CN1989151A - 禾本科来源的变应原性已降低并保持t-细胞反应性的1类变应原的变体 - Google Patents
禾本科来源的变应原性已降低并保持t-细胞反应性的1类变应原的变体 Download PDFInfo
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- CN1989151A CN1989151A CNA2005800244645A CN200580024464A CN1989151A CN 1989151 A CN1989151 A CN 1989151A CN A2005800244645 A CNA2005800244645 A CN A2005800244645A CN 200580024464 A CN200580024464 A CN 200580024464A CN 1989151 A CN1989151 A CN 1989151A
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Abstract
本发明涉及禾本科(白菖蒲)的1类变应原的变体的制备和用途,该1类变应原的特征在于与已知野生型变应原相比,其IgE反应性已降低,同时基本上保持了与T-淋巴细胞的反应性。这些低变应原性的变应原变体可被用于对草花粉变态反应患者的特异性免疫治疗(脱敏作用)或用于对草花粉变态反应的预防性免疫治疗。
Description
本发明涉及禾本科(白菖蒲)的1类变应原的变体的制备和用途,该1类变应原的特征在于与已知野生型变应原相比,其IgE反应性已降低,同时基本上保持了与T-淋巴细胞的反应性。这些低变应原性的变应原变体可被用于对草花粉变态反应患者的特殊免疫治疗(脱敏作用)或用于对草花粉变态反应的预防性免疫治疗。
本发明的一种优选实施方案涉及梯牧草(猫尾草)花粉来源的主要变应原Phlp1的变体。
发明背景
1型变态反应对全世界均有意义。工业化国家有高达20%的人口患有诸如过敏性鼻炎、结膜炎或支气管哮喘的疾病。这些变态反应均是由多种来源,诸如植物花粉、螨、猫或狗释放并存在于空气中的变应原(气源性变应原)引起的。在这些1型变态反应患者中,高达40%的患者依次表现出与草花粉变应原的特异性IgE反应性(Freidhoff etal.,1986,J.Allergy Clin.Immunol.78:1190-2002)。
触发1型变态反应的物质是蛋白质、糖蛋白或多肽。这些变应原通过粘膜被吸收后,可与致敏的人类个体内肥大细胞表面结合的IgE分子反应。2个IgE分子通过1个变应原进行的彼此交联导致效应细胞释放介质(例如组胺、前列腺素)和细胞因子,从而导致了相应的临床症状。
根据单个变应原分子与变态反应患者的IgE抗体反应的相对频率,可区分主要和次要变应原。
以梯牧草(猫尾草)为例,迄今已鉴别的主要变应原为Phl p1(Petersen et al.,1993,J.Allergy Clin.Immunol.92:789-796)、Phl p5(Matthiesen and Lwenstein,1991,Clin.Exp.Allergy 21:297-307;Petersen et al.,1992,Int.Arch.Allergy Immunol.98:105-109)、Phl p6(Petersen et al.,1995,Int.Arch.Allergy Immunol.108,49-54)、Phl p2/3(Dolecek et al.,1993,FEBS 335(3),299-304)、Phl p4(Haavik et al.,1985,Int.Arch.Allergy Appl.Immunol.78:260-268;Valenta et al.,1992,Int.Arch.Allergy Immunol.97:287-294,Fischer et al.,1996,J.Allergy Clin.Immunol.98:189-198)和Phl p13(Suck et al.,2000,Clin.Exp.Allergy30:324-332;Suck et al.,2000,Clin.Exp.Allergy 30:1395-1402)。
梯牧草(猫尾草)内,占优势的主要变应原为Phl p1和Phl p5。由于禾本科成员来源的主要变应原彼此具有高度同源性,因而具有非常相似的生化和免疫学特性,这些相关蛋白被集中分为1类和5类变应原。
1类变应原可与95%以上的草花粉变态反应患者体内的IgE抗体反应,因而是草花粉中占优势的主要变应原。
1类变应原是分子量约为32kDa的糖蛋白,位于花粉粒的细胞质内。花粉粒与上呼吸道粘膜的接触以及花粉粒被雨润湿后均可导致这些变应原的快速释放。亚细胞微粒形式的1类变应原的快速释放能够渗透进入下呼吸道,从而触发严重的哮喘发作。
业已鉴别了猫尾草(Laffer et al.,1994,J.Allergy Clin.Immunol.94:689-698)、黑麦草(Perez et al.,1990,J.Biol.Chem.265:16210-16250)、绒毛草(Schramm et al.,1997,J.Allergy Clin.Immunol.1999:781-787)、草地早熟禾(Sturaro u.Viotti,1998,NCBI GenBank,Acc.No.AJ 131850)、狗牙根(Smith et al.,1996,J.Allergy Clin.Immunol.98:331-343)、喜湿虉草(Suphioglu et al.,1995,Clin.Exp.Allergy 25:853-865)和水稻(Xu et al.,1995,Gene 164:255-259)来源的1类变应原的cDNAs。
除了对这些序列最初的描述,与原始序列在各个位置处存在差异的其它1类变应原序列也已被公开在数据库中。这些同种型也被认为是其它的草花粉变应原。
正因为它们的同源性,白菖蒲(禾本科)的1类变应原与人IgE抗体具有高交叉反应性(Laffer et al.,1996,Mol.Immunology 33:417-426)。该免疫学交叉反应性基于非常相似的氨基酸序列,如梯牧草(猫尾草)的1类变应原Phl p1与图1中选定物种来源的1类分子的序列比较所示。
该禾本科来源的其它1类变应原中的同源序列区同时存在本文在低变应原性变体的构建中所述的Phl p1氨基酸序列缺失的序列区域,及其侧翼序列区域。此外,该禾本科的1类变应原的半胱氨酸的数目以及周围的序列区域不变。正因为这些序列同源性,禾本科的1类变应原被划分在β-苹果青霉素的蛋白质家族内(Cosgrove D.J.,2000Nature 407:321-6)。
有效治疗变态反应的传统方法是特异性免疫疗法或脱敏作用(Fiebig,1995,Allergo J.4(6):336-339:Bousquet et al.,1998,J.AllergyClin.Immunol.102(4):558-562),即将天然变应原提取物以递增剂量皮下注射进患者体内。但该方法却有导致变态反应或者甚至过敏性休克的风险。为了将这些风险减至最小,人们应用了类变应原形式的创新制剂。与上述未经处理的提取物相比,这些制剂是IgE反应性已显著降低,但仍保持了T-细胞反应性的化学改良变应原提取物。这些T-细胞表位在脱敏作用中对上述变应原制剂的治疗作用至关重要(Fiebig,1995,Allergo J.4(7):377-382)。
采用重组法制备的变应原实现更高程度的治疗优化是可能的。通过重组法制备的高纯度变应原的成分明确的混合物,如果与患者的各个致敏模式相符,便可取代天然变应原来源的提取物,因为后者除含有多种变应原外,还含有相当大量具有免疫原性而非变应原性的附属蛋白质。
经过特异性突变的重组变应原,即其中的IgE表位被特异性缺失,而脱敏疗法所必需的T-细胞表位并未受损的重组变应原,使利用重组表达产物可能实现安全脱敏的现实前景得以出现(Schramm et al.,1999,J.Immunol.162:2406-2414)。
对脱敏作用而言,一个不同的概念则基于下述事实,即保护性免疫应答被诱导,尤其是被具有阻断作用的IgG4抗体诱导。依照该假设,业已描述了重组Phl p1片段,并认为其适用于诱导保护性IgG4应答(Ball et al.,1999,FASEB J.13:1277-1290)。该概念与具有降低的IgE反应性并保持T-细胞反应性的低变应原性变应原变体的概念完全不同。
通过治疗影响变态反应患者体内被扰乱的T辅助细胞平衡的另一种可能性是采用编码相关变应原的可表达DNA进行治疗(免疫治疗性DNA接种)。业已通过注射变应原编码DNA,在啮齿动物体内试验证实了其对免疫应答的变应原特异性影响(Hsu et al.,1996,NatureMedicine 2(5):540-544)。
本发明的目标在于提供禾本科的新型1类变应原的变体,并可在蛋白质和DNA水平根据其降低的IgE活性和基本上保持的T-细胞反应性对其进行区分,从而使其适用于治疗和预防性特异免疫疗法和免疫治疗性DNA接种。
附图简述
图1:禾本科物种的Phl p1-同源氨基酸序列(由cDNA序列推导的成熟蛋白序列):草地早熟禾(Poa p)、绒毛草(Holl)、黑麦草(Lolp)、狗牙根 (Cyn d)、水稻(Ory s)和喜湿虉草(Pha a),蛋白质序列根据美国国立生物技术信息中心(NCBI,Bethesda,USA)的“ Gen-Bank”数据库的cDNA序列推导而得,编号方式:成熟蛋白的氨基酸位置,下划线突出显示的:与Phl p1序列不同的氨基酸,黑框:半胱氨酸
图2:已加工的Phl p1野生型蛋白和变体Phl p1 NoCys、Phl p1 Wt(野生型)的氨基酸序列比对:蛋白质序列根据cDNA序列(位于Bethesda的美国国立生物技术信息中心(NCBI)的“GenBank”数据库登录号为Z27090)推导而得,编号方式:成熟蛋白的氨基酸位置,黑色突出显示:蛋白Phl p1 NoCys中丝氨酸对半胱氨酸的氨基酸取代
图3:低变应原性变体Phl p1 NoCys、Phl p1 NoCysΔ213-220和Phl p1 NoCysΔ1-6、115-119、213-220的氨基酸序列比对,作为实例描述,编号方式:氨基酸位置,下划线突出显示的:缺失
图4:对重组变体Phl p1 NoCys、Phl p1 NoCysΔ213-220和Phl p1 NoCysΔ1-6,115-119,213-220的SDS-PAGE和身份检验
A:SDS-PAGE
B:利用aPhl p1抗体(Allergopharma)进行的蛋白质印迹
1.标记蛋白
2.nPhl p1*
3.rPhl p1 Wt(-组氨酸-标志)*
4.标记蛋白
5.Phl p1 NoCys(+组氨酸-标记)
6.Phl p1 NoCys D213-220(+组氨酸-标记)
7.Phl p1 NoCys D1-6,115-119,213-220(+组氨酸-标记)
8.标记蛋白
*还原样品(二硫苏糖醇)
图5:检验Phl p1 NoCys、Phl p1 NoCysΔ213-220和Phl p1 NoCysΔ1-6,115-119,213-220在非变性条件下的IgE结合能力的条带检验
1)rPhl p1 Wt
2)Phl p1 NoCys
3)Phl p1 NoCys D213-220
4)Phl p1 NoCys D1-6,115-119,213-220
5)rPhl p1 Wt
TP:总蛋白着色
P:临床确定为草花粉变态反应患者的血清
图6:通过采用4个草花粉变态反应患者(P)的代表性血清样品进行EAST抑制试验,对Phl p1 NoCys、Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220的降低的IgE反应性的确定
图7:通过采用4个草花粉变态反应患者(P)的嗜碱性粒细胞进行的嗜碱性粒细胞活化试验对Phl p1 NoCys的低变应原性的确定
图8:通过采用4个草花粉变态反应患者(P)的嗜碱性粒细胞进行的嗜碱性粒细胞活化试验对Phl p1 NoCys Δ213-220的低变应原性的确定
图9:通过采用4个草花粉变态反应患者(P)的嗜碱性粒细胞进行的嗜碱性粒细胞活化试验对Phl p1 NoCys Δ1-6,115-119,213-220的低变应原性的确定
得到上述变体的工作是以Phl p1为模型变应原进行的。由图1所示序列比对可清楚地知道由于1类变应原之间的高同源性,即使起点为另一个1类变应原,也可获得相同的结果。
因此,一定也可以假定上下文提供的结果也适用于黑麦来源的Secc1,或言之利用Sec c1也可以获得这些结果,尽管该1类变应原的序列尚未知。
因此,本发明涉及禾本科的1类变应原的变体,其特征在于与已知野生型变应原相比,其IgE反应性已降低,但仍保持了与T-淋巴细胞的反应性。这些1类变应原优选猫尾草、黑麦草、草地早熟禾、绒毛草、狗牙根、水稻和喜湿虉草来源的Phl p1、Poa p1、Hol p1、Lolp1、Cyn d1、Ory s1和Pha a1。更优选的是Phl p1、Poa p1、Holp1、Lol p1或Pha a1,尤其优选的是Phl p1。
构建1类变应原的低变应原性变体的起点是野生型Phl p1的cDNA,其是借助于特异性引物通过聚合酶链反应(PCR)从猫尾草花粉的总cDNA中分离获得(“GenBank”登录号为Z27090;NCBI,Bethesda,USA)(SEQ ID NO 1)。
氨基酸序列SEQ ID NO 2由野生型Phl p1的cDNA序列推导而得。
由240个氨基酸构成的Phl p1以其天然形式被糖基化,与所有的1类变应原一样(参见图1)----特征在于成熟分子中存在7个半胱氨酸。除了Cyn d1和Ory s1,这7个半胱氨酸在所有1类变应原中的氨基酸位置均为41、57、69、72、77、83和139(Petersen et al.,1995,J.Allergy Clin.Immunol 95:987-994)。
业已在大肠杆菌中表达获得非糖基化蛋白形式的Phl p1。该重组野生型蛋白(rPhl p1 wt)可与草花粉变态反应患者来源的IgE抗体反应,该IgE抗体具有与天然纯化Phl p1(nPhl p1)的反应性(Petersenet al.,1998,Clin.Exp.llergy 28:315-321)。
发明详述
低变应原性Phl p1变体的制备和表征
由上述rPhl p1 wt cDNA开始,制备了通过遗传工程修饰的重组Phl p1变体。
重组变体Phl p1 NoCys(SEQ ID NO 4)的氨基酸序列含有7个丝氨酸残基,而不是野生型中存在的7个半胱氨酸(图2)。变体Phl p1NoCys充当了构建多种缺失突变体的起点。这些缺失突变体均已缺失了编码Phl p1 NoCys的cDNA中长度为15-90bp的单个片段,或这些片段的组合,导致在大肠杆菌中表达的蛋白质的多肽链中相应缺失了氨基酸1-6、1-30、92-104、115-119、175-185和213-220,从而获得:Phl p1 NoCys Δ1-6(SEQ ID NO 5和6)、Phl p1 NoCys Δ1-30(SEQ IDNO 7和8)、Phl p1 NoCys Δ92-104(SEQ ID NO 9和10)、Phl p1 NoCysΔ115-119(SEQ ID NO 11和12)、Phl p1 NoCys Δ175-185(SEQ ID NO13和14)、Phl p1 NoCysΔ213-220(SEQ ID NO 15和16),以及Phl p1 NoCys Δ1-6,115-119,213-220(SEQ ID NO 17和18)。
