CN107216373B - Antigen polypeptide pool for detecting mycobacterium tuberculosis infection and application thereof - Google Patents
Antigen polypeptide pool for detecting mycobacterium tuberculosis infection and application thereof Download PDFInfo
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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
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- 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 an antigen polypeptide pool for detecting mycobacterium tuberculosis infection, which specifically stimulates fresh whole blood infected by mycobacterium tuberculosis, can specifically secrete IFN-gamma, and increases detection sensitivity. Experiments prove that the reagent can directly utilize peripheral blood to stimulate antigens without separating peripheral blood mononuclear cells, and experimental data shows that the reagent has higher sensitivity and specificity when used for detecting the mycobacterium tuberculosis infection, is simple and convenient to operate, has lower cost and has higher clinical application value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antigen polypeptide pool for detecting mycobacterium tuberculosis infection and application thereof.
Background
Tuberculosis is one of the important infectious diseases which are mainly transmitted through respiratory tracts and seriously harm China and the whole world. According to the world health organization, 17-20 million people infected with Mycobacterium tuberculosis all over the world die of the disease every year, at least 200 million people die (XieJianping, methodology for functional genomics research of Mycobacterium tuberculosis, microbiological bulletin 2001.28(5): 92-97). Therefore, a method of accurately screening an infected person plays an extremely important role in the prevention and treatment of tuberculosis, and even the eventual eradication.
The existing diagnosis technology is very difficult to diagnose the mycobacterium tuberculosis infected person. In recent decades, the tuberculin test (TST) has been used clinically to diagnose patients with mycobacterium tuberculosis infections. However, the TST active ingredient is a Pure Protein Derivative (PPD), which is a crude extract of M.tuberculosis culture supernatant, contains more than 200 components, and has cross reaction with Bacillus Calmette-Guerin (BCG) inoculation and environmental Mycobacterium infection. The TST detection results can be interfered by the vaccine BCG vaccine immunization widely used in clinic [ P.Andersen, et al.Lancet356(2000) 1099-. Since 1921 and Ming BCG, 35 hundred million people have been inoculated with BCG worldwide. Therefore, the diagnostic value of TST on the infected person is greatly influenced, so that the accuracy of TST diagnosis is low, and therefore, whether the Mycobacterium tuberculosis infection really exists cannot be judged according to the result.
In the diagnosis technology of tuberculosis patients, acid-fast staining of sputum smears or sputum bacteria culture are difficult to culture and long in time consumption; pulmonary x-ray examination of pathological changes in the lungs, also only diagnosed after typical clinical symptoms; other serological and molecular diagnostic methods lack specificity and have a high false positive rate.
The researchers involved point out that the sensitivity and specificity of different diagnostic tests are as follows: mycobacterial culture (73% and 100%, respectively); PCR (42% and 100%, respectively); chest X-ray (67% -77% and 66% -76% respectively); tuberculin test (94% and 20%, respectively); serology (33% and 87%, respectively). It is clear that the sensitivity and specificity of the existing methods for detecting mycobacteria cannot be optimized simultaneously.
After the mycobacterium tuberculosis infects a human body, the mycobacterium tuberculosis is firstly recognized by the immune system of the human body, T cell immune response of the human body is activated, and gamma interferon (IFN-gamma) is secreted and generated, and the gamma interferon plays a role in resisting tuberculosis infection. Part of T cells are converted into immune memory cells, and IFN-gamma is secreted again after meeting the mycobacterium tuberculosis again, so that the anti-tuberculosis infection effect is achieved. The specific stimulation of the antigen to produce IFN-gamma and the production quantity thereof are closely related to whether the organism is infected or diseased. Therefore, if T cells of infected persons and patients are stimulated in vitro with antigen specific to Mycobacterium tuberculosis, these T cells can be induced to produce high levels of IFN-gamma specific to Mycobacterium tuberculosis, while non-Mycobacterium tuberculosis infected persons or non-tuberculosis patients produce IFN-gamma in an amount similar to that produced by non-antigen stimulation, thereby achieving the purpose of diagnosis. Therefore, the finding and discovery of the specific antigen of the mycobacterium tuberculosis have important value and significance for developing a new generation of diagnostic reagent.
