CN116410275A - 重组黄花蒿花粉i型变应原及其制备方法和应用 - Google Patents
重组黄花蒿花粉i型变应原及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及重组黄花蒿花粉I型变应原及其编码基因和表达纯化方法。Arta1是黄花蒿花粉中最重要的过敏原之一,本发明通过不同基因序列优化、不同分泌信号肽、表达载体和菌株的组合得到的Arta1蛋白表达量>200mg/L,纯度>99%,氨基酸序列、二硫键及分子量与天然蛋白一致,在体外与过敏患者血清中特异性抗体的免疫反应活性与天然蛋白相当,具有良好的药用前景。相比天然提取的过敏原产品,重组表达的过敏原分子具有较多优势,如具有规定质量的纯蛋白质/肽、根据GMP规范制造、以可重复的方式生产规定的数量和浓度、预先确定了过敏性及免疫原性和耐受性、用于诊断时可以鉴别真正引起过敏反应的过敏原分子及揭示交叉反应性等、满足现代药品和疫苗的监管要求,可用于蒿属花粉过敏性疾病的脱敏免疫治疗和过敏原诊断。
Description
技术领域
本发明属于生物工程基因领域,涉及一种表征及活性与天然变应原一致的重组黄花蒿花粉I型变应原及其制备方法和应用。
背景技术
花粉是季节性过敏的主要诱因之一,不同于食物过敏,花粉过敏通过空气传播,常常难以避免,可诱发鼻炎、皮炎和哮喘等一系列过敏反应,严重影响患者生活质量。在美国约7%的成人和9%的儿童受花粉过敏的困扰(NIAID,National Institute of Allergyand Infectious Diseases),欧洲的患病率估计高达40%(G.D’Amato,2007)。近年来随着我国退耕还林和绿地面积不断增加,花粉过敏发病率逐年上升,高发病区可达5%。
蒿属(Genus Artemisia)植物花粉是引起夏秋季花粉症的重要过敏原之一。国内一项2008-2010年215210次过敏原特异性IgE检测结果显示,在吸入性过敏原中,阳性率最高的为蒿花粉。杨琼梁等2015年研究结果表明中国北方地区最主要的致敏花粉为蒿属花粉。
蒿属是菊科中物种数量最多的大属之一,全球约有200种蒿属植物,广泛分布于北半球。北艾是研究最深入的致敏花粉之一,在欧洲分布广泛,在中国主要分布于西北地区。黄花蒿可用于提取抗疟疾特效药青蒿素,是我国研究最早的蒿属过敏植物,也是我国最常见的蒿属植物之一。《中国蒿属花粉过敏原分子生物学分析和组分诊断》(浙江大学,付婉艺,2018年)研究结果表明蒿属花粉中黄花蒿为最主要过敏原。我国主要的蒿属花粉还有艾蒿、大籽蒿、茵陈蒿、野艾蒿。不同蒿属花粉的主要致敏蛋白均为I型和III型变应原,其中I型变应原属于防御素蛋白(Defensin-like protein)家族,分子量约12kD,不同蒿属植物的I型变应原蛋白序列保守;III型变应原属于非特异性脂质转运蛋白(nsLTP),在不同蒿属植物花粉中变化度较高。
过敏性疾病主要的预防和治疗措施包括避免接触变应原、对症药物治疗和特异性免疫治疗。特异性免疫疗法是目前唯一一种能够对过敏性疾病达到治疗目的的“对因治疗”方法,其通过使用逐渐增加剂量的过敏原,提高患者对该变应原的耐受性,减轻由暴露于该过敏原而引发的症状,最终达到耐受乃至免疫耐受的目的。
2021年浙江我武用于治疗黄花蒿/艾蒿花粉过敏引起的变应性鼻炎的脱敏药物黄花蒿花粉变应原舌下滴剂获批上市,其专利CN101905022A指出“以蒿属花粉为原料,将花粉脱脂、浸提、浓缩,制得蒿属花粉变应原浸提液”。然而,天然过敏原提取物由于原料来源及生产方法的限制不可避免存在质量方面的问题,比如存在未定义的非过敏性物质、污染物以及过敏原含量和生物活性的高度变异性(Valenta R,et al.Allergen Extracts for invivo diagnosis and treatment of allergy:is there a future[J].Journal ofallergy&Clinical immunology in practice,2018.)。欧洲过敏与临床免疫学会2018年发布的过敏性鼻炎过敏原免疫治疗指南(EAACI Guidelines on allergen immunotherapy:allergic rhinoconjunctivitis(2018))也指出:混合过敏原存在许多潜在缺点,包括稀释效应、由于某些过敏原的酶活性导致的潜在过敏原降解以及充分证明过敏原组合的功效的困难。经EMA、HMA、FDA审批上市的标准化脱敏药物基本都限定了主要致敏蛋白,如治疗尘螨过敏的ODACTRA含有Der P1、Der P2、Der F1、Der F2四种主要致敏蛋白,治疗梯牧草过敏的GRAZAX含有主要致敏蛋白Phl P5,治疗豚草过敏的RAGWIZAX含有主要致敏蛋白Amb a1。
目前还没有重组表达的蒿属花粉过敏原蛋白药物上市或开展临床试验。
发明内容
申请人希望提供一种主要致敏蛋白明确的重组黄花蒿花粉变应原产品,以提升产品质量可控性,保证蒿属花粉过敏性疾病的脱敏免疫治疗药物的精准性和过敏原诊断的准确性。
