CN108918890A - The method for screening the ligandin of the people of selectively targeted mycobacterium tuberculosis - Google Patents
The method for screening the ligandin of the people of selectively targeted mycobacterium tuberculosis Download PDFInfo
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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
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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 the methods of the ligandin for the people for screening selectively targeted mycobacterium tuberculosis.The present invention provides a kind of methods for the human protein that screening can interact with tuberculoprotein, include the following steps：It will hybridize respectively with human protein chip from the mycoprotein of mycobacterium tuberculosis and secretory protein, the human protein to interact with the mycoprotein and/or the secretory protein be determined to according to results of hybridization.The present invention is extracted the mycoprotein and secretory protein of mycobacterium tuberculosis, and further with HuProtTMHuman protein chip is hybridized, and the specific human albumen that can be interacted with tuberculoprotein is obtained：84 people's albumen, these specific human albumen will likely be used for Diagnosis of Tuberculosis and tuberculosis therapy.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to screens the ligand egg of the people of selectively targeted mycobacterium tuberculosis
White method.
Background technique
Tuberculosis is still to seriously threaten human health, popular most one of wide, the highest communicable disease of case fatality rate, often
Year is global, and there are about 10,000,000 new cases, and 1,600,000 people are dead, and death toll occupies first of single infectious disease.As cause lungy
Germ, mycobacterium tuberculosis have been evolved by long-term evolution as a kind of extremely successful pathogenic bacteria intracellular.Mycobacterium tuberculosis
After entering human body by respiratory tract, main parasitic escapes the immune of host cells in macrophage, and by its a variety of strategy
Lethal effect can slowly replicate, long-term surviving in macrophage, finally generate retention-state.When host immunity is low
When, the mycobacterium tuberculosis of suspend mode can reactivate, and leading to body development is active tuberculosis.Mycobacterium tuberculosis from into
Enter host cell until immune response complicated in host is excited during host's morbidity, on the one hand by adjusting place
The balance of main immune system is allowed to be more suitable for it in host's endobiosis, and another aspect under appropriate circumstances again can be fast
Speed breeding causes tuberculosis, or even eventually leads to host's death.However, infection lungy and pathogenic molecular mechanism are still not
It is clear.But accommodating is not doubted, and the process of tuberculosis infection and morbidity is also that complexity occurs between mycobacterium tuberculosis and host
Interaction process, can be with the host protein of mycobacterium tuberculosis direct interaction tuberculosis infection and cause a disease
Bridge and key identify people's albumen of these specificity, it will help the molecular mechanism for deeply disclosing Tuberculosis, it can also
Potential drug target is provided for diagnosing and treating lungy.
Summary of the invention
The object of the present invention is to provide the methods of the ligandin for the people for screening selectively targeted mycobacterium tuberculosis, and
The related application of the human protein screened.
In a first aspect, the side for the human protein that a kind of claimed screening can interact with tuberculoprotein
Method.
The method for the human protein that screening provided by the present invention can interact with tuberculoprotein, it may include following step
Suddenly：It will hybridize respectively with human protein chip from the mycoprotein of mycobacterium tuberculosis and secretory protein, according to miscellaneous
Knot fruit is determined to the human protein to interact with the mycoprotein and/or the secretory protein.
In the method, from the mycoprotein of the mycobacterium tuberculosis and the secretory protein be according to
What the method included the following steps was prepared：
(1) extraction of mycobacterium tuberculosis mycoprotein：The thallus of the mycobacterium tuberculosis is resuspended in PBS, so
By multigelation, ultrasound, centrifugation, gained supernatant is the solution for containing the mycoprotein.
Further, before the thallus of the mycobacterium tuberculosis being resuspended in PBS, may also include the steps of：With
PBS washs the thallus of the mycobacterium tuberculosis (such as washing 2 times).The number of the multigelation can be 3 times.The ultrasound
Condition can be：P=800W, ultrasound 2 seconds, interval 2 seconds, totally 40 minutes.The condition of the centrifugation can be 4 DEG C of 12000rpm centrifugations 10
Minute.
(2) extraction of mycobacterium tuberculosis secretory protein：After the culture supernatant filtration sterilization of the mycobacterium tuberculosis
Ultrafiltration is carried out, the liquid retained in super filter tube is the solution for containing the secretory protein.
