CN103805678B - The screening method of bacterium alkene acyl ACP reductase inhibitor - Google Patents
The screening method of bacterium alkene acyl ACP reductase inhibitor Download PDFInfo
- Publication number
- CN103805678B CN103805678B CN201410051932.9A CN201410051932A CN103805678B CN 103805678 B CN103805678 B CN 103805678B CN 201410051932 A CN201410051932 A CN 201410051932A CN 103805678 B CN103805678 B CN 103805678B
- Authority
- CN
- China
- Prior art keywords
- fabv
- fabi
- acp reductase
- acyl acp
- alkene acyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of screening method of bacterium alkene acyl ACP reductase inhibitor, comprise the following steps: by the fabK of enterococcus faecalis at expression in escherichia coli, suppress with triclosan and do not suppress the FabI of Escherichia coli recombinant strain, building the two dull and stereotyped sensitive difference screening model for FabK; Build fabI gene elmination mutant strain and the fabV gene elmination mutant strain of Pseudomonas aeruginosa, take wild mushroom as contrast, build the three dull and stereotyped sensitive difference screening models for FabI and FabV; The combination of the first two screening model is screened together, and combines and measure activity extract to the 503nhibiting concentration of the alkene acyl ACP reductase enzyme expression strain different with detection alkene acyl ACP reductase enzyme to the secondary screens of the sensitivity differences of activity extract.Usefulness of the present invention is: the efficiency not only increasing screening, more easily screens the inhibitor of wide spectrum, and for the diversity of alkene acyl ACP reductase enzyme also without the need to setting up multi-medicament screening model; Solve the problem that false positive is high and screening efficiency is low simultaneously.
Description
Technical field
The present invention relates to a kind of screening method of inhibitor, be specifically related to a kind of screening method of bacterium alkene acyl ACP reductase inhibitor, belong to technical field of molecular biology.
Background technology
Because bacterium creates resistance to existing microbiotic, and make the health of its still serious threat mankind, even occur the super drug-resistant bacteria making the whole world panic.Although scientific circles have hankered after finding the new antibiotic acting on new target site very much, the result that these effort obtain since the sixties in last century has been limited.Although hundreds of bacterium indispensable protein is identified can be used as potential drug action target spot, minority is wherein only had to become the target spot of clinical treatment medicine.Decades in the past, by carrying out to existing antibiotic skeleton the activity that chemically modified improves them, this strategy Development has gone out effective microbiotic; But the difficulty being developed more effective antibiotics by chemically modified is increasing.In order to adapt to promptly clinical needs, especially overcoming the bacterial drug resistance constantly occurred, finding that the novel chemistry of antibiotics skeleton with mode of action is vital for redemption life.
The synthesis of II type lipid acid is that bacterial cell existence is necessary, and therefore, it is the potential target spot of new antibiotic.Bacterial fatty acid route of synthesis is compared with mammiferous, although biochemical reaction process is similar, the two does not have homology in the enzymatic structure participating in catalyzed reaction and aminoacid sequence, belongs to different fatty acid synthetase systems.The special role that between bacterium and Mammals, the significant difference of lipid acid synthesis system and lipid acid are played the part of in cell, the drug screening target spot this system being become have a great attraction.The alkene acyl ACP reductase enzyme performing circulating reaction the 4th step in fatty acid synthesis pathway is that bacterial fatty acid synthesis is necessary, and it has been proved to be the action target spot of two kinds of antimicrobial triclosans commercially sold (triclosan) and vazadrine (isoniazid).Although other inhibitor many of alkene acyl ACP reductase enzyme are in the news, the new commercial antiseptic-germicide that up to the present market also not to occur with alkene acyl ACP reductase enzyme be drug target.In bacterium, there is diversity in alkene acyl ACP reductase enzyme, and the alkene acyl ACP reductase enzyme had been found that has 4 kinds.And the screening of inhibitor is at present all with single a kind of alkene acyl ACP reductase enzyme for drug target, the diversity for this reason for alkene acyl ACP reductase enzyme needs to set up multi-medicament screening model, and this have impact on the speed of drug screening to a great extent.
Therefore, if can set up a set of for alkene acyl ACP reductase enzyme diversity, namely the screening model being simultaneously drug screening target spot with multiple alkene acyl ACP reductase enzyme, will improve the efficiency of drug screening, this has very important significance to the discovery of the new antibiotic with mode of action.
The reduction of last rate-limiting step trans-2-alkene acyl ACP of alkene acyl ACP reductase enzyme catalysis II type lipid acid synthesis cycle reaction is the important target spot of screening antibacterials.In different bacterium, had been found that 4 kinds of different alkene acyl ACP reductase enzymes, FabI, FabL, FabK and FabV, specifically in table 1.
The diversity of table 1 bacterium alkene acyl ACP reductase enzyme
Note: SDRs is short carbon chain alcohol dehydrogenase/reductase enzyme superfamily member.
