CN100497592C - Mycobactrium tuberculosis protein for diagnosing rifampicin dependent mycobacterium tuberculosis - Google Patents
Mycobactrium tuberculosis protein for diagnosing rifampicin dependent mycobacterium tuberculosis Download PDFInfo
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- CN100497592C CN100497592C CNB2005100574863A CN200510057486A CN100497592C CN 100497592 C CN100497592 C CN 100497592C CN B2005100574863 A CNB2005100574863 A CN B2005100574863A CN 200510057486 A CN200510057486 A CN 200510057486A CN 100497592 C CN100497592 C CN 100497592C
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Abstract
The present invention relates to a kind of specific rifampicin dependent mycobacterium tuberculosis with the preservation number of CGMCC No.1570; rifampicin dependent mycobacterium tuberculosis protein with the amino acid sequence shown in the sequence 1, molecular weight 43894 Da and pI of5.25; one kind of nucleic acid with the nucleotide sequence as shown in the sequence 2; method of artificially inducing rifampicin dependent mycobacterium tuberculosis to obtain high concentration rifampicin dependent mycobacterium tuberculosis protein; the composition and reagent kit containing the rifampicin dependent mycobacterium tuberculosis protein and the application of the reagent kit in the diagnosis of rifampicin dependent mycobacterium tuberculosis type tuberculosis and drug resisting tuberculosis and epidemiological monitoring and surveying.
Description
Technical field
The present invention relates to a kind of mycobacterium tuberculosis protein, and this albumen is in the application of aspects such as the clinical diagnosis of rifampicin dependent mycobacterium tuberculosis and epidemiological surveillance, and this albumen is as the application of drug targets at the tuberculotherapy of anti-multiple medicines new drug development.
Background technology
The whole nation in 2000 is the 4th tuberculosis epidemiological random sampling survey result show: China has 5,000,000 tuberculosis patients approximately, 500,000,000 tuberculosis infection persons, and the tuberculosis epidemic situation is still very serious.WHO estimates, the world today has 2/3 tuberculosis patient to be among the danger that the resistance case takes place approximately.Because resistant organism, continuous generation and the propagation of particularly anti-multiple medicines bacterium are the unmanageable keys of current tuberculosis.
Rifampicin dependent mycobacterium tuberculosis (hereinafter to be referred as according to the R bacterium).According to the R bacterium is that the present invention first contriver is first in clinical lung tubercular sputum specimen, the drug sensitive test of cultivating by mycobacterium Luo Shi bacterium colony is as a result observed and is found and the research report to have the special mycobacterium tuberculosis of rifampicin dependent (R) antitubercular agent growth.Mainly show as according to R bacterium biological characteristics, its bacterial growth is vigorous under suitable Rifampin concentration, leaves the Rifampin poor growth and maybe can not grow.As shown in Figure 1, find that simultaneously in that to have under R and the no R condition under two kinds of conditions the growth protein of bacteria express different, the former has significant high expression level albumen, shown in Fig. 4 (c).
Studies show that in a large number according to the R bacterium is having the aspects such as its form of bacterium, structure, dyeability, virulence and protein expression of growing under R and the no R condition that notable difference is all arranged.And, according to having 74.4%～100% to be anti-multiple medicines tubercule bacillus in the R bacterium.Have the Clinical symptoms of state of an illness weight, anti-multiple medicines, chemotherapy effect difference and prognosis mala according to R bacterium pulmonary tuberculosis, bigger than general resistance case harm.Raise new difficuties for control lungy again according to generation of R bacterium and propagation.The incidence according in the drug sensitivity test of R bacterium after mycobacterium is cultivated the positive of various places report is 6%～17% at present, and this is the country of a tuberculosis high incidence for China, and numerical value is surprising.
At present, Rifampin is that tuberculosis is just controlled the main bactericide in the chemotherapy assembled scheme, is the essential medicine of just controlling tuberculotherapy, and its use is very extensive.Re-use Rifampin if infect the tuberculosis caused according to the R bacterium, not only chemotherapy will fail, and can make that sb.'s illness took a turn for the worse, be one of intractable generation lungy and chemotherapy major reason of failing.Therefore, clinical in all very urgent according to the demand of the early diagnosis lungy of R bacterium and effective treatment.Existing ordinary method from the separation and Culture of mycobacterium to drug sensitivity assay, at least need time about 2 months just can observe pathogenic bacterium and whether have the pharmacological dependence phenomenon, and existing method can only be cultivated the male pulmonary tuberculosis patient at the phlegm bacterium, to negative pulmonary tuberculosis of phlegm bacterium more than half and extrapulmonary tuberculosis patient, conventional bacteriological method is helpless at all.Therefore, seeking a kind of fast and convenient and effective means is used for according to the diagnosis lungy of R bacterium very urgent.
