CN1318103A - Nucleic acids and proteins from streptococcus pneumoniae - Google Patents

Nucleic acids and proteins from streptococcus pneumoniae Download PDF


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CN1318103A CN 99810978 CN99810978A CN1318103A CN 1318103 A CN1318103 A CN 1318103A CN 99810978 CN99810978 CN 99810978 CN 99810978 A CN99810978 A CN 99810978A CN 1318103 A CN1318103 A CN 1318103A
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    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • C07K2319/00Fusion polypeptide


本发明描述了新的肺炎链球菌蛋白质以及编码它们的核酸序列。 The present invention describes a new protein and the S. pneumoniae nucleic acid sequences encoding them. 还描述了它们在疫苗和筛选方法中的用途。 Also described their use in vaccines and screening methods.


肺炎链球菌的核酸和蛋白质 S. pneumoniae nucleic acids and proteins

本发明涉及源自肺炎链球菌(Streptococcus pneumoniae)的蛋白质,编码该蛋白质的核酸分子,该核酸和/或蛋白质作为抗原/免疫原的用途和在检测/诊断中的用途,以及筛选作为潜在抗-微生物靶的蛋白质/核酸序列的方法。 The present invention relates to a protein derived from Streptococcus pneumoniae (Streptococcus pneumoniae), the nucleic acid molecule encoding the protein, the nucleic acid and / or protein as an antigen / immunogen use and use in the detection / diagnosis, as well as the screening of potential anti - the method of microbial target protein / nucleic acid sequence.

肺炎链球菌常被称为肺炎球菌,它是一种重要的致病微生物。 Streptococcus pneumoniae pneumonia often referred to, it is an important pathogenic microorganisms. 在发展中国家和发达国家,肺炎链球菌感染在人类疾病中始终占有重要地位,有关专家已对此进行过评述(Fiber,GR,科学,265:1385-1387(1994))。 In developing and developed countries, Streptococcus pneumoniae infections in human disease has always played an important role, the experts had this been reviewed (Fiber, GR, Science, 265: 1385-1387 (1994)). 据信在世界范围内,该微生物是急性呼吸道感染最常见的致病菌,估计每年可导致1百万名儿童死亡,其中的大多数为发展中国家的儿童(Stansfield,SK,儿科传染病,6:622(1987))。 It is believed in the world, the microbial infection is the most common acute respiratory pathogens, can lead to an estimated 1 million children die each year, most of them children in developing countries (Stansfield, SK, pediatric infectious diseases, 6: 622 (1987)). 在美国,有人提出(Breiman等,Arch.Intern.Med.,150:1401(1990))肺炎球菌仍是细菌性肺炎最常见的致病菌,在儿童,老年人和患有易感染疾病,如无脾症,心脏病,肺病和肾病,糖尿病,酒精中毒的患者,或患有免疫抑制疾病(如AIDS)的患者中,细菌性肺炎的发病率特别高。 In the United States, it was suggested (Breiman, etc., Arch.Intern.Med, 150:. 1401 (1990)) Streptococcus pneumoniae remains the most common pathogen of bacterial pneumonia in children, the elderly and people with susceptible to diseases such as asplenia, heart disease, lung disease and kidney disease, diabetes, alcoholism patients suffering from immunosuppression or disease (e.g., AIDS), the incidence of bacterial pneumonia particularly high. 这些人群患肺炎球菌败血症和(因此患)脑膜炎的风险较高,因此死于肺炎球菌感染的风险较大。 These people suffer from pneumococcal sepsis, and (therefore suffering) higher risk of meningitis, so the risk of death from pneumococcal infection more likely. 肺炎球菌也是中耳炎和鼻窦炎的致病菌,在发达国家的儿童中这两种病一直是流行感染,并且带来很大的花费。 Pneumococcal bacteria also otitis media and sinusitis in children in developed countries in these two diseases has been a popular infection, and a great deal of expense.

最近,青霉素-抗性肺炎球菌的出现使得对有效预防肺炎球菌感染之策略的需要日趋迫切。 Recently, penicillin - resistant pneumococci appeared necessary to make the policy effective in preventing pneumococcal infection is becoming more urgent. 据报道,在美国12个州的13家医院中,6.6%的肺炎球菌分离株对青霉素具有抗性,一些分离株也对其它抗生素,包括第三代环孢菌素具有抗性(Schappert,SM,国家疾病防治中心/健康状况统计中心的人口和健康状况统计数据,214:1(1992))。 It is reported that 13 hospitals in 12 states in the United States, 6.6% of pneumococcal isolates are resistant to penicillin, some of the isolates also to other antibiotics, including third-generation cyclosporine resistant (Schappert, SM population and health statistics statistical Center of the national Center for disease Control and prevention / health, 214: 1 (1992)). 在某些医院,青霉素抗性的比例更高(达20%)(Breiman等,美国医学会志,271:1831(1994))。 In some hospitals, a higher proportion of penicillin-resistant (up 20%) (Breiman like the American Medical Association Chi, 271: 1831 (1994)). 由于肺炎球菌的青霉素抗性是在青霉素保持较好疗效几十年之后的近期突然出现的,因此这些发现令人恐慌。 Because penicillin-resistant pneumococcus is to maintain a good effect in the sudden appearance of penicillin recent decades later, these findings scary.

鉴于上述理由,必须致力于改良预防,控制,诊断或治疗肺炎球菌疾病的方法。 For these reasons, we must be committed to improve the prevention, control, diagnosis or treatment method pneumococcal disease.

已采用多种方法提供预防肺炎球菌感染所用的疫苗。 We have been using a variety of methods to provide vaccine to prevent pneumococcal infection used. 根据微生物周围的多糖荚膜结构可将其分为不同的血清型(至少90种),由此产生多个难题。 The structure of the capsular polysaccharide surrounding the microorganisms can be divided into different serotypes (at least 90 species), thereby producing a plurality of problems. 针对单个血清型的疫苗对其它血清型无效,这意味着疫苗必须包括所有血清型的多糖抗原才能在大多数情况下有效。 On the other serotypes is not valid for a single vaccine serotypes, which means that the vaccine must include all serotypes of polysaccharide antigen to be effective in most cases. 有人发现当荚膜多糖(各自决定着血清型,并且是主要的保护性抗原)被纯化并用作疫苗时,不能在两岁以下的儿童体内可靠地诱导保护性抗体应答,而该年龄组恰恰是侵害性肺炎球菌感染和脑膜炎的发病率最高的人群。 It was found that when the capsular polysaccharide (each determine the serotype and is the major protective antigen) when it is purified and used as a vaccine, can not reliably induce a protective antibody response in children under two years old, and it is precisely this age group invasive pneumococcal infection and meningitis incidence rate was highest among.

使用荚膜抗原方法的改良依靠多糖与蛋白质的缀合,从而特别地通过赋予应答依赖于T-细胞的特征而获得增强的免疫应答。 The method of using a modified antigen conjugated to capsular polysaccharide relies protein, in particular so as to obtain an enhanced immune response is dependent on the response characteristics by imparting a T- cell. 该方法已被用于开发例如抗流感嗜血杆菌(Haemophilus influenzae)的疫苗。 This method has been used to develop such as anti-Hib (Haemophilus influenzae) vaccine. 然而,有关多重多糖疫苗和基于缀合物的疫苗的花费仍是争论的焦点。 However, the costs associated multiple polysaccharide vaccines and conjugate-based vaccine is still a bone of contention.

第三种方法是寻找其它具有成为候选疫苗之潜力的抗原组分,这构成了本发明的基础。 A third method is to look for other antigenic components of the vaccine candidate has the potential, which constitutes the basis of the present invention. 使用特别开发的细菌表达系统,我们已经能鉴定出一组肺炎球菌蛋白质抗原,所述抗原与细菌外膜相结合或者能被分泌。 Specially developed using bacterial expression systems, we have been able to identify a group of pneumococcal protein antigen, the antigen is combined with the bacterial outer membrane or be secreted.

因此,第一方面,本发明提供了肺炎链球菌的蛋白质或多肽,其具有选自表1所示的序列。 Accordingly, a first aspect, the present invention provides a Streptococcus pneumoniae protein or polypeptide having the sequence shown in Table 1 is selected.

第二方面,本发明提供了肺炎链球菌的蛋白质或多肽,其具有选自表2所示的序列。 A second aspect, the present invention provides a Streptococcus pneumoniae protein or polypeptide having the sequence shown in Table 2 is selected.

所提供的本发明的蛋白质或多肽可以是基本上纯的形式。 A protein or polypeptide of the invention may be provided in substantially pure form. 例如,可以是基本上不含其它蛋白质的形式。 For example, the form may be substantially free of other proteins.

如本文所述,本发明的蛋白质和多肽可用作抗原物质。 As described herein, the proteins and polypeptides of the present invention are useful as antigenic material. 所述物质可以是“抗原性的”和/或“免疫原性的”。 The material can be "antigenic" and / or "immunogenic." 通常,“抗原性的”是指蛋白质或多肽能被用于产生抗体,或者实际上能在受试者体内诱导抗体应答。 Generally, "antigenic" refers to a protein or polypeptide can be used to raise antibodies or indeed is capable of inducing an antibody response in a subject. “免疫原性的”是指蛋白质或多肽能在受试者体内引发保护性免疫应答。 "Immunogenic" refers to a protein or polypeptide capable of eliciting a protective immune response in a subject. 因此,在后一种情况下,蛋白质或多肽不仅能产生抗体应答,还能产生不基于抗体的免疫应答。 Thus, in the latter case, the protein or polypeptide not only generate an antibody response, but also to produce antibody-based immune response.

本领域技术人员应懂得本发明蛋白质或多肽的同系物或衍生物也可用于本发明的上下文,即用作抗原性的/免疫原性的物质。 Those skilled in the art should understand homologues or derivatives of the present invention is a protein or polypeptide may also be used in the context of the present invention, i.e. as / immunogenicity of antigenic. 因此,本发明还包括例如具有一个或多个添加,缺失,取代等的蛋白质或多肽。 Accordingly, the present invention further includes, for example, protein or polypeptide having one or more additions, deletions, substitutions and the like. 另外,可以用一种氨基酸取代另一种相似“类型”的氨基酸。 In addition, another substituent may be similar "type" of an amino acid with an amino acid. 例如,用一种疏水氨基酸取代另一种疏水氨基酸。 For example, another hydrophobic amino acid substituted with one hydrophobic amino acid.

可以使用程序(如CLUSTAL程序)比较氨基酸序列。 Program may be used (e.g., CLUSTAL program) to compare amino acid sequences. 该程序可比较氨基酸序列,并通过适当时在任一序列中插入间隔找到最佳序列对比。 The amino acid sequence comparison program may, where appropriate, either by inserting a spacer sequence to find the best sequence alignment. 可以计算最佳序列对比的氨基酸同一性或相似性(同一性加上氨基酸类型的保守性)。 Calculates optimal comparison amino acid sequence identity or similarity (identity plus conservation of amino acid type). 诸如BLASTx的程序会对比最长的一段相似序列并指定符合值。 Such as BLASTx program will compare the longest stretch of similar sequences and assign a value that meets. 因此,可以得到比较结果,在其中可以发现几个相似性区域,它们各自具有不同的评分值。 Thus, it is possible to obtain a comparison result, where several regions of similarity may be found, each having a different score values. 本发明希望进行这两种类型的同一性分析。 The present invention is desirable for both types of identity analysis.

对同系物和衍生物而言,与本文所述蛋白质或多肽的同一性程度较不重要,重要的是同系物或衍生物应该保持原始蛋白质或多肽的抗原性或免疫原性。 For homologues and derivatives, the degree of identity described herein is less important protein or polypeptide, it is important homolog or derivative should maintain the original antigenic protein or polypeptide or immunogenic. 然而,提供与本文所述蛋白质或多肽具有至少60%相似性的同系物或衍生物(如上文所讨论)较为合适,优选提供具有至少70%相似性的同系物或衍生物,更优选提供具有至少80%相似性的同系物或衍生物,最优选提供具有至少90%或甚至95%相似性的同系物或衍生物。 However, providing the protein or polypeptide described herein having at least 60% similarity homologues or derivatives (as discussed above) is appropriate, preferably providing at least 70% similarity homologues or derivatives thereof, and more preferably provide a at least 80% similarity homologues or derivatives thereof, most preferably provided with at least 90% or even 95% similarity homologues or derivatives thereof.

在另一种方法中,同系物或衍生物可以是融合蛋白,其中掺入的组成成分通过例如有效地标记所需蛋白质或多肽而使纯化变得更容易。 In another approach, the homologues or derivatives could be fusion proteins, wherein the components incorporated for example by effectively marking the desired protein or polypeptide purification easier. 必需除去“标记物”,或者,事实上融合蛋白自身应该保留足够的抗原性以使其有用。 Must be removed "label", or, in fact, be a fusion protein itself retains sufficient antigenicity to be useful it.

在本发明的另一方面,提供了本发明蛋白质或多肽,或其同系物或衍生物的抗原性/免疫原性片段。 In another aspect of the present invention, there is provided a protein or polypeptide of the invention, or an antigenic / immunogenic fragment or derivative homologues.

