KR101311886B1 - Composition for diagnosis of pneumoniae comprising secreted protein of streptococcus pneumoniae - Google Patents

Abstract

The present invention relates to a diagnostic marker and diagnostic composition for pneumonia. More specifically, by identifying a protein secreted from a mixed strain of Streptococcus pneumoniae, pneumonia diagnostic composition comprising an antibody specific for the protein and a pneumonia diagnostic composition comprising a primer or probe specific for the nucleic acid encoding the protein It is about and can use usefully for diagnosis of pneumonia.

Description

Composition for diagnosis of pneumonia comprising secreted proteins of Streptococcus pneumoniae {Composition for diagnosis of pneumoniae comprising secreted protein of streptococcus pneumoniae}

The present invention relates to a diagnostic marker for pneumonia and a composition for diagnosing pneumonia. More specifically, the present invention relates to a pneumonia diagnostic composition comprising an antibody specific for a protein secreted from a mixed strain of Streptococcus pneumoniae, and a pneumonia diagnostic composition comprising a primer or probe specific for the nucleic acid encoding the protein.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, and it is a deadly pathogen causing mortality of more than 30% depending on conditions. Streptococcus pneumoniae is also the most common bacterial source of respiratory tract infections, including acute exacerbation of otitis media, sinusitis, chronic bronchitis and bacterial pneumonia. Although powerful antibiotics have been readily available for the treatment of Streptococcus pneumoniae for about 50 years, the morbidity and mortality rates of systemic infections remain almost the same, especially in elderly patients. In addition, Streptococcus pneumoniae infections are commonly recurrent. The treatment of Streptococcus pneumoniae is currently problematic due to the limited efficacy of agents with in vitro activity, antimicrobial resistance, and increased sensitivity to infection.

Therefore, the rapid diagnosis of bacteria is an important factor in the survival rate of patients.

Streptococcus pneumoniae has been diagnosed as a method of culturing and identifying samples. Recently, rapid diagnostic methods for the easy diagnosis of surface antigens of pneumococcal streptococci have been developed using immunochromatography. In the case of the currently used pneumococcal streptococcus diagnostic kit, NOW S. pneumoniae ™ of the US Binax, Inc. uses a polysaccharide as an antigen, but a diagnostic kit derived from proteins has not been commercialized until now.

Therefore, there is a need for the development of markers and compositions that can more accurately diagnose the presence of Streptococcus pneumoniae.

The present inventors have recognized the above problems and necessities, and have made a thorough study on protein markers capable of diagnosing pneumococcal streptococci. The present invention has been completed by confirming that the present invention can be used for such purposes.

Accordingly, an object of the present invention is to provide a marker for diagnosing pneumonia.

Another object of the present invention is to provide a composition for diagnosing pneumonia comprising an antibody specific for the marker or a primer specific for a gene encoding the marker.

Still another object of the present invention is to provide a method for providing information for diagnosing pneumonia.

        The present invention provides a marker for diagnosing pneumonia and a composition for diagnosing pneumonia.

Specifically, the present invention provides cell wall-associated serine proteinase precursor PrtA (gi: 15902605), cell wall surface anchor family protein (gi: 15902119), beta-galactosidase precursor (gi: 15902609), endo-beta-N-acetylglucosaminidase ( gi: 15902484), hyaluronate lyase precursor (hyaluronidase / hyase, gi: 15902330), sialidase A precursor (neuraminidase A, gi: 15903579), ABC transporter substrate-binding protein (gi: 15902127), branched chain amino acid ABC transporter amino acid -binding protein (gi: 15902703), transcriptional regulator (gi: 15903801), ABC transporter substrate-binding protein- oligopeptide transport (gi: 15902371), general stress protein GSP-781 (gi: 15904062), ABC transporter substrate-binding protein -manganese transport (gi: 15903537), foldase protein PrsA (gi: 15902928), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903749), hypothetical protein spr0554 (gi: 15902598), hypothetical protein spr1875 (gi: 15903916) , beta-N-acetylhexosaminidase (gi: 15902 101), ABC transporter substrate-binding protein-amino acid transport (gi: 15902190), hypothetical protein spr0747 (gi: 15902791), iron-compound ABC transporter, iron compound-binding protein (gi: 15902978), hypothetical protein spr0931 (gi : 15902975), peptidyl-prolyl cis-trans isomerase, cyclophilin-type (gi: 15902723), maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein (gi: 15903959), amino acid ABC transporter amino acid-binding protein (gi (15903396), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903425), choline binding protein E (gi: 15902875), choline binding protein A (gi: 15904036), adhesion lipoprotein (gi: 15902950), 1,4 -beta-N-acetylmuramidase (gi: 15903474), sugar ABC transporter, sugar-binding protein (gi: 15903570), zinc ABC transporter zinc-binding protein (gi: 15904016), pneumococcal histidine triad protein E precursor (gi: 15902952) , iron-compound ABC transporter, iron-compound-binding protein (gi: 15903729), Fructose-bisphosphat e aldolase (gene no. spr0530), Serine protease (gene no. spr2045), Glutamyl aminopeptidase (gene no. spr1682), Zinc-containing alcohol dehydrogenase (gene no. spr1866), N-acetylglucosamine-6-phosphate deacetylase (gene no. spr1867), Glucose- 6-phosphate isomerase (gene no.spr1882), Glutamine synthetase, type I (gene no. Spr0444), Pneumococcal histidine triad protein D precursor (gene no. Spr0907), Zinc metalloprotease (gene no. Spr0581), Phosphopyruvate hydratase (gene no spr1036), Aminopeptidase N (gene no. spr0706), Molecular chaperone DnaK (gene no. spr0455), and Endopeptidase O (gene no. spr1491).