0.22 μm of filter can be used when further, by the culture supernatant filtration sterilization of the mycobacterium tuberculosis.It carries out
Centrifugal ultrafiltration pipe (3KD) can be used when the ultrafiltration.It may also include the step of being washed with PBS (such as after carrying out the ultrafiltration
It washes 3 times).
Further, in the mycoprotein to the mycobacterium tuberculosis and before the secretory protein extracts
It may also include the steps of：By the mycobacterium tuberculosis be inoculated into 7H9 fluid nutrient medium (U.S. company BD, article No. 271310,
Containing 10% growth-promoting additive OADC, 0.5% Tween-80, % indicates volumn concentration) in culture to logarithmic growth phase, so
After carry out inactivation treatment, supernatant thallus is collected by centrifugation；The supernatant is the training of the mycobacterium tuberculosis in step (2)
Supernatant is supported, the thallus is the thallus of the mycobacterium tuberculosis in step (1).
More specifically, the mycobacterium tuberculosis is inoculated into the 7H9 fluid nutrient medium and is cultivated to logarithmic growth
Phase can cultivate 3 weeks for 37 DEG C for the mycobacterium tuberculosis to be inoculated into the 7H9 fluid nutrient medium.The inactivation treatment can be
It inactivates within 10 minutes in 90 DEG C of water.The centrifugation can be centrifuged 10 minutes for 2000rpm.
In the method, the mycoprotein and the secretory protein hybridize with human protein chip respectively be
It is carried out according to the method included the following steps：
(1) it closes：The human protein chip is placed in confining liquid, and (the TBST solution containing 5%BSA, % indicate g/
Incubation at room temperature 1.5-2 hours in 100ml), shake (60rpm) on the oscillator；
(2) protein sample prepares：The mycoprotein and the secretory protein are diluted to final concentration 1 with confining liquid respectively
μ g/ml is mixed, is protected from light, saves on ice；
(3) protein sample and chip hybridization：5ml step (2) ready protein sample is added to what step (1) had been closed
On the human protein chip, room temperature, which is shaken, is incubated for 2h, is protected from light；
(4) it develops a film：With TBST solution and ddH2The processed human protein chip of the successive washing step of O (3), keeps away
Light；
(5) dry plate, scanning；
(6) data are analyzed：It scans the image obtained and analyzes acquisition initial data with GenePix Pro software, then with letter
It makes an uproar than (SNR：Net signal value divided by background ratio) level of evaluating the point positive, if SNR>2, then it is assumed that this albumen is sun
Property signaling point.
Further, the human protein chip is HuProtTMHuman protein chip.The mycobacterium tuberculosis is tuberculosis
Mycobacteria laboratory standard strain H37Rv.
In a specific embodiment of the invention, the final resulting mankind's egg that can be interacted with the tuberculoprotein
White totally 84, shown in text specific as follows.
Second aspect, in claimed 84 human proteins it is all or part of in tuberculoprotein interaction
Using.
Wherein, 84 human proteins are OVOL1 (5017), ARID3A (1820), RNF113B (140432), SMAD2
(4087), (4899) NRF1, SRCAP (10847), ZNF699 (374879), SP6 (80320), DPRX (503834), INSM1
(3642), (56242) ZNF253, ZNF410 (57862), SMAD4 (4089), HOXB5 (3215), SRY (6736), KLF8
(11279), (148268) ZNF570, ZNF700 (90592), HOXB9 (3219), ESRRG (2104), ZNF557 (79230),
ZNF23 (7571), ZNF100 (163227), ZNF337 (26152), ZNF343 (79175), IKZF3 (22806), STAT5B
(6777), (5607) MAP2K5, GMEB2 (26205), FOXM1 (2305), SKOR2 (652991), VENTX (27287),
ZBTB12 (221527),
ZNF425 (155054), TSC22D4 (81628), GLIS2 (84662), ZNF136 (7695), ZNF569
(148266), (3214) HOXB4, MTA2 (9219), ZNF541 (84215), KLF11 (8462), ZNF540 (163255), RORA
(6095), (4780) NFE2L2, HNF4A (3172), PCK2 (5106), MBTPS1 (8720), MAPK3 (5595), VPS8
(23355), (9741) LAPTM4A, PPCDC (60490), SGCA (6442), BCL2L14 (79370), SLC39A8 (64116),
SF3B2 (10992), GLO1 (2739), ITM2B (9445), CSRP2BP (57325), NDUFB6 (4712), CYP39A1
(51302), (5984) RFC4, PARVA (55742), CD300LB (124599), GNB1L (54584), CPA1 (1357), LDHA
(3939), (1045) CDX2, SOX4 (6659), ACP1 (52), ZNF70 (7621), MBD2 (8932), BARX2 (8538),
IKZF4 (64375), PPARD (5467), IRF2 (3660), ZFP91 (80829), SOX10 (6663), FOXD3 (27022),
GRM8 (2918), KIAA2018, BAT1, FLJ20366 and C6orf166.More than, the number in bracket is the coding of corresponding albumen
The identifiable unique gene number of the Entrez ID, NCBI of gene.