这些重组蛋白在大肠杆菌内以组氨酸融合蛋白的形式被表达。用该类型的融合蛋白进行免疫学表征。
首先,将重组变体固定在硝酸纤维素膜上后,考察它们被代表性血清集合中IgE抗体结合的能力,和被草花粉变态反应患者来源的个体血清中IgE抗体结合的能力(条带检验)。在该方法中,惊讶地观察到IgE抗体与Phl p1 NoCys的结合减少了。该结果通过IgE抑制试验(EAST)得到证实,该试验考察了未固定蛋白与溶液中的IgE抗体的IgE结合能力。
因此,本发明尤其涉及位于相应于成熟Phl p1蛋白氨基酸位置41、57、69、72、77、83和139上的半胱氨酸已被除去或被另一种氨基酸取代后获得的1类变应原变体。尤其优选的是Phl p1、Poa p1、Hol p1、Lol p1或Pha a1的相应变体,更优选的是Phl p1的变体。
如果上述7个半胱氨酸中的至少2个被除去而未被取代,或被另一种氨基酸取代,便可使上述变体与IgE抗体的结合减少。但优选的是所有7个半胱氨酸均被丝氨酸取代。
在效应细胞上膜结合IgE的交联及其体外活化期间,Phl p1 NoCys的降低的IgE结合能力对功能性作用的影响是通过采用草花粉变态反应患者来源的嗜碱性粒细胞进行的活化试验考察的。与rPhl p1 wt相比,Phl p1 NoCys显示对嗜碱性粒细胞的活化显著较低,因此在功能上其变应原性也降低了。
通过相同方法,根据IgE结合能力(条带检验,EAST)和功能作用(嗜碱性粒细胞活化)考察了以Phl p1 NoCys为基础制备的多种缺失突变体。令人惊讶的是,这些缺失突变体表现出了尤其强的低变应原性特性。
因此,本发明还涉及下述1类变应原变体,即---任选地,除了上述半胱氨酸已被除去或替代后所获变体之外---与野生型变应原相比,在相应于成熟Phl p1蛋白一级序列的氨基酸位置1-6,1-30,92-104,115-119,175-185和213-220的至少一个区域或多个区域的组合缺失后所获得的1类变应原变体。
此处尤其优选的是1类变应原Phl p1、Poa p1、Hol p1、Lol p1和Pha a1的相应缺失突变体。非常优选的是相应的Phl p1变体。
根据上述特定优选性,本发明同样涉及下述1类变应原变体,即仅缺失相应于成熟Phl p1序列的氨基酸213-220或同时还缺失氨基酸1-6和115-119后获得的1类变应原变体。此处更优选的变应原为Phl p1、Poa p1、Hol p1、Lol p1和Pha a1,Phl p1尤其优选。
特异性免疫疗法的功效所基于的低变应原性Phl p1变体的T-细胞反应性是通过采用草花粉变态反应患者来源的Phl p1-特异性T-淋巴细胞进行的增殖试验体外检验的。该修饰变应原显示基本上保持了T-细胞反应性,从而使低变应原性Phl p1变体能够被应用于免疫治疗中。
根据本发明的变应原变体可借助于遗传工程方法从克隆DNA序列开始制备获得。但原理上,也可能涉及到对天然变应原提取物的化学修饰(Fiebig,1995,Allergo J.4(7),377-382)。
在本专利申请所述1类变应原的变异之外,在不同位置上的进一步修饰----例如,为了增强低变应原性-----当然也是可能的。这些修饰可以是,例如,氨基酸插入、缺失和交换、蛋白裂解为片段、蛋白或其片段与其它蛋白或肽的融合。本发明还涉及编码上述变应原变体的DNA分子,含有该DNA分子的重组表达载体以及由该DNA分子或该表达载体转化的宿主生物。合适的宿主生物可以是原核或真核、单或多细胞生物,诸如细菌或酵母。根据本发明,优选宿主生物为大肠杆菌。
本发明还涉及通过培养上述宿主生物并从培养物中分离相应的变应原变体以制备本发明所述变应原变体的方法。
本发明还涉及上述变应原变体、DNA分子和表达载体作为药物的特性。
此外,本发明还涉及被用于治疗变态反应,或用于免疫治疗接种患有变态反应的患者,和/或预防这种变态反应的药物组合物,其中禾本科的1类变应原参与所述变态反应的触发,该药物组合物含有至少一种上述变应原变体或相应的DNA分子或相应的表达载体,并进一步任选地含有活性化合物和/或佐剂。
如果药物组合物属于第二种类型(含有至少一个DNA分子或表达载体),这些组合物优选地还含有氢氧化铝或含有免疫刺激性CpG的寡核苷酸,或二者的组合。
就本发明的用途而言,药物组合物可作为治疗剂被应用在人类或兽医学中。合适的赋形剂是适合肠胃外给药并不与本发明所述1类变应原变体反应的有机或无机物质。适合肠胃外应用的尤其指溶液,优选油或水性溶液,此外还包括悬液、乳浊液或植入物。本发明的变应原变体还可能是冻干的,且所获得的冻干物被用于,例如制备注射用制剂。所述组合物可能是已灭菌的和/或含有佐剂,诸如润滑剂、防腐剂、稳定剂和/或润湿剂、乳化剂、调节渗透压的盐、缓冲物质和/或多种其它活性化合物。
此外,通过例如,使合适配方的本发明变应原变体吸附在氢氧化铝上,可获得缓释制剂。
最后,本发明涉及根据本发明的至少一种变应原变体或DNA分子或表达载体在制备用于治疗变态反应或用于免疫治疗接种患有变态反应的患者和/或预防这种变态反应的药物中的用途,其中禾本科的1类变应原参与所述变态反应的触发。
下文以具有上述遗传工程修饰的低变应原性Phl p1变体为例描述了变体Phl p1 NoCys、Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220(图3)的制备以及它们的免疫学特征。重组Phl p1变体的表达和纯化
这些重组蛋白是作为组氨酸融合蛋白(表达载体pProExHT;Invitrogen,Carlsbad,USA)在大肠杆菌(菌株JM109)中表达的。rPhlp1 wt和变体首先是通过N-末端组氨酸残基与Ni2+螯合基质(固定化金属离子亲和层析,IMAC)的特异性结合并接着通过制备型凝胶过滤(尺寸排阻层析,SEC)而得以纯化的。洗脱蛋白的纯度是通过SDS-PAGE和分析性SEC监测的(图4a)。纯化蛋白的身份是通过与Phl p1-特异性单克隆抗体的结合证实的(图4b)。
对重组Phl p1变体降低的IgE结合性的证实
证实变态反应患者血清来源的特异性IgE在膜结合试验蛋白上的IgE反应性的一种简单试验方法是条带检验。
为实现该目的,使试验物质在非变性条件下以相同的浓度和量并排结合在硝酸纤维素膜上。一组这样的膜条带可平行地与不同变态反应患者来源的血清一起温育。完成洗涤步骤后,通过由抗人IgE/碱性磷酸酶偶联物促进的显色反应,使特异性结合的IgE抗体显现在膜上。
通过以上述修饰Phl p1分子为例,本文描述了利用各草花粉变态反应患者来源的血清针对Phl p1 NoCys、Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220进行条带检验的结果(图5)。
只有从具有抗天然Phl p1的强IgE滴度的变态反应患者来源的血清可被采用。从这些患者获得的IgE抗体同样可与重组的相当的rPhl p1 wt反应。
从结果清楚可知的是与其同野生型变应原Phl p1的结合程度相比,所有患者血清的Phl p1特异性IgE抗体与重组变体Phl p1 NoCys的结合程度均降低了。
正如以变体Phl p1 NoCys Δ213-220为例所述的内容,通过另外除去某些序列片段,还可进一步降低IgE结合能力。与未经修饰的重组野生型蛋白相比,变体Phl p1 NoCys Δ213-220显示其与变态反应患者来源的所有试验血清的IgE结合能力均非常显著地降低了。从利用草花粉变态反应患者血清P14,针对变体Phl p1 NoCys Δ1-6,115-119,213-220试验的结果清楚可知,将许多缺失修饰组合可进一步地使Phl p1与某些血清来源IgE抗体的IgE结合能力降低(图5)。
由上清楚可知取代半胱氨酸和缺失特定序列片段均可降低Phl p1分子的IgE结合能力。
与条带检验形成对比,EAST抑制试验(酶变应原吸附试验)使我们能够考察溶液中的变应原/IgE相互作用,可通过固定化到膜上以基本上排除试验物质的表位被干扰遮蔽的可能性。
EAST抑制试验以如下方法进行。用变应原包被微量滴定板,此处的变应原为nPhl p1。洗涤除去未结合的变应原分子后,用牛血清白蛋白封闭板以阻断随后的非特异性结合。变态反应患者的个体血清或代表性的个体血清集合(血清集合)来源的IgE抗体以合适的稀释度与变应原包被的微量滴定板一起温育。变应原结合的IgE抗体的量是借助于抗hIgE/碱性磷酸酶偶联物与底物的反应以生成有色终产物并通过光度测定法而得以确定的。
IgE抗体的结合被可溶性变应原或待检验物质(重组修饰变应原)以底物特异性方式抑制,该抑制与浓度成函数关系。
本文以上述修饰Phl p1分子为例,对其与参考分子nPhl p1进行比较,显示了针对Phl p1 NoCys、Phl p1 NoCys Δ213-220和Phl p1NoCys Δ1-6,115-119,213-220的试验结果。
图6所示采用草花粉变态反应患者来源的4个个体血清进行的代表性IgE抑制试验显示,即使采用高浓度的变体Phl p1 NoCys(高达5μg/ml),也仅实现未修饰天然变应原nPhl p1的最大抑制作用的大约20-50%。该较低的最大抑制作用说明IgE表位丧失。
与变体Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220对应的曲线证实这些Phl p1变体甚至具有更低的IgE结合能力。采用单个变异不能检测到抑制作用,或仅检测到非常低水平的抑制作用(最大抑制作用的0-20%)。
与条带检验的结果一致,该EAST抑制试验可证实插入另外的特异性缺失可进一步降低Phl p1的IgE结合能力。
通过嗜碱性粒细胞活化试验对重组Phl p1变体的低变应原性的测定
变应原性的功能性降低是通过嗜碱性粒细胞活化试验体外测定的。该嗜碱性粒细胞活化实验是将草花粉变态反应患者的肝素化全血与多个浓度的试验物质一起温育。变应原性物质被嗜碱性粒细胞的FcεRI结合的IgE抗体特异性结合,从而导致了FcεRI分子的交联。
该变应原诱导的,IgE促进的FcεRI交联导致了嗜碱性粒细胞的活化。该活化是这些效应细胞的变应性反应的第一步。后继的信号转导致使效应细胞的脱粒,从而触发体内的变应原性反应。
在体外,变应原诱导的嗜碱性粒细胞活化可通过与IgE受体交联的信号转导偶联的表面蛋白(CD203c)的表达的定量而得以确定(Kahlert et al.,2003,Cli.,Exp.Allergy 33:1266-72)。细胞上表达的CD203c蛋白数量和细胞集合中活化细胞的百分比是通过下述方法以高灵敏度测定的,即借助于荧光标记的单克隆抗体与表面标记的结合并接着通过荧光活化的流式细胞计量术进行分析。此处所应用的参考物质是纯化天然Phl p1(nPhl p1),与试验物质平行。本文以上述修饰Phl p1分子为例显示了与Phl p1 NoCys、Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220对应的试验结果。
图7所示曲线显示了变体Phl p1 NoCys与4个临床确定为变态反应患者来源的嗜碱性粒细胞的代表性试验结果。通过该活化曲线中的偏移便可清楚变体Phl p1 NoCys相对于野生型nPhl p1的变应原性效力的降低。
与条带检验和IgE抑制试验的结果一致,变体Phl p1 NoCysΔ213-220和Phl p1 NoCys Δ1-6,115-119,213-220的试验结果显示了相对变应原性效力的更大程度的降低,如图8和9的代表性曲线所示。
尽管有最大比例的嗜碱性粒细胞已被试验物质浓度范围为100-1000pM的天然变应原活化,采用修饰变应原Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220时仍未或仅检测到极低的嗜碱性粒细胞活化。
正如根据曲线的A50值计算的结果,与参考nPhl p1(A50:已活化嗜碱性粒细胞的数量达到最大数量的50%时的变应原浓度)相比,变体Phl p1 NoCys Δ213-220的变应原性效力降低了约100-1000倍,变体Phl p1 NoCys Δ1-6,115-119,213-220的变应原性效力则被降低了1000倍以上。
低变应原性Phl p1变体的T-细胞反应性
T辅助淋巴细胞可与特定变应原肽片段(大约12-25个氨基酸)反应,该变应原肽片段通过抗原呈递细胞(APCs)中的酶促降解而形成,并在合适的肽被包含进APCs表面上的单个MHC II类分子之后被呈递到T-细胞。T辅助淋巴细胞的变应原特异性活化是增殖和功能性分化(TH1和TH2)的前提。在脱敏期间采用变应原或变应原衍生物的治疗对该变应原特异性T-淋巴细胞的影响被认为是治疗效力的关键。
为考察T细胞反应性,通过采用nPhl p1或rPhl p1 wt分子刺激的常规方法建立了草花粉变态反应患者来源的寡克隆T-细胞系。在增殖试验中,采用了参考变应原nPhl p1和rPhl p1 wt和经过修饰的重组Phl p1变体刺激多个T-细胞系。增殖率是通过常规方法掺入[3H]-胸腺核苷酸测定的。
本文以上述修饰Phl p1分子为例显示了Phl p1 NoCys、Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220的增殖试验结果。
表1所列采用8个草花粉变态反应患者来源T-细胞系的试验结果显示以重组变应原变体刺激T-淋巴细胞增殖是可能的。与未经修饰的天然和重组野生型变应原相比,Phl p1 NoCys、Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220的T-细胞反应性仅轻微降低,这证实了关键T-细胞表位的保留。
表1:通过采用Phl p1反应性T-细胞系(TCLs)进行的增殖试验对Phlp1 NoCys、Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220的T-细胞反应性的测定
刺激指数1 | ||||||
供体2 | TCL | nPhl p1 | rPhl p1Wt | Phl p1NoCys | Phl p1NoCysΔ213- | Phl p1NoCysΔ1-6,115- |
ABCDEFGH | 11.1621.160.51104.810.2741.855.7457.43 | 3.223.53.92.626.42.77.63.1 | 4.713.95.74.627.84.98.44.0 | 3.712.13.94.132.73.54.93.0 | 3.513.04.23.430.83.04.62.6 | 3.912.32.03.031.43.14.52.9 |
1由[3H]测定值计算而得。变应原-刺激的细胞培养物的cpm测定值/未经刺激的细胞培养物的cpm测定值
2供体:临床确定的草花粉变态反应患者
通过遗传工程对低变应原性Phl p1变体的构建
实施例1:Phl p1 NoCys
构建变体Phl p1 NoCys(SEQ ID NO 3和4)时,从rPhl p1 wt的cDNA(“GenBank”登录号为Z27090;NCBI,Bethesda,USA)开始进行6个PCR步骤。点突变是利用特异性PCR引物导入的,该特异性PCR引物含有编码丝氨酸的密码子,而不是编码半胱氨酸的密码子(引物序列参见表2)。
第1步-N-末端DNA片段″Phl p1 [C41S,C57S,C69S](bp1-212)“的制备:含有突变C41S,C57S,C69S的DNA片段是通过PCR扩增长重叠寡核苷酸(P1-63、P49-111、P97-158和P144-212)生成的。