In another aspect, the present invention provides a composition for diagnosing pneumonia, comprising an antibody specifically binding to one or more proteins selected from the group consisting of the above proteins.

In another aspect, the present invention provides a composition for diagnosing pneumonia, comprising a primer or probe specifically binding to a gene encoding one or more proteins selected from the group consisting of the above proteins.

In another aspect, the present invention provides a kit for diagnosing pneumonia comprising a composition consisting of the antibody, a composition consisting of a primer or a probe.

In addition, the present invention provides a method for providing information for diagnosing pneumonia, characterized in that for reacting the kit with a sample to be tested.

The pneumonia is characterized by being infected with pneumococcal streptococci.

Pneumonia diagnostic markers and compositions according to the invention can be used for the identification and diagnosis of pneumococcal streptococci. In addition, the diagnostic composition of the present invention can diagnose pneumonia more accurately and sensitively, thereby enabling early treatment and prevention of pneumonia.

1 is a result of measuring the titer of the antibody produced using the heat treatment cells,
It was confirmed that the antibody activity was higher in pathogenic pneumococcal streptococci than in non-pathogenic pneumococcal streptococci (BBA255). When dot blotting by diluting the antibody to 1: 500, it was confirmed that the antibody reaction occurred up to 50ng.
Figure 2 is a result of measuring the titer of the antibody produced using a secreted protein derived from the mixed culture of three pathogenic clinical pneumococcal streptococci as an antigen, the antibody response up to 1ng when the antibody was diluted 1: 500 to dot blotting Showed. In other words, the antibody response of the secreted protein has a 50-fold sensitivity compared to the heat-treated cells of the pathogenic microorganisms.
FIG. 3 shows the results of two-dimensional electrophoresis using secreted proteins. As a result, 154 proteins in secreted proteins were identified.
FIG. 4 shows Western blots of secretory proteins derived from secretory proteins developed on two-dimensional gels. As a result, 54 strong proteins were detected when an antibody diluted 1: 4,000 was used.

The present invention relates to cell wall-associated serine proteinase precursor PrtA (gi: 15902605), cell wall surface anchor family protein (gi: 15902119), beta-galactosidase precursor (gi: 15902609), endo-beta-N-acetylglucosaminidase (gi: 15902484 ), hyaluronate lyase precursor (hyaluronidase / hyase, gi: 15902330), sialidase A precursor (neuraminidase A, gi: 15903579), ABC transporter substrate-binding protein (gi: 15902127), branched chain amino acid ABC transporter amino acid-binding protein (gi: 15902703), transcriptional regulator (gi: 15903801), ABC transporter substrate-binding protein- oligopeptide transport (gi: 15902371), general stress protein GSP-781 (gi: 15904062), ABC transporter substrate-binding protein-manganese transport (gi: 15903537), foldase protein PrsA (gi: 15902928), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903749), hypothetical protein spr0554 (gi: 15902598), hypothetical protein spr1875 (gi: 15903916), beta- N-acetylhexosaminidase (gi: 15902101), ABC transpo rter substrate-binding protein-amino acid transport (gi: 15902190), hypothetical protein spr0747 (gi: 15902791), iron-compound ABC transporter, iron compound-binding protein (gi: 15902978), hypothetical protein spr0931 (gi: 15902975), peptidyl-prolyl cis-trans isomerase, cyclophilin-type (gi: 15902723), maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein (gi: 15903959), amino acid ABC transporter amino acid-binding protein (gi: 15903396), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903425), choline binding protein E (gi: 15902875), choline binding protein A (gi: 15904036), adhesion lipoprotein (gi: 15902950), 1,4-beta-N -acetylmuramidase (gi: 15903474), sugar ABC transporter, sugar-binding protein (gi: 15903570), zinc ABC transporter zinc-binding protein (gi: 15904016), pneumococcal histidine triad protein E precursor (gi: 15902952), iron-compound ABC transporter, iron-compound-binding protein (gi: 15903729), Fructose-bisphosphate aldolase (gene no. spr0530), Serine protease (gene no. spr2045), Glutamyl aminopeptidase (gene no. spr1682), Zinc-containing alcohol dehydrogenase (gene no. spr1866), N-acetylglucosamine-6-phosphate deacetylase (gene no. spr1867), Glucose- 6-phosphate isomerase (gene no.spr1882), Glutamine synthetase, type I (gene no. Spr0444), Pneumococcal histidine triad protein D precursor (gene no. Spr0907), Zinc metalloprotease (gene no. Spr0581), Phosphopyruvate hydratase (gene no spr1036), Aminopeptidase N (gene no. spr0706), Molecular chaperone DnaK (gene no. spr0455), and Endopeptidase O (gene no. spr1491).

