CN101684148A - 脑膜炎奈瑟氏球菌的新表面蛋白 - Google Patents
脑膜炎奈瑟氏球菌的新表面蛋白 Download PDFInfo
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Abstract
本发明提供了来自脑膜炎奈瑟氏球菌的新的表面多肽以及编码这一蛋白质的核酸和核酸序列同系物。还公开了含有本发明的多肽和核酸的药物组合物以及用于治疗、预防和诊断脑膜炎奈瑟氏球菌感染的方法。
Description
本申请是1998年12月14日提交的,发明名称为“脑膜炎奈瑟氏球菌的新表面蛋白”的中国专利申请98813558.2的分案申请。
发明领域
本发明涉及新多肽,如可得自脑膜炎奈瑟氏球菌(Neisseriameningitidis)的多肽,以及编码此多肽的核苷酸序列,及其在诊断剂,治疗和预防性疫苗中的应用及在设计和/或筛选药物中的应用。
发明背景
脑膜炎奈瑟氏球菌是一种革兰氏阴性菌,且是脑膜炎和败血病的致病因素。其已知的唯一宿主是人,且其可被将近10%的人群无症状地携带(Caugant,D.et al.1994,临床微生物学杂志,32:323-30)。
脑膜炎奈瑟氏球菌可表达多糖荚膜,且其使细菌可根据表达的荚膜的性质进行分类。有至少13个血清群的脑膜炎奈瑟氏球菌:A,B,C,29-E,H,I,K,L,W135,X,Y及Z;其中血清群A,B和C导致90%的脑膜炎疾病(Poolman,J.T.et al.,1995,传染因子及疾病4:13-28)。现可获得抗血清群A和C的疫苗,但血清群B荚膜多糖是低免疫原性的,且在人体不能诱导保护作用。
因而检测其它膜及胞外组分包含在疫苗中是否合适。实例包括1,2,及3(膜孔蛋白)类,及4(Rmp)和5(不透明蛋白质)类外膜蛋白质。但目前,这些候选物无一能诱导完全保护作用,尤其在儿童中(Romero,J.D.,1994,临床微生物学综述,7:559-575;Poolman,J.T.et al,1995前述)。
为产生有效疫苗,需鉴别存在于大多数菌株中并能诱导保护性免疫应答(杀菌抗体)的脑膜炎奈瑟氏球菌的组分。为此可参考Brodeur等(国际公开WO 96/29412)所述,其揭示了一种22kDa表面蛋白,其在99%的全部已知脑膜炎奈瑟氏球菌菌株中是高保守的。注射纯化的重组22kDa表面蛋白,使80%的免疫小鼠抗由脑膜炎奈瑟氏球菌导致的致死感染。尽管揭示了此蛋白质,仍需分离更多的大多数菌株中高保守的,并具有抗脑膜炎奈瑟氏球菌免疫保护分布图,和/或可用于与脑膜炎奈瑟氏球菌的其它组分组合以增强抗此生物体的保护效力的脑膜炎奈瑟氏球菌的表面蛋白。
发明概述
本发明揭示了一种新基因,其存在于所有测试的脑膜炎奈瑟氏球菌菌株中,且其编码推定分子量大约为62kDa的一种新多肽。基于其序列特点及同源性,此多肽被推定是粘附素,且结合试验数据推测其组成一表面蛋白,可用于抗脑膜炎奈瑟氏球菌的治疗性和/或预防性疫苗的生产。
据此,本发明的一方面提供了一分离的多肽或其片段,或其变体或衍生物,所述多肽选自以下组成的一组:
(a)如SEQ ID NO:2的多肽;
(b)如SEQ ID NO:5的多肽;
(c)如SEQ ID NO:7的多肽;
(d)如SEQ ID NO:9的多肽;
(e)如SEQ ID NO:11的多肽;
(f)如SEQ ID NO:13的多肽;
(g)如SEQ ID NO:15的多肽;
(h)如SEQ ID NO:17的多肽;
(i)如SEQ ID NO:19的多肽;和
(j)如SEQ ID NO:21的多肽;
优选地,所述多肽、片段、变体或衍生物表现出抗一或多个选自以下一组的成员的免疫活性:
(i)脑膜炎奈瑟氏球菌;
(ii)所述多肽;
(iii)所述片段;
(iv)所述变体;和
(v)所述衍生物。
本发明另一方面提供了编码前述多肽或其片段,或其变体或衍生物的分离的核酸序列。适当地,所述序列选自
(1)SEQ ID NO:1的核苷酸序列;
(2)SEQ ID NO:3的核苷酸序列;
(3)SEQ ID NO:4的核苷酸序列;
(4)SEQ ID NO:6的核苷酸序列;
(5)SEQ ID NO:8的核苷酸序列;
(6)SEQ ID NO:10的核苷酸序列;
(7)SEQ ID NO:12的核苷酸序列;
(8)SEQ ID NO:14的核苷酸序列;
(9)SEQ ID NO:16的核苷酸序列;
(10)SEQ ID NO:18的核苷酸序列;
(11)SEQ ID NO:20的核苷酸序列;
(12)SEQ ID NO:1,3,4,6,8,10,12,14,16,18及20任一种的核苷酸片段;和
(13)任一上述序列的核苷酸序列同系物。
优选地,所述片段编码表现出抗选自以下一组的一或多个成员的免疫活性的产物:
(i)脑膜炎奈瑟氏球菌;
(ii)第一方面所述的多肽;
(iii)第一方面所述的片段;
(iv)第一方面所述变体;和
(v)第一方面所述衍生物。
本发明再一方面涉及一表达载体,其含有如第二方面所述的核酸序列,其中所述序列可操作地连接于转录及翻译调节核酸。
本发明另一方面提供了一种宿主细胞,其含有如第三方面所述的表达载体。
本发明的另一方面提供了一种生产如第一方面所述的重组多肽的方法,所述方法包括以下步骤:
(A)培养含如第三方面所述的表达载体的宿主细胞,由此所述重组多肽从所述核酸表达;和
(B)分离所述重组多肽。
本发明另一方面提供了一种抗体或其片段,其与选自以下一组的一或多个成员结合:
(i)脑膜炎奈瑟氏球菌;
(ii)第一方面所述多肽;
(iii)第一方面所述片段;
(iv)第一方面所述变体;和
(v)第一方面所述衍生物。
本发明另一方面提供了一种检测怀疑含有脑膜炎奈瑟氏球菌的生物样品中的脑膜炎奈瑟氏球菌的方法,所述方法包括以下步骤:
(A)从病人中分离生物样品;
(B)将上述抗体或片段与生物样品混合成混合物;和
(C)检测混合物中特异结合的抗体或片段,其表明脑膜炎奈瑟氏球菌的存在。
本发明另一方面提供了一种检测怀疑含有脑膜炎奈瑟氏球菌的生物样品中脑膜炎奈瑟氏球菌的方法,所述方法包括以下步骤:
(I)从病人中分离生物样品;
(II)检测所述样品中第二方面所述的核酸序列,其表明所述细菌的存在。
本发明还涉及诊断病人被脑膜炎奈瑟氏球菌感染的方法,所述方法包括以下步骤:
(1)将取自病人的生物样品与本发明的多肽、片段、变体或衍生物相接触;并
(2)测定所述多肽、片段、变体或衍生物与所述样品中脑膜炎奈瑟氏球菌特异性抗体之间复合物的存在与否,其中存在复合物表明有感染。
本发明还涉及如第一方面所述的多肽,如第三方面所述的核酸或上述抗体或抗体片段在检测生物样品中脑膜炎奈瑟氏球菌的试剂盒中的应用。
本发明另一方面提供了一种药物组合物,其含有如第一方面所述的分离的多肽或其片段,或其变体或衍生物。
优选地,所述药物组合物是疫苗。
本发明另一方面提供了一种预防脑膜炎奈瑟氏球菌感染的方法,包括为病人施用药物有效量的上述疫苗。
本发明另一方面提供了一种鉴别如第一方面所述的多肽、变体或衍生物的免疫反应性片段的方法,包括以下步骤:
(a)产生所述多肽、变体或衍生物的片段;
(b)给哺乳动物施用所述片段;
(c)检测所述哺乳动物中的免疫应答,所述应答包括产生与脑膜炎奈瑟氏球菌和/或所述多肽、变体或衍生物特异结合的因子,和/或产生抗脑膜炎奈瑟氏球菌感染的保护作用。
附图简述:
图1描绘了质粒图谱及克隆策略。引物A3A和A3B(分别为SEQID NO:28和SEQ ID NO:29)用于从MC58中扩增在TIGR数据库中鉴定为与AIDI-I同源的区域。将PCR产物克隆以提供pNMAIDA3。引物A3C(SEQ ID NO:30)及A3D(SEQ ID NO:31)用于反向PCR中扩增包括hiaNm的3kbp EagI片段。将此产物克隆以提供piEAGA3。将piEAGA3亚克隆以提供piEaga3.8及piEagA3.9。引物HiaNm:M和HiaNm:P(分别为SEQ ID NO:22和SEQ ID NO:23)用于从MC58扩增邻接区域并亚克隆产物产生pHiaNm。引物Hia-MBPA(SEQ ID NO:24)和Hia-MBPB(SEQ ID NO:25)用于扩增hiaNm的开放读框,并将此产物克隆入pMALC2以产生pMBP-HiaNm。
图2是一些脑膜炎奈瑟氏球菌菌株的基因组DNA的Southern印迹。2A:血清群B菌株,1道PMC28,2道PMC27,3道PMC25,4道PMC24,5道PMC16,6道PMC13,7道PMC12,8道MWt标准物,9道2970,10道1000,11道528,12道SWZ107,13道H41,14道H38,15道NGH36,16道H15,17道NGG40,18道NGF26,19道NGE30,20道NGE28。2B:除B之外其它血清群菌株。1道PMC3,2道PMC17,3道PMC20,4道PMC23,5道PMC8,6道PMC9,7道PMC11,8道PMC14,9道PMC18,10道PMC21,11道PMC29,12道MWt标准物,13道PMC19,14道PMC1,15道PMC6,16道PMC10,17道PMC22,18道PMC26,19道PMC2。分子量标记以千碱基对(kb)表示。基因组DNA与相应于SEQ ID NO:1的276~2054位的探针杂交。
图3示出MBP-HiaNm的Coomassie染色凝胶。示出用IPIG诱导后的含pMALC2(2道)或pMBP-HiaNm(3道)的细胞。1道为分子量标准(KDa)。箭头代表MBP和MBP-HiaNm。
图4是用兔免疫血清培养的MC58及MC58ΔHiaNm蛋白质的Western印迹。1道为分子量标准(KDa),2道为MC58的总细胞蛋白,3道为MC58ΔHiaNm的总细胞蛋白,4道为MC58的OMC制备物,5道为MC58ΔHiaNm的OMC制备物,每道含有A280=3.75的50μL的蛋白质悬浮液。
图5示出平行于图4的Western印迹的凝胶的Coomassie染色凝胶。泳道与图4一致。
图6示出用PILEUP序列对比程序对HiaNm,Hia,Hsf多肽的序列对比。
图7示出用PILEUP程序对来自脑膜炎奈瑟氏球菌10个菌株中HiaNm的多肽序列对比。
发明详述
在本说明书及权利要求书中,除非上下文要求其它含义,术语“包括”是指包括所述的整体或整体组,但不排除任何其它整体或整体组。
多肽序列:
本发明提供一种如SEQ ID NO:2,5,7,9,11,13,15,17,19及21的分离多肽,或其片段,或其变体或衍生物。在一优选的实施方案中,本发明的多肽,片段,变体及衍生物展示抗选自脑膜炎奈瑟氏球菌,所述多肽,所述片段,所述变体及所述衍生物任一种的免疫活性。
SEQ ID NO:2相当于得自脑膜炎奈瑟氏球菌菌株MC58的HiaNm基因的新的大约62kDa表面多肽,在下文充分阐述。SEQ ID NO:5,7,9,11,13,15,17,19及21分别相当于从得自脑膜炎奈瑟氏球菌菌株BZ10,BZ198,EG327,EG329,H15,H38,H41,P20及PMC21的核苷酸序列推导的同源多肽。
为本发明之目的,术语“免疫活性”指上述多肽,片段,变体或衍生物在施用其的哺乳动物中产生应答的能力,其中应答包括产生与脑膜炎奈瑟氏球菌和/或所述多肽,片段,变体或衍生物特异结合的因子,和/或产生抗脑膜炎奈瑟氏球菌感染的保护效应。
“分离的”是指基本上不含在其原始状态正常与其相伴的组分的物质。
“多肽”是指长链肽,包括蛋白质。
本文所用术语“片段”包括缺失变体及小肽,例如长度为至少6个,优选至少10个,更优选至少20个氨基酸,其包括抗原决定簇或表位。一些如此片段可连接在一起。此类肽可通过应用标准重组核酸技术或用常规液相或固相合成技术合成而获得。例如,可参考如由Atherton和Shephard所著《肽合成》第9章及包含于由Nicholson编辑且由Blackwell科学出版公司出版的《合成疫苗》中的液相或固相合成。或者,可通过将本发明的多肽用蛋白酶如endoLys-C,endoArg-C,endoGlu-C及葡萄球菌V8蛋白酶消化而产生肽。此消化的片段可通过如HPLC技术进行纯化。
术语“变体”指一种多肽,其中一或多个氨基酸已经被不同氨基酸置换。本领域熟知一些氨基酸可被其它具有广泛相似性的氨基酸置换,而不改变此多肽的活性性质(保守取代)。根据下表可进行多肽中举例性保守取代。
表1
原始残基 | 举例性取代 |
AlaArgAsnAspCysGlnGluGlyHisIleLeuLysMetPheSerThrTrpTyrVal | SerLysGln,HisGluSerAsnAspProAsn,GlnLeu,ValIle,ValArg,Gln,GluLeu,IleMet,Leu,TyrThrSerTyrTrp,PheIle,Leu |
通过选择比表1所示较低保守的取代进行功能的基本改变。其它置换是非保守取代并相对较少的这些取代是耐受的。一般地,易于产生多肽性质最大变化的取代是那些其中(a)亲水性残基(如Ser或Thr)取代或被疏水性残基(如Ala,Leu,Ile,Phe或Val)取代;(b)半胱氨酸或脯氨酸取代或被任何其它残基取代;(c)具有正电侧链的残基(如Arg,His或Lys)取代或被负电残基(如Glu或Asp)取代;或(d)具有庞大侧链的残基(如Phe或Trp)取代或被具有较小侧链(如Ala,Ser)或无侧链(如Gly)的残基取代。
一般地,变体与例如SEQ ID NO:2,5,7,9,11,13,15,17,19和21所示的基本序列应具有至少75%、更合适至少80%、优选至少85%、最优选至少90%的同源性。同源性的定义是相同或组成如表1所定义的保守取代的氨基酸数的百分数。同源性可用序列比较程序如GAP(Deveraux等,1984,核酸研究12,387-395)确定,该文献引入本文作参考。以这种方式,与本文所述序列相似长度或不同长度的序列可通过将缺口掺入对比序列而比较,这种缺口可通过例如GAP使用的比较算法而确定。可通过常规技术确定合适变体的组成。例如,编码SEQ ID NO:2,5,7,9,11,13,15,17,19和21的多肽的核酸,可用随机诱变发转座子诱变,或用位点直接诱变进行突变。然后将所得DNA片段用常规技术克隆入适当的表达宿主如E.coli中,并检测保持所需活性的克隆。在已用随机诱变衍生的克隆中,将阳性克隆测序以检测突变。术语“变体”也包括天然发生的等位基因变体。
“衍生物”是指通过修饰基本序列而衍生的多肽,如通过本领域已知的与其它化学成分缀合或复合,或通过翻译后修饰。如此衍生物包括如SEQ ID NO:2,5,7,9,11,13,15,17,19和21的多肽或其变体的氨基酸缺失和/或添加,其中所述衍生物保持免疫活性。氨基酸的“添加”可包括多肽或其变体与其它多肽或蛋白质的融合。在这点上应意识到本发明的多肽或变体可掺入较大多肽中,且该较大多肽也预期保持抗如脑膜炎奈瑟氏球菌的免疫活性。如上所述的多肽可融入其它蛋白质中,如非衍生于脑膜炎奈瑟氏球菌的蛋白质。此其它蛋白质例如可有助于蛋白质的纯化。例如聚组氨酸标记或麦芽糖结合蛋白可用于此方面,并如下详述。或者,其可产生有效抗脑膜炎奈瑟氏球菌的免疫应答,或产生抗其它病原体的免疫应答。其它可能的融合蛋白是那些产生免疫调制应答的蛋白质。该蛋白质的特例包括A蛋白或谷胱甘肽S-转移酶(GST)。另外,此多肽可融入基于寡糖的疫苗组分,在此其作为载体蛋白。
其它本发明期望的衍生物包括但非限于侧链修饰,在肽,多肽或蛋白质合成期间掺入非天然氨基酸和/或其衍生物,及应用交联剂和其它对本发明的多肽,片段及变体有构象限制影响的方法。
本发明期望的侧链修饰的实例包括氨基基团的修饰,如通过用乙酐的酰化;用琥珀酸酐及四氢氨甲酰苯甲酸酐对氨基基团酰化;用甲基乙酰亚胺化物的脒化;用氰酸酯对氨基基团的氨甲酰化;用吡哆-5-磷酸酯对赖氨酸吡哆化,随后用NaBH4还原;通过与乙醛反应,随后用NaBH4还原的还原烷化;及用2,4,6-三硝基苯磺酸(TNBS)对氨基基团的三硝基苯化。
羧基基团的修饰可通过经O-酰基异脲的形成碳二亚胺活化,随后衍生为例如相应酰胺而进行。
精氨酸残基的胍基修饰可通过用如2,3-丁二酮,苯基乙二醛及乙二醛等试剂形成杂环缩合产物而进行。
巯基基团修饰可通过如过甲酸氧化成磺基丙氨酸;用4-氯汞基苯基磺酸,4-氯汞基苯甲酸;2-氯汞基-4-硝基苯酚,氯化苯汞及其它汞化合物形成衍生物;用其它巯基化合物形成混合的二硫化物;与马来酰亚胺,马来酸酐或其它取代的马来酰亚胺反应;用碘乙酸或碘乙酰胺羧甲基化;及在碱性pH值用氰酸酯氨甲酰化。
色氨酸残基修饰可通过用2-羟基-5-硝基苄基溴化物或磺酰卤对吲哚环烷基化,或通过用N-溴琥珀酰亚胺氧化。
酪氨酸残基修饰可通过用四硝基甲烷硝化形成3-硝基酪氨酸衍生物进行。
组氨酸残基的咪唑环修饰可通过用焦碳酸二乙酯N-乙酯基化,或用碘乙酸衍生物烷基化进行。
在肽合成期间,掺入的非天然氨基酸及衍生物例如包括但非限于使用4-氨基丁酸,6-氨基己酸,4-氨基-3-羟基-5-苯基戊酸,4-氨基-3-羟基-6-甲基庚酸,叔丁基甘氨酸,正亮氨酸,正缬氨酸,苯基甘氨酸,鸟氨酸,肌氨酸,2-噻吩丙氨酸,和/或氨基酸的D-异构体。本发明期望的非天然氨基酸示于表2。
表2
非常规氨基酸 | 非常规氨基酸 |
α-氨基丁酸α-氨基-α-甲基丁酸酯氨基环丙烷甲酸酯氨基异丁酸氨基降冰片基羧基酯环己基丙氨酸环戊基丙氨酸L-N-甲基异亮氨酸D-丙氨酸D-精氨酸D-天冬氨酸D-半胱氨酸D-谷氨酰胺D-谷氨酸D-组氨酸D-异亮氨酸D-亮氨酸D-赖氨酸D-甲硫氨酸D-鸟氨酸D-苯丙氨酸D-脯氨酸D-丝氨酸D-苏氨酸D-色氨酸D-酪氨酸 | L-N-甲基丙氨酸L-N-甲基精氨酸L-N-甲基天冬酰胺L-N-甲基天冬氨酸L-N-甲基半胱氨酸L-N-甲基谷氨酰胺L-N-甲基谷氨酸L-N-甲基组氨酸L-N-甲基亮氨酸L-N-甲基赖氨酸L-N-甲基甲硫氨酸L-N-甲基正亮氨酸L-N-甲基正缬氨酸L-N-甲基鸟氨酸L-N-甲基苯丙氨酸L-N-甲基脯氨酸L-N-甲基丝氨酸L-N-甲基苏氨酸L-N-甲基色氨酸L-N-甲基酪氨酸L-N-甲基缬氨酸L-N-甲基乙基甘氨酸L-N-甲基叔丁基甘氨酸L-正亮氨酸L-正缬氨酸α-甲基氨基异丁酸酯 |
D-缬氨酸D-α-甲基丙氨酸D-α-甲基精氨酸D-α-甲基天冬酰胺D-α-甲基天冬氨酸D-α-甲基半胱氨酸D-α-甲基谷氨酰胺D-α-甲基组氨酸D-α-甲基异亮氨酸D-α-甲基亮氨酸D-α-甲基赖氨酸D-α-甲基甲硫氨酸D-α-甲基鸟氨酸D-α-甲基苯丙氨酸D-α-甲基脯氨酸D-α-甲基丝氨酸D-α-甲基苏氨酸D-α-甲基色氨酸D-α-甲基酪氨酸L-α-甲基亮氨酸L-α-甲基甲硫氨酸L-α-甲基正缬氨酸L-α-甲基苯丙氨酸L-α-甲基丝氨酸L-α-甲基色氨酸L-α-甲基缬氨酸N-(N-(2,2-二苯基乙基氨甲酰甲基)甘氨酸1-羧基-1-(2,2-二苯基乙基氨基)环丙烷 | α-甲基-γ-氨基丁酸酯α-甲基环己基丙氨酸α-甲基环戊基丙氨酸α-甲基-α-萘丙氨酸α-甲基青霉胺N-(4-氨基丁基)甘氨酸N-(2-氨基乙基)甘氨酸N-(3-氨基丙基)甘氨酸N-氨基-α-甲基丁酸酯α-萘丙氨酸N-苄基甘氨酸N-(2-氨甲酰ediyl)甘氨酸N-(氨甲酰甲基)甘氨酸N-(2-羧乙基)甘氨酸N-(羧甲基)甘氨酸N-环丁基甘氨酸N-环庚基甘氨酸N-环己基甘氨酸N-环癸基甘氨酸L-α-甲基赖氨酸L-α-甲基正亮氨酸L-α-甲基鸟氨酸L-α-甲基脯氨酸L-α-甲基苏氨酸L-α-甲基酪氨酸L-N-甲基高苯丙氨酸N-(N-(2,2-二苯基乙基氨甲酰甲基)甘氨酸 |
本发明还涉及用二硝基苯酚共价修饰本发明的多肽,片段或变体,以使其在人体内是免疫原性的。
优选地,本发明包括选自如SEQ ID NOS:2,5,7,9,11,13,15,17,19及21任一多肽的多肽。
本发明的多肽可通过任何本领域技术人员已知的适当方法制备。例如,多肽可通过含以下步骤的方法制备:
(a)制备一重组核酸,该核酸含有编码如SEQ ID NOS:2,5,7,9,11,13,15,17,19及21任一多肽,或其片段,或其变体或衍生物的核苷酸序列,此核苷酸序列可操纵地与转录及翻译调节核酸连接;
(b)用重组核酸转染或转化适当宿主细胞;
(c)培养宿主细胞,以从所述重组核酸表达重组多肽;
(d)分离重组多肽。
适当的所述核苷酸序列选自SEQ ID NOS:1,3,4,6,8,10,12,14,16,18及20。
“重组多肽”意为用重组技术生产的多肽,即通过重组核酸的表达而生产的多肽。
术语“重组核酸”是指通过将核酸处理为非天然中正常发现的形式的在体外形成的核酸。在此方面,重组核酸优选包括表达载体,其可以是自主复制的染色体外载体如质粒,或整合入宿主基因组的载体。通常地,该表达载体包括可操纵地与所述核苷酸序列连接的转录及翻译调节核酸。
“可操纵地连接”是指转录及翻译调节核酸相对于编码所述多肽、片段、变体或衍生物的核苷酸序列的位置能使转录启动。转录及翻译调节核酸一般适于用于表达的宿主细胞。本领域已知为各种宿主细胞所适用的各类表达载体及调节序列。
典型地,该转录及翻译调节核酸可包括但非限于启动子序列,前导或信号序列,核糖体结合位点,转录起始及终止序列,翻译起始及终止序列,及增强子或激活子序列。
本领域已知的组成型或可诱导启动子是本发明所期望的。该启动子可以是天然发生的启动子,或组合一个以上启动子的杂合启动子。
在一优选的实施方案中,此表达载体含有一可选择标记基因,以选择转化的宿主细胞。本领域已熟知选择基因,可根据所用宿主细胞而变化。
表达载体还可包括融合配偶体(典型地由表达载体提供),以便本发明的重组多肽被表达作具有所述融合配偶体的融合多肽。融合配偶体的主要优点是其有助于所述融合多肽的鉴别和/或纯化。
为表达所述融合多肽,需将本发明的核苷酸序列连接于表达载体中,以便融合配偶体的翻译读框与本发明的核苷酸序列恰好重合。
融合配偶体例如包括但非限于谷胱甘肽S转移酶(GST),人IgG的Fc部分,麦芽糖结合蛋白(MBP)及6组氨酸(HIS6),其尤其用于经亲和层析分离融合多肽。为通过亲和层析纯化融合多肽之目的,亲和层析的相关基质分别是谷胱甘肽缀合的,直链淀粉缀合的,镍或钴缀合的树脂。一些如此基质可以“试剂盒”形式获得,如具有(HIS6)融合配偶体的QIAexpressTM系统(Qiagen),及Pharmacia GST纯化系统。
其它为本领域所熟知的融合配偶体是绿色荧光蛋白(GFP)。此融合配偶体作为荧光“标记”,其使本发明的融合多肽可经荧光显微镜或流式细胞计量术鉴别。当确定本发明融合多肽的亚细胞定位,或分离表达本发明融合多肽的细胞时,GFP标记是有用的。如荧光激活细胞分选(FACS)等流式细胞计量法尤其用于后一应用中。
优选地,此融合配偶体还具有蛋白酶切割位点,如因子Xa或凝血酶,其使相关蛋白酶部分消化本发明的融合多肽,并从而从中释放本发明的重组多肽。然后通过随后的层析分离可将释放的多肽从融合配偶体中分离出。
本发明的融合配偶体还包括“附加表位”,其通常是短肽序列,由此可获得特异抗体。由其可易于获得特异单克隆抗体的公知附加表位例如包括c-myc,流感病毒血凝素及FLAG标记。
生产本发明的重组多肽可通过培养用表达载体转化的宿主细胞进行,该载体含有编码本发明多肽,片段,变体或衍生物的核酸序列。适于蛋白质表达的条件将随选择的表达载体及宿主细胞而变化。这些条件经常规试验将为本领域技术人员所易于确定。
适于表达的宿主细胞可以是原核或真核细胞。一优选的表达本发明多肽的宿主细胞是细菌。所用细胞可以是E.coli。或者,宿主细胞可以是昆虫细胞,如SF9细胞,其可以与杆状病毒表达系统一起利用。
本领域技术人员用标准方案可常规制备重组蛋白质,例如并入本文参考的Sambrook等在分子克隆实验手册(冷泉港实验室出版社,1989)中所述,尤其第16及17节;Ausubel等分子生物学当前方案(John Wiley& Sons,Inc.1994-1998),尤其第10和16章;及Coligan等蛋白质科学当前方案(John Wiley & Sons,Inc.1995-1997),尤其第1,5,6章所述。
核苷酸序列
本发明还提供一核苷酸序列,其编码上述多肽,片段,变体或衍生物。适当的所述序列选自SEQ ID NOS:1,3,4,6,8,10,12,14,16,18及20;SEQ ID NOS:1,3,4,6,8,10,12,14,16,18及20任一核苷酸序列的核苷酸片段;及前述序列的核苷酸序列同系物。优选地,这些序列编码呈现前述免疫活性的产物。
如后文更充分阐述,SEQ ID NO:1相当于得自脑膜炎奈瑟氏球菌菌株MC58的hiaNm基因。此基因编码SEQ ID NO:2的新62kDa(大约)表面多肽。SEQ ID NO:3相当于菌株MC58的hiaNm开放读框序列,HiaNm。SEQ ID NOS:4,6,8,10,12,14,16,18及20相当于分别得自脑膜炎奈瑟氏球菌菌株BZ10,BZ198,EG327,EG329,H15,H38,H41,P20,及PMC21的同源hiaNm开放读框序列。
术语“核苷酸序列”指mRNA,RNA,cRNA,cDNA或DNA。
术语“核苷酸序列同系物”通常指在基本严格条件下与本发明野生型核苷酸序列杂交的核苷酸序列。适当的杂交条件将在后文阐述。
本发明的核苷酸序列同系物可如以下步骤制备:
(i)从适当宿主中获得核酸提取物;
(ii)产生任选地简并的引物,其中每种引物均含有本发明野生型核苷酸序列的一部分;
(iii)通过核酸扩增技术,用所述引物从所述核酸提取物扩增一或多种扩增产物。
适当地,此宿主可以是细菌。优选地,此宿主是奈瑟氏球菌属,更优选地,是脑膜炎奈瑟氏球菌。
优选地,此引物选自以下一组:
(1)5’-TTAGATTCCACGTCCCAGATT-3’(SEQ ID NO.22);
(2)5’-CTTCCCTTCAAACCTTCC-3’(SEQ ID NO:23);
(3)5’-GGTCGCGGATCCATGAACAAAATATACCGCAT-3’(SEQ ID NO.24);
(4)5’-TCACCCAAGCTTAAGCCCTTACCACTGATAAC-3’(SEQ ID NO.25);
(5)5’-CCAAACCCCGATTTAACC-3’(SEQ ID NO.26);
(6)5’-AATCGCCACCCTTCCCTTC-3’(SEQ IDNO.27);
(7)5’-TTTGCAACGGTTCAGGCA-3’(SEQ ID NO.28);
(8)5’-TATTCAGCAGCGTATCGG-3’(SEQ ID NO.29);
(9)5’TGCCTGAACCGTTGCAAA-3’(SEQ ID NO.30);及
(10)5’-CCGATACGCTGCTGAATA-3’(SEQ ID NO.31)。
适当的核酸扩增技术已为本领域技术人员所熟知,包括并入本文参考的如Ausubel等(1994-1998,见前述,第15章)所述的聚合酶链反应(PCR);如U.S.专利No.5.422.252所述的链置换扩增(SDA);如Liu等(1996,J.Am.Chem.Soc.118:1587-1594及国际申请W92/01813)及Lizardi等(国际申请WO97/19193)所述的滚环复制(RCR);如Sooknanan等(1994,Biotechniques 17:1077-1080)所述的基于核酸序列的扩增(NASBA);及如Tyagi等所述的Q-β复制酶扩增(1996,Proc.Natl.Acad.Sci.USA 93:5395-5400)。
本文所用术语“扩增产物”指通过核酸扩增技术产生的核酸产物。
“杂交”指不同核苷酸序列的互补碱基配对,根据碱基配对原则产生DNA-DNA杂交体,DNA-RNA杂交体或RNA-RNA杂交体。
在DNA中,互补碱基是:
(i)A和T;及
(ii)C和G。
在RNA中,互补碱基是:
(i)A和U;及
(ii)C和G。
在RNA-DNA杂交体中,互补碱基是:
(i)A和U;及
(ii)A和T;及
(iii)G和C。
典型地,基本互补的核苷酸序列通过印迹技术鉴别,该技术包括将核苷酸固定在一基质(优选合成膜如硝酸纤维素)上的步骤,杂交步骤及检测步骤。Southern印迹用于鉴别互补DNA序列;Northern印迹用于鉴别互补RNA序列。斑点印迹及狭缝印迹可用于鉴别互补DNA/DNA,DNA/RNA,或RNA/RNA多核苷酸序列。本领域已熟知此类技术,并已如Ausubel等所述(1994-1998,见前述)P2.9.1~P2.9.20。
根据如此方法,Southern印迹包括根据大小经凝胶电泳分离DNA分子,将依大小分离的DNA转移至合成膜,并将结合DNA的膜与放射性标记,酶标记或荧光染色标记的互补核苷酸序列杂交。在斑点及狭缝印迹中,将DNA样品在杂交前直接施加于合成膜。
当鉴别cDNA或基因组文库中互补核苷酸序列时用一另外的印迹步骤,如经噬斑或菌落杂交法。此步骤的一典型实例见于Sambrook等(1989,前述)中第8-12章所述。
典型地,下述通用程序用于确定杂交条件。如上述将核苷酸序列印迹/转移至合成膜。如上述将本发明的野生型核苷酸序列标记,并分析此标记的核苷酸序列与固定的核苷酸序列杂交的能力。
本领域技术人员将意识到:许多因素可影响杂交。放射标记的多核苷酸序列的比活性典型地应高于或等于约108dpm/mg,以提供一可检测信号。比活性108~109dpm/mg的放射标记核苷酸序列可检测大约0.5pg的DNA。本领域熟知足够的DNA必须固定于膜上以容许检测。一般需要有过量的固定的DNA,通常为10μg。在杂交期间加入惰性聚合物如10%(w/v)葡聚糖硫酸酯(MW500,000),或聚乙二醇6000也可提高杂交的敏感性(见Ausubel前述2.10.10)。
为在固定于膜上的核苷酸序列与标记的核苷酸序列之间获得富有意义的杂交结果,必须将足够量的标记的核苷酸序列与固定的核苷酸序列在杂交后洗涤。洗涤可确保标记的核苷酸序列只与具有所需的与标记核苷酸序列互补性程度的固定的核苷酸序列杂交。
本文所用的“严格性”指在杂交期间的温度,离子强度条件及一定有机溶剂的存在与否。严格性越高,固定的核苷酸序列及标记的多核苷酸序列之间互补性程度越高。
“严格条件”是指仅使具有高频率互补碱基的核苷酸序列杂交的那些条件。
典型的严格条件包括如:(1)0.75M磷酸氢二钠/0.5M磷酸二氢钠/1mM乙二胺四乙酸二钠/1%sarkosyl。在约42℃至少30分钟;或(2)6.0M尿素/0.4%十二烷基硫酸钠/0.1×SSC在大约42℃至少30分钟;或(3)0.1×SSC/0.1%SDS在大约68℃至少20分钟;或(4)1×SSC/0.1%SDS在大约55℃大约60分钟;或(5)1×SSC/0.1%SDS在大约62℃大约60分钟;或(6)1×SSC/0.1%SDS在大约68℃大约60分钟;或(7)0.2×SSC/0.1%SDS在大约55℃大约60分钟;或(8)0.2×SSC/0.1%SDS在大约55℃大约60分钟;或(7)0.2×SSC/0.1%SDS在大约55℃大约60分钟;或(8)0.2×SSC/0.1%SDS在大约62℃大约1小时;或(9)0.2×SSC/0.1%SDS在大约68℃大约60分钟。详细论述见并入本文参考的分子生物学当前方案(前述)P.2.10.1~2.10.16,及Sambrook等在分子克隆实验手册(冷泉港实验室出版社,1989)在1.101~1.104节中所述。
尽管严格洗涤典型地在42℃~68℃进行,本领域技术人员将知晓其它温度也许适于严格条件。在形成DNA-DNA杂交体时,最大杂交典型地发生在低于Tm大约20℃~25℃的温度。本领域熟知Tm是解链温度,或两个互补多核苷酸序列解离的温度。估算Tm的方法已为本领域所熟知(见分子生物学当前方案(前述)P.2.10.8)。在DNA-RNA杂交体情况下,最大杂交典型地发生在低于Tm大约10℃~15℃的温度。
本领域熟知其它严格条件。熟练技术人员将意识到可对各种因素进行操纵以使杂交特异性最佳化。最终洗涤的严格性最佳化用于保证高度杂交。