The present invention is by discovering in a large number, but according to the R bacterium in vitro under the inducing of suitable concentration Rifampin high expression level amount (accounting for the 30-50% of total bacterioprotein) produce a kind of bacterioprotein of specific amino acids sequence.And by a large amount of clinical trials confirmations, the bacterioprotein of this specific amino acids sequence has the epidemic disease of exempting from service active function, is used for having according to R bacterium serology quick diagnosis lungy the specificity and the good sensitivity of height.
Mycobacterium tuberculosis protein according to R bacterium specific amino acids sequence of the present invention is not only a kind according to R bacterium diagnosis albumen lungy, also can be used as the drug targets of the new effective tuberculotherapy of anti-multiple medicines of exploitation.Because discover in a large number according to having 74.4%～100% all to be anti-multiple medicines tubercule bacillus in the R bacterium.
Summary of the invention
The invention summary
The purpose of this invention is to provide a kind of specific mycobacterium tuberculosis, the nucleotide sequence of coding mycobacterium tuberculosis protein shown in sequence 2, and induce the method for expressing according to R sclerotium nucleotide sequence according to the R mycobacterium tuberculosis protein with rifampicin dependent (hereinafter to be referred as according to R) growth.The present invention also comprises the test kit of described rifampicin dependent mycobacterium tuberculosis protein and in clinical diagnosis rifampicin dependent mycobacterium tuberculosis tuberculosis, anti-multiple medicines tuberculosis and epidemiological surveillance Application in Survey thereof.
The mycobacterium tuberculosis that the present invention relates to (Mycobacterium tuberculosis), it has the specificity of rifampicin dependent growth, and its preserving number is: CGMCC NO.1570, the preservation time: on December 19th, 2005, the bacterial strain of ginseng certificate number is: 1219.
The rifampicin dependent mycobacterium tuberculosis protein that the present invention relates to, it has the aminoacid sequence shown in sequence 1, and its molecular weight is 43894 dalton, and the pI value is 5.25.
The present invention relates to a kind of nucleic acid, it has the nucleotide sequence shown in sequence 2.
Indication of the present invention according to the expression of nucleic acid of R bacterium mycobacterium tuberculosis protein by the nucleotide sequence shown in the sequence 2, expressed proteic aminoacid sequence is shown in sequence 1.
Mycobacterium tuberculosis protein of the present invention is as follows according to R mycobacterium tuberculosis inductive method by the typical case:
1) isolation identification screen clinical typical rifampicin dependent mycobacterium tuberculosis bacterial strain (preserving number is: CGMCC NO.1570, the bacterial strain of ginseng certificate number is: 1219);
2) in the 7H9 liquid nutrient medium, it is proteic according to the R bacterial strain to be rich in sequence 2 with Rifampin 6～24 μ g/ml as induced concentration cultivation acquisition;
3) or in the L-J solid medium cultivation obtains to be rich in sequence 2 proteic rifampicin dependent mycobacterium tuberculosis strains as induced concentration with Rifampin 50～300 μ g/ml;
4) the rifampicin dependent mycobacterium tuberculosis protein solution after preparation process 2 or 3 is induced;
5) protein isolate, recovery, purifying and renaturation promptly obtain the rifampicin dependent mycobacterium tuberculosis protein.
Mycobacterium tuberculosis protein of the present invention also can utilize the nucleotide sequence shown in the sequence 2, by the albumen of conventional gene engineering method express amino acid sequence shown in sequence 1.
The present invention relates to comprise the test kit of invention mycobacterium tuberculosis protein.
The present invention relates to a kind of new tuberculosis rapid diagnosis method, comprise according to the invention mycobacterium tuberculosis protein of R mycobacterium tuberculosis strain and directly diagnose, and come auxiliary diagnosis according to R bacterium tuberculosis with carry out epidemiology survey etc. with serological test.
Mycobacterium tuberculosis protein according to R mycobacterium tuberculosis specific amino acids sequence of the present invention is not only a kind ofly according to R mycobacterium tuberculosis diagnosis albumen lungy, also can be used as the drug targets of the new effective tuberculotherapy of anti-multiple medicines of exploitation.Because discover in a large number according to having 74.4%～100% all to be anti-multiple medicines tubercule bacillus in the R mycobacterium tuberculosis.
