CN101735321B - Polypeptide composition and application thereof in detecting tuberculosis antibody - Google Patents
Polypeptide composition and application thereof in detecting tuberculosis antibody Download PDFInfo
- Publication number
- CN101735321B CN101735321B CN 200810203532 CN200810203532A CN101735321B CN 101735321 B CN101735321 B CN 101735321B CN 200810203532 CN200810203532 CN 200810203532 CN 200810203532 A CN200810203532 A CN 200810203532A CN 101735321 B CN101735321 B CN 101735321B
- Authority
- CN
- China
- Prior art keywords
- antigen
- mpt64
- esat
- cfp10
- mtb8
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a polypeptide composition which is formed by combining an antigen Mtb16.3, an antigen 38kD, ESAT-6, CFP 10, MPT 64, Mtb 8 and Mtb 8.4. The antigens Mtb 16.3, 38kD, ESAT-6, CFP 10 and MPT 64 have excellent complementarities in serological detection. The sensitivity for diagnosing tuberculosis is obviously promoted by combining antigen Mtb16.3 with at least one of antigen 38kD, ESAT-6, CFP 10 and MPT 64.
Description
Technical field
The present invention relates to a kind of peptide composition, relate in particular to a kind of peptide composition of the Mtb16.3 of comprising specific epitopes antigen peptide section, relate to more specifically a kind of be applied to Mtb16.3 specific epitopes antigen peptide section that tuberculosis antibody detects peptide composition.
Background technology
In recent years, (Tuberculosis TB) becomes public health problem and the social concern that the whole world is paid close attention to white plaque.According to statistics, at present there is pulmonary tuberculosis patient 2,000 ten thousand in the whole world, and New Development patient 1,000 ten thousand, and annual death toll 3,000,000 has surpassed the summation of other transmissible disease.
Pulmonary tuberculosis (Pulmonary tuberculosis) demonstrates global epiphytotics all principal characters, and in worldwide, spreads.Nearly 1/3 people infects tubercule bacillus on the earth, causes annual 8000000 routine active tuberculosis and 3,000,000 people dead.The appearance of the multiple medicine persister of tubercule bacillus and merging HIV infect and make situation become more severe.According to the World Health Organization (WorldHealth Organization, information WHO) is if can not effectively control; In following 10 years, will there be 3,000 ten thousand people to die from white plaque, also have 9; 0,000,000 active tuberculosis new cases, white plaque are becoming adult's No.1 killer.Tuberculosis so powerful popularity and infectivity is a great challenge to public health; People press for the method that addresses these problems; Yet existing some are far from being enough to antiphthisic strategy, especially in diagnostic method, exist more problem.
Though present diagnosis lungy has several different methods, all has such or such problem.Though imaging diagnosis is still main body, it needs other aided diagnosis method; The phlegm smear is simple, but positive rate is low; It is gold standard that phlegm is cultivated, but the cycle is oversize, can not satisfy the needs of quick diagnosis clinically; PCR molecular biology method susceptibility is high, diagnosis speed is fast, detects unsettled defective but exist; The phage splitting method can detect slip-knot nuclear bacillus, can not detect non-mycobacterium tuberculosis infection, is applicable to tubercule bacillus resistance experimental study more; And the serodiagnosis technology has its inherent advantages, as easy and simple to handle, quick, be prone to judged result, good stability, be convenient to promote, need not special precision instrument etc.
The serology that detects for white plaque detects, and seeks the antigenic component of hypersensitivity (Sensitivity) and high specific (Speciality), and the detection method of setting up the high specific hypersensitivity is to solve the early stage key of diagnosis quick and precisely of white plaque.People have dropped into increasing concern to setting up the reliable serology diagnosis of tuberculosis method of specific specificity and susceptibility at present.At diagnostic field lungy; Though use Tuberculins,PPD (purified proteinderivative usually; PPD) in order to judge whether to infect mycobacterium tuberculosis (Mycobacteriumtuberculosis); But the negative findings of tuberculin test can not be got rid of and suffers from possibility lungy (Postgrad.Med.1996,100,201-204).In addition; Since antigen that PPD comprised since for pathogenic mycobacterium (Mycobacterium), environment mycobacterium and BCG-CWS (Mycobacterium.bovis bacillus Calmette-Gu é rin, BCG) common, so the positive findings that in time detects can not clearly be distinguished the BCG immunity, the environment mycobacterium infects and pathogenic mycobacterium tuberculosis infection (Mol.Immuno.; 2002; 39,113-119), thus can not realize the accurately purpose of diagnosis.The PPD source that commercialization provides is different, does not pass through meticulous preparation, and antigen is formed does not have stdn yet, so can produce some skin test reactions (skin test response) (Karger, Basel, 1996,201-211).
Comparative genomics discloses, and has 16 kinds of zones to appear in pathogenic clinical strains mycobacterium tuberculosis and the Mycobacterium bovis (Mycobacterium bovis).These zones be named as RD1-RD16 (region deleted, zone RD) do not appear in the M.bovis BCG vaccine strains (Bacteriology 1996,178,1274-1282; Mol.Microbiol., 1999,32,643-655).The albumen of these regional codes comprises: early stage secreted antigen target protein (early secretoryantigen target 6kDa; ESAT-6), MPT64 (protein of Mycobacteriumtuberculosis), culturing filtrate protein 10 (culture filtrate protein 10, CFP10), MPB83 and KatG or the like.There are some researches show, with these albumen separately or combination use and can play effect (Leukoc.Biol., 1995,57,221-225 infection that animal pattern is resisted mycobacterium tuberculosis; Gen.Microbiol., 1987,133,1431-1435).In these albumen, like ESAT-6, CFP10 and MPT64 etc., just current diagnosis white plaque area research get at most, most important antigen.In addition, comprise that 38kD antigen is all as the mark of serology detected activity property tuberculosis.
38kD antigen is a kind of lipoprotein and main immunogens, is one of diagnosis of tuberculosis antigen the most commonly used at present.38kD antigen is that tubercule bacillus specific specificity proteantigen and content exceed BCG10 doubly.When being used for antitubercle sera detection, this proteic susceptibility and specificity are superior to other single antigen.There is research to confirm that with 38kD Detection of antigen serum of tuberculosis patients, sensitivity and specificity are respectively 65.6% and 95.8% (J.Clin.Microbiol., 1997,35,553-557).
It is active that ESAT-6 and CFP10 have stronger cellular immunization, in the immunne response of resisting tuberculosis infection, play an important role, and be two kinds of antigens very promising in the white plaque immunodiagnosis.According to the gene ORFs (open reading frames, ORF) and the computer program of design has been predicted the RD1 district of the M.tuberculosis special albumen of 14 kinds of length 99-666 amino acid scopes of encoding.Wherein just comprise these two antigens (Mol.Immuno., 2002,39,113-119) of CFP10 (Rv3874) that constitutes by 100 amino acid and the ESAT-6 (Rv3875) that constitutes by 95 amino acid.The RD1 district only is present in the pathogenic tubercle bacillus gene group, all should zone (J.Bacteriol., 1996,178,1274-1282) in all BCG strain gene groups.Through to antigen PPD-M, PPD-A, ESAT-6, Ag85; 38kD, MPB64, MPB70, MPB83; Hsp16.1, hsp65, what the t cell responses of and hsp70 was comprehensive and comprehensive discovers, ESAT-6 is the early stage major antigen (Infect.Immun. of tuberculosis molecule coli infections; 2000,68,2573-2578).RD1 is first zone of in the inferior bacterial strain of BCG pasteur bacterial strain deutero-BCG, not finding, and the ESAT-6 albumen of being encoded by this district has surprising immunodominance (Nature1997,389,133-134) in the immunoreation of human body mycobacterium tuberculosis.ESAT-6 and CFP10 show very strong inducing action to IFN-(interferon gamma, IFN-γ).Comparatively speaking, the inducing action of CFP10 seems more weak, but after itself and ESAT-6 are combined, response capacity is improved.ESAT-6 and CFP10 can form the heterodimer structure, and combined utilization ESAT-6 and CFP10 diagnosis of tuberculosis are found hybrid antigen susceptibility than the former increase of monoclonal antibody, and do not reduce its specificity (Clin.Diagn.Lab.Immunol., 2000,7,155-160).So ESAT-6 and CFP10 are expected to become the specific antigens of the pathogenic mycobacterium tuberculosis infection of diagnosis animal and human.38kD and MPT64 also are parts of forming vaccine not only as diagnostic antigen.