检测与固定的核苷酸序列杂交的标记的核苷酸序列的方法已为本领域技术人员熟知。如此方法包括放射自显影,化学发光,荧光及比色检测。
抗体
本发明还涉及抗前述多肽,片段,变体及衍生物的抗体,这种抗体可包括任何与本发明多肽、片段、变体或衍生物结合或缀合的适当抗体。例如,抗体可包含多克隆抗体。可通过将本发明多肽、片段、变体或衍生物注射入生产物种中,该物种可包括鼠或兔,以获得多克隆抗血清,从而制备抗体。本领域技术人员熟知生产多克隆抗体的方法。可应用的方案例如引入本文作参考的由Coligan等在免疫学当前方案(John Wiley & Sons,Inc.1991),及Ausubel等(1994-1998,前述)尤其在第11章III节中所述。
为代替得自生产物种的多克隆抗血清,可用如Kohler及Milstein(1975,Nature 256,495-497)所述,或对其更新近的修改如Coligan等(1991,前述)通过无限增殖脾或其它抗体生产细胞生产单克隆抗体,该细胞衍生自己用一或多种本发明的多肽,片段,变体或衍生物接种的生产物种。
在其抗体范围内本发明还包括上述多克隆或单克隆抗体的Fc或Fab片段。或者,该抗体可包含抗本发明肽的单链Fv抗体(scFvs)。这种scFvs可根据分别如由美国专利No.5,091,513,欧洲专利No.239,400或上Winter及Milstein的文章(1991,Nature,349,293)所述进行制行,以上文献并入本文参考。
本发明的抗体可用于亲和层析以分离天然或重组脑膜炎奈瑟氏球菌多肽。例如参见Coligan等(1995-1997,前述)在9-5章中所述的免疫亲和层析法。
抗体可用于在表达文库中筛选本发明多肽变体。如后文所述,本发明的抗体还可用于检测脑膜炎奈瑟氏球菌感染。
检测脑膜炎奈瑟氏球菌
患者体内脑膜炎奈瑟氏球菌的存在与否可如下测定:从患者分离生物样品,将上述抗体或抗体片段与生物样品混合形成一混合物,并检测混合物中特异结合的抗体或结合片段,其代表样品中存在脑膜炎奈瑟氏球菌。
本文所用术语“生物样品”是指得自患者的样品,其可以是提取的,未处理的,处理的,稀释的或浓缩的。适当地,生物样品选自全血,血清,血浆,唾液,尿,汗液,腹水,腹膜液,滑液,羊水,脑脊液,皮肤活检等。
可应用任何适当的技术测定复合物的形成。例如,根据本发明的结合标记的抗体或抗体片段可用于免疫分析中。这种免疫分析可包括但非限于放射免疫分析(RIAS),酶联免疫吸附测定(ELISAs),及免疫层析技术(ICTs),这些已为本领域所熟知。例如可参见“免疫学当前方案”(1994,前述),其揭示了根据本发明可应用的各种免疫分析。如本领域所知,免疫分析可包括竞争分析。
与抗体或抗体片段结合的标记可包括:
i.与抗体或抗体片段直接附着的标记;
ii.与抗体或抗体片段非直接附着的标记;即标记与其它随后与抗体或抗体片段结合的分析试剂附着;
iii附着于抗体或抗体片段的随后反应产物的标记。
标记可选自色原,催化剂,酶,荧光团,化学发光分子、镧系离子如铕(Eu34),放射性同位素及直接目视标记。
在直接目视标记情况中,可应用由胶体金属或非金属颗粒,染料颗粒,酶或底物,有机聚合物、乳胶颗粒、脂质体,或其它含信号产生物质的小泡。
大量适用作标记的酶见美国专利U.S.4,366,241、U.S.4,843,000及U.S.4,849,338中所揭示,在此全部并入本文参考。用于本发明的适当酶标记包括碱性磷酸酶,辣根过氧化物酶,荧光素酶、β-半乳糖苷酶,葡糖氧化酶,溶菌酶,苹果酸脱氢酶等。酶标记可单独应用,或与在溶液中的第二种酶组合应用。
适当地,荧光团选自异硫氰酸荧光素(FITC),异硫氰酸四甲基罗丹明(TRITL),或R-藻红蛋白(RPE)。
本发明还涉及检测被脑膜炎奈瑟氏球菌感染的患者的方法,所述方法包括以下步骤:将取自患者的生物样品与本发明的多肽,片段,变体或衍生物接触,并检测所述多肽,片段,变体或衍生物与所述血清中脑膜炎奈瑟氏球菌特异抗体之间复合物的存在与否,其中若存在所述复合物,则表明有所述感染。
在一优选的实施方案中,通过用本领域熟知的适当标记可检测地修饰所述多肽、片段、变体或衍生物,并如上述在适当的免疫分析中应用此修饰的化合物以检测上述复合物。
另一方面,本发明提供了一种在怀疑含有脑膜炎奈瑟氏球菌的生物样品中检测脑膜炎奈瑟氏球菌的方法,所述方法包括以下步骤:从患者分离生物样品,检测所述样品中是否存在如本发明的核酸序列,其代表存在所述细菌。
所述核酸序列的检测可用任何适当技术测定。例如,如本发明的标记的核酸序列可在得自患者的核酸提取物的Southern印迹中用作探针。这已为本领域所熟知,或者,如本发明的标记的核酸序列可在患者RNA提取物的Northern印迹中用作探针。优选地,患者核酸提取物在核酸扩增反应如PCR,或LCR(如并入本文参考的国际申请WO89/09385中所述)中,与相应于本发明核酸序列的有义及反义序列,或其侧翼序列的寡核苷酸引物一起应用。各种自动固相检测技术也是合适的。例如,非常大规模的固定化引物组(VLSIPSTM)用于核酸的检测,例如由Fodor等(1991,Science 251:767-777)及Kazal等(1996,Nature Medicine 2:753-759)所述。以上通用技术为本领域技术人员所熟知。
药物组合物
本发明另一特点是用本发明的多肽,片段,变体或衍生物(“免疫原性制剂”)作为药物组合物中的活性成分,以保护患者抗脑膜炎奈瑟氏球菌感染。适当地,药物组合物含有药物可接受载体。
“药物可接受载体”意为一固体或液体充填剂,稀释剂或胶囊化物质,其可安全地用于系统施用。依于施用的特殊途径,可应用各种本领域熟知药物可接受载体。这些载体可选自糖,淀粉,纤维素及其衍生物、麦芽、明胶、滑石,硫酸钙,植物油,合成油,多元醇,藻酸,磷酸盐缓冲液,乳化剂,等渗盐水及无热原水。
可应用任何适当途径以为患者提供本发明的组合物。例如,可应用口服,直肠、非肠道、舌下、口腔、静脉内,动脉内,肌内,皮内,皮下,吸入,眼内,腹膜内,脑室内,经皮等途径,肌内及皮下注射适用于例如施用免疫药物组合物,疫苗及DNA疫苗。
剂型包括片剂、散剂、悬浮剂、注射剂,溶液,糖浆、锭剂,胶囊、栓剂、烟雾剂、经皮膏药等。这些剂型还可包括注射或植入为此目的特别设计的控制释放装置,或其它修改的以此方式额外发挥作用的植入形式。治疗剂的控制释放可通过用例如包括丙烯酸树脂、蜡、高级脂族醇、聚乳酸及聚乙醇酸和某些纤维素衍生物如羟丙甲基纤维素等疏水聚合物包被而得。另外,可用其它聚合物基质,脂质体和/或微球体进行控释。
适于口服或非肠道施用的本发明药物组合物可以是分立的单位如含预定量一或多种本发明治疗剂的胶囊剂、囊剂(sachets)或片剂,如粉末或颗粒,或如在水性液体,非水液体,水包油乳剂或油包水液态乳剂中的溶液或悬浮液。该组合物可通过任何制药方法制备,但所有方法均包括将如上所述一或多种免疫原性制剂与组成一或多种必需成分的载体结合。总之,组合物通过将本发明的免疫原性制剂与液体载体或细碎的固体载体或二种载体均匀并充分混合,然后如果需要,将产物塑成所要求形式。
上述组合物可以与剂量配方相容的方式,并以预防患者感染脑膜炎奈瑟氏球菌的免疫学有效量施用。根据本发明,施用于患者的剂量应在一定时间内足以在病人中实现有益应答,如降低脑膜炎奈瑟氏球菌水平,或抑制脑膜炎奈瑟氏球菌感染。施用的免疫原性制剂的量可依于受试者的年龄、性别、体重及其一般健康状态而定。为此,需被施用的免疫原性制剂的精确量将依于从业者的判断而定。在确定治疗或预防抗脑膜炎奈瑟氏球菌感染中施用的免疫原性制剂的有效量中,医者可估计循环血浆水平,疾病进程、及抗脑膜炎奈瑟氏球菌抗体的产生。在任何情况中,本发明免疫原性制剂的适当剂量可为本领域技术人员容易地确定。该剂量可以是钠克至毫克级的本发明免疫原性制剂。
上述组合物可用作治疗或预防疫苗。由此,本发明涉及含本发明一或多种免疫原性制剂作为活性成分的疫苗的生产。任何生产这种疫苗的适当方法均是期望的。例如包括并入本文参考的《新一代疫苗》(1997,Levine et al.,Marcel Dakker,Inc.纽约,巴塞尔,香港)中所述方法。
本发明的免疫原性制剂可与其它抗原包括其它抗原的B或T细胞表位混合、缀合或融合。另外,其可与如下述载体缀合。
当使用本发明的半抗原肽时(即与关联抗体反应,但不能自身激发免疫应答的肽),其可与免疫原性载体缀合。本领域熟知实用的载体,包括如甲状腺球蛋白;白蛋白如人血清白蛋白;来自破伤风、白喉,白日咳,假单胞菌,大肠杆菌,葡萄球菌及链球菌的毒素,类毒素或任何毒素交叉反应物(CRM)的变体;聚氨基酸如聚(赖氨酸:谷氨酸);流感病毒;轮状病毒VP6,细小病毒VP1和VP2;乙型肝炎病毒核心蛋白;乙型肝炎病毒重组疫苗等。或者,可使用载体蛋白或其它免疫原性蛋白的片段或表位。例如,本发明的半抗原肽可与细菌毒素、类毒素或CRM的T细胞表位偶联。为此可参考并入本文参考的U.S.专利No.5,785,973。
另外,本发明的多肽、片段,变体或衍生物可在抗奈瑟氏球菌,或抗其它细菌或病毒的疫苗组合物中作为载体蛋白。
本发明的免疫原性制剂可作为与脑膜炎奈瑟氏球菌的抗原,或其它生物体包括病原性细菌H.influenzae,M.catarrhalis,N.gonorrhoeae,E.coli,S.pneumoniae等的抗原组合的多价亚单位疫苗而施用。
疫苗还可含有生理学可接受稀释剂或赋形剂,如水,磷酸缓冲盐水及盐水。
疫苗及免疫原性组合物可包括一佐剂,这已为本领域所熟知。适当的佐剂包括但非限于表面活性剂如十六胺,十八胺,二甲基双十八烷基溴化铵,N,N-双十八烷基-N’,N’-双(2-羟乙基丙二胺),甲氧基十六烷基甘油,及pluronic多元醇;多元胺如吡喃,葡聚糖硫酸酯,聚IC carbopol;肽如胞壁酰二肽及衍生物,二甲甘氨酸,吞噬作用激素;油乳剂;及无机凝胶如磷酸铝,氢氧化铝或矾;淋巴因子,QuilA及免疫刺激复合物(ISCOMS)。
本发明的免疫原性制剂可由减毒的病毒宿主表达。“减毒的病毒宿主”意为天然或已经基本无毒的病毒载体。通过任何适当的物理(如热处理)或化学方式(如甲醛处理)可将病毒变为基本无毒的。“基本无毒的”指其感染性已被破坏的病毒。理想地,病毒的感染性被破坏,而不影响携带病毒免疫原性的蛋白质。由前所述,应意识到减毒的病毒宿主可包括活的病毒或灭活的病毒。
可用于本发明疫苗的减毒的病毒宿主可包含病毒载体,包括腺病毒、巨细胞病毒并优选痘病毒如痘苗(见如Paoletti和Panicali,U.S.专利4,603,112,并入本文参考)及减毒的沙门氏菌菌株(见如并入本文参考的Stocker,U.S.专利4,550,081)。活病毒特别有利,因为它们导致能赋予非常长期免疫性的延长刺激。
多价疫苗可制备自一或多种表达脑膜炎奈瑟氏球菌不同表位(如,脑膜炎奈瑟氏球菌的其它表面蛋白或表位)的微生物。另外,其它病原性微生物的表位可掺入疫苗中。
在一优选的实施方案中,包括构建一重组痘苗病毒,以表达本发明核酸序列。在导入宿主时,此重组痘苗病毒表达免疫原性制剂,并从而激发宿主CTL应答。例如,可参考U.S.专利No.4,722,848,其阐述了痘苗载体,及用于免疫方案的方法。
通过本发明揭示,各种其它与本发明免疫原性制剂用于治疗性施用或免疫接种的载体对本领域技术人员是显而易见的。
在另一实施方案中,核苷酸序列可以本领域已知的“裸DNA”形式用作疫苗。例如,本发明的表达载体可导入哺乳动物中,由此在体内导致一多肽的产生,宿主产生对此多肽的免疫应答,如Barry,M.etal.,(1995,Nature,377:632-635)所述,该文献引入本文作参考。
检测试剂盒
本发明还提供了在生物样品中检测脑膜炎奈瑟氏球菌的试剂盒。这些试剂盒根据所用测试方法的性质含有一或多个上述特定制剂。由此,该试剂盒可包括一或多个本发明多肽,片段,变体,衍生物,抗体,抗体片段或核酸。该试剂盒还可选择地包括适当标记检测试剂,阳性或阴性对照,洗涤溶液,稀释缓冲液等。例如,基于核酸的检测试剂盒可包括(i)本发明的核酸(其可用作阳性对照),(ii)本发明的寡核苷酸引物,及根据所用核酸扩增技术,任选地包括DNA聚合酶,DNA连接酶等。
制备免疫反应性片段
本发明还涉及鉴别本发明多肽、变体或衍生物的免疫反应性片段的方法。该方法主要包括产生多肽、变体或衍生物的片段,将片段施用于哺乳动物;并检测哺乳动物中的免疫应答。这种应答包括特异结合脑膜炎奈瑟氏球菌和/或所述多肽、变体或衍生物的因子的产生,和/或抗脑膜炎奈瑟氏球菌感染的防护效应的产生。
在上述方法中测试特异免疫反应性片段之前,可应用各种预测方法推断一特定片段能否用于获得与天然抗原交叉反应的抗体,这些预测方法可基于氨基末端或羧基末端序列,例如如Ausubel等(1994-1998,前述)第11.14章所述。或者,这些预测方法可基于并入本文参考的如Kyte和Doolittle(1982,J.Mol.Biol.157:105-132),及Hopp和Woods(1983,Mol.Immunol.20:483-489)所述亲水性的预测,或基于如Choo和Fasman(1978,Ann.Rev.Biochem.47:251-276)所述二级结构的预测。
一般地,由10~15个残基组成的肽片段提供最佳结果。小如6个或大如20个残基组成的肽作用也令人满意。然后可将这种肽片段化学偶联于载体分子如KLH或BSA,如Ausubel等(1994-1998前述)在11.14和11.15节所述。
肽可用于如上所述免疫接种动物。抗天然或亲代多肽(由其选择所述的肽)的抗体效价可通过如放射免疫分析或ELISA测定,如Ausubel等(1994-1998,前述)在11.16和114节所述。
然后通过本领域熟知的硫酸铵分级分离,或通过层析将抗体从动物的适当的生物学液体中纯化出。抗体纯化的方案例如由Ausubel等(1994-1998,前述)在10.11及11.13节中所述。
通过任何适当方法如Western印迹可测定抗天然或亲代多肽的抗体的免疫反应性。
功能阻断剂
如SEQ ID NOS:2,5,7,9,11,13,15,17,19和21的多肽被认为具有粘附素性质,事实上它们与是表面抗原的流感嗜血杆菌粘附素具有相似性。具体地,它们与流感嗜血杆菌的Hia蛋白大约67%同源(Barenkamp,S.和St.Geme III,J.1996 Molecular Microbiology 19:1215-1233),并与流感嗜血杆菌的Hsf蛋白74%同源(St.Geme III,J.Et al.,1996 Journal of Bacteriology 178:6281-6287;美国专利5,646,259)。为这些对比,所用GAP程序中缺口重为3,长度重为0.01(Deveraux,1984,前述)。这些蛋白质的对比序列见于图6。这样,这些多肽功能的中断对治疗是明显有益的,由于它们防止脑膜炎奈瑟氏球菌粘附并侵入细胞。可以一些方式阻断功能。
例如,可以施用阻断与如SEQ ID NOS:2,5,7,9,11,13,15,17,19和21的多肽相互作用的细胞表面受体的组分,如化学试剂或多肽,这些组分与感染性微生物竞争受体位点。这种组分可包括如本发明的多肽,尤其是其片段,或功能等价物以及模拟物。
术语“模拟物”指设计为模拟蛋白质或肽的特异功能区的化学物质。也可应用阻断细菌与细胞表面结合的抗上述抗体的抗独特型抗体。或者,与如SEQ ID NOS:2,5,7,9,11,13,15,17,19和21的多肽中的受体结合位点相互作用的组分,可有效地防止脑膜炎奈瑟氏球菌感染细胞,该组分可包括阻断抗体,肽或其它化学试剂。
所有这种组分,它们与药物可接受载体组合的药物组合物,及经施用该组分或组合物使患者免于脑膜炎奈瑟氏球菌感染的方法,构成了本发明的另一方面。
本发明的多肽可用于筛选在上述方法中应用的化合物。例如,本发明的多肽可与一标记组合,并在所测试剂存在下暴露于细胞培养物,然后观测该试剂抑制标记的多肽与细胞表面结合的能力。在此筛选中,标记的多肽可直接用于生物体如E.coli上。或者,可将脑膜炎奈瑟氏球菌自身工程化,以表达修饰的及可检测形式的多肽,此方法中工程化的脑膜炎奈瑟氏球菌菌株的应用是优选的,因为似乎蛋白质的三级结构更近于代表在野生型细菌中表达的蛋白质。
为更易于理解本发明并实施本发明,通过以下非限制性实施例阐述特别优选的实施方案。
实施例1分子克隆及亚克隆及hiaNm突变体构建
用标准方法以PCR扩增初始分离hiaNm基因,概而言之,由于我们预先对E.coli的AIDA-I蛋白同源性的研究(Jennings,M.et al.,1995,Microbial Pathogenesis,19:391-407,Peak,I.et al.,MicrobialPathogenesis,出版中),我们进行了同源性搜索,从来自对MC58¢3基因组测序计划的初步数据(The Institute for Genomic Research,(ftp://ftp.tigr.org/pub/date/n meningitidis/)中鉴别相应的序列,并用寡核苷酸A3A(5’-TTTGCAACGGTTCAGGCA-3’,SEQ ID NO.28)及A3B(5’-TATTCAGCAGCGTATCGG-3’,SEQ ID NO.29)经PCR扩增同源性区域。将所得449碱基对(bp)产物克隆入pT7 Blue中,以产生质粒pNMAIDA3。为克隆全长基因,设计另外的寡核苷酸并用于反向PCR反应。这些寡核苷酸是A3C(SEQ ID NO.30)和A3D(SEQID NO.31),并分别相应于A3A(SEQ ID NO.28)和A3B(SEQ IDNO.31)的互补序列。此反应的模板是MC58的染色体DNA,其已用EagI限制性消化后自身连接。将所得3kbp PCR产物克隆入载体pCR II(Invitrogen)中,产生质粒piEagA3。将其用EagI和EcoRI消化,并将含克隆的DNA的所得1.4kbp和1.6kbp片段克隆入pBluescript SKII,M13minus(Stratagene),得到piEagA3.8和piEagA3.9。用分别相应于SEQ ID NO:1的113~133和2102~2085核苷酸位置(ntp)的寡核苷酸引物HiaNm:P(5’-TTAGATTCCACGTCCCAGATT-3’SEQ ID NO:22)和HiaNm:M(5’-CTTCCCTTCAAACCTTCC-3’,SEQ ID NO:23),经PCR扩增hiaNm和其5’和3’序列并将产物克隆进pT7Blue产生质粒pHiaNm。通过将一卡那霉素抗性盒插入相应于SEQ ID NO:1的680ntp的pHiaNm的单一BglII位点中,产生质粒pHiaNmΔKan。此卡那霉素抗性盒是用BamHI从pUC4Kan(Pharmacia)切下的。通过在补加10%加热马血的脑心浸渍琼脂(Acumedia Manufacturer’s Inc)(“BHI平板”)上,在37℃在5%CO2中将质粒DNA与细菌保温3小时,以将pHiaNmΔKan转化入脑膜炎奈瑟氏球菌菌株MC58中。将单个菌落挑到新鲜选择性培养基上,培养,并用于进一步研究,此突变体菌株被命名为MC58ΔHiaNm。此菌株中hiaNm基因的破坏用相应于SEQ ID NO.1的276~2054ntp的探针,经Southern印迹确定。
实施例2:核苷酸序列分析
用PRISM染料终止子测序试剂盒与Ampli Taq DNA聚合酶FS,或BigDye终止子测序试剂盒,如厂商(Perkin Elmer)的指导,结合373a型自动测序仪(Applied Biosystems),进行核苷酸序列分析,对每种菌株而言,在三种引物HiaNm5′A2:5’-CCAAACCCGATTTAACC-3’(SEQ ID NO:26)和HiaNm3′A:5’-AATCGCCACCCTTCCCTTC-3’(SEQ ID NO:27)的三个独立PCR反应中,hiaNm被扩增,此二种引物如图1所示,并相当于SEQ ID NO.1的230~247及2144~2097ntp,将所得产物纯化并集合。以此用作模板以在双链上直接测序,用GCG程序(Deveraux et al.,(1984)Nucleic Acids Research12,387-395)及AssemblyLIGN(Oxford Molecular)分析数据。为完整序列的需要,产生一些寡核苷酸。10个菌株的hiaNm序列如SEQID NOS:1,3,4,6,8,10,12,14,16,18及20所示,且那些基因推导的氨基酸序列如SEQ ID NOS:2,5,7,9,11,13,15,17,19及21所示。
这些菌株的hiaNm对比表明:它们与MC58的hiaNm呈90-99%的相同性,另外,MC58的hiaNm与流感嗜血杆菌的hia和hsf具有62%及68%的同源性。但在试验的菌株中,hiaNm是1770~1800bp长。这明显不同于分别是3294和7059bp长的hia和hsf。HiaNm的预测的多肽,HiaNm,也与一些其它细菌蛋白呈同源性包括AIDA-I,参与致腹泻E.coli菌株2787(O126:H27)的扩散粘附中的粘附素,HMW1,另一种嗜血杆菌的粘附素,UspA1,Moraxella catarrthalis的高分子量蛋白质,及参与弗氏志贺氏菌(Shigella flexnerir)组织侵染中的SepA(Benz,I和Schmidt,M.A.,1992,分子微生物学6:1539~1546,Barenkamp,S.J.和Leininger,E.1992,感染和免疫性60:1302-1313,Aebi,C.et al.,1997,感染个免疫性65:4367-4377,Benjelloun-Touimi,Z.et al.,1995,分子微生物学17:123-135)。与这些(及一些其它蛋白质)的同源性发生在HiaNm的头50个氨基酸。该序列的分析揭示了在所有测试HiaNm序列的第50位氨基酸具有切割位点的推测的信号序列的存在。该长信号序列在位于革兰氏阴性菌外膜内的蛋白质中是共有的(Henderson I et al.,1998,Trends in Microbiology 6:370-8)。与HiaNm头50个氨基酸同源的上述蛋白质均是“自身转运蛋白”外膜蛋白家族的成员(Henderson,I,前述)。这强有力地提示HiaNm位于脑膜炎奈瑟氏球菌的外膜中。
实施例3:Southern印迹分析
用标准技术进行Southern印迹分析(Sambrook et al.,前述,Ausubelet al.,前述)。概而言之,从几个血清群脑膜炎奈瑟氏球菌的70个菌株中制备基因组DNA,在毛细管转移至尼龙膜之前,限制性消化并在琼脂糖凝胶上经电泳分离。将这些膜与标记的探针杂交。所用探针相应于SEQ ID NO:1的276~2054ntp,包含MC58菌株的hiaNm的全部开放读框,根据厂商(Boehringer Mannheim)的指导,将其用DIG(双氧蛋白)标记。进行严格洗涤(在22℃用2×SSC/0.1%SDS洗涤2次,每次5分钟,随后在68℃,用0.2×SSC/0.1%SDS洗涤2次,每次30分钟)。如厂商的推荐,用氮蓝四唑/溴氯吲哚磷酸(NBT/BCIP)比色检测杂交。在所有测试菌株中,均检测到信号(如图2)。除原型菌株MC58之外,研究了以下菌株:
表3
菌株名 | 来源 | 血清群 | 菌株名 | 来源 | 血清群 |
PMC 3(J1079) | 2A | A | NGF26 | 1 | B |
PMC17(K874)PMC20((H79)PMC23(K750)PMC12(K852)PMC13(K859)PMC16(K873)PMC24(K782)PMC25(K791)PMC27(K816)PMC28(K837)BZ10BZ47BZ83BZ133BZ147BZ163BZ169BZ198BZ232NG3/88NG4/88NG6/88EG327EG329DK353179/8266/84DK24NGH36H38H41NGE28NGE30NGP20 | 22222222221B11111111111111111111111 | AAABBBBBBBBBBPBBBBBBBBBBBBBBBBBBBB | NGG40H15SWZ10752829701000MPJB28MPJB56MPJB88MPJB157MPJB328MPJB627MPJB820MPJB945PMC8(K157)PMC9(K497)PMC11(K848)PMC14(K860)PMC18(K879)PMC21(K656)PMC29(K841)MPJC05MPJC14MPJC154MPJC302MPJC379PMC19MPJW025PMC1(J603)PMC6(K131)PMC10(K526)PMC22(K685)PMC26(K810)PMC2((J1049) | 1111113c333333322222223333323222222 | BBBBBBBBBBBBBBCCCCCCCCCCCCWWXXYYYZ |
A世界卫生组织脑膜炎球菌参比及研究协作中心,挪威奥斯陆
B公共卫生实验室服务脑膜炎球菌参比实验室,英国曼彻斯特
C布里斯班医院,现由M.P.Jennings保存,澳大利亚布里斯班的昆士兰大学微生物学系
实施例4:MBP-HiaNm的表达及部分纯化
构建容许由麦芽糖结合蛋白与HiaNm融合体(MBP-HiaNm)组成的蛋白质表达的质粒载体,用引物HiaNm-MBPA 5’-GGTCGCGGATCCATGAACAAAATATACCGCAT-3’(SEQ ID NO.24)和HiaNm-MBPB 5’-TCACCCAAGCTTAAGCCCTTACCACTGATAAC-3’(SEQ ID NO.25)从MC58中扩增HiaNm以产生质粒pHiaMBP。这些引物包含脑膜炎奈瑟氏球菌菌株MC58的hiaNm的起始和终止密码子以及便于克隆的工程化限制位点。质粒限制图谱及寡核苷酸位置示于图1。将所得PCR产物与Bam HI/Hind III限制消化的质粒pMALC2(New England Biolabs)连接,并将所得质粒pHiaMBP(见图1)再导入E.coli菌株DH5α。诱导含有pHiaMBP的E.coli菌株,以在厂商(New England Biolabs)推荐的条件下表达HiaNm-MBP融合蛋白。将提取自含pHiaMBP的培养物的细胞提取物经10%SDS-PAGE分离,并根据厂商的指导用微凝胶电洗脱仪(BioRad)经洗脱部分纯化融合蛋白。用兔抗MBP血清经Western印迹检测含HiaNm-MBP融合蛋白的部分,经SDS-PAGE随后Coomassie染色确定HiaNm-MBP融合蛋白的纯度,并通过BCA分折(Sigma)或在280nm波长吸收判断回收的蛋白质数量。
实施例5:多克隆血清的产生
将在实施例4中获得的部分纯化的HiaNm-MBP融合蛋白用于在兔体内产生多克隆血清,将洗脱的HiaNm-MBP融合蛋白的样品用无菌磷酸盐缓冲的盐水pH 7.4(PBS)(Sigma)透析。然后将其与佐剂(MPL+TDM+CWS,Sigma)以50-150μg/ml浓度混合,并以两周间隔接种二只新西兰白兔,从这些兔体取血,经在室温下凝血1小时,在4℃培养过夜随后以4000×rpm在4℃离心以提取血清。除去上清液并再离心,将血清等分贮存在-80℃。所得血清用于杀菌分析及Western印迹(如下所示)。
为测试所得血清特异性,进行Western印迹分析,概而言之,将脑膜炎奈瑟氏球菌菌株MC58及MC58ΔHianm的蛋白质,在用Semi-Dry Blotter(BioRad)经电泳转移至硝酸纤维素膜之前,在SDS-PAGE上电泳分离。然后将这些蛋白质在用NBT/BCIP(Sigma)比色检测之前,依次与血清及碱性磷酸酶缀合的抗兔IgG(Sigma)培养。这些试验表明抗体由HiaNm-MBP融合蛋白激发,其特异于MC58而非MC58ΔHiaNm并检测到一条带(见图4)。推导的HiaNm多肽的推断分子量是62.3KDa。由血清检测的条带以超过150KDa的表观MW迁移。文献中报导的至少三种同源“自身转运蛋白”的蛋白质也呈现如此异常迁移:Moraxella catarrhalis的高分子量外膜蛋白UspA1和UspA2分别具有推定分子量62.5KDa和88.3KDa,但分别以85KDa和120KDa迁移,以作为UspA复合物以350KDa~720KDa迁移。(Aebi,C.et al.,1997,Infection and Immunity,65:4367-4377;Klingman,k.L.and Murphy,T.F.,1994,Infiction and Immunity,62:1150-1155)。相似地,流感嗜血杆菌的Hia具有116KDa的预测分子量,但当在噬菌体中表达时,Hia以大于200KDa迁移(Barenkamp,S.and St.Gema III,J.1996 Molecular Microbiology 19:1215-1233)。
为证实HiaNm与脑膜炎奈瑟氏球菌的外膜相关,基本如前述(vander Ley,P.et al.,1991,Infection and Immunity,59:2963-71)制备外膜复合物(omc)。简而言之,在补加10%加热的马血BHI平板的脑心浸液琼脂(Acumedia Manufacturer’s Inc)上将细菌生长过夜,在超声破坏膜之前,将其重悬浮于10mM Tris pH8.0中并热灭活。通过在10,000×g(rcf,相对离心力)离心除去细胞碎片,并将上清液在50,000×g再离心。将粒状沉淀重悬浮于1%Sarkosyl/10mM Tris pH8.4中。将含有外膜蛋白的Sarkosyl可溶组分的等份(50μl A280=3.75)如上所述进行SDS-PAGE及Western印迹,示于图4的结果表明:观测到抗HiaNm MBP抗血清与野生型MC58而非其中hiaNm已灭活的MC58ΔHiaNm的反应性。在MC58培养物中全细胞样品,和含同量总蛋白的omc样品之间观测到的与抗Hia MBP血清反应性的提高,与HiaNm的膜结合性一致。
实施例6:杀菌分析
为测定抗HiaMBP抗血清是否含有特异于HiaNm的杀菌抗体,用野生型MC58和MC58ΔHiaNm进行杀菌分析。该分析通过由Hoogerhout等(1995,Infection and Immunity,63:3473~3478)所述分析加以修改而进行。简而言之,将MC58和MC58HiaNm在37℃在5%CO2中在BHI平板上生长过夜。取自此过夜培养物的细菌在悬浮于1ml PBS中之前,在相同条件下传代培养4-6小时。根据在0.2NNaOH/1%SDS中样品的裂解,以及在波长260nm处的吸收,其中A260=109cfu/ml计算细菌数。将此细菌悬浮液在PBS中调正至近于105cfu/ml。将待测试的兔血清在56℃热灭活45分钟,将取自4周龄新西兰白兔的血清集合,并用作补体来源(Central Animal BreedingHouse,University of Queensland)。在无菌聚苯乙烯平底96孔微量滴定板中进行分析,每孔总体积是24μl∶12μl在PBS中二倍连续稀释的血清,及6μl细菌悬浮液(含300~900个细菌)。在加入6μl 80%的在PBS中的补体(终浓度为20%vol/vol)之前,将血清和细菌在室温培养10分钟。对照是a)PBS,细菌及补体,b)PBS、细菌和血清,在加入所有组分并混合之后,将每个对照孔的7μl等份涂布在BHI平板上,然后将此微量滴定平板在37℃,在5%CO2中培养60分钟,在培养后,将每孔7μl等份涂布在BHI平板上,然后将所有BHI平板在37℃在5%CO2中培养14-18小时,并计数细菌菌落。其中至少90%细菌被杀灭时的最高稀释度倒数被报道为血清杀菌。所用的血清取自如实施例5所述Western印迹试验的相同兔及相同测试血液。这些试验平行示出,与野生型菌株MC58相比,抗MC58ΔHiaNm的杀伤滴定度降低(约3倍,表4),这表明抗HiaMBP抗血清含有特异于HiaNm的杀菌抗体。
