CN102359988A

CN102359988A - Screening method for mycobacterium tuberculosis drug-resistance protein

Info

Publication number: CN102359988A
Application number: CN2011101784120A
Authority: CN
Inventors: 张华蓉; 林标扬
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2011-06-29
Filing date: 2011-06-29
Publication date: 2012-02-22

Abstract

The invention discloses a screening method for a mycobacterium tuberculosis drug-resistance protein. The method comprises the following steps: (1) selecting a mycobacterium tuberculosis strain which has resistance and/or sensitivity to a drug, wherein, the drug is one or more selected from the group consisting of isoniazid, streptomycin, rifampin and ethambutol; (2) boiling the mycobacterium tuberculosis strain in boiling water with a temperature of 100 DEG C for 10 minutes, carrying out inactivation, collecting bacteria liquid, and freezing the bacteria liquid to a temperature of -80 DEG C for subsequent usage; (3) extracting proteins from the bacteria liquid, isolating the proteins by using gel electrophoresis, carrying out in gel digestion, and extracting peptides after sequencing of the proteins; (4) carrying out mass spectrometry on the peptides after the peptides are separated by using liquid chromatogram, comparing and analyzing obtained results of the mass spectrometry with existing mass spectrometric data of proteins, and screening the mycobacterium tuberculosis drug-resistance protein. The method provided in the invention can comprehensively screen mycobacterium tuberculosis drug-resistance proteins which have drug resistance to commonly used tuberculosis drugs at present, and is of important significance to research on novel drugs which are used for treating diseases caused by mycobacterium tuberculosis, e.g. pneumonia and tuberculosis.

Description

The screening technique of Mycobacterium tuberculosis drug-resistant protein

Technical field

The present invention relates to the screening field of drug-resistant protein, be specifically related to a kind of screening technique of Mycobacterium tuberculosis drug-resistant protein.

Background technology

Much's bacillus (M.tuberculosis) abbreviates tubercle bacillus (tubercle bacilli) as.As far back as 1882, (Robert Koch 1843-1910) just proved that Much's bacillus is a pathogen lungy to German bacteriologist Koch.This bacterium can be invaded each histoorgan of whole body, but sees at most with pulmonary infection.Along with the continuous development of antituberculotic and the improvement of health weather, the M & M of tuberculosis once once declined to a great extent.After the eighties in 20th century, because AIDS and the appearance of Much's bacillus antibody-resistant bacterium, the factors such as application, drug abuse, poverty and movement of population of immunodepressant, epidemic situation lungy worsens suddenly in the global range.According to the WHO statistics, per approximately 3 philtrums in the whole world just have 1 people to infect Much's bacillus, and the Much's bacillus carrying rate is up to 80% in some adult of developing country, and wherein about 5%～10% carrier can develop into active tuberculosis.Recent two decades has infected the Much's bacillus carrier of HIV because AIDS popular and since virus damage body's immunological function, the possibility that develops into active tuberculosis is than infected by HIV person is not high 30～50 times, and the course of disease of tuberculosis is developing faster.In addition, in the development process that HIV infects, tuberculosis is a kind of opportunistic infections that take place the earliest, and tuberculosis has increased the weight of the disease burden of HIV the infected or aids patient, and it is dead that it more is prone to.The whole world 800 ten thousand tuberculosis new cases occur every year approximately at present, and causes about 3 million peoples dead.It is about 250,000 more than that China dies from people lungy every year, is that the twice of all kinds of infectious disease death toll summations is many.Therefore, tuberculosis becomes the global hygienic issues that threatens human health again, and becomes some developing countries and regions, particularly AIDS district occurred frequently crowd's the primary cause of the death.

To contain the tubercle bacillus that isoniazid 1g, streptomysin 10g, rifampin 50g can grow be the Mycobacterium tuberculosis drug-resistant bacterium to commonly used in solid medium, and the tubercle bacillus that can not grow is the tubercle bacillus sensitive bacteria.The antibody-resistant bacterium virulence weakens to some extent.The isoniazid can influence the synthetic of mycolic acid in the cell membrane, induces tubercle bacillus to become the L type, and this possibly be a kind of reason of its anti-isoniazid.Drug sensitivity test shows the isoniazid resistance, and still responsive mostly to rifampin and streptomysin.So treatment many opinions isoniazid and rifampin or pyrazinamide drug combination to reduce chemical sproof generation, heighten the effect of a treatment at present.

