CN1952168A - Method for screening mycobacterium tuberculosis multidrug-resistance protein - Google Patents
Method for screening mycobacterium tuberculosis multidrug-resistance protein Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology of screening novel antituberculosis drugs target, vaccinum antigen and testing novel markers. The invention provides a method for screening the protein of Bacillus tuberculosis rifampicine-resistant: First, cultivating multi-drug-resistant strains; Secondly, separating the were isolated strains of multi-drug-resistant and sensitive strains of multi drug-resistant strains and protein from sensitive strains separately; Thirdly, determining the multi-drug-resistant protein by comparing the proteins of multi-drug-resistant strains and sensitive strains; Finally, separating and identifying the multi-drug-resistant protein. This invention provides a method which can accurately separate and identify multi-drug-resistant protein of Bacillus tuberculosis. The invention provides novel way for better understand of the multi-drug-resistant mechanism of Bacillus tuberculosis, rapid detecting of clinical multi-drug-resistant strains and the development of vaccine and drug target.
Description
Technical field
The invention belongs to molecular biology and screen novel antitubercular agent target, vaccine antigen and detection target field, specifically, the invention provides a kind of protein technique that passes through, the method for the multidrug-resistance protein in the screening tuberculosis bar mycobacterium.The present invention also provides this method to detect the evaluation of mark and the application for preparing aspects such as vaccine at the screening of medicine target, the bacterial strain of anti-the multiple medicines.
Background technology
Tuberculosis still constitutes huge threat to the mankind.The global at present population near 32% has infected tubercule bacillus.Have every year 2700000 people to die from tuberculosis approximately, average every day is dead above 7000 people.The World Health Organization announces that in 1993 tuberculosis is the global healthy emergency state.
China's tuberculosis epidemic situation is more serious, is one of the high burden of 22 tuberculosis in whole world country, and the tuberculosis number occupies the second place of the world.The 4th national tuberculosis epidemiological random sampling survey interpretation of result of China in 2000, China's epidemic situation lungy is very severe, is only second to India and occupies the second place of the world in the whole world.The whole nation has 5.5 hundred million people to infect tubercule bacillus at present, 4,510,000 active tuberculosis patients, and wherein the infectivity lunger reaches 2,000,000.Annual because of tuberculosis death person reaches 130,000, be maximum a kind of of death toll in the various transmissible diseases.
WHO and international anti-consumptive disease and tuberculosis federation (IUATLD) have announced the monitoring result to anti-multiple medicines tuberculosis in 72 countries and regions, the whole world in March, 2000, among the new case at least the average anti-multiple medicines rate to a kind of antitubercular agent be 10.7% (1.7%～36.9%).Existing 5.5 hundred million people of China are subjected to tubercle bacillus affection at present, and total anti-multiple medicines rate is 27.8%, and initial anti-multiple medicines rate is 18.6%, and acquired anti-multiple medicines rate is 46.5%-68.4%, and anti-multiple medicines rate is 10.7%, the existing tuberculosis patient of anti-multiple medicines the 55.5 ten thousand.
Anti-multiple medicines tuberculosis (MDR-TB): general definition is meant the anti-tuberculosis (Xiamen meeting in 1997) of antitubercular agent more than two kinds of tubercule bacillus.Modern age, WHO proposed the narrow sense definition about MDR-TB, was meant the tuberculosis of the anti-at least vazadrine of tubercule bacillus, Rifampin two medicines or more antitubercular agents.Because of vazadrine, Rifampin are most important tuberculosis main ingredients, tubercule bacillus all has an immense impact on to clinical and popular control to the anti-multiple medicines of this two medicine.
Before the nineties in 20th century, the anti-multiple medicines tubercule bacillus that is separated to from the tuberculosis new patient is less than 5%.Yet according to the up-to-date a report of survey of the U.S., any in 5 kind of first line antituberculosis drugs (vazadrine, Rifampin, Streptomycin sulphate, ethambutol, pyrazinoic acid amide) had drug-fast tubercule bacillus accounted for 13%, wherein simultaneously vazadrine and Rifampin are had drug-fast tubercule bacillus and accounted for 3.2%.The eruption and prevalence lungy that is caused by the tubercule bacillus with multiple drug resistance has taken place repeatedly in New York city,U.S and Florida State, and the mortality ratio that causes can be up to 72%-89% in the AIDS people; And the time to death of falling ill is also from shortening to 4-16 week more than 1 year.The resistance problem of tubercule bacillus is very serious in the certain areas of China.
Estimate among 2,000,000,000 the infecteds of the whole world, have 5,000 ten thousand people to infect the MTB bacterial strain of anti-the multiple medicines approximately at present according to The World Health Organization (WHO), the 500000 anti-multiple medicines cases of having an appointment every year occur, and wherein have 2/3 tuberculosis patient that the danger that anti-multiple medicines tuberculosis (MDR-TB) takes place is arranged.
