CN101477125B - Composition based on tubercle bacillus antigenic polypeptide for diagnosing tuberculosis - Google Patents
Composition based on tubercle bacillus antigenic polypeptide for diagnosing tuberculosis Download PDFInfo
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Abstract
The invention relates to a composition used for serological diagnosis of tuberculosis, which comprises a combination of a special antigen epitope peptide of tubercle bacillus Mtb16.3 with at least one tubercle bacillus special antigen epitope peptide selected from 38kD, ESAT-6, CFP10andMPT64.
Description
Technical field
The present invention relates to diagnostic field lungy.More specifically relate to the combined diagnosis field lungy of using the multiple antigenic polypeptide that comes from tubercle bacillus.
Background of invention
In recent years, tuberculosis is revivable, becomes public health problem and social concern that the whole world is paid close attention to.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 infectious disease, becomes the No.1 killer of infectious disease.
Pulmonary tuberculosis demonstrates global epiphytotics all principal characters, and in worldwide, spreads.Nearly 1/3 people infects tubercle bacillus on the earth, causes annual 8000000 routine active tuberculosis and 3,000,000 people dead.The appearance of the multiple medicine persister of tubercle bacillus and merging HIV infect and make situation become more severe.If can not effectively control, in following 10 years, therefore nearly 3,000 ten thousand people can get killed, and also have 9,000 ten thousand active tuberculosis new cases.Tuberculosis so powerful popularity and infectiousness 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 goldstandard that phlegm is cultivated, but the cycle is oversize, can not meet clinical needs; PCR method susceptibility is high, diagnosis speed is fast, but has instability; The phage splitting method can detect slip-knot nuclear bacillus, can not detect non-mycobacterium tuberculosis infection, is applicable to tulase resistance to the action of a drug 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 exact instrument etc.
The serology that detects for tuberculosis detects, and seeks the antigenic component of hypersensitivity and high specific, and the detection method of setting up the high specific hypersensitivity is to solve the early stage key of diagnosis quick and precisely of tuberculosis.Present increasing interest is invested species specificity and all reliable serology diagnosis of tuberculosis method of sensitivity set up.The antigen that the purified protein derivative of tuberculin (PPD) that uses is traditionally comprised is that pathogenic mycobacterium, environment mycobacterium and BCG vaccine (BCG) are common, can not clearly distinguish BCG immunity, the infection of environment mycobacterium and pathogenic mycobacterium tuberculosis infection.At present the most important antigen that is used for diagnosis of tuberculosis of research comprises 38kD antigen, ESAT-6, CFP10, MPT64 etc., and these are 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 tubercle bacillus species specificity proteantigen and content are higher than BCG10 doubly.When being used for antitubercle sera detection, the susceptibility of this albumen and specificity are superior to other single antigen.There is research to confirm that sensitivity and specificity are respectively 65.6% and 95.8% (WilKinson etc., J Clin Microbiol, 35 (3): 553-557 (1997)) with 38kD Detection of antigen serum of tuberculosis patients.
Early stage secreted antigen target (ESAT-6) and culturing filtrate protein 10 (CFP10) are two up-and-coming antigens in the tuberculosis immunodiagnosis, and it is active to have stronger cellular immunity, in the immune response of resisting tuberculosis infection, plays an important role.These two antigens are by RD1 district coding, and this district only is present in the pathogenic tubercle bacillus gene group, and all lacking in all BCG strain gene groups should zone (Mahairas GG etc., Bacteriol, 178:1274-1282 (1996)).ESAT-6 and CFP10 can form the heterodimer structure; Combined application ESAT-6 and CFP10 diagnosis of tuberculosis find that hybrid antigen susceptibility is than the former increase of monoclonal antibody; And do not reduce its specificity (van Pinxteren LAH etc., ClinDiagn Lab Immunol, 7 (2): 155-160 (2000)).So ESAT-6 and CFP10 are expected to become the specific antigen of the pathogenic mycobacterium tuberculosis infection of diagnosis animal and human.
