KR101753580B1 - Method for Brucella canis antigen - Google Patents
Method for Brucella canis antigen Download PDFInfo
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- KR101753580B1 KR101753580B1 KR1020150118842A KR20150118842A KR101753580B1 KR 101753580 B1 KR101753580 B1 KR 101753580B1 KR 1020150118842 A KR1020150118842 A KR 1020150118842A KR 20150118842 A KR20150118842 A KR 20150118842A KR 101753580 B1 KR101753580 B1 KR 101753580B1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/23—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Brucella (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
Abstract
The present invention relates to a method for producing Brucella canis antigen, a Brucella canis antigen produced by the method, a composition for diagnosing brucellosis including the antigen, a kit for diagnosing brucellosis, and a method for diagnosing brucellosis. The sensitivity and specificity of the Brucella canis antigen prepared by the method for producing Brucella canis antigen according to the present invention is superior to that of the purified antigen using the conventional HS method and has a very high correlation, have.
Description
The present invention relates to a method for producing Brucella canis antigen, a Brucella canis antigen produced by the method, a composition for diagnosing brucellosis including the antigen, a kit for diagnosing brucellosis, and a method for diagnosing brucellosis.
Brucellosis is a major common infectious disease (human, 4th army infectious disease, livestock, two statutory infectious diseases) and causes public health and economically serious problems both domestically and globally. In recent years, the demand for companion animals such as dogs has increased rapidly in Korea, and there is a possibility of infecting animals or people through them. Therefore, it is urgent to take measures against them.
The brucellosis causing brucellosis are Brucella abortus (bovine), Brucella melitensis ( brucella) melitensis (sheep, goat), Brucella canis ), Brucella ( Brucella) suis ) (pig) are known, and all of the above 4 species can infect the human body. B. abortus and B. canis were isolated from Korea. When the brucellosis is developed, it shows a mortality rate of less than 1 to 3% when it is infected to the human body, and it progresses to serious diseases such as endocarditis, encephalomyelitis and arthritis while showing persistent fever, headache, anorexia and malaise. In addition, symptoms in infected animals are known to cause abortion due to placental salts and endometritis in females without special external symptoms, and in males to cause sterilization due to testisitis and malformed sperm. Because brucellosis is proliferating within the cell and causes onset, antibiotic treatment for several years is needed for a short period of time for human infection. In addition, it is necessary to improve the treatment method because of the sequelae due to the large amount of antibiotics, and even if it is judged as cured, it is often recurred within a few years. However, in the case of brucellosis in an animal, the brucella are continuously released to the outside while maintaining the long-term carrier without any special symptoms except abortion. It is difficult to treat by brucellosis with general antibiotics, and there are not many results on intracellular and proliferative mechanisms. In addition, even when infected in the body, since immunogenicity is low and immune cells are hardly formed, studies on development of vaccine are also ineffective, so that there is a problem that it is very difficult to eradicate the disease.
The diagnosis of brucellosis is screened and monitored using serological diagnosis. The major diagnostic methods used for diagnosis of brucellosis are diagnosis of various coagulation reaction methods using LPS (lipopolysaccharide) -based bacterial antigens. In addition, purified or purified LPS or lipid-depleted O-polysaccharide (OPS) can be used as an antigen for indirect or competitive ELISA (enzyme-linked immunosorbent assay) or FPA (fluorescence polarization assay) can do. But woninche dog Brucellosis is because the lack of an R type strain (rough strain) LPS on the cell surface with B. canis, are experiencing great difficulties in diagnosis of situation can not use most of the diagnostic methods to be applied to existing brucellosis diagnosis. Agar gel immunodiffusion (AGID) method or immunochromatography test kit (ICT) method using an antigen extracted by a rapid slide agglutination test (RSAT) method or a hot saline (HS) method can be used as a known method so far However, since these methods have low sensitivity and low detection efficiency due to a large number of non - specific reactions, development of new diagnostic methods is required.
Therefore, the inventors of the present invention have studied a method for effectively diagnosing brucellosis. As a result, it has been found that when a mixed protein derived from Brucella canis and separated and purified by removing R-LPS is used as an antigen, And thus it is more effective in the diagnosis of brucellosis. Thus, the present invention has been completed.
It is an object of the present invention to provide a method for producing Brucella canis antigen.
It is also an object of the present invention to provide a brucella canis antigen produced by the above method.
It is also an object of the present invention to provide a composition for diagnosing brucellosis comprising the antigen.
It is also an object of the present invention to provide a kit for the diagnosis of brucellosis comprising the above composition.
It is also an object of the present invention to provide a method for diagnosing brucellosis.
