KR20150067058A - Membrane vesicles derived from Streptococcus pneumoniae, and method for the diagnosis of Streptococcus pneumoniae-mediated diseases using the same - Google Patents

Abstract

The present invention relates to a membrane vesicle derived from Streptococcus pneumoniae and a method for diagnosing Streptococcus pneumoniae-mediated disease using the same. More specifically, the membrane vesicle is separated from Streptococcus pneumoniae, which are gram-positive bacteria; and then constituent proteins included in the membrane vesicle are identified. Therefore, the vesicle membrane and the proteins can be advantageously used as biomarkers, which can be used to diagnose Streptococcus pneumoniae-mediated disease or to identify exposure to Streptococcus pneumoniae.

Description

[0001] The present invention relates to a pneumococcal strain-derived membrane envelope and a method for diagnosing pneumococcal streptococcal-mediated disease using the same. More particularly, the present invention relates to a pneumococcal pneumococcal pneumoniae-

The present invention relates to a pneumococcal strain-derived membrane envelope and a method for diagnosing pneumococcal streptococcal disease using the same. More particularly, the present invention relates to a method for diagnosing pneumococcal streptococcal disease in which specific proteins of pneumococcal streptococci are secreted out through a membrane- And a method for confirming the diagnosis of pneumococcal streptococcal disease or exposure to pneumococcal streptococci using the protein contained therein.

Outer Membrane Vesicles (OMV) is one of several bacterial secretion mechanisms that swell up the outer membrane and then form the endoplasmic reticulum from the bacteria. Therefore, it has been known that OMV is secreted by Gram-negative bacteria with outer membrane. OMV is a spherical phospholipid bilayer with a size of 20 to 200 nm and various outer membrane proteins. It is known that OMV plays an important role in promoting bacterial growth, exchanging information with hosts, transmitting pathogenic factors, and delivering genetic material, and this role plays an important role in the pathogenesis of host cells. In addition, most OMVs have recently been shown to induce host immune responses and are likely to be used in vaccine development.

These OMVs have been known to be produced only in Gram-negative bacteria. Gram-positive bacteria have no extracellular membrane, and the cell membrane is surrounded by cell walls and thus it has not been known to secrete membrane endoplasmic reticulum. However, recently, Gram- Membrane Vesicles have been found to be able to secrete membrane vesicles out of the cells even in Gram-positive bacteria.

Furthermore, it has been reported that membrane endoplasmic reticulum is found in secretions of human or experimental animals, feces, or tissue washes. In addition, tissue-derived membrane cell bodies are known to reflect the state of tissues that secrete the endoplasmic reticulum and have been reported to be useful for the diagnosis of diseases. However, no studies have been reported to date on the development of diagnostic techniques for diseases caused by pneumococcal streptococcal infection using pneumococcal streptococcal membrane envelope, or membrane envelope derived from host cells by pneumococcal streptococci.

Streptococcus , on the other hand, is a heterogeneous gram-positive bacterium present in human oral and respiratory tracts. Among them, Streptococcus pneumoniae ( Streptococcus pneumoniae ), which is a major pathogen, is a resident microorganism in the mouth of a person, and 40 to 60% of healthy people are carriers. The majority of carriers have weak bacterial pathogens. It is sporadic and intermittent rather than permanent. Such pneumococcal streptococci are known to spread through the respiratory tract or by the vasculature system and migrate to other parts of the body causing pneumonia, mumps, conjunctivitis, peritonitis, otitis media, endocarditis and sepsis (Suzuki et al., J Clin Microbiol , 43: 4528-4534, 2005). According to the data from the National Statistical Office, the death rate from pneumonia is 14.9 out of 100,000 people, which is the sixth leading cause of death and the first in death due to infection. Pneumococcal streptococcus is considered to be the main cause of illness and death.

Currently, pneumococcal streptococcus is the most commonly used method to measure biochemical reactions or metabolites after culturing bacteria. Once the bacterium is identified as a bacterium, the sensitivity to optochin / bile acid and the reactivity with the antibody to the capsular polysaccharide are measured to confirm that the bacterium is pneumococcal. However, there is a high possibility that the experiment is inappropriately performed or that the bacteria are mistaken for resistance to optokin or anabolic acid due to errors in the interpretation of the results. In addition, serotypes determined according to the capsular polysaccharide of pneumococcal streptococci use polyvalent antiserum or monoclonal antibodies against polysaccharides, but the antiserum induced by such capsular polysaccharide antigens is different from other bacteria other than pneumococcal streptococcus, there is a problem that the reliability of the identification result is low in response to? -streptococcal bacteria.

Therefore, in order to overcome these conventional problems and to provide a rapid and effective method for diagnosing pneumococcal streptococcal disease, a method for identification of a marker which is highly antigenic and secreted outside the cell and stably expressed, and a rapid identification method using the same This is a situation that is desperately required.

Korean Patent Laid-Open No. 10-2011-0025603

It is an object of the present invention to provide a pneumococcal strain-derived membrane envelope and a method for diagnosing pneumococcal streptococcal disease using the same.

In order to achieve the above object, one aspect of the present invention relates to a method for producing Streptococcus pneumoniae- derived membrane vesicles.

According to another aspect of the present invention, there is provided a method of separating a membrane envelope from a biological sample, an environmental sample, and a food sample,

2) confirming the presence of pneumococcal streptococcal-derived proteins in the membrane envelope; And

3) when the presence of any one or more of the proteins of step 2) is detected, it is determined that the disease is caused by pneumococcal streptococci or exposed to pneumococcal streptococci, or Provided is a protein detection method for providing information on the diagnosis of pneumococcal streptococcus exposure.

The pneumococcal streptococcal-derived membrane proteins of the present invention are secreted out of the cells in the form of follicular cells and contained in the vesicular form of membrane vesicles. The pneumonic streptococcal-derived proteins secreted by the vesicle-type membrane vesicles are spatially separated from the external factors such as proteolytic enzymes And can be physically protected by separation. That is, since the proteins derived from Streptococcus pneumoniae can be safely secreted in an intact state by the membrane endoplasmic reticulum, the membrane endoplasmic reticulum and the proteins contained therein can be used for the diseases caused by infection with pneumococcal streptococci, Can be usefully used as a labeling substance capable of promptly and accurately diagnosing the exposure of pneumococcal streptococci to the < RTI ID = 0.0 >

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

FIG. 1 is a flow chart of the pellicle streptococcal-derived membrane envelope separation method.
Fig. 2 is an electron micrograph of a pneumococcal streptococcal membrane vesicle confirmed by Cryo-TEM, and arrows indicate membrane vesicles.
Figure 3 is a plot of eight fractions identified as the result of electrophoresis of pneumococcal streptococcal membrane envelope.
FIG. 4 is a view of six fractions identified by reacting mouse antibody against pneumococcal streptococcal-derived membrane envelope with proteins of the membrane envelope and electrophoresis.
FIG. 5 is a photograph showing pneumococcal streptococcal follicular antibody reacted with sera from pneumonia patients and healthy persons.

First, terms used in the specification of the present invention will be described.

Gram-positive bacteria referred to in the present invention generally refers to bacteria that are stained with Gram stain to purple, have no extracellular membrane as compared with Gram-negative bacteria, and have a large amount of peptidoglycan on the cell wall. Streptococcus , a cocci, It belongs to the representative gram-positive bacteria.

The pneumococcal strain-derived membrane envelope referred to in the present invention refers to an endoplasmic reticulum which is naturally or artificially secreted by pneumococcal streptococci living in the body or the surrounding environment, and can be intermixed with extracellular vesicles.

Diagnosis referred to in the present invention means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to ascertain whether pneumococcal streptococcal mediated disease or pneumococcal streptococcus is exposed.

Hereinafter, the present invention will be described in detail.

One. Streptococcus pneumoniae strain Streptococcus pneumoniae ) Derived membrane envelope ( membrane vesicles )

One aspect of the present invention provides a pneumococcal strain-derived membrane envelope.

The membrane endoplasmic reticulum of the present invention is preferably extracellular vesicles secreted out of pneumococcal streptococcal cells, and is secreted into culture medium of pneumococcal streptococci, naturally secreted or artificially secreted.

The membrane vesicle is preferably separated by the following method, but is not limited thereto. Specifically, the separation method

1) centrifuging the pneumococcal streptococcus culture to obtain a supernatant;

2) primary filtration of the supernatant solution to remove impurities and cells;

3) Secondly filtering the obtained filtrate with a membrane having a molecular weight cutoff of 300 kDa to 1000 kDa, preferably a molecular weight cutoff of 500 kDa, to obtain a fraction having a molecular weight above the cutoff value; And

4) ultracentrifuging the obtained fractions to obtain a precipitate.

After the secondary filtration in the step 3), the step of concentrating the filtrate may be further included.

