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Method for identification of proteins from intracellular bacteria

A protein and bacteria technology, applied in the field of identifying intracellular bacterial proteins, can solve problems such as reducing tryptophan synthesis ability

Inactive Publication Date: 2004-09-22
阿兰·克里斯蒂安·肖 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Truncation or deletion of tryptophan synthase in trachoma-causing serotypes (C. trachomatis A, B, and C) may reduce tryptophan synthesis capacity and render these serotypes more susceptible to IFN-γ-mediated tryptophan The effect of the absence of acid

Method used

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  • Method for identification of proteins from intracellular bacteria
  • Method for identification of proteins from intracellular bacteria
  • Method for identification of proteins from intracellular bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0235] Infection of mammalian cell cultures

[0236] According to the methods described in [19.] and [17.], use Chlamydia pneumoniae VR1310, Chlamydia trachomatis serotype A (HAR-13), D (UW-3 / Cx) or L2. (434 / Bu) (ATCC) ) An inclusion body forming unit (IFU) infected semi-confluent HeLa, HEp-2 or McCoy (ATCC, Rockville, MD, USA) cell monolayer. The infection medium for Chlamydia trachomatis A and D consists of RPMI 1640, 25mM HEPES, 10% FCS, 1% w / v glutamine, and 10mg / ml gentamicin. The infection medium for Chlamydia trachomatis L2 consists of RPMI 1640, 25mM HEPES, 5% FCS, 1% w / v glutamine, 10mg / ml gentamicin.

Embodiment 2

[0238] Pulse marking / tracking analysis

[0239] In order to label the chlamydia protein for a period of two hours, according to the above-mentioned method (Shaw et al., 1999, 2000) [18.] [19.], containing RPMI 1640, 10 mg / ml gentamicin, 40 μg / ml actinomycetes The infected cells were cultured in a ketone, 100 μCi / ml [35S]-methionine / cysteine ​​(Promix, Amersham Pharmacia Biotech, Uppsala, Sweden) medium. After labeling, the labeling medium was replaced with a normal medium by washing twice with normal medium, and the infected cells were collected at different time points after the labeling. Similarly, the labeled EB protein was obtained by culturing the chlamydia for two hours after labeling to 72 hours after infection. The labeled EBs are then collected and purified using two consecutive steps of density gradient ultracentrifugation, which is basically as used for Chlamydia trachomatis (Schacter and Wyrick, 1994) [22.] and pneumonia Chlamydia (Knudsen et al. 1999 [17.]) density g...

Embodiment 3

[0241] Sample preparation

[0242]After [35S]-labeled, the cells were washed twice with PBS and then dissolved in a standard lysis buffer containing 9M urea, 4% w / v 3-[(3-cholamidopropyl)dimethyl Ammonium]-1-propanesulfonate (CHAPS; Roche, Germany), 40 mM Tris Base, 65 mM DTE, and Pharmalyte 3-10 (Amersham Pharmacia Biotech). In order to enrich high molecular weight hydrophobic proteins, 7M urea, 2M thiourea, 4% w / v 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS ; Boehringer Mannheim, Germany), 40mM Tris Base, 65mM dithioerythritol (DTE) and 2% v / vPharmalyte 3-10 (Amersham Pharmacia Biotech), basically in accordance with (Harder et al. 1999 [23.]) The proceeding. Sonicate the sample containing intact fine lysate or purified EB, and centrifuge at 10000×g for 10 minutes. Store the sample at -70°C until use.

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Abstract

The present invention relates to a novel combination of methods that enables identification of proteins secreted from intracellular bacteria regardless of the secretion pathway. The invention further provides proteins that are identified by these methods. Secreted proteins are known to be suitable candidates for inclusion in immunogenic compositions and / or diagnostic purposes. The invention also provides peptide epitopes (T-cell epitopes) from the identified secreted proteins, as well as nucleic acid compounds that encode the proteins. The invention further comprises various applications of the proteins or fragments thereof, such as pharmaceutical and diagnostic applications.

Description

Technical field [0001] The present invention relates to a new combination method capable of identifying proteins secreted by intracellular bacteria in any secretion pathway. The present invention further provides proteins identified by these methods. It is known that secreted proteins are suitable candidates for inclusion in immunogenic compositions and / or diagnostic purposes. The present invention also provides peptide epitopes (T-cell epitopes) derived from the identified secreted protein and nucleic acid materials encoding the protein. The present invention further includes various applications of the protein or fragments thereof, such as pharmaceutical or diagnostic applications. technical background [0002] Chlamydia is an obligate intracellular bacterium that reproduces in eukaryotic host cells and is an important human pathogen. The Chlamydia order includes a family containing one genus (Chlamydia), which is divided into four species: C. trachomatis, C. pneumoniae, C. psi...

Claims

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Application Information

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IPC IPC(8): G01N33/53A61K31/7088A61K38/00A61K39/00A61K39/118A61K39/395A61K48/00A61P7/02A61P15/00A61P27/02A61P31/04C07K14/295C07K16/12C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12P21/02C12P21/08C12Q1/02C12Q1/37C12R1/01G01N33/483G01N33/569G01N33/68
CPCG01N2550/00G01N2333/81A61K2039/53C07K14/295G01N33/6878G01N33/6803A61K2039/505A61K39/00A61P15/00A61P27/02A61P31/04A61P7/02C12Q1/04
Inventor 阿兰·克里斯蒂安·肖布赖恩·伯格·旺达赫尔
Owner 阿兰·克里斯蒂安·肖
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