Detailed Description Of The Invention
The invention discloses a kind of nucleotide sequence shown in sequence 2 of the mycobacterium tuberculosis protein of encoding, the method of the mycobacterium tuberculosis protein preparation of its coding, by the mycobacterium tuberculosis protein that comprises aminoacid sequence shown in the sequence 1 of this method preparation, and comprise this and invent proteic test kit.They are also disclosed simultaneously according to R bacterium diagnosis of tuberculosis and epidemiology survey Application in Monitoring.In addition, mycobacterium tuberculosis protein of the present invention is not only a kind of diagnosis albumen according to the R bacterium, also can be used as the drug targets of the new effective tuberculotherapy of anti-multiple medicines of exploitation.Because discover in a large number according to having 74.4%～100% all to be anti-multiple medicines tubercule bacillus in the R bacterium.
The method that mycobacterium tuberculosis protein of the present invention adopts the typical case manually to induce encoding gene to express outward according to the R thalline can obtain the invention albumen of high expression level amount, such as embodiment 2 narration.This invention albumen can also use albumen synthetic instrument system directly synthetic also by engineered ordinary method expression, recovery and purifying.The preferred artificial induction's method that adopts, because this method is simply effective, cost is low, importantly the albumen of Huo Deing has the excellent function activity.Simultaneously, can avoid genetically engineered ordinary method expressing protein, the antigen that may cause in processes such as expression, recovery and purifying loses with the reduction of product functionally active and the fidelity enhanced may.
One embodiment of the invention relate to the test kit with the mycobacterium tuberculosis protein combination of method for preparing.
Disclosed in this invention according to R bacterium tuberculosis seroimmunity diagnostic kit, be meant the complete detection reagent that comprises mycobacterium tuberculosis protein assembly of the present invention.This test kit is used for according to R bacterium quick auxiliary diagnosis lungy, or is used for according to epidemiology survey lungy of R bacterium and monitoring.The detection mode of this test kit is a single part of check-out console system, and its test kit comprises 20～50 sealing detection plates that assembled.Mycobacterium tuberculosis protein of the present invention is (can 4～8 ℃ of kept dry more than June) on the film of predetermined fixed in check-out console.Other combine detection reagent of this test kit also comprises: the goat anti-human igg antibody of confining liquid, colloid gold label and PBS washings, and positive and negative quality controling serum etc.This test kit is after separating acquisition clinical detection sample, and the time of monocyte sample antibody test only needed to finish in several minutes.Carry out this purpose clinical detection and do not need special plant and instrument.Its method is simple, quick, cost is low, is suitable for different medical unit and applies.
The biological sample that this test kit can detect can comprise that serum, cerebrospinal fluid, urine are tucked in puncture fluid etc. and may contain the anti-clinical sample according to R mycoprotein antibody of serum, preferentially select serum sample for use.
Above-mentionedly all comprise within the scope of the invention according to used all reagent or the test kit that comprises mycobacterium tuberculosis protein of the present invention in the R bacterium tuberculosis seroimmunity diagnostic kit.
Mycobacterium tuberculosis protein of the present invention also can directly pass through mark, as enzyme, fluorescent substance, luminophore and radioactive substance etc., directly detects more micro-specific antibody by the relevant detection instrument.Preferred marker is a luminophore, and as different luminol,3-aminophthalic acid cyclic hydrazide, lucigenin and luminol,3-aminophthalic acid cyclic hydrazide etc., its susceptibility, stability and thoroughness are better.
Mycobacterium tuberculosis protein of the present invention, can also prepare monoclonal antibody by genetically engineered and animal model, development is according to R mycoprotein detection of antigens system and test kit, be used for direct detection according to R bacterium and proteantigen thereof, its detection method can adopt histological chemistry, immune serum and the flow cytometer etc. of various marks, and it detects sample can comprise various clinical samples and cultures such as phlegm, tissue and serum.
Description of drawings
Accompanying drawing 1 is the growth comparison diagram according to the single bacterium colony of R bacterial strain; Among the figure according to the R bacterial strain by Chongqing City Pneumology Hospital---the clinical separation in tuberculosis diagnositc center laboratory, Chongqing City, the L-J substratum adopts the E-test test to obtain; R among the figure
100For being growing state in the 100 μ g/ml culture tubes according to the R bacterial strain containing R concentration, its bacterium colony is typical case's " cauliflower-like " growth in the pipe, and it is vigorous to grow, and easily scrapes and gets.And what reflect in the control tube is according to the growing state of R bacterial strain under no R condition, and its bacterium colony is typical case's " poached egg sample ", and is and embeds growth, is difficult for scraping and gets, and shows as serious poor growth state.