MPT64 is a kind of secretor type negre antigen, also is that a kind of (Extra-pulmonary tuberculosis EPTB) has the diagnostic antigen (Diagnostic Pathology 2007,2,36-34) of specificity and sensitivity to the outer tuberculosis of lung.MPT64 is a growth of bacillus tubercle excretory major protein the earliest, by the RD2 regional code of in BCG pasteur bacterial strain, not finding.Separate in the cavy body that this antigen can be crossed from mycobacterium tuberculosis immunity and obtain (Infect.Immun.2002,70,672-678; Infect Immun.2000,68,214-220).ESAT-6 and MPT64 did not then find in the BCG vaccine by the mycobacterium tuberculosis genes encoding.The natural MPT64 and the MPT64 of reorganization all can make tuberculosis patient or tuberculin positive person produce t cell responses and skin delayed hypersensitivity (delayed-type hypersensitivity, DTH).But because MPT64 only is present among the compound crowd of tubercule bacillus, most of BCG bacterial strains lack the MPT64 genes, BCG inoculator or environment mycobacterium the infected (as: bird mycobacterium) to MPT64 tuerculoderma do not react.There is result of study to show; Though is lower as the positive rate of Detection of antigen tuberculosis antibody with MPT64 albumen in various tubercule bacillus secreted proteins; If but form fusion rotein with other albumen then might have stability (Clin.Diagn.Lab.Immunol., 2000,7 preferably; 155-160), thus MPT64 albumen promise to be one of the candidate of the combined antigen of tuberculosis serological diagnosis.
Research has been found that; Because the individual immunoreation that causes because of white plaque has complicacy, and main histocompatibility complex (major histocompatibility complex, the heredity that MHC) is caused limitation; So a kind of effective subunit vaccine or diagnostic reagent are necessary to comprise multiple epitope; To guarantee that it can be applicable to upward different crowd (Infect.Immun.1999,67,1702-1707 of performance of heredity; Eur.Respir.J.2001,17,537-557).People such as Tavares show the Studies on Diagnosis that 38kD/CFP10,38kD/MPT64, ESAT-6/MPT64, four kinds of fusion rotein antigens of ESAT-6/CFP10 are used for tuberculosis infection; The detection sensitivity basically identical of 38kD/CFP10 and ESAT-6/CFP10; But (tuberculin skin test, the patient who TST) is positive have higher reaction preference (Microbiol.Immunol., 2007 to tuberculin skin test; 51,289-296).With process work of the present invention; Be surprisingly found out that the unique advantage of Mtb16.3 specific epitopes antigen peptide section aspect detection white plaque, it all improves with combined detection sensitivity lungy and the specificity of can making of at least a specific epitopes antigen peptide section that is selected from 38kD, ESAT-6, CFP10 and MPT64.In mouse experiment, to compare with single MPT64, the ESAT-6/MPT64 fusion rotein shows stronger human body and cell immune response, and the white plaque model mice in the experiment is had the certain protection effect.The investigator thinks that this result shows that the ESAT-6/MPT64 fusion rotein is a kind of potential vaccine (Protein Expr.Purif., 2008,59,189-196).
Mtb8.4 (Rv1174c), Mtb8 (Rv0379) and Mtb16.3 (Rv2185c) are though extensively be present in the tubercule bacillus (comprising BCG), and its meaning aspect diagnosis of tuberculosis is still unclear.Mtb8.4 is a kind of LMWP antigen that purifies and separates obtains from tubercule bacillus culture filtrating recently; Scholars such as Coler (J Immunol.; 1998; 161,2356-2364) research further discloses Mtb8.4 and suffers from immunocompetence T cell antigen important in the individuality of the mycobacterium tuberculosis infection of hiding, and in confirming pathogenic mycobacterium tuberculosis infection, has vital role.Mtb8 possibly be a translocator matter; Only at (Clin.Diagno.Lab.Immun. such as Houghton; 2002; 9,883-891) in the research Mtb8 and other several kinds of antigens (Mtb11, Mtb48,38kD etc.) composition fused antigen are used to detect white plaque, show that Mtb8 might strengthen the antigenic reactive behavior of 38kD with other several kinds of antigens.Mtb16.3 is a kind of unknown function protein, there are some researches show Mtb16.3 and Mtb9.7 combination help to improve to the recall rate of the tuberculosis patient of HIV coinfection.
Carry out a large amount of research in the field lungy though scholars detect in serology, still do not found all gratifying single antigen of sensitivity and specificity, fused antigen or its combined antigen at present.
Summary of the invention
One object of the present invention is to provide a kind of peptide composition, and this peptide composition is combined by antigens such as Mtb16.3 and 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4.Mtb16.3 can detect aspect the binding antibody complementaryly mutually with other several peptide species, detects sensitivity lungy and specificity thereby improve serology.
Another object of the present invention is to provide a kind of peptide composition, and this peptide composition is that Mtb16.3 forms mixing of fusion rotein with antigens such as 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4.Aspect the detection tuberculosis antibody, Mtb16.3 can not only be complementary mutually with other several individual antigen, and can also be complementary mutually with multiple antigen, detects sensitivity lungy and specificity thereby further improve serology.
Another purpose of the present invention is to provide a kind of peptide composition, and is combined or constitute the fused antigen remix and form by antigens such as Mtb16.3 and 38kD, ESAT-6, CFP10 and MPT64.It is used to detect the antibody of replying tubercle bacillus antigen and producing, and the application in the white plaque antibody test.
Antigen Mtb16.3,38kD, ESAT-6, CFP10, MPT64, Mtb8 or Mtb8.4; Also comprise and use the protein epitope forecasting software; Like BIOSUN (calculation biology center, Institute of Basic Medical Sciences, Beijing), choose then have one or more epi-positions in the antigen, the corresponding peptides section of advantage epi-position (can excite stronger immunoreactive epi-position) especially; Molecular biology method through routine angles to be got its encoding sequence and gives expression to this antigenic peptide.
Through the antigenic aminoacid sequence of protein epitope forecasting software gained Mtb16.3 specific epitopes is SEQ ID No 1, and its corresponding D NA encoding sequence is SEQ ID No 10.
Through the antigenic aminoacid sequence of protein epitope forecasting software gained 38kD specific epitopes is SEQ ID No 2, and its corresponding D NA encoding sequence is SEQ ID No 11.
Through the antigenic aminoacid sequence of protein epitope forecasting software gained ESAT-6 specific epitopes is SEQ ID No 3, and its corresponding D NA encoding sequence is SEQ ID No 12.
Through the antigenic aminoacid sequence of protein epitope forecasting software gained CFP10 specific epitopes is SEQ ID No 4, and its corresponding D NA encoding sequence is SEQ ID No 13.
Through the antigenic aminoacid sequence of protein epitope forecasting software gained MPT64 specific epitopes is SEQ ID No 5, and its corresponding D NA encoding sequence is SEQ ID No 14.
Through the antigenic aminoacid sequence of protein epitope forecasting software gained Mtb8 specific epitopes is SEQ ID No 6, and its corresponding D NA encoding sequence is SEQ ID No 15.
Through the antigenic aminoacid sequence of protein epitope forecasting software gained Mtb8.4 specific epitopes is SEQ ID No 7, and its corresponding D NA encoding sequence is SEQ ID No 16.
Mtb16.3,38kD, ESAT-6, CFP10, MPT64, Mtb8 and the Mtb8.4 specific epitopes antigen peptide section selected according to computer forecast are carried out suitable modification and are still had the detection effect.For example indivedual amino acid wherein guard replacement, increase or are lacked, amino acid refer to be less than 10,9,8,7,6,5,4,3,2,1 amino acid individually.The concrete mode of conserved amino acid replacement is: the mutual replacement between Ala, Val, Leu and Ile; Mutual replacement between Ser and Thr; Mutual replacement between acidic residues Asp and Glu; Mutual replacement between Asn and Gln; And the mutual replacement between alkaline residue Lys and Arg; The perhaps mutual replacement between aromatic residue Phe and Tyr.These amino acid of replacing mutually have identical/akin biochemical property, as: (" biological chemistry ", Beijing: Higher Education Publishing House, 1990) such as wetting ability, electric charge property, iso-electric points should be regarded as being equal to replacement.The antigen that indivedual amino acid are guarded replacement, increased or lack, its space pleated sheet structure can not change, and epitope does not change yet.
