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Structural and immunochemical characterization of a Neisseria gonorrhoeae epitope defined by a monoclonal antibody 2C7;the antibody recognizes a conserved epitope on specific lipo-oligosaccharides in spite of the presence of human carbohydrate epitopes

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第51期274卷 36550-36558页

作者： Yamasaki, R Koshino, H Kurono, S Nishinaka, Y McQuillen, DP Kume, A Gulati, S Rice, PA Tottori Univ Dept Biochem & Biotechnol Tottori 6808553 Japan Inst Phys & Chem Res Wako Saitama 3510198 Japan Boston Univ Maxwell Finland Lab Infect Dis Evans Mem Dept Clin Res Boston MA 02118 USA Boston Univ Dept Med Boston MA 02118 USA

Lipo-oligosaccharides (LOS) produced by Neisseria gonorrhoeae are important antigenic and immunogenic components of the outer membrane complex. Previously, we showed that murine monoclonal antibody (mAb) 2C7 did not cross-react with human glycosphingolipids but identified the LOS epitope that is widely expressed in vivo and in vitro (Gulati, S., McQuillen, D. P., Mandrell, R. E., Jani, D. E,, and Rice, P. A. (1996) J. Infect. Dis. 174, 1223-1237). In the present study, we analyzed the structure of gonococcal strain WG LOS containing the 2C7 epitope and investigated the structural requirements for expression of the epitope, We determined that the WG LOS components are Hep[1]-elongated forms of 15253 LOS that have a lactose on both Hep[1] and Hep[2] (Yamasaki, a, Kerwood, D. E., Schneider, H., Quinn, a P., Griffiss, J. M., and Mandrell, a E, (1994) J. Biol. Chem. 269, 30345-30351). In addition, we found that expression of the 2C7 epitope within the LOS is blocked when the Hep[2]-lactose is elongated. Eased on the structural data of these LOS and the results obtained from immunochemical analyses, we conclude the following: 1) mAb 2C7 requires both the 15253 OS minimum structure and the N-linked fatty acids in the lipoidal moiety for expression of the epitope;2) mAb 2C7 binds to the LOS that elongates the lactose on Hep[1] of the 15253 OS, but not the one on Hep[2];and 3) the 2C7 epitope is expressed on gonococcal LOS despite the presence of human carbohydrate epitopes such as a lactosamine or its N-acetylgalactosaminylated (globo) form. Our study shows that the conserved epitope defined by mAb 2C7 could potentially be used as a safe site for the development of a vaccine candidate.

关键词：抗体细菌/免疫学抗体单克隆/免疫学抗体特异性抗原细菌/免疫学碳水化合物/免疫学保守序列表位/化学表位/遗传学表位/免疫学脂多糖类/免疫学淋病奈瑟球菌/免疫学人类

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Generation of epitope-tagged proteins by inverse PCR mutagenesis

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BIOTECHNIQUES 1999年第5期26卷 814-+页

作者： Gama, L Breitwieser, GE Johns Hopkins Univ Sch Med Dept Physiol Baltimore MD 21205 USA

No abstract available

关键词：氨基酸序列碱基序列生物技术 DNA引物/遗传学表位/遗传学诱变插入寡肽类肽类/遗传学肽类/免疫学聚合酶链反应/方法蛋白质类/遗传学蛋白质类/免疫学蛋白质类/分离和提纯

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One-step unidirectional cloning of tandem repeats of DNA fragments: An application for fusion protein production

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ANALYTICAL BIOCHEMISTRY 2002年第1期303卷 104-107页

作者： Jobbagy, Z Ward, JL Toan, SV Leung, GPH Tse, CM Johns Hopkins Univ Dept Med Div Gastroenterol Baltimore MD 21205 USA

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Molecular evolution of the thermosensitive PAb1620 epitope of human p53 by DNA shuffling

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JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第39期274卷 28042-28049页

作者： Xirodimas, DP Lane, DP Univ Dundee Inst Med Sci Dept Biochem Canc Res CampaignCell Transformat Grp Dundee DD1 4HN Scotland