Accompanying drawing 2 is according to R bacterium growth curve in the culturing bottle when R concentration is 8 μ g/ml; Reflection is having under the R situation growth vigorous according to the R bacterial strain among the figure, and growth curve is typical parabolic type;
Accompanying drawing 3 is according to the growth curve of R bacterium in no medicine contrast culture bottle; Grow under no R situation poor growth, metabolic condition of reflection R bacterial strain is low among the figure, and growth curve is mild.
Fig. 2 and Fig. 3 show isolation identification in the clinical lung tubercular sputum specimen according to the R bacterium, the growth curve of bacterium under the different condition in 7H9 liquid culture 3D system.Fig. 2 shows that the growth curve that BacT/ALERT 3DSelect system contains in the R:8 μ g/ml culturing bottle is para-curve, its lag phase 8.09 days, and initial reflectance: 1750, maximum reflectivity: 3429, maximum reflectivity absolute value: 1679; Fig. 3 shows that the growth curve of no medicine control bottle is mild oblique line, its lag phase 17.09 days, and initial reflectance: 1750, maximum reflectivity: 2461 maximum reflectivity absolute values: 711.Fig. 2 and Fig. 3 show according to the R bacterium at the growth curve obvious difference that has under R and the no R condition.
[Fig. 2 and Fig. 3 growth curve note]
Transverse axis: incubation time (my god)
The longitudinal axis: reflectivity (bacterial growth produces CO2, and the latex inductor block produces the reflectivity increase that irreversible color change causes system monitoring at the bottom of the airtight culturing bottle)
Growth curve: per 10 minutes readings of system are once monitored reflectivity, the growth response curve (initial reflectance of being set up: 1750)
Inoculation bacterial concentration: 10-4 μ g/ml
Total incubation time: 40 days (39.78)
Accompanying drawing 5 is protein band total ion current mass spectrum of the present invention (1), mass spectrum adopts Q-TOF2 type LC-ESI-MS-MS systems analysis among the figure, wherein: X-coordinate: mass-to-charge ratio (m/z), ordinate zou: mass of ion per-cent (100%), crest: be N peptide section of target protein.
Accompanying drawing 8 is the m/z 656.83 peptide section tandem mass spectrum aminoacid sequence analysis diagrams (2) in Fig. 5 or Fig. 6 total ion current mass spectrum, mass spectrum adopts Q-TOF2 type LC-ESI-MS-MS systems analysis among the figure, wherein: X-coordinate: mass-to-charge ratio (m/z), ordinate zou: mass of ion per-cent (100%), ordinate zou top: be peptide section aminoacid sequence analysis result.
Accompanying drawing 9 is the m/z 656.83 peptide section tandem mass spectrum aminoacid sequence analysis diagrams (3) in Fig. 5 or Fig. 6 total ion current mass spectrum, mass spectrum adopts Q-TOF2 type LC-ESI-MS-MS systems analysis among the figure, wherein: X-coordinate: mass-to-charge ratio (m/z), ordinate zou: mass of ion per-cent (100%), ordinate zou top: be peptide section aminoacid sequence analysis result.
Accompanying drawing 10 is the m/z656.83 peptide section tandem mass spectrum aminoacid sequence analysis diagram (4) in Fig. 5 or Fig. 6 total ion current mass spectrum, mass spectrum adopts Q-TOF2 capillary liquid chromatography-electron spray(ES)-quadrupole-flight time tandom mass spectrometer (LC-ESI-MS/MS among the figure, Q-TOF2) systems analysis, wherein: horizontal seat X-coordinate: mass-to-charge ratio (m/z), ordinate zou: mass of ion per-cent (100%), ordinate zou top: be peptide section aminoacid sequence analysis result.
Accompanying drawing 11 is the m/z656.83 peptide section tandem mass spectrum aminoacid sequence analysis diagram (5) in Fig. 5 or Fig. 6 total ion current mass spectrum, mass spectrum adopts Q-TOF2 type LC-ESI-MS-MS systems analysis among the figure, wherein: X-coordinate: mass-to-charge ratio (m/z), ordinate zou: mass of ion per-cent (100%), ordinate zou top: be peptide section aminoacid sequence analysis result.
Accompanying drawing 12 is protein peptide section aminoacid sequence result for retrieval table of the present invention.
Figure 13 is the encoding gene pcr amplification electrophorogram of Rv0341; The Rv0341 encoding gene DNA cloning fragment electrophoresis result that shows different strains among the figure.Among the figure: M: molecular weight standard, 1:H37Rv reference culture, 2: the bacterial strain of anti-R, 3: according to R bacterial strain, 4: the reagent blank contrast.
