨基酸序列或如权利要求2所述的含OVA表位的融合蛋白；所述固体支持物为连接抗体、蛋白质、多肽、肽和/或多核苷酸的固体表面的任一种。
10.如权利要求1所述的氨基酸序列或如权利要求2所述的含OVA表位的融合蛋白在制备诊断、预防、治疗、抑制过敏性疾病的试剂或药物中的应用。
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