At present, can not in time treat, isolate the patient because the diagnosis of MDR-TB and the cycle of traditional drug sensitive test are long, conventional antitubercular agent is not enough to treat MDR-TB, causes its course of disease prolongation, infectivity to increase, and becomes one of important contagium lungy and major causes of death; MDR-TB increases more than 50 times single case associated cost simultaneously, wastes a large amount of health resourceses.The merging of present anti-multiple medicines tuberculosis and acquired immune deficiency syndrome (AIDS) is popular to bring more severe challenge to tuberculosis control work.Therefore illustrate the anti-multiple medicines molecular mechanism of mycobacterium tuberculosis, the method for setting up rapid detection and monitoring MDR-TB has been problem demanding prompt solution, and it can be early diagnosis, treatment patient, medicine and vaccine development and formulates anti-epidemic measure and establish molecular basis.
But the protein technique high-throughput is described the protein group feature of microorganism, use the method for comparison protein group, people can promptly seek the virulence factor of pathogenic bacteria, clear and definite pathogenesis, make up new generation vaccine, promote novel therapeutic medicine of anti-pathogenic bacterium and developing rapidly of diagnostic reagent.At present, used the comparison protein group and learned a skill the protein expression of mycobacterium composite flora is studied, and the genetic expression of mycobacterium tuberculosis (M.tb) under multiple envrionment conditions is analyzed.But still do not have the full cell of seeing about the clinical bacterial strain of anti-multiple medicines of M.tb and form proteic comparative analysis.
Summary of the invention
One of purpose of the present invention provides a kind of method of screening the mycobacterium tuberculosis multidrug-resistance specific proteins.
Two of purpose of the present invention provides gene of anti-multiple medicines the and protein pool, for novel anti tuberculosis drug development provides the medicine target; For the new technology of developing the clinical detection bacterial strain of anti-the multiple medicines provides gene pool and protein library; Provide candidate gene and albumen library for making up novel tuberculosis control vaccine.
The invention provides a kind of method of screening the mycobacterium tuberculosis multidrug-resistance specific proteins; described medicine is 3-{[(4-methyl isophthalic acid-piperazinyl) imino-] methyl }-rifomycin (RIF; Rifampin); 4-pyridine formyl hydrazine-vazadrine (INH; the vazadrine); { 2R; 2[S-(R*; R*)]-R}-(+) 2; 2 '-(1; 2-second two basic diiminos)-two-1-butanols dihydrochloride-Tibutol (EMB; Tibutol); O-2-methylamino--2-deoxidation-α-L-Glucopyranyl-(1 → 2)-O-5-deoxidation-3-C-formyl radical-α-L-lysol furyl glycosyl-(1 → 4)-N1; N3-diamidino-D-streptamine vitriol-Streptomycin sulphate (SM; Streptomycin sulphate) and Zinamide-pyrazinoic acid amide (PZA; pyrazinoic acid amide), promptly present 5 kinds of clinical line antitubercular agents, this method may further comprise the steps successively:
(1) the screening bacterial strain of anti-multiple medicines the in clinical tubercule bacillus strain isolated is the bacterial strain of anti-the multiple medicines to satisfy the bacterial strain that five kinds of medicine minimum inhibitory concentrations are higher than corresponding critical resistance concentration simultaneously; The critical resistance concentration of five kinds of medicines is respectively: imino-3-{[(4-methyl isophthalic acid-piperazinyl)] methyl }-rifomycin is 16 μ g/ml, 4-pyridine formyl hydrazine-vazadrine is 2 μ g/ml, { 2R, 2[S-(R*, R*)]-R }-(+) 2,2 '-(1,2-second two basic diiminos)-two-1-butanols dihydrochloride-Tibutol is 4 μ g/ml, O-2-methylamino--2-deoxidation-α-L-Glucopyranyl-(1 → 2)-O-5-deoxidation-3-C-formyl radical-α-L-lysol furyl glycosyl-(1 → 4)-N1, N3-diamidino-D-streptamine vitriol-Streptomycin sulphate is 8 μ g/ml, and Zinamide-pyrazinoic acid amide is 16 μ g/ml; The strain of tubercule bacillus standard test, the clinical sensitive strain cultivated with not dosing under the same terms are contrast; (" sensitive strain " is meant that the tolerance to said medicine all is lower than the critical resistant organism concentration of relative medicine)
(2) albumen in difference separation criterion experiment strain, clinical sensitive strain and the bacterial strain of anti-the multiple medicines;
(3) albumen in the bacterial strain of anti-multiple medicines the and standard test strain, the clinical sensitive strain is compared, with the albumen that in the bacterial strain of anti-the multiple medicines, increases or lack, the perhaps albumen of at least 2 times of expression amount increases, perhaps to be reduced by at least 50% albumen be anti-multiple medicines associated protein to expression amount; (be with proteic expression amount in standard test strain or the clinical sensitive strain respectively with the bacterial strain of anti-the multiple medicines in the comparative result of corresponding protein be foundation)
(4) isolation identification multidrug-resistance protein.
Among the present invention, the cultural method of tubercule bacillus can be with reference to conventional cultural method, for example, adopt and add 10%ADC (DIFCO, 0.5% bovine serum albumin, 0.2% glucose, 140mmol/LNaCl) and the Middlebrook7H9Broth (DIFCO of 0.5% glycerine, Bection-Dickinson) substratum carries out liquid culture.
Among the present invention, can only keep basic protein in (2).
Among the present invention, detecting the middle proteic bacterial strain uses therefor of separation and Extraction in minimum inhibitory concentration and (2) in (1) can be the tubercule bacillus that is in logarithmic phase.
