CN1060310A - Be used for immunodiagnosis new 17KDa proteantigen lungy and by the preparation method of its deutero-peptide fragment - Google Patents
Be used for immunodiagnosis new 17KDa proteantigen lungy and by the preparation method of its deutero-peptide fragment Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)
-
- 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
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
Abstract
The application discloses the immuno-chemical property of the amino acid whose 17KDa proteantigen of fractional separation, purifying and sequencing mycobacterium tuberculosis (S II 1).17KDa proteantigen with N-terminal ATTLPVQR (aa1-8) has 3 specific antibody bonded epitopes that are positioned at linear precedence RATYDKRYEVR (aa91-101) and SEFAYGSFVR (aa68-77) peptide at least.The short mitotic 17KDa proteinogen of human body tuberculosis peripheral blood lymphocyte contains the T cell antigen decision position that is positioned at linear precedence SEFAYGSFVR (aa68-77) and AELPGVDPDCDVCITR (aa107-122) peptide of 3 predictions.The 17KDa proteantigen may be used for human body immunodiagnosis lungy, treatment and prevention.
Description
The present invention relates to the 17KDa proteantigen of new mycobacterium tuberculosis (Mycobacterium tuberculosis) (southern India isolate S II 1) and by the some peptide fragment of its deutero-and described antigen with by the application of its deutero-peptide fragment in immunodiagnosis, immunotherapy and the immunoprophylaxis of people and test tuberculosis.The invention still further relates to the antigenic DNA sequence of the described 17KDa of coding, coding by the DNA sequence of 17KDa antigen deutero-peptide fragment with to comprise that the antigenic peptide fragment of the 17KDa antigenic protein of 17KDa in proper order is the DNA and the rna probe of fundamental construction in proper order.Main Application Areas is 17KDa antigen and the application of peptide fragment in immunodiagnosis tuberculosis thereof.The Another application field is 17KDa antigen or the application of its peptide substructure in preparation tuberculosis vaccine.Also having an Application Areas is tuerculoderma or isolated measuring by human body, 17KDa antigen or its substructure peptide is applied to detect the propagation of T cell.The Application Areas in back might treated aspect the human body cancer significant by strengthening cellular immunity.Also having an Application Areas is 17KDa antigen or the application of its substructure peptide aspect Laboratory Production cell growth factor and enzyme.
The human body tuberculosis that is caused by mycobacterium tuberculosis is the important chronic body that extensively the occurs in about 1,600 ten thousand philtrums disease that declines.When developed country and developing country were seriously spread, the infection of the acquired immune deficiency syndrome (AIDS) day after tomorrow (AIDS) had become infectious another difficult problem of the mycobacterium that comprises mycobacterium tuberculosis in developed country at present just when tuberculosis.
Although become a problem, accurately make a definite diagnosis human body tuberculosis and just can utilize effective antibiotic to carry out early treatment in many national resistance.Therefore, the early diagnosis of structure disease means can carry out the effective chemical treatment, thereby eliminates the propagation of live bacilli among the lunger.
Routine diagnosis lungy is decided by clinically to separate with the microscopy of radioexmination result, tubercule bacillus sample and the bacterium of mycobacterium tuberculosis culture.
Because existing available diagnostic method is imperfect, therefore can't global controlling tuberculosis.
Though comprise many countries of India the suspected patient of all X-ray examinations is all carried out the tuberculosis chemotherapy, but many Clinical symptoms lungy not only have specificity to tuberculosis, and the research of carrying out in India (institute of tuberculosis provides by the Bangalore, IND whole nation) illustrates that only the suspected patient of 30% X-ray examination develops into tuberculosis patient at last.
Under the condition in the open air, the tuberculosis sample is carried out microscopy and is not easy, and at least 10
4Bacillus/ml need screen.Many tuberculosis samples as the celiolymph in the tuberculosis meningitis, are not often to contain bacillus.In addition, bacterial cultures will cultivate for 6 to 8 weeks usually, and is really too time-consuming as a kind of diagnostic method.
Widely used tuberculin skin test then lacks susceptibility and specificity, and needs just can finish in about three days.Because the chemotherapy of tuberculosis needs at least 6 months, the patient of many undesired treatments has produced resistance, causes the propagation of live bacilli.Have now found that traditional BCG vaccine inoculation, its degree of protection also changes with the area is different.
