KR20030056628A - Identification of streptococcus pneumoniae using alcohol dehydrogenase derived from streptococcus pneumoniae - Google Patents

Abstract

The present invention relates to a method for identifying pneumococci by using alcohol dehydrogenase (ADH) of pneumococci secreted out of cells and having high retention, or adh gene expressing this protein.

That is, the antibodies induced by pneumococcal ADH not only reacted with cell lysates of other bacteria, but also reacted with various pneumococcal strains secreted out of the cell and isolated from the clinic. In addition, since pneumococcal ADH is induced in a large amount when impacted with alcohol, the reactivity is improved compared to the conventional method, so ADH of pneumococcal ADH can be usefully used in clinical pathology as a rapid and accurate identification method of pneumococcal pneumonia.

Description

Identification method of pneumococci using pneumococcal alcohol dehydrogenase {IDENTIFICATION OF STREPTOCOCCUS PNEUMONIAE USING ALCOHOL DEHYDROGENASE DERIVED FROM STREPTOCOCCUS PNEUMONIAE}

According to the present invention, alcohol dehydrogenase (NAD oxidoreductase, EC 1.1.1.1: ADH), which has been identified as Streptococcus pneumoniae heat shock protein (HSP), is antigenic and secreted out of the cell, As the antibodies raised against the drug reveals a property that does not react with the corresponding protein of other organisms, the present invention relates to a method for quickly and easily identifying Streptococcus pneumoniae using the same.

Pneumococci have a thick polysaccharide capillary outside the cell wall and live in the nasopharnyx of normal people, causing disease when they become less resistant or infected by other pathogens (Atlas, 1995). Antibiotic treatment and 23 polysaccharide vaccines Nevertheless, the mortality rate is about 5-10% (AlonsoDeVelasco et al., 1995). However, infants and the elderly have higher mortality rates, causing more than 4 million deaths from pneumonia each year worldwide (Siber, 1994). It also causes rheumatic heart disease, meningitis and otitis media, and acts as a major causative agent of meningitis in adults (Joklik et al., 1988), but the blood brain barrier (dense cell structure of the meninges) prevents antibiotic penetration. It is difficult for antibiotics to be transported into the meninges, resulting in high mortality and permanent neurological disorders even when cured (Tuomanen, 1993, 1999).

Currently, the most commonly used pneumococcal identification method is to measure biochemical reactions and metabolites after culturing the bacteria. In particular, once the shape of the bacteria is identified as cocci, the sensitivity to optochin / bile acid and reactivity with antibodies to the capsular polysaccharides are measured to determine whether the bacteria are pneumococci. Difficulty in interpreting the results can be mistaken for resistance to optokines or bile acids (Fenoll et al., 1990; Gardam and Miller, 1998; Isenberg, 1995; Mundy et al., 1998). In addition, the serotype determined according to pneumococcal capsular polysaccharide is using a polyvalent antiserum or monoclonal antibody made against polysaccharide (Henrichsen, 1995; Musher, 1995). However, this identification method is not only reliable but also has the disadvantage of not being able to determine serotypes up to 20% of pneumococci (Ridgway et al., 1995; Cima et al., 1996; Fenoll et al., 1997). Antisera specifically induced against pneumococcal capsular polysaccharide antigens and / or C-polysaccharides may also be used to identify pneumococci by reacting with bacteria other than pneumococci, for example α- streptococci (Sørensen, 1994).

Such an immune response can be very useful for emergency clinical examinations because of the short response time (minutes to tens of minutes). However, in order to use the immune response, it is preferable to use antibodies induced against antigens specifically present on the surface of the bacteria rather than the interior (cytoplasm) of the bacteria. This is because some pneumococci are resistant to optokines and bile acids and do not autolyze. In this case, a complete antigen such as a protein is advantageous for generating a high titer of antibody. In addition, by using protein antigens on the cell surface, bacteria can be easily identified by sedimentation or aggregation.