The secreted protein markers of the present invention can be isolated by mixing three strains of pneumococcal isolates from pneumonia patients and culturing the strains, or by separating the markers with strong signals by performing two-dimensional electrophoresis and Westin blot with secreted proteins. Can be.

Genbank No. of protein markers used in the present invention. And the molecular weight and the like are shown in Table 1 of Example 2 and Table 2 of Example 4.

As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether pneumonia develops.

As used herein, the term "pneumoniae" refers to inflammation of the lungs caused by infections caused by microorganisms such as bacteria, viruses, and fungi, and is preferably pneumonia infected by Streptococcus pneumoniae.

Pneumonia diagnosis using a pneumonia diagnostic marker according to the present invention can be performed by confirming the presence of a marker protein or nucleic acid of the present invention in a biological sample.

As a method for confirming the presence of the marker protein of the present invention, various assay methods known in the art may be used. Preferably, the presence of the marker protein of the present invention in a biological sample can be confirmed by measuring the formation of the antigen-antibody complex by contacting the antibody specifically binding to the marker protein of the present invention. As used herein, the term “antigen-antibody complex” refers to the combination of a particular protein in a biological sample with an antibody that specifically recognizes it.

Therefore, the present invention provides cell wall-associated serine proteinase precursor PrtA (gi: 15902605), cell wall surface anchor family protein (gi: 15902119), beta-galactosidase precursor (gi: 15902609), endo-beta-N-acetylglucosaminidase (gi: 15902484), hyaluronate lyase precursor (hyaluronidase / hyase, gi: 15902330), sialidase A precursor (neuraminidase A, gi: 15903579), ABC transporter substrate-binding protein (gi: 15902127), branched chain amino acid ABC transporter amino acid-binding protein (gi: 15902703), transcriptional regulator (gi: 15903801), ABC transporter substrate-binding protein- oligopeptide transport (gi: 15902371), general stress protein GSP-781 (gi: 15904062), ABC transporter substrate-binding protein-manganese transport (gi: 15903537), foldase protein PrsA (gi: 15902928), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903749), hypothetical protein spr0554 (gi: 15902598), hypothetical protein spr1875 (gi: 15903916), beta -N-acetylhexosaminidase (gi: 15902101), A BC transporter substrate-binding protein-amino acid transport (gi: 15902190), hypothetical protein spr0747 (gi: 15902791), iron-compound ABC transporter, iron compound-binding protein (gi: 15902978), hypothetical protein spr0931 (gi: 15902975) , peptidyl-prolyl cis-trans isomerase, cyclophilin-type (gi: 15902723), maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein (gi: 15903959), amino acid ABC transporter amino acid-binding protein (gi: 15903396) , ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903425), choline binding protein E (gi: 15902875), choline binding protein A (gi: 15904036), adhesion lipoprotein (gi: 15902950), 1,4-beta- N-acetylmuramidase (gi: 15903474), sugar ABC transporter, sugar-binding protein (gi: 15903570), zinc ABC transporter zinc-binding protein (gi: 15904016), pneumococcal histidine triad protein E precursor (gi: 15902952), iron- compound ABC transporter, iron-compound-binding protein (gi: 15903729), Fructose-bisphosphate aldo lase (gene no. spr0530), Serine protease (gene no. spr2045), Glutamyl aminopeptidase (gene no. spr1682), Zinc-containing alcohol dehydrogenase (gene no. spr1866), N-acetylglucosamine-6-phosphate deacetylase (gene no. spr1867), Glucose- 6-phosphate isomerase (gene no.spr1882), Glutamine synthetase, type I (gene no. Spr0444), Pneumococcal histidine triad protein D precursor (gene no. Spr0907), Zinc metalloprotease (gene no. Spr0581), Phosphopyruvate hydratase (gene no spr1036), Aminopeptidase N (gene no. spr0706), Molecular chaperone DnaK (gene no. spr0455), and Endopeptidase O (gene no. spr1491). It provides a pneumonia diagnostic composition.

"Antibody" in the present invention means a specific protein molecule directed against an antigenic site. Antibodies used in the present invention include monoclonal or polyclonal antibodies, immunologically active fragments (eg, Fab or (Fab) 2 fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain Fv molecules, Chimeric antibodies and the like.