本发明的一个目的是提供一种用于治疗蒿属花粉过敏的蛋白,其为重组Art a1蛋白。Art a1由N端的防御素结构域和C端的富羟脯氨酸部分组成的糖蛋白。研究表明Art a1蛋白序列高度保守,各亚型与sIgE抗体的结合能力相似,其免疫活性主要由N端防御素结构域决定。付婉艺等2018年发表的论文《中国蒿属花粉过敏原分子生物学分析和组分诊断》结果表明:与国外患者相同,中国蒿属花粉过敏患者对黄花蒿花粉I型变应原(Art a1)的识别程度高于其他组分,为黄花蒿花粉中的主要致敏蛋白。
优选的,该Art a1蛋白的氨基酸序列如SEQ ID NO:4所示。
所述重组Art a1蛋白可用于蒿属花粉过敏性疾病的治疗和诊断,如过敏性鼻炎、哮喘等。
本发明的另一个目的是提供一种编码Art a1蛋白的DNA序列,其碱基序列如SEQID NO:3所示。该序列针对毕赤酵母表达系统进行了密码子优化,其更利于Art a1在毕赤酵母中表达。发明人惊喜地发现,不同基因优化方法制备得到的Art a1基因表达量有较大差异,其中一种基因优化方法构建筛选得到的Art a1克隆表达量为200mg/L,经过纯化的重组Art a1蛋白与天然蛋白相比在体外与过敏患者血清中特异性抗体的免疫反应活性与天然蛋白相当。
本发明的另一个目的是提供一种有利于提高Art a1蛋白表达量,且氨基酸一级序列与天然蛋白完全一致的分泌信号肽,优选的,所述信号肽为酵母α-factor信号肽、黑曲霉素信号肽、酸性磷酸酶信号肽(PHO)、酿酒酵母信号肽(SUC2)或Art a1蛋白自身信号肽,更优选为α-factor信号肽(SEQ ID NO:11)和自身信号肽(SEQ ID NO:12)。
本发明的另一个目的是提供一种含有上述编码优化后Art a1基因的载体,优选的,所述的载体为pAO815,pPIC9,pPIC9K,pPIC3.5,pPIC3.5K,pPICZαA、B、C或pGAPZαA、B、C,更优选为pPICZαA或pGAPZαA。
本发明的另一个目的是提供一种包含上述所述载体的毕赤酵母菌株,优选的,所述毕赤酵母菌株为SMD1168、GS115、KM71、X33或KM71H,更优选为KM71或X33菌株。
发明人惊喜地发现,以上不同分泌信号肽、表达载体和菌株的组合得到的工程菌株Art a1蛋白表达量有较大差异,其中一种组合方式筛选得到的Art a1克隆表达量最高可达200mg/L,经过纯化的重组Art a1蛋白具有与天然蛋白完全一致的氨基酸序列、二硫键及分子量,在体外与过敏患者血清中特异性抗体的免疫反应活性与天然蛋白相当。
本发明的另一个目的是提供一种Art a1蛋白的表达方法,所述方法包含下述步骤:
A.构建含有上述编码Art a1基因的载体;
B.将步骤A的载体线性化后转入毕赤酵母菌株中,并在合适的条件下培养;
C.回收纯化蛋白质。
上述所述载体优选为pPICZαA或pGAPZαA。
上述所述毕赤酵母菌株优选为KM71或X33菌株。
本发明的另一个目的是提供一种重组Art a1蛋白纯化方法,所述纯化方法如下:
A.将Art a1发酵液低温高速离心收集上清,3KD膜包超滤浓缩,25mM PB,pH7.0缓冲液置换,0.45μm滤膜过滤。
B.第一步阳离子层析,用平衡缓冲液平衡层析柱,接着运用纯化系统将步骤A中得到的发酵液通过分离填料,然后运用洗脱缓冲液梯度洗脱,收集洗脱峰;平衡缓冲液为25mMPB,pH7.0,洗脱缓冲液为25mM PB,1.0M NaCl,pH7.0。
C.第二步将B中收集得到的Art a1蛋白峰用平衡缓冲液稀释,平衡缓冲液平衡层析柱,将稀释后的Art a1蛋白溶液上疏水层析填料,收集洗脱峰;平衡缓冲液为1.0M(NH4)2SO4,25mM PB,pH7.0,洗脱缓冲液为25mM PB,pH7.0。
D.第三步将C中收集到的目的蛋白峰超滤置换,缓冲液为25mM PB,pH7.0;过滤除菌后即得到Art a1蛋白原液。
本发明提供的重组Art a1蛋白SEC-HPLC纯度>99%,表达量为200mg/L,且具有与天然蛋白完全一致的氨基酸序列、二硫键及分子量,在体外与过敏患者血清中特异性抗体的免疫反应活性与天然蛋白相当,具有良好的药用前景。相比天然提取的过敏原产品,重组表达的过敏原分子具有较多优势,如避免不同来源的天然花粉主要变应原含量及活性批次间差异、变应原工艺及质量更加稳定可控、避免天然花粉中其他成分相互作用对主要变应原的降解以及产生其他致敏反应等,满足现代生物制品的安全有效质量可控的需求;此外重组变应原蛋白如用于过敏诊断试剂盒的开发可准确鉴别引发机体的变应原蛋白。
附图说明
图1表示优化前后Art a1基因序列对比图。
其中未优化序列对应的为天然Art a1基因核苷酸序列;Art a1-01为第一种优化的核苷酸序列,Art a1-02为第二种优化的核苷酸序列。
图2-a,2-b,2-c为密码子优化前后Art a1基因在毕赤酵母表达系统中平均GC碱基含量分布区域图。
