CN107266582A - 牙龈卟啉单胞菌感染的预防、治疗和诊断 - Google Patents
牙龈卟啉单胞菌感染的预防、治疗和诊断 Download PDFInfo
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12Y—ENZYMES
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Abstract
本发明涉及用于预防和治疗牙龈卟啉单胞菌相关状态和疾病的细胞应答和体液应答的产生和用途。
Description
发明领域
本发明涉及肽和嵌合或融合蛋白并涉及这些蛋白在引起细胞应答和体液应答从而预防和治疗牙龈卟啉单胞菌(P.gingivalis)相关状态和疾病中的用途。
发明背景
慢性牙周炎是牙支持组织炎性疾病,其导致牙槽骨吸收并最终导致牙齿脱落。在所有的社群中,该疾病都是主要的公共健康问题,并且据估计高达15%的成年人群患病,其中5-6%为重症。
慢性牙周炎的发展和进行与龈下菌斑中特定的革兰氏阴性细菌有关。龈下菌斑中存在的牙龈卟啉单胞菌(Porphyromonas gingivalis)与该疾病是显著相关的。
据报道,治疗(刮牙和根面平整术)后牙周炎患者龈下菌斑中牙龈卟啉单胞菌依然存在,这与进行性牙槽骨丧失显著相关。此外,龈下菌斑中牙龈卟啉单胞菌细胞数的增加已证实与通过附着丧失、牙周袋深度和探针出血所检测的疾病严重程度相关。
牙龈卟啉单胞菌的口腔感染已证实在小鼠、大鼠和非人的灵长类动物中诱导牙周骨丧失。此外,牙周疾病和牙龈卟啉单胞菌感染与心血管疾病和某些癌症存在正向的关联。
据报道,多种毒力因子与牙龈卟啉单胞菌的致病性有关,包括:LPS、菌毛、血凝素、溶血素和胞外水解酶(尤其是Arg-X和Lys-X特异性蛋白酶),也被称为“牙龈卟啉单胞菌胰酶样酶”。
公共健康问题的重要性在于需要对牙龈卟啉单胞菌感染提供强保护性应答的抗血清,尤其是特异性抗体,以及提供该抗血清的方法。
现有的一个问题是在需要从大量的毒力因子中进行选择的情况下,不清楚如何获得对于牙龈卟啉单胞菌感染的强保护性应答。
对毒力因子中表位的相对免疫原性以及给定因子上表位的相对免疫原性也不是很了解,特别是在不清楚是否仍需鉴定其他表位的情况下。
一个具体的问题是:很多毒力因子由多个结构域构成并且难以表达以提供与牙龈卟啉单胞菌上发现的构象接近的构象。此外,当这些结构域表达为分离的单元,即与其他毒力因子结构域分离时,它们倾向于折叠成不同于牙龈卟啉单胞菌上所发现的的构象的构象。
此外,在修饰毒力因子的免疫原性的多种不同选择中,不清楚哪一种最可能提供保护性免疫应答。
在进行本发明的工作中,本发明人已经鉴定出具有与构成牙龈卟啉单胞菌胰酶样酶的区的氨基酸序列相同或享有同源性的氨基酸序列的肽,所述区在所述酶中界定了用于在含有Lys或Arg的肽中切割位于Lys或Arg的C端的肽键的位点,并且本发明人将该肽并入嵌合或融合蛋白,当将所述嵌合或融合蛋白用作疫苗时,其比从天然牙龈卟啉单胞菌胰酶样酶形成的纯化的蛋白酶-粘附素复合物或杀死的全细胞提供更好的针对牙周组织破坏的保护。
发明概述
一方面,本发明提供了用于诱导对于牙龈卟啉单胞菌的免疫应答的嵌合或融合蛋白,所述蛋白包含第一肽和第二肽,所述第一肽与第二肽直接连接或通过连接子连接,其中:
(A)所述第一肽包含:
(i)与SEQ ID No:1所示的序列相同或同源的序列的一部分或全部;或
(ii)与SEQ ID No:2所示的序列相同或同源的序列的一部分或全部;以及
(B)所述第二肽包含:
(i)与牙龈卟啉单胞菌Lys-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(ii)与牙龈卟啉单胞菌Arg-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(iii)与牙龈卟啉单胞菌HagA粘附素结构域的序列相同或同源的序列的一部分或全部。
另一方面,本发明提供了用于诱导对于牙龈卟啉单胞菌的免疫应答的嵌合或融合蛋白,所述蛋白包含肽和多肽,所述肽与所述多肽直接连接或通过连接子连接,其中:
(A)所述肽包含:
(i)与SEQ ID No:1所示的序列相同或同源的序列的一部分或全部;或
(ii)与SEQ ID No:2所示的序列相同或同源的序列的一部分或全部;以及
(B)所述多肽包含:
(i)与牙龈卟啉单胞菌Lys-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(ii)与牙龈卟啉单胞菌Arg-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(iii)与牙龈卟啉单胞菌HagA粘附素结构域的序列相同或同源的序列的一部分或全部。
另一方面,本发明提供了用于诱导对于牙龈卟啉单胞菌的免疫应答的肽,所述肽具有下述序列:
(i)与SEQ ID No:64-66中的一项所示的序列相同或同源的序列;以及
(ii)与SEQ ID No:67或68所示的序列相同或同源的序列。
一方面,可以以嵌合或融合蛋白的形式提供具有与SEQ ID No:64-68中的一项所示的序列相同或同源的序列的肽,在所述嵌合或融合蛋白中,所述肽与第二肽直接连接或通过连接子连接,其中所述第二肽包含:
(i)与牙龈卟啉单胞菌Lys-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(ii)与牙龈卟啉单胞菌Arg-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(iii)与牙龈卟啉单胞菌HagA粘附素结构域的序列相同或同源的序列的一部分或全部。
另一方面,本发明提供组合物,诸如抗原组合物,尤其是疫苗组合物,所述组合物包含上文概括描述的嵌合或融合蛋白或肽,任选地与佐剂联合。
在本方面中,本发明还提供了预防或降低个体中牙龈卟啉单胞菌相关状态或疾病的发生率或严重程度的方法,包括给予所述个体上述嵌合或融合蛋白或组合物。
在本方面中,本发明还提供了上述嵌合或融合蛋白或上述组合物在用于预防或降低个体中牙龈卟啉单胞菌相关状态或疾病的发生率或严重程度的药物中的用途或者在制备所述药物中的用途。
另一方面,本发明提供了针对上文概括描述的嵌合或融合蛋白或肽而产生的抗体,尤其是单克隆抗体。
在本方面中,本发明还提供了预防或降低个体中牙龈卟啉单胞菌相关疾病或状态的严重程度的方法,其包括将上述抗体给予所述个体。
在本方面中,本发明还提供了上述抗体在用于预防或降低个体中牙龈卟啉单胞菌相关状态或疾病的发生率或严重程度的药物中的用途或者在制备所述药物中的用途。
另一方面,本发明还提供了核酸分子,其包含编码上文概括描述的嵌合或融合蛋白的核苷酸序列,所述核苷酸序列任选地与至少一种调节元件可操作地连接。
在本方面中,本发明还提供了包含所述核酸分子的载体,以及包含所述核酸分子的原核或真核细胞。
在本方面中,本发明还提供了预防或降低个体中牙龈卟啉单胞菌相关状态或疾病的发生率或严重程度的方法,包括将上述核酸分子、上述载体或上述原核或真核细胞给予所述个体。
在本方面中,本发明还提供了上述核酸分子、上述载体或上述原核或真核细胞在用于预防或降低个体中牙龈卟啉单胞菌相关状态或疾病的严重程度的药物中的用途或者在制备所述药物中的用途。
在其他方面中,本发明提供了诊断或监测个体中牙龈卟啉单胞菌相关状态或疾病的方法,包括将上述嵌合或融合蛋白用于检测来自所述个体的生物样品中抗牙龈卟啉单胞菌抗体。
在本方面中,本发明还提供了上述嵌合或融合蛋白在检测来自个体的生物样品中抗牙龈卟啉单胞菌抗体中的用途。
另一方面,本发明提供了诊断或监测个体中牙龈卟啉单胞菌相关状态或疾病的方法,包括将上述抗体用于检测来自所述个体的生物样品中牙龈卟啉单胞菌的存在。
在本方面中,本发明还提供了上述抗体在检测来自个体的生物样品中牙龈卟啉单胞菌的存在中的用途。
另一方面,本发明提供了具有与牙龈卟啉单胞菌Lys-X-蛋白酶或Arg-X-蛋白酶的序列相同或同源的序列的一部分或全部的肽或编码所述肽的核酸在制备用于诱导对于牙龈卟啉单胞菌的免疫应答的嵌合或融合蛋白中的用途。在本方面中,所述肽可以具有SEQID No:17、18、25或26中的一项所示的序列。
附图简要说明
图1显示了重组Kgp蛋白的SDS-PAGE凝胶考马斯亮蓝染色。泳道1=KAS2-KLA1、泳道2=KLA1、泳道3=KsA1、泳道4=KAS1-KsA1。分子量标记表示为kDa。
图2显示了KAS2肽和福尔马林杀死的牙龈卟啉单胞菌W50细胞的抗体识别。(A)在ELISA中,用针对福尔马林杀死的牙龈卟啉单胞菌W50细胞产生的抗血清(FK-W50)、重组蛋白KAS1-KsA1、KAS2-KLA1和合成的KAS2-DT缀合物以及PBS探测KAS2肽。(B)在ELISA中,用针对福尔马林杀死的牙龈卟啉单胞菌W50细胞产生的抗血清(FK-W50)、重组蛋白KAS1-KsA1、KAS2-KLA1、KLA1和PBS探测福尔马林杀死的牙龈卟啉单胞菌W50细胞。将抗体应答表示为减去背景水平的两倍而获得的ELISA滴度OD415,每个滴度代表3个值的平均值±标准差。
图3显示了牙龈卟啉单胞菌诱导的、用重组蛋白和重组嵌合蛋白、福尔马林杀死的牙龈卟啉单胞菌和单独的佐剂(PBS,IFA)免疫的小鼠或未经口感染的(未激发的)小鼠的上颌骨臼齿的水平骨丧失。在本图中,将KAS2-KLA1表示为AS2-LA1、KLA1表示为LA1、KAS1-KsA1表示为AS1-sA1、KsA1表示为sA1。骨丧失的检测结果是从左和右上颌骨的每个上颌骨臼齿的颊侧的釉质牙骨质界(CEJ)至牙槽嵴(ABC)以平方毫米(mm2)为单位测量的面积的平均值。如Levene方差同质性所检测的,数据为正态分布并表示为平均值(n=12)、单位为mm2,并用单向方差分析和Dunnett T3检验进行分析。*表明该组的骨丧失显著少于(P<0.001)对照组(感染的),表明该组的骨丧失显著多于(P<0.001)AS2-LA1组。
图4显示了牙周炎模型中被免疫的小鼠的血清抗体亚类应答。在ELISA中使用来自用重组蛋白KsA1、KLA1、KAS1-KsA1和KAS2-KLA1和福尔马林杀死的牙龈卟啉单胞菌菌株W50免疫的小鼠(A为口腔接种之前、B为口腔接种之后)的血清,用福尔马林杀死的牙龈卟啉单胞菌菌株W50作为吸附抗原。将抗体应答IgG(黑色柱)、IgG1(灰色柱)、IgG2a(白色柱)、IgG2b(水平条纹的柱)、IgG3(斜条纹的柱)表示为减去背景水平而获得的ELISA滴度(log2),每个滴度代表3个值的平均值±标准差。
图5显示了对于代表KAS2肽序列433-468的重叠肽(overlappingpeptide)的肽特异性抗体反应性的PEPSCAN分析。(A)用KAS1-KsA1(白色柱)抗血清、KAS2-KLA1(黑色柱)抗血清探测的KAS2重叠肽(步移为1,重叠为7)。(B)用KAS2-DT缀合物抗血清探测的KAS2重叠肽(步移,重叠为7)。每个柱显示抗体反应性(在415nm处的光密度[OD])。
图6.嵌合体AS2-LA1在远交系小鼠中诱导识别牙龈卟啉单胞菌全细胞和RgpA-Kgp复合物的抗体应答。用嵌合体AS2-LA1(50mg/小鼠)免疫CD1远交系小鼠,并将收集的血清用在ELISA中,用AS2-LA1(A)、福尔马林杀死的牙龈卟啉单胞菌菌株W50(B)和RgpA-Kgp复合物(C)作为吸附抗原。在本图中,将KAS2-KLA1表示为AS2-LA1。测定对于每种抗原的每种免疫球蛋白同型的滴度,并将数据表示为减去背景水平的两倍而获得的ELISA滴度(‘000),每个滴度代表3个值的平均值±标准差。
图7.Kgp蛋白酶的蛋白模型。KAS2[Asn433-Lys468](A)、KAS4[Asp388-Val395](B)、KAS5[Asn510-Asp516](C)和KAS6[Ile570-Tyr580](D)。
发明的详细描述
应当理解,本说明书中所公开和定义的发明适用于所提及的或从全文或附图中显而易见的两个或更多个单独特征的所有可选的组合。所有这些不同的组合构成本发明各个可选方面。
本发明人已经发现,位于切割肽键的催化位点或活性位点的侧翼或以其他方式界定切割肽键的催化位点或活性位点的牙龈卟啉单胞菌胰酶样酶的区是高免疫原性的,并确实足以提供对牙龈卟啉单胞菌感染的体液应答。特别是已经发现,包含一个或多个这些区域的嵌合或融合蛋白提供对于牙槽骨丧失的保护作用,该保护作用大于针对全细胞和其他免疫原产生的抗血清所观察到的保护作用。该发现尤其让人意想不到的是,到目前为止,牙龈卟啉单胞菌胰酶样酶的催化结构域的已有发现是具有相对弱的免疫原性。
一方面,本发明提供了用于诱导对于牙龈卟啉单胞菌的免疫应答的嵌合或融合蛋白,所述蛋白包含第一肽和第二肽,所述第一肽与第二肽直接连接或通过连接子连接,其中:
(A)所述第一肽包含:
(i)与SEQ ID No:1所示的序列相同或同源的序列的一部分或全部;或
(ii)与SEQ ID No:2所示的序列相同或同源的序列的一部分或全部;以及
(B)所述第二肽包含:
(i)与牙龈卟啉单胞菌Lys-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(ii)与牙龈卟啉单胞菌Arg-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(iii)与牙龈卟啉单胞菌HagA粘附素结构域的序列相同或同源的序列的一部分或全部。
如本文所用的术语“肽”是指多至约40个氨基酸残基、优选为5-40个氨基酸残基的氨基酸序列。
在一个实施方案中,多肽用于指代“第二肽”或换句话说用于代替“第二肽”。术语“多肽”是指至少约40个氨基酸残基的氨基酸序列。
因此,另一方面,提供了用于诱导对于牙龈卟啉单胞菌的免疫应答的嵌合或融合蛋白,所述蛋白包含肽和多肽,所述肽与所述多肽直接连接或通过连接子连接,其中:
(A)所述肽包含:
(i)与SEQ ID No:1所示的序列相同或同源的序列的一部分或全部;或
(ii)与SEQ ID No:2所示的序列相同或同源的序列的一部分或全部;以及
(B)所述多肽包含:
(i)与牙龈卟啉单胞菌Lys-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(ii)与牙龈卟啉单胞菌Arg-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(iii)与牙龈卟啉单胞菌HagA粘附素结构域的序列相同或同源的序列的一部分或全部。
另一方面,本发明提供了用于诱导对于牙龈卟啉单胞菌的免疫应答的肽,所述肽选自:
(i)与SEQ ID No:64-66中的一项所示的序列相同或同源的序列;和
(ii)与SEQ ID No:67或68所示的序列相同或同源的序列。
在本发明的一方面中,当肽具有SEQ ID No:64-68的序列时,可以以嵌合或融合蛋白的形式提供所述肽,其中所述肽与第二肽直接连接或通过连接子连接。在一个实施方案中,所述嵌合或融合蛋白的第二肽包含:
(i)与牙龈卟啉单胞菌Lys-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(ii)与牙龈卟啉单胞菌Arg-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(iii)与牙龈卟啉单胞菌HagA粘附素结构域的序列相同或同源的序列的一部分或全部。
在上述实施方案中,多肽用于指代第二肽或换句话说用于代替第二肽。因此,另一方面,提供了用于诱导对于牙龈卟啉单胞菌的免疫应答的嵌合或融合蛋白,所述蛋白包含肽和多肽,所述肽与所述多肽直接连接或通过连接子连接,其中:
(A)所述肽包含:
(i)与SEQ ID No:64-66中的一项所示的序列相同或同源的序列;或
(ii)与SEQ ID No:67或68所示的序列相同或同源的序列;以及
(B)所述多肽包含:
(i)与牙龈卟啉单胞菌Lys-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(ii)与牙龈卟啉单胞菌Arg-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(iii)与牙龈卟啉单胞菌HagA粘附素结构域的序列相同或同源的序列的一部分或全部。
如本文所用的,当涉及肽或多肽的“同源物”时,是指肽或多肽所具有的氨基酸序列与先前提及的肽或多肽的氨基酸序列享有同源性或是同源的或具有同一性,对于所述同一性,当通过BLAST算法进行比较时,其优选为至少90%的序列同一性,更优选为至少95%,并更优选为至少98%的序列同一性,其中该算法的参数被选择为在各自的序列之间、在各自参考序列的全长上给出最大的匹配。序列同一性是指在两条被比较序列的氨基酸之间的精确匹配。这类同源物可以来自天然存在的变体或牙龈卟啉单胞菌Lys-X-蛋白酶或Arg-X-蛋白酶的分离物。可选择地,它可以是来自牙龈卟啉单胞菌Lys-X-蛋白酶或Arg-X-蛋白酶的肽或多肽的“保守型取代”变体,其中改变了一个或多个氨基酸残基而没有改变所述肽或多肽的总体构象和功能;所述保守型取代包括但不限于用具有相似性质的氨基酸对氨基酸进行取代。具有相似性质的氨基酸在本领域内是公知的。例如,可互换的极性/亲水性氨基酸包括天冬酰胺、谷氨酰胺、丝氨酸、半胱氨酸、苏氨酸、赖氨酸、精氨酸、组氨酸、天冬氨酸和谷氨酸;可互换的非极性/疏水性氨基酸包括甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、酪氨酸、苯丙氨酸、色氨酸和蛋氨酸;可互换的酸性氨基酸包括天冬氨酸和谷氨酸,以及可互换的碱性氨基酸包括组氨酸、赖氨酸和精氨酸。这类保守型取代变体优选有少于20个,更优选少于15个,更优选少于10个,以及最优选少于5个氨基酸改变。
通过本说明书的以下教导,尤其结合图7和实施例9,可以确定界定了酶中切割肽键的位点的牙龈卟啉单胞菌胰酶样酶,尤其是Lys-X-蛋白酶(Kgp)或Arg-X-蛋白酶(RgpA)的区,其中图7和实施例9对预测Lys-X-蛋白酶催化位点在牙龈卟啉单胞菌上展现的三维构象的过程进行了举例说明。实施例10提供了Arg-X-蛋白酶三维构象的建模方法。
在某些实施方案中,嵌合或融合蛋白或其第一肽或第二肽组分由类肽(peptidomimetic)形成。类肽是模拟给定肽的一个或多个诸如构象的特征的分子,并且所述类肽由氨基酸残基组成,其中一些氨基酸残基可以不是天然存在的。