表4
菌株 | 滴定度a |
MC58MC58ΔHiaNm | 12(+/-4.6)3.5(+/-1) |
a四个独立实验的平均值。
讨论
在一些病原菌中,重复DNA与毒力决定簇相关。用此重复DNA基序的Southern印迹揭示在脑膜炎奈瑟氏球菌菌株MC58中含此基序的至少3种基因座的存在(Peak,I.et al.,1996,FEMS MicrobiologyLetters,137:109-114)。将这些基因克隆,并对两个这种重复相关的基因座(nmrep 2和nmrep 3)进行序列分析,揭示有近于670个氨基酸的开放读框(Jennings,M.et al.,1995,Microbial Pathogenesis,19:391-407.Peak,I.et al.,Microbial Pathogenesis,出版中)。它们呈彼此同源,并同源于E.coli粘附素AIDA-I羧基末端。AIDA-I为1286个氨基酸,其羧基末端区组成了推定的外膜转运结构域,成熟代表的氨基末端结构域组成了粘附素结构域。氨基末端结构域通过推定的转运结构域穿过膜并被称为过客结构域。
由于Nmep2和Nmep3与AIDA-I的转运蛋白结构域序列同源,其被认为形成膜孔,Nmrep 2和Nmrep 3大约是AIDA-I大小的一半,并同源于AIDA-I跨膜区。我们假定脑膜炎奈瑟氏球菌中存在与AIDA-I氨基末端同源的基因座,我们在对脑膜炎奈瑟氏球菌菌株MC58¢3测序计划所得数据(TIGR前述)中搜索这种同系物,并发现一与流感嗜血杆菌菌株Rd(HI1732)称为AIDA-I的基因同源的区域,因为它同源于E.coli的AIDA-I(Fleischmann et al.,1995 Science269:496-512)。鉴于以上提及的同源性,本申请人决定进一步研究。
通过将相应于来自脑膜炎奈瑟氏球菌MC58 3的名为gnmaa84r的471碱基对片段的DNA进行PCR扩增而初步分离基因,并确定序列。进一步的PCR试验使较大片段被扩增。将这些产物克隆,并如图1所示进行序列分析,该基因呈与E.coli的AIDA-I的氨基末端区域的同源性,我们称其为aida3,因此其代表脑膜炎奈瑟氏球菌(具有nmrep 2和nmrep 3)中第三个AIDA-I同系物。由此,发现自流感嗜血杆菌的两个另外基因hia和hsf已被公布(Barenkamp,S.and St.Geme III,J.et al.,1996,分子微生物学19:1215-1233,St.Geme III,J.等,1996,细菌学杂志178:6281-6287),aida 3与其更相似。我们因而将之重命名为hiaNm(根据Barenkamp及St.Geme III之报道,HI1732,第一个被鉴别为AIDA-I同系物的流感嗜血杆菌基因,也被重命名为hia)。
通过本发明说明书阐述了本发明优选实施方案,而非限制本发明至任一实施方案或特征的具体组合。本领域技术人员应知:根据本发明揭示,在不偏离本发明范围之内,对例举的实施方案可作各种修改和变动,所有改动均在本发明权利要求范围内。
序列表
<110>Peak,Ian R. (仅美国)
Jennings,Michael P. (仅美国)
Moxom,Edward R. (仅美国)
昆士兰大学 (除了美国)
埃斯埃斯发明有限公司 (除了美国)
<120>新表面抗原
<130>脑膜炎奈瑟氏球菌HiaNm抗原
<140>PCT/AU98/01031
<141>1998-12-14
<150>GB 9726398.2
<151>1997-12-12
<160>31
<170>PatentIn Ver.2.0
<210>1
<211>2308
<212>DNA
<213>脑膜炎奈瑟氏球菌
<220>
<221>CDS
<222>(276)..(2054)
<400>1
gaaaaaccac aggaatttat cagcaaaaac agaaacccca ccgccgtcat tcccgcaaaa 60
gcgggaatcc agacccgtcg gcacggaaaa cttaccgaat aaaacagttt ccttagattc 120
cacgtcccag attcccgcct tcgcggggaa tgacgagatt ttaagttggg ggaatttatc 180
agaaaacccc caacccccaa aaaccgggcg gatgccgcac catccgcccc caaaccccga 240
tttaaccatt caaacaaacc aaaagaaaaa acaaa atg aac aaa ata tac cgc 293
Met Asn Lys Ile Tyr Arg
1 5
atc att tgg aat agt gcc ctc aat gcc tgg gtc gtc gta tcc gag ctc 341
Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp Val Val Val Ser Glu Leu
10 15 20
aca cgc aac cac acc aaa cgc gcc tcc gca acc gtg aag acc gcc gta 389
Thr Arg Asn His Thr Lys Arg Ala Ser Ala Thr Val Lys Thr Ala Val
25 30 35
ttg gcg aca ctg ttg ttt gca acg gtt cag gca agt gct aac aat gaa 437
Leu Ala Thr Leu Leu Phe Ala Thr Val Gln Ala Ser Ala Asn Asn Glu
40 45 50
aga cca aga aag aaa gat tta tat tta gac ccc gta caa cgc act gtt 485
Arg Pro Arg Lys Lys Asp Leu Tyr Leu Asp Pro Val Gln Arg Thr Val
55 60 65 70
gcc gtg ttg ata gtc aat tcc gat aaa gaa ggc acg gga gaa aaa gaa 533
Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly Thr Gly Glu Lys Glu
75 80 85
aaa gta gaa gaa aat tca gat tgg gca gta tat ttc aac gag aaa gga 581
Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr Phe Asn Glu Lys Gly
90 95 100
gta cta aca gcc aga gaa atc acc ctc aaa gcc ggc gac aac ctg aaa 629
Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala Gly Asp Asn Leu Lys
105 110 115
atc aaa caa aac ggc aca aac ttc acc tac tcg ctg aaa aaa gac ctc 677
Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr Ser Leu Lys Lys Asp Leu
120 125 130
aca gat ctg acc agt gtt gga act gaa aaa tta tcg ttt agc gca aac 725
Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu Ser Phe Ser Ala Asn
135 140 145 150
ggc aat aaa gtc aac atc aca agc gac acc aaa ggc ttg aat ttt gcg 773
Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala
155 160 165
aaa gaa acg gct ggg acg aac ggc gac acc acg gtt cat ctg aac ggt 821
Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr Val His Leu Asn Gly
170 175 180
att ggt tcg act ttg acc gat acg ctg ctg aat acc gga gcg acc aca 869
Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr
185 190 195
aac gta acc aac gac aac gtt acc gat gac gag aaa aaa cgt gcg gca 917
Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala
200 205 210
agc gtt aaa gac gta tta aac gct ggc tgg aac att aaa ggc gtt aaa 965
Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys
215 220 225 230
ccc ggt aca aca gct tcc gat aac gtt gat ttc gtc cgc act tac gac 1013
Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp
235 240 245
aca gtc gag ttc ttg agc gca gat acg aaa aca acg act gtt aat gtg 1061
Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val
250 255 260
gaa agc aaa gac aac ggc aag aaa acc gaa gtt aaa atc ggt gtg aag 1109
Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val Lys Ile Gly Val Lys
265 270 275
act tct gtt att aaa gaa aaa gac ggt aag ttg gtt act ggt aaa gac 1157
Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Asp
280 285 290
aaa ggc gag aat ggt tct tct aca gac gaa ggc gaa ggc tta gtg act 1205
Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr
295 300 305 310
gca aaa gaa gtg att gat gca gta aac aag gct ggt tgg aga atg aaa 1253
Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys
315 320 325
aca aca acc gct aat ggt caa aca ggt caa gct gac aag ttt gaa acc 1301
Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr
330 335 340
gtt aca tca ggc aca aat gta acc ttt gct agt ggt aaa ggt aca act 1349
Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser Gly Lys Gly Thr Thr
345 350 355
gcg act gta agt aaa gat gat caa ggc aac atc act gtt atg tat gat 1397
Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile Thr Val Met Tyr Asp
360 365 370
gta aat gtc ggc gat gcc cta aac gtc aat cag ctg caa aac agc ggt 1445
Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly
375 380 385 390
tgg aat ttg gat tcc aaa gcg gtt gca ggt tct tcg ggc aaa gtc atc 1493
Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile
395 400 405
agc ggc aat gtt tcg ccg agc aag gga aag atg gat gaa acc gtc aac 1541
Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn
410 415 420
att aat gcc ggc aac aac atc gag att acc cgc aac ggt aaa aat atc 1589
Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile
425 430 435
gac atc gcc act tcg atg acc ccg cag ttt tcc agc gtt tcg ctc ggc 1637
Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly
440 445 450
gcg ggg gcg gat gcg ccc act ttg agc gtg gat ggg gac gca ttg aat 1685
Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp Gly Asp Ala Leu Asn
455 460 465 470
gtc ggc agc aag aag gac aac aaa ccc gtc cgc att acc aat gtc gcc 1733
Val Gly Ser Lys Lys Asp Asn Lys Pro Val Arg Ile Thr Asn Val Ala
475 480 485
ccg ggc gtt aaa gag ggg gat gtt aca aac gtc gca caa ctt aaa ggc 1781
Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly
490 495 500
gtg gcg caa aac ttg aac aac cgc atc gac aat gtg gac ggc aac gcg 1829
Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala
505 510 515
cgt gcg ggc atc gcc caa gcg att gca acc gca ggt ctg gtt cag gcg 1877
Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu Val Gln Ala
520 525 530
tat ttg ccc ggc aag agt atg atg gcg atc ggc ggc ggc act tat cgc 1925
Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg
535 540 545 550
ggc gaa gcc ggt tac gcc atc ggc tac tcc agt att tcc gac ggc gga 1973
Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Gly Gly
555 560 565
aat tgg att atc aaa ggc acg gct tcc ggc aat tcg cgc ggc cat ttc 2021
Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe
570 575 580
ggt gct tcc gca tct gtc ggt tat cag tgg taa gggctttatc gcctgtctgc 2074
Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp
585 590
tgttgggaca ggcggaaggt ttgaagggaa gggtggcgat ttgccgcctg agacctttgc 2134
aaaatccccc caaaatcccc taaattccca ccaagacatt taggggattt ctcatgagca 2194
ccttcttccg gcaaaccgcg caagccatga ttgccaaaca catcaaccgt ttcccgctat 2254
tgaagttgga ccaagtgatt gattggcagc cgatcgagca gtacctgaac cgtc 2308
<210>2
<211>592
<212>PRT
<213>Neisseria meningitidis
<400>2
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
Ala Ser Ala Asn Asn Glu Arg Pro Arg Lys Lys Asp Leu Tyr Leu Asp
50 55 60
Pro Val Gln Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu
65 70 75 80
Gly Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val
85 90 95
Tyr Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys
100 105 110
Ala Gly Asp Asn Leu Lys Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr
115 120 125
Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys
130 135 140
Leu Ser Phe Ser Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr
145 150 155 160
Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr
165 170 175
Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu
180 185 190
Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp
195 200 205
Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp
210 215 220
Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp
225 230 235 240
Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys
245 250 255
Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu
260 265 270
Val Lys Ile Gly Val Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys
275 280 285
Leu Val Thr Gly Lys Asp Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu
290 295 300
Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys
305 310 315 320
Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln
325 330 335
Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala
340 345 350
Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn
355 360 365
Ile Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn
370 375 380
Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly
385 390 395 400
Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys
405 410 415
Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr
420 425 430
Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe
435 440 445
Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val
450 455 460
Asp Gly Asp Ala Leu Asn Val Gly Ser Lys Lys Asp Asn Lys Pro Val
465 470 475 480
Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn
485 490 495
Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp
500 505 510
Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr
515 520 525
Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile
530 535 540
Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser
545 550 555 560
Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly
565 570 575
Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp
580 585 590
<210>3
<211>1779
<212>DNA
<213>Neisseria meningitidis
<400>3
atgaacaaaa tataccgcat catttggaat agtgccctca atgcctgggt cgtcgtatcc 60
gagctcacac gcaaccacac caaacgcgcc tccgcaaccg tgaagaccgc cgtattggcg 120
acactgttgt ttgcaacggt tcaggcaagt gctaacaatg aaagaccaag aaagaaagat 180
ttatatttag accccgtaca acgcactgtt gccgtgttga tagtcaattc cgataaagaa 240
ggcacgggag aaaaagaaaa agtagaagaa aattcagatt gggcagtata tttcaacgag 300
aaaggagtac taacagccag agaaatcacc ctcaaagccg gcgacaacct gaaaaccaaa 360
caaaacggca caaacttcac ctactcgctg aaaaaagacc tcacacatct gaccaccgtt 420
ggaactgaaa aattatcgtt tagcgcaaac ggcaataaag tcaacatcac aagcgacacc 480
aaaggcttga attttgcgaa agaaacggct gggacgaacg gcgacaccac ggttcacctg 540
aacggtattg gttcgacttt gaccgatacg ctgctgaata ccggaccgac cacaaacgta 600
accaacgaca acgttaccga tgacgagaaa aaacgtgcgg caagccctaa agacccacca 660
aacgctggct ggaacattaa aggcgttaaa cccggtacaa cagcttccga taacgccgat 720
ttcgtccgca cttacgacac agtcgagttc ttgagcgcag atacgaaaac aacgaccgtt 780
aatgtggaaa gcaaagacaa cggcaagaaa accgaagtta aaatccctgt gaagacctct 840
gttattaaag aaaaagacgg taagttggtt actggtaaag acaaacgcga gaatggctct 900
tctacagacg aaggcgaagg cttagtgact gcaaaagaag tgattgatgc agtaaacaag 960
gctggttgga gaatgaaaac aacaaccgct aatggtcaaa caggtcaagc tgacaagttt 1020
gaaaccgtta catcaggcac aaatgtaacc tttgctagtg gtaaaggtac aactccgact 1080
gtaagtaaag atgatcaagg caacatcact gttatgtatg atgtaaatgt cggcgacgcc 1140
ctaaacgtca atcagctgca aaacagcggt tggaatttgg attccaaagc ggttccaggt 1200
tcttcgggca aagtcatcag cggcaatgtt tcgccgagca agggaaagat ggatgaaacc 1260
gtcaacatta atgccggcaa caacatcgag attacccgca acggtaaaaa tatccacatc 1320
gccacttcga tgaccccgca gttttccagc gtttcgctcg gcgcgggggc ggatccgccc 1380
actttgagcg tggatgggga cgcattgaat gtcggcagca agaaggacaa caaacccgtc 1440
cgcattacca atgtcgcccc gggcgttaaa gagggggatg ttacaaacgt cgcacaactt 1500
aaaggcgtgg cgcaaaactt gaacaaccgc atcgacaatg tggacggcaa cgcgcgcgcg 1560
ggcatcgccc aagcgattgc aaccgcaggt ctggttcagg cgtatccgcc cggcaagagt 1620
atgatggcga tcggcggcgg cacttatcgc ggcgaagccg gttaccccat cggctactcc 1680
agtatttccg acggcggaaa ttggattatc aaaggcacgg cttcccgcaa ttcgcccggc 1740
catttcggtg cttccgcatc tgtcggttat cagtggtaa 1779
<210>4
<211>1797
<212>DNA
<213>Neisseria meningitidis
<220>
<221>CDS
<222>(1)..(1797)
<400>4
atg aac aaa ata tcc cgc atc att tgg aat agt gcc ctc aat gcc tcg 48
Met Asn Lys Ile Ser Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
gtc gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
acc gtg gcg acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
gcg aat gct acc gat gac gac gat tta tat tta gaa ccc gta caa cgc 192
Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg
50 55 60
act gct gtc gtg ttg agc ttc cgt tcc gat aaa gaa ggc acg gga gaa 240
Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu
65 70 75 80
aaa gaa ggt aca gaa gat tca aat tgg gca gta tat ttc gac gag aaa 288
Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys
85 90 95
aga gta cta aaa gcc gga gca atc acc ctc aaa gcc ggc gac aac ctg 336
Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu
100 105 110
aaa atc aaa caa aac acc aat gaa aac acc aat gaa aac acc aat gac 384
Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Glu Asn Thr Asn Asp
115 120 125
agt agc ttc acc tac tcc ctg aaa aaa gac ctc aca gat ctg acc agt 432
Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser
130 135 140