In intestinal bacteria, the alkene acyl ACP reductase enzyme FabI catalysis of the also reason NADH dependence of trans-2-alkene acyl ACP, FabI belongs to short carbon chain alcohol dehydrogenase/reductase enzyme (SDRs) superfamily member, and the albumen of its coding comprises conservative Tyr-(Xaa)
6-Lys catalytic active center sequence.FabI is the action target spot of extensive pedigree antibiotic triclosan (triclosan), and triclosan (triclosan) forms one non-covalent ternary complex (FabINAD closely with the oxidized form cofactor of zymoprotein and enzyme active center
+triclosan) FabI is suppressed.Have the homologous gene of fabI in the genome of many bacteriums, and there is not FabI in some bacteriums, as streptococcus pneumoniae, vibrio cholerae etc.Research shows that FabI is the unique action target spot of triclosan (triclosan) in intestinal bacteria, but FabI is not unique action target spot of triclosan (triclosan) in some bacterium.Streptococcus aureus is the same with intestinal bacteria only exists a necessary FabI of cell, but it is using NADPH as coenzyme.Except FabI in subtilis, also there is FabL, the alkene acyl ACP reductase enzyme that a kind of NADPH relies on simultaneously, low with the homology degree of FabI.FabK is alkene acyl ACP reductase enzyme unique in streptococcus pneumoniae, it has a flavine in conjunction with motif, and in conjunction with FMN, it is the reductase enzyme that NADH relies on, not there is homology with FabI, and amino acid alignment finds to have fabI and fabK two kinds of alkene acyl ACP reductase genes in enterococcus faecalis genome, but also there is no relevant experimental data at present.FabV finds for 2008 in vibrio cholerae, and the coenzyme that it utilizes is NADH.In Pseudomonas aeruginosa, then there are two kinds of alkene acyl ACP reductase enzymes, FabI and FabV, they all depend on NADH, and research finds that fabV sudden change makes the pathogenic of Pseudomonas aeruginosa and toxicity significantly reduce.Except FabI, its excess-three kind alkene acyl ACP reductase enzyme is insensitive to triclosan (Triclosan).As can be seen here, alkene acyl ACP reductase enzyme is in bacterium, and particularly there is diversity in pathogenetic bacteria, conservative property is not high, this restrict the range of application of the fungistat of target alkene acyl ACP reductase enzyme.Therefore, this just requires that scientific research personnel searches out corresponding inhibitor for different alkene acyl ACP reductase enzymes.
The alkene acyl ACP reductase inhibitor of current discovery is all for Sites Screening arrives with single alkene acyl ACP reductase enzyme, mainly contain FabI inhibitor and two kinds, FabK inhibitor, except these two kinds of inhibitor of IndoleNaphthyridinones and PhenylimidazoleDerivativesof4-Pyridone can suppress FabI and FabK simultaneously, other inhibitor are all suppress a kind of alkene acyl ACP reductase enzyme FabI or FabK specifically, and do not have inhibit activities to other three kinds of alkene acyl ACP reductase enzymes.But little for the research report of FabV inhibitor, only report that two kinds of 2-pyridone (PT172 and PT173) alkene acyl ACP reductase enzyme FabV to yersinia pestis have restraining effect, therefore in the urgent need to the inhibitor of exploitation for FabV recently.
The screening method of current alkene acyl ACP reductase inhibitor is mainly divided into two kinds: the first sets up external high-throughout alkene acyl ACP reductase inhibitor Screening Platform at molecular level.Whether this method is by being suppressed in vitro detection alkene acyl ACP reductase enzyme catalytic substrate reaction process, and from compound library, directly filter out compound alkene acyl ACP reductase enzyme being had to inhibit activities.This method also can in conjunction with Computer-Aided Drug Design, namely according to the architecture computer assisting sifting of alkene acyl ACP reductase enzyme or design and a series ofly the compound of inhibit activities may be had to form a compound library, then screens from this compound library.The advantage of this method to realize high flux screening.But the inhibitor screened in this way owing to often effectively can not enter cell, and can not demonstrate effective anti-microbial activity, and false positive is higher; In addition, need higher cost and require good experiment condition.
Second method sets up the Screening Platform of the fungistat of efficient target alkene acyl ACP reductase enzyme.First this method will build two kinds of alkene acyl ACP reductase enzymes and express discrepant bacterial strain, then occurs that the difference of upgrowth situation screens antimicrobial substance according to these two kinds of bacterial strains to the susceptibility difference of alkene acyl ACP reductase inhibitor.And be all build the bacterial strain of two kinds of alkene acyl ACP reductase enzyme differential expressions by process LAN alkene acyl ACP reductase enzyme in cell at present.But the screening efficiency of this method is lower.
Summary of the invention
For solving the deficiencies in the prior art, the object of the present invention is to provide a kind of screening efficiency high and the screening method of the bacterium alkene acyl ACP reductase inhibitor that false positive is low.