Among the present invention, can adopt electrophoresis, centrifugal in (2) and (4), saltout, method protein isolate such as filtration or chromatography.Among the present invention, except that particularly pointing out, used experiment condition all adopts routine operation method and experiment condition.
Among the present invention, can pass through methods such as protein electrophoresis atlas analysis, protein sequencing, mass spectroscopy or amino acid composition analysis in (4) and identify albumen.
Among the present invention, the multidrug-resistance protein that (4) are obtained can be used for screening or detects antitubercular agent as the medicine target.Screening or the main thought that detects antitubercular agent be, can with the multidrug-resistance protein binding interactions, perhaps can influence the antitubercular agent that material that multidrug-resistance protein expresses promptly can be used as the candidate.Molecular structure with bioinformatics method simulation multidrug-resistance protein and multidrug-resistance protein, filter out can with the material of multidrug-resistance protein binding interactions, then, further determine its action site and the mechanism of action, simultaneously, can adopt the checking of experimental strain or laboratory animal.
Among the present invention, the encoding gene that (4) can be obtained multidrug-resistance protein suddenlys change, and is used to make up vaccine.After identifying multidrug-resistance protein, can further determine the key amino acid residue that it causes anti-multiple medicines, these amino acid that suddenly change then, and with corresponding protein or its encoding nucleoside acid construct vaccine after suddenling change.
Among the present invention, can detect the mycobacterium tuberculosis multidrug-resistance bacterial strain according to the multidrug-resistance protein that (4) obtain.
The present invention systematically compares proteome analysis to the intracellular protein of the clinical bacterial strain of anti-the multiple medicines first, simultaneously, compare with human-like experimental standard strain H37Rv and clinical complete quick strain, detect the clinical bacterial strain of anti-multiple medicines of 8 strains, clinical complete quick strain of 9 strains and type strain H37Rv be totally 18 strain M.tb bacterial strains, and each sample carries out repeated experiments at least 3 times.Found 15 anti-multiple medicines associated protein of M.tb, Rv0738 (M1), TrpC (Rv1611 such as wherein screened, M2), and OpcA (Rv1446, M5), CysK (Rv2334, M8), DevR (Rv3133c, M11), and nuoB (Rv3146,13#), Rv2624c (M14), and ClpP (Rv2461, M6), (Rv3002 10#) waits 10 gene up-regulated expressions for Rv2629 (M9) and ilvN; Rv0992 (M4), Rv2159 (7#), Rv0927 (M3), PPE51 (Rv3136, M12) and trkA (Rv2691 M15) waits 5 downward modulations to express.Through looking into new no relevant report.
Tuberculosis is maximum a kind of of death toll in the various transmissible diseases.The diagnosis of the anti-multiple medicines tuberculosis cycle loaded down with trivial details and traditional drug sensitive test is grown and makes the patient can not in time isolate, obtain medical treatment, and becomes one of important contagium lungy and major causes of death; Anti-multiple medicines tuberculosis has increased more than 50 times the treatment associated cost simultaneously, wastes a large amount of health resourceses, has also increased the weight of the burden of patient and family thereof simultaneously.Method provided by the invention is the isolation identification mycobacterium tuberculosis multidrug-resistance protein accurately, for the exploitation of the rapid detection of understanding the anti-multiple medicines mechanism of M.tb, clinical anti-multiple medicines strain in depth and vaccine, medicine target provides new approach.
Description of drawings
Fig. 1 is a M.tb H37Rv strain tropina 2-DE collection of illustrative plates (pH4～7).Wherein, 2-DE is a two-dimensional electrophoresis; Be numbered differentially expressed protein site (back together) among the figure such as M1.
Fig. 2 is the clinical full sensitive strain tropina 2-DE collection of illustrative plates of M.tb (pH4～7).
Fig. 3 is the clinical bacterial strain of anti-multiple medicines tropina of a M.tb 2-DE collection of illustrative plates (pH4～7).
Fig. 4 is the comparison (pH4～7) of the bacterial strain of anti-multiple medicines tropina M2 cutting spectrum.Wherein, (a) M.tb H37Rv, (b) clinical full sensitive strain, (c) bacterial strain of anti-multiple medicines the, (d) protein expression quantitative comparison.Arrow indication place is different strains proteic electrophoresis inequality site.
Fig. 5 is for being the comparison (pH4～7) of the bacterial strain of anti-multiple medicines tropina M11 cutting spectrum.Wherein, (a) M.tbH37Rv, (b) clinical full sensitive strain, (c) bacterial strain of anti-multiple medicines the, (d) protein expression quantitative comparison.Arrow indication place is different strains proteic electrophoresis inequality site.
Fig. 6 is a M.tb H37Rv thalline basic protein 2-DE collection of illustrative plates (pH7～10).
Fig. 7 is the clinical bacterial strain of anti-multiple medicines thalline of a M.tb basic protein 2-DE collection of illustrative plates (pH7～10).
Fig. 8 is the comparison (pH7～10) of the bacterial strain of anti-multiple medicines thalline basic protein M4 cutting spectrum.Wherein, (a) M.tbH37Rv, (b) clinical full sensitive strain, (c) bacterial strain of anti-multiple medicines the, (d) protein expression quantitative comparison.Arrow indication place is different strains proteic electrophoresis inequality site.