第2步-C-末端DNA片段″Phl p1[C69S,C72S,C77S,C83S](bp193-720)“的制备:采用引物P193-261和P 703-720 HindIII对Phl p1 wt-cDNA进行PCR。
第3步-编码″Phl p1 [C41S,C57S,C69S,C72S,C77S,C83S](bp1-720)“的DNA的制备:采用引物P1-63和P703-720 HindIII对重叠片段″Phl p1[C41S,C57S,C69S](bp1-212)“和″Phl p1[C69S,C72S,C77S,C83S](bp193-720)“进行PCR。
第4步-N-末端DNA片段″Phl p1[C41S,C57S,C69S,C72S,C77S,C83S,C139S](bp1-428)“的制备:采用引物P1-63和P 406-428对″Phl p1[C41S,C57S,C69S,C72S,C77S,C83S](bp1-720)“的cDNA进行PCR。
第5步-C-末端DNA片段″Phl p1[C139S](bp 406-720)“的制备:采用引物P 406-428s和P703-720 HindIII对rPhl p1 wt的cDNA进行PCR。
第6步-编码Phl p1 NoCys的完整DNA的制备:采用引物P1-63和P703-720 HindIII对重叠片段″Phl p1[C41S,C57S,C69S,C72S,C77S,C83S,C139S](bp1-428)“和″Phl p1[C139S](bp 406-720)”进行PCR。
利用限制酶HindIII消化编码Phl p1 NoCys的DNA,并借助于限制位点EheI和HindIII将其连接进表达载体pProExHT(Invitrogen,Carlsbad,USA)中,接着对其进行完整测序。
表2:制备Phl p1 NoCys、Phl p1 NoCys Δ213-220和Phl p1 NoCys Δ1-6,115-119,213-220应用的PCR引物
引物 | 方向 | SEQID NO | 序列(5′→3′) |
P1-181P1-63P49-111P97-158P144-212P193-261P703-720HindIIIP406-428asP406-428sP22-63(Δ1-18)P250-318P301-384(Δ343-357)P613-720(Δ637-660)HindIII | 有义有义反义有义反义有义反义反义有义有义反义有义反义 | 19202122232425262728293031 | atc ccg aag gtc ccg ccgatc ccg aag gtc ccg ccg ggc ccg aac atc acggcg acc tac ggc gac aag tgg ctg gac gcggtt gtc ctt ggg acc ggc ggc cgt cgg ctt gcc gtacca ggt gct ctt cgc gtc cag cca cttggt ccc aag gac aac ggc ggc gcg agc ggg tacaag gac gtg gac aag ccc ccg ttc agc gggag ccg ct gcc ccg gcc gga ctt gaa gat ggg ggtgtt gcc gga gcc ggt cat gcc gct gaa cgg ggg ctcc ggc cgg ggc agc ggc tcc tcc ttc gag atc aagagc acc aag ccc gag gcc tcc tcc ggc gag cccggt aag ctt tca ctt gga ctc gta ggc ggttcc ggg tac ttg gac ttg acg cgcgc gtc aag tcc aag tac ccg gaccg aac atc acg gcg acc tac ggc gac aag tggctg gac gcggtc gaa gtg gta cgc ggc gat ggg ctc ctc gtt gtcgtc ggt gat gtg gac cac cac ggg ctc gcc ggagcc gcg tac cac ttc gac ctc tcc ggc atc gcg ttcggg tcc gac gag cag aag ctg cgc agc gcc ggcggt aag ctt tca ctt gga ctc gta ggc ggt gtc ggcctt cca gcc ctc ggg gat gac gtc ctt ggc ctc gccgcg gac ggt gaa ggg gcc ctt gag |
1所指出的数字:基于Phl p1野生型蛋白的核苷酸序列(无信号肽;“GenBank”登录号为Z27090;NCBI,Bethesda,USA)的引物位置。某些情况中的引物序列经过密码子优化以适合大肠杆菌。
实施例2:Phl p1 NoCys Δ213-220
编码缺失变体Phl p1 NoCys Δ213-220(SEQ ID NO 15和16)的DNA序列是通过采用被待缺失序列区域特异性缩短的5’-引物P1-18和3′-引物P 613-720(Δ637-660)HindIII对Phl p1 NoCys的DNA进行PCR而得以生成的。
采用限制酶HindIII消化cDNA,借助于限制位点EheI和HindIII将其连接进表达载体pProExHT(Invitrogen,Carlsbad,USA)中,接着对其进行完整测序。
实施例3:通过遗传工程对Phl p1 NoCys Δ1-6,115-119,213-220的构
建
编码缺失变体Phl p1 NoCys Δ1-6,115-119,213-220(SEQ ID NO 5和6)的DNA序列是通过采用经过待缺失序列区域特异性缩短的寡核苷酸进行PCR分三步生成的。
第1步-N-末端DNA片段″Phl p1 NoCys Δ1-6(bp1-300)“的制备:采用引物P22-63(Δ1-18)和P250-318对Phl p1 NoCys的cDNA进行PCR。
第2步-C-末端DNA片段″Phl p1 NoCys Δ115-119,213-220(bp283-663)“的制备:采用引物P 301-384(Δ343-357)和P 613-720(Δ637-660)HindIII对Pbl p1 NoCys进行PCR。
第3步-编码Phl p1 NoCys Δ1-6,115-119,213-220的完整DNA的制备:采用引物P22-63(Δ1-18)和P 703-720 HindIII对重叠片段″Phl p1NoCys Δ1-6(bp1-300)“和″Phl p1 NoCys Δ115-119,213-220(bp 283-663)“进行PCR。
采用限制酶HindIII消化编码Phl p1 NoCys Δ1-6,115-119,213-220的DNA,借助于限制位点EheI和HindIII将其连接进表达载体pProExHT(Invitrogen,Carlsbad,USA)中,接着对其进行完整测序。
相应地制备、克隆变体Phl p1 NoCys Δ1-6(SEQ ID NO 5和6)、Phl p1 NoCys Δ1-30(SEQ ID NO 7和8)、Phl p1 NoCys Δ92-104(SEQID NO 9和10)、Phl p1 NoCys Δ115-119(SEQ ID NO 11和12)、Phl p1NoCys Δ175-185(SEQ ID NO 13和14)的DNA,并测序。
序列表
<110>Merck Patent GmbH
<120>禾本科来源的变应原性已降低并保持T-细胞反应性的1类变应原的变体
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atc ccc aag gtc ccc ccc ggc ccg aac atc acg gcg acc tac ggc gac 48
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
aag tgg ctg gac gcg aag agc acc tgg tac ggc aag ccg acg gcc gcc 96
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
ggt ccc aag gac aac ggc ggc gcg tgc ggg tac aag gac gtg gac aag 144
Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys
35 40 45
ccc ccg ttc agc ggc atg acc ggc tgc ggc aac acc ccc atc ttc aag 192
Pro Pro Phe Ser Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys
50 55 60
tcc ggc cgg ggc tgc ggc tcc tgc ttc gag atc aag tgc acc aag ccc 240
Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro
65 70 75 80
gag gcc tgc tcc ggc gag ccc gtg gtg gtc cac atc acc gac gac aac 288
Glu Ala Cys Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
85 90 95
gag gag ccc atc gcc gcg tac cac ttc gac ctc tcc ggc atc gcg ttc 336
Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
100 105 110
ggg tcc atg gcc aag aag ggc gac gag cag aag ctg cgc agc gcc ggc 384
Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly
115 120 125
gag gtg gag atc cag ttc cgc cgc gtc aag tgc aag tac ccg gag ggc 432
Glu Val Glu Ile Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Glu Gly
130 135 140
acc aag gtc acc ttc cac gtg gag aag ggg tcc aac ccc aac tac ctg 480
Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu
145 150 155 160
gcg ctg ctg gtg aag ttt gtc gcc ggc gac ggc gac gtg gtg gcg gtg 528
Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val Ala Val
165 170 175
gac atc aag gag aag ggc aag gac aag tgg atc gcg ctc aag gag tcg 576
Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser
180 185 190
tgg gga gcc atc tgg agg atc gac acc ccg gag gtg ctc aag ggc ccc 624
Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu Lys Gly Pro
195 200 205
ttc acc gtc cgc tac acc acc gag ggc ggc acc aag ggc gag gcc aag 672
Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Gly Glu Ala Lys
210 215 220
gac gtc atc ccc gag ggc tgg aag gcc gac acc gcc tac gag tcc aag 720
Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
225 230 235 240
tga 723
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Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
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Gly Pro Lys Asp Asn Gly Gly Ala Cys Gly Tyr Lys Asp Val Asp Lys
35 40 45
Pro Pro Phe Ser Gly Met Thr Gly Cys Gly Asn Thr Pro Ile Phe Lys
50 55 60
Ser Gly Arg Gly Cys Gly Ser Cys Phe Glu Ile Lys Cys Thr Lys Pro
65 70 75 80
Glu Ala Cys Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
85 90 95
Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
100 105 110
Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly
115 120 125
Glu Val Glu Ile Gln Phe Arg Arg Val Lys Cys Lys Tyr Pro Glu Gly
130 135 140
Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu
145 150 155 160
Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val Ala Val
165 170 175
Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser
180 185 190
Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu Lys Gly Pro
195 200 205
Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Gly Glu Ala Lys
210 215 220
Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
225 230 235 240
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atc ccg aag gtc ccg ccg ggc ccg aac atc acg gcg acc tac ggc gac 48