In 1996, Stover et al determined the domain of BCG loss during in vitro passaging by subtractive hybridization as a deletion Region (RD) present in M.tuberculosis and absent in BCG and most environmental Mycobacteria [ ChengYu, Limao. 23(6): 342-343.]. Among them, the early differentiating antigen target 6KD (ESAT-6) and culture filtrate protein 10KD (c mu Ltufiltrateprotein 10KD, CFP-10) coded by the RD1 region are two small molecular weight secretory proteins which are one of the most important antigens of T cells and can induce stronger cellular immune response.
For the samples with children or low immunity, the literature reports that only ESAT-6 and CFP-10 or the polypeptide thereof is used as the stimulus, and the sensitivity is about 73.5-88.9% (Sun Lin, the application of ELISPOT detection technology in the diagnosis of children tuberculosis, labeled immunoassay and clinic 2008.6: 349-. For some special samples, such as children, low immunity and newly infected samples, the detection of the Mycobacterium tuberculosis sample by the ESAT-6 polypeptide and CFP-10 polypeptide and derivative kit is still not high, false negative occurs, and the clinical requirement cannot be met.
Disclosure of Invention
Aiming at the defects of the detection rate of the existing detection method for some special samples, such as children, low immunity and recently infected mycobacterium tuberculosis samples, the invention aims to provide the antigen polypeptide pool for detecting mycobacterium tuberculosis infection, the specific antigen polypeptide pool specifically stimulates fresh whole blood infected by mycobacterium tuberculosis, can specifically secrete IFN-gamma, and increases the detection sensitivity.
An antigen polypeptide pool for detecting mycobacterium tuberculosis infection comprises any eight polypeptides or the combination of any eight or more polypeptides, wherein the amino acid sequence of the eight polypeptides is shown in SEQ ID NO. 1-SEQ ID NO. 26.
The pool of antigenic polypeptides as described above, preferably said polypeptides are artificially synthesized or naturally isolated.
The antigen polypeptide pool as described above, preferably, the antigen polypeptide pool further comprises ESAT-6 and CFP-10.
Preferably, the amino acid sequence of the ESAT-6 is shown as SEQ ID NO.27, and the amino acid sequence of the CFP-10 is shown as SEQ ID NO. 28.
The antigen polypeptide pool as described above, preferably, the antigen polypeptide pool further comprises ESAT-6-CFP-10.
Preferably, the amino acid sequence of the ESAT-6-CFP-10 is shown in SEQ ID NO. 29.
The antigen polypeptide pool as described above, preferably, the antigen polypeptide pool comprises polypeptides having 85% homology with any one of the sequences shown in SEQ ID NO. 1-SEQ ID NO. 26.
A kit for detecting mycobacterium tuberculosis infection, the kit comprising a pool of antigenic polypeptides as described above.
A kit for detecting Mycobacterium tuberculosis infection, preferably, the kit further comprises IFN-gamma detection reagent.
The application of the antigen polypeptide pool for detecting the mycobacterium tuberculosis infection adopts the antigen polypeptide pool as a stimulant to stimulate whole blood and detect the generated IFN-gamma.
The antigen polypeptide pool provided by the invention and IFN-gamma specific antigen proteins (ESAT6-CFP10, ESAT6 and CFP10) are used for detecting a whole blood sample infected by mycobacterium tuberculosis, and the antigen polypeptide pool has the following characteristics:
(1) the positive detection rate of the sample is high under the condition of low immunity, and the sensitivity is high. Aiming at the defects of the existing detection method for detecting special samples, such as children, low immunity and newly infected samples or latent infected patients, the invention provides the antigen polypeptide pool for the mycobacterium tuberculosis infection with high sensitivity and specificity, the polypeptide pool combines ESAT-6 and CFP-10 to realize the rapid and specific detection of patients with tuberculosis or early infected patients or latent infected patients (tuberculosis symptoms do not appear), and the antigen polypeptide pool has great significance in blocking the spread and prevalence of tuberculosis and controlling the tuberculosis.