Conformational stability of the p53 protein is an absolute necessity for its physiological function as a tumor suppressor. Recent in vitro studies have shown that wild-type p53 is a highly temperature-sensitive protein at the structural and functional levels. Upon heat treatment at 37 degrees C, p53 loses its wild-type (PAb1620(+)) conformation and its ability to bind DNA, but can be stabilized by different classes of ligands. To further investigate the thermal instability of p53, we isolated p53 mutants resistant to heat denaturation. For this purpose, we applied a recently developed random mutagenesis technique called DNA shuffling and screened for p53 variants that could retain reactivity to the native conformation-specific anti-p53 antibody PAb1620 upon thermal treatment. After three rounds of mutagenesis and screening, mutants were isolated with the desired phenotype. The isolated mutants were translated in vitro in either Escherichia coli or rabbit reticulocyte lysate and characterized biochemically. Mutational analysis identified 20 amino acid residues in the core domain of p53 (amino acids 101-120) responsible for the thermostable phenotype. Furthermore, the thermostable mutants could partially protect the PAb1620(+) conformation of tumor-derived p53 mutants from thermal unfolding, providing a novel approach for restoration of wild-type structure and possibly function to a subset of p53 mutants in tumor cells.

关键词： DNA/遗传学表位/遗传学大肠杆菌进化分子基因 p53 遗传学技术热温度蛋白质生物合成蛋白质构象蛋白质变性网状细胞/代谢热力学转录遗传肿瘤抑制蛋白质p53/化学肿瘤抑制蛋白质p53/遗传学动物人类兔

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Identification of an immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection

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ARCHIVES OF VIROLOGY 2001年第6期146卷 1197-1211页

作者： Truong, C Mahe, D Blanchard, P Le Dimna, M Madec, F Jestin, A Albina, E Agence Francaise Securite Sanit Aliments Unite Virol & Immunol Porcines F-22440 Ploufragan France

Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified disease of pigs linked to the emergence of a new porcine circovirus (PCV2). We report here the characterization of immunorelevant linear B-ci:ll epitopes of the Open Reading Frame 2-encoded protein (Orf2) from PCV2 by an enzyme-linked immunosorbent assay (ELISA) using experimental antisera collected from pigs inoculated with a PCV2 isolate. Two epitopes spanning residues 69 to 83 and 117 to 131 were specific to PCV2. Antibodies to the 117 to 131 epitope (B-133) were detected in 22% and 100% of specific pathogen-free (SPF) pig sera 6 and 11 weeks post inoculation, respectively. Cross-sectional studies performed with field sera collected from PMWS-affected herds showed B-133 antibodies in 5% of 8 to 10 week-old pigs, 38% of 13-14 week-old pigs, 62% of 16 to 19 week-old pi,os, 56% of 20 to 25 week-old pigs and 45% of 26 to 31 week-old pigs. All these data suggest that epitope B-133 is a serological marker of PCV2 infection that could be used for the detection of PCV2 antibody response.

关键词：抗体病毒/血液抗体特异性抗原病毒/遗传学碱基序列生物学标记圆环病毒科感染/免疫学圆环病毒科感染/兽医学圆环病毒科感染/病毒学圆环病毒属/分类圆环病毒属/遗传学圆环病毒属/免疫学 DNA引物/遗传学酶联免疫吸附测定表位/遗传学基因型开放读码框架猪动物

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Three epitopes located on the coat protein amino terminus of viruses in the bean common mosaic potyvirus subgroup

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ARCHIVES OF VIROLOGY 1999年第6期144卷 1173-1189页

作者： Mink, GI Vetten, HJ Wyatt, SD Berger, PH Silbernagel, MJ Univ Idaho Dept Plant Soil & Entomol Sci Moscow ID 83844 USA Washington State Univ Prosser WA USA Inst Biochem & Plant Virol Braunschweig Germany Washington State Univ Dept Plant Pathol Pullman WA 99164 USA ARS USDA Prosser WA USA