其中,图2-a表示Art a1天然基因核苷酸序列在毕赤酵母表达系统中平均GC碱基含量为50.71%;图2-b表示Art a1-01密码子在毕赤酵母表达系统中平均GC碱基含量为58.53%;图2-c表示Art a1-02密码子在毕赤酵母表达系统中平均GC碱基含量为55.48%。
图3为密码子优化后Art a1-01和Art a1-02基因(含自身信号肽)PCR产物的琼脂糖凝胶电泳图。
其中,泳道1为Art a1-01基因PCR产物;泳道2为200bp DNA Ladder;泳道3为Arta1-02基因PCR产物。
图4为密码子优化后Art a1-01和Art a1-02基因(无自身信号肽)PCR产物的琼脂糖凝胶电泳图。
其中,泳道1为200bp DNA Ladder;泳道2为Art a1-01基因PCR产物;泳道3为Arta1-02基因PCR产物。
图5为Art a1-01,02基因在pPIC体系工程菌中的表达鉴定图。
其中,图5-a为pPICZα-Art a1-01工程菌株表达72小时后,菌液上清SDS-PAGE凝胶电泳图。其中泳道1为10-94KD范围的非预染蛋白Marker;泳道2-10为通过Zeocin筛选出来的Art a1-01基因各阳性单克隆宿主工程菌株培养菌液上清。
图5-b为pPICZ-Art a1-02工程菌株表达72小时后,菌液上清SDS-PAGE凝胶电泳图。其中泳道1为10-94KD范围的非预染蛋白Marker;泳道2-10为通过Zeocin筛选出来的Arta1-02基因各阳性单克隆宿主工程菌株培养菌液上清。
图6为Art a1-01,02基因在pGAP体系工程菌中的表达鉴定图。
其中,图6-a为pGAPZα-Art a1-01工程菌株表达48小时后,菌液上清SDS-PAGE凝胶电泳图。其中泳道1为10-94KD范围的非预染蛋白Marker;泳道2-10为通过Zeocin筛选出来的Art a1-01基因单克隆工程菌株培养菌液上清。
图6-b为pGAPZ-Art a1-02工程菌株表达48小时后,菌液上清SDS-PAGE凝胶电泳图。其中泳道1为10-94KD范围的非预染蛋白Marker;泳道2-10为通过Zeocin筛选出来的Arta1-02基因单克隆工程菌株培养菌液上清。
图7为重组Art a1发酵液第一步阳离子层析纯化色谱图及电泳鉴定图。
其中图7-a为重组Art a1发酵液上清第一步阳离子层析纯化色谱图;有两个洗脱峰。
图7-b为重组Art a1发酵液上清第一步阳离子层析电泳图;泳道1为10-94KD非预染蛋白Marker;泳道2为Art a1发酵液第一步阳离子层析纯化洗脱峰1;泳道3为Art a1发酵液第一步阳离子层析纯化洗脱峰2。
图8为重组Art a1蛋白第二步疏水层析纯化色谱图及电泳鉴定图;
其中图8-a为重组Art a1蛋白第二步疏水层析纯化色谱图,仅有一个洗脱峰。
图8-b为重组Art a1蛋白第二步疏水层析电泳鉴定图;其中泳道1为10-94KD非预染蛋白Marker;泳道2为Art a1蛋白第二步疏水层析纯化穿透峰;泳道3为重组Art a1蛋白第二步疏水层析纯化洗脱峰1(F1)。
图9为天然Art a1蛋白阳离子层析色谱图及电泳鉴定结果;
其中图9-a为天然Art a1蛋白阳离子层析色谱图,有5个洗脱峰。
图9-b为天然Art a1蛋白阳离子层析电泳图;泳道1为10-94KD非预染蛋白Marker;泳道2为洗脱峰1;泳道3为洗脱峰2;泳道4为洗脱峰3;泳道5为洗脱峰4;泳道6为洗脱峰5。
图10为Art a1蛋白肽段覆盖率检测结果;
其中图10-a为重组Art a1蛋白肽段覆盖率检测结果。
图10-b为天然Art a1蛋白肽段覆盖率检测结果。
图11为重组Art a1蛋白二硫键鉴定结果;其中图11-a为trypsin单酶切鉴定结果,图11-b为trypsin和chymotrypsin双酶切结果。
图12为重组Art a1蛋白SEC-HPLC纯度检测结果。
具体实施方式
下面结合具体实施例,进一步阐述本发明,应理解,引用实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1Art
a1基因密码子优化
发明人根据NCBI已公开的Art a1的DNA序列(Genbank登录号:KC700033.1,含自身信号肽),如SEQ ID No:1所示,对该基因进行密码子优化后得到两条基因序列:Art a1-01和Art a1-02,核苷酸序列分别如SEQ ID No:2和SEQ ID No:3所示,氨基酸序列如SEQ IDNo:4所示。密码子优化前后碱基序列对比如图1所示。
GC含量会影响基因的表达水平,GC含量理想分布区域为30%-70%,在这个区域外的出现任何峰都会不同程度地影响转录和翻译效率。由图2-a、图2-b和图2-c的Art a1基因的GC碱基平均含量分布区域图对比可知,图2-a中Art a1基因GC碱基平均含量为50.71%,图2-b中优化后Art a1-01的GC碱基平均含量为58.53%,图2-c中优化后Art a1-02的GC碱基平均含量为55.48%;经过优化后GC平均含量得到了提高,Art a1-01、Art a1-02相差不大。