就本文所述蛋白质或多肽,或其同系物或衍生物的片段而言,情况略有不同。 To the protein or polypeptide described herein, or a fragment or derivative homolog, the situation is slightly different. 众所周知,可以筛选抗原蛋白质或多肽以鉴定表位区域,即负责蛋白质或多肽的抗原性或免疫原性的区域。 It is well known antigenic protein or polypeptide can be screened to identify the epitope region, i.e., protein or polypeptide region responsible for antigenic or immunogenic. 进行这种筛选的方法是本领域众所周知的,因此,本发明的片段应该包括一个或多个所述表位区域,或者与所述区域足够相似以保留其抗原性/免疫原性。 Such screening methods are well known in the art, therefore, should be fragment of the invention comprises one or more of the epitope region, the region or similar enough to retain their antigenic / immunogenic. 因此,对于本发明的片段而言,同一性程度可能是不相关的,因为它们可以与本文所述的蛋白质或多肽,同系物或衍生物的特定部分100%相同。 Thus, for fragments according to the present invention, the degree of identity may be irrelevant, since they may be the same as the proteins or polypeptides described herein certain portions of 100% homologue or derivative thereof. 同样,主要的问题是片段必须保留抗原性/免疫原性。 Likewise, the main problem is that fragment must be retained antigenicity / immunogenicity.

因此,对于由蛋白质或多肽衍生得到的同系物,衍生物和片段而言,重要的是它们应具有其所衍生自的所述蛋白质或多肽的至少部分抗原性/免疫原性。 Thus, for obtaining a protein or polypeptide derived from homologues, derivatives and fragments, it is important that they should have the protein from which it is derived at least in part or polypeptide antigenic / immunogenic.

可使用基因克隆技术提供基本上纯的本发明蛋白质。 Gene cloning techniques may be used to provide a substantially pure protein of the invention. 所述技术公开于例如Sambrook等,分子克隆,第2版,冷泉港实验室出版社(1989)。 The technique is disclosed in, for example Sambrook et al, Molecular Cloning, 2nd Ed., Cold Spring Harbor Laboratory Press (1989).

因此,第三方面,本发明提供了核酸分子,其含有下列序列或由下列序列组成:(ⅰ)表1所示的任何DNA序列或其RNA等同物;(ⅱ)与(ⅰ)中的任一序列互补的序列;(ⅲ)与(ⅰ)或(ⅱ)中的序列编码相同蛋白质或多肽的序列;(ⅳ)与(ⅰ),(ⅱ)和(ⅲ)中的任一序列基本上相同的序列;(ⅴ)编码表1所限定蛋白质的同系物,衍生物或片段的序列。 Accordingly, a third aspect, the present invention provides a nucleic acid molecule comprising the following sequences or consist of the following sequences: (i) any DNA sequence table or RNA equivalent as depicted. 1; (ii) any one of (i) the a sequence complementary to sequence; the coding sequence of the sequence (i) or (ii) in the same protein or polypeptide of (iii); (iv) and (ⅰ), (ⅱ) and (iii) any one of the sequences substantially the same sequence; sequence homologs (ⅴ) encoding a protein as defined in table 1, derivatives or fragments.

第四方面,本发明提供了核酸分子,其含有下列序列或由下列序列组成:(ⅰ)表2所示的任何DNA序列或其RNA等同物;(ⅱ)与(ⅰ)中的任一序列互补的序列;(ⅲ)与(ⅰ)或(ⅱ)中的序列编码相同蛋白质或多肽的序列;(ⅳ)与(ⅰ),(ⅱ)和(ⅲ)中的任一序列基本上相同的序列;(ⅴ)编码表2所限定蛋白质的同系物,衍生物或片段的序列。 A fourth aspect, the present invention provides a nucleic acid molecule comprising the following sequences or consist of the following sequences: (i) any DNA sequence table or RNA equivalent as depicted 2; (ii) any of the sequences of (i) the complementary sequence; (iii) a sequence encoding (i) or (ii) in the same protein or polypeptide sequence; (iv) and (ⅰ), (ⅱ) and (iii) any one of the sequences are substantially identical sequence; sequence homologs (ⅴ) encoding a protein as defined in table 2, derivatives or fragments.

本发明的核酸分子包括多个这种序列和/或片段。 Nucleic acid molecule of the present invention includes a plurality of such sequences and / or fragments. 本领域技术人员应懂得本发明包括本文所例举的新的特定核酸分子的新变体。 Those skilled in the art should be understood that the invention includes a new new variants of a particular nucleic acid molecules exemplified herein. 本发明包括这种变体。 The present invention includes such variants. 所述变体可以是天然变体,例如由于菌株变异而产生的变体。 The variants may be naturally occurring variants, for example variants produced due strain variation. 例如,包括添加,取代和/或缺失。 For example, including additions, substitutions and / or deletions. 另外,特别是当使用微生物表达系统时,人们希望通过利用表达所用特定生物体中已知的偏好使用的密码子来改造核酸序列。 In addition, particularly when using the microbial expression systems, it is desirable to transform the nucleic acid sequence of a particular organism using known codon preferences employed by using expression. 因此,本发明的范围内也包括合成的或非-天然的变体。 Therefore, within the scope of the present invention also comprises synthetic or non - natural variants.

上文所用术语“RNA等同物”表示给定的RNA分子具有的序列与给定DNA分子的序列互补(允许在RNA遗传密码中用“U”替代“T”)。 The above represents a given RNA molecule has a sequence of a given DNA molecule complementary to the sequence (using the genetic code allows RNA "U" alternative "T"), the term "RNA equivalent."

当比较核酸序列以测定同源性或同一性程度时,可以使用例如BESTFIT和GAP(皆得自Wisconsin Genetics Computer Group(GCG)软件包)BESTFIT程序比较两个序列,产生最相似区段的最佳序列对比。 When comparing nucleic acid sequences to determine the degree of homology or identity, may be used, for example, BESTFIT and GAP (from Jiede Wisconsin Genetics Computer Group (GCG) software package) BESTFIT program comparison of two sequences, most similar segment to produce the best sequence comparison. GAP能使序列沿着其全长进行对比,并通过适当时在任一序列中插入间隔找到最佳序列对比。 GAP enables sequences were compared along its entire length, by any of the appropriate spacer sequence is inserted to find the best sequence alignment. 在本发明的上下文中,当讨论核酸序列的同一性时,适于通过沿着全长序列进行序列对比来比较序列。 In the context of the present invention when discussing identity of nucleic acid sequences by comparing the sequences is adapted to compare a sequence along the full length sequence.

优选基本上相同的序列与所述序列具有至少50%的序列同一性,更优选具有至少75%的序列同一性,更优选具有至少90%或至少95%的序列同一性。 Preferably substantially identical to the sequence of a sequence having at least 50% sequence identity, more preferably at least 75% sequence identity, more preferably at least 90% or at least 95% sequence identity. 在某些情况下,序列同一性可以是99%或以上。 In some cases the sequence identity may be 99% or more.

优选地,术语“基本上相同”表示:相对于所述序列与现有技术的核酸序列的同一性程度而言,所述序列与本文所述任一序列的同一性程度更高。 Preferably, the term "substantially identical" means: a nucleic acid sequence with respect to the degree of identity with the sequence of the prior art, the high degree of identity with the sequence of any one of the sequences described herein.

然而,应该指出当本发明的核酸序列编码至少部分新基因产物时,本发明范围内包括编码基因产物或其新的部分的所有可能的序列。 However, it should be noted that when a new nucleic acid sequence encoding a gene product of the present invention is at least partially, within the scope of the present invention comprises encoding a gene product or a new portion of all possible sequences.

核酸分子可以是分离的形式或重组的形式。 The nucleic acid molecule may be in the form of isolated or recombinant. 可以将其掺入载体,载体可被掺入宿主。 Which may be incorporated into a vector, the vector can be incorporated into the host. 所述载体和适当的宿主构成本发明的另一方面。 The vectors and suitable host constitutes another aspect of the present invention.

因此,例如,通过使用基于本文所提供的核酸序列的探针,可以鉴定肺炎链球菌的基因。 Thus, for example, by using nucleic acid sequence based probes provided herein, the gene can be identified in Streptococcus pneumoniae. 使用限制性酶可以切下这些基因,将其克隆至载体,再将载体导入适当宿主以进行表达。 We can cut using restriction enzymes these genes, cloned into the vector, and then introducing the vector into an appropriate host for expression.

通过使用与核酸分子的部分序列互补的适当探针,可以从肺炎链球菌中得到本发明的核酸分子。 By appropriately using a probe a partial sequence complementary to a nucleic acid molecule, the nucleic acid molecule of the invention can be obtained from the S. pneumoniae. 可使用限制性酶或超声处理技术得到适当大小的片段以用作探针。 Using restriction enzymes or sonication techniques fragment of appropriate size for use as a probe.

或者,可使用PCR技术扩增所需的核酸序列。 Alternatively, a PCR amplified nucleic acid sequence desired. 因此,可使用本文提供的序列资料设计两个引物用于PCR,从而使包括完整基因或其片段的所需序列被靶向,随即被高水平地扩增。 Thus, the sequence data provided herein can be used to design two primers for the PCR, so that the desired sequence comprises the complete gene or fragment thereof is targeted, then amplified at high levels.

引物一般至少为15至25个核苷酸长。 Primer typically at least 15 to 25 nucleotides long.

另一种方法是使用化学合成,该方法可以实行自动化。 Another approach is to use chemical synthesis, the method may automate. 可以化学合成相对较短的序列,再相互连接形成较长的序列。 It can be synthesized chemically relatively short sequence, and then interconnected to form a longer sequence.

使用本文所述的细菌表达系统已鉴定出另一组得自肺炎链球菌的蛋白质。 Using a bacterial expression system described herein has identified another group of proteins from S. pneumoniae. 它们是肺炎链球菌的已知蛋白质,但以前无人将它们鉴定为抗原蛋白质。 They are known proteins of Streptococcus pneumoniae, but no one before them was identified as a protein antigen. 该组蛋白质的氨基酸序列以及编码它们的DNA序列示于表3。 The group of proteins, and amino acid sequence of the DNA sequences encoding them are shown in Table 3. 这些蛋白质或其同系物,衍生物和/或片段也可用作抗原/免疫原。 These proteins or a homologue, derivative and / or fragment thereof may be used as an antigen / immunogen. 因此,另一方面,本发明提供了具有选自表1-3所示序列之序列的蛋白质或多肽或其同系物,衍生物和/或片段作为免疫原/抗原的用途。 Accordingly, another aspect, the present invention provides a use of a protein or polypeptide sequence selected from Table sequence of 1-3 or a homologue, derivative and / or fragment thereof as an immunogen / antigen.

另一方面,本发明提供了免疫原性/抗原性的组合物,该组合物含有一种或多种具有选自表1-3所示序列之序列的蛋白质或多肽或其同系物或衍生物,和/或它们中的任一个的片段。 Another aspect, the present invention provides an immunogenic / antigenic composition, the composition contains one or more sequences having the sequence shown in Table 1-3 selected protein or polypeptide or a homologue or derivative thereof , and / or fragments of any of them. 在优选的实施方案中,免疫原性/抗原性的组合物是疫苗,或者可用于诊断试验。 In a preferred embodiment, the immunogenic / antigenic composition is a vaccine, or can be used in diagnostic assays.

就疫苗而言,它可包括适当添加的赋形剂,稀释剂,佐剂等。 Case of vaccines, which may include adding suitable excipients, diluents, adjuvants, etc. 多个具体实例是本领域众所周知的。 Numerous specific examples are known in the art.

也可以使用表1-3所示的核酸序列制备所谓的DNA疫苗。 It may also be used in the nucleic acid sequence shown in Table 1-3 prepared in so-called DNA vaccines. 因此,本发明也提供了含有一种或多种本文所定义的核酸序列的疫苗组合物。 Accordingly, the present invention also provides a nucleic acid sequence comprising one or more defined herein vaccine composition. 本领域中已有人描述过DNA疫苗(例见Donnelly等,免疫学年评,15:617-648(1997)),熟练技术人员可使用本领域所述技术生产和使用本发明的DNA疫苗。 Some in the art have been described DNA vaccine (see Example Donnelly et al., Annual Review of immunity, 15: 617-648 (1997)), the skilled person in the art may be used and the use of DNA technology to produce vaccines of the present invention.

如上所述,在检测/诊断肺炎链球菌的方法中,可以使用本文所述的蛋白质或多肽,其同系物或衍生物,和/或它们中的任一个的片段。 As described above, in the detection / diagnosis of S. pneumoniae methods may be used as described herein to a protein or polypeptide, homologue or derivative thereof, and / or a fragment of any of them. 检测受试者体内存在的抗所述蛋白质的抗体,以此为基础建立上述方法。 Antibodies against the protein of detecting the presence of a subject, the method described above in order to establish the basis. 因此,本发明提供了检测/诊断肺炎链球菌的方法,所述方法包括使受试样品与至少一种本文所述的蛋白质或其同系物,衍生物或片段接触的步骤。 Accordingly, the present invention provides a detection / diagnosis of Streptococcus pneumoniae, said method comprising contacting a test sample with at least one kind of a protein or a homologue thereof as described herein, the step of contacting a fragment or derivative. 优选样品为生物样品,如得自待测受试者的组织样品或血样或唾液。 Preferably the sample is a biological sample, such as blood or tissue sample obtained from a subject to be measured or saliva.

在另一种方法中,可使用本文所述的蛋白质,或其同系物,衍生物和/或片段产生抗体,反过来,所述抗体可用于检测抗原,因而可检测肺炎链球菌。 In another approach, a protein may be used as described herein, or a homologue, derivative and / or fragment to produce antibodies, in turn, be used to detect the antigen antibody, thus the detection of Streptococcus pneumoniae. 所述抗体构成本发明的另一方面。 The antibodies constituting another aspect of the present invention. 本发明范围内的抗体可以是单克隆或多克隆抗体。 Antibodies within the scope of the present invention may be a monoclonal or polyclonal antibody.

当将本文所述的蛋白质,或其同系物,衍生物或片段注射至适当动物宿主(如小鼠,大鼠,豚鼠,兔,绵羊,山羊或猴)体内时,通过刺激抗体生成即可使动物产生多克隆抗体。 When the proteins described herein, or a homologue, derivative or fragment thereof is injected into a suitable animal host (e.g., mouse, rat, guinea pig, rabbit, sheep, goat or monkey) in vivo by stimulating antibody production can make animal to produce polyclonal antibodies. 必要时,可同时使用佐剂和蛋白质。 If necessary, adjuvants can be used simultaneously and proteins. 众所周知的佐剂包括弗氏佐剂(完全和不完全佐剂)和氢氧化铝。 Known adjuvants include Freund's adjuvant (complete and incomplete adjuvants) and aluminum hydroxide. 然后,利用抗体与本文所述蛋白质的结合即可纯化抗体。 Then, using an antibody binding to purified antibody to the protein described herein.

单克隆抗体可由杂交瘤产生。 Monoclonal antibodies produced by the hybridoma. 通过融合骨髓瘤细胞和产生所需抗体的脾细胞以形成无限增殖的细胞系即可形成杂交瘤。 By fusing myeloma cells and spleen cells produce the desired antibody to form an immortal cell line to form hybridomas. 因此,可使用众所周知的Kohler&Milstein技术(自然,256(1975))或在此技术的基础上进行改动的技术。 Thus, using the well known Kohler & amp; Milstein technique (Nature, 256 (1975)) or to make modifications in the art based on this technology.

目前,生产与特定多肽/蛋白质结合的单克隆和多克隆抗体的技术已是本领域的成熟技术。 At present, the production of a particular polypeptide / protein binding of monoclonal and polyclonal antibodies is an established technique in the art in the art. 有关讨论可参见标准的免疫学课本,例如Roitt等,免疫学,第2版(1989),Churchill Livingstone,伦敦。 For a discussion can be found in standard immunology textbooks, for example Roitt etc., Immunology, 2nd edition (1989), Churchill Livingstone, London.

除了完整的抗体外,本发明还包括该抗体的衍生物,所述衍生物能与如本文所述的蛋白质等结合。 In addition to intact antibodies, the present invention further includes derivatives of the antibodies, the derivatives can be combined with other proteins as described herein. 因此,本发明包括抗体片段和合成构建体。 Accordingly, the present invention includes antibody fragments and synthetic constructs. 抗体片段和合成构建体的例子可参见Dougall等,Tibtech 12372-379(1994年9月)。 Examples of antibody fragments and synthetic constructs can be found in other Dougall, Tibtech 12372-379 (September 1994).

抗体片段包括例如Fab,F(ab')2和Fv片段。 Antibody fragments include, for example, Fab, F (ab ') 2 and Fv fragments. Fab片段(这些片段在Roitt等[文献同上]中讨论)。 Fab fragments (these fragments in Roitt et al [supra] discussed). 可修饰Fv片段以产生已知为单链Fv(scFv)分子的合成构建体。 Fv fragments can be modified to produce a composite known as single chain Fv (scFv) molecule construct. 该分子包括与Vh和Vl区共价连接的肽接头,它对分子的稳定性有所贡献。 The molecule includes a peptide linker covalently connecting the Vh and Vl regions, contribute to the stability of the molecule. 可以使用的其它合成构建体包括CDR肽,它们是含有抗原-结合决定簇的合成肽。 Other synthetic constructs that can be used include CDR peptides, which are containing the antigen - binding synthetic peptide determinant. 也可使用模拟肽,这些分子通常是构象受限的有机环,它能模拟CDR环的结构,并包括能与抗原相互作用的侧链。 Peptidomimetics may also be used, these molecules are usually conformationally constrained organic ring, it can simulate the structure of CDR loops, and include side chains capable of interacting with the antigen.

合成构建体包括嵌合分子。 Synthetic construct comprising a chimeric molecule. 因此,例如,本发明范围内还包括人源化(或灵长动物化)抗体或其衍生物。 Thus, for example, within the scope of the present invention further comprises a humanized (or primatized) antibodies or derivatives thereof. 人源化抗体的例子是具有人构架区和啮齿动物高变区的抗体。 Examples of the humanized antibody is an antibody having human framework regions and rodent hypervariable regions. 产生嵌合抗体的方法可参见例如Morrison等,PNAS,81,6851-6855(1984)和Takeda等,自然,314,452-454(1985)。 Methods for producing chimeric antibodies can be found, for example, Morrison et al., PNAS, 81,6851-6855 (1984) and the like Takeda, Nature, 314,452-454 (1985).

合成构建体还包括含有其它组成成分的分子,所述其它成分可为分子提供一些除抗原结合特性之外的所需特性。 Synthetic construct further comprises molecules containing other ingredients, other ingredients can provide the desired characteristics in addition to the antigen-binding properties of the molecule. 例如,所述组成成分可以是标记物(例如荧光或放射性标记)。 For example, the composition may be a label (e.g. a fluorescent or radioactive label). 或者,也可以是药物活性剂。 Alternatively, it may be a pharmaceutically active agent.

抗体或其衍生物可用于检测/诊断肺炎链球菌。 Antibody or derivative thereof can be used for the detection / diagnosis of S. pneumoniae. 因此,另一方面,本发明提供了检测/诊断肺炎链球菌的方法,所述方法包括使受试样品与抗体接触的步骤,所述抗体能结合一种或多种本文所述的蛋白质,或其同系物,衍生物和/或片段。 Accordingly, another aspect, the present invention provides a detection / diagnosis of Streptococcus pneumoniae, said test method comprising the step of contacting the sample with an antibody, the antibody capable of binding to one or more of the proteins described herein, or a homologue, derivative and / or fragment thereof.

另外,可使用所谓的“亲和体”。 In addition, you can use the so-called "pro and body." 这些亲和体是选自α-螺旋细菌受体结构域之组合文库的结合蛋白(Nord等)。 These bodies are selected from a combination of the affinity domain of α- helical bacterial receptor libraries of binding protein (Nord, etc.). 因此,可使用组合文库法选择能与不同靶蛋白质特异性结合的小蛋白质结构域。 Thus, the selection of small protein domains capable of specifically binding to different target proteins using the combinatorial library method.

显然,可使用本文所述的核酸序列检测/诊断肺炎链球菌。 Obviously, the detection of nucleic acid sequences may be used herein / diagnosis Streptococcus pneumoniae. 因此,另一方面,本发明提供了检测/诊断肺炎链球菌的方法,所述方法包括使受试样品与至少一种本文所述的核酸序列接触。 Accordingly, another aspect, the present invention provides a detection / diagnosis of Streptococcus pneumoniae, said method comprising contacting a nucleic acid sequence of the test sample with at least one article. 优选样品为生物样品,如得自待测受试者的组织样品或血样或唾液。 Preferably the sample is a biological sample, such as blood or tissue sample obtained from a subject to be measured or saliva. 在用于本发明的方法之前,可对样品进行预处理。 Before the method of the present invention, the sample may be pretreated. 因此,例如,可处理样品以提取DNA。 Thus, for example, the sample may be processed to extract DNA. 然后可使用基于本文所述核酸序列(通常为该序列的片段)的DNA探针检测肺炎链球菌的核酸。 May then be detected using nucleic acid S. pneumoniae DNA probe based on the nucleic acid sequence described herein (typically a fragment of that sequence) is.

在其它方面,本发明提供了:(a)免疫接种受试者以防肺炎链球菌的方法,所述方法包括给受试者施用本发明的蛋白质或多肽,或其衍生物,同系物或片段,或本发明的免疫原性组合物的步骤;(b)免疫接种受试者以防肺炎链球菌的方法,所述方法包括给受试者施用本文所定义的核酸分子的步骤;(c)预防或治疗肺炎链球菌感染的方法,所述方法包括给受试者施用本发明的蛋白质或多肽,或其衍生物,同系物或片段,或本发明的免疫原性组合物的步骤;(d)预防或治疗肺炎链球菌感染的方法,所述方法包括给受试者施用本文所定义的核酸分子的步骤;(e)用于检测/诊断肺炎链球菌感染的试剂盒,其包括一种或多种本发明的蛋白质或多肽,或其同系物,衍生物或片段,或本发明的抗原性组合物;和(f)用于检测/诊断肺炎链球菌感染的试剂盒,其包括一种或多种 In other aspects, the present invention provides: (a) a subject immunized against Streptococcus pneumoniae, said method comprising administering to a subject a protein or polypeptide of the present invention, or a derivative, homologue or fragment thereof or step immunogenic composition of the present invention; (b) a method of immunization against Streptococcus pneumoniae subject, said method comprising the step of administering to a subject a nucleic acid molecule as defined herein; (c) a method for preventing or treating Streptococcus pneumoniae infection, said method comprising administering to a subject a protein or polypeptide of the present invention, or a derivative, homologue or fragment of step, or immunogenic compositions of the present invention; (D ) a method of preventing or treating Streptococcus pneumoniae infection, said method comprising the step of nucleic acid molecules to a subject, as defined herein, administered; (e) for the detection / diagnosis of S. pneumoniae infection in a kit, which comprises one or more protein or polypeptide of the present invention, or a homologue, derivative or fragment thereof or antigenic composition of the present invention; and (f) for the detection / diagnostic kit for the S. pneumoniae infection, which comprises one or multiple 文所定义的核酸分子。 A nucleic acid molecule as defined.

即使我们已鉴定出一组重要的蛋白质,所述蛋白质是抗微生物疗法的潜在靶标。 Even if we have identified an important group of proteins, the protein is an antimicrobial potential target for therapy. 然而仍必需测定每个蛋白质是否是微生物存活所必需的。 Yet still necessary to determine whether each protein is essential for the survival of microorganisms. 因此,本发明也提供了测定本文所述蛋白质或多肽是否为潜在的抗微生物靶标的方法,所述方法包括拮抗,抑制或要不然干扰所述蛋白质的功能或表达,并测定肺炎链球菌是否仍旧存活。 Accordingly, the present invention also provides a Streptococcus pneumoniae protein or polypeptide assay described herein, whether as a potential target, the method comprising the antimicrobial antagonize, inhibit or otherwise interfere with the function or expression of the protein, and determining whether the still survival.

灭活蛋白质的适当方法是实行选定的基因敲除,即阻止蛋白质的表达并测定是否会导致致死性改变。 Appropriate method of inactivating protein is the implementation of the selected gene knockout, that prevent the expression of the protein and determine whether the changes will lead to death. 进行所述基敲除的适当方法描述于Li等,美国国家科学院院报(PNAS),94:13251-13256(1997)和Kolkman等,178:3736-3741(1996)。 Suitable methods for the knockout group are described in Li et al., PNAS USA (PNAS), 94: 13251-13256 (1997) and Kolkman et al., 178: 3736-3741 (1996).

最后一方面,本发明提供了能拮抗,抑制或要不然干扰本发明蛋白质或多肽的功能或表达的药剂用于制备药物的用途,所述药物可用于治疗或预防肺炎链球菌感染。 In a final aspect, the present invention provides a can antagonize, inhibit or otherwise interfere with the function of the present invention is a protein or polypeptide or expression of an agent for the manufacture of a medicament, the medicament useful for the treatment or prevention of S. pneumoniae infection.

如上所述,我们已使用细菌表达系统作为鉴定蛋白质的工具,所述蛋白质是与表面相结合的,被分泌或输出的蛋白质,因此可用作抗原。 As described above, we have used a bacterial expression system as a tool for the identification of protein, the protein is a combination of a surface, secreted or exported proteins are therefore useful as antigens.

已深入研究了细菌中分泌/输出蛋白质所必需的遗传信息。 We studied the genetic information in bacteria secrete / output necessary to have in-depth protein. 在大多数情况下,蛋白质输出需要前体蛋白的N-末端存在信号肽,以将其导向质膜上的转运机器。 In most cases, the precursor protein required for protein export N- terminal signal peptide is present, to transport the machine which guide the plasma membrane. 转运当中或转运之后,通过与膜结合的信号肽酶除去信号肽。 Among the transport or after the transport, the signal peptide is removed by signal peptidase bound to the membrane. 最后,通过序列而不是前导肽本身来确定蛋白质的定位(即蛋白质是被分泌,还是膜内在蛋白质或是结合至细胞壁上)。 Finally, to determine the localization of the protein, rather than by a leader peptide sequence itself (i.e., the protein is secreted, or the protein binding membrane or to the cell wall).

我们对位于表面或被输出的蛋白质特别感兴趣,因为它们可能是抗原,可以用于疫苗,用作诊断试剂或用新的化合物分子进行治疗的靶。 We are particularly interested in the protein located on the surface or output, because they may be an antigen, can be used for vaccines, diagnostic reagents or as therapeutic target molecule with the new compounds. 因此,我们在乳酸乳球菌(Lactococcus lactis)中研究出一种筛选载体-系统,使用该系统可鉴定和分离编码输出蛋白质的基因。 Therefore, we developed a screening vector in L. lactis (Lactococcus lactis) - the system, the system may use the identification and isolation of the protein encoded output. 下文将提供代表性的例子,显示如何鉴定和表征得自肺炎链球菌的给定的新的表面结合蛋白质。 Representative examples will be provided below, show how to identify and obtain from the table given new surface binding protein of Streptococcus pneumoniae. 筛选载体掺入缺乏其自身输出信号的葡萄球菌核酶基因nuc作为分泌报道基因。 Lack of incorporation of its own output signal of the ribozyme gene screening vector aureus nuc as a secreted reporter gene. 葡萄球菌核酶是天然分泌的热-稳定性单体酶,它可以在革兰氏阳性细菌中被有效表达和分泌(Shortle,基因,22:181-189(1983);Kovacevic等,细菌学杂志,162:521-528(1985);Miller等,细菌学杂志,169:3508-3514(1987);Liebl等,细菌学杂志,174:1854-1861(1992);LeLoir等,细菌学杂志,176:5135-5139(1994);Poquet等,细菌学杂志,180:1904-1912(1998))。 Ribozymes are naturally secreted staphylococcal thermal - monomeric enzyme stability, which can be efficiently expressed and secreted (Shortle, Gene, 22 in Gram-positive bacteria: 181-189 (1983); Kovacevic et al., J. Bacteriol. , 162: 521-528 (1985); Miller et al., Journal of bacteriology, 169: 3508-3514 (1987); Liebl et al., Journal of bacteriology, 174: 1854-1861 (1992); LeLoir et al., Journal of bacteriology, 176 : 5135-5139 (1994); Poquet et al., J. Bacteriol., 180: 1904-1912 (1998)).

最近,Poquet等((1998),文献同上)描述了一种筛选载体,其掺入缺乏其自身前导信号的nuc基因作为报道基因,以鉴定革兰氏阳性细菌中的输出蛋白质,该载体已应用于乳酸乳球菌。 Recently, Poquet et ((1998), supra) describes a screening vector which incorporated nuc gene lacking its own preamble signal as a reporter gene to identify the protein output of Gram-positive bacteria, the vector has been applied in Lactococcus lactis. 除了含有促进大肠杆菌和某些其它革兰氏阴性细菌中的复制的ColE1复制子外,该载体(pFUN)还含有在宽宿主范围的革兰氏阳性细菌中起作用的pAMβ1复制子。 In addition to promoting comprising ColE1 replicon-replicating E. coli and some other gram-negative bacteria, but the carrier (pFUN) further contains Gram-positive bacteria in the broad host range replicon function pAMβ1. 可使用载体中存在的唯一克隆位点产生转录和翻译融合物,所述融合物是在克隆的基因组DNA片段和不含其自身信号分泌前导序列的截短nuc基因的开放阅读框之间产生的。 Using the unique cloning site present in the vector to generate fusion transcription and translation, is the fusion between the genomic DNA fragment was cloned with and without its own signal secretion leader sequence of the open reading frame truncated gene generated nuc . nuc基因是理想的报道基因,因为使用简单而敏感的平板试验即可容易地检测到核酶的分泌:分泌核酶的重组菌落产生粉色晕圈,而对照菌落仍为白色(Shortle,1983,文献同上;Le Loir等,1994,文献同上)。 nuc gene is an ideal reporter gene, because of the use of simple and sensitive plate assay can readily detect the ribozyme secretion: secretion produced pink colonies ribozyme recombinant halo, whereas the control remains white colonies (Shortle, 1983, literature supra; Le Loir et al., 1994, supra).

因此,以下将参照代表性的实施例描述本发明,这些实施例将详细描述本文所述的蛋白质,多肽和核酸序列是如何被鉴定为抗原靶的。 Accordingly, the present invention will be described below with reference to representative embodiments, these embodiments will be described in detail a protein, polypeptide and nucleic acid sequences described herein are identified according to how the target is an antigen.

在本文中,我们描述了3个报道载体的构建,以及它们在乳酸乳球菌中用于鉴定和分离编码分泌的或表面结合蛋白质的肺炎链球菌基因组DNA片段的用途。 In this article, we describe the construction of three reporter vector, as well as their identification and isolation of coding for secreted or surface bound use of S. pneumoniae genomic DNA fragments of a protein in L. lactis.

以下将参照实施例描述本发明,不应将实施例看成是以任何方式限制本发明。 The following embodiments described with reference to embodiments of the present invention, as in any way limiting embodiment of the present invention should not be implemented. 实施例中参照的附图为:图1:显示出大量DNA疫苗试验的结果;和图2:显示出另一些DNA疫苗试验的结果。 Example embodiments with reference to the accompanying drawings as follows: Figure 1: shows the results of a large number of DNA vaccine trial; and Figure 2: shows the results of other DNA vaccine trial.

按下述方法构建pTREX载体。 Construction pTREX vector as follows. 通过退火2个互补的寡核苷酸并用Tfl DNA聚合酶延伸,可产生这样的人工DNA片段,所述片段含有推定的RNA稳定化序列,翻译起始区(TIR),用于插入靶基因的多克隆位点和转录终止序列。 Polynucleotide by annealing 2 complementary oligonucleotides and extending with DNA polymerase, TfI, may generate such an artificial DNA fragment that contains putative RNA stabilizing sequence, a translation initiation region (the TIR), for inserting the target gene a multiple cloning site and transcription termination sequence. 有义和反义寡核苷酸在其5'末端分别含有NheⅠ和BamHⅠ的识别位点以便于克隆。 Sense and antisense oligonucleotides recognition site at its 5 'end and each containing NheⅠ BamHⅠ to facilitate cloning. 将该片段克隆至pUC19NT7的XbaⅠ和BamHⅠ位点之间,pUC19NT7是pUC19的衍生物,它含有得自pLET1的T7表达盒(Wells等,应用细菌学杂志,74:629-636(1993)),该表达盒被克隆于EcoRⅠ和HindⅢ位点之间。 This fragment was cloned into the XbaⅠ and between pUC19NT7 BamHⅠ sites, pUC19NT7 is a derivative of pUC19 which contains the T7 expression cassette (Wells et al., Journal of Applied Bacteriology, 74: 629-636 (1993)) is available from pLET1, the expression cassette is cloned between the sites EcoRⅠ and HindⅢ. 所得构建体被称为pUCLEX。 The resulting construct was called pUCLEX. 通过用HindⅢ切割,使其成为平端,再用EcoRⅠ切割以除去pUCLEX的整个表达盒,然后克隆至pIL253的EcoRⅠ和SacⅠ(平端)位点,产生载体pTREX(Wells和Schofield,代谢,遗传学和应用最新进展-NATO ASISeries,H 98:37-62(1996))。 By cutting with HindIII, blunt-ended making it, and then cutting the entire expression cassette EcoRⅠ pUCLEX removed, and then cloned into the SacI and pIL253 the EcoRⅠ (blunted) sites, generating vector pTREX (Wells and Schofield, metabolism, genetics and applications the latest developments -NATO ASISeries, H 98: 37-62 (1996)). 推定的RNA稳定化序列和TIR得自大肠杆菌T7噬菌体序列,并在一个核苷酸位置上被修饰以增强Shine Dalgarno(SD)基元与乳酸乳球菌核糖体16s RNA的互补性(Schofield等私人通信,剑桥大学病理学系)。 Putative RNA stabilizing sequence and TIR sequences derived from the E. coli bacteriophage T7, and is modified to enhance the Shine Dalgarno (SD) motif complementary Milk 16S ribosomal RNA of Lactococcus lactis (Schofield et a nucleotide position in the private communication, Department of pathology, University of Cambridge).

将表现出启动子活性(随后称之为P7)的乳酸乳球菌MG1363染色体DNA片段克隆至表达盒中存在的EcoRⅠ和BglⅡ位点之间,产生pTREX7。 Will exhibit promoter activity (subsequently referred to P7) of Lactococcus lactis MG1363 chromosomal DNA fragments cloned into an expression cassette between the present and EcoRⅠ BglⅡ sites, generating pTREX7. 以前,曾使用启动子探测载体pSB292(Waterfield等,基因,165:9-15(1995))分离出该活性启动子区域。 Previously it had used the promoter probe vector pSB292 (Waterfield et al., Gene, 165: 9-15 (1995)) separating the active promoter regions. 根据厂商的说明,使用VentDNA聚合酶,经PCR扩增启动子片段。 According to the manufacturer's instructions, using VentDNA polymerase promoter fragment was amplified by PCR.

然后按下述构建pTREP1载体。 Then follows pTREP1 vector construct. 通过退火2个重叠的部分互补的合成寡核苷酸,并根据厂商说明用测序酶进行延伸即可产生这样的人工DNA片段,该片段含有转录终止序列,正向pUC测序引物,启动子多克隆位点区域和通用的翻译终止序列。 By annealing two partially complementary overlapping synthetic oligonucleotides, according to the manufacturer's instructions using Sequenase extending to produce such artificial DNA fragment containing a transcription termination sequence, pUC forward sequencing primer, promoter polyclonal site region and universal translation termination sequence. 有义和反义(pTREPF和pTREPR)寡核苷酸在其5'末端分别含有EcoRV和BamHⅠ的识别位点以便于克隆至pTREX7。 Sense and antisense (pTREPF and pTREPR) oligonucleotide at its 5 'end, respectively, and contain EcoRV recognition site to facilitate cloning into the BamHⅠ pTREX7. 转录终止序列源自芽孢杆菌青霉素酶基因,已证实它在乳球菌属中同样有效(Jos等,应用和环境微生物学,50:540-542(1985))。 Transcription termination sequences derived from Bacillus licheniformis penicillinase gene, which has been demonstrated as effective in the Lactococcus genus (Jos et al., Applied and Environmental Microbiology, 50: 540-542 (1985)). 据认为该序列是必需的,因为经观察靶基因在pTREX载体中的表达有缺陷,有人认为这是起始区隐蔽的启动子活性所致(Schofield等,私人通信,剑桥大学病理学系)。 It is believed that the sequence is required because the target gene expression was observed in pTREX defective vector, it was considered covert promoter activity induced initiation region (Schofield et al., Personal communication, Department of Pathology, University of Cambridge). 包括正向pUC引物测序可以直接测定克隆DNA片段的序列。 Including pUC forward sequencing primer sequence may be determined directly cloned DNA fragments. 包括编码3个不同构架中的终止密码子的翻译终止序列可以防止载体基因和克隆DNA片段之间产生翻译融合物。 It comprises three different frame encoding the translation termination codon of the translation termination sequence may prevent fusion between the carrier and the genes cloned DNA fragments. 首先,用EcoRⅠ消化pTREX7载体,根据厂商说明,使用T4 DNA聚合酶(NEB)的5'-3'聚合酶活性使其成为平端。 First, EcoRⅠ pTREX7 digested vector, according to the manufacturer's instructions, using T4 DNA polymerase (NEB) in the 5'-3 'polymerase activity to become blunted. 然后用BglⅡ消化经EcoRⅠ消化并平端化的pTREX7载体,以除去P7启动子。 Was then digested with BglⅡ EcoRⅠ digested and blunted pTREX7 carrier to remove P7 promoter. 用EcoRV和BamHⅠ消化由退火的合成寡核苷酸衍生的人工DNA片段,克隆至经EcoRⅠ(平端)-BglⅡ消化的pTREX7载体,产生pTREP。 Digested with EcoRV and BamHⅠ annealed synthetic oligonucleotides derived artificial DNA fragment, was cloned into EcoRI (blunt end) -BglⅡ pTREX7 digested vector generated pTREP. 然后将被称为P1的乳酸乳球菌MG1363染色体启动子克隆至pTREP表达盒中存在的EcoRⅠ和BglⅡ位点之间,形成pTREP1。 Then will be called Lactococcus lactis MG1363 chromosomal promoter, P1 is cloned into the expression cassette between pTREP present EcoRⅠ and BglⅡ sites formed pTREP1. 使用启动子探测载体pSB292分离该启动子,并通过Waterfield等(1995),文献同上进行鉴定。 Use promoter probe vector pSB292 isolating the promoter and identified by Waterfield supra (1995). 起初,根据厂商说明,使用VentDNA聚合酶经PCR扩增P1启动子片段,并且作为EcoRⅠ-BglⅡ DNA片段被克隆至pTREX。 At first, according to the manufacturer's instructions, using PCR amplified P1 VentDNA polymerase promoter fragment and cloned into pTREX fragment as EcoRⅠ-BglⅡ DNA. 通过限制性酶消化从pTREX1中除去含有EcoRⅠ-BglⅡ P1启动子的片段,并用于克隆至pTREP(Schofield等,私人通信,剑桥大学病理学系)。 Removing the fragment containing the promoter EcoRⅠ-BglⅡ P1 from the pTREX1 by restriction enzyme digestion, and cloned into pTREP (Schofield et al., Personal communication, Department of Pathology, University of Cambridge). (b)PCR扩增金黄色葡萄球菌(S.aureus)nuc基因使用金黄色葡萄球菌nuc基因的核苷酸序列(EMBL数据库登记号为V01281)设计PCR扩增所用的合成寡核苷酸引物。 (B) PCR amplification of S. aureus (of S. aureus) nuc gene nucleotide sequences of the S. aureus nuc gene (EMBL database accession number V01281) design synthetic oligonucleotide primers for PCR amplification were used. 设计引物以扩增nuc基因的成熟形式(被称为nucA),通过用蛋白酶裂解分泌型前肽(被称为Snase B)N-末端的19至21个氨基酸即可产生nucA(Shortle,(1983),文献同上)。 Primers were designed to amplify the mature form of the nuc gene (referred nucA), secreted by cleavage with a protease propeptide (referred Snase B) N- 19 to 21 amino-terminal can generate nucA (Shortle, (1983 ), supra). 设计3个有义引物(nucS1,nucS2和nucS3,附录Ⅰ),每个引物在与nuc基因不同的读码框中具有平端限制性内切核酸酶裂解位点EcoRV或SmaⅠ。 3 design sense primers (nucS1, nucS2 and nucS3, appendix Ⅰ), each primer having a blunt end endonuclease cleavage site of restriction endonuclease EcoRV at SmaⅠ nuc gene or a different reading frame. 另外,分别在有义和反义引物的5'末端掺入BglⅡ和BamHⅠ以便克隆至经BamHⅠ和BglⅡ切割的pTREP1。 Further, there are the 5 'end of the sense and antisense primers to be incorporated BglⅡ BamHⅠ and cloned into BamHⅠ and BglⅡ cut pTREP1. 所有引物的序列示于附录Ⅰ。 All primer sequences are shown in Appendix Ⅰ. 联合使用各个有义引物和上述反义引物,经PCR扩增3个编码核酶基因成熟形式(NucA)的nuc基因DNA片段。 Each joint using the above sense primer and antisense primer, three amplified by PCR ribozyme gene encoding the mature form (NUCA) gene DNA fragment nuc. 使用金黄色葡萄球菌基因组DNA模板,Vent DNA聚合酶(NEB)和厂商推荐的条件,经PCR扩增nuc基因片段。 S. aureus genomic DNA template, Vent DNA polymerase (NEB) and conditions recommended by the manufacturer, the amplified fragment by PCR nuc gene. 起初于93℃变性2分钟,接着以93℃变性45秒,50℃退火45秒和73℃延伸1分钟循环30次,最后于73℃延伸5分钟。 At 93 ℃ initially denatured for 2 min, followed by denaturation at 93 ℃ 45 seconds, 50 deg.] C anneal 45 seconds, and extension 73 ℃ 1 minute cycle 30 times, a final extension at 73 ℃ 5 minutes. 使用Wizard清洗柱(Promega)纯化PCR扩增产物以除去未掺入的核苷酸和引物。 The column was washed using Wizard (Promega) PCR amplification product was purified to remove unincorporated nucleotides and primers. (c)构建pTREP1-nuc载体使用标准条件,用BglⅡ和BamHⅠ消化b部分中所述的纯化的nuc基因片段,连接至经BamHⅠ和BglⅡ切割并去磷酸化的pTREP1,产生pTREP1-nuc1,pTREP1-nuc2和pTREP1-nuc3系列的报道基因载体。 (C) Construction of pTREP1-nuc vector using standard conditions, the purified gene fragments nuc b digested with the portion BamHⅠ BglⅡ and, via connection to the cutting and BglⅡ BamHⅠ and dephosphorylated pTREP1, generating pTREP1-nuc1, pTREP1- nuc2 and pTREP1-nuc3 series of reporter gene vectors. 使用由厂商提供的试剂和缓冲液或使用标准条件进行普通的分子生物学技术(Sambrook和Maniatis,(1989),文献同上)。 Using reagents supplied by the manufacturer using standard conditions and buffer or for general molecular biology techniques (Sambrook and Maniatis, (1989), supra). 在各个pTREP1-nuc载体中,表达盒含有转录终止序列,乳球菌启动子P1,唯一的克隆位点(BglⅡ,EcoRV或SmaⅠ),接着是成熟形式的nuc基因和第二个转录终止序列。 In various pTREP1-nuc vector, the expression cassette comprising a transcription termination sequence, lactococcal promoter P1, a unique cloning site (BglⅡ, EcoRV or SmaI), followed by the mature form of the second nuc gene and a transcription termination sequence. 应注意:在此构建体中故意将nuc基因的翻译和分泌所需序列排除在外。 It should be noted: nuc body deliberately translate the gene and secretion of the desired sequence excluded from this construct. 这类元件只有通过经适当消化的外源DNA片段(相当于靶细菌)来提供,所述片段可被克隆至紧接nuc基因上游的唯一限制性位点。 Such elements only be provided by an appropriately digested foreign DNA fragment (corresponding to target bacterium), the fragment may be cloned into the gene immediately upstream nuc unique restriction sites.

在拥有启动子方面,pTREP1-nuc载体与Poquet等,(1998),文献同上所述的pFUN载体不同,pFUN载体可用于通过直接在乳酸乳球菌中直接筛选Nuc活性来鉴定乳酸乳球菌的输出蛋白质。 In the promoter has, pTREP1-nuc vector and Poquet et al., (1998), supra according pFUN different carrier, pFUN output vectors can be used to identify proteins by direct Lactococcus direct screening of Nuc activity in Lactococcus lactis . 由于pFUN载体在nuc开放阅读框的上游不含有启动子,因此,除了提供Nuc的翻译起始和分泌所需的那些元件外,克隆基因组DNA片段还必须提供转录信号。 Since pFUN vector does not contain a promoter upstream nuc open reading frame, and therefore, in addition to those elements required for translation initiation and secretion of Nuc provide cloned genomic DNA fragments must also provide for transcription signal. 这一限制可防止分离与启动子远离的基因,例如多顺反子操纵子内的基因。 This restriction prevents gene promoter isolated away from, for example, genes within a polycistronic operon. 此外,无法保证得自其它种细菌的启动子能被乳酸乳球菌识别并在其中起作用。 Also, from promoter can not guarantee that other species of bacteria Lactococcus lactis can be identified and played in it. 某些启动子处于天然宿主而不是乳酸乳球菌的严格调控之下。 Some promoters are under strict regulation, rather than the natural host Lactococcus lactis. 相反,pTREP1-nuc系列载体中P1启动子的存在能确保无启动子的DNA片段(或含有在乳酸乳球菌中无活性的启动子序列的DNA片段)仍能被转录。 Rather, there pTREP1-nuc vector series P1 promoter ensures promoterless DNA fragment (containing no or activity in a Lactococcus lactis promoter sequence of the DNA fragment) can still be transcribed. (d)筛选肺炎链球苗中的分泌型蛋白质用限制性酶Tru9Ⅰ消化分离自肺炎链球菌的基因组DNA。 (D) Screening of the pneumococcal vaccine secreted protein was digested with restriction enzymes Tru9Ⅰ isolated from genomic DNA of Streptococcus pneumoniae. 之所以使用识别序列5'-TTAA-3'的此酶是因为它能有效切割富含A/T的基因组,并能产生优选大小范围(通常平均为0.5-1.0kb)内的随机基因组DNA片段。 The reason for using the recognition sequence 5'-TTAA-3 'This enzyme is effective because it can cut rich genome A / T, and the preferred size range can be produced (typically average 0.5-1.0kb) from the random genomic DNA fragments . 之所以优选此大小范围是因为此时P1启动子用于转录新基因序列的可能性更大。 The reason for this size range is preferred because the greater the likelihood of P1 promoters for transcription of the new gene sequence. 然而,在所有的情况下P1启动子不都是必需的,因为很多链球菌的启动子可以在乳酸乳球菌中被识别。 However, in all cases P1 promoter is not necessary, because a lot of streptococcal promoters can be identified in Lactococcus lactis. 从肺炎链球菌基因组DNA的部分Tru9Ⅰ消化物中纯化不同大小范围的DNA片段。 DNA fragments of different size range purified from Streptococcus pneumoniae portion Tru9Ⅰ digest of genomic DNA. 由于Tru9Ⅰ限制性酶产生了交错的末端,因此不得不先使DNA片段成为平端,然后再与经EcoRV或SmaⅠ切割的pTREP1-nuc载体连接。 Since Tru9Ⅰ end restriction enzyme produces a staggered, so that the DNA fragment had to be blunt-ended, and then connected to the pTREP1-nuc vector cut by EcoRV or SmaⅠ. 通过使用Klenow酶的5'-3'聚合酶活性进行部分补平酶反应即可实现此目的。 This object can be achieved by partial filling enzymatic reaction "using Klenow polymerase activity of the enzyme 5'-3. 简单地说,将经Tru9Ⅰ消化的DNA溶解于添加有T4 DNA连接酶缓冲液(New EnglandBiolabs;NEB)(1X)和33μM各种所需dNTP(此处为dATP和dTTP)的溶液中(总体积通常为10-20μl)。 Briefly, the digested DNA was dissolved in Tru9Ⅰ added with T4 DNA ligase buffer (New EnglandBiolabs; NEB) (1X) and various desired 33μM dNTP (dATP here and dTTP) in a solution (total volume usually 10-20μl). 加入Klenow酶(每μg DNA加1个单位Klenow酶(NEB)),25℃保温反应15分钟。 Klenow enzyme was added (plus 1 μg DNA per unit of Klenow enzyme (NEB)), 25 ℃ incubated for 15 min. 通过于75℃保温混合物20分钟以终止反应。 By incubation at 75 deg.] C the mixture for 20 minutes to terminate the reaction. 然后加入经EcoRV或SmaⅠ消化的pTREP-nuc质粒DNA(通常为200-400ng)。 Was then added and EcoRV or SmaⅠ pTREP-nuc digested plasmid DNA (typically 200-400ng). 再在混合物中添加400个单位的T4 DNA连接酶(NEB)和T4 DNA连接酶缓冲液(1X),于16℃保温过夜。 Add 400 units of T4 DNA ligase mixture (NEB) and T4 DNA ligase buffer (1X), incubated overnight at 16 ℃. 直接在100%乙醇和1/10体积的3M醋酸钠(pH5.2)中沉淀连接混合物,并用于转化乳酸乳球菌MG1363(Gasson,1983)。 3M sodium directly in 100% ethanol and 1/10 volume of acetic acid (pH 5.2) ligation mixture was precipitated, and used to transform L. lactis MG1363 (Gasson, 1983). 或者,pTREP-nuc载体的基因克隆位点也含有BglⅡ位点,该位点可用于克隆例如经Sau3AⅠ消化的基因组DNA片段。 Alternatively, the cloning site pTREP-nuc vector also contains BglⅡ site, the site can be used to clone genomic DNA fragment was digested e.g. Sau3AⅠ.

在脑心浸制琼脂上培养乳酸乳球菌的转化子菌落,基本上按Shortle,1983,文献同上和Le Loir等,1994,文献同上所述,通过甲苯胺蓝-DNA-琼脂覆层(0.05M Tris pH9.0,10g琼脂/l,10g NaCl/l,0.1mM CaCl2,0.03%wt/vol鲑精DNA和90mg甲苯胺蓝O染料)检测分泌核酶(Nuc+)的克隆。 Lactococcus lactis cultured on brain heart infusion agar transformant colonies were prepared, essentially as described by Shortle, 1983, supra, and Le Loir et al., 1994, supra said, by toluidine blue -DNA- agar overlay (0.05M ribozymes secreting clones (Nuc +) Tris pH9.0,10g of agar / l, 10g NaCl / l, 0.1mM CaCl2,0.03% wt / vol and salmon sperm DNA, toluidine blue O dye 90mg) detection. 然后将平板置于37℃保温2小时,分泌核酶的克隆显示出容易鉴别的粉色晕圈。 The plate was then incubated for 2 hours at 37 ℃, ribozyme secreting clones showed pink halo readily identified. 从Nuc+重组乳酸乳球菌克隆中分离质粒DNA,使用附录Ⅰ中所述的NucSeq测序引物测定DNA插入片段一条链的序列,所述引物的序列直接穿过DNA插入片段。 Plasmid DNA was isolated from Lactococcus lactis recombinant Nuc + clones using the appendix Ⅰ in NucSeq insert sequencing primer sequence determination of DNA fragment of one strand, the sequence of the primer directly through the insert DNA. 从肺炎链球菌中分离编码输出蛋白质的基因使用核酶筛选系统已鉴定出大量据推测可编码肺炎链球菌的输出蛋白质的基因序列。 A gene encoding a protein isolated from S. pneumoniae output using ribozymes screening system has identified a large number of gene sequences may encode presumably output Streptococcus pneumoniae proteins. 以下将进一步对其进行分析以除去人为假象。 It will be further analyzed to remove artifacts. 使用大量参数分析用筛选系统鉴定出的序列。 Sequence identified by screening a large number of parameters analyzed using the system.

1. 1. 使用软件程序Sequencher(Gene Codes公司)和DNA Strider(Marck,核酸研究,16:1829-1836(1988))分析所有推定的表面蛋白质的前导/信号肽序列。 Using a software program Sequencher (Gene Codes Corporation) and DNA Strider (Marck, Nucleic Acids Res., 16: 1829-1836 (1988)) analysis of the leader / signal peptide sequence for all putative surface protein. 细菌信号肽序列享有共同的设计,其特征在于短的带正电N-末端(N区域)后面紧接着一段疏水残基(中央部分-h区域),接着是含有裂解位点且更具极性的C-末端部分(c-区域)。 Bacterial signal peptide sequences share a common design, which is characterized in that the rear (N region) in a short period immediately positively charged N- terminal hydrophobic residues (-h central portion region), followed by a cleavage site and containing more polar C- terminal part (the C-region). 可以使用能绘制出推定蛋白质的亲水性分布图,并能容易地鉴定前导肽序列代表性的非常与众不同的疏水部分(h-区域)的计算机软件。 It can be used to draw out the hydrophilicity profile of the putative protein, and can be readily identified leader sequence representative very different hydrophobic moieties (H- region) computer software. 另外,需检查序列中是否存在潜在的核糖体结合位点(Shine-Dalgarno基元),该位点是推定的nuc报道基因融合蛋白的翻译起始所必需的。 Further, the need to check whether there is a potential ribosome binding site (Shine-Dalgarno motif) sequence, a translation initiation site of the gene is required for a fusion protein putative nuc it reported.

2. 2. 使用公开的数据库[包括SwissProt和GenBank翻译的OWL-proteins]将所有推定的表面蛋白质序列与所有蛋白质/DNA序列相匹配。 Using public databases [GenBank and SwissProt include translation OWL-proteins] All putative surface protein sequence with all the protein / DNA sequence match. 这样即可鉴定与已知基因或具有一些功能的基因同系物相似的序列。 Thus to identify similar sequences with known genes or gene homologs have some functions. 由此可推测使用LEEP系统鉴定出的一些基因的功能,并且可以毫无疑义地确定该系统可用于鉴定和分离表面结合蛋白质的基因序列。 Whereby LEEP system using estimation of some genes identified functions and may be unambiguously determined that the system may be used to identify and isolate a gene sequence of a protein binding surface. 我们应该也能证实这些蛋白质实际上就是表面结合蛋白质而不是人为假象。 We should be able to confirm that these proteins actually surface binding protein rather than artifacts. LEEP系统已被用于鉴定疫苗和治疗所用的新的基因靶。 LEEP systems have been used to identify new gene targets and therapeutic vaccines used.

3. 3. 一些基因已被鉴定的蛋白质不具有典型的前导肽序列,与数据库中的任何DNA/蛋白质序列也不具有同源性。 Some genes have been identified in the protein does not have a typical leader peptide sequence, with any DNA database / protein sequence having no homology. 实际上,这些蛋白质能显示出本发明筛选方法的主要优点,即分离不典型的表面相关蛋白质,而在所有前述筛选方法或基于序列同源性检索的方法中,却无法做到这一点。 Indeed, these proteins can show the major advantage of the screening methods of the present invention, i.e., separation is not typical surface associated protein, and in all the screening methods based on sequence homology search, or method, but can not do this.

在所有情况下,起初只得到部分基因序列。 In all cases, initially only partly gene sequence. 在所有情况下,参照TIGR肺炎链球菌数据库(可得到全长基因。 In all cases, refer to S. pneumoniae TIGR database ( to obtain full-length gene. 因此,通过使起初得到的部分序列与数据库相匹配,即可鉴定出全长基因序列。 Thus, by matching the first portion so that the sequence obtained with the database, to identify the full length gene sequence. 如本文所述,通过这种方法清楚地鉴定出3组基因,即一组编码以前未曾鉴定的肺炎链球菌蛋白质的基因,另一组编码与得自多种来源的已知蛋白质表现出一些同源性的蛋白质的基因,第三组编码已知的但不是已知抗原的肺炎链球菌蛋白质的基因。 As described herein, this method is clearly identified three groups of genes, i.e. genes encoding a not previously identified set of Streptococcus pneumoniae protein, and another group of encoded proteins obtained from a variety of sources known to exhibit some of the same proteins endogenous gene, the third set of encoding a known but not known antigen protein gene of Streptococcus pneumoniae.

Zhang,D.,Yang,X.,Berry,J.Shen,C.,McClarty,G和Brunham,RC(1997),“用主要外膜蛋白基因进行DNA免疫能诱导针对沙眼衣原体(小鼠肺炎)感染的获得性免疫力”,感染和免疫,176,1035-40。 Zhang, D., Yang, X., Berry, J.Shen, C., McClarty, G and Brunham, RC (1997), "major outer membrane protein gene with DNA immunization can induce Chlamydia trachomatis (mouse pneumonitis) infection acquired immunity ", infection and immunity, 176,1035-40.

Kurar,E和Splitter,GA(1997),“流产布鲁氏菌核糖体L7/L12基因的核酸免疫引发免疫应答”,疫苗,15,1851-57。 Kurar, E and Splitter, GA (1997), "nucleic acid immunization abortion BRUCELLA body L7 / L12 gene of eliciting an immune response" vaccine, 15,1851-57.

Ander,R.,Gao,XM,Papakonstantinopoulou,A.,Roberts,M和Dougan,G(1996),“用编码破伤风毒素C片段的DNA免疫小鼠之后的免疫应答”,感染和免疫,64,3168-3173。 Ander, R., Gao, XM, Papakonstantinopoulou, A., Roberts, M and Dougan, G (1996), "after immunization with the tetanus toxin C fragment of DNA encoding an immune response in mice," Infection and Immunity, 64, 3168-3173. 制备DNA疫苗为使用LEEP系统得到的各个所需基因设计寡核苷酸引物。 Preparation of DNA vaccines using LEEP systems are required for each gene to design oligonucleotide primers. 彻底检查各个基因,如果可能的话,应设计引物使其靶向据认为仅编码基因蛋白质的成熟部分的基因部分。 Each gene thorough examination, if possible, should be designed so that the primers targeted gene is believed to only a portion of the gene encoding the mature part of the protein. 我们希望当在哺乳动物细胞中表达时,表达仅编码靶基因蛋白质的成熟部分的那些序列会有利于蛋白质的正确折叠。 We hope that when expressed in mammalian cells, the expression of those sequences encoding only the mature portion of the target gene protein will facilitate correct protein folding. 例如,在大多数情况下,设计引物以。 For example, in most cases, primers were designed to. 使克隆至pcDNA3.1表达载体的最终扩增产物不含推定的N-末端信号肽序列。 Cloned into pcDNA3.1 expression vector so that the final amplification product does not contain N- terminal putative signal peptide sequence. 信号肽将多肽前体经由蛋白质输出途径导向细胞膜,在此一般通过信号肽酶Ⅰ(如果是脂蛋白,即为信号肽酶Ⅱ)切下信号肽。 The signal peptide precursor polypeptide through the cell membrane protein export pathway guide, typically by this signal peptidase Ⅰ (if lipoproteins, i.e. a signal peptidase Ⅱ) signal peptide is excised. 因此,无论是呈现在细菌表面还是被分泌,信号肽都不构成成熟蛋白质的任一部分。 Thus, both present in the bacterial surface or secreted, signal peptide do not constitute any part of the mature protein. 当N-末端前导肽序列不是立即显而易见时,应设计引物以靶向整个基因序列以在pcDNA3.1中克隆并最终进行表达。 When the N- terminal leader peptide sequence is not immediately apparent, the primers should be designed to target the entire gene sequence and eventually cloned in pcDNA3.1 expression.

然而,已经说过蛋白质的其它特征也能影响可溶性蛋白质的表达和呈递。 However, it has been said that other features of the protein can also affect the soluble protein expression and presentation. 在设计寡核苷酸的过程中,应在编码所需蛋白质的基因中排除编码这些特征的DNA序列。 In designing the oligonucleotides, DNA sequencing the gene encoding the proteins of these features should be excluded in the desired encoding. 这些特征包括:1. These features include: 1. LPXTG细胞壁锚着基元。 LPXTG cell wall anchor motif.

2. 2. LXXC脂蛋白结合位点。 LXXC lipoprotein binding site.

3. 3. 疏水性的C-末端结构域。 Hydrophobic C- terminal domain.

4. 4. 当不存在N-末端信号肽或LXXC时,应除去终止密码子。 When LXXC or N- terminal signal peptide is not present, should be removed the stop codon.

5. 5. 当不存在疏水性C-末端结构域或LPXTG基元时,应除去终止密码子。 When the hydrophobic C- terminal domain or motif LPXTG not present, should be removed the stop codon.

为感兴趣的各个基因设计适当的PCR引物,当设计这些引物时,应从基因中除去编码上述特征的任何和所有区域。 Any and all regions of the respective gene design appropriate PCR primers of interest, when designing these primers, a gene should be removed encoding the above features. 引物被设计成具有适当的限制性酶位点,其后紧接着保守的Kozak核苷酸序列(在大多数情况下(注意,除了在极少数情况下,例如ID59)使用的是GCCACC。Kozak序列有利于真核核糖体识别起始序列)和位于所需基因插入物上游的ATG起始密码子。 Primers were designed to have an appropriate restriction enzyme site, immediately followed by the nucleotide sequence conserved Kozak (in most cases (note, except in rare cases, e.g. ID59) using sequence GCCACC.Kozak facilitate identification of eukaryotic ribosomal initiation sequences), and is at a desired gene inserted upstream of the ATG start codon of the material. 例如,使用BamHⅠ位点的正向引物以GCGGGATCCGCCACCATG起始,后面接着所需基因5'末端的一小段序列。 For example, using site BamHⅠ forward primer GCGGGATCCGCCACCATG start, followed by the gene 5 'terminus of a short sequence desired. 设计反向引物以与正向引物相容,并在大多数情况下与5'末端的NotⅠ限制性位点(该位点是TTGCGGCCGC)相容(注意,除了在极少数情况下,例如ID59,用XhoⅠ位点替代NotⅠ)。 Reverse primer designed to be compatible with the forward primer, and in most cases, be compatible with the 5 'NotⅠ end restriction site (this site is TTGCGGCCGC) (note that, except in rare cases, e.g. ID59, Alternatively NotⅠ with XhoⅠ site). PCR引物下列PCR引物被设计用于扩增所需的截短基因。 PCR primers were designed following primers for PCR amplification of the desired truncated gene. ID5正向引物5′CGGATCCGCCACCATGGGTCTAATTGAAGACTTAAAAAATCAA 3′反向引物5′TTGCGGCCGCCAATGCTAGACTAAACACAAGACTCA 3′ID59正向引物5′CGCGGATCCATGAAAAAAATCTATTCATTTTTAGCA 3′反向引物5′CCCTCGAGGGCTACTTCCGATACATTTTAAACTGTAGG3′ID51正向引物5′CGGATCCGCCACCATGAGTCATGTCGCTGCAAATG 3′反向引物5′TTGCGGCCGCATACCAAACGCTGACATCTACG 3′ID29正向引物5′CGGATCCGCCACCATGCAAAAAGAGCGGTATGGTTATG3′反向引物5′TTGCGGCCGCACCCCCATTCTTAATCCCTT 3′ID50正向引物5′CGGATCCGCCACCATGGAGGTATGTGAAATGTCACGTAAA 3′反向引物5′TTGCGGCCGCTTTTACAAAGTCAAGCAAAGCC 3′克隆从得自国家典型培养物保藏中心的4型肺炎链球菌菌株11886中分离基因组DNA,以此为模板经PCR扩增插入物以及具有上述特征的侧翼序列。 ID5 forward primer 5'CGGATCCGCCACCATGGGTCTAATTGAAGACTTAAAAAATCAA 3 'reverse primer, forward primer 5'TTGCGGCCGCCAATGCTAGACTAAACACAAGACTCA 3'ID59 5'CGCGGATCCATGAAAAAAATCTATTCATTTTTAGCA 3' reverse primer, forward primer 5'CCCTCGAGGGCTACTTCCGATACATTTTAAACTGTAGG3'ID51 5'CGGATCCGCCACCATGAGTCATGTCGCTGCAAATG 3 'reverse primer 5'TTGCGGCCGCATACCAAACGCTGACATCTACG 3' ID29 forward primer 5'CGGATCCGCCACCATGCAAAAAGAGCGGTATGGTTATG3 'reverse primer 5'TTGCGGCCGCACCCCCATTCTTAATCCCTT 3'ID50 forward primer 5'CGGATCCGCCACCATGGAGGTATGTGAAATGTCACGTAAA 3' reverse primer 5'TTGCGGCCGCTTTTACAAAGTCAAGCAAAGCC 3 'derived from cloned from a strain of Streptococcus pneumoniae type 4 type culture Collection national Center 11886 isolated genomic DNA, as a template, and by PCR amplification of the insertion flanking sequence having the above characteristics. 用适当限制性酶切割PCR产物,使用常规分子生物学技术将其克隆至pcDNA3.1的多克隆位点。 PCR product was cut with a suitable restriction enzyme, and cloned into the multiple cloning site of pcDNA3.1 using conventional molecular biology techniques. 培养经适当作图的所需基因克隆,使用Plasmid Mega试剂盒(Qiagen)大规模分离质粒(>1.5mg)。 Culturing a suitable cloning the desired mapping, using the Plasmid Mega kit (Qiagen) large scale plasmids were isolated (> 1.5mg). 通过对各个构建体的每个大规模制品的5'克隆接头的约700个碱基对进行限制性图谱分析和测序,进一步证实基因的成功克隆和维持。 By 5 'cloning junctions approximately 700 bases for each individual article mass to construct restriction map analysis and sequencing confirmed the successful cloning of genes and maintenance. 菌株证实在克隆和攻击的方法中使用4型菌株,该菌株的肺炎链球菌基因组已被测序。 Type 4 strain was confirmed using clonal strains and challenged, the strain S. pneumoniae genome has been sequenced. 4型肺炎链球菌菌株11886的均质实验室菌株冻干安瓿得自国家典型菌种保藏中心。 4 Streptococcus pneumoniae strains 11886 homogeneous laboratory strain obtained from the freeze-dried ampoule country Type Culture Collection. 打开安瓿,用0.5ml胰胨豆胨肉汤(0.5%葡萄糖,5%血液)重新悬浮培养物。 Open ampoule with 0.5ml tryptic soy broth (0.5% glucose, 5% blood) re-suspension cultures. 在10ml胰胨豆胨肉汤(0.5%葡萄糖,5%血液)中传代培养悬浮液,于37℃静止保温过夜。 10ml in tryptic soy broth (0.5% glucose, 5% blood) subcultured suspension was incubated overnight at 37 [deg.] C static. 在5%血液琼脂平板上划线此培养物以检查污染物并证实存活,在血液琼脂斜面上划线,使用其余培养物制备20%甘油原种。 Streaked onto 5% Blood agar plates to check for contamination of this culture was confirmed and survival, streaked on blood agar slant, 20% glycerol stock was prepared using the remaining cultures. 将斜面培养物送至公共卫生实验中心,经证实血清型为4型。 The slant culture to the Public Health Laboratory Center, confirmed serotype type 4.

在5%血液琼脂平板上划线NCTC11886的甘油原种,于37℃在CO2气罐中保温过夜。 NCTC11886 scribing glycerol stocks in 5% blood agar plates, incubated overnight at 37 [deg.] C in CO2 gas tank. 制备新鲜的划线培养物并证实奥普托欣敏感性。 Preparation of a freshly streaked cultures were confirmed optochin sensitivity. 肺炎球菌攻击经小鼠传代肺炎球菌培养物1次,从感染动物的血液中收集肺炎球菌,在肉汤中培养至预定的活菌数约为109cfu/ml,然后冷冻,从而制备出4型肺炎链球菌的标准接种冷冻培养物。 Pneumococcal attack pneumococcal mouse passaged cultures 1, blood collected from pneumococcal infection in animals, grown to a predetermined number of viable cells was about 109cfu / ml in the broth, and then frozen to prepare a 4 pneumonia Streptococcus standard inoculum frozen culture. 制备的流程图如下:划线肺炎球菌培养物并证实其身份↓在上述平板上培养4-5个菌落的过夜培养物↓用动物传代肺炎球菌培养物(腹膜内注射心取血收集) A flow chart of the following: scribing pneumococcal cultures were cultured confirm their identity ↓ 4-5 colonies on the plates with an overnight culture of passage ↓ pneumococcal culture (intraperitoneal injection cardiac blood collection)

↓培养经动物传代的肺炎球菌的过夜培养物↓由经动物传代的过夜培养物培养全天培养物(至预定的光密度)并于-70℃冷冻-这是标准的极限↓融化一等份标准的接种物以进行存活计数↓使用标准接种物以测定有效剂量(称之为毒力检测试验)↓使用有效剂量的标准接种物进行随后所有的攻击用PBS将一等份标准的接种物稀释500倍,并用于接种小鼠。 Animal ↓ pneumococcal culture passaged overnight culture of the overnight culture ↓ Animal day culture passaged culture (to a predetermined optical density) and frozen at -70 ℃ - This is a standard limit of a melting aliquot ↓ standard inoculum viable for all subsequent attacks an aliquot standard inoculum diluted in PBS ↓ counted using standard inoculum to determine the effective dose (called virulence testing) ↓ using an effective dose of standard inoculum 500 times, and used to inoculate mice.

使用卤代烷轻度麻醉小鼠,然后将剂量为1.4×105cfu的肺炎球菌施用于每个小鼠的鼻腔,小鼠的正常呼吸将有利于摄入肺炎球菌,让小鼠背朝下躺着等待恢复。 Mice were lightly anesthetized using an alkyl halide and then a dose of 1.4 × 105cfu pneumococcal administered to the nasal cavity of each mouse, normal breathing will favor uptake mice pneumococcus, so that backs of mice lying WTR . 肺炎链球菌疫苗试验通过给6周龄的CBA/ca小鼠(Harlan,UK)施用DNA而在小鼠中进行疫苗试验。 Pneumococcal vaccine trials by CBA ca to 6 weeks old mice (Harlan, UK) administered / DNA vaccine trials conducted in mice. 将待免疫接种的小鼠分成6组,每组用重组pcDNA3.1+质粒DNA进行免疫接种,所述质粒DNA中含有所需的特定靶基因序列。 Immunized mice to be divided into six groups, each group were immunized with recombinant pcDNA3.1 + plasmid DNA, the plasmid DNA of a specific target gene sequence containing the desired. 将总共100μg溶于Dulbecco PBS(Sigma)中的DNA肌内注射至两腿的前胫骨肌(每条腿50μl)。 A total of 100μg DNA was dissolved intramuscular Dulbecco PBS (Sigma) is injected into the tibialis anterior legs (each leg 50μl). 4周后使用相同的方法进行加强免疫。 Using the same method as booster immunization after 4 weeks. 为了进行比较,在所有疫苗试验中都包括对照组。 For comparison, in all vaccine trials include a control group. 这些对照组或者是未经免疫的动物,或者是按与上述相同的时间进程仅施用了非重组pcDNA3.1+DNA(假免疫)的动物。 The unimmunized control group or animals, or by the same time as the above-described process only the non-administered recombinant pcDNA3.1 + DNA (sham immunized) animals. 第二次免疫接种3周后,用致死剂量的肺炎链球菌血清型4(菌株NCTC11886)鼻内攻击所有小鼠组。 The second vaccination three weeks later with a lethal dose of S. pneumoniae serotype 4 (strain NCTC11886) all groups of mice intranasally. 通过在5%血液琼脂平板上平铺接种经连续稀释的接种物来监测所施用细菌的数目。 The number of the bacteria was monitored by tiling vaccination administered serially diluted inoculum was 5% blood agar plates. 鼻内免疫接种的问题是:在一些小鼠中,接种物从鼻孔中象吹泡似地冒出来,在结果表中已注意到这个问题,并且在计算时已将此问题考虑在内。 Intranasal immunization problem is: in some mice, like the inoculum was blown bubble nostrils emerge Similarly, this problem has been noted in the results table, and this problem has been taken into account in the calculation. 较不明显的问题是每只小鼠可能吞下了一部分接种物。 Less obvious problem is likely to swallow part per mouse inoculum. 假定对每只小鼠而言,被吞下去的量是相同的,在接种过程中将达到平均。 For each mouse is assumed, the amount swallowed is the same, in the average inoculation reached. 然而,所使用的样品较少,这一问题将对一些实验产生显著影响。 However, small sample used, this problem will have a significant impact on a number of experiments. 在注射后3或4天,杀死攻击后仍存活的所有小鼠。 In three or four days after injection, after killing all the mice survived the attack. 在感染过程中,监测被攻击的小鼠中与肺炎链球菌诱导-疾病的发作相关的症状发展情况。 During infection, the mice were monitored attacked with Streptococcus pneumoniae-induced - the onset of disease symptoms associated developments. 典型的症状依次包括立毛,逐渐增加的隆起体态,眼里流出排泄物,嗜睡和不愿动。 Typical symptoms include Li Mao in turn, increasing uplift body, eyes out of excrement, lethargy and do not want to move. 后期的症状通常与濒死状态的产生一致,剔除濒死状态的小鼠以使它们不再遭受痛苦。 Late symptoms consistent with generally produced near-death state, knockout mice dying state so that they no longer suffer. 据认为这些小鼠濒临死亡,使用剔除时间确定存活时间用以进行统计学分析。 It is believed that these mice dying, reject the use of time to determine the survival time for statistical analysis. 当发现小鼠死亡时,存活时间被当成是经监测认为小鼠仍旧活着的最后的时间点。 When the mice were found dead, as is the time of survival was the last time point monitored by the thought of mice still alive. 结果的解释如果被克隆并用于上述攻击实验的任何DNA序列能产生对抗攻击的保护作用,即为阳性结果。 If the interpretation of the results was cloned and used for any DNA sequence capable of producing the above-described challenge experiment protection against attack, the result is positive. 能产生统计学上显著的保护作用(置信水平为95%(p<0.05))的DNA序列,和使用Mann-Whitney发现的勉强或接近显著的保护作用,或显示出一些保护性特征,例如有一只或多只无关的小鼠或因为首次出现死亡的时间延长,被认为是保护作用。 Yield significant protective effect statistically (confidence level of 95% (p <0.05)) DNA sequence, and using the Mann-Whitney found barely or near significant protective effect, or exhibit some protection features, for example, a only unrelated or only because of the time of death of mice or extended for the first time, is considered to be a protective effect. 当我们认为一些结果被使用鼻内感染的相关问题再得模糊不清时,接近显著或非显著的结果被认为是潜在的阳性结果是可以接受的。 When we think are some of the results of the use of intranasal infection related issues and then get blurred, close to the significant non-significant result is considered a potential positive result is acceptable. 结果试验1-6(见图1) 1-6 Test results (see Figure 1)

*-当按此剂量免疫时,有一小部分象吹泡似地被冒出,因此可能未接受全部接种物。 * - Click when immunization dose, a small portion of the image is wildly blowing out, and therefore may not accept all the inoculum. T-在实验结束时终止,没有感染症状。 T- at the end of the termination of the experiment, no symptoms of infection. 括号内的数字-不考虑所采用的不完全剂量的存活时间。 Figures in brackets - do not consider incomplete dose survival time employed. P值1指的是与未经免疫的对照相比的显著性试验。 P value of 1 refers to the significance of the test compared to non-immunized controls. P值2指的是与pcDNA3.1+免疫的对照相比的显著性试验。 2 P value refers to the significance of the test compared to the control immunized pcDNA3.1 +. 统计学分析试验1-其它组无一比对照有显著更长的存活时间。 Statistical Analysis of Trial 1 none other than the control group has a significantly longer survival time. 未经免疫和pcDNA3.1对照组的存活时间没有显著的不同。 Non-immunized control group and pcDNA3.1 survival times were not significantly different. ID5的一个小鼠是无关的结果,ID5的平均存活时间被延长,但不显著。 A mouse is irrelevant ID5 result, the average survival time is extended ID5, but not significantly.

试验2-与未经免疫的对照组相比,ID59免疫组的存活时间显著更长。 Test 2 - as compared to non-immunized controls, survival time ID59 immunized group was significantly longer.

试验5-与对照相比,ID59免疫组的存活时间平均约长10小时,但该结果在统计学上不是很显著。 5- test compared to the control group immunized ID59 survival time average length of about 10 hours, but this result was not statistically significant.

试验6-与未经免疫的对照组相比,ID51免疫组不具有显著更长的存活时间(p=<36.0),然而,经免疫组中有2个无关的小鼠。 Test 6- compared to unimmunized controls, ID51 immunized group does not have a significantly longer survival (p = <36.0), however, the group immunized with 2 independent mouse. 疫苗试验7和8(见图2) Vaccine Trials 7 and 8 (see FIG. 2)

*-当按此剂量免疫时,有一小部分象吹泡似地被冒出,因此可能未接受全部接种物。 * - Click when immunization dose, a small portion of the image is wildly blowing out, and therefore may not accept all the inoculum. T-在实验结束时终止,没有感染症状。 T- at the end of the termination of the experiment, no symptoms of infection. 括号内的数字-不考虑所采用的不完全剂量的存活时间。 Figures in brackets - do not consider incomplete dose survival time employed. P值1指的是与未经免疫的对照相比的显著性试验。 P value of 1 refers to the significance of the test compared to non-immunized controls. 统计学分析试验7-ID29免疫组首次出现死亡的时间延长。 Statistical analysis test 7-ID29 immunohistochemistry extend the time of death for the first time.

试验8-与未经免疫的对照组相比,ID50免疫组具有显著更长的存活时间。 8- test compared with non-immunized control group, ID50 immunohistochemistry have a significantly longer survival time.

附录Ⅰ-寡核苷酸引物nucS1BglⅡ Eco RV5′-cgagatctgatatctcacaaacagataacggcgtaaatag-3′nucS2BglⅡ SmaⅠ5′-gaagatcttccccgggatcacaaacagataacggcgtaaatag-3′nucS3BglⅡ Eco RV5′-cgagatctgatatccatcacaaacagataacggcgtaaatag-3′nucRBamHⅠ5′-cgggatccttatggacctgaatcagcgttgtc-3′NucSeq5′-ggatgctttgtttcaggtgtatc-3′pTREPF5′-catgatatcggtacctcaagctcatatcattgtccggcaatggtgtgggctttttttgttttagcggataagttatccgcta-3'pTREPR5′-gcggatcccccgggcttaattaatgtttaaacactagtcgaagatctcgcgaattctcctgtgtgaaattgttatccgcta-3'pUCF5′-cgccagggttttcccagtcacgac-3′VR5′-tcaggggggcggagcctatg-3′V15′-tcgtatgttgtgtggaattgtg-3′V25′-tccggctcgtatgttgtgtggaattg-3′ Appendix Ⅰ- oligonucleotide primers nucS1BglⅡ Eco RV5'-cgagatctgatatctcacaaacagataacggcgtaaatag-3'nucS2BglⅡ SmaⅠ5'-gaagatcttccccgggatcacaaacagataacggcgtaaatag-3'nucS3BglⅡ Eco RV5'-cgagatctgatatccatcacaaacagataacggcgtaaatag-3'nucRBamHⅠ5'-cgggatccttatggacctgaatcagcgttgtc-3'NucSeq5'-ggatgctttgtttcaggtgtatc-3'pTREPF5 ' -catgatatcggtacctcaagctcatatcattgtccggcaatggtgtgggctttttttgttttagcggataagttatccgcta-3'pTREPR5'-gcggatcccccgggcttaattaatgtttaaacactagtcgaagatctcgcgaattctcctgtgtgaaattgttatccgcta-3'pUCF5'-cgccagggttttcccagtcacgac-3'VR5'-tcaggggggcggagcctatg-3'V15'-tcgtatgttgtgtggaattgtg-3'V25'-tccggctcgtatgttgtgtggaattg-3 '


Claims (20)

1. 1. 肺炎链球菌蛋白质或多肽,其具有选自表1所示序列的序列。 Streptococcus pneumoniae protein or polypeptide having a sequence selected from the sequence shown in Table 1.
2. 2. 肺炎链球菌蛋白质或多肽,其具有选自表2所示序列的序列。 Streptococcus pneumoniae protein or polypeptide having a sequence selected from the sequence shown in Table 2.
3. 3. 权利要求1或2的蛋白质或多肽,它以基本上纯的形式被提供。 Protein or polypeptide according to claim 1 or 2, which is provided in a substantially pure form.
4. 4. 与权利要求1至3中任一项所定义的基本上相同的蛋白质或多肽。 Substantially the same protein or polypeptide as defined in any one of claims 1 to 3.
5. 5. 权利要求1至4中任一项所定义的蛋白质或多肽的同系物或衍生物。 Homologue or derivative as defined in any one of a protein or polypeptide as claimed in claim 1 to 4.
6. 6. 表1-3中所定义的蛋白质或多肽的抗原性和/或免疫原性片段。 A protein or antigenic polypeptide and / or an immunogenic fragment as defined in Tables 1-3.
7. 7. 核酸分子,其含有下列序列或由下列序列组成:(ⅰ)表1所示的任何DNA序列或其RNA等同物;(ⅱ)与(ⅰ)中的任一序列互补的序列;(ⅲ)与(ⅰ)或(ⅱ)中的序列编码相同蛋白质或多肽的序列;(ⅳ)与(ⅰ),(ⅱ)和(ⅲ)中的任一序列基本上相同的序列;(ⅴ)编码表1所定义蛋白质的同系物,衍生物或片段的序列。 A nucleic acid molecule comprising the following sequences or consist of the following sequences: (i) any DNA sequence table or RNA equivalent as depicted. 1; (ii) a sequence complementary to either (i) in a; (iii) and sequence identity sequence encoding a protein or polypeptide (i) or (ii) is; sequence substantially identical to a sequence (iv) and (ⅰ), (ⅱ) and (iii) either; (ⅴ) encoding tABLE 1 protein sequences of homologues, derivatives or fragments as defined above.
8. 8. 核酸分子,其含有下列序列或由下列序列组成:(ⅰ)表2所示的任何DNA序列或其RNA等同物;(ⅱ)与(ⅰ)中的任一序列互补的序列;(ⅲ)与(ⅰ)或(ⅱ)中的序列编码相同蛋白质或多肽的序列;(ⅳ)与(ⅰ),(ⅱ)和(ⅲ)中的任一序列基本上相同的序列;(ⅴ)编码表2所定义蛋白质的同系物,衍生物或片段的序列。 A nucleic acid molecule comprising the following sequences or consist of the following sequences: (i) any DNA sequence table or RNA equivalent as depicted 2; (ii) a sequence complementary to any sequence of (i) the; (iii) and sequence identity sequence encoding a protein or polypeptide (i) or (ii) is; sequence substantially identical to a sequence (iv) and (ⅰ), (ⅱ) and (iii) either; (ⅴ) code table 2 protein sequences of homologues, derivatives or fragments as defined above.
9. 9. 具有选自表1-3所示序列之序列的蛋白质或多肽、或其同系物、衍生物和/或片段用作免疫原和/或抗原的用途。 Protein or polypeptide having a sequence selected from Table 1-3 of the sequence, or a homologue, derivative and / or fragment thereof use as immunogens and / or antigens.
10. 10. 免疫原性和/或抗原性的组合物,该组合物含有一种或多种具有选自表1-3所示序列之序列的蛋白质或多肽或其同系物或衍生物、和/或它们中的任一个的片段。 Immunogenic and / or antigenic composition, the composition comprising one or more sequences having the sequence shown in Table 1-3 selected protein or polypeptide or a homologue or derivative thereof, and / or in which a fragment of any of.
11. 11. 权利要求10所述的免疫原性和/或抗原性组合物,该组合物是疫苗,或者可用于诊断试验。 Immunogenic and / or antigenic composition according to claim 10, which is a vaccine composition, or may be useful in diagnostic assays.
12. 12. 权利要求11所述的疫苗,其含有一种或多种选自赋形剂、稀释剂、佐剂等的其它组分。 Vaccine according to claim 11, which contains one or more further components selected from excipients, diluents, adjuvants and the like.
13. 13. 疫苗组合物,其含有一种或多种如表1-3所定义的核酸序列。 Vaccine composition, containing one or more nucleic acid sequences as defined in Table 1-3.
14. 14. 一种检测/诊断肺炎链球菌的方法,所述方法包括使受试样品与至少一种如表1-3所定义的蛋白质或多肽或其同系物、衍生物或片段接触的步骤。 A method for detecting / diagnosis of S. pneumoniae, said method comprising contacting a test sample with at least one step of contacting a protein or polypeptide or a homologue, derivative or fragment thereof as defined in Table 1-3.
15. 15. 一种抗体,所述抗体能与如表1-3所定义的蛋白质或多肽或其同系物、衍生物或片段结合。 An antibody, the antibody capable of binding to such a protein or polypeptide or a homologue, derivative or fragment thereof as defined in Table 1-3.
16. 16. 权利要求15所定义的抗体,所述抗体是单克隆抗体。 15 defined in claim antibody, the antibody is a monoclonal antibody.
17. 17. 一种检测/诊断肺炎链球菌的方法,所述方法包括使受试样品与至少一种如权利要求15或16所定义的抗体接触的步骤。 A method of detecting / diagnosis of Streptococcus pneumoniae, said method comprising the step of contacting a test sample with an antibody of the contact 15 or 16 as defined in at least one claim.
18. 18. 检测/诊断肺炎链球菌的方法,所述方法包括使受试样品与至少一种如权利要求7或8所定义的核酸序列接触的步骤。 The detection / diagnosis of S. pneumoniae, said method comprising the step of contacting a test sample with at least one nucleic acid sequence as claimed in 7 or 8 defined requirements.
19. 19. 一种测定如表1-3所定义的蛋白质或多肽是否是潜在的抗微生物靶的方法,所述方法包括灭活所述蛋白质或多肽并测定肺炎链球菌是否仍然存活。 An assay such as a protein or polypeptide as defined in Table 1-3 whether the antimicrobial potential target, the method comprising inactivating the protein or polypeptide and determining whether a Streptococcus pneumoniae still alive.
20. 20. 能拮抗、抑制或要不然干扰如表1-3所定义的蛋白质或多肽的功能或表达的药剂用于制备药物的用途,所述药物可用于治疗或预防肺炎链球菌感染。 Could antagonize, inhibit or otherwise interfere with the use of an agent such as a protein or functional polypeptide as defined in Tables 1-3 or expression for the preparation of a medicament, the medicament useful for the treatment or prevention of S. pneumoniae infection.
CN 99810978 1998-07-27 1999-07-27 Nucleic acids and proteins from streptococcus pneumoniae CN1318103A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Families Citing this family (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6800744B1 (en) 1997-07-02 2004-10-05 Genome Therapeutics Corporation Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics
PL204863B1 (en) 1998-07-22 2010-02-26 Stichting Dienst Landbouwkundig Onderzoek Streptococcus suis
US7128918B1 (en) 1998-12-23 2006-10-31 Id Biomedical Corporation Streptococcus antigens
EP1950302B1 (en) * 1998-12-23 2012-12-05 ID Biomedical Corporation of Quebec Streptococcus antigens
CN1191362C (en) * 1998-12-23 2005-03-02 夏尔生化公司 Streptococcus antigens
AT500843T (en) * 1999-06-10 2011-03-15 Medimmune Llc Streptococcus pneumoniae proteins and vaccines
US6887480B1 (en) 1999-06-10 2005-05-03 Medimmune, Inc. Streptococcus pneumoniae proteins and vaccines
DE10012370A1 (en) * 2000-03-14 2001-09-27 Chiron Behring Gmbh & Co Use of oil-in-water emulsion as vaccine adjuvant, particularly for influenza and pneumococcal vaccines, administered at different site from the vaccine
US7074415B2 (en) 2000-06-20 2006-07-11 Id Biomedical Corporation Streptococcus antigens
EP1205552A1 (en) * 2000-11-09 2002-05-15 ID-Lelystad, Instituut voor Dierhouderij en Diergezondheid B.V. Virulence of streptococci, involving ORFs from Streptococcus suis
GB0107658D0 (en) * 2001-03-27 2001-05-16 Chiron Spa Streptococcus pneumoniae
GB0107661D0 (en) 2001-03-27 2001-05-16 Chiron Spa Staphylococcus aureus
GB0108079D0 (en) 2001-03-30 2001-05-23 Microbial Technics Ltd Protein
US7262024B2 (en) 2001-12-20 2007-08-28 Id Biomedical Corporation Streptococcus antigens
WO2004048575A2 (en) * 2002-11-26 2004-06-10 Id Biomedical Corporation Streptococcus pneumoniae surface polypeptides
EP2311988A1 (en) * 2003-04-15 2011-04-20 Intercell AG S. pneumoniae antigens
GB0714963D0 (en) * 2007-08-01 2007-09-12 Novartis Ag Compositions comprising antigens
KR101208778B1 (en) 2008-03-03 2012-12-05 노파르티스 아게 Compounds and compositions as tlr activity modulators
AU2009226949A1 (en) 2008-03-17 2009-09-24 Intercell Ag Peptides protective against S. pneumoniae and compositions, methods and uses relating thereto
AT523204T (en) 2009-02-16 2011-09-15 Karlsruher Inst Technologie Cd44v6 peptides as bacterial infection inhibitors
TR201802380T4 (en) 2009-06-10 2018-03-21 Glaxosmithkline Biologicals Sa Vaccines containing iodine Benzonaftiri.
NZ597858A (en) 2009-06-29 2014-01-31 Genocea Biosciences Inc Vaccines and compositions against streptococcus pneumoniae
EA026401B1 (en) 2009-09-02 2017-04-28 Новартис Аг Immunogenic compositions including tlr activity modulators
JO3257B1 (en) 2009-09-02 2018-09-16 Novartis Ag Compounds and compositions as tlr activity modulators
CN102695523A (en) 2009-09-10 2012-09-26 诺华有限公司 Combination vaccines against respiratory tract diseases
WO2011057148A1 (en) 2009-11-05 2011-05-12 Irm Llc Compounds and compositions as tlr-7 activity modulators
SG10201501980SA (en) 2009-12-15 2015-05-28 Novartis Ag Homogeneous suspension of immunopotentiating compounds and uses thereof
MX341395B (en) 2010-03-23 2016-08-18 Novartis Ag * Compounds (cystein based lipopeptides) and compositions as tlr2 agonists used for treating infections, inflammations, respiratory diseases etc.
WO2012072769A1 (en) 2010-12-01 2012-06-07 Novartis Ag Pneumococcal rrgb epitopes and clade combinations
EP2665490A4 (en) 2011-01-20 2015-07-01 Genocea Biosciences Inc Vaccines and compositions against streptococcus pneumoniae
JP2015510872A (en) 2012-03-07 2015-04-13 ノバルティス アーゲー Enhanced formulation of Streptococcus pneumoniae antigen
JP6411378B2 (en) 2013-02-01 2018-10-24 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム Intradermal delivery of an immunological composition comprising a TOLL-like receptor agonist
CA2900008A1 (en) * 2013-02-07 2014-08-14 Children's Medical Center Corporation Protein antigens that provide protection against pneumococcal colonization and/or disease

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5928900A (en) * 1993-09-01 1999-07-27 The Rockefeller University Bacterial exported proteins and acellular vaccines based thereon
EP0907738A1 (en) * 1996-04-02 1999-04-14 Smithkline Beecham Corporation Novel compounds
JP2000508178A (en) * 1996-05-14 2000-07-04 スミスクライン・ビーチャム・コーポレイション Novel compounds
DK0942983T4 (en) * 1996-10-31 2014-12-01 Human Genome Sciences Inc Streptococcus pneumoniae antigens and vaccines
US6060282A (en) * 1996-12-13 2000-05-09 Eli Lilly And Company Streptococcus pneumoniae gene sequence Dpj
GB9700939D0 (en) * 1997-01-17 1997-03-05 Microbial Technics Limited Therapy

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101155833B (en) 2005-04-08 2011-04-20 惠氏公司 Separation of contaminants from streptococcus pneumoniae polysaccharide by ph manipulation
CN103834667A (en) * 2013-12-31 2014-06-04 李越希 Chemosynthetic extracellular region gene fragment of streptococcus pneumonia PspA protein, and expression and application thereof
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