CN107739736A - 流感病毒核酸分子及由其制备的疫苗 - Google Patents
流感病毒核酸分子及由其制备的疫苗 Download PDFInfo
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Abstract
本文中提供的是编码HA血凝素的新型共有氨基酸序列的核酸序列,以及表达所述序列的基因构建体/载体和疫苗。本文中还提供的是用于使用所提供的疫苗产生抗一种或多种甲型流感病毒血清型的免疫反应的方法。
Description
发明领域
本发明涉及改进的流感病毒疫苗、用于诱导抗流感的免疫反应的改进的方法、用于诊断已接种了和感染了流感病毒的哺乳动物宿主及用于预防性和/或治疗性免疫个体以对抗流感病毒的改进的方法。
发明背景
流感,通常称为流行性感冒,是由正粘病毒科(family Orthomyxoviridae)的RNA病毒引起的传染病。流感或流行性感冒病毒感染鸟类和哺乳动物。正粘病毒科的5个属中的3个属是流感病毒:甲型流感病毒、乙型流感病毒和丙型流感病毒。在这些流感病毒中,甲型流感病毒最常见。
流感病毒通常以咳嗽或打喷嚏产生的气雾形式通过空气传播或通过与含有病毒的体液或已污染的表面直接接触传播。流感的季节性流行在世界范围发生并且导致每年数十万人的死亡。在一些年份,大范围流行病发生并且导致数百人死亡。此外,家畜,特别是禽类和猪也对每年一次的流行病和引起大量动物死亡和金钱损失的偶然的大范围流行病易感。
在结构上,流感病毒是相似的,通常具有约80-120nm的由相似分子组分组成的球状或丝状病毒颗粒。包含病毒蛋白质和病毒RNA的中心核心被由两种不同糖蛋白构成的病毒包膜和来源于其中产生病毒颗粒的细胞的脂质包衣(lipid coat)覆盖。两种额外的不同糖蛋白锚定在病毒包膜内并且包括在表面上向外突出的部分。
流感病毒RNA基因组通常提供为8个不同的单链负义RNA区段,它们一起组成基因组的编码11种蛋白质(HA、NA、NP、M1、M2、NS1、NEP、PA、PB1、PB1-F2、PB2)的11个病毒基因。8个RNA区段为:1)HA,其编码血凝素(产生一个病毒体需要约500个血凝素分子);2)NA,其编码神经氨酸酶(产生一个病毒体需要约100个神经氨酸酶分子);3)NP,其编码核蛋白;4)M,其通过使用来自相同RNA区段的不同读框编码2种基质蛋白(M1和M2)(产生一个病毒体需要约3000个基质蛋白分子);5)NS,其通过使用来自相同RNA区段的不同读框编码两种不同的非结构蛋白(NS1和NEP);6)PA,其编码RNA聚合酶;7)PB1,其通过使用来自相同RNA区段的不同读框编码RNA聚合酶和PB1-F2蛋白(诱导细胞凋亡);和8)PB2,其编码RNA聚合酶。
在这11种蛋白质中,血凝素(HA)和神经氨酸酶(NA)是锚定在病毒包膜中并且存在于病毒颗粒的外表面上的两种大糖蛋白。此类蛋白质用作针对流感病毒的免疫反应的免疫原。HA(其编码介导病毒与靶细胞的结合和病毒基因组进入靶细胞的凝集素)表达为单个基因产物HA0,随后被宿主蛋白酶加工,从而产生两个亚基HA1和HA2,它们一起在流感病毒颗粒的表面上形成复合物。NA参与在感染的细胞中产生的新产生的成熟病毒颗粒的释放。
存在16个已知的HA血清型和9个已知的针对甲型流感病毒的NA血清型。存在病毒颗粒中的不同血清型的鉴定通常用于描述病毒。例如,H1N1是具有HA血清型H1和NA血清型N1的流感病毒;H5N1是具有HA血清型H5和NA血清型N1的流感病毒。只有H1、H2和H3血清型以及N1和N2血清型通常感染人。
流感病毒株通常具有种或属特异性;即可感染猪(猪流感病毒)的流感病毒株通常不感染人或鸟类;可感染鸟类(禽流感病毒)的流感病毒株不感人或猪;以及可感染人(人流感病毒)的流感病毒株不感染鸟类或猪。然而,流感病毒株可突变并且使对于一个物种的感染性变成对于另一个物种的感染性。例如,仅感染猪的病毒株(猪流感病毒)可突变或重组变成可以仅感染人或感染猪和人的病毒株。通常称为“猪流感病毒”的流感病毒为流感病毒株,例如H1N1株,其可感染人并且来源于先前对于猪是特异性的病毒株(即猪流感病毒是猪来源的人流感病毒或猪源性人流感病毒)。通常称为“鸟流感病毒”的流感病毒是流感病毒株,例如H5N1株,其可感染人并且来源于先前对于鸟类是特异性的病毒株(即禽流感病毒是禽类来源人流感病毒或禽类源性人流感病毒)。
季节性地给发达国家的许多人以及有时给家畜接种针对流感的疫苗。使用的疫苗在其保护结果上是有限的,因为由疫苗诱导的免疫反应对于病毒的某些亚型是特异性的。每年基于哪种类型和病毒株的病毒将在给定的年份传播的国际监测和科学家的估计来开发和施用不同的流感疫苗。病毒通过区段的突变、重组和再分布产生显著的改变。因此,一年中提供的疫苗被认为不具有抗下一年广泛传播的季节性病毒株的保护作用。
通常由美国疾病控制和预防中心推广的“流感疫苗注射(flu shot)”通常包括三种杀死的/灭活的流感病毒:一种甲型(H3N2)病毒、一种甲型(H1N1)病毒和一种乙型病毒。因此,很明显接种受限于亚型的预测以及针对该该亚型的特异性疫苗的可获得性(availability)。
直接施用核酸序列以接种抗动物和人疾病的疫苗一直已来都在进行研究并且许多努力集中在核酸递送的有效和高效方式以产生期望抗原的必要表达,从而导致免疫源应答和最终获得该技术的成功。
DNA疫苗具有许多优于更常规的接种方法例如基于活减毒病毒和重组蛋白的疫苗的概念性优势。DNA疫苗是安全、稳定、容易产生的并且在人中能被良好耐受,临床前试验显示几乎无质粒整合的证据[Martin,T.,等人,质粒DNA疟疾疫苗:肌内注射后用于基因组整合的潜能(Plasmid DNA malaria vaccine:the potential for genomic integrationafter intramuscular injection).Hum Gene Ther,1999.10(5):p.759-68;Nichols,W.W.,等人,整合入宿主细胞基因组的潜在DNA疫苗(Potential DNA vaccine integrationinto host cell genome).Ann N Y Acad Sci,1995.772:p.30-9]。此外,DNA疫苗因疫苗的功效不受针对载体的预先存在的抗体滴度影响的事实而十分适合用于重复施用[Chattergoon,M.,J.Boyer和D.B.Weiner,基因免疫:疫苗和免疫治疗的新领域(Geneticimmunization:a new era in vaccines and immune therapeutics).FASEB J,1997.11(10):p.753-63]。然而,DNA疫苗的临床采用的一个主要障碍一直以来是当移至更大的动物时平台的免疫原性的减弱[Liu,M.A.和J.B.Ulmer,质粒DNA疫苗的人临床性状(Humanclinical trials of plasmid DNA vaccines).AdV Genet,2005.55:p.25-40]。DNA疫苗免疫原的工程的最近技术进展,例如密码子最优化、RNA最优化和免疫球蛋白前导序列的添加[Andre,S.,等人,利用具有最优化密码子选择的合成gp120序列的DNA接种引发的增强的免疫反应(Increased immune response elicited by DNA vaccination with a syntheticgp120 sequence with optimized codon usage).J Virol,1998.72(2):p.1497-503;Deml,L.,等人,密码子选择最优化对编码1型人免疫缺陷病毒Gag蛋白的DNA候选疫苗的表达和免疫原性的多个影响(Multiple effects of codon usage optimization onexpression and immunogenicity of DNA candidate vaccines encoding the humanimmunodeficiency virus type 1Gag protein).J Virol,2001.75(22):p.10991-1001;Laddy,D.J.,等人,抗禽流感的基于共有序列的新型DNA疫苗的免疫原性(immunogenicityof novel consensus-based DNA vaccines against avian influenza).Vaccine,2007.25(16):p.2984-9;Frelin,L.,等人,密码子最优化和mRNA扩增有效地增强丙型肝炎病毒非结构3/4基因的免疫原性(Codon optimization and mRNA amplificationeffectively enhances the immunogenicity of the hepatitis Cvirus nonstructural3/4A gene).Gene Ther,2004.11(6):p.522-33]以及质粒递送技术的最近开发的技术例如电穿孔[Hirao,L.A.,等人,利用电穿孔的真皮内/皮下免疫增进了质粒疫苗在猪和恒河猕猴中的递送和潜能(Intradermal/subcutaneous immunization by electroporationimproves plasmid vaccine delivery and potency in pigs and rhesus macaques).Vaccine,2008.26(3):p.440-8;Luckay,A.,等人,质粒DNA疫苗的设计和体内电穿孔对恒河猕猴的所产生的疫苗特异性免疫反应的效应(Effect of plasmid DNA vaccine designand in vivo electroporation on the resulting vaccine-specific immuneresponses in rhesus macaques).J Virol,2007.81(10):p.5257-69;Ahlen,G.,等人,体内电穿孔通过增加的局部DNA摄取、蛋白质表达、炎症和CD3+T细胞的浸润增强丙型肝炎病毒非结构3/4A DNA的免疫原性(In vivo electroporation enhances theimmunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased localDNA uptake,protein expression,inflammation,and infiltration of CD3+T cells).JImmunol,2007.179(7):p.4741-53]已提高了DNA疫苗的表达和免疫原性。此外,研究已表明共有免疫原的使用与单独的天然抗原相比较能够增加细胞免疫反应的广度[Yan,J.,等人,由基于共有序列的工程HIV-1B亚型包膜DNA疫苗引发的增强的细胞免疫反应(Enhancedcellular immune responses elicited by an engineered HIV-1subtype B consensus-based envelope DNA vaccine).Mol Ther,2007.15(2):p.411-21;Rolland,M.,等人,1型祖先树中心人免疫缺陷病毒蛋白的重建和功能(Reconstruction and function ofancestral center-of-tree human immunodeficiency virus type 1proteins).JVirol,2007.81(16):p.8507-14]。
用于递送核酸序列例如质粒DNA的一个方法是电穿孔(EP)技术。所述技术已在人临床试验中被用于递送抗癌药物例如博来霉素以及用于关于许多动物物种的临床前研究。
仍然存在对免疫原性流感病毒共有血凝素蛋白,对编码这样的蛋白质的核酸构建体和对用于诱导抗多株流感病毒的免疫反应的组合物的需要。仍然存在对用于治疗个体的跨许多流感病毒亚型的经济有效的抗流感病毒的有效疫苗的需要。
发明概述
本文中提供的是包含核酸序列的分离的核酸分子,所述核酸序列选自:SEQ IDNO:1;与SEQ ID NO:1具有95%同源性的核酸序列;SEQ ID NO:1的片段;与SEQ ID NO:1的片段具有95%同源性的核酸序列;SEQ ID NO:3;与SEQ ID NO:3具有95%同源性的核酸序列;SEQ ID NO:3的片段;与SEQ ID NO:3的片段具有95%同源性的核酸序列;SEQ ID NO:6;与SEQ ID NO:6具有95%同源性的核酸序列;SEQ ID NO:6的片段;与SEQ ID NO:6的片段具有95%同源性的核酸序列;SEQ ID NO:9;与SEQ ID NO:9具有95%同源性的核酸序列;SEQID NO:9的片段;与SEQ ID NO:9的片段具有95%同源性的核酸序列;SEQ ID NO:11;与SEQID NO:11具有95%同源性的核酸序列;SEQ ID NO:11的片段;与SEQ ID NO:11的片段具有95%同源性的核酸序列;SEQ ID NO:13;与SEQ ID NO:13具有95%同源性的核酸序列;SEQID NO:13的片段;与SEQ ID NO:13的片段具有95%同源性的核酸序列;和SEQ ID NO:15;与SEQ ID NO:15具有95%同源性的核酸序列;SEQ ID NO:15的片段;与SEQ ID NO:15的片段具有95%同源性的核酸序列。
还提供了组合物,其包含:a)选自:SEQ ID NO:1;与SEQ ID NO:1具有95%同源性的核酸序列;SEQ ID NO:1的片段;与SEQ ID NO:1的片段具有95%同源性的核酸序列;SEQID NO:3;与SEQ ID NO:3具有95%同源性的核酸序列;SEQ ID NO:3的片段;与SEQ ID NO:3的片段具有95%同源性的核酸序列;SEQ ID NO:6;与SEQ ID NO:6具有95%同源性的核酸序列;SEQ ID NO:6的片段;与SEQ ID NO:6的片段具有95%同源性的核酸序列;SEQ ID NO:9;与SEQ ID NO:9具有95%同源性的核酸序列;SEQ ID NO:9的片段;与SEQ ID NO:9的片段具有95%同源性的核酸序列;SEQ ID NO:11;与SEQ ID NO:11具有95%同源性的核酸序列;SEQ ID NO:11的片段;与SEQ ID NO:11的片段具有95%同源性的核酸序列;SEQ ID NO:13;与SEQ ID NO:13具有95%同源性的核酸序列;SEQ ID NO:13的片段;与SEQ ID NO:13的片段具有95%同源性的核酸序列;SEQ ID NO:15;与SEQ ID NO:15具有95%同源性的核酸序列;SEQ ID NO:15的片段;与SEQ ID NO:15的片段具有95%同源性的核酸序列中的一种或多种的第一核酸序列;和b)编码蛋白质的第二核酸序列,所述蛋白质选自:甲型流感病毒H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9、乙型流感病毒血凝素、神经氨酸酶及其片段中的一种或多种。
本发明的一些方面提供了诱导免疫反应的方法,其包括步骤:给个体施用此类核酸分子和/或组合物。
本发明的其它方面提供了保护个体免受感染的方法。所述方法包括步骤:给所述个体施用预防有效量的包含此类核酸序列或组合物的核酸分子;其中所述核酸序列在所述个体的细胞中表达并且针对由所述核酸序列编码的蛋白质的保护性免疫反应被诱导。在一些实施方案中,免疫反应是抗猪来源的人流感病毒的保护性免疫反应。
在本发明的一些方面,提供了用于治疗已被流感病毒感染的个体的方法。所述方法包括给所述个体施用治疗有效量的此类核酸分子和/或组合物的步骤。在一些实施方案中,免疫反应是抗猪来源的人流感病毒的免疫反应。
附图简述
图1是2999碱基对主链载体质粒pVAX1(茵维特罗根(Invitrogen),卡尔斯巴德(Carlsbad)加利福尼亚州(CA))的图谱。CMV启动子位于碱基137-724。T7启动子/引发位点位于碱基664-683。多克隆位点位于碱基696-811。牛GH多腺苷酸化信号位于碱基829-1053。卡那霉素抗性基因位于碱基1226-2020。pUC来源位于碱基2320-2993。
基于可从茵维特罗根公司获得的pVAX1的序列,在用作pGX2009的主链的pVAX1的序列中发现下列突变:
C>G 241在CMV启动子中
C>T 1942主链,牛生长激素多腺苷酸化信号(bGHpolyA)的下游
A>-2876主链,卡那霉素基因的下游
C>T 3277在pUC复制起始区(Ori)中,高拷贝数突变(参见Nucleic AcidResearch 1985)
G>C 3753正好在pUC Ori的末端,RNASeH位点的上游
碱基对2、3和4在主链中CMV启动子的上游被从ACT改变成CTG。
图2显示质粒pGX2009的两个图谱,所述质粒也称为pH1HA09。质粒pGX2009的核酸序列(SEQ ID NO:5)包括共有H1蛋白构建体的编码序列(由SEQ ID NO:3编码的氨基酸SEQID NO:4),所述共有H1蛋白包括连接于共有H1氨基酸序列(由SEQ ID NO:1编码的氨基酸SEQ ID NO:2)的N末端的IgE前导(氨基酸SEQ ID NO:17),所述共有H1氨基酸序列在其C末端上连接于HA标签(SEQ ID NO:18)。共有H1蛋白(由SEQ ID NO:3编码的氨基酸SEQ ID NO:4)为标记的SwiHum Con HA和H1HA09。
图3显示质粒pGX2006的图谱。质粒pGX2006的核酸序列(SEQ ID NO:8)包括共有H2蛋白(由SEQ ID NO:6编码的氨基酸SEQ ID NO:7)的编码序列,所述共有H2蛋白为标记的H2HA。
图4显示来自利用经免疫的雪貂的血清进行的血凝抑制测定的数据。
图5显示利用新型H1N1株攻击经免疫的和未经免疫的雪貂的结果。
详述
提供了甲型H1和H2流感病毒的各自的共有氨基酸序列(在本文中分别称为“共有H1”(SEQ ID NO:2)和“共有H2”(SEQ ID NO:7)),以及新型合成杂交体共有甲型H1流感病毒血凝素氨基酸序列(在本文中称为“共有U2”(SEQ ID NO:10))和乙型流感病毒血凝素的共有氨基酸序列(在本文中称为“共有BHA”(SEQ ID NO:13)),其可为哺乳动物提供抗流感病毒的保护作用。此外,提供了包含共有H1氨基酸序列、共有H2氨基酸序列、共有U2氨基酸序列和/或共有BHA氨基酸序列的蛋白质。在一些方面,提供了核酸序列,所述核酸序列编码包含共有H1氨基酸序列(例如(SEQ ID NO:1)或(SEQ ID NO:3))、共有H2氨基酸序列(例如(SEQ ID NO:6))、共有U2氨基酸序列(例如(SEQ ID NO:9)或(SEQ ID NO:11))和/或共有BHA氨基酸序列(例如(SEQ ID NO:13)或(SEQ ID NO:15))的蛋白质。
然而不受科学理论束缚,可用于引发广泛地抗多种流感病毒亚型的免疫反应(体液、细胞或两者)的疫苗可包括如下的一种或多种:1)编码包含共有H1氨基酸序列的蛋白质的核酸序列;2)包含共有H1氨基酸序列的蛋白质;3)编码包含共有H2氨基酸序列的蛋白质的核酸序列;4)包含共有H2氨基酸序列的蛋白质;5)编码包含共有U2氨基酸序列的蛋白质的核酸序列;6)包含共有U2氨基酸序列的蛋白质;7)编码包含共有BHA氨基酸序列的蛋白质的核酸序列;和8)包含共有BHA氨基酸序列的蛋白质。
可进行免疫方法以及可制备使用和/或组合下列组分的两种或更多种的疫苗:1)编码包含共有H1氨基酸序列的蛋白质的核酸序列;2)包含共有H1氨基酸序列的蛋白质;3)编码包含共有H2氨基酸序列的蛋白质的核酸序列,4)包含共有H2氨基酸序列的蛋白质;5)编码包含共有U2氨基酸序列的蛋白质的核酸序列,6)包含共有U2氨基酸序列的蛋白质,7)编码包含共有BHA氨基酸序列的蛋白质的核酸序列,和8)包含共有BHA氨基酸序列的蛋白质。对于基于更广泛的抗流感病毒的治疗,可进行免疫方法,并且可制备疫苗,所述疫苗使用和/或组合一种或多种其它流感病毒蛋白质例如甲型H1-H16流感病毒、甲型N1-N9流感病毒、乙型流感病毒血凝素、乙型流感病毒神经氨酸酶和/或编码此类蛋白质的基因以及如下组分的一种或多种:1)编码包含共有H1氨基酸序列的蛋白质的核酸序列;2)包含共有H1氨基酸序列的蛋白质;3)编码包含共有H2氨基酸序列的蛋白质的核酸序列,4)包含共有H2氨基酸序列的蛋白质;5)编码包含共有U2氨基酸序列的蛋白质的核酸序列,6)包含共有U2氨基酸序列的蛋白质,7)编码包含共有BHA氨基酸序列的蛋白质的核酸序列,和8)包含共有BHA氨基酸序列的蛋白质。
1.定义.