Twenty-seven of 29 strains of viruses in the bean common mosaic virus (BCMV) subgroup of legume-infecting potyviruses reacted strongly with one or more of the monoclonal antibodies (MAbs) which are known to be specific for epitopes located along the 50 amino acids which constitute the N-terminal end of the viral coat protein. Approximately one half of the virus strains reacted with the N-terminal epitope specific (NTES) MAb 4G12 which is specific for epitope E/B4, while the other half reacted with NTES MAbs 4 Al or 4F9 which are specific for epitope E/B3. All but two strains contained at least one of these epitopes while no strain contained both. Competitive assays using five sequential, nonoverlapping, synthetic, 10mer peptides indicated that the amino acids critical for epitope E/B3 reaction were located at positions 5, 7, and 10 from the N-terminal end of the coat protein. By deduction we postulate that the amino acids critical for epitope E/B4 are located at positions 10, 16, and 17. Because epitope E/B3 requires isoleucine at position 10 for expression whereas epitope E/B4 requires valine to be expressed, no one strain can express both epitopes. Two viruses in our tests (azuki mosaic and Dendrobium mosaic viruses) had deletions in this portion of their sequence explaining their failure to react MAbs specific for either epitope. The critical amino acids for a third epitope, E/B3A, were located at positions 16 and 17. We found no correlation between any of the three N-terninal epitopes defined in this study and the presence or absence of any biological property that we could accurately measure: i.e., symptomatology, host range, or pathotype. However, when coat protein sequences were aligned according to epitope type E/B3 or E/B4, we found that sequences within groups had high levels of identity while between group identities were low. We also found that sequences in the 3'-end non-coding region exhibited similar relationships within and between epitope groups. Tw

关键词：氨基酸序列抗原病毒/遗传学抗原病毒/免疫学碱基序列表位/遗传学豆科/病毒学分子序列数据植物药用马铃薯Y病毒属/遗传学马铃薯Y病毒属/免疫学序列比对病毒包膜蛋白质类/遗传学病毒包膜蛋白质类/免疫学

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Pl^A polymorphism of glycoprotein IIIa and risk of adverse events after coronary stent placement

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JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY 2000年第1期36卷 84-89页

作者： Kastrati, A Koch, W Gawaz, M Mehilli, J Böttiger, C Schömig, K von Beckerath, N Schömig, A Deutsch Herzzentrum Munich D-80636 Munich Germany Tech Univ Munich Med Klin Rechts Isar 1 D-8000 Munich Germany

OBJECTIVE We designed this prospective study to test the hypothesis that platelet antigen (P1(A)) polymorphism of glycoprotein (GP) IIIa is associated with an increased risk for adverse events after coronary stent placement. BACKGROUND Platelets play a central role in arterial thrombosis. The P1(A) polymorphism of GP IIIa, a constituent of the fibrinogen receptor, may influence the platelet function and, thereby, the early outcome of patients after coronary stent placement. METHODS The study included 1,759 consecutive patients with stable or unstable angina and successful coronary stent placement. Platelet antigen genotypes were determined by allele-specific restriction enzyme analysis. The end point of the study was a composite of death, myocardial infarction and urgent revascularization during the first 30 days after stent placement. RESULTS The P1(A) genotype of the patients included was: 70.2% were homozygous for platelet antigen 1 (P1(A1)), 2.6% homozygous for platelet antigen 2 (P1(A2)), and 27.2% were heterozygous (p1(A1/A2)). The incidence of the composite end point was 5.5% among P1(A2) carriers and 5.4% in homozygous P1(A1) subjects (p = 0.94). It was 5.4% in p1(A1/A1) patients, 4.8% in p1(A1/A2) patients and 13.0% in P1(A2/A2) patients (P = 0.06). The combined incidence of death or myocardial infarction was 4.3% in P1(A1/A2) p1(A2/A2) patients (p = 0.02). CONCLUSION The isolated presence of the p1(A2) allele in heterozygous patients is not associated with anp detectable increase in the risk for an adverse 30-day outcome after coronary stenting. This study suggests also that an increased risk is likely to be present in homozygous carriers of the P1(A2) allele, but this should be confirmed in a much larger series of patients. (J Am Coil Cardiol 2000;36: 84-9) (C) 2000 by the American College of Cardiology.