(2) The detection time is short, the operation is convenient, and the diagnosis is rapid. Compared with the traditional detection of mycobacterium tuberculosis, the detection of the specific antigen protein combining IFN-gamma by utilizing the polypeptide pool provided by the invention has short time and quick diagnosis. The traditional mycobacterium tuberculosis culture diagnosis needs 1-2 months, TST detection needs 3 days, and the whole detection time can be finished within 1-2 days.
(3) The early detection of the infection of the mycobacterium tuberculosis can be carried out. Once the mycobacterium tuberculosis infects a human body, the mycobacterium tuberculosis is firstly identified by the immune system of the body, the humoral and cellular immune response is activated, and the attack time is 1-2 months after infection, so that the cell immunology detection method for combining the polypeptide pool with the specific antigen protein of the whole blood IFN-gamma can be used for quickly diagnosing whether a sample is infected with the mycobacterium tuberculosis. In the prior art, the X-ray diagnosis can be made only after the lung of the infected person has been significantly changed, which usually takes a long time. Antigen-antibody detection is also only detected when the disease symptoms are in active phase, but mycobacteria are widely present in the environment, and the common antigen of the mycobacteria and the mycobacterium tuberculosis can interfere with the diagnosis result of the experiment. Therefore, the invention has important significance for the early detection of the recent mycobacterium tuberculosis infection or low immunity, and has positive significance for the control and elimination of tuberculosis.
The invention also provides a novel detection reagent for detecting the mycobacterium tuberculosis infection, and experiments prove that the invention can directly utilize peripheral blood to stimulate antigens without separating peripheral blood mononuclear cells, and experimental data shows that the reagent has higher sensitivity and specificity for detecting the mycobacterium tuberculosis infection, effectively avoids false negative, has simple and convenient operation and lower cost and has higher clinical application value.
Detailed Description
In order to achieve the purpose of the invention, the inventor predicts polypeptides according to computer software, comprehensively selects suitable polypeptides, and finally obtains polypeptides with amino acid sequences shown as SEQ ID NO. 1-SEQ ID NO.26 through a large amount of experimental research verification, wherein the screening research of the polypeptides is determined by the following technical means:
1. single polypeptide stimulation effectiveness screening: preparing the preliminarily screened polypeptide into a polypeptide solution with a certain concentration, stimulating fresh whole blood, detecting whether the whole blood is infected with mycobacterium tuberculosis by using a gamma interferon detection kit, and selecting the polypeptide which has a stimulation effect on a positive sample and has no non-specific stimulation on a negative sample.
2. Polypeptide combination stimulation effect: randomly 8-20 effective polypeptides are screened to prepare a mixed polypeptide solution, fresh whole blood is stimulated, incubation is carried out for 20 +/-4 hours at 37 ℃, supernatant is collected centrifugally, the supernatant is added into a human IFN-gamma detection kit (enzyme linked immunosorbent assay) for detection, mixed polypeptides which have stimulation effects on positive samples and have no non-specific stimulation on negative samples are selected, and an effective mixed polypeptide sequence can be determined.
3. Combined ESAT-6 and CFP-10 stimulatory effects: combining the screened effective mixed polypeptide solution with ESAT-6 and CFP-10 to prepare an irritant solution, stimulating fresh whole blood, incubating for 20 +/-4 hours at 37 ℃, centrifugally collecting supernatant, adding the supernatant into a human IFN-gamma detection kit (enzyme linked immunosorbent assay) for detection, selecting mixed polypeptide which has a stimulation effect on a positive sample and has no non-specific stimulation on a negative sample, and determining a mixed polypeptide sequence which can be effectively combined.