In order to solve the above problems, the present invention provides a method for producing Brucella canis- derived mixed protein comprising the steps of 1) treating trichloroacetic acid (TCA) in a culture medium of Brucella canis to obtain a mixed protein derived from Brucella canis ; And 2) separating and purifying the mixed protein of 1) above, and a method for producing Brucella canis antigen.
The present invention also provides a brucella canis antigen produced by the above method.
The present invention also provides a composition for diagnosing brucellosis comprising the antigen.
The present invention also provides a kit for diagnosing brucellosis comprising the above composition.
It is another object of the present invention to provide a method for diagnosing brucellosis comprising the step of performing an antigen-antibody reaction using the antigen from a biological sample to detect the occurrence of brucellosis.
The sensitivity and specificity of the Brucella canis antigen prepared by the method for producing Brucella canis antigen according to the present invention is superior to that of the purified antigen using the conventional HS method and has a very high correlation, have.
Brief Description of the Drawings Fig. 1 is a graph showing the results of confirming the protein molecular weight and the immune response of the TCA antigen of the present invention using 2D SDS-PAGE (A) and western blot (B).
FIG. 2 is a graph showing the results of analysis of the components of the TCA antigen of the present invention by MALDI-TOF MS. FIG.
The present invention provides a method for producing Brucella canis , comprising: 1) treating trichloroacetic acid (TCA) in a culture medium of Brucella canis to obtain a mixed protein derived from Brucella canis ; And 2) separating and purifying the mixed protein of 1) above, and a method for producing Brucella canis antigen.
In the present invention, the term " Brucella " Brucella canis , a rough strain, has been found to be a smooth strain of Brucella arborescens (Brucella arborescens) Brucella abortus , Brucella melitensis ) and smooth-lipopolysaccharide (S-LPS).
The brucella canis may be an M-strain, but is not limited thereto.
The Brucella canis-derived mixed protein includes an
In the present invention, the term "LPS (lipopolysaccharide)" is a lipid polysaccharide and is an important constituent of the outer membrane surrounding the peptide glycans of the Gram-negative bacterial surface layer. It can be broadly divided into O antigen (O antigen or O polysaccharide), core oligosaccharide and lipid A (Lipid A).
In the present invention, the above "R-type-LPS" is a kind of LPS and can be divided into R type-LPS and S type-LPS depending on whether the O antigen of LPS is O-chain. The full length O-chain is S-LPS in presence and R-LPS when the O-chain is deficient or missing.
In the present invention, the "outer membrane protein 31 (omp 31)" is characterized as potential immunogenicity and protective antigen. Brucella Abbott abortus ) has a high degree of gene homology in all brucellosis species except for this part of the gene is defective.
In one embodiment of the present invention, the culture medium of Brucella canis M-strain is sterilized, and the supernatant is recovered by centrifugation to extract the heat-stable antigen, which is then treated with trichloroacetic acid (TCA) Type-LPS was removed. Thereafter, the mixed protein was separated and purified to obtain a protein derived from Brucella cannis, which was named "TCA antigen ". The present inventors analyzed the TCA antigen of the present invention obtained through the above process and found that the outer membrane protein 31 (omp31), the GroES protein, the MerR family transcriptional regulator, 1 (nesprin-1), a DNA-directed RNA polymerase subunit beta and a metal-dependent phosphohydrolase, and has heat-resistant characteristics. In addition, as a result of diagnosing dog brucellosis by performing Indirect ELISA using the TCA antigen of the present invention, sensitivity and specificity of the TCA antigen of the present invention when compared with the HS antigen purified and isolated by the existing HS method HS antigen and highly correlated with brucellosis, it was confirmed that the TCA antigen according to the present invention is effective in the diagnosis of dog brucellosis.
The present invention also provides a brucella canis antigen produced by the above method.
In the present invention, "antigen" is a substance that causes an immune response to a living body, and binds to an antigen and a corresponding antibody.
The present invention also provides a composition for diagnosing brucellosis comprising the antigen.
In the present invention, the term "brucellosis" refers to an infectious disease caused by infection of various brucellosis, and when infected to the human body, it has a mortality rate of less than 1-3%, persistent fever, headache, , Showing signs of weakness, endocarditis, encephalomyelitis and arthritis will progress to severe diseases. Symptoms seen in infected animals have no special external symptoms, but they cause abortion due to placental salts and endometritis in females and infertility due to testisitis and malformed sperm in males.
In the present invention, the "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to ascertain the onset of brucellosis.
The diagnostic composition of the present invention may further include reagents known in the art used for immunological analysis in addition to antigens for brucellosis diagnosis. The reagent may include a label capable of generating a detectable signal, a solubilizer, and a cleaning agent. When the labeling substance is an enzyme, the reagent 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 fluorescent substance include fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, 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, 58 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.