MPT64 is the major protein that growth of bacillus tubercle is secreted the earliest, only in tubercle bacillus and ox type mycobacterium, exists, and does not then contain among the BCG as vaccine.The natural MPT64 and the MPT64 of reorganization all can make tuberculosis patient or tuberculin positive person produce t cell responses and skin delaying type (DTH) hypersensitivity.But because MPT64 only is present among the compound crowd of tubercle bacillus, most of BCG bacterial strains lack the MPT64 genes, BCG inoculator or environment mycobacterium the infected (like bird mycobacterium) to MPT64 Skin-test do not react.There is result of study to show; Though is lower as the positive rate of Detection of antigen approach antibody with MPT64 albumen in various tubercle bacillus secreted proteins; If but form fusion with other albumen then might have stability (van PinxterenLAH etc. preferably; Clin Diagn Lab Immunol, 7 (2): 155-160 (2000)), so MPT64 albumen promises to be one of the candidate of the combined antigen of tuberculosis serological diagnosis.
Mtb8.4 (Rv1174c), Mtb8 (Rv0379) and Mtb16.3 (Rv2185c) extensively are present in the tubercle 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 tubercle bacillus culture filtrating recently; Scholars such as Coler (J Immunol.; 161:2356-2364 (1998)) research further discloses Mtb8.4 and suffers from immunocompetence T cellular antigens important in the individuality of the mycobacterium tuberculosis infection of hiding, and in confirming pathogenic mycobacterium tuberculosis infection, has vital role.Mtb8 is possible transport protein matter; Only at (Clin Diagno Lab Immun such as Houghton; 9 (4): in research 883-891 (2002)) Mtb8 and other several kinds of antigens (Mtb11, Mtb48,38kD etc.) are formed fused antigen and be used to detect tuberculosis, show that Mtb8 might strengthen the reactivity of 38kD antigen 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.
But, also do not find susceptibility and all gratifying combination of specificity aspect detection tuberculosis in this area.Through work of the present invention; Be surprisingly found out that the unique advantage of Mtb16.3 specific epitopes antigenic peptides section aspect detection tuberculosis, it all improves with combined detection sensitivity lungy and the specificity of can making of at least a specific epitopes antigenic peptides section that is selected from 38kD, ESAT-6, CFP10 and MPT64.
The invention summary
The present invention relates to be used to detect the composition of tubercle bacillus, it comprises the specific epitopes antigenic peptides section and the combination that is selected from least a tubercle bacillus differential epitope antigen peptide section of 38kD, ESAT-6, CFP10 and MPT64 of tubercle bacillus Mtb16.3; Wherein Mtb16.3 specific epitopes antigenic peptides section is polypeptide or its functional variety of amino acid sequence shown in the SEQ ID NO:14; 38kD specific epitopes antigenic peptides section is polypeptide or its functional variety of amino acid sequence shown in the SEQ ID NO:2; ESAT-6 specific epitopes antigenic peptides section is polypeptide or its functional variety of amino acid sequence shown in the SEQ ID NO:4; CFP10 specific epitopes antigenic peptides section is polypeptide or its functional variety of amino acid sequence shown in the SEQ ID NO:6, and MPT64 specific epitopes antigenic peptides section is polypeptide or its functional variety of amino acid sequence shown in the SEQ ID NO:8.
In another embodiment of the invention, composition also contains at least a tubercle bacillus differential epitope antigen peptide section that is selected from Mtb8 and Mtb8.4; Wherein Mtb8 specific epitopes antigenic peptides section is polypeptide or its functional variety of amino acid sequence shown in the SEQ ID NO:10, and Mtb8.4 specific epitopes antigenic peptides section is polypeptide or its functional variety of amino acid sequence shown in the SEQ ID NO:12.
In one embodiment of the invention, each specific epitopes antigenic peptides section individualism in composition.
In a preferred embodiment of the invention, some specific epitopes antigenic peptides sections exist with the form of fused protein in composition.In a preferred embodiment, 38kD, ESAT6 and CFP10 specific epitopes antigenic peptides section exist as fused protein.
In a preferred embodiment of the present invention; The form of 38kD, ESAT6 and Duan Yiyi fused protein of CFP10 specific epitopes antigenic peptides exists in composition, and MPT64, Mtb8, Mtb8.4 and Mtb16.3 specific epitopes antigenic peptides section exist with the form of another fused protein.
