CN104789582A - Novel carrier for antibacterial medicine screening - Google Patents
Novel carrier for antibacterial medicine screening Download PDFInfo
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- CN104789582A CN104789582A CN201510176236.5A CN201510176236A CN104789582A CN 104789582 A CN104789582 A CN 104789582A CN 201510176236 A CN201510176236 A CN 201510176236A CN 104789582 A CN104789582 A CN 104789582A
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Abstract
The invention discloses a novel carrier for antibacterial medicine screening. The carrier is pKD-0011, and is mainly used for screening the antibacterial medicine which plays an obvious inhibiting role on pathogenicity and drug tolerance of pseudomonas aeruginosa. The carrier takes a drug tolerance and pathogenicity double related gene PA0011 as a medicine acting target point, and clones the promotor area of the drug tolerance and pathogenicity double related gene PA0011 through PCR amplification and enzyme digestion to a report plasmid pMS402 luciferase operon gene by a plasmid pMS402 with a report gene luxCDABE lack of a promotor, so as to form the promotor-reportor fusing carrier pKD-0011. An electrotransformation technology is utilized to convert a recombinant plasmid into a wild type pseudomonas aeruginosa, and antibacterial substance for inhibiting PA0011 expression can be screened by utilizing the luminous intensity of the carrier pKD-0011. The carrier can realize high flux screening, utilizes related screening equipment, is simple to operate, and is economic and practical.
Description
Technical field
The present invention relates to a kind of carrier for selection of antibacterial, specifically a kind of for screening the screening vector the pathogenic of Pseudomonas aeruginosa and resistance being had to obvious inhibiting antibacterials.
Background technology
Pseudomonas aeruginosa is a class opportunistic pathogen, is the major cause causing nosocomial infection, immune system defect patient such as patient HIV, severe burn patients and pulmonary cyst fibrosis patient in usually cause higher M & M.
In recent years, the tolerance of Pseudomonas aeruginosa to nearly all antibacterials increases all to some extent.Be due to irrational use microbiotic on the one hand, make thalline create adaptability resistance; Because the stronger inherent resistance mechanism that Pseudomonas aeruginosa self exists plays a role on the other hand.The continuous increase of Pseudomonas aeruginosa to antibiotic resistance makes to select suitable therapeutic strategy to become very difficult to the infection for the treatment of Pseudomonas aeruginosa clinically, result in more and more higher mortality ratio, thus find that novel effective antimicrobial substance is extremely urgent.
Chinese medicine demonstrates unique advantage gradually preventing in chronic infection oneself.China has abundant Chinese herbal medicine resource, originate wide, cheap, toxic side effect is little, less there will be resistance and in bacterial cell action pathway variation, can many-sided pharmacological effect be produced, therefore can study as the medicine of potential resisting pseudomonas aeruginosa.
The screening method of new antibacterials is the key factors finding novel antibacterial material.Traditional antibiotic-screening method shortcoming is that action target spot is indefinite, mechanism is unintelligible, insensitive, screening process has randomness.Genetic marker method is one of state-of-the-art technology in current screening method, by mark special gene, finds relevant biological activity thing, advantage be sensitive, poor efficiency thing can be detected, can be used for that chemistry improves, easily screening, high-throughput.
Microbiotic is except traditional antibacterial and germicidal action, also show other active, particularly lower than under minimum inhibition concentration, extraneous antimicrobial substance can have regulating effect to the expression of gene, the expression of virulence factor can be regulated, the resistance of induction pathogenic bacteria, to affect in microbial population intercellular interaction etc.Utilize this characteristic antibiotic, the novel antibacterial material this gene to regulating effect can be screened by the expression vector building specific gene.
Lipoid A is one of important feature of lipopolysaccharides (LPS), forms effective barrier of adventitia with other composition interphase interactions of LPS; When LPS constructional device defect, the structure of LPS will be destroyed, there is breach in LPS layer thereupon, phospholipid molecule will move to fatiscent place from outer film inner layer, repair, namely in adventitia, define phospholipid bilayer spot, cause the barrier action of adventitia impaired, thus hydrophobic microbiotic and objectionable impurities can be made freely penetrating, bacterial antibiotic or objectionable impurities become responsive.In addition, the intracellular toxin component of lipoid A or LPS, intracellular toxin is heat-resisting and stable, is the important morbid substance of bacterium.