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
aag tgg ctg gac gcg aag agc acc tgg tac ggc aag ccg acg gcc gcc 96
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
ggt ccc aag gac aac ggc ggc gcg agc ggg tac aag gac gtg gac aag 144
Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
ccc ccg ttc agc ggc atg acc ggc tcc ggc aac acc ccc atc ttc aag 192
Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys
50 55 60
tcc ggc cgg ggc agc ggc tcc tcc ttc gag atc aag agc acc aag ccc 240
Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
65 70 75 80
gag gcc tcc tcc ggc gag ccc gtg gtg gtc cac atc acc gac gac aac 288
Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
85 90 95
gag gag ccc atc gcc gcg tac cac ttc gac ctc tcc ggc atc gcg ttc 336
Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
100 105 110
ggg tcc atg gcc aag aag ggc gac gag cag aag ctg cgc agc gcc ggc 384
Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly
115 120 125
gag gtg gag atc cag ttc cgc cgc gtc aag tcc aag tac ccg gag ggc 432
Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly
130 135 140
acc aag gtg acc ttc cac gtg gag aag ggg tcc aac ccc aac tac ctg 480
Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu
145 150 155 160
gcg ctg ctg gtg aag ttt gtc gcc ggc gac ggc gac gtg gtg gcg gtg 528
Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val Ala Val
165 170 175
gac atc aag gag aag ggc aag gac aag tgg atc gcg ctc aag gag tcg 576
Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser
180 185 190
tgg gga gcc atc tgg agg atc gac acc ccg gag gtg ctc aag ggc ccc 624
Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu Lys Gly Pro
195 200 205
ttc acc gtc cgc tac acc acc gag ggc ggc acc aag ggc gag gcc aag 672
Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Gly Glu Ala Lys
210 215 220
gac gtc atc ccc gag ggc tgg aag gcc gac acc gcc tac gag tcc aag 720
Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
225 230 235 240
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Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
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Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
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Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys
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Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
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Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
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Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
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Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly
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Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly
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Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu
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Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val Ala Val
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Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser
180 185 190
Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu Lys Gly Pro
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Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Gly Glu Ala Lys
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Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
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ggc ccg aac atc acg gcg acc tac ggc gac aag tgg ctg gac gcg aag 48
Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys
1 5 10 15
agc acc tgg tac ggc aag ccg acg gcc gcc ggt ccc aag gac aac ggc 96
Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala Gly Pro Lys Asp Asn Gly
20 25 30
ggc gcg agc ggg tac aag gac gtg gac aag ccc ccg ttc agc ggc atg 144
Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe Ser Gly Met
35 40 45
acc ggc tcc ggc aac acc ccc atc ttc aag tcc ggc cgg ggc agc ggc 192
Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys Ser Gly Arg Gly Ser Gly
50 55 60
tcc tcc ttc gag atc aag agc acc aag ccc gag gcc tcc tcc ggc gag 240
Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro Glu Ala Ser Ser Gly Glu
65 70 75 80
ccc gtg gtg gtc cac atc acc gac gac aac gag gag ccc atc gcc gcg 288
Pro Val Val Val His Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Ala
85 90 95
tac cac ttc gac ctc tcc ggc atc gcg ttc ggg tcc atg gcc aag aag 336
Tyr His Phe Asp Leu Ser Gly Ile Ala Phe Gly Ser Met Ala Lys Lys
100 105 110
ggc gac gag cag aag ctg cgc agc gcc ggc gag gtg gag atc cag ttc 384
Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly Glu Val Glu Ile Gln Phe
115 120 125
cgc cgc gtc aag tcc aag tac ccg gag ggc acc aag gtg acc ttc cac 432
Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe His
130 135 140
gtg gag aag ggg tcc aac ccc aac tac ctg gcg ctg ctg gtg aag ttt 480
Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Phe
145 150 155 160
gtc gcc ggc gac ggc gac gtg gtg gcg gtg gac atc aag gag aag ggc 528
Val Ala Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly
165 170 175
aag gac aag tgg atc gcg ctc aag gag tcg tgg gga gcc atc tgg agg 576
Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg
180 185 190
atc gac acc ccg gag gtg ctc aag ggc ccc ttc acc gtc cgc tac acc 624
Ile Asp Thr Pro Glu Val Leu Lys Gly Pro Phe Thr Val Arg Tyr Thr
195 200 205
acc gag ggc ggc acc aag ggc gag gcc aag gac gtc atc ccc gag ggc 672
Thr Glu Gly Gly Thr Lys Gly Glu Ala Lys Asp Val Ile Pro Glu Gly
210 215 220
tgg aag gcc gac acc gcc tac gag tcc aag tga 705
Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
225 230
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Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys
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Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala Gly Pro Lys Asp Asn Gly
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Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe Ser Gly Met
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Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys Ser Gly Arg Gly Ser Gly
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Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro Glu Ala Ser Ser Gly Glu
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Pro Val Val Val His Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Ala
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Tyr His Phe Asp Leu Ser Gly Ile Ala Phe Gly Ser Met Ala Lys Lys
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Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly Glu Val Glu Ile Gln Phe
