CN104789582B - A kind of new support for selection of antibacterial - Google Patents
A kind of new support for selection of antibacterial Download PDFInfo
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- CN104789582B CN104789582B CN201510176236.5A CN201510176236A CN104789582B CN 104789582 B CN104789582 B CN 104789582B CN 201510176236 A CN201510176236 A CN 201510176236A CN 104789582 B CN104789582 B CN 104789582B
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Abstract
The invention discloses a kind of new support for selection of antibacterial, the carrier is pKD 0011, is mainly used for screening the antibacterials for having obvious inhibiting effect to the pathogenic and drug resistance of pseudomonas aeruginosa.Carrier is using drug resistance and therapeutic two-phase correlation gene PA0011 as drug target, pass through the plasmid pMS402 of the reporter luxCDABE with deletion promoters, the promoter region of drug resistance and pathogenic two-phase correlation gene PA0011 are constituted into promoter reporter fusion vector pKD 0011 by PCR amplification, enzyme cutting clone to before reporting plasmid pMS402 luciferase operon genes；Recombinant plasmid is transferred in the pseudomonas aeruginosa of wild type using electrotransformation technology, the antibacterial material for the PA0011 expression that is inhibited can be screened using the luminous power of carrier pKD 0011.High flux screening may be implemented in the carrier, easy to operate using related screening installation, economical and practical.
Description
Technical field
It is specifically a kind of for screening to pseudomonas aeruginosa the present invention relates to a kind of carrier for selection of antibacterial
Pathogenic and drug resistance have obvious inhibiting effect antibacterials screening vector.
Background technology
Pseudomonas aeruginosa is a kind of opportunist, is lacked in immune system the main reason for causing inside-hospital infection
Relatively high incidence is often resulted in sunken patient such as patient HIV, severe burn patients and pulmonary cyst fibrosis patient
And the death rate.
In recent years, pseudomonas aeruginosa all increased the tolerance of nearly all antibacterials.On the one hand be due to
Unreasonable uses antibiotic so that thalline produces adaptability drug resistance；On the other hand it is because pseudomonas aeruginosa itself is deposited
It is stronger in play a role in resistance mechanism.Pseudomonas aeruginosa is continuously increased to antibiotic resistance so that clinically
Select suitable therapeutic strategy becomes very difficult to treat the infection of pseudomonas aeruginosa, results in higher and higher death
Rate, thus found that novel effective antibacterial material is extremely urgent.
Oneself gradually shows unique advantage to Chinese medicine in preventing chronic infection.China has abundant Chinese herbal medicine resource,
Source is wide, cheap, toxic side effect is small, it is less will appear drug resistance and in bacterial cell action pathway diversification, can produce
Raw various aspects pharmacodynamics effect, therefore can be studied as the drug of potential resisting pseudomonas aeruginosa.
The screening technique of new antibacterials is the key factor for finding novel antibacterial substance.Traditional antibiotic-screening side
Method is the disadvantage is that action target spot is indefinite, mechanism is unintelligible, insensitive, screening process has randomness.Genetic marker method is current
One of state-of-the-art technology in screening technique, by mark special gene, find relevant biological activity object, advantage be it is sensitive, can
Inefficient object is detected, can be used for being chemically modified, easily screen, is high-throughput.
Antibiotic also shows other activity, especially less than most in addition to traditional antibacterial and bactericidal effect
Under small inhibition concentration, extraneous antibacterial material can have adjustment effect to the expression of gene, can adjust the expression of virulence factor,
The drug resistance for inducing pathogen influences intercellular interaction etc. in micropopulation.It, can using this characteristic of antibiotic
To screen the novel antibacterial substance that there is adjustment effect to the gene by building the expression vector of specific gene.
Lipoid A is one of the important feature of lipopolysaccharides (LPS), and interacting between LPS other compositions forms having for outer membrane
Imitate barrier；When the structure of LPS constructional device defects, LPS will be destroyed, there is breach for LPS layers therewith, phospholipid molecule will be from
Outer film inner layer moves to fatiscent place, is repaired, i.e., forms phospholipid bilayer spot in outer membrane, lead to outer membrane
Barrier action is impaired, so as to keep hydrophobic antibiotic and harmful substance freely penetrating, bacterial antibiotic or nuisance qualitative change
It obtains sensitive.In addition, the endotoxin component of lipoid A or LPS, endotoxin is heat-resisting and stablizes, and is the important causative agent of bacterium
Matter.