In addition, it is more preferable to further purify the precipitated membrane endoplasmic reticulum by sucrose concentration-gradient ultracentrifugation after the step 4), but the present invention is not limited thereto. And suspending the obtained precipitate.

In a specific example of the present invention, in order to isolate membrane endoplasmic reticulum from pneumococcal streptococci as Gram-positive bacteria, the pneumococcal streptococcal culture broth is centrifuged at 8,000 rpm to obtain only the medium component, which is then filtered through a 0.2 탆 filter membrane, The cells and suspension were removed and secondary filtration was performed to obtain only 500 kDa or greater. Then, the resulting solution was subjected to ultra-high-speed centrifugation to recover the deposited membrane filaments, followed by secondary ultrafast centrifugation using a sucrose concentration gradient to remove the mixed proteins, thereby confirming that the membrane filament was more purely isolated 1 and 2).

The membrane envelope of the present invention thus isolated is a pneumococcal streptococcus-derived protein, which contains Hypothetical protein spr0747 (NCBI gene no. Spr0747), ABC transporter substrate-binding protein-manganese transport (NCBI gene no. Spr1494) ABC transporter substrate-binding protein (NCBI gene no. Spr0884), branched chain amino acid ABC transporter amino acid-binding protein (NCBI gene no. Spr0659), ABC transporter substrate- binding protein (NCBI gene no. spr0934), amino acid ABC transporter amino acid-binding protein (NCBI gene no. spr1251), hypothetical protein spr0931 (NCBI gene no. Spr0931), adenylosuccinate synthetase (NCBI gene no. Spr0021), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. Spr1707), hypothetical protein spr0554 (NCBI gene no. Spr0554), peptidyl-prolyl cis -trans isome rase, cyclophilin-type (NCBI gene no. spr0679), surface protein pspA precursor (NCBI gene no. spr0121), lipoprotein (NCBI gene no.spr0147), amino acid ABC transporter amino acid-binding protein (NCBI gene no. spr1353), hypothetical protein spr0688 (NCBI gene no. spr0688), phosphopyruvate hydratase (NCBI gene no. (NCBI gene no. spr1840), zinc ABC transporter zinc binding protein (NCBI gene no. spr1975), hypothetical protein spr1782 (NCBI gene no. spr1782), signal peptidase I (NCBI gene no. spr0364), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1382), hypothetical protein spr1912 (NCBI gene no. spr1912), 30S ribosomal protein S8 (NCBI gene no. spr0203), phosphate ABC transporter phosphate-binding protein ABC transporter substrate-binding protein (NCBI gene no. spr0584), sugar ABC transporter, sugar-binding protein (NCBI gene no. spr1527), hypothetical protein spr0975 (NCBI gene no. , spR2003), 50S ribosomal protein L18 (NCBI gene no. spr0205), L-lactate dehydrogenase (NCBI gene no. (NCBI gene no. spr1270), 30S ribosomal protein S5 (NCBI gene no. spr0206), hypothetical protein spr1370 (NCBI gene no. spr1370), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1962), 3-ketoacyl- (acyl-carrier-protein) reductase (NCBI gene no. spr0381), glycerol uptake (spr1194), hypothetical protein spr1962 promoter protein, putative (NCBI gene no.spr1344), hypothetical protein spr0693 (NCBI gene no.spr0693), hypothetical protein spr1457 (NCBI gene no.spr1457), preprotein translocase subunit SecG (NCBI gene no.spr0877), glyceraldehyde- phosphate dehydrogenase (NCBI gene no. spr1825), sugar ABC transporter, ATP-binding protein (NCBI gene no. spr1437), cytoplasmic membrane protein (NCBI gene no. spr1163), phosphoglycerate kinase (NCBI gene no. spr0441), ABC transporter substrate -binding protein (NCBI gene no. spr1645), phosphoglyceromutase (NCBI gene no. spr 1499), ribosome-binding factor A (NCBI gene no. spR0482, hypoxanthine-guanine phosphoribosyltransferase (NCBI gene no. spr0011), 30S ribosomal protein S7 (NCBI gene no. spr0249), amino acid ABC transporter permease / amino acid-binding protein (NCBI gene no. spr1120), 50S ribosomal protein L22 NCBI gene no. Spr0194), 50S ribosomal protein L5 (NCBI gene no. Spr0201), hypothetical protein spr0174 (NCBI gene no. Spr0174), pyruvate oxidase (NCBI gene no. Spr0642), cell division protein DivIVA (NCBI gene no. ), iron-compound ABC transporter, iron-compound-binding protein (NCBI gene no. spr1687), hypothetical protein spr1112 (NCBI gene no. spr1112), F0F1 ATP synthase subunit delta (NCBI gene no. D-alanine carboxypeptidase (NCBI gene no. Spr0776), large conductance mechanosensitive channel protein MscL (NCBI gene no. Spr0915), PTS system, mannose-specific IIC component (NCBI gene no. Spr0260), hypothetical protein spr0595 (NCBI gene no. spr0595), hypothetical protein spr0615 (NCBI gene no. spr0615), rod shape-determining protein M reC (NCBI gene no. spr2023), adhesion lipoprotein (NCBI gene No. spr0906), transcriptional repressor CodY (NCBI gene no. spr1439), hypothetical protein spr1056 (NCBI gene no. spr1056), 50S ribosomal protein L16 (NCBI gene no. spr0196), F0F1 ATP synthase (NCBI gene no. spr1360), serine protease (NCBI gene no. spr2045), phosphotransferase system, mannose-specific EIID (NCBI gene no. spr0259), 50S ribosomal protein L13 (NCBI gene no. spr0271), ABC transporter ATP -binding protein-unknown substrate (NCBI gene no. spr1559), 50S ribosomal protein L10 (NCBI gene no. spr1212), phosphate ABC transporter ATP-binding protein (NCBI gene no. spr1253), choline transporter (NCBI gene no. ), ABC transporter substrate-binding protein (NCBI gene No. spr0083), peptidoglycan GlcNAc deacetylase (NCBI gene no. Spr1333), lactate oxidase (NCBI gene no. Spr0627), PTS system, fructose specific IIABC components (NCBI gene no. ), Zinc metalloprotease (NCBI gene no. Spr0581), hypothetical protein spr0904 (NCBI gene no. Spr0 904), fructose-bisphosphate aldolase (NCBI gene no. (NCBI gene no. spr1699), ribose-phosphate pyrophosphokinase (NCBI gene no. spr0028), endo-beta-N-acetylglucosaminidase (NCBI gene no. spr0867), transcriptional regulator (NCBI gene no. spr0635), manganese ABC transporter, ATP-binding protein (NCBI gene no. spr1492), PTS system, IIABC components (NCBI gene no. spr0668), glutamine synthetase, type I (NCBI gene no. spr0444), sodium alanine symporter family protein (NCBI gene no. spr0369), sugar ABC transporter, sugar-binding protein (NCBI gene no. spr1712), DNA-directed RNA polymerase subunit beta spr1776), polysaccharide biosynthesis protein, putative (NCBI gene no. spr1150), NADH oxidase (NCBI gene no. spr1323), maltodextrin phosphorylase (NCBI gene no. spr1916), preprotein translocase subunit SecY (NCBI gene no. , beta-galactosidase precursor (NCBI gene no. spr0565), isoleucyl-tRNA synthetase (NCBI gene e no. maltose / maltodextrin ABC transporter, and maltose / maltodextrin-binding protein (NCBI gene no. spr1918).