115 120 125
Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe His
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Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Phe
145 150 155 160
Val Ala Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly
165 170 175
Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg
180 185 190
Ile Asp Thr Pro Glu Val Leu Lys Gly Pro Phe Thr Val Arg Tyr Thr
195 200 205
Thr Glu Gly Gly Thr Lys Gly Glu Ala Lys Asp Val Ile Pro Glu Gly
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Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
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Ala Ala Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val
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gac aag ccc ccg ttc agc ggc atg acc ggc tcc ggc aac acc ccc atc 96
Asp Lys Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile
20 25 30
ttc aag tcc ggc cgg ggc agc ggc tcc tcc ttc gag atc aag agc acc 144
Phe Lys Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr
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Lys Pro Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp
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gac aac gag gag ccc atc gcc gcg tac cac ttc gac ctc tcc ggc atc 240
Asp Asn Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile
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gcg ttc ggg tcc atg gcc aag aag ggc gac gag cag aag ctg cgc agc 288
Ala Phe Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser
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gcc ggc gag gtg gag atc cag ttc cgc cgc gtc aag tcc aag tac ccg 336
Ala Gly Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr Pro
100 105 110
gag ggc acc aag gtg acc ttc cac gtg gag aag ggg tcc aac ccc aac 384
Glu Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn
115 120 125
tac ctg gcg ctg ctg gtg aag ttt gtc gcc ggc gac ggc gac gtg gtg 432
Tyr Leu Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val
130 135 140
gcg gtg gac atc aag gag aag ggc aag gac aag tgg atc gcg ctc aag 480
Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys
145 150 155 160
gag tcg tgg gga gcc atc tgg agg atc gac acc ccg gag gtg ctc aag 528
Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu Lys
165 170 175
ggc ccc ttc acc gtc cgc tac acc acc gag ggc ggc acc aag ggc gag 576
Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Gly Glu
180 185 190
gcc aag gac gtc atc ccc gag ggc tgg aag gcc gac acc gcc tac gag 624
Ala Lys Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu
195 200 205
tcc aag tga 633
Ser Lys
210
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Ala Ala Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val
1 5 10 15
Asp Lys Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile
20 25 30
Phe Lys Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr
35 40 45
Lys Pro Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp
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Asp Asn Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile
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Ala Phe Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser
85 90 95
Ala Gly Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr Pro
100 105 110
Glu Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn
115 120 125
Tyr Leu Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val
130 135 140
Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys
145 150 155 160
Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu Lys
165 170 175
Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Gly Glu
180 185 190
Ala Lys Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu
195 200 205
Ser Lys
210
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atc ccg aag gtc ccg ccg ggc ccg aac atc acg gcg acc tac ggc gac 48
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
aag tgg ctg gac gcg aag agc acc tgg tac ggc aag ccg acg gcc gcc 96
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
ggt ccc aag gac aac ggc ggc gcg agc ggg tac aag gac gtg gac aag 144
Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
ccc ccg ttc agc ggc atg acc ggc tcc ggc aac acc ccc atc ttc aag 192
Pro Pro Phe Ser Gly Met Thr Gly Ser G1y Asn Thr Pro Ile Phe Lys
50 55 60
tcc ggc cgg ggc agc ggc tcc tcc ttc gag atc aag agc acc aag ccc 240
Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
65 70 75 80
gag gcc tcc tcc ggc gag ccc gtg gtg gtc cac ttc gac ctc tcc ggc 288
Glu Ala Ser Ser Gly Glu Pro Val Val Val His Phe Asp Leu Ser Gly
85 90 95
atc gcg ttc ggg tcc atg gcc aag aag ggc gac gag cag aag ctg cgc 336
Ile Ala Phe Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg
100 105 110
agc gcc ggc gag gtg gag atc cag ttc cgc cgc gtc aag tcc aag tac 384
Ser Ala Gly Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr
115 120 125
ccg gag ggc acc aag gtg acc ttc cac gtg gag aag ggg tcc aac ccc 432
Pro Glu Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro
130 135 140
aac tac ctg gcg ctg ctg gtg aag ttt gtc gcc ggc gac ggc gac gtg 480
Asn Tyr Leu Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val
145 150 155 160
gtg gcg gtg gac atc aag gag aag ggc aag gac aag tgg atc gcg ctc 528
Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu
165 170 175
aag gag tcg tgg gga gcc atc tgg agg atc gac acc ccg gag gtg ctc 576
Lys Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu
180 185 190
aag ggc ccc ttc acc gtc cgc tac acc acc gag ggc ggc acc aag ggc 624
Lys Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Gly
195 200 205
gag gcc aag gac gtc atc ccc gag ggc tgg aag gcc gac acc gcc tac 672
Glu Ala Lys Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr
210 215 220
gag tcc aag tga 684
Glu Ser Lys
225
<210>10
<211>227
<212>PRT
<213>猫尾草
<400>10
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys
50 55 60
Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
65 70 75 80
Glu Ala Ser Ser Gly Glu Pro Val Val Val His Phe Asp Leu Ser Gly
85 90 95
Ile Ala Phe Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg
100 105 110
Ser Ala Gly Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr
115 120 125
Pro Glu Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro
130 135 140
Asn Tyr Leu Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val
145 150 155 160
Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu
165 170 175
Lys Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu
180 185 190
Lys Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr Lys Gly
195 200 205
Glu Ala Lys Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr
210 215 220
Glu Ser Lys
225
<210>11
<211>708
<212>DNA
<213>猫尾草
<220>
<221>CDS
<222>(1)..(708)
<223>
<400>11
