CN102174114A - Whole blood INF-gamma specific antigen protein and preparation method and application thereof - Google Patents
Whole blood INF-gamma specific antigen protein and preparation method and application thereof Download PDFInfo
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- CN102174114A CN102174114A CN2011100310790A CN201110031079A CN102174114A CN 102174114 A CN102174114 A CN 102174114A CN 2011100310790 A CN2011100310790 A CN 2011100310790A CN 201110031079 A CN201110031079 A CN 201110031079A CN 102174114 A CN102174114 A CN 102174114A
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Abstract
The invention relates to a whole blood interferon-gamma (INF-gamma) specific antigen protein prepared by a gene engineering method and a preparation method and application thereof, a nucleotide sequence for coding the protein, a recombinant vector and a recon. The antigen protein is a fusion protein obtained by connecting early secreting antigen target 6 (ESAT-6), cultufiltrate protein 10 (CFP-10) and MPB64 in any order through a connecting peptide, wherein the connecting peptide is a short peptide which does not influence the immunogenicity of the ESAT-6, the CFP-10 and the MPB64. The tuberculosis specific antigen quickly diagnoses tuberculosis, has high sensitivity and specificity, and has great significance for the early diagnosis of the tuberculosis.
Description
Technical field
The present invention relates to specific antigen protein, the Preparation Method And The Use of a kind of whole blood INF-γ by gene engineering method preparation, and relate to this proteic nucleotide sequence of coding, recombinant vectors and recon.
Background technology
Tuberculosis is main through one of respiratory infectious, serious harm China and global important transmissible disease.According to the World Health Organization, there is the mycobacterium tuberculosis infection person of 17-20 hundred million in the whole world, and the people died from this disease and (thanked to the Jianping, the methodology of mycobacterium tuberculosis functional genome research, microorganism circular .2001.28 (5): 92-97) every year at least 200 ten thousand.Therefore, accurately screening the infected's method is played the part of important role in control lungy even final elimination.
Existing diagnostic techniques is very difficult to mycobacterium tuberculosis infection person's diagnosis.In recent decades, adopt tuberculin test (TST) to come diagnosing tubercle bacillus the infected clinically.But the TST active ingredient is purified protein derivative (purified protein derivative of tuberculin, PPD), be the crude extract of mycobacterium tuberculosis culture supernatant, contain more than 200 kind of composition, have cross reaction with BCG inoculation and environment mycobacterial infections.The TST detected result can be disturbed [P.Andersen, et al.Lancet356 (2000) 1099-1104.] by clinical widely used vaccine-bacille Calmette-Guerin vaccine immunity now.From nineteen twenty-one and bright bacille Calmette-Guerin vaccine till now, Quan Shan circle has 3,500,000,000 people's bcg vaccinations.Therefore TST is subjected to very big influence to the infected's diagnostic value, cause the accuracy of TST diagnosis lower, so we can't judge whether really to exist m tuberculosis infection according to its result.
Phlegm smear acid-fast stain in tuberculosis patient's the diagnostic techniques or phlegm bacterium are cultivated, cultivate difficulty, consuming time longer; X X-ray test X lung of lung pathological change also only could be diagnosed after having typical clinical symptom; Other serological diagnostic methods and molecular diagnosis method lack specificity, and false positive rate is higher.Relevant researchist points out that the susceptibility and the specificity of different diagnostic test methods are as follows: mycobacterium is cultivated (being respectively 73% and 100%); PCR (being respectively 42% and 100%); Chest x-ray (being respectively 67%～77% and 66%～76%); Tuberculin test (being respectively 94% and 20%); Serology (being respectively 33% and 87%).This shows that it is optimum that the susceptibility of existing diagnostic test method and specificity can't reach simultaneously.Therefore, must study tuberculosis and mycobacterium tuberculosis infection person's early stage, quick, special and responsive diagnostic method as early as possible.