Among the above proteins, the amino acid sequence of the hybrid protein spr0747 (NCBI gene no. Spr0747), ABC transporter substrate-binding protein-manganese transport (NCBI gene no. Spr1494), foldase protein PrsA (NCBI gene no. Spr0884), branched chain amino acid ABC transporter amino acid -binding protein (NCBI gene no. spr0659), ABC transporter substrate-binding protein-amino acid transport (NCBI gene no. spr0146), ABC transporter substrate-binding protein-oligopeptide transport transporter, iron compound-binding protein (NCBI gene no. spr0934), amino acid ABC transporter amino acid-binding protein (NCBI gene no. spr1251), hypothetical protein spr0931 (NCBI gene no. oligopeptide transport (NCBI gene no. spr1707), hypothetical protein spr0554 (NCBI gene no. spr0554), peptidyl-prolyl cis-trans isomerase, cyclophilin type (NCBI gene no. spr0679), lipoprotein (NCBI gene no. acid ABC transporter amino acid-binding protein (NCBI gene no. (NCBI gene no. spr1975), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1382), phosphate ABC transporter phosphate-binding protein (NCBI gene no. ABC transporter substrate-binding protein (NCBI gene no. spr2003), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene No. spr0975), sugar ABC transporter, sugar- ABC transporter substrate-binding protein (NCBI gene no. Spr1645), 50S ribosomal protein L5 (NCBI gene no. Spr0201), hypothetical protein spr0174 (NCBI gene no. Spr0174), iron-compound ABC transporter , iron-compound-binding protein (NCBI gene no. spr1687), D-alanyl-D-alanine carboxypeptidase (NCBI gene no. spr0776), adhesion lipoprotein (NCBI gene no. spr0906), ABC transporter substrate-binding protein No. spr0083), hypothetical protein spr0904 (NCBI gene no. spr0904), endo- beta-N-acetylglucosaminidase (NCBI gene no. maltodextrin-binding protein (NCBI gene no. spr1918) secreted by the transcriptional regulator (NCBI gene no. spr1759), sugar ABC transporter, sugar-binding protein (NCBI gene no. spr1712) and maltose / maltodextrin ABC transporter (NCBI gene no. Spr0012), hypothetical protein spr0688 (NCBI gene no. Spr0688), phosphopyruvate hydratase (NCBI gene no. Spr1036), hypothetical protein spr1912 (NCBI gene no. Spr1912), 30S ribosomal protein S8 (NCBI gene no.spr0203), glucokinase (NCBI gene no.spr0584), 50S ribosomal protein L18 (NCBI gene no.spr0205), L-lactate dehydrogenase (NCBI gene no.spr1100), phosphate transporter PhoU (NCBI gene no.spr1252 ), 30S ribosomal protein S5 (NCBI gene no. Spr0206), hypothetical protein spr1370 (NCBI gene no. Spr1370), 3-ketoacyl- (acyl-carrier-protein) reductase (NCBI gene no. Spr0381), glyceraldehyde- dehydrogenase (NCBI gene no. spr1825), sugar ABC transporter, ATP-binding protein (NCBI gene no. r1437), phosphoglycerate kinase (NCBI gene no. spr0441), phosphoglyceromutase (NCBI gene no. spr1499), ribosome-binding factor A (NCBI gene no. spr0482), hypoxanthine-guanine phosphoribosyltransferase (NCBI gene no. spr0011), 30S ribosomal protein S7 (NCBI gene no. ribosomal protein L22 (NCBI gene no. spr0194), 50S ribosomal protein L5 (NCBI gene no. spr0201), pyruvate oxidase (NCBI gene no. spr0642), cell division protein DivIVA (NCBI gene no. spr1505), F0F1 ATP synthase subunit delta (NCBI gene no. Spr1363), transcriptional repressor CodY (NCBI gene no. Spr1439), hypothetical protein spr1056 (NCBI gene no. Spr1056), 50S ribosomal protein L16 (NCBI gene no. Spr0196), F0F1 ATP synthase subunit beta (NCBI gene no. spr0271), ABC transporter ATP-binding protein-unknown substrate (NCBI gene no. spr1559), 50S ribosomal protein L10 (NCBI gene no. spr1212), phosphate ABC transporter ATP-binding protein (NCBI gene no. Spr1253), choline transporter (NCBI gene no. Spr1678), lactate oxidase gene no. phosphogluconate dehydrogenase (NCBI gene no. spr0335), manganese ABC transporter, ATP-binding protein (NCBI gene no. spr0530), ribose-phosphate pyrophosphokinase (NCBI gene No. spr0028), fructose-bisphosphate aldolase spr1492), glutamine synthetase, type I (NCBI gene no. spr0444), DNA-directed RNA polymerase subunit beta (NCBI gene no. spr1776), NADH oxidase (NCBI gene no. spr1323), maltodextrin phosphorylase (NCBI gene no. spr1122) and the isoleucyl-tRNA synthetase (NCBI gene no. spr1502) are cytoplasmic proteins and the surface protein pspA precursor (NCBI gene no. spr0121), preprotein translocase subunit YajC (NCBI gene no. spr1840), hypothetical protein spr1782 (NCBI gene no. spr1962), glyceral uptake facilitator protein, putative (NCBI gene no. spr1962), signal peptidase I (NCBI gene no. spr0364), hypothetical protein spr1962 No. spr1344), hypothetical protein spr0693 (NCBI gene no. spr0693), hypothetica l protein spr1457 (NCBI gene no. amino acid-binding protein (NCBI gene no. spr1120), hypothetical protein spr1112 (NCBI gene no. spr1163), cytoplasmic membrane protein (NCBI gene no. spr1163), amino acid ABC transporter permease / (NCBI gene no. spr0915), hypothetical protein spr0595 (NCBI gene no. spr0595), hypothetical protein spr0615 (NCBI gene no. (NCBI gene No. spr0615), rod shape-determining protein MreC (NCBI gene no. Spr2023), serine protease (NCBI gene no. Spr2045), phosphotransferase system, mannose-specific EIID (NCBI gene no. Spr0259), peptidoglycan GlcNAc deacetylase (NCBI gene no. Spr1333), PTS system, fructose specific IIABC components (NCBI gene no. Spr0780), Zinc metalloprotease (NCBI gene no. Spr0581), phosphotransferase system, trehalose-specific IIBC component system, IIABC components (NCBI gene no. spr0668), s odium: alanine symporter family protein (NCBI gene no. (NCBI gene no. spr1150) and preprotein translocase subunit SecY (NCBI gene no. spr0209) are cell membrane proteins and the beta-galactosidase precursor (NCBI gene no.

In the present invention, the pneumococcal streptococcal proteins are composed of 28 membrane proteins, 1 cell wall protein, 32 secretory proteins and 43 cytoplasmic proteins identified from the membrane envelope of Streptococcus pneumoniae.

In a specific example of the present invention, in order to identify the proteins present in the pancreatic streptococcal-derived membrane envelope, proteins of the membrane envelope were electrophoresed by SDS-PAGE, in-gel digestion was performed for each band, Proteins were analyzed and identified using S. pneumoniae R6 of NCBI as a database.

As a result, it was confirmed that the proteins were contained in the membrane endoplasmic reticulum, thereby confirming that the proteins could be used as biological molecular markers capable of determining the presence of pneumococcal streptococci (Figs. 3 to 4 and Table 1 Reference).

2. Protein Detection Method to Provide Information on Diagnosis of Pneumonia Streptococcal Disease or Pneumococcal Streptococcal Exposure

Another aspect of the present invention provides a protein detection method for providing information on the diagnosis of a disease caused by pneumococcal streptococci or a pneumococcal streptococcal exposure.

The protein detection method of the present invention

1) separating a membrane vesicle from any one of a biological sample, an environmental sample and a food sample;

2) confirming the presence of pneumococcal streptococcal-derived proteins in the separated membrane envelope; And

3) If the presence of any one or more of the proteins of step 2) is detected, it may be determined that the disease is caused by Streptococcus pneumoniae or exposed to pneumococcal streptococci.

In the protein detection method of the present invention, the membrane envelope separated in the above step 1) can be confirmed by an assay under an electron microscope, but in this method, it is not always necessary to carry out the step of confirming the membrane envelope.

The biological sample of step 1) above is a group consisting of blood, serum, lung lavage fluid, bronchial lavage fluid, urine, ascites, synovial fluid, sputum, saliva, sweat, tears and nasal mucosa taken from patients suspected of being infected or infected with pneumococcal streptococci And the environmental sample is preferably any one selected from the group consisting of drinking water, ground water, river water, livestock wastewater, soil, air and waste, but is not limited thereto.

As for the step of separating the membrane vesicles of step 1) is the same as described in the above 1. pneumoniae Streptococcus aureus (Streptococcus pneumoniae) derived from the endoplasmic reticulum membrane (membrane vesicles) items. Thus this regard the 1 Streptococcus pneumoniae (Streptococcus pneumoniae) derived from the endoplasmic reticulum membrane (membrane vesicles , and detailed description thereof will be omitted, and only the constitution specific to the method for providing the information on the diagnosis of the disease caused by pneumococcal streptococci or the pneumococcal streptococcus through the pancreas streptococcal-derived membrane envelope will be described .

A variety of analytical methods known in the art may be used to confirm the presence of the proteins of step 2). An immunodiagnostic or molecular diagnostic method for detecting the presence of a protein can be used. The antibody can be detected by an antigen-antibody reaction using each antibody that specifically binds to membrane endoplasmic reticulum protein of Streptococcus pneumoniae . The 'antigen-antibody reaction' refers to a binding reaction between a specific protein in a specimen sample and an antibody that specifically recognizes it. Examples of the antigen-antibody binding reaction include immunoblotting, enzyme immunoassay, immunoprecipitation, fluorescent immunoassay, enzyme substrate staining and antigen-antibody aggregation. Specifically, it can be measured by conventional ELISA, radioimmunoassay (RIA), sandwich assay, Western blotting on polyacrylamide gel, immunoblot analysis or immunohistochemical staining. In addition, a biological microchip and an automated microarray system capable of detecting an antigen protein for the antibody by immobilizing an antibody specific to the protein on a biological microchip and reacting with the sample Microarray system can be used to diagnose a large number of samples.

