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WO2008004902A2 - Protéine, ses fragments et leur utilisation - Google Patents

Protéine, ses fragments et leur utilisation Download PDF

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Publication number
WO2008004902A2
WO2008004902A2 PCT/PL2007/000045 PL2007000045W WO2008004902A2 WO 2008004902 A2 WO2008004902 A2 WO 2008004902A2 PL 2007000045 W PL2007000045 W PL 2007000045W WO 2008004902 A2 WO2008004902 A2 WO 2008004902A2
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WO
WIPO (PCT)
Prior art keywords
protein
fragment
producing
application
kda
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/PL2007/000045
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English (en)
Other versions
WO2008004902A3 (fr
Inventor
Andrzej Gamian
Danuta Witkowska
Bernadeta Szostko
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Instytut Immunologii i Terapii Doswiadczalnej PAN
Instytut Przemyslu Farmaceutycznego
Original Assignee
Instytut Immunologii i Terapii Doswiadczalnej PAN
Instytut Przemyslu Farmaceutycznego
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 Instytut Immunologii i Terapii Doswiadczalnej PAN, Instytut Przemyslu Farmaceutycznego filed Critical Instytut Immunologii i Terapii Doswiadczalnej PAN
Publication of WO2008004902A2 publication Critical patent/WO2008004902A2/fr
Publication of WO2008004902A3 publication Critical patent/WO2008004902A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/25—Shigella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the subject of this invention is method of isolation of diagnostic preparation, which is bacterial protein purified from bacterial mass of enterobacteria and also the method of its using in test for the determination of specific anti enterobacterial immunoglobulin deficiencies as an indicator of antibacterial immunity, which play a main role in pathogenesis of infectious diseases, by direct using of this protein preparation for the determination of specific anti-enterobacterial antibodies in human sera, also the method of using of this preparation in affinity chromatography for isolation of specific protective immunoglobulins as a therapeutic agent in immunodeficiencies, and also the method of using this preparation as a protein carrier in conjugate vaccines.
  • Outer membrane proteins (OMP) of Gram-negative bacterial cell wall participate in maintaining of bacterial cell integrity, its adaptation to the environment and in interactions with host cells [Koebnik R. et al. MoI. Microbiol, 2000, 37, 239-253]. These proteins named porins, exposed on the bacterial cell surface, are immunologically important molecules in many Gram-negative bacteria. These proteins may affect the physiological functions of tissues and participate in mechanisms of pathogenicity, progression of infections and in development of inflammatory response [Lin J. et al. Microbes and Infection, 2002, 4, 325-331]. In many inflammation processes caused by Gram-negative as well as Gram-positive bacteria, antibodies against bacterial proteins were found [Biswas T., FEMS Immunol. Med. Microbiol.
  • mice induced with OMP may be transferred passively to nonimmunized animals by the serum of immunized animals [Witkowska D. et al. Arch. Immunol. Ther. Exp. 1986, 34, 499-504]. It was also found that OMPs isolated from Shigella flexneri exhibit immunomodulatory properties, as small doses of OMPs stimulated delayed hypersensitivity to sheep red blood cells, whereas higher doses suppressed this type of immune response. Immunochemical analysis of OMPs from Shigella dysenteriae, S. flexneri, S.
  • Immunoglobulins are important effectors of specific humoral immunity. Different classes and subclasses of immunoglobulins exhibit distinct functions. The predominant subclasses of immunoglobulins G (IgG) elicited during natural infection could influence the capacity of the humoral response to provide an adequate defense of the host. Bacterial proteins are the antigens preferentially inducing IgGl response, with minor contribution of IgG3 and IgG4 [Islam D. et al. Infect. Immun. 1995, 63, 2054-2061].
  • cord plasma antibodies may be protective, because antibacterial protective antibodies are transferred from the mother to fetus. That reactivity was present in all examined children, adolescents and adults sera. Specific antibodies of IgA and IgG classes interacted primarily with 38 kDa protein, in similar way for several studied enterobacterial strains, but different for Pseudomonas aeruginosa from Pseudomonadaceae family. This indicates for the specificity towards Enterobacteriaceae strains, the major components of intestinal microbial flora, which translocate to the circulation and are responsible for the development of antibacterial immunity. Reactivity was then determined of sera of several groups of children with 38 kDa protein from Sh.
