KR101587859B1 - Composition for diagnosing infection of streptococcus pneumoniae comprising N-aceylglucosamine-6-phosphate deacetylase of streptococcus pneumoniae - Google Patents

Abstract

The present invention relates to a composition for diagnosing infection of pneumococcal streptococci comprising Nagase protein of Streptococcus pneumoniae. When the NagA protein of the present invention is used as an antigen, only the pneumococcal streptococci can be selectively detected without detecting other Gram-negative bacteria, and the detection sensitivity of the pneumococcal streptococci is excellent, and the infection of pneumococcal streptococci And the like.

Description

Technical Field [0001] The present invention relates to a composition for diagnosing infection of pneumococcal streptococci comprising NagA protein of pneumococcal streptococci. [0002] Composition for diagnosing infection of streptococcus pneumoniae is,

The present invention relates to a composition for diagnosing infection with Streptococcus pneumoniae comprising NagA protein of Streptococcus pneumoniae.

Streptococcus pneumoniae is a common cause of community-acquired pneumonia, which is a fatal disease causing more than 30% mortality. In addition, pneumococcal streptococci are the most common bacterial source of respiratory tract infections including acute deterioration of otitis media, sinusitis, chronic bronchitis, and bacterial pneumonia. Although strong antibiotics have been used for the treatment of pneumococcal streptococci for about 50 years, the morbidity and mortality of systemic infections remain almost unchanged, especially in the elderly. In addition, pneumococcal streptococcal infections are commonly recurrent. Currently, treatment of pneumococcal streptococci is a problem due to the limited efficacy of drugs with in vitro activity, antimicrobial resistance, and increased susceptibility to infection. Therefore, rapid diagnosis of bacteria is becoming an important factor in the survival rate of patients.

Pneumococcal streptococci have been diagnosed by the method of culturing and identifying the specimens. Recently, a rapid diagnosis method for easily diagnosing surface antigens of pneumococcal streptococci has been developed and used by immunochromatography. In the case of NOW S. pneumoniae (TM) of US Binax, Inc., which is a currently used pneumococcal strain test kit, polysaccharide is used as an antigen, but a diagnostic kit derived from a protein has not been commercialized to date. With the recent development of protein engineering techniques, it has become possible to mass-produce biomarkers derived from proteins. In particular, secreted proteins are recognized as external antigens in the host and can be used as a major biomarker to stimulate the host's immune system to make antibodies. Since the secreted protein is leaked to the outside of the bacteria, it is easy to separate from the cytoplasmic proteins in the pathogen.

On the other hand, nagA (N-acetylglucosamine-6-phosphate deaceylase) is a hydrolase that produces N-acetyl-D-glucosamine 6-phosphate and water as a substrate and D-glucosamine 6-phosphate and acetic acid.

Accordingly, the present inventors confirmed that NagA is excellent in detecting pneumococcal streptococci while continuing to study a protein marker for diagnosing pneumococcal streptococci, and completed the present invention.

It is an object of the present invention to provide a method for measuring the level of a nucleotide of a gene encoding NagA (N-acetylglucosamine-6-phosphate deacetylase), a reagent for measuring the level of a NagA protein, A composition for diagnosing infection of Streptococcus pneumoniae, and a diagnostic kit.

Another object of the present invention is to provide a method for diagnosing pneumococcal streptococcal infection, comprising the step of detecting the NagA protein of the pneumococcal streptococcus, the antibody of the protein or the nucleotide of NagA in the biological sample using the diagnostic composition Method.

The present invention relates to a method for measuring the level of a nucleotide of a gene coding for NagA (N-acetylglucosamine-6-phosphate deacetylase), a preparation for measuring the level of a NagA protein, A composition for diagnosing infection of pneumococcal streptococci and a diagnostic kit comprising the same.

Hereinafter, the present invention will be described in more detail.

When the NagA protein of the pneumococcal streptococcus of the present invention is used as an antigen, only the pneumococcal streptococci can be selectively detected without detecting other Gram-negative bacteria, and the detection sensitivity to pneumococcal streptococci is excellent, It can be used for diagnosis of Streptococcus infection.

In the present invention, "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to ascertain whether or not the otitis media, acute sinusitis, acute exacerbation of chronic bronchitis, bacterial pneumonia, or the likelihood of developing.