Recently, there have been attempts to identify pneumolysin, which is expressed by pneumococci and affects the development of organs. However, pneumolysine is a polypeptide present in the cell and induced by pneumolysin. Even if the antibody can be used, it is difficult to identify pneumococci quickly because it cannot directly interact with pneumolysin in cells without cell lysis treatment.

Pneumococcal surface protein A (PspA), one of the main pathogens of pneumococci, varies in size and has a large difference in amino acid sequence (Briles et al., 1998). For pneumococci, problems may arise in identification.

In addition, US Pat. Nos. 5,981,229 and 5,928,900, which disclose the extracellular secreting proteins of Gram-positive bacteria, in particular pneumococcus, disclose methods for the diagnosis of sterile vaccines and pneumococci based on these proteins.

However, the above-described prior art has a disadvantage in that a large amount of these proteins or identification factors cannot be induced during identification reactions.

In order to provide a rapid and effective diagnosis method of pneumococcal disease while overcoming these problems, the identification of proteins that can be induced by ethanol while being highly antigenic and secreted to the outside of cells, and the identification of pneumococci using the same There is still a need for a rapid identification method.

All organisms express stress proteins (heat shock proteins (HSPs)) to combat heat shock, DNA damaging agents, antibiotics and environmental changes, as well as inhibit the degeneration of stress-induced proteins (Caperone function), as well as transfer of cellular proteins to cell membranes. (Translocation) (Craig et al., 1993; Hendrick and Hartl, 1993). In bacteria other than pneumococci, HSP is induced by alcohol bombardment, especially large amounts of DnaK and GroEL (Bernhardt et al., 1997; Boutibonnes et al., 1991; Boutibonnes et al., 1993; Qoronfleh et al., 1990; Van Bogelen et al., 1987). In pneumococcus, DnaK and GroEL were not induced at all but instead induced large amounts of ADH to 23% of the total protein (Choi et al., 1999). These findings were thought to indicate that alcohol shock-induced ADH, like other bacteria's HSP, inhibits protein denaturation.

In general, ADH is a coenzyme that oxidizes alcohol using NAD (H) or NADP (H) and is a metalloprotein containing mostly zinc ions in the active site (Jeffery et al., 1981; Neale et al., 1986; Reid). And Fewson, 1994). Escherichia coli and Bacillus subtilis (B. subtilis), AdhE, consisting of about 900 amino acids, has ADH activity, acetaldehyde dehydrogenase activity, acetyl CoA conversion to alcohol, as well as inactivation of pyruvate formate lyase in anaerobic conditions. (Kessler et al., 1991). Also, E. coliadhEMutant is lactic acid bacteriaadhEComplementation by genes (Arnau et al., 1998) shows that ADH of Gram-negative and Gram-positive bacteria is very conservative in function.

Therefore, although this protein is generally present in all bacteria and is conserved in its function, ADH protein, which is produced only in pneumococci in large quantities in contrast to other bacteria during alcohol shock, inhibits the degeneration of pneumococcal proteins by alcohol. Based on these findings, the researchers found out which part of pneumococcal ADH protein is present in pneumococcal cells and how it differs from other bacteria. In addition, by making an antibody against this ADH protein and measuring the property that the antibody does not react with other pathogens, we want to determine whether it can be used as a biomarker that can be used to identify pneumococci.

As such, the present inventors concentrated on the proteins that can be used for rapid identification of pneumococci without exhibiting the disadvantages of the prior art described above. It is an object of the present invention to select a protein that can be used quickly and usefully in identifying pneumococci and to provide a method for quickly and accurately identifying pneumococci using the protein.

1 is a comparative experiment of whether pneumococcal ADH and GroEL proteins are secreted out of cells under normal culture temperature and heat shock.

Figure 2 shows the change in the protein pattern induced in pneumococcal when the pneumococcal shock to various stresses.

Figure 3 shows the results of the reactivity test by immunoblot of the antibody of rabbits and the cell lysates of various cells caused by pneumococcal ADH.