In order to realize above-mentioned target, the present invention adopts following technical scheme:
A screening method for bacterium alkene acyl ACP reductase inhibitor, is characterized in that, comprise the following steps:
(1) be, the unique action target spot of triclosan in intestinal bacteria according to FabI, by the fabK of enterococcus faecalis at expression in escherichia coli, suppress with triclosan and do not suppress the FabI of Escherichia coli recombinant strain, building the two dull and stereotyped sensitive difference screening model for FabK;
(2) otherness between the isozyme of alkene acyl ACP reductase enzyme and can be mutually substituting, is utilized, build fabI gene elmination mutant strain and the fabV gene elmination mutant strain of Pseudomonas aeruginosa, take wild mushroom as contrast, build the three dull and stereotyped sensitive difference screening models for FabI and FabV;
(3), by the screening model combination constructed by step () and step (two) screen together, and combine and measure activity extract to the 503nhibiting concentration of the alkene acyl ACP reductase enzyme expression strain different with detection alkene acyl ACP reductase enzyme to the secondary screens of the sensitivity differences of activity extract.
The screening method of aforesaid bacterium alkene acyl ACP reductase inhibitor, it is characterized in that, step () comprises following sub-step:
(1) fabK of pcr amplification enterococcus faecalis, fabI and fabV of Pseudomonas aeruginosa, and be cloned on pET28b carrier respectively, build protein expression and purification carrier, at expression in escherichia coli;
(2) by enterococcus faecalis fabK, Pseudomonas aeruginosa fabI and fabV respectively from pET28b recombinant vectors subclone to pBluescript carrier and transformation of E. coli MG1655, build high expression level enterococcus faecalis fabK, the Escherichia coli recombinant strain MBlueK of Pseudomonas aeruginosa fabI and fabV, MBlueI and MblueV, empty carrier pBluescript is transformed MG1655 and builds Escherichia coli recombinant strain Mblue;
(3) apply manure to the fields pET28b carrier enterococcal fabK and Pseudomonas aeruginosa fabV, on subclone to low copy expression vector pHSG575, and transformation of E. coli wild type strain MG1655, build Escherichia coli recombinant strain MGK and the MGV of low expression enterococcus faecalis FabK and Pseudomonas aeruginosa FabV, be respectively used to build the two dull and stereotyped sensitive difference screening model for FabK and FabV.
The screening method of aforesaid bacterium alkene acyl ACP reductase inhibitor, it is characterized in that, step (two) comprises following sub-step:
With the upstream and downstream sequence of Pseudomonas aeruginosa genomic dna for template difference pcr amplification fabI and fabV, the method of being cut connection by Overlap extension PCR or enzyme builds gene elmination box △ fabI and △ fabV, from plasmid p34s-Gm, enzyme cuts GmR gene, be connected to the side of gene elmination box △ fabI and △ fabV, build gene knockout box △ fabI-GmR and △ fabV-GmR, be cloned on suicide vector pK18mobsacB, transformation of E. coli S17-1 builds recombinant bacterium, and by conjugation will recombinate suicide vector import Pseudomonas aeruginosa, in conjunction with resistance screening, sucrose screening and PCR qualification, obtain the gene elmination mutant strain of fabI and fabV non-resistant selection markers, called after PAO-I and PAO-V respectively, take wild type strain as contrast, build the three dull and stereotyped sensitive difference screening models for FabI and FabV.
The screening method of aforesaid bacterium alkene acyl ACP reductase inhibitor, it is characterized in that, step (three) comprises following sub-step:
(1), elementary screening: extract activity extract from plant and microorganism, extract paper disk method or cylinder plate method carry out Antibacterial Activity;
(2), secondary screens: detect the different expression strain of alkene acyl ACP reductase enzyme to the sensitivity differences of the activity extract that primary dcreening operation obtains, detect the activity extract of primary dcreening operation acquisition to the 503nhibiting concentration of alkene acyl ACP reductase enzyme.
The screening method of aforesaid bacterium alkene acyl ACP reductase inhibitor, it is characterized in that, in aforementioned secondary screens, detect the different expression strain of alkene acyl ACP reductase enzyme to the sensitivity differences of activity extract by the antimicrobial spectrum of activity extract and the mensuration of minimal inhibitory concentration.
The screening method of aforesaid bacterium alkene acyl ACP reductase inhibitor, is characterized in that, in aforementioned secondary screens, the activity extract detecting primary dcreening operation acquisition, to the 503nhibiting concentration of alkene acyl ACP reductase enzyme, comprises the following steps:
(1), the protein expression and purification carrier that will build, at expression in escherichia coli, utilize 6 × his-tag separation and purification of protein technology, the colibacillary FabI of purifying, the FabK of enterococcus faecalis, FabI and FabV of Pseudomonas aeruginosa;
(2), with crotonoyl-CoA or trans-2-decoyl-N-acetylcysteamine for substrate, add zymoprotein, NADH or NADPH and activity extract, reconstruction in vitro alkene acyl ACP reduces enzymatic reduction reaction, measures the absorbancy OD of NADH or NADPH in reaction process
340change, once, record data, keep system temperature to be 37 DEG C to every 10sec reading in reaction process, take time as X-coordinate, OD
340for ordinate zou, curve plotting, simulate equation of linear regression, slope calculations;
(3), activity extract is to the inhibit activities IC(% of alkene acyl ACP reductase enzyme)=100 × [1-(slope of slope/blank when activity extract exists)], with the concentration of activity extract be X-coordinate, inhibit activities for ordinate zou curve plotting, try to achieve IC by curve
50.
