CN107216373A - Detect antigen polypeptide pond and its application of mycobacterium tuberculosis infection - Google Patents
Detect antigen polypeptide pond and its application of mycobacterium tuberculosis infection Download PDFInfo
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- CN107216373A CN107216373A CN201710198835.6A CN201710198835A CN107216373A CN 107216373 A CN107216373 A CN 107216373A CN 201710198835 A CN201710198835 A CN 201710198835A CN 107216373 A CN107216373 A CN 107216373A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
- G01N33/6866—Interferon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
Abstract
The invention discloses a kind of antigen polypeptide pond for being used to detect mycobacterium tuberculosis infection, the specific antigen polypeptide pond, the fresh whole blood of differential stimulus mycobacterium tuberculosis infection can specific secretion IFN γ, increase detection sensitivity.The invention provides a kind of new detection reagent that can be used for mycobacterium tuberculosis infection detection, experiment is proved, the present invention directly can carry out antigenic stimulus using peripheral blood, without separating peripheral blood mononuclear cells, experimental data, which is shown for mycobacterium tuberculosis infection detection, has higher sensitivity and specificity, and it is easy to operate, cost is relatively low, with higher clinical value.
Description
Technical field
The present invention relates to biological technical field, in particular to the antigen polypeptide pond of detection mycobacterium tuberculosis infection
And its application.
Background technology
Tuberculosis is main through respiratory infectious, serious harm one of China and global Important Infectious Diseases.According to generation
There is 17-20 hundred million mycobacterium tuberculosis the infected in boundary's health organization, the whole world, and at least 2,000,000 people die from this disease and (thank and build every year
It is flat, the methodology of mycobacterium tuberculosis functional genome research, microorganism circular .2001.28 (5):92-97).Therefore, accurately
The method of screening the infected plays the part of extremely important effect in preventing and treating lungy or even final elimination.
Diagnosis of the existing diagnostic techniques to mycobacterium tuberculosis the infected is very difficult.In recent decades, clinically use
Tuberculin test (TST) carrys out diagnosis of tuberculosis mycobacterial infections person.But TST active ingredients are purified protein derivatives
(purified protein derivative of tuberc μ Lin, PPD), is the thick of mycobacterium tuberculosis culture supernatant
Extract, containing 200 Multiple components, with BCG vaccine (Bacillus Calmette-Guerin, BCG) inoculation and environment mycobacteria
There is cross reaction in infection.TST testing results can be immunized by present clinical widely used vaccine-BCG vaccine and disturb
[P.Andersen, et al.Lancet356 (2000) 1099-1104.].From nineteen twenty-one and bright BCG vaccine till now, entirely
The existing 3,500,000,000 people's bcg vaccinations in the world.Therefore TST is greatly affected to the diagnostic value of the infected, causes TST to diagnose
Accuracy it is relatively low, therefore we can not judge whether to be truly present mycobacterium tuberculosis infection according to its result.
To in the diagnostic techniques of tuberculosis patient, Sputum smears acid-fast stain or Phlegm incubation, culture are difficult, take longer;
Lung's x-ray checks Pulmonary lesions, also could only be diagnosed after with typical clinical symptoms;Other serodiagnosis sides
Method and molecular diagnosis method lack specificity, and false positive rate is higher.
Relevant researcher points out that the Sensitivity and Specificity of different diagnostic test methods is as follows:Mycobacteria is cultivated
(being respectively 73% and 100%);PCR (being respectively 42% and 100%);Chest x-ray (respectively 67%~77% and 66%~
76%);Tuberculin test (being respectively 94% and 20%);Serology (being respectively 33% and 87%).As can be seen here, it is existing
The Sensitivity and Specificity of mycobacteria detection method can not be optimal simultaneously.
After mycobacterium tuberculosis infection human body, it can be recognized first by the immune system of human body, the T cell of activation body is immunized
Response, secretion produces interferon (IFN-γ), and the latter plays resisting tuberculosis infection effect.Part T cell is converted into immunological memory
Cell, after being met again with mycobacterium tuberculosis, secretion can produce IFN-γ again, play resisting tuberculosis infection effect.Antigen
It is closely related whether special stimulation generation IFN-γ and its yield infect or fall ill with body.Therefore, if using tuberculosis
The special antigen of mycobacteria stimulates the T cell of the infected and patient in vitro, can induce these T cells and produces high-caliber knot
The special IFN-γ of core mycobacteria, and the amount for the IFN-γ that non-tuberculous mycobacteria the infected or non-tuberculosis patient produce with
The amount that unused antigenic stimulus is produced is close, so as to realize the purpose of diagnosis.As can be seen here, find and find that mycobacterium tuberculosis is special
Specific Antigen, has important value and meaning to the diagnostic reagent of development a new generation.