The peptide finger printing that Fig. 9 is ordered for M15.Wherein, X-coordinate is specific charge (m/z), and ordinate zou is protein content (%intensity).Mass spectrum parameter: 4700 reflector spec #1 MC[BP=732.5,17942].
(MSMS is a tandem mass spectrum to 1487.86 MSMS that Figure 10 is ordered for M15,1487.86 molecular weight that polypeptide fragments had for the evaluation of PMF mass spectrum; This peptide Duan Zaijing amino acid degradation carries out further amino acid mass spectroscopy, to determine its amino acid composition and arrangement).Wherein, X-coordinate is specific charge (m/z), and ordinate zou is protein content (%intensity).Mass spectrum parameter: 4700 MS/MS Precursor, 1487.86 spec #1[BP=771.8,216].
1762.02 MSMS that Figure 11 is ordered for M15.Wherein, X-coordinate is specific charge (m/z), and ordinate zou is protein content (%intensity).Mass spectrum parameter: 4700 MS/MS, 1762.02 Precursor spec #1MC[BP=1078.1,137].
Embodiment
The cultivation and the drug sensitive experiment of embodiment 1 Mycobacterium tuberculosis
Mycobacterium tuberculosis type strain Mycobacterium t uberculosis H37Rv (ATCC93009), the clinical bacterial strain of anti-multiple medicines the (8 strain), clinical complete quick strain (9 strain) provides by Shanghai Pulmonary Hospital tuberculosis research department.With inoculation to having added 10%ADC (DIFCO, 0.5% bovine serum albumin, 0.2% glucose, 140mmol/LNaCl) and the Middlebrook 7H9Broth (DIFCO of 0.5% glycerine, Bection-Dickinson) in, leave standstill in 37 ℃ of constant incubators and cultivated 15～20 days, when cell concentration reach to bacterial density be 1 * 10
8～2 * 10
8Or optical density(OD) (OD
600) be to collect thalline between 0.8～1.
80 ℃ of heating in water bath 2h are with killing bacteria.Through 5000rpm, abandon supernatant (supernatant discards earlier) after centrifugal for 4 ℃ behind 121 ℃ of 30min autoclavings, with the PBS damping fluid washing bacterium of pH7.4 2 times, bacterial sediment is put-80 ℃ of cryogenic refrigerators and is stored standby.
Adopt BACTEC MGIT-960 method detection of drugs susceptibility, medicine adopts Sigma company standard sample, and article No. is non-not to be: Rifampin, and R 3501; The vazadrine, I 3377; Tibutol, E 4630; Streptomycin sulphate, S 6501; Pyrazinoic acid amide, P 7136.The clinical complete quick bacterial strain of 9 strains is lower than 0.01 μ g/ml to the 0.5ug/mL of Rifampin to vazadrine MIC value, and Tibutol MIC value is lower than 0.5 μ g/ml, and Streptomycin sulphate MIC value is lower than 0.5 μ g/ml, and pyrazinoic acid amide MIC value is lower than 0.5 μ g/ml.The 8 strain bacterial strains of anti-the multiple medicines are respectively 16ug/mL (1 strain) 32ug/mL (4 strain) and 64ug/mL (3 strain) to the MIC value of Rifampin; MIC value to the vazadrine is respectively 4ug/mL (2 strain) 16ug/mL (2 strain) and 32ug/mL (4 strain); MIC value to Tibutol is respectively 8ug/mL (3 strain) 16ug/mL (5 strain); MIC value to Streptomycin sulphate is respectively 16ug/mL (3 strain) 32ug/mL (2 strain) and 64ug/mL (3 strain); MIC value to pyrazinoic acid amide is respectively 32ug/mL (5 strain) 64ug/mL (3 strain).
Embodiment 2-10 concrete steps are seen patent application " a kind of method of screening Mycobacterium tuberculosis drug-resistant protein " (application number 200610117294.1)
The extraction of embodiment 2 tropinas
The extraction of embodiment 3 basic proteins
Embodiment 6 second is to the SDS-PAGE electrophoresis
Embodiment 7 proteinic silver dye
The analysis of embodiment 8 gel images
Embodiment 9MALDI-TOF-MS peptide fingerprint map analyzing
Method finishing screen of the present invention is chosen 15 albumen, specifically sees Table 1.Wherein have and somely are known proteins and are proved to be with tubercule bacillus close ties are arranged.
PMF is meant that (peptide mass fingerprinting, PMF), this method is a present proteome research identification of protein authentication method comparatively commonly used to peptide quality fingerprinting spectrum.Because the peptide section sequence that aminoacid sequence (primary structure) difference of range protein, protein produce after by enzymolysis is also different, the peptide mixt total mass number is also had a characteristic, is called peptide quality fingerprinting spectrum (PMF), can be used for proteinic evaluation.Usually after the protein of interest site being downcut, through enzyme (often being pancreatin) degraded, after the molecular weight of each enzymatic fragment of mass spectroscopy, according to these information search databases, the peptide fragment that obtains mating.According to the overlapping search of different fragments, determine the protein of primary source, and provide the score value of a coupling.