The term " antibody " refers to a specific protein molecule directed against an antigenic site. An antibody used in the present invention may be a monoclonal or polyclonal antibody, an immunologically active fragment (such as an Fab or (Fab) ₂ fragment), an antibody heavy chain, a humanized antibody, an antibody light chain, a genetically engineered single chain Fv molecule, Chimeric antibody, and the like. The antibody used in the present invention can be produced by a conventional method well known in the immunology field using the pneumococcal streptococcal-derived proteins as an antigen.

A polyclonal antibody can be produced by injecting a protein antigen into an animal and collecting it from an animal to obtain a serum containing the antibody. Such antibodies can be produced using various warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats. Monoclonal antibodies can also be prepared using known fusion methods (Kohler and Milstein, European J. Immunol . , 6: 511-519 1976), recombinant DNA methods (US Patent No. 4,816,567) and phage antibody libraries (Clackson et al. , Nature , 352, 624-628, 1991; Marks et al., J. Mol . Biol . , 222, 58: 1-597, 1991).

The proteins of the pneumococcal streptococci strain to be detected in the step 2) include Hypothetical protein spr0747 (NCBI gene no. Spr0747), ABC transporter substrate-binding protein-manganese transport (NCBI gene no. Spr1494), foldase protein PrsA ABC transporter substrate-binding protein-amino acid transport (NCBI gene no. spr0146), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. (NCBI gene no. Spr0327), iron compound ABC transporter, iron compound-binding protein (NCBI gene no. Spr0934), amino acid ABC transporter amino acid-binding protein (NCBI gene no. Spr1251), hypothetical protein spr0931 (NCBI gene no. spr0021), ABC transporter substrate binding protein-oligopeptide transport (NCBI gene no. spr1707), hypothetical protein spr0554 (NCBI gene no. spr0554), peptidyl-prolyl cis-trans isomerase , cyc lophilin-type (NCBI gene no. spr0679), surface protein pspA precursor (NCBI gene no. spr0121), lipoprotein (NCBI gene no.spr0147), amino acid ABC transporter amino acid-binding protein (NCBI gene no. spr1353), hypothetical protein spr0688 (NCBI gene no. spr0688), phosphopyruvate hydratase (NCBI gene no. (NCBI gene no. spr1840), zinc ABC transporter zinc binding protein (NCBI gene no. spr1975), hypothetical protein spr1782 (NCBI gene no. spr1782), signal peptidase I (NCBI gene no. spr0364), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1382), hypothetical protein spr1912 (NCBI gene no. spr1912), 30S ribosomal protein S8 (NCBI gene no. spr0203), phosphate ABC transporter phosphate-binding protein ABC transporter substrate-binding protein (NCBI gene no. spr0584), sugar ABC transporter, sugar-binding protein (NCBI gene no. spr1527), hypothetical protein spr0975 (NCBI gene no. , spR2003), 50S ribosomal protein L18 (NCBI gene no. spr0205), L-lactate dehydrogenase (NCBI gene no. (NCBI gene no. spr1270), 30S ribosomal protein S5 (NCBI gene no. spr0206), hypothetical protein spr1370 (NCBI gene no. spr1370), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1962), 3-ketoacyl- (acyl-carrier-protein) reductase (NCBI gene no. spr0381), glycerol uptake (spr1194), hypothetical protein spr1962 promoter protein, putative (NCBI gene no.spr1344), hypothetical protein spr0693 (NCBI gene no.spr0693), hypothetical protein spr1457 (NCBI gene no.spr1457), preprotein translocase subunit SecG (NCBI gene no.spr0877), glyceraldehyde- phosphate dehydrogenase (NCBI gene no. spr1825), sugar ABC transporter, ATP-binding protein (NCBI gene no. spr1437), cytoplasmic membrane protein (NCBI gene no. spr1163), phosphoglycerate kinase (NCBI gene no. spr0441), ABC transporter substrate -binding protein (NCBI gene no. spr1645), phosphoglyceromutase (NCBI gene no. spr 1499), ribosome-binding factor A (NCBI gene no. spR0482, hypoxanthine-guanine phosphoribosyltransferase (NCBI gene no. spr0011), 30S ribosomal protein S7 (NCBI gene no. spr0249), amino acid ABC transporter permease / amino acid-binding protein (NCBI gene no. spr1120), 50S ribosomal protein L22 NCBI gene no. Spr0194), 50S ribosomal protein L5 (NCBI gene no. Spr0201), hypothetical protein spr0174 (NCBI gene no. Spr0174), pyruvate oxidase (NCBI gene no. Spr0642), cell division protein DivIVA (NCBI gene no. ), iron-compound ABC transporter, iron-compound-binding protein (NCBI gene no. spr1687), hypothetical protein spr1112 (NCBI gene no. spr1112), F0F1 ATP synthase subunit delta (NCBI gene no. D-alanine carboxypeptidase (NCBI gene no. Spr0776), large conductance mechanosensitive channel protein MscL (NCBI gene no. Spr0915), PTS system, mannose-specific IIC component (NCBI gene no. Spr0260), hypothetical protein spr0595 (NCBI gene no. spr0595), hypothetical protein spr0615 (NCBI gene no. spr0615), rod shape-determining protein M reC (NCBI gene no. spr2023), adhesion lipoprotein (NCBI gene No. spr0906), transcriptional repressor CodY (NCBI gene no. spr1439), hypothetical protein spr1056 (NCBI gene no. spr1056), 50S ribosomal protein L16 (NCBI gene no. spr0196), F0F1 ATP synthase (NCBI gene no. spr1360), serine protease (NCBI gene no. spr2045), phosphotransferase system, mannose-specific EIID (NCBI gene no. spr0259), 50S ribosomal protein L13 (NCBI gene no. spr0271), ABC transporter ATP -binding protein-unknown substrate (NCBI gene no. spr1559), 50S ribosomal protein L10 (NCBI gene no. spr1212), phosphate ABC transporter ATP-binding protein (NCBI gene no. spr1253), choline transporter (NCBI gene no. ), ABC transporter substrate-binding protein (NCBI gene No. spr0083), peptidoglycan GlcNAc deacetylase (NCBI gene no. Spr1333), lactate oxidase (NCBI gene no. Spr0627), PTS system, fructose specific IIABC components (NCBI gene no. ), Zinc metalloprotease (NCBI gene no. Spr0581), hypothetical protein spr0904 (NCBI gene no. Spr0 904), fructose-bisphosphate aldolase (NCBI gene no. (NCBI gene no. spr1699), ribose-phosphate pyrophosphokinase (NCBI gene no. spr0028), endo-beta-N-acetylglucosaminidase (NCBI gene no. spr0867), transcriptional regulator (NCBI gene no. spr0635), manganese ABC transporter, ATP-binding protein (NCBI gene no. spr1492), PTS system, IIABC components (NCBI gene no. spr0668), glutamine synthetase, type I (NCBI gene no. spr0444), sodium alanine symporter family protein (NCBI gene no. spr0369), sugar ABC transporter, sugar-binding protein (NCBI gene no. spr1712), DNA-directed RNA polymerase subunit beta spr1776), polysaccharide biosynthesis protein, putative (NCBI gene no. spr1150), NADH oxidase (NCBI gene no. spr1323), maltodextrin phosphorylase (NCBI gene no. spr1916), preprotein translocase subunit SecY (NCBI gene no. , beta-galactosidase precursor (NCBI gene no. spr0565), isoleucyl-tRNA synthetase (NCBI gene e no. maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein (NCBI gene no. spr1918), foldase protein PrsA (NCBI gene no. spr0884), penicillin (NCBI gene no. spr0329), surface protein pspA precursor (NCBI gene no. spr0121) and maltose / maltodextrin ABC transporter and maltose / maltodextrin-binding protein But is not limited to, one or more proteins.

The disease caused by the pneumococcal streptococci may be any one selected from the group consisting of pneumonia, mumps, conjunctivitis, peritonitis, otitis media, endocarditis, and sepsis. However, the present invention is not limited to this and all diseases caused by infection with pneumococcal streptococci Diagnosis is possible. Streptococcus pneumoniae is a convenient symbiotic organism that generally colonizes the mucosal surface of human nasopharynx. When the host allows access to the organism, a vigorous inflammatory reaction occurs and exudates accumulate in the alveolar space causing curing. This condition is commonly referred to as pneumonia. Severe signs of pneumococcal streptococcal infection include sepsis, meningitis, or bacteremia that can be exacerbated by both sepsis and meningitis, and can cause mumps, conjunctivitis, peritonitis, otitis media, or endocarditis (Suzuki et al., J. Clin . Microbiol . , 43: 4528-4534, 2005).

