KR100891014B1 - Diagnosing Method For Infection Of Vancomycin Resistant Enterococcus Using an Immune Reaction of Specific Antigen - Google Patents

Abstract

The present invention is for diagnosing vancomycin-resistant enterococcus infection, and more specifically, a vancomycin-resistant enterococcus that can more rapidly diagnose an infection of vancomycin-resistant enterococcus by using an antigenic protein that has an immunospecific response to vancomycin-resistant enterococcus. It relates to a method for diagnosing a caustic infection.

The method for diagnosing infection of vancomycin-resistant enterococcus according to the present invention enables the diagnosis of the infection of vancomycin-resistant enterococcus by using an antigen-antibody reaction, and thus the effect of detecting the diagnosis of vancomycin-resistant enterococcus more quickly. To provide.

Vancomycin Resistant Enterococcus, Specific Antigen, Enterococcus picalis, Enterococcus pisces, Enterococcus gallinarum, Enterococcus caselliflavus

Description

Diagnosing Method For Infection Of Vancomycin Resistant Enterococcus Using an Immune Reaction of Specific Antigen}

The present invention is for diagnosing vancomycin-resistant enterococcus infection, and more specifically, a vancomycin-resistant enterococcus that can more rapidly diagnose an infection of vancomycin-resistant enterococcus by using an antigenic protein that has an immunospecific response to vancomycin-resistant enterococcus. It relates to a method for diagnosing a caustic infection.

Enterococci are the normal gut flora of most mammals and birds, and are one of the most common bacteria present in the environment that are excreted from animal and human feces.

Enterococci can form colonies in the oral cavity, biliary tract and vagina of the human body and are responsible for infectious diseases such as endocarditis, intraperitoneal infection, urethral infection and bacteremia. It is emerging as an important cause of infection in hospitals, accounting for 12%.

Enterococci are normal enterobacteriaceae in humans as well as animals, and enterococcus picalis, enterococcus fish, enterococcus gallinarum, enterococcus casselliflavus and enterococcus durans are opportunistic to infect chicken sepsis and bovine mastitis. It also causes bovine and sheep endocarditis and canine urethritis.

As for the treatment for enterococci, various antibiotics are used alone or in combination.

For example, in case of urethritis of enterococci, it is treated with ampicillin, penicillin or vancomycin alone.

Nitroflantoin is also effective for urinary tract infections, and ciprofloxacin and norfloxacin also have no problem in treating enterococcal urethritis, but are effective in treating other site infections or systemic infections other than the urethra. Is insignificant.

Erythromycin and chloramphenicol may temporarily relieve symptoms of endocarditis and meningitis, but like tetracycline, these drugs cannot completely treat enterococci.

Vancomycin alone was effective in patients with enterococcal endocarditis, and teicoplanin was shown to be effective in enterococcal endocarditis rabbits, such as ampicillin, and combined administration with gentamicin (GM) was more effective than administration alone. It is known.

  Antimicrobial therapy recommended for the treatment of opportunistic infections by enterococci in animals is the combination of penicillin with gentamicin or streptomycin, or ampicillin, vancomycin and trimethoprim-sulfamethoxazole. It is administered alone.

However, as enterococci increase, it is known that resistance to beta-lactam and aminoglycoside antibiotics is widespread in enterococci.

In particular, since the first reports of vancomycin-resistant enterococcus pichas and vancomycin-resistant enterococcus fish have been reported in the UK for the first time in the UK, which have been used to treat enterococci that are sensitive to beta-lactam antimicrobial agents or that are resistant to beta-lactam antimicrobials, Vancomycin-resistant Enterococci (VRE) infections have been reported throughout.

In the United States, the proportion of vancomycin-resistant enterococcal infections among hospital-infected enterococci increased sharply from 0.3% in 1989 to 7.9% in 1993, and as the number of vancomycin-resistant enterococcus infections increased, the Center for Disease Control (CDC) has prepared guidelines for the prevention of vancomycin resistant enterococcus transmission.