5 of ESAT-6 specific epitopes antigen peptide sections (SEQ ID No 3) and 34 Asp can replace with Glu individually or simultaneously; 6,24 and 39 Glu can be independent, any two or replace with Asp simultaneously; 26 Tyr can replace with Phe; 2,10 and 21 Ser can be independent, any two or replace with Thr simultaneously; 8,13 and 32 Lys can be independent, any two or replace with Arg simultaneously; 9,27,30 and 31 Gln can independent, any two, any three or replace with Asn simultaneously; 3,4,11,14 and 40 Leu can independent, any two, any three, any four or replace with Ile simultaneously or Ala or Val; 15-17,25,35 and 37 Ala can independent, any two, any three, any four, any five or replace with Ile simultaneously or Leu or Val.These replacement types can be carried out separately, also can carry out with the combination of any replacement type.
11 of CFP10 specific epitopes antigen peptide sections (SEQ ID No 4) and 28 Asp can replace with Glu individually or simultaneously; 1,9,12,29 and 30 Glu can be separately,, any two, any three, any four or replace with Asp simultaneously; 24 Tyr can replace with Phe; 14,25,36 and 37 Ser can independent, any two, any three or replace with Thr simultaneously; 5 and 7 Lys can replace with Arg individually or simultaneously; 18 and 26 Arg can replace with Lys individually or simultaneously; 9,6,8,19,23 and 31-33 Gln can independent, any two, any three, any four, any five, any six or replace with Asn simultaneously; 10 and 35 Leu can replace with Ile or Ala or Val individually or simultaneously; 20,27 and 34 Ala can be independent, any two or replace with Ile simultaneously or Leu or Val.These replacement types can be carried out separately, also can carry out with the combination of any replacement type.
19,22 and 23 Asp of Mtb8 specific epitopes antigen peptide section (SEQ ID No 6) can be independent, any two or replace with Glu simultaneously; 17 and 22 Glu can replace with Asp individually or simultaneously; 5 and 20 Ser can replace with Thr individually or simultaneously; 16,18,26 and 28 Arg can independent, any two, any three or replace with Lys simultaneously; 8 and 15 Gln can replace with Asn individually or simultaneously; 14,21,26 and 29 Val can independent, any two, any three or replace with Ile simultaneously or Ala or Leu; 9-10,11,13,17 and 27 Ala can independent, any two, any three, any four, any five or replace with Ile simultaneously or Leu or Val.These replacement types can be carried out separately, also can carry out with the combination of any replacement type.
These are through the computer software prediction and definite specific epitopes antigen that stems from biological autoantigen (female antigen) can substitute its corresponding female antigen and other antigen is combined, or to substitute female antigenic form mutual combination separately.
Antigen combination refers to that Mtb16.3 mixes each other independently of one another with antigens such as 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 or forms fusion rotein antigen.
One of Mtb16.3 antigen and 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 antigen are combined, or with 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 antigen any several kinds combined.
Concrete, Mtb16.3 antigen mixes with any mol ratio ratio of 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 antigen respectively mutually; Mtb16.3 antigen mixes with 38kD and any mol ratio ratio of ESAT-6 mutually; Mtb16.3 antigen mixes with 38kD and any mol ratio ratio of MPT64 mutually; Mtb16.3 antigen mixes with any mol ratio ratio of 38kD, ESAT-6 and CFP10 mutually; Mtb16.3 antigen mixes with any mol ratio ratio of 38kD, ESAT-6, MPT64 and CFP10 mutually.
Concrete, Mtb16.3 antigen respectively with 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 antigen with any mol ratio composition of proportions fusion rotein; Mtb16.3 antigen merges with any mol ratio ratio with 38kD and ESAT-6 mutually; Mtb16.3 antigen merges with any mol ratio ratio with 38kD and MPT64 mutually; Mtb16.3 antigen merges with any mol ratio ratio with 38kD, ESAT-6 and CFP10 mutually; Mtb16.3 antigen merges with any mol ratio ratio with 38kD, ESAT-6, MPT64 and CFP10 mutually.Further, Mtb16.3 antigen and Mtb8.4, Mtb8 and MPT64 fusion rotein that antigen constitutes are followed successively by Mtb8.4-MPT64-Mtb16.3-Mtb8 from the order of N-terminal to C-terminal.These fusion roteins comprise the multicopy fusion protein of the modular construction that contains same composition.The polypeptid acid sequence that connects between each antigen is preferentially selected SerGlyGlyGlySerSerGlyGlyGlySer.
Being mixed in Mtb16.3 and the antigens such as 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 one or more and forming fused antigens and mix mutually with any mol ratio ratio with the fused antigen that any two or more antigens of Mtb16.3,38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 form again of fusion rotein of the present invention.Described fused antigen comprises the multiple copied fused antigen of the modular construction that contains same composition.This combination can be optionally for one or more form fused antigens and mix mutually with any mol ratio ratio with the fused antigen of any two or more antigens formation of 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 in Mtb16.3 and 38kD, ESAT-6, CFP10, MPT64, Mtb8 and the Mtb8.4 antigen.
Concrete, Mtb16.3 antigen and Mtb8.4, Mtb8 and MPT64 antigen mix with any mol ratio ratio with 38kD, ESAT-6 and CFP10 fused antigen after merging mutually.Further, Mtb16.3 antigen and Mtb8.4, Mtb8 and MPT64 fusion rotein that antigen constitutes are followed successively by Mtb8.4-MPT64-Mtb16.3-Mtb8 from the order of N-terminal to C-terminal; 38kD, ESAT-6 and CFP10 fused antigen are followed successively by 38kD-ESAT-6-CFP10 from the order of N-terminal to C-terminal.These fused antigens comprise the multiple copied fused antigen of the modular construction that contains same composition.
Fusion rotein antigen can make through the genetically engineered mode; Promptly through the proteic DNA of the Target Fusion engineering strain of transduceing/be converted into; As expressing and purifying in: intestinal bacteria (E.coli), the yeast (Saccharomyces cerevisiae), or in mammalian cells such as Chinese hamster ovary (CHO) cell, COS cell, little hamster kidney (BHK) cell, mouse myeloma SP2/O cell, mouse NSO thymoma cell, express and purifying after, obtain wherein a kind of antigens c is terminal; Via amido linkage (amide bond) and the end to end fusion protein product of another kind of antigen N-terminal (Protein Expr.Purif.; 2008,59,189-196).
Clone, expression and purifying that gene engineering method prepares proteantigen can carry out through several different methods.Concrete grammar is referring to " molecular cloning experiment guide " (third edition), Science Press, 2002, the 1217-1270 pages.Suitable prokaryotic expression carrier such as pEGX series prokaryotic expression carrier (Amersham Pharmacia), pET series prokaryotic expression carrier (Novagen) etc.Preferred especially pGEX-4T-2 carrier.
Antigen can also be accomplished through the mode of chemosynthesis.The Fmoc method is adopted in chemosynthesis, and is synthetic through solid phase synthesis technique.The concrete steps of this method are referring to Eur.J.Immunol.1994, and 24,3188-3193; J.Org.Chem.1972,37,3404-3409; Huang Weide, old normal celebrating polypeptide is synthetic, Beijing: Science Press, 1985.
For the antigen that different peptide sections merge, well-known its in this area makes up principle and method.Particularly, need between different peptide sections, add a joint in order not influence the function separately of treating the fusogenic peptide section.Selection about joint specifically can be referring to Protein Eng., 2001,14,529-532.
Concrete; Select polypeptide or protein with the formation fusion rotein that is connected between each antigen; Said polypeptide or protein sequence length are 1-100 amino acid, and selecting length usually is 4-30 amino acid whose polypeptide, and preferentially selecting length is 4-15 amino acid whose polypeptide.Form the organic molecule that these polypeptide and proteinic amino acid refer to have the asymmetric center carbon atom of not only covalently bound amino but also covalently bound carboxyl.