实施例2:含自身信号肽的Art
a1基因表达质粒构建
1、构建至pPIC表达质粒系统
将实施例1中密码子优化后的Art a1-01、02基因在5’端引入EcoR I酶切位点序列,在3’端引入XhoI酶切位点序列,并进行全基因合成,将合成的基因片段,构建到pPICZ质粒(由南京金斯瑞科技有限公司提供)中,得到一种长期保存质粒,分别根据不同优化方式记为pPICZ-Art a1-01和pPICZ-Art a1-02质粒。
2、构建至pGAP表达质粒系统
分别以pPICZ-Art a1-01和pPICZ-Art a1-02质粒为模板,进行PCR扩增,所用引物序列如下:
上游引物5’AOX primer,序列如SEQ ID No:5所示;下游引物3’AOX primer,序列如SEQ ID No:6所示。
反应总体积50μL,其中浓度为10μmol/L引物各加2.5μL,浓度为10mmol/L的dNTP加1μL,所用DNA聚合酶为Q5(购自New England Biolabs公司),2U/μL,加0.5μL。反应条件为98℃5秒、55℃45秒、72℃30秒,25个循环后,产物经1.0%琼脂糖凝胶电泳分析,结果显示产物大小与预期大小(400bp)一致(结果如图3所示)。分别用Xho I(R0146S,购自New EnglandBiolabs公司)和EcoR I(R3101S,购自New England Biolabs公司)双酶切后,1%琼脂糖电泳,得到的基因产物用DNA凝胶回收试剂盒(DP214,北京天根生化科技有限公司)纯化。T4连接酶(M0202S,购自New England Biolabs)连接到pGAPZ A质粒(V205-20,购自Invitrogen公司)中,转化到DH5α感受态细胞(CB101,购自北京天根生化科技有限公司)中,在含有博来霉素(购自Invitrogen公司)的LB固体培养基中37℃培养过夜。第二天挑取阳性克隆菌测序,比对,与预期序列完全一致,即得到Art a1密码子优化后的表达质粒,记为pGAPZ-Arta1-01和pGAPZ-Art a1-02。
实施例3:含酵母α-factor信号肽的Art
a1基因表达质粒构建
以pPICZ-Art a1-01质粒为模板,进行PCR扩增,得到无信号肽的Art a1-01基因序列,所用引物序列:上游引物为SEQ ID No:7;下游引物为SEQ ID No:8。
以pPICZ-Art a1-02质粒为模板,进行PCR扩增,得到无信号肽的Art a1-02基因序列,所用引物序列:上游引物为SEQ ID No:9;下游引物为SEQ ID No:10。
反应总体积50μL,其中浓度为10μmol/L引物各加2.5μL,浓度为10mmol/L的dNTP加1μL,所用DNA聚合酶为Q5(购自New England Biolabs公司),2U/μL,加0.5μL。反应条件为98℃5秒、55℃45秒、72℃30秒,25个循环后,产物经1.0%琼脂糖凝胶电泳分析,结果显示产物大小与预期大小(400bp)一致(结果如图4所示)。分别用Xho I(R0146S,购自New EnglandBiolabs公司)和Xba I(R01445S,购自New England Biolabs公司)双酶切后,1%琼脂糖电泳,得到的基因产物用DNA凝胶回收试剂盒(DP214,北京天根生化科技有限公司)纯化。
1、构建至pPICZα表达质粒系统
T4连接酶(M0202S,购自New England Biolabs)连接到pPICZαA质粒(V205-20,购自Invitrogen公司)中,转化到DH5α感受态细胞(CB101,购自北京天根生化科技有限公司)中,在含有博来霉素(购自Invitrogen公司)的LB固体培养基中37℃培养过夜。第二天挑取阳性克隆菌测序,比对,与预期序列完全一致,即得到Art a1密码子优化后的表达质粒,记为pPICZα-Art a1-01和pPICZα-Art a1-02。
2、构建至pGAPZα表达质粒系统
T4连接酶(M0202S,购自New England Biolabs)连接到pGAPZαA质粒(V205-20,购自Invitrogen公司)中,转化到DH5α感受态细胞(CB101,购自北京天根生化科技有限公司)中,在含有博来霉素(购自Invitrogen公司)的LB固体培养基中37℃培养过夜。第二天挑取阳性克隆菌测序,比对,与预期序列完全一致,即得到Art a1密码子优化后的表达质粒,记为pGAPZα-Art a1-01和pGAPZα-Art a1-02。
实施例4:Art
a1表达质粒转化和工程菌株的筛选
YPDS+Zeocin抗性固体培养基配制:Invitrogen公司Pichia expression vectorsfor constitutive expression and purification of recombinant proteins说明书提供,其中酵母提取物10g/L,蛋白胨20g/L,葡萄糖20g/L,琼脂15g/L,山梨醇182g/L,Zeocin终浓度0.1mg/ml。
