Name: immuno-fluorescence assay of leptospiral surface-exposed proteins
Uploaded: 2011-07-01T12:00:00
Description: Watch this Scientific Journal Video about Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins at JoVE.com

这项研究是支持公共卫生服务补助金的AI - 34431（DAH）由美国国立过敏和传染病研究所和VA医学研究基金（DAH）。

1。 钩端螺旋体固定玻片<ol><li>钩端螺旋体是一种类BSL2病原体。活细胞的工作，它需要适当的处理，如戴手套，实验室外套，并在无菌罩移液步骤。 </li><li>在EMJH medium11成长的钩端螺旋体，辅以1％兔血清在30 ° C，直到他们达到月中下旬数期约6天（5 × 10 8细胞/ ml密度为5 × 10 7） 。 </li><li> 〜2000年7分钟在室温XG离心收获的文化。 </li><li>移除吸上清，轻轻重悬沉淀磷酸盐缓?...

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	<li>Achouak, W., Heulin, T., Pages, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+facets+of+bacterial+porins.">Multiple facets of bacterial porins.</a> FEMS Microbiol Lett. 199, 1-7 (2001).</li><li>Haake, D. A., Matsunaga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+leptospiral+outer+membrane+and+description+of+three+novel+leptospiral+membrane+proteins.">Characterization of the leptospiral outer membrane and description of three novel leptospiral membrane proteins.</a> Infect Immun. 70, 4936-4945 (2002).</li><li>Haake, D. 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A., Trueba, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+outer+membrane+and+secreted+proteins+of+Leptospira+interrogans+serovar+pomona.">Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona.</a> Microb Pathog. 10, 311-322 (1991).</li><li>Cullen, P. A., Haake, D. A., Adler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+proteins+of+pathogenic+spirochetes.">Outer membrane proteins of pathogenic spirochetes.</a> FEMS Microbiol Rev. 28, 291-318 (2004).</li><li>Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spirochaetal+lipoproteins+and+pathogenesis.">Spirochaetal lipoproteins and pathogenesis.</a> Microbiology. 146, 1491-1504 (2000).</li><li>Koebnik, R., Locher, K. P., Gelder, P. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+bacterial+outer+membrane+proteins:+barrels+in+a+nutshell.">Structure and function of bacterial outer membrane proteins: barrels in a nutshell.</a> Mol Microbiol. 37, 239-253 (2000).</li><li>Schulz, G., Benz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The structures of general porins. , (2004).</li><li>Pinne, M., Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+approach+to+identification+of+surface-exposed,+outer+membrane-spanning+proteins+of+Leptospira+interrogans.">A comprehensive approach to identification of surface-exposed, outer membrane-spanning proteins of Leptospira interrogans.</a> PLoS One. 4, e6071-e6071 (2009).</li><li>Johnson, R. C., Rogers, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+leptospires.+II.+The+action+of+8-azaguanine.">Metabolism of leptospires. II. The action of 8-azaguanine.</a> Can J Microbiol. 13, 1621-1629 (1967).</li><li>Cullen, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfaceome+of+Leptospira+spp.">Surfaceome of Leptospira spp.</a> Infect Immun. 73, 4853-4863 (2005).</li><li>Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+sequence+analysis+of+the+gene+encoding+OmpL1,+a+transmembrane+outer+membrane+protein+of+pathogenic+Leptospira+spp.">Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.</a> J Bacteriol. 175, 4225-4234 (1993).</li><li>Hefty, P. S., Jolliff, S. E., Caimano, M. J., Wikel, S. K., Akins, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+temporal+and+spatial+patterns+of+outer+surface+lipoprotein+expression+generate+population+heterogeneity+and+antigenic+diversity+in+the+Lyme+disease+spirochete,+Borrelia+burgdorferi.">Changes in temporal and spatial patterns of outer surface lipoprotein expression generate population heterogeneity and antigenic diversity in the Lyme disease spirochete, Borrelia burgdorferi.</a> Infect Immun. 70, 3468-3478 (2002).</li><li>Noppa, L., &#214;stberg, Y., Lavrinovicha, M., Bergstr&#246;m, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P13+an+integral+membrane+protein+of+Borrelia+burgdorferi,+is+C-terminally+processed+and+contains+surface-exposed+domains.">P13 an integral membrane protein of Borrelia burgdorferi, is C-terminally processed and contains surface-exposed domains.</a> Infect Immun. 69, 3323-3334 (2001).</li><li>Parveen, N., Leong, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+candidate+glycosaminoglycan-binding+adhesin+of+the+Lyme+disease+spirochete+Borrelia+burgdorferi.">Identification of a candidate glycosaminoglycan-binding adhesin of the Lyme disease spirochete Borrelia burgdorferi.</a> Mol Microbiol. 35, 1220-1234 (2000).</li><li>Ristow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+OmpA-like+protein+Loa22+is+essential+for+leptospiral+virulence.">The OmpA-like protein Loa22 is essential for leptospiral virulence.</a> PLoS Pathog. 3, e97-e97 (2007).</li></ol>

我们的表面IFA技术是用来展示各种borrelial 15，16和钩端螺旋体12，17种蛋白质的表面接触的相似。这里描述的，旨在最大限度地减少操纵细胞在努力保持外膜的完整性，同时利用优势钩端螺旋体细胞的倾向，坚持以玻片表面IFA方法的细节。该方法涉及的多聚甲醛浓度较低（2％和4％），外层膜稳定缓冲液（PBS 5毫米氯化镁 2）和更少的洗涤步骤洗衣机比以前公布的协议12，...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>双室玻片</td><td>实验室康</td><td> 177380 </td><td></td></tr><tr><td>兔血清</td><td>罗克兰Immunochemicals </td><td> D209 - 00 - 0100 </td><td></td></tr><tr><td> 钩端螺旋体浓缩EMJH </td><td> BD DIFCO </td><td> 279510 </td><td></td></tr><tr><td>的Alexa Fluor 488山羊抗兔IgG（H + L） </td><td> Invitrogen公司/分子探针</td><td> A - 11034 </td><td></td></tr><tr><td>的Alexa Fluor 488山羊抗小鼠IgG（H + L） </td><td> Invitrogen公司/分子探针</td><td> A - 11029 </td><td></td></tr><tr><td> 4&#39;6 - diamidino - 2 -苯基吲哚盐酸盐（DAPI） </td><td> Invitrogen公司/分子探针</td><td> D1306 </td><td></td></tr><tr><td>延长黄金防褪色的安装介质</td><td> Invitrogen公司/分子探针</td><td> P36930 </td><td>在使用前平衡至室温</td></tr><tr><td> Fisherfinest高级玻璃盖</td><td>费舍尔</td><td> 12-544-14 </td><td></td></tr><tr><td>荧光显微镜</td><td>蔡司</td><td> Axioskop 40 </td><td></td></tr></tbody></table>

immuno-fluorescence assay of leptospiral surface-exposed proteins

细菌表面蛋白与宿主细胞的直接接触和吸收营养物质从环境1的参与。出于这个原因，细胞定位，可以提供细菌蛋白质的功能作用的见解。表面细菌蛋白质的本地化是对识别致病的机制，涉及的致病因素关键步骤。 分馏2-5钩端螺旋体膜的方法可为一定阶级的外膜蛋白（OMPS），如脂蛋白与跨膜外膜蛋白，有选择性的，因此导致的错误分类。这可能是由于结构上的差异，以及它们是如何相关的外膜。脂蛋白与膜通过脂基团之间的N -末端（3脂肪酸）和脂质双层磷脂6，7的疏水相互作用。相比之下，跨膜外膜蛋白通常集成在一个筒状结构，8，9排列的双亲性β-张成脂质双层。此外，在外膜蛋白的存在并不一定能保证，暴露在表面上的蛋白质或它的域。 Spirochetal外膜被称为脆弱，因此必要的方法，涉及温柔操纵子表面蛋白细胞和列入控制评估外膜的完整性。 在这里，我们目前的免疫荧光法（IFA）的方法来直接评估完好leptospires的蛋白质表面暴露。这种方法是基于对钩体表面蛋白抗原特异性抗体识别。在这里，特定抗体OmpL54 10 detetcted aftero约束力的本地人，表面抗原决定簇暴露。抗体反应的比较完整的与透细胞，使细胞分布进行评估，并与否的一种蛋白质是钩体表面选择性。应分摊的外膜的完整性，使用一个或多个地下蛋白质，最好在位于周质抗体。 表面IFA方法可用于分析任何钩端螺旋体特异性抗体的蛋白质表面暴露。的实用性和方法的限制，取决于是否能够绑定到本地的抗原表位的抗体。由于抗体往往提出对重组蛋白抗原表位，原生的，暴露于表面的蛋白质可能无法识别。然而，表面的IFA方法是一个完整的细菌表面成分研究的宝贵工具。这种方法不仅可应用于leptospires而且其他螺旋体和革兰氏阴性菌。更强的外膜蛋白的表面接触的结论，是一个全面的方法，涉及多种细胞定位方法推荐10。

Watch this Scientific Journal Video about Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins at JoVE.com

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

一种有效的方法来评估钩端螺旋体蛋白质表面暴露描述。该方法是专门设计，以避免破坏脆弱的钩体细胞的外膜。这种技术需要雇用几个阴性对照，以评估外膜的完整性和特异性抗体反应。

钩端螺旋体暴露于表面的蛋白免​​疫荧光法

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

Research

JoVE Journal

Immunology and Infection

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

科研

免疫与感染

Plaque Assay for Murine Norovirus

对小鼠诺沃克类病毒的空斑法

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

噬菌体型组学：生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

高灵敏检测方法的沙粒，细胞附着的测量

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Optimization of a Quantitative Micro-neutralization Assay

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

钩端螺旋体暴露于表面的蛋白免​​疫荧光法

钩端螺旋体暴露于表面的蛋白免疫荧光法