A kind of concrete amalgamation mode does, is SEQ ID No 8 through protein epitope forecasting software gained 38kD-ESAT-6-antigenic modular construction aminoacid sequence of CFP10, and its corresponding D NA encoding sequence is SEQ ID No 17.The amino acid length that connects each antigenic polypeptide is 10, and sequence is SerGlyGlyGlySerSerGlyGlyGlySer.
Another kind of concrete amalgamation mode does, is SEQ ID No 9 through protein epitope forecasting software gained Mtb8.4-MPT64-antigenic modular construction aminoacid sequence of Mtb16.3-Mtb8, and its corresponding D NA encoding sequence is SEQ ID No 18.The amino acid length that connects each antigenic polypeptide is 10, and sequence is SerGlyGlyGlySerSerGlyGlyGlySer.
Multiple antigenic connection can also be adopted connexon (space linker), carries out with chemical ways of connecting.This connexon is selected from polypeptide or protein, the organic high molecular polymer (Polymer) of amino acid length 1-500, and molecular weight is 100-2, the organic molecule of 000Da.As: using two ends activatory molecular weight is 2,000-50, the lipid acid (J.Gene.Med., 2005,7,604-612) of 000Da polyoxyethylene glycol (NOF Corporation), two ends activatory C1-C30.The chemistry connection can be that the antigens c end is linked in sequence with another antigen N-terminal from beginning to end, also can will form antigenic some amino acid side chains by connexon and be connected with another antigen.Form amido linkage (amide bond) or urine key (urine bond) between connexon and the antigen.
High molecular polymer is meant to have molecular weight greater than 2, and the compound of 000Da is intermolecularly interconnected via covalent linkage by structure unit (structural unit) or monomer.Said polymkeric substance specifically is selected from polyoxyethylene glycol, polyamino acid, polynucleotide, POLYACTIC ACID, polylysine, gathers imines and 10 saccharans (polysaccharide) that above monose forms through glycosidic link, as: starch and hydrolysate thereof, Mierocrystalline cellulose, chitin or VISOSE.
The disaccharides (disaccharide) (as: sucrose, lactose or SANMALT-S) that organic molecule specifically is selected from each amino acid, all kinds of monose, each biostearin (Vitamin), is formed through glycosidic link by two monosaccharide units, pass through oligosaccharide (oligosaccharide) (as: oligomeric isomaltose, xylooligosaccharides or oligomeric galactose) that glycosidic link forms and molecular weight polymkeric substance at 20-2000Da by 2-10 monose.
Antigen combination or fused antigen also are included in the product that carries out gained behind chemically modified or the bio-modification on its aminoacid sequence skeleton.Described modification comprises Pegylation (PEGylation) (Adv.Drug deliv.Rev., 28,275-299; Adv.Drug deliv.Rev., 54,453-609; Adv.Drug deliv.Rev., 60,1-88), acylations (Acylation) (J.Pharma.Sci., 86,768-773; J.Pharma.Sci., 86,1365-1368), glycosylation (Glycosylation) (J.Pharma.Sci.87,326-332; Adv.Drug deliv.Rev., 6,103-131; Adv.Drug deliv.Rev., 13,251-267) and the organic molecule modification etc.
Pegylation is modified the polyoxyethylene glycol that used polyoxyethylene glycol is end sealing, and blocking groups can be the alkyl of C1-C30, the lipid acid of C1-C30 or glycosyl.Polyoxyethylene glycol is a molecular weight 2,000-100, and 000Da can select 2,000-50, and 000Da generally selects 5,000-50,000Da.
Alkyl and polyoxyethylene glycol one end form ehter bond usually, also can be connected with ester bond.Chain length can be selected the alkyl of C1-C10, preferentially selects methyl, ethyl or propyl group.
Lipid acid and polyoxyethylene glycol one end form ester bond usually, and fatty acid chain length is generally selected the lipid acid of C1-C18, and preferentially selecting chain length is the lipid acid of C10, C12, C16 and C18.
Acylations is used the lipid acid of C1-C30, further selects the lipid acid of C1-C18.
Several kinds of elementary composition molecular weight such as organic molecule refers to have C, H, O or C, H, O, N, P, S are less than 2, the organic molecule of 000Da.Refer to each amino acid, all kinds of monose or polysaccharide, each biostearin (Vitamin), vitamin H and molecular weight polymkeric substance particularly at 20-2000Da, as: polyoxyethylene glycol, polypeptide, oligosaccharide and saccharan (VISOSE, starch and chitin etc.) and nucleosides etc.
Any one blending ratio scope of antigen Mtb16.3 and antigen 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 is 100: 1～1: 100, mol ratio.Said blending ratio scope specifically was selected from 50: 1～1: 50,30: 1～1: 30,20: 1～1: 20,10: 1～1: 10,5: 1～1: 5,3: 1～1: 3,2: 1～1: 2 or 1: 1.
Antigen Mtb16.3 mixes with any several kinds of antigen 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 (two kinds, three kinds, four kinds, five kinds, six kinds), and it accounts for total antigenic mol ratio ratio range is 0.01-0.99.Can select the mol ratio ratio range is 0.05-0.95,0.10-0.90,0.20-0.80,0.30-0.70,0.35-0.65,0.40-0.60,0.45-0.55,0.60-0.40,0.70-0.30,0.80-0.20 or 0.90-0.10.Mol ratio ratio specifically is selected from 0.05,0.1,0.15,0.20,0.25,0.30,0.35,0.40,0.45,0.50,0.60,0.65,0.70,0.75,0.80,0.85 or 0.90.
Any one the ratio of fusion of antigen Mtb16.3 and antigen 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 is 100: 1～1: 100, mol ratio.Can select blending ratio is 50: 1～1: 50,30: 1～1: 30,20: 1～1: 20,10: 1～1: 10,5: 1～1: 5,3: 1～1: 3,2: 1～1: 2 or 1: 1.Fused antigen comprises the multiple copied fused antigen of the modular construction that contains same composition.
Antigen Mtb16.3 and antigen 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 any several kinds (two kinds, three kinds, four kinds, five kinds, six kinds) merge, and it accounts for total antigenic mol ratio ratio range is 0.01-0.99.Can select the mol ratio ratio range is 0.05-0.95,0.10-0.90,0.20-0.80,0.30-0.70,0.35-0.65,0.40-0.60,0.45-0.55,0.60-0.40,0.70-0.30,0.80-0.20 or 0.90-0.10.Concrete selection mol ratio ratio is 0.05,0.1,0.15,0.20,0.25,0.30,0.35,0.40,0.45,0.50,0.60,0.65,0.70,0.75,0.80,0.85 or 0.90.Fused antigen comprises the multiple copied fused antigen of the modular construction that contains same composition.
A kind of concrete amalgamation mode does, Mtb16.3 and antigen MPT64 or Mtb8 or Mtb8.4 merge separately, and various antigens can also can be the multiple copied of its aminoacid sequence for single copy of its aminoacid sequence.It is 0.20,0.25,0.30,0.35,0.40,0.45,0.50,0.60,0.65,0.70,0.75 or 0.80 that Mtb16.3 accounts for total antigenic mol ratio ratio.
Another kind of concrete amalgamation mode does, Mtb16.3 and antigen MPT64 and Mtb8 merge, and various antigens can be singly the copying of its aminoacid sequence, and also can be the multiple copied of its aminoacid sequence, and general formula is expressed as [Mtb16.3] respectively
m, [MPT64]
n[Mtb8]
p, n, m or p be>=1 and≤100 positive integer, value is independent separately.It is 0.20,0.25,0.30,0.35,0.40,0.45,0.50,0.60,0.65,0.70,0.75 or 0.80 that Mtb16.3 accounts for total antigenic mol ratio ratio.As m, n or p when value is 1 simultaneously, its modular construction fusion sequence is selected from Mtb16.3-MPT64-Mtb8, Mtb16.3-Mtb8-MPT64, Mtb8-Mtb16.3-MPT64, Mtb8-MPT64-Mtb16.3, MPT64-Mtb8-Mtb16.3 or MPT64-Mtb16.3-Mtb8.
