CN101477125A - 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 spreads in worldwide.Nearly 1/3 people infects tubercle bacillus on the earth, causes annual 8000000 routine active tuberculosis and 3,000,000 people's death.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 exist more problem in diagnostic method.
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 height, diagnosis speed are fast, but have 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 judged result easy and simple to handle, quick, easy, 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.At present increasing interest is invested species specificity and all reliable serology diagnosis of tuberculosis method of sensitivity set up.The antigen that comprised of the purified protein derivative of tuberculin of Shi Yonging (PPD) is that pathogenic mycobacterium, environment mycobacterium and Bacille Calmette-Guerin (BCG) are common traditionally, can not clearly distinguish the BCG immunity, the environment mycobacterium infects 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 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 better than other single antigen.Studies 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 secretion property antigen target (ESAT-6) and culturing filtrate protein 10 (CFP10) are two up-and-coming antigens in the tuberculosis immunodiagnosis, have stronger cellular immunity activity, play an important role in the immune response of resisting tuberculosis infection.These two antigens are by RD1 district coding, and this district only is present in the pathogenic tubercle bacillus gene group, all lacks this zone (Mahairas GG etc., Bacteriol, 178:1274-1282 (1996)) in all BCG strain gene groups.ESAT-6 and CFP10 can form the heterodimer structure, use in conjunction 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 exists in tubercle bacillus and ox type mycobacterium, 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 group of tubercle bacillus, most of BCG bacterial strains lack the MPT64 genes, BCG inoculator or environment mycobacterium the infected (as 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 low molecular weight protein (LMWP) antigen that purifies and separates obtains from tubercle bacillus culture filtrate 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 has vital role in determining pathogenic mycobacterium tuberculosis infection.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 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 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 the 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 field of tubercle bacillus, also there is not a kind of antigen can high specific and sensitivity Detect to property tubercle bacillus. The antigen group that has complementation in tubercle bacillus serology context of detection It is particularly advantageous closing.
For detecting tubercle bacillus, effectively specific epitopes antigenic peptides section determine can pass through many Kind method is carried out. Exemplary method is for example used the epi-position of software such as BIOSUN predicted protein matter; Choose then and have one or more epi-positions, especially Dominant Epitopes and (can excite stronger immune response Epi-position) the peptide section; Molecular biology method by routine angles to be got its coded sequence and gives expression to that this is anti-Former polypeptide; By ELISA method detectable antigens polypeptide for example to the reactivity of different serum. To tuberculosis Patient's serum has reactivity and the non-tuberculosis patients serum is not had reactive antigen polypeptide Can be used for effective specific antigen polypeptide of the present invention.
Carry out the reactivity inspection by using different specific epitopes antigenic peptides sections for same group of blood serum sample Survey, can find aspect serum reactivity, to have complementary specific epitopes antigenic peptides section. Although, Its mechanism is not yet fully clear, but the combination of this specific epitopes antigenic peptides section is so that the blood of tubercle bacillus Clear specificity and the sensitivity of learning detection improves.
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 replacements, increase or are lacked, individually amino acid refer to be less than 10, 9,8,7,6,5,4,3,2,1 amino acid, the polypeptide that obtains by this type of modification is at this paper In be called functional variety. Example that conserved amino acid is replaced is for example between Ala, Val, Leu and Ile Replace; Replacement between Ser and Thr; Replacement between acidic residues Asp and Glu; Asn and Gln Between replacement; And the replacement between alkaline residue Lys and Arg; Perhaps aromatic residue Phe and Tyr Between replacement.
For the antigen that different peptide sections merge, well-known its methods and principles in this area. Specifically , need between different peptide sections, add one in order not affect the function separately for the treatment of the fusogenic peptide section Joint. About the selection of joint specifically can referring to (AraiR etc., Protein Enginering, 14 (5): 529-532 (2001)).
The clone of antigen protein, expression and purifying can be undertaken by several different methods. The concrete grammar ginseng See " 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 public affairs Department), pET series prokaryotic expression carrier (Novagen company) etc. Especially preferred pGEX-4T-2 carries Body.
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.
Describe the present invention in detail below in conjunction with specific embodiment, but should not constitute qualification the scope of the present invention.
Embodiment
Determining 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 determine 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 BamH I and Xho I.
The primer sequence that is used for amplifying specific epitope antigen peptide section 38kD is as follows:
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 as follows:
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 as follows:
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 as follows:
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 as follows:
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 as follows:
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 as follows:
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 as follows: 95 ℃ of pre-sex change 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 by electrophoresis, shows 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 endonuclease digestion, order-checking is finished 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 precipitation, 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 BamH I and Xho I, all primers are synthetic by the handsome biotech company in Shanghai, its sequence is as follows:
38kD-F：5’-GGGATCCGCGGCGGGCTGTGGCTCGA-3’
38kD-RI：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 increased respectively 38kD, ESAT-6 and CFP10 specific epitopes antigenic peptides section.The pcr amplification condition is as follows: 95 ℃ of pre-sex change 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 by electrophoresis, show the genetic fragment that all obtains corresponding size.Then with genetic fragment with acomplementary connector each other template increase, 38kD, ESAT-6 are connected together with CFP10 antigen gene fragment, make up fusion antigen gene.With digestion with restriction enzyme and connect into the carrier through the pGEX-4T-2 of same restrictions endonuclease digestion, order-checking is finished 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 BamH I and Xho I, all primers are synthetic by the handsome biotech company in Shanghai, its sequence is as follows:
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, 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 can detect whole tuberculosis patients without any a kind of, but is 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 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, though can detect whole tuberculosis patients without any a kind of 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 may be complicated because negre antigen is many, shows different antibody repertoires in host.Therefore, the multiple antigen of use in conjunction 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
As described in embodiment 5, because Mtb16.3 specific epitopes antigenic peptides section has the advantage that other antigenic peptides section is not had, therefore contrived experiment is verified itself and four kinds of antigens commonly used, 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 detection Mtb16.3 specific epitopes antigenic peptides section is mixed the reactivity (S/co) to tuberculosis patient and normal human blood sample with other specific epitopes antigenic peptides section
By table 2 as seen, 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 does not but reduce.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 use in conjunction 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 and Mtb8.4-MPT64-Mtb16.3-Mtb8 fused antigen bag alone or in combination by elisa plate, the effect of fused antigen in detecting tuberculosis patient of having used the indirect ELISA method preliminary assessment.
In Preliminary detection, when applied in any combination 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, wherein 9 examples are strong positive (S/co〉2.0), reach 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
Sequence table
＜110〉Institute of Basic Medical Sciences, Academy of Military Medical Sciences, PLA
＜120〉composition based on tubercle bacillus antigenic polypeptide of diagnosis of tuberculosis
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<170>PatentIn?version?3.3
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＜213〉tubercle bacillus
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＜213〉tubercle bacillus
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＜213〉tubercle bacillus
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＜213〉tubercle bacillus
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＜213〉tubercle bacillus
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＜213〉tubercle bacillus
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＜213〉tubercle bacillus
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＜213〉tubercle bacillus
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＜213〉tubercle bacillus
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＜213〉tubercle bacillus
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<210>14
<211>143
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＜213〉tubercle bacillus
<400>14
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＜213〉artificial sequence
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＜213〉artificial sequence
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Claims (6)
1. be used for serodiagnosis composition lungy, 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 IDNO: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.
2. the composition of claim 1, it 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 IDNO: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.
3. claim 1 or 2 described compositions, wherein each specific epitopes antigenic peptides section individualism in composition.
4. claim 1 or 2 described compositions, wherein some specific epitopes antigenic peptides sections exist with the form of fused protein in composition.
5. claim 1 or 2 described compositions, wherein 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.
6. the described composition of claim 5, one of them 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.
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| CN200810000021A CN101477125B (en) | 2008-01-03 | 2008-01-03 | Composition based on tubercle bacillus antigenic polypeptide for diagnosing tuberculosis |
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| CN200810000021A CN101477125B (en) | 2008-01-03 | 2008-01-03 | Composition based on tubercle bacillus antigenic polypeptide for diagnosing tuberculosis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104678097A (en) * | 2015-03-10 | 2015-06-03 | 中国人民解放军军事医学科学院基础医学研究所 | Mycobacterium tuberculosis combined antigen for diagnosing pulmonary tuberculosis |
| EP2812704A4 (en) * | 2012-02-07 | 2016-03-23 | 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 |
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2008
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2812704A4 (en) * | 2012-02-07 | 2016-03-23 | Intuitive Biosciences Inc | Mycobacterium tuberculosis specific peptides for detection of infection or immunization in non-human primates |
| 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 |
| US10094830B2 (en) | 2012-02-07 | 2018-10-09 | Intuitive Biosciences, Inc. | Mycobacterium tuberculosis specific peptides for detection of infection or immunization in non-human primates |
| EP3415919A1 (en) * | 2012-02-07 | 2018-12-19 | Intuitive Biosciences Inc. | Mycobacterium tuberculosis specific peptides for detection of infection or immunization in non-human primates |
| US10859575B2 (en) | 2012-02-07 | 2020-12-08 | 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 |
| CN104678097A (en) * | 2015-03-10 | 2015-06-03 | 中国人民解放军军事医学科学院基础医学研究所 | Mycobacterium tuberculosis combined antigen for diagnosing pulmonary tuberculosis |
| 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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| CN101477125B (en) | 2012-10-17 |
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