CN107045063A - Pulmonary tuberculosis associated serum identifies albumen TIMP-1 and its application - Google Patents
Pulmonary tuberculosis associated serum identifies albumen TIMP-1 and its application Download PDFInfo
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Abstract
The invention belongs to biochemical immunity and analysis technical field, and in particular to pulmonary tuberculosis associated serum identifies albumen TIMP-1 and its application.The present invention is screened using cell factor and holoprotein chip to host protein, it is albumen substrate TIMP TIMP-1, inhibiting factor TIMP-1 protein sequence such as SEQ ID NO there is provided a kind of detectable pulmonary tuberculosis associated serum biological label albumen：Shown in 1.The inhibiting factor TIMP-1 of the present invention can be used as mark antigen protein application in pulmonary tuberculosis serum diagnostic test agent box is prepared.
Description
Technical field
The invention belongs to biochemical immunity analysis field, and in particular to pulmonary tuberculosis associated serum identifies albumen TIMP-1 and its application.
Background technology
Human tuberculosis are a kind of main chronic debilitating infectious diseases caused by mycobacterium tuberculosis (Mycobacterium tuberculosis, M.tb).Although mycobacterium tuberculosis can encroach on the multiple organ-tissues of human body, pulmonary tuberculosis is topmost tuberculosis type.It is estimated that the whole world is annual about 8,000,000 new cases, and about 2,000,000 deaths, 80% patient is in developing country.Unique vaccine both at home and abroad for tuberculosis prophylaxis is BCG vaccine (BCG) at present.The vaccine has certain protective effect to children, but to the protecting effect extreme difference of adult.And the protecting effect of children is reported all over the world differed, fluctuate very big.Therefore, early diagnosis early treatment is still the important means of tuberculosis prevention and control.
Method currently used for diagnosis of tuberculosis detected except clinically conventional chest x-ray perspective, tracheal endoscope combination biopsy sampling, in addition to the detection method such as sputum smear microscopy and Bacteria Culture, also immunology and molecular biology method.Requirement of the bacteria distribution culture detection generally to biosafety level is very high, and the bacteria growing is slow, and separation and Purification need more than January, waste time and energy, and point bacterium rate is low, therefore, uncomfortable cooperation conventional detection；Molecular biology method has higher requirements to instrument and equipment and operating personnel；Therefore, application of the immunological method in terms of diagnosis of tuberculosis is commonplace.
It is generally believed that immune response feature lungy shows as cellular immunity and humoral immunity separation, cellular immunity dominance is shown as in initial infection, it is dominant that morbidity stage shows as humoral immunity.Traditional intracutaneous abnormal method of tuberculin (PPD) is mainly based upon cellular immunity principle, this method sensitivity is high, has continued to use over one hundred year, but specificity is poor, environment mycobacterial infections person has cross reaction, and immune compromised individuals may occur without reaction；Because BCG and tuberculin have cross reaction, developing country children are generally inoculated with BCG, vaccine immunity reaction interference skin test detection.The detection method sensitivity for discharging IFN-γ as principle design under antigen of mycobacterium tuberculosis stimulated in vitro using PBLC in the market is high, but detection is Infection Status, with pathologic process lungy without directly contacting.Tuberculosis is a high infection rate, the chronic disease of the low incidence of disease, and only 5%-10% the infected will develop into active tuberculosis, and wherein most humans are pulmonary tuberculosis.Therefore, diagnostic marker molecule related to morbidity, especially with pulmonary tuberculosis correlation is found significant to tuberculotherapy and control.
The content of the invention
Present invention aims to overcome that the defect of prior art, the host protein of tuberculosis patient is screened using cell factor and holoprotein chip, a kind of serum biological label albumen related to detection pulmonary tuberculosis is provided, the mark albumen can be used for lunger's haemocyanin biological marker, set up lunger's TIMP ((Metalloproteinase inhibitor1, abbreviation TIMP-1) expression detection method, timely diagnosis for activity and primary pulmonary tuberculosis and treatment provide support.