The application may be either the application (being such as used purely for scientific research) in non-disease diagnoses and treatment, can also examine for disease
Application in disconnected treatment.
The third aspect, in claimed 84 human proteins described previously it is all or part of it is following it is any in
Application：
(A1) tuberculoprotein is detected；
(A2) product for detecting tuberculoprotein is prepared.
The application may be either the application (being such as used purely for scientific research) in non-disease diagnoses and treatment, can also examine for disease
Application in disconnected treatment.
Fourth aspect, in claimed 84 human proteins described previously it is all or part of it is following it is any in
Application：
(B1) mycobacterium tuberculosis is detected；
(B2) product for detecting mycobacterium tuberculosis is prepared.
The application may be either the application (being such as used purely for scientific research) in non-disease diagnoses and treatment, can also examine for disease
Application in disconnected treatment.
5th aspect, in claimed 84 human proteins described previously it is all or part of it is following it is any in
Application：
(C1) tuberculosis is diagnosed and/or treated；
(C2) preparation is for diagnosing and/or treating product lungy.
It, can be by detecting detection of the tuberculoprotein realization to mycobacterium tuberculosis in fourth aspect.In the 5th side
In face, it can be realized by detecting the tuberculoprotein to diagnosis lungy.
In the second to five aspect application, the tuberculoprotein can be for from the thallus egg of mycobacterium tuberculosis
White and/or secretory protein.
It further, can be according to from the mycoprotein of the mycobacterium tuberculosis and/or the secretory protein
Correlation technique described in first aspect is prepared above.
The present invention is extracted the mycoprotein and secretory protein of mycobacterium tuberculosis, and further with HuProtTMMankind's egg
White chip is hybridized, and the specific human albumen that can be interacted with tuberculoprotein is obtained：84 people's albumen, these
Specific human albumen will likely be used for Diagnosis of Tuberculosis and tuberculosis therapy.
Detailed description of the invention
Fig. 1 is that the mycobacterium tuberculosis secretory protein that the present invention extracts and mycoprotein SDS-PAGE running gel coomassie are bright
Indigo plant dyeing.
After mycobacterium tuberculosis secretory protein and mycoprotein and human protein chip hybridization that Fig. 2 extracts for the present invention
As a result.
Fig. 3 is the ligandin with 84 people of mycobacterium tuberculosis protein interaction.A is to secrete egg with mycobacterium tuberculosis
44 ligandins of white and the common interaction of mycoprotein people；B be only with the special interaction of mycobacterium tuberculosis mycoprotein
30 ligandins of people；C is 10 ligandins only with the people of the special interaction of mycobacterium tuberculosis secretory protein.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1, the selectively targeted mycobacterium tuberculosis of screening people ligandin
One, the extraction of mycobacterium tuberculosis mycoprotein and secretory protein
For trying mycobacterium tuberculosis：Mycobacterium tuberculosis laboratory standard strain H37Rv (country of BJ Chest Science Hospital tuberculosis
Clinical labororatory).