In most preferred embodiment of the present invention; A fused protein is followed successively by 38kD, ESAT6 and CFP10 specific epitopes antigenic peptides section from N end to C end, and another fused protein is followed successively by Mtb8.4, MPT64, Mtb16.3 and Mtb8 specific epitopes antigenic peptides section from N end to C end.
The accompanying drawing summary
The epitope analysis of tubercle bacillus protein 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and Mtb16.3 of Fig. 1-7..
The DNA sequences encoding and the amino acid sequence of the specific epitopes antigenic peptides section among the selected tubercle bacillus protein of Fig. 8-14. 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and the Mtb16.3.
Figure 15. DNA sequences encoding and the amino acid sequence of fused antigen peptide section 38kD-ESAT6-CFP10.
Figure 16. DNA sequences encoding and the amino acid sequence of fused antigen peptide section Mtb8.4-MPT64-Mtb16.3-Mtb8.
Detailed Description Of The Invention
At the serology detection range of tubercle bacillus, also do not have a kind of antigen can high specific and susceptibility detect tubercle bacillus.The antigen combination that has complementation in tubercle bacillus serology context of detection is particularly advantageous.
For detecting tubercle bacillus, effectively specific epitopes antigenic peptides section confirms and can carry out through several different methods.Exemplary method is for example used the epi-position of software such as BIOSUN predicted protein matter; Choose then have one or more epi-positions, the peptide section of advantage epi-position (can excite stronger immunoreactive epi-position) especially; Molecular biology method through routine angles to be got its coded sequence and gives expression to this antigen polypeptide; Detect the reactivity of antigen polypeptide through for example ELISA method to different serum.Tuberculosis patient serum is had reactivity and non-tuberculosis patient serum is not had reactive antigen polypeptide is to can be used for effective specific antigen polypeptide of the present invention.
Detect through using different specific epitopes antigenic peptides sections to carry out reactivity, can find aspect serum reactivity, to have complementary specific epitopes antigenic peptides section for same group of blood serum sample.Though its mechanism is not clear fully as yet, the combination of this specific epitopes antigenic peptides section makes SD specificity of tubercle bacillus and sensitivity improve.
Should be pointed out that selected antigenic peptides section is carried out suitable modification still has 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, and the polypeptide that obtains through this type of modification is called functional variety in this article.The embodiment of conserved amino acid replacement such as the replacement between Ala, Val, Leu and Ile; Replacement between Ser and Thr; Replacement between acidic residues Asp and Glu; Replacement between Asn and Gln; And the replacement between alkaline residue Lys and Arg; The perhaps replacement between aromatic residue Phe and Tyr.
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 (AraiR etc., Protein Enginering, 14 (5): 529-532 (2001)).
The clone of antigen protein, expression and purifying 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 or leaves.Suitable prokaryotic expression carrier such as pEGX series prokaryotic expression carrier (Amersham Pharmacia company), pET series prokaryotic expression carrier (Novagen company) etc.Preferred especially pGEX-4T-2 carrier.
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.Preferred indirect enzyme-linked immunosorbent is measured (indirect ELISA) method.
Specify the present invention below in conjunction with specific embodiment, but should not constitute qualification practical range of the present invention.
Embodiment
Confirming of embodiment 1:7 kind tubercle bacillus differential epitope antigen 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 amino acid sequence; Seek the B cell epitope then, gained epi-position distribution plan is shown in Fig. 1-7.Finally confirm specific epitopes antigenic peptides section according to the epi-position distribution plan.Wherein the DNA sequences encoding of the specific epitopes antigenic peptides section of negre antigen 38kD and amino acid sequence are respectively SEQ ID NO:1 and SEQ ID NO:2; The DNA sequences encoding of the specific epitopes antigenic peptides section of negre antigen ESAT-6 and amino acid sequence are respectively SEQ ID NO:3 and SEQ ID NO:4; The DNA sequences encoding of the specific epitopes antigenic peptides section of negre antigen CFP10 and amino acid sequence are respectively SEQ IDNO:5 and SEQ ID NO:6; The DNA sequences encoding of the specific epitopes antigenic peptides section of negre antigen MPT64 and amino acid sequence are respectively SEQ ID NO:7 and SEQ ID NO:8; The DNA sequences encoding of the specific epitopes antigenic peptides section of negre antigen Mtb8 and amino acid sequence are respectively SEQ ID NO:9 and SEQ ID NO:10; The DNA sequences encoding and the amino acid sequence of the specific epitopes antigenic peptides section of negre antigen Mtb8.4 are respectively SEQ ID NO:11 and SEQ IDNO:12; The DNA sequences encoding of the specific epitopes antigenic peptides section of negre antigen Mtb16.3 and amino acid sequence are respectively SEQ ID NO:13 and SEQ ID NO:14.
Embodiment 2: the preparation of 7 kinds of independent tubercle bacillus differential epitope antigen peptide sections
1. the clone of specific epitopes antigenic peptides fragment gene
Nucleotide sequence according to specific epitopes antigenic peptides section has designed and synthesized the upstream and downstream primer, and employed restriction enzyme is respectively BamHI and XhoI.
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 tubercle bacillus bacterial strain H37Rv genomic DNA is template, the genetic fragment of 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and the Mtb16.3 specific epitopes antigenic peptides 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 genetic 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 antigenic peptides 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% glutathione-agarose resin homogenate.Potpourri 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 fibrin ferment 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 antigenic peptides section of purity.
The preparation of embodiment 3:38kD-ESAT6-CFP10 fused antigen
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 BamHI and XhoI, 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 tubercle bacillus bacterial strain H37Rv genomic DNA is template, the genetic fragment of 38kD, ESAT-6 and the CFP10 specific epitopes antigenic peptides 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 genetic fragment that all obtains corresponding size.Then with genetic fragment with acomplementary connector each other template increase, 38kD, ESAT-6 and CFP10 antigen gene fragment are connected together, make up fusion antigen gene.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 fusion antigen gene fragment.
3. antigen preparation
See embodiment 2.
The preparation of embodiment 4:MPT64-Mtb8-Mtb8.4-Mtb16.3 fused antigen
According to the sequences Design of 4 antigen genes the upstream and downstream primer, the restriction enzyme of use is respectively BamHI and XhoI, 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 preparation are seen embodiment 3.
Embodiment 5: indirect ELISA method is estimated the antigenicity of 7 kinds of independent specific epitopes antigenic peptides sections
Be antigen with selected 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and Mtb16.3 specific epitopes antigenic peptides section respectively; The 30 routine tuberculosis negative serum samples of having used the indirect ELISA method Preliminary detection; Mean value+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 approach 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.The susceptibility of 38kD, ESAT-6, CFP10, MPT64, Mtb8, Mtb8.4 and seven kinds of antigens 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 antigenic peptides sections to tuberculosis patient and normal human blood sample
We notice in these seven kinds of antigens, to have no and a kind ofly can detect whole tuberculosis patients, but are surprisingly found out that Mtb16.3 specific epitopes antigenic peptides section has special advantages in detecting tubercle bacillus.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 antigenic peptides Duan Ze presented strong positive (BP19) to the reactivity of this serum.This shows that Mtb16.3 specific epitopes antigenic peptides section and 38kD, ESAT-6, CFP10, four kinds of specific epitopes antigenic peptides of MPT64 section have good complementarity in the serology context of detection.The combination of at least a specific epitopes antigenic peptides section of Mtb16.3 specific epitopes antigenic peptides section and selection 38kD, ESAT-6, CFP10, MPT64 might improve the susceptibility of diagnosis of tuberculosis.
In addition, a kind ofly can detect whole tuberculosis patients though have no in these seven kinds of antigens, the associating recall rate of seven kinds of antigens 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 application detects and might develop high specific and hypersensitivity diagnostic reagent guaranteeing to help to improve detection sensitivity on the specific basis.
Embodiment 6: indirect ELISA method is estimated Mtb16.3 specific epitopes antigenic peptides section and the combined effect in detecting tuberculosis patient of all the other specific epitopes antigenic peptides sections
Of embodiment 5; Because Mtb16.3 specific epitopes antigenic peptides section has the advantage that other antigenic peptides 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 antigenic peptides sections of specific epitopes antigenic peptides section.Experimental result is as shown in table 2.
Table 2 indirect ELISA method detects Mtb16.3 specific epitopes antigenic peptides section and mixes the reactivity (S/co) to tuberculosis patient and normal human blood sample with other specific epitopes antigenic peptides section
Visible by table 2, Mtb16.3 specific epitopes antigenic peptides section and all the other one or more antigenic peptides 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 antigenic peptides section aspect the tuberculosis patient with other specific epitopes antigenic peptides section detecting.The multiple antigen of Combined application can improve recall rate, avoids omission.
Embodiment 7: indirect ELISA method is estimated the effect of fused antigen in detecting tuberculosis patient
Use 38kD-ESAT6-CFP10 alone or in combination and the Mtb8.4-MPT64-Mtb16.3-Mtb8 fused antigen encapsulates elisa plate, the effect of fused antigen in detecting tuberculosis patient of having used the indirect ELISA method preliminary assessment.
In Preliminary detection; When Combination application 38kD-ESAT6-CFP10 and Mtb8.4-MPT64-Mtb16.3-Mtb8 fused antigen; The serum of 20 routine tuberculosis patients only 1 routine testing 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.Susceptibility 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
Claims (1)
1. be used for serodiagnosis composition lungy, it is a kind of in the combination of specific epitopes antigenic peptides section and 38kD tubercle bacillus differential epitope antigen peptide section and ESAT-6 tubercle bacillus differential epitope antigen peptide section and CFP10 tubercle bacillus differential epitope antigen peptide section and MPT64 tubercle bacillus differential epitope antigen peptide section of combination, tubercle bacillus Mtb16.3 of specific epitopes antigenic peptides section and 38kD tubercle bacillus differential epitope antigen peptide section and ESAT-6 tubercle bacillus differential epitope antigen peptide section and CFP10 tubercle bacillus differential epitope antigen peptide section of combination, tubercle bacillus Mtb16.3 of specific epitopes antigenic peptides section and 38kD tubercle bacillus differential epitope antigen peptide section and MPT64 tubercle bacillus differential epitope antigen peptide section of combination, tubercle bacillus Mtb16.3 of specific epitopes antigenic peptides section and 38kD tubercle bacillus differential epitope antigen peptide section and ESAT-6 tubercle bacillus differential epitope antigen peptide section of combination, tubercle bacillus Mtb16.3 of specific epitopes antigenic peptides section and MPT64 tubercle bacillus differential epitope antigen peptide section of combination, tubercle bacillus Mtb16.3 of specific epitopes antigenic peptides section and CFP10 tubercle bacillus differential epitope antigen peptide section of combination, tubercle bacillus Mtb16.3 of specific epitopes antigenic peptides section and ESAT-6 tubercle bacillus differential epitope antigen peptide section of combination, tubercle bacillus Mtb16.3 of specific epitopes antigenic peptides section and the 38kD tubercle bacillus differential epitope antigen peptide section of tubercle bacillus Mtb16.3; Wherein Mtb16.3 specific epitopes antigenic peptides section is the polypeptide of amino acid sequence shown in the SEQ ID NO:14; 38kD specific epitopes antigenic peptides section is the polypeptide of amino acid sequence shown in the SEQ ID NO:2; ESAT-6 specific epitopes antigenic peptides section is the polypeptide of amino acid sequence shown in the SEQ ID NO:4; CFP10 specific epitopes antigenic peptides section is the polypeptide of amino acid sequence shown in the SEQ ID NO:6, and MPT64 specific epitopes antigenic peptides section is the polypeptide of amino acid sequence shown in the SEQ ID NO:8; Each specific epitopes antigenic peptides section individualism in composition wherein.
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| 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 |
| CN105486860A (en) * | 2014-10-09 | 2016-04-13 | 中国人民解放军军事医学科学院基础医学研究所 | Mycobacterium tuberculosis antigen detection method based on specific multi-antibody |
| CN104678097B (en) * | 2015-03-10 | 2017-04-05 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of mycobacterium tuberculosis combined antigen for diagnosis of pulmonary tuberculosis |
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| 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. * |
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