1996, the method that Stover etc. is hybridized by subtrahend determined the domain that BCG is lost in succeeding generations in vitro, fixed
Justice is missing area (Region of Difference, RD), and runt domain is present in mycobacterium tuberculosis, and in BCG and mostly
[the laboratory diagnosis progress foreign medical science clinics of the full mycobacterium tuberculosis of Cheng Yu, Li Mao are raw for missing in the mycobacteria of ring of numbers border
Thing chemistry and ecsomatics fascicle .2002;23(6):342-343.].Wherein, the early stage of RD1 regional codes point antigen target 6KD
(early secreting antigen target 6KD, ESAT-6) and culturing filtrate protein 10 KD (c μ Ltufiltrate
Protein 10KD, CFP-10) it is two kinds of small-molecular-weight secretory proteins, they are one of topmost antigen of T cell, can be lured
Lead stronger cell immune response.
For children or immunocompromised sample, there is document report, thorn is only used as using ESAT-6 and CFP-10 or its polypeptide
Swash original, its sensitivity be about 73.5%-88.9% (Sun Lin, ELISPOT detection technique children tuberculosis diagnosis in application,
Labelling immunoassay and clinic .2008.6:349-353;Zhong Huaqing, interferon-γ release test is in childhood tuberculosis infection
Application value, laboratory medicine .2016.3:176-179;Li Hai, using enzyme-linked immunospot assay quick diagnosis childhood tuberculosis branch
The research of bacillus infection, Chinese mother and child care, 2012.11:1703-1706).It is such as children, immune low for some special samples
Sample is infected down and recently, ESAT-6 and CFP-10 polypeptides and derivative kit mycobacterium tuberculosis sample are detected still not
, there is false negative in height, it is impossible to meet clinical demand.
The content of the invention
For existing detection method for some special samples, recently such as children, immunocompromised and infection tuberculosis branch bar
The recall rate of bacterium sample is not enough, it is an object of the invention to provide a kind of antigen polypeptide pond for detecting mycobacterium tuberculosis infection,
The fresh whole blood of the specific antigen polypeptide pond differential stimulus mycobacterium tuberculosis infection, the specific secretion of gamma-IFN of energy,
Increase detection sensitivity.
A kind of antigen polypeptide pond for detecting mycobacterium tuberculosis infection, the antigen polypeptide pond is included containing amino acid sequence such as
The combination of wantonly eight polypeptides or wantonly more than eight polypeptides shown in SEQ ID NO.1~SEQ ID NO.26.
Antigen polypeptide pond as described above, it is preferable that the polypeptide is artificial synthesized or naturally isolated.
Antigen polypeptide pond as described above, it is preferable that the antigen polypeptide pond also includes ESAT-6 and CFP-10.
Preferably, the amino acid sequence of the ESAT-6 is as shown in SEQ ID NO.27, the amino acid sequence of the CFP-10
Row are as shown in SEQ ID NO.28.
Antigen polypeptide pond as described above, it is preferable that the antigen polypeptide pond also includes ESAT-6-CFP-10.
Preferably, the amino acid sequence of the ESAT-6-CFP-10 is as shown in SEQ ID NO.29.
Antigen polypeptide pond as described above, it is preferable that the antigen polypeptide pond includes and the SEQ ID NO.1~SEQ ID
Any shown sequence has the polypeptide of 85% homology in NO.26.
A kind of kit for detecting mycobacterium tuberculosis infection, the kit includes antigen polypeptide pond as described above.
A kind of kit for detecting mycobacterium tuberculosis infection, it is preferable that the kit also detection including IFN-γ is tried
Agent.
The application in the antigen polypeptide pond of detection mycobacterium tuberculosis infection as described above, is made using the antigen polypeptide pond
For stimulant stimulation of whole, the IFN-γ produced is detected.
The antigen polypeptide pond provided using the present invention and specific antigen protein (ESAT6-CFP10, the ESAT6 of IFN-γ
And CFP10), for detecting the metainfective whole blood sample of mycobacterium tuberculosis, have the characteristics that:
(1) immunocompromised sample positive rate is high, and sensitivity is high.For existing detection method for some special samples
This, the detection of such as children, immunocompromised and recently infection sample or latent infection person are not enough, the present invention is provided to sensitiveness and
The antigen polypeptide pond of the high mycobacterium tuberculosis infection of specificity, the polypeptide pond, which merges ESAT-6 and CFP-10, can realize to tuberculosis
Patient or quick, the special detection of early infection person or latent infection person (not occurring tuberculosis illness), are blocking tuberculosis
The propagation of disease and prevalence, and control lungy is significant.
