CN101368184A - Patina pseudomonas migration ability correlated gene PA5001 and application thereof - Google Patents

Abstract

The invention discloses pseudomonas aeruginosa swimming motility related gene PA5001 which belongs to the technical field of microbial biology. The size of the gene is 957bp and has no international reports on the function of the gene at present. The experiment discovers that the gene participates in the swimming motility of thalli for the first time. The swimming motility (swimming motility) of pseudomonas aeruginosa is one motion mode of flagellum mediate. The experiment discovers that the inactivation of the gene results in the loss of the swimming motility of the pseudomonas aeruginosa. The flagella of the pseudomonas aeruginosa and the flagellum-mediated motion ability thereof are important virulence factors and pathogenic factors of the pseudomonas aeruginosa. The discovery and the study of the gene contribute to the study on the motion mechanism of the flagella of the pseudomonas aeruginosa, reveal the pathogenesis of the pseudomonas aeruginosa, and provide the theoretical basis for the prevention and the treatment of the infection which is caused by the pseudomonas aeruginosa.

Description

Patina pseudomonas migration ability correlated gene PA 5001 and application

[technical field]: the invention belongs to field of microbial biotechnology, particularly a kind of Patina pseudomonas migration ability correlated gene PA 5001.

[background technology]: Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA), belong to Rhodopseudomonas (Pseudomonas), be called Pseudomonas aeruginosa again, Gram-negative bacteria, be a kind of in nature (as soil, water and air) conditioned pathogen widely.Often cause that clinically intractable repeatability infects, wherein the formation of biofilm load is an important factor, bacterium under the barrier protection effect of tunicle, have very strong resistance and anti-ly engulf, anti-chemotaxis.It is reported that 60% above infected by microbes is caused by bacterial biofilm.

Main locomotive organ of flagellum conduct and important virulence factor obtain nutritive substance to Pseudomonas aeruginosa, escape objectionable impurities, transfer to suitable host, find suitable set site and spread propagation to environment to play an important role.Swimming be bacterium under the promotion of single-ended flagellum, the mode of motion of single somatic cells in the high environment of moisture content is a kind of behavior of individuality.The swimming of flagellum mediation plays a part crucial in the early stage and normal biofilm load structure of formation that biofilm load forms.

At present known have at least 35 genes relevant with function with synthesizing of flagellum, and new flagellum genes involved constantly is found.Research to the flagellar movement ability genes involved can be for disclosing the function of flagellar movement mechanism, the new gene of research, explore the mechanism of causing a disease of Pseudomonas aeruginosa, for medicinal design provides new target site, for prevention and the infection that suppresses Pseudomonas aeruginosa provide theoretical foundation.

[summary of the invention]: the present invention seeks to find the new gene relevant,, design new antibacterials, the infection that prevention and treatment Pseudomonas aeruginosa cause as target site with flagellar movement ability.

Patina pseudomonas migration ability correlated gene PA 5001 provided by the invention has nucleotide sequence shown in sequence table sequence 1.

The application of above-mentioned Patina pseudomonas migration ability correlated gene PA 5001, being used for this gene is the medicine that design of new drug target spot and preparation suppress thalline swimming ability, to reduce its virulence and pathogenic, strengthens antibiotic drug effect.

Gene PA5001 preparation method of the present invention is: utilize Mu swivel base recombinant technology, make up transposon and insert the sudden change library, the disabled muton of screening flagellum swimming, extract genomic dna, cut genomic dna with Bam HI enzyme, endonuclease bamhi is connected with the pUC18 carrier, to connect product by the electricity conversion and be transformed into the bacillus coli DH 5 alpha competent cell, be coated in and contain on penbritin and the two anti-LB flat boards of kantlex, cut evaluation through PCR and enzyme, screening positive transformant, order-checking determine that Mu inserts segmental nucleotide sequence in the transformant, determine the gene location that Mu is inserted through NCBI Blast comparison.

Beneficial effect of the present invention:

PA5001 gene M u inserts the mutant strain forfeiture swimming ability of inactivation, illustrate that this gene is relevant with the motion of Pseudomonas aeruginosa flagellum, and this gene is the unknown fully new gene of a function, and this experiment finds that first it is relevant with flagellar movement ability.The discovery of new gene function is to disclosing flagellar movement mechanism, the target site of medicinal design being provided, and the infection of prevention and treatment Pseudomonas aeruginosa all has directive significance

[description of drawings]:

The detection of Fig. 1, Pseudomonas aeruginosa swimming ability

1. wild-type PA68 is as positive control 2, PA5001::Mu mutant strain

The electrophoresis detection of Fig. 2 transformant PCR product, 1.DL2000 2.Mu clones sub-PCR product

With Mu1 and Mu2 is primer, is template with clone's daughter bacteria liquid, can amplify the specific fragment about 700bp in theory, shows in the sub-plasmid of clone and is carrying Mu transposon dna fragmentation.