4 is pneumococcal alcohol dehydrogenase (ADH) and other bacteria, i.e., Staphylococcus aureus, the amino acid sequence of the ADH: (LL L. lactis) (S. aureus:: SA), Escherichia coli (E. coli EC), lactic acid bacteria It is a comparison.

Figures 5 and 6 show the reactivity of pneumococcal lysate with pneumococcal ADH antibodies isolated from clinical trials.

Figure 7 shows the identification results of pneumococci using a part of the pneumococcal adh gene.

The present invention is a protein identified as an indicator for identifying pneumococci, ie, alcohol of pneumococci, which is secreted to the cells of pneumococci without heat shock and can be used for identification of pneumococci quickly and usefully. A dehydrogenase (hereinafter abbreviated as ADH) and a method for identifying pneumococci using this protein are provided.

In one aspect, the present invention provides a method for the detection of an organism complex comprising: i) collecting a biological specimen, ii) contacting the biological specimen with an antibody induced against ADH of pneumococcal, and iii) detecting an immunocomplex formed between the antibody and the specimen. It provides a method for identifying the presence of pneumococcal in the organism, including the step of indicating the presence of pneumococcal in the organism.

Biological specimens include specimens that may contain pathogens, such as blood, cerebrospinal fluid, bile, or specimens taken from the mouth or throat (using cotton swabs). In particular, blood or cerebrospinal fluid is preferred in order to reduce false-positive reactions due to contamination during specimen collection.

The pneumococcal ADH is composed of 883 amino acids and has a molecular weight of about 104,000 (104 kDa, p104), the amino acid sequence of which is known as shown in FIG. 4, but the function and mode of action of the protein are known. There is no bar at all. As described above, based on the report that pneumococcal ADH is induced in 23% of the total protein in pneumococcus instead of DnaK and GroEL of other bacteria, the present inventors have found that pneumococcal ADH, its function and physiology, It is assumed that there will be specificity in biochemical properties. In various experiments demonstrating this presumption, the ADH of pneumococci is isolated after mass expression according to genetic engineering methods conventional in the art or prepared according to the protein isolation and purification method commonly used in the art. Can be. Briefly, the ADH protein of pneumococcal inserts an open reading frame portion of a gene encoding pneumococcal ADH into an E. coli expression vector (e.g., a pET vector containing a codon encoding histidine). Isopropyl thio-galactoside (IPTG), an expression inducing agent, may be added to express ADH in large quantities and then passed through a nickel column for simple purification of ADH. Alternatively, after incubating pneumococci, ethanol may be added to 1%, followed by further incubation for 10 to 30 minutes to induce ADH, followed by SDS-PAGE to cut and isolate an ADH band of 104 kDa.

In order to explore the differences between various ADHs in terms of genetics, we compared the nucleotide sequences of genes encoding ADHs through BLAST research. It showed high similarity with the members of ADH system.E. histolytica) With ADH2L.Lactis (lactis) 60% and 46% identity with ADH and 75% and 63% similarity, respectively. AlsoE. coli(E. coli) 64% ADH, Clostridium acetobutylicum (Clostridium acetobutylicum) 60% with ADH,C.Acetobutyliccum (acetobutylicum) NADPH dependent butanol dehydrogenase and 61%, trichomonas vaginalisTrichomonas vaginalis) 55% with NADPH dependent butanol dehydrogenase,B.Subtilis (subtilis) 49% similarity with ADH.

Antibodies raised against pneumococcal ADH can also be prepared according to antibody production methods commonly used in the art. For example, the pneumococcal ADH antibody can be obtained by injecting the ADH protein obtained as described above into a rabbit or mouse to obtain a polyclonal antibody or a monoclonal antibody. For methods of producing such antibodies, see Sambrook and Russell, 2001; Harlow and Lane, 1988; Ausubel et al., 1999].