关键词：等位基因心绞痛/外科学抗原 CD/遗传学抗原 CD/代谢抗原人血小板/血液抗原人血小板/遗传学冠状血管造影术冠状动脉血栓形成/血液冠状动脉血栓形成/并发症冠状动脉血栓形成/诊断显像冠状动脉血栓形成/遗传学 DNA/分析 DNA引物/化学表位/血液表位/遗传学遗传标记基因型整合素β3 整合素类/血液整合素类/遗传学心肌梗死/血液心肌梗死/诊断显像心肌梗死/病因学比值比血小板膜糖蛋白类/遗传学血小板膜糖蛋白类/代谢聚合酶链反应多态现象遗传预后前瞻性研究危险因素支架/副作用老年人女(雌)性人类男(雄)性中年人

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Failure of highly immunogenic filarial proteins to provide host-protective immunity

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EXPERIMENTAL PARASITOLOGY 1999年第4期91卷 334-340页

作者： Peralta, ME Schmitz, KA Rajan, TV Univ Connecticut Ctr Hlth Dept Pathol Farmington CT 06030 USA Loeb Res Inst Ottawa ON Canada Oakland Univ Rochester MI 48309 USA

In areas that are endemic for lymphatic filariasis, there are individuals who are parasite free and who appear not to have experienced symptoms attributable to filarial infection. These "putatively immune" individuals may recognize immunogens that could be important in host protection. We have immunoscreened expression libraries expressing epitopes encoded by filarial open reading frames and have identified three antigens that are differentially recognized by the two polar clinical groups-endemic normals and asymptomatic microfilaremics. Pre-immunization of susceptible hosts (Meriones unguiculatus) with these antigens revealed that none was able to elicit consistent host protective immunity. Our data are consistent with Waksman's conjecture that highly immunogenic antigens of parasite origin may be inappropriate candidates for prophylactic immunization. (C) 1999 Academic Press.

关键词：抗原蠕虫/遗传学抗原蠕虫/免疫学马来丝虫/免疫学胶原/遗传学胶原/免疫学疾病易感性象皮病丝虫性/免疫学象皮病丝虫性/预防和控制表位/遗传学表位/免疫学沙鼠亚科 HSP70热休克蛋白质类/遗传学 HSP70热休克蛋白质类/免疫学蠕虫蛋白质类/遗传学蠕虫蛋白质类/免疫学肌球蛋白类/遗传学肌球蛋白类/免疫学开放读码框架接种动物女(雌)性男(雄)性

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Immunisation with phage displaying peptides representing single epitopes of the glycoprotein G can give rise to partial protective immunity to HSV-2

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VIROLOGY 2000年第1期269卷 47-53页

作者： Grabowska, AM Jennings, R Laing, P Darsley, M Jameson, CL Swift, L Irving, WL Univ Nottingham Hosp Queens Med Ctr Dept Microbiol Nottingham NG7 2UH England Univ Sheffield Sch Med Sheffield Inst Vaccine Studies Div Med & Mol Genet Sheffield S Yorkshire England Peptide Therapeut Ltd Cambridge England

Filamentous phage displaying peptides representing single epitopes of the glycoprotein G of HSV-2 (gG2) were used as immunogens via the subcutaneous route in Balb/c mice without additional adjuvant. The phage were isolated from a random phage peptide display library and contain 15-mer peptide inserts that mimic epitopes of gG2. In each case, an antibody response to gG2 was generated that was dependent on the dose of phage administered and on the presence of the peptide insert. Phage displaying epitopes of gG2, which map to amino acids 551-570, were the most immunogenic;interestingly, this region of gG2 is frequently recognised by patients infected with HSV-2. The data also provide interesting information as regards choice of peptide mimics for use as immunogens because. surprisingly. the most antigenic of the individual clones was the least immunogenic. In two of the experiments, mice immunised with phage displaying a single epitope of gG2 were protected against challenge with a lethal dose of whole HSV-2. This suggests a possible role for phage-displayed peptides in inducing protective immunity against pathogens and provides a model system for investigating the underlying mechanisms. (C) 2000 Academic Press.