Figure 14 shows immunoblotting Collaurum marking Serum Antibody Detection result, and the left side of figure is that the typical case is anti-according to R bacterium Rv03411 protein antibodies positive findings according to R bacterium lunger serum, and figure the right is the normal human serum negative findings.
Embodiment
Embodiment 1: according to the isolation identification of R bacterium
The method that mycobacterium tuberculosis protein of the present invention adopts the typical case manually to induce encoding gene to express outward according to the R thalline.The cultivation of Luo Shi (L-J) mycobacterium, strain identification and the absolute concentration indirect method drug sensitive experiment that adopts according to the screening of the isolation identification of R bacterium all by " diagnosis of tuberculosis bacteriological analysis rules " requirement carry out [Chinese anti-tuberculosis association. middle national defence consumptive disease magazine, 1996,18:28-31.
]Also can adopt 7H9 liquid 3D culture systems, judge by indexs such as growth curves.
Judging criterion according to the screening of R bacterium is to have in conventional L-J absolute concentration indirect method susceptibility is cultivated, its bacterium colony growth in R culture tube (R250 μ g/ml or R50 μ g/ml) is thick vigorous, the bacterioid that bacterium colony strain identification tiny, poor growth (being the growth of L type) is a mycobacterium tuberculosis in the control tube is judged as the bacterium according to R, as shown in Figure 1.Anti-R bacterium is then in that to contain R pipe identical with the vigorous degree (bacterium colony mode of appearance) of bacterial growth in the control tube.The contrast difference of 7H9 liquid 3D culture systems growth curve also can be used as basis for estimation, as shown in Figures 2 and 3.
Research with the typical case of clinical screening according to R bacterial strain (preserving number: CJMCC No:1570, the bacterial strain of ginseng certificate number is: 1219), its optimum medicine concentration of gradient experiment screening by different Rifampin concentration is as the drug level of inducing this invention mycobacterium tuberculosis protein.The best rifampicin medicine concentration that its Luo Shi cultivates is 100 μ g/ml, and the best rifampicin medicine concentration of 7H9 liquid culture is 8 μ g/ml.
Mycobacterium tuberculosis protein of the present invention adopts the typical clinical of cultivating in 3-4 week according to R bacterial strain (bacterium numbering: 1219, CJMCC No:1570) bacteria suspension (10
-2Mg/ml), be inoculated in that to contain the Rifampin final concentration be that the 7H9 liquid nutrient medium of 8 μ g/ml or the Russell medium that contains 100 μ g/ml are cultivated 3-4 weeks for 35～37 ℃.The high expression level invention albumen that obtains is according to the R bacterial strain, and is frozen with conventional mycobacteria strain store method-700C.
Embodiment 2: the preparation of mycobacterium tuberculosis protein of the present invention
The preparation of mycobacterium tuberculosis protein of the present invention, adopt high expression level invention albumen that embodiment 1 prepares according to the cultivation bacterium liquid (or getting bacterium colony) of R bacterial strain in the PBS damping fluid after Pasteur's deactivation, the centrifugal supernatant that goes, and again with after the PBS damping fluid washing 2 times, every 2mg bacterial precipitation thing adds the granulated glass sphere 2mg of 1 * sample loading buffer (SDS doubled amount), 100 μ l and diameter 200 μ m, ultrasonic 15min in the water-bath ultrasonic apparatus then, 10min in the boiling water, 20 ℃ of 14000 centrifugal 8min of commentaries on classics/min, it is standby to collect supernatant liquor.Then with its supernatant samples with the quantitative protein concentration of ultraviolet spectrophotometer, being controlled between 1～2g/L of electrophoresis sample.The SDS-PAGE deposition condition is: 5% spacer gel, 10% separation gel, sample 15～20 μ l, voltage 100V, electrophoresis 3～4h, running gel Kao Masi light blue dyeing localizing objects albumen.Shown in Fig. 4 right side.
The recovery of mycobacterium tuberculosis protein of the present invention adopts direct location to cut target protein in the above SDS-PAGE glue, with ordinary method reclaim, purifying and renaturation.Simultaneously, also can adopt albumen in the direct transfer printing SDS-PAGE of the conventional electrotransfer technology glue to the PVDV film after, 1 film Kao Masi light blue dyeing of location cutting localizing objects albumen is shown in Fig. 4 left side.Target protein band on the location cutting film is used for analyzing or detecting then.The latter is proteic lose and the influence of functionally active less, cost is lower, can preferably adopt.