gtt gaa act gaa aaa tta tcg ttt ggc gca aac ggt aat aaa gtc aac 480
Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn
145 150 155 160
atc aca agc gac acc aaa ggc ttg aat ttt gcg aaa gaa acg gct ggg 528
Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly
165 170 175
acg aac ggc gac ccc acg gtt cat ctg aac ggt atc ggt tcg act ttg 576
Thr Asn Gly Asp Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu
180 185 190
acc gat acg ctg ctg aat acc gga gcg acc aca aac gta acc aac gac 624
Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp
195 200 205
aac gtt acc gat gac gag aaa aaa cgt gcg gca agc gtt aaa gac gta 672
Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val
210 215 220
tta aac gca ggc tgg aac att aaa ggc gtt aaa ccc ggt aca aca gct 720
Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Tnr Thr Ala
225 230 235 240
tcc gat aac gtc gat ttc gtc cgc act tac gac aca gtc gag ttc ttg 768
Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu
245 250 255
agc gca gat acg aaa aca acg act gtt aat gtg gaa agc aaa gac aac 816
Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn
260 265 270
ggc aag aga acc gaa gtt aaa atc ggt gcg aag act tct gtt att aaa 864
Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys
275 280 285
gaa aaa gac ggt aag ttg gtt act ggt aaa ggc aaa ggc gag aat ggt 912
Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Gly Glu Asn Gly
290 295 300
tct tct aca gac gaa ggc gaa ggc tta gtg act gca aaa gaa gtg att 960
Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile
305 310 315 320
gat gca gta aac aag gct ggt tgg aga atg aaa aca aca acc gct aat 1008
Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn
325 330 335
ggt caa aca gct caa gct gac aag ttt gaa acc gtt aca tca ggc aca 1056
Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr
340 345 350
aaa gta acc ttt gct agt ggt aat ggt aca act gcg act gta agt aaa 1104
Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys
355 360 365
gat gat caa gcc aac atc act gtt aag tat gat gta aat gtc ggc gat 1152
Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp
370 375 380
gcc cta aac gcc aat cag ctg caa aac agc ggt tgg aat ttg gat tcc 1200
Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser
385 390 395 400
aaa gcg gtt gca ggt tct tcg ggc aaa gtc atc agc ggc aat gtt tcg 1248
Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser
405 410 415
ccg agc aag gga aag atg gat gaa acc gtc aac att aat gcc ggc aac 1296
Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn
420 425 430
aac atc gag act acc cgc aac ggc aaa aat atc gac atc gcc act tcg 1344
Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser
435 440 445
atg acc ccg caa ttt tcc agc gtt tcg ctc ggc gcg ggg gcg gat gcg 1392
Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala
450 455 460
ccc act tta agc gtg gat gac gag ggc gcg ttg aat gtc ggc agc aag 1440
Pro Thr Leu Ser Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys
465 470 475 480
gat gcc aac aaa ccc gtc cgc att acc aat gtc gcc ccg ggc gtt aaa 1488
Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys
485 490 495
gag ggg gat gtt aca aac gtc gca caa ctt aaa ggt gtg gcg caa aac 1536
Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn
500 505 510
ttg aac aac cgc atc gac aat gtg gac ggc aac gcg cgc gcg ggt atc 1584
Leu Asn Asn Arc Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile
515 520 525
gcc caa gcg att gca acc gca ggt ttg gct cag gcc tat ttg ccc ggc 1632
Ala Gln Ala Ile Ala Thr Ala Gly Leu Ala Gln Ala Tyr Leu Pro Gly
530 535 540
aag agt atg atg gcg atc ggc ggc ggt act tat cgc ggc gaa gcc ggt 1680
Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly
545 550 555 560
tac gcc atc ggc tac tcg agc att tct gac act ggg aat tgg gtt atc 1728
Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Thr Gly Asn Trp Val Ile
565 570 575
aag ggc acg gct tcc ggc aat tcg cgc ggt cat ttc ggt act tcc gca 1776
Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Thr Ser Ala
580 585 590
tct gtc ggt tat cag tgg taa 1797
Ser Val Gly Tyr Gln Trp
595
<210>5
<211>598
<212>PRT
<213>Neisseria meningitidis
<400>5
Met Asn Lys Ile Ser Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg
50 55 60
Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu
65 70 75 80
Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys
85 90 95
Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu
100 105 110
Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Glu Asn Thr Asn Asp
115 120 125
Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser
130 135 140
Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn
145 150 155 160
Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly
165 170 175
Thr Asn Gly Asp Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu
180 185 190
Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp
195 200 205
Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val
210 215 220
Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala
225 230 235 240
Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu
245 250 255
Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn
260 265 270
Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys
275 280 285
Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Gly Glu Asn Gly
290 295 300
Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile
305 310 315 320
Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn
325 330 335
Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr
340 345 350
Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys
355 360 365
Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp
370 375 380
Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser
385 390 395 400
Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser
405 410 415
Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn
420 425 430
Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser
435 440 445
Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala
450 455 460
Pro Thr Leu Ser Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys
465 470 475 480
Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys
485 490 495
Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn
500 505 510
Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile
515 520 525
Ala Gln Ala Ile Ala Thr Ala Gly Leu Ala Gln Ala Tyr Leu Pro Gly
530 535 540
Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly
545 550 555 560
Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Thr Gly Asn Trp Val Ile
565 570 575
Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Thr Ser Ala
580 585 590
Ser Val Gly Tyr Gln Trp
595
<210>6
<211>1785
<212>DNA
<213>Neisseria meningitidis
<220>
<221>CDS
<222>(1)..(1785)
<400>6
atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
gtc gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
acc gtg gcg acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
gcg aat gct acc gat gac gac gat tta tat tta gaa ccc gta caa cgc 192
Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg
50 55 60
act gct gtc gtg ttg agc ttc cgt tcc gat aaa gaa ggc acg gga gaa 240
Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu
65 70 75 80
aaa gaa ggt aca gaa gat tca aat tgg gca gta tat ttc gac gag aaa 288
Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys
85 90 95
aga gta cta aaa gcc gga gca atc acc ctc aaa gcc ggc gac aac ctg 336
Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu
100 105 110
aaa atc aaa caa aac acc aat gaa aac acc aat gac agt agc ttc acc 384
Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Asp Ser Ser Phe Thr
115 120 125
tac tcc ctg aaa aaa gac ctc aca gat ctg acc agt gtt gaa act gaa 432
Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Glu Thr Glu
130 135 140
aaa tta tcg ttt ggc gca aac ggt aat aaa gtc aac atc aca agc gac 480
Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp
145 150 155 160
acc aaa ggc ttg aat ttt gcg aaa gaa acg gct ggg acg aac ggc gac 528
Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp
165 170 175
ccc acg gtt cat ctg aac ggt atc ggt tcg act ttg acc gat acg ctg 576
Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu
180 185 190
ctg aat acc gga gcg acc aca aac gta acc aac gac aac gtt acc gat 624
Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp
195 200 205
gac gag aaa aaa cgt gcg gca agc gtt aaa gac gta tta aac gca ggc 672
Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly
210 215 220
tgg aac att aaa ggc gtt aaa ccc ggt aca aca gct tcc gat aac gtt 720
Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val
225 230 235 240
gat ttc gtc cgc act tac gac aca gtc gag ttc ttg agc gca gat acg 768
Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr
245 250 255
aaa aca acg act gtt aat gtg gaa agc aaa gac aac ggc aag aaa acc 816
Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr
260 265 270
gaa gtt aaa atc ggt gcg aag act tct gtt att aaa gaa aaa gac ggt 864
Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly
275 280 285
aag ttg gtt act ggt aaa ggc aaa gac gag aat ggt tct tct aca gac 912
Lys Leu Val Thr Gly Lys Gly Lys Asp Glu Asn Gly Ser Ser Thr Asp
290 295 300
gaa ggc gaa ggc tta gtg act gca aaa gaa gtg att gat gca gta aac 960
Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn
305 310 315 320
aag gct ggt tgg aga atg aaa aca aca acc gct aat ggt caa aca gct 1008
Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly
325 330 335
caa gct gac aag ttt gaa acc gtt aca tca ggc aca aat gta acc ttt 1056
Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe
340 345 350
gct agt ggt aaa ggt aca act gcg act gta agt aaa gat gat caa ggc 1104
Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly
355 360 365
aac atc act gtt aag tat gat gta aat gtc ggc gat gcc cta aac gtc 1152
Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val
370 375 380
aat cag ctg caa aac agc ggt tgg aat ttg gat tcc aaa gcg gtt gca 1200
Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala
385 390 395 400
ggt tct tcg ggc aaa gtc atc agc ggc aat gtt tcg ccg agc aag gga 1248
Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly
405 410 415
aag atg gat gaa acc gtc aac att aat gcc ggc aac aac atc gag att 1296
Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile
420 425 430
acc cgc aac ggt aaa aat atc gac atc gcc act tcg atg gcg ccg cag 1344
Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Ala Pro Gln
435 440 445
ttt tcc agc gtt tcg ctc ggt gcg ggg gcg gat gcg ccc act ttg agc 1392
Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser
450 455 460
gtg gat gac gag ggc gcg ttg aat gtc ggc agc aag gat acc aac aaa 1440
Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Thr Asn Lys
465 470 475 480
ccc gtc cgc att acc aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt 1488
Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val
485 490 495
aca aac gtc gca caa ctt aaa ggc gtg gcg caa aac ttg aac aac cgc 1536
Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg
500 505 510
atc gac aat gtg gac ggc aac gcg cgt gcg ggc atc gcc caa gcg att 1584
Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile
515 520 525
gca acc gca ggt cta gtt cag gcg tat ctg ccc ggc aag agt atg atg 1632
Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met
530 535 540
gcg atc ggc ggc gac act tat cgc ggc gaa gcc ggt tac gcc atc ggc 1680
Ala Ile Gly Gly Asp Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly
545 550 555 560
tac tca agt att tcc gac ggc gga aat tgg att atc aaa ggc acg gct 1728
Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala
565 570 575
tcc ggc aat tcg cgc ggc cat ttc ggt gct tcc gca tct gtc ggt tat 1776
Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr
580 585 590
caa tgg taa 1785
Gln Trp
595
<210>7
<211>594
<212>PRT
<213>Neisseria meningitidis
<400>7
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg
50 55 60
Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu
65 70 75 80
Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys
85 90 95
Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu
100 105 110
Lys Ile Lys Gln Asn Thr Ash Glu Asn Thr Asn Asp Ser Ser Phe Thr
115 120 125
Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Glu Thr Glu
130 135 140
Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp
145 150 155 160
Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp
165 170 175
Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu
180 185 190
Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp
195 200 205
Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly
210 215 220
Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val
225 230 235 240
Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr
245 250 255
Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr
260 265 270
Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly
275 280 285
Lys Leu Val Thr Gly Lys Gly Lys Asp Glu Asn Gly Ser Ser Thr Asp
290 295 300
Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn
305 310 315 320
Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly
325 330 335
Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe
340 345 350
Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly
355 360 365
Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val
370 375 380
Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala
385 390 395 400
Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly
405 410 415
Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile
420 425 430
Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Ala Pro Gln
435 440 445
Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser
450 455 460
Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Thr Asn Lys
465 470 475 480
Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val
485 490 495
Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg
500 505 510
Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile
515 520 525
Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met
530 535 540
Ala Ile Gly Gly Asp Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly
545 550 555 560
Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala
565 570 575
Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr
580 585 590
Gln Trp
<210>8
<211>1785
<212>DNA
<213>Neisseria meningitidis
<220>
<221>CDS
<222>(1)..(1785)
<400>8
atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
gtc gcc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96
Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
acc gtg gcg acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
gcg agt act acc gat gac gac gat tta tat tta gaa ccc gta caa cgc 192
Ala Ser Thr Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg
50 55 60
act gct gtc gtg ttg agc ttc cgt tcc gat aaa gaa ggc acg gga gaa 240
Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu
65 70 75 80
aaa gaa gtt aca gaa gat tca aat tgg gga gta tat ttc gac aag aaa 288
Lys Glu Val Thr Glu Asp Ser Asn Trp Gly Val Tyr Phe Asp Lys Lys
85 90 95
gga gta cta aca gcc gga aca atc acc ctc aaa gcc ggc gac aac ctg 336
Gly Val Leu Thr Ala Gly Thr Ile Thr Leu Lys Ala Gly Asp Asn Leu
100 105 110
aaa atc aaa caa aac acc aat gaa aac acc aat gcc agt agc ttc acc 384
Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Ala Ser Ser Phe Thr
115 120 125
tac tcg ctg aaa aaa gac ctc aca gat ctg acc agt gtt gga act gaa 432
Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu
130 135 140
aaa tta tcg ttt agc gca aac agc aat aaa gtc aac atc aca agc gac 480
Lys Leu Ser Phe Ser Ala Asn Ser Asn Lys Val Asn Ile Thr Ser Asp
145 150 155 160
acc aaa ggc ttg aat ttc gcg aaa aaa acg gct gag acc aac ggc gac 528
Thr Lys Gly Leu Asn Phe Ala Lys Lys Thr Ala Glu Thr Asn Gly Asp
165 170 175
acc acg gtt cat ctg aac ggt atc ggt tcg act ttg acc gat acg ctg 576
Thr Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu
180 185 190
ctg aat acc gga gcg acc aca aac gta acc aac gac aac gtt acc gat 624
Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp
195 200 205
gac gag aaa aaa cgt gcg gca agc gtt aaa gac gta tta aac gca ggc 672
Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly
210 215 220
tgg aac att aaa ggc gtt aaa ccc ggt aca aca gct tcc gat aac gtt 720
Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val
225 230 235 240
gat ttc gtc cgc act tac gac aca gtc gag ttc ttg agc gca gat acg 768
Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr
245 250 255
aaa aca acg act gtt aat gtg gaa agc aaa gac aac ggc aag aga acc 816
Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Arg Thr
260 265 270
gaa gtt aaa atc ggt gcg aag act tct gtt atc aaa gaa aaa gac ggt 864
Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly
275 280 285
aag ttg gtt act ggt aaa gac aaa ggc gag aat gat tct tct aca gac 912
Lys Leu Val Thr Gly Lys Asp Lys Gly Glu Asn Asp Ser Ser Thr Asp
290 295 300
aaa ggc gaa ggc tta gtg act gca aaa gaa gtg att gat gca gta aac 960
Lys Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn
305 310 315 320
aag gct ggt tgg aga atg aaa aca aca acc gct aat ggt caa aca ggt 1008
Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly
325 330 335
caa gct gac aag ttt gaa acc gtt aca tca ggc aca aat gta acc ttt 1056
Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe
340 345 350
gct agt ggt aaa ggt aca act gcg act gta agt aaa gat gat caa ggc 1104
Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly
355 360 365
aac atc act gtt atg tat gat gta aat gtc ggc gat gcc cta aac gtc 1152
Asn Ile Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val
370 375 380
aat cag ctg caa aac agc ggt tgg aat ttg gat tcc aaa gcg gtt gca 1200
Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala
385 390 395 400
ggt tct tcg ggc aaa gtc atc agc ggc aat gtt tcg ccg agc aag gga 1248
Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly
405 410 415
aag atg gat gaa acc gtc aac att aat gcc ggc aac aac atc gag att 1296
Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile
420 425 430
acc cgc aac ggc aaa aat atc gac atc gcc act tcg atg acc ccg caa 1344
Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln
435 440 445
ttt tcc agc gtt tcg ctc ggc gcg ggg gcg gat gcg ccc act tta agc 1392
Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser
450 455 460
gtg gat gac gag ggc gcg ttg aat gtc ggc agc aag gat gcc aac aaa 1440
Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys
465 470 475 480
ccc gtc cgc att acc aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt 1488
Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val
485 490 495
aca aac gtc gca caa ctt aaa ggc gtg gcg caa aac ttg aac aac cac 1536
Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn His
500 505 510
atc gac aat gtg gac ggc aac gcg cgt gcg ggc atc gcc caa gcg att 1584
Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile
515 520 525
gca acc gca ggt ctg gtt cag gcg tat ctg ccc ggc aag agt atg atg 1632
Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met
530 535 540
gcg atc ggc ggc ggc act tat cgc ggc gaa gcc ggt tat gcc atc ggc 1680
Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly
545 550 555 560
tac tca agc att tcc gac ggc gga aat tgg att atc aaa ggc acg gct 1728
Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala
565 570 575
tcc ggc aat tcg cgc ggc cat ttc ggt gct tcc gca tct gtc ggt tat 1776
Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr
580 585 590
cag tgg taa 1785
Gln Trp
595
<210>9
<211>594
<212>PRT
<213>Neisseria meningitidis
<400>9
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
Ala Ser Thr Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg
50 55 60
Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu
65 70 75 80
Lys Glu Val Thr Glu Asp Ser Asn Trp Gly Val Tyr Phe Asp Lys Lys
85 90 95
Gly Val Leu Thr Ala Gly Thr Ile Thr Leu Lys Ala Gly Asp Asn Leu
100 105 110
Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Ala Ser Ser Phe Thr
115 120 125
Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu
130 135 140
Lys Leu Ser Phe Ser Ala Asn Ser Asn Lys Val Asn Ile Thr Ser Asp
145 150 155 160
Thr Lys Gly Leu Asn Phe Ala Lys Lys Thr Ala Glu Thr Asn Gly Asp
165 170 175
Thr Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu
180 185 190
Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp
195 200 205
Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly
210 215 220
Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val
225 230 235 240
Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr
245 250 255
Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Arg Thr
260 265 270
Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly
275 280 285
Lys Leu Val Thr Gly Lys Asp Lys Gly Glu Asn Asp Ser Ser Thr Asp
290 295 300
Lys Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn
305 310 315 320
Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly
325 330 335
Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe
340 345 350
Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly
355 360 365
Asn Ile Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val
370 375 380
Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala
385 390 395 400
Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly
405 410 415
Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile
420 425 430
Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln
435 440 445
Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser
450 455 460
Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys
465 470 475 480
Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val
485 490 495
Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn His
500 505 510
Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile
515 520 525
Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met
530 535 540
Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly
545 550 555 560
Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala
565 570 575
Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr
580 585 590
Gln Trp
<210>10
<211>1776
<212>DNA
<213>Neisseria meningitidis
<220>
<221>CDS
<222>(1)..(1776)
<400>10
atg aac gaa ata ttg cgc atc att tgg aat agc gcc ctc aat gcc tgg 48
Met Asn Glu Ile Leu Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
gtc gtt gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
acc gtg aag acc gcc gta ttg gcg act ctg ttg ttt gca acg gtt cag 144
Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
gca agt gct aac aat gaa gag caa gaa gaa gat tta tat tta gac ccc 192
Ala Ser Ala Asn Asn Glu Glu Gln Glu Glu Asp Leu Tyr Leu Asp Pro
50 55 60
gtg cta cgc act gtt gcc gtg ttg ata gtc aat tcc gat aaa gaa ggc 240
Val Leu Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly
65 70 75 80
acg gga gaa aaa gaa aaa gta gaa gaa aat tca gat tgg gca gta tat 288
Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr
85 90 95
ttc aac gag aaa gga gta cta aca gcc aga gaa atc acc ctc aaa gcc 336
Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala
100 105 110
ggc gac aac ctg aaa atc aaa caa aac ggc aca aac ttc acc tac tcg 384
Gly Asp Asn Leu Lys Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr Ser
115 120 125
ctg aaa aaa gac ctc aca gat ctg acc agt gtt gga act gaa aaa tta 432
Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu
130 135 140
tcg ttt agc gca aac ggc aat aaa gtc aac atc aca agc gac acc aaa 480
Ser Phe Ser Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys
145 150 155 160
ggc ttg aat ttt gcg aaa gaa acg gct ggg acg aac ggc gac acc acg 528
Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr
165 170 175
gtt cat ctg aac ggt att ggt tcg act ttg acc gat acg ctg ctg aat 576
Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn
180 185 190
acc gga gcg acc aca aac gta acc aac gac aac gtt acc gat gac gag 624
Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu
195 200 205
aaa aaa cgt gcg gca agc gtt aaa gac gta tta aac gct ggc tgg aac 672
Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn
210 215 220
att aaa ggc gtt aaa ccc ggt aca aca gct tcc gat aac gtt gat ttc 720
Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe
225 230 235 240
gtc cgc act tac gac aca gtc gag ttc ttg agc gca gat acg aaa aca 768
Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr
245 250 255
acg act gtt aat gtg gaa agc aaa gac aac ggc aag aaa acc gaa gtt 816
Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val
260 265 270
aaa atc ggt gcg aag act tct gtt att aaa gaa aaa gac ggt aag ttg 864
Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu
275 280 285
gtt act ggt aaa gac aaa ggc gag aat ggt tct tct aca gac gaa ggc 912
Val Thr Gly Lys Asp Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly
290 295 300
gaa ggc tta gtg act gca aaa gaa gtg att gat gca gta aac aag gct 960
Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala
305 310 315 320
ggt tgg aga atg aaa aca aca acc gct aat ggt caa aca ggt caa gct 1008
Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala
325 330 335
gac aag ttt gaa acc gtt aca tca ggc aca aat gta acc ttt gct agt 1056
Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser
340 345 350
ggt aaa ggt aca act gcg act gta agt aaa gat gat caa ggc aac atc 1104
Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile
355 360 365
act gtt atg tat gat gta aat gtc ggc gat gcc cta aac gtc aat cag 1152
Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln
370 375 380
ctg caa aac agc ggt tgg aat ttg gat tcc aaa gcg gtt gca ggt tct 1200
Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser
385 390 395 400
tcg ggc aaa gtc atc agc ggc aat gtt tcg ccg agc aag gga aag atg 1248
Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met
405 410 415
gat gaa acc gtc aac att aat gcc ggc aac aac atc gag att acc cgc 1296
Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg
420 425 430
aac ggt aaa aat atc gac atc gcc act tcg atg acc ccg cag ttt tcc 1344
Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser
435 440 445
agc gtt tcg ctc ggc gcg ggg gcg gat gcg ccc act ttg agc gtg gat 1392
Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp
450 455 460
ggg gac gca ttg aat gtc ggc agc aag aag gac aac aaa ccc gtc cgc 1440
Gly Asp Ala Leu Asn Val Gly Ser Lys Lys Asp Asn Lys Pro Val Arg
465 470 475 480
att acc aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt aca aac gtc 1488
Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val
485 490 495
gca caa ctt aaa ggc gtg gcg caa aac ttg aac aac cgc acc gac aat 1536
Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn
500 505 510
gtg gac ggc aac gcg cgt gcg ggc atc gcc caa gcg att gca acc gca 1584
Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala
515 520 525
ggt ctg gtt cag gcg tat ttg ccc ggc aag agt atg atg gcg atc ggc 1632
Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly
530 535 540
ggc ggc act tat cgc ggc gaa gcc ggt tac gcc atc ggc tac tcc agt 1680
Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser
545 550 555 560
att tcc gac ggc gga aat tgg att atc aaa ggc acg gct ccc ggc aat 1728
Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly Asn
565 570 575
tcg cgc ggc cat ttc ggt gct tcc gca tct gtc ggt tat cag tgg taa 1776
Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp
580 585 590
<210>11
<211>591
<212>PRT
<213>Neisseria meningitidis
<400>11
Met Asn Glu Ile Leu Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
Ala Ser Ala Asn Asn Glu Glu Gln Glu Glu Asp Leu Tyr Leu Asp Pro
50 55 60
Val Leu Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly
65 70 75 80
Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr
85 90 95
Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala
100 105 110
Gly Asp Asn Leu Lys Ile Lys Gln Asn Gly Thr Asn Phe Thr Tyr Ser
115 120 125
Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu
130 135 140
Ser Phe Ser Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys
145 150 155 160
Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr
165 170 175
Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Leu Asn
180 185 190
Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu
195 200 205
Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn
210 215 220
Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe
225 230 235 240
Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr
245 250 255
Thr Thr Val Asn Val Gln Ser Lys Asp Asn Gly Lys Lys Thr Glu Val
260 265 270
Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu
275 280 285
Val Thr Gly Lys Asp Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly
290 295 300
Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala
305 310 315 320
Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala
325 330 335
Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Asn Val Thr Phe Ala Ser
340 345 350
Gly Lys Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile
355 360 365
Thr Val Met Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln
370 375 380
Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser
385 390 395 400
Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met
405 410 415
Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg
420 425 430
Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser
435 440 445
Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp
450 455 460
Gly Asp Ala Leu Asn Val Gly Ser Lys Lys Asp Asn Lys Pro Val Arg
465 470 475 480
Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val
485 490 495
Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn
500 505 510
Val Asp Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala
515 520 525
Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly
530 535 540
Gly Gly Thr Tyr Arg Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser
545 550 555 560
Ile Ser Asp Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly Asn
565 570 575
Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp
580 585 590
<210>12
<211>1797
<212>DNA
<213>Neisseria meningitidis
<220>
<221>CDS
<222>(1)..1797)
<400>12
atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
gtc gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc ccc tcc gca 96
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
acc gtg gcc acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
gcg aat gct acc gat gac gac gat tta tat tta gaa ccc gta caa cgc 192
Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg
50 55 60
act gct gtc gtg ttg agc ttc cgt tcc gat aaa gaa ggc acg gga gaa 240
Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu
65 70 75 80
aaa gaa ggt aca gaa gat tca aat tgg gca gta tat ttc cac gag aaa 288
Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys
85 90 95
aga gta cta aaa gcc gga gca atc acc ctc aaa gcc ggc cac aac ctg 336
Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu
100 105 110
aaa atc aaa caa aac acc aat gaa aac acc aat gaa aac acc aat gac 384
Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Glu Asn Thr Asn Asp
115 120 125
agt agc ttc acc tac tcc ctg aaa aaa gac ctc aca gat ctg acc agt 432
Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser
130 135 140
gtt gaa act caa aaa tta tcg ttt ggc gca aac ggt aat aaa gtc aac 480
Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn
145 150 155 160
atc aca agc gac acc aaa ggc ttg aat ttt gcc aaa gaa acg gct ggg 528
Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly
165 170 175
acg aac ggc gac ccc acg gtt cat ctg aac ggt atc ggt tcg act ttg 576
Thr Asn Gly Asp Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu
180 185 190
acc gat acg ctg ctg aat acc gga gcg acc aca aac gta acc aac gac 624
Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp
195 200 205
aac gtt acc gat gac gag aaa aaa cgt gcg gca agc gtt aaa gac gta 672
Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val
210 215 220
tta aac gca ggc tgg aac att aaa ggc gtt aaa ccc ggt aca aca gct 720
Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala
225 230 235 240
tcc gat aac gtt gat ttc gtc cgc act tac gac aca gtc gag ttc ttg 768
Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu
245 250 255
agc gca gat acg aaa aca acg act gtt aat gtg gaa agc aaa gac aac 816
Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn
260 265 270
ggc aag aaa acc gaa gtt aaa atc ggt gcg aag act tct gtt att aaa 864
Gly Lys Lys Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys
275 280 285
gaa aaa gac ggt aag ttg gtt act ggt aaa ggc aaa gac gag aat ggt 912
Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Asp Glu Asn Gly
290 295 300
tct tct aca gac gaa ggc gaa ggc tta gtg act gca aaa gaa gtg att 960
Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile
305 310 315 320
gat gca gta aac aag gct ggt tgg aga atg aaa aca aca acc gct aat 1008
Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn
325 330 335
ggt caa aca ggt caa gct gac aag ttt gaa acc gtt aca tca ggc aca 1056
Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr
340 345 350
aaa gta acc ttt gct agt ggt aat ggt aca act gcg act gta agt aaa 1104
Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys
355 360 365
gat gat caa ggc aac atc act gtt aag tat gat gta aat gtc ggc gat 1152
Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp
370 375 380
gcc cta aac gtc aat cag ctg caa aac agc ggt tgg aat ttg gat tcc 1200
Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser
385 390 395 400
aaa gcg gtt gca ggt tct tcg ggc aaa gtc atc agc ggc aat gtt tcg 1248
Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser
405 410 415
ccg agc aag gga aag atg gat gaa acc gtc aac att aat gcc ggc aac 1296
Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn
420 425 430
aac atc gag att acc cgc aac ggc aaa aat atc gac atc gcc act tcg 1344
Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser
435 440 445
atg acc ccg caa ttt tcc agc gtt tcg ctc ggc gcg ggg gcg gat gcg 1392
Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala
450 455 460
ccc act tta agc gtg gat gac gag ggc gcg ttg aat gtc ggc agc aag 1440
Pro Thr Leu Ser Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys
465 470 475 480
gat gcc aac aaa ccc gtc cgc att acc aat gtc gcc ccg ggc gtt aaa 1488
Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys
485 490 495
gag ggg gat gtt aca aac gtc gca caa ctt aaa ggt gtg gcg caa aac 1536
Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn
500 505 510
ttg aac aac cgc atc gac aat gtg gac ggc aac gcg cgc gcg ggt atc 1584
Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile
515 520 525
gcc caa gcg att gca acc gca ggt ttg gct cag gcg tat ttg ccc ggc 1632
Ala Gln Ala Ile Ala Thr Ala Gly Leu Ala Gln Ala Tyr Leu Pro Gly
530 535 540
aag agt atg atg gcg atc ggc ggc ggt act tat cgc ggc gaa gcc ggt 1680
Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly
545 550 555 560
tac gcc atc ggc tac tcg agc att tct gac act ggg aat tgg gtt atc 1728
Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Thr Gly Asn Trp Val Ile
565 570 575
aag ggc acg gct tcc ggc aat tcg cgc ggc cat ttc ggt gct tcc gca 1776
Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala
580 585 590
tct gtc ggt tat cag tgg taa 1797
Ser Val Gly Tyr Gln Trp
595
<210>13
<211>598
<212>PRT
<213>Neisseria meningitidis
<400>13
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
Ala Asn Ala Thr Asp Asp Asp Asp Leu Tyr Leu Glu Pro Val Gln Arg
50 55 60
Thr Ala Val Val Leu Ser Phe Arg Ser Asp Lys Glu Gly Thr Gly Glu
65 70 75 80
Lys Glu Gly Thr Glu Asp Ser Asn Trp Ala Val Tyr Phe Asp Glu Lys
85 90 95
Arg Val Leu Lys Ala Gly Ala Ile Thr Leu Lys Ala Gly Asp Asn Leu
100 105 110
Lys Ile Lys Gln Asn Thr Asn Glu Asn Thr Asn Glu Asn Thr Asn Asp
115 120 125
Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser
130 135 140
Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val Asn
145 150 155 160
Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly
165 170 175
Thr Asn Gly Asp Pro Thr Val His Leu Asn Gly Ile Gly Ser Thr Leu
180 185 190
Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn Asp
195 200 205
Asn Val Thr Asp Asp Glu Lys Lys Arg Ala Ala Ser Val Lys Asp Val
210 215 220
Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr Ala
225 230 235 240
Ser Asp Asn Val Asp Phe Val Arg Thr Tyr Asp Thr Val Glu Phe Leu
245 250 255
Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp Asn
260 265 270
Gly Lys Lys Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile Lys
275 280 285
Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Asp Glu Asn Gly
290 295 300
Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val Ile
305 310 315 320
Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala Asn
325 330 335
Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly Thr
340 345 350
Lys Val Thr Phe Ala Ser Gly Asn Gly Thr Thr Ala Thr Val Ser Lys
355 360 365
Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly Asp
370 375 380
Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser
385 390 395 400
Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val Ser
405 410 415
Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly Asn
420 425 430
Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser
435 440 445
Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp Ala
450 455 460
Pro Thr Leu Ser Val Asp Asp Glu Gly Ala Leu Asn Val Gly Ser Lys
465 470 475 480
Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val Lys
485 490 495
Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln Asn
500 505 510
Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly Ile
515 520 525
Ala Gln Ala Ile Ala Thr Ala Gly Leu Ala Gln Ala Tyr Leu Pro Gly
530 535 540
Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala Gly
545 550 555 560
Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Thr Gly Asn Trp Val Ile
565 570 575
Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser Ala
580 585 590
Ser Val Gly Tyr Gln Trp
595
<210>14
<211>1800
<212>DNA
<213>Neisseria meningitidis
<220>
<221>CDS
<222>(1)..(1800)
<400>14
atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat ccc tgg 48
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
gtc gcc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96
Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
acc gtg aag acc gcc gta ttg gcg acg ctg ttg ttt gca acg gtt cag 144
Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
gcg aat gct acc gat gaa gat gaa gaa gaa gag tta gaa ccc gta gta 192
Ala Asn Ala Thr Asp Glu Asp Glu Glu Glu Glu Leu Glu Pro Val Val
50 55 60
cgc tct gct ctg gtg ttg caa ttc atg atc gat aaa gaa ggc aat gga 240
Arg Ser Ala Leu Val Leu Gln Phe Met Ile Asp Lys Glu Gly Asn Gly
65 70 75 80
gaa aac gaa tct aca gga aat ata ggt tgg agt ata tat tac gac aat 288
Glu Asn Glu Ser Thr Gly Asn Ile Gly Trp Ser Ile Tyr Tyr Asp Asn
85 90 95
cac aac act cta cac ggc gca acc gtt acc ctc aaa gcc ggc gac aac 336
His Asn Thr Leu His Gly Ala Thr Val Thr Leu Lys Ala Gly Asp Asn
100 105 110
ctg aaa atc aaa caa aac acc aat aaa aac acc aat gaa aac acc aat 384
Leu Lys Ile Lys Gln Asn Thr Asn Lys Asn Thr Asn Glu Asn Thr Asn
115 120 125
gac agt agc ttc acc tac tcg ctg aaa aaa gac ctc aca gat ctg acc 432
Asp Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr
130 135 140
agt gtt gaa act gaa aaa tta tcg ttt ggc gca aac ggc aat aaa gtc 480
Ser Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val
145 150 155 160
aac atc aca agc gac acc aaa ggc ttg aat ttc gcg aaa gaa acg gct 528
Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala
165 170 175
ggg acg aac ggc gac acc acg gtt cat ctg aac ggt att ggt tcg act 576
Gly Thr Asn Gly Asp Thr Thr Val His Leu Asn Gly Ile Gly Ser Thr
180 185 190
ttg acc gat acg ctg ctg aat acc gga gcg acc aca aac gta acc aac 624
Leu Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn
195 200 205
gac aac gtt acc gat gac aag aaa aaa cgt gcg gca agc gtt aaa gac 672
Asp Asn Val Thr Asp Asp Lys Lys Lys Arg Ala Ala Ser Val Lys Asp
210 215 220
gta tta aac gca ggc tgg aac att aaa ggc gtt aaa ccc ggt aca aca 720
Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr
225 230 235 240
gct tcc gat aac gtt gat ttc gtc cac act tac gac aca gtc gag ttc 768
Ala Ser Asp Asn Val Asp Phe Val His Thr Tyr Asp Thr Val Glu Phe
245 250 255
ttg agc gca gat acg aaa aca acg act gtt aat gtg gaa agc aaa gac 816
Leu Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp
260 265 270
aac ggc aag aga acc gaa gtt aaa atc ggt gcg aag act tct gtt att 864
Asn Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile
275 280 285
aaa gaa aaa gac ggt aag ttg gtt act ggt aaa ggc aaa ggc gag aat 912
Lys Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Gly Glu Asn
290 295 300
ggt tct tct aca gac gaa ggc gaa ggc tta gtg act gca aaa gaa gtc 960
Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val
305 310 315 320
att gat gca gta aac aag gct ggt tgg aga atg aaa aca aca acc gct 1008
Ile Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala
325 330 335
aat ggt caa aca ggt caa gct gac aag ttt gaa acc gtt aca tca ggc 1056
Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly
340 345 350
aca aat gta acc ttt gct agt ggt aaa ggt aca act gcg act gta agt 1104
Thr Asn Val Thr Phe Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser
355 360 365
aaa gat gat caa ggc aac atc act gtt aag tat gat gta aat gtc ggc 1152
Lys Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly
370 375 380
gat gcc cta aac gtc aat cag ctg caa aac agc ggt tgg aat ttg gat 1200
