CN1796549A - Transfering enzyme of sulfur thiosulfuric acid and as medicament target drone for treating tuberculosis, and application for target drone of developing broadspectrum antibiotic - Google Patents
Transfering enzyme of sulfur thiosulfuric acid and as medicament target drone for treating tuberculosis, and application for target drone of developing broadspectrum antibiotic Download PDFInfo
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- CN1796549A CN1796549A CN 200410081616 CN200410081616A CN1796549A CN 1796549 A CN1796549 A CN 1796549A CN 200410081616 CN200410081616 CN 200410081616 CN 200410081616 A CN200410081616 A CN 200410081616A CN 1796549 A CN1796549 A CN 1796549A
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Abstract
This invention describes a thiosulfuric acid sulfur-transferring enzyme and its applications as the drug for tuberculosis and the developing target for other antibiotics. This invention also provides a process for separating the thiosulfuric acid sulfur-transferring enzyme of tuberculomyces and other bacteria, expressing and purifying the enzyme in colibacillus, streptomycosis and saccharomyces, utilizing the heterogenetic system containing the coding gene of the enzyme for screening of the suppresser or activator for the enzyme, and utilizing the purified enzyme for in vitro screening of the suppresser or activator for the enzyme.
Description
Technical field
The invention belongs to medical microbial and Microbial and Biochemical Pharmacy field, also belong to industrial microorganism and genetically engineered field, relate to separation and purification tubercule bacillus and other pathogenic bacterium thiosulfate sulfurtransferases, and this enzyme coding gene of heterogenous expression and carry out purifying, and utilize the allos system of expressing this enzyme as medicaments sifting model, the method for screening new tuberculotherapy medicine is the method for the Broad spectrum antibiotics of drug targets with the thiosulfate sulfurtransferase with other.
Background technology
Tuberculosis is the important transmissible disease of the serious threat human health that caused by mycobacterium tuberculosis (Mycobacterium tuberculosis is hereinafter to be referred as MTB), its mortality ratio in the whole world single bacterial pathogen infect cause in the disease the highest.Have every year nearly 3,000,000 people to die from tuberculosis in the world, about 1/3 population in the whole world infected mycobacterium tuberculosis.Substance of medicines-resistant branched tubercle bacillus is increasing, and has more increased the difficulty of tuberculotherapy and control with HIV virus coinfection, and it is extremely urgent to develop new tuberculotherapy medicine and diagnostic tool.The mycobacterium tuberculosis genome encoded altogether 4 kinds of thiosulfate sulfurtransferases (CysA2[Rv0815c], CysA3[Rv3117], SseA[Rv3283], SseB[Rv2291]), illustrate that this proteinoid is extremely important to mycobacterium.
Summary of the invention
The object of the present invention is to provide the new tuberculotherapy medicine of exploitation target, based on the tuberculotherapy drug development system of target and other new be the microbiotic exploitation system of target with the thiosulfate sulfurtransferase.
The basic fundamental route of realizing above-mentioned purpose is:
1. the separation and purification of thiosulfate sulfurtransferase: the biochemical separation method that utilizes biochemical field those skilled in the art to be familiar with (is seen Higher Education Publishing House, volumes such as Zhang Longxiang, " biochemical test technology "), comprise cytoclasis, ammonium sulfate precipitation, acetone precipitation, technology separation and purification such as ion-exchange, chromatography, SDS-PAGE obtain thiosulfate sulfurtransferase.
2. clone the encoding gene of thiosulfate sulfurtransferase, the gene of thiosulfate sulfurtransferase can freely obtain on the related web site of public database such as NCBI (www.ncbi.nlm.nih.gov) and SANGER (www.sanger.ac.uk) center and Pasteur Institut, the general networking person of being familiar with can both download, and the method that adopts genetically engineered field those skilled in the art to be familiar with (is seen Hunan science and technology press, 1999, Yuan Hanying, Xie Yi, Wang Honghai etc. write " study course of genetically engineered experimental technique ") comprise gene clone, heterogenous expression, abduction delivering, the thiosulfate sulfurtransferase that technical points such as gene engineering product downstream purification are expressed from purifying gene engineering.
3. the heterogenous expression host that will contain the thiosulfate sulfurtransferase encoding gene adds testing compound as screening model, and detection system is to the reaction of testing compound, thereby whether definite these compounds are suitable as guide's thing of tuberculotherapy medicine.
4. (concrete grammar is seen " international microbiotic magazines " 22 (2) in 2003 such as thanking to the Jianping: 172-174) the rough determination compounds effective to be added clinical separation tubercule bacillus, measure the effect of inhibition tubercule bacillus He other pathogenic bacterium of these compounds, and carry out in the body and experimentation on animals external compounds effective.
The invention effect: the present invention utilizes 8M urea cracking bacterium and genetic engineering bacterium, and dialysis makes enzyme folding again then.Inclusion body utilizes cinnyl to dissolve at pH11.In differing temps, utilize different inductor abduction deliverings respectively, then adopt IPTG as intestinal bacteria, then adopt methyl alcohol as pichia yeast, streptomycete is then adopted the constructive expression.Ammonium sulfate precipitation
Embodiment:
Embodiment
1.1 material
1.1.1 bacterial strain and cultivation: vazadrine resistance MTB is provided by clinical laboratory of Shanghai Pulmonary Hospital mycobacterium testing laboratory, and going down to posterity through flat board shows inheritance stability.Bacterium is added OADC (0.5% bovine serum albumin, 0.2% glucose, 0.06% olein, 37 ℃ of cultivations in liquid nutrient medium 140mmol/LNaCl) at Middlebrook 7H9 (Difco).Inoculation adds up to 2 * 10 in the 200ml Middlebrook7H9 substratum
8The resistance mycobacterium tuberculosis, cultivated 6～8 days for 37 ℃, to bacterial density be 1 * 10
8～2 * 10
8Or optical density(OD) (OD
600) be between 0.8～1.
1.2 method
1.2.1 clone and functional expression: genomic dna separates from SDS handles the bacterium of boiling, and comprises tubercule bacillus.Phenol chloroform extracting then, ethanol sedimentation DNA.Nucleotide sequence with BLAST relatively.
1.2.2 the preparation of cell protein sample: 4000r/min, 4 ℃ of centrifugal 15min precipitums, the PBS damping fluid washing precipitation bacterium of usefulness pH7.4 2 times.With suspend again precipitum and change 1.5ml EP pipe over to of 1mlPBS.1000r/min, 4 ℃ of centrifugal 15min precipitums.Dissolve with 1mlMilliQ water (containing 1mmol/L PMSF, 10mmol/L EDTA).Ultrasonication cracking bacterium 15 minutes, amplitude 70%, ultrasonic 1min stops 10s.Slowly to final concentration 9mol/L, dithiothreitol (DTT) (DTT) to final concentration is 70mmol/L to adding urea to the ultrasonic degradation thing.Add respectively Bio-lyte and OG to final concentration be 2%.Sample places room temperature 30min, at interval vibration.10000r/min, 20 ℃ of centrifugal 30min collect supernatant liquor, and-70 ℃ are frozen.The Bradford method is surveyed protein concn
[3]
2 results
Thiosulfate sulfurtransferase is a kind of albumen of similar rhodanese (rhodanese)
[7], extensively be present in the various organisms, comprise bacterium, plant and Mammals
[8]Its function is that the sulphur in the negatively charged ion of the similar thiosulfate anion of sulfur-bearing is transferred in other sulphur acceptor molecules, transfers to cyanate radical usually, forms thiocyanate ion.In Mammals, the anaerobic metabolism of this enzyme and halfcystine, methionine(Met), the detoxifcation of prussiate is transcribed sulfuration and the immunostimulant of back tRNA and all is correlated with
[9]4 kinds of thiosulfate sulfurtransferases and the mycobacterium tuberculosis genome has been encoded altogether (CysA2[Rv0815c], CysA3[Rv3117], SseA[Rv3283] and, SseB[Rv2291]), illustrate that this proteinoid is extremely important to mycobacterium.At present also few to the research of mycobacterium tuberculosis thiosulfate sulfurtransferase.The intestinal bacteria thiosulfate sulfurtransferase plays an important role in synthetic iron-sulphur bunch (4Fe-4S).Iron-sulphur bunch is the biosensor of oxygen and iron picked-up.Under the hypoxia condition, bacterium need strengthen the metabolism of unstable sulfide and iron-sulphur in the cell bunch
[10]Iron is the indispensable metallic element of mycobacterium tuberculosis.Therefore infer that thiosulfate sulfurtransferase is also probably relevant with the picked-up of MTB iron.This remains further to be studied.Under the hypoxia condition, the CysA2 of BCG and CysA3 express obviously and raise
[11]This illustrates that this enzyme may also have effect in the bacterium persistent infection.
MTB thiosulfate sulfurtransferase after the effect of this research employing Radix Ranunculi Ternati extract, suppress the expression of thiosulfate sulfurtransferase, illustrate that it may be by the inhibition to this enzyme, amino acid metabolism to mycobacterium tuberculosis, transcriptional level, aspects such as the detoxifcation of prussiate and sulphur and iron transfer exert an influence, and this may be that Radix Ranunculi Ternati extract is treated a kind of mechanism lungy.Radix Ranunculi Ternati extract may be as new tuberculosis medicine guide thing.
Claims (3)
1. the separation purification method of thiosulfate sulfurtransferase is characterized in that the gene of clones coding thiosulfate sulfurtransferase, heterogenous expression in comprising intestinal bacteria, streptomycete and yeast, and purifying.
2. the suitable medicament sifting motion system that contains the thiosulfate sulfurtransferase gene is characterized in that containing suitable reporter gene and promotor, is beneficial to measure the action effect of testing compound to the enzyme gene.
3. the method that suppresses its compound with the thiosulfate sulfurtransferase in-vitro screening of purifying is characterized in that the enzyme and the testing compound effect of purifying, measures the characteristic change of enzyme.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410081616 CN1796549A (en) | 2004-12-27 | 2004-12-27 | Transfering enzyme of sulfur thiosulfuric acid and as medicament target drone for treating tuberculosis, and application for target drone of developing broadspectrum antibiotic |
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| CN 200410081616 CN1796549A (en) | 2004-12-27 | 2004-12-27 | Transfering enzyme of sulfur thiosulfuric acid and as medicament target drone for treating tuberculosis, and application for target drone of developing broadspectrum antibiotic |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584962A (en) * | 2012-03-16 | 2012-07-18 | 河南中医学院 | Preparation method and application of mycobacterium tuberculosis Rv3117 recombinant protein |
-
2004
- 2004-12-27 CN CN 200410081616 patent/CN1796549A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584962A (en) * | 2012-03-16 | 2012-07-18 | 河南中医学院 | Preparation method and application of mycobacterium tuberculosis Rv3117 recombinant protein |
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Open date: 20060705 |