Antibodies used in the present invention can be prepared by conventional methods well known in the art of immunology using the marker proteins as antigens. Proteins used as antigens of the antibodies according to the invention can be extracted or synthesized in nature and can be prepared by recombinant methods based on DNA sequences. When using a recombinant technique, a nucleic acid encoding a protein is inserted into an appropriate expression vector, the host cell is cultured so that the target protein is expressed in the transformant transformed with the recombinant expression vector, and then It can be obtained by recovering the protein. For example, polyclonal antibodies can be produced by the method of injecting a protein antigen into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such antibodies can be prepared using various warm blooded animals such as horses, cattle, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats. Monoclonal antibodies are also known as fusion methods (Kohler and Milstein, European J. Immnunol. 6: 511-519 1976), recombinant DNA methods (US Pat. No. 4816567), and phage antibody libraries (Clackson et al., Nature , 352, 624-628, 1991; Marks et al., J. Mol. Biol. 222, 58: 1-597, 1991).

The diagnostic composition of the present invention may further include reagents known in the art for use in immunological analysis in addition to the antibody specific for the protein. Immunological analysis in the above may include any method that can measure the binding of the antigen and the antibody. Such methods are known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunosorbent assay (ELISA), immunoblotting, and PAR assays. Farr assay), immunoprecipitation, latex aggregation, erythrocyte aggregation, non-turbidity, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion, immunochromatography, protein chips and immunofluorescence.

Reagents used in immunological analysis include labels, solubilizers, and detergents capable of producing a detectable signal. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. The label capable of generating a detectable signal enables qualitatively or quantitatively measuring the formation of an antigen-antibody complex, examples of which include enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and redox molecules. And radioisotopes can be used. Enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycosidase, hexokinase, malate dehydrogenase, and glucose-6 Phosphate dehydrogenase, invertase and the like can be used. As the fluorescent substance, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, fluorine isothiocyanate and the like can be used. Ligands include biotin derivatives, and luminescent materials include acridinium esters, luciferin, and luciferase. The microparticles include colloidal gold and colored latex, and the redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone and hydroquinone. Radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. However, any of those that can be used in immunological assays other than those exemplified above may be used.

On the other hand, the diagnostic composition of the present invention can be provided in a fixed state using a variety of methods known on a suitable carrier or support to increase the speed and convenience of diagnosis (Antibodies: A Labotory Manual, Harlow & Lane Cold Spring Harbor, 1988). Examples of suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamides, polyesters, companion rocks, filter papers, ion exchange resins, plastic films, plastic tubes , Glass, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylons, cups, flat packs and the like. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible form, for example spherical (bead), cylindrical (test tube or inner surface of the well), planar (sheet, test strip).

Preferably, the pneumonia diagnostic composition of the present invention may be provided in the form of a diagnostic kit, microarray, protein chip. The diagnostic kit may be provided, for example, in the form of a lateral flow assay kit based on immunochromatography to detect a specific protein in a sample. The lateral flow assay kit includes a sample pad applied to the sample, a release pad coated with a detection antibody, a developing membrane (e.g., nitro) in which the sample is moved and separated and an antigen-antibody reaction occurs. Cellulose) or strip, and an absorption pad.

In addition, the present invention provides cell wall-associated serine proteinase precursor PrtA (gi: 15902605), cell wall surface anchor family protein (gi: 15902119), beta-galactosidase precursor (gi: 15902609), endo-beta-N-acetylglucosaminidase (gi: 15902484), hyaluronate lyase precursor (hyaluronidase / hyase, gi: 15902330), sialidase A precursor (neuraminidase A, gi: 15903579), ABC transporter substrate-binding protein (gi: 15902127), branched chain amino acid ABC transporter amino acid-binding protein (gi: 15902703), transcriptional regulator (gi: 15903801), ABC transporter substrate-binding protein- oligopeptide transport (gi: 15902371), general stress protein GSP-781 (gi: 15904062), ABC transporter substrate-binding protein-manganese transport (gi: 15903537), foldase protein PrsA (gi: 15902928), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903749), hypothetical protein spr0554 (gi: 15902598), hypothetical protein spr1875 (gi: 15903916), beta -N-acetylhexosaminidase (gi: 15902101), ABC transporter substrate-binding protein-amino acid transport (gi: 15902190), hypothetical protein spr0747 (gi: 15902791), iron-compound ABC transporter, iron compound-binding protein (gi: 15902978), hypothetical protein spr0931 (gi: 15902975), peptidyl-prolyl cis-trans isomerase, cyclophilin-type (gi: 15902723), maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein (gi: 15903959), amino acid ABC transporter amino acid-binding protein (gi: 15903396), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903425), choline binding protein E (gi: 15902875), choline binding protein A (gi: 15904036), adhesion lipoprotein (gi: 15902950), 1,4-beta-N -acetylmuramidase (gi: 15903474), sugar ABC transporter, sugar-binding protein (gi: 15903570), zinc ABC transporter zinc-binding protein (gi: 15904016), pneumococcal histidine triad protein E precursor (gi: 15902952), iron-compound ABC transporter, iron-compound-binding protein (gi: 15903729), Fructose-bisphosphate aldolas e (gene no. spr0530), Serine protease (gene no. spr2045), Glutamyl aminopeptidase (gene no. spr1682), Zinc-containing alcohol dehydrogenase (gene no. spr1866), N-acetylglucosamine-6-phosphate deacetylase (gene no. spr1867), Glucose- 6-phosphate isomerase (gene no.spr1882), Glutamine synthetase, type I (gene no. Spr0444), Pneumococcal histidine triad protein D precursor (gene no. Spr0907), Zinc metalloprotease (gene no. Spr0581), Phosphopyruvate hydratase (gene no spr1036), Aminopeptidase N (gene no. spr0706), Molecular chaperone DnaK (gene no. spr0455), and Endopeptidase O (gene no. spr1491), specifically binding to a gene encoding one or more proteins It provides a pneumonia diagnostic composition comprising a primer or a probe.