If can filter out the drug-resistant protein that present treatment pneumonia and common drug lungy isoniazid (INH), streptomysin (SM), rifampin (RFP) and ethambutol (EMB) is had chemical sproof Mycobacterium tuberculosis drug-resistant bacterial strain; Can seek the virulence factor of pathogen rapidly; Study corresponding newtype drug; Reduce the drug resistance of medicine, improve patient's cure rate.

Summary of the invention

The invention provides a kind of screening technique of Mycobacterium tuberculosis drug-resistant protein, can screen comprehensively present medicine for tuberculosis commonly used is had chemical sproof Mycobacterium tuberculosis drug-resistant protein.

A kind of screening technique of Mycobacterium tuberculosis drug-resistant protein may further comprise the steps:

(1) chooses the tubercle bacillus bacterial strain that medicine is had drug resistance and/or susceptibility;

Described medicine is one or more in isoniazid, streptomysin, rifampin, the ethambutol;

(2) the tubercle bacillus bacterial strain was boiled 10 minutes in 100 ℃ of boiling water, bacterium liquid is collected in deactivation, freezes in-80 ℃ subsequent use;

(3) from bacterium liquid, extract albumen, adopt the gel electrophoresis protein isolate, carry out enzymolysis in the glue, extracting the peptide section behind the protein sequencing;

(4) with carrying out mass spectrophotometry after the separation of peptide section employing liquid chromatography, mass spectrum result and existing protein spectrum data are compared analysis, filter out Mycobacterium tuberculosis drug-resistant protein.

In the step (1), choose the method that medicine is had the tubercle bacillus bacterial strain of drug resistance and/or susceptibility, can adopt existing dosing cultural method to cultivate and obtain.

In order to reach better invention effect, preferably:

In the step (3), described gel electrophoresis is sodium dodecyl sulfate-polyacrylamide gel electrophoresis method (a SDS-PAGE gel electrophoresis).

In the step (3), the method for enzymolysis comprises step in the described glue:

The albumen adhesive tape of gel electrophoresis gained is cut into several bands according to the albumen situation from top to bottom, is cut into micelle again, the 200ml destainer takes off to colourless fully; Separating obtained micelle uses final concentration to reduce 10 minutes 60 ℃ of water-baths as the three carboxyethyl phosphorus WS of 50mmol/L, carries out the halfcystine alkylation again and uses indole-3-acetic acid lucifuge condition incubated at room 1 hour, and separating obtained micelle adds the 200ul destainer and washes twice; Each 15 minutes; In micelle, add the 50ul acetonitrile then, room temperature was placed 15 minutes, discarded acetonitrile; Centrifugal revolve dried; The micelle that revolves after doing adds a 10ul order-checking level trypsin solution, and micelle incubated at room 15 minutes in order-checking level trypsin solution adds the 25mmol/LNH of 20ul ₄HCO ₃Damping fluid, shaken overnight in 37 ℃ of water-baths, enzymolysis is more than 18 hours, and the micelle behind the pancreatin enzymolysis adds 50ul peptide section extract at every turn; Incubated at room 30 minutes is extracted the peptide section, repeats 3 times; The peptide section solution that extracts is merged, centrifugally revolve driedly, dry powder is subsequent use-80 ℃ of preservations.

Described destainer is acetonitrile-ammonium bicarbonate aqueous solution, and wherein the acetonitrile concentration expressed in percentage by volume is 50%, and ammonium bicarbonate concentration is 25mmol/L.

The compound method of described order-checking level trypsin solution is: trypsase is used 25mmol/LNH ₄HCO ₃Damping fluid dissolving is mixed with the mother liquor that trypsinase concentration is 1ug/ul, is 1: 50 according to trypsase with the albumen quality ratio during use, uses 25mmol/L NH ₄HCO ₃Damping fluid is diluted to required concentration.