在鉴定出催化位点的免疫原区之后,本发明人测定了可以引起体液应答的各种肽免疫原的序列。具体而言,已经将位于催化位点侧翼的或以其他方式界定催化位点的“六个”区定义如下:KAS1/RAS1、KAS2/RAS2、KAS3/RAS3、KAS4/RAS4、KAS5/RAS5和KAS6(参见表1)。通过该信息,本发明人能查询蛋白序列数据库,从而确定与构成位于催化位点侧翼的区的氨基酸序列享有同源性并因此代表见于牙龈卟啉单胞菌上的免疫原性表位的肽。通过下述结构式鉴定这些肽的序列:
表1位于Kgp和RgpA活性位点侧翼的序列
本发明人已经发现包含这些肽的嵌合蛋白具有多种功效。例如,如本文所述,一些嵌合蛋白产生体液应答,其对于治疗或预防慢性牙周炎中所观察到的骨丧失是具有高度保护性的。所述肽也可以用在诊断分析中,其中所述肽能检测或监测个体血清中的特异性,从而指示所述个体是否被感染,如果被感染了,是否需要治疗或如果已提供治疗,治疗是否有效。
应当理解,界定了酶中切割位于Lys或Arg的C端的肽键的位点的牙龈卟啉单胞菌胰酶样酶的区不包含Lys-X-蛋白酶或Arg-X-蛋白酶的完整序列。
本文所用的术语“异源蛋白”或“嵌合或融合蛋白”是指由功能单元、结构域、氨基酸的序列或区所组成的蛋白,所述功能单元、结构域、氨基酸的序列或区可以来自不同来源,或来自相同来源并进行组装,从而所具有的结构不同于所述单元、结构域、序列或区所来自的分子或相关的分子中所观察到的结构。本发明嵌合或融合蛋白的共有特征是:它们含有至少一个这样的肽,所述肽具有与界定了切割肽键的催化位点的、牙龈卟啉单胞菌胰酶样酶的序列相同或享有同源性的氨基酸序列。
在优选的实施方案中,当第一肽包含来自Kgp[432-468]区的肽时,其优选为(i)包含选自VSFANYT和VGFANYT的序列,更优选地包含选自GVSFANYT、GVGFANYT、VSFANYTA和VGFANYTA的序列的肽;或(ii)包含选自ETAWAD、ETSWAD、TAWADP和TSWADP的序列,优选地包含选自SETAWAD、SETSWAD、ETAWADP、ETSWADP、TAWADPL和TSWADPL的序列,更优选地包含选自GSETAWAD、GSETSWAD、SETAWADP、SETSWADP、ETAWADPL、ETSWADPL、TAWADPLL和TSWADPLL的序列的肽。更优选地,该肽选自表1所示的KAS1[432-454]肽、KAS2[433-468]肽和KAS3[436-455]肽。可选择地,所述第一肽可以是PAS1K[432-453]肽,也被称为PAS1(K48),其在国际专利申请第PCT/AU98/00311(WO 98/049192)号中公开。这些肽所对应的序列标识符显示在表3中。
同样,在另一个优选的实施方案中,当第一肽包含来自RgpA[426-462]区的肽时,该肽优选地选自表1所示的RAS1[426-448]肽、RAS2[427-462]肽和RAS3[430-449]肽。可选择地,所述第一肽可以是PAS1R[426-446]肽,也被称为PAS1(R45),其在国际专利申请第PCT/AU98/00311(WO98/049192)号中公开。
在本发明的嵌合或融合蛋白中,所述第二肽可以是来自诸如Lys-X-蛋白酶(Kgp)或Arg-X-蛋白酶(RgpA)或HagA的牙龈卟啉单胞菌胰酶样酶的粘附素结构域的肽(参见表2)。这些结构域有时也被称为血凝素。在Lys-X-蛋白酶中,优选的结构域是如表2鉴定出的KA1、KA2、KA3、KA4、KA5。在Arg-X-蛋白酶中,优选的结构域是如表2鉴定出的RA1、RA2、RA3和RA4。在HagA中,优选的结构域是HagA1、HagA1*和HagA1**。
表2.Kgp和RgpA蛋白酶的粘附素结构域
当将诸如KAS1、KAS2、KAS3、KAS4、KAS5和KAS6或RAS1、RAS2和RAS3、RAS4和RAS5的本发明的肽包括在嵌合或融合蛋白中时,可以增强对于所述本发明的肽的体液应答,除此之外,所述粘附素结构域还含有免疫原性表位,因此导致多特异性的产生,从而引起保护性免疫原性应答。以下发现是未预计到的:当将粘附素结构域的免疫原性表位提供在本发明的嵌合或融合蛋白中时,所述免疫原性表位保留的形式与其在牙龈卟啉单胞菌胰酶样酶中的形式接近。
应当理解,在本发明的这些实施方案中,嵌合或融合蛋白可以含有选自KAS1/RAS1、KAS2/RAS2、KAS3/RAS3、KAS4/RAS4、KAS5/RAS5和KAS6/RAS6的肽中的任意一个或多个,以及任意一个或多个牙龈卟啉单胞菌胰酶样酶的粘附素结构域,尤其是Lys-X-蛋白酶粘附素结构域(KA1、KA2、KA3、KA4和KA5)或Arg-X-蛋白酶粘附素结构域(RA1、RA2、RA3和RA4)或HagA结构域HagA1、HagA1*和HagA1**中的任意一个或多个。
还应当理解,所述粘附素结构域未必是如牙龈卟啉单胞菌胰酶样酶中所观察到的完整结构域。例如,所述粘附素结构域可以是此类结构域的片段,尤其优选的片段是Lys-X-蛋白酶A1结构域的KsA1和KLA1结构域片段(参见表2)。当所述结构域是粘附素结构域片段时,其通常含有一个或多个粘附素结构域特异性表位。
粘附素相关肽所对应的序列标识符显示在表3中。
在一个实施方案中,所述第二肽或多肽包含SEQ ID No:69-79中的一项或多项或SEQ ID No:83-85中的一项或多项所示的序列。
本发明的嵌合或融合蛋白还可以包含一个或多个选自Lys-X-蛋白酶的Kgp[432-468]区的其他的肽和/或一个或多个选自Arg-X-蛋白酶的RgpA[426-462]区的其他的肽。
在本发明的优选的实施方案中,嵌合或融合蛋白包含KAS1、KAS2、KAS3、KAS4、KAS5和KAS6的一种或多种或RAS1、RAS2、RAS3、RAS4和RAS5的一种或多种,以及KsA1或KLA1。
因此,在某些实施方案中,嵌合或融合蛋白可以包含至少一种其他的肽,其中所述其他的肽包含:
(i)与SEQ ID No:1所示的序列相同或同源的序列的一部分或全部;或
(ii)与SEQ ID No:2所示的序列相同或同源的序列的一部分或全部;或
(iii)与牙龈卟啉单胞菌Lys-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(iv)与牙龈卟啉单胞菌Arg-X-蛋白酶的粘附素结构域的序列相同或同源的序列的一部分或全部;或
(v)与牙龈卟啉单胞菌HagA粘附素结构域的序列相同或同源的序列的一部分或全部。
本文所述的嵌合或融合蛋白中可以包含的结构域、单元、序列或区域的其他实例包括用于与受体或配体结合的结构域,例如Fc结合区或Fc受体;用于改善半寿期的结构域,例如白蛋白;或用于促进嵌合或融合蛋白的表达或纯化的结构域。
在本发明的嵌合或融合蛋白中,第一肽的C末端残基可以与粘附素结构域多肽的N末端残基共价连接,或第一肽的N末端残基可以与粘附素结构域多肽的C末端残基共价连接。在该排列中,所述第一肽和粘附素结构域多肽被认为是“直接连接”或“紧邻”。
在其他的实施方案中,嵌合或融合蛋白包含用于连接第一肽和粘附素结构域多肽的连接子。所述连接子可以是能将肽与多肽连接的任意连接子,包括氨基酸和非氨基酸连接子。优选地,所述连接子是非免疫原性的。合适的连接子的长度可以多至15个氨基酸,但是优选少于5个氨基酸。所述连接子的功能可以是使第一肽和粘附素结构域多肽的空间排列比在牙龈卟啉单胞菌胰酶样酶中正常观察到的更接近。可选择地,所述连接子可以将第一肽与粘附素结构域多肽隔开。
可以通过重组表达系统(诸如重组DNA技术)或通过化学合成(诸如固相肽合成)产生本发明的嵌合或融合蛋白。这些技术在本领域内是公知的。
异源或嵌合蛋白特别有优势,因为与单独使用嵌合或融合蛋白的第一或第二肽组分所获得的体液应答相比,它增强了所获得的体液应答。
本发明人已经发现包含这些肽的嵌合蛋白具有多种功效。例如,如本文所述,一些嵌合蛋白产生体液应答,其对于治疗或预防慢性牙周炎中所观察到的骨丧失是具有高度保护性的。所述肽也可以用在诊断分析中,其中所述肽能检测或监测个体血清中的特异性,从而指示所述个体是否被感染,如果被感染了,是否需要治疗或如果已提供治疗,治疗是否有效。
在一个实施方案中,所述嵌合或融合蛋白诱导保护性免疫应答,通常为至少最小化或限制以其他方式与牙龈卟啉单胞菌感染有关的结缔组织损伤的应答。在一个实施方案中,所述保护性应答至少最小化或限制牙龈卟啉单胞菌诱导的骨丧失。本文讨论了用于测定牙龈卟啉单胞菌感染介导的骨丧失的模型系统。通常,所述保护性免疫应答主要为体液应答。在某些实施方案中,所述保护性免疫应答还包括细胞应答。
本发明还提供了包含上文概括描述的嵌合或融合蛋白的组合物。所述组合物通常是抗原性的或免疫原性的。更具体而言,本发明提供了适于引起对于牙龈卟啉单胞菌感染的保护性或治疗性免疫应答的组合物,所述组合物包含所述嵌合或融合蛋白并任选地与佐剂结合。这类组合物还可以包含用于调节或增强免疫应答的另一组分。在一个实施方案中,所述组合物采用疫苗的形式。
各种佐剂已知可以与疫苗组合物联合使用。所述佐剂协助调节免疫应答,并且有助于使用比单独给予疫苗抗原更小量的疫苗抗原或更少的剂量来获得更持久和更高的免疫力水平。佐剂的实例包括弗氏不完全佐剂(IFA)、佐剂65(含有花生油、二缩甘露醇一油酸和单硬脂酸铝)、油乳化剂、Ribi佐剂、复合多元醇、多胺、阿夫立定(Avridine)、Quil A、皂苷、MPL、QS-21、诸如铝盐和钙盐的矿物质凝胶、诸如羟磷灰石、磷酸钙、铝盐、糖低聚物的纳米颗粒、和诸如甘露聚糖、壳聚糖的聚合物。其他的实例包括水包油乳化剂,诸如SAF-1、SAF-0、MF59、Seppic ISA720和其他的颗粒佐剂,诸如ISCOMsTM和ISCOM基质TM。佐剂的其他实例的广泛但不完全的列表见于Cox and Coulter 1992[In:Wong WK(ed.)Animalsparasite control utilizing technology(动物寄生虫控制实用技术).Bocca Raton;CRCpress,1992;49-112]。除了佐剂之外,疫苗组合物可以视情况包含常规的药学可接受的载体、赋形剂、填料、缓冲液或稀释剂。可以预防性地给予一个或多个剂量的含有佐剂的疫苗组合物以预防牙周炎,或治疗性地给予一个或多个剂量的含有佐剂的疫苗组合物以治疗已患的牙周炎。
在优选的组合物中,嵌合或融合蛋白与粘膜佐剂组合,并且通过口腔、颊或鼻腔途径进行给药。粘膜佐剂的实例是纳米颗粒、霍乱毒素和热不稳定的大肠杆菌毒素、这些毒素的非毒性B亚基、这些毒素的具有降低的毒性的遗传突变体。可以用来口腔/颊/鼻腔递送抗原性蛋白的其他方法包括将蛋白通过微囊化作用合并在或吸附在可生物降解的聚合物(诸如丙烯酸酯或聚酯)颗粒或纳米颗粒(诸如羟磷灰石)的内部或上面,从而有助于微球从胃肠道或其他粘膜表面的摄取并保护蛋白免于降解。脂质体、ISCOMsTM、水凝胶是其他可能方法的实例,通过并入诸如LTB、CTB或凝集素的用于将抗原性蛋白递送至粘膜免疫系统的靶分子可以进一步增强这些方法。除了抗原性蛋白和粘膜佐剂或递送系统之外,所述疫苗组合物可以视情况包含常规的药学可接受的载体、赋形剂、填料、包被、分散介质、抗细菌剂或抗真菌剂和缓冲液或稀释剂。
在本方面中,本发明还提供了预防或降低个体中牙龈卟啉单胞菌相关状态或疾病的发生率或严重程度的方法,其包括给予所述个体上述嵌合或融合蛋白或上述组合物。
所述个体可以是人或其他动物个体,且优选为人。
通常,牙龈卟啉单胞菌相关状态或疾病是慢性牙周炎,然而也可以是骨丧失,尤其是牙槽骨丧失或冠状动脉疾病。
已知许多方法可以用于将疫苗组合物给予人或动物个体,所述方法包括但不限于皮内给药、肌肉内给药、腹膜内给药、静脉内给药、皮下给药、鼻内给药、舌下给药、颊给药和口服给药。这些给药途径尤其适用于疫苗接种。
另一方面,本发明提供了针对上文概括描述的嵌合或融合蛋白所产生的抗体,优选为单克隆抗体。
可以通过标准技术产生这些抗体,并且可以将这些抗体用于个体的被动免疫。因此,在本方面中,本发明还提供了预防或降低个体中牙龈卟啉单胞菌相关疾病或状态的严重程度的方法,包括给予所述个体上述抗体。
另一方面,本发明提供了包含编码上文概括描述的嵌合或融合蛋白的核苷酸序列的核酸分子,所述核苷酸序列任选地与至少一种调节元件可操作地连接。在一个实施方案中,以分离的或基本上纯化的形式提供所述核酸。
例如,可以将所述核酸分子插入合适的表达载体中,通过将所述表达载体插入原核或真核宿主细胞,可以用于产生作为重组蛋白的嵌合蛋白。重组蛋白的成功表达需要表达载体含有转录和翻译所必需的调节元件,所述调节元件与用于表达的具体宿主细胞系统相容并可以被所述宿主细胞系统识别。各种宿主细胞系统可以用来表达重组蛋白,包括但不限于,用噬菌体载体、质粒载体或粘粒DNA转化的细菌;含有酵母载体的酵母;含有真菌载体的真菌;用病毒(例如,杆状病毒)感染的昆虫细胞系;和用质粒或病毒表达载体转染的或用重组病毒(例如,牛痘病毒、腺病毒、腺相关病毒、逆转录病毒等)感染的哺乳动物细胞系。
使用分子生物学领域内已知的方法,可以将各种启动子和增强子合并入表达载体,从而增加重组蛋白的表达,只要增加的氨基酸序列表达与所用的具体宿主细胞系统相容(例如,对所述宿主细胞系统没有毒性)。
启动子的选择取决于所用的表达系统。启动子在强度上,即在促进转录的能力上可以不同。通常,为了获得编码核苷酸序列的高水平转录和重组蛋白的高水平表达,使用强启动子是理想的。例如,本领域内已知的、通过其可以在包括大肠杆菌在内的宿主细胞系统中观察到高水平转录的细菌、噬菌体或质粒启动子包括lac启动子、trp启动子、recA启动子、核糖体RNA启动子、PR与PL启动子、lacUV5、ompF、bla、lpp等,并可以用于提供插入的编码氨基酸序列的核苷酸序列的转录。
用于高效转录或翻译的其他控制元件包括增强子和调节信号。增强子序列是表现为增加转录效率的DNA元件,其作用方式相对不依赖于它们相对于邻近的编码核苷酸序列的位置或方向。因此,根据所用的宿主细胞表达载体系统,可以将增强子置于插入的编码序列的上游或下游来增加转录效率。诸如转录或翻译起始信号的其他调节位点可以用来调节编码序列的表达。
在另一个实施方案中,载体可以是病毒疫苗载体或细菌疫苗载体,且用来提供重组病毒疫苗、重组细菌疫苗、重组减毒细菌疫苗或灭活重组病毒疫苗。牛痘病毒是本领域内最熟知的感染性病毒的实例,对其进行基因工程以表达源于其他生物体的疫苗抗原。将重组活牛痘病毒用来免疫宿主,所述重组活牛痘病毒被减毒过或以其他方式处理过以使其自身不会导致疾病。在宿主中,随后的重组病毒的复制提供了疫苗抗原对免疫系统的连续刺激,由此提供长效免疫。
其他活疫苗载体包括:腺病毒、巨细胞病毒,并优选诸如牛痘[PaolettiandPanicali,美国专利第4,603,112号]的痘病毒和减毒沙门氏菌(Salmonella)菌株[Stockeret al,美国专利第5,210,035号、第4,837,151号和第4,735,801号和Curtiss et al,1988,Vaccine 6:155-160]。活疫苗特别具有优势,因为它们持续刺激免疫系统,这能赋予基本上长效的免疫力。当免疫应答对于后续牙龈卟啉单胞菌感染具有保护作用时,活疫苗自身可以用在针对牙龈卟啉单胞菌的预防性疫苗中。特别是,活疫苗可以基于口腔的共生物细菌。可以用携带重组嵌合蛋白的载体转化该细菌,然后将其用来在口腔中定殖,尤其是口腔粘膜。一旦在口腔粘膜中定殖,所述重组蛋白的表达将刺激粘膜相关淋巴组织,从而产生中和抗体。为了进一步说明本实施方案,利用本领域内公知的分子生物学技术,可以将编码本发明嵌合蛋白的核苷酸序列插入牛痘病毒基因组DNA,所插入的位点允许表位的表达但不会对牛痘病毒载体的生长或复制产生不利影响。所得重组病毒可以用作疫苗制剂中的免疫原。同样的方法可以用来构建灭活重组病毒疫苗制剂,区别仅在于,所述重组病毒在用作免疫原之前例如通过本领域内已知的化学方法进行灭活,但这基本上不影响表达的免疫原的免疫原性。为了增强对疫苗抗原的免疫应答,可以将灭活重组疫苗与合适的佐剂一起配制。
本发明还提供了将包含编码本发明嵌合或融合蛋白的核苷酸序列的核酸分子直接作为疫苗制剂的用途。可以将可操作地连接于一个或多个调节元件的编码嵌合蛋白的核苷酸序列直接引入(“直接的基因转移”),从而对个体进行针对牙龈卟啉单胞菌病原菌株的疫苗接种。将基因直接转移进接种的个体会导致被接种个体的诸如血管内皮细胞的细胞以及主要器官组织中遗传物质的表达,这已经通过本领域内的技术得到证实,诸如通过静脉内注射表达质粒:阳离子脂质体复合物[Zhu et al,1993,Science 261:209-211]。将载体DNA递送进靶细胞的其他有效的方法在本领域内是已知的。在一个实例中,将含有病毒基因的纯化的重组质粒DNA用于接种疫苗(肠胃外免疫、粘膜免疫或通过基因枪进行免疫),以诱导保护性免疫应答[Fynan et al.1993,Proc Natl Acad Sci USA 90:11478-11482]。在另一个实例中,可以通过本领域内已知的标准方案对从个体移除的细胞进行转染或电穿孔,这导致重组载体DNA被引入靶细胞内。然后,可以用本领域内已知的方法选择含有重组载体DNA的细胞,诸如通过使用载体中表达的选择标记,然后,可以将选择的细胞再引入个体,以表达重组蛋白。
在本方面中,本发明还提供了预防或降低个体中牙龈卟啉单胞菌相关状态或疾病的发生率或严重程度的方法,所述方法包括给予所述个体上述核酸分子、上述载体或上述原核或真核细胞。
在其他的实施方案中,提供了包含上述的嵌合或融合蛋白或抗体的药物组合物。所述组合物还可以包含稀释液、赋形剂或载体或用于治疗牙龈卟啉单胞菌相关状态或疾病的化疗剂,并且所述组合物可以被调整为适于口腔给药。可将本发明的组合物合并在锭剂或口香糖或其他产品中,例如,通过将其搅拌进温热胶基或包被胶基的外表面,所述胶基的实例为节路顿胶(jelutong)、橡胶乳胶、乙烯基树脂等,理想情况伴有常规的增塑剂或软化剂、糖或其他甜味剂或诸如葡萄糖、山梨醇等。
可以制备和使用适于口腔的各种形式的、含有上述药物组合物的本发明的口腔组合物,例如包括牙膏、牙粉和液体洁牙剂的洁牙剂、漱口剂、锭剂、口香糖、牙科用糊剂、齿龈按摩乳膏、漱口片剂、乳制品和其他食品。根据具体口腔组合物的类型和形式,本发明的口腔组合物还可以包含其他公知的组分。
在本发明的某些优选形式中,所述口腔组合物的性质可以基本为液体,诸如漱口剂或清洗剂。在这类制剂中,介质通常是水-醇混合物,理想情况包含下述湿润剂。通常,水与醇的重量比为约1:1至约20:1。在该类制剂中,水-醇混合物的总量通常为所述制剂重量的约70%-约99.9%。所述醇通常是乙醇或异丙醇。优选乙醇。