Fig. 3. clone sub-enzyme and cut the product electrophorogram

1, DL15000 2, plasmid 3, plasmid enzyme restriction

[embodiment]:

Embodiment 1: use the suddenly change foundation in library of Mu swivel base recombinant technology

1. from the single bacterium colony of PA68 of picking on the fresh flat board of 37 ℃ of cultivation 16～20h, forward in the test tube that contains 5mL LB liquid nutrient medium, 37 ℃, 200rpm, overnight shaking is cultivated 14～18h.

2. be transferred in the triangular flask that contains 100mL LB liquid nutrient medium by 1: 100 next day, 37 ℃, 200rpm, the about 3-5h of shaking culture uses the absorbancy of 752 spectrophotometric determination bacterial culturess at the 540nm place, at OD ₅₄₀=0.5～0.6 o'clock, stop to cultivate.

3. under aseptic condition, thalline is transferred in the 50mL centrifuge tube of an aseptic precooling, placed 10min, make culture be cooled to 0 ℃ on ice.

4. in 4 ℃, 6000rpm, centrifugal 10min collects thalline.

5. pour out nutrient solution, with the resuspended thalline of 300mM sucrose of 100mL precooling, 4 ℃, 6000rpm, centrifugal 10min.

6. repeat (5) resuspended with the 300mM sucrose solution of 50mL, 20mL precooling successively, washing thalline.

7. at last cell is dissolved in the 1mL300mM sucrose solution, is distributed into 100 μ L/ pipe, prepare the competent cell suspension, ice bath is preserved standby.

8. get 1 μ LMu swivel base mixture, join in the 100 μ L competent cell suspensions mixing, the 0.2cm electricity that withdraw mix joins precooling transforms in the cup, uses the Bio-Rad electroporation apparatus, and voltage 2.6kv is set, shock by electricity, note electric shock used voltage and time.

9. add in the transformation system after electric shock in the 900 μ LSOC substratum of 37 ℃ of temperature baths in advance, 37 ℃, 200rpm, recovery 1h.

10. the transformation system of getting after an amount of recovery is coated on the LB flat board that contains 50 μ g/mL kantlex.37C cultivates 16-18h, and negative control does not add in transformation system the Mu swivel base mixture, and all the other processes and sample are identical.If no bacterium colony grows on the negative control flat board, the transformant that grows on resistant panel further screens.

Do template 11. get 2 μ l bacterium liquid, do primer with the distinguished sequence Mu1 on the artificial Mu transposon (5 '-GCAACTGTCCATACTCTGA-3 ') and Mu2 (5 '-CGCTGGGTTTATCGTCGA-3 '), with PCR method amplification Mu transposon fragment.Do feminine gender and positive control with pseudomonas aeruginosa strains and plasmid pHTH2 respectively simultaneously.Amplification condition is, earlier at 94 ℃ of pre-sex change 15min, then, and 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, and totally 30 circulations are extended 10min at 72 ℃ again.

12.PCR product is electrophoresis detection in 0.8% (W/V) sepharose, observations and stay photo under the ultraviolet lamp of EB dyeing back, and the transformant with 700bp kalamycin resistance gene has the Mu transposon.

Embodiment 2: the detection of flagellum swimming ability

1. layer overlay swimming ability detects substratum, dried overnight under the room temperature in the aseptic plastic culture dish.Detect the substratum of flagellum swimming ability: 5g/LNaCl, the 10g/L Tryptones, 0.3% (W/V) agarose (Spanish packing), the pH value transfers to about 7.2.

2. from Mu transposon mutant library, activate mutant strain.

3. with the Mu swivel base muton of the PA68 of aseptic toothpick picking incubated overnight on the LB flat board, dibbling is to the surface of swimming substratum.30 ℃, just putting and cultivating 16～24h.

4. swimming ability detects: it is the center of circle growth of swimming towards periphery on the surface of phenotype detection substratum with the inoculation point that bacterium can rely on flagellar movement, forms an annulus, the muton that filters out the swimming Disability or weaken (Fig. 1).