Such as peptide finger printing of the present invention coupling use PepIdent (
Www.expasy.org/tools/peptident. Pl).Parameter is set to: source of species-bacterium; Mw-apparent molecular weight ± 30%; The pI-system of not limiting; Enzyme-pancreatin; Allow the restriction enzyme site-1 ignored; Modified types-iodacetylization, score value greater than 56 can be under the probability of P＜0.05 definite target protein matter.The M1 result as a result that table 1 obtains, the PMF score value is---160 (4/5), refer to that promptly 5 mass spectrum fragments are searched library searching after, 4 degradation fragments of determining position Rv0738 wherein, it is 160 that original segments is defined as this proteic score value.
Whole 15 proteic functions are sorted out in the his-and-hers watches 1, the functional classification standard of institute's foundation (http://genolist.pasteur.fr/TubercuList/).0: virulence, detoxifcation adapts to; 1: the metabolism of lipoid (chemical combination) thing; 2: signal path; 3: cell walls and cell processing; 5: insertion sequence and antibiotic; 6:PE/PPE; 7: intermediary metabolism and breathing; 8: the unknown; 9: regulate albumen; 10: conservative false albumen.
The function of most of anti-multiple medicines associated protein concentrates on the 7th class and the 10th class, and the function of this two proteinoid belongs to metabolism and respiratory intermediate product and conservative putative protein, and is in the majority with the former.As seen, after bacterium produces anti-multiple medicines, mainly be that some participate in metabolism and respiratory albumen changes, thereby by regulating the resistance of metabolism influence to multiple antitubercular agent.
15 differential proteins relevant with many resistances are identified out, wherein 9 albumen change jointly with Rifampin list resistance, its function no longer is repeated in this description, and sees inventor's " a kind of method of screening Mycobacterium tuberculosis drug-resistant protein " (application number 200610117294.1) for details.
M1, Rv0738 is a kind of conservative protein, Unknown Function.Adopt the ICAT method that M.tb genome encoding albumen is identified, find that this is a membranin, but its function is not explanation [Schmidt F., Donahoe S., Hagens K., Mattow J., Schaible U.E., Kaufmann S.H.E., Aebersold R., Jungblut P.R..Complementary Analysis of the Mycobacterium tuberculosis Proteome byTwo-dimensional Electrophoresis and Isotope Coded Affinity Tag Technology[J] .Molecular ﹠amp; Cellular Proteomics, 2004,3 (1): 24-42].The variation of inferring this proteinoid can cause the change of cell membrane function, thereby changes the perviousness of bacterium to medicine, stops drug absorption or promotes pumping of medicine.In clinical full Resistant strain and sensitive strain, the synchronous up-regulated expression of this albumen.
M2, TrpC is indole-3-glycerolphosphate synthase (IGPS).Catalysis the 4th step reaction [Hu D.S.J. in the tryptophane biosynthesis pathway, Hood D.W., HeidstraR., Hodgson D.A..The Expressionof the TrpD, TrpC and TrpBA genes of Streptomyces coelicolor A3 (2) is Regulatedby Growth Rate and Growth Phase but not by Feedback Repression[J] .MolecularMicrobiology, 1999,32 (4): 869-880].Proved that by the transposition sudden change this is the constitutive enzyme in M.tb and M.bovis.Its aminoacid sequence and Mammals are without any homology.Therefore, IGPS can be used as candidate's medicine target site preferably, screen effective tuberculosis reagent [Yanping Yang in view of the above, Min Zhang, HongmeiZhang, Jianqiang Lei, Ruiliang Jin, Shengfeng Xu, Jialing Bao, Lu Zhang, andHonghai Wang.Purification and Characterization of Mycobacterium tuberculosisIndole-3-glycerol Phosphate Synthase[J]. Biochemistry (Moscow), 2006,71 (1): 38-43].The same with Rv0738, this albumen is also only relevant with the full resistance of M.tb, compare with H37Rv, up-regulated expression in persister is constant in the complete quick strain.
M4, Rv0992 is the discrepancy of finding in full Resistant strain basic protein detects, and changes not obvious in single Resistant strain.Expression amount descends in the full resistance.Though this albumen is expressed, do not see so far about the information of its function.
M5, OpcA can mediate thalline to endotheliocyte with epithelially stick, locate in intrusion and the born of the same parents.Although about few known to this proteic concrete function, find in our research, different resistance patterns, protein expression mode are different fully.In Rifampin list Resistant strain, this albumen downward modulation is expressed, and is similar to clinical complete quick bacterial strain, but is obvious up-regulated expression in full Resistant strain.The diametrically opposite expression pattern of this same albumen is reminded us, and the expression of OpcA may depend on the resistance of M.tb to medicine, and different anti-liquid medicine is flat, this proteic regulative mode difference.Simultaneously, may there be different mechanism in the many resistances of M.tb with single resistance.
M6, the P1 subunit of ClpP, ClpP is a casein hydrolysis proteolytic enzyme, belongs to the serine stretch protein enzyme family.Strong [the Smith of homology in bacterium, C.K., Baker, T.A., and Sauer, R.T.Lon and Clp FamilyProteases and Chaperones Share Homologous Substrate-recognition Domains[J] .Proc Natl Acad, 1999,96:6678-6682:Wu, W.F., Zhou, Y., and Gottesman, S..Redundant in vivo Proteolytic Activities of Escherichia coli Ion and the ClpYQ (HslUV) Protease[J] .J Bacteriol, 1999,181 (12): 3681-3687].A large amount of studies have shown that pathogenic bacteria need could be caused a disease by the dependent proteolytic enzyme of this ATP, so be that its virulence is necessary., false folding, location of mistake destroyed or when just merely not bringing into play katalysis, the intravital protein of pathogenic bacteria can be degraded usually as ClpP.Pathogenic bacteria is resisted host's defense system [Susan M.Butler by this way, RichardA.Festa, Michael J.Pearce, K.Heran Darwin.Self-compartmentalized bacterialproteases and pathogenesis[J] .Molecular Microbiology, 2006,60 (3): 553-562].