本发明的这类液体和其他制剂的pH一般为约5至约9,且通常为约5.0-7.0。可以用酸(例如,柠檬酸或苯甲酸)或碱(例如,氢氧化钠)或缓冲剂(如用柠檬酸钠、安息香酸钠、碳酸钠或碳酸氢钠、磷酸氢二钠、磷酸二氢钠等)控制pH。
在本发明的其他理想形式中,所述药物组合物的性质可以基本为固体或糊状,例如牙粉、牙科用片剂或牙膏(牙科用乳膏)或凝胶洁牙剂。这类固体或糊状口腔制剂的介质通常含有牙科上可接受的磨光材料。
在牙膏中,液体介质可包含水和湿润剂,它们的量通常为所述制剂重量的约10%-约80%。甘油、丙二醇、山梨醇和聚丙二醇是合适的湿润剂/载体的实例。水、甘油和山梨醇的液体混合物也是有利的。在透明凝胶中,因为折射指数是重要的考虑因素,所以优选采用约2.5-30%w/w的水、0-约70%w/w的甘油和约20-80%w/w的山梨醇。
牙膏、乳膏和凝胶通常含有天然的或合成的增稠剂或胶凝剂,其比例为约0.1%-约10%w/w,优选为约0.5%-约5%w/w。合适的增稠剂是合成的锂蒙脱石,例如LaporteIndustries Limited销售的名为Laponite(例如,CP、SP 2002、D)的合成胶体镁碱金属硅酸盐复合物粘土。以重量计Laponite D大约为58.00%SiO2、25.40%MgO、3.05%Na2O、0.98%Li2O以及一些水和微量金属。它的真比重为2.53,并且在8%湿度时,它有1.0g/ml的表观松装密度。
其他合适的增稠剂包括爱尔兰藓、iota-角叉胶、黄蓍胶、淀粉、聚乙烯吡咯烷酮、羟乙基丙基纤维素、羟丁基甲基纤维素、羟丙基甲基纤维素、羟乙基纤维素(例如,商业名Natrosol)、羧甲基纤维素钠和硅胶,例如精细研磨的Syloid(例如,244)。还可以包含增溶剂,例如,湿润剂多元醇,例如丙二醇、二丙二醇和己二醇;溶纤剂,例如甲基溶纤剂和乙基溶纤剂;在直链中含有至少约12个碳的植物油和蜡,例如橄榄油、蓖麻油和矿脂;以及酯类,例如乙酸戊酯、乙酸乙酯和苯甲酸苄酯。
应当理解,常规情况下,口腔制剂通常是在合适的带标签的包装中进行销售或分销。因此,瓶装口腔清洗剂会带有在本质上作为口腔清洗剂或漱口剂对其进行描述的标签,并且所述标签写有它的使用说明;通常,牙膏、乳膏或凝胶在通常为铝、铅衬或塑料的收缩管,或用于剂量内容物的其他挤压器、泵取或加压分配器中,所述收缩管或分配器带有在本质上作为牙膏、凝胶或牙科用乳膏对其进行描述的标签。
在本发明的组合物中可以使用有机表面活性剂,以达到增强的预防作用,协助实现活性剂彻底、完全地分散在整个口腔中,并且使本发明的组合物更适合化妆品应用。所述有机表面活性物质的性质优选为阴离子的、非离子的或两性的,且优选不与所述活性剂相互作用。优选地将去垢剂物质作为表面活性剂,所述去垢剂物质赋予组合物去垢和发泡性质。阴离子表面活性剂的合适实例是高级脂肪酸单硫酸甘油单脂的水溶性盐,例如氢化椰子油脂肪酸的单硫酸甘油单脂的钠盐;高级烷基硫酸盐,例如月桂基磺酸钠;烷基芳基磺酸盐,例如十二烷基苯磺酸钠、高级烷基磺基醋酸盐、1,2-二羟基丙磺酸盐的高级脂肪酸酯;以及低级脂肪族氨基羧酸化合物的基本饱和的高级脂肪族酰胺,诸如在脂肪酸、烷基或酰基中具有12-16个碳的那些化合物等。以上提到的酰胺的实例是N-月桂酰肌氨酸、以及N-月桂酰肌氨酸、N-肉豆蔻酰肌氨酸或N-棕榈酰肌氨酸的钠盐、钾盐和乙醇胺盐,以上应当基本脱离于肥皂或相似的高级脂肪酸材料。适于使用的水溶性非离子表面活性剂的实例是环氧乙烷和可与其发生反应的各种反应性含氢化合物的缩合产物,所述含氢化合物具有长疏水性链(例如,约12-20个碳原子的脂肪族链),该缩合产物("ethoxamers")含有亲水性聚氧乙烯部分,所述缩合产生例如聚(环氧乙烯)与脂肪酸、脂肪醇、脂肪酰胺、多羟基醇(例如,山梨醇酐单硬脂酸酯)和聚环氧丙烷(例如,普流尼克(Pluronic)材料)的缩合产物。
表面活性剂的量通常为以重量计约0.1%-5%。值得注意的是,表面活性剂可以促进本发明活性剂的溶解,因而减少所需的增溶湿润剂的量。
在本发明的口腔制剂中可以掺入各种其他材料,诸如增白剂、防腐剂、硅酮、叶绿素复合物和/或诸如尿素、磷酸氢二铵的含胺材料,以及以上的混合物。这些佐剂如果存在,则掺入制剂的佐剂的量应当基本上不会对所需的性质和特征产生不利影响。
还可以使用任何合适的调味材料或增甜材料。合适的调味组分的实例是调味油和水杨酸甲酯,所述调味油例如,绿薄荷油、薄荷油、鹿蹄草油、檫木油、丁香油、鼠尾草油、桉树油、媒墨角兰油、肉桂油、柠檬油和橙油。合适的增甜剂包括蔗糖、乳糖、麦芽糖、山梨醇、木糖醇、环氨酸钠、紫苏亭、AMP(天冬酰胺苯丙氨酸酯甲酯)、糖精等。适当地,调味剂和增甜剂可以单独或共同占制剂的约0.1%至5%或更多。
可以按照任何本领域内已知的制备药物组合物的方法来制备意图用于口腔的组合物,并且为了提供满足药学上外观和味觉的制剂,该组合物可以含有一种或多种选自以下的试剂:增甜剂、调味剂、着色剂和防腐剂。片剂含有与适于制备片剂的无毒的药学可接受赋形剂混合的活性组分。这些赋形剂可以是,例如,惰性稀释剂,例如碳酸钙、碳酸钠、乳糖、磷酸钙或磷酸钠;成粒剂和崩解剂,例如,玉米淀粉或褐藻酸;粘合剂,例如,淀粉、明胶或阿拉伯树胶;和润滑剂,例如硬脂酸镁、硬脂酸或滑石。片剂可以是无包被的或可以用已知的技术进行包被以延迟在胃肠道或牙周袋中的崩解和吸收,从而在更长的时期提供持续的作用。例如,可以使用时间延迟材料,诸如单硬脂酸甘油酯或双硬脂酸甘油酯。
用于口腔的制剂还可以以硬明胶胶囊的形式提供,其中活性组分与诸如碳酸钙、磷酸钙或高岭土的惰性固体稀释剂混合;用于口腔的制剂还可以以软明胶胶囊的形式提供,其中活性组分与水或诸如花生油、液体石蜡或橄榄油的油介质混合。
水性悬液含有活性物质与适于制备水性悬液的赋形剂的混合物。这类赋形剂是悬浮剂,例如羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、藻酸钠、聚乙烯吡咯烷酮、黄蓍胶和阿拉伯树胶。分散剂或润湿剂可以是天然存在的磷脂,例如,卵磷脂;或环氧烷烃与脂肪酸的缩合物,例如聚氧乙烯硬脂酸酯;或环氧乙烷与长链脂肪醇的缩合物,例如十七乙烯氧基十六烷醇(heptadecaethyleneoxycetanol);或环氧乙烷与来自脂肪酸和己糖醇的部分酯的缩合物,例如聚氧乙烯山梨醇单油酸酯;或环氧乙烷与来自脂肪酸和己糖醇酐的部分酯的缩合物,例如聚乙烯脱水山梨聚醇单油酸酯。
水性悬液还可以含有一种或多种防腐剂或抗微生物剂,例如苯甲酸酯,诸如乙基或n-丙基p-羟基苯甲酸酯;另一个实例是葡萄糖酸氯己定;水性悬液还可以含有一种或多种着色剂、一种或多种调味剂和一种或多种增甜剂,诸如蔗糖或糖精。
可以通过将活性组分悬浮于植物油或矿物油中来配制油性悬液,所述植物油例如花生油、橄榄油、芝麻油或椰子油;所述矿物油例如液体石蜡。油性悬液可以含有增稠剂,例如蜂蜡、硬石蜡或鲸蜡醇。可以添加调味剂和诸如上文列出的增甜剂,以提供符合味觉需要的口腔制剂。通过添加诸如抗坏血酸的抗氧化剂可以使这些组合物防腐。
在其他方面,本发明提供了诊断或监测个体中牙龈卟啉单胞菌相关状态或疾病的方法,包括使用上述的嵌合或融合蛋白检测来自所述个体的生物样品中的抗牙龈卟啉单胞菌抗体。
另一方面,本发明提供了诊断或监测个体中牙龈卟啉单胞菌相关状态或疾病的方法,包括使用上述的抗体检测来自所述个体的生物样品中牙龈卟啉单胞菌的存在。
另一方面,本发明提供了用于诱导对于牙龈卟啉单胞菌的免疫应答的肽,所述肽包括SEQ ID No:17、18、25和26中的一项所示的序列。在一个实施方案中,所述肽具有与SEQID No:17、18、25和26中的一项同源的序列。所述肽的长度可以为5-40个氨基酸。
另一方面,本发明提供了编码具有SEQ ID No:17、18、25和26中的一项所示序列的肽的核酸。
另一方面,本发明提供了具有SEQ ID No:17、18、25和26中的一项所示序列的肽或编码具有SEQ ID No:17、18、25和26中的一项所示序列的肽的核酸在制备用于诱导对于牙龈卟啉单胞菌的免疫应答的嵌合或融合蛋白中的用途。
另一方面,本发明提供了具有SEQ ID No:17、18、25和26中的一项所示序列的肽或编码具有SEQ ID No:17、18、25和26中的一项所示序列的肽的核酸在诱导对于牙龈卟啉单胞菌的免疫应答中的用途。在一个实施方案中,所述肽与第二肽同时给药或相继给药,所述第二肽包含:
(i)与牙龈卟啉单胞菌Lys-X-蛋白酶粘附素结构域的序列相同或同源的序列的一部分或全部;或
(ii)与牙龈卟啉单胞菌Arg-X-蛋白酶粘附素结构域的序列相同或同源的序列的一部分或全部;或
(iii)与牙龈卟啉单胞菌HagA粘附素结构域的序列相同或同源的序列的一部分或全部。
表3
通过下述实施例对本发明进行进一步说明,下述实施例是为了举例说明而不是为了限制本发明。
实施例
实施例1
方法和材料
细菌菌株和生长条件
使牙龈卟啉单胞菌W50的冻干培养物于37℃下、在补充了5μg/ml血晶素、0.5μg/ml半胱氨酸的裂解马血琼脂平板上厌氧生长(HB琼脂,<10代)。3-4天后,将集落接种含有5μg/ml血晶素、0.5μg/ml半胱氨酸的脑心浸液培养基(1)。使批量培育物在MK3厌氧工作站(DonWhitleyScientific Ltd.,Adelaide,Australia)中厌氧生长。在指数生长期中,通过离心收集细胞(7500g,30分钟,4℃),并且在厌氧工作站中用PG缓冲液(50mM Tris-HCl、150mMNaCl、5mM CaCl2和5mM半胱氨酸-HCl,pH 8.0)洗涤两次。用分光光度计(型号295E,Perkin-Elmer)在650nm处监测批量培养物的生长。按照Slots(2)所述,通过革兰氏染色、显微镜检查并使用各种生化测试常规检测培养物纯度。
含有粘附素序列和N末端添加了Kgp蛋白酶序列的粘附素序列的pET28构建体的构建
使用pET表达载体(Novagen),将代表活性位点(AS)与KgpA1粘附素(A1)结构域的肽和嵌合肽的Kgp残基在大肠杆菌中过表达为带有6×His标签的重组(r)蛋白。所表达的r-蛋白是rKAS2和rKLA1,r-嵌合蛋白是rKAS2-KLA1、rKAS1-KsA1和rKAS4-KAS3-KAS5-KAS6-KLA1(也被称为“多KAS-KLA1”)。代表各种A1和AS结构域的氨基酸序列描述于表1和表2中。
使用表4列出的引物、Taq DNA聚合酶(Invitrogen)和PC-960热循环仪(CorbettResearch Technologies)分别从pNS1(pUC18中的3.5kb BamHIlys片段)或牙龈卟啉单胞菌基因组DNA扩增kgp基因的各种KAS和KA1结构域。分别使用引物对KAS2-FOR与KAS2-REV和KLA1-FOR与KLA1-REV来产生编码KAS2和KLA1的PCR片段,使用下述条件进行反应:94℃、3分钟;每个循环为94℃、45秒(变性),62℃、40秒(退火)和72℃、20秒(延伸),进行28个循环;72℃、5分钟进行最后1个循环。
按照下述,通过利用重叠延伸(SOEing)的基因剪接产生KAS2-KLA1嵌合PCR产物:使用引物对KAS2-FOR与KAS2-KLA1-嵌合体-REV和KAS2-KLA1-嵌合体-FOR与KLA1-REV、按上述条件产生PCR产物。然后,将该PCR产物退火,并用引物KAS2-FOR和KLA1-REV进行最后的PCR(94℃、2分钟;每个循环为94℃、30秒,50℃、30秒和72℃、40秒,进行28个循环;72℃、5分钟进行最后1个循环)。
对于KAS1-KsA1PCR产物的制备,进行两次连续PCR,该PCR中使用KAS1-KsA1-REV引物并且相继使用KAS1-KsA1-FOR引物1和2中的每个(反应条件:94℃、2分钟;每个循环为94℃、15秒,63℃、30秒和72℃、2分钟,进行35个循环),从而产生KAS1-KsA1PCR产物。KAS1-KsA1-FOR1和KAS1-KsA1-FOR2引物含有与之前PCR产物的5'重叠的3'延伸。
对于多KAS-KLA1PCR片段的制备,进行四次连续PCR,该PCR中使用多REV引物并且相继使用多FOR引物1、2、3和4中的每个(反应条件:95℃、2分钟;每个循环为95℃、20秒,68℃、1.5分钟,进行35个循环),从而产生多KAS-KLA1PCR产物。每个多FOR引物含有与之前PCR产物的5'重叠的3'延伸。
按照生产商的方法,用PCR纯化柱(Qiagen)纯化编码KAS2、KLA1、KAS2-KLA1、KAS1-KsA1和多KAS-KLA1的所有PCR片段,将它们连接到TA克隆载体pGem-T Easy(Promega),并转化进大肠杆菌JM109。用Ncol和Xhol消化纯化的重组pGemT-Easy构建体,定向克隆进Ncol/Xhol消化的pET28b(Novagen),并转化进非表达宿主大肠杆菌JM109[DH5α]。按照生产商的说明,纯化重组pET28构建体,将其转化进大肠杆菌表达宿主BL21(DE3)[HMS174(DE3)](Novagen)中,并在含有50μg卡那霉素的LB上进行选择。通过DNA序列分析确认每个插入物的完整性。
寡核苷酸引物(表4)被设计为合并了限制性酶位点、终止密码子,并且如果需要的话合并6×His标签。用于rKAS2、rKLA1和rKAS2-KLA1的引物被设计为将r-蛋白中所包含的无关编码序列限制为不多于3个氨基酸外加6×His标签。rKAS1和rKLA1被设计为分别在N末端和C末端含有6×His标签,使得可以将它们与在N末端和C末端都具有6×His标签的rKAS2-KLA1直接比较。在rKAS1-KsA1和r多KAS-KLA1中,His标签见于C末端。
表4.用于扩增编码Kgp A1和AS的不同片段
和嵌合体的核苷酸序列的寡核苷酸引物
*来自序列登录号为U75366的赖氨酸特异性半胱氨酸蛋白酶基因的核苷酸(nt)序列编号
重组蛋白的表达和纯化
通过使用异丙基β-D-硫代半乳糖苷酶(IPTG)进行诱导,从pET28::KLA1(KAS2、KAS2-LA1、KAS1-SA1、多KAS-KLA1)构建体表达重组蛋白。产生的所有重组蛋白为6×His标签融合蛋白,在变性条件下使用NI-NTA纯化系统(Invitrogen)将其纯化。简单而言,将大肠杆菌(DE3)单一集落转化体接种至20mL含有50μg/ml卡那霉素的Luria-Bertani(LB)培养基,于37℃振荡过夜。然后用该接种物接种至1L含有50μg/ml卡那霉素的LB中。允许该培养物的OD600达到0.5-0.7(对数中期),然后用0.1mM异丙基IPTG、于37℃、200rpm振荡下诱导蛋白表达2小时。收获细胞(7,500g),并在变性结合缓冲液(8M尿素、pH 8.0的20mM磷酸钠和500mM NaCl)中重悬,并利用Branson Sonifer 250细胞破碎仪(Branson UltronicsCorporation,Danbury,CT)、使用装置3上的微尖端在冰上超声破碎细胞,每次15秒,共3次,每次间隔30秒,然后于4℃下,39,000g离心30分钟。通过上样至预先平衡的Ni-NTA琼脂糖柱从上清中纯化重组蛋白,然后用变性洗涤缓冲液(8M尿素、pH 6.0的20mM磷酸钠和500mMNaCl)进行洗涤以洗脱未结合的蛋白。然后用10倍体积的结合缓冲液B洗涤柱,并用变性洗脱缓冲液(8M尿素、pH 6.0的20mM磷酸钠、500mM NaCl和0.5M咪唑)洗脱重组蛋白。用2M尿素的PBS透析纯化的蛋白,并保存于-80℃。
通过SDS-PAGE分析重组蛋白样品,并用在线ProtParam(http://au.expasy.org/ tools/protparam.html)确定它们的分子量。通过Bio-Rad蛋白测定、使用BSA作为标准测定所有样品的蛋白浓度。
免疫和小鼠牙周炎模型
按照之前的报道所述(3)进行小鼠牙周炎实验,并且该实验由墨尔本大学动物实验道德规范委员会(University of Melbourne Ethics Committeefor AnimalExperimentation)准许。6-8周龄的BALB/c小鼠(每组12只小鼠)饲养在微型隔离间中,用50μg的重组蛋白或RgpA-Kgp复合物之一、2×109福尔马林杀死的牙龈卟啉单胞菌菌株W50细胞或PBS对上述小鼠进行皮下免疫(s.c.100μl);用弗氏不完全佐剂(IFA)乳化每种抗原。30天后,用抗原对小鼠进行加强免疫(s.c.注射,IFA乳化),并且在12天后从眼球后血管丛取血。第二次免疫后4天,给予小鼠去离子水配制的1mg/ml卡那霉素(Sigma-Aldrich,NewSouth Wales,Australia),使小鼠自由摄取持续7天。抗生素治疗后3天(取血后2天),用1×1010活牙龈卟啉单胞菌W50(25μl)对小鼠口腔接种4次,每次间隔2天,所述活牙龈卟啉单胞菌W50在含有2%(重量/体积)羧甲基纤维素(CMC;Sigma-Aldrich,NewSouth Wales,Australia)的PG缓冲液(50mM Tris-HCl、150mM NaCl、5mMCaCl2和5mM半胱氨酸-HCl,pH8.0)中,并且仅使用含有2%(重量/体积)CMC的PG缓冲液对对照组进行假感染。在厌氧室中制备接种物,然后立即将其施用于上颌臼齿的齿龈边缘。两周后,小鼠再接受4次给药的(每次间隔2天),每次给药为含有2%(重量/体积)CMC的PG缓冲液中的1×1010活牙龈卟啉单胞菌W50细胞(25μl)。通过血琼脂上的计数验证每个接种物的活细菌数。用软的粉状食物(Barastock,Australia)喂养小鼠,并将小鼠饲养在配备有加高的丝网底部的笼子中以防止接触到垫料。最后给药后4周,从小鼠眼球后血管丛取血并处死小鼠,取出上颌骨并切成两半,将一半(右侧)用于牙槽骨丧失检测,另一半(左侧)用于实时PCR。
将右半上颌骨在去离子水中沸煮(1分钟),机械去肉,并浸入2%(重量/体积)氢氧化钾中(16小时、25℃)。然后洗涤该半块上颌骨(用去离子水洗涤两次),并将其浸入3%(重量/体积)过氧化氢中(6小时、25℃)。洗涤该半块上颌骨(用去离子水洗涤两次)后,用0.1%(重量/体积)的亚甲基蓝水溶液对其进行染色,并用安装在解剖显微镜上的Olympus DP12数码相机捕获每个半块上颌骨的颊侧的数码图像,用OLYSIA BioReport软件3.2版(Olympus Australia Pty Ltd.,New South Wales,Australia)评价水平骨丧失。水平骨丧失是发生在水平面上、垂直于牙槽嵴(ABC)的丧失,其导致嵴高度的降低。将每个半块上颌骨排列,使的每颗齿的图像的臼齿颊尖侧和舌尖侧相叠,并且捕获的图像在框内标有测微计标尺,使得可以将每个图像的测量结果标准化。用OLYSIA BioReport软件3.2版成像软件测量了每个臼齿的从釉质牙骨质界至ABC的面积。由一个检测者使用随机化的并且盲法的方案测定骨丧失检测结果两次。