Detection of immunocomplexes can be carried out by a variety of methods well known to those skilled in the art, including immunofluorescence (IF), immunoprecipitation, immunoaggregation or Western immunoblot. For example, briefly describing Western immunoblot analysis, incubation of antibodies and organisms raised against selected proteins is followed by incubation with anti-human IgG Ab conjugated with alkaline phosphatase. The formed immune complexes are then detected by color reaction using NBT (nitroblue tetrazolium) / BCIP (5-bromo 4-chloro 3-indolyl phosphate). The detection of such immunocomplexes suggests the possibility of the presence of pneumococci in the specimen of the organism.

In addition, as a genetic identification method instead of the above-described immunological identification method, the present invention provides a nucleic acid isolated from an organism specimen using a portion of the adh gene expressing pneumococcal ADH as a primer or using all or a portion of the adh gene as a marker. Provided are methods for identifying the presence of pneumococci in organisms characterized by reacting with components. That is, a part of adh gene is used as a primer and chromosomal DNA or RNA isolated from bacteria is used as a template to amplify pneumococcal adh gene in a conventional manner, or a constant matrix as a marker for all or part of adh gene And immobilized to detect the hybridization with pneumococcal adh gene.

In this embodiment, the biological specimen includes those mentioned in the above-mentioned embodiment.

In a specific embodiment, the present invention provides a process for i) collecting a biological specimen, ii) isolating and colonizing various microorganisms present in the specimen, iii) selecting cocci from colonized clones if necessary, iv) Collecting and suspending colonies of isolated cultures or selected colonies, and v) suspending them in a homolysis buffer, and v) adding the part of the adh gene expressing pneumococcal ADH to the suspension, and in vitro DNA under conditions of suitable polymerization. It provides a method for identifying the presence of pneumococcal in the organism, including amplifying by a chain polymerization reaction and determining whether the amplified gene is present in the organism.

In another specific embodiment, the present invention provides a method for identifying the presence of pneumococci in living organisms by immobilizing adh gene expressing pneumococcal ADH protein as a marker and detecting DNA hybridized thereto. Ie all or part of the adh gene as an indicator, immobilized in an appropriate matrix (e.g. nylon membrane) in a conventional manner (Sambrook and Russell, 2001), and then hybridized with chromosomal DNA or RNA isolated from bacteria, It provides a method for identifying the presence of pneumococcal in the organism, including detecting the resulting complex.

Isolation and culture of the various microorganisms present in the organism specimen can be carried out by a method well known to those skilled in the art. To exemplify a specific method, the culture is cultured in blood agar medium, Todd-Hewitt broth or Brain heart infusion broth with 0.5% yeast extract.

In each microorganism isolated alone in this manner, the selection of cocci can be carried out by microscopy under an optical microscope. However, this method does not have to be performed to identify the cocci.

For the PCR amplification reaction to identify whether the selected or cultured pneumococci are pneumococci, primers with the following sequence can be used: p104yj02 (5'-CGG AAT TCA CTG ATA AAA AAA CTGT-3 ' ) And p104yj04 (5'-GCC AAG CTT AAT TAT TTA CGG CGT CC-3 '). This primer can be easily produced by requesting a DNA synthesizer or nucleic acid synthesis company. In addition, since there are many parts having low similarity between the pneumococcal adh gene and other bacteria adh genes, a part of such a low similarity may be used as a primer.

Suitable reaction conditions for the PCR reaction were first denatured at 94 ° C. for 5 minutes, re-annealed the primer and chromosomal DNA for 1 minute at 44 ° C., and then polymerized at 72 ° C. for 2 minutes, followed by 1 at 94 ° C. for DNA amplification. 30 cycles of denaturation for 1 minute at 58 ° C., resuspension recovery at 2 ° C., and polymerization at 72 ° C. for 2 minutes are repeated. Finally, incubation at 94 ° C. for 1 minute, 58 ° C. for 1 minute, and 72 ° C. for 12 minutes is performed. The reaction conditions at this time can be sufficiently changed by those skilled in the art.

The amplified DNA can be detected by electrophoresis or the like. If the amplified DNA band is confirmed, it can be considered that pneumococcal exists in the specimen of the organism.

The presence or absence of pneumococci identified by the identification method of the above-mentioned embodiments suggests the presence of pneumococcal induced diseases in the organism and / or the possibility of progression to pneumococcal related diseases.