In Pseudomonas aeruginosa, PA0011 genes encoding 2-OH-acyltransferase participates in the building-up process of lipoid A; team is found by early-stage Study: PA0011 has a very important role for the biochemical functions of Pseudomonas aeruginosa, resistance and pathogenecity; after PA0011 afunction, mycin received to tsiklomitsin, gentamicin, polymyxin, Rifampin, Pyocianil, card; the resistance of carbapenem antibiotic all obviously reduces; and cause Pseudomonas aeruginosa to reduce the invasiveness of HeLa cell, the lethality rate of fruit bat is also obviously reduced.Such as, and also have similar discovery in other bacterial strains, in Salmonellas, htrB is the homologous gene of Pseudomonas aeruginosa PA0011, the mutant activity of endotoxin of htrB reduces, and mutant is pathogenic to be also affected.Therefore, PA0011 can as a good drug target, for screening novel antibacterial material.Medication treatment for other pathogenic bacterium also has reference function.
Summary of the invention
The object of the present invention is to provide a kind of carrier of selection of antibacterial, specifically with the drug resistant gene of in Pseudomonas aeruginosa for target spot, build the detection carrier of the promotor-reporter of this gene, to solve the problem proposed in above-mentioned background technology.
For achieving the above object, the invention provides following technical scheme:
For a carrier for selection of antibacterial, described carrier is pKD-PA0011, and being mainly used in screening has obvious inhibiting antibacterials to the pathogenic of Pseudomonas aeruginosa and resistance.The construction process of described carrier is: using resistance and pathogenic two-phase correlation gene PA0011 as drug target, with the plasmid pMS402 of the reporter gene luxCDABE with deletion promoters for carrier, by the promoter region of PA0011 gene through pcr amplification, enzyme cutting clone to report plasmid pMS402 with luciferase luxCDABE operon before, form promotor-reporter fusion vector pKD-0011; Utilize electric transformation technology to proceed in the Pseudomonas aeruginosa of wild-type by recombinant plasmid pKD-0011, screened the antimicrobial substance obtaining and suppress PA0011 to express by the luminous situation detecting pKD-0011.
As the further scheme of the present invention: the primer sequence comprising the drug resistant gene PA0011 of promotor is:
Upstream primer is 5 '-TAT
ctcgagcAGGACTGCGACCGTTACG-3 ',
Downstream primer is 5 '-CTC
ggatcccACATCAGCCAGCCTATG-3 '.
Compared with prior art, the invention has the beneficial effects as follows: the present invention constructs one from " overriding resistance " angle and utilizes luciferase gene (luxCDABE) as the high-throughput genetic expression detection technique of reporter, screens and has inhibiting antimicrobial substance to the promotor of the important resistance of in Pseudomonas aeruginosa and pathogenic two-phase correlation gene PA0011.This carrier can realize high flux screening; Utilize relevant screening installation, simple to operate, economical and practical; Screen the pathogenic and resistance of the effective Pseudomonas aeruginosa of medicine energy obtained, and mechanism of action clear and definite.
Accompanying drawing explanation
Fig. 1 is carrier pKD-0011 schematic diagram;
Fig. 2 is that the aqueous extract of different pharmaceutical affects schematic diagram to pKD-0011 luminescence, and luminescence more weak showing suppresses degree higher to described carrier;
Fig. 3 is the control group of the substratum not adding any medicine and adds substratum drug treating group comparison diagram pKD-0011 being had to inhibiting Herba Lophatheri aqueous extract;
Fig. 4 is the drug treating group fruit bat Survival curves figure of control group and Herba Lophatheri aqueous extract in Fig. 3;
Fig. 5 is that Chinese cabbage infection model detects the drug treating group of Herba Lophatheri aqueous extract and pathogenic without Pseudomonas aeruginosa in the control group of drug treating.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described.Obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
The structure of novel antibacterial drug screening carrier pKD-0011: refer to Fig. 1.Containing luciferase gene luxCDABE in pMS402 plasmid, but deletion promoters before luciferase gene sequence, and therefore when not connecting promotor, pMS402 plasmid can not fluoresce.Based on this characteristic of pMS402 plasmid, before the promotor of resistance and pathogenic two-phase correlation gene PA0011 is connected to the luxCDABE of pMS402 plasmid, can effectively start luxCDABE gene, thus successfully build PA0011 genetic expression report carrier.
Refer to Fig. 1, first, design packet contains promotor at interior PA0011 gene primer:
Upstream primer is 5 '-TATctcgagCAGGACTGCGACCGTTACG-3 ',
Downstream primer is 5 '-CTCggatccCACATCAGCCAGCCTATG-3 ',
After pcr amplification, carry out enzyme with XhoI and BamHI cut, XhoI and the BamHI restriction enzyme site before pMS402 luciferase gene reporter is connected to after purifying, to spend the night connection 16 hours, adopt the method that electricity transforms, bacterium liquid is coated on the LB solid medium containing 50ug/mL kantlex, cultivating some mono-clonals of picking after 12 hours carries out streak culture, through PCR checking, is finally verified correct promotor-reporter fusion vector and is overriding resistance screening vector pKD-0011.
Embodiment 2
Adopt overriding resistance screening vector pKD-0011, can realize by porous plate microplate reader BioTeksynergy2 high flux screening resistance and pathogenic two-phase correlation gene PA0011 being had to obvious inhibiting antibacterials.