The detection of a specific nucleic acid using a primer may be performed by amplifying the sequence of the gene of interest using an amplification method such as PCR, and then checking whether the gene is amplified by a method known in the art. In addition, the detection of a particular nucleic acid using a probe may be performed by contacting a sample nucleic acid with a probe under suitable conditions and confirming the presence of the hybridizing nucleic acid.

The "primer" refers to a nucleic acid sequence having a short free hydroxyl group, which can form a base pair with a complementary template, and serves as a starting point for template strand copying. Primers of the invention can be chemically synthesized using methods known in the art, such as, for example, phosphoramidite solid support methods.

The "probe" refers to a nucleic acid fragment such as RNA or DNA consisting of several to several hundred bases that can specifically bind to mRNA and is labeled to confirm the presence or absence of a specific mRNA. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, and the like, and can be labeled with biotin, FITC, rhodamine, DIG, or the like, or radioisotopes.

The probe may also be labeled with a detectable substance, for example, a radiolabel that provides a suitable signal and has sufficient half-life. Labeled probes can be hybridized to nucleic acids on a solid support as known from Sam et al., Molecular Cloning, A Laboratory Mannual, 1989.

Primers or probes of the invention may be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Further, it can be modified by methylation, capping or the like by a known method.

The pneumonia diagnostic composition of the present invention may further include a reagent generally used in the method for detecting the above-described nucleic acid. For example, metal ion salts such as deoxynulceotide triphosphate (dNTP), a heat resistant polymerase, and magnesium chloride required for a PCR reaction may be included, and may include dNTP, a sequencease, etc. required for sequencing. Can be.

The present invention also provides a pneumonia diagnostic kit comprising the pneumonia diagnostic composition.

In another aspect, the present invention relates to a method for providing information for diagnosing pneumonia, characterized in that for reacting the pneumonia diagnostic kit with a sample to be tested. The method is preferably for diagnosing pneumonia caused by Streptococcus pneumoniae.

Hereinafter, the process of separating secretion proteins and discovering protein-derived biomarkers with high antibody reactivity will be described in detail through Examples. The following examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples according to the gist of the present invention to those skilled in the art. Will be self-evident.

Example  1: Streptococcus pneumoniae mixed strain culture and ammonium  through sulfate precipitation Secretory protein  Separation tablet

In order to isolate the common secreted proteins of pathogenic pneumococcal streptococci, strains were cultured and secreted proteins were isolated by mixing various species (three species). The strain was added 0.5% yeast extract (THY) to 500ml Thdd-Hewitt broth (BD, USA) and then cultured up to OD 0.9 at 37 ℃, 5% CO2 conditions. The grown bacteria were centrifuged at 8,000 rpm for 20 minutes at 4 ° C to separate media components and cells. The ammonium sulfate was mixed up to 80% while the medium component was mixed with a stirrer. This was centrifuged at 14,000 rpm for 20 minutes at 4 ℃ to remove the supernatant and collected the pellet (pellet). Ammonium sulfate in the precipitate was removed by dialysis using 20 mM Tris-HCl (pH 8.0). After dialysis, proteins in the dialysis membrane were concentrated by centrifugation at 40 ° C. at 4 ° C. and 3,500 rpm using a 5,000 Da Viva spin, followed by analysis.

Example  2: Secreted protein  Identification through electrophoresis and mass spectrometry

After secretion protein was developed using 12% SDS-PAGE, in-gel digestion was performed with trypsin. Each peptide was analyzed using LCQ DECA XP plus (Thermo Finnigan. USA) and FT-ICR (Thermo Finnigan. USA). The protein was identified using MASCOT software (ver.2.2), and the genetic databases required were S. pneumoniae R6 and D-39 from NCBI. Protein quantitation was calculated by the emPAI calculation method, and the characteristics of the identified proteins were analyzed using a program such as Signal P, TMHMM, SecretomeP. 33 cell wall proteins and extracellular proteins were identified in the secreted protein using three pathogenic pneumococcal streptococci as mixed strains, which are shown in Table 1. In addition, about 30 membrane proteins and about 200 cytoplasmic proteins were identified.