Embodiment 3: the aminoacid sequence of mycobacterium tuberculosis protein of the present invention is identified
Q-TOF2 type LC-ESI-MS-MS instrument system is adopted in the evaluation of Argine Monohydrochloride sequence of the present invention.Provide service by National Center of Blomedical Analysls of Military Medical Science Institute mass spectrum chamber.Its basic skills is the invention protein band that cutting embodiment 2 obtains, and after reclaiming purifying, carries out a peptide mixt mass spectroscopy earlier, shown in Fig. 5-6; Again 5 peptide sections are wherein carried out tandem mass spectrum (sequence label), parse the aminoacid sequence of each peptide section, shown in Fig. 7-11; Retrieval comparison then, result for retrieval as shown in figure 12.Be that mycobacterium tuberculosis protein of the present invention is accredited as mycobacterium tuberculosis H through aminoacid sequence
37Rv hypothetical protein Rv0341, relative molecular mass: 43894, iso-electric point (PI) 5.25, score 183, aminoacid sequence is shown in sequence 2.
Embodiment 3: mycobacterium tuberculosis protein coding gene sequence of the present invention is analyzed
The amplification of mycobacterium tuberculosis protein encoding gene of the present invention is the sequence data that provides according to Genbank, by computer aided design (CAD) embodiment 2 results' mycobacterium tuberculosis H
37The segmental amplimer I of Rv hypothetical protein Rv0341 encoding gene (409360-410799 sequence) 1440bp (5 '-CGATTACATCCTGAGCCTGTT-3 ') and primer I I (3 '-GGTAGTCCGAAAAGGCCGTA-5 ').DNA of bacteria is extracted: adopt U.S. Promega company DNA extraction test kit (lot number: 178886), extract experimental strain DNA by its specification sheets method.The condition preparation routinely of pcr amplification system
[10], amplification condition is: behind 95 ℃ of 5min, circulate 35 times with 95 ℃ of 1min, 52 ℃ of 1min and 72 ℃ of 2min, 72 ℃ are extended 10min, 4 ℃ of preservations.
The dna sequence analysis of Rv0341 protein coding gene amplified fragments is encoding gene to be carried out the order-checking of segmentation dna sequence dna, cross section and two ends design primer in addition and repeat order-checking, is as the criterion to repeat consistent result.The relevant primer that order-checking is used, except that the encoding gene amplimer, also have the target fragment upstream termination to extend sequencing primer (5 '-GTATTGGGACGGGTCTGG-3 ' and 3 '-ATGAACCCGCCAGTGAC-5 '), stage casing 290bp and oppositely extend primer (3 '-GCTAGCCAGATCGGTGTC-5 '), and target fragment downstream end extension sequencing primer (5 '-ACATTGGCGACGTCCTG-3 ' and 3 '-ATGAACCCGCCAGTGAC-5 ').The purifying of pcr amplification product, determined dna sequence (the relevant primer design is synthetic) and result's ratio equity provide service by Jikang Biotechnology Co Ltd, Shanghai.
In order to the pcr amplification according to R bacterial strain, anti-R bacterial strain and R sensitive strain each 10 strains carry out Rv0341 encoding gene of last method to randomly drawing from embodiment 1, the result all obtains 1440bp fragment (Figure 13).Its fragment after segmentation order-checking, intersection and two-end part repeat sequencing result and all can repeat, splicing and go up the public database compare of analysis again with NCBI.The Rv0341 encoding gene 1440bp fragment sequence of the different clinical strains of result and the corresponding sequence of gene pool mycobacterium tuberculosis are identical, promptly do not find the sudden change of DNA.Its result confirms the Rv0341 albumen high expression level according to the R bacterial strain, is not due to the sudden change of its encoding gene DNA, but is controlled by the concentration of Rifampin.
Embodiment 4: mycobacterium tuberculosis protein of the present invention is used for the clinical trial according to the direct diagnosis of R bacterium
According to clinical screening of R bacterial strain and judging criterion: have in L-J absolute concentration indirect method susceptibility is cultivated, its bacterium is at Rifampin (R) culture tube (R
250μ g/ml or R
50μ g/ml) colony growth is thick vigorous in, and the mycobacterium tuberculosis of tiny, the poor growth of bacterium colony in the control tube (being the growth of L type) is judged as according to R bacterium (Fig. 1,2).Anti-R bacterium is then in that to contain R pipe identical with the vigorous degree (bacterium colony mode of appearance) of bacterial growth in the control tube.