4. Combined ESAT-6-CFP-10 stimulation effects: combining the screened effective mixed polypeptide solution with ESAT-6-CFP-10 to prepare an irritant solution, stimulating fresh whole blood, incubating for 20 +/-4 hours at 37 ℃, centrifugally collecting supernatant, adding the supernatant into a human IFN-gamma detection kit (enzyme linked immunosorbent assay) for detection, selecting mixed polypeptide which has a stimulation effect on a positive sample and has no non-specific stimulation on a negative sample, and determining a mixed polypeptide sequence which can be effectively combined.
The polypeptide screened by the invention can specifically stimulate the secretion of IFN-gamma of the fresh whole blood specificity of a patient infected by mycobacterium tuberculosis through verification, and specifically is a specific protein of pathogenic mycobacterium tuberculosis: 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), and the screened proteins can be used for aiming at the polypeptides of the binding site of CD8+ lymphocytes, through repeated clinical verification experiments, the polypeptides are combined with ESAT-6 and CFP-10 to improve the detection sensitivity, the combined stimulant can specifically act on lymphocyte epitopes in whole blood of a mycobacterium tuberculosis infection sample, and the cytokine IFN-gamma released in the whole blood is detected, so that whether the organism is infected with the mycobacterium tuberculosis or not is indirectly reflected. The method improves the detection rate of the mycobacterium tuberculosis infection sample, particularly the detection sensitivity of a newly infected sample, children and a mycobacterium tuberculosis infection sample under low immunity, and effectively avoids the problems of missed detection and wrong detection which are easy to occur in the prior detection technology.
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof. The experimental methods in the following examples, in which specific conditions are not specified, were according to the conventional experimental methods; the reagents used are commercially available products unless otherwise specified.
Example 1 Single polypeptide stimulation effectiveness screening
The polypeptide sequence of the present invention has the amino acid sequence shown in SEQ ID NO. 1-SEQ ID NO.26, which may be artificially synthesized or naturally isolated, and the polypeptides used in this and the following examples are artificially 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 prepared polypeptide was dissolved in sterile low endotoxin (endotoxin should be less than 2.5 EU/. mu.g) PBS to a concentration of 250. mu.g/mL.
A commercially available gamma interferon detection kit (Jili biotechnology, Wuhan Hai) was used to detect whether the patients were infected with Mycobacterium tuberculosis by taking fresh blood samples from 10 clinically confirmed tuberculosis patients and fresh blood samples from 10 healthy patients without tuberculosis symptoms (the same below). And meanwhile, taking 20 mu L of polypeptide solution to be placed in a sterile culture tube (the final concentration of the polypeptide is 100 mu g/mL), adding 1mL of fresh collected whole blood into each tube, uniformly mixing, then placing in an oven at 37 ℃ for 20 +/-4 hours, mixing the blood, centrifuging at 6000rpm for 1min, and taking the supernatant to detect the content of the gamma interferon.
The result shows that the polypeptide shown in SEQ ID NO. 1-SEQ ID NO.26 designed by the invention has stimulation effect on positive samples and has no non-specific stimulation on negative samples. It should be noted that the polypeptide having 85% homology with any one of the polypeptides of SEQ ID NO.1 to SEQ ID NO.26 can also achieve the above object.
Example 2 Combined stimulation Effect
Combining any 8 polypeptide solutions with ESAT-6 and CFP-10 to prepare a stimulant solution, wherein the ESAT-6 and the CFP-10 are obtained by recombinant expression of escherichia coli genetic engineering bacteria, and the sequence of the ESAT-6 is SEQ ID NO. 27: MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA, respectively; CFP-10 sequence is SEQ ID NO. 28: MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF are provided. In this example, the experimental results of only some of the polypeptides are shown as numbers of stimulator 1 to stimulator 4 (stimulator 1: SEQ ID NO.1 to SEQ ID NO.8+ ESAT-6+ CFP-10; stimulator 2: SEQ ID NO.6 to SEQ ID NO.13+ ESAT-6+ CFP-10; stimulator 3: SEQ ID NO.11 to SEQ ID NO.18+ ESAT-6+ CFP-10; stimulator 4: SEQ ID NO.19 to SEQ ID NO.26+ ESAT-6+ CFP-10), and positive and non-infected negative samples of Mycobacterium tuberculosis are taken for testing.