In M.tb, scavenger cell can produce NO, and NO can suppress to invade the growth of M.tb, and in secular interaction process, Mycobacterium tuberculosis has also produced NO-resistant gene mpa and pafA in born of the same parents.The Mpa AAAATPase that encodes, can form 6 aggressiveness structure [the Nancianne Knipfers similar to the ClpP-ATP enzyme, Anjali Seth, Stanislav G.Roudiak, Thomas E.Shrader.Species Variation in ATP-dependentProtein Degradation:Protease Profiles Differ Between Mycobacteria and ProteaseFunctions Differ Between Mycobacterium smegmatis and Escherichia coli[J] .Gene, 1999,231:95-104].Therefore as can be seen, in comprising the pathogenic bacteria of M.tb, ClpP is that thalline opposing host defense system is necessary, M.tb can eliminate the destruction of NO to tropina by the effect of this enzyme, also can directly or indirectly regulate thalline antioxidant effect and toxicity Expression of Related Genes.
ClpP albumen is not expressed in H37Rv, clinical full sensitivity and Rifampin list Resistant strain, but in full Resistant strain, occur, the appearance that full resistance proterties is described is than single resistance proterties complexity, the NO resistivity that host's scavenger cell is produced is also stronger, and therefore harm and the propagation potentiality to the host are also bigger.
M13, NuoB, basic protein find that relatively this proteic expression fluctuation is very big, but and asynchronous.Disappear in the Rifampin list Resistant strain, but the expression amount in the full Resistant strain rises.The resistance that M.tb is described is relevant with its respiratory variation, but different resistance patterns is different to respiratory influence, may various Resistant strain have specific resistance mechanism.
M14, Rv2624 belongs to ubiquity pressure albumen (USP) family in the Mycobacterium tuberculosis body.USP can adjust most protein metabolism process in the thalline, thereby guarantee the influence [Ronan O ' Toole of body opposing such as heat, nutritional deficiency, heavy metal, reductive agent, microbiotic etc., Huw D.Williams.Universal Stress Proteinsand Mycobacterium tuberculosis[J] .Research in Microbiology, 2003,154:387-392].Under low-oxygen environment, detect discovery Rv2624 at M.tb by gene chip, M.bovis, whole up-regulated expression [Sherman D.R. among the M.lepars, Voskuil M., Schnappinger D., Liao R., Harrell M.I., Schoolnik G.K..Regulation of the Mycobacterium tuberculosisHypoxic Response Gene Encoding Alpha-crystallin[J] .Proc.Natl.Acad.Sci., 2001,98:7534-7539], illustrate that this albumen is significant to mycobacterium resistance, in close relations with the latent infection of M.tb.This albumen is constant in single resistance and complete quick bacterial strain, but expression amount increases in full Resistant strain.
M15, CeoB, one of Rv2691 coding Potassium Absorption albumen TrkA family member CeoB albumen.Knock out this gene by homologous recombination, found that mutant strain heightens to the absorbed dose of K+, growth velocity rises significantly, and meanwhile, body also descends to antibiotic tolerance.So this is the albumen of the outer heat-extraction system of a medicine, play an important role in the natural and acquired resistance mycobacterium.Find that in our research this albumen disappears in persister, this result is not corresponding with its function, and concrete mechanism is still needed and wanted further experimental verification.
Differential protein in comprehensive Rifampin list resistance and the anti-multiple medicines tubercule bacillus bacterial strain, we find, in these albumen of being identified, BfrB (Rv3841), OpcA (Rvl446), Wag31 (Rv2145), Rv2629, DevR (Rv3133c), six albumen of Rv2624 are different with expression pattern in the Resistant strain full sensitive strain, and the expression of clinical full sensitive strain more approaches the pattern of laboratory standard bacterial strain H37Rv.Therefore can think that these six albumen very likely are distinctive differential proteins in the Resistant strain, directly related with Rifampin resistance or the many resistances of bacterium.
BfrB, OpcA, Wag31 together with Rv2159, Rv0738, belong to Mycobacterium tuberculosis membranin and embrane-associated protein.People extremely pay close attention to the research of membranin all the time, not only because these albumen play crucial effect in cell activities, are membranin because in all potential medicine targets the albumen above 50% is arranged more; Membranin is positioned at the outside of bacterium in addition, discerned by host immune system easilier, so these membranins also is very helpful to the diagnosis of tuberculosis.Distinctive differential protein has 3 to belong to membranin in 6 Resistant strain, pointing out us M.tb may perhaps stop medicine to enter cell by changing the saturating property of cytolemma to medicine, perhaps promotes medicine to efflux in born of the same parents, the final effect that reduces microbiotic to thalline causes chemical sproof appearance.