Behind the m tuberculosis infection human body, at first can be activated the T cellullar immunologic response of body by the immune system recognition of human body, secretion produces IFN-γ, and the latter brings into play the resisting tuberculosis infection effect.Part T cell transformation is an immunological memory cell, and after meeting with tubercule bacillus once more, secretion produces IFN-γ once more, the effect of performance resisting tuberculosis infection.Whether the IFN-γ generation of antigen-specific and body infect or fall ill closely related.Therefore, if the antigen that adopts tubercle bacillus differential is at stimulated in vitro the infected and patient's T cell, can induce these T cells to produce the IFN-γ of high-caliber tuberculosis bar Tong antigen-specific, but not the amount of the IFN-γ that mycobacterium tuberculosis infection person or non-tuberculosis patient produce is close with the amount that does not produce with antigenic stimulation, thereby realizes the purpose of diagnosis.This shows, seek and find tubercle bacillus specific antigen, the diagnostic reagent that develops a new generation is had important value and meaning.
1996, Stover etc. have determined the territory that BCG loses by the method for subtrahend hybridization in the subculture in vitro separately process, be defined as disappearance district (Region of Difference, RD), runt domain is present in the mycobacterium tuberculosis, and in BCG and most of environment mycobacterium, lack [Cheng Yu, Li Maoquan. the laboratory diagnosis progress of mycobacterium tuberculosis. foreign medical science clinical biochemistry and ecsomatics fascicle .2002; 23 (6): 342-343.].Wherein, early stage minute antigen target 6KD of RD1 regional code (early secreting antigen target 6KD, ESAT-6) and culturing filtrate protein 10 KD (cultufiltrate protein 10KD, CFP-10) be two kinds of small molecular weight secretory proteins, they are one of topmost antigen of T cell, can induce stronger cell immune response.In addition, MPB64 albumen is the protein of mycobacterium tuberculosis excretory 24KDa molecular weight in growth and breeding, is a secretory protein that special role is arranged, and can cause the intensive delayed type hypersensitivity.MPB64 albumen mainly is present in the mycobacterium tuberculosis composite flora, the non-tuberculous mycobacteria only a few occurs positive, M.flavescens is a weak positive [happy army, Liang Li, Li Suhui, Deng. the clinical value [J] of enzyme linked immunological spot test quick diagnosis m tuberculosis infection. Chinese laboratory medicine magazine, 2006,29 (11): 1005-1008.].Yet because the proteinic molecular weight of these tuberculosis specific diagnostics is less, directly separation and purification is comparatively difficult from the culture of tubercule bacillus, and production cost is very high; Although can adopt peptide synthetic method to produce, peptide synthetic cost is very high, and the cost of the clinical application test kit of Jian Liing increases greatly thus.In addition, by the above-mentioned antigenic epi-position of the independent acquisition of genetic engineering technique, because external and intravital difference, expressed products also easily because formation forgive not loses activity of proteins as reporting both at home and abroad.
Summary of the invention
Deficiency at existing diagnostic method, one of purpose of the present invention is to provide a kind of susceptibility and the high diagnosis of tuberculosis antigen protein of specificity, this antigen protein can be realized quick, the special diagnosis to tuberculosis patient or early infection person, blocking propagation lungy and popular, and control lungy is being significant.
In order to realize above-mentioned purpose of the present invention, the contriver finally obtains following technical scheme by a large amount of experimental studies:
The specific antigen protein of a kind of whole blood INF-γ, it is following any fusion rotein that is connected with biological label: ESAT-6-connection peptides-CFP-10, CFP-10-connection peptides-ESAT-6, ESAT-6-connection peptides-MPB64, MPB64-connection peptides-ESAT-6, CFP-10-connection peptides-MPB64, MPB64-connection peptides-CFP-10, ESAT-6-connection peptides-CFP-10-connection peptides-MPB64, ESAT-6-connection peptides-MPB64-connection peptides-CFP-10, CFP-10-connection peptides-ESAT-6-connection peptides-MPB64, CFP-10-connection peptides-MPB64-connection peptides-ESAT-6, MPB64-connection peptides-CFP-10-connection peptides-ESAT-6 and MPB64-connection peptides-ESAT-6-connection peptides-CFP-10, wherein said connection peptides is not for influencing ESAT-6, the immunogenic small peptide of CFP-10 and MPB64.