Based on these factors, the many researchists and the World Health Organization are all thought, should be considered as early diagnosis lungy the field of preferential research and development.
Mycobacterium is used for the immune auxiliaries of booster immunization reaction, verified it be effective antigens to humans and animals.Now know, can impel the generation of tuberculosis patient specific antibody and fertile lymphocytic formation (Ivanyi et al., 1988) and experimental animal model by many antigens of mycobacterium tuberculosis generation.The detection of antibody response can be applicable to early diagnosis in the tuberculosis patient.About the research to the mycobacterium tuberculosis of T lymphocyte specific, the development by tuerculoderma and protectiveness vaccine equally also can be applicable to early diagnosis.
About the center of the above-mentioned each side research of tuberculosis combines in the evaluation of mycobacterium tuberculosis specific antigens and synthetic.
Many researchists have identified the proteantigen of the mycobacterium tuberculosis with immunodiagnosis or immune protection application possibility.The interior amino-acid sequence of many above-mentioned antigenic N-terminal is delivered, and existing wherein some antigenic N terminal amino acid is listed in table 1 in proper order.
Table 1: through the antigenic N terminal amino acid order of the mycobacterium tuberculosis protein of identifying
Investigator's antigen and N-terminal
Shinnick et al,1987 65 kDa:RGCRHPV
Yamaguchi et al,1987 MPB 57:MAKFNIKPL
Pattorroyo et al,1987 13 kDa:AKVNI
18 kDa:GDLVGPGAE
23 kDa:APKTY
30 kDa:FSXPGL
68 kDa:WMTMT
77 kDa:GKXIAYDGAA
Matsuo et al,1988 30 kDa:FSRPGLP
Ashbridge et al,1989 19 kDa:EHRVKRGLTV
Baird et al,1989 10 kDa:AKVNIPKP
Garcia et al,1989 70 kDa:FQRITRQDLL
Borremans et al,1989 32 kDa:FSRPGLP
As far as we know; none can be as the test products of immunodiagnosis in the above-mentioned antigen; wherein the 65KDa homologue of two antigen 10KDa that is (1989) such as Baird and Shinnick etc. (1987) after measured; it is used for the protectiveness extreme difference (U.S. Wei Sikangxin university D.W.Smith, private communication) of the vaccine of experimental animal model to mycobacterium tuberculosis.
The A60 diagnostic antigen ELISA box lungy that use is present in all mycobacteriums has been used for ANDA diagnosis (France), but this mensuration also not only has specificity to human body tuberculosis.
The other method of diagnosis of tuberculosis just is to use dna probe.The gene probe box (1988) that obtains by commercial sources has been used to identify the composition that is separated the mycobacterium tuberculosis mixture that obtains by culture, but has not been directly used in clinical sample.These mensuration can not be got rid of other mycobacteriums and obtain positive result.(J.Clin.Microbiol.1988 Dec.) has been used for 1000 bases to the dna probe that is designed by Enzo Biochem or longer specific DNA order and requirement has higher specificity.Subject matter based on all probes of DNA is whether can obtain real positive result with patient's sample, thereby avoids complicated bacillus to cultivate.
Separate in the mycobacterium tuberculosis bacterial strain that obtains (Naganathan et al., 1987) in southern India, known virose phenotypic variation, its molecular basis is not clear.(1988) such as Abou Zeid find that the 13KDa proteantigen is present in the phage II type toxicity mycobacterium tuberculosis, but do not exist in phage I type south India hypotoxicity tubercule bacillus.And the relation between this antigen and toxicity is not also studied in great detail so far.
Have now found that, in the isolate of mycobacterium tuberculosis, have the 17KDa proteantigen, the invention discloses its immunochemistry feature.
Therefore, the present invention relates to following all respects:
1. the 17KDa proteantigen of mycobacterium tuberculosis as described below (S II 1) and some substructures (peptide) thereof.
2. the antigenic DNA sequence of 17KDa in coding mycobacterium tuberculosis (S II 1).