The present invention is stimulated whole blood using mycobacterium tuberculosis differential protein CFP10/ESAT6 simulation mycobacterium tuberculosis, post-stimulatory supernatant is detected using cell factor chip (516 points of RayBiotech), 510 kinds of cell factors are analyzed altogether.After being stimulated through CE albumen, there are 67 kinds of cytokine-expressing amounts to increase more than 1.9 times, 43 kinds have been lowered at least 2.0 times.Including inflammatory factor, immune-regulating factor, chemotactic factor (CF), growth factor, angiogenic factor, soluble recepter, adhesion factor etc..Simultaneously, analyzed (656 points of SpringBio chips) using protein chip, direct detection has been carried out to 651 kinds of albumen in active tuberculosis patient, latent infection person, lung cancer patient and normal healthy controls human plasma, as a result more than 1.9 times of albumen of expression rise has 21 kinds only in active tuberculosis human plasma, and the albumen that more than 2.0 times of downward has 72 kinds.Include keratin, NF-M, with gene repair GAP-associated protein GAP, heat shock protein etc. in the albumen of up-regulation.
Implement further checking to TIMP-1,125 parts of active tuberculosis cases are have detected using the ELISA based on TIMP-1 antigens and 90 parts of normal healthy controls person's serum checkings find TIMP-1 to the detection sensitivity of active tuberculosis patient up to 100%, specificity reachable 92%.
Present invention firstly discovers that TIMP-1 can be used as an important biological indicator, in pulmonary tuberculosis clinical diagnosis, antidiastole and the effect effectively in observation with mark albumen, research for later tuberculosis patient haemocyanin provides technical foundation, and to provide new technical thought from molecular level diagnosis of tuberculosis, with great theory significance and potential use value.
The present invention is achieved through the following technical solutions：
A kind of screening and detection of the haemocyanin related to pulmonary tuberculosis, comprise the following steps：
1. stimulate expressing protein using cell factor chip detection peripheral blood
Chip detection, including 510 kinds of cell factors are carried out to the post-stimulatory tuberculosis patients of Specific Antigen of Mycobacterium Tuberculosis CFP10/ESAT6 and normal healthy controls person's peripheral blood blood plasma using RayBiotech cytokine antibodies chip (516 points of RayBiotech).Obtain up-regulation and the cell factor lowered.
2. protein chip detects plasma protein
Tuberculosis patient and collator's plasma protein are directly detected using protein chip (656 points of SpringBio chips)：651 kinds of albumen in active tuberculosis patient, latent infection person, lung cancer patient and normal healthy controls human plasma have been carried out with direct detection, the protein only expressed and be significantly raised and lowered in active tuberculosis human plasma is obtained.
Application in 3.TIMP-1 discriminating detection pulmonary tuberculosis
The testing result of two kinds of chips shows that TIMP-1 expression is related to pulmonary tuberculosis.Therefore the ELISA method of TIMP-1 detections is established, checking detection further is carried out to 125 parts of active tuberculosis cases and 90 parts of normal healthy controls person's serum, discovery TIMP-1 is to the sensitiveness of active tuberculosis up to 100%, and specificity is up to 92%；And primary tuberculosis and secondary tuberculosis case can be distinguished.It is thus determined that TIMP-1 can be used for the serum label protein that consumptive diagnoses.
Biological function checking shows that pulmonary tuberculosis associated serum protein biology marker TIMP-1 of the invention can be applied in pulmonary tuberculosis serum solution detection kit is prepared.
Effect of the invention is that：Find first and screen a kind of haemocyanin TIMP TIMP-1 as an important serum solution Biological Detection index, played a role in observing in the diagnosis of pulmonary tuberculosis, antidiastole and effectively, research for pulmonary tuberculosis haemocyanin from now on provides foundation, and new direction is provided from serum solution protein molecular level diagnosis pulmonary tuberculosis, with important theory value and potential Practical significance.
Brief description of the drawings
Sequence table SEQ ID NO：1 is the protein sequence for the haemocyanin TIMP TIMP-1 that the present invention is cloned.
Fig. 1：Differential expressions of the haemocyanin TIMP-1 in lunger and normal healthy controls person.
Fig. 2：Differential expressions of the haemocyanin TIMP-1 in supernatant after lunger and normal healthy controls person's whole blood ESAT6/CFP10 stimulate.
Fig. 3：Expression differences of the haemocyanin TIMP-1 in the human macrophage system THP-1 that M.bovis and BCG infects.
Fig. 4：SpringBio antibody high flux chip cardinal principle figures.Description of reference numerals：The protein sample marked with Biotin, is hybridized with chip, and the antibody for adding Streptavidin with being fluorescently labeled after protein binding in sample is recognized.
Embodiment
Embodiment 1
Below by way of the discovery embodiment of pulmonary tuberculosis correlated identities albumen, the present invention is described in detail.
1. the collection and preparation of blood sample
(1) sample is collected
Tuberculosis patient blood sample (anticoagulated whole blood or serum) sample comes from Wuhan City medical treatment center, according to various means such as clinical diagnosis, X images, Phlegm incubations, it is defined as active tuberculosis disease, obvious TB focus or cavity are there are, and excludes the new admitted patient of the complication such as AIDS, disease of urinary tract.In 111 active tuberculosis patients, the people of male 71, the people of women 40；58 parts of Sputum smears positive case, negative 20 parts, remaining case does not correspond to result；13 parts of Phlegm incubation positive case, negative 23 parts, remaining is without correspondence result；46 parts of PPD skin test positives case, negative 12 parts, remaining is without correspondence result；74 patients have obvious focus.
284 normal healthy controls are university student volunteer.All volunteers detect by chest x-ray, are showed no focus or cavity.PPD skin test reaction detections, positive 35 people, negative 11 people, including the people of male 30, the people of women 16 are carried out to wherein 46 people to volunteer through specialist.
(2) blood sample is handled
Anticoagulant heparin whole blood 5mL is extracted, by whole blood and RPMI-1640 complete mediums (being purchased from Hyclone companies) using volume ratio as 1:1 mixing, the amount by cumulative volume 2mL/ per hole is transferred in 24 porocyte culture plates respectively.20 μ g phytohemagglutin phytolectin (PHA) is added in the 1st hole, positive control is used as；20 μ g mycobacterium tuberculosis differential protein CFP10/ESAT6 fusion proteins are added in the 2nd hole), as instrument connection, 10 μ l physiological saline are added in the 3rd hole, negative control is used as.Each reagent in hole is gently mixed with blood, 37 DEG C is put in and contains 5%CO2Incubator in cultivate 14~16h.CFP10/ESAT6 antigen-4 fusion protein genes come from bacillus tuberculosis typus humanus (M.tuberculosis) H37RV strain genes group (GenBan sequence numbers：GenBank:AL123456.3), the bacterial strain is given by Beijing Tuberculosis and Thoracic Tumor Research Institute professor Li Chuanyou.Clone and Bacillus coli expression and fusion protein purification carry out [1] according to a conventional method.
2. cell factor chip is detected
(1) sample prepares
After IFN-γ release in vitro method [2] detection sample, 5 parts of stimulation liquid supernatants of the positive value highest of IFN-γ method for releasing are filtered out in 111 parts of active tuberculosis cases, stimulate each draw of every part of supernatant (negative control) to carry out the detection of RayBiotech cytokine antibodies chip after 20 μ L are sufficiently mixed in supernatant physiological saline after mycobacterium tuberculosis differential protein ESAT6/CFP10 stimulations.
(2) chip is detected
RayBiotech cytokine antibodies chip prepares and completed detection by upper Haikang into bioengineering Co., Ltd, and reagent needed for detection is provided by the said firm.Predominantly detect step as follows：
1) cytokine antibodies chip film is closed in confining liquid (RayBio).
2) sample that 100 μ L are handled well is added, 4 DEG C of effects are stayed overnight.
3) antibody (deriving from chip matched reagent) marked is added, 1h is acted at room temperature.
4) add after chemical luminous substrate, chip is subjected to chemiluminescence detection, X-ray film is exposed on film and shows testing result.