In order to further facilitate the scale operation of recombinant protein, add the biological label of some purifying on the basis of above-mentioned series protein matter, as biological labels commonly used such as histidine-tagged, GST label, Trx labels, these labels can be in proteinic N end and C end, the use of label can make things convenient for the scale operation of recombinant protein, can significantly reduce the test kit cost, reduce the clinical detection expense.
The specific antigen protein of above-mentioned whole blood INF-γ, the aminoacid sequence of wherein said connection peptides is SEQ ID:No.3.
The specific antigen protein of above-mentioned whole blood INF-γ, the preferred amino acid sequence is the antigen protein of SEQ ID:No.1.
Second purpose of the present invention has been to provide the nucleotide sequence of the specific antigen protein of coding whole blood INF-γ.It is the nucleotide sequence SEQ ID NO:2 of SEQ ID:No.1 that encoding amino acid sequence especially is provided.
The 3rd purpose of the present invention has been to provide the expression vector that contains above-mentioned polynucleotide, and wherein carrier is pET28b.
The 4th purpose of the present invention has been to provide the microorganism that contains above-mentioned expression vector, and wherein said microorganism is an e. coli bl21.
It is the preparation method of the antigen protein of SEQ ID:No.1 that the 5th purpose of the present invention has been to provide aminoacid sequence.
The 5th purpose of the present invention realizes by technique means once:
Aminoacid sequence is the preparation method of the antigen protein of SEQ ID:No.1, comprise the steps: that SEQ ID NO:2 sequence is stored among the DH5 α, with NcoI, XhoI restriction endonuclease respectively enzyme cut DH5 α and pET28b expression vector, reclaim SEQ ID NO:2 sequence fragment and pET28b expression vector, at T
4Connect under the dna ligase effect, be transformed in the e. coli bl21 (DE3), transformant is cultivated in liquid LB substratum, adding final concentration is the penbritin cultivation of 50 μ g/ml, adding final concentration is the IPTG of 0.5mM, 37 ℃, and 200rpm, abduction delivering 8 hours, with expressed recombinant protein collection, purifying promptly.
The composition that contains above-mentioned antigen protein provided by the invention can quick, specific diagnosis of tuberculosis.
The present invention has further studied whole blood INF-γ and has discharged analytical procedure, and the optimal stimulus time is 37 ℃, and 10 hours-30 hours, more excellent stimulation time was 16 hours-24 hours.
The specific antigen protein of whole blood INF-γ provided by the invention has following progressive;
(1) compare with traditional tubercule bacillus diagnosis, utilize the specific antigen protein diagnosis of whole blood INF-γ provided by the invention to take weak point, diagnosis fast.The tradition tubercule bacillus is cultivated diagnosis and takes time the 1-2 month, and TST detects needs 3 days, and the present invention can finish in 1-2 days whole detection times.
(2) can carry out early diagnosis to tubercule bacillus.Because in a single day tubercule bacillus infects human body, at first just discerned by the immunity system of body, activate body fluid and cellullar immunologic response, individual month of time of origin 1-2 after infection, therefore, utilize the cellular immunology detection method of the specific antigen protein of whole blood INF-γ provided by the invention can make diagnosis fast.The diagnosis of X-ray sheet only just can be made diagnosis after tangible pathological change appears in an infection name lung, this needs long time usually.Antigen-antibody only detects also under disease symptoms is in the situation of active period and detects, but mycobacterium extensively exists in the environment, the common antigen of itself and tubercule bacillus, but the diagnostic result of interference experiment.
(3) susceptibility is strong, specificity is high.Because what adopt is the antigen of tubercle bacillus specific, only with the mycobacterium tuberculosis infection human body after the immunological memory T cell that produces react, thereby the Interferon, rabbit that produces is the specific IFN-γ of negre antigen.Adopt antigen of the present invention to stimulate, the susceptibility of the whole blood IFN-gamma diagnosis of tuberculosis antigen specific is 100%, and specificity has good diagnostic accordance rate up to 95.7%.