The diagnosis of pneumococcal streptococcal infection using the diagnostic composition according to the present invention can be performed by confirming the presence of the marker protein of the present invention in a biological sample. As the method for confirming the presence of the marker protein of the present invention, various assay methods known in the art can be used. Preferably, the presence or absence of the marker protein of the present invention can be confirmed in a biological sample by contacting the antibody with the antibody specifically binding to the marker protein of the present invention to determine whether the antigen-antibody complex is formed. As used herein, the term "antigen-antibody complex" refers to a combination of a specific protein in a biological sample and an antibody specifically recognizing it. In the present invention, the term "Lt; / RTI > Antibodies used in the present invention may be monoclonal or polyclonal antibodies, immunologically active fragments (e.g., Fab or (Fab) 2 fragments), antibody heavy chains, humanized antibodies, antibody light chains, engineered single chain Fv molecules, Chimeric antibody, and the like. The antibody used in the present invention can be prepared by a conventional method well known in the immunology field using the marker protein as an antigen. The protein used as the antigen of the antibody according to the present invention can be extracted or synthesized in nature and can be prepared by a recombinant method based on the DNA sequence. When a gene recombination technique is used, a nucleic acid encoding a protein is inserted into an appropriate expression vector, a host cell is cultured so that a protein of interest is expressed in the transformant transformed with the recombinant expression vector, Can be obtained by recovering the protein. For example, a polyclonal antibody can be produced by injecting a protein antigen into an animal and collecting it from the 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. 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 NagA protein is preferably an amino acid sequence represented by SEQ ID NO: 2. Further, the gene encoding the NagA protein is preferably the nucleotide sequence shown in SEQ ID NO: 1. In the present invention, the term "nucleotide" is a concept including RNA and DNA.

Agents that measure the nucleotide level of the gene encoding NagA include, but are not limited to, sense primers, antisense primers, and probes complementary to the NagA gene sequence.

Agents that measure the level of the NagA protein include, but are not limited to, antibodies, peptides, peptide mimetics, substrate analogs and aptamers that specifically recognize NagA.

The detection of a specific nucleic acid using the primer of the present invention can be carried out by amplifying the sequence of the target gene using an amplification method such as PCR and confirming amplification of the gene by a method known in the art. Further, the detection of a specific nucleic acid using a probe can be performed by contacting a sample nucleic acid with a probe under suitable conditions, and then confirming the presence or absence of a nucleic acid to be hybridized.

The term "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. The primers of the present invention can be chemically synthesized using methods known in the art such as, for example, the phosphoramidite solid support method.

The term "probe" means a nucleic acid fragment such as RNA or DNA consisting of several to several hundred bases capable of specifically binding to mRNA and is labeled to confirm the presence or absence of a specific mRNA. The probe can be produced in the form of an oligonucleotide probe, a short-chain DNA probe, a double-stranded DNA probe, an RNA probe, etc., and can be labeled with biotin, FITC, rhodamine, DIG or the like or labeled with radioisotope.

The probe can also be labeled with a detectable substance, for example, a radioactive label that provides a suitable signal and has a sufficient half-life. Labeled probes can be hybridized to nucleic acids on a solid support as known in the literature (Sambook et al., Molecular Cloning, A Laboratory Manual, 1989).

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Further, it can be modified by methylation, capping or the like by a known method. The composition for diagnosing pneumonia according to the present invention may further comprise a reagent generally used in the above-described method for detecting nucleic acid. For example, it may include metal ion salts such as dNTP (deoxynucleotide triphosphate), heat-resistant polymerase, and magnesium chloride required for the PCR reaction, and may include dNTPs, sequenase, and the like required for sequencing .

As the antibody specifically binding to NagA, it is preferable to use a polyclonal antibody or a monoclonal antibody. The antibody specifically binding to NagA may be prepared by a known method known to those skilled in the art, and commercially available NagA antibodies can be purchased and used. The antibody may be prepared by injecting an immunogenigenic NagA protein into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, and rabbits. The immunogen may be injected intramuscularly, intraperitoneally or by subcutaneous injection, and may generally be administered with an adjuvant to increase the antigenicity. Blood can be collected periodically from the external host and the antibody formed can be collected by collecting sera showing specificity to the antigen and the titer formed.