Accordingly, the present invention provides, as another aspect, a method for identifying, monitoring and / or diagnosing the progression to pneumococcal-induced diseases and / or pneumococcal related diseases using the above-described method for identifying pneumococci.

In still another aspect, the present invention provides a kit for identifying pneumococci, including an easily detectable labeled pneumococcal ADH antibody and a coloring reagent.

Hereinafter, the physiological, biochemical and immunological characteristics of pneumococcal ADH are examined through Examples to explain the present invention in detail, and examples of the method of using pneumococcal ADH protein for the identification of pneumococci based on the results are illustrated. It was. This example is merely illustrative of the present invention and is not intended to limit the present invention thereto.

The pneumococcal ADH used in this example was obtained by mass expression and purification in Escherichia coli using genetic engineering methods as described above, and the ADH antibody induced against this ADH was subcutaneously subcutaneously treated with rabbit 6 times in ADH. Serum produced by injection was collected and prepared.

Example

I. Biochemical Characterization of Pneumococcal Alcohol Dehydrogenase (ADH)

Example 1 Extracellular Secretion of Pneumococcal ADH

To investigate whether pneumococcal ADH protein is secreted out of cells by thermal shock, the non-pathogenic R-type pneumococcal CP1200 strain [Choi et al., 1999; Rhee and Morrison, 1988] were incubated at 30 ° C. using CAT medium using casein hydrolysates as described in the literature, and at the same time incubated at 42 ° C. for 30 minutes for thermal shock reactions, followed by centrifugation of each culture. Bacteria and culture supernatants were separated and developed on 12.5% acrylamide gel and reacted with ADH antibody. As a positive control, the bacterial lysates after heat shock were developed together in lane 1. In addition, the comparison solution was presented by reacting the homolysate after the thermal shock with the GroEL antibody to the GroEL protein known as the thermal shock expression protein. As a result, as shown in FIG. 1, unlike the GroEL protein expressed only in the cell after the thermal shock, the ADH protein was secreted out of the cell even at the normal culture temperature regardless of the thermal shock. In FIG. 1, lane 1 shows the bacterial lysate after applying a thermal shock, lane 2 shows the bacterial culture supernatant at a normal culture temperature, and lane 3 shows the bacterial culture supernatant after applying a thermal shock.

Example 2 Effects of Various Stress on the Expression of Pneumococcal ADH

Each culture was treated under various stresses to observe the protein patterns induced by pneumococci when pneumococci were impacted by various stresses. In this case, in order to easily observe the change in protein expression pattern, the culture medium was cultured until the absorbance was 0.1 in CAT medium, and then replaced with a labeling medium containing a small amount of methionine [Choi et al., 1999]. 1 μl of [ ³⁵ S] -methionine (555 MBq / ml) was added.

In order to apply thermal shock, the pneumococcal CP1200 strain was incubated as in Example 1 above, and added to the pneumococcal culture so that the final concentration of ethanol was 1% in order to apply alcohol shock and then incubated at 37 ° C. for 30 minutes. . In order to measure the stress caused by the salt concentration change, sodium chloride was added to the final concentration of 0.3M in the pneumococcal CP1200 culture medium, and hydrogen peroxide was added to the final concentration of 400μM to give an oxidative shock. Methane sulfonate and ethyl methane sulfonate were added at a final concentration of 0.01% and 0.1%, and incubated at 37 ° C for 30 minutes.

After incubation as described above, only 20 µl of the cells were incubated with a buffer solution (5 mM Tris-HCl, pH 8.0, 30 mM ethylenediamine-N, N, N ', N'-tetraacetic acid [EDTA], 0.1% Triton X). The cycle of suspension in -100, 0.25 mg / ml phenylmethanesulfonic acid fluoride [PMSF], 1 mM dithiothreitol) followed by rapid freezing and dissolving in ice water was repeated until the bacteria completely dissolved. The bacterial lysates were centrifuged to carry out 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) of the supernatant containing the protein in the cytoplasm, and autoradiography was performed to identify the proteins induced by stress. At this time, unlike pneumococcal stress response, major stress proteins such as DnaK and GroEL were not induced by ethanol shock, but ADH was induced significantly.