Asp Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp
385 390 395 400
tcc aaa gcg gtt gca ggt tct tcg ggc aaa gtc atc agc ggc aat gtt 1248
Ser Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val
405 410 415
tcg ccg agc aag gga aag atg gat gaa acc gtc aac att aat gcc ggc 1296
Ser Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly
420 425 430
aac aac atc gag att acc cgc aac ggt aaa aat atc gac atc gcc act 1344
Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr
435 440 445
tcg atg acc ccg cag ttt tcc agc gtt tcg ctc ggc gcg ggg gcg gat 1392
Ser Mat Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp
450 455 460
gcg ccc act ttg agc gtg gat gac aag ggc gcg ttg aat gtc ggc agc 1440
Ala Pro Thr Leu Ser Val Asp Asp Lys Gly Ala Leu Asn Val Gly Ser
465 470 475 480
aag gat gcc aac aaa ccc gtc cgc att acc aat gtc gcc ccg ggc gtt 1488
Lys Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val
485 490 495
aaa gag ggg gat gtt aca aac gtc gca caa ctt aaa ggc gtg gcg caa 1536
Lys Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln
500 505 510
aac ttg aac aac cgc atc gac aat gtg gac ggc aac gcg cgt gcc ggc 1584
Asn Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly
515 520 525
atc gcc caa gcg att gca acc gca ggt ctg gtt cag gcg tat ctc ccc 1632
Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro
530 535 540
ggc aag agt atg atg gcg atc ggc ggc ggc act tat cgc ggc gaa gcc 1680
Gly Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala
545 550 555 560
ggt tac gcc atc ggc tac tcc agt att tcc gac ggc gga aat tgg att 1728
Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile
565 570 575
atc aaa ggc acg gct tcc ggc aat tcg cgc ggt cat ttc ggt gct tcc 1776
Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser
580 585 590
gca tct gtc ggt tat cag tgg taa 1800
Ala Ser Val Gly Tyr Gln Trp
595 600
<210>15
<211>599
<212>PRT
<213>Neisseria meningitidis
<400>15
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Aln Ser Ala
20 25 30
Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
Ala Asn Ala Thr Asp Glu Asp Glu Glu Glu Glu Leu Glu Pro Val Val
50 55 60
Arg Ser Ala Leu Val Leu Gln Phe Met Ile Asp Lys Glu Gly Asn Gly
65 70 75 80
Glu Asn Glu Ser Thr Gly Asn Ile Gly Trp Ser Ile Tyr Tyr Asp Asn
85 90 95
His Asn Thr Leu His Gly Ala Thr Val Thr Leu Lys Ala Gly Asp Asn
100 105 110
Leu Lys Ile Lys Gln Asn Thr Asn Lys Asn Thr Asn Glu Asn Thr Asn
115 120 125
Asp Ser Ser Phe Thr Tyr Ser Leu Lys Lys Asp Leu Thr Asp Leu Thr
130 135 140
Ser Val Glu Thr Glu Lys Leu Ser Phe Gly Ala Asn Gly Asn Lys Val
145 150 155 160
Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn Phe Ala Lys Glu Thr Ala
165 170 175
Gly Thr Asn Gly Asp Thr Thr Val His Leu Asn Gly Ile Gly Ser Thr
180 185 190
Leu Thr Asp Thr Leu Leu Asn Thr Gly Ala Thr Thr Asn Val Thr Asn
195 200 205
Asp Asn Val Thr Asp Asp Lys Lys Lys Arg Ala Ala Ser Val Lys Asp
210 215 220
Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys Pro Gly Thr Thr
225 230 235 240
Ala Ser Asp Asn Val Asp Phe Val His Thr Tyr Asp Thr Val Glu Phe
245 250 255
Leu Ser Ala Asp Thr Lys Thr Thr Thr Val Asn Val Glu Ser Lys Asp
260 265 270
Asn Gly Lys Arg Thr Glu Val Lys Ile Gly Ala Lys Thr Ser Val Ile
275 280 285
Lys Glu Lys Asp Gly Lys Leu Val Thr Gly Lys Gly Lys Gly Glu Asn
290 295 300
Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu Val Thr Ala Lys Glu Val
305 310 315 320
Ile Asp Ala Val Asn Lys Ala Gly Trp Arg Met Lys Thr Thr Thr Ala
325 330 335
Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe Glu Thr Val Thr Ser Gly
340 345 350
Thr Asn Val Thr Phe Ala Ser Gly Lys Gly Thr Thr Ala Thr Val Ser
355 360 365
Lys Asp Asp Gln Gly Asn Ile Thr Val Lys Tyr Asp Val Asn Val Gly
370 375 380
Asp Ala Leu Asn Val Asn Gln Leu Gln Asn Ser Gly Trp Asn Leu Asp
385 390 395 400
Ser Lys Ala Val Ala Gly Ser Ser Gly Lys Val Ile Ser Gly Asn Val
405 410 415
Ser Pro Ser Lys Gly Lys Met Asp Glu Thr Val Asn Ile Asn Ala Gly
420 425 430
Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys Asn Ile Asp Ile Ala Thr
435 440 445
Ser Met Thr Pro Gln Phe Ser Ser Val Ser Leu Gly Ala Gly Ala Asp
450 455 460
Ala Pro Thr Leu Ser Val Asp Asp Lys Gly Ala Leu Asn Val Gly Ser
465 470 475 480
Lys Asp Ala Asn Lys Pro Val Arg Ile Thr Asn Val Ala Pro Gly Val
485 490 495
Lys Glu Gly Asp Val Thr Asn Val Ala Gln Leu Lys Gly Val Ala Gln
500 505 510
Asn Leu Asn Asn Arg Ile Asp Asn Val Asp Gly Asn Ala Arg Ala Gly
515 520 525
Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu Val Gln Ala Tyr Leu Pro
530 535 540
Gly Lys Ser Met Met Ala Ile Gly Gly Gly Thr Tyr Arg Gly Glu Ala
545 550 555 560
Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser Asp Gly Gly Asn Trp Ile
565 570 575
Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg Gly His Phe Gly Ala Ser
580 585 590
Ala Ser Val Gly Tyr Gln Trp
595
<210>16
<211>1779
<212>DNA
<213>Neisseria meningitidis
<220>
<221>CDS
<222>(1)..(1779)
<400>16
atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
gtc gcc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96
Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
acc gtg aag acc gcc gta ttg gcg aca ctg ttg ttt gca acg gtt cag 144
Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
gcg aat gct acc gat gaa gat gaa gaa gaa gag tta gaa tcc gta caa 192
Ala Asn Ala Thr Asp Glu Asp Glu Glu Glu Glu Leu Glu Ser Val Gln
50 55 60
cgc tct gtc gta ggg agc att caa gcc agt atg gaa ggc agc gtc gaa 240
Arg Ser Val Val Gly Ser Ile Gln Ala Ser Met Glu Gly Ser Val Glu
65 70 75 80
ttg gaa acg ata tca tta tca atg act aac gac agc aag gaa ttt gta 288
Leu Glu Thr Ile Ser Leu Ser Met Thr Asn Asp Ser Lys Glu Phe Val
85 90 95
gac cca tac ata gta gtt acc ctc aaa gcc ggc gac aac ctg aaa atc 336
Asp Pro Tyr Ile Val Val Thr Leu Lys Ala Gly Asp Asn Leu Lys Ile
100 105 110
aaa caa aac acc aat gaa aac acc aat gcc agt agc ttc acc tac tcg 384
Lys Gln Asn Thr Asn Glu Asn Thr Asn Ala Ser Ser Phe Thr Tyr Ser
115 120 125
ctg aaa aaa gac ctc aca ggc ctg atc aat gtt gaa act gaa aaa tta 432
Leu Lys Lys Asp Leu Thr Gly Leu Ile Asn Val Glu Thr Glu Lys Leu
130 135 140
tcg ttt ggc gca aac ggc aag aaa gtc aac atc ata agc gac acc aaa 480
Ser Phe Gly Ala Asn Gly Lys Lys Val Asn Ile Ile Ser Asp Thr Lys
145 150 155 160
ggc ttg aat ttc gcg aaa gaa acg gct ggg acg aac ggc gac acc acg 528
Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr
165 170 175
gtt cat ctg aac ggt atc ggt tcg act ttg acc gat atg ctg ctg aat 576
Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Met Leu Leu Asn
180 185 190
acc gga gcg acc aca aac gta acc aac gac aac gtt acc gat gac gag 624
Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu
195 200 205
aaa aaa cgt gcg gca agc gtt aaa gac gta tta aac gca ggc tgg aac 672
Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn
210 215 220
att aaa ggc gtt aaa ccc ggt aca aca gct tcc gat aac gtt gat ttc 720
Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe
225 230 235 240
gtc cgc act tac gac aca gtc gag ttc ttg agc gca gat acg aaa aca 768
Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr
245 250 255
acg act gtt aat gtg gaa agc aaa gac aac ggc aag aaa acc gaa gtt 816
Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val
260 265 270
aaa atc ggt gcg aag act tct gtt att aaa gaa aaa gac ggt aag ttc 864
Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu
275 280 285
gtt act ggt aaa ggc aaa ggc gag aat ggt tct tct aca gac gaa ggc 912
Val Thr Gly Lys Gly Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly
290 295 300
gaa ggc tta gtg act gca aaa gaa gtg att gat gca gta aac aag gct 960
Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala
305 310 315 320
ggt tgg aga atg aaa aca aca acc gct aat ggt caa aca ggt caa gct 1008
Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala
325 330 335
gac aag ttt gaa acc gtt aca tca ggc aca aaa gta acc ttt gct agt 1056
Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Lys Val Thr Phe Ala Ser
340 345 350
ggt aat ggt aca act gcg act gta agt aaa gat gat caa ggc aac atc 1104
Gly Asn Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile
355 360 365
act gtt aag tat gat gta aat gtc ggc gat gcc cta aac gtc aat cag 1152
Thr Val Lys Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln
370 375 380
ctg caa aac agc ggt tgg aat ttg gat tcc aaa gcg gtt gca ggt tct 1200
Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser
385 390 395 400
tcg ggc aaa gtc atc agc ggc aat gtt tcg ccg agc aag gga aac atc 1248
Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met
405 410 415
gat gaa acc gtc aac att aat gcc ggc aac aac atc gag att acc cgc 1296
Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg
420 425 430
aac ggc aaa aat atc gac atc gcc act tcg atg acc ccg caa ttt tcc 1344
Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser
435 440 445
agc gtt tcg ctc ggc gcg ggg gcg gat gcg ccc act tta agc gtg gat 1392
Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp
450 455 460
gac gag ggc gcg ttg aat gtc ggc agc aag gat gcc aac aaa ccc gtc 1440
Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys Pro Val
465 470 475 480
cgc att acc aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt aca aac 1488
Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn
485 490 495
gtc gcg caa ctt aaa ggt gtg gcg caa aac ttg aac aac cgc atc gac 1536
Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp
500 505 510
aat gtg aac ggc aac gcg cgt gcg ggc atc gcc caa gcg att gca acc 1584
Asn Val Asn Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr
515 520 525
gca ggt ctg gtt cag gcg tat ctg ccc ggc aag agt atg atg gcg atc 1632
Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile
530 535 540
ggc ggc ggc act tat ctc ggc gaa gcc ggt tat gcc atc ggc tac tca 1680
Gly Gly Gly Thr Tyr Leu Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser
545 550 555 560
agc att tcc gcc ggc gga aat tgg att atc aaa ggc acg gct tcc ggc 1728
Ser Ile Ser Ala Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly
565 570 575
aat tcg cgc ggc cat ttc ggt gct tcc gca tct gtc ggt tat cag tcc 1776
Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp
580 585 590
taa 1779
<210>17
<211>592
<212>PRT
<213>Neisseria meningitidis
<400>17
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
Val Ala Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
Ala Asn Ala Thr Asp Glu Asp Glu Glu Glu Glu Leu Glu Ser Val Gln
50 55 60
Arg Ser Val Val Gly Ser Ile Gln Ala Ser Met Glu Gly Ser Val Gln
65 70 75 80
Leu Glu Thr Ile Ser Leu Ser Met Thr Asn Asp Ser Lys Glu Phe Val
85 90 95
Asp Pro Tyr Ile Val Val Thr Leu Lys Ala Gly Asp Asn Leu Lys Ile
100 105 110
Lys Gln Asn Thr Asn Glu Asn Thr Asn Ala Ser Ser Phe Thr Tyr Ser
115 120 125
Leu Lys Lys Asp Leu Thr Gly Leu Ile Asn Val Glu Thr Glu Lys Leu
130 135 140
Ser Phe Gly Ala Asn Gly Lys Lys Val Asn Ile Ile Ser Asp Thr Lys
145 150 155 160
Gly Leu Asn Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Thr Thr
165 170 175
Val His Leu Asn Gly Ile Gly Ser Thr Leu Thr Asp Met Leu Leu Asn
180 185 190
Thr Gly Ala Thr Thr Asn Val Thr Asn Asp Asn Val Thr Asp Asp Glu
195 200 205
Lys Lys Arg Ala Ala Ser Val Lys Asp Val Leu Asn Ala Gly Trp Asn
210 215 220
Ile Lys Gly Val Lys Pro Gly Thr Thr Ala Ser Asp Asn Val Asp Phe
225 230 235 240
Val Arg Thr Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr
245 250 255
Thr Thr Val Asn Val Glu Ser Lys Asp Asn Gly Lys Lys Thr Glu Val
260 265 270
Lys Ile Gly Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu
275 280 285
Val Thr Gly Lys Gly Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly
290 295 300
Glu Gly Leu Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala
305 310 315 320
Gly Trp Arg Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala
325 330 335
Asp Lys Phe Glu Thr Val Thr Ser Gly Thr Lys Val Thr Phe Ala Ser
340 345 350
Gly Asn Gly Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile
355 360 365
Thr Val Lys Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln
370 375 380
Leu Gln Asn Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser
385 390 395 400
Ser Gly Lys Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met
405 410 415
Asp Glu Thr Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg
420 425 430
Asn Gly Lys Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser
435 440 445
Ser Val Ser Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp
450 455 460
Asp Glu Gly Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys Pro Val
465 470 475 480
Arg Ile Thr Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn
485 490 495
Val Ala Gln Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp
500 505 510
Asn Val Asn Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr
515 520 525
Ala Gly Leu Val Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile
530 535 540
Gly Gly Gly Thr Tyr Leu Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser
545 550 555 560
Ser Ile Ser Ala Gly Gly Asn Trp Ile Ile Lys Gly Thr Ala Ser Gly
565 570 575
Asn Ser Arg Gly His Phe Gly Ala Ser Ala Ser Val Gly Tyr Gln Trp
580 585 590
<210>18
<211>1770
<212>DNA
<213>Neisseria meningitidis
<220>
<221>CDS
<222>(1)..(1770)
<400>18
atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gcc tgg 48
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
gta gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
acc gtg gcg acc gcc gta ttg gcg aca ctg ctg tcc gca acg gtt cag 144
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Ser Ala Thr Val Gln
35 40 45
gcg aat gct acc gat acc gat gaa gat gaa gag tta gaa tcc gta gca 192
Ala Asn Ala Thr Asp Thr Asp Glu Asp Glu Glu Leu Glu Ser Val Ala
50 55 60
cgc tct gct ctg gtg ttg caa ttc atg atc gat aaa gaa ggc aat gga 240
Arg Ser Ala Leu Val Leu Gln Phe Met Ile Asp Lys Glu Gly Asn Gly
65 70 75 80
gaa atc gaa tct aca gga gat ata ggt tgg agt ata tat tac gac gat 288
Glu Ile Glu Ser Thr Gly Asp Ile Gly Trp Ser Ile Tyr Tyr Asp Asp
85 90 95
cac aac act cta cac ggc gca acc gtt acc ctc aaa gcc ggc gac aac 336
His Asn Thr Leu His Gly Ala Thr Val Thr Leu Lys Ala Gly Asp Asn
100 105 110
ctg aaa atc aaa caa agc ggc aaa gac ttc acc tac tcg ctg aaa aaa 384
Leu Lys Ile Lys Gln Ser Gly Lys Asp Phe Thr Tyr Ser Leu Lys Lys
115 120 125
gag ctg aaa gac ctg acc agt gtt gaa act gaa aaa tta tcg ttt ggc 432
Glu Leu Lys Asp Leu Thr Ser Val Glu Thr Glu Lys Leu Ser Phe Gly
130 135 140
gca aac ggt aat aaa gtc aac atc aca agc gac acc aaa ggc ttg aat 480
Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn
145 150 155 160
ttt gcg aaa gaa acg gct ggg acg aac ggc gac ccc acg gtt cat ctg 528
Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Pro Thr Val His Leu
165 170 175
aac ggt atc ggt tcg act ttg acc gat acg ctt gcg ggt tct tct gct 576
Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Ala Gly Ser Ser Ala
180 185 190
tct cac gtt gat gcg ggt aac caa agt aca cat tac act cgt gca gca 624
Ser His Val Asp Ala Gly Asn Gln Ser Thr His Tyr Thr Arg Ala Ala
195 200 205
agt att aag gat gtg ttg aat gcg ggt tgg aat att aag ggt gtt aaa 672
Ser Ile Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys
210 215 220
act ggc tca aca act ggt caa tca gaa aat gtc gat ttc gtc cgc act 720
Thr Gly Ser Thr Thr Gly Gln Ser Glu Asn Val Asp Phe Val Arg Thr
225 230 235 240
tac gac aca gtc gag ttc ttg agc gca gat acg aaa aca acg act gtt 768
Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr Thr Thr Val
245 250 255
aat gtg gaa agc aaa gac aac ggc aag aga acc gaa gtt aaa atc ggt 816
Asn Val Glu Ser Lys Asp Asn Gly Lys Arg Thr Glu Val Lys Ile Gly
260 265 270
gcg aag act tct gtt att aaa gaa aaa gac ggt aag ttg gtt act ggt 864
Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Val Thr Gly
275 280 285
aaa ggc aaa ggc gag aat ggt tct tct aca gac gaa ggc gaa ggc tta 912
Lys Gly Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu
290 295 300
gtg act gca aaa gaa gtg att gat gca gta aac aag gct ggt tgg aga 960
Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala Gly Trp Arg
305 310 315 320
atg aaa aca aca acc gct aat ggt caa aca ggt caa gct gac aag ttt 1008
Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe
325 330 335
gaa acc gtt aca tca ggc aca aaa gta acc ttt gct agt ggt aat ggt 1056
Glu Thr Val Thr Ser Gly Thr Lys Val Thr Phe Ala Ser Gly Asn Gly
340 345 350
aca act gcg act gta agt aaa gat gat caa ggc aac atc act gtt aag 1104
Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile Thr Val Lys
355 360 365
tat gat gta aat gtc ggc gat gcc cta aac gtc aat cag ctg caa aac 1152
Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln Leu Gln Asn
370 375 380
agc ggt tgg aat ttg gat tcc aaa gcg gtt gca ggt tct tcg ggc aaa 1200
Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser Ser Gly Lys
385 390 395 400
gtc atc agc ggc aat gtt tcg ccg agc aag gga aag atg gat gaa acc 1248
Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met Asp Glu Thr
405 410 415
gtc aac att aat gcc ggc aac aac atc gag att acc cgc aac ggc aaa 1296
Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys
420 425 430
aat atc gac atc gcc act tcg atg acc ccg caa ttt tcc agc gtt tcg 1344
Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser Ser Val Ser
435 440 445
ctc ggc gcg ggg gcg gat gcg ccc act tta agc gtg gat gac gag ggc 1392
Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp Asp Glu Gly
450 455 460
gcg ttg aat gtc ggc agc aag gat gcc aac aaa ccc gtc cgc att acc 1440
Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys Pro Val Arg Ile Thr
465 470 475 480
aat gtc gcc ccg ggc gtt aaa gag ggg gat gtt aca aac gtc gca caa 1488
Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val Ala Gln
485 490 495
ctt aaa ggt gtg gcg caa aac ttg aac aac cgc atc gac aat gtg aac 1536
Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn Val Asn
500 505 510
ggc aac gcg cgc gcg ggt atc gcc caa gcg att gca acc gca ggt ttg 1584
Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu
515 520 525
gct cag gcc tat ttg ccc ggc aag agt atg atg gcg atc ggc ggc ggt 1632
Ala Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly Gly Gly
530 535 540
act tat ctc ggc gaa gcc ggt tac gcc atc ggc tac tcg agc att tct 1680
Thr Tyr Leu Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser
545 550 555 560
gac act ggg aat tgg gtt atc aag ggc acg gct tcc ggc aat tcg cgc 1728
Asp Thr Gly Asn Trp Val Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg
565 570 575
ggt cat ttc ggt act tcc gca tct gtc ggt tat cag tgg taa 1770
Gly His Phe Gly Thr Ser Ala Ser Val Gly Tyr Gln Trp
580 585 590
<210>19
<211>589
<212>PRT
<213>Neisseria meningitidis
<400>19
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Ash Ala Trp
1 5 10 15
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
Thr Val Ala Thr Ala Val Leu Ala Thr Leu Leu Ser Ala Thr Val Gln
35 40 45
Ala Asn Ala Thr Asp Thr Asp Glu Asp Glu Glu Leu Glu Ser Val Ala
50 55 60
Arg Ser Ala Leu Val Leu Gln Phe Met Ile Asp Lys Glu Gly Asn Gly
65 70 75 80
Glu Ile Glu Ser Thr Gly Asp Ile Gly Trp Ser Ile Tyr Tyr Asp Asp
85 90 95
His Asn Thr Leu His Gly Ala Thr Val Thr Leu Lys Ala Gly Asp Asn
100 105 110
Leu Lys Ile Lys Gln Ser Gly Lys Asp Phe Thr Tyr Ser Leu Lys Lys
115 120 125
Glu Leu Lys Asp Leu Thr Ser Val Glu Thr Glu Lys Leu Ser Phe Gly
130 135 140
Ala Asn Gly Asn Lys Val Asn Ile Thr Ser Asp Thr Lys Gly Leu Asn
145 150 155 160
Phe Ala Lys Glu Thr Ala Gly Thr Asn Gly Asp Pro Thr Val His Leu
165 170 175
Asn Gly Ile Gly Ser Thr Leu Thr Asp Thr Leu Ala Gly Ser Ser Ala
180 185 190
Ser His Val Asp Ala Gly Asn Gln Ser Thr His Tyr Thr Arg Ala Ala
195 200 205
Ser Ile Lys Asp Val Leu Asn Ala Gly Trp Asn Ile Lys Gly Val Lys
210 215 220
Thr Gly Ser Thr Thr Gly Gln Ser Glu Asn Val Asp Phe Val Arg Thr
225 230 235 240
Tyr Asp Thr Val Glu Phe Leu Ser Ala Asp Thr Lys Thr Thr Thr Val
245 250 255
Asn Val Glu Ser Lys Asp Asn Gly Lys Arg Thr Glu Val Lys Ile Gly
260 265 270
Ala Lys Thr Ser Val Ile Lys Glu Lys Asp Gly Lys Leu Val Thr Gly
275 280 285
Lys Gly Lys Gly Glu Asn Gly Ser Ser Thr Asp Glu Gly Glu Gly Leu
290 295 300
Val Thr Ala Lys Glu Val Ile Asp Ala Val Asn Lys Ala Gly Trp Arg
305 310 315 320
Met Lys Thr Thr Thr Ala Asn Gly Gln Thr Gly Gln Ala Asp Lys Phe
325 330 335
Glu Thr Val Thr Ser Gly Thr Lys Val Thr Phe Ala Ser Gly Asn Gly
340 345 350
Thr Thr Ala Thr Val Ser Lys Asp Asp Gln Gly Asn Ile Thr Val Lys
355 360 365
Tyr Asp Val Asn Val Gly Asp Ala Leu Asn Val Asn Gln Leu Gln Asn
370 375 380
Ser Gly Trp Asn Leu Asp Ser Lys Ala Val Ala Gly Ser Ser Gly Lys
385 390 395 400
Val Ile Ser Gly Asn Val Ser Pro Ser Lys Gly Lys Met Asp Glu Thr
405 410 415
Val Asn Ile Asn Ala Gly Asn Asn Ile Glu Ile Thr Arg Asn Gly Lys
420 425 430
Asn Ile Asp Ile Ala Thr Ser Met Thr Pro Gln Phe Ser Ser Val Ser
435 440 445
Leu Gly Ala Gly Ala Asp Ala Pro Thr Leu Ser Val Asp Asp Glu Gly
450 455 460
Ala Leu Asn Val Gly Ser Lys Asp Ala Asn Lys Pro Val Arg Ile Thr
465 470 475 480
Asn Val Ala Pro Gly Val Lys Glu Gly Asp Val Thr Asn Val Ala Gln
485 490 495
Leu Lys Gly Val Ala Gln Asn Leu Asn Asn Arg Ile Asp Asn Val Asn
500 505 510
Gly Asn Ala Arg Ala Gly Ile Ala Gln Ala Ile Ala Thr Ala Gly Leu
515 520 525
Ala Gln Ala Tyr Leu Pro Gly Lys Ser Met Met Ala Ile Gly Gly Gly
530 535 540
Thr Tyr Leu Gly Glu Ala Gly Tyr Ala Ile Gly Tyr Ser Ser Ile Ser
545 550 555 560
Asp Thr Gly Asn Trp Val Ile Lys Gly Thr Ala Ser Gly Asn Ser Arg
565 570 575
Gly His Phe Gly Thr Ser Ala Ser Val Gly Tyr Gln Trp
580 585
<210>20
<211>1776
<212>DNA
<213>Neisseria meningitidis
<220>
<221>CDS
<222>(1)..(1776)
<400>20
atg aac aaa ata tac cgc atc att tgg aat agt gcc ctc aat gca tgg 48
Met Asn Lys Ile Tyr Arg Ile Ile Trp Asn Ser Ala Leu Asn Ala Trp
1 5 10 15
gtc gtc gta tcc gag ctc aca cgc aac cac acc aaa cgc gcc tcc gca 96
Val Val Val Ser Glu Leu Thr Arg Asn His Thr Lys Arg Ala Ser Ala
20 25 30
acc gtg aag acc gcc gta ttg gcg act ctg ttg ttt gca acg gtt cag 144
Thr Val Lys Thr Ala Val Leu Ala Thr Leu Leu Phe Ala Thr Val Gln
35 40 45
gca agt gct aac aat gaa gag caa gaa gaa gat tta tat tta gac ccc 192
Ala Ser Ala Asn Asn Glu Glu Gln Glu Glu Asp Leu Tyr Leu Asp Pro
50 55 60
gta caa cgc act gtt gcc gtg ttg ata gtc aat tcc gat aaa gaa ggc 240
Val Gln Arg Thr Val Ala Val Leu Ile Val Asn Ser Asp Lys Glu Gly
65 70 75 80
acg gga gaa aaa gaa aaa gta gaa gaa aat tca gat tgg gca gta tat 288
Thr Gly Glu Lys Glu Lys Val Glu Glu Asn Ser Asp Trp Ala Val Tyr
85 90 95
ttc aac gag aaa gga gta cta aca gcc aga gaa atc acc ctc aaa gcc 336
Phe Asn Glu Lys Gly Val Leu Thr Ala Arg Glu Ile Thr Leu Lys Ala
100 105 110
ggc gac aac ctg aaa atc aaa caa aac ggc aca aac ttc acc tac tcg 384
Gly Asp Asn Leu Lys I1e Lys G1n Asn Gly Thr Asn Phe Thr Tyr Ser
115 120 125
ctg aaa aaa gac ctc aca gat ctg acc agt gtt gga act gaa aaa tta 432
Leu Lys Lys Asp Leu Thr Asp Leu Thr Ser Val Gly Thr Glu Lys Leu
130 135 140
tcg ttt agc gca aac ggc aat aaa gtc aac atc aca agc gac acc aaa 480
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580 585 590
<210>21
<211>591
<212>PRT
<213>Neisseria meningitidis
<400>21
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<210>22
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<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:
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<210>23
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<212>DNA
<213>人工序列
<220>
<223>人工序列的描述:
用于PCR的3′寡核苷酸引物
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<223>人工序列的描述:
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Claims (34)
1、一种分离的多肽或其片段,或它们的变体或衍生物,所述多肽选自:
(a)如SEQ ID NO:2的多肽;
(b)如SEQ ID NO:5的多肽;
(c)如SEQ ID NO:7的多肽;
(d)如SEQ ID NO:9的多肽;
(e)如SEQ ID NO:11的多肽;
(f)如SEQ ID NO:13的多肽;
(g)如SEQ ID NO:15的多肽;
(h)如SEQ ID NO:17的多肽;
(i)如SEQ ID NO:19的多肽;和
(j)如SEQ ID NO:21的多肽。
2、如权利要求1的多肽,片段,变体或衍生物,具有抗以下一或多个成员的免疫学活性:
(i)脑膜炎奈瑟氏球菌;
(ii)所述多肽;
(iii)所述片段;
(iv)所述变体;及
(v)所述衍生物。
3、如权利要求1的多肽、片段、变体或衍生物,呈现抗脑膜炎奈瑟氏球菌的免疫学活性。
4、一种编码一种多肽或其片段,或它们的变体或衍生物的分离的核酸序列,所述多肽选自:
(a)如SEQ ID NO:2的多肽;
(b)如SEQ ID NO:5的多肽;
(c)如SEQ ID NO:7的多肽;
(d)如SEQ ID NO:9的多肽;
(e)如SEQ ID NO:11的多肽;
(f)如SEQ ID NO:13的多肽;
(g)如SEQ ID NO:15的多肽;
(h)如SEQ ID NO:17的多肽;
(i)如SEQ ID NO:19的多肽;和
(j)如SEQ ID NO:21的多肽。
5、如权利要求4的分离的核酸序列,编码呈现抗选自以下一或多个成员的免疫学活性的产物:
(i)脑膜炎奈瑟氏球菌;
(ii)所述多肽;
(iii)所述片段;
(iv)所述变体;及
(v)所述衍生物。
6、如权利要求4的分离的核酸序列,编码呈抗脑膜炎奈瑟氏球菌免疫学活性的产物。
7、一种分离的核酸序列,选自:
(1)如SEQ ID NO:1的核苷酸序列;
(2)如SEQ ID NO:3的核苷酸序列;
(3)如SEQ ID NO:4的核苷酸序列;
(4)如SEQ ID NO:6的核苷酸序列;
(5)如SEQ ID NO:8的核苷酸序列;
(6)如SEQ ID NO:10的核苷酸序列;
(7)如SEQ ID NO:12的核苷酸序列;
(8)如SEQ ID NO:14的核苷酸序列;
(9)如SEQ ID NO:16的核苷酸序列;
(10)如SEQ ID NO:18的核苷酸序列;
(11)如SEQ ID NO:20的核苷酸序列;
(12)SEQ ID NOS:1,3,4,6,8,10,12,14,16,18及20任一序列的核苷酸序列片段;及
(13)前述任一序列的核苷酸序列同系物。
8、如权利要求7的核酸序列,编码呈抗选自以下一或多个成员的免疫学活性的产物:
(i)脑膜炎奈瑟氏球菌;
(ii)所述多肽;
(iii)所述片段;
(iv)所述变体;及
(v)所述衍生物。
9、如权利要求7的核酸序列,编码呈抗脑膜炎奈瑟氏球菌免疫学活性的产物。
10、如权利要求7的核酸序列,其中所述同系物得自奈瑟氏球菌属。
11、如权利要求5或7的核酸序列,其中所述同系物得自脑膜炎奈瑟氏球菌的一个菌株。
12、一种获得核苷酸序列同系物的方法,包括以下步骤:
(i)从适当宿主获得核酸提取物;
(ii)产生任选地简并的引物,其中每个引物均包含如权利要求5或7的核酸序列的一部分;和
(iii)用所述引物通过核酸扩增技术从所述核酸提取物中扩增一或多个扩增产物。
13、如权利要求12的方法,其中所述核酸提取物得自奈瑟氏球菌属。
14、如权利要求12的方法,其中所述核酸提取物得自脑膜炎奈瑟氏球菌的一个菌株。
15、如权利要求12的方法,其中所述引物选自SEQ ID NOS:22,23,24,25,26,27,28,29,30及31。
16、如权利要求12的方法,其中核酸扩增技术是PCR。
17、一种含有如权利要求4或7的核酸序列的表达载体,其中所述序列可操纵地与转录或翻译调节核酸连接。
18、一种用含如权利要求4或7的核酸序列的表达载体转染或转化的宿主细胞,其中所述序列可操纵地与转录或翻译调节核酸连接。
19、一种生产重组多肽的方法,包括以下步骤:
(A)培养如权利要求18的宿主细胞,从而从所述核酸表达所述重组多肽;和
(B)分离所述重组多肽。
20、一种结合以下一或多个成员的抗体或抗体片段:
(1)脑膜炎奈瑟氏球菌;
(2)如权利要求1的多肽;
(3)所述多肽的片段;
(4)所述多肽或所述片段的变体;和
(5)所述多肽或所述片段的衍生物。
21、如权利要求20的抗体,其中所述的抗体或抗体片段结合脑膜炎奈瑟氏球菌。
22、一种检测怀疑含有脑膜炎奈瑟氏球菌的生物学样品中脑膜炎奈瑟氏球菌的方法,所述方法包括以下步骤:
(A)从患者分离生物学样品;
(B)将权利要求20或21的抗体或抗体片段与生物学样品混合,形成混合物;
(C)检测混合物中的特异结合的抗体或结合的片段,其表明脑膜炎奈瑟氏球菌的存在。
23、一种检测怀疑含有脑膜炎奈瑟氏球菌的生物学样品中脑膜炎奈瑟氏球菌的方法,所述方法包括以下步骤:
(I)从患者分离生物学样品;
(II)检测样品中的核酸序列,其表明脑膜炎奈瑟氏球菌的存在。
24、一种诊断患者感染脑膜炎奈瑟氏球菌的方法,包括:
(1)将取自患者的生物样品与权利要求1的多肽、片段、变体或衍生物接触;
(2)确定所述样品中,所述多肽、片段、变体或衍生物与脑膜炎奈瑟氏球菌特异抗体间复合物的存在与否,存在所述复合物代表有所述感染。
25、如权利要求1的多肽、片段、变体或衍生物在制备用于检测或诊断人体内的脑膜炎奈瑟氏球菌感染的试剂盒中的应用。
26、如权利要求4或7的核酸序列在制备用于检测或诊断人体内的脑膜炎奈瑟氏球菌感染的试剂盒中的应用。
27、选自SEQ ID NOS 22,23,24,25,26,27,28,29,30及31的一或多个寡核苷酸引物,和任选地热稳定聚合酶在用于检测或诊断人体内的脑膜炎奈瑟氏球菌感染的试剂盒中的应用。
28、权利要求20或21的抗体或抗体片段在制备应用检测或诊断人体内的脑膜炎奈瑟氏球菌感染的试剂盒中的应用。
29、药物学有效量的如权利要求1的多肽、片段、变体或衍生物在预防或治疗人体内脑膜炎奈瑟氏球菌感染中的应用。
30、药物学有效量的如权利要求20或21的抗体或抗体片段在预防或治疗人体内脑膜炎奈瑟氏球菌感染中的应用。
31、一种药物组合物,含有如权利要求1的分离的多肽或其片段,或它们的变体或衍生物。
32、权利要求31的药物,其是疫苗。
33、一种预防或治疗被脑膜炎奈瑟氏球菌感染的患者的方法,包括为其施用药物学有效量的如权利要求32的疫苗。
34、一种鉴别权利要求1的多肽,变体或衍生物的免疫反应性片段的方法,包括以下步骤:
(a)产生所述多肽,变体或衍生物的片段;
(b)将所述片段给予哺乳动物;和
(c)检测所述哺乳动物中的免疫应答,所述应答包括特异结合脑膜炎奈瑟氏球菌和/或所述多肽,变体或衍生物的因子产生,和/或抗脑膜炎奈瑟氏球菌感染的防护效应的产生。
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GBGB9726398.2A GB9726398D0 (en) | 1997-12-12 | 1997-12-12 | Polypeptide and coding sequences |
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CN98813558A Pending CN1284965A (zh) | 1997-12-12 | 1998-12-14 | 脑膜炎奈瑟氏球菌的新表面蛋白 |
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CN (2) | CN101684148A (zh) |
AU (1) | AU747742B2 (zh) |
BR (1) | BR9814276A (zh) |
CA (1) | CA2314319C (zh) |
CZ (1) | CZ299646B6 (zh) |
GB (1) | GB9726398D0 (zh) |
HU (1) | HUP0100094A3 (zh) |
IL (2) | IL136684A0 (zh) |
NO (1) | NO20002990L (zh) |
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GB9726398D0 (en) | 1997-12-12 | 1998-02-11 | Isis Innovation | Polypeptide and coding sequences |
CA2317815A1 (en) * | 1998-01-14 | 1999-07-22 | Chiron S.P.A. | Neisseria meningitidis antigens |
FR2776928B1 (fr) * | 1998-04-03 | 2000-06-23 | Merial Sas | Vaccins adn adjuves |
ES2304065T3 (es) | 1998-05-01 | 2008-09-01 | Novartis Vaccines And Diagnostics, Inc. | Antigenos y composiciones de neisseria meningitidis. |
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