Another concrete amalgamation mode does, Mtb16.3 and antigen MPT64, Mtb8 and Mtb8.4 merge, and various antigens can also can be the multiple copied of its aminoacid sequence for single copy of its aminoacid sequence, and general formula is expressed as [Mtb16.3] respectively
m, [MPT64]
n, [Mtb8]
p[Mtb8.4]
q, m, n, p or q be>=1 and≤100 positive integer, value is independent separately.It is 0.20,0.25,0.30,0.35,0.40,0.45,0.50,0.60,0.65,0.70,0.75 or 0.80 that Mtb16.3 accounts for total antigenic mol ratio ratio.As m, n, p or q when value is 1 simultaneously, its modular construction fusion sequence is selected from Mtb16.3-MPT64-Mtb8-Mtb8.4, Mtb16.3-Mtb8-MPT64-Mtb8.4, Mtb8-Mtb16.3-MPT64-Mtb8.4, Mtb8-Mtb8.4-Mtb16.3-MPT64, MPT64-Mtb8.4-Mtb16.3-Mtb8, MPT64-Mtb16.3-Mtb8-Mtb8.4 or Mtb8.4-MPT64-Mtb16.3-Mtb8.
The fused antigen of one or more formation mixes with the fused antigen that any two or more antigens of Mtb16.3,38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4 form mutually again in Mtb16.3 and the antigens such as 38kD, ESAT-6, CFP10, MPT64, Mtb8 and Mtb8.4.Blending ratio between the fused antigen is 100: 1～1: 100, mol ratio.Can select blending ratio is 50: 1～1: 50,30: 1～1: 30,20: 1～1: 20,10: 1～1: 10,5: 1～1: 5,3: 1～1: 3,2: 1～1: 2 or 1: 1, mol ratio.
Mtb16.3 antigen merges with Mtb8.4, Mtb8 and MPT64 antigen mutually, and it accounts for total antigenic mol ratio ratio is 0.01-0.99.Can select mol ratio ratio is 0.05-0.95,0.10-0.90,0.20-0.80,0.30-0.70,0.35-0.65,0.40-0.60,0.45-0.55,0.60-0.40,0.70-0.30,0.80-0.20 or 0.90-0.10.Concrete selection mol ratio ratio is 0.05,0.1,0.15,0.20,0.25,0.30,0.35,0.40,0.45,0.50,0.60,0.65,0.70,0.75,0.80,0.85 or 0.90.Fused antigen comprises the multiple copied fused antigen of the modular construction that contains same composition.
38kD, ESAT-6 and CFP10 fused antigen are combined, and mol ratio is 1: 1: 1.
A kind of concrete embodiment is that Mtb16.3 antigen and Mtb8.4, Mtb8 and MPT64 fusion rotein that antigen constitutes are followed successively by Mtb8.4-MPT64-Mtb16.3-Mtb8 from the order of N-terminal to C-terminal; 38kD, ESAT-6 and CFP10 fused antigen are followed successively by 38kD-ESAT-6-CFP10 from the order of N-terminal to C-terminal.These fused antigens comprise the multiple copied fused antigen of the modular construction that contains same composition, and general formula is respectively [Mtb8.4-MPT64-Mtb16.3-Mtb8]
x[38kD-ESAT-6-CFP10]
y, x or y be>=1 and≤1000 positive integer, value is independent separately.The blending ratio of two kinds of antigen modular construction Mtb8.4-MPT64-Mtb16.3-Mtb8 and 38kD-ESAT-6-CFP10 is 1: 3,1: 2,1: 1,2: 1 or 3: 1.
Another kind of concrete embodiment does, various antigens with its separately the multiple copied mode of aminoacid sequence merge, general formula is expressed as [Mtb16.3] respectively
m, [MPT64]
n, [Mtb8]
p[Mtb8.4]
q, m, n, p or q be>=1 and≤100 positive integer, value is independent separately.Mtb16.3 antigen and Mtb8.4, Mtb8 and MPT64 fusion rotein that antigen constitutes are followed successively by [Mtb8.4] from the order of N-terminal to C-terminal
q-[MPT64]
n-[Mtb16.3]
m-[Mtb8]
p38kD, ESAT-6 and CFP10 fused antigen are followed successively by 38kD-ESAT-6-CFP10 from the order of N-terminal to C-terminal, and general formula is expressed as [38kD] respectively
u, [ESAT-6]
v[CFP10]
w, u, v or w be>=1 and≤100 positive integer, value is independent separately.These fused antigens comprise the multiple copied fused antigen of the modular construction that contains same composition, and general formula is respectively [Mtb8.4-MPT64-Mtb16.3-Mtb8]
x[38kD-ESAT-6-CFP10]
y, x or y be>=1 and≤1000 positive integer, value is independent separately.The blending ratio of two kinds of antigen modular construction Mtb8.4-MPT64-Mtb16.3-Mtb8 and 38kD-ESAT-6-CFP10 is 1: 3,1: 2,1: 1,2: 1 or 3: 1.
Combined or constitute the peptide composition that fused antigen remix mode obtains and be used to detect the antibody of replying tubercle bacillus antigen and producing by antigens such as Mtb16.3 and 38kD, ESAT-6, CFP10 and MPT64.Particularly, this is combined in the application in the white plaque antibody test.
Detection method can have multiple, for example indirect enzyme-linked immunosorbent determination techniques, double antigens sandwich enzyme immunoassay technique, gold mark Fast Detection Technique, immunity percolation detection technique, protein chip detection technique etc.The concrete indirect enzyme-linked immunosorbent of selecting is measured, i.e. indirect ELISA method.
The indirect ELISA method ultimate principle is: polypeptide (antigen) is coated on the enzyme plate surface, and serum to be checked after the dilution and control are added in the reaction plate hole, if having antibody in the seized serum; Behind incubation, then specific antibody combines with antigenic peptide in the reaction plate hole in the serum, forms solid phase antigen-antibody complex; Other composition in the unconjugated serum of flush away; The anti-human IgG antibody who adds enzyme labelling, incubation, solid phase antigen (polypeptide) bonded antibody combines with the anti-human IgG antibody of enzyme labelling again; The unconjugated enzymic-labelled antibody composition of flush away; Add enzyme substrates, and be catalyzed into and be coloured product, add the stop buffer termination reaction at last.According to control, but antagonist carries out quantitatively or qualitative test.Its concrete grammar can be referring to Infect.Immun.1998,66,3936-3940 with Protein Expr.Purif., 2008,59,189-196.
The beneficial effect that the present invention realizes:
Mtb16.3 antigen has special advantages in detecting tubercule bacillus.Four kinds of antigens such as Mtb16.3 antigen and 38kD, ESAT-6, CFP10 and MPT64 have good complementarity in the serology context of detection.At least a antigenic combination of Mtb16.3 antigen and selection 38kD, ESAT-6, CFP10 and MPT64 can significantly improve the sensitivity of diagnosis of tuberculosis.
Mtb16.3 specific epitopes antigen peptide section (SEQ ID No 1) has more special advantages in detecting tubercule bacillus.When four kinds of specific epitopes antigens; Be 38kD (SEQ ID No 2), ESAT-6 (SEQ ID No 3), CFP10 (SEQ ID No 4) and MPT64 (SEQ ID No 5); When total overall reaction property was negative, Mtb16.3 antigen peptide Duan Ze presented strong positive (BP19) to the reactivity of this serum.This shows that Mtb16.3 specific epitopes antigen peptide section and four kinds of specific epitopes antigen peptide sections such as 38kD, ESAT-6, CFP10 and MPT64 have good complementarity in the serology context of detection.The combination of at least a specific epitopes antigen peptide section of Mtb16.3 specific epitopes antigen peptide section and selection 38kD, ESAT-6, CFP10 and MPT64 can significantly improve the sensitivity of diagnosis of tuberculosis.
Indirect ELISA method detects; The corresponding separately specific epitopes polypeptide fused antigen of Combination application 38kD-ESAT6-CFP10 and Mtb8.4-MPT64-Mtb16.3-Mtb8 fused antigen or its is respectively 95% and 100% to detection sensitivity lungy and specificity, shows better detection effect.
Description of drawings
Fig. 1 representes the epitope analysis of tubercule bacillus protein 38kD;
Fig. 2 representes the epitope analysis of tubercule bacillus protein ESAT-6;
Fig. 3 representes the epitope analysis of tubercule bacillus protein C FP10;
Fig. 4 representes the epitope analysis of tubercule bacillus protein MPT64;
Fig. 5 representes the epitope analysis of tubercule bacillus protein Mtb8;
Fig. 6 representes the epitope analysis of tubercule bacillus protein Mtb8.4;
Fig. 7 representes the epitope analysis of tubercule bacillus protein Mtb16.3.