In addition, vancomycin-resistant enterococcus has been isolated not only in hospitals but also in sewage, animal waste, meat and feces of healthy people, suggesting the possibility of vancomycin-resistant enterococcus infections caused by other causes than hospitals.

Avoparcin, a glycopeptides antibiotic used in animal feed additives in Europe and other countries, has been cross-resistant with vancomycin until recently. The selective frequency of avoparcin used in animal feed additives increased the frequency of vancomycin-resistant enterococcus in carnivores, which may also be a source of human vancomycin-resistant enterococcus. Is being raised.

As described above, researches on how to diagnose vancomycin-resistant enterococcus as well as how to cope with vancomycin-resistant enterococcus that can be transmitted through various paths are being actively conducted in various fields.

As described above, in order to effectively control and screen vancomycin-resistant enterococcus, which is resistant to conventional antibiotics and can be infected by animals and humans through various routes, conventional bacterial isolation and identification methods, a method of measuring minimum growth inhibitory concentration of vancomycin, Specific gene detection methods by multiplex PCR take a long time, or there is a problem that the method is complicated.

The present invention is to provide a method for diagnosing the infection of vancomycin-resistant enterococcus quickly and effectively to solve such problems.

The present invention is to provide a method for diagnosing the infection of vancomycin-resistant enterococcus quickly and effectively to solve such problems.

In order to achieve the above object, the present invention,

A method for diagnosing infection of vancomycin resistant enterococcus, which is characterized by using an antigen-antibody reaction with a specific antigen of vancomycin resistant enterococcus.

In other words, the method for diagnosing vancomycin-resistant enterococcus infection according to the present invention finds a specific antigen in a cell membrane protein or a cytoplasmic protein of vancomycin-resistant enterococcus, and performs an antigen-antibody reaction with the specific antigen to positive the specific antigen. If you do, you can easily diagnose that you have been infected with vancomycin-resistant enterococcus.

The method for diagnosing infection of vancomycin-resistant enterococcus according to the present invention enables the diagnosis of the infection of vancomycin-resistant enterococcus using an antigen-antibody response.

It is possible to confirm whether the infection of vancomycin-resistant enterococcus is diagnosed more quickly, and to cope with the control of vancomycin-resistant enterococcus more quickly.

Next, it will be described in more detail for the practice of the present invention.

First, the mechanism by which enterococcus becomes resistant to vancomycin and the genotypes causing resistance will be described.

In general, the cell envelope of enterococci is composed of peptidoglycan (PG) and cytoplasmic membrane (CM) to which various proteins are attached.

On the other hand, vancomycin-resistant enterococcus has a second D-alanine (a-amino acid), instead of D-lactate (a-hydroxy acid) among the peptidoglycan precursors, and vancomycin is D-alanine-D-alanine ( Adhesion to D-alanyl-D-lactate (D-Ala-D-Lac) is 1000 times weaker than that of D-Ala-D-Ala), so that the resistant vancomycin-resistant Enterococcus survives at 1000-fold higher vancomycin concentrations. You can do it.

At this time, the D-alanyl-D-lactate (D-Ala-D-Lac) is synthesized by VanA and VanB, D-Ala ligase related enzymes.

As a genotype resistant to vancomycin, the C-terminus of the peptidoglycan precursor, a binding site of vancomycin, is made into D-alanyl-D-lactate (D-Ala-D-Lac) to reduce the affinity of vancomycin. There are vanA and vanB genotypes that cause resistance, and in the vancomycin resistant enterococcus, these D-Ala ligase-related enzymes are present with D-alanine: D-dlalanine ligase (Ddl).

In particular, for enterococcus gallinarum, the C-terminus of the precursor of peptidoglycan is synthesized by VanC1 to D-alanyl-D-serine (D-Ala-D-Ser) to show resistance to vancomycin.