In bacterium, there is multiple isozyme in alkene acyl ACP reductase enzyme, there are two kinds of isozymes in some pathogenetic bacteria even simultaneously, as there is FabI, FabV and FabI, FabK respectively in Pseudomonas aeruginosa and enterococcus faecalis simultaneously, the otherness between the multiple isozyme of alkene acyl ACP reductase enzyme and can the substituting highly efficient depressor Screening Platform building cell levels mutually therefore can be utilized.But yet there are no relevant report up till now.
Usefulness of the present invention is: the present invention is directed to bacterium alkene acyl ACP reductase enzyme diversity, based on alkene acyl ACP reductase enzyme isozyme between otherness and can substituting and FabI be the theoretical research foundation of the unique action target spot of triclosan in intestinal bacteria mutually, Protocols in Molecular Biology is utilized to build with three kinds of alkene acyl ACP reductase enzyme FabI, FabK and FabV is the novel alkene acyl ACP reductase inhibitor cell screening model of target spot, not only increase the efficiency of screening, more easily screen the inhibitor of wide spectrum, and for the diversity of alkene acyl ACP reductase enzyme also without the need to setting up multi-medicament screening model, in addition, the present invention is the screening based on cell levels, solves the shortcoming that the false positive of prior art one is high, solves the low shortcoming of the screening efficiency of prior art two based on the multifarious screening of alkene acyl ACP reductase enzyme.
Accompanying drawing explanation
Fig. 1 is the main flow figure of screening method of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, concrete introduction is done to the present invention.
With reference to Fig. 1, the screening method of bacterium alkene acyl ACP reductase inhibitor of the present invention, comprises the following steps:
The first step: be the unique action target spot of triclosan in intestinal bacteria according to FabI, by the fabK of enterococcus faecalis at expression in escherichia coli, suppress with triclosan and do not suppress the FabI of Escherichia coli recombinant strain, building the two dull and stereotyped sensitive difference screening model for FabK.
In order to improve the sensitivity of screening, express FabK, such as pET28b carrier with low copy expression vector.
Concrete, this first step comprises following sub-step:
1, the fabK of pcr amplification enterococcus faecalis, fabI and fabV of Pseudomonas aeruginosa, and be cloned on pET28b carrier respectively, build protein expression and purification carrier, at expression in escherichia coli, for purifying FabI, FabK and FabV albumen.
2, by enterococcus faecalis fabK, Pseudomonas aeruginosa fabI and fabV respectively from pET28b recombinant vectors subclone to pBluescript carrier and transformation of E. coli MG1655, build high expression level enterococcus faecalis fabK, the Escherichia coli recombinant strain MBlueK of Pseudomonas aeruginosa fabI and fabV, MBlueI and MblueV, empty carrier pBluescript is transformed MG1655 and builds Escherichia coli recombinant strain Mblue.
3, apply manure to the fields pET28b carrier enterococcal fabK and Pseudomonas aeruginosa fabV, on subclone to low copy expression vector pHSG575, and transformation of E. coli wild type strain MG1655, build Escherichia coli recombinant strain MGK and the MGV of low expression enterococcus faecalis FabK and Pseudomonas aeruginosa FabV, be respectively used to build the two dull and stereotyped sensitive difference screening model for FabK and FabV.
Second step: utilize the otherness between the isozyme of alkene acyl ACP reductase enzyme and can be mutually substituting, build fabI gene elmination mutant strain and the fabV gene elmination mutant strain of Pseudomonas aeruginosa, take wild mushroom as contrast, build the three dull and stereotyped sensitive difference screening models for FabI and FabV.
In addition, more flourishing for Pseudomonas aeruginosa discharging system, stronger to the resistance of medicine, in order to more effectively screen the inhibitor of FabV, the method provided according to the first step by the fabV of Pseudomonas aeruginosa with low copy expression vector at expression in escherichia coli, build the two dull and stereotyped sensitive difference screening model for FabV.