(4) make things convenient for scale operation, significantly reduce the patient diagnosis expense.
Therefore, tuberculosis special antigen provided by the invention is significant to early diagnosis lungy, thereby control lungy and elimination are had positive meaning.
Description of drawings
Fig. 1: contain ESAT6-CFP10 Expression of Fusion Protein carrier pET28b structural representation
Fig. 2: the ESAT6-CFP10 fusion rotein efficiently expresses the electrophorogram with purifying SDS-PAGE in intestinal bacteria
1, protein molecular weight standard 11,17,26,34,43,55,72,95,130,170Da
2, induce preceding bacterium
3, induce the back bacterium
4, supernatant behind the induced ultrasonic
5, induced ultrasonic postprecipitation
Fig. 3: the ESAT6-CFP10 fusion rotein efficiently expresses the electrophorogram with purifying WB in intestinal bacteria
1, protein molecular weight standard 11,17,26,34,43,55,72,95,130,170Da
2, induce preceding bacterium
3, induce the back bacterium
4, supernatant behind the induced ultrasonic
5, induced ultrasonic postprecipitation
Embodiment
Below be specific embodiments of the invention, technical scheme of the present invention is done further the description, but protection scope of the present invention is not limited to these embodiment.Every do not deviate from the change of the present invention design or be equal to substitute include within protection scope of the present invention.
The structure of the synthetic and expression vector of the goal gene of embodiment 1ESAT6-CFP10 fusion rotein
The dna sequence dna of the ESAT6-CFP10 of synthetic is stored among the DH5 α, again with NcoI, XhoI restriction endonuclease respectively enzyme cut DH5 α and pET28b expression vector, reclaim purpose segment and expression vector, at T
4The dna ligase effect connects 16 hours for following 4 ℃, transforms in the e. coli bl21 (DE3) 37 ℃ of overnight incubation with the heat-shocked method.Picking in the flat board growth preferably, bacterium colony with typical intestinal bacteria feature is cultivated in liquid LB substratum, adding final concentration simultaneously in substratum is the kantlex of 30 μ g/ml, 37 ℃, 220rpm, cultivated 3 hours, and extracted plasmid and carry out enzyme evaluations of cutting and check order, enzyme figure and check order collection of illustrative plates and expection are consistent.Vector construction figure sees Fig. 1.
Picking in the flat board growth preferably, bacterium colony with typical intestinal bacteria feature is cultivated in liquid LB substratum, the adding final concentration is that the penbritin of 50 μ g/ml was cultivated 6 hours, adding final concentration is the IPTG of 0.5mM, 37 ℃, 200rpm, abduction delivering 8 hours, collect thalline, SDS-PAGE electrophoresis qualification result is seen Fig. 2.
The placed in-line purifying of embodiment 3 recombinant protein ESAT6-CFP10
Protein purification is undertaken by the Ni-NTA standard operating procedure, concise and to the point step is as follows, get the engineering bacteria of the centrifugal collection abduction delivering of 50ml bacterium liquid, with ultrasonic wave with the large intestine broken cell, the centrifuging and taking supernatant, supernatant is in Tris (50mM, pH8.0) dialysed overnight in the damping fluid, the centrifuging and taking supernatant directly is splined on and has used Tris (50mM, pH8.0) in the damping fluid equilibrated Ni-NTA resin, (50mM pH8.0) after the buffer solution elution balance, uses the imidazoles gradient elution of 0-1M again with Tris, collect elution peak, elution peak is merged, in Tris (50mM, pH8.0) dialysed overnight in the damping fluid, dialyzate freeze-drying in vacuum freeze drier is the ESAT6-CFP10 lyophilized powder, and-20--40 ℃ of preservation is standby.The SDS-PAGE of the reorganization ESAT6-CFP10 of purifying identifies Fig. 2 and 3, and the molecular weight of reorganization ESAT6-CFP10 is 21.8Kda, and purity is up to more than 95%.Detect through BCA, organic efficiency reaches 200mg/L.
The monoclonal antibody that the present invention screens anti-INF γ by standard monoclonal antibody screening method, obtained the monoclonal antibody of two strains at the different epitope clusters of INF γ, one of them antibody is pressed the finite concentration coated elisa plate, 4 ℃ are spent the night, again 37 ℃ 1-2 hour the sealing after, after the enzyme plate lyophilize, be commerce and use enzyme plate, an other strain antibody is according to standard method mark vitamin H.Interferon-gamma standard (1 μ g/ml) is diluted to the typical curve contrast on request.According to requirement of experiment, get suitable ELIAS strip, no enzyme plate or put into the enzyme plate bag, 4 ℃-8 ℃ are continued to preserve, (the interferon-gamma standard: 1ng/ml), blank and sample to be checked (supernatant after fresh blood is cultivated) of adding standard, positive control in corresponding aperture, every hole 50 μ l, add biotin labeling antibody again, every hole 50 μ l mix, stick sealing compound, hatched 30 minutes in 37 ℃.20 times of concentrated cleaning solutions are diluted to the washing working fluid for 20 times with purified water or distilled water.Discard liquid in each hole, every hole adds washing working fluid 250 μ l, discards after leaving standstill the several seconds, repeats 5 times, pats dry.Every hole adds avidin mark horseradish peroxidase 100 μ l, sticks sealing compound, puts 37 ℃ of incubations 30 minutes.Repeat to wash the plate step.Every hole adds substrate solution 100 μ l, and mixing was put 37 ℃ of lucifuge incubations 15 minutes.After colour developing finished, every hole added stop buffer 50 μ l, and the vibration mixing is put microplate reader wavelength 450nm (with the blank well zeroing) or dual wavelength 450nm/630nm immediately and measured the OD value down.Sensitivity is 1pg/ml.
The monoclonal antibody that the present invention screens anti-INF γ by standard monoclonal antibody screening method, obtained the monoclonal antibody of two strains at the different epitope clusters of INF γ, one of them antibody is pressed the finite concentration coated elisa plate, 4 ℃ are spent the night, again 37 ℃ 1-2 hour the sealing after, after the enzyme plate lyophilize, be commerce and use enzyme plate, an other strain antibody is according to standard method mark vitamin H.Interferon-gamma standard (1 μ g/ml) is diluted to the typical curve contrast on request.According to requirement of experiment, get suitable ELIAS strip, no enzyme plate or put into the enzyme plate bag, 4 ℃-8 ℃ are continued to preserve, (the interferon-gamma standard: 1ng/ml), blank and sample to be checked (supernatant after fresh blood is cultivated) of adding standard, positive control in corresponding aperture, every hole 50 μ l, add biotin labeling antibody again, every hole 50 μ l mix, stick sealing compound, hatched 30 minutes in 37 ℃.20 times of concentrated cleaning solutions are diluted to the washing working fluid for 20 times with purified water or distilled water.Discard liquid in each hole, every hole adds washing working fluid 250 μ l, discards after leaving standstill the several seconds, repeats 5 times, pats dry.Every hole adds avidin mark horseradish peroxidase 100 μ l, sticks sealing compound, puts 37 ℃ of incubations 30 minutes.Repeat to wash the plate step.Every hole adds chemical luminous substrate liquid 50 μ l, mixing, 2min stablize in the chemiluminescence detector the inside, survey 425nm light intensity, detection time 1s.Sensitivity is 0.1pg/ml.
Embodiment 6 whole blood INF γ detection kit-radioimmunologies
The monoclonal antibody that the present invention screens anti-INF γ by standard monoclonal antibody screening method, obtained the monoclonal antibody of two strains at the different epitope clusters of INF γ, one of them antibody is pressed the finite concentration coated elisa plate, 4 ℃ are spent the night, again 37 ℃ 1-2 hour the sealing after, after the enzyme plate lyophilize, be commerce and use enzyme plate, an other strain antibody is according to standard method mark I
125Interferon-gamma standard (1 μ g/ml) is diluted to the typical curve contrast on request.According to requirement of experiment, get suitable ELIAS strip, no enzyme plate or put into the enzyme plate bag, 4 ℃-8 ℃ are continued to preserve, (the interferon-gamma standard: 1ng/ml), blank and sample to be checked (supernatant after fresh blood is cultivated) of adding standard, positive control in corresponding aperture, every hole 50 μ l add I again
125Traget antibody, every hole 50 μ l mix, and stick sealing compound, after appropriate time is hatched.Detect data with gamma counter.Sensitivity is 0.1pg/ml.
Embodiment 7 test kit sensitivity and specific detection
Gather 50 examples and be diagnosed as tuberculosis patient and the healthy not fresh anticoagulant heparin venous blood 3ml of tuberculosis infected students of 20 examples, respectively getting the 1ml whole blood cultivates in the sterile culture plate, (concentration is that 1-50 μ g/ml carries out INF γ stimulation to add reorganization ESAT6-CFP10 in 1 hole, another hole contrast, hatched 20 hours for 37 ℃, centrifugal collection supernatant, supernatant can be preserved several weeks in 4 ℃ of conditions, can preserve 1 year for-20 ℃, with on get 100 μ l and add in the people INF γ detection kit (enzyme linked immunosorbent assay) of our company's exploitation and detect, the susceptibility of data presentation diagnosis tuberculosis infected students is 100%, specificity is up to 95.7%, with the clinical diagnosis result relatively, this test kit shows good diagnosis consistence.Therefore, the method for the tuberculosis early diagnosis that the present invention proposes will be significant on tuberculosis and mycobacterium tuberculosis infection person's diagnostic use, has good social benefit.
Sequence table
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Claims (10)
1. the specific antigen protein of whole blood INF-γ, it is characterized in that: it is following any fusion rotein that is connected with biological label: ESAT-6-connection peptides-CFP-10, CFP-10-connection peptides-ESAT-6, ESAT-6-connection peptides-MPB64, MPB64-connection peptides-ESAT-6, CFP-10-connection peptides-MPB64, MPB64-connection peptides-CFP-10, ESAT-6-connection peptides-CFP-10-connection peptides-MPB64, ESAT-6-connection peptides-MPB64-connection peptides-CFP-10, CFP-10-connection peptides-ESAT-6-connection peptides-MPB64, CFP-10-connection peptides-MPB64-connection peptides-ESAT-6, MPB64-connection peptides-CFP-10-connection peptides-ESAT-6 and MPB64-connection peptides-ESAT-6-connection peptides-CFP-10, wherein said connection peptides is not for influencing ESAT-6, the immunogenic small peptide of CFP-10 and MPB64.
2. the specific antigen protein of whole blood INF-γ as claimed in claim 1 is characterized in that: described biological label is histidine-tagged, GST label or Trx label.
3. the specific antigen protein of whole blood INF-γ as claimed in claim 1 is characterized in that: the aminoacid sequence of described connection peptides is SEQ ID:No.3.
4. the specific antigen protein of whole blood INF-γ as claimed in claim 1 is characterized in that: aminoacid sequence is SEQ ID:No.1.
5. the nucleotide sequence of the specific antigen protein of coding claim 1 described whole blood INF-γ.
6. the nucleotide sequence SEQID NO:2 of the specific antigen protein of coding claim 4 described whole blood INF-γ.
7. the carrier that comprises the described nucleotide sequence of claim 6, wherein carrier is pET28b.
8. the microorganism that comprises the described carrier of claim 7, wherein microorganism is an e. coli bl21.