(2) the detection used time is short, easy to operate, and diagnosis is quick.Compared with traditional mycobacterium tuberculosis is detected, the present invention is utilized
The specific antigen protein detection used time that the polypeptide pond of offer merges IFN-γ is short, and diagnosis is quick.Traditional mycobacterium tuberculosis training
Foster diagnosis is taken time the 1-2 months, and TST detections need 3 days, and present invention whole detection time can be completed in 1-2 days.
(3) early detection can be carried out to infection mycobacterium tuberculosis.Because mycobacterium tuberculosis once infects human body, first
Just recognized by the immune system of body, activation humoral and cellular immune response response, disease time 1-2 months after infection, therefore,
The polypeptide pond provided using the present invention merges the cellular immunology detection method of the specific antigen protein of whole blood IFN-gamma
Diagnosis quickly is made, is diagnosed to be whether sample infects mycobacterium tuberculosis.And in existing technology, X-ray diagnosis is only in sense
Ran Zhe lungs can just make diagnosis after there is obvious pathological change, and this usually requires long time.Antigen and antibody
Only detected in the case where disease symptomses are in active stage, but mycobacteria is widely present in environment, itself and tuberculosis branch bar
The common antigen of bacterium, may interfere with the diagnostic result of experiment.It can be seen that, the present invention infects or immunocompromised mycobacterium tuberculosis recently
The early detection of person is significant, has positive meaning to control lungy and elimination.
Present invention also offers a kind of new detection reagent that can be used for mycobacterium tuberculosis infection detection, it is demonstrated experimentally that
The present invention directly can carry out antigenic stimulus using peripheral blood, and without separating peripheral blood mononuclear cells, experimental data, which is shown, to be used for
Mycobacterium tuberculosis infection detection has higher sensitivity and specificity, is prevented effectively from false negative, and easy to operate, cost compared with
It is low, with higher clinical value.
Embodiment
In order to realize the purpose of the present invention, inventor carries out polypeptide prediction according to computer software, comprehensive preferably suitable
Polypeptide, is studied by lot of experiments and verified, the final amino acid sequence that obtains is as shown in SEQ IDNO.1~SEQ ID NO.26
The screening study of polypeptide, wherein these polypeptides is determined by following technological means:
1st, single polypeptide stimulates validity to screen:The polypeptide tentatively sieved is configured to certain density polypeptide solution, stimulated
Fresh whole blood, detects whether it infects mycobacterium tuberculosis with interferon detection kit, and selection has stimulation to positive sample
Effect, and to polypeptide of the negative sample without nonspecific stimulation.
2nd, polypeptides in combination effect of stimulation:Effective polypeptide random 8-20 will be screened and be configured to mixed polypeptide solution, pierced
Swash fresh whole blood, 37 DEG C are incubated 20 ± 4 hours, and supernatant is collected by centrifugation, it is (enzyme-linked to exempt from that supernatant is added into people's IFN-γ detection kit
Epidemic disease method) in detected, select have effect of stimulation to positive sample, it is and many to mixing of the negative sample without nonspecific stimulation
Peptide, it is determined that effective mixed polypeptide sequence can be combined.
3rd, ESAT-6 and CFP-10 effect of stimulation is merged:Effective mixed polypeptide solution of screening is merged into ESAT-6 and CFP-
10 are configured to stimulate agent solution, stimulate fresh whole blood, and 37 DEG C are incubated 20 ± 4 hours, and supernatant is collected by centrifugation, supernatant is added into people
Detected in IFN-γ detection kit (ELISA), selection has effect of stimulation to positive sample, and to negative sample
Mixed polypeptide without nonspecific stimulation, it is determined that effective mixed polypeptide sequence can be combined.
4th, ESAT-6-CFP-10 effect of stimulation is merged:Effective mixed polypeptide solution of screening is merged into ESAT-6-CFP-10
It is configured to stimulate agent solution, stimulates fresh whole blood, 37 DEG C is incubated 20 ± 4 hours, and supernatant is collected by centrifugation, and supernatant is added into people IFN-
Detected in γ detection kits (ELISA), selection has effect of stimulation to positive sample, and to negative sample nothing but
The mixed polypeptide of differential stimulus, it is determined that effective mixed polypeptide sequence can be combined.