The concrete operations of second step are as follows:
With the upstream and downstream sequence of Pseudomonas aeruginosa genomic dna for template difference pcr amplification fabI and fabV, the method of being cut connection by Overlap extension PCR or enzyme builds gene elmination box △ fabI and △ fabV, from plasmid p34s-Gm, enzyme cuts GmR gene, be connected to the side of gene elmination box △ fabI and △ fabV, build gene knockout box △ fabI-GmR and △ fabV-GmR, be cloned on suicide vector pK18mobsacB, transformation of E. coli S17-1 builds recombinant bacterium, and by conjugation will recombinate suicide vector import Pseudomonas aeruginosa, in conjunction with resistance screening, sucrose screening and PCR qualification, obtain the gene elmination mutant strain of fabI and fabV non-resistant selection markers, called after PAO-I and PAO-V respectively, take wild type strain as contrast, build the three dull and stereotyped sensitive difference screening models for FabI and FabV.
3rd step: the screening model combination constructed by step () and step (two) is screened together, and combine and measure activity extract to the 503nhibiting concentration of the alkene acyl ACP reductase enzyme expression strain different with detection alkene acyl ACP reductase enzyme to the secondary screens of the sensitivity differences of activity extract.Secondary screens can improve the accuracy of screening.
1, elementary screening process is as follows: from plant and microorganism, extract activity extract, and extract paper disk method or cylinder plate method carry out Antibacterial Activity.
Test organisms flat board has seven kinds, is divided into three groups, and they are LB+MGK and LB+MGK+ triclosan (triclosan) two flat board respectively, LB+MGV and LB+MGV+ triclosan (triclosan) two flat board, and LB+PAO-I, LB+PAO-V and LB+PAO1 tri-is dull and stereotyped.
After cultivation, if the inhibition zone on the flat board of correspondence varies in size, showing may containing the meta-bolites suppressing alkene acyl ACP reductase enzyme in extract, thus can activity extract be decided to be, this is because the character of alkene acyl ACP reductase enzyme contained between each pair of test strain and expression amount exist different and cause them different to the susceptibility of the secondary metabolite suppressing alkene acyl ACP reductase enzyme, the size of the inhibition zone of formation is also just different.
If the size of inhibition zone does not have difference in same group of flat board, this show antibacterial substance not with alkene acyl ACP reductase enzyme for target spot.
The active substance that specificity to suppress in FabI, FabK and FabV these three kinds of alkene acyl ACP reductase enzymes one or two can be filtered out by this screening system.
2, secondary screens process is as follows:
(1) the different expression strain of alkene acyl ACP reductase enzyme is detected to the sensitivity differences of the activity extract that primary dcreening operation obtains.
The different expression strain of alkene acyl ACP reductase enzyme can be detected to the sensitivity differences of activity extract by the mensuration of the antimicrobial spectrum of activity extract and minimal inhibitory concentration.
Minimal inhibitory concentration (MIC) measures concrete grammar: by activity extract with continuous print twice dilution in bacterial concentration for 10
5in the bacterium bacterium liquid to be measured of CFU/ml, continue under proper temperature to cultivate 20h.Minimal inhibitory concentration (MIC) is defined as the minimum concentration of the activity extract of complete bacteria growing inhibiting.Detection of active extract to the minimal inhibitory concentration (MIC) of the pathogenic strainss such as intestinal bacteria, Pseudomonas aeruginosa, vibrio cholerae, streptococcus aureus, enterococcus faecalis and streptococcus pneumoniae, and compares by following aspect:
FabI inhibitor: compare MG1655(pBluescript-fabI) and MG1655 (pBluescript) to the sensitivity differences of activity extract;
FabK inhibitor: triclosan compares MG1655(pHSG575-fabK under suppressing intestinal bacteria FabI prerequisite) and MG1655 (pBluescript-fabK) to the sensitivity differences of activity extract;
FabV inhibitor: triclosan compares MG1655(pHSG575-fabV under suppressing intestinal bacteria FabI prerequisite) and MG1655 (pBluescript-fabV) to the sensitivity differences of activity extract.
(2) activity extract of primary dcreening operation acquisition is detected to the 503nhibiting concentration (IC of alkene acyl ACP reductase enzyme
50).Can operate in accordance with the following steps:
A, the protein expression and purification carrier that will have built, at expression in escherichia coli, utilize 6 × his-tag separation and purification of protein technology, the alkene acyl ACP reductase enzymes such as the colibacillary FabI of purifying, the FabK of enterococcus faecalis, FabI and FabV of Pseudomonas aeruginosa.
B, with crotonoyl-CoA or trans-2-decoyl-N-acetylcysteamine for substrate, add zymoprotein, NADH or NADPH and activity extract, reconstruction in vitro alkene acyl ACP reduces enzymatic reduction reaction, measures the absorbancy OD of NADH or NADPH in reaction process
340change, once, record data, often organize Data duplication 3 times, in reaction process, keep system temperature to be 37 DEG C, take time as X-coordinate to every 10sec reading, OD
340for ordinate zou, curve plotting, simulate equation of linear regression, slope calculations.
C, activity extract are to the inhibit activities IC(% of alkene acyl ACP reductase enzyme)=100 × [1-(slope of slope/blank when activity extract exists)], IC
50then represent the concentration of extract when inhibit alkene acyl ACP reductase 50 % active.