Extraneous antibacterials lower than under minimum inhibition concentration namely in sub-inhibition concentration time, do not affect the growth of thalline, regulating effect can be had to the expression of gene simultaneously.Embodiment is for the sub-inhibition concentration of each medicine, the concentration of baicalin, forulic acid is respectively 30mg/ml and 50mg/ml, the concentration of testing other Chinese medical extract used is 100mg/ml, studies various Chinese medicine material under the condition not affecting bacterial growth to the impact that resistance and pathogenic two-phase correlation gene PA0011 are expressed.
Genetic expression detection is carried out: will detect that bacterial strain is rule on LB solid medium, 37 DEG C of incubated overnight especially by enzyme mark detector; Picking mono-clonal is inoculated in white 96 orifice plate containing 100 μ LLB substratum, 37 DEG C, 200rpm cultivates 12h; Then be transferred in fresh LB by 5% inoculum size, activation 3h, get 95 μ L and contain sub-inhibition concentration or not containing in transparent bottom sterilized black as a control group 96 orifice plates of the fresh LB of pharmaceutical compositions, add simultaneously 5 μ L activate after bacterium liquid; Last every hole 50 μ L mineral oil cover, and are placed in multi-functional microplate reader, measure its CPS value and OD600 value, survey 24h altogether every 0.5h.Test preliminary screening 20 kinds of traditional Chinese medicine ingredients, the impact of each medicine on pKD-0011 is as shown in table 2:
The each medicine of table 2 is on the impact of pKD-0011
"-" represents medicine effect hypothallus luminescent decay; " 0 " represents medicine to thalline luminescence not impact
Experiment finds baicalin, the expression of Herba Lophatheri aqueous extract to the overriding resistance gene PA0011 of Pseudomonas aeruginosa has obvious restraining effect, as Fig. 2; Obvious inhibiting Chinese medicine material is not had with Fig. 2 Wild Chrysanthemum aqueous extract example to the expression of the overriding resistance gene PA0011 of Pseudomonas aeruginosa.
Embodiment 3
Mainly illustrate that the antibacterials utilizing described vector selection to obtain are on the resistance of Pseudomonas aeruginosa and pathogenic impact for Herba Lophatheri aqueous extract.
The antibiotic concentration of 3.1 preparation is respectively: than Ya Peinan (Biapenem, Bip) 50 μ g/mL, gentamicin (Gentamicin, Gm) 50mg/mL, kantlex (Kanamycin, Kan) 10mg/mL, tsiklomitsin (tetracycline, Tc) 15mg/mL.
By the fresh PAO1 colony inoculation of incubated overnight in 5mLLB liquid nutrient medium, incubator overnight is cultivated; By both OD
6000.8 is adjusted to fresh LB liquid nutrient medium; Get 100 μ L dilute after bacterium liquid add in the sterile solid substratum dissolved respectively, substratum temperature lower than 50 DEG C, one group of medicine adding sub-inhibition concentration, another group does not add the medicine of sub-inhibition concentration, is down flat plate after mixing; Put in Bechtop by cool for flat board, after slightly dry, its surface arrangement places the aseptic filter paper sheet that specification is D=6mm uniformly, adds 10 μ L microbiotic, be inverted incubated overnight in 37 DEG C of incubators on each filter paper, observes inhibition zone size.
3.2 have obvious restraining effect owing to screening the overriding resistance gene PA0011 of medicine to Pseudomonas aeruginosa obtained, and PA0011 has important effect to the pathogenic of Pseudomonas aeruginosa, screen the overriding resistance material that obtains act on impact on host's pathogenecity after Pseudomonas aeruginosa to study, with reference to Sibley, the drosophila melanogaster model that the people such as C.D set up, fruit bat is infected with the PAO1 bacterium liquid adding sub-inhibition concentration medicine by feeding respectively with the PAO1 bacterium liquid and control group that do not add drug treating, the lethality rate observing control group and drug treating group fruit bat detects the impact of this medicine on host's pathogenecity.
First picking PAO1 mono-clonal is in the aseptic LB liquid nutrient medium of 20mL, and 10-12h cultivated by 37 DEG C of shaking tables; Dilution bacterium liquid regulates OD
600value is 2.0, gets 15mLOD
600the bacterium liquid of ≈ 2.0, collected by centrifugation, adds 1mL5% sucrose solution respectively to control group and drug treating group, and careful pressure-vaccum makes bacterium liquid suspend wherein; Get 1.5mL after being dissolved by 5% sucrose solids substratum to add in sterilized 24 orifice plates, solidify and coolly to put in super clean bench a moment, the filter paper that every hole layer overlay is aseptic afterwards; The bacterium liquid be suspended in 5% sucrose solution is got 100 μ L to add on the filter paper in each hole respectively, is aseptically dried up by the bacterium liquid of its surface, 4-6h consuming time; The often Male Drosophila 15 of pipe picking 3-5d size, freezing after placing 2-3h swoons is added in 24 orifice plates, and 26 DEG C of cultivations, add up the fruit bat number of each hole survival every day.