(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE) technology is to cultivating the mycobacterium tuberculosis H in 3-4 week to adopt conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis
37Rv type strain, R sensitive strain, anti-R strain with result according to the comparative analysis of the tropina scanning spectra of R strain are: the protein graphical spectrum band number of different strains is similar substantially with the position.Wherein 84% (41/49) according to R bacterial strain tropina expressed in abundance in containing the R pipe, and an identical high expression level band is arranged in the lower end with respect to standard substance molecular mass 43 000; Through full automatic gel imaging system content analysis, this protein band accounts for 30%～50% of total tropina.86% (72/84) non-according to R bacterial strain (30 strain R sensitive organisms and the anti-R bacterium of 54 strains) and H37Rv reference culture to contain R pipe similar with the protein graphical spectrum of control tube, and do not have corresponding high expression level phenomenon.According to the SDS-PAGE collection of illustrative plates of bacterial strains such as R bacterium as shown in figure 14.Its common high expression level protein band according to the R bacterial strain is mycobacterium tuberculosis H
37Rv hypothetical protein Rv0341, its aminoacid sequence such as sequence 2, authentication method such as embodiment 3.
Experimental result shows: Rifampin is to having obvious rise effect (inducing action) according to the proteic expression of R bacterium Rv0341, and it raises Rv0341 albumen with relevant according to the R bacterium, can be considered associated protein or mark according to the R bacterium.
Embodiment 5: mycobacterium tuberculosis protein of the present invention is used for the clinical trial according to R strain tuberculosis serological diagnosis
[method] adopts immunoblotting Collaurum marking Serum Antibody Detection method.Press embodiment 2 preparations mycobacterium tuberculosis protein of the present invention, be fixed on the PVDV film.Location and installation is in the reacting hole of test panel, and after the quality examination, 4～8 ℃ of kept dry are standby.
[test kit composition] test kit is formed and is comprised: test panel, confining liquid, washings, gold marked reagent and positive and negative quality controlled serum.
[detection step]
1. in the reacting hole of test panel, add 1 of confining liquid, treat that it permeates in film fully.
2. get fresh test serum 50 μ l in the reacting hole center, treat that it permeates in film fully.
3. add 3 of washingss, treat that it permeates in film fully.Repeated washing 1 time.
4. add 1 of gold marked reagent, treat that it permeates in film fully.
5. add 3 of washingss, treat that it permeates in film the visual inspection result fully.
[result's judgement]
Positive: as to occur the punctation band on the film in reacting hole.As Figure 14 left side.
Negative: as not have the punctation band on the film in reacting hole or the band vestige is arranged.As Figure 14 right side.
[quality control] each batch test kit is furnished with positive and negative character control serum, is used for monitoring reagent box quality.During positive and negative character control serum reliable results, can be used for the detection of clinical sample.
[methodology experimental result]
Anti-according to the proteic antibody positive serum of R bacterium Rv0341 from the typical case according to successfully obtaining the R bacterium lunger.30 parts of normal human serum antibody tests are all negative.Crowd interior parallel repeated experiments result of 30 parts of negative serums and 30 parts of positive serums is all identical.The repeated experiments result is all negative between 3 times batches of 10 parts of negative serums; The repeated experiments result is all positive between 3 times batches of 10 parts of positive serums, and its positive degree has certain difference, and the positive degree for preparing Rv0341 protein band film with new cultivation bacterial strain is stronger.
[detected results of 327 parts of serum antibodies]
327 parts of serum come from Chongqing City Pneumology Hospital---tuberculosis diagnositc center laboratory, Chongqing City Serum Bank.Detected result sees Table 1.
The detected result of 327 parts of serum antibodies of table 1
Detected result shows: the immunoblotting colloid gold label Serum Antibody Detection method that mycobacterium tuberculosis protein of the present invention is set up, and have and criticize repeatability between interior batch preferably, its method is more stable.This method is respectively 100% (30/30), 93.5% (29/30), 83.3% (30/36) and 97.6% (40/41), average out to 93.5% (129/138) in the specificity of normal people's group, anti-R bacterium pulmonary tuberculosis group, R sensitive organism pulmonary tuberculosis group and non-tuberculosis pulmonary disorder group.Be 86.7% (26/30) according to the susceptibility in the R bacterium pulmonary tuberculosis R treatment group.12 examples of not using in the recent period R treatment group only detect 3 examples positive (25.0%) according to R bacterium lunger's serum antibody, and its result conforms to experiment in vitro substantially.Promptly do not having poor growth under the condition of R according to the R bacterium, target protein can not carry out effective expression, the anti-time of keeping in vivo according to R mycoprotein antibody, depends on derivative according to the amount of R mycoprotein and the time of keeping in vivo.In cultivating negative tuberculosis group, obtained 19.7% (29/147) positive rate, wherein answer refractory case 13 examples, just control 16 examples (comprising the tuberculosis of cervical lymph nodes 2 examples, tuberculous peritonitis 2 examples and the blood property broadcast tuberculosis 1 example), and 29 routine patients all use the treatment plan that contains R, have wherein treated the 3-6 month, and 6 examples of unsatisfactory curative effect, 3 examples that the state of an illness increases the weight of, clinical and experimental result all point out these patients may be for according to R bacterium tuberculosis.In addition, whether exist problem values must confirm that (known research and clinical effectiveness all point out the intravital mycobacterium tuberculosis of same case to have unhomogeneity simultaneously in the body of the responsive pulmonary tuberculosis case of the R of 6 routine antibody positives according to the R bacterium, the bacterial strain that promptly may have different resistance situations simultaneously), the follow of the clinical efficacy of its case and bacteriology data carries out.