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with accompanying drawing.Should be noted that; Embodiment of the present invention only is used to explain technical scheme of the present invention and is unrestricted; Although with reference to preferred embodiment the present invention is specified, those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement the technical scheme of invention; And not breaking away from the spirit and the scope of technical scheme of the present invention, it all should be encompassed in the claim scope of the present invention.
The used reagent of the present invention is not if clearly indicate, then all available from Sigma-aldrich (Sigma-Aldrich).
Confirming of 17 kinds of tubercle bacillus differential epitope antigens of embodiment peptide section
Utilize BIOSUN biological analysis software to analyze the epi-position distribution situation of 7 kinds of negre antigen 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and Mtb16.3 respectively; At first import its aminoacid sequence; Seek the B cell epitope then, gained epi-position distribution plan is shown in Fig. 1-7.According to the epi-position distribution plan, finally confirm specific epitopes antigen peptide section.Wherein the aminoacid sequence of the specific epitopes antigen peptide section of negre antigen 38kD and DNA sequences encoding are respectively SEQ IDNO 2 and SEQ ID NO 11; The aminoacid sequence of the specific epitopes antigen peptide section of negre antigen ESAT-6 and DNA sequences encoding are respectively SEQ ID NO 3 and SEQ ID NO 12; The aminoacid sequence of the specific epitopes antigen peptide section of negre antigen CFP10 and DNA sequences encoding are respectively SEQ ID NO 4 and SEQ ID NO 13; The aminoacid sequence of the specific epitopes antigen peptide section of negre antigen MPT64 and DNA sequences encoding are respectively SEQ ID NO 5 and SEQ ID NO14; The aminoacid sequence of the specific epitopes antigen peptide section of negre antigen Mtb8 and DNA sequences encoding are respectively SEQ ID NO 6 and SEQ ID NO 15; The aminoacid sequence of the specific epitopes antigen peptide section of negre antigen Mtb8.4 and DNA sequences encoding are respectively SEQ ID NO 7 and SEQID NO 16; The aminoacid sequence of the specific epitopes antigen peptide section of negre antigen Mtb16.3 and DNA sequences encoding are respectively SEQ ID NO 1 and SEQ ID NO 10.
The preparation of 7 kinds of tubercle bacillus differential epitope antigen peptide sections that embodiment 2 is independent
1, the clone of specific epitopes antigen peptide fragment gene
Nucleotide sequence according to specific epitopes antigen peptide section has designed and synthesized the upstream and downstream primer, and employed restriction enzyme is respectively BamH I and Xho I.
The primer sequence that is used for amplifying specific epitope antigen peptide section 38kD is following:
38kD-F：5’-GGGATCCGCGGCGGGCTGTGGCTCGA-3’
38kD-R：5’-GCTCGAGGCTGGAAATCGTCGCGA-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section ESAT6 is following:
ESAT6-F：5’-GGGATCCCATTCCCTCCTTGACGA-3’
ESAT6-R：5’-GCTCGAGGTTCAGCTCGGTAGCCGT-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section CFP10 is following:
CFP10-F：5’-CGGATCCGAAGCAGCCAATAAGCAG-3’
CFP10-R：5’-GCTCGAGCGAGGACAGCGCCTGCTG-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section MPT64 is following:
MPT64-F：5’-GGGATCCCCCAAGACCTACTGCGA-3’
MPT64-R：5’-GCTCGAGCGGCGCTATCGATACCT-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section Mtb8 is following:
Mtb8-F：5’-GGGATCCACCAGCCCCACATCCT-3’
Mtb8-R：5’-GCTCGAGAATGACCCGAGCGACGCGGA-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section Mtb8.4 is following:
Mtb8.4-F：5’-GGGATCCCCGGGGTCGCCTCCGCA-3’
Mtb8.4-R：5’-GCTCGAGTTGCGCGGCCATGGCA-3’
The primer sequence that is used for amplifying specific epitope antigen peptide section Mtb16.3 is following:
Mtb16.3-F：5’-GGGATCCCGGACAAGACGACACAGA-3’
Mtb16.3-R：5’-GCTCGAGGCCCTCGACTCGTTTCTT-3’
With tubercule bacillus bacterial strain H37Rv (the national Reference Lab of Chinese tuberculosis prevention and treatment clinical center provides) genomic dna is template, the gene fragment of 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and the Mtb16.3 specific epitopes antigen peptide section of having increased respectively.The pcr amplification condition is following: in advance 95 ℃ of sex change are 2 minutes, 94 ℃ of sex change 30 seconds; 58 ℃ of renaturation 30 seconds; Extend 72 ℃ of 30-90 second (different and different) with mrna length, 32 circulations of increasing, 72 ℃ were extended 7 minutes again.
The fragment of pcr amplification gained is identified through electrophoresis, is shown the gene fragment that all obtains corresponding size.
2, the structure of expression vector
With digestion with restriction enzyme and connect into the carrier through the pGEX-4T-2 of same restrictions property endonuclease digestion, order-checking is accomplished by the sincere Bioisystech Co., Ltd of Li Jiafu with the PCR product.
3, the expression and purification of specific epitopes antigen peptide section in bacillus coli DH 5 alpha
With resulting recombinant plasmid transformed coli strain DH5 α, PCR is identified that positive colony carries out IPTG and induces.Thalline after inducing carries out ultrasonication, supernatant is mixed room temperature jog 30 minutes with an amount of 50% gsh-agarose resin homogenate.Mixture in 4 ℃ with 500g centrifugal 5 minutes, carefully remove supernatant, in deposition, add the PBS of 10 times of column volumes, put upside down centrifuge tube for several times, flush away not with the albumen of resin-bonded.In 4 ℃ with 500g centrifugal 5 minutes.Repeated washing process 2 times.Add the zymoplasm that 50 units are dissolved in 1ml PBS at every milliliter of resin, put upside down centrifuge tube mixing for several times, vibrated under the room temperature 10～16 hours.In 4 ℃ with 500g centrifugal 5 minutes, supernatant carefully is transferred in the new pipe.Carry out SDS-PAGE and analyze, the result shows and has obtained the higher specific epitopes antigen peptide section of purity.
The preparation of embodiment 3 38kD-ESAT6-CFP10 fused antigens
1, design of primers
According to the sequences Design of three antigen genes and joint the upstream and downstream primer, the restriction enzyme of use is respectively BamH I and Xho I, all primers are synthetic by the handsome biotech company in Shanghai, its sequence is following:
38kD-F：5’-GGGATCCGCGGCGGGCTGTGGCTCGA-3’
38kD-RL：5’-ACTACCGCCACCAGAGCTGGAAATCGTCGC-3’
ESAT6-FL：5’-AGCGGCGGCGGTAGCCATTCCCTCCTTGAC-3’
ESAT6-RL：5’-AGAGCCACCGCCACTGTTCAGCTCGGTAGC-3’
CFP10-FL：5’-CTTCCGGTGGTGGCTCTGAAGCAGCCAATAA-3’
CFP10-R：5’-GCTCGAGCGAGGACAGCGCCTGCTG-3’
2, vector construction
With tubercule bacillus bacterial strain H37Rv genomic dna is template, the gene fragment of 38kD, ESAT-6 and the CFP10 specific epitopes antigen peptide section of having increased respectively.The pcr amplification condition is following: in advance 95 ℃ of sex change are 2 minutes, 94 ℃ of sex change 30 seconds; 58 ℃ of renaturation 30 seconds; Extend 72 ℃ of 30-90 second (different and different) with mrna length, 32 circulations of increasing, 72 ℃ were extended 7 minutes again.Identify through electrophoresis, show the gene fragment that all obtains corresponding size.Then with gene fragment with acomplementary connector each other template increase, 38kD, ESAT-6 and CFP10 antigen gene fragment are connected together, make up fusion antigen gene.The fusion antigen gene fragment is connected into the carrier through the pGEX-4T-2 of same restrictions property endonuclease digestion after with digestion with restriction enzyme, and order-checking is accomplished by the sincere Bioisystech Co., Ltd of Li Jiafu.
3, antigen prepd
Referring to embodiment 2.