3. the DNA sequence of the 17KDa antigen substructure (peptide) in coding mycobacterium tuberculosis (S II 1).
4. described 17KDs albumen or described substructure (peptide) are used to prepare mono-clonal or the polyclonal antiserum that reacts with described 17KDa proteantigen or its substructure.Described antibody can be used for polyclonal such as mouse, rabbit and sheep one class Mammals and produce being used for monoclonal mouse.
5. the 17KDa proteantigen of described substructure (peptide) is used for detecting the antibody of the humans and animals sample of immunodiagnosis.Detection method is methods known in the art, for example ELISA, radioimmunoassay (RIA) and passive reverse hemaglutination methods such as (RPHA).
6.17KDa proteantigen or its substructure (peptide) application in treatment tuberculosis.
7.17KDa proteantigen or its substructure (peptide) application in preparation tuberculosis vaccine.
8.17KDa proteantigen or its substructure (peptide) are used for the application of tuerculoderma immunodiagnosis reagent lungy in preparation.
9. the protein order with 17KDa antigen or its substructure (peptide) is DNA or the rna probe that fundamental construction is used for diagnosis of tuberculosis.This class probe can make up and mark by means known in the art, for example mixes radio isotope or carries out the nonradioactive labeling with vitamin H.
10. by monoclonal antibody reactive such as body fluid such as the serum of waiting to diagnose the patient, CSF and Pleural fluid and 3 described 17KDa antigens or its substructure (peptide), diagnosis human body method lungy.
11. by the body fluid (for example serum) and 5 described 17KDa albumen tests of waiting to diagnose the patient, diagnosis human body method lungy.
12. by the body fluid (for example phlegm, serum, CSF and Pleural fluid) and 9 described DNA or rna probe reaction, diagnosis human body method lungy of waiting to diagnose the patient.
13. vitro detection human body method lungy, this method comprise that patient's body fluid (for example phlegm, CSF, Pleural fluid or serum) sample contacts with 10 described monoclonal antibodies through mark.
14. vitro detection human body method lungy, this method comprise that patient's body fluid (for example phlegm, CSF, Pleural fluid or serum) sample contacts with 10 described monoclonal antibodies through mark.
15. vitro detection human body method lungy, this method comprise that patient's body fluid (for example phlegm, CSF, Pleural fluid or serum) sample contacts with 3 described polyclonal antibodies through mark.
Implement immunodiagnosis medicine box lungy 16. use the antigenic monoclonal antibody of 3 described 17KDa.
Implement immunodiagnosis medicine box lungy 17. use 5 described 17KDa antigens or peptide substructure.
Implement the medicine box of diagnosis of tuberculosis 18. use 9 described DNA or rna probe.
19. express the microorganism of 1 described 17KDa albumen or its substructure.
20. with 1 described 17KDa antigen or its substructure peptide tuberculosis vaccine that is foundation development.This class vaccine can be the product of the organism (for example salmonella and vaccinia virus etc.) handled by genetic engineering method.
The present invention diagnosis, treatment and the prevention by exemplifying disease, the particularly diagnosis of mycobacterium tuberculosis transmissible disease are illustrated, but are not to limit the present invention with these embodiment.The disclosed content of the application, its application facet can comprise the deagnostic test of infectious agents such as prevailing disease inspection, legal medical expert's investigation, food contamination mensuration, public health inspection, prophylactic agent and farming animals animal husbandry.
Antigenic fractional separation of embodiment 1:17KDa and purifying
Sonic treatment antigen raw product under 37 ℃, places the Kirchner substratum to cultivate for 2 weeks mycobacterium tuberculosis, and the bacillus of collecting killed through 18 hours down and in the cold acetone at 4 ℃.Bacillus is used salt water washing 3 times, salt solution [5ml] suspension of getting the 10mg bacillus with Branson ultrasonoscope micro detector under 40 watts of output ratings through sonic treatment, under 20000 * g centrifugal 30 minutes then, measure in its supernatant liquor behind the protein content, with the Lowry method again-70 ℃ of following chilled storages.