(1) mycobacterium tuberculosis is inoculated into 7H9 fluid nutrient medium (U.S. company BD, article No. 271310, containing 10% growth-promoting
Long additive OADC, 0.5% Tween-80, % indicate volumn concentration) afterwards 37 DEG C cultivate 3 weeks (in logarithmic growth phase), in
It inactivates within 10 minutes in 90 DEG C of water, 2000rpm is centrifuged 10 minutes, collects bacterium, collects supernatant thallus respectively.In order to utmostly obtain
Mycobacterium tuberculosis all protein, we carry out secretory protein and bacterium to mycobacterium tuberculosis culture supernatant and its thallus respectively
The extraction of body protein.
(2) extraction of mycobacterium tuberculosis mycoprotein：Thallus is washed 2 times with PBS, is resuspended in PBS, multigelation 3
After secondary, ultrasonic on ice (P=800W, ultrasound 2 seconds, interval 2 seconds) totally 40 minutes.Liquid after ultrasound is centrifuged in 4 DEG C of 12000rpm
10 minutes, supernatant was mycoprotein solution.
(3) extraction of mycobacterium tuberculosis secretory protein：Collect the culture supernatant of mycobacterium tuberculosis, 0.22 μm of filter mistake
Bacterium is filtered out, ultrafiltration is carried out using centrifugal ultrafiltration pipe (3KD), removes medium component and other small-molecule substances, and wash 3 with PBS
Time.The liquid retained in super filter tube is secretory protein solution.
The mycobacterium tuberculosis secretory protein and mycoprotein SDS-PAGE running gel coomassie brilliant blue staining such as Fig. 1 of extraction
It is shown.As seen from the figure：Secretory protein total amount height is higher, and mainly based on the albumen of 70kDa, mycoprotein is higher because extracting difficulty
And total amount is less, and mainly based on the albumen of 80kDa.
Two, the human protein of the protein-protein interaction experimental study based on human protein chip and tuberculoprotein interaction
HuProtTMHuman protein chip：CDI Products, article No. MA-005.Contain 19000 mankind on the chip
Protein.
1, the preservation of protein chip：
HuProt will be housedTMThe plastic core film magazine of human protein chip is stored in -80 DEG C of refrigerators or is placed on dry ice.Core
Piece should be directly placed into confining liquid after taking out, and what it is with bar code is the front of chip on one side.
2, it closes：
5.0ml confining liquid is added in every hole in 4 orifice plates (5%BSA/TBS-T, % indicate g/100ml).Carefully use tack
Tweezers take out chip from the plastic casing being placed on dry ice, are immediately placed in four orifice plates added with confining liquid, just face
On, it is incubated at room temperature 1.5-2 hours, gently shakes (60rpm) on the oscillator.
3, the preparation of sample：
Mycoprotein and secretory protein equivalent that step 1 is extracted are diluted to 1 μ g/ml of final concentration with confining liquid, mixed,
It is protected from light, saves on ice.
4, sample and chip hybridization：
Confining liquid in 4 orifice plates is sopped up from angle, is carefully added into the ready protein sample of step 3 (5.0ml), is protected
Hold chip front side upward.Notice that sample loading gun is first not touch chip surface surely.Room temperature, which is shaken, is incubated for 2h, is protected from light.
5, it develops a film：
After incubation, the liquid in 4 orifice plates is sopped up from angle, 5.0ml TBS-T, short rinse three are added in every hole
It is secondary.Then 5.0ml TBS-T is added in every hole, 10 minutes (60rpm) of washing is shaken in room temperature, in triplicate.Use ddH2O is quick
Rinsing three times, pays attention to being protected from light in experimentation.
6, dry plate：
Blotting paper is layered on to the bottom of Glass carrier box, it is also possible to which the centrifuge tube of 50ml, each Glass carrier box can fill multiple
Chip, but each centrifuge tube may only fill a chip.
Chip is taken out from four orifice plates with blunt-ended forceps, is placed on blotting paper vertically, is drawn from chip edge remaining
Moisture is careful not to touch the surface of chip！Chip is placed in Glass carrier box or in 50ml centrifuge tube vertically, is centrifuged,
800rpm, 3 minutes (low-speed centrifugal is had in attention, in order to avoid make chip rupture).Chip is transferred to after centrifugation new clean
In Glass carrier box.
7, it scans and saves：
If the time allows, dried chip can be scanned at once, chip can also be placed in the Glass carrier box being protected from light
In, -20 DEG C of preservations.Pay attention to：Chip must be scanned within three days after the test.
8, data are analyzed：
It scans the image obtained and analyzes acquisition initial data with GenePix Pro software, then use signal-to-noise ratio (SNR：Net letter
Number value divided by background ratio) level of evaluating the point positive, if SNR>2, then it is assumed that this albumen is positive signal point.
9, result
(1) protein chip results of hybridization such as Fig. 2 institute of the mycobacterium tuberculosis secretory protein and mycoprotein and people that extract
Show.As seen from the figure：In addition to positive control point, there is the positive on chip after mycobacterium tuberculosis secretory protein and mycoprotein hybridization
Protein site shows screening to corresponding interaction albumen.
(2) collected all signals are analyzed with GenePix Pro software and obtains initial data (.gpr file), then
With signal-to-noise ratio (SNR：Net signal value divided by background ratio), the level of the point positive is evaluated, if SNR>2, then it is assumed that this egg
White is positive signal point.Filter out ligandin totally 84 of the people with mycobacterium tuberculosis secretory protein and mycoprotein interaction
(Fig. 3).Wherein, with mycobacterium tuberculosis secretory protein and the ligandin of the people of the common interaction of mycoprotein 44 (in Fig. 3
A)；The only ligandin 30 (B in Fig. 3) with the people of the special interaction of mycobacterium tuberculosis mycoprotein；Only with tuberculosis branch bar
The ligandin 10 (C in Fig. 3) of the people of the special interaction of bacterium secretory protein.
The present invention uses people's holoprotein chip technology, screening and mycobacterium tuberculosis protein (secretory protein and bacterium for the first time
Body protein) interaction people ligandin.It is finally obtained the mycobacterium tuberculosis ligand egg of 84 selectively targeted people
It is white, including OVOL1 (5017), ARID3A (1820), RNF113B (140432), SMAD2 (4087), NRF1 (4899), SRCAP
(10847), (374879) ZNF699, SP6 (80320), DPRX (503834), INSM1 (3642), ZNF253 (56242),
ZNF410 (57862), SMAD4 (4089), HOXB5 (3215), SRY (6736), KLF8 (11279), ZNF570 (148268),
ZNF700 (90592), HOXB9 (3219), ESRRG (2104), ZNF557 (79230), ZNF23 (7571), ZNF100
(163227), (26152) ZNF337, ZNF343 (79175), IKZF3 (22806), STAT5B (6777), MAP2K5 (5607),
GMEB2 (26205), FOXM1 (2305), SKOR2 (652991), VENTX (27287), ZBTB12 (221527),
ZNF425 (155054), TSC22D4 (81628), GLIS2 (84662), ZNF136 (7695), ZNF569
(148266), (3214) HOXB4, MTA2 (9219), ZNF541 (84215), KLF11 (8462), ZNF540 (163255), RORA
(6095), (4780) NFE2L2, HNF4A (3172), PCK2 (5106), MBTPS1 (8720), MAPK3 (5595), VPS8
(23355), (9741) LAPTM4A, PPCDC (60490), SGCA (6442), BCL2L14 (79370), SLC39A8 (64116),
SF3B2 (10992), GLO1 (2739), ITM2B (9445), CSRP2BP (57325), NDUFB6 (4712), CYP39A1
(51302), (5984) RFC4, PARVA (55742), CD300LB (124599), GNB1L (54584), CPA1 (1357), LDHA
(3939), (1045) CDX2, SOX4 (6659), ACP1 (52), ZNF70 (7621), MBD2 (8932), BARX2 (8538),
IKZF4 (64375), PPARD (5467), IRF2 (3660), ZFP91 (80829), SOX10 (6663), FOXD3 (27022),
GRM8 (2918), KIAA2018, BAT1, FLJ20366 and C6orf166.More than, the number in bracket is the coding of corresponding albumen
The identifiable unique gene number of the Entrez ID, NCBI of gene.
Claims (10)
1. a kind of method for the human protein that screening can interact with tuberculoprotein, includes the following steps：It will be from knot
The mycoprotein and secretory protein of core mycobacteria are hybridized with human protein chip respectively, are determined to according to results of hybridization
The human protein to interact with the mycoprotein and/or the secretory protein.
2. according to the method described in claim 1, it is characterized in that：From the mycoprotein of the mycobacterium tuberculosis
It is prepared by a method comprising the following steps with the secretory protein：
(1) extraction of mycobacterium tuberculosis mycoprotein：The thallus of the mycobacterium tuberculosis is resuspended in PBS, is then passed through
Multigelation, ultrasound, centrifugation, gained supernatant are the solution for containing the mycoprotein；
(2) extraction of mycobacterium tuberculosis secretory protein：It will be carried out after the culture supernatant filtration sterilization of the mycobacterium tuberculosis
Ultrafiltration, the liquid retained in super filter tube are the solution for containing the secretory protein.
3. method according to claim 1 or 2, it is characterised in that：In step (1), the number of the multigelation is 3
It is secondary；And/or
The condition of the ultrasound is：P=800W, ultrasound 2 seconds, interval 2 seconds, totally 40 minutes；And/or
The condition of the centrifugation is that 4 DEG C of 12000rpm are centrifuged 10 minutes；
And/or
In step (2), 0.22 μm of filter will be used when the culture supernatant filtration sterilization of the mycobacterium tuberculosis；And/or
It carries out using 3KD centrifugal ultrafiltration pipe when the ultrafiltration；And/or
Further include the steps that being washed with PBS after carrying out the ultrafiltration；
And/or
To the mycobacterium tuberculosis the mycoprotein and the secretory protein extract before further include following steps：
The mycobacterium tuberculosis is inoculated into culture in 7H9 fluid nutrient medium then to carry out inactivation treatment to logarithmic growth phase, be centrifuged
Collect supernatant thallus；The supernatant is the culture supernatant of the mycobacterium tuberculosis in step (2), and the thallus is
The thallus of the mycobacterium tuberculosis in step (1)；
And/or
The human protein chip is HuProtTMHuman protein chip；
And/or
The mycobacterium tuberculosis is mycobacterium tuberculosis type strain H37Rv.
In 4.84 human proteins it is all or part of with the application in tuberculoprotein interaction；
84 human proteins be ZNF70, MBD2, BARX2, IKZF4, PPARD, IRF2, ZFP91, SOX10, FOXD3,
GRM8、OVOL1、ARID3A、KIAA2018、RNF113B、SMAD2、NRF1、SRCAP、ZNF699、SP6、DPRX、INSM1、
ZNF253、ZNF410、SMAD4、HOXB5、SRY、KLF8、ZNF570、ZNF700、HOXB9、ESRRG、ZNF557、ZNF23、
ZNF100、ZNF337、ZNF343、IKZF3、STAT5B、MAP2K5、GMEB2、FOXM1、SKOR2、VENTX、ZBTB12、
ZNF425、TSC22D4、ZNF136、ZNF569、HOXB4、MTA2、ZNF541、KLF11、ZNF540、GLIS2、RORA、
NFE2L2、HNF4A、PCK2、MBTPS1、MAPK3、VPS8、BAT1、LAPTM4A、PPCDC、SGCA、BCL2L14、SLC39A8、
FLJ20366、SF3B2、GLO1、ITM2B、CSRP2BP、NDUFB6、CYP39A1、RFC4、PARVA、CD300LB、GNB1L、
CPA1, C6orf166, LDHA, CDX2, SOX4 and ACP1.
In 5.84 human proteins it is all or part of it is following it is any in application：
(A1) tuberculoprotein is detected；
(A2) product for detecting tuberculoprotein is prepared；
84 human proteins be ZNF70, MBD2, BARX2, IKZF4, PPARD, IRF2, ZFP91, SOX10, FOXD3,
GRM8、OVOL1、ARID3A、KIAA2018、RNF113B、SMAD2、NRF1、SRCAP、ZNF699、SP6、DPRX、INSM1、
ZNF253、ZNF410、SMAD4、HOXB5、SRY、KLF8、ZNF570、ZNF700、HOXB9、ESRRG、ZNF557、ZNF23、
ZNF100、ZNF337、ZNF343、IKZF3、STAT5B、MAP2K5、GMEB2、FOXM1、SKOR2、VENTX、ZBTB12、
ZNF425、TSC22D4、ZNF136、ZNF569、HOXB4、MTA2、ZNF541、KLF11、ZNF540、GLIS2、RORA、
NFE2L2、HNF4A、PCK2、MBTPS1、MAPK3、VPS8、BAT1、LAPTM4A、PPCDC、SGCA、BCL2L14、SLC39A8、
FLJ20366、SF3B2、GLO1、ITM2B、CSRP2BP、NDUFB6、CYP39A1、RFC4、PARVA、CD300LB、GNB1L、
CPA1, C6orf166, LDHA, CDX2, SOX4 and ACP1.
In 6.84 human proteins it is all or part of it is following it is any in application：
(B1) mycobacterium tuberculosis is detected；
(B2) product for detecting mycobacterium tuberculosis is prepared；
84 human proteins be ZNF70, MBD2, BARX2, IKZF4, PPARD, IRF2, ZFP91, SOX10, FOXD3,
GRM8、OVOL1、ARID3A、KIAA2018、RNF113B、SMAD2、NRF1、SRCAP、ZNF699、SP6、DPRX、INSM1、
ZNF253、ZNF410、SMAD4、HOXB5、SRY、KLF8、ZNF570、ZNF700、HOXB9、ESRRG、ZNF557、ZNF23、
ZNF100、ZNF337、ZNF343、IKZF3、STAT5B、MAP2K5、GMEB2、FOXM1、SKOR2、VENTX、ZBTB12、
ZNF425、TSC22D4、ZNF136、ZNF569、HOXB4、MTA2、ZNF541、KLF11、ZNF540、GLIS2、RORA、
NFE2L2、HNF4A、PCK2、MBTPS1、MAPK3、VPS8、BAT1、LAPTM4A、PPCDC、SGCA、BCL2L14、SLC39A8、
FLJ20366、SF3B2、GLO1、ITM2B、CSRP2BP、NDUFB6、CYP39A1、RFC4、PARVA、CD300LB、GNB1L、
CPA1, C6orf166, LDHA, CDX2, SOX4 and ACP1.
In 7.84 human proteins it is all or part of it is following it is any in application：
(C1) tuberculosis is diagnosed and/or treated；
(C2) preparation is for diagnosing and/or treating product lungy；
84 human proteins be ZNF70, MBD2, BARX2, IKZF4, PPARD, IRF2, ZFP91, SOX10, FOXD3,
GRM8、OVOL1、ARID3A、KIAA2018、RNF113B、SMAD2、NRF1、SRCAP、ZNF699、SP6、DPRX、INSM1、
ZNF253、ZNF410、SMAD4、HOXB5、SRY、KLF8、ZNF570、ZNF700、HOXB9、ESRRG、ZNF557、ZNF23、
ZNF100、ZNF337、ZNF343、IKZF3、STAT5B、MAP2K5、GMEB2、FOXM1、SKOR2、VENTX、ZBTB12、
ZNF425、TSC22D4、ZNF136、ZNF569、HOXB4、MTA2、ZNF541、KLF11、ZNF540、GLIS2、RORA、
NFE2L2、HNF4A、PCK2、MBTPS1、MAPK3、VPS8、BAT1、LAPTM4A、PPCDC、SGCA、BCL2L14、SLC39A8、
FLJ20366、SF3B2、GLO1、ITM2B、CSRP2BP、NDUFB6、CYP39A1、RFC4、PARVA、CD300LB、GNB1L、
CPA1, C6orf166, LDHA, CDX2, SOX4 and ACP1.
8. application according to claim 6 or 7, it is characterised in that：It is by detecting the tuberculosis egg in claim 6
It is white to realize to the detection of mycobacterium tuberculosis；
It is to be realized by detecting the tuberculoprotein to diagnosis lungy in claim 7.
9. according to the application any in claim 4-8, it is characterised in that：In the application, the tuberculoprotein is next
Derived from the mycoprotein and/or the secretory protein of the mycobacterium tuberculosis.
10. application according to claim 9, it is characterised in that：From the thallus egg of the mycobacterium tuberculosis
The white and/or described secretory protein is prepared according to method as stated in claim 2.
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