1、pPIC体系表达质粒转化和工程菌株筛选
按照Invitrogen公司Easy SelectPichia Expression Kit说明书的方法制备成电感受态细胞。将实施例2步骤1和实施例3步骤1得到的质粒pPICZ-Art a1-01、pPICZ-Arta1-02、pPICZα-Art a1-01和pPICZα-Art a1-02,分别用Sac I限制性内切酶(购自NewEngland Biolabs)酶切线性化,乙醇沉淀后将线性化载体,电转化毕赤酵母X33感受态细胞中,涂布于YPDS固体培养基,30培培养直到转化子长出。
2、pGAP体系表达质粒转化和工程菌株筛选
按照Pichia expression vectors for constitutive expression andpurification of recombinant proteins说明书的方法制备成电转化感受态细胞。将实施例2步骤2和实施例3步骤2得到的质粒pGAPZ-Art a1-01、pGAPZ-Art a1-02、pGAPZα-Arta1-01和pGAPZα-Art a1-02,分别用Avr II限制性内切酶(R0174S,购自New EnglandBiolabs)酶切线性化,乙醇沉淀后将线性化载体,电转化毕赤酵母X33感受态细胞中,涂布于YPDS固体培养基,30℃培养直到转化子长出。
实施例5:Art
a1基因工程菌株诱导表达及鉴定
1、pPIC体系克隆筛选鉴定
挑取实施例4步骤1中获得的宿主单克隆工程菌于5mL BMGY培养基中,于50mL无菌离心管中30℃,至OD600=1.0-2.0时,将菌液4000rpm离心10分钟,用BMMY培养基重悬后诱导表达,每隔24h补加甲醇至终浓度为1%,220rpm培养72小时,离心收集菌液上清,通过SDS-PAGE凝胶电泳分析,观察表达产物条带亮度,图5-a、b分别为表达量最低的pPICZα-Arta1-01和表达量最高的pPICZ-Art a1-02工程菌株诱导表达鉴定图;其余构建方式表达鉴定图未列出,表达量结果见实施例8表1,Art a1蛋白在不同构建方式的工程菌株中均得到了表达,其中表达量最高的pPICZ-Art a1-02菌株为100mg/L,最低的pPICZα-Art a1-01菌株表达量仅20mg/L,pPICZ-Art a1-02菌株为pPICZα-Art a1-01菌株表达量的5倍。
BMGY+zeocin培养基配制:Invitrogen公司Easy SelectPichia Expression Kit说明书提供,其中酵母提取物10g/L,蛋白胨20g/L,K2HPO4 3g/L,KH2PO4 11.8g/L,YNB13.4g/L,生物素4×10-4g/L,甘油10g/L,Zeocin终浓度0.1mg/ml。
BMMY+Zeocin培养基配制:Invitrogen公司Easy SelectPichia Expression Kit说明书提供,其中酵母提取物10g/L,蛋白胨20g/L,K2HPO4 3g/L,KH2PO4 11.8g/L,YNB13.4g/L,生物素4×10-4g/L,甲醇5mL/L,Zeocin终浓度0.1mg/ml。
2、pGAP体系克隆筛选鉴定
挑取实施例4步骤2中获得的宿主单克隆工程菌于5mL YPD培养基中,于50mL无菌离心管中30℃,220rpm培养48小时,离心收集菌液上清,通过SDS-PAGE凝胶电泳分析,观察表达产物条带亮度,图6-a、b分别为表达量最低的pGAPZα-Art a1-01和表达量最高的pGAPZ-Art a1-02工程菌株诱导表达鉴定图;其余构建方式表达鉴定图未列出,表达量结果见实施例8表1,Art a1蛋白在不同构建方式的工程菌株中均得到了表达,其中pGAPZ-Arta1-02构建方式菌株最高表达量为200mg/L,最低的pGAPZα-Art a1-01菌株表达量仅50mg/L,pGAPZ-Art a1-02菌株为pPICZα-Art a1-01菌株表达量的4倍。
YPD+Zeocin抗性培养基配制:Invitrogen公司Pichia expression vectors forconstitutive expression and purification of recombinant proteins说明书提供,其中酵母提取物10g/L,蛋白胨20g/L,葡萄糖20g/L,Zeocin终浓度0.1mg/ml。
实施例6:重组Art
a1蛋白的纯化
利用实施例5中筛选到的表达克隆,利用实施例5中培养方法扩大培养规模至1L,制备得到发酵液,采用离子交换和疏水层析进行样品纯化。层析填料选择为Hitrap SP HP,Hitrap Phenyl HP,具体步骤如下:
1.发酵液预处理:将发酵液低温高速离心收集上清,3KD膜包超滤浓缩,25mMpH7.0的PB缓冲液超滤置换,0.45μm滤膜过滤。