本文中使用的术语仅为了描述特定的实施方案并且不旨在限制。如本说明书中和所附权利要求中所用,除非上下文中另外明确指定,否则单数形式“一种/一个(a)”、“一种/一个(an)”和“该(the)”包括复数指示物。
对于本文的数字范围的引述,明确地包括具有相同精度的介于其间的每个介入数字。例如,对于6-9的范围,除了6和9外还包括数字7和8,对于范围6.0-7.0,明确地包括数字6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9和7.0。
a.佐剂
如本文中所用,“佐剂”意指添加至本文中描述的DNA质粒疫苗以增强由所述DNA质粒和下文中描述的编码核酸序列编码的抗原的免疫原性。
b.抗体
如本文中所用,″抗体″意指种类IgG、IgM、IgA、IgD或IgE的抗体及其片段或衍生物,包括Fab、F(ab′)2、Fd和单链抗体、双链抗体(diabody)、双特异性抗体、双功能抗体及其衍生物。抗体可以是从哺乳动物的血清样品分离的抗体、多克隆抗体、亲和纯化的抗体或显示对于期望的表位或从其衍生的序列的充分结合特异性的其混合物。
c.编码序列
如本文中所用,“编码序列”或“编码核酸”意指包含编码蛋白质的核苷酸序列的核酸(RNA或DNA分子)。编码序列还可包括有效地连接于调控元件(包括能够在已对其施用了所述核酸的个体或哺乳动物的细胞中指导表达的启动子和多腺苷酸化信号)的起始和终止信号。
d.互补
如本文中所用,“互补”或“互补的”意指可进行核酸分子的核苷酸或核苷酸类似物之间的沃尔森-克里克(例如,A-T/U和C-G)或Hoogsteen碱基配对的核酸。
e.共有区或共有序列
如本文中所用,“共有区”或“共有序列”意指基于多个亚型的特定流感病毒抗原的比对的分析的多肽序列。可制备编码共有多肽序列的核酸序列。包含含有共有序列的蛋白质和/或编码此类蛋白质的疫苗可用于诱导抗多个亚型或血清型的特定流感病毒抗原的广泛免疫。共有流感病毒抗原可包括甲型流感病毒共有血凝素氨基酸序列(包括例如共有H1、共有H2)或乙型流感病毒共有血凝素氨基酸序列。
f.恒定电流
如本文中所用,“恒定电流”意指由组织或定义所述组织的细胞在递送至相同组织的电脉冲的持续过程中接收或经历的电流。从本文中描述的电穿孔设备递送电脉冲。该电流在电流冲的寿命过程中在所述组织中保持恒定的安培数,因为本文中提供的电穿孔设备具有反馈元件(优选具有瞬时值反馈(instantaneous feedback))。所述反馈元件可测量整个脉冲持续过程中组织(或细胞)的电阻并且引起电穿孔设备改变其电能输出(例如,升高电压),以便相同组织中的电流在整个电泳冲(在微秒级上)中以及电脉冲之间保持恒定。在一些实施方案中,反馈元件包括控制器。
g.电流反馈或反馈
“电流反馈”或“反馈”可互换使用并且意指提供的电穿孔设备的主动反应,其包括测量电极之间的组织中的电流和相应地改变由EP设备递送的能量输出以使电流维持在恒定水平。该恒定水平是在起始脉冲序列或电处理之前由用户预先设定的。反馈可通过电穿孔组件例如电穿孔设备的控制器来实现,因为本文中的电路能够连续监测电极之间的组织中的电流并且将该监测的电流(或组织内的电流)与预设电流相比较,以及连续调整能量输出以将监测的电流维持在预设水平。反馈环可以是瞬时的,因为其是模拟闭环反馈。
h.分散电流
如本文中所用,“分散电流”意指来源于本文中描述的电穿孔设备的各种针电极阵列的电流的模式,其中所述模式最小化或优选消除电穿孔相关的热应激在被电穿孔的组织的任何区域上的发生。
i.电穿孔
如本文中可互换使用的,“电穿孔”、“电透化(electro-permeabilization)”或“电动力增强(electro-kinetic enhancement)”(“EP”)意指跨膜电场脉冲在生物膜中诱导极微小通道(孔)的用途;它们的存在允许生物分子例如质粒、寡核苷酸、siRNA、药物、离子和水从细胞膜的一侧通过至另一侧。
j.反馈机制
如本文中所用,“反馈机制”意指通过软件或硬件(或固件)进行的过程,所述过程(在递送能量脉冲之前、期间和/或之后)接收期望组织的阻抗并且将其与预设值(优选电流)相比较,然后调整所递送的能量脉冲以达到预设值。可通过模拟闭合电路进行反馈机制。
k.片段
如本文中所用,关于核酸序列的“片段”意指核酸序列或其部分,所述核酸序列或其部分编码能够引起哺乳动物的免疫反应的多肽,所述多肽与全长野生型病毒株的流感病毒抗原(包括例如甲型H1流感病毒血凝素、甲型H2流感病毒血凝素或乙型流感病毒血凝素)交叉反应。片段可以是选自编码共有氨基酸序列的多个核苷酸序列中的至少一个和包含这样的序列(包括SEQ ID NO:1、3、6、9、11、13和15)的构建体的DNA片段。DNA片段可包括免疫球蛋白前导序列例如IgE或IgG前导序列的编码序列。DNA片段在长度上可以为30或更多个核苷酸,45或更多个,60或更多个,75或更多个,90或更多个,120或更多个,150或更多个,180或更多个,210或更多个,240或更多个,270或更多个,300或更多个,360或更多个,420或更多个,480或更多个,540或更多个,600或更多个,660或更多个,720或更多个,780或更多个,840或更多个,900或更多个,960或更多个,1020或更多个,1080或更多个,1140或更多个,1200或更多个,1260或更多个,1320或更多个,1380或更多个,1440或更多个,1500或更多个,1560或更多个,1620或更多个,1680或更多个,1740或更多个,1800或更多个,1860或更多个,1820或更多个,1880或更多个,1940或更多个,2000或更多个,2600或更多个,2700或更多个,2800或更多个,2900或更多个,2910或更多个,2920或更多个,2930或更多个,2931或更多个,2932或更多个,2933或更多个,2934或更多个,2935或更多个,2936或更多个,2937或更多个或2938或更多个核苷酸。DNA片段可少于10个核苷酸,少于20个,少于30个,少于40个,少于50个,少于60个,少于75个,少于90个,少于120个,少于150个,少于180个,少于210个,少于240个,少于270个,少于300个,少于360个,少于420个,少于480个,少于540个,少于600个,少于660个,少于720个,少于780个,少于840个,少于900个,少于960个,少于1020个,少于1080个,少于1140个,少于1200个,少于1260个,少于1320个,少于1380个,少于1440个,少于1500个,少于1560个,少于1620个,少于1680,或少于1740个核苷酸,少于1800,少于1860个,少于1820,少于1880个,少于1940个,少于2000个,少于2600个,少于2700个,少于2800个,少于2900个,少于2910个,少于2920个,少于2930个,少于2931个,少于2932个,少于2933个,少于2934个,少于2935个,少于2936个,少于2937个或少于2938个核苷酸。
关于多肽序列的“片段”意指能够引发哺乳动物的免疫反应的多肽,所述多肽与全长野生型病毒株流感病毒抗原例如甲型H1流感病毒血凝素、甲型H2流感病毒血凝素或乙型流感病毒血凝素交叉反应。片段可以是选自本发明的多个多肽序列(包括SEQ ID NO:2、4、7、10、12、14和16)中的至少一个的多肽片段。可分析多肽片段以接触至少一个由公共可获得的数据库例如洛斯阿拉莫斯国家实验室的HA(Los Alamos National Laboratory’s HA)序列数据库提供的抗原表位。多肽HA片段还可包含免疫球蛋白前导序列(例如IgE或IgG前导序列)的氨基酸序列。多肽片段在长度上可以为30或更多个氨基酸,在长度上为45或更多个,60或更多个,75或更多个,90或更多个,120或更多个,150或更多个,180或更多个,210或更多个,240或更多个,270或更多个,300或更多个,360或更多个,420或更多个,480或更多个,540或更多个,600或更多个,660或更多个或710个氨基酸或更多个氨基酸。多肽片段在长度上可少于10个氨基酸,少于20个,少于30个,少于40个,少于50个,少于60个,少于75个,少于90个,少于120个,少于150个,少于180个,少于210个,少于240个,少于270个,少于300个,少于360个,少于420个,少于480个,少于540个,少于600个,少于660个,少于700个,少于701个,少于702个,少于703个,少于704个,少于705个,少于706个,少于707个,少于708个,少于709个或少于710个氨基酸。
l.基因构建体
如本中所用,术语“基因构建体”是指包含编码蛋白质的核苷酸序列的DNA或RNA分子。编码序列包括有效地连接于调控元件(包括能够在已对其施用了核酸分子的个体的细胞中指导表达的启动子和多腺苷酸化信号)的起始和终止信号。如本文中所用,术语“可表达形式”是指基因构建体,所述基因构建体包含有效地连接于编码蛋白质的编码序列的必需调控元件,以便当存在于个体的细胞中时编码序列可被表达。
m.同一的
如本文中所用,在两个或更多个核酸或多肽序列的上下文中,″同一的″或″同一性″意指序列具有指定的在指定的区域范围中为相同的残基的百分比。可如下计算百分比:最佳地对齐两个序列,在指定的区域范围内比较两个序列,确定在其上相同残基存在于两个序列中的位置的数目以产生匹配位置的数目,将匹配位置的数目除以指定区域中位置的总数,将结果乘以100以产生序列同一性的百分比。在其中两个序列具有不同长度或比对产生一个或多个交错末端并且指定的比较区域只包括单个序列的情况下,单个序列的残基包括在计算的分母中而非分子中。当比较DNA与RNA时,胸腺嘧啶(T)和尿嘧啶(U)可以被认为是等同的。可人工或通过使用计算机序列算法例如BLAST或BLAST 2.0进行同一性。
n.阻抗
当讨论反馈机制时可使用“阻抗”,其可按照欧姆定律被转化成电流值,从而使得能够与预设电流相比较。
o.免疫反应
如本文中所用,“免疫反应”意指宿主的免疫系统,例如哺乳动物的免疫系统响应抗原例如流感病毒血凝素共有抗原的引入的激活。免疫反应可以以细胞反应或体液反应或两者的形式存在。
p.核酸
本文中所用的“核酸”或“寡核苷酸”或“多核苷酸”意指共价连接在一起的至少两种核苷酸。单链的描述也确定互补的链的序列。因此,核酸还包括描述的单链的互补链。核酸的许多变体可用于与给定的核酸相同的目的。因此,核酸还可包括大体上相同的核酸和其互补序列。单链提供了可在严格杂交条件下与靶序列杂交的探针。因此,核酸还包括在严格杂交条件下杂交的探针。
核酸可以是单链或双链的,或可包含双链和单链序列的部分。核酸可以是DNA,基因组和eDNA、RNA或杂交体,其中核酸可包含脱氧核糖和核糖-核酸苷酸的组合以及碱基(包括尿嘧啶、腺嘌呤、胸腺嘧啶、胞嘧啶、鸟嘌呤、肌苷、黄嘌呤、次黄嘌呤、异胞嘧啶和异鸟嘌呤)的组合。核酸可通过化学合成法或通过重组法获得。
q.有效连接的
如本文中所用,“有效连接的”意指基因的表达处于与其空间上连接的启动子的控制之下。启动子可定位在处于其控制之下的基因的5′(上游)或3′(下游)。启动子与基因之间的距离可与在该启动子所源自的基因中该启动子与其控制的基因之间的距离大致相同。如本领域中是已知的,该距离的变化可被容纳而不丧失启动子功能。
r.启动子
如本文中所用“启动子”意指能够在细胞中赋予核酸的表达、激活或增强所述表达的合成或天然来源的分子。启动子可以包括一个或多个特异性转录调控序列以进一步增强所述基因的表达和/或改变所述基因的空间表达和/或时间表达。启动子还可包括远端增强子或阻遏物元件,所述元件可位于远离转录起始位点多达数千个碱基对。启动子可来源于包括病毒、细菌、真菌、植物、昆虫和动物的来源。启动子对于其中表达发生的组织或器官,对于表达发生所处的发育阶段或响应外部刺激例如生理应激、病原体、金属离子或诱导剂,可组成型地或差异地调控基因组件的表达。启动子的代表性实例包括噬菌体T7启动子、噬菌体T3启动子、SP6启动子、lac操纵子-启动子、tac启动子、SV40晚期启动子、SV40早期启动子、RSV-LTR启动子,CMV IE启动子、SV40早期启动子或SV40晚期启动子和CMV IE启动子。
s.严格杂交条件
如本文中所用,“严格杂交条件”意指在其下第一核酸序列(例如,探针)将与第二核酸序列(例如,靶)例如在核酸的复杂混合物中杂交的条件下。严格条件是序列依赖性的并且在不同的环境中是不同的。严格条件可经选择比特定离子强度pH下特定序列的热解链温度(thermal melting point)(Tm)低约5-10℃。Tm可以是(在确定的离子强度、pH和核酸浓度下)50%的与靶互补的探针与靶序列平衡杂交时所处的温度(当靶序列过量存在时,在Tm下,50%的探针被平衡地占据)。严格条件可以是这样的条件,其中在pH 7.0至8.3下盐浓度低于约1.0M钠离子,例如约0.01-1.0M的钠离子浓度(或其它盐)并且温度为至少约30℃(对于短探针,例如约10-50个核苷酸)和至少约60℃(对于长探针,例如大于至少约50个核苷酸)。严格条件还可通过添加去稳定剂例如甲酰胺来获得。对于选择性或特异性杂交,阳性信号可为本底杂交的至少2至10倍。示例性严格杂交条件包括下列条件:在42℃下于50%甲酰胺、5x SSC和1% SDS中温育,或在65℃下于5x SSC,1% SDS中温育,和在65℃下于0.2x SSC和0.1% SDS中洗涤。
t.大体上互补
如本文中所用,“大体上互补”意指第一序列与第二序列的互补序列在8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、180、270、360、450、540、630、720、810、900、990、1080、1170、1260、1350、1440、1530、1620、1710、1800、1890、1980、2070或更多个核苷酸或氨基酸的区域范围内具有至少60%、65%、70%、75%、80%、85%、90%、95%、97%、98%或99%的同一性,或两个序列在严格杂交条件下杂交。
u.大体同一的
如本文中所用,“大体上同一的”意指第一序列与第二序列在8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、180、270、360、450、540、630、720、810、900、990、1080、1170、1260、1350、1440、1530、1620、1710、1800、1890、1980、2070或更多个核苷酸或氨基酸的区域范围内具有至少60%、65%、70%、75%、80%、85%、90%、95%、97%、98%或99%的同一性,或就核酸而言,如果第一序列与第二序列的互补序列大体上互补,则第一序列与第二序列大体上同一。
v.亚型或血清型
“亚型”或“血清型”:如本文中所用,可互换,并且当提及流感病毒时,意指流感病毒的遗传性变型(genetic variant),以便一个亚型免疫系统识别而与不同亚型相区分。
w.变体
如本文中所用,关于核酸的“变体”意指(i)参照核苷酸序列的部分或片段;(ii)参照核苷酸序列或其部分的互补序列;(iii)与参照核酸或其互补序列大体上同一的核酸;或(iv)在严格条件下与参照核酸、其互补序列或与其大体上同一的序列杂交的核酸。
“变体”是指在氨基酸序列上相异在于氨基酸的插入、缺失或保守置换但保持至少一种生物活性的肽或多肽。变体还可以指其氨基酸序列与参考的蛋白质的氨基酸序列大体上同一的蛋白质,其保留至少一种生物活性。氨基酸的保守置换,即用具有相似性质(例如,亲水性、带电荷区域的程度和分布)的不同氨基酸替代氨基酸,在本领域通常被认为包括小的变化。这些小的变化可部分地通过考虑氨基酸的亲水指数来鉴定,如本领域中所理解的。Kyte等人,J.Mol.Biol.157:105-132(1982)。氨基酸的亲水指数基于其疏水性和电荷的考虑。在本领域中已知具有相似亲水指数的氨基酸可被置换并且仍然保留蛋白质功能。在一个方面,具有±2的亲水指数的氨基酸被置换。氨基酸的亲水性还可用于揭示可产生保留生物功能的蛋白质的置换。在肽的上下文中氨基酸的亲水性的考虑允许计算该肽的最大局部平均亲水性,已报道与抗原性和免疫原性相关的有用的测量。美国专利No.4,554,101,通过引用完全并入本文。具有相似亲水性值的氨基酸的置换可产生保留生物活性例如免疫源性的肽,这在本领域中是被理解的。可利用具有在±2内的亲水性值的氨基酸彼此进行置换。氨基酸的疏水性指数和亲水性值受该氨基酸的具体侧链影响。与该观察一致,应理解与生物功能相容的氨基酸置换依赖于氨基酸的相似性,特别地所述氨基酸的侧链的相似性,如通过疏水性、亲水性、电荷、大小和其它性质显示的。
x.载体
如本文中所用,″载体″意指包含复制起始位点的核酸序列。载体可以是载体、噬菌体、细菌人工染色体或酵母人工染色体。载体可以是DNA或RNA载体。载体可以自主复制的染色体外载体,优选地为DNA质粒。
2.流感病毒抗原
本文中提供的是能够在引发哺乳动物中的抗一个或多个流感病毒血清型的免疫反应的抗原。所述抗原能够引发哺乳动物中的抗一个或多个流感病毒血清型的免疫反应,包括抗一个或多个大范围流行病的病毒株,例如2009H1N1猪源的流感病毒。所述抗原能够引发哺乳动物中的抗一个或多个流感病毒血清型的免疫反应,包括抗猪来源的人流感病毒的一个或多个病毒株。抗原可包含表位,所述表位可使它们成为可针对其诱导抗流感病毒的免疫反应的特别有效的免疫原。
抗原可包括全长翻译产物HA0、亚基HA1、亚基HA2、其变体、其片段或其组合。流感病毒血凝素抗原可以是来源于甲型流感病毒血清型H1的多个病毒株的共有序列,来源于甲型流感病毒血清型H2的多个病毒株的共有序列,包含来源于不同组的甲型流感病毒血清型H1的多个病毒株的两个不同共有序列的部分的杂交体序列或来源于乙型流感病毒的多个病毒株的共有序列。流感病毒血凝素抗原可来自乙型流感病毒。抗原可包含至少一个抗原性表位,所述表位对于可针对其诱导免疫反应的特定流感病毒免疫原是有效的。抗原可提供存在于完整流感病毒中的免疫原性位置和表位的完全库。抗原可以是共有血凝素抗原序列,所述序列可来源于来自一个血清型的多个甲型流感病毒株例如血清型H1或血清型H2的多个甲型流感病毒株的血凝素抗原序列。抗原可以是杂交共有血凝素抗原序列,其可通过组合两个不同的共有血凝素抗原序列或其部分衍生而来。两个不同共有血凝素抗原序列的每一个可来源于不同组的一个血清型的多个甲型流感病毒株例如血清型H1的多个甲型流感病毒株。抗原可以是可来源于来自多个乙型流感病毒株的血凝素抗原序列的共有血凝素抗原序列。
共有血凝素抗原可以是包含SEQ ID NO:2(共有H1氨基酸序列)的蛋白质,其中氨基酸1-343相应于前体HA0共有H1氨基酸序列的HA1亚基并且氨基酸344-566相应于HA0共有H1氨基酸序列的HA2亚基。共有血凝素抗原可以是包含SEQ ID NO:7(共有H2氨基酸序列)的蛋白质。共有血凝素抗原可以是合成杂交共有H1序列,其包含各自来源于彼此不同的组的序列的两个不同共有H1序列。作为合成杂交共有H1蛋白的共有HA抗原的实例是包含SEQ IDNO:10(U2氨基酸序列)的蛋白质。共有血凝素抗原可以是来源于乙型流感病毒株的血凝素序列的共有血凝素蛋白,例如包含SEQ ID NO:14(共有BHA氨基酸序列)的蛋白质。
共有血凝素抗原还可包含一种或多种额外的氨基酸序列元件。共有血凝素抗原还可在其N末端上包含IgE或IgG的前导氨基酸序列。IgE前导氨基酸序列可以是SEQ ID NO:17。共有血凝素抗原还可包含作为可通过容易获得的抗体检测的独特免疫原性表位的免疫原性标签。这样的免疫原性标签的实例为9个氨基酸的流感病毒HA标签,其可连接于共有血凝素的C端。HA标签氨基酸序列可以是SEQ ID NO:18。在一些实施方案中,共有血凝素抗原还可在其N末端上包含IgE或IgG前导氨基酸序列并且其C末端上包含HA标签。
共有血凝素抗原可以是由共有流感病毒氨基酸序列或其片段和变体组成的共有血凝素蛋白。共有血凝素抗原可以是包含非-流感病毒蛋白质序列和流感病毒蛋白质序列或其片段和变体的共有血凝素蛋白。
共有H1蛋白的实例包括可由共有H1氨基酸序列(SEQ ID NO:2)组成的所述蛋白质或还包含额外元件例如IgE前导序列或HA标签或IgE前导序列和HA标签两者的所述蛋白质。包括IgE前导序列和HA标签的共有H1蛋白的实例为SEQ ID NO:4,其包含在其N末端连接于IgE前导氨基酸序列(SEQ ID NO:17)并且在其C末端连接于HA标签(SEQ ID NO:18)的共有H1氨基酸编码序列(SEQ ID NO:2)。
共有H2蛋白的实例包括可由共有H2氨基酸序列(SEQ ID NO:7)组成的所述蛋白质或还包含IgE前导序列或HA标签或IgE前导序列和HA标签的所述蛋白质。
杂交共有H1蛋白的实例包括可由共有U2氨基酸序列(SEQ ID NO:10)组成的所述蛋白质或还包含IgE前导序列、HA标签或IgE前导序列和HA标签的所述蛋白质。共有U2蛋白的实例为SEQ ID NO:12,其包含在其N末端连接于IgE前导氨基酸序列(SEQ ID NO:17)和在其C末端连接于HA标签(SEQ ID NO:18)的共有U2氨基酸序列(SEQ ID NO:10)。
杂交共有B型流感病毒凝血素蛋白的实例包括可由共有BHA氨基酸序列(SEQ IDNO:14)组成的所述蛋白质,或其可包含IgE前导序列或HA标签或IgE前导序列和HA标签。共有BHA蛋白的实例为SEQ ID NO:16,其包含在其N末端连接于IgE前导氨基酸序列(SEQ IDNO:17)和在其C末端连接于HA标签(SEQ ID NO:18)的共有BHA氨基酸序列(SEQ ID NO:14)。
共有血凝素蛋白可由共有血凝素核酸、其变体或片段编码。与可以是来源于来自不同株和变体的多个不同的血凝素序列的共有序列的共有血凝素蛋白不同,共有血凝素核酸是指编码共有蛋白质序列的核酸序列,并且所使用的编码序列可与用于编码所述共有血凝素蛋白序列所源自的多个不同血凝素序列中特定氨基酸序列的编码序列不同。共有核酸序列可以进行密码最优化和/或RNA最优化。共有血凝素核酸序列可在5’非翻译区中包含Kozak’s序列。共有血凝素核酸序列可包含编码前导序列的核酸序列。N末端前导序列的编码序列为血凝素编码序列的5’。N末端前导序列可促进分泌。N前导序列可以是IgE前导序列或IgG前导序列。共有血凝素核酸序列可包含编码免疫原性标签的核酸序列。免疫原性标签可在蛋白质的C端上并且编码其的序列为HA编码序列的3’。免疫原性标签提供了可容易获得针对其的抗体的独特表位,以便此类抗体可用于检测和确认蛋白质的表达的测定。免疫原性标签可以是蛋白质的C端上的H标签。
共有血凝素核酸可具有编码包含SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:10或SEQID NO:14的氨基酸序列的多核苷酸序列。编码SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:10或SEQ ID NO:14的共有血凝素核酸可以分别为SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:9或SEQ ID NO:13。共有血凝素核酸还可包含编码IgE前导氨基酸序列的多核苷酸或编码HA标签氨基酸序列的多核苷酸或两者。SEQ ID NO:17为IgE前导多肽序列。SEQ ID NO:18为HA标签多肽序列。还包含编码IgE前导序列和HA标签的多核苷酸序列的血凝素共有核酸的实例包括核酸分子,所述核酸分子编码包括SEQ ID NO:4、SEQ ID NO:12或SEQ ID NO:16的氨基酸序列的蛋白质。编码SEQ ID NO:4、SEQ ID NO:12或SEQ ID NO:16的共有血凝素核酸可以分别为SEQ ID NO:3、SEQ ID NO:11或SEQ ID NO:15。
3.基因构建体和质粒
本文中提供的是可包含编码血凝素抗原的核酸序列的基因构建体。基因构建体可以以包含编码血凝素抗原的核酸的功能性染色体外分子的形式存在于细胞中。包含编码血凝素抗原的核酸的基因构建体可以是包含着丝粒、端粒的线性微型染色体或质粒或粘粒。
基因构建体还可以重组病毒载体(包括重组腺病毒、重组腺病毒相关病毒和重组牛痘)的基因组的部分。基因构建体可以是存在于细胞中的减毒活微生物的遗传物质或重组微生物载体的部分。
基因构建体可包含用于血凝素核酸的基因表达的调控元件。调控元件可以是启动子、增强子、起始密码子、终止密码子或多腺苷酸化信号。
组合物可包含第一核酸序列(其编码选自:甲型流感病毒共有血凝素H1抗原、甲型流感病毒共有血凝素H2抗原、甲型流感病毒共有血凝素U2抗原和乙型流感病毒共有血凝素蛋白BHA中的一种或多种的血凝素共有抗原),并且还可包含一种或多种额外的核酸序列(所述核酸序列编码选自:甲型流感病毒血凝素蛋白H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、甲型流感病毒神经氨酸酶N1、N2、N3、N4、N5、N6、N7、N8、N9、乙型流感病毒血凝素(BHA)和乙型流感病毒神经氨酸酶(BNA)中的一种或多种蛋白质)。第一和额外的核酸序列可存在于相同核酸分子或不同核酸分子上。第一和额外的核酸序列可处于在人细胞中起作用的调控元件的控制之下。额外的编码序列可编码一种或多种来自一个或多个流感病毒株的H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9、BHA和BNA,或为来源于具有相同血清型的多个病毒株的共有序列,或为包括来自两种或更多种共有序列的序列的杂交体。
核酸序列可组成可以是载体的基因构建体。载体可以能够在哺乳动物的细胞中以有效地引发哺乳动物的免疫反应的量表达共有血凝素抗原。载体可以是重组的。载体可包含编码共有血凝素抗原的异源核酸。载体可以是质粒。载体对于用编码共有血凝素抗原的核酸转染细胞是有用的,将所述转染的宿主细胞在其中共有血凝素抗原的表达发生的条件下进行培养和维持。
载体包含编码共有血凝素抗原的异源核酸并且还可包含起始密码子(其可位于共有血凝素编码序列的上游)和终止密码子(其可位于共有血凝素编码序列的下游)。起始和终止密码子可以与共有血凝素编码序列存在于读框中。载体还可包含有效地连接于共有血凝素编码序列的启动子。有效地连接于共有血凝素编码序列的启动子可以是来自猴病毒40(SV40)的启动子、小鼠乳房肿瘤病毒(MMTV)启动子、人免疫缺陷病毒(HIV)启动子例如牛免疫缺陷病毒(BIV)长末端重复(LTR)启动子、莫洛尼病毒启动子、禽类自血病病毒(ALV)启动子、巨细胞病毒(CMV)启动子例如CMV立即早期启动子、EB病毒(Epstein Barr virus)(EBV)启动子或Rous肉瘤病毒(RSV)启动子。启动子还可以是来自人基因例如人肌动蛋白、人肌球蛋白、人血红蛋白、人肌肉肌酸或人金属硫蛋白的启动子。启动子还可以是组织特异性启动子,例如肌肉或皮肤特异性启动子,天然或合成的。此类启动子的实例描述于美国专利申请公布no.US20040175727(将其内容通过引用整体并入本文)中。
载体还可包含多腺苷酸化信号,其可位于HA编码序列的下游。多腺苷酸化信号可以为SV40多原苷酸信号、LTR多腺苷酸信号、牛生长激素(bGH)多腺苷酸化信号或人β-珠蛋白多腺苷酸化信号。SV40多腺苷酸化信号可以是来自pCEP4载体(茵维特罗根公司,圣地亚哥市,加利福尼亚州)的多腺苷酸化信号。
载体还可在共有血凝素编码序列的上游包含增强子。增强子可以是DNA表达所必需的。增强子可以是人肌动蛋白、人肌球蛋白、人血红蛋白、人肌肉肌酸或病毒增强子例如来自CMV、HA、RSV或EBV的增强子。多腺苷酸功能增强描述于美国专利No.5,593,972、5,962,428和WO 94/016737,将所述专利和申请的每一个的内容通过引用整体并入本文。
载体还可包含哺乳动物复制起始位点以在染色体外维持载体并且在细胞中产生多个拷贝的载体。载体可以是来自茵维特罗根公司(圣地亚哥市,加利福尼亚州)的pVAX1(图1)、pCEP4或pREP4,其可包含EB病毒复制起始位点和核抗原EBNA-1编码区,其可产生高拷贝附加型复制而无整合。载体可以是具有变化例如参考上文中附图简述部分的图1的段落中描述的变化的pVAX1。载体的主链可以是pAV0242。载体可以是复制缺陷型腺病毒5型(Ad5)载体。
载体还可包含调控序列,其可以非常适用于在已将载体施入其中的哺乳动物或人细胞中进行基因表达。共有血凝素编码序列可包含密码子,所述密码子可允许编码序列在宿主细胞中的更高效转录。
载体可以是pSE420(茵维特罗根公司,圣地亚哥市,加利福尼亚州),其可用于在大肠杆菌(Escherichia coli)(E.coli)中产生蛋白质。载体还可以是pYES2(茵维特罗根),圣地亚哥市,加利福尼亚州),其可用于在酵母的酿酒酵母(Saccharomyces cerevisiae)菌株中产生蛋白质。载体还可具有MAXBACTM完全杆状病毒表达系统(茵维特罗根,圣地亚哥市,加利福尼亚州),其可用于在昆虫细胞中产生蛋白质。载体还可以是pcDNA I或pcDNA3(茵维特罗根,圣地亚哥市,加利福尼亚州),其可用于在哺乳动物细胞例如中国仓鼠卵巢(CHO)细胞中产生蛋白质。载体可以是利用常规技术和可容易获得的起始材料(包括Sambrook等人,分子克隆和实验室手册,第2版,冷泉港(Cold Spring Harbor)(1989)(将其通过引用并入本文))来产生蛋白质的表达载体或系统。
载体可以是pGX2009或pGX2006,其可用于表达共有血凝素抗原。载体pGX2009(4739bp,图2;SEQ ID NO:5)为经修饰的pVAX1质粒,其具有编码共有H1蛋白(由SEQ ID NO:3编码的氨基酸SEQ ID NO:4)酸的核酸序列,所述共有H1蛋白包含连接于共有H1氨基酸序列(由SEQ ID NO:1编码的氨基酸SEQ ID NO:2)的IgE前导序列(由SEQ ID NO:11编码的氨基酸SEQ ID NO:12)。载体pGX2006(4628bp;图3,SEQ ID NO:8)为pVAX1质粒,其具有编码共有H2蛋白(由SEQ ID NO:6编码的氨基酸SEQ ID NO:7)的核酸序列的质粒。
本文中公开的包括共有血凝素编码序列的基因构建体和组分可用于表达其它流感病毒蛋白质例如甲型流感病毒H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9、乙型流感病毒血凝素或神经氨酸酶蛋白,由此包括替代共有血凝素编码序列的甲型流感病毒蛋白H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9、乙型流感病毒血凝素或神经氨酸酶蛋白的编码序列。
4.药物组合物
本文中提供的是根据本发明的药物组合物,其包含约1纳克至约10mg的DNA。在一些实施方案中,根据本发明的药物组合物包含:1)至少10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95或100纳克,或至少1、5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、405、410、415、420、425、430、435、440、445、450、455、460、465、470、475、480、485、490、495、500、605、610、615、620、625、630、635、640、645、650、655、660、665、670、675、680、685、690、695、700、705、710、715、720、725、730、735、740、745、750、755、760、765、770、775、780、785、790、795、800、805、810、815、820、825、830、835、840、845、850、855、860、865、870、875、880、885、890、895.900、905、910、915、920、925、930、935、940、945、950、955、960、965、970、975、980、985、990、995或1000微克,或至少1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5或10mg或更多;和2)达到并且包括15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95或100纳克,或达到并且包括1、5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95,100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、405、410、415、420、425、430、435、440、445、450、455、460、465、470、475、480、485、490、495、500、605、610、615、620、625、630、635、640、645、650、655、660、665、670、675、680、685、690、695、700、705、710、715、720、725、730、735、740、745、750、755、760、765、770、775、780、785、790、795、800、805、810、815、820、825、830、835、840、845、850、855、860、865、870、875、880、885、890、895. 900、905、910、915、920、925、930、935、940、945、950、955、960、965、970、975、980、985、990、995或1000微克,或达到并且包括1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5或10mg。在一些实施方案中,根据本发明的药物组合物包含约5纳克至约10mg的DNA。在一些实施方案中,根据本发明的药物组合物包含约25纳克至约5mg的DNA。在一些实施方案中,药物组合物包含约50纳克至约1mg的DNA。在一些实施方案中,药物组合物包含约0.1至约500微克的DNA。在一些实施方案中,药物组合物包含约1至约350微克的DNA。在一些实施方案中,药物组合物包含约5至约250微克的DNA。在一些实施方案中,药物组合物包含约10至约200微克的DNA。在一些实施方案中,药物组合物包含约15至约150微克的DNA。在一些实施方案中,药物组合物包含约20至约100微克的DNA。在一些实施方案中,药物组合物包含约25至约75微克的DNA。在一些实施方案中,药物组合物包含约30至约50微克的DNA。在一些实施方案中,药物组合物包含约35至约40微克的DNA。在一些实施方案中,药物组合物包含约100至约200微克DNA。在一些实施方案中,药物组合物包含约10微克至约100微克的DNA。在一些实施方案中,药物组合物包含约20微克至约80微克的DNA。在一些实施方案中,药物组合物包含约25微克至约60微克的DNA。在一些实施方案中,药物组合物包含约30纳克至约50微克的DNA。在一些实施方案中,药物组合物包含约35纳克至约45微克的DNA。在一些优选实施方案中,药物组合物包含约0.1至约500微克的DNA。在一些优选实施方案中,药物组合物包含约1至约350微克的DNA。在一些优选实施方案中,药物组合物包含约25至约250微克的DNA。在一些优选实施方案中,药物组合物包含约100至约200微克DNA.
根据待使用的施用模式配制根据本发明的药物组合物。在其中药物组合物是可注射的药物组合物的情况下,它们是无菌、无热原和无颗粒的。优选使用等渗制剂。通常,用于等渗性的添加剂可包括氯化钠、葡萄糖、甘露糖、山梨醇和乳糖。在一些情况下,等渗溶液例如磷酸缓冲盐溶液是优选的。稳定剂包括明胶和白蛋白。在一些实施方案中,将血管添加剂添加至制剂。
优选药物组合物是疫苗,更优选是DNA疫苗。
本文中提供的是能够在哺乳动物中产生抗一种或多种流感病毒血清型的免疫反应。疫苗可包含上述的基因构建体。疫苗可包含多种载体,所述载体各自针对一种或多种甲型流感病毒血清型例如H1-H16乙型流感病毒血凝素或其组合。疫苗可包含编码一种或多种共有血凝素抗原的一个或多个核酸序列。当疫苗包含超过一种共有血凝素核酸序列时,所有此类序列可存在于单个核酸分子上或每一种这样的序列可存在于不同的核酸分子上。或者,包含超过一个的共有血凝素核酸序列的疫苗可包含具有单个共有血凝素核酸序列的核酸分子和具有超过一个的共有血凝素核酸序列的核酸分子。此外,包含一个或多个共有血凝素核酸序列的疫苗还可包含一种或多种选自H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9和乙型流感病毒神经氨酸酶的蛋白质的编码序列。
在一些实施方案中,疫苗可包含蛋白质。一些疫苗可包含一种或多种共有血凝素抗原例如H1、H2、U2和BHA。疫苗可包含一种或多种选自HI、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9和乙型流感病毒神经氨酸酶的其它蛋白质。疫苗可包含与一种或多种其它蛋白质组合的一种或多种共有血凝素抗原,所述其它蛋白质选自H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9、乙型流感病毒血凝素和神经氨酸酶。
疫苗可以是DNA疫苗。DNA疫苗可包含多个相同或不同的包含一个或多个共有血凝素核酸序列的质粒。DNA疫苗可包含一个或多个编码一种或多种共有血凝素抗原的核酸序列。当DNA疫苗包含超过一个的共有血凝素核酸序列时,所有此类序列可存在于单个质粒上,或每一个此类序列可存在于不同质粒上,或一些质粒可包含单个共有血凝素核酸序列,然而其它质粒具有超过一个的共有血凝素核酸序列。此外,DNA疫苗还可包含一种或多种蛋白质的一个或多个共有编码序列,所述蛋白质选自甲型流感病毒H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9、乙型流感病毒血凝素和神经氨酸酶。此类额外的编码序列彼此可存在于相同或不同的质粒上和来自包含一种或多种共有血凝素核酸序列的质粒。
在一些实施方案中,疫苗可包含编码与流感病毒抗原组合的流感病毒抗原的核酸序列。在一些实施方案中,核酸序列编码一种或多种共有血凝素抗原例如H1、H2、U2和BHA。在一些实施方案中,核酸序列编码一种或多种选自甲型流感病毒H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9、乙型流感病毒血凝素和神经氨酸酶的蛋白质。在一些实施方案中,疫苗包含一种或多种共有血凝素抗原例如H1、H2、U2和BHA。在一些实施方案中,疫苗包含一种或多种选自甲型流感病毒H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9、乙型流感病毒血凝素和神经氨酸酶的其它蛋白质。
在一些实施方案中,疫苗包含3个或更多个共有血凝素核酸序列(包括编码H1、H2、U2和BHA中的一种或多种的核酸序列)的组合。在一些实施方案中,疫苗包含3个或更多个血凝素核酸序列(包括编码共有U2、共有BHA和H3血凝素的核酸序列)的组合。在一些实施方案中,疫苗包含3个或更多个血凝素核酸序列(包括编码共有BHA、H1血凝素和H3血凝素的核酸序列)的组合。在一些实施方案中,疫苗包含一个或更多个编码美国系列No.12/375,518(将所述美国系列申请通过引用并入本文)和/或美国系列No.12/269,824(将所述美国系列申请通过引用并入本文)中公开的一种或多种流感病毒抗原的核酸序列。在一些实施方案中,疫苗包含编码疫苗包含编码SEQ ID NO:20(其为美国系列No.12/375,518中公开的H1血凝素,在该美国系列申请中分别为SEQ ID NO:36和SEQ ID NO:37)的核酸序列SEQ ID NO:19和/或编码SEQ ID NO:22(其为在美国系列No.12/269,824中公开的H1血凝素,在该美国系列申请中分别为SEQ ID NO:9和SEQ ID NO:10)的核酸序列SEQ ID NO:21。在一些实施方案中,疫苗包含编码SEQ ID NO:24(其为美国系列No.12/269,824中公开的H3血凝素,在该美国系列申请中分别为SEQ ID NO:11和SEQ ID NO:12)的核酸序列SEQ ID NO:23。
在一些实施方案中,疫苗包含3种或更多种共有血凝素蛋白(包括H1、H2、U2和BHA的一种或多种)的组合。在一些实施方案中,疫苗包含3种或更多种血凝素蛋白(包括共有U2、共有BHA和H3血凝素)的组合。在一些实施方案中,疫苗包含3种或更多种血凝素蛋白(包括共有BHA、H1血凝素和H3血凝素)的组合。在一些实施方案中,疫苗包含1种或更多种来自美国系列No.12/375,518和/或美国系列No.12/269,824的抗原。在一些实施方案中,疫苗包含SEQ ID NO:20和/或SEQ ID NO:22和/或SEQ ID NO:24。
在一些实施方案中,疫苗包含1)共有血凝素U2蛋白和/或编码所述共有血凝素U2蛋白的核酸序列,2)共有血凝素BHA蛋白和/或编码所述共有血凝素BHA蛋白的核酸序列,和3)SEQ ID NO:24中公开的血凝素H3蛋白的组合。
在一些实施方案中,疫苗包含1)共有血凝素BHA蛋白和/或编码所述共有血凝素BHA蛋白的核酸序列,2)具有SEQ ID NO:20和/或SEQ ID NO:22的血凝素H1蛋白和/或血凝素H1蛋白编码核酸序列SEQ ID NO19和/或SEQ ID NO:21,和3)具有SEQ ID NO:24的血凝素H3蛋白和/或编码在其中的核酸序列SEQ ID NO:23的血凝素H3蛋白的组合。
DNA疫苗公开于美国专利No.5,593,972、5,739,118、5,817,637、5,830,876、5,962,428、5,981,505、5,580,859、5,703,055和5,676,594(将所述专利通过引用整体并入本文)中。DNA疫苗还可包含抑制其整合入染色体的元件或试剂。疫苗可以是血凝素抗原的RNA。可将RNA疫苗引入细胞。
疫苗可以是包含上述的基因构建体或抗原的重组疫苗。疫苗还可包含以一种或多种蛋白质亚基的形式存在的一种或多种共有血凝素抗原,包含一种或多种共有血凝素抗原的一种或多种杀死的流感病毒颗粒或包含一种或多种共有血凝素抗原的一种或多种减毒的流感病毒颗粒。减毒的疫苗可以是减毒的活疫苗、杀死的疫苗和使用重组载体递送编码一种或多种共有血凝素抗原的外源基因的疫苗,以及亚基和糖蛋白疫苗。减毒的活疫苗、使用重组载体递送外来抗原的疫苗、亚基疫苗和糖蛋白疫苗描述于美国专利No.:4,510,245;4,797,368;4,722,848;4,790,987;4,920,209;5,017,487;5,077,044;5,110,587;5,112,749;5,174,993;5,223,424;5,225,336;5,240,703;5,242,829;5,294,441;5,294,548;5,310,668;5,387,744;5,389,368;5,424,065;5,451,499;5,453,364;5,462,734;5,470,734;5,474,935;5,482,713;5,591,439;5,643,579;5,650,309;5,698,202;5,955,088;6,034,298;6,042,836;6,156,319和6,589,529(将所述专利各自通过引用并入本文)中。
疫苗可包含针对来自世界特定地区例如亚洲的甲型流感病毒血清型的载体和/或蛋白质。疫苗还可抗目前感染人的猪来源的甲型流感病毒血清型。疫苗可包含针对来自世界特定地区的乙型流感病毒的载体和/或蛋白质。疫苗还可抗感染人的乙型流感病毒。疫苗可包含针对甲型和/或乙型流感病毒的一个或多个病毒株的一种或多种载体和/或一种或多种蛋白质。
提供的疫苗还可用于诱导免疫反应,包括治疗性或预防性免疫反应。可产生针对共有血凝素抗原,并且还广泛地跨多个亚型的流感病毒的抗体和/或杀伤T细胞。可分离此类抗体和细胞。
疫苗还可包含药学上可接受的赋形剂。药学上可接受的赋形剂可以是功能性分子如媒介物、佐剂、载体或稀释剂。药学上可接受的赋形剂可以是转染促进剂(transfectionfacilitating agent),其可包括表面活性剂例如免疫刺激复合物(ISCOMS)、弗氏不完全佐剂、LPS类似物(包括单磷酰脂质A、胞壁酰肽、醌类似物、囊泡例如角鲨烯和角鲨烯、透明质酸)、脂质、脂质体、钙离子、病毒蛋白质、聚阴离子、聚阳离子或纳米颗粒或其它已知的转染促进剂。
转染促进剂为聚阴离子、聚阳离子,包括聚-L-谷氨酸盐(LGS)或脂质。转染促进剂为聚-L-谷氨酸,和更优选,聚-L-谷氨酸以低于6mg/ml的浓度存在于疫苗中。转染促进剂还可包括表面活性剂例如免疫刺激复合物(ISCOMS)、弗氏不完全佐剂、LPS类似物(包括单磷酰脂质A、胞壁酰肽、醌类似物及囊泡例如角鲨烯和角鲨烯和透明质酸),还可将所转染促进剂与基因构建体结合施用。在一些实施方案中,DNA载体疫苗还可包括转染促进剂例如脂质、脂质体(包括卵磷脂脂质体或本领域内已知的其它脂质体,如DNA-脂质体混合物(参见例如W09324640))、钙离子、病毒蛋白质、聚阴离子、聚阳离子或纳米颗粒或其它已知的转染促进剂。优选,转染促进剂为聚阴离子、聚阳离子(包括聚-L-谷氨酸盐(LGS)或脂质)。疫苗中转染剂的浓度低于4mg/ml,低于2mg/ml,低于1mg/ml,低于0.750mg/ml,低于0.500mg/ml,低于0.250mg/ml,低于0.100mg/ml,低于0.050mg/ml或低于0.010mg/ml。
药学上可接受的赋形剂可以是佐剂。佐剂可以是在替代性质粒中表达的或以蛋白质形式与疫苗中的上述质粒组合地递送的其它基因。佐剂可选自:α-干扰素(IFN-α)、β-干扰素(IFN-β)、γ-干扰素、血小板衍生生长因子PDGF)、TNFα、TNFβ、GM-CSF、表皮生长因子(EGF)、皮肤T细胞吸引趋化因子(CTACK)、上皮胸腺表达趋化因子(TECK)、粘膜相关上皮趋化因子(MEC)、IL-12、IL-15、MHC、CD80、CD86(包括缺失信号序列和任选地包括来自IgE的信号肽的IL-15)。佐剂可以是IL-12、IL-15、IL-28、CTACK、TECK、血小板衍生生长因子(PDGF)、TNFα、TNFβ、GM-CSF、表皮生长因子(EGF)、IL-1、IL-2、IL-4、IL-5、IL-6、IL-10、IL-12、IL-18或其组合。
可以是有用的佐剂的其它基因包括编码如下物质的基因:MCP-1、MIP-1a、MIP-1p、IL-8、RANTES、L-选择蛋白、P-选择蛋白、E-选择蛋白、CD34、GlyCAM-1、MadCAM-1、LFA-1、VLA-1、Mac-1、p150.95、PECAM、ICAM-1、ICAM-2、ICAM-3、CD2、LFA-3、M-CSF、G-CSF、IL-4、IL-18的突变体形式、CD40、CD40L、血管生长因子、成纤维细胞生长因子、IL-7、神经生长因子、血管内皮生长因子、Fas、TNF受体、Flt、Apo-1、p55、WSL-1、DR3、TRAMP、Apo-3、AIR、LARD、NGRF、DR4、DR5、KILLER、TRAIL-R2、TRICK2、DR6、天胱蛋白酶ICE、Fos、c-jun、Sp-1、Ap-1、Ap-2、p38、p65Rel、MyD88、IRAK、TRAF6、IkB、灭活的NIK、SAP K、SAP-1、JNK、干扰素反应基因、NFkB、Bax、TRAIL、TRAILrec、TRAILrecDRC5、TRAIL-R3、TRAIL-R4、RANK、RANK LIGAND、Ox40、Ox40LIGAND、NKG2D、MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、TAP1、TAP2及其功能片段。
疫苗还可包含1994年4月1日提交的美国系列No.021,579(将其通过引用并入本文)中描述的基因疫苗促进剂。
5.递送的方法
本文中提供的是用于递送药物制剂(优选疫苗),以用来提供包含表位的血凝素抗原的基因构建体和蛋白质的方法,所述表位使得所述血凝素抗原成为可对其诱导针对流感病毒感染的免疫反应的特别有效的免疫原。可提供递送疫苗或接种的方法以诱导治疗和/或预防性免疫反应。接种方法可在哺乳动物中产生抗多个流感病毒亚型(包括H1N1血清型例如2009猪源的H1N1)或其它季节性和/或大范围流行病的变种的免疫反应。可将疫苗递送至个体以调节哺乳动物的免疫系统的活性和增强免疫反应。疫苗的递送可以转染以核酸分子的形式在细胞中表达并且被递送至细胞表面的HA抗原,免疫系统可识别所述HA抗原并且诱导细胞、体液或细胞及体液反应。疫苗的递送可用于通过给哺乳动物施用本文中论述的疫苗来诱导或引发哺乳动物中的抗多种流感病毒的免疫反应。
在将疫苗递送至哺乳动物,从而将载体递送入哺乳动物的细胞后,转染的细胞将表达和分泌相应的流感病毒蛋白质,包括共有抗原中的至少一种,优选地H1、H2、U2和BHA。此类分泌的蛋白质或合成抗原将被免疫系统识别为外来物,其将引发免疫反应,包括:针对抗原产生的抗体和特异性针对抗原的T细胞反应。在一些实例中,用本文中论述的疫苗接种的哺乳动物将有已接触抗原的免疫系统并且当用流感病毒株攻击时,已接触抗原的免疫系统将使得能够快速消除随后的流感病毒,无论是通过体液、细胞还是通过两者。可将疫苗递送至个体以调节个体的免疫系统的活性,从而增强免疫反应。
可以DNA疫苗的形式递送疫苗,递送DNA疫苗的方法描述于美国专利No.4,945,050和5,036,006(将所述两个专利通过引用整体并入本文)中。
可给哺乳动物施用疫苗以引发哺乳动物中的免疫反应。哺乳动物可以为人、非人灵长类动物、牛、猪、绵羊、山羊、羚羊、野牛、水牛、牛科动物、鹿、刺猬、象、骆马、羊驼、小鼠、大鼠或鸡、并且优选地为人、牛、猪或鸡。
a.联合治疗
可将药物组合物(优选疫苗)与一种或多种其它流感病毒蛋白质或编码甲型流感病毒H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15、H16、N1、N2、N3、N4、N5、N6、N7、N8、N9、乙型流感病毒血凝素和神经氨酸酶的基因联合施用。可将疫苗与蛋白质或编码佐剂的基因联合施用,所述佐剂包括:α-干扰素(IFN-α)、β-干扰素(IFN-β)、γ-干扰素、IL-12、IL-15、IL-28、CTACK、TECK、血小板衍生生长因子(PDGF)、TNFα、TNFβ、GM-CSF、表皮生长因子(EGF)、IL-1、IL-2、IL-4、IL-5、IL-6、IL-10、IL-12、IL-18、MCP-1、MIP-1a、MIP-1p、IL-8、RANTES、L-选择蛋白、P-选择蛋白、E-选择蛋白、CD34、GlyCAM-1、MadCAM-1、LFA-1、VLA-1、Mac-1、p150.95、PECAM、ICAM-1、ICAM-2、ICAM-3、CD2、LFA-3、M-CSF、G-CSF、IL-4、IL-18的突变体形式、CD40、CD40L、血管生长因子、成纤维细胞生长因子、IL-7、神经生长因子、血管内皮生长因子、Fas、TNF受体、Flt、Apo-1、p55、WSL-1、DR3、TRAMP、Apo-3、AIR、LARD、NGRF、DR4、DR5、KILLER、TRAIL-R2、TRICK2、DR6、天胱蛋白酶ICE、Fos、c-jun、Sp-1、Ap-1、Ap-2、p38、p65Re1、MyD88、IRAK、TRAF6、IkB、灭活的NIK、SAP K、SAP-1、JNK、干扰素反应基因、NFkB、Bax、TRAIL、TRAILrec、TRAILrecDRC5、TRAIL-R3、TRAIL-R4、RANK、RANK LIGAND、Ox40、Ox40LIGAND、NKG2D、MICA、MICB、NKG2A、NKG2B、NKG2C、NKG2E、NKG2F、TAP1或TAP2或其功能片段。
b.施用途径
可通过不同途径包括口服、胃肠外、舌下、经皮肤、经直肠、经粘膜、局部、通过吸入、通过颊部施用、胸膜内、静脉内、动脉内、腹膜内、皮下、肌内、鼻内、鞘内以及关节内或其组合施用疫苗。对于兽医学使用,可按照正常的兽医学操作将组合物作为适当地可接受的制剂施用。兽医可容易地确定最适合于特定动物的给药方案和施用途径。可利用常规注射器、无针注射设备、″微粒轰击基因枪(microprojectile bombardment gone gun)″或其它物理方法例如电穿孔(“EP”)、“水动力学法”或超声施用疫苗。
可利用几种公知的技术包括DNA注射(也称为DNA接种)(利用和不利用体内电穿孔)、脂质体介导的、纳米颗粒促进的、重组载体例如重组腺病毒、重组腺病毒相关病毒和重组牛痘将疫苗的载体递送至哺乳动物。可通过DNA注射连同体内电穿孔一起递送HA抗原。
c.电穿孔
可使用电穿孔设备来实现通过疫苗的质粒的电穿孔施用疫苗,所述电穿孔设备可被构造成将有效地引起可逆的孔在细胞膜上形成的能量脉冲递送至哺乳动物的期望的组织,并且优选地能量脉冲是与由用户预设的电流输入相似的恒定电流。电穿孔设备可包括电穿孔组件和电极部件或操作部件(handle assembly)。电穿孔组件可包括和整合电穿孔设备的各种元件中的一个或多个元件,包括:控制器、电流波形发生器、阻抗检测器、波形记录器、输入元件、状态报告元件、通信端口、存储器组件(memory component)、电源和电源开关。电穿孔可使用体内电穿孔设备例如EP系统(VGX制药公司(VGXPharmaceuticals),布鲁贝尔市(Blue Bell),宾夕法尼亚州)或Elgen电穿孔仪(electroporator)(Genetronics,圣地亚哥市,加利福尼亚州)来实现以有利于质粒对细胞的转染。
电穿孔组件可用作电穿孔设备的一个元件,并且其它元件是与电穿孔组件是与与穿孔组件连通的独立元件(或组件)。电穿孔组件可用作电穿孔设备的超过一个的元件,其可与电穿孔设备的与电穿孔组件分开的其它元件连通。作为一个电机或机器设备的部件存在的电穿孔设备的元件可以不受限制,因为所述元件可用作一个设备或用作彼此连通的独立元件。电穿孔组件可以能够将递送在期望的组织中产生恒定电流并且包括反馈机制的能量脉冲。电极部件可包括在空间排列中具有多个电极的电极阵列,其中电极装配件接收来自电穿孔组件的能量脉冲并且将所述能量脉冲通过电极递送至期望的组织。多个电极的至少一个在能量脉冲的递送过程中是中性的并且测量期望组织的阻抗,将阻抗通信传送至电穿孔组件。反馈机制可接收测量的阻抗并且可调整通过电穿孔组件递送的能量脉冲以维持恒定电流。
多个电极可以以分散模式递送能量脉冲。多个电极可通过在编程序列(programmed sequence)下控制电极来以分散模式递送能量脉冲,并且所述编程序列由用户输入至电穿孔组件。编程序列可包括多个按顺序递送的泳冲,其中利用至少两个有源电极(具有一个测量阻抗的中性电极)递送多个脉冲的每一个脉冲,并且其中利用至少两个有源电极(具有一个测量阻抗的中性电极)中的不同电极递送多个脉冲的后续脉冲。
反馈机制可利用硬件或软件来进行。反馈机制可通过模拟闭环电路来进行。反馈每50μs、20μs、10μs或1μs发生一次,但优选实时反馈或瞬时反馈(即,大体上同时,如通过用于测定反应时间的可获得的技术测量的)。中性电极可测量期望的组织中的阻抗和将阻抗通信传送至反馈机制,并且反馈机制对阻抗作出反应并且调节能量脉冲以将恒定电流维持在与预设电流相似的值上。反馈机制可在能量脉冲的递送过程中连续和即时地维持恒定电流。
可帮助本发明的DNA疫苗递送的电穿孔设备和电穿孔方法的实例包括Draghia-Akli等人的美国专利No.7,245,963、由Smith等人提交的美国专利公布2005/0052630(将所述专利和专利公布的公开内容通过引用整体并入本文)中描述的电穿孔设备和方法。可用于帮助DNA疫苗的递送的其它电穿孔设备和电穿孔方法包括2007年10月17日提交的共同未决和共同拥有的美国专利申请系列No.11/874072(其根据35USC 119(e)要求2006年10月17日提交的美国专利申请系列No.60/852,149和2007年10月10日提交的美国专利申请系列No.60/978,982的利益)(将所述申请均通过引用整体并入本文)中提供的电穿孔设备和电穿孔方法。
Draghia-Akli等人的美国专利No.7,245,963描述了标准电极系统及它们用于帮助将生物分子引入身体或植物中的选定组织的细胞的用途。标准电极系统可包括多个针电极;皮下针头;提供从可编程的恒定电流脉冲控制器至多个针电极的导电油墨的电接插件(electrical connector);和电源。操作者可抓住安装在支持结构上的多个针电极,并且将它们牢固地插入身体或植物的选定组织。随后通过皮下针头将生物分子递送入选定组织。激活可编程的恒定电流脉冲控制器,并且将恒定电流电脉冲施加至多个针电极。施加的恒定电流电脉冲帮助将生物分子引入多个电极之间的细胞。将美国专利No.7,245,963的全部内容通过引用并入本文。
由Smith等人提交的美国专利公布2005/0052630描述了可用于有效地帮助将生物分子引入身体或植物的选定组织的细胞内的电穿孔设备。电穿孔设备包括其操作由软件或固件指定的电动设备(″EKD设备″)。EKD设备基于用户控制和脉冲参数的输入在阵列中的电极之间产生一系列可编程的恒定电流脉冲模式,并且允许贮存和获取电流波形数据。电穿孔设备还包括具有针电极的阵列的可替换电极盘、用于注射针头的中央注射通道和可移动的导盘。将美国专利公布2005/0052630的全部内容通过引用并入本文。
可将美国专利No.7,245,963和美国专利公布2005/0052630中描述的电极阵列和方法改造以适用于不仅深入穿透组织例如肌肉,而且还深入穿透其它组织或器官。由于电极阵列的构型的原因,因此也将注射针(递送选择的生物样品)完全插入靶器官,并且在用电极预先界定的区域中垂直于靶组织进行注射。美国专利No.7,245,963和美国专利公布2005/005263中描述的电极优选为20mm长并且为21规。
此外,在一些实施方案中涉及整合电穿孔设备及其用途,存在为下列专利中描述的设备的电穿孔设备:1993年12月28日发布的美国专利5,273,525、2000年8月29日发布的美国专利6,110,161、2001年7月17日发布的6,261,281以及2005年10月25日发布的6,958,060和2005年9月6日发布的美国专利6,939,862。此外,在本文中涉及覆盖2004年2月24日发布的美国专利6,697,669(其涉及使用多种设备的任一设备递送DNA)和2008年2月5日提交的美国专利7,328,064(涉及注射DNA的方法)中提供的主题的专利。将上述专利通过引用整体并入本文。
d.制备疫苗的方法
本文中提供的是用于制备包含本文中论述的DNA疫苗的DNA质粒的方法。在最终亚克隆入哺乳动物表达质粒的步骤后,可使用本领域已知的方法将DNA质粒用于在大规模发酵罐中接种细胞培养物。
可使用已知设备和技术的组合配制或制备与本发明的EP设备一起使用的DNA质粒,但优选使用在2007年5月23日提交的批准的共同未决的美国临时申请美国系列No.60/939,792中描述的最优化质粒制备技术制备它们。在一些实施例中,可以以大于或等于10mg/mL的浓度配制这些研究中使用的DNA质粒。除了美国系列No.60/939792中描述的设备和方案(包括在2007年7月3日发布的批准的专利美国专利No.7,238,522中描述的设备和方案)外,制备技术还包括或整合对于本领域技术人员来说通常是已知的各种设备和方案。通过将上述申请和专利(分别地美国系列No.60/939,792和美国专利No.7,238,522)通过引用整体并入本文。
实施例
还在下列实施例中对本发明进行了举例说明。应当理解,这些实施例虽然表示本发明的优选实施方案,但仅以举例说明的方式给出。根据上文中的论述和这些实施例,本领域技术人员可确定本发明的基本特征,并且在不背离其精神和范围下,可对本发明进行任何改变和变动以使之适合于各种用法和条件。因此,根据上述说明,除了本文中显示和描述的变动外的本发明的各种变动对于本领域技术人员来说是很显然的。此类变动也意欲落在所附权利要求的范围内。
实施例1
pGX2009(pH1HA09)-编码2009H1N1流感病毒(猪流感病毒)血凝素抗原的质粒
pGX2009(H1HA09)的主链为处于巨细胞病毒立即早期(CMV)启动子控制之下的经修饰的表达载体pVAX1(茵维特罗根公司,卡尔斯巴德,加利福尼亚州)。原始pVAX1购自茵维特罗根公司(目录编号V260-20)并且于-20℃下维持。如上文中所指出的,序列分析显示用作pGX2009的主链的pVAX1的序列与可从茵维特罗根公司获得的pVAX1序列之间的差异。差异示于上文中。
质粒pGX2009(也称为pHlHA09)包含编码共有2009H1N1流感病毒(猪流感病毒)血凝素分子的核酸序列。用于产生共有序列的79个基本序列选自流感病毒序列数据库。
编码各种甲型流感病毒血凝素H1蛋白的核苷酸序列以及由所述核苷酸序列编码的氨基酸序列的登录号在GenBank数据库中相应于下列登录号。不在括号中的登录号公开了核苷酸序列和由它们编码的额外列的氨基酸序列。括号内的登录号表示GenBank的蛋白质数据库中的相应氨基酸序列的条目。
登录号如下:GQ323579.1(ACS72657.1)、GQ323564.1(ACS72654.1)、GQ323551.1(ACS72652.1)、GQ323530.1(ACS72651.1)、GQ323520.1(ACS72650.1)、GQ323495.1(ACS72648.1)、GQ323489.1(ACS72647.1)、GQ323486.1(ACS72646.1)、GQ323483.1(ACS72645.1)、GQ323455.1(ACS72641.1)、GQ323451.1(ACS72640.1)、GQ323443.1(ACS72638.1)、GQ293077.1(ACS68822.1)、GQ288372.1(ACS54301.1)、GQ287625.1(ACS54262.1)、GQ287627.1(ACS54263.1)、GQ287623.1(ACS54261.1)、GQ287621.1(ACS54260.1)、GQ286175.1(ACS54258.1)、GQ283488.1(ACS50088.1)、GQ280797.1(ACS45035.1)、GQ280624.1(ACS45017.1)、GQ280121.1(ACS45189.1)、GQ261277.1(ACS34968.1)、GQ253498.1(ACS27787.1)、GQ323470.1(ACS72643.1)、GQ253492.1(ACS27780.1)、FJ981613.1(ACQ55359.1)、FJ971076.1(ACP52565.1)、FJ969540.1(ACP44189.1)、FJ969511.1(ACP44150.1)、FJ969509.1(ACP44147.1)、GQ255900.1(ACS27774.1)、GQ255901.1(ACS27775.1)、FJ966974.1(ACP41953.1)、GQ261275.1(ACS34967.1)、FJ966960.1(ACP41935.1)、FJ966952.1(ACP41926.1)、FJ966082.1(ACP41105.1)、GQ255897.1(ACS27770.1)、CY041645.1(ACS27249.1)、CY041637.1(ACS27239.1)、CY041629(ACS27229.1)、GQ323446.1(ACS72639.1)、CY041597.1(ACS27189.1)、CY041581.1(ACS14726.1)、CY040653.1(ACS14666.1)、CY041573.1(ACS14716.1)、CY041565.1(ACS14706.1)、CY041541.1(ACS14676.1)、GQ258462.1(ACS34667.1)、CY041557.1(ACS14696.1)、CY041549.1(ACS14686.1)、GQ283484.1(ACS50084.1)、GQ283493.1(ACS50095.1)、GQ303340.1(ACS71656.1)、GQ287619.1(ACS54259.1)、GQ267839.1(ACS36632.1)、GQ268003.1(ACS36645.1)、CY041621.1(ACS27219.1)、CY041613.1(ACS27209.1)、CY041605.1(ACS27199.1)、FJ966959.1(ACP41934.1)、FJ966982.1(ACP41963.1)、CY039527.2(ACQ45338.1)、FJ981612.1(ACQ55358.1)、FJ981615.1(ACQ55361.1)、FJ982430.1(ACQ59195.1)、FJ998208.1(ACQ73386.1)、GQ259909.1(ACS34705.1)、GQ261272.1(ACS34966.1)、GQ287621.1(ACS54260.1)、GQ290059.1(ACS66821.1)、GQ323464.1(ACS72642.1)、GQ323473.1(ACS72644.1)、GQ323509.1(ACS72649.1)、GQ323560.1(ACS72653.1)、GQ323574.1(ACS72655.1)和GQ323576.1(ACS72656.1)。从NCBI序列数据库下载氨基酸序列,使用Clustal X产生比对和共有序列。将高效前导序列(IgE前导序列)在读框内融合至起始密码子的上游以帮助表达。为了具有高水平的表达,使该融合基因的密码子使用(codon usage)适合于智人基因的密码子偏性。此外,也进行RNA最优化:避免具有极高(>80%)或极低(<30%)GC含量的区域和顺式作用序列基序例如内部TATA盒、chi位点和核糖进入位点的区域。在Geneart(雷根斯堡市(Regensburg),德国)合成产生完整序列。合成的工程化H1HA09基因在长度上为1818bp(SEQ ID NO:1)并且由Geneart将其在BamHI和XhoI位点克隆入pVAX1(图2)。
实施例2
利用A/Mexico/InDRE4487/2009攻击流感病毒pGX2009免疫的雪貂
使用雪貂(优选的流感模型)进行攻击实验。使用质粒pGX2009免疫雪貂。
动物:4组x 5只动物/组+一个具有4只动物的对照组=总共24只雪貂(雄性)
持续时间:18周(包括攻击)
剂量:2mg质粒
方案概述:将雪貂随机分配至DNA疫苗组中。在研究的第0天、第28天和第56天免疫动物。按照批准的麻醉方案用氯胺酮/咪达唑仑混合物、异氟醚或等同物麻醉动物,利用流感病毒DNA疫苗组合进行IM接种。使用适应性恒定电流电穿孔(EP)设备以0.5Amp,52毫秒的脉冲,脉冲之间0.2秒,4秒的点火延迟(firing delay),总共3个脉冲,对组1和2立即进行电穿孔。对照动物是首次接触实验的对照(无质粒,无EP)。使雪貂在它们的笼中从麻醉恢复,密切监控其24小时以确保完全恢复。
在整个实验期间食物和水可随意获得。在第84天,通过利用1ml MX10(A/Mexico/InDRE4487/2009;5x 105PFU/ml)的鼻内感染攻击动物。使用已建立和批准的评分表每天监测动物的临床体征(体重、温度等)。在转染后第1、3、6、9和15天进行鼻洗涤,收集直肠拭子。在第15天收集肺。将样品贮存在RNAlater中(以在进行实时PCR时,用于病毒上样),贮存在培养基中(用于感染性病毒(TCDI50))和贮存在福尔马林中(以当适当时进行组织学分析)。
图4显示利用经免疫的雪貂(3次免疫)的血清进行的血凝抑制测定。大于1∶40的滴度被认为是“阳性的”。虚线表示1∶40标志。所有动物在3次免疫后高于1∶40标志。图5显示利用新型H1N1株MX10(A/Mexico/InDRE4487/2009)攻击经免疫的和未经免疫的雪貂的结果。全部经免疫的雪貂都存活,然而75%的首次接触实验的雪貂在15天的时间内死亡。
序列表
<110> 宾夕法尼亚大学托管会(The Trustees of the University ofPennsylvania)
D.B.韦纳(David, Weiner B.)
严健(Yan, Jian)
M.P.莫罗(Morrow, Matthew P.)
<120> 流感病毒核酸分子及由其制备的疫苗
<130> 133172.03002
<150> 12/694,238
<151> 2010-01-26
<160> 24
<170> PatentIn 3.5版
<210> 1
<211> 1695
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒H1 DNA序列
<400> 1
atgaaggcta tcctcgtcgt gctgctgtac accttcgcca ccgccaacgc cgataccctg 60
tgcatcggct accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac 120
gtgaccgtga cccacagcgt gaacctgctg gaagataagc acaacggcaa gctgtgcaag 180
ctgagaggcg tggcccctct gcacctgggc aagtgcaata tcgccggctg gattctgggc 240
aaccccgagt gcgagagcct gtctaccgct agctcctggt cctacatcgt ggagacaagc 300
agcagcgaca acggcacctg ttaccccggc gacttcatcg actacgagga actgcgggag 360
cagctgagca gcgtgtccag cttcgagcgg ttcgagatct tccccaagac cagctcctgg 420
cccaaccacg acagcaacaa gggcgtgacc gccgcctgtc ctcacgctgg cgccaagagc 480
ttctacaaga acctgatctg gctggtcaag aagggcaaca gctaccccaa gctgagcaag 540
agctacatca acgacaaggg caaagaggtc ctcgtcctct ggggcatcca ccaccctagc 600
accagcgccg accagcagag cctgtaccag aacgccgacg cctacgtgtt cgtgggctca 660
tctcggtaca gcaagaagtt caagcccgag atcgccatca gacccaaagt gcgggaccag 720
gaaggccgga tgaactacta ctggaccctg gtggagcccg gcgacaagat caccttcgag 780
gccaccggca atctggtggt gcccagatac gccttcgcca tggaaagaaa cgccggcagc 840
ggcatcatca tcagcgacac ccccgtgcac gactgcaaca ccacctgtca gacccccaag 900
ggcgccatca acaccagcct gcccttccag aacatccacc ccatcaccat cggcaagtgc 960
cctaagtacg tgaagtccac taagctcaga ctggccaccg gcctgagaaa cgtgcccagc 1020
atccagagca gaggcctgtt tggcgccatt gccggcttta tcgagggcgg ctggaccgga 1080
atggtggacg ggtggtacgg ctaccaccac cagaatgagc agggcagcgg ctacgccgcc 1140
gacctgaagt ccacacagaa cgccatcgac gagatcacca acaaagtgaa cagcgtgatc 1200
gagaagatga acacccagtt caccgccgtg ggcaaagagt tcaaccacct ggaaaagcgg 1260
atcgagaacc tgaacaagaa ggtggacgac ggcttcctgg acatctggac ctacaacgcc 1320
gagctgctgg tgctgctgga aaacgagcgg accctggact accacgactc caacgtgaag 1380
aatctgtacg agaaagtgcg gagccagctg aagaacaacg ccaaagagat cggcaacggc 1440
tgcttcgagt tctaccacaa gtgcgacaac acctgtatgg aaagcgtgaa gaacggcacc 1500
tacgactacc ccaagtacag cgaggaagcc aagctgaacc gggaagagat cgacggcgtg 1560
aagctggaaa gcacccggat ctaccagatc ctggccatct actctactgt ggccagctca 1620
ctggtgctgg tggtgtccct gggcgccatc tccttttgga tgtgctccaa cggcagcctg 1680
cagtgccgga tctgc 1695
<210> 2
<211> 566
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒蛋白H1序列
<400> 2
Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn
1 5 10 15
Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr
20 25 30
Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn
35 40 45
Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg Gly Val
50 55 60
Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly
65 70 75 80
Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile
85 90 95
Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe
100 105 110
Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe
115 120 125
Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp
130 135 140
Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser
145 150 155 160
Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser Tyr Pro
165 170 175
Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val Leu Val
180 185 190
Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu
195 200 205
Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser
210 215 220
Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln
225 230 235 240
Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys
245 250 255
Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe
260 265 270
Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr Pro
275 280 285
Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala Ile Asn
290 295 300
Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly Lys Cys
305 310 315 320
Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg
325 330 335
Asn Val Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly
340 345 350
Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr
355 360 365
His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser
370 375 380
Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile
385 390 395 400
Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn His
405 410 415
Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe
420 425 430
Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
435 440 445
Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu
450 455 460
Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly
465 470 475 480
Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val
485 490 495
Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu
500 505 510
Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg Ile Tyr
515 520 525
Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Val
530 535 540
Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu
545 550 555 560
Gln Cys Arg Ile Cys Ile
565
<210> 3
<211> 1818
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> IgE-H1-HAT抗原DNA序列
<400> 3
ttaattaagg atccgccacc atggactgga cctggattct gttcctggtg gctgctgcca 60
ctagagtgca cagcatgaag gctatcctcg tcgtgctgct gtacaccttc gccaccgcca 120
acgccgatac cctgtgcatc ggctaccacg ccaacaacag caccgacacc gtggataccg 180
tgctggaaaa gaacgtgacc gtgacccaca gcgtgaacct gctggaagat aagcacaacg 240
gcaagctgtg caagctgaga ggcgtggccc ctctgcacct gggcaagtgc aatatcgccg 300
gctggattct gggcaacccc gagtgcgaga gcctgtctac cgctagctcc tggtcctaca 360
tcgtggagac aagcagcagc gacaacggca cctgttaccc cggcgacttc atcgactacg 420
aggaactgcg ggagcagctg agcagcgtgt ccagcttcga gcggttcgag atcttcccca 480
agaccagctc ctggcccaac cacgacagca acaagggcgt gaccgccgcc tgtcctcacg 540
ctggcgccaa gagcttctac aagaacctga tctggctggt caagaagggc aacagctacc 600
ccaagctgag caagagctac atcaacgaca agggcaaaga ggtcctcgtc ctctggggca 660
tccaccaccc tagcaccagc gccgaccagc agagcctgta ccagaacgcc gacgcctacg 720
tgttcgtggg ctcatctcgg tacagcaaga agttcaagcc cgagatcgcc atcagaccca 780
aagtgcggga ccaggaaggc cggatgaact actactggac cctggtggag cccggcgaca 840
agatcacctt cgaggccacc ggcaatctgg tggtgcccag atacgccttc gccatggaaa 900
gaaacgccgg cagcggcatc atcatcagcg acacccccgt gcacgactgc aacaccacct 960
gtcagacccc caagggcgcc atcaacacca gcctgccctt ccagaacatc caccccatca 1020
ccatcggcaa gtgccctaag tacgtgaagt ccactaagct cagactggcc accggcctga 1080
gaaacgtgcc cagcatccag agcagaggcc tgtttggcgc cattgccggc tttatcgagg 1140
gcggctggac cggaatggtg gacgggtggt acggctacca ccaccagaat gagcagggca 1200
gcggctacgc cgccgacctg aagtccacac agaacgccat cgacgagatc accaacaaag 1260
tgaacagcgt gatcgagaag atgaacaccc agttcaccgc cgtgggcaaa gagttcaacc 1320
acctggaaaa gcggatcgag aacctgaaca agaaggtgga cgacggcttc ctggacatct 1380
ggacctacaa cgccgagctg ctggtgctgc tggaaaacga gcggaccctg gactaccacg 1440
actccaacgt gaagaatctg tacgagaaag tgcggagcca gctgaagaac aacgccaaag 1500
agatcggcaa cggctgcttc gagttctacc acaagtgcga caacacctgt atggaaagcg 1560
tgaagaacgg cacctacgac taccccaagt acagcgagga agccaagctg aaccgggaag 1620
agatcgacgg cgtgaagctg gaaagcaccc ggatctacca gatcctggcc atctactcta 1680
ctgtggccag ctcactggtg ctggtggtgt ccctgggcgc catctccttt tggatgtgct 1740
ccaacggcag cctgcagtgc cggatctgca tctaccccta cgacgtgccc gactacgcct 1800
gatgactcga ggcgcgcc 1818
<210> 4
<211> 593
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> IgE-H1-HAT抗原氨基酸序列
<400> 4
Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val
1 5 10 15
His Ser Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr
20 25 30
Ala Asn Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
35 40 45
Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser
50 55 60
Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Arg
65 70 75 80
Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile
85 90 95
Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser
100 105 110
Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly
115 120 125
Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser
130 135 140
Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn
145 150 155 160
His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala
165 170 175
Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn Ser
180 185 190
Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly Lys Glu Val
195 200 205
Leu Val Leu Trp Gly Ile His His Pro Ser Thr Ser Ala Asp Gln Gln
210 215 220
Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe Val Gly Ser Ser Arg
225 230 235 240
Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg
245 250 255
Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly
260 265 270
Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr
275 280 285
Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp
290 295 300
Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly Ala
305 310 315 320
Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile Thr Ile Gly
325 330 335
Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly
340 345 350
Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile
355 360 365
Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr
370 375 380
Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu
385 390 395 400
Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser
405 410 415
Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe
420 425 430
Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp
435 440 445
Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu
450 455 460
Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser Asn Val Lys Asn Leu
465 470 475 480
Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly
485 490 495
Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu
500 505 510
Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala
515 520 525
Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Thr Arg
530 535 540
Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val
545 550 555 560
Leu Val Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly
565 570 575
Ser Leu Gln Cys Arg Ile Cys Ile Tyr Pro Tyr Asp Val Pro Asp Tyr
580 585 590
Ala
<210> 5
<211> 4739
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pGX2009
<400> 5
gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60
atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120
acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180
aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240
gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300
ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360
atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420
gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480
tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540
aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600
ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660
aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720
accgagctcg gatccgccac catggactgg acctggattc tgttcctggt ggctgctgcc 780
actagagtgc acagcatgaa ggctatcctc gtcgtgctgc tgtacacctt cgccaccgcc 840
aacgccgata ccctgtgcat cggctaccac gccaacaaca gcaccgacac cgtggatacc 900
gtgctggaaa agaacgtgac cgtgacccac agcgtgaacc tgctggaaga taagcacaac 960
ggcaagctgt gcaagctgag aggcgtggcc cctctgcacc tgggcaagtg caatatcgcc 1020
ggctggattc tgggcaaccc cgagtgcgag agcctgtcta ccgctagctc ctggtcctac 1080
atcgtggaga caagcagcag cgacaacggc acctgttacc ccggcgactt catcgactac 1140
gaggaactgc gggagcagct gagcagcgtg tccagcttcg agcggttcga gatcttcccc 1200
aagaccagct cctggcccaa ccacgacagc aacaagggcg tgaccgccgc ctgtcctcac 1260
gctggcgcca agagcttcta caagaacctg atctggctgg tcaagaaggg caacagctac 1320
cccaagctga gcaagagcta catcaacgac aagggcaaag aggtcctcgt cctctggggc 1380
atccaccacc ctagcaccag cgccgaccag cagagcctgt accagaacgc cgacgcctac 1440
gtgttcgtgg gctcatctcg gtacagcaag aagttcaagc ccgagatcgc catcagaccc 1500
aaagtgcggg accaggaagg ccggatgaac tactactgga ccctggtgga gcccggcgac 1560
aagatcacct tcgaggccac cggcaatctg gtggtgccca gatacgcctt cgccatggaa 1620
agaaacgccg gcagcggcat catcatcagc gacacccccg tgcacgactg caacaccacc 1680
tgtcagaccc ccaagggcgc catcaacacc agcctgccct tccagaacat ccaccccatc 1740
accatcggca agtgccctaa gtacgtgaag tccactaagc tcagactggc caccggcctg 1800
agaaacgtgc ccagcatcca gagcagaggc ctgtttggcg ccattgccgg ctttatcgag 1860
ggcggctgga ccggaatggt ggacgggtgg tacggctacc accaccagaa tgagcagggc 1920
agcggctacg ccgccgacct gaagtccaca cagaacgcca tcgacgagat caccaacaaa 1980
gtgaacagcg tgatcgagaa gatgaacacc cagttcaccg ccgtgggcaa agagttcaac 2040
cacctggaaa agcggatcga gaacctgaac aagaaggtgg acgacggctt cctggacatc 2100
tggacctaca acgccgagct gctggtgctg ctggaaaacg agcggaccct ggactaccac 2160
gactccaacg tgaagaatct gtacgagaaa gtgcggagcc agctgaagaa caacgccaaa 2220
gagatcggca acggctgctt cgagttctac cacaagtgcg acaacacctg tatggaaagc 2280
gtgaagaacg gcacctacga ctaccccaag tacagcgagg aagccaagct gaaccgggaa 2340
gagatcgacg gcgtgaagct ggaaagcacc cggatctacc agatcctggc catctactct 2400
actgtggcca gctcactggt gctggtggtg tccctgggcg ccatctcctt ttggatgtgc 2460
tccaacggca gcctgcagtg ccggatctgc atctacccct acgacgtgcc cgactacgcc 2520
tgatgactcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct 2580
agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 2640
actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt 2700
cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat 2760
agcaggcatg ctggggatgc ggtgggctct atggcttcta ctgggcggtt ttatggacag 2820
caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag 2880
taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca agctctgatc 2940
aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc 3000
cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct 3060
ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg 3120
acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca 3180
cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc 3240
tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga 3300
aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc 3360
cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc 3420
ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg 3480
ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct 3540
gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc 3600
tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc 3660
ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc 3720
agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac gcttacaatt 3780
tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atcaggtggc 3840
acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 3900
atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca cgtgctaaaa 3960
cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 4020
atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 4080
tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 4140
ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 4200
ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac 4260
cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 4320
gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 4380
gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 4440
acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 4500
gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 4560
agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 4620
tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 4680
agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttctt 4739
<210> 6
<211> 1719
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒H2抗原DNA序列
<400> 6
ggtaccaagc ttgccaccat ggccatcatc tacctgatcc tgctgttcac cgccgtgcgg 60
ggcgaccaga tctgcatcgg ctaccacgcc aacaacagca ccgagaaggt ggacaccatc 120
ctggaacgga acgtgaccgt gacccacgcc aaggacatcc tggaaaagac ccacaacggc 180
aagctgtgca agctgaacgg catccccccc ctggaactgg gcgactgcag cattgccggc 240
tggctgctgg gcaaccccga gtgcgaccgg ctgctgtccg tgcccgagtg gagctacatc 300
atggaaaaag agaacccccg ggacggcctg tgctaccccg gcagcttcaa cgactacgag 360
gaactgaagc acctgctgtc cagcgtgaag cacttcgaga aggtgaaaat cctgcccaag 420
gaccggtgga cccagcacac caccaccggc ggcagcagag cctgtgccgt gagcggcaac 480
cccagcttct tccggaacat ggtgtggctg accaagaagg gcagcaacta ccccgtggcc 540
aagggcagct acaacaacac ctccggagaa cagatgctga tcatctgggg cgtgcaccac 600
cccaacgacg agacagagca gcggaccctg taccagaacg tgggcaccta cgtgagcgtg 660
ggcaccagca ccctgaacaa gcggagcacc cccgagatcg ccacccggcc caaggtgaac 720
ggcctgggca gccggatgga attcagctgg accctgctgg acatgtggga caccatcaac 780
ttcgagagca ccggcaacct gatcgccccc gagtacggct tcaagatcag caagcggggc 840
agcagcggca tcatgaaaac cgagggcacc ctggaaaact gcgagacaaa gtgccagacc 900
cccctgggcg ccatcaacac caccctgccc ttccacaacg tgcaccccct gaccatcggc 960
gagtgcccca agtacgtgaa gagcgagaag ctggtgctgg ccaccggcct gcggaacgtg 1020
ccccagatcg agagcagggg cctgttcggc gccattgccg gattcatcga gggcggctgg 1080
cagggcatgg tggacgggtg gtacggctac caccacagca acgaccaggg cagcggctac 1140
gccgccgaca aagagagcac ccagaaggcc ttcgacggca tcaccaacaa ggtgaacagc 1200
gtgatcgaga agatgaacac ccagttcgag gccgtgggca aagagttcag caacctggaa 1260
cggcggctgg aaaacctgaa caagaaaatg gaagatggct tcctggacgt gtggacctac 1320
aacgccgagc tgctggtgct gatggaaaac gagaggaccc tggacttcca cgacagcaac 1380
gtgaagaacc tgtacgacaa agtgcggatg cagctgcggg acaacgtgaa agagctgggc 1440
aacggctgct tcgagttcta ccacaagtgc gacgacgagt gcatgaactc cgtgaagaac 1500
ggcacctacg actaccctaa gtacgaggaa gagtccaagc tgaaccggaa cgagatcaag 1560
ggcgtgaagc tgtccagcat gggcgtgtac cagatcctgg ccatctacgc caccgtggcc 1620
ggcagcctga gcctggctat tatgatggct ggcatcagct tttggatgtg cagcaacggc 1680
agcctgcagt gccggatctg catctgatga ctcgagctc 1719
<210> 7
<211> 562
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒H2氨基酸序列
<400> 7
Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp
1 5 10 15
Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp
20 25 30
Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu
35 40 45
Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro
50 55 60
Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro
65 70 75 80
Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu
85 90 95
Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp
100 105 110
Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys
115 120 125
Val Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly
130 135 140
Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn
145 150 155 160
Met Val Trp Leu Thr Lys Lys Gly Ser Asn Tyr Pro Val Ala Lys Gly
165 170 175
Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val
180 185 190
His His Pro Asn Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val
195 200 205
Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr
210 215 220
Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Leu Gly Ser Arg Met
225 230 235 240
Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu
245 250 255
Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys
260 265 270
Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys
275 280 285
Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro
290 295 300
Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val
305 310 315 320
Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln
325 330 335
Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
340 345 350
Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn
355 360 365
Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala
370 375 380
Phe Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn
385 390 395 400
Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Ser Asn Leu Glu Arg Arg
405 410 415
Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp
420 425 430
Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu
435 440 445
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met
450 455 460
Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe
465 470 475 480
Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr
485 490 495
Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu
500 505 510
Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala
515 520 525
Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Met Ala
530 535 540
Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile
545 550 555 560
Cys Ile
<210> 8
<211> 4628
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pGX2006 DNA序列
<400> 8
gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60
atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120
acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180
aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240
gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300
ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360
atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420
gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480
tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540
aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600
ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660
aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttgcc 720
accatggcca tcatctacct gatcctgctg ttcaccgccg tgcggggcga ccagatctgc 780
atcggctacc acgccaacaa cagcaccgag aaggtggaca ccatcctgga acggaacgtg 840
accgtgaccc acgccaagga catcctggaa aagacccaca acggcaagct gtgcaagctg 900
aacggcatcc cccccctgga actgggcgac tgcagcattg ccggctggct gctgggcaac 960
cccgagtgcg accggctgct gtccgtgccc gagtggagct acatcatgga aaaagagaac 1020
ccccgggacg gcctgtgcta ccccggcagc ttcaacgact acgaggaact gaagcacctg 1080
ctgtccagcg tgaagcactt cgagaaggtg aaaatcctgc ccaaggaccg gtggacccag 1140
cacaccacca ccggcggcag cagagcctgt gccgtgagcg gcaaccccag cttcttccgg 1200
aacatggtgt ggctgaccaa gaagggcagc aactaccccg tggccaaggg cagctacaac 1260
aacacctccg gagaacagat gctgatcatc tggggcgtgc accaccccaa cgacgagaca 1320
gagcagcgga ccctgtacca gaacgtgggc acctacgtga gcgtgggcac cagcaccctg 1380
aacaagcgga gcacccccga gatcgccacc cggcccaagg tgaacggcct gggcagccgg 1440
atggaattca gctggaccct gctggacatg tgggacacca tcaacttcga gagcaccggc 1500
aacctgatcg cccccgagta cggcttcaag atcagcaagc ggggcagcag cggcatcatg 1560
aaaaccgagg gcaccctgga aaactgcgag acaaagtgcc agacccccct gggcgccatc 1620
aacaccaccc tgcccttcca caacgtgcac cccctgacca tcggcgagtg ccccaagtac 1680
gtgaagagcg agaagctggt gctggccacc ggcctgcgga acgtgcccca gatcgagagc 1740
aggggcctgt tcggcgccat tgccggattc atcgagggcg gctggcaggg catggtggac 1800
gggtggtacg gctaccacca cagcaacgac cagggcagcg gctacgccgc cgacaaagag 1860
agcacccaga aggccttcga cggcatcacc aacaaggtga acagcgtgat cgagaagatg 1920
aacacccagt tcgaggccgt gggcaaagag ttcagcaacc tggaacggcg gctggaaaac 1980
ctgaacaaga aaatggaaga tggcttcctg gacgtgtgga cctacaacgc cgagctgctg 2040
gtgctgatgg aaaacgagag gaccctggac ttccacgaca gcaacgtgaa gaacctgtac 2100
gacaaagtgc ggatgcagct gcgggacaac gtgaaagagc tgggcaacgg ctgcttcgag 2160
ttctaccaca agtgcgacga cgagtgcatg aactccgtga agaacggcac ctacgactac 2220
cctaagtacg aggaagagtc caagctgaac cggaacgaga tcaagggcgt gaagctgtcc 2280
agcatgggcg tgtaccagat cctggccatc tacgccaccg tggccggcag cctgagcctg 2340
gctattatga tggctggcat cagcttttgg atgtgcagca acggcagcct gcagtgccgg 2400
atctgcatct gatgactcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact 2460
gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 2520
gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 2580
agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 2640
gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctac tgggcggttt 2700
tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc 2760
cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa 2820
gctctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 2880
caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 2940
tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 3000
tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt 3060
ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 3120
gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 3180
ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 3240
ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 3300
aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 3360
aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg 3420
gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 3480
gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 3540
ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 3600
ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg 3660
cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 3720
tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 3780
cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac 3840
gtgctaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 3900
atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 3960
atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 4020
aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 4080
aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 4140
ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 4200
ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 4260
tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 4320
ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 4380
acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 4440
gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 4500
cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 4560
aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 4620
atgttctt 4628
<210> 9
<211> 1695
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒U2 DNA序列
<400> 9
aaggccaagc tgctggtgct gctgtgcacc ttcgccgcca ccaacgccga caccatctgc 60
atcggctacc acgccaacaa cagcaccgac accgtggata ccgtgctgga aaagaacgtg 120
accgtgaccc acagcgtgaa cctgctggaa gataagcaca acggcaagct gtgcaagctg 180
aagggaatcg cccccctgca gctgggcaag tgcaatatcg ccggctggat tctgggcaac 240
cccgagtgcg agagcctgag cagcaagagc agctggtcct acatcgtgga aacccccaac 300
agcgagaacg gcacctgtta ccccggcgac ttcgccgact acgaggaact gcgcgagcag 360
ctgagcagcg tgtccagctt cgagagattc gagatcttcc ccaagaccag cagctggccc 420
aaccacgacg tgaccaaggg cgtgaccgct agctgtagcc acgcaggcgc cagcagcttc 480
tacaagaacc tgctgtggct gaccaagaag aacggcagct accccaagct gagcaagagc 540
tacatcaaca acaaagaaaa agaggtgctg gtcctctggg gcgtccacca ccccagcaca 600
atcgccgacc agcagagcct gtaccagaac gagaacgcct acgtgtccgt gggcagcagc 660
cactacagcc ggaagttcac ccccgagatc gccaagcggc ccaaagtgcg ggaccaggaa 720
ggccggatca actactactg gaccctgctg gaacccggcg acaccatcat cttcgaggcc 780
aacggcaacc tgatcgcccc cagatacgcc ttcgccctga gcagaggctt cggcagcggc 840
atcatcatca gcaacgcccc catgcacgac tgcgacacca agtgccagac ccctcagggc 900
gccatcaaca gcagcctgcc cttccagaac atccaccccg tgaccatcgg cgagtgcccc 960
aaatacgtgc ggagcaccaa gctgcggatg gccaccggcc tgcggaacat ccccagcatc 1020
cagagcagag gcctgttcgg cgccattgcc ggcttcatcg agggcggctg gaccggaatg 1080
gtggacgggt ggtacggcta ccaccaccag aatgagcagg gcagcggcta cgccgccgac 1140
cagaagtcca cccagaacgc catcgacggc atcaccaaca aagtgaacag cgtgatcgag 1200