Cell wall proteins and extracellular proteins identified in Streptococcus pneumoniae strains SignalP Accession No. Protein name
Excavating protein Cell locat by LocateP
Intracellular location NN TMM MW
Molecular Weight pI
Isoelectric point emPAI gi | 15902605 cell wall-associated serine proteinase precursor PrtA Cell wall Y Y 240290 5.84 0.134 gi | 15902119 cell wall surface anchor family protein Cell wall Y Y 123326 5.36 0.376 gi | 15902609 beta-galactosidase precursor Cell wall N Y 246632 5.81 0.025 gi | 15902484 endo-beta-N-acetylglucosaminidase, putative Cell wall Y Y 182996 5.24 0.032 gi | 15902330 hyaluronate lyase precursor (hyaluronidase / hyase) Cell wall Y Y 122101 5.77 0.038 gi | 15903579 sialidase A precursor (neuraminidase A) Cell wall N Y 114671 5.98 0.050 gi | 15902127 ABC transporter substrate-binding protein Extracellular Y Y 57190 5.94 0.092 gi | 15902703 branched chain amino acid ABC transporter amino acid-binding protein Extracellular Y Y 40389 5.29 0.628 gi | 15903801 transcriptional regulator Extracellular Y Y 37543 5.11 0.147 gi | 15902371 ABC transporter substrate-binding protein-oligopeptide transport Extracellular Y Y 72958 5 0.377 gi | 15904062 general stress protein GSP-781 Extracellular Y Y 41672 5.85 0.449 gi | 15903537 ABC transporter substrate-binding protein-manganese transport. Extracellular Y Y 34573 5.3 1.360 gi | 15902928 foldase protein PrsA Extracellular Y Y 34419 5.04 2.384 gi | 15903749 ABC transporter substrate-binding protein-oligopeptide transport Extracellular Y Y 72460 4.95 0.961 gi | 15902598 hypothetical protein spr0554 Extracellular Y Y 26522 5.13 0.581 gi | 15903916 hypothetical protein spr1875 Extracellular Y Y 40541 4.29 0.410 gi | 15902101 beta-N-acetylhexosaminidase Extracellular Y Y 144695 5.09 0.116 gi | 15902190 ABC transporter substrate-binding protein-amino acid transport Extracellular Y Y 30609 5.14 0.715 gi | 15902791 hypothetical protein spr0747 Extracellular N Y 39481 5.46 2.412 gi | 15902978 iron-compound ABC transporter, iron compound-binding protein Extracellular Y Y 37483 5.38 0.147 gi | 15902975 hypothetical protein spr0931 Extracellular Y Y 35377 5.46 0.705 gi | 15902723 peptidyl-prolyl cis-trans isomerase, cyclophilin-type Extracellular Y Y 28991 5.47 0.335 gi | 15903959 maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein Extracellular Y Y 45339 5.1 0.564 gi | 15903396 amino acid ABC transporter amino acid-binding protein Extracellular Y Y 31000 4.83 0.185 gi | 15903425 ABC transporter substrate-binding protein-oligopeptide transport Extracellular Y Y 72517 5.24 0.153 gi | 15902875 choline binding protein E Extracellular Y Y 72073 5.29 0.084 gi | 15904036 choline binding protein A Extracellular Y Y 79049 7.53 0.096 gi | 15902950 adhesion lipoprotein Extracellular Y Y 34678 5.27 0.168 gi | 15903474 1,4-beta-N-acetylmuramidase Extracellular Y Y 58644 6.07 0.136 gi | 15903570 sugar ABC transporter, sugar-binding protein Extracellular Y Y 48273 5.31 0.123 gi | 15904016 zinc ABC transporter zinc-binding protein Extracellular Y Y 56237 5.05 0.214 gi | 15902952 pneumococcal histidine triad protein E precursor Extracellular Y Y 114555 5.55 0.047 gi | 15903729 iron-compound ABC transporter, iron-compound-binding protein Extracellular Y Y 34841 5.55 0.142

Example  3: Secretion protein  Antibody Production and Antibody Titer Measurement

3-1. Antibody Production and Serum Isolation

Antibodies were prepared using proteins and heat-treated cells as antigens. The amount of antigen (secretory protein, heat treated cells) was used more than 1mg. Primary injections were mixed 1: 1 with Freund's complete adjuvant (SIGMA, USA) and secondary injections were 1: 1 mixed with incomplete adjuvant. The second injection was performed 14 days after the first injection, and after 14 days, blood was collected from rabbit ears, and then titer of the antibody was measured using dot-blotting. The collected blood was hardened by reaction at 37 ° C. for 1 hour, and then overnight at 4 ° C. to ensure clotting. Centrifugation was performed at 4,000 rpm to remove blood clots and serum was obtained.