The peptide mimetics bind to the binding domain of the NagA protein to confirm the presence of the NagA protein. Peptide mimetics may be peptides or non-peptides and may include amino acids linked by non-peptide bonds, such as psi bonds (Benkirane, N., et al. J. Biol. Chem., 271: 33218-33224, Lt; / RTI > Also included within the scope of the present invention are "conformationally constrained" peptides, cyclic mimetics, at least one exocyclic domain, a binding moiety (binding amino acid) Can be. Peptide mimetics are structurally similar to the secondary structural features of the NAgA protein and are characterized by antibodies (Park, BW et al. Nat Biotechnol 18, 194-198, 2000) or water soluble receptors (Takasaki, W. et al. Nat Biotechnol 15, 1266- (Wrighton, NC et al., Nat Biotechnol 15, 1261-1265, 1997), which can mimic the inhibitory properties of gigantic molecules such as, for example, .

The aptamer is a single-chain DNA or RNA molecule, which has high affinity for a specific chemical molecule or biological molecule by an evolutionary method using an oligonucleotide library called SELEX (systematic evolution of ligands by exponential enrichment) (J. Tuerand L. Gold, Science 249, 505-510, 2005; AD Ellington and JW Szostak, Nature 346, 818-822, 1990; M. Famulok, et al. DS Wilson and Szostak, Annu Rev. Biochem., 68, 611-647, 1999). An aptamer can specifically bind to a target and detect the target.

 The diagnostic composition of the present invention may further comprise reagents known in the art which are used for immunological analysis in addition to the NagA antigen protein. Immunological analysis may include any method capable of measuring the binding of an antigen to an antibody. Such methods are well known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunoabsorbent assays (ELISA), immunoblotting, Farr assay, immunoprecipitation, latex agglutination, erythrocyte agglutination, agglutination, immunodiffusion, counter-current electrophoresis, single-radical immunodiffusion, immunochromatography, protein chip and immunofluorescence.

Reagents used in immunoassays include labels, solubilizers, and detergents that can generate a detectable signal. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring the enzyme activity and a reaction terminator. A marker capable of generating a detectable signal in the above enables the formation of an antigen-antibody complex to be qualitatively or quantitatively measurable, and examples thereof include an enzyme, a fluorescent substance, a ligand, a luminescent material, a microparticle, a redox molecule And radioactive isotopes. Examples of the enzymes include? -Glucuronidase,? -D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycosidoxidase, hexokinase, malate dehydrogenase, glucose-6 Phosphoric acid dehydrogenase, invertase and the like can be used. Examples of the minerals include fluorescein, isothiocyanate, rhodamine, picoeriterine, phycocyanin, allophycocyanin, fluorine synthase, and the like. Examples of the ligand include biotin derivatives, and examples of the luminescent material include acridinium ester, luciferin, and luciferase. Examples of the fine particles include colloidal gold and colored latex. Examples of redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone and the like. Radioactive isotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re. However, in addition to those exemplified above, any of them can be used as long as they can be used for immunological assays.

On the other hand, the diagnostic composition of the present invention may be provided in an immobilized state using various methods known on a suitable carrier or supporter to increase the speed and convenience of diagnosis (Antibodies: A Laboratory Manual, Harlow &Lane; Cold Spring Harbor, 1988). Examples of suitable carriers or supports are agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethylcellulose, polyacrylamide, polysterine, gabbro, filter paper, ion exchange resin, plastic film, plastic Polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, polyvinylpyrrolidone, toluene, glass, polyamine-methylvinylether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (test tube or well inner surface), planar (sheet, test strip). Preferably, the diagnostic composition of the present invention may be provided in the form of a diagnostic kit, a microarray, or a 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 comprises a sample pad applied to the sample, a releasing pad coated with the detection antibody, a developing membrane where the sample migrates and separates and an antigen-antibody reaction takes place (for example, nitro Cellulose) or strip, and an absorption pad.

The diagnostic kit may be prepared using a method commonly used in the art. These diagnostic kits include not only NagA antigen proteins but also tools, reagents and the like commonly used in the art used for immunological analysis. Such tools / reagents include, but are not limited to, suitable carriers, labeling agents capable of producing a detectable signal, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose and combinations thereof.