Rv2629 is the significant albumen that bacterium enters latent infection, and great expression in the persister illustrates that entering latent state is the drug-fast a kind of mechanism of M.tb.DevR is the earliest mediation in the hypoxia signal among the M.tb, and the genetic expression that nearly all hypoxia condition is relevant all needs inducing of DevR, is that M.tb is essential when the persistent infection of hypoxemia latent.In the clinical drug-resistant bacterial strain, the whole up-regulated expressions of DevR can promote thalline to enter the non-resistance state that duplicates.Rv2624 belongs to ubiquity pressure albumen (USP) family in the Mycobacterium tuberculosis body.By adjusting the influence of the opposing of protein metabolism process assurance body such as heat, nutritional deficiency, heavy metal, reductive agent, microbiotic etc. in the thalline.Significant to mycobacterium resistance, in close relations with the latent infection of M.tb.This albumen increases at expression amount in the bacterial strain of anti-the multiple medicines.These 3 proteic discovery explanations, after the resistance Mycobacterium tuberculosis enters body, by reducing viability, enter latent state and resist extraneous poor environment, or reduce functioning efficiency with medicine in this mode that enters latent state, thereby reduce or the opposing microbiotic to its killing action.Changing appears in the expression of the katalaze enzyme in numerous metabolism and the respiratory, enters this growth conditions with thalline and adapts.
The qualification result of table 1 mycobacterium tuberculosis multidrug-resistance bacterial strain differential protein
| Sequence number | Albumen | Among the NCBI gi number | The seat label | The PMF score value | Tandem mass spectrum (MS/MS) | PI (theoretical value/experimental value) | MW (theoretical value/experimental value) | The expression of clinical strains |
| M1 M2 M3 M4 M5 M6 | Hypothetic al protein TrpC Hypothetic al protein Hypothetic al protein OpcA ClpP | gi|15607878 gi|15608749 gi|15608067 gi|15608132 gi|15608584 gi|15609598 | Rv0738 Rv1611 Rv0927 Rv0992 Rv1446 Rv2461 | 160(4/5) 92(6/15) 132(6/11) 58(5) 143(6/18) 157(6/9) | Parent ion: 1359.7378MH+ peptide section: AQDAFAALLANVR score value: 37 parent ions: 1540.823MH+ peptide section: ERPPADQLAAFLGR score value: 27 parent ions: 1594.9314MH+ peptide section: LPLGEVPGQVFIGLR score value: 50 parent ions: 1601.807MH+ peptide section: DFVVQPYQIHEAR score value: 28 parent ions: 1418.7122MH+ peptide section: TSSELDAVAEQIR score value: 71 parent ions: 2056.1548MH+ peptide section: GASLRGAAIALAFAQAGADVLI score value: 16 parent ions: 1212.731MH+ peptide section: AVGELKVELVR score value: 29 parent ions: 1360.7218MH+ peptide section: VGADAGAGEFVVLR score value: 38 parent ions: 1237.7262MH+ peptide section: TGKPDALVPLAR score value: 15 parent ions: 1374.7012MH+ peptide section: LNAEFTGQPIER score value: 69 parent ions: 1689.8805MH+ | 5.21/5.29 4.86/5.07 5.6/5.49 9.36/9.45 4.98/5.2 4.54/4.79 | 19115.61/ 19249.8 28023.03/ 28005.8 26745.62/ 26729.1 21412.88/ 21570.5 32717.49/ 32697.4 21675.58/ 21693.8 | Increasing disappears descends increases new the appearance |
| 7 |
Hypothetic al protein CysK Hypothetic al protein Rv2629 ilvN DevR PPE nuoB HyPothetic al protein | gi|15609296 gi|15609471 gi|15609766 gi|15610139 gi|15610269 gi|15610272 gi|15610282 gi|15609761 | Rv2159 Rv2334 Rv2629 Rv3002 Rv3133c Rv3136 Rv3146 Rv2624c | 189(5/13) 91(6/12) 187(4/15) 70(2/3) 90 73(6/7) 55(4) 130(6/14) | ：IIFLGSEVNDEIANR ：58 ：1515.7914MH+ ：IGTWIGAAAEGQVSR ：12 ：2147.1455MH+ ：LAAALRLTGLAPHQVTDDDVA ：70 ：1356.7885 MH+ ：LIVVVLPDFGER ：32 ：2028.0085MH+ ：YFVPQQFENPANPAIHR ：17 ：1194.7205MH+ ：IAPLDGVGALLR ：14 ：1801.933MH+ ：LVDAADPEVVFVSGEVR ：10 ：1275.7054MH+ ：SQVIEAVNLFR ：38 ：1477.8485MH+ ：VPAARPDVAVLDVR ：21 ：1559.7886MH+ ：MDFALLPPEVNSAR ：62 ：1619.9227MH+ ：QAPILALGGRPEELR ：16 ：1765.806MH+ ：MTDAPDNEAVLEYAAR ：55 | 7.82/7.21 4.93/5.2 5.01/5.2 8.52/7.93 5.45/5.62 4.11/4.37 7.95/7.68 6.44/6.01 | 36376.38/ 36353.9 32752.62/ 32732.3 40839.76/ 40900 18186.89/ 18175.8 23261.87/ 23279.3 37979.75/ 37956.2 20186.78/ 20433.5 29400.37/ 29382.2 | Descending increases the new increase that disappears that occurs newly occurring increasing |
| M15 | trkA | gi|15609828 | Rv2691 | 283(6/24) | Parent ion: 1487.8328MH+ peptide section: VLGQGFDRDVLLR score value: 29 parent ions: 1761.9857MH+ peptide section: TVLQAGDKVYIAAISGR score value: 34 parent ions: 1883.9722MH+ peptide section: IGHEVAIIDRDSAAFNR score value: 31 parent ions: 2043.998MH+ peptide section: AGAAERAAAIAALPPSEDFES score value: 38 parent ions: 2292.1213MH+ peptide section: ASGNIIIQSGAARDAFAAVSSGDN score value: 21 | 6.29/5.97 | 24239.44/ 24224.7 | Disappear |
Claims (8)
1. method of screening mycobacterium tuberculosis multidrug-resistance protein, it is characterized in that, described medicine is 3-{[(4-methyl isophthalic acid-piperazinyl) imino-] methyl }-rifomycin, 4-pyridine formyl hydrazine-vazadrine, { 2R, 2[S-(R*, R*)]-R}-(+) 2,2 '-(1,2-second two basic diiminos)-two-1-butanols dihydrochloride-Tibutol, 0-2-methylamino--2-deoxidation-α-L-Glucopyranyl-(1 → 2)-0-5-deoxidation-3-C-formyl radical-α-L-lysol furyl glycosyl-(1 → 4)-N1, N3-diamidino-D-streptamine vitriol-Streptomycin sulphate and Zinamide-pyrazinoic acid amide; This method may further comprise the steps:
(1) the screening bacterial strain of anti-multiple medicines the in clinical tubercule bacillus strain isolated is the bacterial strain of anti-the multiple medicines five kinds of medicines are satisfied simultaneously the bacterial strain that minimum inhibitory concentration is higher than corresponding critical resistance concentration; The critical resistance concentration of five kinds of medicines is respectively: imino-3-{[(4-methyl isophthalic acid-piperazinyl)] methyl }-rifomycin is 16 μ g/ml, 4-pyridine formyl hydrazine-vazadrine is 2 μ g/ml, { 2R, 2[S-(R*, R*)]-R}-(+) 2,2 '-(1,2-second two basic diiminos)-two-1-butanols dihydrochloride-Tibutol is 4 μ g/ml, 0-2-methylamino--2-deoxidation-α-L-Glucopyranyl-(1 → 2)-0-5-deoxidation-3-C-formyl radical-α-L-lysol furyl glycosyl-(1 → 4)-N1, N3-diamidino-D-streptamine vitriol-Streptomycin sulphate is 8 μ g/ml, and Zinamide-pyrazinoic acid amide is 16 μ g/ml; The strain of tubercule bacillus standard test, the clinical sensitive strain cultivated with not dosing under the same terms are contrast;
(2) albumen in difference separation criterion experiment strain, clinical sensitive strain and the bacterial strain of anti-the multiple medicines;
(3) with in the bacterial strain of anti-the multiple medicines, with respect to standard test strain and clinical sensitive strain, increase or the albumen of disappearance, the perhaps albumen of at least 2 times of expression amount increases, perhaps to be reduced by at least 50% albumen be multidrug-resistance protein to expression amount;
(4) isolation identification multidrug-resistance protein.
2. the method for claim 1 is characterized in that, step only keeps basic protein in (2).
3. the method for claim 1 is characterized in that, detects the middle proteic bacterial strain uses therefor of separation and Extraction of minimum inhibitory concentration and step (2) in the step (1) and is the tubercule bacillus that is in logarithmic phase.
4. the method for claim 1 is characterized in that, adopts electrophoresis, centrifugal in step (2) and the step (4), saltouts, filtration or chromatography method protein isolate.
5. the method for claim 1 is characterized in that, the method by protein electrophoresis atlas analysis, protein sequencing, mass spectroscopy or amino acid composition analysis in the step (4) is identified albumen.
6. the application of method according to claim 1 is characterized in that, the multidrug-resistance protein that step (4) is obtained is as the medicine target, screening or detect antitubercular agent.
7. the application of method according to claim 1 is characterized in that, the gene that step (4) is obtained multidrug-resistance protein suddenlys change, and is used to make up vaccine.
8. the application of method according to claim 1 is characterized in that, the multidrug-resistance protein that obtains according to step (4) detects the mycobacterium tuberculosis multidrug-resistance bacterial strain.
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| CN103122364A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Antitubercular inhibitor screened target gene Rv2498c and application thereof |
| CN104502610A (en) * | 2014-12-25 | 2015-04-08 | 中山大学 | Method for screening antibiotics binding proteins |
| CN111471632A (en) * | 2020-02-28 | 2020-07-31 | 中国科学院广州生物医药与健康研究院 | Construction method and application of recombinant drug-resistant BCG strain |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122364A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Antitubercular inhibitor screened target gene Rv2498c and application thereof |
| CN104502610A (en) * | 2014-12-25 | 2015-04-08 | 中山大学 | Method for screening antibiotics binding proteins |
| CN111471632A (en) * | 2020-02-28 | 2020-07-31 | 中国科学院广州生物医药与健康研究院 | Construction method and application of recombinant drug-resistant BCG strain |
| CN111471632B (en) * | 2020-02-28 | 2022-03-08 | 中国科学院广州生物医药与健康研究院 | Construction method and application of recombinant drug-resistant BCG strain |
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