Fractional separation and purifying: get the raw product of 500 μ g, by Hunkapi-ller and Lujan(1986 through sonic treatment) fractional separation on 12.5% polyacrylamide gel introduced.Simply dye with Coomassie brilliant blue, can be visual to protein band.With 0.1%SDS and 0.05M bicarbonate of ammonia 17KDa antigen is carried out electroelution, use 0.02%SDS and the electrodialysis of 0.01M bicarbonate of ammonia then, the albumen chloroform-methanol extraction of wash-out is removed SDS, with drying precipitate.
Get 5 these throw outs of μ g, carry out HPLC with Lichrosorb RP18 post (LKB) and detect, analyze the purity of eluted protein.At 45%B with located the peak value that eluate presents in 26 minutes, contain immune response antigen.Fig. 1 represents the antigenic HPLC figure of 17KDa.
The antigenic amino-acid sequence analysis of embodiment 2:17DKa
About the antigenic peptide mapping of 17KDa, this paper provides trypsinase figure and V8 proteolytic enzyme figure.
Trypsinase figure: get the 17KDa albumen of 30 μ g, under 37 ℃, in 0.1M ammonium bicarbonate buffers (pH7.8), using the trypsin enzyme-to-substrate ratio of handling through TPCK is 1: 50) digested 5 hours.At the RP18 post of crossing through solvent orange 2 A (the 0.1%TFA aqueous solution) balance (0.46 * 25cm) goes up with HPLC fractional separation tryptic digestion thing, and contains 70% acetonitrile solution of 0.085%TFA with the gradient solvent B(of 0-65%) with peptide wash-out 60 minutes.Trypsinase is illustrated in Fig. 2.
V8 proteolytic enzyme figure: get the 17KDa antigen of 30 μ g, under 37 ℃, in 0.07% ammonia, use staphylococcus V8 protease treatment 48 hours.The mol ratio of enzyme-to-substrate is 1: 25.Under the condition that is used for proteolytic enzyme figure, the various peptides on the HPLC post in the purifying enzyme digest.Peptide is illustrated in Fig. 3.
The antigenic sequence analysis of 17KDa: use that (Applied Bios-ystems Inc., the PTH amino acid analyzer that USA) is connected carries out the amino-acid sequence analysis of albumen and peptide with 477A type protein order instrument.Sample is dissolved in 10% formic acid, was fixed on through what TFA handled and scribbles Polybre-ne(1mg) fiberglass disc on, be used for sequencing.
Measure preceding 18 amino acid of N end with adequate proteins.According to the amino-acid sequence of the zymolytic peptide section of Trypsin, select the V8 protease-producing can with the zymolytic peptide section of Trypsin eclipsed peptide.The zymolytic peptide section of trypsinase and V8 albumen is linked to be straight line, obtains the antigenic complete sequence of 17KDa.The eclipsed details is shown in Fig. 4.
The antigenic amino acid profiles of 17KDa: protein has A9, C3, D11, E10, F9, G8, H2, I7, K4, L11, M2, P9, Q2, R12, S8, T9, V11 and Y4.Obviously wherein there are not tryptophane and l-asparagine.Because this proteinic acidic amino acid (D+E=21) is Duoed 5 than basic aminoacids sum (R+K=16), so be acid.This albumen has 131 amino acid, and molecular weight is 14762.
The antigenic immunoreactivity of embodiment 3:17KDa
In the mycobacterium tuberculosis bacterial strain, present the antigenic immundominance of 17KDa: mycobacterium tuberculosis (M.tuberculosis) ATCC 27294, black mycobacterium (M.avium intracellulare) and scrofulaceum mycobacterium (M.scrofulace-um) were cultivated for 2 weeks in the Kirchner substratum in Mycobacterium phlei (M.phlei), M. smegmatics (M.smegmatis), Kanasasii mycobacterium (M.kanasasii), the born of the same parents, placed cold acetone to kill the bacillus of collecting.It is described to press embodiment 1, by the antigen of each strain preparation through sonic treatment, in 12.5% gel these antigens is carried out SDS PAGE then and analyzes.Prove that through the blue stained gel of coomassie 17KDa antigen exists only in the mycobacterium tuberculosis bacterial strain.In the Western photolithography, the rabbit anti-serum of mycobacterium tuberculosis S II 1 also is used to detect these sonic treatment things.In the Weste-rn photolithography.Present 17KDa dominance band.
In experimental animal, obtain 17KDs antibody: with isopyknic Fu Luodeshi Freund (FICA) emulsification, and be used for the intraperitoneal immunity of 10BALB/C mouse through the 17KDa of electroelution antigen (10 μ g are in 100 μ l salt solution).After the immunity 30 days, from these mouse, collect serum, the 17KDa band of identification in mycobacterium tuberculosis S II 1 antigen that sonic treatment is crossed.
This tests affirmation, in the mouse of identification antigenic protein structure of 17KDa and substructure (peptide) thereof, can produce polyclone or monoclonal antibody.This antibody-like, particularly monoclonal antibody be as the ELISA method, can be used for the antigen detection method of 17KDa antigen in the human body tuberculosis sample or its substructure, thereby can carry out immunodiagnosis lungy.
The proof of 17KDa antigen and human tuberculosis patients serum reaction: the serum that obtains from the people of 24 health and 20 cultures that turned out to be tuberculosis patient carries out titration with 17KDa antigen as described below.Under 22 ℃, applied PVC Dynatech plate 24 hours through the antigenic PBS of the 17KDa of electroelution with 1 μ g/ml.The plate blockaded of PBS-BSA is used in 2.5 hours serum of 22 ℃ of following incubations and repeats (1/200) diluent titration then.The washing plate obtained anti-human IgG HRP binding substances in 1.5 hours.The washing plate is with O-Phenylene Diamine substrate mensuration and at 492nm place reading.The table II provides the antigenic susceptibility of 17KDa and reaches 70%, and specificity reaches 85%.
Table II: ELISA microdetermination 17KDa antigen on the serum of tuberculosis patient and contrast
Serum n ELISA+ ELISA-susceptibility specificity
Healthy people 24 4 20-85%
Susceptibility: known tuberculosis patient is positive
Specificity: known normal healthy controls group is negative
ELISA+:OD 492 nm>=0.3(1/200 dilution) normal healthy controls group=average+2SDOD 492 nm, n=24).
Therefore, this test has affirmed that 17KDa antigen in the tuberculosis mycobacterium strain can be used in immunodiagnosis human body micro-ELISA lungy system.
The proof of the antibody epitope of its qualification of 17KDa proteantigen: (embodiment 2, and the peptide fragment that 17KDa antigen Fig. 2) produces is used the serum titration of healthy people and tuberculosis patient respectively by the above by tryptic digestion.In 14 determined peptides, have RATYDK, YEVR, LEDEMK, LMR, the peptide of DFDGR and SEFAYGSFVR order shows that the susceptibility of antibody binding activity is 17-36%.Whether form linearity or conformation antibody epi-position in order to measure these peptides, measure another peptide, promptly carry out the inhibition of ELISA and measure with the mouse resisting anteserum of 17KDa antibody and with reference to other 5 peptides.Two peptides of YEVR and ATYDK suppress mutually, thereby illustrate that they are somes of complete antibody epi-position, and press embodiment 2 and Fig. 4 described, the mensuration by the antigenic complete structure of 17KDa makes an affirmation.The explanation of antagonist epi-position, other four peptides all are linear peptides, and may be the conformation peptide.In above-mentioned 6 antibody epitopes of band peptide, the peptide of following amino-acid sequence is the solid phase method synthetic by Merrifi-eld, and contains the antibody binding activity of specificity and susceptibility:
RATYDKRYEVR: susceptibility 65%, specificity 95%
SEFAYGSFVR: susceptibility 66%, specificity 95%
Aforesaid antibody epitope figure spectrometry has illustrated that the antigenic substructure of 17KDa or the peptide that limit can be synthesized, and adopts micro-ELISA method, can use it for immunodiagnosis human body tuberculosis.
Order has been synthesized for the peptide of RATYDKRYEVRDFDGRAEL, and when the CSF sample of the culture that is proved to be tuberculosis patient and contrast was measured, its susceptibility was 86.6, and specificity is 100%.Therefore this peptide is suitable for the 17KDa antigen that replaces in the ELISA mensuration especially.