The results are shown in FIG. In Figure 2 lane 1 is normal culture temperature, lane 2 is thermal shock, lane 3 is 1% ethanol, lane 4 is 0.3M NaCl, lane 5 is 0.4M hydrogen peroxide, lane 6 is 0.01% methyl methane sulfonate, lane 7 is 0.1 Protein expression pattern upon impact with% ethyl methane sulfonate is shown. Here, p104 represents pneumococcal ADH protein, p84 represents ClpL protein, p73 represents DnaK protein, and p65 represents GroEL protein.

II. Immunological Characterization of Pneumococcal ADH

Example 3: Antigen Specificity of Pneumococcal ADH Under Normal Culture Conditions

For CAT liquid medium (1 L), pneumococcal CP1200 (Choi et al., 1999), Staphylococcus aureus ATCC 6538P, Bacillus subtilis Marburg strain (Boylan et al., 1988), Escherichia coli MG1655 strain (Hanahan, 1983), human lung cancer cell line A549 (ATCC) CCL 185), typhoid bacteria ATCC 27870 strain, lactobacillus ATCC 11454 strain, yeast ATCC 287 strain was prepared by culturing the medium according to the instructions of the supplier or published in the literature, and then the Staphylococcus aureus adsorbed ADH antibody as follows React with That is, after culturing the Staphylococcus aureus Cowan 1 (ATCC 12598) strain according to the supplier's instructions, the protein A secreted outside the cells is fixed according to the previously reported immobilization method (Kronwall, 1973). In other words, Staphylococcus aureus secretes protein A outside the cell and binds to the immunoglobulin Fc part. Therefore, when the pneumococcal ADH antibody is reacted with Staphylococcus aureus, the Fc part of the ADH antibody is on the surface of Staphylococcus aureus. Binds to protein A. When reacted with pneumococci containing / secreting ADH, ADH binds to the Fab portion of the Staphylococcus aureus surface and aggregates. In this case, the immobilized Staphylococcus aureus may be first reacted with E. coli JM105 cell lysate to increase reaction specificity, thereby removing components that react with Protein A of Staphylococcus aureus nonspecifically. In detail, some of the cultured bacteria or colonies were suspended in 25 μl of phosphate-buffered saline (PBS) containing 0.05% Triton X-100, pH 7.3, and incubated at 37 ° C. for 20 minutes, followed by a desktop centrifuge. After centrifugation for 5 minutes, only the supernatant was dropped onto 25 µl of Staphylococcus aureus solution adsorbed with ADH antibody and slide glass, and left and right mixed for a while to observe the aggregation reaction visually within 3 minutes. As a result, only pneumococci aggregated while reacting with the Staphylococcus aureus solution, and other bacteria did not aggregate at all. To make it easier to observe the aggregation reaction, one drop of commonly used dyeing reagents may be dropped.

Example 4 Antigen Specificity of Pneumococcal ADH Under Alcohol Shock

The colonies obtained after incubating pneumococci, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, human lung cancer cell lines A549, Typhoid bacteria, Lactobacillus, and yeast as in Example 3 were suspended in 25 µl of medium containing 1% ethanol or PBS 37 Incubate for 10 minutes at ℃. Treatment of 1% ethanol induces ADH, resulting in 10 times more sensitive aggregation. 25 μl of phosphate-buffered saline (PBS) containing 0.15% Triton X-100, pH 7.3 was added thereto, followed by incubation at 37 ° C. for 20 minutes, followed by centrifugation for 5 minutes using a tabletop centrifuge, and then only the supernatant. When pneumococcal coagulation reaction was observed by visual observation within 3 minutes when mixed with 25 µl of Staphylococcus aureus solution adsorbed on ADH antibody and shaken from side to side for a while after mixing on the slide glass. Not shown.

