CN107522788A - 包含流感嗜血杆菌蛋白e和菌毛蛋白a的融合蛋白和组合疫苗 - Google Patents

包含流感嗜血杆菌蛋白e和菌毛蛋白a的融合蛋白和组合疫苗 Download PDF

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CN107522788A
CN107522788A CN201710656150.1A CN201710656150A CN107522788A CN 107522788 A CN107522788 A CN 107522788A CN 201710656150 A CN201710656150 A CN 201710656150A CN 107522788 A CN107522788 A CN 107522788A
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N.布莱斯
S.拉贝
J.普尔曼
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Abstract

本发明涉及包含流感嗜血杆菌蛋白E和菌毛蛋白A的组合物。更具体地，本申请涉及包含蛋白E和PilA的融合蛋白和免疫原性组合物、包含所述免疫原性组合物的疫苗和其治疗用途。

Description

包含流感嗜血杆菌蛋白E和菌毛蛋白A的融合蛋白和组合疫苗
本申请是以下申请的分案申请：申请日：2012年4月12日；申请号：201280018032.3(PCT/CA2012/050236)；发明名称：同上。
本申请主张2011年4月13日提交的美国专利申请号61/474779和2011年9月13日提交的美国专利申请号61/534012的优先权。
发明领域
本发明涉及包含流感嗜血杆菌(Haemophilus influenzae，H. influenzae)蛋白E和菌毛蛋白A的组合物。更具体地，本申请涉及包含蛋白E和菌毛蛋白A的融合蛋白和免疫原性组合物、包含所述免疫原性组合物的疫苗和其治疗用途。
发明背景
蛋白E(PE)是具有粘附特性的外膜脂蛋白。其在无法分型的流感嗜血杆菌(NTHi)粘附至/侵袭上皮细胞中发挥作用。(J. Immunology 183: 2593-2601 (2009); The Journalof Infectious Diseases 199:522-531 (2009), Microbes and Infection 10:87-96(2008))。其在有荚膜的流感嗜血杆菌和无法分型的流感嗜血杆菌二者中高度保守，且具有保守的上皮结合结构域。(The Journal of Infectious Diseases 201:414-419 (2010))。当与作为参考菌株的流感嗜血杆菌Rd比较时，已在不同嗜血杆菌物种中描述了13种不同点突变。在对数生长期和静止期细菌中都观察到其表达。(WO2007/084053)。
蛋白E也经由结合玻连蛋白而参与人补体抗性。(Immunology 183: 2593-2601(2009))。PE通过结合结构域PKRYARSVRQ YKILNCANYH LTQVR (SEQ ID NO. 1，对应于SEQID NO. 4的氨基酸84-108)结合玻连蛋白，所述玻连蛋白是末端补体途径的重要抑制剂。(J. Immunology 183:2593-2601 (2009))。
菌毛蛋白A(PilA)可能是参与蹭行运动(twitching motility)的IV型流感嗜血杆菌菌毛(Tfp)的主要菌毛蛋白亚单位(Infection and Immunity, 73: 1635-1643(2005))。NTHi PilA是在体内表达的保守粘附素。已显示其参与NTHi粘附、定殖和生物膜形成。(Molecular Microbiology 65: 1288-1299 (2007))。
无法分型的流感嗜血杆菌是引起婴儿和儿童中耳炎的重要且常见的呼吸病原体。NTHi仅次于肺炎链球菌(Streptococcus pneumoniae)，是儿童急性中耳炎的最常见原因(J. Immunology 183: 2593-2601 (2009), Pediatrics 113:1451-1465 (2004))。其是儿童和成人鼻窦炎的重要原因。(Current Infectious Disease Reports 11:177-182(2009))。其与成人慢性阻塞性肺病(COPD)的恶化风险增加有关。(Journal of ChronicObstructive Pulmonary Disease 3:109-115 (2006))。此外，无法分型的流感嗜血杆菌引起成人社区获得性肺炎，且可在发展中国家中引起儿童肺炎。(Current InfectiousDisease Reports 11:177-182 (2009))。
当前需要NTHi疫苗。
发明概述
作为第一方面，本发明提供式(I)的融合蛋白
其中：
X是信号肽或MHHHHHH (SEQ ID NO. 2)；
m是0或1；
R1是一个氨基酸；
n是0、1、2、3、4、5或6；
A是来自流感嗜血杆菌的蛋白E或其免疫原性片段、或来自流感嗜血杆菌的PilA或其免疫原性片段；
Y选自GG、SG、SS、GGG和(G)h，其中h是4、5、6、7、8、9、或10；
o是0或1；
B是来自流感嗜血杆菌的PilA或其免疫原性片段、或来自流感嗜血杆菌的蛋白E或其免疫原性片段；
Z是GGHHHHHH (SEQ ID NO. 3)；且
p是0或1。
作为第二方面，本发明提供包含式(I)的融合蛋白的免疫原性组合物。该组合物可进一步包含药学可接受的佐剂。该组合物可包含赋形剂。
在第三方面，本发明提供用于治疗或预防完全或部分由流感嗜血杆菌引起的状况或疾病的方法。该方法包括将治疗有效量的式(I)的融合蛋白施用于需要其的受试者。
在第四方面，本发明提供用于治疗或预防中耳炎的方法。该方法包括将治疗有效量的式(I)的融合蛋白施用于需要其的受试者。
在第五方面，本发明提供用于治疗或预防慢性阻塞性肺病恶化的方法。该方法包括将治疗有效量的式(I)的融合蛋白施用于需要其的受试者。
在第六方面，本发明提供用于治疗或预防肺炎的方法。该方法包括将治疗有效量的式(I)的融合蛋白施用于需要其的受试者。
在第七方面，本发明提供包含式(I)的融合蛋白的药物组合物，其用于治疗或预防完全或部分由流感嗜血杆菌引起的状况或疾病。药物组合物可进一步包含药学可接受的佐剂。
在第八方面，本发明提供编码本发明蛋白的核酸。
在第九方面，本发明提供产生本发明核酸的方法。
本发明的其他方面在下文的特定实施方案、实施例和权利要求的详细说明中予以描述。
附图简要说明
图1. 融合蛋白构建体LVL291、LVL268和LVL269的诱导的细菌提取物的SDS-PAGE。对于LVL291、LVL268和LVL269，在诱导(ind)之前和之后上样不可溶级分(I)、可溶级分(S)和培养基级分(M)。
图2. 融合蛋白构建体LVL291、LVL268和LVL269的纯化提取物相关的SDS-PAGE和Western印迹。对于LVL291、LVL268和LVL269的纯化，上样流通物级分(Ft)、洗涤级分(W)和洗脱级分(E)。使用抗his标签来探测提取物。
图3. 融合蛋白构建体LVL291和LVL315的诱导的细菌和纯化提取物的SDS-PAGE。对于LVL291和LVL315，上样培养基级分(M)、可溶级分(Sol)、不可溶级分(Ins)、流通物级分(Ft)、洗涤级分#1(W1)、洗涤级分#2(W2)和洗脱级分(E)。
图4. 融合蛋白构建体LVL312的诱导的细菌和纯化提取物的SDS-PAGE。对于LVL312，上样培养基级分(M)、可溶级分(Sol)、不可溶级分(Ins)、流通物级分(Ft)、洗涤级分#1(W1)、洗涤级分#2(W2)和洗脱级分(E)。
图5. 融合蛋白构建体LVL317的诱导(1 mM和10μM IPTG)细菌提取物的SDS-PAGE。诱导之前(NI)和诱导之后(In)的提取物、可溶级分(S)、不可溶级分(I)。
图6. 融合蛋白构建体LVL318的诱导(1 mM和10μM IPTG)细菌提取物的SDS-PAGE。诱导之前(NI)和诱导之后(In)的提取物、培养基级分(M)、可溶级分(S)、不可溶级分(I)。
图7. PE、PilA和PE-PilA融合蛋白的CD谱。
图8. PE与PilA CD谱的组合。
图9. PilA热变性曲线。
图10. PE变性曲线。
图11. PE-PilA融合蛋白热变性曲线。
图12. 典型SP SepharoseTM快速流动层析图。
图13. 典型Q SepharoseTM快速流动层析图。
图14. 来自PE-PilA融合蛋白的纯化过程的过程中样品的SDS-PAGE。
图15. 来自PE-PilA融合蛋白的纯化过程的过程中样品的Western印迹。使用兔多克隆抗PE实施印迹。
图16. 来自PE-PilA融合蛋白的纯化过程的过程中样品的Western印迹。使用兔多克隆抗大肠杆菌(BLR)实施印迹。
图17. PE-PilA融合蛋白和PE和PilA蛋白的热转化。曲线：PilA (1)，蛋白E(ProtE，PE) (2)，PE-PilA纯化的容积物未稀释，737µg/ml (3)，和PE-PilA纯化的容积物，稀释至最终容器浓度60µg/ml (4)。
图18. Balb/c小鼠模型中针对LVL291 PE-PilA融合蛋白和针对单价PE和PilA的抗体应答。
图19. PE-PilA融合蛋白接种对小鼠鼻咽中NTHi菌株86-028NP细菌清除的影响。
图20. PE-PilA融合蛋白接种对小鼠鼻咽中NTHi菌株3224A细菌清除的影响。
图21. PilA接种对小鼠鼻咽中细菌清除的影响。
图22. PE接种对小鼠鼻咽中细菌清除的影响。
图23. (a)结合至玻连蛋白的LVL317 PE-PilA融合蛋白；和(b)结合至玻连蛋白的LVL317和LVL735 PE-PilA融合蛋白。
图24. 针对PE-PilA融合蛋白的多克隆抗体对玻连蛋白结合的抑制。
图25. 融合蛋白构建体LVL291、LVL702、LVL736、LVL737、LVL738、LVL739、LVL740和pET26b载体(阴性对照)的诱导的细菌提取物的可溶级分的SDS-PAGE。(a)实验1(b)实验2(c)实验3。PE-PilA融合蛋白由箭头指示。
图26. 来自实验1、2和3的可溶级分中融合蛋白的平均条带百分比。
图27. 针对LVL317和LVL735的PE和PilA抗体应答。
图28. LVL735和LVL317接种对无法分型的流感嗜血杆菌鼻咽定殖的小鼠模型中的细菌清除的影响。
发明详述
除非另有解释或本文定义，否则本文使用的所有技术和科学术语具有本公开所属领域普通技术人员通常理解的相同含义。例如，分子生物学的常见术语的定义可参见 BenjaminLewin, Genes V, Oxford University Press出版, 1994 (ISBN 0-19-854287-9);Kendrew等人(编), The Encyclopedia of Molecular Biology, Blackwell Science Ltd出版，1994 (ISBN 0-632-02182-9)；以及Robert A. Meyers (编), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc.出版,1995 (ISBN 1-56081-569-8)。
单数词语“一(a)”、“一(an)”和“该(the)”包括复数的对象，除非上下文明确另有说明。类似地，单词“或”意味着包括“和”，除非上下文明确另有说明。还要进一步理解的是，对于核酸或多肽给出的所有碱基大小或氨基酸大小和所有分子量或分子质量值是近似的，且提供用于说明。另外，给出的有关物质(诸如抗原)的浓度或水平的数值限制可以是近似的。因此，如果指明浓度为(例如)近似200 pg，则是指此浓度包括稍大于或稍小于(“约”或“~”) 200 pg的值。
虽然类似于或等同于本文所述方法和材料的方法和材料可用于实施或检验本公开，但下面描述了合适的方法和材料。
术语“包含(comprises)”是指“包括(includes)”。因此，除非上下文另有要求，否则词语“包含(comprises)”和各种变化诸如“包含(comprise)”和“包含(comprising)”应理解为表示含有规定的化合物或组合物(例如核酸、多肽、抗原)或步骤，或一组化合物或一组步骤，但非排除任何其他的化合物、组合物、步骤或其组。缩写词“e.g.”源于拉丁文exempligratia，本文用来表示非限制性实例。因此，缩写词“e.g.”是术语“例如”的同义词。
为了协助对本公开各种实施方案的概述，提供以下术语解释。在本公开的上下文中提供其他术语和解释。
如本文使用，“受试者”是哺乳动物，包括人、非人灵长类动物和非灵长类哺乳动物，诸如啮齿类动物成员(包括但不限于小鼠和大鼠)和兔形目(Lagomorpha)成员(包括但不限于兔)。
如本文使用，“蛋白E(Protein E)”、“蛋白E(protein E)”、“Prot E”和“PE”意指来自流感嗜血杆菌的蛋白E。蛋白E可由以下氨基酸序列组成或包含以下氨基酸序列：
以及在整个长度上与SEQ ID NO. 4至少或确切地75%、77%、80%、85%、90%、95%、97%、99%或100%相同的序列。来自流感嗜血杆菌的53个蛋白E序列(表1，SEQ IDNO. 5-SEQ ID NO. 57)的比较显示与如SEQ ID NO. 4中所述的蛋白E具有约77%至约100%同一性。例如，在蛋白E的氨基酸序列中，氨基酸#20可以是异亮氨酸(I)或苏氨酸(T)；氨基酸#23可以是丙氨酸(A)或缬氨酸(V)；氨基酸#24可以是赖氨酸(K)或谷氨酸(E)；氨基酸#31可以是丙氨酸(A)或苏氨酸(T)；氨基酸#32可以是脯氨酸(P)或丙氨酸(A)；氨基酸#34可以是苏氨酸(T)或丙氨酸(A)；氨基酸#37可以是精氨酸(R)或谷氨酰胺(Q)；氨基酸#47可以是缬氨酸(V)或丙氨酸(A)；氨基酸#57可以是色氨酸(W)或可不存在(-)；氨基酸#70可以是丙氨酸(A)或苏氨酸(T)；氨基酸#93可以是谷氨酰胺(Q)或不存在(-)；氨基酸#109可以是苏氨酸(T)或异亮氨酸(I)；氨基酸#119可以是甘氨酸(G)或丝氨酸(S)；氨基酸#153可以是谷氨酸(E)或赖氨酸(K)；氨基酸#156可以是丝氨酸(S)或亮氨酸(L)；氨基酸#160可以是赖氨酸(K)或天冬酰胺(N)；氨基酸#161可以是赖氨酸(K)、异亮氨酸(I)或不存在(-)；氨基酸#162- #195 可不存在，或如SEQ ID NO. 15 中所述(其中(-)指示氨基酸#166不存在)或如SEQID NO. 16 中所述；或其任何组合。
蛋白E可由选自下列的任一或多个氨基酸处与SEQ ID NO. 4不同的氨基酸序列组成或包含该氨基酸序列：氨基酸#20、氨基酸#23、氨基酸#24、氨基酸#31、氨基酸#32、氨基酸#34、氨基酸#37、氨基酸#47、氨基酸#57、氨基酸#70、氨基酸#93、氨基酸#109、氨基酸#119、氨基酸#153、氨基酸#156、氨基酸#160、氨基酸#161和氨基酸#162-#195，其中氨基酸#20是苏氨酸(T)；氨基酸#23是缬氨酸(V)；氨基酸#24是赖氨酸(K)；氨基酸#31是苏氨酸(T)；氨基酸#32是丙氨酸(A)；氨基酸#34是丙氨酸(A)；氨基酸#37是谷氨酰胺(Q)；氨基酸#47是丙氨酸(A)；氨基酸#57不存在(-)；氨基酸#70是苏氨酸(T)；氨基酸#93不存在(-)；氨基酸#109是异亮氨酸(I)；氨基酸#119是丝氨酸(S)；氨基酸#153是赖氨酸(K)；氨基酸#156是亮氨酸(L)；氨基酸#160是天冬酰胺(N)；氨基酸#161是赖氨酸(K)或异亮氨酸(I)；或氨基酸#162- #195是如SEQ ID NO. 15中所述(其中(-)指示氨基酸#166不存在)或如SEQ ID NO. 16中所述。
表1：来自53株流感嗜血杆菌的蛋白E氨基酸序列(SEQ ID NO. 5 - SEQ ID NO.57)。-指示氨基酸不存在。
蛋白E可以是来自以下流感嗜血杆菌菌株的蛋白E：
蛋白E可以是如SEQ ID NO. 5-SEQ ID NO. 57中的任一个中所述的蛋白E。
蛋白E可以是在整个长度上与SEQ ID NO. 4-SEQ ID NO. 57中的任一个具有至少95%同一性的序列。蛋白E可以是在整个长度上与表1中SEQ ID NO. 5-SEQ ID NO. 57所述序列中的任一个具有至少95%同一性的序列。
蛋白E的免疫原性片段包含SEQ ID NO. 4的至少7个、10个、15个、20个、25个、30个或50个连续氨基酸的免疫原性片段。所述免疫原性片段可引发可结合SEQ ID NO. 4 的抗体。
蛋白E的免疫原性片段可包含SEQ ID NO. 4 – SEQ ID NO. 57中的任一个的至少7个、10个、15个、20个、25个、30个或50个连续氨基酸的免疫原性片段。所述免疫原性片段可引发可结合衍生出该片段的全长序列的抗体。
蛋白E的免疫原性片段包含SEQ ID NO. 5 – SEQ ID NO. 57的至少7个、10个、15个、20个、25个、30个或50个连续氨基酸的免疫原性片段。所述免疫原性片段可引发可结合衍生出该片段的全长序列的抗体。
如本文使用，“PilA”意指来自流感嗜血杆菌的菌毛蛋白A。PilA可由以下蛋白序列组成或包含以下蛋白序列：SEQ ID NO. 58的蛋白序列
以及与SEQ ID NO. 58具有80%至100%同一性的序列。例如，PilA可与SEQ ID NO. 58至少80%、85%、90%、95%、97%或100%相同。来自流感嗜血杆菌的64个PilA序列(表2，SEQ ID NO. 58 – SEQ ID NO. 121)的全长比较显示与如SEQ ID NO.58中所述的PilA约80%至100%同一性。例如，在PilA的氨基酸序列中，氨基酸#6可以是谷氨酰胺(Q)或亮氨酸(L)；氨基酸#7可以是谷氨酰胺(Q)或苏氨酸(T)；氨基酸#37可以是谷氨酰胺(Q)或赖氨酸(K)；氨基酸#44可以是丙氨酸(A)或丝氨酸(S)；氨基酸#57可以是丙氨酸(A)或丝氨酸(S)；氨基酸#67可以是天冬酰胺(N)或甘氨酸(G)；氨基酸#68可以是谷氨酸(E)或赖氨酸(K)；氨基酸#69可以是苏氨酸(T)或脯氨酸(P)；氨基酸#71可以是赖氨酸(K)、天冬酰胺(N)、丝氨酸(S)或苏氨酸(T)；氨基酸#73可以是苏氨酸(T)、丝氨酸(S)或甲硫氨酸(M)；氨基酸#76可以是赖氨酸(K)、丝氨酸(S)或天冬酰胺(N)；氨基酸#84可以是苏氨酸(T)或赖氨酸(K)；氨基酸#86可以是丙氨酸(A)或缬氨酸(V)；氨基酸#91可以是赖氨酸(K)或丙氨酸(A)；氨基酸#94可以是苏氨酸(T)、异亮氨酸(I)或赖氨酸(K)；氨基酸#96可以是丝氨酸(S)或谷氨酰胺(Q)；氨基酸#97可以是天冬酰胺(N)或丝氨酸(S)；氨基酸#99可以是丙氨酸(A)或甘氨酸(G)；氨基酸#103可以是丙氨酸(A)或赖氨酸(K)；氨基酸#109可以是天冬氨酸(D)、丙氨酸(A)或苏氨酸(T)；氨基酸#110可以是甘氨酸(G)、天冬酰胺(N)或精氨酸(R)；氨基酸#112可以是丝氨酸(S)或谷氨酸(E)；氨基酸#114可以是苏氨酸(T)或异亮氨酸(I)；氨基酸#116可以是苏氨酸(T)或谷氨酰胺(Q)；氨基酸#118可以是谷氨酸(E)、苏氨酸(T)、丙氨酸(A)、赖氨酸(K)或丝氨酸(S)；氨基酸#121可以是丝氨酸(S)或丙氨酸(A)；氨基酸#122可以是丙氨酸(A)或苏氨酸(T)；氨基酸#123可以是赖氨酸(K)、苏氨酸(T)或丙氨酸(A)；氨基酸#128可以是赖氨酸(K)或苏氨酸(T)；氨基酸#135可以是天冬氨酸(D)或谷氨酸(E)；氨基酸#136可以是丙氨酸(A)或苏氨酸(T)；氨基酸#145可以是甘氨酸(G)或精氨酸(R)；氨基酸#149可以是谷氨酰胺(Q)或赖氨酸(K)；或其任何组合。
Pil A可由在选自下列的任一或多个氨基酸处与SEQ ID NO. 58不同的氨基酸序列组成或包含该氨基酸序列：氨基酸#6、氨基酸#7、氨基酸#37、氨基酸#44、氨基酸#57、氨基酸#67、氨基酸#68、氨基酸#69、氨基酸#71、氨基酸#73、氨基酸#76、氨基酸#84、氨基酸#86、氨基酸#91、氨基酸#94、氨基酸#96、氨基酸#97、氨基酸#99、氨基酸#103、氨基酸#109、氨基酸#110、氨基酸#112、氨基酸#114、氨基酸#116、氨基酸#118 氨基酸#121、氨基酸#122、氨基酸#123、氨基酸#128、氨基酸#135、氨基酸#136、氨基酸#145和氨基酸#149，其中氨基酸#6是亮氨酸(L)；氨基酸#7是苏氨酸(T)；氨基酸#37是赖氨酸(K)；氨基酸#44是丝氨酸(S)；氨基酸#57是丝氨酸(S)；氨基酸#67是甘氨酸(G)；氨基酸#68是赖氨酸(K)；氨基酸#69是脯氨酸(P)；氨基酸#71是赖氨酸(K)、丝氨酸(S)或苏氨酸(T)；氨基酸#73是丝氨酸(S)或甲硫氨酸(M)；氨基酸#76是丝氨酸(S)或天冬酰胺(N)；氨基酸#84是赖氨酸(K)；氨基酸#86是缬氨酸(V)；氨基酸#91是丙氨酸(A)；氨基酸#94是异亮氨酸(I)或赖氨酸(K)；氨基酸#96是谷氨酰胺(Q)；氨基酸#97是丝氨酸(S)；氨基酸#99是甘氨酸(G)；氨基酸#103是丙氨酸(A)；氨基酸#109是天冬氨酸(D)或苏氨酸(T)；氨基酸#110是甘氨酸(G)或精氨酸(R)；氨基酸#112是丝氨酸(S)；氨基酸#114是苏氨酸(T)；氨基酸#116是苏氨酸(T)；氨基酸#118是谷氨酸(E)、丙氨酸(A)、赖氨酸(K)或丝氨酸(S)；氨基酸#121是丝氨酸(S)；氨基酸#122是苏氨酸(T)；氨基酸#123是赖氨酸(K)或丙氨酸(A)；氨基酸#128是赖氨酸(K)；氨基酸#135是谷氨酸(E)；氨基酸#136是苏氨酸(T)；氨基酸#145是精氨酸(R)；氨基酸#149是赖氨酸(K)。
表2：来自64株流感嗜血杆菌的菌毛蛋白A氨基酸序列(SEQ ID NO. 58 – SEQ IDNO. 121)。
PilA可以是来自以下流感嗜血杆菌菌株的PilA：
来自流感嗜血杆菌菌株D204CD的PilA的氨基酸序列描述于SEQ ID NO. 106中，其中位置#116上的X是谷氨酰胺(Q)或亮氨酸(L)；关于位置#116上的氨基酸的不明确性可通过技术解析编码氨基酸#116的第二核苷酸来澄清，从而明确菌株D204CD的PilA序列。PilA可以是如SEQ ID NO. 58-SEQ ID NO. 121中任一个所述的PilA。
PilA可以是在整个长度上与SEQ ID NO. 58-SEQ ID NO. 121 (如表2中所述)中任一个具有至少95%同一性的序列。
PilA的免疫原性片段包含SEQ ID NO. 58 – SEQ ID NO. 121的至少7个、10个、15个、20个、25个、30个或50个连续氨基酸的免疫原性片段。所述免疫原性片段可引发可结合衍生出该片段的全长序列的抗体。
例如，PilA的免疫原性片段包含SEQ ID NO. 58的至少7个、10个、15个、20个、25个、30个或50个连续氨基酸的免疫原性片段。所述免疫原性片段可引发可结合SEQ ID NO.58的抗体。
多肽间的同一性可通过多种算法来计算。例如，可使用来自EMBOSS软件包(免费软件；EMBOSS: The European Molecular Biology Open Software Suite (2000). Trendsin Genetics 16(6): 276—277)的Needle程序和来自GCG® 软件包(Accelrys公司)的Gap程序。此Gap程序是描述于Needleman, S. B. 和Wunsch, C. D. (1970) J. Mol. Biol.48, 443-453中的Needleman-Wunsch算法的执行。已使用BLOSUM62评分矩阵，且空位开放和延伸罚分分别为8和2。
查看所计算的比对，可观察到两个比较序列间的相同残基。可通过以下来计算同一性百分比：(1)计算相同残基数除以比对长度，乘以100(例如，对于Needle程序分析)，(2)计算相同残基数除以最长序列长度，乘以100，(3)计算相同数除以最短序列长度，乘以100，或(4)计算相同数除以比对残基数，乘以100(如果一个残基在另一残基之前，则该残基为比对残基)(例如，对于Gap程序分析)。
如本文使用，“佐剂”意指当与疫苗、免疫治疗剂或其他含有抗原或免疫原的组合物一起施用于受试者时增加或增强受试者对施用的抗原或免疫原的免疫应答(与佐剂不存在的情况下获得的免疫应答相比)的化合物或物质。这不同于National Cancer Instituteof the United States Institutes of Health定义的在癌症治疗背景中作为初步治疗后给予的额外治疗以降低癌症复发的风险的“佐剂治疗”。
保守取代是众所周知的，并且通常设置为序列比对计算机程序中的默认评分矩阵。这些程序包括PAM250(Dayhoft M.O.等人, (1978), “A model of evolutionarychanges in proteins”, In“Atlas of Protein sequence and structure”5(3) M.O.Dayhoft (编), 345-352), 国家生物医学研究基金会, 华盛顿, 和Blosum 62 (StevenHenikoft and Jorja G. Henikoft (1992), “Amino acid substitution matrices fromprotein blocks”), Proc. Natl. Acad. Sci. USA 89 (Biochemistry): 10915-10919。本发明进一步提供含有保守氨基酸取代的式(I)的融合蛋白。例如，式(I)的融合蛋白可含有如本文所述序列中的任一个中所描述的流感嗜血杆菌PE或PilA(例如，描述于SEQ IDNO. 4-SEQ ID NO. 57中的任一PE序列和/或描述于SEQ ID NO. 58-SEQ ID NO.121中的任一PilA序列)中的任一氨基酸的保守取代。
如本文使用，“信号肽”是指存在于前体蛋白上(通常在N末端)且通常在成熟蛋白中不存在的短(小于60个氨基酸，例如，3至60个氨基酸)多肽。信号肽(sp)通常富含疏水氨基酸。信号肽指导翻译蛋白转运穿过膜和/或翻译蛋白分泌。信号肽也可称为靶向信号、转送肽、定位信号或信号序列。例如，信号序列可以是共翻译或翻译后信号肽。
异源信号肽可在蛋白转运或分泌期间或之后通过信号肽肽酶从融合蛋白构建体切割。例如，信号肽肽酶是信号肽肽酶I。“异源”信号肽是当存在于自然界中时不与所述蛋白结合的信号肽。
如本文使用，“治疗”意指预防受试者状况或疾病的症状的发生、预防受试者状况或疾病的症状的复发、延迟受试者状况或疾病的症状的复发、降低受试者状况或疾病的症状的严重度或频率、减缓或消除受试者状况进展和部分或完全消除受试者疾病或状况的症状。
如本文使用，“任选地”意指随后描述的事件可发生或可不发生，且包括发生的事件和不发生的事件。
NTHi引起的疾病的发病以鼻咽定殖开始。在鼻咽微环境内粘附和保持长期停留的机制视为NTHi的“毒力决定因素”。(Vaccine 28：279-289 (2010))。
NTHi能够粘附至人宿主的黏膜上皮表面的重要性反映在由NTHi表达的粘附素的多样性。例如，一些NTHi表达菌毛。其他粘附结构属于蛋白的自转运蛋白家族；这些包括Hap、HMW1/HMW2和Hia/Hsf蛋白。已描述其他外膜蛋白，诸如P2蛋白、P5蛋白和OapA作为流感嗜血杆菌的粘附素。(Cellular Microbiology 4:191-200 (2002), Microbes andInfection 10: 87-96 (2008), Vaccine 28: 279-289 (2010))。
中耳炎是80%的所有小于3岁儿童发病的主要原因。(Expert Rev. Vaccines 5:517-534 (2006))。超过90%的儿童在7岁之前会发生中耳炎(Current Opinion inInvestigational Drugs 4:953-958 (2003))。在2000年，在美国有1600万次由于中耳炎而拜访诊所医生(office-based physician)，且分发了约1300万个抗细菌处方。(Pediatrics113:1451-1465 (2004))。在欧洲国家中，报导的急性中耳炎比率范围为每个儿童每年(perchild-year)0.125至1.24。(Expert Review of Vaccines 8:1479-1500 (2009))。中耳炎是花费高的感染且是儿童接受抗生素的最常见原因。(Current Infectious DiseaseReports 11:177-182 (2009))。约70%的急性中耳炎病例由细菌引起，其中肺炎链球菌、无法分型的流感嗜血杆菌和卡他莫拉菌 (Moraxella catarrhalis)作为主要的致病物占主导 (Expert Review of Vaccines 5:517-534 (2006))。一个亚组的儿童经历复发性和慢性中耳炎，且这些易患耳炎的儿童长期具有中耳渗出液，这与听力损失和言语和语言发育延迟有关。(Current Infectious Disease Reports 11:177-182 (2009))。
继在许多国家引入七价肺炎球菌疫苗后，一些研究已证实由流感嗜血杆菌引起的急性中耳炎的比例显著增加，其中流感嗜血杆菌成为主要的病原体。(PediatricInfectious Disease Journal 23:824-828; Pediatric Infectious Disease Journal23:829-833 (2004))。
由于中耳炎是多因素疾病，所以使用接种策略来预防中耳炎的可行性一直受到质疑。(Current Infectious Disease Reports 11:177-182 (2009))。然而，来自一项研究的结果表明，抗原可诱导至少部分对抗无法分型的流感嗜血杆菌的保护。(Lancet 367:740-748 (2006))。一种开发疫苗抗原的途径是使用遗传上异源但大量表达的表面分子的抗原性保守区域。另一种方法是鉴定显示序列或功能表位保守的表面蛋白。疫苗抗原的第三种考虑可以是选择在感染和定殖期间在人宿主中表达的抗原。Murphy (Curr. Infect.Disease Reports 11:177-182 (2009)陈述，尽管存在几种潜在的无法分型的流感嗜血杆菌候选抗原，但不能确定地预测候选抗原是否将有效。(Current Infectious DiseaseReports 11:177-182 (2009))。描述为潜在疫苗抗原的一些蛋白是：嗜血杆菌粘附素蛋白(Hap)、高分子量(HMW)蛋白1和2、流感嗜血杆菌粘附素(Hia)、D15蛋白、HtrA热休克蛋白、P2表面蛋白、脂蛋白D、P5丝束蛋白衍生肽、外膜蛋白P4、外膜蛋白(OMP)26(OMP26)、P6蛋白、蛋白E、IV型菌毛、脂寡糖和磷酰胆碱。(Current Infectious Disease Reports 11:177-182(2009); Expert Review of Vaccines 5:517-534 (2006))。
毛丝鼠属(chinchilla)模型是中耳炎和其预防的稳健且经验证的动物模型(Expert Review of Vaccines 8:1063-1082 (2009))。尽管毛丝鼠属模型可模拟人感染的自然过程，但其他人已提出，毛丝鼠属模型的结果在各实验室间可不同。(Current Opinionin Investigational Drugs 4:953-958 (2003))。
也已使用多种其他啮齿类动物来诱导中耳炎，且概述于Vaccine 26:1501-1524(2008)中。在中耳炎研究中通常研究鼠动物模型。
杀菌性抗体的存在与防止发生由无法分型的流感嗜血杆菌引起的中耳炎有关。(Current Opinion in Infectious Disease 16:129-134 (2003))。然而，免疫应答有效对抗NTHi无需具有杀菌性。仅仅与NTHi表面粘附素反应的抗体可减轻或消除毛丝鼠属中耳炎。(Current Opinion in Investigational Drugs 4:953-958 (2003))。
慢性阻塞性肺病是慢性炎性肺部疾病且是全世界患病和死亡的主要原因。在2005年，在美国，20例死亡中约有1例以COPD作为根本原因。(Drugs and Aging 26:985-999(2009))。据预计，在2020年，COPD将上升成为残疾改变生命年数(disability adjustedlife years)的第五主要原因，慢性致病残性疾病(chronic invalidating diseases)，且成为死亡的第三重要原因 (Lancet 349:1498-1504 (1997))。
COPD病程的特征在于气流受限的进行性恶化和肺功能的衰退。COPD可由于频繁且反复急性恶化(AE)而变得复杂，所述急性恶化会造成庞大的健康护理花费和高患病率。(Proceedings of the American Thoracic Society 4:554–564 (2007))。一项研究表明，在COPD中约50%症状的急性恶化是由无法分型的流感嗜血杆菌、黏膜炎莫拉菌、肺炎链球菌和铜绿假单胞菌(Pseudomonas aeruginosa)引起。(Drugs and Aging 26:985-999(2009))。在20-30%的COPD恶化中发现流感嗜血杆菌；在10-15%的COPD恶化中发现肺炎链球菌；且在10-15%的COPD恶化中发现黏膜炎莫拉菌。(New England Journal of Medicine359:2355-2365 (2008))。已显示，流感嗜血杆菌、肺炎链球菌和黏膜炎莫拉菌是香港、南韩和菲律宾支气管炎急性恶化的主要病原体，而克雷伯菌属(Klebsiella spp.)、铜绿假单胞菌和不动杆菌属(Acinetobacter spp.)占其他亚洲国家/地区(包括印度尼西亚、泰国、马来西亚和台湾)病原体的大部分(Respirology, (2011) 16, 532-539; doi:10.1111/j.1440.1843.2011.01943.x)。在孟加拉国国，20%的COPD患者显示假单胞菌属、克雷伯菌属、肺炎链球菌和流感嗜血杆菌的阳性痰培养物，而65%的AECOPD患者显示假单胞菌属、克雷伯菌属、不动杆菌属、肠杆菌属、黏膜炎莫拉菌和其组合物的阳性培养物。(MymensinghMedical Journal 19:576-585 (2010))。然而，已提出，两种最重要的预防COPD恶化的措施是主动免疫和长期维持药物治疗。(Proceedings of the American Thoracic Society 4:554–564 (2007))。
当前需要有效对抗NTHi的疫苗。使用可在不同发病步骤起作用的抗原可提高疫苗的功效。本发明人已发现，PilA和PE可作为融合蛋白有益地存在于本发明免疫原性组合物中。
本发明涉及式(I)的融合蛋白。
其中：
X是信号肽或MHHHHHH (SEQ ID NO. 2)；
m是0或1；
R1 是一个氨基酸；
n 是0、1、2、3、4、5或6；
A 是来自流感嗜血杆菌的蛋白E或其免疫原性片段、或来自流感嗜血杆菌的PilA或其免疫原性片段；
Y选自GG、SG、SS和(G)h，其中h是4、5、6、7、8、9或10；
o 是0或1；
B 是来自流感嗜血杆菌的PilA或其免疫原性片段、或来自流感嗜血杆菌的蛋白E或其免疫原性片段；
Z是GGHHHHHH (SEQ ID NO: 3)；且
p是0或1。
在一个实施方案中，式(I)的融合蛋白定义为其中X选自来自下列的信号序列：CcmH(细胞色素C膜蛋白H)、DsbA(周质蛋白二硫化物异构酶I)、DsbB(二硫键膜蛋白B)、FlgI(鞭毛肽多糖环蛋白)、FocC(F1c陪伴分子(Chaperone)蛋白)、MalE(麦芽糖转运蛋白亚单位E)、NadA(喹啉酸合酶亚单位A)、NikA(镍ABC转运蛋白组分A)、NspA(奈瑟球菌属(Neisserial)表面蛋白A)、Omp26(外膜蛋白26)、OmpA(外膜蛋白A)、OspA(外表面蛋白A)、pelB(果胶酸裂解酶B)、PhoA(细菌碱性磷酸酶)、PhtD(肺炎球菌组氨酸三联蛋白D)、PhtE(肺炎球菌组氨酸三联蛋白E)、SfmC(周质菌毛蛋白陪伴分子)、Sip1(表面免疫原性蛋白)、TolB(Tol-Pal细胞荚膜复合物组分B)、TorA(三甲胺N-氧化物还原酶系统亚单位A)、TorT(三甲胺N-氧化物还原酶系统周质蛋白T)和Yral(推定的周质菌毛蛋白陪伴分子)；或其任何亚群。在一个实施实施方案中，X是共翻译信号肽或翻译后信号肽。在一个实施方案中，X是来自FlgI的信号序列(flgI sp)。在另一个特定实施方案中，X是来自 pelB的信号序列(pelB sp)。在另一个实施方案中，X是翻译后信号肽。在另一个实施方案中，X选自来自FlgI、NadA和pelB的信号序列。
在一个实施方案中，式(I)的融合蛋白定义为其中m是1。在另一个实施方案中，m是0。
在一个特定实施方案中，R1和n定义为其中(R1)n是富含小的通常亲水氨基酸的1至6个氨基酸。亲水氨基酸包括谷氨酸(E)、天冬氨酸(D)和天冬酰胺(N)。
在一个实施方案中，式(I)的融合蛋白定义为其中n选自0、1、2和6。在一个特定实施方案中，R1和n定义为其中(R1)n选自D、E、ATNDDD(SEQ ID NO. 178)和MD或其任何亚组。
在一个特定实施方案中，n选自1、2和6。在一个特定实施方案中，n是0。
在一个实施方案中，式(I)的融合蛋白定义为其中A是来自流感嗜血杆菌的蛋白E。在另一个实施方案中，式(I)的融合蛋白定义为其中A是由选自下列的氨基酸序列编码的蛋白E：
或SEQ ID NO. 5至SEQ ID NO. 57的任何亚组。在另一个实施方案中，式(I)的融合蛋白定义为其中A是蛋白E，其中蛋白E与SEQ ID NO：4中所述的蛋白E氨基酸序列约75%至100%相同。在另一个实施方案中，A是蛋白E，其中蛋白E与SEQ ID NO：4中所述的蛋白E氨基酸序列约90%至100%相同。在另一个实施方案中，A是蛋白E，其中蛋白E与SEQ ID NO：4中所述的蛋白E氨基酸序列至少95%相同。在额外的实施方案中，A是蛋白E，其中蛋白E与SEQ ID NO.4 – SEQ ID NO. 57的任一个中所述的蛋白E至少95%相同。在一个特定实施方案中，A是具有SEQ ID NO：4中所述的氨基酸序列的蛋白E。
在另一个实施方案中，式(I)的融合蛋白定义为其中A是来自流感嗜血杆菌的蛋白E的免疫原性片段。在另一个实施方案中，A是蛋白E的免疫原性片段，其中蛋白E具有选自下列的氨基酸序列：
或SEQ ID NO. 4至SEQ ID NO. 57的任何亚组。在另一个实施方案中，A是蛋白E的免疫原性片段，其中蛋白E与SEQ ID NO：4中所述的氨基酸序列约75%至100%相同。在另一个实施方案中，A是蛋白E的免疫原性片段，其中蛋白E与SEQID NO：4约90%至100%相同。在额外的实施方案中，A是蛋白E的免疫原性片段，其中蛋白E与SEQ ID NO. 4 – SEQ ID NO. 57中的任一个至少95%相同。更具体而言，在一个实施方案中，A是蛋白E的免疫原性片段，其中蛋白E与SEQ ID NO. 124 93%至100%相同。在一个特定实施方案中，A是蛋白E的免疫原性片段，其中蛋白E是SEQ ID NO. 4。