Patient's fresh whole blood of the polypeptide empirical tests screened in the present invention energy differential stimulus mycobacterium tuberculosis infection
Specific secretion of gamma-IFN, the specific protein for the mycobacterium tuberculosis that specially causes a disease:Ag85B (SEQ ID NO.1~
SEQ ID NO.6), Mpt649 (SEQ ID NO.7~SEQ ID NO.10), RV1985C (SEQ ID NO.11~SEQ ID
NO.16), RV1989c (SEQ ID NO.17~SEQ ID NO.21), Rv3425 (SEQ ID NO.22~SEQ ID
NO.26), these protein of screening can be directed to the polypeptide of the binding site of CD8+ lymphocytes, pass through clinical verification repeatedly
Experiment, these polypeptides merge ESAT-6 and CFP-10 can improve detection sensitivity, the merging stimulant can specific effect in knot
Lymphocyte epitopes in the whole blood of core mycobacterial infections sample, the cell factor IFN-γ discharged by detecting in whole blood,
So as to which whether reaction body infected mycobacterium tuberculosis indirectly.This method improves the detection that mycobacterium tuberculosis infects sample
Rate, particularly infects sample, children, the Sensitivity of immunocompromised infection mycobacterium tuberculosis sample, is prevented effectively from recently
The missing inspection that easily occurs in existing detection technique, false retrieval problem.
The invention will be further described with reference to embodiments, but protection scope of the present invention is not limited to these realities
Apply example.Every change or equivalent substitute without departing substantially from present inventive concept is included within protection scope of the present invention.Following reality
The experimental method for applying unreceipted actual conditions in example is routinely experimental method;Agents useful for same is commercially available purchase unless otherwise instructed
Buy product.
The single polypeptide of embodiment 1 stimulates validity to screen
The peptide sequence of the present invention, its amino acid sequence is as shown in SEQ ID NO.1~SEQ ID NO.26, and these can lead to
Cross artificial synthesized or naturally isolated, the polypeptide in the present embodiment and following examples is by artificial synthesized.
SEQ ID NO.1:RLWVYCGNGTPNEL
SEQ ID NO.2:LMIGTAAAVVLPGL
SEQ ID NO.3:VQFQSGGNNSPAVY
SEQ ID NO.4:MPVGGQSSFYSDWY
SEQ ID NO.5:SAAIGLSMAGSSAM
SEQ ID NO.6:PAFEWYYQSGLSIV
SEQ ID NO.7:YQSAIPPRGTQAVV
SEQ ID NO.8:IQMSDPAYNINISL
SEQ ID NO.9:KSLENYIAQTRDKF
SEQ ID NO.10:MLVTAVVLLCCSGV
SEQ ID NO.11:RKAFRRAITRPTHF
SEQ ID NO.12:MVDPQLDGPQLAAL
SEQ ID NO.13:KLDSPIIARITDTV
SEQ ID NO.14:RLAAQTALLESEAL
SEQ ID NO.15:ITIAVNADSMATWF
SEQ ID NO.16:REKPCRATTAGIPL
SEQ ID NO.17:LLFPAIYLADSAQA
SEQ ID NO.18:AVLDLTTPQAREAV
SEQ ID NO.19:KMLEAAYRLHTIDV
SEQ ID NO.20:TAYEQRTRPGQLQL
SEQ ID NO.21:GRWNPPLLFPAIYL
SEQ ID NO.22:RELAYSVETTAESL
SEQ ID NO.23:VSWTRSALSDLPRW
SEQ ID NO.24:LSDLPRWREPPQIY
SEQ ID NO.25:SLFFASGQLRELAY
SEQ ID NO.26:ALSDLPRWREPPQI
The polypeptide of preparation is dissolved into concentration with sterile low endotoxin (endotoxin should be less than 2.5EU/ μ g) PBS for 250 μ g/
mL。
Take the fresh blood sample of 10 clinical definite tuberculosis symptom crowds, 10 fresh bloods without tuberculosis symptom healthy population
Whether sample, (similarly hereinafter), it is detected with commercially available interferon detection kit (Wuhan Hygiea Bioscience Co., Ltd.)
Infect mycobacterium tuberculosis.The μ L of polypeptide solution 20 (final concentration of 100 μ g/mL of polypeptide) in sterile culture pipe are taken simultaneously, often
Pipe adds the fresh collection whole bloods of 1mL, is well mixed after after 37 DEG C of baking ovens 20 ± 4 hours, mixing blood, 6000rpm centrifugations
1min, takes supernatant to detect interferon content.
As a result show, the polypeptide as shown in SEQ ID NO.1~SEQ ID NO.26 designed by the present invention is to positive sample
Originally there is effect of stimulation, and to negative sample without nonspecific stimulation.It should be noted that with SEQ ID NO.1~SEQ ID
The polypeptide that any polypeptide has 85% homology in NO.26 can also realize above-mentioned purpose.
Embodiment is 2-in-1 and effect of stimulation
It will appoint and take 8 polypeptide solutions merging ESAT-6 and CFP-10 to be configured to stimulate agent solution, wherein, ESAT-6 and CFP-
10 recombinantly express acquisition by Recombinant organism, and ESAT-6 sequences are SEQ ID NO.27:
MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTIS
EAGQAMASTEGNVTGMFA;CFP-10 sequences are SEQ ID NO.28:
MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIR
QAGVQYSRADEEQQQALSSQMGF.The experimental result of part of polypeptide is only enumerated in the present embodiment, numbering is stimulant 1~thorn
Swash (the stimulant 1 of agent 4:SEQ ID NO.1~SEQ ID NO.8+ESAT-6+CFP-10;Stimulant 2:SEQ ID NO.6~SEQ
ID NO.13+ESAT-6+CFP-10;Stimulant 3:SEQ ID NO.11~SEQ ID NO.18+ESAT-6+CFP-10;Stimulate
Agent 4:SEQ ID NO.19~SEQ ID NO.26+ESAT-6+CFP-10), take the positive and do not feel that mycobacterium tuberculosis infects
The negative sample of dye is tested.
20 whole blood samples in be the same as Example 1 are taken, detect whether it infects tuberculosis point with interferon detection kit
Branch bacillus.Each 20 μ L of 1~stimulant of stimulant 4 (final concentration of 100 μ g/mL of each polypeptide) in sterile culture pipe are taken simultaneously,
Often pipe adds the fresh collection whole bloods of 1mL, is well mixed after after 37 DEG C of baking ovens 20 ± 4 hours, mixing blood, 6000rpm centrifugations
1min, takes supernatant interferon detection kit to detect, detection method and reagent be the same as Example 1, and testing result is shown in Table 1, its
In, commercial reagent box detects for the mycobacterium tuberculosis specific cell immunoreaction of Wuhan Hygiea Bioscience Co., Ltd.
Kit (ELISA), similarly hereinafter.
The mixed polypeptide of table 1. merges ESAT-6 and CFP-10 results of stimulation statistics
Commercial reagent box | Stimulant 1 | Stimulant 2 | Stimulant 3 | Stimulant 4 | |
10 positive samples detect number of cases | 8 | 9 | 9 | 10 | 9 |
10 negative samples detect number of cases | 10 | 10 | 10 | 10 | 10 |
As a result illustrate that polypeptide pond prepared by the present invention combines ESAT-6 and CFP-10 for detecting that mycobacterium tuberculosis infects
Whole blood, improve detection sensitivity, be prevented effectively from prior art detection false negative and missing inspection.
Embodiment 3 merges effect of stimulation
It will appoint and take 8 polypeptide solutions merging ESAT-6-CFP-10 to be configured to stimulate agent solution, wherein, ESAT-6-CFP-10
Recombinantly expressed and obtained by Recombinant organism, ESAT-6-CFP-10 sequences are SEQ ID NO.29:
MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTIS
EAGQAMASTEGNVTGMFANVAMAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVV
RFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF, numbering is the (stimulant 5 of 5~stimulant of stimulant 8:
SEQ ID NO.1~SEQ ID NO.8+ESAT-6-CFP-10;Stimulant 6:SEQ ID NO.6~SEQ ID NO.13+
ESAT-6-CFP-10;Stimulant 7:SEQ ID NO.11~SEQ ID NO.18+ESAT-6-CFP-10;Stimulant 8:SEQ ID
NO.19~SEQ ID NO.26+ESAT-6-CFP-10), take mycobacterium tuberculosis is positive to be tested with negative sample, prepare
Into agent solution is stimulated, 20 whole blood samples in be the same as Example 1 are taken, detect whether it infects knot with interferon detection kit
Core mycobacteria.Take each 20 μ L of 5~stimulant of stimulant 8 (final concentration of 100 μ g/ of each polypeptide in sterile culture pipe simultaneously
ML), often pipe adds the fresh collection whole bloods of 1mL, is well mixed after after 37 DEG C of baking ovens 20 ± 4 hours, mixing blood, 6000rpm
1min is centrifuged, takes supernatant interferon detection kit to detect.Detection method be the same as Example 2, testing result is shown in Table 2.
The mixed polypeptide of table 2. merges ESAT-6-CFP-10 results of stimulation
Commercial reagent box | Stimulant 5 | Stimulant 6 | Stimulant 7 | Stimulant 8 | |
10 positive samples detect number of cases | 8 | 9 | 9 | 10 | 10 |
10 negative samples detect number of cases | 10 | 10 | 10 | 10 | 10 |
As a result illustrate that polypeptide pond prepared by the present invention combines ESAT-6-CFP-10 for detecting mycobacterium tuberculosis infection
Whole blood, improves detection sensitivity, is prevented effectively from false negative and the missing inspection of prior art detection.
Embodiment 4 merges mixed polypeptide detection sensitivity and specificity
Sample:The fresh blood sample of 20 clinical definite tuberculosis symptom crowds is taken, 20 without the new of tuberculosis symptom healthy population
Blood sample.
The whole blood sample of above-mentioned sample is gathered, respectively with stimulant 9 (ESAT-6 (10 μ g/mL) and CFP-10 (10 μ g/mL)
+ mixed polypeptide (SEQ ID NO.6~SEQ ID NO.13, each 100 μ g/mL)), (ESAT-6-CFP-10 (the 10 μ g/ of stimulant 10
ML)+mixed polypeptide (SEQ ID NO.6~SEQ ID NO.13, each 100 μ g/mL)), (ESAT-6 (10 μ g/mL) of stimulant 11
With CFP-10 (10 μ g/mL)), stimulant 12 (ESAT-6-CFP-10 (10 μ g/mL)) and (the commercial reagent box of stimulant 13:Wuhan
The mycobacterium tuberculosis specific cell immunoreaction detection kit (ELISA) of Hai Jili bio tech ltd
Each 20 μ L are in sterile culture pipe, and often pipe adds the fresh collection whole bloods of 1mL and stimulated, and is well mixed small after 37 DEG C of baking ovens 20 ± 4
When, mix after blood, 6000rpm centrifugation 1min take supernatant interferon detection kit to detect.It the results are shown in Table 3.
Table 3. merges mixed polypeptide sensitivity and specificity testing result statistics
As a result illustrate that polypeptide pond prepared by the present invention is tied with reference to ESAT-6 and CFP-10 or ESAT-6-CFP-10 for detecting
The whole blood of core mycobacterial infections, its detection sensitivity is up to 95%, and sensitivity and specificity are stronger, is prevented effectively from prior art
The false negative of detection and missing inspection.
The sensitivity and specificity detection of the present invention of embodiment 5
Sample:The fresh blood sample of 100 clinical definite tuberculosis symptom crowds is taken, wherein 65 years old age and sample above 21
Example, less than 6 years old age sample number 18,51, HIV samples, man 56, female 44;50 without the new of tuberculosis symptom healthy population
Blood sample, wherein 65 years old age and sample above 20, less than 6 years old age sample number 7,23, HIV samples, man 26, female
24.
Gather the whole blood sample of above-mentioned sample, respectively with stimulant (ESAT-6 (10 μ g/mL) and CFP-10 (10 μ g/mL)+
Mixed polypeptide (SEQ ID NO.11~SEQ ID NO.18, each 100 μ g/mL)) and commercial reagent box (Wuhan Hai Jili biologies section
The mycobacterium tuberculosis specific cell immunoreaction detection kit (ELISA) of skill Co., Ltd) each 20 μ L of stimulant
In sterile culture pipe, often pipe adds the fresh collection whole bloods of 1mL and stimulated, and is well mixed after 37 DEG C of baking ovens 20 ± 4 hours, mixing
After blood, 6000rpm centrifugation 1min take supernatant to be detected with commercially available interferon detection kit, commercial reagent
Box is operated by its specification.Testing result is shown in Table 4 and table 5.
The ELISA testing result statistics of the mycobacterium tuberculosis of table 4. infection sample
The ELISA testing result statistics of the healthy sample of table 5.
As a result illustrate that polypeptide pond prepared by the present invention can improve special to some with reference to ESAT-6 and CFP-10 and its derivative
Different sample, such as children, immunocompromised and recently infection sample recall rate, sensitivity and specificity are stronger, are prevented effectively from existing skill
The false negative of art detection and missing inspection.Mycobacterium tuberculosis is infected the present invention recently or the early detection of immunocompromised person has important
Meaning, has positive meaning to control lungy and elimination.
SEQUENCE LISTING
<110>Wuhan Hygiea Bioscience Co., Ltd.
<120>Detect antigen polypeptide pond and its application of mycobacterium tuberculosis infection
<130>
<160> 29
<170> PatentIn version 3.5
<210> 1
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 1
Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu
1 5 10
<210> 2
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 2
Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro Gly Leu
1 5 10
<210> 3
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 3
Val Gln Phe Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr
1 5 10
<210> 4
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 4
Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr
1 5 10
<210> 5
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 5
Ser Ala Ala Ile Gly Leu Ser Met Ala Gly Ser Ser Ala Met
1 5 10
<210> 6
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 6
Pro Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val
1 5 10
<210> 7
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 7
Tyr Gln Ser Ala Ile Pro Pro Arg Gly Thr Gln Ala Val Val
1 5 10
<210> 8
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 8
Ile Gln Met Ser Asp Pro Ala Tyr Asn Ile Asn Ile Ser Leu
1 5 10
<210> 9
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 9
Lys Ser Leu Glu Asn Tyr Ile Ala Gln Thr Arg Asp Lys Phe
1 5 10
<210> 10
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 10
Met Leu Val Thr Ala Val Val Leu Leu Cys Cys Ser Gly Val
1 5 10
<210> 11
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 11
Arg Lys Ala Phe Arg Arg Ala Ile Thr Arg Pro Thr His Phe
1 5 10
<210> 12
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 12
Met Val Asp Pro Gln Leu Asp Gly Pro Gln Leu Ala Ala Leu
1 5 10
<210> 13
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 13
Lys Leu Asp Ser Pro Ile Ile Ala Arg Ile Thr Asp Thr Val
1 5 10
<210> 14
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 14
Arg Leu Ala Ala Gln Thr Ala Leu Leu Glu Ser Glu Ala Leu
1 5 10
<210> 15
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 15
Ile Thr Ile Ala Val Asn Ala Asp Ser Met Ala Thr Trp Phe
1 5 10
<210> 16
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 16
Arg Glu Lys Pro Cys Arg Ala Thr Thr Ala Gly Ile Pro Leu
1 5 10
<210> 17
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 17
Leu Leu Phe Pro Ala Ile Tyr Leu Ala Asp Ser Ala Gln Ala
1 5 10
<210> 18
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 18
Ala Val Leu Asp Leu Thr Thr Pro Gln Ala Arg Glu Ala Val
1 5 10
<210> 19
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 19
Lys Met Leu Glu Ala Ala Tyr Arg Leu His Thr Ile Asp Val
1 5 10
<210> 20
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 20
Thr Ala Tyr Glu Gln Arg Thr Arg Pro Gly Gln Leu Gln Leu
1 5 10
<210> 21
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 21
Gly Arg Trp Asn Pro Pro Leu Leu Phe Pro Ala Ile Tyr Leu
1 5 10
<210> 22
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 22
Arg Glu Leu Ala Tyr Ser Val Glu Thr Thr Ala Glu Ser Leu
1 5 10
<210> 23
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 23
Val Ser Trp Thr Arg Ser Ala Leu Ser Asp Leu Pro Arg Trp
1 5 10
<210> 24
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 24
Leu Ser Asp Leu Pro Arg Trp Arg Glu Pro Pro Gln Ile Tyr
1 5 10
<210> 25
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 25
Ser Leu Phe Phe Ala Ser Gly Gln Leu Arg Glu Leu Ala Tyr
1 5 10
<210> 26
<211> 14
<212> PRT
<213>It is artificial synthesized
<400> 26
Ala Leu Ser Asp Leu Pro Arg Trp Arg Glu Pro Pro Gln Ile
1 5 10
<210> 27
<211> 95
<212> PRT
<213> ESAT-6
<400> 27
Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser
1 5 10 15
Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly
20 25 30
Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser
35 40 45
Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu
50 55 60
Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly
65 70 75 80
Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala
85 90 95
<210> 28
<211> 100
<212> PRT
<213> CFP-10
<400> 28
Met Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu Ala Gly
1 5 10 15
Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp Gln Val
20 25 30
Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala Ala Gly
35 40 45
Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala Asn Lys
50 55 60
Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln Ala Gly
65 70 75 80
Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu Ser Ser
85 90 95
Gln Met Gly Phe
100
<210> 29
<211> 198
<212> PRT
<213> ESAT-6-CFP-10
<400> 29
Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser
1 5 10 15
Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly
20 25 30
Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser
35 40 45
Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu
50 55 60
Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly
65 70 75 80
Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Asn
85 90 95
Val Ala Met Ala Glu Met Lys Thr Asp Ala Ala Thr Leu Ala Gln Glu
100 105 110
Ala Gly Asn Phe Glu Arg Ile Ser Gly Asp Leu Lys Thr Gln Ile Asp
115 120 125
Gln Val Glu Ser Thr Ala Gly Ser Leu Gln Gly Gln Trp Arg Gly Ala
130 135 140
Ala Gly Thr Ala Ala Gln Ala Ala Val Val Arg Phe Gln Glu Ala Ala
145 150 155 160
Asn Lys Gln Lys Gln Glu Leu Asp Glu Ile Ser Thr Asn Ile Arg Gln
165 170 175
Ala Gly Val Gln Tyr Ser Arg Ala Asp Glu Glu Gln Gln Gln Ala Leu
180 185 190
Ser Ser Gln Met Gly Phe
195
Claims (10)
1. a kind of antigen polypeptide pond for detecting mycobacterium tuberculosis infection, it is characterised in that the antigen polypeptide pond includes containing ammonia
The combination of wantonly eight polypeptide or wantonly more than eight polypeptide of the base acid sequence as shown in SEQ ID NO.1~SEQ ID NO.26.
2. antigen polypeptide pond as claimed in claim 1, it is characterised in that the polypeptide is artificial synthesized or naturally isolated
's.
3. antigen polypeptide pond as claimed in claim 1, it is characterised in that the antigen polypeptide pond also includes ESAT-6 and CFP-
10。
4. antigen polypeptide pond as claimed in claim 3, it is characterised in that the amino acid sequence of the ESAT-6 such as SEQ ID
Shown in NO.27, the amino acid sequence of the CFP-10 is as shown in SEQ ID NO.28.
5. antigen polypeptide pond as claimed in claim 1, it is characterised in that the antigen polypeptide pond also includes ESAT-6-CFP-
10。
6. antigen polypeptide pond as claimed in claim 5, it is characterised in that the amino acid sequence of the ESAT-6-CFP-10 is such as
Shown in SEQ ID NO.29.
7. antigen polypeptide pond as claimed in claim 1, it is characterised in that the antigen polypeptide pond includes and the SEQ ID
Any shown sequence has the polypeptide of 85% homology in NO.1~SEQ ID NO.26.
8. a kind of kit for detecting mycobacterium tuberculosis infection, it is characterised in that the kit is included as in claim 1-7
Any described antigen polypeptide pond.
9. kit as claimed in claim 8, it is characterised in that the kit also includes the detection reagent of IFN-γ.
10. the application in the antigen polypeptide pond of the detection mycobacterium tuberculosis infection as described in claim 1-7 is any, its feature exists
In the IFN-γ produced using described antigen polypeptide pond as stimulant stimulation of whole, detection.
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Cited By (5)
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CN107144694A (en) * | 2017-03-29 | 2017-09-08 | 武汉海吉力生物科技有限公司 | Antigen, kit and application for detecting tuberculosis infection T cell |
CN107219362A (en) * | 2017-03-29 | 2017-09-29 | 武汉海吉力生物科技有限公司 | Antigen, kit and application for detecting tuberculosis infection T cell |
CN110423279A (en) * | 2019-06-20 | 2019-11-08 | 扩增生物科技(北京)有限公司 | Mycobacterium tuberculosis recombinant fusion protein EECC and its preparation method and application |
CN111518165A (en) * | 2020-05-08 | 2020-08-11 | 宁夏大学 | Polypeptide specifically binding to mycobacterium tuberculosis, coding gene and application thereof |
WO2023078438A1 (en) * | 2021-11-08 | 2023-05-11 | 成都可恩生物科技有限公司 | Mycobacterium tuberculosis fusion protein, preparation method therefor and use thereof |
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CN103804499A (en) * | 2014-01-22 | 2014-05-21 | 上海交通大学医学院 | Reagent for detecting specific immune response of mycobacterium tuberculosis and use thereof |
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CN107144694A (en) * | 2017-03-29 | 2017-09-08 | 武汉海吉力生物科技有限公司 | Antigen, kit and application for detecting tuberculosis infection T cell |
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CN107219362B (en) * | 2017-03-29 | 2019-08-09 | 武汉海吉力生物科技有限公司 | For detecting the antigen, kit and application of tuberculosis infection T cell |
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CN110423279B (en) * | 2019-06-20 | 2021-07-27 | 扩增生物科技(北京)有限公司 | Mycobacterium tuberculosis recombinant fusion protein EECC and preparation method and application thereof |
CN111518165A (en) * | 2020-05-08 | 2020-08-11 | 宁夏大学 | Polypeptide specifically binding to mycobacterium tuberculosis, coding gene and application thereof |
CN111518165B (en) * | 2020-05-08 | 2021-10-01 | 宁夏大学 | Polypeptide specifically binding to mycobacterium tuberculosis, coding gene and application thereof |
WO2023078438A1 (en) * | 2021-11-08 | 2023-05-11 | 成都可恩生物科技有限公司 | Mycobacterium tuberculosis fusion protein, preparation method therefor and use thereof |
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