Described peptide section extract for the acetonitrile concentration expressed in percentage by volume be 50% with the formic acid concentration expressed in percentage by volume be acetonitrile-aqueous formic acid of 5%.

Compared with prior art, the present invention has following advantage:

The inventive method can be screened comprehensively has chemical sproof Mycobacterium tuberculosis drug-resistant protein to present medicine for tuberculosis commonly used, and the newtype drug of the disease that research treatment pneumonia, tuberculosis etc. are caused because of tubercle bacillus has great importance.

Description of drawings

Fig. 1 is desirable protein SDS-PAGE figure;

Fig. 2 is the protein SDS-PAGE figure of tubercle bacillus sample of the present invention.

Embodiment

Embodiment 1

1. instrument and reagent:

Ettan multidimensional liquid chromatograph (Ettan MDLC System) is available from AM General company (GE Healthcare, Pittsburgh, PA USA).The liquid chromatography Trap post and the upgrading reverse chromatograms post (Nanocolumn) of receiving are that fill voluntarily in the laboratory; Trap column packing C18AQ is Michrom Bioresouces; Inc Company products (article No. is PM5/61200/00); 5 μ m;

is filled to column cap with 5um silicon earlier, burns the silicon fusing is blocked, and again filler is filled to 15 * 0.1mm I.D. post.The upgrading reverse chromatograms column packing C18AQ that receives is Michrom Bioresouces; Inc Company products (article No. is PM5/61100/00); 5 μ m;

pulls into quartz capillary head burning pointed earlier; Then head is cut flatly, again filler is filled to 15 * 0.075mm I.D. post.

The LTQ-Orbitrap mass spectrometer is that (Thermo Finnigan, Bremen Germany), are furnished with the upgrading electron spray ionisation source of receiving to U.S. power & light company product.

S peedvac Labconco Centrivap Concentrator traditional vacuum drying system is Lab conco product (Labconco, Kansas City, MO USA).

The Amicon Ultra-15 ultrafiltration pipe that the Ultracel-3 ultra filtration membrane is housed available from Millipore company (Millipore Inc., USA).

Experimental water is the ultrapure water that resistivity is 18.2MQ.cm, and (High Wycombe, UK) purifying obtains by ELGA LabWater purification system.

HPLC level acetonitrile available from Burdick&Jackson company (SK Chemie, Ulson, Korea).Ammonium bicarbonate available from FLUKA company (Sigma-Aaldrich Chemic GmbH, Japan).Protease inhibitor cocktail (Protease inhibitors cocktail) available from Roche Holding Ag (Roche Diagnostics GmbH, Mannheim, Germany).Three carboxyethyl phosphorus (TCEP), Coomassie brilliant blue G-250 and iodoacetamide (IAA) available from Sigma company (Sigma-Aldrich, St.Louis, MO, USA).SDS, extra-pure grade acrylic amide and bisacrylamide are all available from Bio Basic company (Canada).Order-checking level trypsase available from Promega company (Madison, WI, USA).

2. albumen in the extraction bacterial strain

Get 1-8 tubercle bacillus bacterial strain sample as shown in table 1,100 ℃ of boiling water boiled 10 minutes, deactivation, and bacterium liquid is collected in the centrifuge tube, freezes in-80 ℃ of refrigerators, is used for the mass spectrum experiment

Table 1

Numbering	SM	RFP	INH	EMB
					1	R	R	R	S
2	R	R	R	R
					3	R	R	R	R
4	R	R	R	R
					5	S	S	S	S
6	S	S	S	S
					7	S	S	S	S
8	S	S	S	S

In the table 1, R representes drug resistance, and S representes susceptibility.