2.阳离子层析:用平衡缓冲液平衡SP HP层析柱,接着用纯化系统将上一步中已超滤的发酵液通过分离填料,然后用洗脱缓冲液洗脱,收集洗脱峰;平衡缓冲液为25mM PB,pH7.0,洗脱缓冲液为25mM PB,1.0M NaCl,pH7.0;如图7所示,目的蛋白主要在洗脱峰2.
3.疏水层析:将上一步中收集得到的Art a1蛋白峰用平缓冲液稀释,将Art a1蛋白溶液上phenyl HP疏水层析填料,平衡缓冲液为1.0M(NH4)2SO4,25mM PB,pH7.0,洗脱缓冲液为25mM PB,pH7.0,收集洗脱峰;如图8所示,仅有一个洗脱峰,目的蛋白在洗脱峰中。
4.超滤置换:收集疏水层析目的蛋白峰,将缓冲液置换为pH7.0,25mM PB;通过以上纯化步骤,最终产量为90mg/L,收率为45%。
实施例7:天然Art
a1蛋白的纯化
1.花粉脱脂:称取黄花蒿花粉,按照w/v=1:5加入乙醚,低温下脱脂24-48小时;去除乙醚,旋蒸去除残留溶剂。
2.粗提液制:配制pH7.0,50mM PB溶液,按照w/v=1:10加入PB溶液,低温下提取48-72小时;低温4000rpm离心收集上清即得到粗提液。
3.层析纯化:将步骤2中收集得到的粗提液上SP FF阳离子层析填料,平衡缓冲液为25mM PB,pH7.0,洗脱缓冲液为25mM PB,1.0M NaCl,pH7.0,收集洗脱峰电泳鉴定;如图9-b所示,天然Art a1蛋白主要在洗脱峰5。
4.超滤置换:合并步骤3中洗脱峰5,将样品浓缩,缓冲液置换为pH7.4 PBS溶液,冻存于-20℃以下待用。
实施例8:LC-MS检测Art
a1蛋白N氨基酸序列和分子量
LC-MS分子量可以精确反映生物大分子一级序列是否正确,包括N、C端序列是否缺失,是否存在糖基化、氧化和脱酰胺等翻译后修饰,是生物大分子最重要的一种分析手段之一;通过对纯化后的不同构建方式的重组Art a1蛋白进行LC-MS分子量进行分析,表1结果显示pGAPZ-Art a1-02构建方式表达量最高,且N端氨基酸序列与理论一致。
表1.不同构建方式表达纯化的重组Art a1蛋白LC-MS分子量
实施例9:Art
a1蛋白肽质量图谱分析
肽质量图谱是蛋白质研究中最重要的鉴定手段之一,理论上每个蛋白消化后都有不同肽段,这些肽段的质量就是这个蛋白的肽图谱,然后将测得的氨基酸序列与已知序列进行比对,就可以知道分析的蛋白的氨基酸一级结构是否正确,将实施例8中pGAPZ-Arta1-02构建方式得到的蛋白和实施例7中天然蛋白进行肽段分析,结果由图10所示,重组Arta1蛋白与天然蛋白一致,与理论序列的覆盖率均为100%,说明我们构建和重组表达的Arta1蛋白具有与天然蛋白完全一致的一级结构。
实施例10:Art
a1蛋白二硫键检测
二硫键是否能正确配对对蛋白质等生物大分子的高级结构和活性的保持至关重要;利用本公司Thermo Scientific Q Exactive LC-MS系统对纯化后的重组Art a1蛋白进行了二硫键确定,结果如图11显示,其中图11-a为利用trypsin单酶切时得到的二硫键信息,结果只鉴定出C17/C37一对二硫键,然后利用trypsin和chymotrypsin双酶切,图11-b显示鉴定到C6/C53、C26/C49、C22/C47三对二硫键,综合两种酶切处理方式,4对理论二硫键均能被鉴定到。
实施例11:Art
a1蛋白纯度HPLC检测
纯化后样品电泳纯度鉴定:采用Agilient 1260型HPLC,色谱柱Sepax Zenix SEC-80,流动相20mM PB+300mM NaCl(pH7.0)缓冲液,流速0.5ml/min,等度洗脱,柱温25.0℃,280nm检测样品SEC-HPLC纯度;图12结果显示经纯化后的重组Art a1蛋白SEC-HPLC纯度为99.72%,纯度达到药用标准。
实施例:12:Art
a1蛋白活性检测
1.将实施例6纯化的重组Art a1蛋白和实施例7纯化制备的天然Art a1蛋白分别用1×CB碳酸盐缓冲液稀释至10μg/ml,100μl每孔,4℃包被过夜;阴性对照不加蛋白,仅为CB缓冲液。
2.次日取出ELISA板,PBST洗涤3次,每孔加200μl 1%BSA/PBST溶液,37℃封闭2h。
3.封闭结束后弃去封闭液,每孔加入100μl阳性血清(血清用1%BSA/PBST溶液稀释10倍),轻微震荡,37℃孵育1h。
4.PBST洗涤3次,每孔加入1:1500稀释的鼠抗人IgE-HRP二抗,100μl每孔,37℃孵育1h。
5.PBST洗涤3次,每孔加入100μl TMB I号显色液,37℃反应10min后每孔加入50μl终止液(2M H2SO4),OD450nm立即检测。
6.结果分析:如表2所示,重组Art a1与天然Art a1蛋白相比,检测值略高于天然Art a1蛋白,说明重组蛋白在体外与过敏患者血清中特异性抗体的免疫反应活性与天然蛋白相当。
表2重组Art a1蛋白与天然Art a1蛋白活性比较
序列表