3-2. Antibody Isolation and Purification

In order to separate IgG from serum, the mixture was mixed with 20 mM Tris-HCl (pH 7.0) at a ratio of 1: 1, and centrifuged at 12,000 rpm for 10 minutes at 4 ° C. The supernatant was passed through a HiTrap PROTEIN G column (GE, USA), and then IgG was attached to the column using 0.1M glycine-HCl (pH 2.7) buffer. The IgG fraction thus separated was purified by buffer exchange using Viva spin (10 kDa).

3-3. Antibody Potency  Measure

Antibodies were diluted 1: 500 and 1: 2,000, respectively, and analyzed by dot blotting. The secreted protein-derived antibody was confirmed to have a 50-fold sensitivity compared to the heat-treated cell-derived antibody (Fig. 1, Fig. 2). This confirmed that the secreted protein contained a protein with sensitive antibody reactivity.

Example  4: Secretion protein  Two-dimensional Electrophoresis and Western Utilization blotting Detection of Antigen Proteins with Strong Sensitivity

4-1. Secretion protein  Two-dimensional Electrophoresis Using

Two-dimensional electrophoresis was performed to determine the isolation and antibody reactivity of each unit protein from secreted proteins (Yun et al., J Microbiol. 2006). 1-DE was performed using IPGphor ™ (Amersham Biosciences Co.) using 100 μg of secreted protein, and 2-DE was performed using DALT 2-D electrophoresis system (Hoefer Co.). As a result, 154 proteins were identified in secreted proteins (Figure 3).

4-2. 2D gel of western blotting

The secreted protein developed on the two-dimensional gel was transferred to PVDF membrane and subjected to western blotting using secreted protein-derived antibodies. As a result of Western blotting, proteins with strong signals were selected and identified. Identification of the excavated protein was performed using MALDI TOF / TOF after in-gel digestion. As a result of Western blotting, when the antibody diluted to 1: 4,000 was used, 54 proteins with strong signals were identified and selected for identification. The results are shown in Table 2 below.

Spot No Gene No. Description of identified protein
(Extracted protein) MW (Da)
(Molecular Weight) pI
(Electric point) SignalP Location
(Intracellular location) One spr0530 Fructose-bisphosphate aldolase 31382 5 N Cytoplasmic 2 spr2045 Serine protease 42262 6.15 Y Membrane 3 spr1682 Glutamyl aminopeptidase 37995 5.46 N Cytoplasmic 4 spr1866 Zinc-containing alcohol dehydrogenase 38067 5.24 N Cytoplasmic 5 spr1867 N-acetylglucosamine-6-phosphate deacetylase 41671 5.11 N Cytoplasmic 6 spr2021 General stress protein GSP-781 41672 5.85 Y Extracellular 7 spr1882 Glucose-6-phosphate isomerase 49847 5 N Cytoplasmic 8 spr0444 Glutamine synthetase, type I 50290 4.93 N Cytoplasmic 9 spr1707 ABC transporter substrate-binding protein-oligopeptide transport 72460 4.95 Y Extracellular 10 spr0907 Pneumococcal histidine triad protein D precursor 95168 5.22 Y Membrane 11 spr0581 Zinc metalloprotease 210928 5.3 Y Membrane 12 spr0530 Fructose-bisphosphate aldolase 31382 5 N Cytoplasmic 15 spr2045 Serine protease 42262 6.15 Y Membrane 16 spr1036 Phosphopyruvate hydratase 47074 4.7 N Cytoplasmic 17 spr1882 Glucose-6-phosphate isomerase 49847 5 N Cytoplasmic 18 spr0444 Glutamine synthetase, type I 50290 4.93 N Cytoplasmic 19 spr0444 Glutamine synthetase, type I 50290 4.93 N Cytoplasmic 20 spr0706 Aminopeptidase N 95291 4.79 N Cytoplasmic 21 spr0455 Molecular chaperone DnaK 64772 4.63 N Cytoplasmic 22 spr1491 Endopeptidase O 71870 4.72 N Cytoplasmic 23 spr0581 Zinc metalloprotease 210928 5.3 Y Membrane

Claims (6)