In sum, with the proteic test kit that comprises indication of the present invention when the clinical diagnosis rifampicin dependent mycobacterium tuberculosis tuberculosis, better repeatability, specificity and susceptibility are not only arranged, can also make detection system simple to operate fast, do not need special instrument and condition, be beneficial to and apply.Among the preparation method, adopt the Rifampin that in conventional substratum, adds specific concentrations to cultivate, need invariably under the situation of specific installation, induce cheaply according to the R bacterium and produce albumen of the present invention according to the R bacterium.Be extensive use of in present clinical tuberculotherapy scheme under the situation of Rifampin, employing comprises the proteic test kit of the present invention and can be used as the quick screening method of serology auxiliary diagnosis according to R bacterium tuberculosis, particularly concerning major part the cloudy diagnosis lungy of the external bacterium that can not obtain the bacteriology index, clinical value is more arranged.
Sequence table
＜110〉Chongqing City Pneumology Hospital
＜120〉be used for diagnosing rifampicin dependent mycobacterium tuberculosis mycobacterium tuberculosis protein lungy
<130>Hypothetical?protein?Rv0341[Mycobacterium?tuberculosis?H37Rv]
<160>4
<210>1
<211>479
<212>PRT
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: inductive
<400>1
<210>2
<211>1440
<212>DNA
＜213〉mycobacterium tuberculosis H37Rv[Mycobacterium tuberculosis H37Rv lungy]
<220>
<400>1
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: upstream primer
<400>3
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: downstream primer
<400>3
Claims (2)
1, a kind of mycobacterium tuberculosis (Mycobacterium tuberculosis), it has the specificity of rifampicin dependent growth and Rv0341 albumen high expression level, and its preserving number is: CGMCC NO.1570.
2, the application of the albumen of rifampicin dependent mycobacterium tuberculosis as claimed in claim 1 in preparation clinical diagnosis rifampicin dependent mycobacterium tuberculosis detection reagent lungy.
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| CN1603415A (en) * | 2004-10-21 | 2005-04-06 | 复旦大学 | Fusion expression method of mycobacterium tuberculosis ESAT-6 protein in pichia |
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| CN1603415A (en) * | 2004-10-21 | 2005-04-06 | 复旦大学 | Fusion expression method of mycobacterium tuberculosis ESAT-6 protein in pichia |
Non-Patent Citations (9)
| Title |
|---|
| BACTEC系统对依赖利福平结核分支杆菌生长与耐药性的分析. 王易伟，钟敏.中国防痨杂志，第24卷第3期. 2002 |
| BACTEC系统对依赖利福平结核分支杆菌生长与耐药性的分析. 王易伟，钟敏.中国防痨杂志，第24卷第3期. 2002 * |
| BACTEC系统对依赖利福平结核分枝杆菌生长与耐药性的分析. 王易伟，钟敏.中国防痨杂志，第24卷第3期. 2002 |
| BACTEC系统对依赖利福平结核分枝杆菌生长与耐药性的分析. 王易伟，钟敏.中国防痨杂志，第24卷第3期. 2002 * |
| NCBI Genbank Acession No.DQ056350. Zhang,M., Yue,J.,,全文,NCBI Genbank Database. 2005 NCBI Genbank Acession No.ZP_00880229. Birren,B., Lander,E.,全文,NCBI Genbank Database. 2005 NCBI NO.DQ056350. ZHANG,M等,全文. 2005 NCBI NO.ZP_00880229. BIRREN,B等,全文. 2005 |
| NCBI Genbank Acession No.DQ056350. Zhang,M., Yue,J.,,全文,NCBI Genbank Database. 2005 * |
| NCBI Genbank Acession No.ZP_00880229. Birren,B., Lander,E.,全文,NCBI Genbank Database. 2005 * |
| NCBI NO.DQ056350. ZHANG,M等,全文. 2005 * |
| NCBI NO.ZP_00880229. BIRREN,B等,全文. 2005 * |
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| CN1821384A (en) | 2006-08-23 |
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