atc ccg aag gtc ccg ccg ggc ccg aac atc acg gcg acc tac ggc gac 48
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
aag tgg ctg gac gcg aag agc acc tgg tac ggc aag ccg acg gcc gcc 96
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
ggt ccc aag gac aac ggc ggc gcg agc ggg tac aag gac gtg gac aag 144
Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
ccc ccg ttc agc ggc atg acc ggc tcc ggc aac acc ccc atc ttc aag 192
Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys
50 55 60
tcc ggc cgg ggc agc ggc tcc tcc ttc gag atc aag agc acc aag ccc 240
Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
65 70 75 80
gag gcc tcc tcc ggc gag ccc gtg gtg gtc cac atc acc gac gac aac 288
Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
85 90 95
gag gag ccc atc gcc gcg tac cac ttc gac ctc tcc ggc atc gcg ttc 336
Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
100 105 110
ggg tcc gac gag cag aag ctg cgc agc gcc ggc gag gtg gag atc cag 384
Gly Ser Asp Glu Gln Lys Leu Arg Ser Ala Gly Glu Val Glu Ile Gln
115 120 125
ttc cgc cgc gtc aag tcc aag tac ccg gag ggc acc aag gtg acc ttc 432
Phe Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe
130 135 140
cac gtg gag aag ggg tcc aac ccc aac tac ctg gcg ctg ctg gtg aag 480
His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys
145 150 155 160
ttt gtc gcc ggc gac ggc gac gtg gtg gcg gtg gac atc aag gag aag 528
Phe Val Ala Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys
165 170 175
ggc aag gac aag tgg atc gcg ctc aag gag tcg tgg gga gcc atc tgg 576
Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp
180 185 190
agg atc gac acc ccg gag gtg ctc aag ggc ccc ttc acc gtc cgc tac 624
Arg Ile Asp Thr Pro Glu Val Leu Lys Gly Pro Phe Thr Val Arg Tyr
195 200 205
acc acc gag ggc ggc acc aag ggc gag gcc aag gac gtc atc ccc gag 672
Thr Thr Glu Gly Gly Thr Lys Gly Glu Ala Lys Asp Val Ile Pro Glu
210 215 220
ggc tgg aag gcc gac acc gcc tac gag tcc aag tga 708
Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
225 230 235
<210>12
<211>235
<212>PRT
<213>猫尾草
<400>12
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys
50 55 60
Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
65 70 75 80
Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
85 90 95
Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
100 105 110
Gly Ser Asp Glu Gln Lys Leu Arg Ser Ala Gly Glu Val Glu Ile Gln
115 120 125
Phe Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe
130 135 140
His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys
145 150 155 160
Phe Val Ala Gly Asp Gly Asp Val Val Ala Val Asp Ile Lys Glu Lys
165 170 175
Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp
180 185 190
Arg Ile Asp Thr Pro Glu Val Leu Lys Gly Pro Phe Thr Val Arg Tyr
195 200 205
Thr Thr Glu Gly Gly Thr Lys Gly Glu Ala Lys Asp Val Ile Pro Glu
210 215 220
Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
225 230 235
<210>13
<211>690
<212>DNA
<213>猫尾草
<220>
<221>CDS
<222>(1)..(690)
<223>
<400>13
atc ccg aag gtc ccg ccg ggc ccg aac atc acg gcg acc tac ggc gac 48
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
aag tgg ctg gac gcg aag agc acc tgg tac ggc aag ccg acg gcc gcc 96
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
ggt ccc aag gac aac ggc ggc gcg agc ggg tac aag gac gtg gac aag 144
Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
ccc ccg ttc agc ggc atg acc ggc tcc ggc aac acc ccc atc ttc aag 192
Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys
50 55 60
tcc ggc cgg ggc agc ggc tcc tcc ttc gag atc aag agc acc aag ccc 240
Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
65 70 75 80
gag gcc tcc tcc ggc gag ccc gtg gtg gtc cac atc acc gac gac aac 288
Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
85 90 95
gag gag ccc atc gcc gcg tac cac ttc gac ctc tcc ggc atc gcg ttc 336
Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
100 105 110
ggg tcc atg gcc aag aag ggc gac gag cag aag ctg cgc agc gcc ggc 384
Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly
115 120 125
gag gtg gag atc cag ttc cgc cgc gtc aag tcc aag tac ccg gag ggc 432
Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly
130 135 140
acc aag gtg acc ttc cac gtg gag aag ggg tcc aac ccc aac tac ctg 480
Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu
145 150 155 160
gcg ctg ctg gtg aag ttt gtc gcc ggc gac ggc gac gtg gtg tgg atc 528
Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val Trp Ile
165 170 175
gcg ctc aag gag tcg tgg gga gcc atc tgg agg atc gac acc ccg gag 576
Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu
180 185 190
gtg ctc aag ggc ccc ttc acc gtc cgc tac acc acc gag ggc ggc acc 624
Val Leu Lys Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr
195 200 205
aag ggc gag gcc aag gac gtc atc ccc gag ggc tgg aag gcc gac acc 672
Lys Gly Glu Ala Lys Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr
210 215 220
gcc tac gag tcc aag tga 690
Ala Tyr Glu Ser Lys
225
<210>14
<211>229
<212>PRT
<213>猫尾草
<400>14
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys
50 55 60
Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
65 70 75 80
Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
85 90 95
Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
100 105 110
Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly
115 120 125
Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly
130 135 140
Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu
145 150 155 160
Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val Trp Ile
165 170 175
Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu
180 185 190
Val Leu Lys Gly Pro Phe Thr Val Arg Tyr Thr Thr Glu Gly Gly Thr
195 200 205
Lys Gly Glu Ala Lys Asp Val Ile Pro Glu Gly Trp Lys Ala Asp Thr
210 215 220
Ala Tyr Glu Ser Lys
225
<210>15
<211>699
<212>DNA
<213>猫尾草
<220>
<221>CDS
<222>(1)..(699)
<223>
<400>15
atc ccg aag gtc ccg ccg ggc ccg aac atc acg gcg acc tac ggc gac 48
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
aag tgg ctg gac gcg aag agc acc tgg tac ggc aag ccg acg gcc gcc 96
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
ggt ccc aag gac aac ggc ggc gcg agc ggg tac aag gac gtg gac aag 144
Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
ccc ccg ttc agc ggc atg acc ggc tcc ggc aac acc ccc atc ttc aag 192
Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys
50 55 60
tcc ggc cgg ggc agc ggc tcc tcc ttc gag atc aag agc acc aag ccc 240
Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
65 70 75 80
gag gcc tcc tcc ggc gag ccc gtg gtg gtc cac atc acc gac gac aac 288
Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
85 90 95
gag gag ccc atc gcc gcg tac cac ttc gac ctc tcc ggc atc gcg ttc 336
Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
100 105 110
ggg tcc atg gcc aag aag ggc gac gag cag aag ctg cgc agc gcc ggc 384
Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly
115 120 125
gag gtg gag atc cag ttc cgc cgc gtc aag tcc aag tac ccg gag ggc 432
Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly
130 135 140
acc aag gtg acc ttc cac gtg gag aag ggg tcc aac ccc aac tac ctg 480
Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu
145 150 155 160
gcg ctg ctg gtg aag ttt gtc gcc ggc gac ggc gac gtg gtg gcg gtg 528
Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val Ala Val
165 170 175
gac atc aag gag aag ggc aag gac aag tgg atc gcg ctc aag gag tcg 576
Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser
180 185 190
tgg gga gcc atc tgg agg atc gac acc ccg gag gtg ctc aag ggc ccc 624
Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu Lys Gly Pro
195 200 205
ttc acc gtc cgc ggc gag gcc aag gac gtc atc ccc gag ggc tgg aag 672
Phe Thr Val Arg Gly Glu Ala Lys Asp Val Ile Pro Glu Gly Trp Lys
210 215 220
gcc gac acc gcc tac gag tcc aag tga 699
Ala Asp Thr Ala Tyr Glu Ser Lys
225 230
<210>16
<211>232
<212>PRT
<213>猫尾草
<400>16
Ile Pro Lys Val Pro Pro Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp
1 5 10 15
Lys Trp Leu Asp Ala Lys Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala
20 25 30
Gly Pro Lys Asp Asn Gly Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys
35 40 45
Pro Pro Phe Ser Gly Met Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys
50 55 60
Ser Gly Arg Gly Ser Gly Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro
65 70 75 80
Glu Ala Ser Ser Gly Glu Pro Val Val Val His Ile Thr Asp Asp Asn
85 90 95
Glu Glu Pro Ile Ala Ala Tyr His Phe Asp Leu Ser Gly Ile Ala Phe
100 105 110
Gly Ser Met Ala Lys Lys Gly Asp Glu Gln Lys Leu Arg Ser Ala Gly
115 120 125
Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser Lys Tyr Pro Glu Gly
130 135 140
Thr Lys Val Thr Phe His Val Glu Lys Gly Ser Asn Pro Asn Tyr Leu
145 150 155 160
Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly Asp Val Val Ala Val
165 170 175
Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile Ala Leu Lys Glu Ser
180 185 190
Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu Val Leu Lys Gly Pro
195 200 205
Phe Thr Val Arg Gly Glu Ala Lys Asp Val Ile Pro Glu Gly Trp Lys
210 215 220
Ala Asp Thr Ala Tyr Glu Ser Lys
225 230
<210>17
<211>666
<212>DNA
<213>猫尾草
<220>
<221>CDS
<222>(1)..(666)
<223>
<400>17
ggc ccg aac atc acg gcg acc tac ggc gac aag tgg ctg gac gcg aag 48
Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys
1 5 10 15
agc acc tgg tac ggc aag ccg acg gcc gcc ggt ccc aag gac aac ggc 96
Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala Gly Pro Lys Asp Asn Gly
20 25 30
ggc gcg agc ggg tac aag gac gtg gac aag ccc ccg ttc agc ggc atg 144
Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe Ser Gly Met
35 40 45
acc ggc tcc ggc aac acc ccc atc ttc aag tcc ggc cgg ggc agc ggc 192
Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys Ser Gly Arg Gly Ser Gly
50 55 60
tcc tcc ttc gag atc aag agc acc aag ccc gag gcc tcc tcc ggc gag 240
Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro Glu Ala Ser Ser Gly Glu
65 70 75 80
ccc gtg gtg gtc cac atc acc gac gac aac gag gag ccc atc gcc gcg 288
Pro Val Val Val His Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Ala
85 90 95
tac cac ttc gac ctc tcc ggc atc gcg ttc ggg tcc gac gag cag aag 336
Tyr His Phe Asp Leu Ser Gly Ile Ala Phe Gly Ser Asp Glu Gln Lys
100 105 110
ctg cgc agc gcc ggc gag gtg gag atc cag ttc cgc cgc gtc aag tcc 384
Leu Arg Ser Ala Gly Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser
115 120 125
aag tac ccg gag ggc acc aag gtg acc ttc cac gtg gag aag ggg tcc 432
Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser
130 135 140
aac ccc aac tac ctg gcg ctg ctg gtg aag ttt gtc gcc ggc gac ggc 480
Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly
145 150 155 160
gac gtg gtg gcg gtg gac atc aag gag aag ggc aag gac aag tgg atc 528
Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile
165 170 175
gcg ctc aag gag tcg tgg gga gcc atc tgg agg atc gac acc ccg gag 576
Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu
180 185 190
gtg ctc aag ggc ccc ttc acc gtc cgc ggc gag gcc aag gac gtc atc 624
Val Leu Lys Gly Pro Phe Thr Val Arg Gly Glu Ala Lys Asp Val Ile
195 200 205
ccc gag ggc tgg aag gcc gac acc gcc tac gag tcc aag tga 666
Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
210 215 220
<210>18
<211>221
<212>PRT
<213>猫尾草
<400>18
Gly Pro Asn Ile Thr Ala Thr Tyr Gly Asp Lys Trp Leu Asp Ala Lys
1 5 10 15
Ser Thr Trp Tyr Gly Lys Pro Thr Ala Ala Gly Pro Lys Asp Asn Gly
20 25 30
Gly Ala Ser Gly Tyr Lys Asp Val Asp Lys Pro Pro Phe Ser Gly Met
35 40 45
Thr Gly Ser Gly Asn Thr Pro Ile Phe Lys Ser Gly Arg Gly Ser Gly
50 55 60
Ser Ser Phe Glu Ile Lys Ser Thr Lys Pro Glu Ala Ser Ser Gly Glu
65 70 75 80
Pro Val Val Val His Ile Thr Asp Asp Asn Glu Glu Pro Ile Ala Ala
85 90 95
Tyr His Phe Asp Leu Ser Gly Ile Ala Phe Gly Ser Asp Glu Gln Lys
100 105 110
Leu Arg Ser Ala Gly Glu Val Glu Ile Gln Phe Arg Arg Val Lys Ser
115 120 125
Lys Tyr Pro Glu Gly Thr Lys Val Thr Phe His Val Glu Lys Gly Ser
130 135 140
Asn Pro Asn Tyr Leu Ala Leu Leu Val Lys Phe Val Ala Gly Asp Gly
145 150 155 160
Asp Val Val Ala Val Asp Ile Lys Glu Lys Gly Lys Asp Lys Trp Ile
165 170 175
Ala Leu Lys Glu Ser Trp Gly Ala Ile Trp Arg Ile Asp Thr Pro Glu
180 185 190
Val Leu Lys Gly Pro Phe Thr Val Arg Gly Glu Ala Lys Asp Val Ile
195 200 205
Pro Glu Gly Trp Lys Ala Asp Thr Ala Tyr Glu Ser Lys
210 215 220
<210>19
<211>18
<212>DNA
<213>猫尾草
<400>19
atcccgaagg tcccgccg 18
<210>20
<211>63
<212>DNA
<213>猫尾草
<400>20
atcccgaagg tcccgccggg cccgaacatc acggcgacct acggcgacaa gtggctggac 60
gcg 63
<210>21
<211>63
<212>DNA
<213>猫尾草
<400>21
gttgtccttg ggaccggcgg ccgtcggctt gccgtaccag gtgctcttcg cgtccagcca 60
ctt 63
<210>22
<211>62
<212>DNA
<213>猫尾草
<400>22
ggtcccaagg acaacggcgg cgcgagcggg tacaaggacg tggacaagcc cccgttcagc 60
gg 62
<210>23
<211>69
<212>DNA
<213>猫尾草
<400>23
gagccgctgc cccggccgga cttgaagatg ggggtgttgc cggagccggt catgccgctg 60
aacgggggc 69
<210>24
<211>69
<212>DNA
<213>猫尾草
<400>24
tccggccggg gcagcggctc ctccttcgag atcaagagca ccaagcccga ggcctcctcc 60
ggcgagccc 69
<210>25
<211>30
<212>DNA
<213>猫尾草
<400>25
ggtaagcttt cacttggact cgtaggcggt 30
<210>26
<211>23
<212>DNA
<213>猫尾草
<400>26
tccgggtact tggacttgac gcg 23
<210>27
<211>23
<212>DNA
<213>猫尾草
<400>27
cgcgtcaagt ccaagtaccc gga 23
<210>28
<211>42
<212>DNA
<213>猫尾草
<400>28
ccgaacatca cggcgaccta cggcgacaag tggctggacg cg 42
<210>29
<211>69
<212>DNA
<213>猫尾草
<400>29
gtcgaagtgg tacgcggcga tgggctcctc gttgtcgtcg gtgatgtgga ccaccacggg 60
ctcgccgga 69
<210>30
<211>69
<212>DNA
<213>猫尾草
<400>30
gccgcgtacc acttcgacct ctccggcatc gcgttcgggt ccgacgagca gaagctgcgc 60
agcgccggc 69
<210>31
<211>96
<212>DNA
<213>猫尾草
<400>31
ggtaagcttt cacttggact cgtaggcggt gtcggccttc cagccctcgg ggatgacgtc 60
cttggcctcg ccgcggacgg tgaaggggcc cttgag 96
Claims (26)
1.禾本科的1类变应原的变体,其特征在于与已知野生型变应原相比,其IgE反应性降低,并基本保持了T-淋巴细胞反应性。
2.权利要求1的变应原变体,选自猫尾草、黑麦草、草地早熟禾、绒毛草、狗牙根、水稻和喜湿虉草。
3.权利要求1-2的一项或多项的变应原变体,其特征在于相应于成熟Phl p1蛋白的氨基酸位置41、57、69、72、77、83和139上的半胱氨酸缺失。
4.权利要求1-2的一项或多项的变应原变体,其特征在于相应于成熟Phl p1蛋白的氨基酸位置41、57、69、72、77、83和139上的半胱氨酸被另一种氨基酸取代。
5.权利要求4的变应原变体,其特征在于相应于成熟Phl p1蛋白的氨基酸位置41、57、69、72、77、83和139上的半胱氨酸被丝氨酸取代。
6.选自成熟Phl p1、Poa p1、Hol p1、Lol p1或Pha a1的变应原变体,其中存在权利要求5所述的序列修饰。
7.权利要求6的Phl p1变体,其特征在于成熟蛋白中位于氨基酸41、57、69、72、77、83和139位上的半胱氨酸被丝氨酸取代(SEQ IDNO 4的变体Phl p1 NoCys)。
8.权利要求1-7的一项或多项的变应原变体,其特征在于与野生型变应原相比,相应于成熟Phl p1蛋白一级序列中氨基酸1-6、1-30、92-104、115-119、175-185和213-220的至少一个区域或多个区域的组合缺失。
9.权利要求1-8的一项或多项的变应原变体,其中相应于成熟Phl p1序列的氨基酸213-220缺失。
10.选自成熟Phl p1、Poa p1、Hol p1、Lol p1或Pha a1的变应原变体,其中存在权利要求8或9所述的序列修饰。
11.权利要求10的Phl p1变体,其中成熟蛋白中位于41、57、69、72、77、83和139位上的半胱氨酸被丝氨酸取代,并且氨基酸213-220缺失(SEQ ID NO 16的变体Phl p1 NoCys Δ213-220)。
12.权利要求8的变应原变体,其中相应于成熟Phl p1序列的氨基酸1-6、115-119和213-220缺失。
13.选自成熟Phl p1、Poa p1、Hol p1、Lol p1或Pha a1的变应原变体,其中存在权利要求12所述的序列修饰。
14.权利要求13的Phl p1变体,其中成熟蛋白中氨基酸41、57、69、72、77、83和139位上的半胱氨酸被丝氨酸取代,并且氨基酸1-6、115-119和213-220缺失(SEQ ID NO 18的变体Phl p1 NoCys Δ1-6,115-119和213-220)。
15.权利要求1-14的变应原变体,其特征在于它们通过重组遗传工程方法获得。
16.编码权利要求1-15的一项或多项的变应原变体的DNA分子。
17.重组表达载体,含有与表达控制序列功能上连接的权利要求16的DNA分子。
18.由权利要求16的DNA分子或权利要求15的表达载体转化的宿主生物。
19.制备权利要求1-15的一项或多项的变应原变体的方法,包括培养权利要求18的宿主生物,并从培养物中分离相应的变应原变体。
20.权利要求1-15的一项或多项的变应原变体,其作为药物。
21.用于预防和治疗变态反应的药物组合物,含有权利要求1-15的一项或多项的至少一种变应原变体,并任选地进一步含有活性化合物和/或佐剂,其中禾本科物种的1类变应原参与所述变态反应的触发。
22.权利要求1-15的一项或多项的至少一种变应原变体在制备用于预防和治疗变态反应的药物中的用途,其中禾本科物种来源的1类变应原参与所述变态反应的触发。
23.权利要求16的DNA分子,其作为药物。
24.权利要求17的重组表达载体,其作为药物。
25.用于对患有变态反应的患者进行免疫治疗性DNA接种和/或预防这种变态反应的药物组合物,其中禾本科的1类变应原参与所述变态反应的触发,所述组合物含有权利要求16的至少一种DNA分子或权利要求24的至少一种表达载体,并任选地进一步含有活性化合物和/或佐剂。
26.权利要求16的至少一种DNA分子或权利要求24的至少一种表达载体在制备用于对患有变态反应的患者进行免疫治疗性DNA接种和/或预防这种变态反应的药物中的用途,其中禾本科的1类变应原参与所述变态反应的触发。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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DE102004035337.9 | 2004-07-21 | ||
DE102004035337A DE102004035337A1 (de) | 2004-07-21 | 2004-07-21 | Varianten der Gruppe 1-Allergene aus Poaceae mit reduzierter Allergenität und erhaltener T-Zellreaktivität |
PCT/EP2005/007481 WO2006008018A1 (de) | 2004-07-21 | 2005-07-11 | Varianten der gruppe 1-allergene aus poaceae mit reduzierter allergenität und erhaltener t-zellreaktivität |
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CN2013104392657A Division CN103467583A (zh) | 2004-07-21 | 2005-07-11 | 禾本科来源的变应原性已降低并保持t-细胞反应性的1类变应原的变体 |
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CN1989151A true CN1989151A (zh) | 2007-06-27 |
CN1989151B CN1989151B (zh) | 2014-08-27 |
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CN200580024464.5A Expired - Fee Related CN1989151B (zh) | 2004-07-21 | 2005-07-11 | 禾本科来源的变应原性已降低并保持t-细胞反应性的1类变应原的变体 |
CN2013104392657A Pending CN103467583A (zh) | 2004-07-21 | 2005-07-11 | 禾本科来源的变应原性已降低并保持t-细胞反应性的1类变应原的变体 |
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US (1) | US8263090B2 (zh) |
EP (2) | EP1768996B1 (zh) |
JP (1) | JP5149002B2 (zh) |
CN (2) | CN1989151B (zh) |
AT (1) | ATE432944T1 (zh) |
AU (1) | AU2005263477B2 (zh) |
BR (1) | BRPI0513504B1 (zh) |
CA (2) | CA2903726A1 (zh) |
DE (2) | DE102004035337A1 (zh) |
ES (1) | ES2328050T3 (zh) |
PT (1) | PT1768996E (zh) |
RU (1) | RU2409589C2 (zh) |
WO (1) | WO2006008018A1 (zh) |
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AT503690A1 (de) | 2006-06-09 | 2007-12-15 | Biomay Ag | Hypoallergene moleküle |
GB2469894B (en) * | 2009-02-05 | 2011-09-07 | Circassia Ltd | Peptides for use in treating allergy to grass pollen |
CN105001315A (zh) * | 2010-01-14 | 2015-10-28 | 默克专利股份公司 | 由于脯氨酸残基突变而具有降低的致敏性的禾本科的第6组过敏原的变异体 |
CN108728450B (zh) * | 2018-06-11 | 2021-06-01 | 中国科学院武汉植物园 | 狗牙根中一个受低温显著诱导的基因CdERF1及其应用 |
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EP0787150A1 (en) * | 1994-10-25 | 1997-08-06 | United Biomedical, Inc. | SYNTHETIC IgE MEMBRANE ANCHOR PEPTIDE IMMUNOGENS FOR THE TREATMENT OF ALLERGY |
AR020102A1 (es) * | 1998-07-30 | 2002-04-10 | Ucb Sa | Compuesto para la prevencion y/o tratamiento de la alergia; composicion farmaceutica, composicion cosmetica, composicion en forma de bebida, alimento y/oalimento para animales domesticos que lo comprende y uso de dicho compuesto o dicha composicion farmaceutica para la fabricacion de un alimento |
AUPR779201A0 (en) * | 2001-09-20 | 2001-10-11 | University Of Melbourne, The | Immunotherapeutic and immunoprophylactic reagents |
JP4668616B2 (ja) * | 2002-08-19 | 2011-04-13 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング | オオアワガエリからの主要アレルゲンPhlp1の変異体 |
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- 2005-07-11 JP JP2007521849A patent/JP5149002B2/ja not_active Expired - Fee Related
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- 2005-07-11 BR BRPI0513504A patent/BRPI0513504B1/pt not_active IP Right Cessation
- 2005-07-11 DE DE502005007428T patent/DE502005007428D1/de active Active
- 2005-07-11 CN CN200580024464.5A patent/CN1989151B/zh not_active Expired - Fee Related
- 2005-07-11 RU RU2007106170/10A patent/RU2409589C2/ru not_active IP Right Cessation
- 2005-07-11 AT AT05761824T patent/ATE432944T1/de active
- 2005-07-11 WO PCT/EP2005/007481 patent/WO2006008018A1/de active Application Filing
- 2005-07-11 PT PT05761824T patent/PT1768996E/pt unknown
- 2005-07-11 US US11/572,370 patent/US8263090B2/en not_active Expired - Fee Related
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Also Published As
Publication number | Publication date |
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US8263090B2 (en) | 2012-09-11 |
AU2005263477A1 (en) | 2006-01-26 |
PT1768996E (pt) | 2009-09-07 |
WO2006008018A1 (de) | 2006-01-26 |
BRPI0513504B1 (pt) | 2016-08-16 |
DE502005007428D1 (de) | 2009-07-16 |
EP1768996B1 (de) | 2009-06-03 |
CA2574449A1 (en) | 2006-01-26 |
AU2005263477B2 (en) | 2011-12-08 |
EP2028189A2 (de) | 2009-02-25 |
ES2328050T3 (es) | 2009-11-06 |
ATE432944T1 (de) | 2009-06-15 |
JP5149002B2 (ja) | 2013-02-20 |
EP2028189A3 (de) | 2009-07-08 |
RU2007106170A (ru) | 2008-08-27 |
JP2008507262A (ja) | 2008-03-13 |
US20080267985A1 (en) | 2008-10-30 |
CN103467583A (zh) | 2013-12-25 |
CA2574449C (en) | 2015-12-01 |
BRPI0513504A (pt) | 2008-05-06 |
CN1989151B (zh) | 2014-08-27 |
CA2903726A1 (en) | 2006-01-26 |
DE102004035337A1 (de) | 2006-03-16 |
RU2409589C2 (ru) | 2011-01-20 |
EP1768996A1 (de) | 2007-04-04 |
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