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Secreted chlamydia polypeptides, polynucleotides coding therefor, therapeutic and diagnostic uses thereof

A polynucleotide, chlamydia technology, applied in the direction of chlamydia antigen components, medical preparations containing active ingredients, peptides, etc., can solve problems such as inability to identify proteins

Inactive Publication Date: 2006-11-01
INST PASTEUR +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the genomes of some Chlamydia species are known, secreted proteins cannot be identified based on their amino acid sequences

Method used

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  • Secreted chlamydia polypeptides, polynucleotides coding therefor, therapeutic and diagnostic uses thereof
  • Secreted chlamydia polypeptides, polynucleotides coding therefor, therapeutic and diagnostic uses thereof
  • Secreted chlamydia polypeptides, polynucleotides coding therefor, therapeutic and diagnostic uses thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Materials and methods

[0096] Strains and Reagents

[0097]Strain M90T is a pathogenic, wild-type strain of S. flexneri 5 (Sansonetti et al., 1982). Strains SF401 and SF620 (deposited in C.N.C.M., deposit number is I-2594) are derivatives of M90T, wherein mxiD and ipaB genes are respectively inactivated (Allaoui, A., P.J.Sansonetti, and C.Parsot.(1993).MxiD , an outer membrane protein necessary for the secretion of the Shigella flexneri Ipa invasins.Mol.Microbiol.7:59-68; Ménard, R., P.Sansonetti and C.Parsot.(1994).These secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. EMBO J. 13:5293-302). E. coli strain TG1 (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989) was used to construct plasmids. Shigella flexneri and E. coli strains were grown in Luria-Bertani (LB) medium. Ampicillin was used at a concentration of 0.1 mg / ml. A monoclonal antibody against calmodu...

Embodiment 2

[0130] Chlamydia genes were cloned by PCR to express full-length Chlamydia proteins with a histidine tag at the carboxyl terminus. The forward and reverse primers contained additional NcoI and KpnI sites, respectively, to enable cloning of the PCR fragment into the pQE-TriSystem expression vector (Qiagen) between the NcoI and KpnI sites. The sequences of the primers are listed in Table 2. The recombinant plasmid was amplified in E.coli TG1 and sequenced to check the sequence.

[0131] M90T derivatives SF401 and SF620 strains were transformed with plasmids, and the mxiD and ipaB genes in SF401 and SF620 had been inactivated, respectively (Allaoui et al., 1993; Ménard et al., 1993). Transformed colonies were isolated in Petri dishes containing 100 μg / ml ampicillin.

[0132] Secreted proteins were analyzed as previously described (Allaoui et al., 1993). Briefly, 1 ml of an overnight grown culture (30°C) was inoculated in 30 ml of LB and incubated at 37°C for 3 hours. Bacteria...

Embodiment 3

[0136] The Psi0705 and Psi0710 proteins with a carboxy-terminal histidine tag were expressed in E. coli and the proteins were purified by standard divalent metal column chromatography according to the manufacturer's (Qiagen) instructions. Rabbits were immunized with the purified protein, thereby obtaining specific antibodies against Psi0705 and Psi0710. These antibodies were used to study the localization of Psi0705 and Psi0710 during Chlamydia infection.

[0137] A: The inventors confirmed by immunofluorescence that Psi0710 is associated with inclusion body membranes in HeLa cells infected with the GPIC strain of Chlamydia psittaci ( FIG. 4 ). The inventors confirmed that the pre-immune sera as a control did not label infected cells, and antibodies against the Major Outer Membrane Protein of Chlamydia did not label inclusion body membranes.

[0138] B: The inventors confirmed by Western blot that Psi0705 was associated with cytoplasmic components in HeLa cells infected with ...

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Abstract

The present invention relates to secreted Chlamydia polypeptides, which may be expressed by a Gram-negative bacterial strain and secreted by the type III secretion pathway of said bacterial strain. The present invention also relates to polynucleotides coding for these polypeptides, as well as to the therapeutic and vaccination uses of these secreted Chlamydia polypeptides.

Description

technical field [0001] The present invention relates to secreted Chlamydia polypeptides. More specifically, the present invention relates to secreted Chlamydia polypeptides expressed by Gram-negative bacterial strains and secreted by the type III secretion pathway of said strains. The invention also relates to polynucleotides encoding these secretory Chlamydia polypeptides, and to therapeutic (including immunization) and diagnostic uses of these secretory Chlamydia polypeptides. Background technique [0002] Chlamydia are Gram-negative bacteria that can only proliferate in eukaryotic host cells. Three human pathogenic types, Chlamydia trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae, cause a variety of diseases including trachoma, sexually transmitted diseases, pelvic inflammatory disease, respiratory Diseases such as bronchitis, pneumonia and their sequelae (Gregory, D.W. and W.Schaffner. (1997). Psittacosis. Seminars in Respiratory Infections. 12: 7-11; Kuo, C.-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/295A61K38/00A61K39/00A61K39/118C07K16/12C12N1/21C12N15/11C12N15/31C12Q1/68G01N33/53
CPCA61K38/00A61K39/00A61K2039/505C07K14/295A61P31/04
Inventor 艾丽斯·多特里-瓦尔萨特阿加特·叙布蒂-桑兹
Owner INST PASTEUR
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