通过ELISA确定亚类抗体
为了确定小鼠血清的亚类抗体应答,按照以下一式三份地进行酶联免疫吸附测定(ELISA):用含有5μg/ml的福尔马林杀死的牙龈卟啉单胞菌W50溶液包被平底聚乙烯微量滴定板的孔(Dynatech Laboratories,McLean,VA),所述牙龈卟啉单胞菌W50配制在pH 7.0、含有0.1%(体积/体积)吐温20的磷酸盐缓冲盐水(PBS)(0.01M Na2HPO4、1.5mMKH2PO4、0.15M NaCl)(PBST)中。去除包被溶液后,将含有2%(重量/体积)脱脂奶粉的PBST加至孔中,于室温封闭未包被的塑料1小时。用PBST洗涤孔4次,将含有0.5%(重量/体积)脱脂乳的PBST(SK-PBST)中的小鼠血清系列稀释液添加至每孔,并于室温孵育16小时。用PBST洗涤孔6次后,添加SK-PBST中的针对小鼠IgM、IgA、IgG1、IgG2a、IgG2b或IgG3(Sigma,New SouthWales,Australia)的羊IgG的1/2000稀释液,并允许室温结合2小时。用PBST洗涤板6次,将SK-PBST中的辣根过氧化物酶偶联兔抗羊免疫球蛋白(Sigma,New South Wales,Australia)的1/5000稀释液添加至每孔,并于室温孵育1小时。用PBST洗涤孔6次后,通过将100μl ABTS底物[含有0.005%(体积/体积)过氧化氢的80mM柠檬酸中的0.9mM 2,2'-联氮-双(3-乙基苯-噻唑啉-6)磺酸,pH 4.0]添加至每孔来检测结合的抗体。用酶标仪(Bio-Rad酶标仪,型号450)检测415nm处的光密度。
SDS-PAGE凝胶电泳和Western印迹
用XCeII surelock Mini-Cell电泳系统分析重组蛋白(10μg)。将重组蛋白混于20μl还原样品缓冲液(10%[重量/体积]SDS、0.05%[重量/体积]溴酚蓝、25%[体积/体积]甘油和0.05%[体积/体积]2-巯基乙醇)中。用1.5M Tris-HCl将pH调至pH 8.0,然后将溶液于100℃加热5分钟。将重组蛋白(10μg/道)上样至Novex 12%(重量/体积)Tris-甘氨酸预制小型凝胶上,并用Novex电泳系统(Novex,San Diego,CA)、使用30-50mA的电流和125V的电压进行电泳。用0.25%w/v考马斯亮蓝R250将蛋白染色。
Kgp蛋白酶活性位点肽(KAS-2)序列的表位分析
通过在多中心(multipin)肽合成系统(Chiron Technologies,Melbourne,Australia)上、使用Fmoc化学的标准固相肽合成方法合成N末端生物素化的重叠的8个残基的肽(1个残基偏移,7个残基重叠)确定Lys特异性蛋白酶活性位点肽KAS2(433-468SEQ IDNo:28)的抗体结合位点。将pH7.4的0.1M PBS中的生物素化的肽(5μg/ml)与卵白素包被的平板结合,于4℃过夜(Nunc,NSW Australia)。用PBST对孔洗涤4次后,用1:1000稀释在1%w/v脱脂奶粉中的的小鼠血清、按照Chiron Technologies的操作说明、通过ELISA进行平板结合肽的表位定位,所述脱脂奶粉配制在含有0.1%v/v吐温20、pH 7.4的0.1M PBS中(SK-PBST)。用PBST将孔洗涤6次后,将抗小鼠IgG的羊IgG(Sigma,New South Wales,Australia)的1/2,000稀释液添加于SK-PBST中,并允许室温下结合2小时。用PBST将平板洗涤6次,并将SK-PBST中的辣根过氧化物酶偶联兔抗羊免疫球蛋白(Sigma,New South Wales,Australia)的1/5,000稀释液添加至每孔,并于室温孵育1小时。用PBST将孔洗涤6次后,通过将100μl ABTS底物[含有0.005%(体积/体积)过氧化氢的80mM柠檬酸中的0.9mM 2,2'-联氮-双(3-乙基苯-噻唑啉-6)磺酸,pH 4.0]添加至每孔来检测结合的抗体。用酶标仪(Bio-Rad酶标仪,型号450)测定415nm处的光密度。
统计学分析
用单因素方差分析(ANOVA)和Dunnett's T3检验(适用于Windows的SPSS,版本12)对骨丧失数据进行统计学分析。使用SPSS软件(适用于Windows的SPSS,版本12)、利用Student's t检验对IgA、IgM和IgG亚类抗体滴度进行统计学分析。
实施例2
重组蛋白(KsA1、KLA1、KAS1-KsA1和KAS2-KLA1)的表征和纯化
为了表征Kgp粘附素A1结构域片段和嵌合体Kgp蛋白酶与Kgp粘附素A1结构域片段对抗牙龈卟啉单胞菌感染的保护能力,我们表达并纯化了以下重组蛋白:KsA1、KLA1、KAS1-KsA1和KAS2-KLA1。使用镍螯合亲和层析从包涵体纯化重组蛋白(KsA1和KLA1)和重组嵌合蛋白(KAS1-KsA1和KAS2-KLA1),并通过SDS-PAGE分析所述纯化的蛋白(图1)。每一种纯化的重组蛋白由对应于KAS2-KLA1、KLA1、KsA1和KAS1-KsA1的分子量为40、36、31和32kDa的一个主要蛋白条带组成,并且这些分子量与用ProtParam计算出的His标签重组蛋白的分子量相对应。为了表征重组蛋白的免疫原性,将KsA1、KLA1、KAS1-KsA1和KAS2-KLA1用来免疫小鼠,并将血清用来探测用KAS2肽包被的平板和用福尔马林杀死的牙龈卟啉单胞菌W50细胞包被的平板(图2)。发现重组嵌合蛋白KAS1-KsA1和KAS2-KLA1抗血清以与KAS2特异性抗血清(KAS2-白喉类毒素缀合物)相似的水平识别KAS2肽(图2A),而且也识别福尔马林杀死的牙龈卟啉单胞菌W50细胞(图2B)。然而,针对重组蛋白KLA1的抗血清仅识别杀死的牙龈卟啉单胞菌W50细胞(图2B)。
实施例3
用重组蛋白(KsA1、KLA1、KAS1-KsA1和KAS2-KLA1)进行的免疫对小鼠牙周炎模型中牙龈卟啉单胞菌诱导的牙槽骨丧失的作用
使用基于Baker et al(4)的报道而改进的牙周骨丧失的小鼠模型,将重组蛋白KsA1、KLA1、KAS1-KsA1和KAS2-KLA1、福尔马林杀死的牙龈卟啉单胞菌菌株W50和RgpA-Kgp复合物用于确定和比较对牙龈卟啉单胞菌诱导的牙槽骨丧失所产生的保护。用重组蛋白KsA1、KLA1、KAS1-KsA1或KAS2-KLA1、RgpA-Kgp复合物或福尔马林杀死的牙龈卟啉单胞菌菌株W50(FK-W50)细胞或单独的PBS佐剂免疫小鼠(第0天和第30天),然后用活牙龈卟啉单胞菌W50口腔激发小鼠。用所有重组抗原、RgpA-Kgp复合物和FK-W50细胞进行的免疫保护BALB/c小鼠抵抗牙龈卟啉单胞菌诱导的牙槽骨丧失,因为这些动物表现出显著(p<0.001)少于PBS免疫组的骨丧失(图3)。然而,用KAS2-KLA1免疫的小鼠的骨丧失显著少于用KLA1(p<0.01)、KsA1(p<0.001)、RgpA-Kgp复合物(p<0.001)、FK-W50细胞免疫的小鼠(p<0.001)和未激发的小鼠(p<0.001)。用KAS2-KLA1和KAS1-KsA1免疫的小鼠的骨丧失之间不存在显著差异。此外,用KAS1-KsA1免疫的小鼠的骨丧失显著少于未激发的小鼠(p<0.01)和用RgpA-Kgp复合物免疫的小鼠(p<0.05),但与用KsA1、KLA1和FK-W50免疫的小鼠不存在显著差异。用KsA1、KLA1、RgpA-Kgp复合物和FK-W50免疫小鼠的骨丧失之间不存在显著差异。
实施例4
用重组蛋白(KsA1、KLA1、KAS1-KsA1和KAS2-KLA1)进行免疫在小鼠牙周炎模型中所诱导的抗体亚类应答
在用活牙龈卟啉单胞菌细胞口腔接种激发之前和之后,对小鼠进行取血并通过离心收集血清。图4表明在小鼠牙周炎模型中,对于每种免疫原(KsA1、KLA1、KAS1-KsA1或KAS2-KLA1或福尔马林杀死的牙龈卟啉单胞菌菌株W50(FK-W50)细胞),对福尔马林杀死的牙龈卟啉单胞菌W50细胞的抗体亚类反应性。所有保护性免疫原诱导对FK-W50的高IgG抗体滴度。此外,每种保护性免疫原所诱导的主要抗体亚类是IgG1,仅伴随弱免疫反应性的IgG2a、IgG2b和IgG3FK-W50特异性抗体(图4)。在口腔接种之前(图4A)和之后(图4B),每种免疫原所诱导的主要抗体亚类都是IgG1。
实施例5
KAS2(433-468)的表位定位
合成了KAS2(433-468)的重叠的生物素化的8个残基的肽(1个残基偏移,7个残基重叠),并将其用来包被链霉亲和素包被的平板。然后利用来自KAS1-KsA1、KAS2-KLA1和KAS2-白喉类毒素缀合物免疫的小鼠的抗血清来鉴定抗体结合表位(图5)。光密度(415nm)高于背景2倍被认为是阳性抗体应答(阈值OD)。所述抗血清识别来自SEQ ID No.28的下述肽序列,即KAS1-KsA1识别肽435-442、436-443、445-452、446-453和447-454(阈值OD=0.07,图5A),而KAS2-KLA1识别肽435-442、447-454和448-455(阈值ID=0.07,图5A)。这提示多个最小表位的识别,即肽436-442(VSFANYT及其变体VGFANYT)、肽447-452(ETAWAD及其变体ETSWAD)和肽448-453(TAWADP及其变体TSWADP)。包括肽436-442表位的肽包括GVSFANYT、GVGFANYT、VSFANYTA和VGFANYTA。包括肽447-452和/或448-453表位的肽包括SETAWAD、SETSWAD、ETAWADP、ETSWADP、TAWADPL和TSWADPL,更具体是GSETAWAD、GSETSWAD、SETAWADP、SETSWADP、ETAWADPL、ETSWADPL、TAWADPLL和TSWADPLL。
实施例6
用于与蛋白缀合的KAS和RAS肽的合成
手动或用CEM微波肽合成仪来合成肽。整个过程中使用Fmoc化学的标准固相肽合成方案。使用Rink-linker来源的AM-sure树脂(AAPPTEC,KY,USA)将肽组装为羧酰胺形式。用4当量Fmoc-氨基酸与6当量DIPEA通过HBTU/HOBt活化完成连接。Fmoc基团由含20%哌啶的1MHOBt/DMF移除。
使携带KAS或RAS肽的树脂在DMF中膨胀,由含有2%v/v哌啶的DMF中的2%v/v DBU将N末端Fmoc基团去除。然后,用5当量SAMA-OPfp和5当量HOBt使N末端氨基衍生带有S-乙酰巯基乙酸(SAMA)基团。通过三硝基苯磺酸(TNBSA)测试监测所述反应。当得到阴性TNBSA测试时,洗涤树脂(5×DMF、3×DCM和3×乙醚)。然后真空干燥树脂。用TFA:苯酚:TIPS:EDT:水(92:2:2:2:2)切割混合物从树脂载体切割肽,根据肽的精氨酸含量,切割进行2.5小时或4小时。切割后,通过过滤去除树脂,并在氮气流下将滤出液浓缩至约1mL。将肽产物在冷的醚中沉淀后,将它们离心并洗涤3次。将肽沉淀物溶解在5-10mL含有0.1%v/v TFA的水中,并通过离心去除不溶的残留物。通过RP-HPLC纯化肽。
多种不同的化学部分可以用来对肽进行衍生以用于与蛋白缀合,可以引入反应性基团,诸如卤化物(溴代、氯代和碘代)、马来酰亚胺、琥珀酰亚胺基、肼基、肟基、硫醇,然后可以将其用于使衍生的肽与诸如KgpA1的蛋白缀合,所述缀合通过蛋白的天然半胱氨酸残基或经过衍生而具有的允许进行化学连接而形成肽-蛋白缀合物的互补反应性基团。
SAMA-肽与KA1的缀合
向含有10mg/mL重组KA1或RgpA-Kgp复合物的其他粘附素结构域的磷酸盐缓冲盐溶液(0.1M磷酸钠、0.9%NaCl,pH 7.4)中添加0.1mL的含有1%w/v马来酰亚胺基苯甲酰-N-羟基琥珀酰亚胺酯(MBS)的DMF溶液。30分钟后,去除未反应的MBS,并使用缀合缓冲液(0.1M磷酸钠、5mM EDTA,pH 6.0)中平衡的PD10柱(Pharmacia,NSW,Australia)、通过凝胶过滤收集MBS修饰的KA1。将纯化的SAMA-肽(1.3μmol)溶解于200μL含有0.5M Tris、2mM EDTA,pH6.0的6M盐酸胍中,用800μL MilliQ水稀释,并通过添加溶解于MilliQ水中的25μL 2MNH2OH(40当量)原位脱保护。将收集的MBS-KA1立即与脱保护的SAMA-肽反应,并于室温搅拌1小时。使用pH 7.4的PBS平衡的PD10柱、通过凝胶过滤将肽-KA1缀合物与未反应的肽分离并冻干。用Ellmans测试监测所述反应。
实施例7
抗体制备
通过用蛋白进行皮下免疫在小鼠中产生针对重组蛋白的多克隆抗血清。在第0天时用配制在弗氏不完全佐剂中的25μg蛋白免疫小鼠,并在第30天时用配制在弗氏不完全佐剂中的25μg蛋白免疫小鼠。用标准方法进行免疫。获得了具有针对蛋白的高滴度的多克隆抗血清。如果需要,可以使用标准方法获得特异性靶向于重组蛋白的单克隆抗体。
实施例8
用于产生抗体的免疫
使用在弗氏不完全佐剂(IFA)中乳化的50μg KAS2-LA1嵌合体和抗原对6-8周龄的BALB/c小鼠或CD1(瑞士远交系小鼠)(每组10只小鼠)进行皮下免疫(s.c.100μL)。30天后,用抗原(s.c.注射,用IFA乳化)加强;12天后处死小鼠并进行心脏取血以收集血清。
通过ELISA确定亚类抗体
为了确定小鼠血清的亚类抗体应答,如下一式三份地进行酶联免疫吸附测定(ELISA):用配制在含有0.1%(体积/体积)的吐温20的pH 7.0的磷酸盐缓冲盐溶液(PBS)(0.01M Na2HPO4、1.5mM KH2PO4、0.15M NaCl)(PBST)中的5-μg/ml KAS2-LA1嵌合体或福尔马林杀死的牙龈卟啉单胞菌W50或RgpA-Kgp复合物溶液包被平底聚乙烯微量滴定板孔(DynatechLaboratories,McLean,VA)。去除包被溶液后,将含有2%(重量/体积)脱脂奶粉的PBST加至孔中,于室温封闭未包被的塑料1小时。用PBST将孔洗涤4次后,将含有0.5%(重量/体积)脱脂乳的PBST(SK-PBST)中的小鼠血清连续稀释液添加至每孔,并于室温孵育16小时。用PBST将孔洗涤6次后,将抗小鼠IgM、IgA、IgG1、IgG2a、IgG2b或IgG3的羊IgG(Sigma,New South Wales,Australia)的1/2,000稀释液添加至SK-PBST,并允许室温结合2小时。用PBST将平板洗涤6次,并将SK-PBST中的辣根过氧化物酶偶联兔抗羊免疫球蛋白(Sigma,NewSouth Wales,Australia)的1/5,000稀释液添加至每孔,并于室温孵育1小时。用PBST将孔洗涤6次后,通过将100μl ABTS底物[含有0.005%(体积/体积)过氧化氢的80mM柠檬酸中的0.9mM 2,2'-联氮-双(3-乙基苯-噻唑啉-6)磺酸,pH 4.0]添加至每孔来检测结合的抗体。用酶标仪(Bio-Rad酶标仪,型号450)检测415nm处的光密度。
远交系(CD1,瑞士)小鼠中用重组蛋白KAS2-KLA1进行免疫所诱导的抗体亚类应答
用KAS2-LA1嵌合体对CD1(瑞士)小鼠进行免疫,对小鼠进行取血并通过离心收集血清。图6表明对KAS2-LA1嵌合体、福尔马林杀死的牙龈卟啉单胞菌W50细胞和RgpA-Kgp复合物的抗体亚类反应。KAS2-LA1嵌合体诱导强IgG抗体,主要为识别KAS2-LA1嵌合体并与福尔马林杀死的牙龈卟啉单胞菌W50细胞和RgpA-Kgp复合物发生强交叉反应的IgG1抗体应答(图6)。此外,KAS2-LA1嵌合体仅诱导弱的免疫反应性IgG2a、IgG2b和IgG3抗原特异性抗体(图6)。
实施例9
Kgp结构模型的开发和活性位点表面可接近序列的鉴定
我们的工作已经表明:Kgp蛋白酶活性位点肽是高免疫原性的,并诱导针对牙龈卟啉单胞菌诱导的骨丧失的高水平保护作用。在试图鉴定其它蛋白酶活性位点肽作为疫苗候选者时,使用Sybyl7.3的Orchestrar程序套装开发了Kgp催化结构域的模型(图7)。该模型是基于来自牙龈卟啉单胞菌的RgpB蛋白酶的PDB结构1crv。该蛋白具有23.58%的配对同一性,且Z评分是25.09(高度可信模型)。Meta-PPisp蛋白相互作用服务器预测了Kgp的两个蛋白-蛋白相互作用表面:底物结合表面(如RgpB中)和Kgp特有的第二表面。RgpB和Kgp模型间的主要差异在于使第二相互作用表面和19个残基的空位(Val526至Phe545)处于框内的环,在落入第二相互作用表面中的Kgp中不能模拟该19个残基的空位。图7显示的是Kgp模型,较粗的带表示Kgp的蛋白酶活性位点周围的表面可接近序列,发现所述表面可接近序列是Asp388-Gln394、Leu421-Ala423、Ala443-Glu447加Ala451、Asn510-Trp513和ILe570-Gly577加Tyr580。从所述模型(图6)可明显观察到,除KAS2(A)之外,三个其他序列KAS4(Asp388-Val395)(B)、KAS5(Asn510-Asp516)(C)和KAS6(ILe570-Tyr580)(D)是重要的并具有成为疫苗靶标的足够长度。因此,可以依次产生具有这些肽中每一个的重组嵌合蛋白,并将其连接至KLA1的N末端,以产生多KAS-KLA1,该多KAS-KLA1可以被用来诱导免疫应答,并从而对于牙龈卟啉单胞菌相关疾病或状态进行保护。
实施例10
模拟Arg-X-蛋白酶从而鉴定位于催化位点侧翼的免疫原性区的过程
按照Eichinger A、Beisel HG、Jacob U、Huber R、Medrano FJ、BanbulaA、PotempaJ、Travis J、Bode W,Crystal structure of gingipain R:anArg-specific bacterialcysteine proteinasewith a caspase-like fold(牙龈卟啉蛋白酶R的晶体结构:具有半胱天冬酶样折叠的Arg-特异性细菌半胱氨酸蛋白酶)EMBO J.1999年10月15日;18(20):5453-62的方法确定Arg-X-蛋白酶的三维结构。
实施例11
下述是含有抗体的牙膏配方的实施例
实施例12
下述是牙膏配方的实施例
实施例13
下述是牙膏配方的实施例.
实施例14
下述是牙膏配方的实施例.
实施例15
下述是液体牙膏配方的实施例
实施例16
下述是漱口剂配方的实施例
实施例17
下述是漱口剂配方的实施例
实施例18
下述是锭剂配方的实施例
实施例19
下述是牙龈按摩乳膏配方的实施例
实施例20
下述是口香糖配方的实施例
实施例21
下述是药物配方的实施例
组分
%w/w
人源化特异性单克隆抗体 10
无菌磷酸盐缓冲盐水 90
实施例22
下述是牙周凝胶配方的实施例
应当理解,尽管在本文中对本发明进行了详细描述,但是这些实施例仅为示例性目的。对分子生物学、牙科诊断学和相关学科领域的技术人员显而易见的对本发明实施方案的其他改动也意图包括在本发明的范围内。
参考文献
1.McKee,A.S.,A.S.McDermid,A.Baskerville,A.B.Dowsett,D.C.Ellwood,andP.D.Marsh.1986.Effect of hemin on the physiology and virulence of Bacteroidesgingivalis W50(血红素对龈拟杆菌W50的生理学和毒力的影响).Infect.Immun.52:349-355.
2.Slots,J.1982.Importance of black-pigmented Bacteroides in humanperiodontal disease(黑色素拟杆菌在人牙周病中的重要性).Host parasiteinteractions in periodontal diseases(牙周病中宿主寄生虫的相互作用).AmericanSociety for Microbiology.
3.O'Brien-Simpson,N.M.,R.Pathirana,R.A.Paolini,Y.-Y.Chen,P.D.Veith,T.V.,R.N.Pike,N.Alley,and E.C.Reynolds.2005.An immune response directed toproteinase and adhesin functional epitopes protects against Porphyromonasgingivalis-induced bone loss(针对蛋白酶和粘附素功能表位的免疫应答防止牙龈卟啉单胞菌诱导的骨丧失).Journal of Immunology 175:3980-3989.
4.Baker,P.J.,R.T.Evans,and D.C.Roopenian.1994.Oral infection withPorphyromonas gingivalis and induced alveolar bone loss in immunocompetentand severe combined immunodeficient mice(免疫活性小鼠和重度联合免疫缺陷小鼠中牙龈卟啉单胞菌口腔感染及诱导的牙槽骨丧失).Arch Oral Biol 39:1035-1040.
序列表
<110> 口腔健康澳洲私人有限公司
<120> 牙龈卟啉单胞菌感染的预防、治疗和诊断
<130> D17C11432CN
<150> AU2008904476
<151> 2008-08-29
<150> AU2008905483
<151> 2008-10-23
<150> US61/151,132
<151> 2009-02-09
<150> AU2009903052
<151> 2009-06-30
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20 25 30
Ala Ser Ser Asn Leu Tyr Ser Ala Asn Phe Glu Tyr Leu Ile Pro Ala
35 40 45
Asn Ala Asp Pro Val Val Thr Thr Gln Asn Ile Ile Val Thr Gly Gln
50 55 60
Gly Glu Val Val Ile Pro Gly Gly Val Tyr Asp Tyr Cys Ile Thr Asn
65 70 75 80
Pro Glu Pro Ala Ser Gly Lys Met Trp Ile Ala Gly Asp Gly Gly Asn
85 90 95
Gln Pro Ala Arg Tyr Asp Asp Phe Thr Phe Glu Ala Gly Lys Lys Tyr
100 105 110
Thr Phe Thr Met Arg Arg Ala Gly Met Gly Asp Gly Thr Asp Met Glu
115 120 125
Val Glu Asp Asp Ser Pro Ala Ser Tyr Thr Tyr Thr Val Tyr Arg Asp
130 135 140
Gly Thr Lys Ile Lys Glu Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp
145 150 155 160
Gly Val Ala Ala Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr
165 170 175
Ala Gly Val Ser Pro Lys Val Cys Lys Asp Val Thr Val Glu Gly Ser
180 185 190
Asn Glu Phe Ala Pro Val Gln Asn Leu Thr Gly Ser Ser Val Gly Gln
195 200 205
Lys Val Thr Leu Lys Trp Asp Ala Pro Asn Gly Thr Pro Asn Pro Asn
210 215 220
Pro Asn Pro Asn Pro Asn Pro Gly Thr Thr Leu Ser Glu Ser Phe Glu
225 230 235 240
Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly
245 250 255
His Gly Trp Lys Pro Gly Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser
260 265 270
Asn Gly Cys Val Tyr Ser Glu Ser Phe Gly Leu Gly Gly Ile Gly Val
275 280 285
Leu Thr Pro Asp Asn Tyr Leu Ile Thr Pro Ala Leu Asp Leu Pro Asn
290 295 300
Gly Gly
305
<210> 38
<211> 362
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 38
Ser Gly Gln Ala Glu Ile Val Leu Glu Ala His Asp Val Trp Asn Asp
1 5 10 15
Gly Ser Gly Tyr Gln Ile Leu Leu Asp Ala Asp His Asp Gln Tyr Gly
20 25 30
Gln Val Ile Pro Ser Asp Thr His Thr Leu Trp Pro Asn Cys Ser Val
35 40 45
Pro Ala Asn Leu Phe Ala Pro Phe Glu Tyr Thr Val Pro Glu Asn Ala
50 55 60
Asp Pro Ser Cys Ser Pro Thr Asn Met Ile Met Asp Gly Thr Ala Ser
65 70 75 80
Val Asn Ile Pro Ala Gly Thr Tyr Asp Phe Ala Ile Ala Ala Pro Gln
85 90 95
Ala Asn Ala Lys Ile Trp Ile Ala Gly Gln Gly Pro Thr Lys Glu Asp
100 105 110
Asp Tyr Val Phe Glu Ala Gly Lys Lys Tyr His Phe Leu Met Lys Lys
115 120 125
Met Gly Ser Gly Asp Gly Thr Glu Leu Thr Ile Ser Glu Gly Gly Gly
130 135 140
Ser Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu
145 150 155 160
Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn
165 170 175
His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys
180 185 190
Val Cys Lys Asp Val Thr Val Glu Gly Ser Asn Glu Phe Ala Pro Val
195 200 205
Gln Asn Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys Trp
210 215 220
Asp Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Asn Pro Asn Pro Asn
225 230 235 240
Pro Asn Pro Gly Thr Thr Thr Leu Ser Glu Ser Phe Glu Asn Gly Ile
245 250 255
Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly His Gly Trp
260 265 270
Lys Pro Gly Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser Asn Gly Cys
275 280 285
Val Tyr Ser Glu Ser Phe Gly Leu Gly Gly Ile Gly Val Leu Thr Pro
290 295 300
Asp Asn Tyr Leu Ile Thr Pro Ala Leu Asp Leu Pro Asn Gly Gly Lys
305 310 315 320
Leu Thr Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser Glu His
325 330 335
Tyr Ala Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser Asn Phe Thr
340 345 350
Asn Ala Leu Leu Glu Glu Thr Ile Thr Ala
355 360
<210> 39
<211> 141
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 39
Asp Asp Tyr Val Phe Glu Ala Gly Lys Lys Tyr His Phe Leu Met Lys
1 5 10 15
Lys Met Gly Ser Gly Asp Gly Thr Glu Leu Thr Ile Ser Glu Gly Gly
20 25 30
Gly Ser Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys
35 40 45
Glu Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly
50 55 60
Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro
65 70 75 80
Lys Val Cys Lys Asp Val Thr Val Glu Gly Ser Asn Glu Phe Ala Pro
85 90 95
Val Gln Asn Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys
100 105 110
Trp Asp Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Asn Pro Asn Pro
115 120 125
Asn Pro Asn Pro Gly Thr Thr Thr Leu Ser Glu Ser Phe
130 135 140
<210> 40
<211> 119
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 40
Ala Asp Phe Thr Glu Thr Phe Glu Ser Ser Thr His Gly Glu Ala Pro
1 5 10 15
Ala Glu Trp Thr Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu
20 25 30
Cys Leu Ser Ser Gly Gln Leu Asp Trp Leu Thr Ala His Gly Gly Ser
35 40 45
Asn Val Val Ser Ser Phe Ser Trp Asn Gly Met Ala Leu Asn Pro Asp
50 55 60
Asn Tyr Leu Ile Ser Lys Asp Val Thr Gly Ala Thr Lys Val Lys Tyr
65 70 75 80
Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly Asp His Tyr Ala Val Met
85 90 95
Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr Val Val Phe Glu
100 105 110
Glu Thr Pro Asn Gly Ile Asn
115
<210> 41
<211> 133
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 41
Pro Gln Ser Val Trp Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr
1 5 10 15
Lys Tyr Val Ala Phe Arg His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile
20 25 30
Leu Leu Asp Asp Ile Gln Phe Thr Met Gly Gly Ser Pro Thr Pro Thr
35 40 45
Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly
50 55 60
Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His
65 70 75 80
Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Lys
85 90 95
Cys Val Asn Val Thr Val Asn Ser Thr Gln Phe Asn Pro Val Gln Asn
100 105 110
Leu Thr Ala Glu Gln Ala Pro Asn Ser Met Asp Ala Ile Leu Lys Trp
115 120 125
Asn Ala Pro Ala Ser
130
<210> 42
<211> 120
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 42
Ala Glu Val Leu Asn Glu Asp Phe Glu Asn Gly Ile Pro Ala Ser Trp
1 5 10 15
Lys Thr Ile Asp Ala Asp Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro
20 25 30
Pro Pro Gly Gly Ser Ser Phe Ala Gly His Asn Ser Ala Ile Cys Val
35 40 45
Ser Ser Ala Ser Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro Asp Asn
50 55 60
Tyr Leu Val Thr Pro Glu Leu Ser Leu Pro Gly Gly Gly Thr Leu Thr
65 70 75 80
Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser Glu His Tyr Ala
85 90 95
Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser Asn Phe Ala Asn Ala
100 105 110
Leu Leu Glu Glu Val Leu Thr Ala
115 120
<210> 43
<211> 185
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 43
Thr Val Val Thr Ala Pro Glu Ala Ile Arg Gly Thr Arg Ala Gln Gly
1 5 10 15
Thr Trp Tyr Gln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys Tyr Val
20 25 30
Ala Phe Arg His Phe Gly Cys Thr Asp Phe Phe Trp Ile Asn Leu Asp
35 40 45
Asp Val Val Ile Thr Ser Gly Asn Ala Pro Ser Tyr Thr Tyr Thr Ile
50 55 60
Tyr Arg Asn Asn Thr Gln Ile Ala Ser Gly Val Thr Glu Thr Thr Tyr
65 70 75 80
Arg Asp Pro Asp Leu Ala Thr Gly Phe Tyr Thr Tyr Gly Val Lys Val
85 90 95
Val Tyr Pro Asn Gly Glu Ser Ala Ile Glu Thr Ala Thr Leu Asn Ile
100 105 110
Thr Ser Leu Ala Asp Val Thr Ala Gln Lys Pro Tyr Thr Leu Thr Val
115 120 125
Val Gly Lys Thr Ile Thr Val Thr Cys Gln Gly Glu Ala Met Ile Tyr
130 135 140
Asp Met Asn Gly Arg Arg Leu Ala Ala Gly Arg Asn Thr Val Val Tyr
145 150 155 160
Thr Ala Gln Gly Gly His Tyr Ala Val Met Val Val Val Asp Gly Lys
165 170 175
Ser Tyr Val Glu Lys Leu Ala Val Lys
180 185
<210> 44
<211> 119
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 44
Ala Asp Phe Thr Glu Thr Phe Glu Ser Ser Thr His Gly Glu Ala Pro
1 5 10 15
Ala Glu Trp Thr Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu
20 25 30
Cys Leu Ser Ser Gly Gln Leu Asp Trp Leu Thr Ala His Gly Gly Thr
35 40 45
Asn Val Val Ser Ser Phe Ser Trp Asn Gly Met Ala Leu Asn Pro Asp
50 55 60
Asn Tyr Leu Ile Ser Lys Asp Val Thr Gly Ala Thr Lys Val Lys Tyr
65 70 75 80
Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly Asp His Tyr Ala Val Met
85 90 95
Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr Val Val Phe Glu
100 105 110
Glu Thr Pro Asn Gly Ile Asn
115
<210> 45
<211> 131
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 45
Pro Gln Ser Val Trp Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr
1 5 10 15
Lys Tyr Val Ala Phe Arg His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile
20 25 30
Leu Leu Asp Asp Ile Gln Phe Thr Met Gly Gly Ser Pro Thr Pro Thr
35 40 45
Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly
50 55 60
Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His
65 70 75 80
Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Lys
85 90 95
Cys Val Asn Val Thr Val Asn Ser Thr Gln Phe Asn Pro Val Lys Asn
100 105 110
Leu Lys Ala Gln Pro Asp Gly Gly Asp Val Val Leu Lys Trp Glu Ala
115 120 125
Pro Ser Ala
130
<210> 46
<211> 275
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 46
Ala Asn Glu Ala Lys Val Val Leu Ala Ala Asp Asn Val Trp Gly Asp
1 5 10 15
Asn Thr Gly Tyr Gln Phe Leu Leu Asp Ala Asp His Asn Thr Phe Gly
20 25 30
Ser Val Ile Pro Ala Thr Gly Pro Leu Phe Thr Gly Thr Ala Ser Ser
35 40 45
Asp Leu Tyr Ser Ala Asn Phe Glu Ser Leu Ile Pro Ala Asn Ala Asp
50 55 60
Pro Val Val Thr Thr Gln Asn Ile Ile Val Thr Gly Gln Gly Glu Val
65 70 75 80
Val Ile Pro Gly Gly Val Tyr Asp Tyr Cys Ile Thr Asn Pro Glu Pro
85 90 95
Ala Ser Gly Lys Met Trp Ile Ala Gly Asp Gly Gly Asn Gln Pro Ala
100 105 110
Arg Tyr Asp Asp Phe Thr Phe Glu Ala Gly Lys Lys Tyr Thr Phe Thr
115 120 125
Met Arg Arg Ala Gly Met Gly Asp Gly Thr Asp Met Glu Val Glu Asp
130 135 140
Asp Ser Pro Ala Ser Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys
145 150 155 160
Ile Lys Glu Gly Leu Thr Glu Thr Thr Tyr Arg Asp Ala Gly Met Ser
165 170 175
Ala Gln Ser His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val
180 185 190
Ser Pro Lys Val Cys Val Asp Tyr Ile Pro Asp Gly Val Ala Asp Val
195 200 205
Thr Ala Gln Lys Pro Tyr Thr Leu Thr Val Val Gly Lys Thr Ile Thr
210 215 220
Val Thr Cys Gln Gly Glu Ala Met Ile Tyr Asp Met Asn Gly Arg Arg
225 230 235 240
Leu Ala Ala Gly Arg Asn Thr Val Val Tyr Thr Ala Gln Gly Gly Tyr
245 250 255
Tyr Ala Val Met Val Val Val Asp Gly Lys Ser Tyr Val Glu Lys Leu
260 265 270
Ala Ile Lys
275
<210> 47
<211> 49
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 47
gaccatggct catcaccatc accatcacaa taccggagtc agctttgca 49
<210> 48
<211> 36
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 48
gactcgagtt atttgtcctt attagtgagt gctttc 36
<210> 49
<211> 31
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 49
gaccatggct tggggagaca atacgggtta c 31
<210> 50
<211> 27
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 50
gactcgagac ctccgttagg caaatcc 27
<210> 51
<211> 41
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 51
ccgtattgtc tccccatttg tccttattag tgagtgcttt c 41
<210> 52
<211> 37
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 52
cactaataag gacaaatggg gagacaatac gggttac 37
<210> 53
<211> 51
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 53
catggatctg agaccgcatg ggctgatcca cttttcttgt tggatgccga t 51
<210> 54
<211> 62
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 54
ccatggcttt gaataccgga gtcagctttg caaactatac agcgcatgga tctgagaccg 60
ca 62
<210> 55
<211> 25
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 55
ctcgaggaat gattcggaaa gtgtt 25
<210> 56
<211> 53
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 56
ccatggctga ttatagctgg aattcccagg tagtcagctt tgcaaactat aca 53
<210> 57
<211> 52
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 57
ctttgcaaac tatacagcgc atggatctga gaccgcatgg gctgatccac tt 52
<210> 58
<211> 52
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 58
atgggctgat ccacttctga attcttattg gggcgagatc ggcaatatta cc 52
<210> 59
<211> 52
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 59
gatcggcaat attacccata ttggtgctca ttacgcttgg ggagacaata cg 52
<210> 60
<211> 52
<212> DNA
<213> 牙龈卟啉单胞菌
<400> 60
ctcgagacct ccgttaggca aatccaatgc cggtgttatc agatagttgt ca 52
<210> 61
<211> 1706
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 61
Met Lys Asn Leu Asn Lys Phe Val Ser Ile Ala Leu Cys Ser Ser Leu
1 5 10 15
Leu Gly Gly Met Ala Phe Ala Gln Gln Thr Glu Leu Gly Arg Asn Pro
20 25 30
Asn Val Arg Leu Leu Glu Ser Thr Gln Gln Ser Val Thr Lys Val Gln
35 40 45
Phe Arg Met Asp Asn Leu Lys Phe Thr Glu Val Gln Thr Pro Lys Gly
50 55 60
Ile Gly Gln Val Pro Thr Tyr Thr Glu Gly Val Asn Leu Ser Glu Lys
65 70 75 80
Gly Met Pro Thr Leu Pro Ile Leu Ser Arg Ser Leu Ala Val Ser Asp
85 90 95
Thr Arg Glu Met Lys Val Glu Val Val Ser Ser Lys Phe Ile Glu Lys
100 105 110
Lys Asn Val Leu Ile Ala Pro Ser Lys Gly Met Ile Met Arg Asn Glu
115 120 125
Asp Pro Lys Lys Ile Pro Tyr Val Tyr Gly Lys Thr Tyr Ser Gln Asn
130 135 140
Lys Phe Phe Pro Gly Glu Ile Ala Thr Leu Asp Asp Pro Phe Ile Leu
145 150 155 160
Arg Asp Val Arg Gly Gln Val Val Asn Phe Ala Pro Leu Gln Tyr Asn
165 170 175
Pro Val Thr Lys Thr Leu Arg Ile Tyr Thr Glu Ile Thr Val Ala Val
180 185 190
Ser Glu Thr Ser Glu Gln Gly Lys Asn Ile Leu Asn Lys Lys Gly Thr
195 200 205
Phe Ala Gly Phe Glu Asp Thr Tyr Lys Arg Met Phe Met Asn Tyr Glu
210 215 220
Pro Gly Arg Tyr Thr Pro Val Glu Glu Lys Gln Asn Gly Arg Met Ile
225 230 235 240
Val Ile Val Ala Lys Lys Tyr Glu Gly Asp Ile Lys Asp Phe Val Asp
245 250 255
Trp Lys Asn Gln Arg Gly Leu Arg Thr Glu Val Lys Val Ala Glu Asp
260 265 270
Ile Ala Ser Pro Val Thr Ala Asn Ala Ile Gln Gln Phe Val Lys Gln
275 280 285
Glu Tyr Glu Lys Glu Gly Asn Asp Leu Thr Tyr Val Leu Leu Ile Gly
290 295 300
Asp His Lys Asp Ile Pro Ala Lys Ile Thr Pro Gly Ile Lys Ser Asp
305 310 315 320
Gln Val Tyr Gly Gln Ile Val Gly Asn Asp His Tyr Asn Glu Val Phe
325 330 335
Ile Gly Arg Phe Ser Cys Glu Ser Lys Glu Asp Leu Lys Thr Gln Ile
340 345 350
Asp Arg Thr Ile His Tyr Glu Arg Asn Ile Thr Thr Glu Asp Lys Trp
355 360 365
Leu Gly Gln Ala Leu Cys Ile Ala Ser Ala Glu Gly Gly Pro Ser Ala
370 375 380
Asp Asn Gly Glu Ser Asp Ile Gln His Glu Asn Val Ile Ala Asn Leu
385 390 395 400
Leu Thr Gln Tyr Gly Tyr Thr Lys Ile Ile Lys Cys Tyr Asp Pro Gly
405 410 415
Val Thr Pro Lys Asn Ile Ile Asp Ala Phe Asn Gly Gly Ile Ser Leu
420 425 430
Ala Asn Tyr Thr Gly His Gly Ser Glu Thr Ala Trp Gly Thr Ser His
435 440 445
Phe Gly Thr Thr His Val Lys Gln Leu Thr Asn Ser Asn Gln Leu Pro
450 455 460
Phe Ile Phe Asp Val Ala Cys Val Asn Gly Asp Phe Leu Phe Ser Met
465 470 475 480
Pro Cys Phe Ala Glu Ala Leu Met Arg Ala Gln Lys Asp Gly Lys Pro
485 490 495
Thr Gly Thr Val Ala Ile Ile Ala Ser Thr Ile Asn Gln Ser Trp Ala
500 505 510
Ser Pro Met Arg Gly Gln Asp Glu Met Asn Glu Ile Leu Cys Glu Lys
515 520 525
His Pro Asn Asn Ile Lys Arg Thr Phe Gly Gly Val Thr Met Asn Gly
530 535 540
Met Phe Ala Met Val Glu Lys Tyr Lys Lys Asp Gly Glu Lys Met Leu
545 550 555 560
Asp Thr Trp Thr Val Phe Gly Asp Pro Ser Leu Leu Val Arg Thr Leu
565 570 575
Val Pro Thr Lys Met Gln Val Thr Ala Pro Ala Gln Ile Asn Leu Thr
580 585 590
Asp Ala Ser Val Asn Val Ser Cys Asp Tyr Asn Gly Ala Ile Ala Thr
595 600 605
Ile Ser Ala Asn Gly Lys Met Phe Gly Ser Ala Val Val Glu Asn Gly
610 615 620
Thr Ala Thr Ile Asn Leu Thr Gly Leu Thr Asn Glu Ser Thr Leu Thr
625 630 635 640
Leu Thr Val Val Gly Tyr Asn Lys Glu Thr Val Ile Lys Thr Ile Asn
645 650 655
Thr Asn Gly Glu Pro Asn Pro Tyr Gln Pro Val Ser Asn Leu Thr Ala
660 665 670
Thr Thr Gln Gly Gln Lys Val Thr Leu Lys Trp Asp Ala Pro Ser Thr
675 680 685
Lys Thr Asn Ala Thr Thr Asn Thr Ala Arg Ser Val Asp Gly Ile Arg
690 695 700
Glu Leu Val Leu Leu Ser Val Ser Asp Ala Pro Glu Leu Leu Arg Ser
705 710 715 720
Gly Gln Ala Glu Ile Val Leu Glu Ala His Asp Val Trp Asn Asp Gly
725 730 735
Ser Gly Tyr Gln Ile Leu Leu Asp Ala Asp His Asp Gln Tyr Gly Gln
740 745 750
Val Ile Pro Ser Asp Thr His Thr Leu Trp Pro Asn Cys Ser Val Pro
755 760 765
Ala Asn Leu Phe Ala Pro Phe Glu Tyr Thr Val Pro Glu Asn Ala Asp
770 775 780
Pro Ser Cys Ser Pro Thr Asn Met Ile Met Asp Gly Thr Ala Ser Val
785 790 795 800
Asn Ile Pro Ala Gly Thr Tyr Asp Phe Ala Ile Ala Ala Pro Gln Ala
805 810 815
Asn Ala Lys Ile Trp Ile Ala Gly Gln Gly Pro Thr Lys Glu Asp Asp
820 825 830
Tyr Val Phe Glu Ala Gly Lys Lys Tyr His Phe Leu Met Lys Lys Met
835 840 845
Gly Ser Gly Asp Gly Thr Glu Leu Thr Ile Ser Glu Gly Gly Gly Ser
850 855 860
Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly
865 870 875 880
Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His
885 890 895
Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Val
900 905 910
Cys Lys Asp Val Thr Val Glu Gly Ser Asn Glu Phe Ala Pro Val Gln
915 920 925
Asn Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys Trp Asp
930 935 940
Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Asn Pro Asn Pro Asn Pro
945 950 955 960
Asn Pro Gly Thr Thr Thr Leu Ser Glu Ser Phe Glu Asn Gly Ile Pro
965 970 975
Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly His Gly Trp Lys
980 985 990
Pro Gly Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser Asn Gly Cys Val
995 1000 1005
Tyr Ser Glu Ser Phe Gly Leu Gly Gly Ile Gly Val Leu Thr Pro
1010 1015 1020
Asp Asn Tyr Leu Ile Thr Pro Ala Leu Asp Leu Pro Asn Gly Gly
1025 1030 1035
Lys Leu Thr Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser
1040 1045 1050
Glu His Tyr Ala Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser
1055 1060 1065
Asn Phe Thr Asn Ala Leu Leu Glu Glu Thr Ile Thr Ala Lys Gly
1070 1075 1080
Val Arg Ser Pro Glu Ala Met Arg Gly Arg Ile Gln Gly Thr Trp
1085 1090 1095
Arg Gln Lys Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala
1100 1105 1110
Phe Arg His Phe Gln Ser Thr Asp Met Phe Tyr Ile Asp Leu Asp
1115 1120 1125
Glu Val Glu Ile Lys Ala Asn Gly Lys Arg Ala Asp Phe Thr Glu
1130 1135 1140
Thr Phe Glu Ser Ser Thr His Gly Glu Ala Pro Ala Glu Trp Thr
1145 1150 1155
Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu Cys Leu Ser
1160 1165 1170
Ser Gly Gln Leu Asp Trp Leu Thr Ala His Gly Gly Thr Asn Val
1175 1180 1185
Val Ser Ser Phe Ser Trp Asn Gly Met Ala Leu Asn Pro Asp Asn
1190 1195 1200
Tyr Leu Ile Ser Lys Asp Val Thr Gly Ala Thr Lys Val Lys Tyr
1205 1210 1215
Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly Asp His Tyr Ala Val
1220 1225 1230
Met Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr Val Val
1235 1240 1245
Phe Glu Glu Thr Pro Asn Gly Ile Asn Lys Gly Gly Ala Arg Phe
1250 1255 1260
Gly Leu Ser Thr Glu Ala Asp Gly Ala Lys Pro Gln Ser Val Trp
1265 1270 1275
Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala
1280 1285 1290
Phe Arg His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile Leu Leu Asp
1295 1300 1305
Asp Ile Gln Phe Thr Met Gly Gly Ser Pro Thr Pro Thr Asp Tyr
1310 1315 1320
Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu
1325 1330 1335
Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His
1340 1345 1350
Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys
1355 1360 1365
Lys Cys Val Asn Val Thr Val Asn Ser Thr Gln Phe Asn Pro Val
1370 1375 1380
Lys Asn Leu Lys Ala Gln Pro Asp Gly Gly Asp Val Val Leu Lys
1385 1390 1395
Trp Glu Ala Pro Ser Ala Lys Lys Thr Glu Gly Ser Arg Glu Val
1400 1405 1410
Lys Arg Ile Gly Asp Gly Leu Phe Val Thr Ile Glu Pro Ala Asn
1415 1420 1425
Asp Val Arg Ala Asn Glu Ala Lys Val Val Leu Ala Ala Asp Asn
1430 1435 1440
Val Trp Gly Asp Asn Thr Gly Tyr Gln Phe Leu Leu Asp Ala Asp
1445 1450 1455
His Asn Thr Phe Gly Ser Val Ile Pro Ala Thr Gly Pro Leu Phe
1460 1465 1470
Thr Gly Thr Ala Ser Ser Asp Leu Tyr Ser Ala Asn Phe Glu Ser
1475 1480 1485
Leu Ile Pro Ala Asn Ala Asp Pro Val Val Thr Thr Gln Asn Ile
1490 1495 1500
Ile Val Thr Gly Gln Gly Glu Val Val Ile Pro Gly Gly Val Tyr
1505 1510 1515
Asp Tyr Cys Ile Thr Asn Pro Glu Pro Ala Ser Gly Lys Met Trp
1520 1525 1530
Ile Ala Gly Asp Gly Gly Asn Gln Pro Ala Arg Tyr Asp Asp Phe
1535 1540 1545
Thr Phe Glu Ala Gly Lys Lys Tyr Thr Phe Thr Met Arg Arg Ala
1550 1555 1560
Gly Met Gly Asp Gly Thr Asp Met Glu Val Glu Asp Asp Ser Pro
1565 1570 1575
Ala Ser Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys
1580 1585 1590
Glu Gly Leu Thr Glu Thr Thr Tyr Arg Asp Ala Gly Met Ser Ala
1595 1600 1605
Gln Ser His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val
1610 1615 1620
Ser Pro Lys Val Cys Val Asp Tyr Ile Pro Asp Gly Val Ala Asp
1625 1630 1635
Val Thr Ala Gln Lys Pro Tyr Thr Leu Thr Val Val Gly Lys Thr
1640 1645 1650
Ile Thr Val Thr Cys Gln Gly Glu Ala Met Ile Tyr Asp Met Asn
1655 1660 1665
Gly Arg Arg Leu Ala Ala Gly Arg Asn Thr Val Val Tyr Thr Ala
1670 1675 1680
Gln Gly Gly Tyr Tyr Ala Val Met Val Val Val Asp Gly Lys Ser
1685 1690 1695
Tyr Val Glu Lys Leu Ala Ile Lys
1700 1705
<210> 62
<211> 1732
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 62
Met Arg Lys Leu Leu Leu Leu Ile Ala Ala Ser Leu Leu Gly Val Gly
1 5 10 15
Leu Tyr Ala Gln Ser Ala Lys Ile Lys Leu Asp Ala Pro Thr Thr Arg
20 25 30
Thr Thr Cys Thr Asn Asn Ser Phe Lys Gln Phe Asp Ala Ser Phe Ser
35 40 45
Phe Asn Glu Val Glu Leu Thr Lys Val Glu Thr Lys Gly Gly Thr Phe
50 55 60
Ala Ser Val Ser Ile Pro Gly Ala Phe Pro Thr Gly Glu Val Gly Ser
65 70 75 80
Pro Glu Val Pro Ala Val Arg Lys Leu Ile Ala Val Pro Val Gly Ala
85 90 95
Thr Pro Val Val Arg Val Lys Ser Phe Thr Glu Gln Val Tyr Ser Leu
100 105 110
Asn Gln Tyr Gly Ser Glu Lys Leu Met Pro His Gln Pro Ser Met Ser
115 120 125
Lys Ser Asp Asp Pro Glu Lys Val Pro Phe Val Tyr Asn Ala Ala Ala
130 135 140
Tyr Ala Arg Lys Gly Phe Val Gly Gln Glu Leu Thr Gln Val Glu Met
145 150 155 160
Leu Gly Thr Met Arg Gly Val Arg Ile Ala Ala Leu Thr Ile Asn Pro
165 170 175
Val Gln Tyr Asp Val Val Ala Asn Gln Leu Lys Val Arg Asn Asn Ile
180 185 190
Glu Ile Glu Val Ser Phe Gln Gly Ala Asp Glu Val Ala Thr Gln Arg
195 200 205
Leu Tyr Asp Ala Ser Phe Ser Pro Tyr Phe Glu Thr Ala Tyr Lys Gln
210 215 220
Leu Phe Asn Arg Asp Val Tyr Thr Asp His Gly Asp Leu Tyr Asn Thr
225 230 235 240
Pro Val Arg Met Leu Val Val Ala Gly Ala Lys Phe Lys Glu Ala Leu
245 250 255
Lys Pro Trp Leu Thr Trp Lys Ala Gln Lys Gly Phe Tyr Leu Asp Val
260 265 270
His Tyr Thr Asp Glu Ala Glu Val Gly Thr Thr Asn Ala Ser Ile Lys
275 280 285
Ala Phe Ile His Lys Lys Tyr Asn Asp Gly Leu Ala Ala Ser Ala Ala
290 295 300
Pro Val Phe Leu Ala Leu Val Gly Asp Thr Asp Val Ile Ser Gly Glu
305 310 315 320
Lys Gly Lys Lys Thr Lys Lys Val Thr Asp Leu Tyr Tyr Ser Ala Val
325 330 335
Asp Gly Asp Tyr Phe Pro Glu Met Tyr Thr Phe Arg Met Ser Ala Ser
340 345 350
Ser Pro Glu Glu Leu Thr Asn Ile Ile Asp Lys Val Leu Met Tyr Glu
355 360 365
Lys Ala Thr Met Pro Asp Lys Ser Tyr Leu Glu Lys Val Leu Leu Ile
370 375 380
Ala Gly Ala Asp Tyr Ser Trp Asn Ser Gln Val Gly Gln Pro Thr Ile
385 390 395 400
Lys Tyr Gly Met Gln Tyr Tyr Tyr Asn Gln Glu His Gly Tyr Thr Asp
405 410 415
Val Tyr Asn Tyr Leu Lys Ala Pro Tyr Thr Gly Cys Tyr Ser His Leu
420 425 430
Asn Thr Gly Val Ser Phe Ala Asn Tyr Thr Ala His Gly Ser Glu Thr
435 440 445
Ala Trp Ala Asp Pro Leu Leu Thr Thr Ser Gln Leu Lys Ala Leu Thr
450 455 460
Asn Lys Asp Lys Tyr Phe Leu Ala Ile Gly Asn Cys Cys Ile Thr Ala
465 470 475 480
Gln Phe Asp Tyr Val Gln Pro Cys Phe Gly Glu Val Ile Thr Arg Val
485 490 495
Lys Glu Lys Gly Ala Tyr Ala Tyr Ile Gly Ser Ser Pro Asn Ser Tyr
500 505 510
Trp Gly Glu Asp Tyr Tyr Trp Ser Val Gly Ala Asn Ala Val Phe Gly
515 520 525
Val Gln Pro Thr Phe Glu Gly Thr Ser Met Gly Ser Tyr Asp Ala Thr
530 535 540
Phe Leu Glu Asp Ser Tyr Asn Thr Val Asn Ser Ile Met Trp Ala Gly
545 550 555 560
Asn Leu Ala Ala Thr His Ala Gly Asn Ile Gly Asn Ile Thr His Ile
565 570 575
Gly Ala His Tyr Tyr Trp Glu Ala Tyr His Val Leu Gly Asp Gly Ser
580 585 590
Val Met Pro Tyr Arg Ala Met Pro Lys Thr Asn Thr Tyr Thr Leu Pro
595 600 605
Ala Ser Leu Pro Gln Asn Gln Ala Ser Tyr Ser Ile Gln Ala Ser Ala
610 615 620
Gly Ser Tyr Val Ala Ile Ser Lys Asp Gly Val Leu Tyr Gly Thr Gly
625 630 635 640
Val Ala Asn Ala Ser Gly Val Ala Thr Val Ser Met Thr Lys Gln Ile
645 650 655
Thr Glu Asn Gly Asn Tyr Asp Val Val Ile Thr Arg Ser Asn Tyr Leu
660 665 670
Pro Val Ile Lys Gln Ile Gln Val Gly Glu Pro Ser Pro Tyr Gln Pro
675 680 685
Val Ser Asn Leu Thr Ala Thr Thr Gln Gly Gln Lys Val Thr Leu Lys
690 695 700
Trp Glu Ala Pro Ser Ala Lys Lys Ala Glu Gly Ser Arg Glu Val Lys
705 710 715 720
Arg Ile Gly Asp Gly Leu Phe Val Thr Ile Glu Pro Ala Asn Asp Val
725 730 735
Arg Ala Asn Glu Ala Lys Val Val Leu Ala Ala Asp Asn Val Trp Gly
740 745 750
Asp Asn Thr Gly Tyr Gln Phe Leu Leu Asp Ala Asp His Asn Thr Phe
755 760 765
Gly Ser Val Ile Pro Ala Thr Gly Pro Leu Phe Thr Gly Thr Ala Ser
770 775 780
Ser Asn Leu Tyr Ser Ala Asn Phe Glu Tyr Leu Ile Pro Ala Asn Ala
785 790 795 800
Asp Pro Val Val Thr Thr Gln Asn Ile Ile Val Thr Gly Gln Gly Glu
805 810 815
Val Val Ile Pro Gly Gly Val Tyr Asp Tyr Cys Ile Thr Asn Pro Glu
820 825 830
Pro Ala Ser Gly Lys Met Trp Ile Ala Gly Asp Gly Gly Asn Gln Pro
835 840 845
Ala Arg Tyr Asp Asp Phe Thr Phe Glu Ala Gly Lys Lys Tyr Thr Phe
850 855 860
Thr Met Arg Arg Ala Gly Met Gly Asp Gly Thr Asp Met Glu Val Glu
865 870 875 880
Asp Asp Ser Pro Ala Ser Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr
885 890 895
Lys Ile Lys Glu Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val
900 905 910
Ala Ala Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly
915 920 925
Val Ser Pro Lys Val Cys Lys Asp Val Thr Val Glu Gly Ser Asn Glu
930 935 940
Phe Ala Pro Val Gln Asn Leu Thr Gly Ser Ser Val Gly Gln Lys Val
945 950 955 960
Thr Leu Lys Trp Asp Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Asn
965 970 975
Pro Asn Pro Asn Pro Gly Thr Thr Leu Ser Glu Ser Phe Glu Asn Gly
980 985 990
Ile Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly His Gly
995 1000 1005
Trp Lys Pro Gly Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser Asn
1010 1015 1020
Gly Cys Val Tyr Ser Glu Ser Phe Gly Leu Gly Gly Ile Gly Val
1025 1030 1035
Leu Thr Pro Asp Asn Tyr Leu Ile Thr Pro Ala Leu Asp Leu Pro
1040 1045 1050
Asn Gly Gly Lys Leu Thr Phe Trp Val Cys Ala Gln Asp Ala Asn
1055 1060 1065
Tyr Ala Ser Glu His Tyr Ala Val Tyr Ala Ser Ser Thr Gly Asn
1070 1075 1080
Asp Ala Ser Asn Phe Thr Asn Ala Leu Leu Glu Glu Thr Ile Thr
1085 1090 1095
Ala Lys Gly Val Arg Ser Pro Lys Ala Ile Arg Gly Arg Ile Gln
1100 1105 1110
Gly Thr Trp Arg Gln Lys Thr Val Asp Leu Pro Ala Gly Thr Lys
1115 1120 1125
Tyr Val Ala Phe Arg His Phe Gln Ser Thr Asp Met Phe Tyr Ile
1130 1135 1140
Asp Leu Asp Glu Val Glu Ile Lys Ala Asn Gly Lys Arg Ala Asp
1145 1150 1155
Phe Thr Glu Thr Phe Glu Ser Ser Thr His Gly Glu Ala Pro Ala
1160 1165 1170
Glu Trp Thr Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu
1175 1180 1185
Cys Leu Ser Ser Gly Gln Leu Asp Trp Leu Thr Ala His Gly Gly
1190 1195 1200
Ser Asn Val Val Ser Ser Phe Ser Trp Asn Gly Met Ala Leu Asn
1205 1210 1215
Pro Asp Asn Tyr Leu Ile Ser Lys Asp Val Thr Gly Ala Thr Lys
1220 1225 1230
Val Lys Tyr Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly Asp His
1235 1240 1245
Tyr Ala Val Met Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe
1250 1255 1260
Thr Val Val Phe Glu Glu Thr Pro Asn Gly Ile Asn Lys Gly Gly
1265 1270 1275
Ala Arg Phe Gly Leu Ser Thr Glu Ala Asn Gly Ala Lys Pro Gln
1280 1285 1290
Ser Val Trp Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr Lys
1295 1300 1305
Tyr Val Ala Phe Arg His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile
1310 1315 1320
Leu Leu Asp Asp Ile Gln Phe Thr Met Gly Gly Ser Pro Thr Pro
1325 1330 1335
Thr Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys
1340 1345 1350
Glu Gly Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr
1355 1360 1365
Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val
1370 1375 1380
Ser Pro Lys Lys Cys Val Asn Val Thr Val Asn Ser Thr Gln Phe
1385 1390 1395
Asn Pro Val Gln Asn Leu Thr Ala Glu Gln Ala Pro Asn Ser Met
1400 1405 1410
Asp Ala Ile Leu Lys Trp Asn Ala Pro Ala Ser Lys Arg Ala Glu
1415 1420 1425
Val Leu Asn Glu Asp Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys
1430 1435 1440
Thr Ile Asp Ala Asp Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro
1445 1450 1455
Pro Pro Gly Gly Ser Ser Phe Ala Gly His Asn Ser Ala Ile Cys
1460 1465 1470
Val Ser Ser Ala Ser Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro
1475 1480 1485
Asp Asn Tyr Leu Val Thr Pro Glu Leu Ser Leu Pro Gly Gly Gly
1490 1495 1500
Thr Leu Thr Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser
1505 1510 1515
Glu His Tyr Ala Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser
1520 1525 1530
Asn Phe Ala Asn Ala Leu Leu Glu Glu Val Leu Thr Ala Lys Thr
1535 1540 1545
Val Val Thr Ala Pro Glu Ala Ile Arg Gly Thr Arg Ala Gln Gly
1550 1555 1560
Thr Trp Tyr Gln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys Tyr
1565 1570 1575
Val Ala Phe Arg His Phe Gly Cys Thr Asp Phe Phe Trp Ile Asn
1580 1585 1590
Leu Asp Asp Val Val Ile Thr Ser Gly Asn Ala Pro Ser Tyr Thr
1595 1600 1605
Tyr Thr Ile Tyr Arg Asn Asn Thr Gln Ile Ala Ser Gly Val Thr
1610 1615 1620
Glu Thr Thr Tyr Arg Asp Pro Asp Leu Ala Thr Gly Phe Tyr Thr
1625 1630 1635
Tyr Gly Val Lys Val Val Tyr Pro Asn Gly Glu Ser Ala Ile Glu
1640 1645 1650
Thr Ala Thr Leu Asn Ile Thr Ser Leu Ala Asp Val Thr Ala Gln
1655 1660 1665
Lys Pro Tyr Thr Leu Thr Val Val Gly Lys Thr Ile Thr Val Thr
1670 1675 1680
Cys Gln Gly Glu Ala Met Ile Tyr Asp Met Asn Gly Arg Arg Leu
1685 1690 1695
Ala Ala Gly Arg Asn Thr Val Val Tyr Thr Ala Gln Gly Gly His
1700 1705 1710
Tyr Ala Val Met Val Val Val Asp Gly Lys Ser Tyr Val Glu Lys
1715 1720 1725
Leu Ala Val Lys
1730
<210> 63
<211> 2164
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 63
Met Arg Lys Leu Asn Ser Leu Phe Ser Leu Ala Val Leu Leu Ser Leu
1 5 10 15
Leu Cys Trp Gly Gln Thr Ala Ala Ala Gln Gly Gly Pro Lys Thr Ala
20 25 30
Pro Ser Val Thr His Gln Ala Val Gln Lys Gly Ile Arg Thr Ser Lys
35 40 45
Ala Lys Asp Leu Arg Asp Pro Ile Pro Ala Gly Met Ala Arg Ile Ile
50 55 60
Leu Glu Ala His Asp Val Trp Glu Asp Gly Thr Gly Tyr Gln Met Leu
65 70 75 80
Trp Asp Ala Asp His Asn Gln Tyr Gly Ala Ser Ile Pro Glu Glu Ser
85 90 95
Phe Trp Phe Ala Asn Gly Thr Ile Pro Ala Gly Leu Tyr Asp Pro Phe
100 105 110
Glu Tyr Lys Val Pro Val Asn Ala Asp Ala Ser Phe Ser Pro Thr Asn
115 120 125
Phe Val Leu Asp Gly Thr Ala Ser Ala Asp Ile Pro Ala Gly Thr Tyr
130 135 140
Asp Tyr Val Ile Ile Asn Pro Asn Pro Gly Ile Ile Tyr Ile Val Gly
145 150 155 160
Glu Gly Val Ser Lys Gly Asn Asp Tyr Val Val Glu Ala Gly Lys Thr
165 170 175
Tyr His Phe Thr Val Gln Arg Gln Gly Pro Gly Asp Ala Ala Ser Val
180 185 190
Val Val Thr Gly Glu Gly Gly Asn Glu Phe Ala Pro Val Gln Asn Leu
195 200 205
Gln Trp Ser Val Ser Gly Gln Thr Val Thr Leu Thr Trp Gln Ala Pro
210 215 220
Ala Ser Asp Lys Arg Thr Tyr Val Leu Asn Glu Ser Phe Asp Thr Gln
225 230 235 240
Thr Leu Pro Asn Gly Trp Thr Met Ile Asp Ala Asp Gly Asp Gly His
245 250 255
Asn Trp Leu Ser Thr Ile Asn Val Tyr Asn Thr Ala Thr His Thr Gly
260 265 270
Asp Gly Ala Met Phe Ser Lys Ser Trp Thr Ala Ser Ser Gly Ala Lys
275 280 285
Ile Asp Leu Ser Pro Asp Asn Tyr Leu Val Thr Pro Lys Phe Thr Val
290 295 300
Pro Glu Asn Gly Lys Leu Ser Tyr Trp Val Ser Ser Gln Glu Pro Trp
305 310 315 320
Thr Asn Glu His Tyr Gly Val Phe Leu Ser Thr Thr Gly Asn Glu Ala
325 330 335
Ala Asn Phe Thr Ile Lys Leu Leu Glu Glu Thr Leu Gly Ser Gly Lys
340 345 350
Pro Ala Pro Met Asn Leu Val Lys Ser Glu Gly Val Lys Ala Pro Ala
355 360 365
Pro Tyr Gln Glu Arg Thr Ile Asp Leu Ser Ala Tyr Ala Gly Gln Gln
370 375 380
Val Tyr Leu Ala Phe Arg His Phe Gly Cys Thr Gly Ile Phe Arg Leu
385 390 395 400
Tyr Leu Asp Asp Val Ala Val Ser Gly Glu Gly Ser Ser Asn Asp Tyr
405 410 415
Thr Tyr Thr Val Tyr Arg Asp Asn Val Val Ile Ala Gln Asn Leu Thr
420 425 430
Ala Thr Thr Phe Asn Gln Glu Asn Val Ala Pro Gly Gln Tyr Asn Tyr
435 440 445
Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Val Cys Lys
450 455 460
Asp Val Thr Val Glu Gly Ser Asn Glu Phe Ala Pro Val Gln Asn Leu
465 470 475 480
Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys Trp Asp Ala Pro
485 490 495
Asn Gly Thr Pro Asn Pro Asn Pro Gly Thr Thr Thr Leu Ser Glu Ser
500 505 510
Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly
515 520 525
Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro Pro Gly Gly Ser Ser Phe
530 535 540
Ala Gly His Asn Ser Ala Ile Cys Val Ser Ser Ala Ser Tyr Ile Asn
545 550 555 560
Phe Glu Gly Pro Gln Asn Pro Asp Asn Tyr Leu Val Thr Pro Glu Leu
565 570 575
Ser Leu Pro Asn Gly Gly Thr Leu Thr Phe Trp Val Cys Ala Gln Asp
580 585 590
Ala Asn Tyr Ala Ser Glu His Tyr Ala Val Tyr Ala Ser Ser Thr Gly
595 600 605
Asn Asp Ala Ser Asn Phe Ala Asn Ala Leu Leu Glu Glu Val Leu Thr
610 615 620
Ala Lys Thr Val Val Thr Ala Pro Glu Ala Ile Arg Gly Thr Arg Val
625 630 635 640
Gln Gly Thr Trp Tyr Gln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys
645 650 655
Tyr Val Ala Phe Arg His Phe Gly Cys Thr Asp Phe Phe Trp Ile Asn
660 665 670
Leu Asp Asp Val Glu Ile Lys Ala Asn Gly Lys Arg Ala Asp Phe Thr
675 680 685
Glu Thr Phe Glu Ser Ser Thr His Gly Glu Ala Pro Ala Glu Trp Thr
690 695 700
Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu Cys Leu Ser Ser
705 710 715 720
Gly Gln Leu Gly Trp Leu Thr Ala His Gly Gly Thr Asn Val Val Ala
725 730 735
Ser Phe Ser Trp Asn Gly Met Ala Leu Asn Pro Asp Asn Tyr Leu Ile
740 745 750
Ser Lys Asp Val Thr Gly Ala Thr Lys Val Lys Tyr Tyr Tyr Ala Val
755 760 765
Asn Asp Gly Phe Pro Gly Asp His Tyr Ala Val Met Ile Ser Lys Thr
770 775 780
Gly Thr Asn Ala Gly Asp Phe Thr Val Val Phe Glu Glu Thr Pro Asn
785 790 795 800
Gly Ile Asn Lys Gly Gly Ala Arg Phe Gly Leu Ser Thr Glu Ala Asn
805 810 815
Gly Ala Lys Pro Gln Ser Val Trp Ile Glu Arg Thr Val Asp Leu Pro
820 825 830
Ala Gly Thr Lys Tyr Val Ala Phe Arg His Tyr Asn Cys Ser Asp Leu
835 840 845
Asn Tyr Ile Leu Leu Asp Asp Ile Gln Phe Thr Met Gly Gly Ser Pro
850 855 860
Thr Pro Thr Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile
865 870 875 880
Lys Glu Gly Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr
885 890 895
Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser
900 905 910
Pro Lys Glu Cys Val Asn Val Thr Val Asp Pro Val Gln Phe Asn Pro
915 920 925
Val Gln Asn Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys
930 935 940
Trp Asp Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Gly Thr Thr Thr
945 950 955 960
Leu Ser Glu Ser Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile
965 970 975
Asp Ala Asp Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro Pro Gly
980 985 990
Gly Thr Ser Phe Ala Gly His Asn Ser Ala Ile Cys Val Ser Ser Ala
995 1000 1005
Ser Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro Asp Asn Tyr Leu
1010 1015 1020
Val Thr Pro Glu Leu Ser Leu Pro Asn Gly Gly Thr Leu Thr Phe
1025 1030 1035
Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser Glu His Tyr Ala
1040 1045 1050
Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser Asn Phe Ala Asn
1055 1060 1065
Ala Leu Leu Glu Glu Val Leu Thr Ala Lys Thr Val Val Thr Ala
1070 1075 1080
Pro Glu Ala Ile Arg Gly Thr Arg Val Gln Gly Thr Trp Tyr Gln
1085 1090 1095
Lys Thr Val Gln Leu Pro Ala Gly Thr Lys Tyr Val Ala Phe Arg
1100 1105 1110
His Phe Gly Cys Thr Asp Phe Phe Trp Ile Asn Leu Asp Asp Val
1115 1120 1125
Glu Ile Lys Ala Asn Gly Lys Arg Ala Asp Phe Thr Glu Thr Phe
1130 1135 1140
Glu Ser Ser Thr His Gly Glu Ala Pro Ala Glu Trp Thr Thr Ile
1145 1150 1155
Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu Cys Leu Ser Ser Gly
1160 1165 1170
Gln Leu Asp Trp Leu Thr Ala His Gly Gly Thr Asn Val Val Ala
1175 1180 1185
Ser Phe Ser Trp Asn Gly Met Ala Leu Asn Pro Asp Asn Tyr Leu
1190 1195 1200
Ile Ser Lys Asp Val Thr Gly Ala Thr Lys Val Lys Tyr Tyr Tyr
1205 1210 1215
Ala Val Asn Asp Gly Phe Pro Gly Asp His Tyr Ala Val Met Ile
1220 1225 1230
Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr Val Val Phe Glu
1235 1240 1245
Glu Thr Pro Asn Gly Ile Asn Lys Gly Gly Ala Arg Phe Gly Leu
1250 1255 1260
Ser Thr Glu Ala Asn Gly Ala Lys Pro Gln Ser Val Trp Ile Glu
1265 1270 1275
Arg Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala Phe Arg
1280 1285 1290
His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile Leu Leu Asp Asp Ile
1295 1300 1305
Gln Phe Thr Met Gly Gly Ser Pro Thr Pro Thr Asp Tyr Thr Tyr
1310 1315 1320
Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu Thr Glu
1325 1330 1335
Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His Glu Tyr
1340 1345 1350
Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Glu Cys
1355 1360 1365
Val Asn Val Thr Val Asp Pro Val Gln Phe Asn Pro Val Gln Asn
1370 1375 1380
Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys Trp Asp
1385 1390 1395
Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Gly Thr Thr Thr Leu
1400 1405 1410
Ser Glu Ser Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile
1415 1420 1425
Asp Ala Asp Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro Pro
1430 1435 1440
Gly Gly Thr Ser Phe Ala Gly His Asn Ser Ala Ile Cys Val Ser
1445 1450 1455
Ser Ala Ser Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro Asp Asn
1460 1465 1470
Tyr Leu Val Thr Pro Glu Leu Ser Leu Pro Asn Gly Gly Thr Leu
1475 1480 1485
Thr Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser Glu His
1490 1495 1500
Tyr Ala Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser Asn Phe
1505 1510 1515
Ala Asn Ala Leu Leu Glu Glu Val Leu Thr Ala Lys Thr Val Val
1520 1525 1530
Thr Ala Pro Glu Ala Ile Arg Gly Thr Arg Val Gln Gly Thr Trp
1535 1540 1545
Tyr Gln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys Tyr Val Ala
1550 1555 1560
Phe Arg His Phe Gly Cys Thr Asp Phe Phe Trp Ile Asn Leu Asp
1565 1570 1575
Asp Val Glu Ile Lys Ala Asn Gly Lys Arg Ala Asp Phe Thr Glu
1580 1585 1590
Thr Phe Glu Ser Ser Thr His Gly Glu Ala Pro Ala Glu Trp Thr
1595 1600 1605
Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu Cys Leu Ser