<110> 江苏众红生物工程创药研究院有限公司
<120> 重组黄花蒿花粉Ⅰ型变应原及其制备方法和应用
<130> 重组黄花蒿花粉Ⅰ型变应原及其制备方法和应用
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 396
<212> DNA
<213> Artemisia annua
<400> 1
atggcaaagt gttcatatgt tttttgtgcg gttcttctga ttttcgtact tgctatcgga 60
gaaatagagg ccgctggttc aaagctgtgt gaaaagacaa gcaagacgtg gtccggtaag 120
tgcgacaaca agaaatgtga caaaaagtgt atagagtggg agaaagcaca acatggtgct 180
tgtcacaaga gagaagccgg taaagaaagt tgcttttgct actttgactg ttccaaatcg 240
cctcctggag cgacaccagc gcctcctgga gcggctcctc ccccagctgc tggtggctct 300
ccaccacctc ccgctgatgg tggctcacca cctcctccag ctgatggtgg atctcctcct 360
gccgatggtg gctctccacc tcctccgtcc gctcac 396
<210> 2
<211> 396
<212> DNA
<213> Artemisia annua
<400> 2
atggcaaagt gttcatatgt tttttgtgcg gttcttctga ttttcgtact tgctatcgga 60
gaaatagagg ccgctggtag caaattatgt gaaaagactt cgaagacatg gagtggaaag 120
tgtgataaca agaaatgtga caaaaagtgc atagagtggg agaaggccca acatggcgcc 180
tgccacaaaa gggaagctgg gaaggaaagt tgtttctgct acttcgactg ctcgaaatcc 240
ccgccaggag cgacgccggc tccacctggg gccgccccgc cacccgcggc aggcgggtct 300
cccccgcctc cagccgatgg gggatcacct ccgccccccg cagacggtgg atcacctccc 360
gcggatggtg gctccccgcc tcctccaagc gctcac 396
<210> 3
<211> 396
<212> DNA
<213> Artemisia annua
<400> 3
atggcaaagt gttcatatgt tttttgtgcg gttcttctga ttttcgtact tgctatcgga 60
gaaatagagg ccgctggttc taaattgtgt gaaaaaactt ctaagacctg gtctggtaaa 120
tgtgataaca agaagtgtga taaaaagtgt atcgaatggg aaaaagctca acatggtgct 180
tgtcataaaa gagaagctgg taaagaatct tgcttctgtt acttcgattg ttctaagtct 240
ccaccaggtg ctactcctgc tcctccaggt gctgctccac ctccagctgc tggtggttct 300
ccacctccac cagctgatgg tggttctcct ccaccaccag ctgacggtgg ttctccccct 360
gctgatggtg gttccccacc accaccttct gctcat 396
<210> 4
<211> 108
<212> PRT
<213> Artemisia annua
<400> 4
Ala Gly Ser Lys Leu Cys Glu Lys Thr Ser Lys Thr Trp Ser Gly Lys
1 5 10 15
Cys Asp Asn Lys Lys Cys Asp Lys Lys Cys Ile Glu Trp Glu Lys Ala
20 25 30
Gln His Gly Ala Cys His Lys Arg Glu Ala Gly Lys Glu Ser Cys Phe
35 40 45
Cys Tyr Phe Asp Cys Ser Lys Ser Pro Pro Gly Ala Thr Pro Ala Pro
50 55 60
Pro Gly Ala Ala Pro Pro Pro Ala Ala Gly Gly Ser Pro Pro Pro Pro
65 70 75 80
Ala Asp Gly Gly Ser Pro Pro Pro Pro Ala Asp Gly Gly Ser Pro Pro