  • the subject of this invention is bacterial surface protein beneficially recognized by antibodies of healthy human without active infection, what indicates that correct level of protective antibodies is very important.
  • the OMP preparations were obtained from Shigella flexneri strain and other Gram-negative strains, and reactivity of human sera was determined with 38 kDa protein isolated from Shigella flexneri strain 3a.
  • the presented invention permits beneficially to prepare the affinity column with chemically linked OMP 38 kDa protein and using this column enables beneficially purifying the specific antibodies from animal and human origin of anti enterobacterial specificity. Such antibodies have diagnostic and therapeutic value.
  • Preparations of human immunoglobulins may be applied to the affinity column, thus producing beneficially the fraction of specific anti enterobacterial antibodies which bind to the affinity gel and also beneficially the fraction of non bound immunoglobulins with total value activity, for example antiviral, for further therapeutic applications.
  • the column is used beneficially for several times. The use of substitutions with high doses of immunoglobulins may be harmful [Bernatowska E., Post. Hig.
  • the present invention enables beneficially also to apply the 38 kDa OMP protein as carrier for conjugate vaccines, useful and safe for its use as active for inducing the formation of protective antibodies.
  • the cell envelope sediment was extracted twice with 10 mM Tris-HCl, pH 7.6, containing 10 mM MgSO4 and 2% Triton X-100 at room temperature to dissolve the cytoplasmic membrane. After centrifugation at 150,000 x g, the sediment was extracted twice with the same buffer containing 2% Triton X-100 and 5 mM EDTA. The supernatant obtained after centrifugation at 160,000 x g contained OMPs, which were precipitated with 2 volumes of 95% ethanol. The OMP fraction was analysed by SDS-PAGE which showed to contain about 20 proteins. Fractions OMP contained less than 5% lipopolysaccharide (LPS), as calculated from 3-deoxyoctulosonic acid measurement.
  • LPS lipopolysaccharide
  • the preparative electrophoresis of the outer membrane proteins was performed beneficially using Prep Cell 491 apparatus (BioRad) with a 37-mm ID tube and 80 ml of 10% or 12.5% resolving gel and 20 ml of 5% stacking gel and buffer containing 25 mM Tris, 0.192 M glycine, and 1% SDS, pH 8.3, for both electrophoresis and elution.
  • electrophoresis was run at the maximum setting of 260 V and 109 mA. Elution was started when the bromophenol blue indicator band reached the base of the separating gel.
  • Fractions of 1.4 ml were collected according to the protein, continuously monitored with an UV detector set at 280 nm, and checked with SDS-PAGE and immunoblotting. Fractions beneficially with the appropriate proteins were dialyzed against water, pooled, and concentrated using vacuum centrifugation. The OMP fractions were characterized in polyacrylamide gel at reducing conditions using 10% or 12,5% gels with standard methods.
  • the level of protective antibodies against enterobacteria was determined in human serum with ELISA test on plates coated with protein antigen.
  • the polystyrene 96-wells plates were coated with a solution of the 38 kDa OMP from Shigella flexneri 3a cell wall (1 ⁇ g/100 ⁇ l antigen) in carbonate buffer, pH 9.6, at 37 0 C for 3 h and then at 4 0 C overnight.
  • the plates were then blocked with 1% BSA in water. After blocking, wells were washed three times using 250 ⁇ l TBS-T per well (TBS-T is a solution of 20 mM Tris-HCl, 50 mM NaCl buffer, pH 7, containing 0.05% Tween 20).
  • the affinity chromatography column was prepared, namely with bound 38 kDa protein.
  • Agarose gel for example Sepharose 4B (Pharmacia) (10-20 ml) was activated with CNBr (Fluka) according to the standard procedure, preferentially for about 30 min at room temperature at a constant pH of 11. The excess of cyanogen bromide was washed with water and 0.1 M NaHCO 3 of pH 8.2. To the activated gel the protein 38 kDa OMP was added (30 mg) in 5 ml 0.1 M NaHCO 3 of pH 8.2 and gel was incubated for 2 hours with gentle rotation.
  • the pellet was solubilized in 10 ml PBS and dialyzed to PBS at 4 0 C with several changes of buffer.
  • the salted-out antibodies were concentrated by ultrafiltration to a volume of 3 ml Then the salted-out antibodies were fractionated of the affinity column with immobilized protein. Antibodies were applied onto the gel and column was washed with PBS without Mg 2+ , Ca 2+ . Fractions 2 ml were collected and checked for the protein content by measuring the absorbance at 280 nm. After washing out the proteins not bound with gel, the antibodies of low affinity to the antigen were eluted with 1 M NaCl in PBS without Mg 2+ , Ca 2+ .
  • Antigen or hapten for example carbohydrate (2-4 mg) and bacterial protein OMP 38 kDa as protein carrier (1-2 mg) were mixed together in water (about 0.2ml), lyophilized, and then heated at a temperature up to 130°C for several tens minutes, beneficially for 15-

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La protéine isolée faisant l'objet de la présente invention, présente dans l'extrait de protéines de membrane externe bactérienne, ainsi que ses fragments, sont appropriés à une utilisation en médicine et en pharmacie, particulièrement pour la production de vaccins et de tests diagnostiques.
PCT/PL2007/000045 2006-07-04 2007-07-04 Protéine, ses fragments et leur utilisation Ceased WO2008004902A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PL380105A PL380105A1 (pl) 2006-07-04 2006-07-04 Białko, jego fragment oraz ich zastosowania
PLP380105 2006-07-04

Publications (2)

Publication Number Publication Date
WO2008004902A2 true WO2008004902A2 (fr) 2008-01-10
WO2008004902A3 WO2008004902A3 (fr) 2008-07-10

Family

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Family Applications (1)

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PCT/PL2007/000045 Ceased WO2008004902A2 (fr) 2006-07-04 2007-07-04 Protéine, ses fragments et leur utilisation

Country Status (2)

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PL (1) PL380105A1 (fr)
WO (1) WO2008004902A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014073998A1 (fr) * 2012-11-07 2014-05-15 Wrocławskie Centrum Badań Eit+ Sp. Z O.O. Épitope et son utilisation

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT1352245E (pt) * 2001-01-18 2007-01-31 Univ Newcastle Ventures Ltd Biossensor com proteínas transmembranares ligadas covalentemente
CA2519696A1 (fr) * 2003-04-11 2004-10-28 Cedars-Sinai Medical Center Procedes pour faire une estimation du phenotype d'un patient souffrant de la maladie de crohn par la reaction serologique i2, ompc et asca

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
DATABASE Geneseq [Online] 13 January 2005 (2005-01-13), "Outer membrane protein c, OmpC, precursor, SEQ ID 10." XP002478621 retrieved from EBI accession no. GSP:ADT50378 Database accession no. ADT50378 & WO 2004/091372 A (CEDARS SINAI MEDICAL CENTER [US]; TARGAN STEPHAN R [US]; VASILIAUSKAS) 28 October 2004 (2004-10-28) *
DATABASE Geneseq [Online] 15 October 2002 (2002-10-15), "E coli outer membrane protein OmpF." XP002478623 retrieved from EBI accession no. GSP:AAO18573 Database accession no. AAO18573 & WO 02/057780 A (UNIV NEWCASTLE VENTURES LTD [GB]; LAKEY JEREMY HUGH [GB]) 25 July 2002 (2002-07-25) *
DATABASE Geneseq [Online] 20 December 1999 (1999-12-20), "E. coli wild-type ompC protein." XP002478622 retrieved from EBI accession no. GSP:AAY42549 Database accession no. AAY42549 *
DATABASE UniProt [Online] 1 June 2003 (2003-06-01), "Outer membrane protein 1a (Ia;b;F)." XP002478625 retrieved from EBI accession no. UNIPROT:Q83RY4 Database accession no. Q83RY4 -& JIN QI ET AL: "Genome sequence of Shigella flexneri 2a: Insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157." NUCLEIC ACIDS RESEARCH, vol. 30, no. 20, 15 October 2002 (2002-10-15), pages 4432-4441, XP002478617 ISSN: 0305-1048 *
DATABASE UniProt [Online] 1 March 2003 (2003-03-01), "Outer membrane protein C precursor (Porin ompC) (Outer membrane protein 1B)." XP002478619 retrieved from EBI accession no. UNIPROT:Q8CVW1 Database accession no. Q8CVW1 *
DATABASE UniProt [Online] 1 November 1996 (1996-11-01), "Outer membrane protein C precursor (Porin ompC) (Porin ompk36)." XP002478627 retrieved from EBI accession no. UNIPROT:Q48473 Database accession no. Q48473 & ALBERTI SEBASTIAN ET AL: "A porin from Klebsiella pneumoniae: Sequence homology, three-dimensional model, and complement binding" INFECTION AND IMMUNITY, vol. 63, no. 3, 1995, pages 903-910, ISSN: 0019-9567 *
DATABASE UniProt [Online] 15 December 1998 (1998-12-15), "Outer membrane protein F precursor (Porin ompF) (Outer membrane protein S3)." XP002478624 retrieved from EBI accession no. UNIPROT:Q56113 Database accession no. Q56113 *
DATABASE UniProt [Online] 15 March 2005 (2005-03-15), "Outer membrane protein C precursor (Porin ompC)." XP002478626 retrieved from EBI accession no. UNIPROT:P0A263 Database accession no. P0A263 & NEGM R S ET AL: "The porin OmpC of Salmonella typhimurium mediates adherence to macrophages." CANADIAN JOURNAL OF MICROBIOLOGY AUG 1999, vol. 45, no. 8, August 1999 (1999-08), pages 658-669, XP002478618 ISSN: 0008-4166 *
DATABASE UniProt [Online] 6 December 2005 (2005-12-06), "Outer membrane protein 1b." XP002478620 retrieved from EBI accession no. UNIPROT:Q32I16 Database accession no. Q32I16 -& YANG FAN ET AL: "Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery." NUCLEIC ACIDS RESEARCH 2005, vol. 33, no. 19, 2005, pages 6445-6458, XP002478616 ISSN: 1362-4962 *
MOLLOY MARK P ET AL: "Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis" ELECTROPHORESIS, vol. 19, no. 5, May 1998 (1998-05), pages 837-844, XP002478715 ISSN: 0173-0835 *
WITKOWSKA DANUTA ET AL: "Enterobacterial 38-kDa outer membrane protein is an age-dependent molecular marker of innate immunity and immunoglobulin deficiency as results from its reactivity with IgG and IgA antibody" FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY, vol. 48, no. 2, November 2006 (2006-11), pages 205-214, XP002478615 ISSN: 0928-8244 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014073998A1 (fr) * 2012-11-07 2014-05-15 Wrocławskie Centrum Badań Eit+ Sp. Z O.O. Épitope et son utilisation
US9890194B2 (en) 2012-11-07 2018-02-13 Wroclawskie Centrum Badan Eit+ Sp. Z O.O. Epitope and its use

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Publication number Publication date
WO2008004902A3 (fr) 2008-07-10
PL380105A1 (pl) 2008-01-07

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