With the concentration of activity extract be X-coordinate, inhibit activities for ordinate zou curve plotting, can IC be tried to achieve by curve
50.
As can be seen here, screening method of the present invention has following features:
(1) be the unique action target spot of triclosan in intestinal bacteria according to FabI, suppress with triclosan and do not suppress the FabI of Escherichia coli recombinant strain, build the two dull and stereotyped sensitive difference screening model for FabK target spot, and with low copy expression vector at expression in escherichia coli FabK, improve the sensitivity of screening.
(2) there is multifarious feature in directed toward bacteria alkene acyl ACP reductase enzyme, utilize the otherness between the isozyme of alkene acyl ACP reductase enzyme and can be mutually substituting, the three dull and stereotyped sensitive difference screening models for FabI and FabV target spot that to build with the fabI mutant strain of Pseudomonas aeruginosa, fabV mutant strain and wild mushroom be strain subject.Simultaneously more flourishing for Pseudomonas aeruginosa discharging system, stronger to the resistance of medicine, by the fabV of Pseudomonas aeruginosa with low copy expression vector at expression in escherichia coli, build the two dull and stereotyped sensitive difference screening model for FabV target spot, more effectively can screen the inhibitor of FabV.
(3) by the elementary screening of paper disk method or cylinder plate method with measure IC
50, the secondary screens of expression strain to the sensitivity differences of activity extract that detect alkene acyl ACP reductase enzyme different combine, and substantially increases the accuracy of screening.
(4) present method utilizes the diversity of bacterium alkene acyl ACP reductase enzyme to carry out the screening of inhibitor, once can carry out inhibitor screening for multiple target spot (in FabI, FabK and FabV one or two), improve the efficiency of screening.
In addition, because the present invention is the screening based on cell levels, solve the shortcoming that the false positive of prior art one is high, the present invention simultaneously is also based on the multifarious screening of alkene acyl ACP reductase enzyme, solves the shortcoming that the screening efficiency of prior art two is low.
It should be noted that, above-described embodiment does not limit the present invention in any form, the technical scheme that the mode that all employings are equal to replacement or equivalent transformation obtains, and all drops in protection scope of the present invention.
Claims (4)
1. the screening method of bacterium alkene acyl ACP reductase inhibitor, is characterized in that, comprise the following steps:
(1) be, the unique action target spot of triclosan in intestinal bacteria according to FabI, by the fabV of the fabK of enterococcus faecalis and Pseudomonas aeruginosa respectively at expression in escherichia coli, suppress with triclosan and do not suppress the FabI of Escherichia coli recombinant strain, build the two dull and stereotyped sensitive difference screening model for FabK and FabV, specifically comprise following sub-step:
(1) fabK of pcr amplification enterococcus faecalis, fabI and fabV of Pseudomonas aeruginosa, and be cloned on pET28b carrier respectively, build protein expression and purification carrier, at expression in escherichia coli;
(2) by enterococcus faecalis fabK, Pseudomonas aeruginosa fabI and fabV respectively from pET28b recombinant vectors subclone to pBluescript carrier and transformation of E. coli MG1655, build high expression level enterococcus faecalis fabK, the Escherichia coli recombinant strain MBlueK of Pseudomonas aeruginosa fabI and fabV, MBlueI and MblueV, empty carrier pBluescript is transformed MG1655 and builds Escherichia coli recombinant strain Mblue;
(3) apply manure to the fields pET28b carrier enterococcal fabK and Pseudomonas aeruginosa fabV, on subclone to low copy expression vector pHSG575, and transformation of E. coli wild type strain MG1655, build Escherichia coli recombinant strain MGK and the MGV of low expression enterococcus faecalis FabK and Pseudomonas aeruginosa FabV, be respectively used to build the two dull and stereotyped sensitive difference screening model for FabK and FabV, described two flat board is that LB+MGK and LB+MGK+ triclosan is two dull and stereotyped, or LB+MGV and LB+MGV+ triclosan is two dull and stereotyped;
(2) otherness between the isozyme of alkene acyl ACP reductase enzyme and can be mutually substituting, is utilized, build fabI gene elmination mutant strain and the fabV gene elmination mutant strain of Pseudomonas aeruginosa, with Pseudomonas aeruginosa wild mushroom PAO1 for contrast, build the three dull and stereotyped sensitive difference screening models for FabI and FabV, described three flat boards are that LB+PAO-I, LB+PAO-V and LB+PAO1 tri-is dull and stereotyped
Wherein, the fabI gene elmination mutant strain and the fabV gene elmination mutant strain that build Pseudomonas aeruginosa specifically comprise the following steps:
With the upstream and downstream sequence of Pseudomonas aeruginosa wild mushroom PAO1 genomic dna for template difference pcr amplification fabI and fabV, the method of being cut connection by Overlap extension PCR or enzyme builds gene elmination box △ fabI and △ fabV, from plasmid p34s-Gm, enzyme cuts GmR gene, be connected to the side of gene elmination box △ fabI and △ fabV, build gene knockout box △ fabI-GmR and △ fabV-GmR, be cloned on suicide vector pK18mobsacB, transformation of E. coli S17-1 builds recombinant bacterium, and by conjugation will recombinate suicide vector import Pseudomonas aeruginosa, in conjunction with resistance screening, sucrose screening and PCR qualification, obtain the gene elmination mutant strain of fabI and fabV non-resistant selection markers, called after PAO-I and PAO-V respectively,
(3), by the screening model combination constructed by step () and step (two) screen together, and combine and measure activity extract to the 503nhibiting concentration of the alkene acyl ACP reductase enzyme expression strain different with detection alkene acyl ACP reductase enzyme to the secondary screens of the sensitivity differences of activity extract.