Further, the present invention provides a method for providing information for diagnosing infection of Streptococcus pneumoniae comprising the steps of: detecting the NagA protein of the pneumococcal streptococcus, the antibody of the protein or the nucleotide of NagA in the biological sample using the composition; do.

The biological sample includes, but is not limited to, cells, blood, urine, saliva, tissue, and the like.

When the NagA protein of the pneumococcal streptococcus of the present invention is used as an antigen, only the pneumococcal streptococci can be selectively detected without detecting other Gram-negative bacteria, and the detection sensitivity to pneumococcal streptococci is excellent, It can be used for diagnosis of Streptococcus infection.

Brief Description of the Drawings Fig. 1 is a diagram showing the process of producing recombinant NagA from Nagase pneumoniae strain BAA-255 and the expression of NagA.
FIG. 2 is a view showing the reactivity between different species of the niger protein of the present invention against an antibody.
FIG. 3 is a graph showing the detection of antibodies against NagA in mouse sera infected with pneumococcal streptococci using NagA of the present invention.

Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

[ Example  1] recombinant pneumococcal streptococcal protein NagA Manufacturing

To obtain NagA protein of pneumococcal streptococci, NagA gene of BAA-255 strain was obtained by PCR method using primers of SEQ ID NOS: 3 and 4. The obtained gene (the NagA gene sequence is shown in SEQ ID NO: 1) was ligated to the expression vector (pET28a) and inserted into E. coli. Thereafter, the NagA protein expressed by the SDS-PAGE method (represented by SEQ ID NO: 2 of the NagA protein) was confirmed. The process for producing the recombinant NagA and the purification result for NagA are shown in FIG.

As shown in Fig. 1, it was confirmed that the Nagase protein of 47.52 Kda was purified.

[ Example  2] nagA  Antibody Interspecific  Check reactivity

2.1 Recombination NagA ≪ / RTI >

The recombinant Nag A protein prepared in Example 1 was used as an antigen to prepare an antibody. Specifically, 600 mg of NagA protein was injected into the rabbit four times to obtain an antibody against NagA. Then, serum was mixed with 20 mM Tris-HCl (pH 7.0) at a ratio of 1: 1 to separate IgG from the rabbit serum, 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 the IgG attached to the column was separated using 0.1 M glycine-HCl (pH 2.7) buffer. The separated IgG fractions were purified by buffer exchange using Viva spin (10 kDa).

2.2 Recombination NagA Other antibodies against Interspecific  Check reactivity

The reactivity of BAA-255 over-expressing nagA antibody was used to confirm the reactivity between the different species. Pneumoniae clinical strain 521, s.pneumoniae clinical strain 521, s.pneumoniae clinical strain 6613, s.pneumoniae clinical strain 7663, K. pneumoniae, A. baumannii DU202 and P. putida KT2440 secreted proteins Or cytoplasmic proteins were separated, followed by Western blotting using antibodies. The results are shown in Fig.

As shown in Fig. 2, it was confirmed that nagA was detected in secretory proteins and cytoplasmic proteins isolated from three pneumococcal streptococci (s.pneumoniae clinical strain 521, s.pneumoniae clinical strain 6613 and s.pneumoniae clinical strain 7663) Respectively.

However, it was confirmed that nagA was not detected in both secretory protein and cytoplasmic protein by using three representative strains of Gram-negative bacteria (K. pneumoniae, A.baumannii DU202 and P. putida KT2440) other than Gram-positive pneumococcal streptococci Respectively.

These results indicate that nagA can be identified in pneumococcal streptococci, which are the same species, but not in other species, confirming that nagA can be used as a biomarker to identify pneumococcal streptococci.

[ Example  3] In infected mice nagA  Check for antibody formation

Five groups of mice were divided into five groups. BAA-255 cells were subcutaneously injected into PBS at 1 × ^{10 2} , 1 × 10 ⁴ , 1 × 10 ⁶ , and 1 × ^{10 8} , respectively. Infected. The other group received intraperitoneal injection of PBS as a control. Three injections were made every two weeks and blood was drawn 2 weeks after the last injection and diluted 1: 500. Thereafter, dot blotting was performed using NagA prepared in Example 1 as an antigen. The results are shown in Fig.