The preparation of embodiment 4 MPT64-Mtb8-Mtb8.4-Mtb16.3 fused antigens
According to the sequences Design of 4 antigen genes the upstream and downstream primer, the restriction enzyme of use is respectively BamH I and Xho I, all primers are synthetic by the handsome biotech company in Shanghai, its sequence is following:
Mtb8.4-F：5’-GGGATCCCCGGGGTCGCCTCCGCA-3’
Mtb8.4-RL：5’-GCTACCACCGCCGGATTGCGCGGCCATGGCA-3’
MPT64-FL：5’-GGTGGCGGCTCCCCCAAGACCTACT-3’
MPT64-RL：5’-CTACCGCCACCAGACGGCGCTATCGATA-3’
Mtb16.3-FL：5’-GCGGCGGCGGTAGCGCGGACAAGACGACAC-3’
Mtb16.3-RL：5’-GAGCCACCGCCACTGCCCTCGACTCGTTTCT-3’
Mtb8-FL：5’-CCGGTGGTGGCTCTGCCGGGGTCGCCTCCG-3’
Mtb8-R：5’-GCTCGAGAATGACCCGAGCGACGCGGA-3’
Vector construction and antigen prepd are referring to embodiment 3.
Embodiment 5 indirect ELISA methods are estimated the antigenicity of 7 kinds of independent specific epitopes antigen peptide sections
Be antigen with selected 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and Mtb16.3 specific epitopes antigen peptide section respectively; The 30 routine tuberculosis negative serum samples of having used the indirect ELISA method Preliminary detection; MV+2SD with its OD value is the cutoff value, and the cutoff value that calculates 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and Mtb16.3 is respectively 0.55,0.2,0.15,0.29,0.17,0.18 and 0.30.Then; Detected the tuberculosis antibody of 20 routine tuberculosis patient serum and 10 routine normal human serums more respectively with these seven kinds of antigens; The ratio (S/co=sample OD value/cutoff value) that has shown the OD value and the cutoff value of institute's test sample in the table 1; Ratio is judged to be the positive greater than 1, and ratio is judged to be strong positive greater than 2, and ratio is judged to be feminine gender less than 1.38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and seven kinds of antigenic susceptibility of Mtb16.3 and specificity are respectively as follows in Preliminary detection: 38kD is 75% and 90%, ESAT-6 is 45% and 90%, CFP10 is 80% and 100%, MPT64 is 50% and 90%, Mtb8 is respectively 55% and 90%; Mtb8.4 is respectively 40% and 90%, and Mtb16.3 is respectively 55% and 80%.
Table 1 indirect ELISA method detects the reactivity (S/co) of seven kinds of specific epitopes antigen peptide sections to tuberculosis patient and normal human blood sample
Find in these seven kinds of antigens, to have no a kind ofly can detect whole tuberculosis patients, but be surprisingly found out that Mtb16.3 specific epitopes antigen peptide section has special advantages in detecting tubercule bacillus by table 1 data.When four kinds of commonly used antigens, i.e. 38kD, ESAT-6, CFP10, MPT64, when total overall reaction property was negative, Mtb16.3 specific epitopes antigen peptide Duan Ze presented strong positive (BP19) to the reactivity of this serum.This shows that Mtb16.3 specific epitopes antigen peptide section and 38kD, ESAT-6, CFP10, four kinds of specific epitopes antigen peptide of MPT64 section have good complementarity in the serology context of detection.The sensitivity that the combination of at least a specific epitopes antigen peptide section of Mtb16.3 specific epitopes antigen peptide section and selection 38kD, ESAT-6, CFP10, MPT64 might improve diagnosis of tuberculosis.
In addition, a kind ofly can detect whole tuberculosis patients though have no in these seven kinds of antigens, seven kinds of antigenic associating recall rates can reach 100%, and this complementarity between the antigen is very important for the clinical diagnosis of tuberculosis patient.With the same patients serum of different Detection of antigen, its reactivity is different, and has certain complementarity between each antigen.This possibly be complicated because negre antigen is many, in host, shows different antibody repertoires.Therefore, the multiple antigen of combined utilization detects and can guarantee effectively to improve detection sensitivity on the specific basis, and develops high specific and highly sensitive detection white plaque antibody reagent.
Embodiment 6 indirect ELISA methods are estimated Mtb16.3 specific epitopes antigen peptide section and the combined effect in detecting tuberculosis patient of all the other specific epitopes antigen peptide sections
Of embodiment 5; Because Mtb16.3 specific epitopes antigen peptide section has the advantage that other antigen peptide section is not had; Therefore itself and four kinds of antigens commonly used of contrived experiment checking; Be 38kD, ESAT-6, CFP10, MPT64, the combined effect in detecting tuberculosis patient of one or more antigen peptide sections of specific epitopes antigen peptide section.Antigen mol ratio ratio in the various combinations is 1.Experimental result is as shown in table 2.
Table 2 indirect ELISA method detects the reactivity (S/co) that Mtb16.3 specific epitopes antigen peptide section and other specific epitopes antigen peptide section are mixed blood sample
Visible by table 2, Mtb16.3 specific epitopes antigen peptide section and all the other one or more antigen peptide sections combined in detecting tuberculosis patient better effects if, recall rate obviously improves, specificity is not reduction but.This shows that really there be complementarity in Mtb16.3 specific epitopes antigen peptide section aspect the tuberculosis patient with other specific epitopes antigen peptide section detecting.The multiple antigen of combined utilization can improve recall rate, avoids omission.
Embodiment 7 indirect ELISA methods are estimated the effect of fused antigen in detecting tuberculosis patient
Use 38kD-ESAT6-CFP10 and Mtb8.4-MPT64-Mtb16.3-Mtb8 fused antigen alone or in combination and encapsulate elisa plate, the effect of fused antigen in detecting tuberculosis patient of having used the indirect ELISA method preliminary assessment.The mol ratio ratio of each component in the fused antigen is 1, and the mol ratio ratio of antigen combination is 1.
In Preliminary detection; During Combination application 38kD-ESAT6-CFP10 and Mtb8.4-MPT64-Mtb16.3-Mtb8 fused antigen; The serum of 20 routine tuberculosis patients only 1 routine detected result is negative; Other 19 example is all positive, and wherein 9 examples are strong positive (S/co>2.0), reaches 95% with the accordance of clinical effectiveness.10 routine healthy blood donor serum are all negative in the Preliminary detection.Sensitivity and specificity that definite application indirect ELISA method detects fused antigen are respectively 95% and 100% (seeing table 3).It is better that above result shows that 7 antigens all merge the effect of (3 merge, in addition 4 merge).