The method of the 3.3 plant model detection P. aeruginosa bacteria pathogenics set up with reference to people such as Rahme, L.G, tests and proves that the antimicrobial substance utilizing described vector selection to obtain is on the impact of Pseudomonas aeruginosa virulence further by Chinese cabbage infection model.Be inoculated in by the mono-clonal of wild-type Pseudomonas aeruginosa in 5 milliliters of LB nutrient solutions, 37 DEG C, 8000rpm, 3min collected by centrifugation thalline after 200 turns of incubated overnight, with the MgSO of 10mM
4after washing bacterial sediment, after bacterium mud is diluted to OD600=1.0, adding isopyknic sterilized water and Herba Lophatheri aqueous extract obtains control group and drug treating group bacterium liquid; With the hydrogen peroxide wiping Chinese cabbage stem surface of 0.1%, be placed in culture dish inside, place one piece bottom culture dish in advance and infiltrated 10mMMgSO
4aseptic filter paper sheet; Draw 10 μ l control groups and drug treating group bacterium liquid respectively, put in Chinese cabbage stem, place 6 days in room temperature; The rotten level that observation control group and drug treating group cause, proves that the medicine utilizing described vector selection to obtain is on the impact of P. aeruginosa bacteria pathogenic.
Experimental result according to 3.1 and 3.2 can find, with Herba Lophatheri aqueous extract for explanation, as shown in Figure 3, Figure 4, in Fig. 4, " ■ " represents in substratum and with the addition of Herba Lophatheri medicine aqueous extract, " ▲ " represents the control group added without medicine, after making treated with medicaments, Pseudomonas aeruginosa obviously increases antibiotic susceptibility, and its toxic action also obviously reduces.The Chinese cabbage infection model of experiment 3.3 also finds, obviously comparatively control group is low for the rotten level that drug treating group causes Chinese cabbage.Prove to utilize this carrier energy Effective selection to suppress Pseudomonas aeruginosa antibiotics resistance and pathogenic antibacterials.
Principle of work of the present invention is: the power of usually genetic expression depends on the power of the promotor of gene own, before the promotor of drug resistant gene is connected to promoterless reporter gene luciferase gene luxCDABE, thus starts the expression of reporter gene.If the promotor of candidate drug to virulence factor has restraining effect, the expression of reporter gene also can weaken, i.e. thalline luminescent decay; Otherwise, if having activation to promotor, then thalline luminescence enhancement.By the change known candidate drug of observing thalline luminescence situation is affected on expression of drug resistance genes.The drug resistant gene of detection system of institute's application is that the expression of PA0011, PA0011 causes Pseudomonas aeruginosa to play very important effect to antibiotic tolerance and pathogenic course.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.Any Reference numeral in claim should be considered as the claim involved by limiting.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (2)
1. for a carrier for selection of antibacterial, described carrier is pKD-0011, and being mainly used in screening all has obvious inhibiting antibacterials to the pathogenic of Pseudomonas aeruginosa and resistance.It is characterized in that, the construction process of described carrier is: carrier is using resistance and pathogenic two-phase correlation gene PA0011 as drug target, with the plasmid pMS402 of the reporter gene luxCDABE with deletion promoters for carrier, by the promoter region of PA0011 before pcr amplification, enzyme cutting clone to the luciferase operon gene luxCDABE of plasmid pMS402, form promotor-reporter fusion vector pKD-0011, i.e. selection of antibacterial carrier; Utilize electric transformation technology to proceed in the Pseudomonas aeruginosa of wild-type by recombinant plasmid, screen by the luminescence power detecting pKD-0011 the antimicrobial substance obtaining and suppress PA0011 to express.The antimicrobial substance that screening obtains obviously can reduce the resistance of Pseudomonas aeruginosa and pathogenic simultaneously.
2. the carrier for selection of antibacterial according to claim 1, is characterized in that: the primer sequence comprising the drug resistant gene PA0011 of promotor is:
Upstream primer is 5 '-TAT
ctcgagcAGGACTGCGACCGTTACG-3 ',
Downstream primer is 5 '-CTC
ggatcccACATCAGCCAGCCTATG-3 '.
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| CN112266928A (en) * | 2020-09-21 | 2021-01-26 | 西北大学 | Method for detecting multiple drug-resistant plasmid horizontal transfer |
| CN112852700A (en) * | 2021-02-08 | 2021-05-28 | 中国人民解放军军事科学院军事医学研究院 | Construction and application of serratia marcescens with fluorescent tracing |
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