In the bacterium liquid of 1-8 sample, get half bacterium liquid respectively and be used for leach protein, 4 ℃ in 13000 rev/mins centrifugal, deposition adds 200ul lysate (lysate prescription: 50mM three carboxymethylamino methane (Tris) (pH 7.4); 150mM NaCl (sodium chloride), 1% Triton X-100 (Triton X-100), 1% deoxysodium cholate (sodium deoxycholate); 0.1% sodium dodecylsulphonate (SDS), 0.5mM sodium vanadate (sodium orthovanadate), 50mM sodium fluoride (sodium fluoride); 0.5mM ethylenediamine tetraacetic acid (EDTA), bright tryptic peptide (leupeptin) adds phenylmethylsulfonyl fluoride (PMSF) before using; Make that the PMSF ultimate density is 5mM in the lysate; MM is mmol/L, and percentage is the quality percentage composition), adopt the pulsed frequency ultrasonic degradation one hour of 1s on ice; 4 ℃ 13000 rev/mins centrifugal 1 hour, get supernatant.

(Centriprep Centrifugal Filter Unit with Ultracel-3membrane, millipore) concentrated supernatant obtains 1-8 sample concentration liquid respectively with 3kD ultrafiltration pipe.

3. mensuration protein concentration

3.1 measure the many protein concentrations (BCA quantification of protein kit, thermo scientific, article No. 23225) of 1-8 sample concentration liquid respectively with the BCA method, each is measured three results that average and sees table 2.The result can find out from table 2, though through concentrating, protein concentration is still very low, and the total amount of albumen is not high yet, the work that therefore can't remove high-abundance proteins.Therefore, adopt the SDS-PAGE gel electrophoresis to analyze.

Table 2 determination of protein concentration result

Numbering	1	2	3	4	5	6	7	8
									Concentration/(ug/ul)	1.766	1.202	2.841	1.214	1.412	1.8464	1.6288	1.3672

3.2SDS-PAGE gel electrophoresis

1-8 sample concentration liquid is respectively got the 50ug albuminous degeneration; 12% (mass percent) SDS-PAGE carries out the albumen one dimension to be separated, and parameter is set to: concentrate glue voltage 60V and ran 30 minutes, ran until concentrated glue; Sample concentration becomes a line; Strengthen voltage and run separation gel to 150V, about 2 hours of time is up to sample-loading buffer (1mol/l three carboxymethylamino methane hydrochloride (pH8.8) 0.6ml; 10% sodium dodecylsulphonate (SDS) 2ml; 50% glycerine 5ml; 2 mercapto ethanol 0.5ml; 1% bromjophenol blue 1ml; Water 0.9ml, percentage are the quality percentage composition) almost run out of glue and stop.With PAGE glue dyeing 20 minutes, use destainer (methyl alcohol: acetate: water=4: 1: 5, volume ratio) decolouring then, with 2% (mass percent) Coomassie brilliant blue till the glue background color is very shallow.Gel imaging system scanning.Desirable proteins gel electrophoresis figure should be as shown in Figure 1, and the proteins gel electrophoresis that obtains is as shown in Figure 2, not ideal enough.Possibly be lower, due to the albumen sample more complicated by protein content.

3.3 enzymolysis in the glue

According to the albumen situation glue is cut into 8 bands from top to bottom.Each band is cut into about 1mm ³The micelle of size, and the 200ml destainer (acetonitrile-ammonium bicarbonate aqueous solution, wherein the acetonitrile volume ratio is 50%; Ammonium bicarbonate concentration is 25mM) take off fully to colourless, separating obtained micelle uses final concentration to reduce 10 minutes 60 ℃ of water-baths as the TCEP WS of 50mM, carries out the halfcystine alkylation again and uses IAA (iodoacetamide) lucifuge condition incubated at room 1 hour; Separating obtained micelle adds the 200ul destainer and washes twice, each 15 minutes, in micelle, adds the 50ul acetonitrile then; Room temperature was placed 15 minutes; To remove the water in the micelle, discard acetonitrile, with Speedvac (Labconco Centrivap Concentrator; Kansas City, MO USA) centrifugal revolve dried.Revolve micelle after doing add a 10ul order-checking level trypsase (Promega, Madison, WI, USA) solution, the compound method of order-checking level trypsin solution is: trypsase is used 25mM NH ₄HCO ₃Damping fluid dissolving, being mixed with trypsinase concentration is the mother liquor packing of 1ug/ul, is 1: 50 according to trypsase with the albumen quality ratio during use, uses 25mM NH ₄HCO ₃Damping fluid is diluted to required concentration.Micelle incubated at room 15 minutes in order-checking level trypsin solution adds the 25mM NH of 20ul ₄HCO ₃Damping fluid, shaken overnight in 37 ℃ of water-baths, enzymolysis is more than 18 hours.Micelle behind the pancreatin enzymolysis add at every turn 50ul peptide section extract (the acetonitrile concentration expressed in percentage by volume be 50% with the formic acid concentration expressed in percentage by volume be acetonitrile-aqueous formic acid of 5%); Incubated at room 30 minutes; Extract the peptide section, repeat 3 times, the peptide section solution that extracts is merged; Revolve driedly with Speedvac is centrifugal, dry powder is used for follow-up mass spectrophotometry-80 ℃ of preservations.