As shown in FIG. 3, it was confirmed that an antibody against NagA, which can detect up to 20 ng, was formed in the mouse serum of the infected group except for the control group.

These results indicate that nagA is sensitive to the antibodies produced in the host infected with Streptococcus pneumoniae.

As a result, it was confirmed that past infections and active infections can be detected without depending on the amount of the cells through experiments using a mouse.

<110> Korea Institute of Science and Technology information <120> Composition for diagnosing infection of streptococcus pneumoniae          comprising N-aceylglucosamine-6-phosphate deacetylase of          streptococcus pneumoniae <130> P-133 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1152 <212> DNA <213> Artificial Sequence <220> <223> It is NagA nucleotide sequence of streptococcus pneumoniae <400> 1 ttatgcttga taacgtttta cgccatctag gtaggttgct accaattcca aatctttatc 60 taatacgatg aagtcagcgt cgtagccttc acggatttgg ccacagacgt catcgatgtg 120 aacggattta gctgggttga agctggccat catgactgct tcatgcggat tcgcaattcc 180 ccattcgacc acattcttca aaccatcttt gagtttgagg atagaacctg ccaagttacc 240 tgtcgatttg aggcgtgcag ttccatttgc aacgacaact gggaattctc ccaacatata 300 atctccgtct tccaatcccc cagctgtcat acagtctgtg ataagagcga tgttttctgt 360 tcctttttgt ttgataagaa tttcgcaagc ctttggatct acgtggtgac catcacagat 420 caactctgca taggtatgtg gcaattggta catggctcca accatcccca attcacggtg 480 agtcaaccca cgcattccat tgtaggcatg cacccaaaca ctcgctccag catcgactgc 540 ttttttggct tcatcaaaag tcgcgtttga atgtccaaga gcaaccgtca caccttcgcc 600 cgtaactgta cgaacaaagt cttccacccc atcacgttct ggtgcaaggg caattttatt 660 aagcaagcca tttgccgctt tttgccaaga atgaaactcc tcaacacccg ggtctctcat 720 ataagttgga ttttgtgccc ccttaaaagt ttctgtgaaa tatggacctt cataataaat 780 cccacgaatc ttagcacctg ttgcttcttt ataatggttt ccaagatttt cagtgactgc 840 aagcaattgc tcataagtgg ctgttaaagt tgtgggtaag aaactggtaa caccggtact 900 aagaagtcct tcactcatag tatgcaatgt accttcaatg ttgttgtcca tcacatctac 960 acctgcatat ccatgaatat gagtatccac aagacctggg gcaatgctat aacctgtata 1020 gtcaatcacc tcagcccctt caggaatctg ctctacatgt ttcccaaact tgccgtccac 1080 aagttccaag taaccacctc gacgaactcc gtgtgggtag aaaaactgat ccgctttaat 1140 atagttaggc at 1152 <210> 2 <211> 383 <212> PRT <213> Artificial Sequence <220> <223> It is NagA amino acid sequence of streptococcus pneumoniae <400> 2 Met Pro Asn Tyr Ile Lys Ala Asp Gln Phe Phe Tyr Pro His Gly Val   1 5 10 15 Arg Arg Gly Gly Tyr Leu Glu Leu Val Asp Gly Lys Phe Gly Lys His              20 25 30 Val Glu Gln Ile Pro Glu Gly Ala Glu Val Ile Asp Tyr Thr Gly Tyr          35 40 45 Ser Ile Ala Pro Gly Leu Val Asp Thr His Ile His Gly Tyr Ala Gly      50 55 60 Val Asp Val Met Asp Asn Asn Ile Glu Gly Thr Leu His Thr Met Ser  65 70 75 80 Glu Gly Leu Leu Ser Thr Gly Val Thr Ser Phe Leu Pro Thr Thr Leu                  85 90 95 Thr Ala Thr Tyr Glu Gln Leu Leu Ala Val Thr Glu Asn Leu Gly Asn             100 105 110 His Tyr Lys Glu Ala Thr Gly Ala Lys Ile Arg Gly Ile Tyr Tyr Glu         115 120 125 Gly Pro Tyr Phe Thr Glu Thr Phe Lys Gly Ala Gln Asn Pro Thr Tyr     130 135 140 Met Arg Asp Pro Gly Val Glu Glu Phe His Ser Trp Gln Lys Ala Ala 145 150 155 160 Asn Gly Leu Leu Asn Lys Ile Ala Leu Ala Pro Glu Arg Asp Gly Val                 165 170 175 Glu Asp Phe Val Arg Thr Val Thr Gly Glu Gly Val Thr Val Ala Leu             180 185 190 Gly His Ser Asn Ala Thr Phe Asp Glu Ala Lys Lys Ala Val Asp Ala         195 200 205 Gly Ala Ser Val Trp Val His Ala Tyr Asn Gly Met Arg Gly Leu Thr     210 215 220 His Arg Glu Leu Gly Met Val Gly Ala Met Tyr Gln Leu Pro His Thr 225 230 235 240 Tyr Ala Glu Leu Ile Cys Asp Gly His His Val Asp Pro Lys Ala Cys                 245 250 255 Glu Ile Leu Ile Lys Gln Lys Gly Thr Glu Asn Ile Ala Leu Ile Thr             260 265 270 Asp Cys Met Thr Ala Gly Gly Leu Glu Asp Gly Asp Tyr Met Leu Gly         275 280 285 Glu Phe Pro Val Val Ala Asn Gly Thr Ala Arg Leu Lys Ser Thr     290 295 300 Gly Asn Leu Ala Gly Ser Ile Leu Lys Leu Lys Asp Gly Leu Lys Asn 305 310 315 320 Val Val Glu Trp Gly Ile Ala Asn Pro His Glu Ala Val Met Met Ala                 325 330 335 Ser Phe Asn Pro Ala Lys Ser Val His Ile Asp Asp Val Cys Gly Gln             340 345 350 Ile Arg Glu Gly Tyr Asp Ala Asp Phe Ile Val Leu Asp Lys Asp Leu         355 360 365 Glu Leu Val Ala Thr Tyr Leu Asp Gly Val Lys Arg Tyr Gln Ala     370 375 380 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> It is forward primer for NagA gene <400> 3 agaagaacat atgcctaact atattaaagc ggatca 36 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> It is reverse primer for the NagA gene <400> 4 gaactcgagt gcttgataac gttttacgcc a 31