17KDa antigen can make lymphocytic hyperplasia: the peripheral blood lymphocyte (PBL) of healthy donors and tuberculosis patient is carried out fractional separation, get 2 * 10
6Cell was cultivated 3 days under the 17KDa antigen condition that contains or do not contain (contrast) 1 μ g in containing the RPMl1640 substratum of 10% autoserum.Collecting culture preceding 24 hours, the 3H thymidine with 1 μ ci washes earlier.The explanation of table III, 17KDa antigen can make the lymphocytic lymphocytic hyperplasia (only 2 patient's data) of tuberculosis patient.
Table III: with 17KDa antigen measuring lymphocytic hyperplasia
The PBL source adds 3H thymidine count per minute, and 3 are repeated the average of culture
Healthy donors contrast: 90 antigens: 121
Tuberculosis patient contrast: 110 antigens: 650
17KDa antigen is except having the character that makes lymphocytic hyperplasia, and the method (Rothbard and Taylor, 1988) of prediction T cell stimulatory epitopes also can be used for drawing the possible site in the 17KDa antigenic structure.These t cell epitopes are positioned on the peptide of SEFAYGSFVR and AELPGVDPDCDVCITR order.
Therefore, this tests explanation, and 17KDa antigen or substructure (peptide) can be used in the stimulation human peripheral blood lymphocyte.Because the lymphocyte of irriate can produce some cell growth and differentiation factors of the vaccine effect that helps 17KDa antigen or its substructure, so 17KDa antigen at first can be used as Vaccinum Calmette-Guerini, but also can strengthen cellular immunization non-specificly.
Fig. 5 represents to contain the antigenic primary structure of 17KDa of the mycobacterium tuberculosis of biologically active zone, but does not need to get rid of similar activity in not marking the district.
The discussion of measurement result and summary: the present invention has introduced the immunochemistry character of 17KDa proteantigen new in the mycobacterium tuberculosis (S II 1 bacterial strain).Mycobacterium tuberculosis has caused worldwide 1,600 ten thousand people's tuberculosis.Because the defective that existing diagnostic method exists, present this disease also fails to be eliminated.Because traditional BCG vaccine only can partly prevent tuberculosis, detect early stage immune diagnostic method lungy and identify suitable epidemic practice so research in recent years concentrates on to carry out.
The research explanation that the present invention introduced, the new 17KDa antigen that is produced by knot persimmon mycobacterium has antigenic activity, and its antigenic property and antigen chemistry are studied.
The biological activity protein of proteantigen is provided, has studied early detection immune diagnostic method lungy.
Therefore, found to have two kinds of peptides that 131 amino acid whose 17KDa proteantigens contain RATYDKRYE-VR and SEFAYGSFVR order, had the antibodies epi-position of diagnosis of tuberculosis.In addition, two kinds of peptides that also contain band prediction T cytositimulation district in SEFAYGSFVR and AELPGVDPDCDVCITR order.The two kinds of peptides in back may increase the antigenic T cytositimulation of whole 17KDa of the present invention character.The T cytositimulation character of 17KDa antigen and substructure peptide thereof means that they can be used for treatment lungy and prevention.
Description of drawings is as follows:
Fig. 1: the HPLC of the 17KDa proteantigen of the mycobacterium tuberculosis behind electroelution (S II 1) analyzes.
The HPLC condition: RP18 post (LKB, 10 μ m apertures), the A=0.1% TFA aqueous solution, the acetonitrile of B:0.085%TFA (70%) solution, gradient: 0-65%, B, 40 minutes, susceptibility: 0.08,220nm.
The tryptic peptide of the 17KDa proteantigen of HPLC fractional separation mycobacterium tuberculosis (S II 1) Fig. 2:, 10 μ m apertures) through RP18(LKB.
Fractional separation: the A:0.1%TFA aqueous solution, 70% acetonitrile solution of B:0.085%TFA, gradient: 0-65%B, 60 minutes, susceptibility: 0.08.220nm.
Amino-acid sequence: provide the order that each peptide is measured.
The V8 mmp polypeptide of the 17KDa proteantigen of the mycobacterium tuberculosis (S II 1) of HPLC fractional separation Fig. 3:, aperture 10 μ m) through RP18(LKB.