Table 3 indirect ELISA method detects the reactivity (S/co) of fused antigen to tuberculosis patient and normal human blood sample
Sequence table
< 110>ShangHai RongSheng Biology Pharmacy Co., Ltd
< 120>peptide composition and the application in the white plaque antibody test thereof
<130>0811571
<160>18
<170>PatentIn?version?3.3
<210>SEQ?ID?No?1
<211>143
<212>PRT
< 213>aminoacid sequence of Mtb16.3 specific epitopes antigen peptide section
<400>1
<210>SEQ?ID?No?2
<211>354
<212>PRT
< 213>aminoacid sequence of 38kD specific epitopes antigen peptide section
<400>2
<210>SEQ?ID?No?3
<211>41
<212>PRT
< 213>aminoacid sequence of ESAT-6 specific epitopes antigen peptide section
<400>3
<210>SEQ?ID?No?4
<211>37
<212>PRT
< 213>aminoacid sequence of CFP10 specific epitopes antigen peptide section
<400>4
<210>SEQ?ID?No?5
<211>154
<212>PRT
< 213>aminoacid sequence of MPT64 specific epitopes antigen peptide section
<400>5
<210>SEQ?ID?No?6
<211>30
<212>PRT
< 213>aminoacid sequence of Mtb8 specific epitopes antigen peptide section
<400>6
<210>SEQ?ID?No?7
<211>64
<212>PRT
< 213>aminoacid sequence of Mtb8.4 specific epitopes antigen peptide section
<400>7
<210>SEQ?ID?No?8
<211>452
<212>PRT
< 213>aminoacid sequence of 38kD-ESAT6-CFP10 fused antigen
<400>8
<210>SEQ?ID?No?9
<211>421
<212>PRT
< 213>aminoacid sequence of Mtb8.4-MPT64-Mtb16.3-Mtb8 fused antigen
<400>9
<210>SEQ?ID?No?10
<211>429
<212>DNA
< 213>DNA sequences encoding of Mtb16.3 specific epitopes antigen peptide section
<400>10
<210>SEQ?ID?No?11
<211>1062
<212>DNA
< 213>DNA sequences encoding of 38kD specific epitopes antigen peptide section
<400>11
<210>SEQ?ID?No?12
<211>123
<212>DNA
< 213>DNA sequences encoding of ESAT-6 specific epitopes antigen peptide section
<400>12
<210>SEQ?ID?No?13
<211>111
<212>DNA
< 213>DNA sequences encoding of CFP10 specific epitopes antigen peptide section
<400>13
<210>SEQ?ID?No?14
<211>462
<212>DNA
< 213>DNA sequences encoding of MPT64 specific epitopes antigen peptide section
<400>14
<210>SEQ?ID?No?15
<211>90
<212>DNA
< 213>DNA sequences encoding of Mtb8 specific epitopes antigen peptide section
<400>15
<210>SEQ?ID?No?16
<211>192
<212>DNA
< 213>DNA sequences encoding of Mtb8.4 specific epitopes antigen peptide section
<400>16
<210>SEQ?ID?No?17
<211>1356
<212>DNA
< 213>DNA sequences encoding of 38kD-ESAT6-CFP10 fused antigen
<400>17
<210>SEQ?ID?No?18
<211>1263
<212>DNA
< 213>DNA sequences encoding of Mtb8.4-MPT64-Mtb16.3-Mtb8 fused antigen
<400>18
Claims (2)
1. fused antigen 38kD-ESAT-6-CFP10, its aminoacid sequence is shown in the SEQ ID No 8.
2. peptide composition; It is characterized in that; Mix mutually with fused antigen Mtb8.4-MPT64-Mtb16.3-Mtb8 by fused antigen 38kD-ESAT-6-CFP10; Wherein the mixing mol ratio of two kinds of fused antigens is 1:1, and wherein the aminoacid sequence of fused antigen 38kD-ESAT-6-CFP10 is shown in the SEQ ID No 8, and the aminoacid sequence of fused antigen Mtb8.4-MPT64-Mtb16.3-Mtb8 is shown in the SEQ ID No 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810203532 CN101735321B (en) | 2008-11-27 | 2008-11-27 | Polypeptide composition and application thereof in detecting tuberculosis antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810203532 CN101735321B (en) | 2008-11-27 | 2008-11-27 | Polypeptide composition and application thereof in detecting tuberculosis antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101735321A CN101735321A (en) | 2010-06-16 |
| CN101735321B true CN101735321B (en) | 2012-12-26 |
Family
ID=42459396
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200810203532 Active CN101735321B (en) | 2008-11-27 | 2008-11-27 | Polypeptide composition and application thereof in detecting tuberculosis antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101735321B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106432508B (en) * | 2016-08-30 | 2017-12-05 | 深圳市伯劳特生物制品有限公司 | A kind of PLA2R THSD7A fusion proteins and its application and kit |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175875B (en) * | 2011-01-27 | 2013-06-05 | 武汉海吉力生物科技有限公司 | Detection kit for diagnosing tuberculosis |
| US9404923B2 (en) | 2012-02-07 | 2016-08-02 | Intuitive Biosciences, Inc. | Mycobacterium tuberculosis specific peptides for detection of infection or immunization in non-human primates |
| CN103304646B (en) * | 2012-03-09 | 2015-02-25 | 中国医学科学院病原生物学研究所 | Mycobacterium tuberculosis candidate antigen polypeptide and application thereof |
| CN102608333B (en) * | 2012-03-30 | 2013-06-05 | 中国科学院微生物研究所 | Tuberculosis diagnostic composition and application thereof |
-
2008
- 2008-11-27 CN CN 200810203532 patent/CN101735321B/en active Active
Non-Patent Citations (6)
| Title |
|---|
| diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10;LAURENS等;《Clinical and Diagnostic Laboratory Immunology》;20000331;第7卷(第2期);第155-160页 * |
| LAURENS等.diagnosis of tuberculosis based on the two specific antigens ESAT-6 and CFP10.《Clinical and Diagnostic Laboratory Immunology》.2000,第7卷(第2期),第155-160页. |
| 一种结核杆菌融合抗原的克隆表达及活性测定;宋晓国等;《中国实验诊断学》;20080531;第12卷(第5期);第569页摘要部分 * |
| 冯晓燕等.结核分枝杆菌4种抗原的基因克隆、表达、纯化和抗原性初步鉴定.《生物技术通讯》.2006,第17卷(第6期),第865-867页. |
| 宋晓国等.一种结核杆菌融合抗原的克隆表达及活性测定.《中国实验诊断学》.2008,第12卷(第5期),第569-573页. |
| 结核分枝杆菌4种抗原的基因克隆、表达、纯化和抗原性初步鉴定;冯晓燕等;《生物技术通讯》;20061130;第17卷(第6期);第865页摘要部分 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106432508B (en) * | 2016-08-30 | 2017-12-05 | 深圳市伯劳特生物制品有限公司 | A kind of PLA2R THSD7A fusion proteins and its application and kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101735321A (en) | 2010-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cheng et al. | An insertion unique to SARS-CoV-2 exhibits superantigenic character strengthened by recent mutations | |
| CN102836425B (en) | The Vaccinum Calmette-Guerini of antigen expressed during comprising the latent infection stage | |
| CN101735321B (en) | Polypeptide composition and application thereof in detecting tuberculosis antibody | |
| CN102004155B (en) | Kit and method for detecting mycobacterium tuberculosis infection and application | |
| EP1278769A2 (en) | Tuberculosis antigens and methods of use thereof | |
| MXPA01004469A (en) | Tuberculosis diagnostic test. | |
| CN101235090B (en) | Specificity fusion protein applied to tuberculosis rapid diagnosis and its construction method | |
| Angeletti et al. | Plasmodium falciparum rosetting epitopes converge in the SD3-loop of PfEMP1-DBL1α | |
| CN107011418A (en) | Detect antigen polypeptide pond and its application of mycobacterium tuberculosis infection | |
| CN1388378A (en) | Tubercle mycobaterium detecting reagent | |
| CN107216373A (en) | Detect antigen polypeptide pond and its application of mycobacterium tuberculosis infection | |
| Kanagavel et al. | B-cell-specific peptides of Leptospira interrogans LigA for diagnosis of patients with acute leptospirosis | |
| CN101493454A (en) | Tuberculosis antigen specific whole blood IFN-gamma diagnosis kit, method for producing the same and method for using same | |
| CN107141341A (en) | Detect antigen polypeptide pond and the application of mycobacterium tuberculosis infection | |
| Blount et al. | Serologic responses to recombinant Pneumocystis jirovecii major surface glycoprotein among Ugandan patients with respiratory symptoms | |
| KR102305770B1 (en) | Diagnostic reagents for improved in vivo or in vitro cell-mediated immunological diagnosis of tuberculosis | |
| CN101477125B (en) | Composition based on tubercle bacillus antigenic polypeptide for diagnosing tuberculosis | |
| CN101293919A (en) | Syphilis spirochete membrane antigen with shorten expression and uses thereof | |
| NZ522191A (en) | Antigenic peptide fragments of VapA protein, and uses thereof | |
| CN101805397A (en) | Mycobacterium tuberculosis ESAT-6 recombinant dipolymer, preparation method and application thereof | |
| Chua-Intra et al. | T-cell recognition of mycobacterial GroES peptides in Thai leprosy patients and contacts | |
| CN102276700B (en) | Swine hepatitis E virus antigen epitope and application thereof | |
| CN101580540B (en) | Mouse HCV (hepatitis C virus) polypeptide epitope combined with MHC-I molecule and application thereof | |
| CN107530414A (en) | Distinguish antigen of mycobacterium tuberculosis and its application of active tuberculosis and latent tuberculosis infection | |
| Grąźlewska et al. | Epitope Mapping of BmpA and BBK32 Borrelia burgdorferi Sensu Stricto Antigens for the Design of Chimeric Proteins with Potential Diagnostic Value |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |