CN107045063B - Pulmonary tuberculosis associated serum identifies albumen TIMP-1 and its application - Google Patents

Abstract

The invention belongs to biochemical immunities and analysis technical field, and in particular to pulmonary tuberculosis associated serum identifies albumen TIMP-1 and its application.The present invention screens host protein using cell factor and holoprotein chip, a kind of detectable i.e. albumen substrate metalloproteinase inhibitor TIMP-1 of pulmonary tuberculosis associated serum biological identification albumen is provided, the protein sequence of inhibiting factor TIMP-1 is as shown in SEQ ID NO:1.Inhibiting factor TIMP-1 of the invention can be in preparation pulmonary tuberculosis serum diagnostic test agent box as mark antigen protein application.

Description

Pulmonary tuberculosis associated serum identifies albumen TIMP-1 and its application

Technical field

The invention belongs to biochemical immunity analysis fields, and in particular to pulmonary tuberculosis associated serum identifies albumen TIMP-1 and its answers With.

Background technique

Human tuberculosis are mainly one caused by mycobacterium tuberculosis (Mycobacterium tuberculosis, M.tb) The chronic debilitating infectious disease of kind.Although mycobacterium tuberculosis can encroach on the multiple organ-tissues of human body, pulmonary tuberculosis is most important Tuberculosis type.It is estimated that there are about 8,000,000 new cases, about 2,000,000 deaths in the whole world every year, 80% patient is being developed Middle country.Being used for unique vaccine of tuberculosis prophylaxis both at home and abroad at present is BCG vaccine (BCG).The vaccine has centainly children Protective effect, but it is very poor to adult protecting effect.And the protecting effect of children is reported all over the world it is different, fluctuation very Greatly.Therefore, early diagnosis early treatment is still the important means of tuberculosis prevention and control.

Method currently used for diagnosis of tuberculosis is in addition to clinically common chest x-ray perspective, tracheal endoscope are combined and lived It examines except the detection methods such as sample detection, sputum smear microscopy and Bacteria Culture, there are also immunologys and molecular biology method.Carefully It is usually very high to the requirement of biosafety level that bacterium is separately cultured detection, and bacterium growth is slow, separation and Purification need January with On, it is time-consuming and laborious, divide bacterium rate low, therefore, discomfort cooperation conventional detection；Molecular biology method is to instrument and equipment and operator Have higher requirements；Therefore, immunological method in terms of diagnosis of tuberculosis using commonplace.

It is generally believed that immune response feature lungy shows as cellular immunity and humoral immunity separation, in initial infection Cellular immunity dominance is shown as, morbidity stage shows as humoral immunity dominance.Traditional tuberculin (PPD) intradermal change State method is mainly based upon cellular immunity principle, and this method sensitivity is high, has continued to use over one hundred year, but specific poor, environment Mycobacterial infections person has cross reaction, and immune compromised individuals may not react；It is handed over since BCG and tuberculin have Fork reaction, developing country children are generally inoculated with BCG, vaccine immunity reaction interference skin test detection.Currently on the market with peripheral blood Lymphocyte discharges the detection method high sensitivity that IFN-γ designs for principle under antigen of mycobacterium tuberculosis stimulated in vitro, but Detection is Infection Status, with pathologic process lungy without directly contacting.Tuberculosis be a high infection rate, low disease incidence it is slow Property disease, only the infected of 5%-10% will develop into active tuberculosis, and wherein most humans are pulmonary tuberculosis. Therefore, finding diagnostic marker molecule related to morbidity, especially relevant with pulmonary tuberculosis has weight to tuberculotherapy and control Want meaning.

Summary of the invention

Present invention aims to overcome that the defect of the prior art, using cell factor and holoprotein chip to tuberculosis patient Host protein is screened, and a kind of serum biological identification albumen relevant to detection pulmonary tuberculosis is provided, which can be used for Lunger's haemocyanin biological marker, establishes lunger's matrix metalloproteinase inhibitors (detection method of (Metalloproteinase inhibitor 1, abbreviation TIMP-1) expression is activity and primary pulmonary The timely diagnosing and treating of tuberculosis provides support.

The present invention pierces whole blood using mycobacterium tuberculosis differential protein CFP10/ESAT6 simulation mycobacterium tuberculosis Swash, post-stimulatory supernatant is detected using cell factor chip (516 points of RayBiotech), analyzes altogether 510 kinds thin Intracellular cytokine.After the stimulation of CE albumen, there are 67 kinds of cytokine-expressing amounts to increase 1.9 times or more, 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 glues Attached factor etc..Meanwhile (656 points of SpringBio chip) is analyzed using protein chip, to active tuberculosis patient, latent infection 651 kinds of albumen have carried out direct detection in person, lung cancer patient and normal healthy controls human plasma, as a result only in active tuberculosis patient's blood The albumen that expression increases 1.9 times or more in slurry has 21 kinds, lowers 2.0 times or more of albumen and has 72 kinds.It is wrapped in the albumen of up-regulation Include keratin, neurofilament protein, with gene repair GAP-associated protein GAP, heat shock protein etc..

Further verifying is implemented to TIMP-1, has detected 125 parts of active tuberculosis using the ELISA based on TIMP-1 antigen Case and 90 parts of normal healthy controls person's serum verifying discovery TIMP-1 are reachable to the detection sensitivity of active tuberculosis patient 100%, specificity is up to 92%.

Present invention firstly discovers that TIMP-1 can be used as an important biological indicator, pulmonary tuberculosis clinical diagnosis, Have the function of identifying albumen in antidiastole and effectively observation, the research for later tuberculosis patient haemocyanin provides skill Art basis, and to provide new technical thought from molecular level diagnosis of tuberculosis, with great theory significance and potential use Value.

The invention is realized by the following technical scheme:

A kind of screening and detection of haemocyanin relevant to pulmonary tuberculosis, comprising the following steps:

1. utilizing cell factor chip detection peripheral blood stimulation expression albumen

Using RayBiotech cytokine antibodies chip (516 points of RayBiotech) to mycobacterium tuberculosis specificity The post-stimulatory tuberculosis patient of antigens c FP10/ESAT6 and normal healthy controls person's peripheral blood blood plasma carry out chip detection, including 510 kinds thin Intracellular cytokine.The cell factor for obtaining up-regulation and lowering.

2. protein chip detects plasma protein

Tuberculosis patient and collator's plasma protein are directly detected using protein chip (656 points of SpringBio chip): to work 651 kinds of albumen have carried out direct detection in dynamic property tuberculosis patient, latent infection person, lung cancer patient and normal healthy controls human plasma, obtain It obtains and only expresses the protein being significantly raised and lowered in active tuberculosis human plasma.

Application in the identification detection pulmonary tuberculosis of 3.TIMP-1

The testing result of two kinds of chips shows that TIMP-1 expression is related to pulmonary tuberculosis.Therefore TIMP-1 detection is established ELISA method, verifying detection further is carried out to 125 parts of active tuberculosis cases and 90 parts of normal healthy controls person's serum, find TIMP-1 is to the sensibility of active tuberculosis up to 100%, and specificity is up to 92%；And can distinguish primary tuberculosis and Secondary tuberculosis case.It is thus determined that TIMP-1 can be used for the serum label protein of consumptive's diagnosis.

Biological function verifying shows that pulmonary tuberculosis associated serum protein biology marker TIMP-1 of the invention can prepared It is applied in pulmonary tuberculosis serum solution detection kit.

Effect of the invention is that: it finds for the first time and screens a kind of haemocyanin matrix metalloproteinase inhibitors Serum solution Biological Detection index TIMP-1 important as one, in the diagnosis of pulmonary tuberculosis, antidiastole and effectively observation In play a role, provide foundation for the research of pulmonary tuberculosis haemocyanin from now on, and diagnose from serum solution protein molecular level Pulmonary tuberculosis provides new direction, has important theoretical value and potential Practical significance.

Detailed description of the invention

Sequence table SEQ ID NO:1 is the haemocyanin matrix metalloproteinase inhibitors TIMP-1 that the present invention clones Protein sequence.

Fig. 1: the haemocyanin TIMP-1 differential expression in lunger and normal healthy controls person.

Fig. 2: haemocyanin TIMP-1 after lunger and normal healthy controls person's whole blood ESAT6/CFP10 stimulation in supernatant Differential expression.

Fig. 3: the haemocyanin TIMP-1 expression in the human macrophage system THP-1 that M.bovis and BCG infects is poor It is different.

Fig. 4: SpringBio antibody high throughput chip cardinal principle figure.Description of symbols: the albumen marked with Biotin Sample is hybridized with chip, and the antibody being fluorescently labeled after protein binding in Streptavidin and sample is added and identifies.

Specific 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 collection

Tuberculosis patient blood sample (anticoagulated whole blood or serum) sample is from Wuhan City medical treatment center, according to clinic The various means such as diagnosis, X image, Phlegm incubation are determined as active tuberculosis disease, there are obvious TB focus or cavity, And exclude the new admitted patient of the complication such as AIDS, disease of urinary tract.In 111 active tuberculosis patients, male 71 People, 40 people of women；58 parts of sputum smear positive case, 20 parts negative, remaining case does not correspond to result；Phlegm incubation positive case 13 parts, 23 parts negative, remaining is without corresponding result；46 parts of PPD skin test positive case, 12 parts negative, remaining is not correspond to As a result；74 patients have obvious lesion.

284 normal healthy controls are university student volunteer.All volunteers pass through chest x-ray detection, are showed no lesion Or cavity.PPD skin test reaction detection carried out to wherein 46 people to volunteer through specialist, positive 35 people, negative 11 people, wherein Including 30 people of male, 16 people of women.

(2) blood sample is handled

Anticoagulant heparin whole blood 5mL is extracted, by whole blood and RPMI-1640 complete medium (being purchased from Hyclone company) with body Product is transferred in 24 porocyte culture plates than being that 1:1 is mixed by the amount in the every hole total volume 2mL/ respectively.It is added in the 1st hole The phytohemagglutin phytolectin (PHA) of 20 μ g, as positive control；20 μ g mycobacterium tuberculosis differential proteins are added in the 2nd hole CFP10/ESAT6 fusion protein), as instrument connection, 10 μ l physiological saline are added in the 3rd hole, as negative control.It will be in hole Each reagent is gently mixed with blood, is put in 37 DEG C containing 5%CO₂Incubator in cultivate 14~16h.CFP10/ESAT6 merges egg White gene from bacillus tuberculosis typus humanus (M.tuberculosis) H37RV strain gene group (GenBan sequence number: GenBank:AL123456.3), which is given by Beijing Tuberculosis and Thoracic Tumor Research Institute professor Li Chuanyou.It clones and big Enterobacteria expression and fusion protein purification carry out [1] according to a conventional method.

2. cell factor chip detects

(1) sample prepares

After detecting sample with IFN-γ release in vitro method [2], IFN- is filtered out in 111 parts of active tuberculosis cases The γ method for releasing positive is worth highest 5 parts of stimulations liquid supernatant, by supernatant after mycobacterium tuberculosis differential protein ESAT6/CFP10 stimulation It is anti-that RayBiotech cell factor is carried out after being sufficiently mixed with every part of 20 μ L of each absorption of physiological saline stimulation supernatant (negative control) The detection of body chip.

(2) chip detects

RayBiotech cytokine antibodies chip is prepared and is completed by upper Haikang to detect at bioengineering Co., Ltd, inspection Reagent needed for surveying is provided by the said firm.Predominantly detect that steps are as follows:

1) closing in confining liquid (RayBio) by cytokine antibodies chip film.

2) sample that 100 μ L are handled well is added, 4 DEG C of effects are overnight.

3) antibody (from chip matched reagent) marked is added, acts on 1h at room temperature.

4) after chemiluminescent substrate being added, chip is subjected to chemiluminescence detection, X-ray film is exposed on film and is shown Show testing result.

Using RayBiotech cytokine antibodies chip have detected altogether ESAT6/CFP10 stimulation after supernatant and negative control Each 510 kinds of cell factors.2 groups are obtained after exposure as a result, each 516 signals of every group of result (including 6 control points). Cell factor difference compared with negative control is more significant in supernatant after ESAT6/CFP10 stimulation.The expression of 67 kinds of cell factors Amount increases 1.9 times or more, including inflammatory factor, immune-regulating factor, chemotactic factor (CF), growth factor, vasogenic because Son, soluble recepter, adhesion factor etc..Opposite, 43 kinds of cell factors are through mycobacterium tuberculosis differential protein ESAT6/CFP10 At least 2.0 times have been lowered after stimulation.The variation multiple of other cell factors is between 0.6~1.0 or 1.0~1.8 times.

3. protein chip detects

(1) sample prepares

After the detection of IFN-γ release in vitro method, IFN-γ method for releasing is filtered out in 111 parts of active tuberculosis cases The positive is worth highest 5 parts of blood plasma, is sufficiently mixed after every part of 20 μ L of absorption spare.It is minimum to choose 5 parts of IFN-γ detections, while antibody Detect minimum, PPD reaction is negative healthy volunteer's blood plasma, is sufficiently mixed after every part of 20 μ L of absorption spare.Choose 5 parts of IFN- Healthy volunteer's blood plasma of γ test positive is sufficiently mixed spare as latent infection person's blood plasma after every part of 20 μ L of absorption.Choosing 5 parts of lung cancer patient blood plasma are taken, are sufficiently mixed after every part of 20 μ L of absorption spare.It is anti-that every part of pooled plasma carries out SpringBio respectively The detection of body high throughput chip.

(2) chip detects

SpringBio antibody high throughput chip (656 points, wherein 651 protein sites, 4 control points) is by upper Haikang Cheng Sheng The preparation of object Engineering Co., Ltd and completion detection, reagent needed for detecting are provided by the said firm.Predominantly detect that steps are as follows:

1) by antibody chip film as closing in confining liquid (SpringBio).

2) sample marked with Biotin is added, is hybridized with chip, 4 DEG C of effects are overnight.

3) protein binding in Streptavidin, with sample is added.

4) antibody of fluorescent marker is added, acts on 1h at room temperature.

5) scanner scanning fluorescence signal.(see Fig. 4)

SpringBio antibody high throughput chip has 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 1.9 times or more in active tuberculosis human plasma, wherein Including keratin, neurofilament protein, with gene repair GAP-associated protein GAP, heat shock protein etc..116 kinds of expressing quantities lower 2.0 Times or more.In lung cancer patient blood plasma, the albumen of 1.9 times of up-regulation or more has 132 kinds, and lowering 2.0 times or more albumen has 119 kinds.

3.TIMP-1 albumen is in the phthisical verifying 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 method, further determines that it in the application established in pulmonary tuberculosis blood testing.

(1) differential expression verifying 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 TIMP-1 ELISA operating procedure it is as follows:

As depicted in figs. 1 and 2, the present invention using commercially available TIMP-1ELISA kit (be purchased from RayBiotech company, USA) supernatant after 125 consumptives and the stimulation of 90 normal healthy controls person's serum and whole blood ESAT6/CFP10 is verified The differential expression of TIMP-1 albumen, sensitivity are 100.0% (95%CI:97,100), specificity for 92% (95%CI:86, 96), area under the curve (AUC) is 0.782 (P < 0.0001,95%CI, 0.688-0.876).

(2) differential expression verifying of the TIMP-1 in infection macrophage

It is limited by this Laboratory biosafety, the present invention is tested in Hua Zhong Agriculture University's agromicrobiology state key It (is studied by Beijing's tuberculosis breast tumor in the laboratory of room virus locellus with Mycobacterium bovis (M.bovis) AF2122/97 Institute professor Li Chuanyou give) mode bacterium has detected the correlation experiment that TIMP-1 expression is infected with mycobacterium tuberculosis.Test institute Source of people macrophage system THP-1 cell (ATCC TIB-202) (passes friend by Beijing Tuberculosis and Thoracic Tumor Research Institute Lee Professor give) Mycobacterium bovis and vaccine strain mycobacterium bovis BCG Tokyo strain (ATCC 35737) are infected (by Beijing Tuberculosis breast tumor research institute of city professor Li Chuanyou give) it is mode bacterium.By THP-1 cell inoculation in 145mm cell culture It in ware, is added PMA (macrophage inducer), final concentration of 40ng/mL, mixing is put in CO₂12h is induced in incubator.Removal Culture medium (contains PMA), is washed twice with the culture medium of incubation, spare.Cell becomes adherent by original suspended state, stretches out pseudo- Foot starts the effect for exercising macrophage.Scattered bacterium is added in Tissue Culture Dish, ratio is infected are as follows: 10:1, mutually Make 12 hours, supernatant (containing bacterium) is collected and is compareed as extracellular bacteria, is washed twice with fresh culture, is completely removed and do not enter The bacterium of cell collected bacterium intracellular and cell sample in 24 hours after 12 hours.Then it is total to be infected front and back macrophage The extraction and quality testing (for conventional method) of RNA, using quantitative fluorescent PCR (for conventional method) detection TIMP-1 at different groups Differential expression (P < 0.05) (referring to Fig. 3) in not.

The present invention obtains the host protein that TIMP-1 has potential mark meaning, while having carried out difference to TIMP-1 albumen Analysis.The expression of TIMP-1 haemocyanin has differences between the two groups as the result is shown.

Leading reference:

[1] Zhang Shuhuan, Wu Bo, Yan Bangfen, Cao Sha, Chen Yingyu, Chao Yanjie, Ling Jieyu, Tan Yadi, Chen Huanchun, Guo like treasure The amalgamation and expression and correlation Analysis China Zoonosis of Mycobacterium bovis antigen MPB70, MPB83, CFP-10 and ESAT-6 Sick magazine, 2007,23 (12): 38-43.

[2] foundation of Chen Ying treasure people IFN-γ release in vitro detection method and its application [J] the life in diagnosis of tuberculosis Object engineering journal .2008,24 (9): 1-6.

Claims (1)

1. prepared by the haemocyanin matrix metalloproteinase inhibitors TIMP-1 of the sequence as shown in sequence table SEQ ID NO:1 Detect the application in pulmonary tuberculosis serum detection kit as unique identification antigen protein.