在另一个实施方案中，A 是选自下列的来自流感嗜血杆菌的蛋白E的免疫原性片段：SEQ ID NO. 4的氨基酸17-160(SEQ ID NO. 122)、SEQ ID NO. 4的氨基酸18- 160(SEQID NO. 123)、SEQ ID NO. 4的氨基酸19-160(SEQ ID NO. 124)、SEQ ID NO. 4的氨基酸20-160(SEQ ID NO. 125)和SEQ ID NO. 4的氨基酸22-160(SEQ ID NO. 126)。在另一个实施方案中，A 是选自下列的来自流感嗜血杆菌的蛋白E的免疫原性片段：SEQ ID NO. 4 的氨基酸17-160(SEQ ID NO. 122)、SEQ ID NO. 4的氨基酸18-160(SEQ ID NO. 123)、SEQID NO. 4的氨基酸19-160(SEQ ID NO. 124)、SEQ ID NO. 4的氨基酸20-160(SEQ ID NO.125)、SEQ ID NO. 4的氨基酸22-160(SEQ ID NO. 126)、SEQ ID NO. 4 的氨基酸23-160(SEQ ID NO. 179)和SEQ ID NO. 4的氨基酸24-160(SEQ ID NO. 180)。在进一步实施方案中，A是选自下列的来自流感嗜血杆菌的蛋白E的免疫原性片段：SEQ ID NO. 4的氨基酸17-160(SEQ ID NO. 122)、SEQ ID NO. 4的氨基酸18-160(SEQ ID NO. 123)、SEQ ID NO. 4的氨基酸20-160(SEQ ID NO. 125)、SEQ ID NO. 4的氨基酸22-160(SEQ ID NO. 126)、SEQID NO. 4的氨基酸23-160(SEQ ID NO. 179)和SEQ ID NO. 4的氨基酸24-160(SEQ ID NO.180)。更具体而言，在一个实施方案中，A是SEQ ID NO. 124 ，即SEQ ID NO. 4的氨基酸19-160。在额外的实施方案中，A是SEQ ID NO. 125，即SEQ ID NO. 5的氨基酸20-160。在另一个实施方案中，A是选自下列的来自流感嗜血杆菌的蛋白E的免疫原性片段：SEQ ID NO. 4的氨基酸23-160(SEQ ID NO. 179)和SEQ ID NO. 4的氨基酸24-160(SEQ ID NO. 180)。
蛋白 E - SEQ ID NO. 4
来自SEQ ID NO. 4的蛋白E 的氨基酸17-160 - SEQ ID NO. 122
来自SEQ ID NO. 4的蛋白E 的氨基酸18-160 - SEQ ID NO. 123
来自SEQ ID NO. 4的蛋白E 的氨基酸19-160 - SEQ ID NO. 124
来自SEQ ID NO. 4的蛋白E 的氨基酸20-160 - SEQ ID NO. 125
来自SEQ ID NO. 4的蛋白E 的氨基酸22-160 - SEQ ID NO. 126
来自SEQ ID NO. 4的蛋白E 的氨基酸23-160 - SEQ ID NO. 179
来自SEQ ID NO. 4的蛋白E 的氨基酸24-160 - SEQ ID NO. 180
在另一个实施方案中，式(I)的融合蛋白定义为其中A是来自流感嗜血杆菌的PilA。在另一个实施方案中，式⑴的融合蛋白定义为其中A是具有选自下列的氨基酸序列的流感嗜血杆菌的PilA：
或SEQ ID NO. 58至SEQ ID NO. 121的任何亚组。在另一个实施方案中，A是PilA，其中PilA与SEQ ID NO. 58约80%至100%相同。在另一个实施方案中，A是PilA，其中PilA与SEQID NO. 58-SEQ ID NO. 121 中的任一个至少95%相同。在一个特定实施方案中，A是SEQ IDNO. 58的PilA。
在另一个实施方案中，式(I)的融合蛋白定义为其中A是来自流感嗜血杆菌的PilA的免疫原性片段。在另一个实施方案中，A是PilA的免疫原性片段，其中PilA与SEQ ID NO.58 约80%至100%相同。例如，A是PilA的免疫原性片段，其中PilA具有选自下列的氨基酸序列：
或 SEQ ID NO. 58 至 SEQ ID NO. 121的任何亚组。在额外的实施方案中，A是PilA的免疫原性片段，其中PilA与SEQ ID NO. 58-SEQ ID NO. 121中的任一个至少95%相同。在一个特定实施方案中，A是来自流感嗜血杆菌菌株86-028NP的PilA的免疫原性片段，其中PilA是SEQ ID NO. 58。
来自流感嗜血杆菌菌株86-028NP的PilA-SEQ ID NO. 58
在另一个实施方案中，A是与SEQ ID NO. 127约75%至100%相同的PilA的免疫原性片段。更具体而言，在一个实施方案中，A是SEQ ID NO. 127，即由SEQ ID NO. 58的氨基酸40-149组成的片段。
来自流感嗜血杆菌菌株86-028NP的PilA的氨基酸40-149-SEQ ID NO. 127
在另一个实施方案中，A是由SEQ ID NO. 58-SEQ ID NO. 121中的任一个的氨基酸40-149组成的PilA的免疫原性片段。在一个额外的实施方案中，A是与SEQ ID NO. 58-SEQ ID NO. 121中的任一个的氨基酸40-149至少95%相同的免疫原性片段。
在一个实施方案中，式(I)的融合蛋白定义为其中Y选自GG、SG和SS。在另一个实施方案中，式(I)的融合蛋白定义为其中Y是GG或SG。在一个特定实施方案中，Y是GG。
在一个实施方案中，式(I)的融合蛋白定义为其中o是1。在另一个实施方案中，o是0。
在一个实施方案中，式(I)的融合蛋白定义为其中当A是来自流感嗜血杆菌的蛋白E或来自流感嗜血杆菌的蛋白E的免疫原性片段时，B是来自流感嗜血杆菌的PilA或来自流感嗜血杆菌的PilA的免疫原性片段。例如，B是来自流感嗜血杆菌菌株86-028NP的PilA。在另一个实施方案中，B是具有选自下列的氨基酸序列的来自流感嗜血杆菌的PilA：
或SEQ ID NO. 58至SEQ ID NO. 121的任何亚组。在另一个实施方案中，B是PilA，其中PilA与SEQ ID NO. 58约80%至100%相同。在另一个实施方案中，B是PilA，其中PilA与SEQID NO. 58-SEQ ID NO. 121 中的任一个至少95%相同。在一个特定实施方案中，B是SEQ IDNO. 58的PilA。
在另一个实施方案中，B是PilA，其中PilA与SEQ ID NO. 58- SEQ ID NO. 121中的任一个至少95%相同，且A是PE，其中PE与SEQ ID NO. 4-SEQ ID NO. 57 中的任一个至少95%相同。
在另一个实施方案中，式(I)的融合蛋白定义为其中当A是来自流感嗜血杆菌的蛋白E的免疫原性片段时，B是来自流感嗜血杆菌的PilA的免疫原性片段。例如，B是来自流感嗜血杆菌菌株86-028NP的PilA的免疫原性片段。在另一个实施方案中，B是PilA的免疫原性片段，其中PilA与SEQ ID NO：58约80%至100%相同。在另一个实施方案中，B是PilA的免疫原性片段，其中PilA具有选自下列的氨基酸：
或SEQ ID NO. 58至SEQ ID NO. 121的任何亚组。在另一个实施方案中，B是PilA的免疫原性片段，其中PilA与SEQ ID NO. 58-SEQ ID NO. 121中的任一个至少95%相同。在一个特定实施方案中，B是来自流感嗜血杆菌的PilA的免疫原性片段，其中PilA具有SEQ ID NO.58中所述的氨基酸序列。在另一个实施方案中，B是PilA的由SEQ ID NO. 58-SEQ ID NO.121中的任一个的氨基酸40-149组成的免疫原性片段。更具体而言，在一个实施方案中，B是如SEQ ID NO. 127中所述的PilA片段。在额外的实施方案中，B是与SEQ ID NO. 58-SEQ IDNO. 121中的任一个的氨基酸40-149至少95%相同的免疫原性片段。
在一个特定实施方案中，B是如SEQ ID NO. 127中所述的PilA片段且A是选自下列的蛋白E的免疫原性片段：SEQ ID NO. 122、SEQ ID NO. 124、SEQ ID NO.125 和 SEQ IDNO. 126。更具体地，B是如SEQ ID NO. 127中所述的PilA片段且A是如SEQ ID NO. 124 (来自SEQ ID NO. 4的蛋白E的氨基酸19-160)中所述的蛋白E片段。在另一个实施方案中，B是如SEQ ID NO. 127中所述的PilA片段且A是如SEQ ID NO. 125中所述的蛋白E片段。
在另一个实施方案中，B是PilA的免疫原性片段，其中PilA与SEQ ID NO.58-SEQID NO. 121 中的任一个至少95%相同，且A是PE的免疫原性片段，其中PE与SEQ ID NO. 4-SEQ ID NO. 57中的任一个至少95%相同。
在另一个实施方案中，式(I)的融合蛋白定义为其中当A是来自流感嗜血杆菌的PilA时，B是来自流感嗜血杆菌的蛋白E。例如，B是具有选自下列的氨基酸序列的蛋白E：
或SEQ ID NO. 4 至SEQ ID NO. 57的任何亚组。在另一个实施方案中，式(I)的融合蛋白定义为其中B是蛋白E，其中蛋白E与SEQ ID NO：4中所述的蛋白E氨基酸序列约75%至100%相同。在另一个实施方案中，B是蛋白E，其中蛋白E与SEQ ID NO：4中所述的蛋白E氨基酸序列约90%至100%相同。例如，B是蛋白E，其中蛋白E与SEQ ID NO：4中所述的蛋白E至少95%相同。在另一个实施方案中，B是蛋白E，其中蛋白E与SEQ ID NO. 4 – SEQ ID NO. 57中的任一个至少95%相同。在一个特定实施方案中，B是具有SEQ ID NO：4中所述的氨基酸序列的蛋白E。
在另一个实施方案中，式(I)的融合蛋白定义为其中当A是来自流感嗜血杆菌的PilA的免疫原性片段时，B是来自流感嗜血杆菌的蛋白E的免疫原性片段。例如，B是蛋白E的免疫原性片段，其中蛋白E具有选自下列的氨基酸序列：
或SEQ ID NO. 4至SEQ ID NO. 57的任何亚组。在另一个实施方案中，式(I)的融合蛋白定义为其中B是蛋白E的免疫原性片段，其中蛋白E与SEQ ID NO. 4中所述的蛋白E氨基酸序列约75%至100%相同。在另一个实施方案中，B是蛋白E的免疫原性片段，其中蛋白E与SEQID NO. 4中所述的蛋白E氨基酸序列约90%至100%相同。在一个特定实施方案中，B是具有SEQ ID NO. 4中所述的氨基酸序列的蛋白E的免疫原性片段。在额外的实施方案中，B是蛋白E的免疫原性片段，其中蛋白E与SEQ ID NO. 4-SEQ ID NO. 57 中的任一个至少95%相同。
在另一个实施方案中，B是选自下列的来自流感嗜血杆菌的蛋白E片段：SEQ IDNO. 4的氨基酸17-160 (SEQ ID NO. 122)、SEQ ID NO. 4的氨基酸18-160(SEQ ID NO.123)、SEQ ID NO. 4的氨基酸19-160(SEQ ID NO. 124)、SEQ ID NO. 4的氨基酸20-160(SEQ ID NO. 125)和SEQ ID NO. 4的氨基酸22-160(SEQ ID NO. 126)。在另一个实施方案中，B是选自下列的来自流感嗜血杆菌的蛋白E的免疫原性片段：SEQ ID NO. 4的氨基酸17-160 (SEQ ID NO. 122)、SEQ ID NO. 4的氨基酸18-160(SEQ ID NO. 123)、SEQ ID NO. 4的氨基酸19-160(SEQ ID NO. 124)、SEQ ID NO. 4 的氨基酸20-160(SEQ ID NO. 125)、SEQ ID NO. 4的氨基酸22-160(SEQ ID NO. 126)、SEQ ID NO. 4的氨基酸23-160(SEQ IDNO. 179)和SEQ IDNO. 4的氨基酸24-160(SEQ ID NO. 180)。更具体而言，在一个实施方案中，B是如SEQ ID NO. 123 (SEQ ID NO. 4的氨基酸18-160)中所述的蛋白E片段。
在一个特定实施方案中，当A是如SEQ ID NO. 127中所述的PilA的免疫原性片段时，B是如SEQ ID NO. 123 (SEQ ID NO. 4的氨基酸18-160)中所述的蛋白E的免疫原性片段。
在一个实施方案中，式(I)的融合蛋白定义为其中p是0。在另一个实施方案中，式(I)的融合蛋白定义为其中p是1。
在一个实施方案中，式(I)的融合蛋白选自下列：
或其任何亚组。在另一个实施方案中，式(I)的融合蛋白与
中的任一个约95%相同。
式(I)的融合蛋白可在受试者诸如哺乳动物(尤其人)中用作免疫原。具体而言，式(I)的融合蛋白可用于在受试者(尤其人)中诱导对抗流感嗜血杆菌的免疫应答。更具体而言，式(I)的融合蛋白可用于治疗或预防中耳炎和/或AECOPD和/或肺炎。
本发明涉及包含来自流感嗜血杆菌的蛋白E(或其免疫原性片段)和来自流感嗜血杆菌的PilA(或其免疫原性片段)的免疫原性组合物、以及包含来自流感嗜血杆菌的蛋白E(或其免疫原性片段)和来自流感嗜血杆菌的PilA(或其免疫原性片段)的融合蛋白的免疫原性组合物。本发明也涉及包含所述免疫原性组合物的疫苗和其治疗用途。
在一个实施方案中，所述免疫原性组合物包含来自流感嗜血杆菌的蛋白E(或其免疫原性片段)和来自流感嗜血杆菌的PilA(或其免疫原性片段)。蛋白E可以是SEQ ID NO. 4或与SEQ ID NO. 4至少75%、80%、85%、90%、95%、96%、97%、98%或99%相同的蛋白E序列。
蛋白E的免疫原性片段可以是SEQ ID NO. 122、SEQ ID NO. 123、SEQ ID NO.124、SEQ ID NO. 125 或SEQ ID NO. 126、或与SEQ ID NO. 122、SEQ ID NO. 123、SEQ IDNO. 124、SEQ ID NO. 125 或SEQ ID NO. 126 中的任一个具有至少90%、95%、96%、97%、98%、99%序列同一性的序列。蛋白E的免疫原性片段可以是SEQ ID NO.122、SEQ ID NO.123、SEQ ID NO. 124、SEQ ID NO. 125、SEQ ID NO. 126、SEQ ID NO. 179或SEQ ID NO.180 或与SEQ ID NO. 122、SEQ ID NO. 123、SEQ ID NO. 124、SEQ ID NO. 125、SEQ IDNO. 126、SEQ ID NO. 179 或SEQ ID NO. 180中的任一个具有至少90%、95%、96%、97%、98%、99%序列同一性的序列。已描述当与作为参考菌株的来自流感嗜血杆菌Rd的蛋白E进行比较时来自不同嗜血杆菌种类的蛋白E的氨基酸差异。Microbes & Infection(“Identification of a novel Haemophilus influenzae protein important foradhesion to epithelia cells”勘误 [Microbes Infect. 10 (2008) 87-97], 2010年7月6日可在线获得，“Article in Press”)提供了来自流感嗜血杆菌菌株772的蛋白E的序列。WO2002/28889提供了来自流感嗜血杆菌菌株12085的蛋白E的序列。
蛋白E含有上皮细胞结合区域(PKRYARSVRQ YKILNCANYH LTQVR, SEQ ID NO.128)，已报导该结合区域在超过100种临床NTHi分离株、有荚膜的流感嗜血杆菌和所分析的培养物收藏中心菌株中是保守的(Singh等人，J. Infect. Dis. 201(3):414-9 (2010))。Singh等人报导，蛋白E在NTHi和有荚膜的流感嗜血杆菌中高度保守(96.9% – 100%同一性，无信号肽)。在一个实施方案中，蛋白E的片段包含SEQ ID NO. 128 (PKRYARSVRQYKILNCANYH LTQVR)的结合区域。
PilA是在体内表达的保守粘附素。来自流感嗜血杆菌的64个PilA序列的全长比较显示约80%至100%同一性。
在另一个实施方案中，免疫原性组合物包含如通过式(I)所定义的融合蛋白。
在一个实施方案中，本发明免疫原性组合物可与来自流感嗜血杆菌的其他抗原一起施用。例如，PE和PilA或式(I)的融合蛋白可与来自流感嗜血杆菌的蛋白D一起施用。蛋白D可如WO91/18926中所述。在另一个实施方案中，免疫原性组合物可包括式(I)的融合蛋白和来自流感嗜血杆菌的蛋白D。
在另一个实施方案中，本发明的免疫原性组合物可与来自已知引起中耳炎、AECOPD或肺炎的其他细菌种类的其他抗原一起施用。
实现期望治疗或生物效应所需要的免疫原性组合物的量将取决于多个因素，诸如其预期用途、施用方式、受体和待治疗状况的类型和严重度，且最终将由主治医师或兽医自行决定。通常，例如，用于治疗人完全或部分由流感嗜血杆菌引起的状况的典型剂量预期可介于约0.003mg至约0.090mg范围内。更具体地，用于治疗人完全或部分由流感嗜血杆菌引起的状况的典型剂量可介于约0.01 mg至约0.03 mg融合蛋白范围内。免疫原性组合物可含有额外抗原；对于每种额外抗原，用于治疗人完全或部分由流感嗜血杆菌引起的状况的典型剂量可介于约0.01 mg至约0.03 mg范围内。该剂量可作为单一单位剂量施用。也可施用几种单独单位剂量。例如，单独单位剂量可在生命中第一年内作为单独引发剂量施用或每隔一定时间(例如，每1年、5年或10年)作为单独加强剂量施用。
包含本发明免疫原性组合物的制剂可配合合适途径施用，例如，通过肌内、舌下、经皮、皮内或鼻内途径。所述制剂可通过本领域已知的任何方法来制备。
本发明免疫原性组合物可额外包含佐剂。当术语”佐剂”用于本说明书中时，其是指与免疫原性组合物一起施用以加强患者对组合物的免疫原性组分的免疫应答的物质。
合适的佐剂包括铝盐诸如氢氧化铝凝胶或磷酸铝或明矾，但是也可以是钙盐、镁盐、铁盐或锌盐，或者可以是酰化酪氨酸或酰化糖、阳离子或阴离子衍生糖或聚磷腈的不溶悬浮液。在一个实施方案中，融合蛋白、PE或PilA可以被吸附在磷酸铝上。在另一个实施方案中，融合蛋白、PE或PilA可以被吸附在氢氧化铝上。在第三个实施方案中，也可以使用明矾作为佐剂。
促进Th1优势应答的合适佐剂系统包括：脂质A的无毒衍生物，单磷酰脂质A(MPL)或其衍生物，特别是3-脱-O-酰化单磷酰脂质A(3D-MPL)(它的制备参见GB 2220211 A)；和单磷酰脂质A的组合，优选3-脱-O-酰化单磷酰脂质A连同铝盐(例如磷酸铝或氢氧化铝)或水包油乳状液。在这样的组合中，抗原和3D-MPL被包含在同一颗粒结构中，以便能够用于抗原性和免疫刺激性信号的更有效递送。研究已经表明3D-MPL能够进一步增强明矾吸附的抗原的免疫原性(Thoelen等人Vaccine (1998) 16:708-14；EP 689454-B1)。
AS01是含有MPL(3-O-去酰基-4’-单磷酰脂质A)、QS21(奎拉雅属皂树(QuillajaSaponaria Molina)，部分21)，Antigenics, New York, NY, USA)和脂质体的佐剂系统。AS01B是含有MPL、QS21和脂质体(50 μg MPL 和 50 μg QS21)的佐剂系统。AS01E是含有MPL、QS21和脂质体(25 μg MPL和25 μg QS21)的佐剂系统。在一个实施方案中，免疫原性组合物或疫苗包含AS01。在另一个实施方案中，免疫原性组合物或疫苗包含AS01B或AS01E。在一个特定实施方案中，免疫原性组合物或疫苗包含AS01E。
AS03是含有油/水(o/w)乳状液中的α-生育酚和角鲨烯的佐剂系统。AS03A是含有o/w乳状液中的α-生育盼和角鲨烯(11.86 mg生育酚)的佐剂系统。AS03B是含有o/w乳状液中的α-生育酚和角鲨烯(5.93 mg生育酚)的佐剂系统。AS03C是含有o/w乳状液中的α-生育酚和角鲨烯(2.97 mg 生育酚)的佐剂系统。在一个实施方案中，免疫原性组合物或疫苗包含AS03。
AS04是含有吸附于铝盐(500 µg Al3+)上的MPL(50 µg MPL)的佐剂系统。在一个实施方案中，免疫原性组合物或疫苗包含AS04。
涉及使用QS21和3D-MPL的系统公开于WO 94/00153中。其中用胆固醇淬灭了QS21的组合物公开于WO 96/33739中。涉及水包油乳状液中的QS21、3D-MPL和生育酚的的额外的佐剂制剂描述于WO 95/17210中。在一个实施方案中，免疫原性组合物额外包含皂苷，其可以是QS21。制剂也可包含水包油乳状液和生育酚(WO 95/17210)。含有未甲基化CpG的寡核苷酸(WO 96/02555)和其他免疫调节性寡核苷酸(WO 0226757和WO 03507822)也是TH1应答的优先诱导物，并且适合用于本发明中。
额外的佐剂是选自下列的那些：金属盐、水包油乳状液、Toll样受体激动剂(特别是Toll样受体2激动剂、Toll样受体3激动剂、Toll样受体4激动剂、Toll样受体7激动剂、Toll样受体8激动剂和Toll样受体9激动剂)、皂苷、或其组合。
本发明提供制备免疫原性组合物的方法，其包括组合式(I)的融合蛋白与佐剂。
本发明进一步提供含有本发明免疫原性组合物和药学可接受的赋形剂的疫苗。
可能的赋形剂包括精氨酸、普朗尼克酸(pluronic acid)和/或聚山梨酯。在优选的实施方案中，使用聚山梨酯80(例如，TWEEN® 80)。在进一步的实施方案中，使用约0.03%至约0.06%的最终浓度。具体而言，可使用约0.03%、0.04%、0.05%或0.06%聚山梨酯80(w/v)的最终浓度。
本发明提供制备免疫原性组合物或疫苗的方法，其包括组合式(I)的融合蛋白与药学可接受的赋形剂。
本发明也提供编码本发明蛋白的核酸。术语“核酸”是指核苷酸的聚合形式。核苷酸可以是核糖核苷酸、脱氧核糖核苷酸、或核糖核苷酸或脱氧核糖核苷酸的修饰形式。该术语包括单链和双链形式的DNA。所述核酸优选基本上不含其他核酸。
本发明提供产生本发明核酸的方法。本发明核酸可通过本领域技术人员已知的方法来制备。例如，本发明核酸可部分或完全合成。所述核酸可通过消化较长氨基酸或连接较短氨基酸来制备。
以下实施例仅意欲用于说明，且并不意欲以任何方式限制本发明的范围。
在所述实施例中，下列术语具有以下所指示含义：
6xhis = 6个组氨酸；
xg = 离心力(重力数)；
ATP = 三磷酸腺苷；
BCA = 二喹啉甲酸；
BSA = 牛血清白蛋白；
℃ = 摄氏度；
CaCl2 = 氯化钙；
CV = 管柱体积；
DNA = 脱氧核糖核酸；
DSC = 差示扫描量热法；
DTT = 二硫苏糖醇；
dNTP = 三磷酸脱氧核苷；
EDTA = 乙二胺四乙酸；
FT = 流通物(flow through)；
HCl = 氯化氢；
His = his = 组氨酸；
HEPES = 4-(2-羟基乙基)-l-哌嗪乙磺酸；
IMAC =固定金属亲和层析；
IPTG = 异丙基β-D-1-硫代吡喃半乳糖苷；
KCl = 氯化钾；
K2HPO4 = 磷酸氢二钾；
KH2PO4 = 磷酸二氢钾；
LDS = 十二烷基硫酸锂；
L = 升；
MES = 2-(N-吗啉基)乙磺酸；
MgCl2 = 氯化镁；
ml = 毫升；
RPM = 每分钟转数；
min =分钟；
mM = 毫摩尔浓度；
μL = 微升；
NaCl = 氯化纳；
Na2HPO4 = 磷酸氢二钠；
NaH2PO4 = 磷酸二氢钠；
ng = 纳克；
nm = 纳米；
O/N = 过夜；
PBS = 磷酸盐缓冲盐水；
PCR = 聚合酶链式反应；
SB = 样品缓冲液；
sec =秒；
w/v = 重量/体积。
实施例
实施例1：融合蛋白
产生具有不同信号肽和氨基酸接头序列的融合蛋白。这些融合蛋白允许分泌蛋白E和PilA(或其片段)，而不限于单一细菌菌株。该融合蛋白在通过信号肽肽酶移除异源信号肽后释放至周质中。从细菌纯化的融合蛋白不含异源信号肽。从细菌移除“纯化”蛋白且其缺乏信号肽。
下表描述制备的融合蛋白构建体。
表3：含有PilA和蛋白E的融合蛋白构建体。
sp=信号肽；A.A.=氨基酸。
下文描述了表3中列出的信号肽和质粒中的每一种的DNA和氨基酸序列。
信号序列：
pelB信号肽(DNA) – SEQ ID NO. 129:
pelB信号肽(氨基酸) - SEQ ID NO. 130:
FlgI信号肽(DNA) - SEQ ID NO. 131:
FlgI信号肽(氨基酸) - SEQ ID NO. 132:
NadA信号肽(DNA) - SEQ ID NO. 133:
NadA信号肽(氨基酸) - SEQ ID NO. 134:
。
融合蛋白构建体序列：
氨基酸序列的单一下划线部分来自流感嗜血杆菌菌株86-028NP的PilA。氨基酸序列的加粗下划线部分衍生自流感嗜血杆菌菌株772的蛋白E。
从其获得上述序列的PE和PilA的全长序列分别获得且描述于SEQ ID NO. 4 (PE)和SEQ ID NO. 58 (PilA)中。
实施例2：载体构建和转化
用于扩增来自流感嗜血杆菌菌株772的PE的引物是基于流感嗜血杆菌菌株Hi Rd的序列来设计的。5’引物序列与NTHi 772序列相比含有1个核苷酸差异，与目前报导的NTHi 772基因组序列相比在位置24上引入一个氨基酸差异。融合蛋白构建体中的氨基酸#24是E(谷氨酸)而非如NTHi 772中所见的K(赖氨酸)。
来自流感嗜血杆菌菌株Rd的PE的DNA序列- SEQ ID NO. 151
来自流感嗜血杆菌菌株Rd的PE的蛋白序列- SEQ ID NO. 152
来自流感嗜血杆菌菌株772的PE的DNA序列 (如以下中描述：Microbes & Infection, “Identification of a novel Haemophilus influenzae protein important for adhesion to epithelia cells”的勘误 [Microbes Infect. 10 (2008) 87-97]，2010年7月6日可在线获得，“Article in Press”)) - SEQ ID NO. 153
来自流感嗜血杆菌菌株772的PE的蛋白序列(如以下中描述：Microbes & Infection, “Identification of a novel Haemophilus influenzae protein important for adhesion to epithelia cells”的勘误[Microbes Infect. 10 (2008) 87-97]，2010年7月6日可在线获得，“Article in Press”)) - SEQ ID NO. 154
载体构建:
为了产生LVL312、LVL291、LVL268、LVL269、LVL270、LVL702、LVL735、LVL778、LVL779、LVL780、LVL781 和LVL782，制备以下组分的聚合酶链式反应(PCR)制剂(具体组分随后例举)：配制36.6 μl去离子水、5 μl缓冲液#1 10X、5 μl dNTPs 2mM、2 μl MgCl2 25 mM、0.4μl引物#1 (50 μM)、0.4 μl引物#2 (50 μM)、0.5 μl模板(100 ng/μl)和0.4 μl KOD HiFiDNA聚合酶，2.5单位/μl (NOVAGEN®)。聚合酶链式反应涉及25个循环的98℃下变性15秒、55℃下退火2秒和72℃下引物延伸20秒。使用QIAQUICK® PCR纯化试剂盒(QIAGEN®)来纯化PCR产物。在供货商推荐的条件下使用该产物：向1体积PCR制剂中添加5体积的QIAQUICK® PCR纯化试剂盒中提供的缓冲液PB。随后通过涡旋将PCR制剂与缓冲液PB混合。将QIAQUICK®管柱放置于2ml收集管中。为了使PCR制剂中的DNA结合至管柱，将混合样品施加至QIAQUICK ®管柱中并在14 000 RPM下离心30-60秒。弃去流通物，并将QIAQUICK ®管柱放置回相同管中。为了洗涤结合的DNA，将0.75 ml QIAQUICK ® PCR纯化试剂盒中提供的缓冲液PE添加至QIAQUICK ®管柱中，并将管柱在14 000 RPM下离心30-60秒。弃去流通物，并将QIAQUICK ®管柱放置回相同管中。将QIAQUICK ®管柱于2ml收集管中再一次离心1分钟以移除残留的洗涤缓冲液。将每一QIAQUICK ®管柱放置在干净的1.5 ml微型离心管中。为了洗脱DNA，向QIAQUICK ®膜中心添加33 μl水，并将管柱在14 000 RPM下离心1分钟。限制性酶和相关缓冲液从New England BioLabs获得。例如，将约5 μl pET26b载体(100 ng/μl)、2 μl NE缓冲液2 (New England Biolabs, 1X NE缓冲液2:50 mM NaCl、10 mM Tris-HCl、10 mM MgCl2、1 mM二硫苏糖醇, pH 7.9 在25℃)、1 μl NdeI (20 000单位/ml)、1 μl HindIII (20 000单位/ml)和11 μl去离子水混合，并在37℃下孵育2小时以进行DNA消化。随后，使用QIAQUICK ® PCR纯化试剂盒(QIAGEN®)用上文所述的程序实施第二步骤的纯化。
使用来自New England BioLabs的Quick T4 DNA连接酶和快速连接反应缓冲液实施连接。例如，将10μl去离子水中的约10 ng载体和30 ng插入物与10 μl 2X快速连接反应缓冲液(New England Biolabs, 132 mM Tris-HCl, 20 mM MgCl2, 2mM二硫苏糖醇, 2 mMATP, 15%聚乙二醇，在25℃下pH 7.6)和1 μl Quick T4 DNA连接酶(New EnglandBiolabs)混合。将酶促反应在室温下孵育5分钟，随后转化。
为了产生LVL315、LVL317、LVL318、LVL736、LVL737、LVL738、LVL739 和LVL740，制备以下组分的PCR制剂：配制40 μl去离子水、5 μl反应缓冲液10X、1 μl dNTPs混合物、1 μl引物#1 (10 μM)、1 μl引物#2 (10 μM)、1 μl模板(25 ng/μl)和1 μl PfuUltra High-Fidelity DNA聚合酶2.5单位/μl (QuikChange II定点诱变试剂盒，AgilentTechnologies, Stratagene Division)。聚合酶链式反应涉及1个循环的95℃下变性30sec，18个循环的95℃下变性30sec、55℃下退火1min和68℃下引物延伸5min30sec。将PCR产物在37℃下使用1 μl Dpnl限制性酶消化1小时，随后转化。
用于扩增的PCR引物序列的详细列表在表4中说明。
为了产生pRIT16711，通过PCR从NTHi菌株772的基因组DNA扩增编码SEQ ID NO. 4的氨基酸22-160的PE基因片段(不包括编码其对应分泌信号的序列)。基于所获得的菌株HiRd序列(在那时，772序列尚未知)来设计扩增引物。5’引物序列与NTHi 772序列(序列如现在可获得)相比含有1个突变，在PE编码序列中在位置24上引入1个氨基酸差异，谷氨酸(E)代替赖氨酸(K)。PCR扩增后，使用BamHI和XhoI限制性位点将插入物克隆于pET-26(+)表达载体(NOVAGEN®)中。
为了产生pRIT16671，从NTHi菌株86-028NP的基因组DNA扩增编码PilA基因片段(SEQ ID NO. 58的氨基酸40-149，SEQ ID NO. 127)的DNA片段(不包括其前导肽以及预测的疏水α螺旋的部分)，并克隆至pET15表达载体中。使用载体pRIT16790(含有来自NTHi菌株86-028NP的氨基酸40-149)作为模板来产生载体pRIT16671。通过PCR使用载体PRIT16790和引物MDES PILA-3和MDES PILA-4 来扩增PilA基因片段。使用NdeI / XhoI限制性位点将PilA片段克隆至pET-26表达载体中。将编码6个组氨酸(his)氨基酸的DNA序列并入5'引物中，以在PilA序列的N末端(MDES PILA-3)添加6个组氨酸(6xhis)。
为了产生LVL312(FlgI信号肽-E-PilA片段-GG-PE片段-GGHHHHHH)，使用pRIT16671载体作为模板使用引物CAN534和CAN537实施聚合酶链式反应，以扩增PilA基因(氨基酸40-149/菌株86-028NP)。将对应于FlgI信号肽(sp)和谷氨酸(E)氨基酸的DNA序列并入5'引物(CAN534)中。为了使PilA序列连接至PE序列，将对应于两个甘氨酸(GG)氨基酸接头和N末端PE氨基酸的DNA序列并入3'引物(CAN537)中。使用pRIT16711载体作为模板使用引物CAN536和CAN538实施另一个聚合酶链式反应，以扩增PE基因(氨基酸18-160)。将对应于C末端PilA氨基酸和GG氨基酸的DNA序列并入5'引物中，以使pilA连接至PE序列(CAN536)。将对应于GG氨基酸接头和6xhis氨基酸的DNA序列并入3'引物(CAN538)中。最后，为了产生LVL312，实施第三个聚合酶链式反应以扩增PilA和PE基因，以便与N末端的FlgI信号肽、FlgI与pilA之间的谷氨酸(E)氨基酸、PilA与PE序列之间的GG接头和C末端的PE与6xhis氨基酸之间的GG接头融合。为了实现该扩增，使用上文所述的两个聚合酶链式反应的产物作为模板且使用引物CAN534和CAN538。将对应于NdeI限制性位点的DNA序列并入5'引物中，且将HindIII限制性位点并入3'引物中。随后将产生的PCR产物插入至pET-26b(+)克隆载体(NOVAGEN®)中。
为了产生LVL291(pelB信号肽-PE片段-GG-PilA片段-GG-6xhis)，使用pRIT16711载体作为模板，使用引物CAN544和CAN546实施聚合酶链式反应，以扩增PE基因(氨基酸19-160)。将对应于pelB信号肽(sp)氨基酸的DNA序列并入5'引物(CAN544)中。为了使PilA序列连接至PE序列，将对应于GG氨基酸接头和N末端PilA氨基酸的DNA序列并入3’引物(CAN546)中。使用pRIT16671载体作为模板，使用引物CAN545和CAN535实施另一个聚合酶链式反应，以扩增PilA基因(SEQ ID NO. 58的氨基酸40-149，SEQ ID NO. 127)。将对应于C末端PE氨基酸和GG氨基酸的DNA序列并入5'引物(CAN545)中，以使pilA序列连接至PE序列。将对应于接头GG氨基酸和6xhis氨基酸的DNA序列并入3'引物(CAN535)中。最后，为了产生LVL291，实施第三个聚合酶链式反应，以扩增PE和PilA基因，以便与N末端的pelB信号肽、PE与PilA序列之间的GG接头和PilA与C末端的6xhis氨基酸之间的GG接头融合。为了实现该扩增，使用上文所述的两个聚合酶链式反应的产物作为模板且使用引物CAN544和CAN535。将对应于NdeI限制性位点的DNA序列并入5'引物中，且将HindIII限制性位点并入3'引物中。随后将产生的PCR产物插入至pET-26b(+)克隆载体(NOVAGEN®)中。
为了产生LVL268(pelB信号肽-D-PE片段-GG-PilA片段-GG-6xhis)，使用pRIT16711载体作为模板，使用引物CAN547和CAN546实施聚合酶链式反应，以扩增PE基因(氨基酸20-160)。将对应于pelB信号肽(sp)氨基酸和天冬氨酸(D)氨基酸的DNA序列并入5'引物(CAN547)中。为了使PilA序列连接至PE序列，将对应于GG氨基酸接头和N末端PilA氨基酸的DNA序列并入3’引物(CAN546)中。使用pRIT16671载体作为模板，使用CAN545和CAN535实施另一个聚合酶链式反应，以扩增PilA基因(氨基酸40-149/NTHi菌株86-028NP)。将对应于C末端PE氨基酸和GG氨基酸的DNA序列并入5'引物(CAN545)中，以使pilA序列连接至PE序列。将对应于接头GG氨基酸和6xhis氨基酸的DNA序列并入3'引物(CAN535)中。最后，为了产生LVL268，实施第三个聚合酶链式反应，以扩增PE和PilA基因，以便与N末端的pelB信号肽、pelB信号肽与PE之间的D氨基酸、PE与pilA序列之间的GG接头和PilA与C末端的6xhis氨基酸之间的GG接头融合。为了实现该扩增，使用上文所述的两个聚合酶链式反应的产物作为模板且使用引物CAN547和CAN535。将对应于NdeI限制性位点的DNA序列并入5'引物中，且将HindIII限制性位点并入3'引物中。随后将产生的PCR产物插入至pET-26b(+)克隆载体(NOVAGEN®)中。
为了产生LVL269(NadA信号肽-ATNDDD-PE片段-GG-PilA片段-GG-6xhis)，使用pRIT16711载体作为模板，使用引物CAN548和CAN546实施聚合酶链式反应，以扩增PE基因(SEQ ID NO. 4的氨基酸22-160)。将对应于pelB信号肽(sp)氨基酸和ATNDDD氨基酸的DNA序列并入5'引物(CAN548)中。为了使PilA序列连接至PE序列，将对应于GG氨基酸接头和N末端PilA氨基酸的DNA序列并入3’引物(CAN546)中。使用pRIT16671载体作为模板，使用引物CAN545和CAN535实施另一个聚合酶链式反应，以扩增PilA基因(SEQ ID NO. 58的氨基酸40-149，SEQ ID NO. 127)。将对应于C末端PE氨基酸和GG氨基酸的DNA序列并入5'引物中，以使pilA序列连接至PE序列(CAN545)。将对应于接头GG氨基酸和6xhis氨基酸的DNA序列并入3'引物(CAN535)中。最后，为了产生LVL269，实施第三个聚合酶链式反应以扩增PE和PilA基因，以便与N末端的NadA信号肽、pelB信号肽与PE之间的ATNDDD氨基酸、PE与pilA序列之间的GG接头和PilA与C末端的6xhis氨基酸之间的GG接头融合。为了实现该扩增，使用上文所述的两个聚合酶链式反应的产物作为模板且使用引物CAN548和CAN535。将对应于NdeI限制性位点的DNA序列并入5'引物中，且将HindIII限制性位点并入3'引物中。随后将产生的PCR产物插入至pET-26b(+)克隆载体(NOVAGEN®)中。
为了产生LVL270(M-6xHis-PE片段-GG-PilA片段)，使用pRIT16711载体作为模板，使用引物CAN540和CAN542实施聚合酶链式反应，以扩增PE基因(氨基酸17-160)。将对应于6xhis氨基酸的DNA序列并入5'引物(CAN540)中。为了使PilA序列连接至PE序列，将对应于GG氨基酸接头和N末端PilA氨基酸的DNA序列并入3’引物(CAN542)中。使用pRIT16671载体作为模板，使用引物CAN541和CAN543实施另一个聚合酶链式反应，以扩增PilA基因(氨基酸40-149/NTHi菌株86-028NP)。将对应于C末端PE氨基酸和GG氨基酸的DNA序列并入5'引物(CAN541)中，以使pilA连接至PE序列。最后，为了产生LVL270，实施第三个聚合酶链式反应以扩增6-his-PE-GG-PilA基因以实现融合。为了实现该扩增，使用上文所述的两个聚合酶链式反应的产物作为模板且使用引物CAN540和CAN543。将对应于NdeI限制性位点的DNA序列并入5'引物中，且将HindIII限制性位点并入3'引物中。随后将产生的PCR产物插入至pET-26b(+)克隆载体(NOVAGEN®)中。
为了产生LVL315(pelB信号肽-MD-PE片段-GG-PilA片段-GG-6xhis)，使用LVL291作为模板使用引物CAN670和CAN671且使用QuikChange II定点诱变试剂盒(AgilentTechnologies, Stratagene Division)实施定点诱变，以使N末端PE氨基酸序列从QIQ改变为MD。
为了产生LVL317(pelB信号肽-PE片段-GG-pilA片段)，使用LVL291作为模板使用引物CAN678和CAN679且使用QuikChange II定点诱变试剂盒(Agilent Technologies,Stratagene Division)实施定点诱变，以便在PilA基因与对应于GGHHHHHH氨基酸残基(SEQID NO: 3)的DNA序列之间引入终止密码子。
为了产生LVL318(pelB信号肽-MD-PE-GG-PilA)，使用LVL315作为模板使用引物CAN678和CAN679且使用QuikChange II定点诱变试剂盒(Agilent Technologies,Stratagene Division)实施定点诱变，以便在PilA基因与对应于GGHHHHHH氨基酸残基(SEQID NO: 3)的DNA序列之间引入终止密码子。