9. the method for preparing the described specific antigen protein of claim 4, comprise the steps: that SEQ ID NO:2 sequence is stored among the DH5 α, use NcoI, XhoI restriction endonuclease enzyme is respectively cut DH5 α and pET28b expression vector, reclaim SEQ ID NO:2 sequence fragment and pET28b expression vector, under the effect of T4DNA ligase enzyme, connect, be transformed in the e. coli bl21 (DE3), transformant is cultivated in liquid LB substratum, adding final concentration is the penbritin cultivation of 50 μ g/ml, adding final concentration is the IPTG of 0.5mM, 37 ℃, and 200rpm, abduction delivering 8 hours is collected expressed recombinant protein, purifying promptly.
10. the arbitrary described antigen protein of claim 1-4 is diagnosed in conjunction with the purposes in the medicine in preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103257236A (en) * | 2012-02-20 | 2013-08-21 | 南京市疾病预防控制中心 | Application of specific CTL cell epitope peptide of HLA-A24 restrictive Mycobacterium tuberculosis |
| CN108440656A (en) * | 2018-03-08 | 2018-08-24 | 中国医学科学院病原生物学研究所 | The application of mycobacterium tuberculosis PKA albumen and its fusion protein in auxiliary diagnosis pulmonary tuberculosis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101455847A (en) * | 2007-12-12 | 2009-06-17 | 中国人民解放军总医院第二附属医院 | Use of ESAT6and CFP10 protein in preparing medicine for diagnosing tuberculosis |
| CN101492497A (en) * | 2008-01-21 | 2009-07-29 | 范雄林 | Expression purification process for tubercle bacillus differential recombinant protein and uses thereof |
| CN101684160A (en) * | 2009-08-03 | 2010-03-31 | 广东大华农动物保健品股份有限公司 | Recombinant protein for diagnosing bovine tuberculosis and application thereof |
-
2011
- 2011-01-27 CN CN 201110031079 patent/CN102174114B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101455847A (en) * | 2007-12-12 | 2009-06-17 | 中国人民解放军总医院第二附属医院 | Use of ESAT6and CFP10 protein in preparing medicine for diagnosing tuberculosis |
| CN101492497A (en) * | 2008-01-21 | 2009-07-29 | 范雄林 | Expression purification process for tubercle bacillus differential recombinant protein and uses thereof |
| CN101684160A (en) * | 2009-08-03 | 2010-03-31 | 广东大华农动物保健品股份有限公司 | Recombinant protein for diagnosing bovine tuberculosis and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| W. R. WATERS ET AL.: "Use of Recombinant ESAT-6:CFP-10 Fusion Protein for Differentiation of Infections of Cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis", 《CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY》, 31 July 2004 (2004-07-31), pages 729 - 735, XP003009922, DOI: doi:10.1128/CDLI.11.4.729-735.2004 * |
| 娄培安等: "结核分枝杆菌分泌蛋白ESAT-6-CFP10 的融合表达及纯化", 《热带病与寄生虫学》, 31 December 2005 (2005-12-31) * |
| 沈颖: "ESAT-6/CFP-10融合蛋白的抗原性分析及其对诊断结核的临床应用价值", 《中国优秀硕士学位论文全文数据库》, 31 December 2009 (2009-12-31) * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103257236A (en) * | 2012-02-20 | 2013-08-21 | 南京市疾病预防控制中心 | Application of specific CTL cell epitope peptide of HLA-A24 restrictive Mycobacterium tuberculosis |
| CN103257236B (en) * | 2012-02-20 | 2015-04-15 | 南京市疾病预防控制中心 | Application of specific CTL cell epitope peptide of HLA-A24 restrictive Mycobacterium tuberculosis |
| CN108440656A (en) * | 2018-03-08 | 2018-08-24 | 中国医学科学院病原生物学研究所 | The application of mycobacterium tuberculosis PKA albumen and its fusion protein in auxiliary diagnosis pulmonary tuberculosis |
| CN108440656B (en) * | 2018-03-08 | 2020-04-17 | 中国医学科学院病原生物学研究所 | Application of mycobacterium tuberculosis PKA protein and fusion protein thereof in auxiliary diagnosis of pulmonary tuberculosis |
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