PA0011 gene codes 2-OH- acyltransferases participate in the building-up process of lipoid A, team in pseudomonas aeruginosa
It is found by early-stage study：PA0011 has the biochemical functions, drug resistance and pathogenecity of pseudomonas aeruginosa
Highly important effect, after PA0011 afunction to tetracycline, gentamicin, polymyxins, rifampin, carbenicillin,
Card receives mycin, and the resistance of carbapenem antibiotic is all substantially reduced, and pseudomonas aeruginosa is caused to invade HeLa cells
Contaminating power reduces, and is significantly reduced to the lethality of drosophila.Moreover, also having similar discovery in other bacterial strains, for example, Salmonella
HtrB is the homologous gene of pseudomonas aeruginosa PA0011 in bacterium, and the mutant activity of endotoxin of htrB reduces, and mutant causes a disease
Property is also affected.Therefore, PA0011 can be used as a good drug target, for screening novel antibacterial substance.For
The medication treatment of other pathogenic bacteria also has reference function.
Invention content
The purpose of the present invention is to provide a kind of carriers of selection of antibacterial, specifically with one in pseudomonas aeruginosa
A drug resistant gene is target spot, the detection carrier of promoter-reporter of the gene is built, to solve to propose in above-mentioned background technology
The problem of.
To achieve the above object, the present invention provides the following technical solutions：
A kind of carrier for selection of antibacterial, the carrier are pKD-PA0011, are mainly used for screening to verdigris vacation
The pathogenic and drug resistance of monad has the antibacterials of obvious inhibiting effect.The construction method of the carrier is：With drug resistance and
Two-phase correlation gene PA0011 cause a disease as drug target, with the plasmid of the reporter luxCDABE with deletion promoters
PMS402 is carrier, by the promoter region of PA0011 genes through PCR amplification, enzyme cutting clone to report plasmid pMS402 institutes band
Before luciferase luxCDABE operons, promoter-reporter fusion vector pKD-0011 is constituted；It will be weighed using electrotransformation technology
Group plasmid pKD-0011 is transferred in the pseudomonas aeruginosa of wild type, is obtained by detecting the luminous situation of pKD-0011 to screen
Inhibit the antibacterial material of PA0011 expression.
As a further solution of the present invention：Including the primer sequence of the drug resistant gene PA0011 including promoter is：
Sense primer is 5 '-TATctcgagCAGGACTGCGACCGTTACG-3 ',
Downstream primer is 5 '-CTCggatccCACATCAGCCAGCCTATG-3’。
Compared with prior art, the beneficial effects of the invention are as follows：The present invention constructs a kind of utilization from " overriding resistance " angle
High-throughput gene expression detection technology of the luciferase gene (luxCDABE) as reporter is screened in pseudomonas aeruginosa
An important drug resistance and the promoter of pathogenic two-phase correlation gene PA0011 have the antibacterial material of inhibiting effect.The carrier can be with
Realize high flux screening；It is easy to operate using related screening installation, it is economical and practical；Screening obtained drug can effective verdigris
The pathogenic and drug resistance of pseudomonad, and mechanism of action clear and definite.
Description of the drawings
Fig. 1 is carrier pKD-0011 schematic diagrames；
Fig. 2 is the influence schematic diagram that the water extract of different pharmaceutical shines to pKD-0011, and shine weaker show to the load
Body inhibition level is higher；
Fig. 3, which is the control group of the culture medium to be not added with any drug, has pKD-0011 with addition the lophatherum gracile of inhibiting effect
The culture medium drug-treated group comparison diagram of water extract；
Fig. 4 is the drug-treated group drosophila Survival curves figure of control group and lophatherum gracile water extract in Fig. 3；
Fig. 5 is that Chinese cabbage infection model detects copper in the drug-treated group of lophatherum gracile water extract and the control group without drug-treated
Green pseudomonad it is pathogenic.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
The structure of novel antibacterial drug screening carrier pKD-0011：It please refers to Fig.1.Contain luciferase in pMS402 plasmids
Gene luxCDABE, but deletion promoters before luciferase gene sequence, therefore in not connected promoter, pMS402 plasmids
It cannot fluoresce.This characteristic based on pMS402 plasmids connects drug resistance and the promoter of pathogenic two-phase correlation gene PA0011
Before the luxCDABE of pMS402 plasmids, can effectively start luxCDABE genes, to successfully build PA0011 bases
Because carrier is reported in expression.
Referring to Fig. 1, first, design includes the PA0011 gene primers including promoter：
Sense primer is 5 '-TATctcgagCAGGACTGCGACCGTTACG-3 ',
Downstream primer is 5 '-CTCggatccCACATCAGCCAGCCTATG-3 ',
Digestion is carried out with XhoI and BamHI after PCR amplification, is connected to pMS402 luciferase gene reporters after purification
Preceding XhoI and BamHI restriction enzyme sites connect 16 hours, using the method for electrotransformation, bacterium solution are coated on containing 50ug/mL overnight
On the LB solid mediums of kanamycins, some monoclonals of picking carry out scribing line culture after culture 12 hours, are verified by PCR,
It is overriding resistance screening vector pKD-0011 to finally obtain the correct promoter-reporter fusion vector of verification.
Embodiment 2
Using overriding resistance screening vector pKD-0011, may be implemented to resistance to by porous plate microplate reader BioTeksynergy2
Medicine and pathogenic two-phase correlation gene PA0011 have the high flux screening of the antibacterials of obvious inhibiting effect.
Extraneous antibacterials do not influence the growth of thalline, together when less than being in sub- inhibition concentration under minimum inhibitory concentration
When can to the expression of gene have adjustment effect.Embodiment is by taking the sub- inhibition concentration of each drug as an example, scutelloside, ferulic acid
Concentration be respectively 30mg/ml and 50mg/ml, test a concentration of 100mg/ml for the other Chinese medical extracts used, research is each
The influence that medicinal substances express drug resistance and pathogenic two-phase correlation gene PA0011 under conditions of not influencing bacterial growth in kind.
Gene expression detection is carried out especially by enzyme mark detector：Will detection bacterial strain cross on LB solid mediums, 37
It DEG C is incubated overnight；Picking monoclonal is inoculated into 96 orifice plate of white containing 100 μ LLB culture mediums, 37 DEG C, 200rpm cultures 12h；
Then be transferred in fresh LB by 5% inoculum concentration, activate 3h, take 95 μ L contain sub- inhibition concentration or without containing drug at
Part fresh LB as a control group in 96 transparent orifice plates of sterilized black bottom, while after 5 μ L activation are added
Bacterium solution；It is last to be covered with 50 μ L mineral oil per hole, it is placed on multi-function microplate reader, its CPS value and OD600 is measured every 0.5h
Value is surveyed for 24 hours altogether.It is as shown in table 2 to test preliminary screening 20 kinds of traditional Chinese medicine ingredients, influence of each drug to pKD-0011：
Influence of the 2 each drug of table to pKD-0011
"-" represents drug effect hypothallus luminescent decay；" 0 ", which represents drug and shines on thalline, not to be influenced
Experiment finds scutelloside, lophatherum gracile water extract to the table Da Youming of the overriding resistance gene PA0011 of pseudomonas aeruginosa
Aobvious inhibiting effect, such as Fig. 2；To the no apparent inhibiting effect of expression of the overriding resistance gene PA0011 of pseudomonas aeruginosa
Middle medicinal substances are with Fig. 2 wild chrysanthemum water extract examples.
Embodiment 3
The antibacterials that mainly explanation is obtained using the vector selection by taking lophatherum gracile water extract as an example are to P. aeruginosa
The drug resistance of bacterium and pathogenic influence.
3.1 preparation antibiotic concentration be respectively：Than 50 μ g/mL of Ya Peinan (Biapenem, Bip), gentamicin
(Gentamicin, Gm) 50mg/mL, kanamycins (Kanamycin, Kan) 10mg/mL, tetracycline (tetracycline, Tc)
15mg/mL。
By in the fresh PAO1 colony inoculations to 5mLLB fluid nutrient mediums being incubated overnight, incubator overnight culture；By the two
OD600It is adjusted to 0.8 with fresh LB liquid medium；Bacterium solution is separately added into the sterile solid culture dissolved after taking 100 μ L dilutions
In base, culture medium temperature is less than 50 DEG C, the drug of one group of addition Asia inhibition concentration, another group of medicine for being added without sub- inhibition concentration
Object is down flat plate after mixing；Set tablet is cool in superclean bench, it is slightly dry after the placement specification that is evenly distributed of its surface be
The aseptic filter paper piece of D=6mm adds 10 μ L antibiotic on each filter paper, is inverted and is incubated overnight in 37 DEG C of incubators, observation suppression
Bacterium circle size.
3.2 have apparent inhibition to make the overriding resistance gene PA0011 of pseudomonas aeruginosa due to screening obtained drug
With, and PA0011 has important role to the pathogenic of pseudomonas aeruginosa, makees to study the overriding resistance substance that screening obtains
For the influence to host's pathogenecity after pseudomonas aeruginosa, with reference to the Drosophila melanogaster model that Sibley, C.D et al. are established,
Respectively with the PAO1 bacterium solutions i.e. control group for being not added with drug-treated and the PAO1 bacterium solutions that sub- inhibition concentration drug is added by feeding come
Drosophila is infected, observes the lethality of control group and drug-treated group drosophila to detect influence of the drug to host's pathogenecity.
First in picking PAO1 monoclonals to the sterile LB liquid mediums of 20mL, 37 DEG C of shaking table culture 10-12h；Dilute bacterium
Liquid adjusts OD600Value is 2.0, takes 15mLOD600The bacterium solution of ≈ 2.0, is collected by centrifugation, and respectively plus 1mL5% sucrose solutions extremely compare
Group and drug-treated group, careful pressure-vaccum make bacterium solution suspend wherein；1.5mL is taken to be added after 5% sucrose solids culture medium is dissolved
It is cool after solidification to set a moment in super-clean bench in 24 orifice plates of sterilizing, per the sterile filter paper of hole layer overlay；5% will be suspended in
Bacterium solution in sucrose solution takes 100 μ L to be separately added on the filter paper in each hole, aseptically by the bacterium solution of surface
Drying takes 4-6h；The often Male Drosophila of pipe picking 3-5d sizes 15, place freeze after 2-3h it is dizzy be added in 24 orifice plates, 26 DEG C
Culture counts the drosophila number of each hole survival daily.
3.3 with reference to Rahme, the method for the plant model detection P. aeruginosa bacteria pathogenic that L.G et al. is established, and experiment is logical
Crossing Chinese cabbage infection model further proves the antibacterial material obtained using the vector selection to pseudomonas aeruginosa pathogenicity
It influences.The monoclonal of wild type pseudomonas aeruginosa is inoculated in 5 milliliters of LB culture solutions, 37 DEG C, after 200 turns are incubated overnight
Thalline were collected by centrifugation by 8000rpm, 3min, with the MgSO of 10mM4After washing thalline precipitation, after bacterium mud is diluted to OD600=1.0
Control group and drug-treated group bacterium solution are obtained in the sterile water and lophatherum gracile water extract for being added isometric；With 0.1% peroxidating
Hydrogen wipes Chinese cabbage stem surface, is placed in inside culture dish, culture dish bottom is pre-placed one piece and has infiltrated 10mMMgSO4It is sterile
Filter paper；10 μ l control groups and drug-treated group bacterium solution are drawn respectively, and point is placed 6 days in Chinese cabbage stem, room temperature；Observation control
Rotten level caused by group and drug-treated group, it was demonstrated that caused a disease to pseudomonas aeruginosa using the drug that the vector selection obtains
The influence of property.
According to 3.1 and 3.2 experimental result it can be found that with lophatherum gracile water extract be illustrate, as shown in Figure 3, Figure 4, Fig. 4
In " ■ " represent and be added to lophatherum gracile drug water extract in culture medium, " ▲ " represents the control group added without drug, is using medicine
After object processing, pseudomonas aeruginosa obviously increases the sensibility of antibiotic, and its toxic effect is significantly reduced.Experiment
3.3 Chinese cabbage infection model is, it was also found that drug-treated group rotten level caused by Chinese cabbage is obviously low compared with control group.It proves to utilize
The carrier energy Effective selection inhibits pseudomonas aeruginosa antibiotic resistance and pathogenic antibacterials.
The present invention operation principle be：The power of usual gene expression depends on the power of gene promoter itself, drug resistance
The promoter of gene is connected to before promoterless reporter luciferase gene luxCDABE, to start reporter
Expression.If candidate drug has inhibiting effect, the expression of reporter that can also weaken the promoter of virulence factor, i.e., thalline is sent out
Light weakens；If conversely, having activation, thalline luminescence enhancement to promoter.It is waited for known to the variation luminous by observing thalline
Survey influence situation of the drug to expression of drug resistance genes.The drug resistant gene of the detecting system of research institute's application is PA0011, PA0011
Expression to cause pseudomonas aeruginosa to play the role of the tolerance and pathogenic course of antibiotic very important.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Profit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent requirements of the claims
Variation is included within the present invention.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiment being appreciated that.
Claims (2)
1. a kind of method for reducing pseudomonas aeruginosa wide spectrum drug resistance and infective economic benefits and social benefits drug screening, feature exists
In including the following steps：
1) it using the drug resistance of pseudomonas aeruginosa PAO1 and pathogenic two-phase correlation gene PA0011 as drug target, is opened with carrying missing
The plasmid pMS402 of the reporter luxCDABE of mover is carrier, by the promoter region of PA0011 genes through PCR amplification, enzyme
It cuts before being cloned into the luciferase luxCDA BE operons of report plasmid pMS402 institutes band, constitutes promoter-reporter fusion and carry
Body pKD-0011；Recombinant plasmid pKD-0011 is transferred in pseudomonas aeruginosa PAO1 using electrotransformation technology, it is false to form verdigris
The overriding resistance screening vector pKD-0011 of monad PAO1；
2) antibacterial material for inhibiting PA0011 expression is obtained to screen by detecting the luminous situation of pKD-0011, high throughput sieve can
The drug resistance of pseudomonas aeruginosa and pathogenic economic benefits and social benefits drug are reduced, the specific steps are：It is anti-using pseudomonas aeruginosa PAO1
Drug resistance screening vector pKD-0011, by porous plate microplate reader BioTek synergy2 to drug resistance and pathogenic two-phase correlation gene
PA0011 has the high flux screening of the antibacterials of obvious inhibiting effect, specific method to be：Gene is carried out by enzyme mark detector
Detection of expression：Will detection bacterial strain cross on LB solid mediums, 37 DEG C be incubated overnight；Picking monoclonal is inoculated into containing 100 μ L
In 96 orifice plate of white of LB culture mediums, 37 DEG C, 200rpm cultures 12h；Then it is transferred to fresh LB by 5% inoculum concentration
In, 3h is activated, 95 μ L is taken to contain or not contain the fresh LBs of sub- inhibition concentration pharmaceutical compositions as a control group in having gone out
In 96 transparent orifice plates of the black bottom of bacterium, while the bacterium solution after 5 μ L activation is added；It is last to be covered with 50 μ L mineral oil per hole,
It is placed on multi-function microplate reader, its CPS value and OD600 values is measured every 0.5h, survey for 24 hours, obtained to drug resistance and pathogenic two-phase altogether
Correlation gene PA0011 has the antibacterials of obvious inhibiting effect.
2. according to claim 1 for reducing pseudomonas aeruginosa wide spectrum drug resistance and infective economic benefits and social benefits drug screening
Method, it is characterised in that：The primer sequence of the promoter region of PA0011 genes is：
Sense primer is 5 '-TATctcgagCAGGACTGCGACCGTTACG-3 ',
Downstream primer is 5 '-CTCggatccCACATCAGCCAGCCTATG-3’。
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| CN103592167A (en) * | 2013-11-18 | 2014-02-19 | 华中农业大学 | Detection method of fluoroquinolone drug residues based on luciferase labelled engineered bacterium |
| CN104232588A (en) * | 2014-09-12 | 2014-12-24 | 中国医学科学院医药生物技术研究所 | Construction and application of anti-hepatic fibrosis drug high-throughput screening cell model |
| CN104498526A (en) * | 2015-01-09 | 2015-04-08 | 西北大学 | Method for rapidly and efficiently screening rhamnolipid producing bacteria nutrition system |
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| CN101643741A (en) * | 2009-09-08 | 2010-02-10 | 南开大学 | Pseudomonas aeruginosa elastin hydrolysis ability-related gene PA5022 and application thereof |
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| CN103592167A (en) * | 2013-11-18 | 2014-02-19 | 华中农业大学 | Detection method of fluoroquinolone drug residues based on luciferase labelled engineered bacterium |
| CN104232588A (en) * | 2014-09-12 | 2014-12-24 | 中国医学科学院医药生物技术研究所 | Construction and application of anti-hepatic fibrosis drug high-throughput screening cell model |
| CN104498526A (en) * | 2015-01-09 | 2015-04-08 | 西北大学 | Method for rapidly and efficiently screening rhamnolipid producing bacteria nutrition system |
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