3.4 the upgrading Ultra Performance Liquid Chromatography of receiving is united the esi-msn analysis

Before the appearance, it is resuspended with the WS that 20ul contains 0.1% (percent by volume) formic acid to revolve dried peptide hydrolysis on carrying out liquid chromatography mass, and centrifugal 15 minutes of 15000g gets supernatant and carries out the liquid chromatography mass analysis.

Adopt Ettan multidimensional liquid chromatography to analyze.The 2ul sample with the WS that contains 0.1% (percent by volume) formic acid uses flow velocity as appearance on the speed of 15ul/min also with enrichment in 4 minutes to the trap post.Mobile phase A is for containing the WS of 0.1% (percent by volume) formic acid and 5% (percent by volume) acetonitrile, and Mobile phase B is the WS that contains 0.1% (percent by volume) formic acid and 95% (percent by volume) acetonitrile.The peptide section was separated gradient elution under the use 150-250nl/min flow velocity: at 0-60 minute; Mobile phase B (B liquid) volume ratio is elevated to 35% from 3%; In 60-75 minute, be elevated to 85% from 35%, kept 85%B liquid 10 minutes, within 5 minutes, make B liquid get back to 3% then.Each scanning of the mass spectrum finishes the initial 3%B liquid balance of back chromatographic column adopting 20 minutes.Twice of every sample repetition running.The peptide section of chromatographic resolution directly gets into mass spectrum analysis, mass spectrometer be the Thermo scientific company LTQ-Orbitrap tandem mass spectrometer that has the nano-ESI source (Thermo scientific, Bremen, Germany).Before the scanning of the mass spectrum, proofread and correct with the correcting fluid of thermo company, with the accuracy of ensuring the quality of products.In the scope of 300-2000Da, positive ion mode adopts down Orbitrap to carry out full scan, during resolution 400Da 60000, selects 5 ions the highest to carry out the second order ms analysis after each full scan, the record experimental data.

3.5 the foundation of tubercle bacillus albumen database

Download the database of different tubercle bacillus strains at NCBI and other websites,, go redundancy, obtain the albumen database that new form is fasta, called after mix.fasta its merging.In addition one of them database 25946.fasta is also carried out roving commission.

3.6 mass spectrometric data retrieval

Using Bioworks browser is that (Thermo scientific, Bremen Germany) retrieves sequest software 3.2 versions.Adopt the new albumen database that makes up voluntarily, the raw mass spectrum data that obtain are retrieved.Search argument is provided with as follows: the quality error of peptide section and fragmention is set to acquiescence; Restriction enzyme site is the arginine and the lysyl oxidase butt formula of pancreatin, allows to lack a restriction enzyme site in the peptide section that identifies; The Search Results error rate is no more than 4%; In fixing the modification, because IAA alkylation halfcystine alkylation (Cys_CAM) therefore adds 57Da molecular weight (halfcystine in each peptide section all increases the 57Da molecular weight) on halfcystine; Because albumen is prepared through one dimension poly acrylamide gel electrophoresis (SDS-PAGE); Halfcystine possibly generate (Cys_PAM) with the acrylamide monomer reaction; Therefore the variable modification that in data search, adds a 14Da molecular weight, the variable modification of halfcystine (quality of Cys-PAM and Cys-CAM differs 14Da); In data search, the methionine oxidative modification (+16Da) be considered to another variable modification; Identify that a required minimum fragmention number of peptide section is 3, the required minimum fragmention number of albumen is 7; The maximum mass number of albumen is set to be no more than 25,000Da.

3.7TPP analyze

Download Trans-Proteomics Pipeline (TPP) software from http://tools.proteomecenter.org/TPP.php, install (version is 4.3.1), the mass spectrometric data of gained is carried out confluence analysis.Earlier SEQUEST result is changed OUT and DTA file, convert it into the pep.xml file again; Mass spectrum RAW file conversion is become the mzXML file; With pep.xml running paper PeptideProphet, obtain a total pep.XML file; This pepXML running paper ProteinProphet with gained obtains final albumen result.

3.8 false positive rate analysis

Make up any storehouse, itself and original protein database are merged, the albumen database when searching for as false positive rate, the result carries out TPP and analyzes, and calculates false positive rate, is lower than 5%.

3.9 mass spectrum result

Adopt the detected albumen of TPP behind two data library searchings from sensitive strain and persister; Can find out from the result who identifies; In sensitive strain and persister tubercle bacillus, all detect the albumen more than 1000, but possibility reduces greatly but than higher albumen number.When adopting the 25946.fasta database, sensitive strain have only 14 possibilities 0.9 or more and identify two reach more than the albumen of unique corresponding polypeptide.

To in sensitive strain, adopt the 25946.fasta database retrieval to 137 albumen identifying of 14 albumen and persister compare discovery; Having only 5 albumen is that persister and sensitive strain are owned together, and all the other albumen all only detect at persister or sensitive strain.Equally, the albumen that adopts the mix.fasta database to identify is also compared, wherein 3 albumen are all detected in two groups of samples, all the other albumen also only therein one group detect.With all these Identification of Fusion Protein to the number of times sum (total num peps) that occurs of unique corresponding peptide section be less than or equal to 10 all be normalized to and 10 calculate; Selection has the albumen conduct albumen truly of difference more than 2 times; The albumen of these differences has 6, and the albumen of these 6 differences is exactly the relevant albumen of Mycobacterium tuberculosis drug-resistant property.The result sees table 3.

The albumen that has 2 times of differences in table 3 persister and the sensitive strain

Difference	Albumen (protein)	?tot_num_r	tot_num_s	Ratio (r/s)	Describe
						Only_s	sp\|A5TZG6\|CH601_MYCTA	?10	104	0.09615385
Only_s	sp\|P66004\|DLDH_MYCTU	?10	29	0.34482759
						Only_s	sp\|A5TZ77\|DNAK_MYCTA	?10	29	0.34482759
Only_r	tr\|A1UJ44\|A1UJ44_MYCSK	?45	10	4.5	Mkms_3658
						Only_r	tr\|A1UMV8\|A1UMV8_MYCSK	?23	10	2.3	Mkms_4977
Only_r	tr\|A1UMF1\|A1UMF1_MYCSK	?20	10	2	Mkms_4819

In the table 3; Only_s representes only to appear in the sensitive bacteria; Only_r representes only to appear in the drug-fast bacteria, and tot_num_r representes the number of times that the corresponding unique peptide section of albumen occurs in drug-fast bacteria, and tot_num_s representes the number of times that the corresponding unique peptide section of albumen occurs in sensitive bacteria.

The albumen of above-mentioned 6 differences is in the numbering of lane database, following to the note and the amino acid whose sequence of albumen:

＞sp|A5TZG6|CH601_MYCTA?60kDa?chaperonin?1?OS＝Mycobacteriumtuberculosis(strain?ATCC?25177/H37Ra)GN＝groL1