2. the screening method of bacterium alkene acyl ACP reductase inhibitor according to claim 1, it is characterized in that, step (three) comprises following sub-step:
(1), elementary screening: extract activity extract from plant and microorganism, extract paper disk method or cylinder plate method carry out Antibacterial Activity;
(2), secondary screens: detect the different expression strain of alkene acyl ACP reductase enzyme to the sensitivity differences of the activity extract that primary dcreening operation obtains, detect the activity extract of primary dcreening operation acquisition to the 503nhibiting concentration of alkene acyl ACP reductase enzyme.
3. the screening method of bacterium alkene acyl ACP reductase inhibitor according to claim 2, it is characterized in that, in described secondary screens, detect the different expression strain of alkene acyl ACP reductase enzyme to the sensitivity differences of activity extract by the antimicrobial spectrum of activity extract and the mensuration of minimal inhibitory concentration.
4. the screening method of bacterium alkene acyl ACP reductase inhibitor according to claim 2, is characterized in that, in described secondary screens, the activity extract detecting primary dcreening operation acquisition, to the 503nhibiting concentration of alkene acyl ACP reductase enzyme, comprises the following steps:
(1), the protein expression and purification carrier that will build, at expression in escherichia coli, utilize 6 × his-tag separation and purification of protein technology, the FabK of purifying enterococcus faecalis, FabI and FabV of Pseudomonas aeruginosa;
(2), with crotonoyl-CoA or trans-2-decoyl-N-acetylcysteamine for substrate, add zymoprotein, NADH or NADPH and activity extract, reconstruction in vitro alkene acyl ACP reduces enzymatic reduction reaction, measures the absorbancy OD of NADH or NADPH in reaction process
340change, once, record data, keep system temperature to be 37 DEG C to every 10sec reading in reaction process, take time as X-coordinate, OD
340for ordinate zou, curve plotting, simulate equation of linear regression, slope calculations;
(3), activity extract is to inhibit activities IC%=100 × [1-(slope of slope/blank when activity extract exists)] of alkene acyl ACP reductase enzyme, with the concentration of activity extract be X-coordinate, inhibit activities for ordinate zou curve plotting, try to achieve IC by curve
50.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410051932.9A CN103805678B (en) | 2014-02-14 | 2014-02-14 | The screening method of bacterium alkene acyl ACP reductase inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410051932.9A CN103805678B (en) | 2014-02-14 | 2014-02-14 | The screening method of bacterium alkene acyl ACP reductase inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103805678A CN103805678A (en) | 2014-05-21 |
| CN103805678B true CN103805678B (en) | 2016-04-06 |
Family
ID=50703063
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410051932.9A Expired - Fee Related CN103805678B (en) | 2014-02-14 | 2014-02-14 | The screening method of bacterium alkene acyl ACP reductase inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103805678B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2509159B (en) * | 2012-12-21 | 2017-07-26 | Oxoid Ltd | Triclosan derivatives and uses thereof |
| CN107557321B (en) * | 2017-10-18 | 2020-11-17 | 延安大学 | Streptomyces jujubae JL-1, active matter thereof, preparation method and application |
| KR102667208B1 (en) | 2018-11-14 | 2024-05-20 | 스마일바이오텍 주하이 리미티드 | Animal models, screening methods, and treatment methods for intraocular diseases or conditions |
| CN111249313A (en) * | 2018-11-14 | 2020-06-09 | 珠海岐微生物科技有限公司 | Application of microorganism in screening of eye disease treatment drugs |
| CN117987443A (en) * | 2024-02-27 | 2024-05-07 | 华南农业大学 | Bacillus subtilis non-resistance expression system with fabL gene as resistance marker, construction method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6613553B1 (en) * | 2000-02-04 | 2003-09-02 | St. Jude Children's Research Hospital | Enoyl reductases and methods of use thereof |
| WO2009011651A1 (en) * | 2007-07-16 | 2009-01-22 | Rvc Enterprise | Mutually suppressive gene/inhibitor combinations for non-antibiotic selection of recombinant strains |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040024068A1 (en) * | 1998-01-23 | 2004-02-05 | Trustees Of Tufts College | Antimicrobial compounds |
-
2014
- 2014-02-14 CN CN201410051932.9A patent/CN103805678B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6613553B1 (en) * | 2000-02-04 | 2003-09-02 | St. Jude Children's Research Hospital | Enoyl reductases and methods of use thereof |
| WO2009011651A1 (en) * | 2007-07-16 | 2009-01-22 | Rvc Enterprise | Mutually suppressive gene/inhibitor combinations for non-antibiotic selection of recombinant strains |
Non-Patent Citations (3)
| Title |
|---|
| An array of Escherichia Coli clone over-expression essential proteins: a new strategy of identifying cellular targets of potent antibacterial compounds;H.Howard Xu, et al;《Biochem Biophys Res Commun》;20061103;第39卷(第4期);1250-1257 * |
| Discovery of FabH/FabF inhibitors from Natural Products;Katherine Young,et al;《Antimicrobial agents and Chemotherapy》;20060228;第50卷(第2期);519-526 * |
| Triclosan Resistance of Pseudomonas aeruginosa PAO1 is due to FabV, a Triclosan-Resistant Enoyl-Acyl Carrier Protein Reductase;Lei Zhu, et al;《 Antimicrobial agents and Chemotherapy》;20100228;第54卷(第2期);689-698 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103805678A (en) | 2014-05-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tang et al. | Metagenomic approaches to understanding bacterial communication during the anammox reactor start-up | |
| Pratscher et al. | Unravelling the identity, metabolic potential and global biogeography of the atmospheric methane‐oxidizing upland soil cluster α | |
| Guo et al. | Development and characterization of a halo-thermophilic bacterial consortium for decolorization of azo dye | |
| CN103805678B (en) | The screening method of bacterium alkene acyl ACP reductase inhibitor | |
| Nunes et al. | Living with sulfonamides: a diverse range of mechanisms observed in bacteria | |
| Chen et al. | Identification of a hotdog fold thioesterase involved in the biosynthesis of menaquinone in Escherichia coli | |
| Zhang et al. | Paracoccus versutus KS293 adaptation to aerobic and anaerobic denitrification: Insights from nitrogen removal, functional gene abundance, and proteomic profiling analysis | |
| Meng et al. | Changes in nitrogen removal and microbiota of anammox biofilm reactors under tetracycline stress at environmentally and industrially relevant concentrations | |
| Guo et al. | Decolorization and detoxification of azo dye by halo-alkaliphilic bacterial consortium: Systematic investigations of performance, pathway and metagenome | |
| Goswami et al. | Bacillus megaterium adapts to acid stress condition through a network of genes: Insight from a genome-wide transcriptome analysis | |
| Kasalický et al. | Aerobic anoxygenic photosynthesis is commonly present within the genus Limnohabitans | |
| Thakur et al. | Degradation of sulphonated azo dye red HE7B by Bacillus sp. and elucidation of degradative pathways | |
| Rathakrishnan et al. | Isolation and characterization of halophilic isolates from Indian salterns and their screening for production of hydrolytic enzymes | |
| Asimakoula et al. | Phenol degradation by Pseudarthrobacter phenanthrenivorans Sphe3 | |
| Akita et al. | Enterobacter oligotrophica sp. nov., a novel oligotroph isolated from leaf soil | |
| Ogasawara et al. | Identification of tirandamycins as specific inhibitors of the futalosine pathway | |
| Shelton et al. | Flexible cobamide metabolism in Clostridioides (Clostridium) difficile 630 Δ erm | |
| Lauritsen et al. | Identification and differentiation of Pseudomonas species in field samples using an rpoD amplicon sequencing methodology | |
| Zhang et al. | Comprehensive metagenomic and enzyme activity analysis reveals the inhibitory effects and potential toxic mechanism of tetracycline on denitrification in groundwater | |
| Zhang et al. | Geomesophilobacter sediminis gen. nov., sp. nov., Geomonas propionica sp. nov. and Geomonas anaerohicana sp. nov., three novel members in the family Geobacterecace isolated from river sediment and paddy soil | |
| Yadav et al. | Genomic analysis of family UBA6911 (group 18 Acidobacteria) expands the metabolic capacities of the phylum and highlights adaptations to terrestrial habitats | |
| Ma et al. | Influence of organic loading rate on purified terephthalic acid wastewater treatment in a temperature staged anaerobic treatment (TSAT) system: Performance and metagenomic characteristics | |
| Zhu et al. | Hymenobacter cavernae sp. nov., isolated from a karst cave | |
| Trang et al. | Discovery of α-glucosidase inhibitors from marine microorganisms: Optimization of culture conditions and medium composition | |
| Kumaran et al. | In vitro and in silico analysis of Brilliant Black degradation by Actinobacteria and a Paraburkholderia sp. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160406 Termination date: 20170214 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |