WO2005007683A1 - Antigen epitopes of the regulatory protein of virulence factor in staphylococcus aureus and their mimotopes and use - Google Patents

Antigen epitopes of the regulatory protein of virulence factor in staphylococcus aureus and their mimotopes and use Download PDF

Info

Publication number
WO2005007683A1
WO2005007683A1 PCT/CN2003/000827 CN0300827W WO2005007683A1 WO 2005007683 A1 WO2005007683 A1 WO 2005007683A1 CN 0300827 W CN0300827 W CN 0300827W WO 2005007683 A1 WO2005007683 A1 WO 2005007683A1
Authority
WO
WIPO (PCT)
Prior art keywords
epitope
sequence
trap
staphylococcus aureus
amino acid
Prior art date
Application number
PCT/CN2003/000827
Other languages
English (en)
French (fr)
Other versions
WO2005007683A8 (fr
Inventor
Ningsheng Shao
Guang Yang
Chuan Liu
Yaping Gao
Jie Dong
Hongmei Ding
Beifen Shen
Original Assignee
Institute Of Basic Medical Sciences, Academy Of Military Medical Sciences
Hainan Gt-Uniput Pharmaceutical Co. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute Of Basic Medical Sciences, Academy Of Military Medical Sciences, Hainan Gt-Uniput Pharmaceutical Co. Ltd. filed Critical Institute Of Basic Medical Sciences, Academy Of Military Medical Sciences
Priority to EP03756419.2A priority Critical patent/EP1719520B1/en
Priority to DK03756419.2T priority patent/DK1719520T3/en
Priority to AU2003304343A priority patent/AU2003304343A1/en
Priority to ES03756419.2T priority patent/ES2535777T3/es
Publication of WO2005007683A1 publication Critical patent/WO2005007683A1/zh
Publication of WO2005007683A8 publication Critical patent/WO2005007683A8/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • Antigen epitope its mimetic epitope and uses
  • the invention relates to an antigenic epitope and a mimetic epitope, in particular to two antigenic epitopes of a Staphylococcus aureus (S. aureus) virulence factor regulating protein and their mimetic epitopes.
  • the present invention also relates to the use of polypeptides with the same amino acid sequence as these epitopes in the preparation of anti-S. Aureus infection drugs or vaccines. Background technique
  • Staphylococcus aureus is a common class of Gram-positive pathogens, and it is one of the main microorganisms that cause fatal diseases such as burns and war wounds infection, pneumonia, endocarditis, sepsis, and toxic shock. More than millions of S. aureus infections occur in hospitals each year. At present, the treatment of Staphylococcus aureus is mostly combined with antibiotics, but the effect is not satisfactory. Because Staphylococcus aureus easily develops drug resistance and there is no good solution, many commonly used antibiotics are not effective. Controlling S. aureus infection is one of the problems to be solved in clinical medicine.
  • RNAIII activates gene transcription of virulence factors and regulates translation of virulence factors through base complementation.
  • RNAIII activating protein RNA III activating protein
  • RAP protein activating protein
  • Staphylococcus aureus virulence stimulating factor Staphylococcus aureus continues to secrete RAP, and it only activates virulence factors when RAP reaches a certain concentration. Staphylococcus aureus without RAP is not pathogenic in itself.
  • New research finds that RAP activates RNAIII transcription A 21KD protein called TRAP (Target of RNAIII activating protein) was mediated.
  • TRAP is composed of 167 amino acids and has His kinase activity. TRAP protein begins to phosphorylate in the early stages of S. aureus growth and reaches its maximum level in the middle of logarithmic growth. After RAP, signal transduction is performed through autophosphorylation, which mediates the rise of intracellular RNAIII levels and accelerates the secretion of S. aureus exotoxin (Naomi B, et al, J. Biol. Chem 2001, 276: 2658-2667 ). It can be seen that TRAP protein also plays a key role in the regulation of toxin expression in S. aureus. A 2001 study found that antibodies to TRAP can effectively reduce the secretion of S. aureus exotoxin (Oleny V, et al. Peptides 2001, 22: 1621-1627).
  • the epitope as the target structure recognized by immune cells and the material basis for stimulating specific immune responses, is of great significance for related research on immune response.
  • the emergence of the concept of "mimotope” not only provided clues for the analysis of antigenic epitopes, but also opened up a new idea for the development of vaccine research. Mimotope, usually refers to a polypeptide structure capable of mimicking an epitope of an antigen.
  • Phage display peptide library The display technology displays randomly arranged exogenous peptides (usually composed of 6 to 15 amino acid residues) on the surface of a phage to form a random peptide library with a diversity of 10 7 or more.
  • target peptides such as monoclonal antibodies, polyclonal antibodies
  • binding peptides are screened from the phage peptide library, and the binding peptides are compared with natural antigens, some of which are related to natural epitopes Highly homologous, some are completely different from natural antigens, but all have similar antigenicity and immunogenicity to natural antigens.
  • binding peptides are called "mimetic epitopes”.
  • phage display technology has strong advantages. It is not only simple and fast, but also has a wide range of applications.
  • the screening process using polyclonal antibodies or antisera as targets is more difficult, but has obvious advantages. It is manifested as follows: First, the target is easy to obtain, compared with the preparation of monoclonal antibodies, it takes less time, costs are lower, and the operation is simpler. Second, multiple epitopes can be obtained, and the screening efficiency is high, which is conducive to the preparation of highly immunogenic antibodies. Compound vaccine. In this experiment, the antiserum was purified by immunoaffinity chromatography, and the non-specific antibodies were removed to obtain highly specific sorting molecules, which is conducive to sorting into specific phage clones and reducing the enrichment of non-specific clones. Summary of the invention
  • the object of the present invention is to provide two antigenic epitopes of staphylococcus aureus virulence factor regulating protein (TRAPC Target of RNAIII activating protein) and their mimetic epitopes.
  • TRAPC Target of RNAIII activating protein staphylococcus aureus virulence factor regulating protein
  • Another object of the present invention is to provide the application of the above epitopes or polypeptides corresponding to the amino acid sequences of these epitopes in the preparation of drugs or vaccines against S. aureus infection.
  • the TRAP polyclonal antibody as the target and obtained two sets of TRAP protein mimic epitopes through linear peptide library selection.
  • the sequences of the two families can be found on the expressed sequence, that is, the two epitopes of the TRAP protein itself: the 21-34 amino acid sequence and the 156-167 amino acid sequence.
  • Their amino acid sequence is as follows: Epitope 1: Corresponding to the 21-34 amino acid sequence of the TRAP protein:
  • Epitope 2 amino acid sequence corresponding to positions 156-167 of the TRAP protein: 156 SYFERYLYPI E 167
  • the two antigen mimic epitopes of the S. aureus virulence factor regulatory protein TRAP of the present invention are polypeptides having the following amino acid sequence structural pattern:
  • Antigen Mimic Epitope 1 XPXHHQHXTGFT
  • the uppercase English letters respectively represent twenty-one known natural L-type amino acid residues or one of its D-type isomers, that is, A represents an alanine residue R, arginine residue, N for asparagine residue, D for aspartic acid residue, Q for glutamine residue, E for glutamic acid residue, and H for histidine residue , W represents tryptophan residue, Y represents tyrosine residue, F represents phenylalanine residue, T represents threonine residue, S represents serine residue, L represents leucine residue, and G represents Glycine residues, P for proline residues, V for valine residues, K for lysine residues, M for methionine residues, I for isoleucine residues, and X for twenty A known natural L-type amino acid residue or any one of its D-isomers.
  • amino acids in the above epitope can be replaced with each other according to the similarity of amino acids, such as glutamine residue (Q), glutamic acid residue (E), aspartic acid residue (D) or asparagine Residues (N) can be replaced with each other; tryptophan residues (W), tyrosine residues (Y) or phenylalanine residues (F) can be replaced with each other; lysine residues ( K) and arginine residue (R) can be replaced with each other; serine residue (S) and threonine residue (T) can be replaced with each other; alanine residue (A) and glycine Acid residues (G) can be replaced with each other; leucine residues (L) and methionine residues (M) can be replaced with each other.
  • amino acids such as glutamine residue (Q), glutamic acid residue (E), aspartic acid residue (D) or asparagine Residues (N) can be replaced with each other; tryp
  • the small molecule polypeptide of the present invention can be prepared by chemical synthesis or recombinant expression by genetic engineering.
  • the main reason for the resistance of traditional antibiotic treatment is that after the treatment, the bacteria produce an inducing enzyme that breaks down the effective groups in the antibiotic under the pressure of survival.
  • the TRAP antigen epitope and the polypeptide corresponding to the mimic epitope utilized by the present invention are highly conserved among different S. aureus strains TRAP proteins, and can stimulate the body to produce antibodies against TRAP and inhibit the activity of TRAP, so a vaccine can be prepared Fight against Staphylococcus aureus infection without losing the pathogenicity of the bacteria without killing the bacteria. It is applicable to all drug-resistant and non-resistant strains. This is a common and frequent cause of eradication of drug-resistant Staphylococcus aureus infections that have been plagued clinically. And deadly diseases find new ways. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 shows the electrophoresis diagram of TRAP polyclonal affinity purification.
  • FIG. 2 is a binding diagram of a competition rap protein and an antibody detected by a competition ELISA method for detecting a phage clone of an antigenic epitope sequence 1.
  • the antigenic epitope and the simulated epitope of the present invention lay a foundation for the research on the vaccine against S. aureus infection, and open a new way for the treatment of S. aureus infection, which has wide application value and broad market prospect. detailed description The present invention will be further described in detail by taking the antigen mimic epitope 1 as an example.
  • Example 1 Preparation and purification of TRAP protein monoclonal antibodies
  • Polyclonal antibodies were prepared by immunizing rabbits with purified TRAP protein.
  • Anti-TRAP IgG was isolated from polyclonal antibody serum using an affinity column coupled with TRAP protein.
  • the SDS-PAGE electrophoresis results showed that the purity of the purified IgG was> 90%, and the ELISA results showed that the titer was above 1x10 s (see Figure 1, Figure 1 represents the low molecular weight protein standard, and 2 represents the purified TRAP polyclonal antibody).
  • Example 2 Screening of TRAP Polyclonal Antigen Mimic Epitope
  • TRAP polyclonal antibody IgG 100 ⁇ l of purified TRAP polyclonal antibody IgG was coated on the enzyme-linked plate and placed in 4 ports overnight. After blocking with 2% gelatin for 1 h, add phage dodecapeptide library, incubate for 1 h at room temperature, wash non-specifically bound phages with TBST (50mmol / L Tris-HCl, 0.1% TWEEN20, pH7.5), and then use 0.2mmol / L L-glycine-HC1 pH2.2 elutes the specifically bound phage, and the eluate is neutralized with 1mmol / L Tris-HCl pH9.0.
  • TBST 50mmol / L Tris-HCl, 0.1% TWEEN20, pH7.5
  • the titer of phage in the eluate was measured, and the titer of the eluted phage of the uncoated target protein was used as a control to determine the input-output ratio.
  • the eluted phage bound to the TRAP antibody IgG was amplified, and its lower titer was measured for the next round of screening. After three rounds of screening, the measured input and output have improved significantly.
  • the enriched phage clones were identified by ELISA, and 24 positive clones were randomly selected for sequencing. After analysis, the peptide sequences of the above two families containing the epitope sequence 1 of the antigen were obtained.
  • Example 3 Comparison of the screened antigen mimic epitope sequence 1 and the expressed TRAP sequence
  • sequence of the epitope sequence 1 of the antigen is similar to the amino acid sequence of positions 21-34 of the TRAP protein:
  • TRAP protein primary sequence 21 NP HO HOFOFSASDT 34
  • Antigen mimic epitope sequence 1 Phage clone competes for binding of TRAP protein to antibody
  • a phage clone was selected which amplifies the mimic epitope sequence 1 of the antigen and tested whether it can compete with the binding of TRAP to the TRAP polyclonal antibody.
  • the TRAP antibody was coated, and a mixture of different amounts of the TRAP protein and the phage (10 9 ) was added.
  • the phage competed for the binding of the TRAP protein to the antibody by using a monoclonal antibody against M13 to detect the phage.
  • the experimental results show that the phage clone of the antigen mimic epitope sequence 1 can compete for the binding of the TRAP protein to its antibody.
  • Figure 1 in Figure 1 represents 9 antigen mimic epitope sequence 1 phage clone
  • 2 represents 5 ⁇ TRAP + 10 9 antigen mimic epitope sequence 1 phage clone
  • 3 represents 10 gTRAP + 10 9 antigen mimic epitope sequence 1 phage clone
  • Example 5 Inhibition of TRAP polyclonal antibody activity by phage clones of the antigen mimic epitope sequence 1
  • TRAP antigen mimic epitope sequence 1 100 RN6390B was inoculated, and the samples were added to CY medium and co-cultured with S. aureus for 6 hours. The secretion level of S. aureus exotoxin was detected by MDBK cytotoxicity model.
  • the experimental results show that the TRAP polyclonal antibody can reduce the level of S. aureus exotoxin, and the phage clone of the antigen mimic epitope sequence 1 can inhibit the function of the antibody, which can reduce the effect of the TRAP polyclonal antibody by about 25%. This indicates that the polypeptide of the antigen mimic epitope sequence 1 can inhibit the action of the antibody by specifically binding the antibody to the TRAP binding site.
  • Example 6. TRAP antigen mimic epitope sequence 1 displayed on bacterial flagella and immune motility Effect observation

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Oncology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Communicable Diseases (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Description

金葡菌毒力因子调控蛋白的
抗原表位及其模拟表位和用途 技术领域
本发明涉及抗原表位及模拟表位， 具体涉及金黄色葡萄球菌 (金葡菌） 毒力因子调控蛋白的两个抗原表位及其模拟表位。 本 发明还涉及与这些表位氨基酸序列一致的多肽在制备抗金葡菌感 染药物或疫苗等方面中的应用。 背景技术
金葡菌是一类常见的革兰氏阳性致病菌， 是引起烧伤及战伤 感染、 肺炎、 心内膜炎、 败血症、 中毒性休克等致命性疾病的主 要微生物之一。 每年仅医院内感染金葡菌的人数就超过数百万。 目前临床上对金葡菌的治疗多采用联合使用抗生素的办法， 但是 效果并不理想。 由于金葡菌极易产生耐药性且无好的解决方法， 常用的许多抗生素对之无效， 控制金葡菌感染是临床医学殛待解 决的问题之一。
金葡菌的主要致病物质是毒素， 包括溶血毒素、杀白细胞素、 肠毒素等。 最新研究表明， 金葡菌这些毒力因子的合成是受一种 可调节 RNA分子， 及 RNAIII控制的。 RNAIII激活毒力因子的 基因转录， 通过碱基互补调节毒力因子的翻译。 在细菌生长的对 数早期其 RNAIII 水平低， 但到对数晚期 RNAIII水平会增加 40 倍， 而 RNAIII的水平是由金葡菌自身分泌的蛋白 RAP (RNA III activating protein) 即 RNAIII 激活蛋白调节的， 故因子 RAP又 称为金葡菌毒力刺激因子。 金葡菌持续分泌 RAP, 在 RAP达到 一定浓度后才有激活毒力因子产生的作用。没有 RAP产生的金葡 菌本身并不致病。 最新研究发现， RAP激活 RNAIII的转录是通 过一个 21KD的蛋白 TRAP (Target of RNAIII activating protein) 介导的， 当 TRAP蛋白的编码基因被突变失活后， RAP不能够激 活 RNAIII的转录。 TRAP由 167个氨基酸组成， 具有 His激酶活 性。 TRAP 蛋白在金葡菌生长的早期开始磷酸化， 在对数生长中 期达到最大水平。 在 RAP作用后， 通过自身磷酸化来进行信号传 导， 从而介导细胞内 RNAIII水平上升， 加速金葡菌外毒素的分 泌 (Naomi B， et al, J.Biol. Chem 2001, 276:2658-2667). 由此可见 TRAP蛋白在金葡菌的毒素表达调控也起着关键性的作用。 2001 的研究发现 TRAP 的抗体可以有效的降低金葡菌外毒素的分泌 (Oleny V, et al. Peptides 2001， 22:1621-1627).
抗原表位作为免疫细胞识别的靶结构和激发特异性免疫应 答的物质基础， 对于免疫反应的相关研究具有重要意义。 抗原缺 失突变、 合成肽段扫描（PEPSCAN )、 X射线晶体衍射等技术的 应用， 使"抗原表位"的确定成为可能， 但这些方法费用高昂， 需 耗费大量人力、 物力， 而且不适于复杂表位的研究， 因而其应用 受到限制。 "模拟表位"（mimotope ) 这一概念的出现， 不仅为抗 原表位的分析提供了线索， 也为疫苗研究的发展开辟了一条新的 思路。 模拟表位（ mimotope ), 通常指能够模拟抗原表位的多肽 结构， 它具有与天然抗原相似的反应原性， 与适当的载体偶联后， 还可能具有相似的免疫原性，（但在天然抗原中可能并无与之相同 或相似的序列或空间结构）。 对于一些难以获得或尚不确定的抗 原， 人们很难甚至无法确定其抗原表位， 使相关的研究难以进行。 而通过获得"模拟表位，，这一径， 便有可能解决上述问题。 不仅为 抗原表位的分析提供了线索， 也为疫苗研究的发展开辟了新思路， 尤其是推动了构象型表位和非蛋白抗原表位的研究。
推动模拟表位研究的一项关鍵技术，是 1985年由 Dr. Smith建立 并发展的噬菌体展示技术。 噬菌体展示肽库的构建是运用噬菌体 展示技术， 将随机排列的外源肽（通常 6 ~ 15个氨基酸残基組成） 展示在噬菌体表面， 构成多样性为 107以上的随机肽库。 通过亲和 纯化的生物淘选过程， 从噬菌体肽库中筛选出靶分子（如单克隆 抗体、 多克隆抗体） 的结合肽， 将结合肽与天然抗原进行比较，其 中有的与天然抗原表位高度同源， 有的与天然抗原完全不同， 但 都具有与天然抗原相似的抗原性和免疫原性， 这些结合肽便被称 为"模拟表位"。 与分析抗原表位的传统方法相比， 噬菌体展示技 术具有很强的优势， 不仅简便、 快捷， 而且应用范围广泛， 适于 构象型表位、 非蛋白抗原表位以及尚不明确的抗原研究 (Zhong G, et al. J Biol Chem 1994, 269:24183-24188; Mottic et al. Gene 1994 146:191-198; Rodriguez L， et al. J Gen Virol 1999, 80(Pt3):727-738)o
与单克隆抗体相比， 以多克隆抗体或抗血清为靶标的筛选过 程， 难度大， 但有明显的优势。 表现在： 首先， 靶标容易获得， 较制备单克隆抗体时间短、 成本低、 操作简单； 其次， 可获得多 个抗原表位的模拟表位， 筛选效率高， 有利于制备具有强免疫原 性的复合疫苗。 本实验将抗血清进行了免疫亲和层析纯化， 去除 了非特异抗体， 以获得特异性高的歸选分子， 有利于歸选到特异 的噬菌体克隆， 减少非特异克隆的富集。 发明内容
本发明的目的是提供金葡菌毒力因子调控蛋白 TRAPC Target of RNAIII activating protein ) 的两个抗原表位及其模拟表位。
本发明的另一目的是提供上述表位或与这些表位氨基酸序列 一致的多肽在制备抗金葡菌感染药物或疫苗等方面中的应用。
为实现本发明的第一个目的， 我们以 TRAP多克隆抗体为靶 标， 通过线性肽库歸选获得了两组 TRAP蛋白的抗原模拟表位， 通过与 TRAP蛋白序列比较， 发现两个家族的序列都可以在表达 序列上找到一致序列， 即 TRAP蛋白本身的两个抗原表位： 21-34 位氨基酸序列和 156-167位氨基酸序列。 它们的氨基酸序列如下： 抗原表位 1: 对应于 TRAP蛋白的 21-34位氨基酸序列：
21 NPTHQLFQFSASDT 34
抗原表位 2: 对应于 TRAP蛋白的 156-167位氨基酸序列： 156 SYFERYLYPI E 167
本发明的金葡菌毒力因子调控蛋白 TRAP的两个抗原模拟表 位是具有以下氨基酸序列结构模式的多肽：
抗原模拟表位 1: XPXHHQHXTGFT
抗原模拟表位 2: SWFDXXLYPXXX
上迷抗原表位和抗原模拟表位结构中， 大写英文字母分别代 表二十一种已知天然 L-型氨基酸残基或其 D-型异构体的一种，即 A代表丙氨酸残基， R代表精氨酸残基， N代表天冬酰胺残基， D 代表天冬氨酸残基， Q代表谷氨酰胺残基， E代表谷氨酸残基， H代表組氨酸残基， W代表色氨酸残基， Y代表酪氨酸残基， F 代表苯丙氨酸残基， T 代表苏氨酸残基， S 代表丝氨酸残基， L 代表亮氨酸残基， G代表甘氨酸残基， P代表脯氨酸残基， V代 表缬氨酸残基， K代表赖氨酸残基， M代表甲硫氨酸残基， I代 表异亮氨酸残基， X代表二十一种已知天然 L-型氨基酸残基或其 D-型异构体的任意一种。
上述表位中的有些氨基酸可以根据氨基酸的相似性进行相互 替代， 如谷氨醜胺残基（Q ), 谷氨酸残基（E ), 天冬氨酸残基（D ) 或天冬酰胺残基（N )之间可以相互替代； 色氨酸残基（W )， 酪 氨酸残基（ Y )或苯丙氨酸残基（F )之间可以相互替代； 赖氨酸 残基（ K )和精氨酸残基（ R )之间可以相互替代； 丝氨酸残基（ S ) 和苏氨酸残基（T )之间可以相互替代； 丙氨酸残基（A )和甘氨 酸残基（G )之间可以相互替代； 亮氨酸残基（L )和甲硫氨酸残 基（M )之间可以相互替代。
本发明的小分子多肽可通过化学合成或用基因工程重組表达 的方法制得。
实验证明， 具有上述结构模式的多肽能够与可以特异竟争
TRAP与其多抗的结合， 有效地抑制抗体的功能， 与合适的载体 耦联后具有与 TRAP相似的免疫原性。 这就为抗金葡菌感染疫苗 的研究打下良好的基础， 从而得以实现本发明的第二个目的。
传统抗生素治疗产生抗药性的原因主要是用药后细菌在生存 压力下产生分解抗生素中有效基团的诱导酶。 本发明利用的 TRAP抗原表位及其模拟表位所对应的多肽在不同的金葡菌菌株 TRAP蛋白之间高度保守， 并且可以刺激人体产生针对 TRAP的 抗体，抑制 TRAP的活性， 因此可以制备疫苗对抗金葡菌的感染， 不杀灭细菌而使细菌的致病性丧失， 适用于所有耐药及非耐药菌 株， 这就为根除一直困扰临床的抗药性金葡菌感染这一常见、 多 发且有致命性的疾病找到新的出路。 附图说明
图 1为 TRAP多抗亲和純化电泳图。
图 2为竟争 ELISA方法检测抗原模拟表位序列 1噬菌体克隆 竟争 TRAP蛋白与抗体的结合图。 本发明的抗原表位及其模拟表位为抗金葡菌感染疫苗方面的 研究奠定了基础， 为治疗金葡菌感染开辟了新的途径， 具有广泛 的应用价值及广阔的市场前景。 具体实施方式 下面以抗原模拟表位 1 为实施例对本发明作进一步详细说 明。 实施例 1. TRAP蛋白多抗的制备及纯化
将纯化的 TRAP蛋白免疫家兔制备多克隆抗体。 利用偶联了 TRAP 蛋白的亲和柱从多抗血清中分离抗 TRAP 的 IgG。 SDS-PAGE电泳结果表明所纯化的 IgG純度 >90%, ELISA结果 表明效价在 1x10s以上（见附图 1,图中 1表示低分子量蛋白标准， 2表示純化后的 TRAP多抗）。 实施例 2. TRAP多抗抗原模拟表位的筛选
首先用 ΙΟΟμΙ纯化的 TRAP多抗 IgG包被于酶联板， 置 4口 过夜。 经过 2%的明胶封闭 lh后， 加入噬菌体十二肽库， 室温孵 育 lh，用 TBST(50mmol/L Tris-HCl,0.1 % TWEEN20，pH7.5)洗涤 非特异结合的噬菌体，再用 0.2mmol/L甘氨酸 -HC1 pH2.2洗脱特 异结合的噬菌体，洗脱液用 lmmol/L Tris-HCl pH9.0中和。按照试 剂盒提供的方法，测定洗脱液中的噬菌体的滴度，以未包被的靶蛋 白的洗脱噬菌体的滴度作为对照， 测定投入产出比。 同时将洗脱 的与 TRAP抗体 IgG结合的噬菌体扩增， 并测定其下滴度， 用于 下一轮的筛选。 经过 3轮筛选后， 测定的投入产出有了明显的提 高。 将富集的噬菌体克隆进行 ELISA鉴定， 随机挑取 24个阳性 克隆进行测序。 分析后得到包含抗原模拟表位序列 1的上述 2个 家族的多肽序列。 实施例 3. 筛选到的抗原模拟表位序列 1和表达的 TRAP序列进 行比较
将筛选的序列和表达的 TRAP 蛋白的原始序列进行分析发 现， 抗原模拟表位序列 1的序列与 TRAP蛋白的 21-34位氨基酸 序列相似：
抗原模拟表位序列 1 NPLHHEHA TGWT
TRAP蛋白一级序列 21 ΝΡΎ HO FOFSASDT 34 实施例 4. 抗原模拟表位序列 1噬菌体克隆竟争 TRAP蛋白与抗 体的结合
选择扩增抗原模拟表位序列 1的噬菌体克隆， 检测其是否能 够竟争 TRAP与 TRAP多抗的结合。 包被 TRAP抗体， 加入不同 量 TRAP蛋白和噬菌体（109 ) 的混和物， 通过酶标抗 M13的单 克隆抗体检测噬菌体竟争 TRAP蛋白与抗体的结合。 实验结果表 明抗原模拟表位序列 1的噬菌体克隆可以竟争 TRAP蛋白与其抗 体的结合， 随着 TRAP蛋白量的逐步提高， 与抗体结合的噬菌体 数目在逐步减少（附图 2, 图中 1表示 109抗原模拟表位序列 1噬 菌体克隆， 2表示 5μ TRAP+109抗原模拟表位序列 1噬菌体克隆， 3表示 lO gTRAP +109抗原模拟表位序列 1噬菌体克隆）。 实施例 5. 抗原模拟表位序列 1的噬菌体克隆对 TRAP 多抗活性 的抑制作用
1： 100接种 RN6390B, 将样品加入 CY 培养基与金葡菌共 培养 6h， 通过 MDBK细胞毒模型检测金葡菌外毒素的分泌水平。 实验结果表明 TRAP多抗可以降低金葡菌外毒素的水平， 而抗原 模拟表位序列 1的噬菌体克隆可以抑制抗体的功能， 能使 TRAP 多抗的作用降低 25%左右。 这表明抗原模拟表位序列 1的多肽可 以通过特异结合抗体与 TRAP结合部位而抑制抗体的作用。 实施例 6. TRAP抗原模拟表位序列 1 展示在细菌鞭毛上及免疫动 物的效果观察
利用鞭毛的在细菌表面的高拷贝数（每个细菌约二万个拷贝） 的性质， 我们将 TRAP抗原模拟表位序列 1对应的多肽展示在大 肠杆菌 GI826鞭毛蛋白的硫氧环蛋白区， 将重組菌免疫小鼠， 检 测特异抗体的产生， 经过两次免疫后。 ELISA和 Westen Blot实 验結果显示产生的抗血清能够特异结合 TRAP蛋白， 并且抗血清 的效价大于 5000， 表明我们获得的模拟肽与合适的载体耦联后具 有与 TRAP相似的免疫原性。 这就为抗金葡菌感染疫苗的研究打 下良好的基础。

Claims

1. 金葡菌毒力因子调控蛋白的的抗原表位， 其特征在于具有 序列表中序列 1所示的氨基酸序列。
2. 金葡菌毒力因子调控蛋白的的抗原表位， 其特征在于具有 序列表中序列 2所示的氨基酸序列。
3. 权利要求 1抗原表位的模拟表位， 其特征在于具有下式所 示的氨基酸序列：
XPXHHQHXTGFT
式中， 字母 X代表二十一种已知天然 L-型氨基酸残基或其 D-型异构体的任意一种。
4. 权利要求 1抗原表位的模拟表位， 其特征在于具有下式所 示的氨基酸序列：
SWFDXXLYPXXX
式中， 字母 X代表二十一种已知天然 L-型氨基酸残基或其 D-型异构体的任意一种。
5. 金葡菌毒力因子调控蛋白的的抗原表位， 其特征在于其氨 基酸序列是根据氨基酸的相似性对权利要求 1或 2序列中的有些 氨基酸进行替代而成。
6. 金葡菌毒力因子调控蛋白的的抗原模拟表位， 其特征在于 其氨基酸序列是根据氨基酸的相似性对权利要求 3或 4序列中的 有些氨基酸进行替代而成。
7. 权利要求 1至 6中的任意一种序列的多肽以任何形式在制 备抗金葡菌感染药物或疫苗中的应用。
PCT/CN2003/000827 2003-07-21 2003-09-27 Antigen epitopes of the regulatory protein of virulence factor in staphylococcus aureus and their mimotopes and use WO2005007683A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP03756419.2A EP1719520B1 (en) 2003-07-21 2003-09-27 Antigen epitopes of the regulatory protein of virulence factor in staphylococcus aureus and their mimotopes and use
DK03756419.2T DK1719520T3 (en) 2003-07-21 2003-09-27 ANTIGENE EPITOPES OF REGULATION PROTEIN OF VIRULENS FACTOR IN STAPHYLOCOCCUS AUREUS AND MIMOTOPES THEREOF AND USE
AU2003304343A AU2003304343A1 (en) 2003-07-21 2003-09-27 Antigen epitopes of the regulatory protein of virulence factor in staphylococcus aureus and their mimotopes and use
ES03756419.2T ES2535777T3 (es) 2003-07-21 2003-09-27 Epítopos antigénicos de la proteína reguladora del factor de virulencia en Staphylococcus aureus y sus mimótopos y uso

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN03150203.2 2003-07-21
CNA031502032A CN1569893A (zh) 2003-07-21 2003-07-21 金葡菌毒力因子调控蛋白的抗原表位及其模拟表位和用途

Publications (2)

Publication Number Publication Date
WO2005007683A1 true WO2005007683A1 (en) 2005-01-27
WO2005007683A8 WO2005007683A8 (fr) 2006-06-15

Family

ID=34069991

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2003/000827 WO2005007683A1 (en) 2003-07-21 2003-09-27 Antigen epitopes of the regulatory protein of virulence factor in staphylococcus aureus and their mimotopes and use

Country Status (6)

Country Link
EP (1) EP1719520B1 (zh)
CN (1) CN1569893A (zh)
AU (1) AU2003304343A1 (zh)
DK (1) DK1719520T3 (zh)
ES (1) ES2535777T3 (zh)
WO (1) WO2005007683A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2011508A1 (en) * 2006-04-17 2009-01-07 Institute of Basic Medical Sciences Academy of Military Medical Sciences Use of trap protein as active ingredient for manufacturing a medicament for the treatment of staphylococcus aureus infection
US8889150B2 (en) 2010-03-17 2014-11-18 SOCPRA—Sciences et Génie, s.e.c. Bacterial vaccine components from Staphylococcus aureus and uses thereof
US11324815B2 (en) 2016-10-21 2022-05-10 Socpra—Sciences et Genie, S.E.C. Vaccine constructs and uses thereof against Staphylococcus infections

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101066463B (zh) * 2007-05-24 2010-09-15 上海大学 金黄色葡萄球菌DNA疫苗pcDNA3.1(+)-Minigene及其制备方法
CN105254719B (zh) * 2015-10-21 2018-07-13 黑龙江八一农垦大学 金黄色葡萄球菌trap蛋白的cd4+t细胞表位鉴定及其重组表位疫苗
CN110156887B (zh) * 2018-02-12 2023-01-13 中国人民解放军军事科学院军事医学研究院 人vasn蛋白抗原表位、抗原模拟表位及其用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020102271A1 (en) * 1998-09-15 2002-08-01 Naomi Balaban Target of RNAIII activating protein (TRAP)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020102271A1 (en) * 1998-09-15 2002-08-01 Naomi Balaban Target of RNAIII activating protein (TRAP)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BALABAN N. ET AL: "Regulation of Staphylococcus aureus Pathogenesis via Target of RNAIII-activatingProtein (TRAP).", JOURNAL OF BIOLOGICAL CHEMISTRY, 26 January 2001 (2001-01-26), pages 2658 - 2667, XP002984048 *
See also references of EP1719520A4 *
WANG Y.: "Phage Display Technology and its Appplication in research of antigenic epitope", CHINESE JOURNAL OF VETERINARY MEDICINE, vol. 39, no. 4, April 2003 (2003-04-01), pages 34 - 37, XP008100491 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2011508A1 (en) * 2006-04-17 2009-01-07 Institute of Basic Medical Sciences Academy of Military Medical Sciences Use of trap protein as active ingredient for manufacturing a medicament for the treatment of staphylococcus aureus infection
JP2009533478A (ja) * 2006-04-17 2009-09-17 海南通用同盟▲約▼▲業▼有限公司 黄色ブドウ球菌感染症の治療用薬剤の製造のための活性成分としてｔｒａｐタンパク質自体の使用
EP2011508A4 (en) * 2006-04-17 2010-11-03 Hainan Gt Unipul Pharmaceutica USE OF TRAP PROTEIN AS AN ACTIVE INGREDIENT FOR THE MANUFACTURE OF A MEDICINAL PRODUCT FOR THE TREATMENT OF STAPHYLOCOCCAL INFECTION
KR101443233B1 (ko) * 2006-04-17 2014-09-22 인스티튜트 오브 베이직 메디컬 사이언시즈, 아카데미오브 밀리터리 메디컬 사이언시즈 황색포도상구균 감염 치료용 의약을 제조하기 위한 활성 성분으로서의 ｔｒａｐ 자체의 용도
US8889150B2 (en) 2010-03-17 2014-11-18 SOCPRA—Sciences et Génie, s.e.c. Bacterial vaccine components from Staphylococcus aureus and uses thereof
US9566322B2 (en) 2010-03-17 2017-02-14 SOCPRA—Sciences et Génie, s.e.c. Bacterial vaccine components and uses thereof
US10029004B2 (en) 2010-03-17 2018-07-24 SOCPRA—Sciences et Génie, s.e.c. Bacterial vaccine components and uses thereof
US10576139B2 (en) 2010-03-17 2020-03-03 SOCPRA—Sciences et Génie, s.e.c. Bacterial vaccine components and uses thereof
US11065322B2 (en) 2010-03-17 2021-07-20 Socpra—Sciences et Genie, S.E.C. Bacterial vaccine components and uses thereof
US11129884B2 (en) 2010-03-17 2021-09-28 Socpra—Sciences et Genie, S.E.C. Bacterial vaccine components and uses thereof
US11324815B2 (en) 2016-10-21 2022-05-10 Socpra—Sciences et Genie, S.E.C. Vaccine constructs and uses thereof against Staphylococcus infections

Also Published As

Publication number Publication date
EP1719520A4 (en) 2009-06-24
ES2535777T3 (es) 2015-05-14
EP1719520B1 (en) 2015-04-15
AU2003304343A8 (en) 2005-02-04
AU2003304343A1 (en) 2005-02-04
DK1719520T3 (en) 2015-05-11
EP1719520A1 (en) 2006-11-08
CN1569893A (zh) 2005-01-26
WO2005007683A8 (fr) 2006-06-15

Similar Documents

Publication Publication Date Title
Li et al. rOmpA is a critical protein for the adhesion ofRickettsia rickettsiito host cells
JP5215275B2 (ja) 機能活性抗体を誘発する髄膜炎菌性ｂエピトープの分子模倣物
Lyerly et al. Vaccination against lethal Clostridium difficile enterocolitis with a nontoxic recombinant peptide of toxin A
US20040047880A1 (en) Component for vaccine
JP2012515551A5 (zh)
Zhao et al. Neutralizing monoclonal antibody against anthrax lethal factor inhibits intoxication in a mouse model
Zeng et al. Screening and identification of the mimic epitope of the adhesion protein of Mycoplasma genitalium
Leitner et al. A mimotope defined by phage display inhibits IgE binding to the plant panallergen profilin
Ferdosian et al. Identification of immunotopes against Mycobacterium leprae as immune targets using PhDTm-12mer phage display peptide library
CN102757481A (zh) 金黄色葡萄球菌肠毒素b的功能表位、与其特异性结合的单克隆抗体及其应用
CA2098296C (en) Pseudomonas peptide composition and method for producing the same
WO2005007683A1 (en) Antigen epitopes of the regulatory protein of virulence factor in staphylococcus aureus and their mimotopes and use
CN101348521B (zh) 人b淋巴细胞刺激因子受体的氨基酸模拟表位及其应用
Mertens et al. Selection of phage-displayed peptides recognised by monoclonal antibodies directed against the lipopolysaccharide of Brucella
Yang et al. A novel peptide isolated from phage library to substitute a complex system for a vaccine against staphylococci infection
CN109369809B (zh) 多表位抗原及其制备方法与在制备防治鹦鹉热衣原体感染的药物中的应用
KR20080068048A (ko) 녹농균의 외막 단백질 ｐａ5158
Cai et al. Identification of the amino acid residues in trichosanthin crucial for IgE response
Li et al. Production of mouse monoclonal antibodies against Helicobacter pylori Lpp20 and mapping the antigenic epitope by phage display library
Cardoso et al. Peptide mimicking antigenic and immunogenic epitope of neuwiedase from Bothrops neuwiedi snake venom
CN111948387B (zh) 结核分枝杆菌抗原蛋白Rv1485在制备结核疫苗中的应用
CN105254719B (zh) 金黄色葡萄球菌trap蛋白的cd4+t细胞表位鉴定及其重组表位疫苗
Li et al. Identification and characterization of antigenic epitope of Staphylococcus aureus ClfA adhesin
Li et al. Klebsiella pneumoniae MrkD adhesin-mediated immunity to respiratory infection and mapping the antigenic epitope by phage display library
EP3892298A1 (en) Epitopes having sequence homology to coronavirus spike protein subunit and uses thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WRT Later publication of a revised version of an international search report translation
WWE Wipo information: entry into national phase

Ref document number: 2003756419

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2003756419

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: JP