Further, the diagnostic composition of the present invention may be provided in an immobilized state using various methods known on a suitable carrier or supporter in order 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, liposomes, carboxymethylcellulose, polyacrylamide, polysterine, gabapentin, filter paper, ion exchange resins, , Glass, polyamine-methyl vinyl ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, cup, flat packs and the like. 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).
The present invention also provides a kit for diagnosing brucellosis comprising the above composition.
The kits of the invention may additionally comprise reagents known in the art for use in immunological assays. In addition, the kit of the present invention may further include a user guide describing an optimum performing condition. The handbook is printed to describe how to use the kit, for example, the reaction conditions presented. The brochure includes instructions on the surface of the package including the brochure or leaflet in the form of a brochure, a label attached to the kit, and a kit. In addition, the brochure includes information that is disclosed or provided through an electronic medium such as the Internet.
It is another object of the present invention to provide a method for diagnosing brucellosis comprising the step of performing an antigen-antibody reaction using the antigen from a biological sample to detect the occurrence of brucellosis.
The diagnostic methods may include, but are not limited to, indirect ELISA, direct ELISA, sandwich ELISA, competitive ELISA, radioimmunoassays, Farr assay, immunoprecipitation, latex coagulation, erythrocyte aggregation, The immunoassay method, the diffusion method, the counter-current electrophoresis method, the single-radical immunodiffusion method, the immunochromatography method, the protein chip and the immunofluorescence method. Preferably, the indirect ELISA is used.
Hereinafter, the present invention will be described in detail by way of examples. It is to be understood that the following examples are illustrative only and are not intended to limit the scope of the invention in any way to the scope of the invention as defined by the appended claims. It will be obvious.
Example 1. Brucella Canis ( Brucella canis )of Protein extraction and purification
In order to obtain a purified protein from which R-LPS (rough-lipopolysaccharide) was removed from the culture broth of Brucella canis and to obtain purified antigen, the following experiment was conducted.
Specifically, the brucella canis M-strain was cultured in brucella agar supplemented with 5% bovine serum for 48 hours, and the bacteria were recovered with 0.85% saline. Then, it was centrifuged three times at 8,000 g, washed, diluted with DW (5 ml per wet g), autoclaved, and centrifuged at 15,000 g for 20 minutes to collect the supernatant, ≪ / RTI >
The supernatant obtained by filtration was purified by two different methods to evaluate its antigenicity. The first method is a known hot saline (HS) method. The supernatant is centrifuged again at 100,000 g for 6 hours and the precipitated protein is concentrated to 1/10. Then, the homogenate was floated with DW and homogenized with an ultrasonic disintegrator (sonicator) was used as HS antigen. The second method is a method of removing R-type LPS. The remaining protein is precipitated by adding trichloroacetic acid (TCA) at a concentration of about 5% to the supernatant, and centrifuged at 15,000 g for 20 minutes The R-LPS was removed by removing the supernatant. Thereafter, the precipitate was recovered with the original volume (5 ml per wet g), and the TCA was removed by dialysis (MWCO 10,000) for 48 to 72 hours while exchanging 3 to 5 times with about 100 times the volume of PBS, Protein from which R-type LPS was removed was recovered and used as a TCA antigen.
Example 2. New TCA Analysis of components of antigens
The components of the TCA antigen obtained in Example 1 were analyzed.
Specifically, TCA antigen was developed by performing 2D SDS-PAGE (pH 4-7 nonlinear gradient strip, 9-16% linear gradient polyacrylamide gels) and stained with CBB G-250 to observe protein molecular weight of TCA antigen 1 (A)). The TCA antigen was also transferred to a PVDF membrane (GE healthcare, USA), treated with 2% skim milk and reacted with positive or negative sera. After that, anti-dog IgG conjugate (KPL, USA) was added and treated with West-Q Chemiluminescent Substrate Plus Kit (Gendepot, USA) Q, USA) was used to analyze the immune response (Fig. 1 (B)).
Through the above analysis, a spot having an immune response was compared with an antigen of a stained gel, and a total of 23 protein spots were extracted and analyzed by MALDI-TOF MS to identify the protein (FIG. 2). The results are shown in Fig. 1 and Fig.
As shown in Fig. 1, it was confirmed that a total of 23 protein spots had immunogenicity.
Also, as shown in Fig. 2, it was confirmed that the approximate molecular weight of 17 spots of the 23 spots having immunogenicity in Fig. 1 was 25.2 kDa and the pI value was 5.22, and the components of the TCA antigen of the present invention were Omp31 Outer membrane protein 31) was the major antigen. In addition, the present inventors have also found that the present invention can be applied to a variety of proteins such as GroES protein, MerR family transcriptional regulator, nesprin-1, DNA-directed RNA polymerase subunit beta, Components such as a metal-dependent phosphohydrolase were identified.