Example 5: Antigen Specificity of Pneumococcal ADH against Different Bacterial Lysis Solutions

As described in Example 3, the cells obtained by culturing pneumococcus, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, human lung cancer cell line A549, typhoid fever, lactic acid bacteria, and yeast were respectively lysed to obtain a buffer solution (5 mM Tris-HCl [ pH 8.0], 30 mM ethyldiamine-N, N, N ', N'-tetraacetic acid [EDTA], 0.1% Triton X-100, 0.025% [w / v] PhenylMethyl Sulfonic fluoride [ PMSF], 1 mM dithiothreitol [DTT]), incubated at 37 ° C. for 30 minutes, and centrifuged to develop a supernatant on a 10% acrylamide gel and reacted with ADH antibody. . As a result, only pneumococcal ADH reacted with the antibody and ADH of other bacteria or organisms did not cross-react.

This specificity is the same when the bacteria are reacted with the obtained bacterial solution after completely dissolving the bacteria for 6 seconds using a sonicator (Vibra Cell Sony, Sonics & Materials Co., Ltd.) for 6 seconds and 9 seconds. Was obtained (see FIG. 3). Lane 1 in Figure 3 is pneumococcal (Streptococcus pneumoniae; S.P); Lane 2 is Staphylococcus aureus (S.A); Lane 3 is Bacillus subtilis (Bacillus subtilis; B.S); Lane 4 is E. coli (E.C); Lane 5 is lactic acid bacteria (L.L); Lane 6 is human HeLa cell line (H.C); Lane 7 is typhoid (Salmonella typhi; S.T); Lane 8 is yeast (Saccharomyces cerevisiae; S.C).

Example 6: Antigen Specific Conservation Test of Pneumococcal ADH against Various Pneumococcal Strains

In order to investigate whether the pneumococcal ADH antibody of the present invention is immunoreactive with various pneumococci isolated from clinical trials, pneumococcal sp. SKP 3001-3008 and SKP3011-3020 (separated from Seoul Samsung Hospital) were centrifuged to separate bacteria and culture supernatant, and the bacteria were disrupted by sonication and developed on 10% acrylamide gel, followed by ADH antibody and Reacted.

As a result, the ADH antibody of the present invention was shown to react with all of the pneumococcal lysate isolated in the clinic (see FIGS. 5 and 6). These results suggest that pneumococcal ADH antibodies have high retention in amino acid sequences.

III. Genetic Identification of Pneumococci

Example 7: Identification of Bacteria Using adh Gene Expressing Pneumococcal ADH Protein

Pneumococcal buffer (5 mM) containing Triton X-100 by collecting pneumococcus, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, human lung cancer cell line A549, typhoid fever, lactic acid bacteria and yeast colonies cultured as described in Example 4. Tris-HCl [pH 8.0], 30 mM EDTA, 0.1% Triton X-100, 0.025% [w / v] PMSF, 1 mM DTT) and suspended in 5 μl 10 × Taq buffer, 4 μl dNTP, MgCl ₂ 3 μl, 2 μl chromosomal DNA solution, 1 μl each of primers p104yj02 (5'-CGG AAT TCA TGG CTG ATA AAA AAA CTGT-3 ') and p104yj04 (5'-GCC AAG CTT AAT TAT TTA CGG CGT CC-3') Add 0.5 μl of Taq polymerase and distilled water to make 50 μl in total. First, the first PCR reaction was denatured at 94 ° C. for 5 minutes, and primer and chromosomal DNA were re-cooled at 44 ° C. for 1 minute, and then polymerized at 72 ° C. for 2 minutes. In order to amplify DNA, 30 cycles of denaturation at 94 ° C. for 1 minute, re-cooling at 58 ° C. for 1 minute, and polymerization at 72 ° C. for 2 minutes were repeated 30 times. Incubated for 12 minutes at ℃. To confirm the amplified DNA in vitro, electrophoresis was performed on 0.8% agarose gel, followed by autoradiographing. As a result, in the case of pneumococci, a DNA band of 2.6 Kb was observed (lane 1 in Fig. 7), but DNA bands were observed at all in Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Lactobacillus, human lung cancer cell line A549, Typhoid bacteria, Yeast, etc. (Corresponding to lanes 2 to 8 of FIG. 7, respectively).