1610 1615 1620
Ser Gly Gln Leu Gly Trp Leu Thr Ala His Gly Gly Thr Asn Val
1625 1630 1635
Val Ala Ser Phe Ser Trp Asn Gly Met Ala Leu Asn Pro Asp Asn
1640 1645 1650
Tyr Leu Ile Ser Lys Asp Val Thr Gly Ala Thr Lys Val Lys Tyr
1655 1660 1665
Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly Asp His Tyr Ala Val
1670 1675 1680
Met Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr Val Val
1685 1690 1695
Phe Glu Glu Thr Pro Asn Gly Ile Asn Lys Gly Gly Ala Arg Phe
1700 1705 1710
Gly Leu Ser Thr Glu Ala Asn Gly Ala Lys Pro Gln Ser Val Trp
1715 1720 1725
Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala
1730 1735 1740
Phe Arg His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile Leu Leu Asp
1745 1750 1755
Asp Ile Gln Phe Thr Met Gly Gly Ser Pro Thr Pro Thr Asp Tyr
1760 1765 1770
Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu
1775 1780 1785
Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His
1790 1795 1800
Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys
1805 1810 1815
Glu Cys Val Asn Val Thr Ile Asn Pro Thr Gln Phe Asn Pro Val
1820 1825 1830
Gln Asn Leu Thr Ala Glu Gln Ala Pro Asn Ser Met Asp Ala Ile
1835 1840 1845
Leu Lys Trp Asn Ala Pro Ala Ser Lys Arg Ala Glu Val Leu Asn
1850 1855 1860
Glu Asp Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile Asp
1865 1870 1875
Ala Asp Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro Pro Gly
1880 1885 1890
Gly Ser Ser Phe Ala Gly His Asn Ser Ala Ile Cys Val Ser Ser
1895 1900 1905
Ala Ser Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro Asp Asn Tyr
1910 1915 1920
Leu Val Thr Pro Glu Leu Ser Leu Pro Gly Gly Gly Thr Leu Thr
1925 1930 1935
Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser Glu His Tyr
1940 1945 1950
Ala Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser Asn Phe Ala
1955 1960 1965
Asn Ala Leu Leu Glu Glu Val Leu Thr Ala Lys Thr Val Val Thr
1970 1975 1980
Ala Pro Glu Ala Ile Arg Gly Thr Arg Val Gln Gly Thr Trp Tyr
1985 1990 1995
Gln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys Tyr Val Ala Phe
2000 2005 2010
Arg His Phe Gly Cys Thr Asp Phe Phe Trp Ile Asn Leu Asp Asp
2015 2020 2025
Val Val Ile Thr Ser Gly Asn Ala Pro Ser Tyr Thr Tyr Thr Ile
2030 2035 2040
Tyr Arg Asn Asn Thr Gln Ile Ala Ser Gly Val Thr Glu Thr Thr
2045 2050 2055
Tyr Arg Asp Pro Asp Leu Ala Thr Gly Phe Tyr Thr Tyr Gly Val
2060 2065 2070
Lys Val Val Tyr Pro Asn Gly Glu Ser Ala Ile Glu Thr Ala Thr
2075 2080 2085
Leu Asn Ile Thr Ser Leu Ala Asp Val Thr Ala Gln Lys Pro Tyr
2090 2095 2100
Thr Leu Thr Val Val Gly Lys Thr Ile Thr Val Thr Cys Gln Gly
2105 2110 2115
Glu Ala Met Ile Tyr Asp Met Asn Gly Arg Arg Leu Ala Ala Gly
2120 2125 2130
Arg Asn Thr Val Val Tyr Thr Ala Gln Gly Gly His Tyr Ala Val
2135 2140 2145
Met Val Val Val Asp Gly Lys Ser Tyr Val Glu Lys Leu Ala Val
2150 2155 2160
Lys
<210> 64
<211> 8
<212> PRT
<213> 牙龈卟啉单胞菌
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> X可以是S或Y
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> X可以是Y或S
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> X可以是P或S
<220>
<221> MISC_FEATURE
<222> (7)..(7)
<223> X可以是K或Q
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> X可以是I或V
<400> 64
Asp Xaa Xaa Trp Asn Xaa Xaa Xaa
1 5
<210> 65
<211> 7
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 65
Asn Ser Tyr Trp Gly Glu Asp
1 5
<210> 66
<211> 11
<212> PRT
<213> 牙龈卟啉单胞菌
<220>
<221> MISC_FEATURE
<222> (4)..(4)
<223> X可以是V或I
<400> 66
Ile Gly Asn Xaa Thr His Ile Gly Ala His Tyr
1 5 10
<210> 67
<211> 8
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 67
Glu Gly Gly Pro Ser Ala Asp Asn
1 5
<210> 68
<211> 7
<212> PRT
<213> 牙龈卟啉单胞菌
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> X可以是N或D
<220>
<221> MISC_FEATURE
<222> (3)..(3)
<223> X可以是S或Y
<220>
<221> MISC_FEATURE
<222> (6)..(6)
<223> X可以是S或P
<400> 68
Xaa Gln Xaa Trp Ala Xaa Pro
1 5
<210> 69
<211> 23
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 69
Pro Val Ser Asn Leu Thr Ala Thr Thr Gln Gly Gln Lys Val Thr Leu
1 5 10 15
Lys Trp Asp Ala Pro Ser Thr
20
<210> 70
<211> 23
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 70
Pro Val Ser Asn Leu Thr Ala Thr Thr Gln Gly Gln Lys Val Thr Leu
1 5 10 15
Lys Trp Glu Ala Pro Ser Ala
20
<210> 71
<211> 23
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 71
Pro Val Gln Asn Leu Thr Gly Ser Ser Val Gly Gln Lys Val Thr Leu
1 5 10 15
Lys Trp Asp Ala Pro Ser Thr
20
<210> 72
<211> 23
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 72
Pro Val Gln Asn Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu
1 5 10 15
Lys Trp Asp Ala Pro Asn Gly
20
<210> 73
<211> 23
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 73
Pro Val Lys Asn Leu Lys Ala Gln Pro Asp Gly Gly Asp Val Val Leu
1 5 10 15
Lys Trp Glu Ala Pro Ser Ala
20
<210> 74
<211> 23
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 74
Pro Val Gln Asn Leu Thr Ala Glu Gln Ala Pro Asn Ser Met Asp Ala
1 5 10 15
Ile Leu Lys Trp Asn Ala Pro
20
<210> 75
<211> 24
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 75
Pro Val Gln Asn Leu Thr Gln Trp Ser Val Ser Gly Gln Thr Val Thr
1 5 10 15
Leu Thr Trp Gln Ala Pro Ala Ser
20
<210> 76
<211> 27
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 76
Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu
1 5 10 15
Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala
20 25
<210> 77
<211> 27
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 77
Tyr Thr Tyr Thr Val Tyr Arg Asp Asn Val Val Ile Ala Gln Asn Leu
1 5 10 15
Thr Ala Thr Thr Phe Asn Gln Glu Asn Val Ala
20 25
<210> 78
<211> 28
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 78
Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu
1 5 10 15
Thr Ala Glu Thr Thr Phe Glu Glu Asp Gly Val Ala
20 25
<210> 79
<211> 16
<212> PRT
<213> 牙龈卟啉单胞菌
<220>
<221> MISC_FEATURE
<222> (6)..(7)
<223> X可以是NP或NPNP或NPNPNP或NPNPNPNP或NPNPNPNPNP或
NPNPNPNPNPNP
<400> 79
Pro Asn Gly Thr Pro Xaa Xaa Gly Thr Thr Thr Leu Ser Glu Ser Phe
1 5 10 15
<210> 80
<211> 325
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 80
Gly Gly Pro Lys Thr Ala Pro Ser Val Thr His Gln Ala Val Gln Lys
1 5 10 15
Gly Ile Arg Thr Ser Lys Ala Lys Asp Leu Arg Asp Pro Ile Pro Ala
20 25 30
Gly Met Ala Arg Ile Ile Leu Glu Ala His Asp Val Trp Glu Asp Gly
35 40 45
Thr Gly Tyr Gln Met Leu Trp Asp Ala Asp His Asn Gln Tyr Gly Ala
50 55 60
Ser Ile Pro Glu Glu Ser Phe Trp Phe Ala Asn Gly Thr Ile Pro Ala
65 70 75 80
Gly Leu Tyr Asp Pro Phe Glu Tyr Lys Val Pro Val Asn Ala Asp Ala
85 90 95
Ser Phe Ser Pro Thr Asn Phe Val Leu Asp Gly Thr Ala Ser Ala Asp
100 105 110
Ile Pro Ala Gly Thr Tyr Asp Tyr Val Ile Ile Asn Pro Asn Pro Gly
115 120 125
Ile Ile Tyr Ile Val Gly Glu Gly Val Ser Lys Gly Asn Asp Tyr Val
130 135 140
Val Glu Ala Gly Lys Thr Tyr His Phe Thr Val Gln Arg Gln Gly Pro
145 150 155 160
Gly Asp Ala Ala Ser Val Val Val Thr Gly Glu Gly Gly Asn Glu Phe
165 170 175
Ala Pro Val Gln Asn Leu Gln Trp Ser Val Ser Gly Gln Thr Val Thr
180 185 190
Leu Thr Trp Gln Ala Pro Ala Ser Asp Lys Arg Thr Tyr Val Leu Asn
195 200 205
Glu Ser Phe Asp Thr Gln Thr Leu Pro Asn Gly Trp Thr Met Ile Asp
210 215 220
Ala Asp Gly Asp Gly His Asn Trp Leu Ser Thr Ile Asn Val Tyr Asn
225 230 235 240
Thr Ala Thr His Thr Gly Asp Gly Ala Met Phe Ser Lys Ser Trp Thr
245 250 255
Ala Ser Ser Gly Ala Lys Ile Asp Leu Ser Pro Asp Asn Tyr Leu Val
260 265 270
Thr Pro Lys Phe Thr Val Pro Glu Asn Gly Lys Leu Ser Tyr Trp Val
275 280 285
Ser Ser Gln Glu Pro Trp Thr Asn Glu His Tyr Gly Val Phe Leu Ser
290 295 300
Thr Thr Gly Asn Glu Ala Ala Asn Phe Thr Ile Lys Leu Leu Glu Glu
305 310 315 320
Thr Leu Gly Ser Gly
325
<210> 81
<211> 260
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 81
Ala Pro Ala Pro Tyr Gln Glu Arg Thr Ile Asp Leu Ser Ala Tyr Ala
1 5 10 15
Gly Gln Gln Val Tyr Leu Ala Phe Arg His Phe Gly Cys Thr Gly Ile
20 25 30
Phe Arg Leu Tyr Leu Asp Asp Val Ala Val Ser Gly Glu Gly Ser Ser
35 40 45
Asn Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Asn Val Val Ile Ala Gln
50 55 60
Asn Leu Thr Ala Thr Thr Phe Asn Gln Glu Asn Val Ala Pro Gly Gln
65 70 75 80
Tyr Asn Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys
85 90 95
Val Cys Lys Asp Val Thr Val Glu Gly Ser Asn Glu Phe Ala Pro Val
100 105 110
Gln Asn Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys Trp
115 120 125
Asp Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Gly Thr Thr Thr Leu
130 135 140
Ser Glu Ser Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile Asp
145 150 155 160
Ala Asp Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro Pro Gly Gly
165 170 175
Ser Ser Phe Ala Gly His Asn Ser Ala Ile Cys Val Ser Ser Ala Ser
180 185 190
Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro Asp Asn Tyr Leu Val Thr
195 200 205
Pro Glu Leu Ser Leu Pro Asn Gly Gly Thr Leu Thr Phe Trp Val Cys
210 215 220
Ala Gln Asp Ala Asn Tyr Ala Ser Glu His Tyr Ala Val Tyr Ala Ser
225 230 235 240
Ser Thr Gly Asn Asp Ala Ser Asn Phe Ala Asn Ala Leu Leu Glu Glu
245 250 255
Val Leu Thr Ala
260
<210> 82
<211> 258
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 82
Pro Gln Ser Val Trp Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr
1 5 10 15
Lys Tyr Val Ala Phe Arg His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile
20 25 30
Leu Leu Asp Asp Ile Gln Phe Thr Met Gly Gly Ser Pro Thr Pro Thr
35 40 45
Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly
50 55 60
Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His
65 70 75 80
Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Glu
85 90 95
Cys Val Asn Val Thr Val Asp Pro Val Gln Phe Asn Pro Val Gln Asn
100 105 110
Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys Trp Asp Ala
115 120 125
Pro Asn Gly Thr Pro Asn Pro Asn Pro Gly Thr Thr Thr Leu Ser Glu
130 135 140
Ser Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp
145 150 155 160
Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro Pro Gly Gly Thr Ser
165 170 175
Phe Ala Gly His Asn Ser Ala Ile Cys Val Ser Ser Ala Ser Tyr Ile
180 185 190
Asn Phe Glu Gly Pro Gln Asn Pro Asp Asn Tyr Leu Val Thr Pro Glu
195 200 205
Leu Ser Leu Pro Asn Gly Gly Thr Leu Thr Phe Trp Val Cys Ala Gln
210 215 220
Asp Ala Asn Tyr Ala Ser Glu His Tyr Ala Val Tyr Ala Ser Ser Thr
225 230 235 240
Gly Asn Asp Ala Ser Asn Phe Ala Asn Ala Leu Leu Glu Glu Val Leu
245 250 255
Thr Ala
<210> 83
<211> 15
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 83
Pro Tyr Gln Pro Val Ser Asn Leu Thr Ala Thr Thr Gln Gly Gln
1 5 10 15
<210> 84
<211> 15
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 84
Glu Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val Ala Ala
1 5 10 15
<210> 85
<211> 17
<212> PRT
<213> 牙龈卟啉单胞菌
<400> 85
Gly Thr Pro Asn Pro Asn Pro Asn Pro Asn Pro Asn Pro Asn Pro Gly
1 5 10 15
Thr
Claims (15)
1.用于诱导对于牙龈卟啉单胞菌(P.gingivalis)的免疫应答的嵌合或融合蛋白,所述蛋白由直接连接或通过连接子连接的第一肽和第二多肽组成,其中:
所述第一肽包含与选自SEQ ID No:27和28的序列相同或至少90%同源的序列;以及所述第二多肽包含与选自SEQ ID No:35、36和37的序列相同或至少90%同源的序列。
2.如权利要求1所述的嵌合或融合蛋白,其中所述第一肽由与选自SEQ ID No:27和28的序列相同或至少90%同源的序列组成,并且所述第二多肽由与选自SEQ ID No:36和37的序列相同或至少90%同源的序列组成。
3.如权利要求1所述的嵌合或融合蛋白,其中所述第一肽具有与SEQ ID No:28相同或至少90%同源的氨基酸序列,并且所述第二多肽具有与SEQ ID No:37相同或至少90%同源的氨基酸序列。
4.如权利要求1所述的嵌合或融合蛋白,其中所述第一肽具有由SEQ ID No:28组成的氨基酸序列,并且所述第二多肽具有由SEQ ID No:37组成的氨基酸序列。
5.如权利要求1所述的嵌合或融合蛋白,其中所述第一肽具有与SEQ ID No:27相同或至少90%同源的氨基酸序列,并且所述第二多肽具有与SEQ ID No:36相同或至少90%同源的氨基酸序列。
6.如权利要求1所述的嵌合或融合蛋白,其中所述第一肽具有由SEQ ID No:27组成的氨基酸序列,并且所述第二多肽具有由SEQ ID No:36组成的氨基酸序列。
7.如权利要求1-6中任一项所述的嵌合或融合蛋白,其中所述第一肽的C末端残基与所述第二多肽的(a)N末端残基或(b)C末端残基直接共价连接或通过连接子共价连接,所述连接子的长度为(i)多至15个氨基酸或(ii)少于5个氨基酸。
8.如权利要求1-6中任一项所述的嵌合或融合蛋白,其中所述第一肽的N末端残基与所述第二多肽的(a)N末端残基或(b)C末端残基直接共价连接或通过连接子共价连接,所述连接子的长度为(i)多至15个氨基酸或(ii)少于5个氨基酸。
9.如权利要求1-8中任一项所述的嵌合或融合蛋白,其用于检测来自个体的生物样品中的抗牙龈卟啉单胞菌抗体。
10.包含权利要求1-9中任一项所述的嵌合或融合蛋白的组合物。
11.如权利要求10所述的组合物,其还包含佐剂。
12.权利要求1-9中任一项所述的嵌合或融合蛋白或权利要求10-11中任一项所述的组合物在制备用于预防或降低个体中牙龈卟啉单胞菌(P.gingivalis)相关状态或疾病的发生率或严重程度的药物中的用途。
13.权利要求1-8中任一项所述的嵌合或融合蛋白在制备用于检测来自个体的生物样品中的抗牙龈卟啉单胞菌(P.gingivalis)抗体的试剂中的用途。
14.包含编码权利要求1-8中任一项所述的嵌合或融合蛋白的序列的核酸分子。
15.如权利要求14所述的核酸分子,其中所述序列与至少一个调节元件可操作地连接。
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
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AU2008904476A AU2008904476A0 (en) | 2008-08-29 | Prevention, treatment and diagnosis of P.gingivalis infection | |
AU2008904476 | 2008-08-29 | ||
AU2008905483A AU2008905483A0 (en) | 2008-10-23 | Prevention, treatment and diagnosis of P.gingivalis infection | |
AU2008905483 | 2008-10-23 | ||
US15113209P | 2009-02-09 | 2009-02-09 | |
US61/151,132 | 2009-02-09 | ||
AU2009903052A AU2009903052A0 (en) | 2009-06-30 | Prevention, treatment and diagnosis of P.gingivalis infection | |
AU2009903052 | 2009-06-30 | ||
CN2009801423053A CN102203121A (zh) | 2008-08-29 | 2009-08-28 | 牙龈卟啉单胞菌感染的预防、治疗和诊断 |
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CN2009801423053A Pending CN102203121A (zh) | 2008-08-29 | 2009-08-28 | 牙龈卟啉单胞菌感染的预防、治疗和诊断 |
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