Example 3. Indirect ELISA TCA Diagnosis of Brucellosis in Antigen
Indirect ELISA was performed in order to compare the diagnostic ability of brucella with the HS antigen purified by the conventional HS method of Example 1 and the TCA antigen purified by the novel method of the present invention.
Specifically, according to Example 1, the HS antigen purified by the conventional HS method and the TCA antigen purified by the novel method of the present invention were each diluted 1: 1000 with a coating buffer. Then, 100 ul was added to the wells, adhered overnight at 4 캜, and blocked with 2% skim milk. Then, diluted serum was added at a ratio of 1: 100, an anti-dog IgG conjugate and an ABTS substrate were added, and the OD value was measured at 405 nm after 10 minutes. Each step was washed 3-4 times with PBS containing 0.05
As shown in Table 1, the TCA antigen of the present invention has a sensitivity of 86.5% and a specificity of 99.2%. In addition, the TCA antigen of the present invention had a coincidence rate of 0.96, indicating whether Brucella sp. It is confirmed that there is a very high correlation. Therefore, it was confirmed that the TCA antigen of the present invention in which R-type-LPS was removed from the HS antigen purified by the conventional method is effective for the diagnosis of brucellosis.
Claims (10)
2) isolating and purifying the mixed protein of 1); preparing a Brucella canis antigen; And
3) Performing an antigen-antibody reaction using the antigen to confirm whether or not Brucella canis infection is present.
A method for providing information on diagnosis of a Brucella canis infection, characterized in that the Brucella canis of 1) is an M strain.
The mixed protein of the above 2) may be selected from the group consisting of outer membrane protein 31, omp31, GroES protein, MerR family transcriptional regulator, nesprin-1, DNA- A method for providing information on the diagnosis of Brucella canis infection characterized in that it comprises DNA-directed RNA polymerase subunit beta and metal dependent phosphohydrolase.
The antigen-antibody reaction can be detected by indirect ELISA, direct ELISA, sandwich ELISA, competitive ELISA, radioimmunoassays, Farr assay, immunoprecipitation, latex agglutination, erythrocyte agglutination, Information on the diagnosis of Brucella canis infection characterized by using at least one assay selected from the group consisting of immunodiffusion, counter-current electrophoresis, single-radical immunodiffusion, immunochromatography, protein chip and immunofluorescence. How to provide.
The method of claim 1, wherein the mixed protein of 2) has heat resistance.
2) isolating and purifying the mixed protein of 1); preparing a Brucella canis antigen; Wherein the Brucella cannis antigen is produced by a method comprising the steps < RTI ID = 0.0 > of: < / RTI >
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| CN108226496A (en) * | 2018-01-29 | 2018-06-29 | 南方医科大学 | A kind of method for detecting brucella |
| CN109490541A (en) * | 2018-09-28 | 2019-03-19 | 河北科技师范学院 | A kind of method for building up for the indirect ELISA detecting Brucella abortus HSP70 |
| KR102124259B1 (en) * | 2018-10-19 | 2020-06-26 | 대한민국(관리부서 질병관리본부장) | Brucellosis diagnostic detection kit using rapid immunochromatography, its specific antibody and antibody-producing cell lines |
| CN109507433A (en) * | 2018-11-16 | 2019-03-22 | 天津出入境检验检疫局动植物与食品检测中心 | The Rapid detection test strip of Brucella antibody |
| CN109507434A (en) * | 2018-11-16 | 2019-03-22 | 天津出入境检验检疫局动植物与食品检测中心 | Detect the ELISA kit of brucella antibody |
| CN109283342A (en) * | 2018-11-16 | 2019-01-29 | 天津出入境检验检疫局动植物与食品检测中心 | A kind of ELISA kit detecting brucella antibody |
| CN111825764B (en) * | 2020-08-17 | 2022-02-01 | 中国兽医药品监察所 | Brucella canicola monoclonal antibody and application thereof |
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| US6582699B2 (en) * | 1999-12-15 | 2003-06-24 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of National Defence In Her Brittanic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland | Combination vaccine for enhancing immunity against brucellosis |
| JP2007089571A (en) | 2005-08-30 | 2007-04-12 | Obihiro Univ Of Agriculture & Veterinary Medicine | Method for serologically diagnosing brucellosis of dog |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6582699B2 (en) * | 1999-12-15 | 2003-06-24 | Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of National Defence In Her Brittanic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland | Combination vaccine for enhancing immunity against brucellosis |
| JP2007089571A (en) | 2005-08-30 | 2007-04-12 | Obihiro Univ Of Agriculture & Veterinary Medicine | Method for serologically diagnosing brucellosis of dog |
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| 논문1;J BACTERIOL |
| 논문2;AM J VET RES* |
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