delete cell wall-associated serine proteinase precursor PrtA (gi: 15902605), cell wall surface anchor family protein (gi: 15902119), beta-galactosidase precursor (gi: 15902609), endo-beta-N-acetylglucosaminidase (gi: 15902484), hyaluronate lyase precursor (hyaluronidase / hyase, gi: 15902330), sialidase A precursor (neuraminidase A, gi: 15903579), ABC transporter substrate-binding protein (gi: 15902127), branched chain amino acid ABC transporter amino acid-binding protein (gi: 15902703), transcriptional regulator (gi: 15903801), ABC transporter substrate-binding protein- oligopeptide transport (gi: 15902371), general stress protein GSP-781 (gi: 15904062), ABC transporter substrate-binding protein-manganese transport (gi: 15903537), foldase protein PrsA (gi: 15902928), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903749), hypothetical protein spr0554 (gi: 15902598), hypothetical protein spr1875 (gi: 15903916), beta-N-acetylhexosaminidase (gi: 15902101), ABC transporter substrate -binding protein-amino acid transport (gi: 15902190), hypothetical protein spr0747 (gi: 15902791), iron-compound ABC transporter, iron compound-binding protein (gi: 15902978), hypothetical protein spr0931 (gi: 15902975), peptidyl- prolyl cis-trans isomerase, cyclophilin-type (gi: 15902723), maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein (gi: 15903959), amino acid ABC transporter amino acid-binding protein (gi: 15903396), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903425), choline binding protein E (gi: 15902875), choline binding protein A (gi: 15904036), adhesion lipoprotein (gi: 15902950), 1,4-beta-N-acetylmuramidase (gi: 15903474), sugar ABC transporter, sugar-binding protein (gi: 15903570), zinc ABC transporter zinc-binding protein (gi: 15904016), pneumococcal histidine triad protein E precursor (gi: 15902952), iron-compound ABC transporter , iron-compound-binding protein (gi: 15903729), Fructose-bisphosphate aldolase (gene no. spr0530), Serine protease (gene no. spr2045), Glutamyl aminopeptidase (gene no. spr1682), Zinc-containing alcohol dehydrogenase (gene no. spr1866), N-acetylglucosamine-6-phosphate deacetylase (gene no. spr1867), Glucose- 6-phosphate isomerase (gene no.spr1882), Glutamine synthetase, type I (gene no. Spr0444), Pneumococcal histidine triad protein D precursor (gene no. Spr0907), Zinc metalloprotease (gene no. Spr0581), Phosphopyruvate hydratase (gene no spr1036), Aminopeptidase N (gene no. spr0706), Molecular chaperone DnaK (gene no. spr0455), and a composition for diagnosing pneumonia comprising an antibody specifically binding to the Endopeptidase O (gene no. spr1491) protein. cell wall-associated serine proteinase precursor PrtA (gi: 15902605), cell wall surface anchor family protein (gi: 15902119), beta-galactosidase precursor (gi: 15902609), endo-beta-N-acetylglucosaminidase (gi: 15902484), hyaluronate lyase precursor (hyaluronidase / hyase, gi: 15902330), sialidase A precursor (neuraminidase A, gi: 15903579), ABC transporter substrate-binding protein (gi: 15902127), branched chain amino acid ABC transporter amino acid-binding protein (gi: 15902703), transcriptional regulator (gi: 15903801), ABC transporter substrate-binding protein- oligopeptide transport (gi: 15902371), general stress protein GSP-781 (gi: 15904062), ABC transporter substrate-binding protein-manganese transport (gi: 15903537), foldase protein PrsA (gi: 15902928), ABC transporter substrate-binding protein-oligopeptide transport (gi: 15903749), hypothetical protein spr0554 (gi: 15902598), hypothetical protein spr1875 (gi: 15903916), beta-N-acetylhexosaminidase (gi: 15902101), ABC transporter substrate-bi nding protein-amino acid transport (gi: 15902190), hypothetical protein spr0747 (gi: 15902791), iron-compound ABC transporter, iron compound-binding protein (gi: 15902978), hypothetical protein spr0931 (gi: 15902975), peptidyl-prolyl cis-trans isomerase, cyclophilin-type (gi: 15902723), maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein (gi: 15903959), amino acid ABC transporter amino acid-binding protein (gi: 15903396), ABC transporter substrate -binding protein-oligopeptide transport (gi: 15903425), choline binding protein E (gi: 15902875), choline binding protein A (gi: 15904036), adhesion lipoprotein (gi: 15902950), 1,4-beta-N-acetylmuramidase ( gi: 15903474), sugar ABC transporter, sugar-binding protein (gi: 15903570), zinc ABC transporter zinc-binding protein (gi: 15904016), pneumococcal histidine triad protein E precursor (gi: 15902952), iron-compound ABC transporter, iron-compound-binding protein (gi: 15903729), Fructose-bisphosphate aldolase (gene no. spr0530), Serine protease (gene no. spr2045), Glutamyl aminopeptidase (gene no. spr1682), Zinc-containing alcohol dehydrogenase (gene no. spr1866), N-acetylglucosamine-6-phosphate deacetylase (gene no. spr1867), Glucose- 6-phosphate isomerase (gene no.spr1882), Glutamine synthetase, type I (gene no. Spr0444), Pneumococcal histidine triad protein D precursor (gene no. Spr0907), Zinc metalloprotease (gene no. Spr0581), Phosphopyruvate hydratase (gene no spr1036), pneumonia comprising a primer or probe that specifically binds to a gene encoding Aminopeptidase N (gene no. spr0706), Molecular chaperone DnaK (gene no. spr0455), and Endopeptidase O (gene no. spr1491) protein Diagnostic composition.
A diagnostic kit for pneumonia, comprising the composition of claim 2 or 3.
A method of providing information for diagnosing pneumonia, which comprises reacting the kit of claim 4 with a sample to be tested.
The method of claim 5, wherein the pneumonia is infected with pneumococcal streptococcus.

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