In a specific example of the present invention, by confirming that pneumococcal streptococci, which are gram-positive bacteria existing in the body or present in the surrounding environment, secrete the membrane endoplasmic reticulum out of the cells, it is possible to use the membrane endoplasmic reticulum to express pneumococcal streptococcal- And to develop a method for diagnosing exposure. Specifically, membranous endoplasmic reticulum was isolated and confirmed from a pneumococcal streptococcal culture (see FIGS. 1 and 2). Proteins usable as the membrane endoplasmic marker markers were confirmed by electrophoresis and mass spectrometry. As a result, 28 membrane proteins, One cell wall protein, 32 secretory proteins and 43 cytoplasmic proteins were identified (see Table 1 and Figure 3). Of these, 38 proteins were found to be proteins involved in transporting substances such as ABC transporters or PTS systems.

In particular, the mouse serum antigen and the proteins were immunoreacted against pneumococcal streptococcal membrane filamentous fungi, resulting in foldase protein PrsA (NCBI gene no. Spr0884), penicillin-binding protein 1A (NCBI gene no. Spr0329), surface protein pspA precursor (NCBI gene no. Spr0121), maltose / maltodextrin ABC transporter, and maltose / maltodextrin-binding protein (NCBI gene no. Thus, the presence or absence of the membrane endoplasmic reticulum proteins identified in the present invention can be efficiently diagnosed in a biological sample, an environmental sample, a food sample, or the like, which can be easily collected, such as a bronchopneumonia wash solution.

Hereinafter, the present invention will be described in detail with reference to examples.

It is to be understood, however, that the following examples are intended to assist the understanding of the present invention and are not intended to limit the scope of the present invention.

≪ Example 1 >

Streptococcus pneumoniae strain origin Membrane endoplasmic reticulum  detach

<1-1> Culture and recovery of pneumococcal streptococci

(THA) was added to the culture medium (Thdd-Hewitt broth, BD, USA) and sterilized by autoclaving at 37 ° C under 5% CO _2. and stably cultured until the optical density was 0.9. Then, the cultured bacteria were centrifuged at 4,000 rpm for 30 minutes at 8,000 rpm to separate the culture supernatant and the cells.

<1-2> Of Membrane Vesicles (MVs)  Detach and verify

The supernatant recovered in Example <1-1> was filtered with a 0.2 μm filter to remove the cells and suspension contained in the medium, and then the substances of 500 kDa or less were removed using a QUIXStand ^™ system (GE, USA) kDa or more was obtained and it was concentrated to 200 ml or less. The resulting solution was centrifuged using an ultrahigh-speed centrifuge, and the membrane envelope that had settled on the bottom was recovered. Then, a concentration gradient was made using sucrose to remove the proteins contained in the recovered membrane vesicles, and a second ultra-high speed centrifugation was performed to recover pure membrane vesicles. In order to identify the membrane vesicles thus recovered, the membrane vesicles isolated using an electron microscope (Cryo-TEM) were visually confirmed (see Fig. 1).

As a result, it was confirmed that the pneumococcal strain-derived membrane envelope was isolated from the medium component of the pneumococcal streptococcal culture (see FIG. 2).

&Lt; Example 2 >

Streptococcus pneumoniae strain origin In membrane envelope  Identification of included proteins

<2-1> Through electrophoresis and mass analysis Membrane endoplasmic reticulum  Protein identification

After loading 15 막 of membrane envelope into 12% SDS-PAGE and electrophoresing, the gel was cut into 8 fractions and in-gel digestion was performed using trypsin (see Figure 3) . Each of the peptides thus obtained was analyzed using LCQ DECA XP plus (Thermo Finnigan, USA). The results of the analysis were used to identify each specific protein using MASCOT software (ver. 2.4) and S. pneumoniae R6 of the NCBI gene database.

<2-2> Membrane vesicle  Analysis of proteins

The concentration of each protein was determined by the emPAI calculation method and the location of each protein in the cells was determined using the LocateP database as described above and in Example <2-1>.

As a result, 28 membrane proteins, 1 cell wall protein, 32 secretory proteins, and 43 cytoplasmic proteins were identified. Of these, it was confirmed that only 12 membrane proteins were identified by the sodium carbonate precipitation method, and 38 proteins were confirmed to be protein related to substance transport such as ABC transporter or PTS system (see Table 1).

Gene No. Protein name MW
(Molecular Weight) pI
(Isoelectric point) Gene name Intracellular location Average% spr0747 hypothetical protein spr0747 39481 5.46 - Extracellular
(Extracellular) 9.388 spr1494 ABC transporter substrate-binding protein - manganese transport. 34573 5.3 psaA Extracellular 8.669 spr0884 foldase protein PrsA 34419 5.04 prsA Extracellular 6.588 spr1918 maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein 45339 5.1 malX Extracellular 6.124 spr0659 branched chain amino acid ABC transporter amino acid-binding protein 40389 5.29 livJ Extracellular 4.398 spr0146 ABC transporter substrate-binding protein - amino acid transport 30609 5.14 ABC-SBP Extracellular 3.453 spr0327 ABC transporter substrate-binding protein - oligopeptide transport 72958 5 alia Extracellular 3.241 spr0934 iron-compound ABC transporter, iron compound-binding protein 37483 5.38 ABC-SBP Extracellular 3.175 spr1251 amino acid ABC transporter amino acid-binding protein 29246 4.96 glnH Extracellular 2.973 spr0931 hypothetical protein spr0931 35377 5.46 - Extracellular 2.494 spr0021 adenylosuccinate synthetase 47541 5.49 purA Cytoplasmic
(cytoplasm) 2.279 spr1707 ABC transporter substrate-binding protein - oligopeptide transport 72460 4.95 amia Extracellular 2.218 spr0554 hypothetical protein spr0554 26522 5.13 - Extracellular 2.010 spr0679 peptidyl-prolyl cis-trans isomerase, cyclophilin-type 28991 5.47 ppiA Extracellular 1.936 spr0121 surface protein pspA precursor 68563 5.12 pspA Membrane
(membrane) 1.518 spr0147 lipoprotein 31128 5.46 ABC-SBP Extracellular 1.507 spr1353 amino acid ABC transporter amino acid-binding protein 31000 4.83 glnH Extracellular 1.486 spr0688 hypothetical protein spr0688 12929 6.97 - Cytoplasmic 1.409 spr1036 포피opyruvate hydratase 47074 4.7 eno Cytoplasmic 1.340 spr1840 preprotein translocase subunit YajC 11040 5.39 yajC Membrane 1.290 spr1975 zinc ABC transporter zinc-binding protein 56237 5.05 adcA Extracellular 1.142 spr1782 hypothetical protein spr1782 37514 5.26 - Membrane 1.070 spr0364 signal peptidase I 23483 5.84 spi Membrane 1.050 spr1382 ABC transporter substrate-binding protein - oligopeptide transport 72517 5.24 aliB Extracellular 0.990 spr1912 hypothetical protein spr1912 11376 6.6 - Cytoplasmic 0.976 spr0203 30S ribosomal protein S8 14745 9.58 rpsH Cytoplasmic 0.884 spr1257 phosphate ABC transporter phosphate-binding protein 31182 4.77 pstS Extracellular 0.883 spr0584 glucokinase 34164 4.95 glcK Cytoplasmic 0.880 spr0975 hypothetical protein spr0975 36417 7.74 ABC-SBP Extracellular 0.877 spr1527 sugar ABC transporter, sugar-binding protein 48273 5.31 ABC-SBP Extracellular 0.850 spr2003 ABC transporter substrate-binding protein 37753 5.41 - Extracellular 0.836 spr0205 50S ribosomal protein L18 12859 10.4 rplR Cytoplasmic 0.771 spr1100 L-lactate dehydrogenase 35333 5.09 ldh Cytoplasmic 0.757 spr1252 phosphate transporter PhoU 25013 4.73 phoU Cytoplasmic 0.756 spr0206 30S ribosomal protein S5 17046 9.52 rpsE Cytoplasmic 0.737 spr1370 hypothetical protein spr1370 60776 5.07 - Cytoplasmic 0.733 spr1194 ABC transporter substrate-binding protein - oligopeptide transport 61169 5.86 appA Extracellular 0.725 spr1962 hypothetical protein spr1962 33573 5.62 - Membrane 0.666 spr0329 penicillin-binding protein 1A 79652 5.41 pbpA Membrane 0.651 spr0381 3-ketoacyl- (acyl-carrier-protein) reductase 25753 5.49 fabG Cytoplasmic 0.647 spr1344 glycerol uptake facilitator protein, putative 30652 9.02 glpF Membrane 0.543 spr0693 hypothetical protein spr0693 41839 5.12 - Membrane 0.532 spr1457 hypothetical protein spr1457 16787 5.03 - Membrane 0.522 spr0877 preprotein translocase subunit SecG 8545 9.69 secG Membrane 0.521 spr1825 glyceraldehyde-3-phosphate dehydrogenase 38739 5.78 gapA Cytoplasmic 0.494 spr1437 sugar ABC transporter, ATP-binding protein 41808 5.83 msmK Cytoplasmic 0.493 spr1163 cytoplasmic membrane protein 20621 5.66 LeMa Membrane 0.472 spr0441 포스 포스 라이cerate kinase 41913 4.92 pgk Cytoplasmic 0.468 spr1645 ABC transporter substrate-binding protein 39178 5.61 ABC-SBP Extracellular 0.450 spr1499 포스 가스ceromutase 26035 5.14 gpmA Cytoplasmic 0.429 spr0482 ribosome-binding factor A 14456 9.36 rbfA Cytoplasmic 0.418 spr0011 hypoxanthine-guanine phosphoribosyltransferase 20199 5.26 hgt Cytoplasmic 0.416 spr0249 30S ribosomal protein S7 17745 10.29 rpsG Cytoplasmic 0.383 spr1120 amino acid ABC transporter permease / amino acid-binding protein 78318 8.39 glnP Membrane 0.367 spr0194 50S ribosomal protein L22 12193 10.76 rplV Cytoplasmic 0.347 spr0201 50S ribosomal protein L5 19762 9.04 rplE Cytoplasmic 0.339 spr0174 hypothetical protein spr0174 21027 5.83 - Extracellular 0.333 spr0642 파르르 비이트 옥시시세 65214 5.03 spxB Cytoplasmic 0.329 spr1505 cell division protein DivIVA 30724 4.7 divIVA Cytoplasmic 0.328 spr1687 iron-compound ABC transporter, iron-compound-binding protein 34841 5.55 fatB Extracellular 0.328 spr1112 hypothetical protein spr1112 20260 10.38 - Membrane 0.323 spr1363 F0F1 ATP synthase subunit delta 20528 7.77 atpH Cytoplasmic 0.323 spr0776 D-alanyl-D-alanine carboxypeptidase 45192 4.87 dacA Extracellular 0.322 spr0915 large conductance mechanosensitive channel protein MscL 13608 9.78 mscL Membrane 0.315 spr0260 PTS system, mannose-specific IIC component 27213 4.84 manM Membrane 0.302 spr0595 hypothetical protein spr0595 14568 10.05 - Membrane 0.293 spr0615 hypothetical protein spr0615 14431 8.76 - Membrane 0.293 spr2023 rod shape-determining protein MreC 29721 5.87 mreC Membrane 0.293 spr0906 adhesion lipoprotein 34678 5.27 lmb Extracellular 0.287 spr1439 transcriptional repressor cody 29738 5.55 codY Cytoplasmic 0.277 spr1056 hypothetical protein spr1056 25660 6.34 - Cytoplasmic 0.273 spr0196 50S ribosomal protein L16 15426 10.66 rplP Cytoplasmic 0.271 spr1360 F0F1 ATP synthase subunit beta 50872 4.85 atpD Cytoplasmic 0.271 spr2045 serine protease 42262 6.15 sphtra Membrane 0.265 spr0259 phosphotransferase system, mannose-specific EIID 33764 8.79 mann Membrane 0.263 spr0271 50S ribosomal protein L13 16133 9.95 rplM Cytoplasmic 0.261 spr1559 ABC transporter ATP-binding protein - unknown substrate 26664 5.71 ABC-NBD Cytoplasmic 0.246 spr1212 50S ribosomal protein L10 17469 5.08 rplJ Cytoplasmic 0.239 spr1253 phosphate ABC transporter ATP-binding protein 27936 5.36 pstB Cytoplasmic 0.235 spr1678 choline transporter 27082 4.97 proV Cytoplasmic 0.220 spr0083 ABC transporter substrate-binding protein 57190 5.94 ABC-SBP Extracellular 0.210 spr1333 peptidoglycan GlcNAc deacetylase 52642 5.45 pgdA Membrane 0.207 spr0627 lactate oxidase 41463 5.67 lctO Cytoplasmic 0.206 spr0780 PTS system, fructose specific IIABC components 66892 5.31 fruA Membrane 0.205 spr0581 Zinc metalloprotease 210928 5.3 zmpB Membrane 0.198 spr0904 hypothetical protein spr0904 21348 7.59 - Extracellular 0.195 spr0530 fructose-bisphosphate aldolase 31382 5 fba Cytoplasmic 0.186 spr1699 phosphotransferase system, trehalose-specific IIBC component 75669 5.28 treP Membrane 0.176 spr0028 ribose-phosphate pyrophosphokinase 37379 5.39 prsA Cytoplasmic 0.169 spr0867 endo-beta-N-acetylglucosaminidase 84238 5.46 lytB Extracellular 0.165 spr1759 가사 37543 5.11 lytR Extracellular 0.157 spr0335 6-phosphogluconate dehydrogenase 52546 4.92 gnd Cytoplasmic 0.152 spr1492 manganese ABC transporter, ATP-binding protein 26813 7.05 psaB Cytoplasmic 0.152 spr0668 PTS system, IIABC components 77972 5.94 ptsG Membrane 0.140 spr0444 glutamine synthetase, type I 50290 4.93 glnA Cytoplasmic 0.126 spr0369 sodium: alanine symporter family protein 46692 8.67 dagA Membrane 0.124 spr1712 sugar ABC transporter, sugar-binding protein 46632 5.13 msmE Extracellular 0.124 spr1776 DNA-directed RNA polymerase subunit beta ~ 136877 6.8 rpoC Cytoplasmic 0.119 spr1150 polysaccharide biosynthesis protein, putative 56482 9.44 - Membrane 0.110 spr1323 NADH oxidase 50180 4.99 nox Cytoplasmic 0.108 spr1916 maltodextrin phosphorylase 85101 5.03 malP Cytoplasmic 0.098 spr0209 preprotein translocase subunit SecY 47323 9.68 secY Membrane 0.087 spr0565 beta-galactosidase precursor 246632 5.81 bgaA Cell Wall 0.063 spr1502 isoleucyl-tRNA synthetase 105409 4.93 ileS Cytoplasmic 0.059

&Lt; Example 3 >

Streptococcus pneumoniae strain derived from antibody Membrane vesicle  Identification of proteins

In order to confirm the usefulness of membrane endoplasmic reticulum-derived membrane endoplasmic reticulum proteins identified through Example 2 as biomarkers, mice were injected three times with 10 ⁸ S. pneumoniae BAA-255 strains, and then sera were obtained from the mice . Then, membrane endoplasmic reticulum proteins shown in Table 1 were electrophoresed, treated with the serum at a ratio of 1: 500, and subjected to Western blotting.

As a result, particularly strong signals were detected in six bands. Among these six proteins, foldase protein PrsA (NCBI gene no. Spr0884), penicillin-binding protein 1A (NCBI gene no. Spr0329), surface protein pspA precursor maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein (NCBI gene no. spr1918) protein (see Fig.

<Example 4>

Origin of pneumococcal streptococcus using patient-derived samples Membrane vesicle  Identification of proteins

Dot blotting was performed to evaluate the presence or absence of papillomavirus streptococcal membrane vesicle protein in the blood of patients with pneumonia. The experimental group consisted of four sera from pneumonia patients who were identified as pneumococcal streptococci in the clinical pathology department and one healthy human serum was used as a control group. 5 μl of each specimen (pneumonia patient serum including the control group) was loaded onto a nitrocellulose membrane (NC membrane; GE, USA) as shown in FIG. 5. When the specimen was adsorbed onto the nitrocellulose membrane, a Tris-buffered saline ) With 5% skim milk (WAKO, Japan) for 30 minutes. After that, the membrane-annuid antibody was mixed with a solution containing 3% skim milk in Tris buffered saline at a ratio of 1: 5,000 and 1: 10,000 for 4 hours. Then, TTBS (Tris-buffered saline with 0.1% tween- For 10 minutes. To this, anti-rabbit IgG antibody was diluted 1: 5,000 in 3% skim milk solution in Tris buffered saline, treated for 1 hour, and then washed 3 times with TTBS for 10 minutes. ECL solution (Electrochemiluminescence solution; GE, USA) was applied to develop color.

As a result, as shown in FIG. 5, when the pneumococcal streptococcal follicular antibody was used to react with sera from pneumonia patients and healthy persons, most of the sera from patients with pneumonia showed reactivity with the membrane endoplasmic reticulum antibody. The serum of the patient was collected from a patient who had been infected with pneumococcal streptococci at the first stage and not from other viral pneumonia patients. Therefore, the above results indicate that the serum of the pneumococcal pneumonia patients (sera) Antibody-based diagnosis was possible.

Referring to FIG. 5, in the control group No. 1, the response to the membrane-enveloped antibody was not observed, and the response was strong in the second to third (pneumococcal sera a to b). In addition, 4 (Phenotypic patient serum c) showed a weak but detectable reaction, and 5 (Phenotypic patient's serum d) showed weak reactivity and can not be visually confirmed. I could confirm. However, the reactivity in the patient sample with the dilution ratio of the membrane ER antibody did not show a large difference.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the invention is not limited to the disclosed exemplary embodiments. It will be possible to change it appropriately.

Claims (14)

Streptococcus pneumoniae- derived membrane vesicles.
The membrane envelope of claim 1, wherein said membrane envelope is secreted out of pneumococcal streptococcal cells.
The membrane according to claim 1,
1) centrifuging the pneumococcal streptococcus culture to obtain a supernatant;
2) primary filtration of the supernatant solution to remove impurities and cells;
3) Secondly filtering the obtained filtrate with a membrane having a molecular weight cutoff of 300 kDa to 1000 kDa to obtain a fraction having a molecular weight of not less than the cut-off value; And
And 4) ultracentrifuging the obtained fractions to obtain a precipitate.
The membrane envelope according to claim 3, further comprising, after the step 4), pure separation of the precipitated membrane filament by ultra-centrifuging the precipitate by sucrose concentration.
4. The membrane envelope of claim 3, wherein the molecular weight cutoff of step 3) is 500 kDa.
The membrane envelope of claim 1, wherein the membrane envelope is selected from the group consisting of Hypothetical protein spr0747 (NCBI gene no. Spr0747), ABC transporter substrate-binding protein-manganese transport (NCBI gene no. Spr1494), foldase protein PrsA amino acid ABC transporter amino acid-binding protein (NCBI gene no. spr0659), ABC transporter substrate-binding protein-amino acid transport (NCBI gene no. spr0146), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. ), iron compound ABC transporter, iron compound-binding protein (NCBI gene no. spr0934), amino acid ABC transporter amino acid-binding protein (NCBI gene no. spr1251), hypothetical protein spr0931 (NCBI gene no. (NCBI gene no. spr0021), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1707), hypothetical protein spr0554 (NCBI gene no. spr0554), peptidyl-prolyl cis- trans isomerase, cyclophilin-type gene no. spr0679), surface pr otein pspA precursor (NCBI gene no. spr0121), lipoprotein (NCBI gene no.spr0147), amino acid ABC transporter amino acid-binding protein (NCBI gene no. spr1353), hypothetical protein spr0688 (NCBI gene no. spr0688), phosphopyruvate hydratase (NCBI gene no. (NCBI gene no. spr1840), zinc ABC transporter zinc binding protein (NCBI gene no. spr1975), hypothetical protein spr1782 (NCBI gene no. spr1782), signal peptidase I (NCBI gene no. spr0364), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1382), hypothetical protein spr1912 (NCBI gene no. spr1912), 30S ribosomal protein S8 (NCBI gene no. spr0203), phosphate ABC transporter phosphate-binding protein ABC transporter substrate-binding protein (NCBI gene no. spr0584), sugar ABC transporter, sugar-binding protein (NCBI gene no. spr1527), hypothetical protein spr0975 (NCBI gene no. , spR2003), 50S ribosomal protein L18 (NCBI gene no. spr0205), L-lactate dehydrogenase (NCBI gene no. (NCBI gene no. spr1270), 30S ribosomal protein S5 (NCBI gene no. spr0206), hypothetical protein spr1370 (NCBI gene no. spr1370), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1962), 3-ketoacyl- (acyl-carrier-protein) reductase (NCBI gene no. spr0381), glycerol uptake (spr1194), hypothetical protein spr1962 promoter protein, putative (NCBI gene no.spr1344), hypothetical protein spr0693 (NCBI gene no.spr0693), hypothetical protein spr1457 (NCBI gene no.spr1457), preprotein translocase subunit SecG (NCBI gene no.spr0877), glyceraldehyde- phosphate dehydrogenase (NCBI gene no. spr1825), sugar ABC transporter, ATP-binding protein (NCBI gene no. spr1437), cytoplasmic membrane protein (NCBI gene no. spr1163), phosphoglycerate kinase (NCBI gene no. spr0441), ABC transporter substrate -binding protein (NCBI gene no. spr1645), phosphoglyceromutase (NCBI gene no. spr 1499), ribosome-binding factor A (NCBI gene no. spR0482, hypoxanthine-guanine phosphoribosyltransferase (NCBI gene no. spr0011), 30S ribosomal protein S7 (NCBI gene no. spr0249), amino acid ABC transporter permease / amino acid-binding protein (NCBI gene no. spr1120), 50S ribosomal protein L22 NCBI gene no. Spr0194), 50S ribosomal protein L5 (NCBI gene no. Spr0201), hypothetical protein spr0174 (NCBI gene no. Spr0174), pyruvate oxidase (NCBI gene no. Spr0642), cell division protein DivIVA (NCBI gene no. ), iron-compound ABC transporter, iron-compound-binding protein (NCBI gene no. spr1687), hypothetical protein spr1112 (NCBI gene no. spr1112), F0F1 ATP synthase subunit delta (NCBI gene no. D-alanine carboxypeptidase (NCBI gene no. Spr0776), large conductance mechanosensitive channel protein MscL (NCBI gene no. Spr0915), PTS system, mannose-specific IIC component (NCBI gene no. Spr0260), hypothetical protein spr0595 (NCBI gene no. spr0595), hypothetical protein spr0615 (NCBI gene no. spr0615), rod shape-determining protein M reC (NCBI gene no. spr2023), adhesion lipoprotein (NCBI gene No. spr0906), transcriptional repressor CodY (NCBI gene no. spr1439), hypothetical protein spr1056 (NCBI gene no. spr1056), 50S ribosomal protein L16 (NCBI gene no. spr0196), F0F1 ATP synthase (NCBI gene no. spr1360), serine protease (NCBI gene no. spr2045), phosphotransferase system, mannose-specific EIID (NCBI gene no. spr0259), 50S ribosomal protein L13 (NCBI gene no. spr0271), ABC transporter ATP -binding protein-unknown substrate (NCBI gene no. spr1559), 50S ribosomal protein L10 (NCBI gene no. spr1212), phosphate ABC transporter ATP-binding protein (NCBI gene no. spr1253), choline transporter (NCBI gene no. ), ABC transporter substrate-binding protein (NCBI gene No. spr0083), peptidoglycan GlcNAc deacetylase (NCBI gene no. Spr1333), lactate oxidase (NCBI gene no. Spr0627), PTS system, fructose specific IIABC components (NCBI gene no. ), Zinc metalloprotease (NCBI gene no. Spr0581), hypothetical protein spr0904 (NCBI gene no. Spr0 904), fructose-bisphosphate aldolase (NCBI gene no. (NCBI gene no. spr1699), ribose-phosphate pyrophosphokinase (NCBI gene no. spr0028), endo-beta-N-acetylglucosaminidase (NCBI gene no. spr0867), transcriptional regulator (NCBI gene no. spr0635), manganese ABC transporter, ATP-binding protein (NCBI gene no. spr1492), PTS system, IIABC components (NCBI gene no. spr0668), glutamine synthetase, type I (NCBI gene no. spr0444), sodium alanine symporter family protein (NCBI gene no. spr0369), sugar ABC transporter, sugar-binding protein (NCBI gene no. spr1712), DNA-directed RNA polymerase subunit beta spr1776), polysaccharide biosynthesis protein, putative (NCBI gene no. spr1150), NADH oxidase (NCBI gene no. spr1323), maltodextrin phosphorylase (NCBI gene no. spr1916), preprotein translocase subunit SecY (NCBI gene no. , beta-galactosidase precursor (NCBI gene no. spr0565), isoleucyl-tRNA synthetase (NCBI gene e no. spr1502), maltose / maltodextrin ABC transporter, and maltose / maltodextrin-binding protein (NCBI gene no. spr1918).
1) separating a membrane vesicle from any one of a biological sample, an environmental sample and a food sample;
2) confirming the presence of pneumococcal streptococcal-derived proteins in the separated membrane envelope; And
3) when the presence of any one or more of the proteins of step 2) is detected, it is determined that the disease is caused by pneumococcal streptococci or exposed to pneumococcal streptococci, or Protein detection method to provide information on pneumococcal streptococcus exposure diagnosis.
The method of claim 7, wherein the biological sample of step 1) is a blood sample, serum, lung lavage fluid, bronchial lavage fluid, urine, ascites, synovial fluid, sputum, saliva, sweat, tears, and runny nose from a patient suspected of being infected with pneumococcal streptococci Wherein the method comprises the steps of: (a) detecting a pathogen-associated pathogenic staphylococcal disease or pneumococcal streptococcal infection;
[Claim 7] The environmental sample according to claim 7, wherein the environmental sample in step 1) is any one selected from the group consisting of drinking water, ground water, river water, livestock wastewater, soil, air and waste, or a disease caused by pneumococcal streptococci, A protein detection method for providing information on exposure diagnosis.
8. The method of claim 7, wherein separating the membrane envelope of step 1)
1) centrifuging the pneumococcal streptococcus culture to obtain a supernatant;
2) primary filtration of the supernatant solution to remove impurities and cells;
3) Secondly filtering the obtained filtrate with a membrane having a molecular weight cut-off of 300 kDa to 1000 kDa to obtain a fraction having a molecular weight above the cut-off value; And
4) Extraction of the fraction obtained by ultracentrifugation to obtain a precipitate. A protein detection method for providing information on diagnosis of disease or pneumococcal streptococcal exposure by streptococcal streptococci.
[Claim 7] The method according to claim 7, wherein the presence or absence of the proteins in step 2) is detected by detecting an antigen-antibody reaction using each antibody that specifically binds to the proteins in step 2) A protein detection method for providing information on a disease caused by streptococcal disease or diagnosis of streptococcal streptococcus exposure.
The method according to claim 7, wherein the pneumococcal streptococcal proteins of step 2) are selected from the group consisting of foldase protein PrsA (NCBI gene no. Spr0884), penicillin-binding protein 1A (NCBI gene no. Spr0329), surface protein pspA precursor (NCBI gene no. ) And maltose / maltodextrin ABC transporter, and maltose / maltodextrin-binding protein (NCBI gene no. Spr1918). A protein detection method for providing information.
The method according to claim 7, wherein the pneumococcal streptococcal proteins of step 2) are selected from the group consisting of Hypothetical protein spr0747 (NCBI gene no. Spr0747), ABC transporter substrate-binding protein-manganese transport (NCBI gene no. Spr1494) ABC transporter substrate-binding protein-amino acid transport (NCBI gene no. spr0146), ABC transporter substrate-binding protein-oligopeptide transport protein (NCBI gene no. spr0327), iron compound ABC transporter, iron compound-binding protein (NCBI gene no. spr0934), amino acid ABC transporter amino acid-binding protein (NCBI gene no. spr1251), hypothetical protein spr0931 (NCBI gene no. spr0021), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1707), hypothetical protein spr0554 (NCBI gene no. spr0554), peptidyl-prolyl cis-trans isomerase, cyclophil in-type (NCBI gene no. spr0679), surface protein pspA precursor (NCBI gene no. spr0121), lipoprotein (NCBI gene no.spr0147), amino acid ABC transporter amino acid-binding protein (NCBI gene no. spr1353), hypothetical protein spr0688 (NCBI gene no. spr0688), phosphopyruvate hydratase (NCBI gene no. (NCBI gene no. spr1840), zinc ABC transporter zinc binding protein (NCBI gene no. spr1975), hypothetical protein spr1782 (NCBI gene no. spr1782), signal peptidase I (NCBI gene no. spr0364), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1382), hypothetical protein spr1912 (NCBI gene no. spr1912), 30S ribosomal protein S8 (NCBI gene no. spr0203), phosphate ABC transporter phosphate-binding protein ABC transporter substrate-binding protein (NCBI gene no. spr0584), sugar ABC transporter, sugar-binding protein (NCBI gene no. spr1527), hypothetical protein spr0975 (NCBI gene no. , spR2003), 50S ribosomal protein L18 (NCBI gene no. spr0205), L-lactate dehydrogenase (NCBI gene no. (NCBI gene no. spr1270), 30S ribosomal protein S5 (NCBI gene no. spr0206), hypothetical protein spr1370 (NCBI gene no. spr1370), ABC transporter substrate-binding protein-oligopeptide transport (NCBI gene no. spr1962), 3-ketoacyl- (acyl-carrier-protein) reductase (NCBI gene no. spr0381), glycerol uptake (spr1194), hypothetical protein spr1962 promoter protein, putative (NCBI gene no.spr1344), hypothetical protein spr0693 (NCBI gene no.spr0693), hypothetical protein spr1457 (NCBI gene no.spr1457), preprotein translocase subunit SecG (NCBI gene no.spr0877), glyceraldehyde- phosphate dehydrogenase (NCBI gene no. spr1825), sugar ABC transporter, ATP-binding protein (NCBI gene no. spr1437), cytoplasmic membrane protein (NCBI gene no. spr1163), phosphoglycerate kinase (NCBI gene no. spr0441), ABC transporter substrate -binding protein (NCBI gene no. spr1645), phosphoglyceromutase (NCBI gene no. spr 1499), ribosome-binding factor A (NCBI gene no. spR0482, hypoxanthine-guanine phosphoribosyltransferase (NCBI gene no. spr0011), 30S ribosomal protein S7 (NCBI gene no. spr0249), amino acid ABC transporter permease / amino acid-binding protein (NCBI gene no. spr1120), 50S ribosomal protein L22 NCBI gene no. Spr0194), 50S ribosomal protein L5 (NCBI gene no. Spr0201), hypothetical protein spr0174 (NCBI gene no. Spr0174), pyruvate oxidase (NCBI gene no. Spr0642), cell division protein DivIVA (NCBI gene no. ), iron-compound ABC transporter, iron-compound-binding protein (NCBI gene no. spr1687), hypothetical protein spr1112 (NCBI gene no. spr1112), F0F1 ATP synthase subunit delta (NCBI gene no. D-alanine carboxypeptidase (NCBI gene no. Spr0776), large conductance mechanosensitive channel protein MscL (NCBI gene no. Spr0915), PTS system, mannose-specific IIC component (NCBI gene no. Spr0260), hypothetical protein spr0595 (NCBI gene no. spr0595), hypothetical protein spr0615 (NCBI gene no. spr0615), rod shape-determining protein M reC (NCBI gene no. spr2023), adhesion lipoprotein (NCBI gene No. spr0906), transcriptional repressor CodY (NCBI gene no. spr1439), hypothetical protein spr1056 (NCBI gene no. spr1056), 50S ribosomal protein L16 (NCBI gene no. spr0196), F0F1 ATP synthase (NCBI gene no. spr1360), serine protease (NCBI gene no. spr2045), phosphotransferase system, mannose-specific EIID (NCBI gene no. spr0259), 50S ribosomal protein L13 (NCBI gene no. spr0271), ABC transporter ATP -binding protein-unknown substrate (NCBI gene no. spr1559), 50S ribosomal protein L10 (NCBI gene no. spr1212), phosphate ABC transporter ATP-binding protein (NCBI gene no. spr1253), choline transporter (NCBI gene no. ), ABC transporter substrate-binding protein (NCBI gene No. spr0083), peptidoglycan GlcNAc deacetylase (NCBI gene no. Spr1333), lactate oxidase (NCBI gene no. Spr0627), PTS system, fructose specific IIABC components (NCBI gene no. ), Zinc metalloprotease (NCBI gene no. Spr0581), hypothetical protein spr0904 (NCBI gene no. Spr0 904), fructose-bisphosphate aldolase (NCBI gene no. (NCBI gene no. spr1699), ribose-phosphate pyrophosphokinase (NCBI gene no. spr0028), endo-beta-N-acetylglucosaminidase (NCBI gene no. spr0867), transcriptional regulator (NCBI gene no. spr0635), manganese ABC transporter, ATP-binding protein (NCBI gene no. spr1492), PTS system, IIABC components (NCBI gene no. spr0668), glutamine synthetase, type I (NCBI gene no. spr0444), sodium alanine symporter family protein (NCBI gene no. spr0369), sugar ABC transporter, sugar-binding protein (NCBI gene no. spr1712), DNA-directed RNA polymerase subunit beta spr1776), polysaccharide biosynthesis protein, putative (NCBI gene no. spr1150), NADH oxidase (NCBI gene no. spr1323), maltodextrin phosphorylase (NCBI gene no. spr1916), preprotein translocase subunit SecY (NCBI gene no. , beta-galactosidase precursor (NCBI gene no. spr0565), isoleucyl-tRNA synthetase (NCBI gene e no. maltodextrin-binding protein (NCBI gene no. spr1918), and maltose / maltodextrin ABC transporter, maltose / maltodextrin-binding protein / RTI &gt;
[Claim 7] The protein detection method according to claim 7, wherein the disease caused by pneumococcal streptococci is any one selected from the group consisting of pneumonia, mumps, conjunctivitis, peritonitis, otitis media, endocarditis and sepsis.

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