关键词：氨基酸序列抗体单克隆抗体病毒/生物合成抗原病毒/投药和剂量抗原病毒/遗传学抗原病毒/免疫学剂量效应关系免疫表位/投药和剂量表位/遗传学表位/免疫学遗传载体疱疹生殖器/免疫学疱疹生殖器/死亡率疱疹生殖器/预防和控制疱疹生殖器/病毒学疱疹病毒2型人/遗传学疱疹病毒2型人/免疫学疱疹病毒2型人/生理学注射皮下丝杆病毒属/遗传学丝杆病毒属/免疫学小鼠近交BALBC 分子序列数据肽碎片/投药和剂量肽碎片/遗传学肽碎片/免疫学肽库时间因素接种疫苗合成/遗传学疫苗合成/免疫学病毒包膜蛋白质类/投药和剂量病毒包膜蛋白质类/遗传学病毒包膜蛋白质类/免疫学病毒疫苗/投药和剂量病毒疫苗/遗传学病毒疫苗/免疫学动物小鼠

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Formation of immunogenic virus-like particles by inserting epitopes into surface-exposed regions of hamster polyomavirus major capsid protein

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VIROLOGY 2000年第1期273卷 21-35页

作者： Gedvilaite, A Frömmel, C Sasnauskas, K Micheel, B Özel, M Behrsing, O Staniulis, J Jandrig, B Scherneck, S Ulrich, R Humboldt Univ Charite Med Sch Inst Virol D-10098 Berlin Germany Max Delbruck Ctr Mol Med D-13092 Berlin Germany Inst Bot LT-2021 Vilnius Lithuania Robert Koch Inst D-13353 Berlin Germany Univ Potsdam Inst Biochem & Mol Physiol D-14943 Luckenwalde Germany Humboldt Univ Charite Med Sch Inst Biochem D-10098 Berlin Germany Inst Biotechnol LT-2028 Vilnius Lithuania

We generated highly immunogenic virus-like particles that are based on the capsid protein VP1 of the hamster polyomavirus (HaPV-VP1) and harbor inserted foreign epitopes. The HaPV-VP1 regions spanning amino acids 81-88 (position 1), 222/223 (2), 244-246 (3), and 289-294 (4) were predicted to be surface exposed. An epitope of the pre-Si region of the hepatitis B virus (designated S1;amino acid sequence DPAFR) was introduced into the predicted positions of VP1. All VP1/S1 fusion proteins were expressed in yeast and generated virus-like particles. Immunoassays using the S1-specific monoclonal antibody MA18/7 and immunization of C57B16 mice with different VP1/S1 constructs showed a pronounced reactivity and a strong S1-specific antibody response for particles carrying the insert in position 1, 2, 1+2, and 1+3, Our results suggest that HaPV-VP1 represents a highly flexible carrier moiety for the insertion of foreign sequences offering a broad range of potential uses, especially in vaccine development. (C) 2000 Academic Press.

关键词：氨基酸序列抗体单克隆/免疫学抗体病毒/免疫学抗原病毒/化学抗原病毒/遗传学抗原病毒/免疫学抗原病毒/代谢病毒壳体/化学病毒壳体/遗传学病毒壳体/免疫学病毒壳体/代谢衣壳蛋白质类仓鼠亚科酶多种免疫测定技术表位/化学表位/遗传学表位/免疫学表位/代谢遗传载体/遗传学遗传载体/免疫学肝炎表面抗原乙型/化学肝炎表面抗原乙型/遗传学肝炎表面抗原乙型/免疫学肝炎表面抗原乙型/代谢小鼠近交C57BL 显微镜检查免疫电子模型分子分子序列数据诱变插入/遗传学肽碎片/化学肽碎片/遗传学肽碎片/免疫学肽碎片/代谢多瘤病毒属/化学多瘤病毒属/遗传学多瘤病毒属/免疫学多瘤病毒属/代谢蛋白质构象蛋白质前体/化学蛋白质前体/遗传学蛋白质前体/免疫学蛋白质前体/代谢重组融合蛋白质类/化学重组融合蛋白质类/遗传学重组融合蛋白质类/免疫学重组融合蛋白质类/代谢酵母菌酿酒/遗传学序列比对疫苗合成/化学疫苗合成/遗传学疫苗合成/免疫学动物小鼠

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