为了产生LVL702(LVL291 ΔQ)，使用LVL291载体作为模板使用引物CAN1517和CAN1518实施聚合酶链式反应。将对应于LVL291序列上的位置23上的氨基酸Q的三个核苷酸的缺失并入5’引物。LVL702与LVL291之间的唯一差异是LVL291序列上的位置23的氨基酸Q的缺失。将NdeI和HindIII限制性位点分别并入5’和3’引物中。随后将产生的PCR产物插入至pET-26b(+)克隆载体(NOVAGEN®)中。
为了产生LVL735 (LVL317 ΔQ)，使用LVL317载体作为模板使用引物CAN1517和CAN1519实施聚合酶链式反应。将对应于LVL317序列上的位置23上的氨基酸Q的三个核苷酸的缺失并入5’引物。LVL7035与LVL317之间的唯一差异是LVL317序列上的位置23的氨基酸Q的缺失。将NdeI和HindIII限制性位点分别并入5’和3’引物中。随后将产生的PCR产物插入至pET-26b(+)克隆载体(NOVAGEN®)中。
为了产生LVL736 (LVL291 + SA)，实施定点诱变以便在LVL291序列上的氨基酸22与23之间添加氨基酸S和A。使用LVL291作为模板使用引物CAN1531和CAN1532且使用QuikChange II定点诱变试剂盒(Agilent Technologies, Stratagene Division)。
为了产生LVL737 (LVL291 + A)，实施定点诱变以便在LVL291序列上的氨基酸22与23之间添加氨基酸A。使用LVL291作为模板使用引物CAN1529和CAN1530且使用QuikChange II定点诱变试剂盒(Agilent Technologies, Stratagene Division)。
为了产生LVL738 (LVL291 ΔQIQ)，实施定点诱变以便在LVL291序列上的位置23至25缺失氨基酸Q、I和Q。使用LVL291作为模板使用引物CAN1523和CAN1524且使用QuikChange II定点诱变试剂盒(Agilent Technologies, Stratagene Division)。
为了产生LVL739 (LVL291 ΔQIQK)，实施定点诱变以便在LVL291序列上的位置23至26缺失氨基酸Q、I、Q和K。使用LVL291作为模板使用引物CAN1525和CAN1526且使用QuikChange II定点诱变试剂盒(Agilent Technologies, Stratagene Division)。
为了产生LVL740 (LVL291 ΔQIQKA)，实施定点诱变以便在LVL291序列上的位置23至27缺失氨基酸Q、I、Q、K和A。使用LVL291作为模板使用引物CAN1527和CAN1528且使用QuikChange II定点诱变试剂盒(Agilent Technologies, Stratagene Division)。
为了产生LVL778 (LVL736 Δ6xHis标签)、LVL779 (LVL737 Δ6xHis标签)、LVL780 (LVL738 Δ6xHis标签)、LVL781 (LVL739 Δ6xHis标签)和LVL782 (LVL740 Δ6xHis标签)，分别使用LVL736、LVL737、LVL738、LVL739和LVL740载体作为模板使用引物CAN1669和CAN543实施聚合酶链式反应。6xHis标签的缺失对应于C末端序列的氨基酸序列GGHHHHHH (SEQ ID NO. 3)。将该缺失并入3'引物。将NdeI和HindIII限制性位点分别并入5’和3’引物中。随后将产生的PCR产物插入至pET-26b(+)克隆载体(NOVAGEN®)中。
表4：用于PE、PilA和PE-PilA扩增的PCR引物序列
转化
将大肠杆菌(Escherichia coli)BLR (DE3)或大肠杆菌HMS (DE3)细胞根据标准方法用CaCl2处理的细胞使用质粒DNA来转化。(Hanahan D. « Plasmid transformation bySimanis. » In Glover, D. M. (编辑), DNA cloning. IRL Press London. (1985): 第109-135页)。简而言之，将BLR(DE3)或HMS174(DE3)感受态细胞在冰上慢慢解冻。使用50-100μl感受态细胞混合约4μl质粒(10-100ng)。此后，将该制剂在冰上孵育30min。为了实施转化反应，将制剂在42℃下脉冲加热45秒，随后在冰上孵育2分钟。向转化细胞中添加约0.5mlSOC培养基(含有分解代谢抑制物的超级最佳培养液(Super Optimal broth withCatabolite repression))，并将细胞培养物在37℃下孵育1小时，随后铺板于含有50 ug/ml卡那霉素的Luria-Bertani(LB)琼脂上。铺板约100 μl转化细胞培养物并在37℃下孵育过夜。
BLR (DE3): BLR是BL21 (F–ompT hsdSB(rB– mB–) gal dcm (DE3)的recA – 衍生物。用于表达重组蛋白的该大肠杆菌菌株提高质粒单体产率，且可有助于稳定含有重复序列或其产物可引起DE3原噬菌体损失的靶质粒。(Studier, F.W. (1991) J. Mol. Biol. 219: 37–44)。大肠杆菌BLR(DE3)的详细基因型已由NOVAGEN®公开。(F- ompT hsdSB (rB-mB-) gal dcm Δ(srl-recA)306::Tn10 (TetR) (DE3)。
HMS174 (DE3): HMS174菌株在K-12背景中提供recA突变。与BLR—样，这些菌株可稳定某些其产物可引起DE3原噬菌体损失的靶基因。大肠杆菌HMS174 (DE3)的详细基因型已由NOVAGEN®公开。(F–recA1 hsdR(rK12– mK12+) (DE3) (Rif R )。
使用BLR(DE3)产生His标签的构建体和所述构建体的表征描述于实施例3至实施 例6中
实施例3：使用摇瓶表达蛋白
通常，将接种经重组质粒转化的大肠杆菌BLR(DE3)的一块汇合的琼脂板剥离，重悬浮于培养基中并用于接种800 ml LB培养液(Becton, Dickinson and Company)±l%(重量/体积，w/v)葡萄糖(Laboratoire MAT，目录号：GR-0101)和50µg/ml卡那霉素(Sigma)，以获得0.1与0.2之间的O.D.600nm。将培养物在37℃下在250RPM搅动下孵育，以达到~0.8的O.D.600nm。
随后收集1ml每种培养物，在14000 RPM下离心5分钟，并将上清液和沉淀在-20℃下单独冷冻。
在O.D.600nm ~0.8时，将BLR(DE3)培养物冷却(-20℃下20分钟或4℃下1小时，优选4℃下1小时)，随后通过添加1 mM异丙基β-D-1-硫代吡喃半乳糖苷(IPTG; EMD ChemicalsInc.，目录号：5815)并在250 RPM搅动下在16℃、22℃和30℃下孵育过夜或在37℃下孵育3小时(优选在22℃下孵育过夜)来诱导重组蛋白的表达。在诱导期后，将培养物在14000RPM下离心5分钟或在6000 RPM下离心15分钟，并将上清液(培养基级分样品)和沉淀(含有可溶和不可溶级分)在-20℃下单独冷冻。
使用这些条件来实施周质蛋白表达。
实施例4：使用摇瓶、细胞糊状物、His标签的构建体来纯化蛋白
将在诱导后获得的每种细菌沉淀重悬浮于20mM含有500 mM NaCl、10 mM咪唑和RocheCOMPLETE®蛋白酶抑制剂混合物(1个片剂/50 ml含有500 mM NaCl的HEPES缓冲液，RocheCOMPLETE® ULTRA片剂，Roche Diagnostics公司)的4-(2-羟基乙基)-1-哌嗪乙磺酸(HEPES)缓冲液(pH 8.0)中。
或者，可使用20-50 mM N,N-二羟乙基甘氨酸缓冲液来代替含有NaCl的HEPES缓冲液。例如，可使用20 mM N,N-二羟乙基甘氨酸缓冲液。使用Constant System 1.1 KW 2 X30 000 PSI(磅/平方英寸)来裂解细菌。通过在20 000 g 下在4℃下离心20 min将可溶(上清液)和不可溶(沉淀)组分分开。
6-His标签的蛋白在天然条件下在固定金属亲和层析(IMAC)上使用PROFINIATM蛋白纯化方案(Bio-Rad Laboratories, Inc.)来纯化。将可溶组分上样于5 ml His Trap 管柱(Bio-Rad Laboratories, Inc.)上，该管柱经用于细菌重悬浮的相同缓冲液预平衡；以至多5ml/min添加可溶组分(产生“流通物级分”)。上样于管柱上之后，用10管柱体积的相同缓冲液以10 ml/min的速率洗涤管柱(产生“洗涤级分1”)。使用20 mM N,N-二羟乙基甘氨酸缓冲液或20 mM HEPES缓冲液(pH 8.0)(含有500 mM NaCl 和20 mM咪唑)实施第二次洗涤，从而产生“洗涤级分2”。使用2管柱体积的20 mM HEPES缓冲液或50 mM N,N-二羟乙基甘氨酸缓冲液(pH 8.0)(含有500 mM NaCl和250 mM咪唑)以10 ml/min的速率实施洗脱，从而产生“洗脱级分”。
为了提高蛋白的纯度，合并来自IMAC的阳性洗脱级分，并上样于大小排阻层析(SEC)管柱(HILOADTM SUPERDEXTM 200 26/60，来自GE Healthcare)上，该管柱在不含钙或镁的磷酸盐缓冲盐水(NaCl 137 mM, KCl 2.7 mM, Na2HPO4 8.1 mM, KH2PO4 1.47 mM, pH7.4)中预平衡。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)来分析洗脱级分样品。使用Centricon 10 000 MW (Millipore)浓缩样品。
使用分光计测定蛋白浓度。
实施例5：His标签的构建体的SDS-PAGE和 Western印迹分析以及未his标签的 LVL317和LVL318构建体的SDS-PAGE分析
可溶和不可溶级分制备
例如，将1 ml诱导后的培养物(参见例如上文实施例3)在14 000 RPM下离心2 min。将沉淀使用40 μl BUGBUSTER®蛋白提取试剂(NOVAGEN®, EMD4 Biosciences, Merck)再溶解，从而产生细胞悬浮液。将细胞悬浮液在旋转平台上在室温下孵育10 min。随后将细胞悬浮液在14 000 RPM下离心2 min以分离可溶级分。将所得沉淀(不可溶级分)使用70 μl去离子水、5 μl二硫苏糖醇(DTT)1M和25 μl NUPAGE® LDS(十二烷基硫酸锂)样品缓冲液4X(INVITROGENTM)再溶解。将可溶级分(来自再溶解沉淀的细胞悬浮液的上清液)添加至30 μl去离子水、5 μl DTT 1M和25 μl 4xLDS样品缓冲液中。
培养基级分制备
例如，为了制备培养基级分，通过添加500 μl RC试剂I (Bio-Rad Laboratories,Inc.)将100 μl来自离心后的经诱导的全细胞培养物(参见例如上文实施例3)的上清液浓缩；混合样品并在室温下孵育1 min。随后，向样品中添加500 μl试剂II(Bio-RadLaboratories, Inc.)并混合。将该制剂在14 000 RPM下离心10 min。将沉淀使用28 μl去离子水、2 μl DTT 1M和10 μl LDS SB 4X再溶解。
纯化级分制备
例如，通过添加70 μl样品、5 μl DTT 1M和25 μl 4xLDS样品缓冲液来制备纯化蛋白(例如，如实施例4中所述来获得)，以用于SDS-PAGE分析。
SDS-PAGE分析和转移至硝酸纤维素膜
根据制造商推荐(Invitrogen)使用NUPAGE® Bis-Tris 4-12%凝胶来实施SDS-PAGE分析和转移至硝酸纤维素膜。在供货商推荐的条件下实施样品、缓冲液和迁移条件的准备。
在一个实施例中，将凝胶上样20 ul来自包含70 μl纯化蛋白级分、5 μl DTT 1M和25 μl 4xLDS SB的混合母液的样品。
在NUPAGE® Bis-Tris 4-12%凝胶上运行样品之后，将蛋白转移至硝酸纤维素膜。
将硝酸纤维素膜在37℃、60 RPM下使用3%奶/PBS 1X新鲜溶液封闭30分钟。封闭孵育后，添加以1:1000稀释于3%奶/ PBS 1X新鲜溶液中的一抗(6X His标签®抗体，AbcamPLC，目录号：ab9108)，并在37℃、60 RPM 下保持1小时。此后，将膜在室温下使用0.02%聚山梨酯20(例如，TWEENTM 20) / PBS 1X洗涤三次，每次5分钟。添加使用3%奶/ PBS 1X新鲜溶液以1:14 000稀释的二抗(碱性磷酸酶(AP)兔抗IgG(H+L)兔，Jackson ImmunoResearchLaboratories, Inc.)。将膜在37℃、60 RPM下孵育1小时。此后，将膜在室温下使用0.02%聚山梨酯20(例如，TWEENTM 20) / PBS 1X洗涤三次，每次5分钟，随后将膜暴露于5-溴-4-氯-3-吲哚基磷酸盐/硝基蓝四唑(例如，BCIP®/NBT，来自Sigma-Aldrich®，1个片剂/10 ml水)。
融合蛋白构建体LVL291、LVL268和LVL269的诱导的细菌提取物的SDS-PAGE参见图1。对于LVL291、LVL268和LVL269，在诱导(ind)之前和之后上样不可溶级分(I)、可溶级分(S)和培养基级分(M)。
融合蛋白构建体LVL291、LVL268和LVL269的纯化提取物相关的SDS-PAGE和Western印迹参见图2。对于LVL291、LVL268和LVL269的纯化，上样流通物级分(Ft)、洗涤级分(W)和洗脱级分(E)。使用抗his标签来探测提取物。
融合蛋白构建体LVL291和LVL315的诱导的细菌和纯化提取物的SDS-PAGE参见图3。对于LVL291和LVL315，上样培养基级分(M)、可溶级分(Sol)、不可溶级分(Ins)、流通物级分(Ft)、洗涤级分#1(W1)、洗涤级分#2(W2)和洗脱级分(E)。
融合蛋白构建体LVL312的诱导的细菌和纯化提取物的SDS-PAGE参见图4。对于LVL312，上样培养基级分(M)、可溶级分(Sol)、不可溶级分(Ins)、流通物级分(Ft)、洗涤级分#1(W1)、洗涤级分#2(W2)和洗脱级分(E)。
融合蛋白构建体LVL291、LVL702、LVL736、LVL737、LVL738、LVL739、LVL740 和pET26b载体(阴性对照)的诱导的细菌提取物的可溶级分的SDS-PAGE参见图25。(a)实验1(b)实验2(c)实验3。PE-PilA融合蛋白由箭头指示。
来自实验1、2和3的可溶级分中融合蛋白的平均条带百分比参见图26。
分别用于图5和图6中的SDS-PAGE分析的LVL317和LVL318细菌提取物通常如上文所述来制备。
图5.融合蛋白构建体LVL317的诱导(1 mM和10 IPTG)细菌提取物的SDS-PAGE。诱导之前(NI)和诱导之后(In)的提取物、可溶级分(S)、不可溶级分(I)。
图6.融合蛋白构建体LVL318的诱导(1 mM和10 IPTG)细菌提取物的SDS-PAGE。诱导之前(NI)和诱导之后(In)的提取物、培养基级分(M)、可溶级分(S)、不可溶级分(I)。
将通过SDS-PAGE分离的蛋白转移至Immobilon-P膜。剪下考马斯蓝(CoomassieBlue)染色的蛋白条带并置于测序仪反应器中。根据制造商方案使用Applied BiosystemsPROCISE®蛋白测序仪(型号为494-cLC)来实施测序。
表5：融合蛋白构建体的摇瓶蛋白表达概况和信号肽切割。
So = 可溶级分。In=不可溶级分。Se=分泌于培养基级分中的蛋白。Nt=未测试。以下排序基于目测(考马斯)+: 低表达；++:中等表达；+++ :高表达；- ：未表达。
实施例6：LVL291融合蛋白表征
LVL291的物理性质：LVL291中的PE和PilA的折叠和熔点
圆二色谱:
二级结构的分析
利用圆二色谱(CD)通过测量由于结构不对称而引起的左旋偏振光相比于右旋偏振光的吸收差异来测定蛋白的二级结构组成。蛋白是否呈现β-片层、α-螺旋或随机卷曲结构，远UV区域(190-250 nm)中CD谱的形状和强度不同。可通过与参考谱进行比较来计算给定蛋白样品中各二级结构类型的相对丰度。
远UV谱使用0.01cm的光程从178 nm至250 nm以1 nm分辨率和带宽在Jasco J-720分光偏振计上进行测量。通过Peltier恒温控制RTE-111单元块将单元温度维持在23℃。测量期间维持10L/min的氮气流动。
结果：
所获得的PE(来自构建体pRIT16762)、PilA(来自构建体pRIT16790)和PE-PilA蛋白的远UV CD谱具有含有α和β结构的混合物的折叠蛋白的特征，但与PilA和PE-PilA相比，PE显著富含α螺旋(图7，PE、PilA和PE-PilA融合蛋白的CD谱)。
为了评价一起结合于嵌合蛋白中之后PE和PilA个别蛋白的折叠的完整性且随后验证二者之间的可能相互作用，计算不同谱。
·当将PE和PilA远UV谱组合时，所得谱与PE-PilA嵌合体的谱重合(图8，PE和PilACD谱的组合)。该结果表明，PE-PilA嵌合体含有在个别组分中所检测到的所有二级结构。其还表明，蛋白的融合对个别组分的二级结构没有较大影响，且因此，无论蛋白是单独的还是融合的，PE和PilA的折叠没有显著不同。
熔点评价：
为了评价融合时的表达是否对个别蛋白的热力学性质具有影响，通过圆二色谱监测α螺旋随温度的去折叠来评价PE、PilA和PE-PilA的熔点。
α螺旋存在的特征在于222 nm下的圆二色谱信号极低，因此温度增加期间222 nm下的CD信号显著增加指示蛋白变性。测定蛋白经历二级结构损失的温度使得能够测定熔点(Tm)，其对应于一半蛋白失去其结构的温度。
熔点可通过鉴定从温度对CD 222 nm图获得的热变性曲线的拐点来测定。
·通过远UV CD所测定的PilA和PE的熔点分别为52℃和68℃(图9，PilA热变性曲线；图10，PE热变性曲线)。
· PE-PilA融合蛋白呈现两个不同的Tm，即48℃和71℃(图11，PE-PilA融合蛋白的热变性曲线)。那些值指示，PE和PilA蛋白在结合成嵌合体后仍独立地折叠，且无论其是分离的还是融合的，其在相似温度下去折叠。PilA部分在48℃下的去折叠没有引起沉淀或影响PE部分的Tm(71℃)的观察结果强烈指示，融合体内PE与PilA之间的相互作用极小，且其彼此间没有较大的可观察到的影响。蛋白的熔点对各种外部条件敏感，包括缓冲液组成或相互作用分子的存在；PE与PilA融合后没有观察到较大变化强烈指示，当PE和PilA结合在一起时，该二者的大部分结构和性质得以保持。
实施例7：发酵过程
本发明的融合蛋白可通过本领域技术人员已知的方法来制备。
实施例8：PE、PilA和LVL317的蛋白纯化
从pRIT16762纯化PE蛋白：
为了产生pRIT 16762表达载体，使用BamHI和NcoI限制性酶来消化PRIT16711载体，以缺失信号序列(pelB)与PE之间的6个氨基酸残基。所获得的载体命名为pRIT16712。在该载体中，在信号序列pelB与PE之间存在3个氨基酸：MDP。在第二个步骤中，使用pRIT16712作为模板使用引物MnoNTHi-44和MnoNTHi-45 (描述于表4中)且使用QuikChange II定点诱变试剂盒(Agilent Technologies, Stratagene Division)来实施定点诱变，以使氨基酸序列从MDP改变为QIQ。
将含有PE QIQ (来自pRIT16762构建体)的大肠杆菌BLR(DE3)的工作菌种(working seed)从-80℃解冻，并用于通过在37℃下在215 RPM下搅动下过夜孵育来制备100 ml LB液体培养基中的预培养物。过夜孵育后，给8个含有800 ml LB APS的烧瓶接种12.5 ml预培养物，且测量OD600为约0.06。将培养物在37℃下在振荡下孵育3h。当OD600约0.9时，添加1mM IPTG以开始诱导。诱导期间，将培养物在22℃下在振荡下孵育19h。诱导后，OD600为约2.2。将细胞培养物转移至1 L离心袋中，置于1 L瓶内并在4℃下在6,000xg下离心30分钟，并弃去上清液。保存1ml诱导前和诱导后的培养物和上清液的等分试样，用于将来的分析。
PE QIQ诱导的BLR (DE3)的裂解
将离心袋从离心瓶取出，打开，并将沉淀从袋排出到烧杯中。将8份沉淀聚集在一起，并重悬浮于100 ml结合缓冲液(20 mM Hepes、10 mM咪唑、500 mM NaCl，pH 8.01)中。用来自Constant Systems Ltd.的TS Series Bench Top细胞破碎器(1x30 kPsi; 1x15kPsi)使含有PE QIQ构建体的大肠杆菌BLR (DE3)破碎。将裂解物在6000 RPM、4℃下离心30分钟。保留上清液并上样于IMAC管柱上。
PE QIQ的IMAC纯化
IMAC管柱(BioRad, Bio-Scale Mini Profinity IMAC液筒5ml)用5CV的结合缓冲液(20 mM HEPES、10 mM咪唑、500 mM NaCl，pH 8.01)以5 ml/min 进行平衡。将100 ml裂解物上清液以2.5 mL/min上样于IMAC上。以50 ml部分收集流通物，用于将来的分析。用3CV的结合缓冲液洗涤管柱以移除未结合的蛋白。将含有未结合蛋白的样品收集在50 ml管中的一份15 ml的等分试样中。用2CV的洗涤缓冲液(20 mM HEPES、20 mM咪唑、500 mM NaCl，pH8.01)洗涤管柱，以2 ml部分收集在96孔板中。随后将结合的蛋白用6CV的100%洗脱缓冲液(20 mM HEPES、250 mM咪唑、500 mM NaCl，pH 8.01)洗脱。将所洗脱的蛋白以2 ml部分收集在96孔板中。以5 ml/min实施洗涤和洗脱。
PE QIQ的IMAC合并物的大小排阻层析(SEC)
SEC管柱(GE healthcare, HILOADTM 26/60 SUPERDEXTM 75制备级，60 cm高，约319 ml体积)用3CV的SEC缓冲液(20 mM HEPES、150 mM NaCl，pH 8.49)进行平衡。将11 ml IMAC洗脱物以2.5 ml/min的流速上样至管柱上。从0.3CV至0.9CV收集2 ml部分。实施两次运行，随后通过SDS-PAGE对各部分进行分析。将来自两次运行的含有Prot E蛋白的部分合并在一起(“SEC合并物”，总体积约为48ml)。向SEC合并物中添加500 mM精氨酸。
上述SEC方案中产生的PE QIQ合并样品的定量
用来自Bio-Rad RC DCTM试剂盒的RCDC(还原剂和去污剂相容(Reducing Agent andDetergent Compatible))方法遵循制造商方案来给SEC合并物定量：
对于每种测试样品和标准品，将25 μL以一式两份分配于微量离心机管中。向每管中添加125 μl Bio-Rad RC试剂I；使每管涡漩并在室温下孵育1分钟。向每管中添加125 μlBio-Rad RC试剂II；使每管涡旋，并随后在14,000xg下离心5分钟。通过将所述管倒置在干净的吸水纸上从而使液体能够完全从管排出来弃去上清液。向每管中添加25.4μl试剂A(已经通过每1 ml试剂A混合20 μl试剂S来制备)；使每管涡漩并在室温下孵育5分钟，或直至沉淀完全溶解。使其涡漩，随后进行下一步骤。向每管中添加200μL DC试剂B，并立即使其涡漩。在室温下孵育15分钟。将所有样品转移至96孔板中，并在750 nm下读取吸光度，以测定各未知蛋白样品的蛋白浓度。
ProtE浓度为1.069 mg/ml。
His-标签的PilA蛋白的纯化：
遵循以下通用程序来纯化PilA：
将含有编码PilA或其片段的构建体的大肠杆菌细胞悬浮于BUGBUSTER®和BENZONASE®核酸酶(NOVAGEN®)(例如10 ml BUGBUSTER®和10 ul BENZONASE®核酸酶)中。将细胞裂解物在室温下在旋转平台上混合(例如)15分钟。将细胞裂解物在4℃下在(例如)16，000g下离心20分钟。将含有蛋白的上清液添加至含有Ni NTA HIS ·BIND®树脂的Ni NTA管柱中，并在4℃下混合(例如)1小时。该管柱可由2 ml Ni NTA HIS ·BIND®树脂(NOVAGEN®)和10 ml 1x结合缓冲液(来自NOVAGEN®的Ni-NTA缓冲液试剂盒)组成。随后收集管柱流通物。将树脂用1x洗涤缓冲液洗涤两次，该洗涤缓冲液含有(例如)300 mM NaCl、50 mMNaH2PO4、25 mM咪唑，pH8.0。通过重力流动来收集洗涤液。蛋白用1X洗脱缓冲液(例如，300mM NaCl、50 mM NaH2PO4、250 mM咪唑，pH 8.0)从管柱洗脱。可通过用结合缓冲液透析和如上文所述再次流过Ni NTA管柱对蛋白实施进一步纯化。
PilA的凝血酶切割。
随后将PilA与凝血酶(以1/50稀释)在室温下一起孵育16h，以移除组氨酸标签。
凝血酶切割的PilA的大小排阻层析(SEC)。
SEC管柱(GE healthcare, HILOADTM 26/60 SUPERDEXTM 75制备级，60 cm高，约319 ml体积)用5CV的SEC缓冲液(20 mM HEPES、150 mM NaCl，pH8.52)进行平衡。将约10 ml经切割的PilA以2.5 ml/min的流速上样至管柱上。从0.3CV至0.9CV收集2 ml部分。实施两次运行，随后通过SDS-PAGE对各部分进行分析。将来自两次运行的含有切割的PilA蛋白的部分合并在一起(“EC合并物”，总体积约为52ml)。
PilA SEC合并物的定量。
该SEC合并物用如上所述的RCDC方法来定量。经切割的PilA的浓度为5.37 mg/ml。
用PBS 1x pH 7.4透析PilA SEC合并物(透析因子=1600)并通过RCDC定量
通过RCDC测定的透析后浓度为3.0 mg/ml。
LVL317的纯化
渗透压休克
由于LVL31 7融合蛋白在细菌周质中表达和加工，所以通过渗透压休克来提取蛋白。
将来自4 L发酵罐培养物的含有LVL317的冷冻(-20℃)收获的大肠杆菌B2448细胞糊状物合并，并重悬浮于由24 mM Tris-HCl、16% (w/v)蔗糖、9.9%(w/v)葡萄糖、10 mMEDTA组成的高渗缓冲液(pH 8.0)中，直至4L的最终体积。将悬浮液在室温下使用安装于RW16基础搅拌器上的3-叶片推进器以中等速度轻轻混合30 min。将悬浮液在室温下在15,900xg下离心30分钟。保留上清液(SN1)以用于凝胶分析。
将所得沉淀重悬浮于高渗溶液(38 mM MgCl2)中，并在室温下混合30 min。将混合物在室温下在15,900 x g下离心30分钟，并在上清液(SN2)中回收抗原。
通过以600 ml/min的流速过滤通过0.45/0.2 µm聚醚砜Sartorius Sartopore 2MidiCap滤器来实施SN2的澄清。
用20 mM NaH2PO4 -Na2HPO4(pH 7.0)以1：3稀释SN2，如果需要将pH调节至7.0，且通过以600 ml/min过滤通过0.45/0.2 µm聚醚砜Sartorius Sartopore 2 MidiCap滤器来实施另一次澄清。
SP SEPHAROSE™快速流动(SP FF)层析
上样经稀释/过滤的SN2，并捕获于在14 cm ID(内径) x 20 cm长管柱(管柱体积3100ml)中的强阳离子交换树脂(SP SEPHAROSE™ FF - GE Healthcare)上，该管柱用2CV的20 mM NaH2PO4 / Na2HPO4缓冲液(pH 7.0)平衡。用5CV的 20 mM NaH2PO4 / Na2HPO4缓冲液(pH 7.0)洗涤管柱后，通过将相同洗涤缓冲液中的NaCl的浓度增加至高达100 mM使抗原(包含在LVL317内)洗脱。
典型SP SEPHAROSETM快速流动层析图见图12。
Q SEPHAROSE™快速流动(Q FF)层析
将存在于SP FF洗脱物中的抗原用20 mM Tris pH 8.5以1：4进行稀释，如果需要将pH调节至8.5，并通过在14 cm ID x 11.8 cm长管柱(管柱体积1800 ml)中的强阴离子交换树脂(Q SEPHAROSE™ FF - GE Healthcare)，该管柱用2CV的20 mM Tris缓冲液(pH 8.5)进行平衡。在流通物级分中回收抗原。
典型Q SEPHAROSETM快速流动层析图见图13。
浓缩、渗滤、聚山梨酯80添加和无菌过滤
将含有抗原的Q FF流通物基于层析图UV浓缩至高达0.7-0.8 mg/ml，并用5DV的10 mMKH2PO4 / K2HPO4缓冲液(pH 6.5)使用Pellicon-2™ 10 kDa截止膜(Millipore)渗滤。
使用5%储存溶液将聚山梨酯80(例如，TWEENTM 80)添加至超滤保留物中，并在4℃下用磁力搅拌器在130 rpm下搅动30分钟。聚山梨酯80的最终浓度为0.04%。通过过滤通过0.45/0.2 μm乙酸纤维素膜(Sartobran 300, Sartorius)将超滤保留物灭菌。将纯化的容积物储存在-20℃或-80℃下。通过AAA(氨基酸分析)测量的绝对蛋白浓度为0.737 mg/ml。
实施例9：聚山梨酯80的用途
滴定实验指示，在无菌过滤之前向纯化的容积物中添加聚山梨酯80(具体而言，TWEENTM 80)至0.04%(w/v)的最终浓度会减少丝状颗粒形成和聚集。
根据DSC分析，TWEENTM 80降低在冷冻/解冻循环后在-20℃下储存后和在4℃、-20℃和-80℃和37℃下储存4天后所见到的结构变化的程度(30-45℃)。
实施例10：LVL317的SDS-PAGE和 Western印迹分析
SDS-PAGE和 Western印迹分析：
如下文所述上样NUPAGE®, Bis-Tris 4-12%凝胶与10µg含有50 mM DTT并在95℃下加热5 min的NUPAGE® LDS样品缓冲液中的样品(对于具有低浓度的样品，上样20µL样品)。迁移：在200伏特下在室温(RT)下在NUPAGE® MES运行缓冲液中35分钟。在速溶蓝(Instantblue)(Novexin目录号：ISB01L)中凝胶染色2小时，并在水中脱色过夜。
泳道内容物：
1: MW标准品(10µL) 2: 开始(总级分) (10µg) 3: SN1未经过滤(10µg)
4: SN2未经过滤(10µg) 5: 未经提取(10µg) 6: 上样SP FF (10µg)
7: 流通物SP FF (6.9µg) 8: 洗涤SP FF (20µL) 9: 洗脱SP FF (10µg)
10: 剥离SP FF (10µg) 11: 上样Q FF (8.9µg) 12: 洗脱Q FF (9.8µg)
13: 剥离Q FF (4.8µg) 14: 掺入0.04% TWEENTM 80之前的TFF保留物(10µg)
15: 经纯化容积物，未经过滤，掺入0.04% TWEENTM 80(10µg)
16: 经纯化容积物，经无菌过滤，掺入0.04% TWEENTM 80(10µg)
17: 经纯化容积物，经无菌过滤，掺入0.04% TWEENTM 80(20μg +掺入的大肠杆菌细胞裂解物Rix (1µg))
18: 大肠杆菌细胞裂解物Rix (2µg)
19: 大肠杆菌细胞裂解物Rix (1µg)
20: 大肠杆菌细胞裂解物Rix (0.5µg)。
来自PE-PilA融合蛋白的纯化过程的过程中样品的SDS-PAGE见图14。
对于 Western印迹，将蛋白在4℃下在30伏特下在NUPAGE®转移缓冲液+20%甲醇、0.1% SDS中过夜转移到硝酸纤维素膜上。将膜用50 mM Tris、150 mM NaCl pH 7.4+5%无脂奶粉封闭1小时，在稀释于封闭缓冲液中的兔多克隆一抗(抗Prot-E 1/50 000和抗大肠杆菌(BLR)1/1000)中孵育2小时，在50 mM Tris pH 7.4 + 0.05% Tween 20中洗涤3 x 5分钟，在二抗(缀合至碱性磷酸酶的山羊抗兔抗体，以1/5000稀释于封闭缓冲液中)中孵育1小时，在洗涤缓冲液中洗涤3x5分钟，并在BCIP/NBT底物(1个片剂/每10 ml)中显色。所有孵育均以25ml/每膜实施。
来自PE-PilA融合蛋白的纯化过程的过程中样品的Western印迹见图15。使用兔多克隆抗PE实施印迹。
泳道内容物：
1: MW标准品(10µL) 2: 开始(总级分) (10µg) 3: SN1未经过滤(10µg)
4: SN2未经过滤(10µg) 5: 未经提取(10µg) 6: 上样SP FF (10µg)
7: 流通物SP FF (6.9µg) 8: 洗涤SP FF (20µL) 9: 洗脱SP FF (10µg)
10: 剥离SP FF (10µg) 11: 上样Q FF (8.9µg) 12: 洗脱Q FF (9.8µg)
13: 剥离Q FF (4.8µg) 14: 掺入0.04% TWEENTM 80之前的TFF保留物(10µg)
15: 经纯化容积物，未经过滤，掺入0.04% TWEENTM 80 (10µg)
16: 经纯化容积物，经无菌过滤，掺入0.04% TWEENTM 80 (10µg)
17: 经纯化容积物，经无菌过滤，掺入0.04% TWEENTM 80 (20μg +掺入的大肠杆菌细胞裂解物Rix (1µg))
18: 大肠杆菌细胞裂解物Rix (2µg)
19: 大肠杆菌细胞裂解物Rix (1µg)
20: 大肠杆菌细胞裂解物Rix (0.5µg)。
来自PE-PilA融合蛋白的纯化过程的过程中样品的Western印迹见图16。使用兔多克隆抗大肠杆菌(BLR)实施印迹。
泳道内容物：
1: MW标准品(10µL) 2: 开始(总级分) (10µg) 3: SN1未经过滤(10µg)
4: SN2未经过滤(10µg) 5: 未经提取(10µg) 6: 上样SP FF (10µg)
7: 流通物SP FF (6.9µg) 8: 洗涤SP FF (20µL) 9: 洗脱SP FF (10µg)
10: 剥离SP FF (10µg) 11: 上样Q FF (8.9µg) 12: 洗脱Q FF (9.8µg)
13: 剥离Q FF (4.8µg) 14: 掺入0.04% TWEENTM 80之前的TFF保留物(10µg)
15: 经纯化容积物，未经过滤，掺入0.04% TWEENTM 80 (10µg)
16: 经纯化容积物，经无菌过滤，掺入0.04% TWEENTM 80 (10µg)
17: 经纯化容积物，经无菌过滤，掺入0.04% TWEENTM 80 (20μg +掺入的大肠杆菌细胞裂解物Rix (1µg))
18: 大肠杆菌细胞裂解物Rix (2µg)
19: 大肠杆菌细胞裂解物Rix (1µg)
20: 大肠杆菌细胞裂解物Rix (0.5µg)。
SDS-PAGE和 Western印迹图评价：PE-PilA融合蛋白以30 kDa迁移。通过渗透压休克的提取提取在细菌周质中表达和加工的融合蛋白，并减少来自细菌的污染。在高渗处理期间会有少量融合蛋白损失(泳道3)。小部分没有通过高渗处理提取出来，且仍然与细胞结合(泳道5)。在SP FF流通物(泳道7)中和在两个管柱的剥离部分(泳道10和13)中会有少量损失。由于剥离部分的总体积较小，所以融合蛋白的损失不明显。在剥离部分中可以看到降解条带，但在最终产物中看不到。在纯化的容积物中没有明显的来自大肠杆菌宿主细胞蛋白的污染(泳道16)。
对LVL735和LVL778的分析产生与LVL317类似的概况。
实施例11：PE、PilA和LVL317的熔点数据
将无His标签蛋白的PE-PilA融合蛋白(LVL317)的热转化与如上文所述纯化的His标签的PE(如实施例8中所述)和切割的PilA(如实施例8中所述)蛋白的热转化进行比较。
在DSC之前，将PE和PilA在10mM K2HPO4/KH2PO4 pH 6.5 + 0.04% Tween 80 (1:250的样品：缓冲液体积比)中透析过夜，以使其存在于与融合蛋白相同的缓冲液中。透析后，通过BCA测量蛋白浓度，并调节至300µg/ml (PE)和500µg/ml (PilA)。
在来自MicroCal, LLC (GE Healthcare的部分)的VPTM-DSC上实施分析。使用最终的透析缓冲液作为参考并从扫描中减除。DSC扫描速率为90℃/小时。为了评价测量配制后最终容器(FC)中热转化的能力，将融合蛋白稀释至FC浓度(60µg/ml)。最终容器数据未显示。
结果：
PE-PilA融合蛋白和PE和PilA蛋白的热转化见图17。曲线：PilA (1)，蛋白E(Prot E，PE) (2)，PE-PilA PB未稀释，737µg/ml (3)，和PE-PilA PB稀释至FC浓度60µg/ml (4)。
1–PilATm: 53℃
2–蛋白ETm: 63
3 – PE-PilA PB (经纯化容积物)，未稀释，737µg/ml Tm1 : 53.7℃和Tm2 : 66.1℃
4 – PE-PilA PB稀释至FC浓度60µg/ml Tm1: 53.2℃和Tm2: 67.6℃。
对于纯化的融合蛋白(LVL3 17)，检测到两种转化(曲线3和4)。
PE-PilA融合蛋白的Tm1 (53.7℃)与PilA转化(53℃)类似。
与PE转化(63℃)相比，PE-PilA的Tm2明显变化(66.1℃)。两个结构域的融合似乎会使PE片段稳定。
稀释的融合蛋白与未稀释的相比Tm2的偏移是由急剧下降斜率引起的浓度假像，此是浓度依赖性聚集的典型特征。
LVL735和LVL778的抗原折叠分析与LVL317类似。
实施例12：Balb/c小鼠中PE-PilA融合蛋白构建体LVL291的抗PilA免疫原性应答
在Balb/c小鼠中评价针对配制于AS03A中的纯化的LVL291 PE-PilA融合蛋白(无异源信号肽的LVL291融合蛋白)的免疫应答。在第0、14和28天通过肌内途径用10 µg PE(来自载体pRIT16762)、PilA(来自载体pRIT16790)或PE-PilA(每种均配制在AS03A中)免疫动物(20只小鼠/组)。对照组仅接种AS03A。在第42天采集的个别血清中测定针对每种抗原的抗体应答。使用阴性对照未获得抗体应答。如图18中所示，与用单价PilA免疫的小鼠中的抗体应答相比，用PE-PilA融合物免疫的小鼠中的针对PilA的抗体应答较强。用融合蛋白免疫的小鼠与用单价PE免疫的小鼠中的针对PE的抗体应答类似。GMT=几何平均滴度。收集数据，并用在WINDOWS® (Microsoft)下运行的SOFTMAX® Pro软件(Molecular Devices)进行分析；使用四参数逻辑对数函数来计算标准曲线。四参数逻辑对数函数以高精确度描述参考血清的曲线，当以光密度-对-浓度(log)比例描述时显示明显的S形。通过标准曲线内插来计算每个小鼠血清样品稀释度下的抗体浓度。质量对照血清和未知血清样品中的抗体通过取落在参考稀释曲线的工作范围(10-80%)内的所有稀释度的值的平均值来获得。
结果显示于图18中，其图示Balb/c小鼠模型中对LVL291 PE-PilA融合蛋白和对单价PE和PilA的抗体应答。
实施例13：鼠鼻咽定殖模型。用PE-PilA免疫。用NTHi菌株86-028NP和NTHi 菌株 3224A攻击。
Balb/c雌性小鼠(20只/组)在第0和14天用6µg与LT(大肠杆菌的热不稳定毒素)配制在一起的纯化的PE-PilA融合蛋白(LVL291，用86-028NP攻击；LVL317，用菌株3224A攻击)且在第28天用6 µg在磷酸盐缓冲盐水(PBS)中的纯化的PE-PilA融合蛋白鼻内免疫。对照小鼠(20只/组)仅接种LT。随后用5 x 106个CFU (菌落形成单位)的同源NTHi菌株86-028NP和异源NTHi菌株3224A鼻内攻击小鼠。同源性和异源性通过参考用以免疫小鼠的NTHi菌株来确定。计数在攻击后1天和2天取出的鼻腔内容物中的细菌菌落。Dl=第1天。D2=第2天。
PE-PilA接种使得攻击后第1天和第2天鼻咽中NTHi菌株86-028NP和菌株3224A的清除增加。
对于用NTHi菌株86-028NP实施的实验：使用计数的logl0值作为应答、以组(4个水平)和天数(2个水平)为固定因子来实施2因子固定ANOVA。排除方差异质性假定，并将异质性方差模型拟合至数据。两个因子之间未检测到明显相互作用。与对照组(LT)相比，融合PE-PilA组(6 µg/每只小鼠)明显减少CFU；几何平均比等于0.06，95%置信区间为0.01，0.25。
对于用NTHi菌株3224A进行的实验：使用logl0值作为应答、以组、天数和实验为固定因子来实施3因子固定ANOVA。Shapiro-Wilk和Levene检验不排除正态和方差同质性假定。在任何两个因子之间或在三个因子之间没有检测到明显的相互作用，且在分析中仅保留主要因子。与对照组相比，PE-PilA / LT显著减少CFU；几何平均比等于0.11，95%置信区间为0.02，0.61。
PE-PilA融合蛋白接种对小鼠鼻咽中NTHi菌株86-028NP细菌清除的影响见图19。
PE-PilA融合蛋白接种对小鼠鼻咽中NTHi菌株3224A细菌清除的影响见图20。
实施例14：鼠鼻咽定殖模型。用PilA免疫。用NTHi菌株3219C攻击。
雌性OF1小鼠(20只小鼠/组)在第0和14天用3µg用LT配制的PilA(来自载体16790)且在第28天用3 µg在PBS中的PilA鼻内免疫。对照小鼠仅接种LT。随后用5 x 106个CFU的NTHi菌株3219C鼻内攻击小鼠。计数在攻击后3天和4天取出的鼻腔内容物中的细菌菌落。D3=第3天。D4=第4天。
PilA接种对小鼠鼻咽中细菌清除的影响见图21。
实施例15：鼠鼻咽定殖模型。用PE免疫。用NTHi菌株3224A攻击。
Balb/c雌性小鼠(20只小鼠/组)在第0和14天用3µg用LT配制的PE (来自载体pRIT16762)且在第28天用3 µg在PBS中的PE鼻内免疫。对照小鼠仅接种LT。随后用5 x 106个CFU的NTHi菌株3224A鼻内攻击小鼠。计数在攻击后3天和4天取出的鼻腔内容物中的细菌菌落。在第3天(D3)检查10只小鼠。在第4天(D4)检查10只小鼠。使用用于统计学分析的Dunn检验，PE接种使得攻击后第4天鼻咽中NTHi的清除显著增加(图22)。
PE接种对小鼠鼻咽中细菌清除的影响见图22。
实施例16：玻连蛋白结合。LVL317 & LVL735 PE-PilA融合蛋白对玻连蛋白结合的 抑制。
评价纯化的LVL317 PE-PilA融合蛋白构建体中的PE结合玻连蛋白的能力。用PE(来自载体pRIT16762)或纯化的LVL317 PE-PilA融合蛋白(10 µg/ml)包被微量滴定板(POLYSORPTM, Nunc, Thermo Fisher Scientific)。用150 mM NaCl-0.05%聚山梨酯20(例如，TWEENTM 20)洗涤板四次，并用1% PBS-BSA封闭1至2小时。洗涤四次后，添加玻连蛋白(玻连蛋白来自人血浆，SIGMA-ALDRICH®)(10 µg/ml)，两倍稀释(12次稀释)，并将该板在室温下孵育lh。随后将该板用150 mM NaCl-0.05%聚山梨酯20(例如，TWEENTM 20)洗涤四次。洗涤后，使用过氧化物酶绵羊抗人玻连蛋白(US Biological)检测结合的玻连蛋白，随后添加邻苯二胺/H2O2底物。显色与固定至玻连蛋白的抗体的量成正比。
见图23：(a)结合至玻连蛋白的LVL317 PE-PilA融合蛋白。PilA = 来自NTHi菌株86-028NP的PilA(如对于pRIT16790所述)；PE=蛋白E(如对于pRIT16762所述)；和(b)结合至玻连蛋白的LVL317和LVL735PE-PilA融合蛋白。
实施例17：玻连蛋白结合。针对LVL291 PE-PilA融合蛋白的抗体对玻连蛋白结合 的抑制。
用PE(来自载体pRIT16762)或纯化的PE-PilA融合蛋白(10 µg/ml)包被微量滴定板(POLYSORPTM, Nunc, Thermo Fisher Scientific)。用150 mM NaCl-0.05%聚山梨酯20(例如，TWEENTM 20)洗涤板四次，并用1% PBS-BSA封闭2小时。洗涤后，添加50µg/ml玻连蛋白(玻连蛋白来自人血浆，SIGMA-ALDRICH®)，并将纯化的抗体抗PE-PilA(内部产生和纯化)两倍连续稀释，并在室温下孵育1h。随后将该板用150 mM NaCl-0.05%聚山梨酯20(例如，TWEENTM 20)洗涤四次。洗涤四次后，使用过氧化物酶绵羊抗玻连蛋白(US Biological)检测结合的玻连蛋白，随后添加邻苯二胺/H2O2底物。显色与固定至玻连蛋白的抗体的量成正比。
观察到针对PE-PilA的多克隆抗体抑制玻连蛋白结合至PE。
针对PE-PilA融合蛋白的多克隆抗体对玻连蛋白结合的抑制见图24。
实施例18：LVL291 PE-PilA融合蛋白的抗原性。ELISA。
在抗原性测试中以单价蛋白作为对照来验证纯化的LVL291 PE-PilA融合蛋白。在夹心ELISA中测试融合蛋白，该ELISA用针对编码SEQ ID NO:4的氨基酸22至160的PE基因片段(如对于pRIT16711所述)或针对NTHi菌株86-028NP的PilA (来自载体pRIT16790)产生的多克隆抗体(兔和豚鼠)开发。
添加100 ng/ml PilA或PE，并连续两倍稀释。孵育30分钟并洗涤后，通过在用PE或PilA免疫后获得的兔多克隆血清来检测结合的抗原。使用过氧化物酶抗兔Ig(JacksonImmunoResearch Laboratories, Inc.)来检测结合的抗体，随后添加邻苯二胺/H2O2底物。显色与存在的抗原量成正比。使用用于微量滴定板的分光光度计来测量吸光度读数。样品的抗原性通过与全长PE或全长PilA参考抗原的曲线进行比较来测定，且以ug/ml表示。参考代表100%抗原性。
如在表6中所观察到：与单价PE和PilA抗原相比，使用纯化的LVL291 PE-PilA融合蛋白观察到抗原性。
表6：在抗原性测试中使用纯化的LVL291 PE-PilA融合蛋白获得的相对抗原性。
实施例19：LVL735 PE-PilA融合蛋白的免疫原性。
雌性Balb/c小鼠(n=34)在第0、14和28天用50 µl疫苗制剂通过肌内途径免疫，该疫苗制剂含有1、0.2或0.04 µg配制在AS01E或AlPO4(磷酸铝)内的PE-PilA融合蛋白LVL317或LVL735。在第42天采集的个别血清中测定对PE和PilA的抗体应答，并测量针对PE和PilA的IgG水平，且以µg /ml表示。
对LVL317和LVL735的PE和PilA抗体应答见图27。GMC=几何平均浓度。GMT=几何平均滴度。IC=置信区间。
实施例20：在无法分型的流感嗜血杆菌鼻咽定殖的小鼠模型中LVL735和LVL317融 合蛋白的保护效力。
雌性Balb/c小鼠在第0和14天用10 µl含有 5.8 µg LVL735或LVL317与0.5 µg大肠杆菌热不稳定毒素(LT)的混合物的疫苗制剂鼻内免疫。在第28天施用5.8 µg不含佐剂的LVL735或LVL317加强剂量。对照小鼠仅在第0 和14天接种LT和在第28天接种PBS。在第42天，用5 x 106个cfu的NTHi 3224A菌株鼻内攻击动物。计数在攻击后1天和2天取出的鼻腔内容物中的细菌菌落(n=10/时间点)。将鼻腔内容物在培养基中均质化，并实施细菌定量。结果以CFU/ml表示。
LVL735和LVL317接种对无法分型的流感嗜血杆菌鼻咽定殖的小鼠模型中的细菌清除的影响见图28。
序列表
<110> POOLMAN, JAN
BLAIS, NORMAND
LABBE, STEVE
<120> 包含流感嗜血杆菌蛋白E和菌毛蛋白A的融合蛋白和组合疫苗
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<150> 61/474779
<151> 2011-04-13
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Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
Ile Lys Lys Ile Cys Thr Leu Ile Ser Leu Asn Phe Ile Gln Leu Leu
165 170 175
Gly Cys Arg Glu Tyr Ser Ile Phe Leu Gln Leu Leu Leu Phe Tyr Cys
180 185 190
Trp His Phe
195
<210> 17
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 17
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Met Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 18
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 18
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 19
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 19
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 20
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 20
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Val Glu Gln Asn Asp Val Lys Leu Thr Ala
20 25 30
Pro Thr Asp Val Arg Ser Gly Phe Val Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Leu Val Asp Lys Lys
145 150 155 160
<210> 21
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 21
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 22
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 22
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Ile Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 23
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 23
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 24
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 24
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Val Glu Gln Asn Asp Val Lys Leu Thr Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Val Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 25
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 25
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 26
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 26
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Val Glu Gln Asn Asp Val Lys Leu Thr Ala
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Val Arg Leu Val Lys Asn Ala Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 27
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 27
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Ile Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 28
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 28
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 29
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 29
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Thr Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 30
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 30
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 31
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 31
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 32
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 32
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 33
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 33
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Val Glu Gln Asn Asp Val Lys Leu Thr Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Val Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 34
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 34
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 35
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 35
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 36
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 36
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 37
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 37
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Ile Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 38
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 38
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Val Glu Gln Asn Asp Val Lys Leu Thr Ala
20 25 30
Pro Ala Asp Val Arg Ser Gly Tyr Val Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 39
<211> 159
<212> PRT
<213> 流感嗜血杆菌
<400> 39
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Tyr Lys Ile Leu
85 90 95
Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp
100 105 110
Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys
115 120 125
His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln
130 135 140
Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155
<210> 40
<211> 159
<212> PRT
<213> 流感嗜血杆菌
<400> 40
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Tyr Lys Ile Leu
85 90 95
Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp
100 105 110
Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys
115 120 125
His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln
130 135 140
Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155
<210> 41
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 41
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 42
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 42
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 43
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 43
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 44
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 44
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 45
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 45
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Thr Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Ser Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 46
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 46
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Val Glu Gln Asn Asp Val Lys Leu Thr Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Val Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Val Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 47
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 47
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Val Glu Gln Asn Asp Val Lys Leu Thr Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Val Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Val Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 48
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 48
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 49
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 49
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Thr Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 50
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 50
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Thr Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 51
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 51
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Thr Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 52
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 52
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 53
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 53
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Thr Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 54
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 54
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Thr Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Ile Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 55
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 55
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 56
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 56
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 57
<211> 159
<212> PRT
<213> 流感嗜血杆菌
<400> 57
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Met Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Val Asp Asn Gln Glu Pro Gln Ile
50 55 60
Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr
65 70 75 80
Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu
85 90 95
Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp
100 105 110
Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys
115 120 125
His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln
130 135 140
Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155
<210> 58
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 58
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 59
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 59
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Lys Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Ala Gly Asn Gly Thr Leu Asp Gly Met Ser
100 105 110
Tyr Thr Leu Thr Ala Glu Gly Asp Ser Ala Lys Gly Val Thr Trp Lys
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 60
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 60
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 61
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 61
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Lys Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Ser Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Ile Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Ala Gly Asn Ala Ala Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 62
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 62
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 63
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 63
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ser Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Val Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Ala Gly Asn Gly Thr Leu Asp Gly Met Ser
100 105 110
Tyr Thr Leu Thr Ala Glu Gly Asp Ser Ala Lys Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 64
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 64
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 65
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 65
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 66
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 66
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 67
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 67
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Lys Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Ile Thr Asn Cys Met Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Ile Thr Gln
145
<210> 68
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 68
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ser Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 69
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 69
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Ser Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Lys Thr Ala Lys Gly Tyr Val Ala Ser Val Ile Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Ala Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Lys
145
<210> 70
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 70
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Ser Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Ser Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 71
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 71
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Ser Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Lys Thr Ala Lys Gly Tyr Val Ala Ser Val Ile Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Ala Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Lys
145
<210> 72
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 72
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 73
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 73
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ser Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ser Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 74
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 74
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 75
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 75
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Glu Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 76
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 76
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ser Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 77
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 77
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 78
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 78
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 79
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 79
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Glu Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 80
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 80
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Glu Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 81
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 81
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ser Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Ala Gly Asn Gly Thr Leu Asp Gly Met Ser
100 105 110
Tyr Thr Leu Thr Ala Glu Gly Asp Ser Ala Lys Gly Val Thr Trp Lys
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Lys
145
<210> 82
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 82
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Ala Thr Lys Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Ser Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 83
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 83
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Ala Thr Lys Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Ser Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 84
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 84
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 85
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 85
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Ile Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ser Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 86
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 86
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Glu Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 87
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 87
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 88
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 88
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ser Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Val Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Ala Gly Asn Gly Thr Leu Asp Gly Met Ser
100 105 110
Tyr Thr Leu Thr Ala Glu Gly Asp Ser Ala Lys Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 89
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 89
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 90
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 90
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 91
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 91
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Ala Gly Asn Gly Thr Leu Asp Arg Met Ser
100 105 110
Tyr Thr Leu Thr Ala Glu Gly Asp Ser Ala Lys Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 92
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 92
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Ser Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Ser Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 93
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 93
Met Lys Leu Thr Thr Gln Thr Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ser Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Ile Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Thr Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Ile Thr Gln
145
<210> 94
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 94
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Asn Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Lys Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 95
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 95
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Ser Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Ser Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 96
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 96
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Ala Thr Lys Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Ser Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Thr Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 97
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 97
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 98
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 98
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 99
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 99
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 100
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 100
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 101
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 101
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 102
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 102
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 103
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 103
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Ala Thr Lys Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Ser Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 104
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 104
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 105
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 105
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 106
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<220>
<221> 变体
<222> (1)...(149)
<223> Xaa = 谷氨酰胺或亮氨酸
<400> 106
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Xaa Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 107
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 107
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 108
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 108
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 109
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 109
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Asn Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Lys Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 110
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 110
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Asn Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Lys Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 111
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 111
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Ser Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Ala Gly Asn Gly Thr Leu Asp Gly Met Ser
100 105 110
Tyr Thr Leu Thr Ala Glu Gly Asp Ser Ala Lys Gly Val Thr Trp Lys
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 112
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 112
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Ser Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Ala Gly Asn Gly Thr Leu Asp Gly Met Ser
100 105 110
Tyr Thr Leu Thr Ala Glu Gly Asp Ser Ala Lys Gly Val Thr Trp Lys
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 113
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 113
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Asn Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Lys Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 114
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 114
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Asn Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Lys Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 115
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 115
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Asn Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Lys Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 116
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 116
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Glu Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 117
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 117
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Asn Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Lys Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 118
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 118
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 119
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 119
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Asn Glu Ala Thr Lys Cys Thr Gly Gly Lys Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
85 90 95
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Ser Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 120
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 120
Met Lys Leu Thr Thr Leu Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ala Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Gly Ser Val Thr Gln
145
<210> 121
<211> 149
<212> PRT
<213> 流感嗜血杆菌
<400> 121
Met Lys Leu Thr Thr Gln Gln Thr Leu Lys Lys Gly Phe Thr Leu Ile
1 5 10 15
Glu Leu Met Ile Val Ile Ala Ile Ile Ala Ile Leu Ala Thr Ile Ala
20 25 30
Ile Pro Ser Tyr Gln Asn Tyr Thr Lys Lys Ala Ser Val Ser Glu Leu
35 40 45
Leu Gln Ala Ser Ala Pro Tyr Lys Ser Asp Val Glu Leu Cys Val Tyr
50 55 60
Ser Thr Gly Lys Pro Ser Thr Cys Ser Gly Gly Ser Asn Gly Ile Ala
65 70 75 80
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Ala Ser Val Lys Thr Gln
85 90 95
Ser Gly Gly Ile Thr Val Lys Gly Asn Gly Thr Leu Ala Asn Met Glu
100 105 110
Tyr Ile Leu Gln Ala Lys Gly Asn Ala Thr Ala Gly Val Thr Trp Thr
115 120 125
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
130 135 140
Arg Ser Val Thr Lys
145
<210> 122
<211> 144
<212> PRT
<213> 流感嗜血杆菌
<400> 122
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
1 5 10 15
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
20 25 30
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
35 40 45
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
50 55 60
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
65 70 75 80
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
85 90 95
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
100 105 110
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
115 120 125
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
130 135 140
<210> 123
<211> 143
<212> PRT
<213> 流感嗜血杆菌
<400> 123
Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro Pro
1 5 10 15
Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr
20 25 30
Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile
35 40 45
Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr
50 55 60
Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu
65 70 75 80
Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp
85 90 95
Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys
100 105 110
His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln
115 120 125
Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
130 135 140
<210> 124
<211> 142
<212> PRT
<213> 流感嗜血杆菌
<400> 124
Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro Pro Thr
1 5 10 15
Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr Tyr
20 25 30
Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile Val
35 40 45
His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr Pro
50 55 60
Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn
65 70 75 80
Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp Glu
85 90 95
Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys His
100 105 110
Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln Ile
115 120 125
Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
130 135 140
<210> 125
<211> 141
<212> PRT
<213> 流感嗜血杆菌
<400> 125
Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro Pro Thr Asp
1 5 10 15
Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr Tyr Ile
20 25 30
Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile Val His
35 40 45
Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr Pro Glu
50 55 60
Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys
65 70 75 80
Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp Glu Phe
85 90 95
Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys His Thr
100 105 110
Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln Ile Ile
115 120 125
Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
130 135 140
<210> 126
<211> 139
<212> PRT
<213> 流感嗜血杆菌
<400> 126
Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro Pro Thr Asp Val Arg
1 5 10 15
Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser
20 25 30
Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile Val His Phe Asp
35 40 45
Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys
50 55 60
Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn
65 70 75 80
Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly
85 90 95
Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys His Thr Leu Ser
100 105 110
Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln Ile Ile Cys Ala
115 120 125
Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
130 135
<210> 127
<211> 110
<212> PRT
<213> 流感嗜血杆菌
<400> 127
Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser Ala Pro Tyr
1 5 10 15
Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu Thr Thr Asn
20 25 30
Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr Thr Ala Lys
35 40 45
Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile Thr Val Lys
50 55 60
Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln Ala Thr Gly
65 70 75 80
Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys Gly Thr Asp
85 90 95
Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val Thr Gln
100 105 110
<210> 128
<211> 25
<212> PRT
<213> 流感嗜血杆菌
<400> 128
Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys
1 5 10 15
Ala Asn Tyr His Leu Thr Gln Val Arg
20 25
<210> 129
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> pelB信号肽
<400> 129
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggcc 66
<210> 130
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> pelB信号肽
<400> 130
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala
20
<210> 131
<211> 57
<212> DNA
<213> 人工序列
<220>
<223> FlgI信号肽
<400> 131
atgattaaat ttctctctgc attaattctt ctactggtca cgacggcggc tcaggct 57
<210> 132
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> FlgI信号肽
<400> 132
Met Ile Lys Phe Leu Ser Ala Leu Ile Leu Leu Leu Val Thr Thr Ala
1 5 10 15
Ala Gln Ala
<210> 133
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> NadA信号肽
<400> 133
atgaaacact ttccatccaa agtactgacc acagccatcc ttgccacttt ctgtagcggc 60
gcactggca 69
<210> 134
<211> 23
<212> PRT
<213> 人工序列
<220>
<223> NadA信号肽
<400> 134
Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala Thr
1 5 10 15
Phe Cys Ser Gly Ala Leu Ala
20
<210> 135
<211> 852
<212> DNA
<213> 人工序列
<220>
<223> LVL312
<400> 135
atgattaaat ttctctctgc attaattctt ctactggtca cgacggcggc tcaggctgag 60
actaaaaaag cagcggtatc tgaattactg caagcgtcag cgccttataa ggctgatgtg 120
gaattatgtg tatatagcac aaatgaaaca acaaactgta cgggtggaaa aaatggtatt 180
gcagcagata taaccacagc aaaaggctat gtaaaatcag tgacaacaag caacggtgca 240
ataacagtaa aaggggatgg cacattggca aatatggaat atattttgca agctacaggt 300
aatgctgcaa caggtgtaac ttggacaaca acttgcaaag gaacggatgc ctctttattt 360
ccagcaaatt tttgcggaag tgtcacacaa ggcggcgcgc agattcagaa ggctgaacaa 420
aatgatgtga agctggcacc gccgactgat gtacgaagcg gatatatacg tttggtaaag 480
aatgtgaatt attacatcga tagtgaatcg atctgggtgg ataaccaaga gccacaaatt 540
gtacattttg atgcagtggt gaatttagat aagggattgt atgtttatcc tgagcctaaa 600
cgttatgcac gttctgttcg tcagtataag atcttgaatt gtgcaaatta tcatttaact 660
caagtacgaa ctgatttcta tgatgaattt tggggacagg gtttgcgggc agcacctaaa 720
aagcaaaaga aacatacgtt aagtttaaca cctgatacaa cgctttataa tgctgctcag 780
attatttgtg cgaactatgg tgaagcattt tcagttgata aaaaaggcgg ccaccaccac 840
caccaccact aa 852
<210> 136
<211> 283
<212> PRT
<213> 人工序列
<220>
<223> LVL312
<400> 136
Met Ile Lys Phe Leu Ser Ala Leu Ile Leu Leu Leu Val Thr Thr Ala
1 5 10 15
Ala Gln Ala Glu Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala
20 25 30
Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn
35 40 45
Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile
50 55 60
Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala
65 70 75 80
Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu
85 90 95
Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys
100 105 110
Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val
115 120 125
Thr Gln Gly Gly Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys
130 135 140
Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys
145 150 155 160
Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln
165 170 175
Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly
180 185 190
Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln
195 200 205
Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr
210 215 220
Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys
225 230 235 240
Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr
245 250 255
Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val
260 265 270
Asp Lys Lys Gly Gly His His His His His His
275 280
<210> 137
<211> 855
<212> DNA
<213> 人工序列
<220>
<223> LVL291
<400> 137
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggcccaga ttcagaaggc tgaacaaaat gatgtgaagc tggcaccgcc gactgatgta 120
cgaagcggat atatacgttt ggtaaagaat gtgaattatt acatcgatag tgaatcgatc 180
tgggtggata accaagagcc acaaattgta cattttgatg cagtggtgaa tttagataag 240
ggattgtatg tttatcctga gcctaaacgt tatgcacgtt ctgttcgtca gtataagatc 300
ttgaattgtg caaattatca tttaactcaa gtacgaactg atttctatga tgaattttgg 360
ggacagggtt tgcgggcagc acctaaaaag caaaagaaac atacgttaag tttaacacct 420
gatacaacgc tttataatgc tgctcagatt atttgtgcga actatggtga agcattttca 480
gttgataaaa aaggcggcac taaaaaagca gcggtatctg aattactgca agcgtcagcg 540
ccttataagg ctgatgtgga attatgtgta tatagcacaa atgaaacaac aaactgtacg 600
ggtggaaaaa atggtattgc agcagatata accacagcaa aaggctatgt aaaatcagtg 660
acaacaagca acggtgcaat aacagtaaaa ggggatggca cattggcaaa tatggaatat 720
attttgcaag ctacaggtaa tgctgcaaca ggtgtaactt ggacaacaac ttgcaaagga 780
acggatgcct ctttatttcc agcaaatttt tgcggaagtg tcacacaagg cggccaccac 840
caccaccacc actaa 855
<210> 138
<211> 284
<212> PRT
<213> 人工序列
<220>
<223> LVL291
<400> 138
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val
20 25 30
Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val
35 40 45
Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn
50 55 60
Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys
65 70 75 80
Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg
85 90 95
Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg
100 105 110
Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro
115 120 125
Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu
130 135 140
Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser
145 150 155 160
Val Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu
165 170 175
Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser
180 185 190
Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala
195 200 205
Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn
210 215 220
Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr
225 230 235 240
Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr
245 250 255
Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly
260 265 270
Ser Val Thr Gln Gly Gly His His His His His His
275 280
<210> 139
<211> 852
<212> DNA
<213> 人工序列
<220>
<223> LVL268
<400> 139
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccgata ttcagaaggc tgaacaaaat gatgtgaagc tggcaccgcc gactgatgta 120
cgaagcggat atatacgttt ggtaaagaat gtgaattatt acatcgatag tgaatcgatc 180
tgggtggata accaagagcc acaaattgta cattttgatg cagtggtgaa tttagataag 240
ggattgtatg tttatcctga gcctaaacgt tatgcacgtt ctgttcgtca gtataagatc 300
ttgaattgtg caaattatca tttaactcaa gtacgaactg atttctatga tgaattttgg 360
ggacagggtt tgcgggcagc acctaaaaag caaaagaaac atacgttaag tttaacacct 420
gatacaacgc tttataatgc tgctcagatt atttgtgcga actatggtga agcattttca 480
gttgataaaa aaggcggcac taaaaaagca gcggtatctg aattactgca agcgtcagcg 540
ccttataagg ctgatgtgga attatgtgta tatagcacaa atgaaacaac aaactgtacg 600
ggtggaaaaa atggtattgc agcagatata accacagcaa aaggctatgt aaaatcagtg 660
acaacaagca acggtgcaat aacagtaaaa ggggatggca cattggcaaa tatggaatat 720
attttgcaag ctacaggtaa tgctgcaaca ggtgtaactt ggacaacaac ttgcaaagga 780
acggatgcct ctttatttcc agcaaatttt tgcggaagtg tcacacaagg cggccaccac 840
caccaccacc ac 852
<210> 140
<211> 284
<212> PRT
<213> 人工序列
<220>
<223> LVL268
<400> 140
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Asp Ile Gln Lys Ala Glu Gln Asn Asp Val
20 25 30
Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val
35 40 45
Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn
50 55 60
Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys
65 70 75 80
Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg
85 90 95
Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg
100 105 110
Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro
115 120 125
Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu
130 135 140
Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser
145 150 155 160
Val Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu
165 170 175
Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser
180 185 190
Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala
195 200 205
Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn
210 215 220
Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr
225 230 235 240
Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr
245 250 255
Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly
260 265 270
Ser Val Thr Gln Gly Gly His His His His His His
275 280
<210> 141
<211> 867
<212> DNA
<213> 人工序列
<220>
<223> LVL269
<400> 141
atgaaacact ttccatccaa agtactgacc acagccatcc ttgccacttt ctgtagcggc 60
gcactggcag ccacaaacga cgacgataag gctgaacaaa atgatgtgaa gctggcaccg 120
ccgactgatg tacgaagcgg atatatacgt ttggtaaaga atgtgaatta ttacatcgat 180
agtgaatcga tctgggtgga taaccaagag ccacaaattg tacattttga tgcagtggtg 240
aatttagata agggattgta tgtttatcct gagcctaaac gttatgcacg ttctgttcgt 300
cagtataaga tcttgaattg tgcaaattat catttaactc aagtacgaac tgatttctat 360
gatgaatttt ggggacaggg tttgcgggca gcacctaaaa agcaaaagaa acatacgtta 420
agtttaacac ctgatacaac gctttataat gctgctcaga ttatttgtgc gaactatggt 480
gaagcatttt cagttgataa aaaaggcggc actaaaaaag cagcggtatc tgaattactg 540
caagcgtcag cgccttataa ggctgatgtg gaattatgtg tatatagcac aaatgaaaca 600
acaaactgta cgggtggaaa aaatggtatt gcagcagata taaccacagc aaaaggctat 660
gtaaaatcag tgacaacaag caacggtgca ataacagtaa aaggggatgg cacattggca 720
aatatggaat atattttgca agctacaggt aatgctgcaa caggtgtaac ttggacaaca 780
acttgcaaag gaacggatgc ctctttattt ccagcaaatt tttgcggaag tgtcacacaa 840
ggcggccacc accaccacca ccactaa 867
<210> 142
<211> 288
<212> PRT
<213> 人工序列
<220>
<223> LVL269
<400> 142
Met Lys His Phe Pro Ser Lys Val Leu Thr Thr Ala Ile Leu Ala Thr
1 5 10 15
Phe Cys Ser Gly Ala Leu Ala Ala Thr Asn Asp Asp Asp Lys Ala Glu
20 25 30
Gln Asn Asp Val Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr
35 40 45
Ile Arg Leu Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile
50 55 60
Trp Val Asp Asn Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val
65 70 75 80
Asn Leu Asp Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala
85 90 95
Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu
100 105 110
Thr Gln Val Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu
115 120 125
Arg Ala Ala Pro Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro
130 135 140
Asp Thr Thr Leu Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly
145 150 155 160
Glu Ala Phe Ser Val Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val
165 170 175
Ser Glu Leu Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu
180 185 190
Cys Val Tyr Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn
195 200 205
Gly Ile Ala Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val
210 215 220
Thr Thr Ser Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala
225 230 235 240
Asn Met Glu Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val
245 250 255
Thr Trp Thr Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala
260 265 270
Asn Phe Cys Gly Ser Val Thr Gln Gly Gly His His His His His His
275 280 285
<210> 143
<211> 792
<212> DNA
<213> 人工序列
<220>
<223> LVL270
<400> 143
atgcaccacc accaccacca cagcgcgcag attcagaagg ctgaacaaaa tgatgtgaag 60
ctggcaccgc cgactgatgt acgaagcgga tatatacgtt tggtaaagaa tgtgaattat 120
tacatcgata gtgaatcgat ctgggtggat aaccaagagc cacaaattgt acattttgat 180
gcagtggtga atttagataa gggattgtat gtttatcctg agcctaaacg ttatgcacgt 240
tctgttcgtc agtataagat cttgaattgt gcaaattatc atttaactca agtacgaact 300
gatttctatg atgaattttg gggacagggt ttgcgggcag cacctaaaaa gcaaaagaaa 360
catacgttaa gtttaacacc tgatacaacg ctttataatg ctgctcagat tatttgtgcg 420
aactatggtg aagcattttc agttgataaa aaaggcggca ctaaaaaagc agcggtatct 480
gaattactgc aagcgtcagc gccttataag gctgatgtgg aattatgtgt atatagcaca 540
aatgaaacaa caaactgtac gggtggaaaa aatggtattg cagcagatat aaccacagca 600
aaaggctatg taaaatcagt gacaacaagc aacggtgcaa taacagtaaa aggggatggc 660
acattggcaa atatggaata tattttgcaa gctacaggta atgctgcaac aggtgtaact 720
tggacaacaa cttgcaaagg aacggatgcc tctttatttc cagcaaattt ttgcggaagt 780
gtcacacaat aa 792
<210> 144
<211> 263
<212> PRT
<213> 人工序列
<220>
<223> LVL270
<400> 144
Met His His His His His His Ser Ala Gln Ile Gln Lys Ala Glu Gln
1 5 10 15
Asn Asp Val Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile
20 25 30
Arg Leu Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp
35 40 45
Val Asp Asn Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn
50 55 60
Leu Asp Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg
65 70 75 80
Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr
85 90 95
Gln Val Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg
100 105 110
Ala Ala Pro Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp
115 120 125
Thr Thr Leu Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu
130 135 140
Ala Phe Ser Val Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser
145 150 155 160
Glu Leu Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys
165 170 175
Val Tyr Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly
180 185 190
Ile Ala Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr
195 200 205
Thr Ser Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn
210 215 220
Met Glu Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr
225 230 235 240
Trp Thr Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn
245 250 255
Phe Cys Gly Ser Val Thr Gln
260
<210> 145
<211> 852
<212> DNA
<213> 人工序列
<220>
<223> LVL315
<400> 145
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccatgg ataaggctga acaaaatgat gtgaagctgg caccgccgac tgatgtacga 120
agcggatata tacgtttggt aaagaatgtg aattattaca tcgatagtga atcgatctgg 180
gtggataacc aagagccaca aattgtacat tttgatgcag tggtgaattt agataaggga 240
ttgtatgttt atcctgagcc taaacgttat gcacgttctg ttcgtcagta taagatcttg 300
aattgtgcaa attatcattt aactcaagta cgaactgatt tctatgatga attttgggga 360
cagggtttgc gggcagcacc taaaaagcaa aagaaacata cgttaagttt aacacctgat 420
acaacgcttt ataatgctgc tcagattatt tgtgcgaact atggtgaagc attttcagtt 480
gataaaaaag gcggcactaa aaaagcagcg gtatctgaat tactgcaagc gtcagcgcct 540
tataaggctg atgtggaatt atgtgtatat agcacaaatg aaacaacaaa ctgtacgggt 600
ggaaaaaatg gtattgcagc agatataacc acagcaaaag gctatgtaaa atcagtgaca 660
acaagcaacg gtgcaataac agtaaaaggg gatggcacat tggcaaatat ggaatatatt 720
ttgcaagcta caggtaatgc tgcaacaggt gtaacttgga caacaacttg caaaggaacg 780
gatgcctctt tatttccagc aaatttttgc ggaagtgtca cacaaggcgg ccaccaccac 840
caccaccact aa 852
<210> 146
<211> 283
<212> PRT
<213> 人工序列
<220>
<223> LVL315
<400> 146
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Met Asp Lys Ala Glu Gln Asn Asp Val Lys
20 25 30
Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys
35 40 45
Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln
50 55 60
Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly
65 70 75 80
Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln
85 90 95
Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr
100 105 110
Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys
115 120 125
Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr
130 135 140
Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val
145 150 155 160
Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln
165 170 175
Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr
180 185 190
Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp
195 200 205
Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly
210 215 220
Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile
225 230 235 240
Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr
245 250 255
Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser
260 265 270
Val Thr Gln Gly Gly His His His His His His
275 280
<210> 147
<211> 831
<212> DNA
<213> 人工序列
<220>
<223> LVL317
<400> 147
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggcccaga ttcagaaggc tgaacaaaat gatgtgaagc tggcaccgcc gactgatgta 120
cgaagcggat atatacgttt ggtaaagaat gtgaattatt acatcgatag tgaatcgatc 180
tgggtggata accaagagcc acaaattgta cattttgatg cagtggtgaa tttagataag 240
ggattgtatg tttatcctga gcctaaacgt tatgcacgtt ctgttcgtca gtataagatc 300
ttgaattgtg caaattatca tttaactcaa gtacgaactg atttctatga tgaattttgg 360
ggacagggtt tgcgggcagc acctaaaaag caaaagaaac atacgttaag tttaacacct 420
gatacaacgc tttataatgc tgctcagatt atttgtgcga actatggtga agcattttca 480
gttgataaaa aaggcggcac taaaaaagca gcggtatctg aattactgca agcgtcagcg 540
ccttataagg ctgatgtgga attatgtgta tatagcacaa atgaaacaac aaactgtacg 600
ggtggaaaaa atggtattgc agcagatata accacagcaa aaggctatgt aaaatcagtg 660
acaacaagca acggtgcaat aacagtaaaa ggggatggca cattggcaaa tatggaatat 720
attttgcaag ctacaggtaa tgctgcaaca ggtgtaactt ggacaacaac ttgcaaagga 780
acggatgcct ctttatttcc agcaaatttt tgcggaagtg tcacacaata a 831
<210> 148
<211> 276
<212> PRT
<213> 人工序列
<220>
<223> LVL317
<400> 148
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val
20 25 30
Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val
35 40 45
Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn
50 55 60
Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys
65 70 75 80
Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg
85 90 95
Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg
100 105 110
Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro
115 120 125
Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu
130 135 140
Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser
145 150 155 160
Val Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu
165 170 175
Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser
180 185 190
Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala
195 200 205
Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn
210 215 220
Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr
225 230 235 240
Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr
245 250 255
Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly
260 265 270
Ser Val Thr Gln
275
<210> 149
<211> 828
<212> DNA
<213> 人工序列
<220>
<223> LVL318
<400> 149
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccatgg ataaggctga acaaaatgat gtgaagctgg caccgccgac tgatgtacga 120
agcggatata tacgtttggt aaagaatgtg aattattaca tcgatagtga atcgatctgg 180
gtggataacc aagagccaca aattgtacat tttgatgcag tggtgaattt agataaggga 240
ttgtatgttt atcctgagcc taaacgttat gcacgttctg ttcgtcagta taagatcttg 300
aattgtgcaa attatcattt aactcaagta cgaactgatt tctatgatga attttgggga 360
cagggtttgc gggcagcacc taaaaagcaa aagaaacata cgttaagttt aacacctgat 420
acaacgcttt ataatgctgc tcagattatt tgtgcgaact atggtgaagc attttcagtt 480
gataaaaaag gcggcactaa aaaagcagcg gtatctgaat tactgcaagc gtcagcgcct 540
tataaggctg atgtggaatt atgtgtatat agcacaaatg aaacaacaaa ctgtacgggt 600
ggaaaaaatg gtattgcagc agatataacc acagcaaaag gctatgtaaa atcagtgaca 660
acaagcaacg gtgcaataac agtaaaaggg gatggcacat tggcaaatat ggaatatatt 720
ttgcaagcta caggtaatgc tgcaacaggt gtaacttgga caacaacttg caaaggaacg 780
gatgcctctt tatttccagc aaatttttgc ggaagtgtca cacaataa 828
<210> 150
<211> 275
<212> PRT
<213> 人工序列
<220>
<223> LVL318
<400> 150
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Met Asp Lys Ala Glu Gln Asn Asp Val Lys
20 25 30
Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys
35 40 45
Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln
50 55 60
Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly
65 70 75 80
Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln
85 90 95
Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr
100 105 110
Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys
115 120 125
Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr
130 135 140
Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val
145 150 155 160
Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln
165 170 175
Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr
180 185 190
Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp
195 200 205
Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly
210 215 220
Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile
225 230 235 240
Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr
245 250 255
Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser
260 265 270
Val Thr Gln
275
<210> 151
<211> 483
<212> DNA
<213> 流感嗜血杆菌
<400> 151
atgaaaaaaa ttattttaac attatcactt gggttactta ccgcttgttc tgctcaaatc 60
caaaaggctg aacaaaatga tgtgaagctg gcaccgccga ctgatgtacg aagcggatat 120
atacgtttgg taaagaatgt gaattattac atcgatagtg aatcgatctg ggtggataac 180
caagagccac aaattgtaca ttttgatgct gtggtgaatt tagatagggg attgtatgtt 240
tatcctgagc ctaaacgtta tgcacgttct gttcgtcagt ataagatttt gaattgtgca 300
aattatcatt taactcaaat acgaactgat ttctatgatg aattttgggg acagggtttg 360
cgggcagcac ctaaaaagca aaagaaacat acgttaagtt taacacctga tacaacgctt 420
tataatgctg ctcagattat ttgtgcaaat tatggtaaag cattttcagt tgataaaaaa 480
taa 483
<210> 152
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 152
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Arg Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Ile Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Lys Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 153
<211> 480
<212> DNA
<213> 流感嗜血杆菌
<400> 153
atgaaaaaaa ttattttaac attatcactt gggttactta ctgcctgttc tgctcaaatc 60
caaaaggcta aacaaaatga tgtgaagctg gcaccgccga ctgatgtacg aagcggatat 120
atacgtttgg taaagaatgt gaattattac atcgatagtg aatcgatctg ggtggataac 180
caagagccac aaattgtaca ttttgatgca gtggtgaatt tagataaggg attgtatgtt 240
tatcctgagc ctaaacgtta tgcacgttct gttcgtcagt ataagatctt gaattgtgca 300
aattatcatt taactcaagt acgaactgat ttctatgatg aattttgggg acagggtttg 360
cgggcagcac ctaaaaagca aaagaaacat acgttaagtt taacacctga tacaacgctt 420
tataatgctg ctcagattat ttgtgcgaac tatggtgaag cattttcagt tgataaaaaa 480
<210> 154
<211> 160
<212> PRT
<213> 流感嗜血杆菌
<400> 154
Met Lys Lys Ile Ile Leu Thr Leu Ser Leu Gly Leu Leu Thr Ala Cys
1 5 10 15
Ser Ala Gln Ile Gln Lys Ala Lys Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
<210> 155
<211> 91
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 155
cacacacata tgattaaatt tctctctgca ttaattcttc tactggtcac gacggcggct 60
caggctgaga ctaaaaaagc agcggtatct g 91
<210> 156
<211> 66
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 156
tgtgtgaagc ttttagtggt ggtggtggtg gtggccgcct tgtgtgacac ttccgcaaaa 60
atttgc 66
<210> 157
<211> 58
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 157
tttgcggaag tgtcacacaa ggcggcgcgc agattcagaa ggctgaacaa aatgatgt 58
<210> 158
<211> 58
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 158
acatcatttt gttcagcctt ctgaatctgc gcgccgcctt gtgtgacact tccgcaaa 58
<210> 159
<211> 59
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 159
tgtgtgaagc ttttagtggt ggtggtggtg gtggccgcct tttttatcaa ctgaaaatg 59
<210> 160
<211> 65
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 160
cacacacata tgcaccacca ccaccaccac agcgcgcaga ttcagaaggc tgaacaaaat 60
gatgt 65
<210> 161
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 161
cattttcagt tgataaaaaa ggcggcacta aaaaagcagc ggtatc 46
<210> 162
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 162
gataccgctg cttttttagt gccgcctttt ttatcaactg aaaatg 46
<210> 163
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 163
tgtgtgaagc ttttattgtg tgacacttcc gcaaa 35
<210> 164
<211> 104
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 164
cacacacata tgaaatacct gctgccgacc gctgctgctg gtctgctgct cctcgctgcc 60
cagccggcga tggcccagat tcagaaggct gaacaaaatg atgt 104
<210> 165
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 165
gcattttcag ttgataaaaa aggcggcact aaaaaagcag cggtatctg 49
<210> 166
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 166
cagataccgc tgctttttta gtgccgcctt ttttatcaac tgaaaatgc 49
<210> 167
<211> 104
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 167
cacacacata tgaaatacct gctgccgacc gctgctgctg gtctgctgct cctcgctgcc 60
cagccggcga tggccgatat tcagaaggct gaacaaaatg atgt 104
<210> 168
<211> 115
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 168
cacacacata tgaaacactt tccatccaaa gtactgacca cagccatcct tgccactttc 60
tgtagcggcg cactggcagc cacaaacgac gacgataagg ctgaacaaaa tgatg 115
<210> 169
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 169
gccggcgatg gccatggata aggctgaaca aaatg 35
<210> 170
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 170
cattttgttc agccttatcc atggccatcg ccggc 35
<210> 171
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 171
ggaagtgtca cacaataagg cggccaccac cacc 34
<210> 172
<211> 34
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 172
ggtggtggtg gccgccttat tgtgtgacac ttcc 34
<210> 173
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 173
gaattccata tgcaccatca ccatcaccat actaaaaaag cagcggtatc tgaa 54
<210> 174
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 174
gcgccgctcg agtcattgtg tgacacttcc gc 32
<210> 175
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 175
gcccagccgg cgatggccca gatccagaag gctgaacaaa atg 43
<210> 176
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 176
cattttgttc agccttctgg atctgggcca tcgccggctg ggc 43
<210> 177
<211> 254
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 177
Gln Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro Pro Thr
1 5 10 15
Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr Tyr
20 25 30
Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile Val
35 40 45
His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr Pro
50 55 60
Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn
65 70 75 80
Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp Glu
85 90 95
Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys His
100 105 110
Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln Ile
115 120 125
Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys Gly Gly
130 135 140
Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser Ala Pro Tyr
145 150 155 160
Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu Thr Thr Asn
165 170 175
Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr Thr Ala Lys
180 185 190
Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile Thr Val Lys
195 200 205
Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln Ala Thr Gly
210 215 220
Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys Gly Thr Asp
225 230 235 240
Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val Thr Gln
245 250
<210> 178
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 合成构建体
<400> 178
Ala Thr Asn Asp Asp Asp
1 5
<210> 179
<211> 138
<212> PRT
<213> 流感嗜血杆菌
<400> 179
Ala Glu Gln Asn Asp Val Lys Leu Ala Pro Pro Thr Asp Val Arg Ser
1 5 10 15
Gly Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu
20 25 30
Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile Val His Phe Asp Ala
35 40 45
Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg
50 55 60
Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr
65 70 75 80
His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln
85 90 95
Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys His Thr Leu Ser Leu
100 105 110
Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn
115 120 125
Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
130 135
<210> 180
<211> 137
<212> PRT
<213> 流感嗜血杆菌
<400> 180
Glu Gln Asn Asp Val Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly
1 5 10 15
Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser
20 25 30
Ile Trp Val Asp Asn Gln Glu Pro Gln Ile Val His Phe Asp Ala Val
35 40 45
Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr
50 55 60
Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His
65 70 75 80
Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly
85 90 95
Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr
100 105 110
Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr
115 120 125
Gly Glu Ala Phe Ser Val Asp Lys Lys
130 135
<210> 181
<211> 849
<212> DNA
<213> 人工序列
<220>
<223> LVL702
<400> 181
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccattc agaaggctga acaaaatgat gtgaagctgg caccgccgac tgatgtacga 120
agcggatata tacgtttggt aaagaatgtg aattattaca tcgatagtga atcgatctgg 180
gtggataacc aagagccaca aattgtacat tttgatgcag tggtgaattt agataaggga 240
ttgtatgttt atcctgagcc taaacgttat gcacgttctg ttcgtcagta taagatcttg 300
aattgtgcaa attatcattt aactcaagta cgaactgatt tctatgatga attttgggga 360
cagggtttgc gggcagcacc taaaaagcaa aagaaacata cgttaagttt aacacctgat 420
acaacgcttt ataatgctgc tcagattatt tgtgcgaact atggtgaagc attttcagtt 480
gataaaaaag gcggcactaa aaaagcagcg gtatctgaat tactgcaagc gtcagcgcct 540
tataaggctg atgtggaatt atgtgtatat agcacaaatg aaacaacaaa ctgtacgggt 600
ggaaaaaatg gtattgcagc agatataacc acagcaaaag gctatgtaaa atcagtgaca 660
acaagcaacg gtgcaataac agtaaaaggg gatggcacat tggcaaatat ggaatatatt 720
ttgcaagcta caggtaatgc tgcaacaggt gtaacttgga caacaacttg caaaggaacg 780
gatgcctctt tatttccagc aaatttttgc ggaagtgtca cacaaggcgg ccaccaccac 840
caccaccac 849
<210> 182
<211> 283
<212> PRT
<213> 人工序列
<220>
<223> LVL702
<400> 182
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Ile Gln Lys Ala Glu Gln Asn Asp Val Lys
20 25 30
Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys
35 40 45
Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln
50 55 60
Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly
65 70 75 80
Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln
85 90 95
Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr
100 105 110
Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys
115 120 125
Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr
130 135 140
Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val
145 150 155 160
Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln
165 170 175
Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr
180 185 190
Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp
195 200 205
Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly
210 215 220
Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile
225 230 235 240
Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr
245 250 255
Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser
260 265 270
Val Thr Gln Gly Gly His His His His His His
275 280
<210> 183
<211> 858
<212> DNA
<213> 人工序列
<220>
<223> LVL736
<400> 183
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccagcg cccagattca gaaggctgaa caaaatgatg tgaagctggc accgccgact 120
gatgtacgaa gcggatatat acgtttggta aagaatgtga attattacat cgatagtgaa 180
tcgatctggg tggataacca agagccacaa attgtacatt ttgatgcagt ggtgaattta 240
gataagggat tgtatgttta tcctgagcct aaacgttatg cacgttctgt tcgtcagtat 300
aagatcttga attgtgcaaa ttatcattta actcaagtac gaactgattt ctatgatgaa 360
ttttggggac agggtttgcg ggcagcacct aaaaagcaaa agaaacatac gttaagttta 420
acacctgata caacgcttta taatgctgct cagattattt gtgcgaacta tggtgaagca 480
ttttcagttg ataaaaaagg cggcactaaa aaagcagcgg tatctgaatt actgcaagcg 540
tcagcgcctt ataaggctga tgtggaatta tgtgtatata gcacaaatga aacaacaaac 600
tgtacgggtg gaaaaaatgg tattgcagca gatataacca cagcaaaagg ctatgtaaaa 660
tcagtgacaa caagcaacgg tgcaataaca gtaaaagggg atggcacatt ggcaaatatg 720
gaatatattt tgcaagctac aggtaatgct gcaacaggtg taacttggac aacaacttgc 780
aaaggaacgg atgcctcttt atttccagca aatttttgcg gaagtgtcac acaaggcggc 840
caccaccacc accaccac 858
<210> 184
<211> 286
<212> PRT
<213> 人工序列
<220>
<223> LVL736
<400> 184
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn
20 25 30
Asp Val Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg
35 40 45
Leu Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val
50 55 60
Asp Asn Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu
65 70 75 80
Asp Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser
85 90 95
Val Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln
100 105 110
Val Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala
115 120 125
Ala Pro Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr
130 135 140
Thr Leu Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala
145 150 155 160
Phe Ser Val Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu
165 170 175
Leu Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val
180 185 190
Tyr Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile
195 200 205
Ala Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr
210 215 220
Ser Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met
225 230 235 240
Glu Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp
245 250 255
Thr Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe
260 265 270
Cys Gly Ser Val Thr Gln Gly Gly His His His His His His
275 280 285
<210> 185
<211> 855
<212> DNA
<213> 人工序列
<220>
<223> LVL737
<400> 185
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccgccc agattcagaa ggctgaacaa aatgatgtga agctggcacc gccgactgat 120
gtacgaagcg gatatatacg tttggtaaag aatgtgaatt attacatcga tagtgaatcg 180
atctgggtgg ataaccaaga gccacaaatt gtacattttg atgcagtggt gaatttagat 240
aagggattgt atgtttatcc tgagcctaaa cgttatgcac gttctgttcg tcagtataag 300
atcttgaatt gtgcaaatta tcatttaact caagtacgaa ctgatttcta tgatgaattt 360
tggggacagg gtttgcgggc agcacctaaa aagcaaaaga aacatacgtt aagtttaaca 420
cctgatacaa cgctttataa tgctgctcag attatttgtg cgaactatgg tgaagcattt 480
tcagttgata aaaaaggcgg cactaaaaaa gcagcggtat ctgaattact gcaagcgtca 540
gcgccttata aggctgatgt ggaattatgt gtatatagca caaatgaaac aacaaactgt 600
acgggtggaa aaaatggtat tgcagcagat ataaccacag caaaaggcta tgtaaaatca 660
gtgacaacaa gcaacggtgc aataacagta aaaggggatg gcacattggc aaatatggaa 720
tatattttgc aagctacagg taatgctgca acaggtgtaa cttggacaac aacttgcaaa 780
ggaacggatg cctctttatt tccagcaaat ttttgcggaa gtgtcacaca aggcggccac 840
caccaccacc accac 855
<210> 186
<211> 285
<212> PRT
<213> 人工序列
<220>
<223> LVL737
<400> 186
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp
20 25 30
Val Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu
35 40 45
Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp
50 55 60
Asn Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp
65 70 75 80
Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val
85 90 95
Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val
100 105 110
Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala
115 120 125
Pro Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr
130 135 140
Leu Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe
145 150 155 160
Ser Val Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu
165 170 175
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
180 185 190
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
195 200 205
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
210 215 220
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
225 230 235 240
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
245 250 255
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
260 265 270
Gly Ser Val Thr Gln Gly Gly His His His His His His
275 280 285
<210> 187
<211> 843
<212> DNA
<213> 人工序列
<220>
<223> LVL738
<400> 187
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccaagg ctgaacaaaa tgatgtgaag ctggcaccgc cgactgatgt acgaagcgga 120
tatatacgtt tggtaaagaa tgtgaattat tacatcgata gtgaatcgat ctgggtggat 180
aaccaagagc cacaaattgt acattttgat gcagtggtga atttagataa gggattgtat 240
gtttatcctg agcctaaacg ttatgcacgt tctgttcgtc agtataagat cttgaattgt 300
gcaaattatc atttaactca agtacgaact gatttctatg atgaattttg gggacagggt 360
ttgcgggcag cacctaaaaa gcaaaagaaa catacgttaa gtttaacacc tgatacaacg 420
ctttataatg ctgctcagat tatttgtgcg aactatggtg aagcattttc agttgataaa 480
aaaggcggca ctaaaaaagc agcggtatct gaattactgc aagcgtcagc gccttataag 540
gctgatgtgg aattatgtgt atatagcaca aatgaaacaa caaactgtac gggtggaaaa 600
aatggtattg cagcagatat aaccacagca aaaggctatg taaaatcagt gacaacaagc 660
aacggtgcaa taacagtaaa aggggatggc acattggcaa atatggaata tattttgcaa 720
gctacaggta atgctgcaac aggtgtaact tggacaacaa cttgcaaagg aacggatgcc 780
tctttatttc cagcaaattt ttgcggaagt gtcacacaag gcggccacca ccaccaccac 840
cac 843
<210> 188
<211> 281
<212> PRT
<213> 人工序列
<220>
<223> LVL738
<400> 188
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Lys Ala Glu Gln Asn Asp Val Lys Leu Ala
20 25 30
Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val
35 40 45
Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro
50 55 60
Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr
65 70 75 80
Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys
85 90 95
Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe
100 105 110
Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln
115 120 125
Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala
130 135 140
Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys
145 150 155 160
Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser
165 170 175
Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu
180 185 190
Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr
195 200 205
Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile
210 215 220
Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln
225 230 235 240
Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys
245 250 255
Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val Thr
260 265 270
Gln Gly Gly His His His His His His
275 280
<210> 189
<211> 840
<212> DNA
<213> 人工序列
<220>
<223> LVL739
<400> 189
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccgctg aacaaaatga tgtgaagctg gcaccgccga ctgatgtacg aagcggatat 120
atacgtttgg taaagaatgt gaattattac atcgatagtg aatcgatctg ggtggataac 180
caagagccac aaattgtaca ttttgatgca gtggtgaatt tagataaggg attgtatgtt 240
tatcctgagc ctaaacgtta tgcacgttct gttcgtcagt ataagatctt gaattgtgca 300
aattatcatt taactcaagt acgaactgat ttctatgatg aattttgggg acagggtttg 360
cgggcagcac ctaaaaagca aaagaaacat acgttaagtt taacacctga tacaacgctt 420
tataatgctg ctcagattat ttgtgcgaac tatggtgaag cattttcagt tgataaaaaa 480
ggcggcacta aaaaagcagc ggtatctgaa ttactgcaag cgtcagcgcc ttataaggct 540
gatgtggaat tatgtgtata tagcacaaat gaaacaacaa actgtacggg tggaaaaaat 600
ggtattgcag cagatataac cacagcaaaa ggctatgtaa aatcagtgac aacaagcaac 660
ggtgcaataa cagtaaaagg ggatggcaca ttggcaaata tggaatatat tttgcaagct 720
acaggtaatg ctgcaacagg tgtaacttgg acaacaactt gcaaaggaac ggatgcctct 780
ttatttccag caaatttttg cggaagtgtc acacaaggcg gccaccacca ccaccaccac 840
<210> 190
<211> 280
<212> PRT
<213> 人工序列
<220>
<223> LVL739
<400> 190
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser Ala
165 170 175
Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu Thr
180 185 190
Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr Thr
195 200 205
Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile Thr
210 215 220
Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln Ala
225 230 235 240
Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys Gly
245 250 255
Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val Thr Gln
260 265 270
Gly Gly His His His His His His
275 280
<210> 191
<211> 837
<212> DNA
<213> 人工序列
<220>
<223> LVL740
<400> 191
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccgaac aaaatgatgt gaagctggca ccgccgactg atgtacgaag cggatatata 120
cgtttggtaa agaatgtgaa ttattacatc gatagtgaat cgatctgggt ggataaccaa 180
gagccacaaa ttgtacattt tgatgcagtg gtgaatttag ataagggatt gtatgtttat 240
cctgagccta aacgttatgc acgttctgtt cgtcagtata agatcttgaa ttgtgcaaat 300
tatcatttaa ctcaagtacg aactgatttc tatgatgaat tttggggaca gggtttgcgg 360
gcagcaccta aaaagcaaaa gaaacatacg ttaagtttaa cacctgatac aacgctttat 420
aatgctgctc agattatttg tgcgaactat ggtgaagcat tttcagttga taaaaaaggc 480
ggcactaaaa aagcagcggt atctgaatta ctgcaagcgt cagcgcctta taaggctgat 540
gtggaattat gtgtatatag cacaaatgaa acaacaaact gtacgggtgg aaaaaatggt 600
attgcagcag atataaccac agcaaaaggc tatgtaaaat cagtgacaac aagcaacggt 660
gcaataacag taaaagggga tggcacattg gcaaatatgg aatatatttt gcaagctaca 720
ggtaatgctg caacaggtgt aacttggaca acaacttgca aaggaacgga tgcctcttta 780
tttccagcaa atttttgcgg aagtgtcaca caaggcggcc accaccacca ccaccac 837
<210> 192
<211> 279
<212> PRT
<213> 人工序列
<220>
<223> LVL740
<400> 192
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Glu Gln Asn Asp Val Lys Leu Ala Pro Pro
20 25 30
Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr
35 40 45
Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile
50 55 60
Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr
65 70 75 80
Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu
85 90 95
Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp
100 105 110
Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys
115 120 125
His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln
130 135 140
Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys Gly
145 150 155 160
Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser Ala Pro
165 170 175
Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu Thr Thr
180 185 190
Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr Thr Ala
195 200 205
Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile Thr Val
210 215 220
Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln Ala Thr
225 230 235 240
Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys Gly Thr
245 250 255
Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val Thr Gln Gly
260 265 270
Gly His His His His His His
275
<210> 193
<211> 825
<212> DNA
<213> 人工序列
<220>
<223> LVL735
<400> 193
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccattc agaaggctga acaaaatgat gtgaagctgg caccgccgac tgatgtacga 120
agcggatata tacgtttggt aaagaatgtg aattattaca tcgatagtga atcgatctgg 180
gtggataacc aagagccaca aattgtacat tttgatgcag tggtgaattt agataaggga 240
ttgtatgttt atcctgagcc taaacgttat gcacgttctg ttcgtcagta taagatcttg 300
aattgtgcaa attatcattt aactcaagta cgaactgatt tctatgatga attttgggga 360
cagggtttgc gggcagcacc taaaaagcaa aagaaacata cgttaagttt aacacctgat 420
acaacgcttt ataatgctgc tcagattatt tgtgcgaact atggtgaagc attttcagtt 480
gataaaaaag gcggcactaa aaaagcagcg gtatctgaat tactgcaagc gtcagcgcct 540
tataaggctg atgtggaatt atgtgtatat agcacaaatg aaacaacaaa ctgtacgggt 600
ggaaaaaatg gtattgcagc agatataacc acagcaaaag gctatgtaaa atcagtgaca 660
acaagcaacg gtgcaataac agtaaaaggg gatggcacat tggcaaatat ggaatatatt 720
ttgcaagcta caggtaatgc tgcaacaggt gtaacttgga caacaacttg caaaggaacg 780
gatgcctctt tatttccagc aaatttttgc ggaagtgtca cacaa 825
<210> 194
<211> 275
<212> PRT
<213> 人工序列
<220>
<223> LVL735
<400> 194
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Ile Gln Lys Ala Glu Gln Asn Asp Val Lys
20 25 30
Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys
35 40 45
Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln
50 55 60
Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly
65 70 75 80
Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln
85 90 95
Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr
100 105 110
Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys
115 120 125
Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr
130 135 140
Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val
145 150 155 160
Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln
165 170 175
Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr
180 185 190
Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp
195 200 205
Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly
210 215 220
Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile
225 230 235 240
Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr
245 250 255
Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser
260 265 270
Val Thr Gln
275
<210> 195
<211> 834
<212> DNA
<213> 人工序列
<220>
<223> LVL778
<400> 195
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccagcg cccagattca gaaggctgaa caaaatgatg tgaagctggc accgccgact 120
gatgtacgaa gcggatatat acgtttggta aagaatgtga attattacat cgatagtgaa 180
tcgatctggg tggataacca agagccacaa attgtacatt ttgatgcagt ggtgaattta 240
gataagggat tgtatgttta tcctgagcct aaacgttatg cacgttctgt tcgtcagtat 300
aagatcttga attgtgcaaa ttatcattta actcaagtac gaactgattt ctatgatgaa 360
ttttggggac agggtttgcg ggcagcacct aaaaagcaaa agaaacatac gttaagttta 420
acacctgata caacgcttta taatgctgct cagattattt gtgcgaacta tggtgaagca 480
ttttcagttg ataaaaaagg cggcactaaa aaagcagcgg tatctgaatt actgcaagcg 540
tcagcgcctt ataaggctga tgtggaatta tgtgtatata gcacaaatga aacaacaaac 600
tgtacgggtg gaaaaaatgg tattgcagca gatataacca cagcaaaagg ctatgtaaaa 660
tcagtgacaa caagcaacgg tgcaataaca gtaaaagggg atggcacatt ggcaaatatg 720
gaatatattt tgcaagctac aggtaatgct gcaacaggtg taacttggac aacaacttgc 780
aaaggaacgg atgcctcttt atttccagca aatttttgcg gaagtgtcac acaa 834
<210> 196
<211> 278
<212> PRT
<213> 人工序列
<220>
<223> LVL778
<400> 196
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Ser Ala Gln Ile Gln Lys Ala Glu Gln Asn
20 25 30
Asp Val Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg
35 40 45
Leu Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val
50 55 60
Asp Asn Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu
65 70 75 80
Asp Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser
85 90 95
Val Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln
100 105 110
Val Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala
115 120 125
Ala Pro Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr
130 135 140
Thr Leu Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala
145 150 155 160
Phe Ser Val Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu
165 170 175
Leu Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val
180 185 190
Tyr Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile
195 200 205
Ala Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr
210 215 220
Ser Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met
225 230 235 240
Glu Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp
245 250 255
Thr Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe
260 265 270
Cys Gly Ser Val Thr Gln
275
<210> 197
<211> 831
<212> DNA
<213> 人工序列
<220>
<223> LVL779
<400> 197
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccgccc agattcagaa ggctgaacaa aatgatgtga agctggcacc gccgactgat 120
gtacgaagcg gatatatacg tttggtaaag aatgtgaatt attacatcga tagtgaatcg 180
atctgggtgg ataaccaaga gccacaaatt gtacattttg atgcagtggt gaatttagat 240
aagggattgt atgtttatcc tgagcctaaa cgttatgcac gttctgttcg tcagtataag 300
atcttgaatt gtgcaaatta tcatttaact caagtacgaa ctgatttcta tgatgaattt 360
tggggacagg gtttgcgggc agcacctaaa aagcaaaaga aacatacgtt aagtttaaca 420
cctgatacaa cgctttataa tgctgctcag attatttgtg cgaactatgg tgaagcattt 480
tcagttgata aaaaaggcgg cactaaaaaa gcagcggtat ctgaattact gcaagcgtca 540
gcgccttata aggctgatgt ggaattatgt gtatatagca caaatgaaac aacaaactgt 600
acgggtggaa aaaatggtat tgcagcagat ataaccacag caaaaggcta tgtaaaatca 660
gtgacaacaa gcaacggtgc aataacagta aaaggggatg gcacattggc aaatatggaa 720
tatattttgc aagctacagg taatgctgca acaggtgtaa cttggacaac aacttgcaaa 780
ggaacggatg cctctttatt tccagcaaat ttttgcggaa gtgtcacaca a 831
<210> 198
<211> 277
<212> PRT
<213> 人工序列
<220>
<223> LVL779
<400> 198
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Ala Gln Ile Gln Lys Ala Glu Gln Asn Asp
20 25 30
Val Lys Leu Ala Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu
35 40 45
Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp
50 55 60
Asn Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp
65 70 75 80
Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val
85 90 95
Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val
100 105 110
Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala
115 120 125
Pro Lys Lys Gln Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr
130 135 140
Leu Tyr Asn Ala Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe
145 150 155 160
Ser Val Asp Lys Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu
165 170 175
Leu Gln Ala Ser Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr
180 185 190
Ser Thr Asn Glu Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala
195 200 205
Ala Asp Ile Thr Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser
210 215 220
Asn Gly Ala Ile Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu
225 230 235 240
Tyr Ile Leu Gln Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr
245 250 255
Thr Thr Cys Lys Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys
260 265 270
Gly Ser Val Thr Gln
275
<210> 199
<211> 819
<212> DNA
<213> 人工序列
<220>
<223> LVL780
<400> 199
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccaagg ctgaacaaaa tgatgtgaag ctggcaccgc cgactgatgt acgaagcgga 120
tatatacgtt tggtaaagaa tgtgaattat tacatcgata gtgaatcgat ctgggtggat 180
aaccaagagc cacaaattgt acattttgat gcagtggtga atttagataa gggattgtat 240
gtttatcctg agcctaaacg ttatgcacgt tctgttcgtc agtataagat cttgaattgt 300
gcaaattatc atttaactca agtacgaact gatttctatg atgaattttg gggacagggt 360
ttgcgggcag cacctaaaaa gcaaaagaaa catacgttaa gtttaacacc tgatacaacg 420
ctttataatg ctgctcagat tatttgtgcg aactatggtg aagcattttc agttgataaa 480
aaaggcggca ctaaaaaagc agcggtatct gaattactgc aagcgtcagc gccttataag 540
gctgatgtgg aattatgtgt atatagcaca aatgaaacaa caaactgtac gggtggaaaa 600
aatggtattg cagcagatat aaccacagca aaaggctatg taaaatcagt gacaacaagc 660
aacggtgcaa taacagtaaa aggggatggc acattggcaa atatggaata tattttgcaa 720
gctacaggta atgctgcaac aggtgtaact tggacaacaa cttgcaaagg aacggatgcc 780
tctttatttc cagcaaattt ttgcggaagt gtcacacaa 819
<210> 200
<211> 273
<212> PRT
<213> 人工序列
<220>
<223> LVL780
<400> 200
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Lys Ala Glu Gln Asn Asp Val Lys Leu Ala
20 25 30
Pro Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val
35 40 45
Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro
50 55 60
Gln Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr
65 70 75 80
Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys
85 90 95
Ile Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe
100 105 110
Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln
115 120 125
Lys Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala
130 135 140
Ala Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys
145 150 155 160
Lys Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser
165 170 175
Ala Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu
180 185 190
Thr Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr
195 200 205
Thr Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile
210 215 220
Thr Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln
225 230 235 240
Ala Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys
245 250 255
Gly Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val Thr
260 265 270
Gln
<210> 201
<211> 816
<212> DNA
<213> 人工序列
<220>
<223> LVL781
<400> 201
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccgctg aacaaaatga tgtgaagctg gcaccgccga ctgatgtacg aagcggatat 120
atacgtttgg taaagaatgt gaattattac atcgatagtg aatcgatctg ggtggataac 180
caagagccac aaattgtaca ttttgatgca gtggtgaatt tagataaggg attgtatgtt 240
tatcctgagc ctaaacgtta tgcacgttct gttcgtcagt ataagatctt gaattgtgca 300
aattatcatt taactcaagt acgaactgat ttctatgatg aattttgggg acagggtttg 360
cgggcagcac ctaaaaagca aaagaaacat acgttaagtt taacacctga tacaacgctt 420
tataatgctg ctcagattat ttgtgcgaac tatggtgaag cattttcagt tgataaaaaa 480
ggcggcacta aaaaagcagc ggtatctgaa ttactgcaag cgtcagcgcc ttataaggct 540
gatgtggaat tatgtgtata tagcacaaat gaaacaacaa actgtacggg tggaaaaaat 600
ggtattgcag cagatataac cacagcaaaa ggctatgtaa aatcagtgac aacaagcaac 660
ggtgcaataa cagtaaaagg ggatggcaca ttggcaaata tggaatatat tttgcaagct 720
acaggtaatg ctgcaacagg tgtaacttgg acaacaactt gcaaaggaac ggatgcctct 780
ttatttccag caaatttttg cggaagtgtc acacaa 816
<210> 202
<211> 272
<212> PRT
<213> 人工序列
<220>
<223> LVL781
<400> 202
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Ala Glu Gln Asn Asp Val Lys Leu Ala Pro
20 25 30
Pro Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn
35 40 45
Tyr Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln
50 55 60
Ile Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val
65 70 75 80
Tyr Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile
85 90 95
Leu Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr
100 105 110
Asp Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys
115 120 125
Lys His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala
130 135 140
Gln Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys
145 150 155 160
Gly Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser Ala
165 170 175
Pro Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu Thr
180 185 190
Thr Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr Thr
195 200 205
Ala Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile Thr
210 215 220
Val Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln Ala
225 230 235 240
Thr Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys Gly
245 250 255
Thr Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val Thr Gln
260 265 270
<210> 203
<211> 813
<212> DNA
<213> 人工序列
<220>
<223> LVL782
<400> 203
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggccgaac aaaatgatgt gaagctggca ccgccgactg atgtacgaag cggatatata 120
cgtttggtaa agaatgtgaa ttattacatc gatagtgaat cgatctgggt ggataaccaa 180
gagccacaaa ttgtacattt tgatgcagtg gtgaatttag ataagggatt gtatgtttat 240
cctgagccta aacgttatgc acgttctgtt cgtcagtata agatcttgaa ttgtgcaaat 300
tatcatttaa ctcaagtacg aactgatttc tatgatgaat tttggggaca gggtttgcgg 360
gcagcaccta aaaagcaaaa gaaacatacg ttaagtttaa cacctgatac aacgctttat 420
aatgctgctc agattatttg tgcgaactat ggtgaagcat tttcagttga taaaaaaggc 480
ggcactaaaa aagcagcggt atctgaatta ctgcaagcgt cagcgcctta taaggctgat 540
gtggaattat gtgtatatag cacaaatgaa acaacaaact gtacgggtgg aaaaaatggt 600
attgcagcag atataaccac agcaaaaggc tatgtaaaat cagtgacaac aagcaacggt 660
gcaataacag taaaagggga tggcacattg gcaaatatgg aatatatttt gcaagctaca 720
ggtaatgctg caacaggtgt aacttggaca acaacttgca aaggaacgga tgcctcttta 780
tttccagcaa atttttgcgg aagtgtcaca caa 813
<210> 204
<211> 271
<212> PRT
<213> 人工序列
<220>
<223> LVL782
<400> 204
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Glu Gln Asn Asp Val Lys Leu Ala Pro Pro
20 25 30
Thr Asp Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr
35 40 45
Tyr Ile Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile
50 55 60
Val His Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr
65 70 75 80
Pro Glu Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu
85 90 95
Asn Cys Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp
100 105 110
Glu Phe Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys
115 120 125
His Thr Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln
130 135 140
Ile Ile Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys Gly
145 150 155 160
Gly Thr Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser Ala Pro
165 170 175
Tyr Lys Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu Thr Thr
180 185 190
Asn Cys Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr Thr Ala
195 200 205
Lys Gly Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile Thr Val
210 215 220
Lys Gly Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln Ala Thr
225 230 235 240
Gly Asn Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys Gly Thr
245 250 255
Asp Ala Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val Thr Gln
260 265 270
<210> 205
<211> 95
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 205
gatatacata tgaaatacct gctgccgacc gctgctgctg gtctgctgct cctcgctgcc 60
cagccggcga tggccattca gaaggctgaa caaaa 95
<210> 206
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 206
ggccgcaagc ttttagtggt ggtggtggtg gtggccgcc 39
<210> 207
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 207
ggccgcaagc ttttattgtg tgacacttcc 30
<210> 208
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 208
gctgcccagc cggcgatggc caaggctgaa caaaatgatg tg 42
<210> 209
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 209
cacatcattt tgttcagcct tggccatcgc cggctgggca gc 42
<210> 210
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 210
gctgcccagc cggcgatggc cgctgaacaa aatgatgtga agc 43
<210> 211
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 211
gcttcacatc attttgttca gcggccatcg ccggctgggc agc 43
<210> 212
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 212
gctgcccagc cggcgatggc cgaacaaaat gatgtgaagc tgg 43
<210> 213
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 213
ccagcttcac atcattttgt tcggccatcg ccggctgggc agc 43
<210> 214
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 214
gctgcccagc cggcgatggc cgcccagatt cagaaggctg aac 43
<210> 215
<211> 43
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 215
gttcagcctt ctgaatctgg gcggccatcg ccggctgggc agc 43
<210> 216
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 216
gctgcccagc cggcgatggc cagcgcccag attcagaagg ctgaac 46
<210> 217
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 217
gttcagcctt ctgaatctgg gcgctggcca tcgccggctg ggcagc 46
<210> 218
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 218
cacacacata tgaaatacct gctgccgacc 30
<210> 219
<211> 253
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白
<400> 219
Ile Gln Lys Ala Glu Gln Asn Asp Val Lys Leu Ala Pro Pro Thr Asp
1 5 10 15
Val Arg Ser Gly Tyr Ile Arg Leu Val Lys Asn Val Asn Tyr Tyr Ile
20 25 30
Asp Ser Glu Ser Ile Trp Val Asp Asn Gln Glu Pro Gln Ile Val His
35 40 45
Phe Asp Ala Val Val Asn Leu Asp Lys Gly Leu Tyr Val Tyr Pro Glu
50 55 60
Pro Lys Arg Tyr Ala Arg Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys
65 70 75 80
Ala Asn Tyr His Leu Thr Gln Val Arg Thr Asp Phe Tyr Asp Glu Phe
85 90 95
Trp Gly Gln Gly Leu Arg Ala Ala Pro Lys Lys Gln Lys Lys His Thr
100 105 110
Leu Ser Leu Thr Pro Asp Thr Thr Leu Tyr Asn Ala Ala Gln Ile Ile
115 120 125
Cys Ala Asn Tyr Gly Glu Ala Phe Ser Val Asp Lys Lys Gly Gly Thr
130 135 140
Lys Lys Ala Ala Val Ser Glu Leu Leu Gln Ala Ser Ala Pro Tyr Lys
145 150 155 160
Ala Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu Thr Thr Asn Cys
165 170 175
Thr Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr Thr Ala Lys Gly
180 185 190
Tyr Val Lys Ser Val Thr Thr Ser Asn Gly Ala Ile Thr Val Lys Gly
195 200 205
Asp Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln Ala Thr Gly Asn
210 215 220
Ala Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys Gly Thr Asp Ala
225 230 235 240
Ser Leu Phe Pro Ala Asn Phe Cys Gly Ser Val Thr Gln
245 250

Claims (10)

1.式I的融合蛋白：
(X)m-(R1)n-A-(Y)o-B-(Z)p(式1)
其中：
X是信号肽或MHHHHHH(SEQ ID NO.2)；
m是0或1；
R1是一个氨基酸；
n是0、1、2、3、4、5或6；
A是来自流感嗜血杆菌的蛋白E或其免疫原性片段、或来自流感嗜血杆菌的PilA或其免疫原性片段；
Y选自GG、SG、SS、GGG和(G)h，其中h是4、5、6、7、8、9、或10；
o是0或1；
B是来自流感嗜血杆菌的PilA或其免疫原性片段、或来自流感嗜血杆菌的蛋白E或其免疫原性片段；
Z是GGHHHHHH(SEQ ID NO.3)；且
p是0或1。
2.融合蛋白，其选自SEQ ID NO.136，SEQ ID NO.
138，SEQ ID NO.140，SEQ ID NO.142，SEQ ID NO.144，SEQ ID NO.146，SEQ ID NO148，SEQ ID NO.150，SEQ ID NO.182，SEQ ID NO.184，SEQ ID NO.186，SEQ ID NO.188，SEQID NO.190，SEQ ID NO.192，SEQ ID NO.194，SEQ ID NO.196，SEQ ID NO.198，SEQ IDNO.200，SEQ ID NO.202和SEQ ID NO.204。
3.权利要求2的融合蛋白，其中信号肽已经移除。
4.SEQ ID NO.148的融合蛋白，其中信号肽已经移除，所述融合蛋白具有
5.SEQ ID NO.194的融合蛋白，其中信号肽已经移除，所述融合蛋白具有
6.免疫原性组合物，其包含来自流感嗜金杆菌的蛋白E和来自流感嗜血杆菌的PilA。
7.权利要求6的免疫原性组合物，其中蛋白E是SEQ ID NO.4的多肽、包含在整个长度上与SEQ ID NO.4具有至少75％、77％、80％、85％、90％、95％、97％、99％或100％同一性的序列的多肽，或者是包含SEQ ID NO.4的至少7个、10个、15个、20个、25个、30个或50个邻接氨基酸的免疫原性片段的多肽。
8.疫苗，其包含权利要求1-5中任一项的融合蛋白或权利要求6-7中任一项的免疫原性组合物。
9.用于产生融合蛋白的周质表达的方法，其中所述方法包括诱导含有信号肽的蛋白的表达。
10.权利要求9的方法，其中所述信号肽来自FlgI。
CN201710656150.1A 2011-04-13 2012-04-12 包含流感嗜血杆菌蛋白e和菌毛蛋白a的融合蛋白和组合疫苗 Pending CN107522788A (zh)

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Cited By (2)

* Cited by examiner, † Cited by third party
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