Example 8 Identification of Bacteria Using Homology of Pneumococcal adh Gene

Pneumococci, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Human lung cancer cell line A549, Typhoid bacteria, Lactobacillus and yeast colonies cultured as described in Example 4 were respectively collected and placed on a nylon membrane according to a conventional method (e.g., Alkaline Lysis Method) DNA was isolated and then fixed (Sambrook and Russell, 2001) and primers as in Example 7 were labeled with biotin and hybridized. When reacted with streptavidin it binds to biotin. However, streptavidin can bind to several biotin, so when the biotin-bound alkaline phosphatase and its substrate are added to the reaction solution, purple color appears while binding to streptavidin. Part of the pneumococcal fixation appeared purple, but no response from other organisms (not shown). This method can be miniaturized using DNA chips and used to detect the presence of pneumococcal adh genes. In other words, by immobilizing a primer corresponding to a part of the adh gene on a DNA chip by a conventional method (Schena, 2000) and detecting the DNA hybridized with the chromosomal DNA isolated from the bacterium, whether or not the presence of pneumococcal in the organism It can also be used to identify.

As described above, pneumococcal ADH, which is composed of 883 amino acids and has a molecular weight of about 104,000 (104-kDa, p104), whose amino acid sequence is known (see FIG. 4), has no known function and mechanism of action. Is secreted out of cells at normal incubation temperature without thermal shock, induced secretion by more than 10 times in ethanol impact, and antibodies induced against pneumococcal ADH do not react with ADH such as human lung cancer cell line, lactic acid bacteria, Staphylococcus aureus, etc. It was confirmed that pneumococcal ADH has different biochemical and immunological specificities from other bacteria. In addition, ADH antibody showed a high conserved amino acid sequence by reacting with all pneumococci isolated from the clinic. Therefore, the pneumococcal ADH and ADH antibody or adh gene of the present invention as a marker, and the method for the diagnosis and progression of pneumococcal disease according to the method for the rapid and accurate in terms of the rapid identification of pneumococci It is believed that the contribution to this required field of medical pathology is large.

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Claims (6)

i) collecting the biological specimen, ii) contacting the biological specimen with the antibody induced against pneumococcal alcohol dehydrogenase (ADH), and iii) detecting an immunocomplex formed between the antibody and the specimen. A method for identifying the presence of pneumococcal in living organisms using ADH or ADH antibodies of pneumococcal, including the presence of pneumococcal. The method of claim 1, wherein the biological specimen comprises blood, cerebrospinal fluid, bile, or a specimen taken from the oral cavity or throat of animals and humans. A method for identifying the presence of pneumococcal in living organisms, characterized in that the adh gene expressing pneumococcal ADH protein is reacted with the gene of the biological specimen. 4. The method of claim 3, wherein the biological specimen comprises blood, cerebrospinal fluid, bile, or a specimen taken from the oral cavity or throat of animals and humans. 4. The method of claim 3, wherein a portion of the adh gene expressing pneumococcal ADH protein is used as a primer, in which case i) collecting a biological specimen, ii) isolating and colonizing various microorganisms present in the specimen, iii) if necessary, selecting cocci from colonized clones, iv) collecting the isolated cultured colonies or colonies of the selected cocci and suspending them in the solubilization buffer, and v) placing a portion of the adh gene in this suspension. Adding a primer to amplify the in vitro DNA chain polymerization under suitable polymerization conditions and determining the presence of pneumococci in the organism by confirming gene amplification. How to sympathize. 4. The method of claim 3, wherein the adh gene expressing pneumococcal ADH protein is used in its entirety or in part as a marker, in which case the adh gene is immobilized on an appropriate matrix, and hybridized with chromosomal DNA or RNA isolated from bacteria. And detecting the presence of the resulting complex.