Fractional separation: the A:0.1%TFA aqueous solution; 70% acetonitrile solution of B:0.085%TFA, gradient: 0-65%, 60 minutes, susceptibility: 0.082,220nm.
Fig. 4: the antigenic primary structure of 17KDa of the mycobacterium tuberculosis (S II 1) that expression peptide straight line connects.Trp: trypsinase, V8: streptococcus aureus V8 proteolytic enzyme.Be marked on the amino-acid sequence that top arrow represents that adequate proteins obtains.
(one-letter code is used for amino acid).
Fig. 5; Provide the antigenic primary structure of 17KDa of the mycobacterium tuberculosis (S II 1) of biologically active zone.
AA68-77: expression antibody and t cell epitope.
AA91-101: expression antibody epitope.
AA107:122: the epi-position of 2 T cells of expression.
Reference
1. A.Hopps and E.J.Woods,Proc.Natl.Acad.Sciences,(1981)78,3429
2. Hunkapiller,M,W and Lujan E,In,Methods in Protein Microcharacterization,Ed.J.E.Shiveley,Humana Press(1986)pp.89
3. Naganathan,N,etal.Tubercle(1986)67,261
4. Shinnick,F,et al,J.Bacteriol,(1987)169,1080
5. Yamaguchi,Y,etal,FEBS Letters,(1987)240,115
6. Pattorroyo G,et al,Modern Biotechnology in Health,Academic press,(1987),pp.110.
7. Ivanyi,J et al,Springers Seminars Immunopathol,(1988)10 279
8. Abou-Zeid etal,J.Gen Microbiol(1988)134,531
9. Matsuo M etal,J.Bacteriol,(1988)170,3847
10. Rothbard,J,B and Taylor,W,EMBO J(1988)7,93
11. Ashbridge S,etal,Nucleic Acids Res(1989)17,1249
12. Boremans et al,Infection and Immunity,(1989)57,3123
13. Garcia E,etal(1989)Infection and Immunity(1989),57,204
14. Baird et al,J.Gen Microbiol,(1989)135,931.
Claims (26)
1, the albumen of following array structure:
1 40
ATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRELM
30
RSIQITIKLEDEMKGIYLPVAKHGELRSEFAYGSFVRTVS
131
LPVGADEDDIRATYDKRYEVRDFDGRAELPGVDPDCDVCITRGILTVSVCV
2, the peptide of following array structure and the proteic fragment of claim 1:
RATYDKRYEVRDFDGRAEL
AELPGVDPDCDVCITR
The order that comprises one or more these peptides.
3, the proteic DNA sequence of coding claim 1 definition.
4, the DNA sequence of the peptide of coding claim 2 definition.
5, DNA or the RNA hybridization probe that makes up according to the albumen or the DNA sequence of peptide or claim 3 or 4 of claim 1 or 2 definition, described probe can carry out mark arbitrarily.
6, according to claim 1 or the albumen of 2 definition or the antibody of peptide.
7, according to the antibody of claim 6, it is polyclone or monoclonal antibody, and described antibody can carry out mark arbitrarily.
8, implement immunodiagnosis medicine box lungy, it comprises the albumen or the peptide of claim 1 or 2.
9, implement immunodiagnosis medicine box lungy, it comprises the monoclonal antibody of claim 7.
10, enforcement is based on the medicine box of the diagnosis of tuberculosis of DNA, and it comprises the hybridization probe of claim 5.
11, can express the microorganism of the albumen or the peptide of claim 1 or 2.
12, Vaccinum Calmette-Guerini comprises the albumen or the peptide of claim 1 or 2.
13, diagnosis human tuberculosis's method, this method comprises patient's body fluid, serum for example reacts to each other with the albumen or the peptide of claim 1 or 2.
14, diagnosis human tuberculosis's method, this method comprises patient's body fluid, for example phlegm, CSF, Pleural fluid or serum react to each other with monoclonal antibody through the claim 7 of mark.
15, diagnosis human tuberculosis's method, this method comprises patient's body fluid, for example phlegm, CSF, Pleural fluid or serum react to each other with dna probe through the claim 5 of mark.
16, vitro detection human tuberculosis's method, this method comprises patient's humoral sample, for example phlegm, CSF, Pleural fluid or serum contact with the polyclonal antibody of the claim 7 of passing through mark.