aagatgaaca cccagttcac cgccgtgggc aaagagttca acaagctgga aaagcggatg 1260
gaaaacctga acaagaaggt ggacgacggc ttcctggaca tctggaccta caacgccgaa 1320
ctgctcgtgc tgctggaaaa cgagcggacc ctggacttcc acgacagcaa cgtgaagaac 1380
ctgtacgaga aagtgaagtc ccagctgaag aacaacgcca aagagatcgg caacggctgc 1440
ttcgagttct accacaagtg caacaacgag tgcatggaaa gcgtgaagaa cggaacctac 1500
gactacccca agtacagcga ggaaagcaag ctgaaccggg aagagatcga cggcgtgaag 1560
ctggaatcca tgggcgtgta ccagatcctg gccatctaca gcaccgtggc tagcagcctg 1620
gtgctgctgg tgtccctggg cgccatctcc ttttggatgt gctccaacgg cagcctgcag 1680
tgccggatct gcatc 1695
<210> 10
<211> 565
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒U2氨基酸序列
<400> 10
Lys Ala Lys Leu Leu Val Leu Leu Cys Thr Phe Ala Ala Thr Asn Ala
1 5 10 15
Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr Val
20 25 30
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn Leu
35 40 45
Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Lys Gly Ile Ala
50 55 60
Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly Asn
65 70 75 80
Pro Glu Cys Glu Ser Leu Ser Ser Lys Ser Ser Trp Ser Tyr Ile Val
85 90 95
Glu Thr Pro Asn Ser Glu Asn Gly Thr Cys Tyr Pro Gly Asp Phe Ala
100 105 110
Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe Glu
115 120 125
Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp Val
130 135 140
Thr Lys Gly Val Thr Ala Ser Cys Ser His Ala Gly Ala Ser Ser Phe
145 150 155 160
Tyr Lys Asn Leu Leu Trp Leu Thr Lys Lys Asn Gly Ser Tyr Pro Lys
165 170 175
Leu Ser Lys Ser Tyr Ile Asn Asn Lys Glu Lys Glu Val Leu Val Leu
180 185 190
Trp Gly Val His His Pro Ser Thr Ile Ala Asp Gln Gln Ser Leu Tyr
195 200 205
Gln Asn Glu Asn Ala Tyr Val Ser Val Gly Ser Ser His Tyr Ser Arg
210 215 220
Lys Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp Gln Glu
225 230 235 240
Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile
245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Arg Tyr Ala Phe Ala
260 265 270
Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Ile Ser Asn Ala Pro Met
275 280 285
His Asp Cys Asp Thr Lys Cys Gln Thr Pro Gln Gly Ala Ile Asn Ser
290 295 300
Ser Leu Pro Phe Gln Asn Ile His Pro Val Thr Ile Gly Glu Cys Pro
305 310 315 320
Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Ala Thr Gly Leu Arg Asn
325 330 335
Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe
340 345 350
Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His
355 360 365
His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr
370 375 380
Gln Asn Ala Ile Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu
385 390 395 400
Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu
405 410 415
Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu
420 425 430
Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu
435 440 445
Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys
450 455 460
Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys
465 470 475 480
Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser Val Lys
485 490 495
Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn
500 505 510
Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr Gln
515 520 525
Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val
530 535 540
Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln
545 550 555 560
Cys Arg Ile Cys Ile
565
<210> 11
<211> 1809
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> IgE-U2-HAT抗原DNA序列
<400> 11
ggtaccggat ccgccaccat ggactggacc tggattctgt tcctggtcgc cgctgctacc 60
cgggtgcact ctaaggccaa gctgctggtg ctgctgtgca ccttcgccgc caccaacgcc 120
gacaccatct gcatcggcta ccacgccaac aacagcaccg acaccgtgga taccgtgctg 180
gaaaagaacg tgaccgtgac ccacagcgtg aacctgctgg aagataagca caacggcaag 240
ctgtgcaagc tgaagggaat cgcccccctg cagctgggca agtgcaatat cgccggctgg 300
attctgggca accccgagtg cgagagcctg agcagcaaga gcagctggtc ctacatcgtg 360
gaaaccccca acagcgagaa cggcacctgt taccccggcg acttcgccga ctacgaggaa 420
ctgcgcgagc agctgagcag cgtgtccagc ttcgagagat tcgagatctt ccccaagacc 480
agcagctggc ccaaccacga cgtgaccaag ggcgtgaccg ctagctgtag ccacgcaggc 540
gccagcagct tctacaagaa cctgctgtgg ctgaccaaga agaacggcag ctaccccaag 600
ctgagcaaga gctacatcaa caacaaagaa aaagaggtgc tggtcctctg gggcgtccac 660
caccccagca caatcgccga ccagcagagc ctgtaccaga acgagaacgc ctacgtgtcc 720
gtgggcagca gccactacag ccggaagttc acccccgaga tcgccaagcg gcccaaagtg 780
cgggaccagg aaggccggat caactactac tggaccctgc tggaacccgg cgacaccatc 840
atcttcgagg ccaacggcaa cctgatcgcc cccagatacg ccttcgccct gagcagaggc 900
ttcggcagcg gcatcatcat cagcaacgcc cccatgcacg actgcgacac caagtgccag 960
acccctcagg gcgccatcaa cagcagcctg cccttccaga acatccaccc cgtgaccatc 1020
ggcgagtgcc ccaaatacgt gcggagcacc aagctgcgga tggccaccgg cctgcggaac 1080
atccccagca tccagagcag aggcctgttc ggcgccattg ccggcttcat cgagggcggc 1140
tggaccggaa tggtggacgg gtggtacggc taccaccacc agaatgagca gggcagcggc 1200
tacgccgccg accagaagtc cacccagaac gccatcgacg gcatcaccaa caaagtgaac 1260
agcgtgatcg agaagatgaa cacccagttc accgccgtgg gcaaagagtt caacaagctg 1320
gaaaagcgga tggaaaacct gaacaagaag gtggacgacg gcttcctgga catctggacc 1380
tacaacgccg aactgctcgt gctgctggaa aacgagcgga ccctggactt ccacgacagc 1440
aacgtgaaga acctgtacga gaaagtgaag tcccagctga agaacaacgc caaagagatc 1500
ggcaacggct gcttcgagtt ctaccacaag tgcaacaacg agtgcatgga aagcgtgaag 1560
aacggaacct acgactaccc caagtacagc gaggaaagca agctgaaccg ggaagagatc 1620
gacggcgtga agctggaatc catgggcgtg taccagatcc tggccatcta cagcaccgtg 1680
gctagcagcc tggtgctgct ggtgtccctg ggcgccatct ccttttggat gtgctccaac 1740
ggcagcctgc agtgccggat ctgcatctac ccctacgacg tgcccgacta cgcctgatga 1800
ctcgagctc 1809
<210> 12
<211> 592
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> IgE-U2-HAT抗原氨基酸序列
<400> 12
Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val
1 5 10 15
His Ser Lys Ala Lys Leu Leu Val Leu Leu Cys Thr Phe Ala Ala Thr
20 25 30
Asn Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
35 40 45
Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val
50 55 60
Asn Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys Lys Leu Lys Gly
65 70 75 80
Ile Ala Pro Leu Gln Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu
85 90 95
Gly Asn Pro Glu Cys Glu Ser Leu Ser Ser Lys Ser Ser Trp Ser Tyr
100 105 110
Ile Val Glu Thr Pro Asn Ser Glu Asn Gly Thr Cys Tyr Pro Gly Asp
115 120 125
Phe Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser
130 135 140
Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His
145 150 155 160
Asp Val Thr Lys Gly Val Thr Ala Ser Cys Ser His Ala Gly Ala Ser
165 170 175
Ser Phe Tyr Lys Asn Leu Leu Trp Leu Thr Lys Lys Asn Gly Ser Tyr
180 185 190
Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asn Lys Glu Lys Glu Val Leu
195 200 205
Val Leu Trp Gly Val His His Pro Ser Thr Ile Ala Asp Gln Gln Ser
210 215 220
Leu Tyr Gln Asn Glu Asn Ala Tyr Val Ser Val Gly Ser Ser His Tyr
225 230 235 240
Ser Arg Lys Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp
245 250 255
Gln Glu Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp
260 265 270
Thr Ile Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Arg Tyr Ala
275 280 285
Phe Ala Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Ile Ser Asn Ala
290 295 300
Pro Met His Asp Cys Asp Thr Lys Cys Gln Thr Pro Gln Gly Ala Ile
305 310 315 320
Asn Ser Ser Leu Pro Phe Gln Asn Ile His Pro Val Thr Ile Gly Glu
325 330 335
Cys Pro Lys Tyr Val Arg Ser Thr Lys Leu Arg Met Ala Thr Gly Leu
340 345 350
Arg Asn Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala
355 360 365
Gly Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly
370 375 380
Tyr His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys
385 390 395 400
Ser Thr Gln Asn Ala Ile Asp Gly Ile Thr Asn Lys Val Asn Ser Val
405 410 415
Ile Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn
420 425 430
Lys Leu Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly
435 440 445
Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu
450 455 460
Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr
465 470 475 480
Glu Lys Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn
485 490 495
Gly Cys Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser
500 505 510
Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys
515 520 525
Leu Asn Arg Glu Glu Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val
530 535 540
Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
545 550 555 560
Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser
565 570 575
Leu Gln Cys Arg Ile Cys Ile Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
580 585 590
<210> 13
<211> 1749
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> BHA DNA序列
<400> 13
aaggccatca tcgtgctgct gatggtggtc acaagcaacg ccgaccggat ctgcaccggc 60
atcaccagca gcaacagccc ccacgtggtc aaaaccgcca cccagggcga agtgaacgtg 120
accggcgtga tccccctgac caccaccccc accaagagcc acttcgccaa cctgaagggc 180
accaagaccc ggggaaagct gtgccccaag tgcctgaact gcaccgacct ggacgtggcc 240
ctgggcagac ctatgtgcgt gggcaccacc cctagcgcca aggccagcat cctgcacgaa 300
gtgcggcccg tgaccagcgg ctgcttcccc atcatgcacg accggaccaa gatccggcag 360
ctccccaacc tgctgcgggg ctacgagaac atccggctga gcacccagaa cgtgatcaac 420
gccgagaagg cccctggcgg cccttacaga ctgggcacaa gcggctcttg ccccaacgcc 480
accagcaaga gcggcttttt cgccacaatg gcctgggccg tgcccaagga caacaacaag 540
accgccacca accccctgac cgtggaagtg ccctacatct gcaccgaggg cgaggaccag 600
atcaccgtgt ggggcttcca cagcgataac aagacccaga tgaagaacct gtacggcgac 660
agcaaccccc agaagttcac cagctccgcc aacggcgtga ccacccacta cgtgtcccag 720
atcggcggct tccccgacca gacagaggat ggcggcctgc cccagagcgg cagaatcgtg 780
gtggactaca tggtgcagaa gcccggcaag accggcacca tcgtgtacca gcggggcatc 840
ctgctgcccc agaaagtgtg gtgcgccagc ggccggtcca aagtgatcaa gggcagcctg 900
cctctgatcg gcgaggccga ttgcctgcac gagaagtacg gcggcctgaa caagagcaag 960
ccctactaca ccggcgagca cgccaaagcc atcggcaact gccccatctg ggtcaaaacc 1020
cccctgaagc tggccaacgg caccaagtac cggcctcccg ccaagctgct gaaagagcgg 1080
ggcttcttcg gcgctatcgc cggctttctg gaaggcggct gggagggcat gatcgccggc 1140
tggcacggct acacatctca cggcgctcat ggcgtggccg tggccgctga tctgaagtcc 1200
acccaggaag ccatcaacaa gatcaccaag aacctgaaca gcctgagcga gctggaagtg 1260
aagaatctgc agcggctgag cggcgccatg gacgagctgc acaacgagat cctggaactg 1320
gacgagaagg tggacgacct gcgggccgac accatctcca gccagatcga gctggccgtg 1380
ctgctgtcca acgagggcat catcaacagc gaggacgagc atctgctggc cctggaacgg 1440
aagctgaaga agatgctggg ccctagcgcc gtggacatcg gcaacggctg cttcgagaca 1500
aagcacaagt gcaaccagac ctgcctggac cggatcgctg ccggcacctt caacgccggc 1560
gagttcagcc tgcccacctt cgacagcctg aacatcaccg ccgccagcct gaacgacgac 1620
ggcctggaca accacaccat cctgctgtac tacagcaccg cagcctccag cctggccgtg 1680
accctgatga tcgccatctt catcgtgtac atggtgtctc gggacaacgt gtcctgcagc 1740
atctgcctg 1749
<210> 14
<211> 583
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> BHA氨基酸序列
<400> 14
Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp Arg
1 5 10 15
Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys Thr
20 25 30
Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr Thr
35 40 45
Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys Thr Arg
50 55 60
Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp Val Ala
65 70 75 80
Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys Ala Ser
85 90 95
Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile Met
100 105 110
His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly Tyr
115 120 125
Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asn Ala Glu Lys Ala
130 135 140
Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn Ala
145 150 155 160
Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val Pro Lys
165 170 175
Asp Asn Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val Pro Tyr
180 185 190
Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His Ser
195 200 205
Asp Asn Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro Gln
210 215 220
Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser Gln
225 230 235 240
Ile Gly Gly Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro Gln Ser
245 250 255
Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly Lys Thr Gly
260 265 270
Thr Ile Val Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val Trp Cys
275 280 285
Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly
290 295 300
Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys
305 310 315 320
Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile
325 330 335
Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro
340 345 350
Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly
355 360 365
Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr
370 375 380
Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser
385 390 395 400
Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser
405 410 415
Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp Glu
420 425 430
Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg
435 440 445
Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser Asn
450 455 460
Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg
465 470 475 480
Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly Asn Gly
485 490 495
Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg Ile
500 505 510
Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp
515 520 525
Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn
530 535 540
His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val
545 550 555 560
Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp Asn
565 570 575
Val Ser Cys Ser Ile Cys Leu
580
<210> 15
<211> 1865
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> IgE-BHA-HAT抗原DNA序列
<400> 15
ggtaccggat ccgccaccat ggactggacc tggattctgt tcctggtggc cgctgccaca 60
cgggtgcaca gcaaggccat catcgtgctg ctgatggtgg tcacaagcaa cgccgaccgg 120
atctgcaccg gcatcaccag cagcaacagc ccccacgtgg tcaaaaccgc cacccagggc 180
gaagtgaacg tgaccggcgt gatccccctg accaccaccc ccaccaagag ccacttcgcc 240
aacctgaagg gcaccaagac ccggggaaag ctgtgcccca agtgcctgaa ctgcaccgac 300
ctggacgtgg ccctgggcag acctatgtgc gtgggcacca cccctagcgc caaggccagc 360
atcctgcacg aagtgcggcc cgtgaccagc ggctgcttcc ccatcatgca cgaccggacc 420
aagatccggc agctccccaa cctgctgcgg ggctacgaga acatccggct gagcacccag 480
aacgtgatca acgccgagaa ggcccctggc ggcccttaca gactgggcac aagcggctct 540
tgccccaacg ccaccagcaa gagcggcttt ttcgccacaa tggcctgggc cgtgcccaag 600
gacaacaaca agaccgccac caaccccctg accgtggaag tgccctacat ctgcaccgag 660
ggcgaggacc agatcaccgt gtggggcttc cacagcgata acaagaccca gatgaagaac 720
ctgtacggcg acagcaaccc ccagaagttc accagctccg ccaacggcgt gaccacccac 780
tacgtgtccc agatcggcgg cttccccgac cagacagagg atggcggcct gccccagagc 840
ggcagaatcg tggtggacta catggtgcag aagcccggca agaccggcac catcgtgtac 900
cagcggggca tcctgctgcc ccagaaagtg tggtgcgcca gcggccggtc caaagtgatc 960
aagggcagcc tgcctctgat cggcgaggcc gattgcctgc acgagaagta cggcggcctg 1020
aacaagagca agccctacta caccggcgag cacgccaaag ccatcggcaa ctgccccatc 1080
tgggtcaaaa cccccctgaa gctggccaac ggcaccaagt accggcctcc cgccaagctg 1140
ctgaaagagc ggggcttctt cggcgctatc gccggctttc tggaaggcgg ctgggagggc 1200
atgatcgccg gctggcacgg ctacacatct cacggcgctc atggcgtggc cgtggccgct 1260
gatctgaagt ccacccagga agccatcaac aagatcacca agaacctgaa cagcctgagc 1320
gagctggaag tgaagaatct gcagcggctg agcggcgcca tggacgagct gcacaacgag 1380
atcctggaac tggacgagaa ggtggacgac ctgcgggccg acaccatctc cagccagatc 1440
gagctggccg tgctgctgtc caacgagggc atcatcaaca gcgaggacga gcatctgctg 1500
gccctggaac ggaagctgaa gaagatgctg ggccctagcg ccgtggacat cggcaacggc 1560
tgcttcgaga caaagcacaa gtgcaaccag acctgcctgg accggatcgc tgccggcacc 1620
ttcaacgccg gcgagttcag cctgcccacc ttcgacagcc tgaacatcac cgccgccagc 1680
ctgaacgacg acggcctgga caaccacacc atcctgctgt actacagcac cgcagcctcc 1740
agcctggccg tgaccctgat gatcgccatc ttcatcgtgt acatggtgtc tcgggacaac 1800
gtgtcctgca gcatctgcct gtacccctac gacgtgcccg actacgctga tgactcgagc 1860
tcctc 1865
<210> 16
<211> 610
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> IgE-BHA-HAT抗原氨基酸序列
<400> 16
Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val
1 5 10 15
His Ser Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala
20 25 30
Asp Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val
35 40 45
Lys Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu
50 55 60
Thr Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Lys
65 70 75 80
Thr Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp
85 90 95
Val Ala Leu Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys
100 105 110
Ala Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro
115 120 125
Ile Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg
130 135 140
Gly Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asn Ala Glu
145 150 155 160
Lys Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro
165 170 175
Asn Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp Ala Val
180 185 190
Pro Lys Asp Asn Asn Lys Thr Ala Thr Asn Pro Leu Thr Val Glu Val
195 200 205
Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe
210 215 220
His Ser Asp Asn Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn
225 230 235 240
Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val
245 250 255
Ser Gln Ile Gly Gly Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro
260 265 270
Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly Lys
275 280 285
Thr Gly Thr Ile Val Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val
290 295 300
Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu
305 310 315 320
Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys
325 330 335
Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys
340 345 350
Pro Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr
355 360 365
Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile
370 375 380
Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His
385 390 395 400
Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu
405 410 415
Lys Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser
420 425 430
Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met
435 440 445
Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp
450 455 460
Leu Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu
465 470 475 480
Ser Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu
485 490 495
Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Asp Ile Gly
500 505 510
Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp
515 520 525
Arg Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr
530 535 540
Phe Asp Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu
545 550 555 560
Asp Asn His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu
565 570 575
Ala Val Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg
580 585 590
Asp Asn Val Ser Cys Ser Ile Cys Leu Tyr Pro Tyr Asp Val Pro Asp
595 600 605
Tyr Ala
610
<210> 17
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> IgE前导序列氨基酸序列
<400> 17
Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val
1 5 10 15
His Ser
<210> 18
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> HA标签氨基酸序列
<400> 18
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 19
<211> 1707
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒H1 DNA序列
<400> 19
atggaaaaga tcgtgctgct gttcgccatc gtgagcctgg tgaagagcga ccagatctgc 60
atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga aaaaaacgtg 120
accgtgaccc acgcccagga catcctggaa aagacccaca acggcaagct gtgcgacctg 180
gacggcgtga agcccctgat cctgcgggac tgcagcgtgg ccggctggct gctgggcaac 240
cccatgtgcg acgagttcat caacgtgccc gagtggagct acatcgtgga gaaggccaac 300
cccgtgaacg acctgtgcta ccccggcgac ttcaacgact acgaggaact gaagcacctg 360
ctgtcccgga tcaaccactt cgagaagatc cagatcatcc ccaagagcag ctggtccagc 420
cacgaggcca gcctgggcgt gagcagcgcc tgcccatacc agggcaagtc cagcttcttc 480
cggaacgtgg tgtggctgat caagaagaac agcacctacc ccaccatcaa gcggagctac 540
aacaacacca accaggaaga tctgctggtc ctgtggggca tccaccaccc caacgacgcc 600
gccgagcaga ccaagctgta ccagaacccc accacctaca tcagcgtggg caccagcacc 660
ctgaaccagc ggctggtgcc ccggatcgcc acccggtcca aggtgaacgg ccagagcggc 720
cggatggaat tcttctggac catcctgaag cccaacgatg ccatcaactt cgagagcaac 780
ggcaacttca tcgcccccga gtacgcctac aagatcgtga agaagggcga cagcaccatc 840
atgaagagcg agctggaata cggcaactgc aacaccaagt gccagacccc catgggcgcc 900
atcaacagca gcatgccctt ccacaacatc caccccctga ccatcggcga gtgccccaag 960
tacgtgaaga gcaacaggct ggtgctggcc accggcctgc ggaacagccc ccagcgggag 1020
cggcgggccg ccgcccgggg cctgttcggc gccatcgccg gcttcatcga gggcggctgg 1080
cagggcatgg tggacgggtg gtacggctac caccacagca atgagcaggg cagcggctac 1140
gccgccgaca aagagagcac ccagaaggcc atcgacggcg tcaccaacaa ggtgaacagc 1200
atcatcgaca agatgaacac ccagttcgag gccgtgggcc gggagttcaa caacctggaa 1260
cggcggatcg agaacctgaa caagaaaatg gaagatggct tcctggacgt gtggacctac 1320
aacgccgagc tgctggtgct gatggaaaac gagcggaccc tggacttcca cgacagcaac 1380
gtgaagaacc tgtacgacaa agtgcggctg cagctgcggg acaacgccaa agagctgggc 1440
aacggctgct tcgagttcta ccacaagtgc gacaacgagt gcatggaaag cgtgcggaac 1500
ggcacctacg actaccccca gtacagcgag gaagcccggc tgaagcggga ggaaatcagc 1560
ggcgtgaaac tggaaagcat cggcatctac cagatcctga gcatctacag caccgtggcc 1620
agcagcctgg ccctggccat catggtggcc ggcctgagcc tgtggatgtg cagcaacggc 1680
agcctgcagt gccggatctg catctag 1707
<210> 20
<211> 568
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒蛋白H1序列
<400> 20
Met Glu Lys Ile Val Leu Leu Phe Ala Ile Val Ser Leu Val Lys Ser
1 5 10 15
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30
Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile
35 40 45
Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys
50 55 60
Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn
65 70 75 80
Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95
Glu Lys Ala Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn
100 105 110
Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu
115 120 125
Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser
130 135 140
Leu Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser Phe Phe
145 150 155 160
Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile
165 170 175
Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp
180 185 190
Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr Lys Leu Tyr Gln
195 200 205
Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg
210 215 220
Leu Val Pro Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly
225 230 235 240
Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn
245 250 255
Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile
260 265 270
Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly
275 280 285
Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser
290 295 300
Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
305 310 315 320
Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335
Pro Gln Arg Glu Arg Arg Ala Ala Ala Arg Gly Leu Phe Gly Ala Ile
340 345 350
Ala Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
355 360 365
Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys
370 375 380
Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser
385 390 395 400
Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415
Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp
420 425 430
Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met
435 440 445
Glu Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
450 455 460
Tyr Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly
465 470 475 480
Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu
485 490 495
Ser Val Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala
500 505 510
Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
515 520 525
Ile Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala
530 535 540
Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly
545 550 555 560
Ser Leu Gln Cys Arg Ile Cys Ile
565
<210> 21
<211> 1728
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒DNA H1序列
<400> 21
ggtaccaagc ttgccaccat gaaggtgaaa ctgctggtgc tgctgtgcac cttcaccgcc 60
acctacgccg acaccatctg catcggctac cacgccaaca acagcaccga caccgtggat 120
accgtgctgg aaaagaacgt gaccgtgacc cacagcgtga acctgctgga agatagccac 180
aacggcaagc tgtgcctgct gaaaggcatc gcccccctgc agctgggcaa ctgcagcgtg 240
gccggctgga tcctgggcaa ccccgagtgc gagctgctga tttccaaaga aagctggtcc 300
tacatcgtgg agacccccaa ccccgagaac ggcacctgct accccggcta cttcgccgac 360
tacgaggaac tgcgggagca gctgtccagc gtgagcagct tcgagcggtt cgagatcttc 420
cccaaagaga gcagctggcc caaccacacc gtgaccggcg tgagcgccag ctgctcccac 480
aatggcaaga gcagcttcta ccggaacctg ctgtggctga ccggcaagaa cggcctgtac 540
cccaacctga gcaagagcta cgccaataac aaagaaaagg aagtgctggt gctgtggggc 600
gtgcaccacc cccccaacat cggcgaccag cgggccctgt accacaccga gaacgcctac 660
gtgagcgtgg tgtccagcca ctacagccgg cggttcaccc ccgagatcgc caagcggccc 720
aaagtgcggg accaggaagg ccggatcaac tactactgga ccctgctgga acccggcgac 780
accatcatct tcgaggccaa cggcaacctg atcgccccca gatacgcctt cgccctgagc 840
cggggcttcg gcagcggcat catcaccagc aacgccccca tggacgagtg cgacgccaag 900
tgccagaccc ctcagggagc tattaacagc agcctgccct tccagaacgt gcaccccgtg 960
accatcggcg agtgccccaa gtacgtgcgg agcgccaagc tgcggatggt gaccggcctg 1020
cggaacatcc ccagcatcca gagcaggggc ctgttcggcg ccatcgccgg cttcatcgag 1080
ggcggctgga ccggcatggt ggacgggtgg tacggctacc accaccagaa cgagcagggc 1140
agcggctacg ccgccgacca gaagagcacc cagaacgcca tcaacggcat caccaacaag 1200
gtgaacagcg tgatcgagaa gatgaacacc cagttcaccg ccgtgggcaa agagttcaac 1260
aagctggaac ggcggatgga aaacctgaac aagaaggtgg acgacggctt cctggacatc 1320
tggacctaca acgccgagct gctggtgctg ctggaaaacg agcggaccct ggacttccac 1380
gacagcaacg tgaagaacct gtacgagaag gtgaaaagcc agctgaagaa caacgccaaa 1440
gagatcggca acggctgctt cgagttctac cacaagtgca acgacgagtg catggaaagc 1500
gtgaagaatg gcacctacga ctaccccaag tacagcgagg aaagcaagct gaaccgggag 1560
aagatcgacg gcgtgaagct ggaaagcatg ggcgtgtacc agatcctggc catctacagc 1620
accgtcgctt ccagcctcgt cctgctcgtg tccctgggcg ccatctcctt ttggatgtgc 1680
agcaacggca gcctgcagtg ccggatctgc atctgatgac tcgagctc 1728
<210> 22
<211> 565
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒蛋白H1序列
<400> 22
Met Lys Val Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr
1 5 10 15
Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr
20 25 30
Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn
35 40 45
Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu Leu Lys Gly Ile
50 55 60
Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly
65 70 75 80
Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile
85 90 95
Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe
100 105 110
Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe
115 120 125
Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr
130 135 140
Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser Ser Phe
145 150 155 160
Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro Asn
165 170 175
Leu Ser Lys Ser Tyr Ala Asn Asn Lys Glu Lys Glu Val Leu Val Leu
180 185 190
Trp Gly Val His His Pro Pro Asn Ile Gly Asp Gln Arg Ala Leu Tyr
195 200 205
His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg
210 215 220
Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp Gln Glu
225 230 235 240
Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile
245 250 255
Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Arg Tyr Ala Phe Ala
260 265 270
Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Pro Met
275 280 285
Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly Ala Ile Asn Ser
290 295 300
Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile Gly Glu Cys Pro
305 310 315 320
Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn
325 330 335
Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe
340 345 350
Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His
355 360 365
His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr
370 375 380
Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu
385 390 395 400
Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu
405 410 415
Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Leu
420 425 430
Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu
435 440 445
Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys
450 455 460
Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys
465 470 475 480
Phe Glu Phe Tyr His Lys Cys Asn Asp Glu Cys Met Glu Ser Val Lys
485 490 495
Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn
500 505 510
Arg Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr Gln
515 520 525
Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val
530 535 540
Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln
545 550 555 560
Cys Arg Ile Cys Ile
565
<210> 23
<211> 1731
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒DNA H3序列
<400> 23
ggtaccaagc ttgccaccat gaaaaccatc atcgccctga gctacatcct gtgcctggtg 60
ttcgcccaga agctgcccgg caacgacaac agcaccgcca ccctgtgtct gggccaccac 120
gccgtgccca acggcaccat cgtgaaaaca atcaccaacg accagatcga ggtgaccaac 180
gccaccgagc tggtgcagag cagcagcacc ggcggcatct gcgacagccc ccaccagatc 240
ctggacggcg agaactgcac cctgatcgac gccctgctgg gcgaccctca gtgcgacggc 300
ttccagaaca aaaagtggga cctgttcgtg gagcggagca aggcctacag caactgctac 360
ccctacgacg tgcccgacta cgccagcctg cggagcctgg tggccagcag cggcaccctg 420
gaattcaaca acgagagctt caactggacc ggcgtgaccc agaacggcac cagcagcgcc 480
tgcaagcggc ggagcaacaa cagcttcttt tccagactga actggctgac ccacctgaag 540
ttcaagtacc ccgccctgaa cgtgaccatg cccaacaatg agaagttcga caagctgtac 600
atctggggcg tgcaccaccc cggcaccgac aatgaccaga tcagcctgta cgcccaggcc 660
agcggccgga tcaccgtgag caccaagcgg agccagcaga ccgtgatccc caacatcggc 720
agccggccca gagtgagaga catccccagc cggatcagca tctactggac aatcgtgaag 780
cccggcgaca tcctgctgat caactccacc ggcaacctga tcgcccccag gggctacttc 840
aagatcagaa gcggcaagag cagcatcatg cggagcgacg cccccatcgg caagtgcaac 900
agcgagtgca tcacccccaa tggcagcatc cccaacgaca agcccttcca gaacgtgaac 960
cggatcacct acggcgcctg ccccagatac gtgaagcaga acaccctgaa gctggccacc 1020
ggcatgcgga acgtgcccga gaagcagacc cggggcatct tcggcgccat cgccggcttc 1080
atcgagaacg gctgggaggg catggtggac gggtggtacg gcttccggca ccagaactcc 1140
gagggcatcg gccaggccgc cgacctgaag agcacccagg ccgccatcaa ccagatcaac 1200
ggcaagctga accggctgat cggcaagacc aacgagaagt tccaccagat cgaaaaagaa 1260
ttcagcgagg tggagggccg gatccaggac ctggaaaagt acgtggagga caccaagatc 1320
gacctgtgga gctacaacgc cgagctgctg gtcgccctgg aaaaccagca caccatcgac 1380
ctgaccgaca gcgagatgaa caagctgttc gagcggacca agaagcagct gcgggagaac 1440
gccgaggaca tgggcaacgg ctgctttaag atctaccaca agtgcgacaa cgcctgcatc 1500
ggcagcatcc ggaacggcac ctacgaccac gacgtgtacc gggacgaggc cctgaacaac 1560
cggttccaga tcaagggcgt ggagctgaag agcggctaca aggactggat cctgtggatc 1620
agcttcgcca tcagctgctt tctgctgtgc gtggccctgc tgggattcat catgtgggcc 1680
tgccagaagg gcaacatccg ctgcaacatc tgcatctgat gactcgagct c 1731
<210> 24
<211> 566
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 流感病毒蛋白H3序列
<400> 24
Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala
1 5 10 15
Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly
20 25 30
His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp
35 40 45
Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr
50 55 60
Gly Gly Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly Glu Asn Cys
65 70 75 80
Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys Asp Gly Phe Gln
85 90 95
Asn Lys Lys Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn
100 105 110
Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu Val
115 120 125
Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe Asn Trp Thr
130 135 140
Gly Val Thr Gln Asn Gly Thr Ser Ser Ala Cys Lys Arg Arg Ser Asn
145 150 155 160
Asn Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr His Leu Lys Phe Lys
165 170 175
Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys
180 185 190
Leu Tyr Ile Trp Gly Val His His Pro Gly Thr Asp Asn Asp Gln Ile
195 200 205
Ser Leu Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val Ser Thr Lys Arg
210 215 220
Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser Arg Pro Arg Val Arg
225 230 235 240
Asp Ile Pro Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly
245 250 255
Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly
260 265 270
Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala
275 280 285
Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile
290 295 300
Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala
305 310 315 320
Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met
325 330 335
Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala
340 345 350
Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly
355 360 365
Phe Arg His Gln Asn Ser Glu Gly Ile Gly Gln Ala Ala Asp Leu Lys
370 375 380
Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys Leu Asn Arg Leu
385 390 395 400
Ile Gly Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser
405 410 415
Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
420 425 430
Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu
435 440 445
Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe
450 455 460
Glu Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn
465 470 475 480
Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly Ser
485 490 495
Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu
500 505 510
Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys
515 520 525
Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys
530 535 540
Val Ala Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile
545 550 555 560
Arg Cys Asn Ile Cys Ile
565
Claims (39)
1.一种分离的核酸分子,包含一个或多个选自以下所述的核酸序列:
a)选自以下所述的核酸序列:SEQ ID NO:13、与SEQ ID NO:13具有95%同源性的核酸序列、含至少60个核苷酸的SEQ ID NO:13的片段、以及与含至少60个核苷酸的SEQ ID NO:13的片段具有95%同源性的核酸序列;
b)选自以下所述的核酸序列:SEQ ID NO:15、与SEQ ID NO:15具有95%同源性的核酸序列、含至少60个核苷酸的SEQ ID NO:15的片段、以及与含至少60个核苷酸的SEQ ID NO:15的片段具有95%同源性的核酸序列;
c)选自以下所述的核酸序列:SEQ ID NO:6、与SEQ ID NO:6具有95%同源性的核酸序列、含至少60个核苷酸的SEQ ID NO:6的片段、以及与含至少60个核苷酸的SEQ ID NO:6的片段具有95%同源性的核酸序列。
2.如权利要求1所述的分离的核酸分子,含有选自SEQ ID NO:6、SEQ ID NO:13和SEQID NO:15的核酸序列。
3.如权利要求1所述的分离的核酸分子,含有选自以下所述的核酸序列:与SEQ ID NO:6具有95%同源性的核酸序列、与SEQ ID NO:13具有95%同源性的核酸序列、以及与SEQ IDNO:15具有95%同源性的核酸序列。
4.如权利要求1所述的分离的核酸分子,含有选自以下所述的核酸序列:与SEQ ID NO:6具有98%同源性的核酸序列、与SEQ ID NO:13具有98%同源性的核酸序列、以及与SEQ IDNO:15具有98%同源性的核酸序列。
5.如权利要求1所述的分离的核酸分子,含有选自SEQ ID NO:6、SEQ ID NO:13、SEQ IDNO:15的核酸序列,还含有一段编码IgE前导序列的核酸序列。
6.一种表达载体,包含权利要求1所述的核酸序列,所述核酸序列有效地连接于调控元件。
7.一种表达载体,包含权利要求1所述的核酸序列,所述核酸序列有效地连接于在人细胞中具有功能的调控元件。
8.如权利要求7所述的表达载体,所述表达载体是质粒。
9.如权利要求8所述的表达载体,所述表达载体是pGX2009(SEQ ID NO:8)。
10.一种组合物,包含:
a)多种一个或多个核酸分子,包含一个或多个选自以下所述的核酸序列:
i)选自以下所述的核酸序列:SEQ ID NO:13、与SEQ ID NO:13具有95%同源性的核酸序列、含至少60个核苷酸的SEQ ID NO:13的片段、以及与含至少60个核苷酸的SEQ ID NO:13的片段具有95%同源性的核酸序列;
ii)选自以下所述的核酸序列:SEQ ID NO:15、与SEQ ID NO:15具有95%同源性的核酸序列、含至少60个核苷酸的SEQ ID NO:15的片段、以及与含至少60个核苷酸的SEQ ID NO:15的片段具有95%同源性的核酸序列;和
iii)选自以下所述的核酸序列:SEQ ID NO:6、与SEQ ID NO:6具有95%同源性的核酸序列、含至少60个核苷酸的SEQ ID NO:6的片段、以及与含至少60个核苷酸的SEQ ID NO:6的片段具有95%同源性的核酸序列;和
b)一个或多个编码一种或多种蛋白质的其他核酸序列,所述蛋白质选自如下的一种或多种:甲型流感病毒血凝素H1、甲型流感病毒血凝素H2、甲型流感病毒血凝素H3、甲型流感病毒H4、甲型流感病毒血凝素H5、甲型流感病毒血凝素H3、甲型流感病毒血凝素H5、A血凝素N1、甲型流感病毒血凝素H6、甲型流感病毒血凝素H7、甲型流感病毒血凝素H5、甲型流感病毒血凝素H6、甲型流感病毒血凝素H7、甲型流感病毒血凝素H8、甲型流感病毒血凝素H9、甲型流感病毒血凝素H10、甲型流感病毒血凝素H11、甲型流感病毒血凝素H12、甲型流感病毒血凝素H13、甲型流感病毒血凝素H14、甲型流感病毒血凝素H15、甲型流感病毒血凝素H16、甲型流感病毒神经氨酸酶N1、甲型流感病毒神经氨酸酶N2、甲型流感病毒神经氨酸酶N3、甲型流感病毒神经氨酸酶N4、甲型流感病毒神经氨酸酶N5、甲型流感病毒神经氨酸酶N6、甲型流感病毒神经氨酸酶N7、甲型流感病毒神经氨酸酶N8、甲型流感病毒神经氨酸酶N9、乙型流感病毒血凝素和乙型流感病毒神经氨酸酶。
11.如权利要求10所述的组合物,所述一个或多个其他核酸序列存在于多种一个或多个不同的核酸分子上,所述不同的核酸分子来自a)中所述的多种核酸分子。
12.如权利要求10所述的组合物,a)中所述多种核酸分子包含一个或多个选自SEQ IDNO:6、SEQ ID NO:13和SEQ ID NO:15的核酸序列。
13.如权利要求10所述的组合物,a)中所述多种核酸分子包含一个或多个选自以下所述的核酸序列:与SEQ ID NO:6具有95%同源性的核酸序列、与SEQ ID NO:13具有95%同源性的核酸序列、以及与SEQ ID NO:15具有95%同源性的核酸序列。
14.如权利要求10所述的组合物,a)中所述多种核酸分子包含一个或多个选自以下所述的核酸序列:与SEQ ID NO:6具有98%同源性的核酸序列、与SEQ ID NO:13具有98%同源性的核酸序列、以及与SEQ ID NO:15具有98%同源性的核酸序列。
15.如权利要求10所述的组合物,a)和b)中所述核酸序列各自有效地连接于调控元件。
16.如权利要求10所述的组合物,a)和b)中所述核酸序列各自有效地连接于在人细胞中具有功能的调控元件。
17.如权利要求10所述的组合物,a)和b)中所述核酸序列是一种或多种表达载体的部分。
18.如权利要求10所述的组合物,所述一种或多种表达载体是质粒。
19.如权利要求10所述的组合物,包含pGX2009和/或pGX2006。
20.如权利要求10所述的组合物,包含:
包含SEQ ID NO:1的核酸序列;并可选地包含以下一项或多项:
包含SEQ ID NO:6的核酸序列;
包含SEQ ID NO:13的核酸序列;
包含SEQ ID NO:15的核酸序列;
编码甲型流感病毒血凝素H1的核酸序列;和编码甲型流感病毒血凝素H3的核酸序列。
21.如权利要求10所述的组合物,所述编码甲型流感病毒血凝素H1的核酸序列包含SEQID NO:21,所述编码甲型流感病毒血凝素H3的核酸序列包含SEQ ID NO:23。
22.如权利要求20所述的组合物,包含处于质粒内的:
包含SEQ ID NO:6的核酸分子;
包含SEQ ID NO:13的核酸分子;和
包含SEQ ID NO:15的核酸分子。
23.如权利要求22所述的组合物,其中
包含SEQ ID NO:6的核酸分子是质粒;
包含SEQ ID NO:13的核酸分子是质粒;且包含SEQ ID NO:15的核酸分子是质粒。
24.权利要求1所述包含核酸序列的核酸分子用于制备药物的用途,所述药物用于诱导个体内免疫反应。
25.权利要求10所述组合物用于制备药物的用途,所述药物用于诱导个体内免疫反应。
26.权利要求23所述组合物用于制备药物的用途,所述药物用于诱导个体内免疫反应。
27.以下所述核酸分子用于制备药物的用途,所述药物用于保护个体免受猪来源的人甲型流感病毒株的感染,所述核酸分子包含选自以下所述的核酸序列:
SEQ ID NO:13,
与SEQ ID NO:13具有95%同源性的核酸序列,
SEQ ID NO:13的片段,
与SEQ ID NO:13的片段具有95%同源性的核酸序列,
SEQ ID NO:15,
与SEQ ID NO:15具有95%同源性的核酸序列,
SEQ ID NO:15的片段,和
与SEQ ID NO:15的片段具有95%同源性的一段核酸序列;
SEQ ID NO:6,
与SEQ ID NO:6具有95%同源性的核酸序列,
SEQ ID NO:6的片段,和
与SEQ ID NO:6的片段具有95%同源性的核酸序列;
其中所述核酸序列在所述个体的细胞中表达并且诱导了针对所述蛋白质的免疫反应,所述免疫反应是抗猪来源的人甲型流感病毒的保护性免疫反应。
28.以下所述组合物用于制备药物的用途,所述药物用于保护个体免受猪来源的人甲型流感病毒株的感染,所述组合物包含:a)选自以下所述的第一核酸序列:
SEQIDNO:13,
与SEQ ID NO:13具有95%同源性的核酸序列,
SEQ ID NO:13的片段,和
与SEQ ID NO:13的片段具有95%同源性的核酸序列;和
SEQ ID NO:l5,
与SEQ ID NO:15具有95%同源性的核酸序列,
SEQ ID NO:15的片段,
与SEQ ID NO:15的片段具有95%同源性的核酸序列,
SEQIDNO:6,
与SEQ ID NO:6具有95%同源性的核酸序列,
SEQ ID NO:6的片段,和
与SEQ ID NO:6的片段具有95%同源性的核酸序列;和
b)一个或多个编码一种或多种蛋白质的其他核酸序列,所述蛋白质选自如下的一种或多种:甲型流感病毒血凝素H1、甲型流感病毒血凝素H2、甲型流感病毒血凝素H3、甲型流感病毒H4、甲型流感病毒血凝素H5、甲型流感病毒血凝素H3、甲型流感病毒血凝素H5、A血凝素N1、甲型流感病毒血凝素H6、甲型流感病毒血凝素H7、甲型流感病毒血凝素H5、甲型流感病毒血凝素H6、甲型流感病毒血凝素H7、甲型流感病毒血凝素H8、甲型流感病毒血凝素H9、甲型流感病毒血凝素H10、甲型流感病毒血凝素H11、甲型流感病毒血凝素H12、甲型流感病毒血凝素H13、甲型流感病毒血凝素H14、甲型流感病毒血凝素H15、甲型流感病毒血凝素H16、甲型流感病毒神经氨酸酶N1、甲型流感病毒神经氨酸酶N2、甲型流感病毒神经氨酸酶N3、甲型流感病毒神经氨酸酶N4、甲型流感病毒神经氨酸酶N5、甲型流感病毒神经氨酸酶N6、甲型流感病毒神经氨酸酶N7、甲型流感病毒神经氨酸酶N8、甲型流感病毒神经氨酸酶N9、乙型流感病毒血凝素和乙型流感病毒神经氨酸酶;
其中所述第一核酸序列在所述个体的细胞中表达并且诱导了针对所述第一蛋白质的免疫反应,所述免疫反应是抗猪来源的人甲型流感病毒的保护性免疫反应,所述一个或多个其他的核酸序列在所述个体的细胞中表达并且诱导了抗所述一种或多种第二蛋白质的免疫反应。
29.如权利要求28所述的用途,所述组合物包含含有以下所述项的核酸序列:
SEQ ID NO:1;和可选的以下一项或多项:
包含SEQ ID NO:6的核酸序列;
包含SEQ ID NO:13的核酸序列;
包含SEQ ID NO:15的核酸序列;
编码甲型流感病毒血凝素H1的核酸序列;和
编码甲型流感病毒血凝素H3的核酸序列。
30.如权利要求28所述的用途,所述编码甲型流感病毒血凝素H1的核酸序列包含SEQID NO:21,所述编码甲型流感病毒血凝素H3的核酸序列包含SEQ ID NO:23。
31.如权利要求28所述的用途,所述组合物包含以下一项或多项:
包含SEQ ID NO:6的核酸分子;
包含SEQ ID NO:13的核酸分子;
包含SEQ ID NO:15的核酸分子;
和包含SEQ ID NO:23的核酸分子。
32.如权利要求31所述的用途,其中,
所述包含SEQ ID NO:6的核酸分子是质粒;
所述包含SEQ ID NO:13的核酸分子是质粒;
所述包含SEQ ID NO:15的核酸分子是质粒;以及
所述包含SEQ ID NO:23的核酸分子是质粒。
33.以下所述核酸分子用于制备药物的用途,所述药物用于治疗已被猪来源的人甲型流感病毒株感染的个体,所述核酸分子包含选自以下所述的核酸序列:
SEQ ID NO:13,
与SEQ ID NO:13具有95%同源性的核酸序列;
SEQ ID NO:13的片段,
与SEQ ID NO:13的片段具有95%同源性的核酸序列;
SEQ ID NO:15,
与SEQ ID NO:15具有95%同源性的核酸序列;
SEQ ID NO:15的片段,
与SEQ ID NO:15的片段具有95%同源性的核酸序列;
SEQ ID NO:6,
与SEQ ID NO:6具有95%同源性的核酸序列;
SEQ ID NO:6的片段,和
与SEQ ID NO:6的片段具有95%同源性的核酸序列;
其中所述核酸序列在所述个体的细胞中表达并且诱导了针对所述蛋白质的免疫反应,所述免疫反应是抗猪来源的人甲型流感病毒的保护性免疫反应。
34.以下所述组合物用于制备药物的用途,所述药物用于治疗已被猪来源的人甲型流感病毒株感染的个体,所述组合物包含:
a)第一核酸序列,选自:
SEQ ID NO:13,
与SEQ ID NO:13具有95%同源性的核酸序列;
SEQ ID NO:13的片段,
与SEQ ID NO:13的片段具有95%同源性的核酸序列;
SEQ ID NO:l5,
与SEQ ID NO:l5具有95%同源性的核酸序列;
SEQ ID NO:15的片段,
与SEQ ID NO:15的片段具有95%同源性的核酸序列;
SEQIDNO:6,
与SEQ ID NO:6具有95%同源性的核酸序列;
SEQ ID NO:6的片段,和
与SEQ ID NO:6的片段具有95%同源性的核酸序列;和
b)一个或多个编码一种或多种蛋白质的其他核酸序列,所述蛋白质选自如下的一种或多种:甲型流感病毒血凝素H1、甲型流感病毒血凝素H2、甲型流感病毒血凝素H3、甲型流感病毒H4、甲型流感病毒血凝素H5、甲型流感病毒血凝素H3、甲型流感病毒血凝素H5、A血凝素N1、甲型流感病毒血凝素H6、甲型流感病毒血凝素H7、甲型流感病毒血凝素H5、甲型流感病毒血凝素H6、甲型流感病毒血凝素H7、甲型流感病毒血凝素H8、甲型流感病毒血凝素H9、甲型流感病毒血凝素H10、甲型流感病毒血凝素H11、甲型流感病毒血凝素H12、甲型流感病毒血凝素H13、甲型流感病毒血凝素H14、甲型流感病毒血凝素H15、甲型流感病毒血凝素H16、甲型流感病毒神经氨酸酶N1、甲型流感病毒神经氨酸酶N2、甲型流感病毒神经氨酸酶N3、甲型流感病毒神经氨酸酶N4、甲型流感病毒神经氨酸酶N5、甲型流感病毒神经氨酸酶N6、甲型流感病毒神经氨酸酶N7、甲型流感病毒神经氨酸酶N8、甲型流感病毒神经氨酸酶N9、乙型流感病毒血凝素和乙型流感病毒神经氨酸酶;
其中所述第一核酸序列在所述个体的细胞中表达并且诱导了针对所述第一蛋白质的免疫反应,所述免疫反应是抗猪来源的人甲型流感病毒的保护性免疫反应,所述一个或多个其他的核酸序列在所述个体的细胞中表达并且诱导了抗所述一种或多种第二蛋白质的免疫反应。
35.如权利要求34所述的用途,所述组合物包含含有以下所述项的核酸序列:
SEQ ID NO:1;和可选的以下一项或多项:
包含SEQ ID NO:6的核酸序列;
包含SEQ ID NO:13的核酸序列;
包含SEQ ID NO:15的核酸序列;
编码甲型流感病毒血凝素Hl的核酸序列;和编码甲型流感病毒血凝素H3的核酸序列。
36.如权利要求34所述的用途,所述编码甲型流感病毒血凝素H1的核酸序列包含SEQID NO:21,所述编码甲型流感病毒血凝素H3的核酸序列包含SEQ ID NO:23。
37.如权利要求34所述的用途,所述组合物包含以下一项或多项:
包含SEQ ID NO:6的核酸分子;
包含SEQ ID NO:13的核酸分子;
包含SEQ ID NO:15的核酸分子;和
包含SEQ ID NO:23的核酸分子。
38.如权利要求37所述的用途,其中
所述包含SEQ ID NO:6的核酸分子是质粒;
所述包含SEQ ID NO:13的核酸分子是质粒;
所述包含SEQ ID NO:15的核酸分子是质粒;
所述包含SEQ ID NO:23的核酸分子是质粒。
39.如权利要求1所述分离的核酸分子,编码选自以下至少其一的氨基酸序列:
a)选自SEQ ID NO:7和与SEQ ID NO:7具有95%同源性的氨基酸序列的氨基酸序列;
b)选自SEQ ID NO:14和与SEQ ID NO:14具有95%同源性的氨基酸序列的氨基酸序列;
c)选自SEQ ID NO:16和与SEQ ID NO:16具有95%同源性的氨基酸序列的氨基酸序列。
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WO2012036993A1 (en) | 2010-09-14 | 2012-03-22 | University Of Pittsburgh-Of The Commonwealth System Of Higher Education | Computationally optimized broadly reactive antigens for influenza |
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AU2012273039B2 (en) | 2011-06-20 | 2016-12-01 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Computationally optimized broadly reactive antigens for H1N1 influenza |
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US10040828B2 (en) | 2012-04-10 | 2018-08-07 | The Trustees Of The University Of Pennsylvania | Human respiratory syncytial virus consensus antigens, nucleic acid constructs and vaccines made therefrom, and methods of using same |
AU2013352179B2 (en) | 2012-11-27 | 2017-12-21 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Computationally optimized broadly reactive antigens for H1N1 influenza |
KR20150093834A (ko) | 2012-12-13 | 2015-08-18 | 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 | Dna 항체 작제물 및 그 이용 방법 |
CN104812401B (zh) * | 2012-12-13 | 2017-10-13 | 宾夕法尼亚大学理事会 | Wt1疫苗 |
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EP2969005B1 (en) | 2013-03-15 | 2019-10-16 | The Research Foundation for The State University of New York | Attenuated influenza viruses and vaccines |
WO2015023461A2 (en) * | 2013-08-06 | 2015-02-19 | The Trustees Of The University Of Pennsylvania | Influenza nucleic acid molecules and vaccines made therefrom |
JP6649252B2 (ja) * | 2013-10-07 | 2020-02-19 | ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア | アジュバントとしてのインターロイキン−33を有するワクチン |
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CA2969214A1 (en) | 2014-12-01 | 2016-06-09 | The Trustees Of The University Of Pennsylvania | Dna antibody constructs and method of using same |
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