85 90 95
Ala Asp Gly Gly Ser Pro Pro Pro Pro Ser Ala His
100 105
<210> 5
<211> 21
<212> DNA
<213> 人工引物()
<400> 5
gactggttcc aattgacaag c 21
<210> 6
<211> 21
<212> DNA
<213> 人工引物()
<400> 6
gcaaatggca ttctgacatc c 21
<210> 7
<211> 29
<212> DNA
<213> 人工引物()
<400> 7
ccgctcgaga aaagagctgg tagcaaatt 29
<210> 8
<211> 29
<212> DNA
<213> 人工引物()
<400> 8
gctctagatt atcagtgagc gcttggagg 29
<210> 9
<211> 29
<212> DNA
<213> 人工引物()
<400> 9
ccgctcgaga aaagagctgg ttctaaatt 29
<210> 10
<211> 29
<212> DNA
<213> 人工引物()
<400> 10
gctctagatt atcaatgagc agaaggtgg 29
<210> 11
<211> 89
<212> PRT
<213> α-factor signal peptide
<400> 11
Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
1 5 10 15
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln
20 25 30
Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60
Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val
65 70 75 80
Ser Leu Glu Lys Arg Glu Ala Glu Ala
85
<210> 12
<211> 24
<212> PRT
<213> Artemisia annua 1.0101 signal peptide
<400> 12
Met Ala Lys Cys Ser Tyr Val Phe Cys Ala Val Leu Leu Ile Phe Val
1 5 10 15
Leu Ala Ile Gly Glu Ile Glu Ala
20
Claims (9)
1.一种用于治疗蒿属花粉过敏的蛋白,其为重组黄花蒿花粉I型变应原蛋白,所述重组黄花蒿花粉I型变应原蛋白的氨基酸序列、二硫键及分子量与天然蛋白一致,在体外与过敏患者血清中特异性抗体的免疫反应活性与天然蛋白相当。
2.权利要求1所述的用于治疗蒿属花粉过敏的蛋白,氨基酸序列如SEQ ID NO:4所示。
3.编码权利要求2所述的用于治疗蒿属花粉过敏的蛋白的核苷酸,碱基序列如SEQ IDNO:3所示。
4.含有权利要求3所述的用于治疗蒿属花粉过敏蛋白的核苷酸的载体,所述的载体为pAO815,pPIC9,pPIC9K,pPIC3.5,pPIC3.5K,pPICZαA、B、C或pGAPZαA、B、C。
5.包含权利要求4所述载体的毕赤酵母菌株,所述毕赤酵母菌株为SMD1168、GS115、KM71、X33或KM71H。
6.权利要求1或2所述治疗蒿属花粉过敏的蛋白的表达方法,所述方法包含下述步骤:
A.构建含有权利要求4所述编码Art a1基因的载体;
B.将步骤A的载体线性化后转入毕赤酵母菌株中,并在合适的条件下培养;
C.回收纯化蛋白质。
7.权利要求1或2所述治疗蒿属花粉过敏的蛋白的纯化方法,所述纯化方法如下:
A.将Art a1发酵液低温高速离心收集上清,3KD膜包超滤浓缩,25mM PB,pH7.0缓冲液置换,0.45μm滤膜过滤;
B.第一步阳离子层析,用平衡缓冲液平衡层析柱,接着运用纯化系统将步骤A中得到的发酵液通过分离填料,然后运用洗脱缓冲液梯度洗脱,收集洗脱峰,平衡缓冲液为25mM PB,pH7.0,洗脱缓冲液为25mM PB,1.0M NaCl,pH7.0;
C.第二步将B中收集得到的Art a1蛋白峰用平衡缓冲液稀释,平衡缓冲液平衡层析柱,将稀释后的Art a1蛋白溶液上疏水层析填料,收集洗脱峰,平衡缓冲液为1.0M(NH4)2SO4,25mM PB,pH7.0,洗脱缓冲液为25mM PB,pH7.0;
D.第三步将C中收集到的目的蛋白峰超滤置换,缓冲液为25mM PB,pH7.0,过滤除菌后即得到Art a1蛋白原液。
8.权利要求1或2的蛋白在制备治疗蒿属花粉过敏性疾病的药物中的应用。
9.权利要求1或2的蛋白在制备检测蒿属花粉过敏原的诊断试剂中的应用。
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