17, vitro detection human tuberculosis's method, this method comprises patient's humoral sample, for example phlegm, CSF, Pleural fluid or serum contact with the monoclonal antibody of the claim 7 of passing through mark.
18, according to the albumen or the peptide of claim 1 or 2, its in the method for treatment human or animal body, be applied to the human tuberculosis immunodiagnosis, treat or inoculate against.
19, according to the albumen or the peptide of claim 1 or 2, it is applied to produce the mammiferous polyclonal antibody or the monoclonal antibody of the immune diagnostic method that is used for picture ELISA method in the mouse body in the method for treatment human or animal body, this method can detect the albumen as claimed in claim 1 among the human tuberculosis.
20, according to the albumen or the peptide of claim 1 or 2, it by early detection serum solution method lungy, is used for detecting the antibody of tuberculosis sample in the method for treatment human or animal body.
21, according to the albumen or the peptide of claim 1 or 2, it for by skin acquisition immunodiagnosis or in order to obtain the means lungy that inoculate against, is used for detecting the propagation of tuberculosis sample T cell in the method for treatment human or animal body.
22, claim 1 or 2 albumen are used for human tuberculosis's immunodiagnosis, treatment and preventive vaccination.
23, claim 1 or 2 albumen or peptide are used to develop the reagent by the propagation treatment tuberculosis infection of T cell.
24, claim 1 or 2 albumen or peptide are applied to the cell proliferating determining by T, produce growth and differentiation factor.
25, the DNA of claim 5 or rna probe are used for detecting the mycobacterium tuberculosis DNA of people's tuberculosis sample, in order to obtaining early detection tuberculosis.
26, the DNA of claim 5 or rna probe are applied to identify mycobacterium DNA and the laboratory study in the culture of isolated thing.
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SE9001105A SE9001105D0 (en) | 1990-03-27 | 1990-03-27 | NEW METHODS FOR DIAGNOSIS OR TUBERCULOSIS |
SE9001105 | 1990-03-27 |
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SE (1) | SE9001105D0 (en) |
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US7300660B2 (en) | 1993-11-23 | 2007-11-27 | The Regents Of The University Of California | Abundant extracellular products and methods for their production and use |
US6752993B1 (en) | 1993-11-23 | 2004-06-22 | The Regents Of The University Of California | Abundant extracellular product vaccines and methods for their production and use |
WO1995014713A2 (en) | 1993-11-23 | 1995-06-01 | The Regents Of The University Of California | Abundant extracellular products and methods for their production and use |
US6290969B1 (en) | 1995-09-01 | 2001-09-18 | Corixa Corporation | Compounds and methods for immunotherapy and diagnosis of tuberculosis |
US6458366B1 (en) | 1995-09-01 | 2002-10-01 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
US6338852B1 (en) | 1995-09-01 | 2002-01-15 | Corixa Corporation | Compounds and methods for diagnosis of tuberculosis |
US6592877B1 (en) | 1995-09-01 | 2003-07-15 | Corixa Corporation | Compounds and methods for immunotherapy and diagnosis of tuberculosis |
US6013660A (en) * | 1996-10-02 | 2000-01-11 | The Regents Of The University Of California | Externally targeted prophylactic and chemotherapeutic method and agents |
US6465633B1 (en) | 1998-12-24 | 2002-10-15 | Corixa Corporation | Compositions and methods of their use in the treatment, prevention and diagnosis of tuberculosis |
CN103063836B (en) * | 2011-10-18 | 2016-03-30 | 复旦大学附属华山医院 | Detect the reagent of mycobacterial infections, method and kit |
CN111518165B (en) * | 2020-05-08 | 2021-10-01 | 宁夏大学 | Polypeptide specifically binding to mycobacterium tuberculosis, coding gene and application thereof |
CN112125954B (en) * | 2020-09-28 | 2023-02-28 | 宁夏医科大学总医院 | Heptapeptide specifically combined with BCG (bacillus calmette guerin), coding gene, preparation method and application |
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US4952395A (en) * | 1987-02-26 | 1990-08-28 | Scripps Clinic And Research Foundation | Mycobacterial recombinants and peptides |
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