MAKTIAYDEEARRGLERGLNALADAVKVTLGPKGRNVVLEKKWGAPTITNDGVSIAKEIE

LEDPYEKIGAELVKEVAKKTDDVAGDGTTTATVLAQALVREGLRNVAAGANPLGLKRGIE

KAVEKVTETLLKGAKEVETKEQIAATAAISAGDQSIGDLIAEAMDKVGNEGVITVEESNT

FGLQLELTEGMRFDKGYISGYFVTDPERQEAVLEDPYILLVSSKVSTVKDLLPLLEKVIG

AGKPLLIIAEDVEGEALSTLVVNKIRGTFKSVAVKAPGFGDRRKAMLQDMAILTGGQVIS

EEVGLTLENADLSLLGKARKVVVTKDETTIVEGAGDTDAIAGRVAQIRQEIENSDSDYDR

EKLQERLAKLAGGVAVIKAGAATEVELKERKHRIEDAVRNAKAAVEEGIVAGGGVTLLQA

APTLDELKLEGDEATGANIVKVALEAPLKQIAFNSGLEPGVVAEKVRNLPAGHGLNAQTG

VYEDLLAAGVADPVKVTRSALQNAASIAGLFLTTEAVVADKPEKEKASVPGGGDMGGMDF

＞sp|P66004|DLDH_MYCTU?Dihydrolipoyl?dehydrogenaseOS＝Mycobacterium?tuberculosis?GN＝lpd

MTHYDVVVLGAGPGGYVAAIRAAQLGLSTAIVEPKYWGGVCLNVGCIPSKALLRNAELVH

IFTKDAKAFGISGEVTFDYGIAYDRSRKVAEGRVAGVHFLMKKNKITEIHGYGTFADANT

LLVDLNDGGTESVTFDNAIIATGSSTRLVPGTSLSANVVTYEEQILSRELPKSIIIAGAG

AIGMEFGYVLKNYGVDVTIVEFLPRALPNEDADVSKEIEKQFKKLGVTILTATKVESIAD

GGSQVTVTVTKDGVAQELKAEKVLQAIGFAPNVEGYGLDKAGVALTDRKAIGVDDYMRTN

VGHIYAIGDVNGLLQLAHVAEAQGVVAAETIAGAETLTLGDHRMLPRATFCQPNVASFGL

TEQQARNEGYDVVVAKFPFTANAKAHGVGDPSGFVKLVADAKHGELLGGHLVGHDVAELL

PELTLAQRWDLTASELARNVHTHPTMSEALQECFHGLVGHMINF

＞sp|A5TZ77|DNAK_MYCTA?Chaperone?protein?dnaKOS＝Mycobacterium?tuberculosis(strain?ATCC?25177/H37Ra)GN＝dnaK

MARAVGIDLGTTNSVVSVLEGGDPVVVANSEGSRTTPSIVAFARNGEVLVGQPAKNQAVT

NVDRTVRSVKRHMGSDWSIEIDGKKYTAPEISARILMKLKRDAEAYLGEDITDAVITTPA

YFNDAQRQATKDAGQIAGLNVLRIVNEPTAAALAYGLDKGEKEQRILVFDLGGGTFDVSL

LEIGEGVVEVRATSGDNHLGGDDWDQRVVDWLVDKFKGTSGIDLTKDKMAMQRLREAAEK

AKIELSSSQSTSINLPYITVDADKNPLFLDEQLTRAEFQRITQDLLDRTRKPFQSVIADT

GISVSEIDHVVLVGGSTRMPAVTDLVKELTGGKEPNKGVNPDEVVAVGAALQAGVLKGEV

KDVLLLDVTPLSLGIETKGGVMTRLIERNTTIPTKRSETFTTADDNQPSVQIQVYQGERE

IAAHNKLLGSFELTGIPPAPRGIPQIEVTFDIDANGIVHVTAKDKGTGKENTIRIQEGSG

LSKEDIDRMIKDAEAHAEEDRKRREEADVRNQAETLVYQTEKFVKEQREAEGGSKVPEDT

LNKVDAAVAEAKAALGGSDISAIKSAMEKLGQESQALGQAIYEAAQAASQATGAAHPGGE

PGGAHPGSADDVVDAEVVDDGREAK

＞tr|A1UJ44|A1UJ44_MYCSK?Putative?GAF?sensor?proteinOS＝Mycobacterium?sp.(strain?KMS)GN＝Mkms_3658

MTGFDEWLNRLLEDCARDAGEDVVSYVARAVASQMVADLRRTERASIDELMAHLNECRVF

AETEMPSVESEITDPDRLQALYATGLLDSPPDEAYDRITRAAAAALDAPSAAMSLIDVDR

QFFKSSVGMGDDERQTPLDRSVCQYAVANGSALLLEDARTDPVFRNHPAVRDGTMVAYLG

IPLTDKDDNAVGTLCVFDVKPRLWGTGHVRILSDLAQLAAEKMFGSRAGQQ

＞tr|A1UMV8|A1UMV8_MYCSK?Aspartate-semialdehyde?dehydrogenaseOS＝Mycobacterium?sp.(strain?KMS)GN＝Mkms_4977

MVNIGVVGATGQVGQVMRTLLEERDFPATSVRFFASARSQGRKLPFRGQEIEVEDAGTAD

PSGLDIALFSAGATMSRVQAPRFAAAGAVVVDNSSAWRKDPDVPLVVSEVNFERDAGTRP

KGIIANPNCTTMAAMPVLKPLHDEAGLVRMIASTYQAVSGSGLAGVEELFGQASAVVSGS

RDLVHDGGAVDFPAPNKYVAPIAFNVVPLAGSLVDDGSGETDEDQKLRNESRKILGIPEL

LVSGTCVRVPVYTGHSLSINAEFAQPLSVDRAKELLAAAPGVKLVDVPTPLAAAGIDESL

VGRIRQDPGAPEGRGLALFISGDNLRKGAALNTIQIAELLAAQL

＞tr|A1UMF1|A1UMF1_MYCSK?Putative?uncharacterized?proteinOS＝Mycobacterium?sp.(strain?KMS)GN＝Mkms_4819

MQRFDVPAAGPDSALTVTWAGVTTLLVDDGSSALMTDGFFSRPGLASVGLRRIGPSAARV

DGCLARLRVDRLDAVLPVHTHFDHALDSALVADRAGAVLIGGASAALLGRGHGLPESRIS

TVTSGATITAGAYDLTLVESGHCPPDRFPGAITTPVTPPARASAYRCGEAWSVLVRHRPT

AHTVLIVGSAGFVPGALHGTRADVVYLGVGQLGRQPEAYLRDYWEQTVRAVGARRVVLIH

WDDFFRPLHRPLRALPYVADDLDATMRGLSRLAEIDGVPLHLPQLWQRADPWR

Claims

1. the screening technique of a Mycobacterium tuberculosis drug-resistant protein may further comprise the steps:

(4) with carrying out mass spectrophotometry after the separation of peptide section employing liquid chromatography, mass spectrum result and existing albumen database are compared analysis, filter out Mycobacterium tuberculosis drug-resistant protein.

2. the screening technique of Mycobacterium tuberculosis drug-resistant protein according to claim 1 is characterized in that, in the step (3), described gel electrophoresis is the sodium dodecyl sulfate-polyacrylamide gel electrophoresis method.

3. the screening technique of Mycobacterium tuberculosis drug-resistant protein according to claim 1 is characterized in that, in the step (3), the method for enzymolysis comprises step in the described glue:

4. the screening technique of Mycobacterium tuberculosis drug-resistant protein according to claim 3 is characterized in that, described destainer is acetonitrile-ammonium bicarbonate aqueous solution, and wherein the acetonitrile concentration expressed in percentage by volume is 50%, and ammonium bicarbonate concentration is 25mmol/L.

5. the screening technique of Mycobacterium tuberculosis drug-resistant protein according to claim 3 is characterized in that, the compound method of described order-checking level trypsin solution is: trypsase is used 25mmol/L NH ₄HCO ₃Damping fluid dissolving is mixed with the mother liquor that trypsinase concentration is 1ug/ul, is 1: 50 according to trypsase with the albumen quality ratio during use, uses 25mmol/L NH ₄HCO ₃Damping fluid is diluted to required concentration.

6. the screening technique of Mycobacterium tuberculosis drug-resistant protein according to claim 3 is characterized in that, described peptide section extract for the acetonitrile concentration expressed in percentage by volume be 50% with the formic acid concentration expressed in percentage by volume be acetonitrile-aqueous formic acid of 5%.