The whole blood samples of 20 cases in example 1 were sampled and tested for infection with Mycobacterium tuberculosis using the interferon gamma assay kit. Meanwhile, 20 mu L of each of 1 to 4 stimulants is taken and put into a sterile culture tube (the final concentration of each polypeptide is 100 mu g/mL), 1mL of fresh collected whole blood is added into each tube, the whole blood is uniformly mixed and put into an oven at 37 ℃ for 20 +/-4 hours, the blood is mixed and centrifuged at 6000rpm for 1min, the supernatant is detected by a gamma interferon detection kit, the detection method and the reagents are the same as in example 1, the detection result is shown in Table 1, wherein the commercially available kit is a mycobacterium tuberculosis specific cell immunoreaction detection kit (enzyme linked immunosorbent assay) of the GmbH, the same as below.
TABLE 1 Mixed polypeptide Combined ESAT-6 and CFP-10 stimulation results statistics
| Commercial kit | Irritant 1 | Irritant 2 | Irritant 3 | Irritant 4 | |
| Number of cases of 10 positive samples | 8 | 9 | 9 | 10 | 9 |
| Number of negative 10 specimens detected | 10 | 10 | 10 | 10 | 10 |
The result shows that the polypeptide pool prepared by the invention is combined with ESAT-6 and CFP-10 to be used for detecting the whole blood infected by the mycobacterium tuberculosis, the detection sensitivity is improved, and the false negative and the omission of the detection in the prior art are effectively avoided.
Example 3 Combined stimulation Effect
Combining any 8 polypeptide solutions with ESAT-6-CFP-10 to prepare a stimulant solution, wherein the ESAT-6-CFP-10 is obtained by recombinant expression of escherichia coli genetic engineering bacteria, and the sequence of the ESAT-6-CFP-10 is SEQ ID NO. 29: MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFANVAMAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF, numbered as stimulator 5-stimulator 8 (stimulator 5: SEQ ID NO. 1-SEQ ID NO.8+ ESAT-6-CFP-10; stimulator 6: SEQ ID NO. 6-SEQ ID NO.13+ ESAT-6-CFP-10; stimulator 7: SEQ ID NO. 11-SEQ ID NO.18+ ESAT-6-CFP-10; stimulator 8: SEQ ID NO. 19-SEQ ID NO.26+ ESAT-6-CFP-10), taking positive and negative samples of Mycobacterium tuberculosis, testing to prepare a stimulator solution, taking 20 samples of the same example as that in example 1, and using a gamma interferon detection kit to detect whether the positive and negative samples of Mycobacterium tuberculosis are infected with Mycobacterium tuberculosis. Meanwhile, 20 mu L of each of 5 to 8 stimulants are taken and put into a sterile culture tube (the final concentration of each polypeptide is 100 mu g/mL), 1mL of fresh collected whole blood is added into each tube, the whole blood is uniformly mixed and put into an oven at 37 ℃ for 20 +/-4 hours, the mixture is centrifuged at 6000rpm for 1min after the blood is mixed, and the supernatant is taken and detected by a gamma interferon detection kit. The detection method is the same as that of example 2, and the detection results are shown in Table 2.
TABLE 2 Mixed polypeptide-Combined ESAT-6-CFP-10 stimulation results
| Commercial kit | Irritant 5 | Irritant 6 | Irritant 7 | Irritant 8 | |
| Number of cases of 10 positive samples | 8 | 9 | 9 | 10 | 10 |
| Number of negative 10 specimens detected | 10 | 10 | 10 | 10 | 10 |
The result shows that the polypeptide pool prepared by the invention is combined with ESAT-6-CFP-10 to be used for detecting the whole blood infected by the mycobacterium tuberculosis, the detection sensitivity is improved, and the false negative and the omission of the detection in the prior art are effectively avoided.
Example 4 combining Mixed polypeptide detection sensitivity and specificity
Sample preparation: fresh blood samples of 20 clinically confirmed tuberculosis patients and fresh blood samples of 20 healthy tuberculosis-free patients are taken.
Collecting whole blood sample of the above samples, and separately using 20. mu.L each of stimulants 9(ESAT-6 (10. mu.g/mL) and CFP-10 (10. mu.g/mL) + mixed polypeptide (SEQ ID NO.6 to SEQ ID NO.13, each 100. mu.g/mL)), stimulants 10(ESAT-6-CFP-10 (10. mu.g/mL) + mixed polypeptide (SEQ ID NO.6 to SEQ ID NO.13, each 100. mu.g/mL)), stimulants 11(ESAT-6 (10. mu.g/mL) and CFP-10 (10. mu.g/mL)), stimulants 12(ESAT-6-CFP-10 (10. mu.g/mL)) and stimulants 13 (commercial kits: Mycobacterium tuberculosis-specific cellular immune response detection kits (enzyme linked immunosorbent assay) of Tsugawa Jili Biotech Co., Ltd.) in a sterile culture tube, adding 1mL of fresh collected whole blood into each tube for stimulation, uniformly mixing, placing in an oven at 37 ℃ for 20 +/-4 hours, mixing the blood, centrifuging at 6000rpm for 1min, and taking the supernatant to detect by using a gamma interferon detection kit. The results are shown in Table 3.
TABLE 3 statistics of pooled mixed polypeptide sensitivity and specificity detection results
The result shows that the polypeptide pool prepared by the invention is combined with ESAT-6 and CFP-10 or ESAT-6-CFP-10 to be used for detecting the whole blood infected by the mycobacterium tuberculosis, the detection sensitivity can reach 95 percent, the sensitivity and the specificity are stronger, and the false negative and the omission of the detection in the prior art are effectively avoided.
Example 5 sensitive and specific detection of the invention
Sample preparation: taking 100 samples of fresh blood of people with clinically confirmed tuberculosis symptoms, wherein 21 samples are taken at age 65 and above, 18 samples are taken at age below 6, 51 HIV samples are taken at age 56 for men, and 44 women; fresh blood samples from 50 healthy persons without tuberculosis symptoms, 20 samples at age 65 and above, 7 samples at age 6 or below, 23 HIV samples, 26 men, and 24 women.
Collecting whole blood samples of the samples, respectively using 20 mu L of stimulants (ESAT-6(10 mu g/mL) and CFP-10(10 mu g/mL) + mixed polypeptides (SEQ ID NO. 11-SEQ ID NO.18, each 100 mu g/mL)) and commercially available kits (Mycobacterium tuberculosis specific cellular immunoreaction detection kit (enzyme linked immunosorbent assay) of GmbH, Wuhanhai Jili Biotech, Inc.) to stimulate each tube, adding 1mL of freshly collected whole blood into each tube, uniformly mixing, drying in an oven at 37 ℃ for 20 +/-4 hours, mixing the blood, centrifuging at 6000rpm for 1min, taking supernatant, detecting by using a commercially available gamma interferon detection kit, and operating according to the instruction of the commercially available kits. The results are shown in tables 4 and 5.
TABLE 4 statistics of ELISA detection results of M.tuberculosis infection samples
TABLE 5 statistics of ELISA assay results for healthy samples
The result shows that the polypeptide pool prepared by the invention can improve the detection rate of special samples such as children, hypoimmunity and newly infected samples by combining ESAT-6, CFP-10 and derivatives thereof, has stronger sensitivity and specificity, and effectively avoids false negative and missed detection of the prior art detection. The invention has important significance for early detection of the recent mycobacterium tuberculosis infection or low immunity, and has positive significance for controlling and eliminating tuberculosis.
SEQUENCE LISTING
<110> Wuhan sea Jili Biotech limited
<120> antigen polypeptide pool for detecting mycobacterium tuberculosis infection and application thereof
<130>
<160>29
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<210>19
<211>14
<212>PRT
<213> Artificial Synthesis
<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> Artificial Synthesis
<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> Artificial Synthesis
<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> Artificial Synthesis
<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> Artificial Synthesis
<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> Artificial Synthesis
<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> Artificial Synthesis
<400>25
Ser Leu Phe PheAla Ser Gly Gln Leu Arg Glu Leu Ala Tyr
1 5 10
<210>26
<211>14
<212>PRT
<213> Artificial Synthesis
<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 (3)
1. An antigen polypeptide pool for detecting mycobacterium tuberculosis infection, which is characterized in that the antigen polypeptide pool comprises any one of the following eight cases:
(1) polypeptide with amino acid sequence shown as SEQ ID NO. 1-SEQ ID NO.8, ESAT-6 and CFP-10;
(2) polypeptide with amino acid sequence shown as SEQ ID NO. 6-SEQ ID NO.13, ESAT-6 and CFP-10;
(3) polypeptide with amino acid sequence shown as SEQ ID NO. 11-SEQ ID NO.18, ESAT-6 and CFP-10;
(4) polypeptide with amino acid sequence shown as SEQ ID NO. 19-SEQ ID NO.26, ESAT-6 and CFP-10;
(5) polypeptide with amino acid sequence shown as SEQ ID NO. 1-SEQ ID NO.8 and ESAT-6-CFP-10;
(6) polypeptide with amino acid sequence shown as SEQ ID NO. 6-SEQ ID NO.13 and ESAT-6-CFP-10;
(7) polypeptide with amino acid sequence shown as SEQ ID NO. 11-SEQ ID NO.18 and ESAT-6-CFP-10;
(8) polypeptide with amino acid sequence shown as SEQ ID NO. 19-SEQ ID NO.26 and ESAT-6-CFP-10;
in the above case, the amino acid sequence of ESAT-6 is shown as SEQ ID NO.27, the amino acid sequence of CFP-10 is shown as SEQ ID NO.28, and the amino acid sequence of ESAT-6-CFP-10 is shown as SEQ ID NO. 29.
2. A kit for detecting mycobacterium tuberculosis infection, the kit comprising a pool of antigenic polypeptides according to claim 1.
3. The kit of claim 2, further comprising an IFN- γ detection reagent.
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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 |
| CN107144694B (en) * | 2017-03-29 | 2019-06-14 | 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 |
| 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 |
| CN111518165B (en) * | 2020-05-08 | 2021-10-01 | 宁夏大学 | Polypeptide specifically binding to mycobacterium tuberculosis, coding gene and application thereof |
| CN113980145B (en) * | 2021-11-08 | 2022-08-12 | 成都可恩生物科技有限公司 | Mycobacterium tuberculosis fusion protein and preparation method and application thereof |
Citations (4)
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|---|---|---|---|---|
| CN102004155A (en) * | 2010-02-12 | 2011-04-06 | 复旦大学附属华山医院 | Kit and method for detecting mycobacterium tuberculosis infection and application |
| CN102175875A (en) * | 2011-01-27 | 2011-09-07 | 武汉海吉力生物科技有限公司 | Detection kit for diagnosing tuberculosis |
| 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 |
| CN105954521A (en) * | 2016-07-08 | 2016-09-21 | 广州华弘生物科技有限公司 | MTB (Mycobacterium Tuberculosis) infection diagnosis kit |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102004155A (en) * | 2010-02-12 | 2011-04-06 | 复旦大学附属华山医院 | Kit and method for detecting mycobacterium tuberculosis infection and application |
| CN102175875A (en) * | 2011-01-27 | 2011-09-07 | 武汉海吉力生物科技有限公司 | Detection kit for diagnosing tuberculosis |
| 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 |
| CN105954521A (en) * | 2016-07-08 | 2016-09-21 | 广州华弘生物科技有限公司 | MTB (Mycobacterium Tuberculosis) infection diagnosis kit |
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