Supernatant and each 510 kinds of cell factors of negative control after ESAT6/CFP10 is stimulated are have detected altogether using RayBiotech cytokine antibodies chip.2 groups of results, each 516 signals of every group of result (including 6 control points) are obtained after exposure.Cell factor difference compared with negative control is more notable in supernatant after ESAT6/CFP10 is stimulated.The expression quantity of 67 kinds of cell factors increases more than 1.9 times, including inflammatory factor, immune-regulating factor, chemotactic factor (CF), growth factor, angiogenic factor, soluble recepter, adhesion factor etc..Opposite, 43 kinds of cell factors have lowered at least 2.0 times after being stimulated through mycobacterium tuberculosis differential protein ESAT6/CFP10.The change multiple of other cell factors is between 0.6~1.0 or 1.0~1.8 times.
3. protein chip is detected
(1) sample prepares
After the detection of IFN-γ release in vitro method, filtered out in 111 parts of active tuberculosis cases be sufficiently mixed after the positive value 5 parts of blood plasma of highest of IFN-γ method for releasing, every part of 20 μ L of absorption it is standby.Choose the detection of 5 parts of IFN-γs minimum, while antibody test is minimum, PPD reactions are negative healthy volunteer's blood plasma, every part draw be sufficiently mixed after 20 μ L it is standby.Healthy volunteer's blood plasma of 5 parts of IFN-γ test positive is chosen as latent infection person's blood plasma, every part draw be sufficiently mixed after 20 μ L it is standby.It is sufficiently mixed after 5 parts of lung cancer patient blood plasma of selection, every part of 20 μ L of absorption standby.Every part of pooled plasma carries out the detection of SpringBio antibody high fluxs chip respectively.
(2) chip is detected
SpringBio antibody high fluxs chip (656 points, wherein 651 protein sites, 4 control points) prepares and completed detection by upper Haikang into bioengineering Co., Ltd, and reagent needed for detection is provided by the said firm.Predominantly detect step as follows：
1) antibody chip film is closed as in confining liquid (SpringBio).
2) sample marked with Biotin is added, is hybridized with chip, 4 DEG C of effects are stayed overnight.
3) protein binding in Streptavidin, with sample is added.
4) antibody of fluorescence labeling is added, 1h is acted at room temperature.
5) scanner scanning fluorescence signal.(see Fig. 4)
SpringBio antibody high flux chips have detected each 651 kinds of albumen in different samples, scans fluorescence signal, obtains three groups of results.Compared with normal healthy controls, 53 kinds of expressing quantities raise more than 1.9 times in active tuberculosis human plasma, including keratin, NF-M, with gene repair GAP-associated protein GAP, heat shock protein etc..116 kinds of expressing quantities lower more than 2.0 times.In lung cancer patient blood plasma, the albumen that more than 1.9 times of up-regulation has 132 kinds, and lowering more than 2.0 times albumen has 119 kinds.3.TIMP-1 albumen is in the phthisical checking of antidiastole
The testing result of two kinds of chips shows that TIMP-1 expression is related to pulmonary tuberculosis.Therefore applicant establishes TIMP-1 detection ELISA methods, further determines that its application in pulmonary tuberculosis blood testing is set up.
(1) differential expression checkings of the TIMP-1 in lunger's blood
Newly collect 125 consumptives and 166 normal healthy controls person's serum.Collection standard is the same.Detect that TIMP-1 ELISA operating procedures are as follows：
As depicted in figs. 1 and 2, the present invention (is purchased from RayBiotech companies using commercially available TIMP-1ELISA kits, USA supernatant verifies the differential expression of TIMP-1 albumen after) stimulating 125 consumptives and 90 normal healthy controls person's serum and whole blood ESAT6/CFP10, and sensitivity is 100.0% (95%CI:97,100), specificity is 92% (95%CI:86,96), TG-AUC (AUC) is 0.782 (P<0.0001,95%CI, 0.688-0.876).
(2) differential expression checkings of the TIMP-1 in infection macrophage
Limited by this Laboratory biosafety, the present invention have detected TIMP-1 expression with Mycobacterium bovis (M.bovis) AF2122/97 (professor Li Chuanyou give by Beijing Tuberculosis and Thoracic Tumor Research Institute) pattern bacterium in the laboratory of the viral locellus of state Key Laboratory of Agricultural Microbiology and be tested with the correlation that mycobacterium tuberculosis infects.Experiment people source macrophage system THP-1 cells (ATCC TIB-202) (professor Li Chuanyou give by Beijing Tuberculosis and Thoracic Tumor Research Institute) used infect Mycobacterium bovis and vaccine strain mycobacterium bovis BCG Tokyo strain (ATCC 35737) (professor Li Chuanyou give by Beijing Tuberculosis and Thoracic Tumor Research Institute) are pattern bacterium.THP-1 cells are inoculated in 145mm Tissue Culture Dish, PMA (macrophage derivant) is added, final concentration of 40ng/mL, mixing is put in CO212h is induced in incubator.Culture medium (containing PMA) is removed, is washed twice with the culture medium of incubation, it is standby.Cell becomes adherent by original suspended state, stretches out pseudopodium, starts to exercise the effect of macrophage.Scattered bacterium is added in Tissue Culture Dish, infection ratio is：10:1, supernatant (containing bacterium) is collected and compareed as extracellular bacteria, washed with fresh culture twice, thoroughly remove the bacterium for being introduced into cell by interaction 12 hours, after 12 hours, collects intracellular bacterium and cell sample within 24 hours.Then extraction and quality testing (being conventional method) of front and rear macrophage total serum IgE are infected, differential expression (P of quantitative fluorescent PCR (being conventional method) the detection TIMP-1 in different groups is utilized<0.05) (referring to Fig. 3).
The present invention, which obtains TIMP-1, has the host protein of potential mark meaning, while having carried out variance analysis to TIMP-1 albumen.As a result show that the expression of TIMP-1 haemocyanins has differences between the two groups.
Leading reference：
[1] Zhang Shuhuan, Wu Bo, Yan Bangfen, Cao Sha, Chen Yingyu, Chao Yanjie, Ling Jieyu, Tan Yadi, Chen Huanchun, Guo's love treasure's Mycobacterium bovis antigens MPB70, MPB83, CFP-10 and ESAT-6 amalgamation and expression and the Chinese Amphixenosis's magazines of correlation Analysis, 2007,23 (12):38-43.
[2] foundation of Chen Ying treasures people IFN-γ release in vitro detection method and its application [J] bioengineering journal .2008,24 (9) in diagnosis of tuberculosis：1-6.
Claims (2)
1. a kind of haemocyanin TIMP TIMP-1, its protein sequence such as SEQ ID NO：Shown in 1.
2. the haemocyanin TIMP TIMP-1 described in claim 1 is preparing detection pulmonary tuberculosis blood
The application of mark antigen protein is used as in clear detection kit.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102667486A (en) * | 2009-11-25 | 2012-09-12 | 霍洛吉克股份有限公司 | Detection of intraamniotic infection |
| CN103065065A (en) * | 2012-11-18 | 2013-04-24 | 浙江大学 | Active tuberculosis differential expression protein profile model and building method thereof |
| CN103237901A (en) * | 2010-03-01 | 2013-08-07 | 卡里斯生命科学卢森堡控股有限责任公司 | Biomarkers for theranostics |
| WO2013177502A1 (en) * | 2012-05-24 | 2013-11-28 | The Broad Institute, Inc. | Methods and devices for tuberculosis diagnosis using biomarker profiles |
| WO2013175459A2 (en) * | 2012-05-25 | 2013-11-28 | Stellenbosch University | Method for diagnosing tuberculosis disease |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102667486A (en) * | 2009-11-25 | 2012-09-12 | 霍洛吉克股份有限公司 | Detection of intraamniotic infection |
| CN103237901A (en) * | 2010-03-01 | 2013-08-07 | 卡里斯生命科学卢森堡控股有限责任公司 | Biomarkers for theranostics |
| WO2013177502A1 (en) * | 2012-05-24 | 2013-11-28 | The Broad Institute, Inc. | Methods and devices for tuberculosis diagnosis using biomarker profiles |
| WO2013175459A2 (en) * | 2012-05-25 | 2013-11-28 | Stellenbosch University | Method for diagnosing tuberculosis disease |
| CN103065065A (en) * | 2012-11-18 | 2013-04-24 | 浙江大学 | Active tuberculosis differential expression protein profile model and building method thereof |
Non-Patent Citations (1)
| Title |
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| LIN S J,ET AL.: "NP_003245.1", 《NCBI》 * |
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