Claims (9)

An N-acetylglucosamine-6-phosphate deacetylase (NagA) protein represented by the amino acid sequence of SEQ ID NO: 2 of pneumococcal streptococci, an agent for measuring the nucleotide level of the gene encoding NagA represented by the nucleotide sequence of SEQ ID NO: 1, Wherein the composition comprises at least one member selected from the group consisting of an agent for measuring the level of the protein and a preparation for measuring the level of the protein. delete delete 2. The method according to claim 1, wherein the agent for measuring the nucleotide level of the gene encoding NagA is at least one selected from the group consisting of a sense primer complementary to the NagA gene sequence, an antisense primer and a probe, Composition for the diagnosis of infection. The method according to claim 1, wherein the agent for measuring the protein level of NagA is at least one selected from the group consisting of an antibody specifically recognizing NagA, a peptide, a peptide mimetic, a substrate analog, and an aptamer. A composition for diagnosing infection of a bacterium. An N-acetylglucosamine-6-phosphate deacetylase (NagA) protein represented by the amino acid sequence of SEQ ID NO: 2 of pneumococcal streptococci, an agent for measuring the nucleotide level of the gene encoding NagA represented by the nucleotide sequence of SEQ ID NO: 1, Wherein the test kit comprises at least one member selected from the group consisting of an agent for measuring the level of the protein and a preparation for measuring the level of the protein. An N-acetylglucosamine-6-phosphate deacetylase (NagA) protein represented by the amino acid sequence of SEQ ID NO: 2 of pneumococcal streptococci, an agent for measuring the nucleotide level of the gene encoding NagA represented by the nucleotide sequence of SEQ ID NO: 1, A microarray for infection of pneumococcal streptococci comprising at least one member selected from the group consisting of agents for measuring the level of a protein. Detecting the Nagase protein of pneumococcal streptococcus, the antibody of said protein, or the nucleotide of NagA in the biological sample using the composition of claim 1. 12. A method of providing information for diagnosis of infection of pneumococcal streptococci, 9. The method according to claim 8, wherein the biological sample is at least one selected from the group consisting of cells, blood, urine, saliva, and tissues.

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