Similar ligases, VanC2 and VanC3, are also present in Enterococcus caselliflavus and Enterococcus flavescens.

Next, in order to explain a method for diagnosing vancomycin-resistant enterococcus infection according to an embodiment of the present invention, strains and culture conditions used for detecting a specific antigen, and guinea pig antiserum production conditions for performing an antigen-antibody reaction with the specific antigen, specificity Extraction methods of cell membrane proteins and intracellular proteins for finding antigens, SDS-PAGE electrophoresis conditions and Western blotting conditions for analyzing the results will be described in more detail.

[ Use strain and  Culture conditions]

Vancomycin-resistant Enterococcus bacterium of VanA, VanB, VanC1 and VanC2 phenotypes, Enterococcus p. B7641 (VanA), Enterococcus picas V583 (VanB), Enterococcus Galinarum GS (VanC1) and Enterococcus Kassellibus ATCC 25788 (Van C2),

Dog-derived enterococcus fish, bovine-derived enterococcus picalis and enterococcus casselliflavus were used, and all cultures were performed with BHI broth (Difco) at 37 ° C. for 18 hours.

[ Guinea pig  Antiserum production conditions]

In order to confirm the immune response of specific antigens according to the present invention, guinea pig antiserum against E. coli was produced according to the method of Liddell and Cryer et al. (1991). (Liddel, JE and Cryer, A., A practical guide to monoclonal antibodies. John Wiley & Sons press, New York, NY.pp. 42-45, 1991)

More specifically, each enterococci, namely Enterococcus fish, Enterococcus picalis, Enterococcus gallinarum, Enterococcus casselliflavus, were each incubated in BHI broth (Difco) for 18 hours at 37 ° C., followed by formalin ( formalin) was added at 0.5%, and the mixture was left at 18 ° C for 18 hours.

Next, after inactivation was confirmed by incubation, 10000 × g, inactivated cells centrifuged for 10 minutes were resuspended in sterile saline, and bicinchoninic acid (BCA) protein assay kit (protein assay kit). Protein concentration was measured by (Pierce, Rockford, Illinois, USA).

The first inoculation was mixed with Sigma's complete Freund's adjuvant in equal amounts with micro-emulsifying needles so that the protein concentration was 100 µg / ml, and then per head of guinea pig. 0.5 ml each was subcutaneously inoculated.

The second inoculation was mixed well with Sigma's incomplete Freund's adjuvant in the same manner as the first inoculation three weeks after the first inoculation, and subcutaneously inoculated 0.5 ml per guinea pig.

After 1 week of inoculation, 1 ml of guinea pig blood was collected, and the antibody titer of inactivated cells was measured using an enzyme immunoassay kit (ELISA). When the antibody titer was 512 times or more, whole blood was collected. Was reinoculated in the same manner as the second inoculation.

The control serum was immunized with guinea pig serum. Antisera and control serum were inactivated at 56 ° C. for 30 minutes and then stored at −20 ° C.

[Cell membrane protein ( Cell envelope protein )and Intracellular  protein( cytoplasmic protein ) extraction]

Extraction of membrane protein (CE) and cytoplasmic protein (CP) from enterococci followed the method of Chart (1994) (Chart, H., Bacterial fractionation and membrane protein characterization., In Chart, H. ed. Methods in practical laboratory bacteriology, 1st ed.CRC press, Boca Raton, FL.pp. 1-10, 1994)

More specifically, the cells cultured in BHI borth were centrifuged at 10000 × g for 10 minutes at 4 ° C. with Beckman Coulter Avanti J-20-Xp (Beckman, Fullerton, California, USA) to obtain the cells, and cold 25 mM Tris-HCl w / 1mM EDTA-Na2 (Ph 7.4) suspended in Vibra cell 500W (Sonic & Materials, Newtown, Connecticut, USA) and the ultrasonic fluid was pulverized 150W on ice for 8 minutes, then cold 25mM Tris-HCl (Ph 7.4) ) Was added to Union 5KR (Hanil, Incheon, Korea), and the supernatant was centrifuged at 5000 ° C for 30 minutes at 4 ° C with OptimaTM XL-100K (Beckman) at 45000 ° C at 4 ° C for 1 hour. Transparent pellets were used as membrane protein (CE),

Intracellular protein (CP) was used as a supernatant of one-time centrifugal precipitation under the same conditions.

[ SDS - PAGE Wow Weston Blotting ]

SDS-PAGE followed the method of Ausubel et al. (1995) and Western blotting followed the method of Bollag and Edelstein (1991) to confirm the immune response of specific antigens according to the present invention. (Bollag, DM and Edelstein, SJ, Immunoblotting.In Protein Methods., A John Wiley & Sons, Inc., New York, NY.pp. 181-211, 1991)

In more detail, 5 × sample buffer was added to the prepared antigen, boiled for 3 minutes, centrifuged at 12000 rpm for 1 second, and loaded onto an SDS-PAGE gel.

After electrophoresis for 80 minutes under conditions of 100V, 80mA, the gel was stained with Coomassie blue and decolorized with 10% methanol / acetic acid to identify the protein.

Western blotting was performed by electrophoresis of the antigen prepared on the SDS-PAGE gel, and then dissolved in 0.75 g of SDS in a solution of 600 ml of transfer buffer and 150 ml of methanol, and the buffer was stirred using a magnetic bar for 1 hour at 100 V and 400 mA. The protein was transferred from the SDS-PAGE gel to a nitrocellulose membrane (Sigma).

Protein-transcribed nitrocellulose (NC) membranes were washed with PBS (Ph 7.4) for 3 minutes, and then 1% skim milk (Sigma) was added to the PBS (Ph 7.4) for 30 minutes at 37 ° C. Blocking

Next, the blocked nitrocellulose (NC) membrane was washed three times with PBS (Ph 7.4) three times for three minutes, and then PBS (Sigma) with 1% skimmed milk (Sigma) was added to guinea pig antiserum and mouse serum. Ph 7.4) was diluted 1: 2000 and 1: 100, respectively, and reacted at 37 ° C. for 1 hour.

Next, the reacted nitrocellulose (NC) membrane was washed, and then reacted with a 3,3-diaminobenzidine (DAB) (Sigma) substrate solution for 5 minutes, and then washed three times with distilled water for 3 minutes to stop the reaction.

Thus, the size of the protein shown in the SDS-PAGE electrophoresis image and Western blotting was analyzed by Bio1D ver 99.03 (VILBER LOURMAT, France) based on the protein size of the protein marker (Sigma).

Next, the immune response of the specific antigen according to the present invention will be described in more detail.

Example 1

1. Cell membrane protein SDS - PAGE  analysis

Cell membrane proteins (CE) of Enterococcus psium, Enterococcus picalis, Enterococcus gallinaum and Enterococcus casselliflavus were analyzed by SDS-PAGE and showed similar protein patterns for each species. It showed an aspect.

1 is a diagram showing the results of electrophoretic analysis of cell membrane protein (CE) of vancomycin resistant enterococcus using a 10% SDS-polyacrylamid gel according to the present invention.

Lane 1 is Molecular weight marker by electrophoresis analysis of Sigma wide marker, Lanes 2 to 9 are Enterococcus pseum B7641 (van A) and Enterococcus casselliflavus isolate 2 (vanC-2), respectively. ), Enterococcus picalis V583 (vanB), Enterococcus picalis (VSE) isolated from cattle, Enterococcus gallinarum GS (vanC-1), Enterococcus picium (VSE) isolated from dogs, Enterococcus caselifolia The electrophoretic analysis of Enterococcus casselliflavus isolate 1 (vanC-2) isolated from Labus ATCC25788 (vanC-2) and cattle.

Vancomycin Resistant Enterococcus faecium (VREFM), unlike vancomycin Sensitive Enterococcus faecium (VSEFM), specifically observed 34 kDa and 40 kDa proteins (Fig. 1). 2, lane 7),

In the Enterococcus casselliflavus, the two strains had the same protein pattern (lane 8 and lane 9 of FIG. 1), but one strain showed a different protein pattern (lane 3 of FIG. 1).

2. Cell membrane protein Weston Blotting  Specific Antigen Analysis

One) Enterococcus Fishium  For cell membrane proteins Weston Blotting  analysis

Figure 2 shows the results of Western blotting analysis for enterococcus fish cell membrane protein using anti-serum of guinea pigs.

For reference, lanes 1 and 2 in FIG. 2 show Western blotting results of guinea pig antiserum and negative serum for cell membrane protein (CE) of Enterococcus p. B7641 (VREFM), respectively. Western blotting results of guinea pig antiserum and negative serum on the isolated membrane proteins of vancomycin sensitive enterococcus fish (VSEFM).

As shown in FIG. 2, Western blotting results for cell membrane proteins (CE) using guinea pig antiserum showed that 24, 44, 54, 60, 107, 129 and 137 kDa (kilo) for guinea pig antiserum in the case of vancomycin resistant enterococcus fish. Daltons) and negative sera responded with 27, 70, 54, 65, 107 and 137 kDa.

In the case of vancomycin-sensitive enterococcus fish, guinea pig antisera responded with 7, 24, 40, 44, 60, 70 and 112 kDa antigens, and negative serum contained 27, 40, 70, 80, 112 and 155 kDa antigens. Reacted.

As a result, the antigens commonly responding to enterococcus were 24 kDa and 44 kDa antigens, the specific antigen of vancomycin-resistant enterococcus fish (VREFM) was 129 kDa, and the vancomycin sensitive enterococcus fish (VSEFM). Specific antigens of each appeared as 7 kDa antigen.

2) Enterococcus Picalis  For cell membrane proteins Weston Blotting  analysis

Figure 3 shows the results of Western blotting analysis on Enterococcus picalis cell membrane protein using anti-serum of guinea pigs.

For reference, in Fig. 3, lane 1 1 and lane 2 show western blotting results of guinea pig antiserum and negative serum for cell membrane protein (CE) of Enterococcus picalis V583 (VREFS), respectively. Western blotting results of guinea pig antiserum and negative serum on the isolated membrane proteins of vancomycin sensitive enterococcus picalis (VSEFS).

As shown in FIG. 3, Western blotting of cell membrane proteins (CE) using guinea pig antiserum showed that 22, 49, 53 and 107 kDa antigens were reacted to guinea pig antiserum in the case of vancomycin-resistant enterococcus picas (VREFS). , Negative serum reacted with 17, 22, 35, 49, 116, 152 and 163 kDa antigens.

Vancomycin-sensitive Enterococcus picalis (VSEFS) responded with 12, 17, 22, 49, 53, 107, 116 and 163 kDa antigens to guinea pig antiserum and 22, 35 and 49 kDa antigens to negative serum. It was.

As a result, the antigen commonly responding to Enterococcus picalis was an antigen of 54 kDa, and no specific antigen of vancomycin-resistant Enterococcus picas (VREFS) was identified, and the specific antigen of vancomycin-sensitive Enterococcus picas (VSEFS). Were shown as antigens of 12 and 16 kDa, respectively.

3) Enterococcus Galina Rum Enterococcus Kasselliverse  For cell membrane proteins Weston Blotting  analysis

Figure 4 shows the results of Western blotting analysis for Enterococcus gallinarum and Enterococcus caseliflava membrane proteins using antisera from Guinea Pigs.

For reference, in Fig. 4, lane 1 1 and lane 2 show western blotting results of guinea pig antiserum and negative serum for cell membrane protein (CE) of Enterococcus gallinarum GS (VREG), respectively. Western blotting results of guinea pig antiserum and negative serum of the cell membrane protein (CE) of the caseliflavus ATCC25788 (VREC), the cell membrane protein of Enterococcus Kassellibus isolate 1 isolated from cattle Western blotting results of guinea pig antiserum and negative serum for (CE), and lane 7 and lane 8 are guinea pig antiserum and negative serum for cell membrane protein (CE) of Enterococcus caselliflavus isolate 2 isolated from cattle. Shows the results of Western blotting.

As shown in FIG. 4, Western blotting of the membrane protein (CE) using guinea pig antiserum showed that 30, 46 and 57 kDa antigens were reacted to guinea pig antiserum in the case of vancomycin-resistant enterococcus gallinarum (VREG). The serum reacted with 11, 20, 31, 36, 47, 51, 57, 82, 97 and 117 kDa antigens.

Enterococcus Kassellibus ATCC25788 (VREC) reacted with 31, 37, 40, 50 and 70 kDa antigens to guinea pig antiserum and 12, 20, 26, 31, 50, 70, 89, 117 to guinea pig antiserum. , 189 and 197 kDa antigens reacted.

In Enterococcus Kassellibus isolate 1, guinea pig antisera responded with 23, 26, 40, 50, 58, 189 and 197 kDa antigens, and negative sera 17, 23, 37, 50, 58, 89, 117 , 189 and 197 kDa antigens reacted.

In Enterococcus caselliflavus isolate 2, 13, 22, 28, 34, 40, 45, 50, 54, 70, 89, 120, and 197 kDa of antigens responded to guinea pig antisera and 28, 34 to negative serum. , 50, 103, 120 and 189 kDa antigens reacted.

As a result, the specific antigen that commonly responds to Enterococcus caselliflavus was a 40 kDa antigen.

Example 2

One. Intracellular  protein( Cytoplasmic protein , CP )of SDS - PAGE analysis

The cytoplasmic protein (CP) of Enterococcus psium, Enterococcus picalis, Enterococcus gallinaum, and Enterococcus casselliflavus were analyzed by SDS-PAGE and showed similar protein patterns for each species. It showed a protein aspect.

5 is a diagram showing the results of electrophoretic analysis of cytoplasmic protein (CP) of vancomycin-resistant enterococcus using a 10% SDS-polyacrylamid gel according to the present invention.

Lane 1 is Molecular weight marker by electrophoresis analysis of Sigma wide marker, Lanes 2 to 9 are Enterococcus pseum B7641 (van A) and Enterococcus casselliflavus isolate 2 (vanC-2), respectively. ), Enterococcus picalis V583 (vanB), Enterococcus picalis (VSE) isolated from cattle, Enterococcus gallinarum GS (vanC-1), Enterococcus picium (VSE) isolated from dogs, Enterococcus caselifolia The electrophoretic analysis of Enterococcus casselliflavus isolate 1 (vanC-2) isolated from Labus ATCC25788 (vanC-2) and cattle.

Vancomycin Resistant Enterococcus faecium (VREFM), unlike vancomycin Sensitive Enterococcus faecium (VSEFM), specifically observed 39 kDa and 48 kDa proteins (Figure 5). 2, lane 7),

In Enterococcus casselliflavus, two strains had the same protein pattern (lane 8 and lane 9 of FIG. 5), but one strain showed a different protein pattern (lane 3 of FIG. 5).

In other words, enterococcal species and different patterns were similar to those analyzed using membrane protein (CE).

2. Intracellular  Protein Weston Blotting  Specific Antigen Analysis

One) Enterococcus Fishium Intracellular  For protein Weston Blotting  analysis

Figure 6 shows the results of Western blotting analysis for proteins in Enterococcus fish cytoplasm using anti-serum of guinea pigs.

For reference, in Fig. 6, lanes 1 and 2 show western blotting results of guinea pig antiserum and negative serum for cytoplasmic protein (CP) of Enterococcus p. B7641 (VREFM), respectively. Figure 2 shows the results of Western blotting of anti-serum and negative serum of guinea pigs against intracellular proteins of vancomycin-sensitive enterococcus fish (VSEFM) isolated from.

As shown in FIG. 6, Western blotting of intracellular protein (CP) using guinea pig antiserum showed that 21, 44, 56 and 61 kDa antigens were reacted to guinea pig antiserum in the case of vancomycin-resistant enterococcus fish. The serum reacted with 40, 44, 56 and 61 kDa antigens.

In the case of vancomycin-sensitive enterococcus fish, 44, 48, 56, and 61 kDa antigens responded to guinea pig antiserum and 44, 48, and 100 kDa antigens to negative serum.

As a result, no antigens that commonly respond to enterococcus fish or antigens that specifically react to vancomycin-sensitive enterococcus fish (VSEFM) were identified, and the specific antigen of vancomycin-resistant enterococcus fish (VREFM) was 21. appeared as an antigen of kDa.

2) Enterococcus Picalis  For the cytoplasm Weston Blotting  analysis

Figure 7 shows the results of Western blotting analysis for proteins in Enterococcus picalis cytoplasm using anti-serum of guinea pigs.

For reference, lanes 1 and 2 in FIG. 7 show western blotting results of guinea pig antiserum and negative serum for cell membrane protein (CE) of Enterococcus picalis V583 (VREFS), respectively. Figure 2 shows the results of Western blotting of guinea pig antiserum and negative serum on the isolated cytoplasmic proteins of vancomycin sensitive enterococcus picalis (VSEFS).

As shown in FIG. 7, Western blotting of intracellular protein (CP) using guinea pig antiserum showed that 22, 28, 35, 47, 49, and 53 kDa for vancomycin resistant enterococcus picalis (VREFS). Antigens reacted, and 22, 47, 49, and 53 kDa antigens responded to negative sera.

In the case of vancomycin sensitive enterococcus picalis (VSEFS), guinea pig antisera responded with 6, 17, 22, 47, 53, 57 and 90 kDa antigens, and negative sera responded with 22, 47, 53 and 57 kDa antigens. It was.

As a result, antigens that commonly respond to enterococcus picas were not identified as enterococcus picium, and the specific antigen of vancomycin resistant enterococcus picas (VREFS) was 28 kDa antigen and vancomycin sensitive enterococcus picas ( Specific antigens of VSEFS) appeared as antigens of 6, 17 and 90 kDa.

3) Enterococcus Galina Rum Enterococcus Kasselliverse  For the cytoplasm Weston Blotting  analysis

Figure 8 shows the results of Western blotting analysis for enterococcus gallinarum cytoplasm using anti-serum of guinea pigs.

For reference, in FIG. 8, lane 1 1 and lane 2 show western blotting results of guinea pig antiserum and negative serum for cytoplasmic protein (CP) of Enterococcus gallinarum GS (VREG), respectively. Western blotting of anti-serum and negative serum of guinea pigs against intracellular protein (CP) of the Caucus casellifolius ATCC25788 (VREC). Western blotting results of guinea pig antiserum and negative serum for intracellular protein (CP), lanes 7 and 8 are the guinea pigs for intracellular protein (CP) of Enterococcus caselliflavus isolate 2 isolated from cattle. Western blotting results of antiserum and negative serum.

As shown in FIG. 8, Western blotting of intracellular protein (CP) using guinea pig antiserum resulted in 19, 30, 37, 46, and 61 kDa in guinea pig antiserum in the case of vancomycin resistant enterococcus gallinarum (VREG). Antigens responded, and 37, 46 and 60 kDa antigens responded to negative sera.

In the case of Enterococcus Kaseliflavus ATCC25788 (VREC), 37, 45, and 58 kDa antigens were reacted to guinea pig antiserum and 37, 45, 58 and 60 kDa antigens to negative serum.

Enterococcus caselliflavus isolate 1 responded with 24, 37, 45 and 58 kDa antigens to guinea pig antiserum and 37, 45, 58 and 60 kDa antigens to negative serum.

In the enterococcus caselliflavus isolate 2, 15, 21, 37, 45, 58, and 60 kDa of antigens responded to guinea pig antiserum, and 37, 45, 49, and 58 kDa of antigens responded to negative serum.

In addition, no specific antigens in common with Enterococcus caselliflavus were identified.

As described above, the specific antigen used in the method for diagnosing vancomycin-resistant enterococcus infection using the specific antigen according to an embodiment of the present invention may be represented as shown in Table 1 below.

In addition, the method for diagnosing vancomycin-resistant enterococcus infection according to an embodiment of the present invention may be used in animals.

The present invention described above is not limited to the above-described embodiment and the accompanying drawings, it is conventional in the art that various substitutions, modifications and changes are possible without departing from the technical spirit of the present invention. It will be evident to those who have knowledge of.

1 is a view showing the results of electrophoresis analysis of cell membrane protein (CE) of vancomycin resistant enterococcus according to the present invention.

Figure 2 shows the results of Western blotting analysis for Enterococcus psium membrane protein using antisera of Guinea Pig.

Figure 3 shows the results of Western blotting analysis for Enterococcus picalis cell membrane protein using antiserum of Guinea pig.

Figure 4 shows the results of Western blotting analysis for Enterococcus gallinarum and Enterococcus caseliflava cell membrane proteins using antisera from Guinea Pig.

Figure 5 shows the results of electrophoresis analysis of intracellular protein (CP) of vancomycin resistant enterococcus according to the present invention.

Figure 6 shows the results of Western blotting analysis for proteins in the enterococcus fish cytoplasm using anti-serum of guinea pigs.

Fig. 7 shows the results of Western blotting analysis on proteins in Enterococcus picalis cytoplasm using antiserum of guinea pigs.

Figure 8 shows the results of Western blotting analysis of the proteins in enterococcus gallinarum and enterococcus caselliflavus cytoplasm using antiserum of guinea pigs.

Claims (6)

Using an antigen-antibody reaction with a specific antigen of vancomycin resistant enterococcus, the vancomycin resistant enterococcus is selected from vancomycin resistant enterococcus fish (VREFM), vancomycin resistant enterococcus picalis (VREFS), vancomycin resistant enterococcus gallinalum ( VREG), vancomycin-resistant enterococcus In a method for diagnosing infection of vancomycin-resistant enterococcus, which is one of Kassellivers, In the case where the vancomycin resistant enterococcus is vancomycin resistant enterococcus fish (VREFM), the vancomycin resistant enterotrope is characterized by using 129 kilodaltons of cell membrane protein (CE) or 21 kilodaltons of intracellular protein (CP) as a specific antigen. How to diagnose infection with the caucasus. delete delete The method of claim 1, When the vancomycin resistant enterococcus is vancomycin resistant enterococcus picalis (VREFS), a 28 kilodalton intracellular protein (CP) is used as a specific antigen. The method of claim 1, When the vancomycin resistant enterococcus is vancomycin resistant enterococcus gallinarum (VREG), the specific antigen is 30 kilodaltons of cell membrane protein (CE), 19 kilodaltons of intracellular protein (CP), 30 kilodaltons of intracellular protein ( CP) A method for diagnosing infection of vancomycin-resistant enterococcus, characterized by using any one of them. The method of claim 1, When the vancomycin resistant enterococcus is a vancomycin resistant enterococcus caselliflavus, a 40 kilodalton cell membrane protein (CE) is used as a specific antigen.

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