Embodiment 3: the clone of Pseudomonas aeruginosa swimming ability genes involved

The extraction of wild-type and defective pnca gene group DNA

1. picking list bacterium colony from the fresh streak culture LB solid plate is inoculated in the 5mL L-broth liquid nutrient medium, and 37 ℃, 200rpm, incubated overnight;

2. be transferred in the 10mLL-broth liquid nutrient medium in 1: 100 ratio next day, 37 ℃, 200rpm cultivates 14-16h;

3. bacterium liquid is transferred in the 11mL centrifuge tube, 4 ℃, 12000rpm, centrifugal 1min.Supernatant discarded is collected thalline;

4. in centrifuge tube, add 1.1mL lysis buffer (20mMTrisHCl pH8.0,10 mMEDTA, 10mM NaCl, the 20% sucrose) thalline that suspends again, add 50 μ LRNase (10mg/ml) again, 37 ℃ of water-bath 1h;

5. 2% SDS that adds 1.1mL fully vibrates to the solution clarification, adds 11 μ L Proteinase Ks (20mg/ml) again, and 37 ℃ of water-baths are spent the night, and often shake centrifuge tube therebetween, makes fully protolysate of Proteinase K;

6. the saturated phenol/chloroform 1:1 mixed solution of Tris that adds 2.2mL, thermal agitation, 12000rpm, centrifugal 20min;

7. water is transferred in another new centrifuge tube on the absorption upper strata, repeats with phenol/chloroform extracting 3 times, and operation steps is the same, to the albumen extracting clean till;

8. the absorption supernatant adds about 4mL dehydrated alcohol, stirs out precipitation with glass stick or rifle head, places a new 11mL centrifuge tube, and 70% ethanol is washed once, takes liquid, vacuum-drying less out of as far as possible;

9. add the heavy molten DNA of 1.5mlTES solution (500mMTrisHCl, pH8.0,5mM EDTA, 50mM NaCl) among the DNA after draining, add 15 μ lRNase (10mg/ml) afterwards, 37 ℃ of water-bath 1h;

10. add the saturated phenol/chloroform extracting of 1.5ml Tris albumen, 12000rpm, centrifugal 15min gets supernatant; Repeat extracting once, get supernatant; Adding 1.5ml chloroform extracting is more once got supernatant;

11. add about 4mL dehydrated alcohol, place 0.5h, 4 ℃, 12000rpm, centrifugal 10min for-20 ℃;

12. supernatant discarded, precipitation 1mL70% washing with alcohol 2 times heavily are dissolved among an amount of TE after the vacuum-drying.0.8% agarose gel electrophoresis roughly detects quality and the concentration of DNA.

The a small amount of of plasmid is extracted, and is also improved with reference to the method for " Molecular Cloning " (Sambrook et al., 1989).

1. picking list colony inoculation is in 5mL LB liquid nutrient medium, and 37 ℃, 180rpm, overnight shaking is cultivated;

2. get 1-1.5mL bacterium liquid and be transferred in the 1.5mL centrifuge tube, 12000rpm, 1min, centrifugal collection thalline;

3. supernatant discarded, thalline control are as far as possible done, and place on ice, add the solution I of 100 μ L precoolings, resuspended precipitation, ice bath 5min;

4. add 200 μ L solution II (1%SDS, 0.2mol/L NaOH), gently mix limpid and thickness to solution becomes, ice bath 5min notices that solution II wants matching while using;

5. add 150 μ L solution III, mixing gently, ice bath 5min;

6.4 ℃, 12000rpm, centrifugal 10min draws supernatant and is transferred in another new pipe;

7. add 8 μ LRnase (10mg/mL), place 45min in 37 ℃ of water-baths;

8. add about 400 μ L the saturated phenol/chloroform of Tris/primary isoamyl alcohol mixed solution (phenol: chloroform: extracting primary isoamyl alcohol=25: 24: 1), 4 ℃, 12000rpm, centrifugal 10min draws supernatant and is transferred in another new pipe;

9. add the dehydrated alcohol of about 1mL precooling, mix ,-20 ℃, place 20min;

10.4 ℃, 12000rpm, centrifugal 10min, supernatant discarded;

11. once with 1mL70% washing with alcohol precipitation, vacuum-drying, precipitation is dissolved among an amount of (the 10-50 μ L) TE (pH=8.0) or sterilized water.

The amount of the plasmid of agarose gel electrophoresis Detection and Extraction 12.0.8% is a dna molecular amount standard with DL15000.

The enzyme of carrier pUC18 plasmid is cut and dephosphorylation is handled

Cut processing pUC18 plasmid with restriction enzyme BamH I enzyme, the method that enzyme is cut is referring to TaKaRa company product description.This enzyme is cut product can directly cut the result with the electrophoresis detection enzyme, if enzyme cuts entirely, then termination reaction adopts heat treated (60 ℃ of 15min) to make the restriction enzyme enzyme deactivation.

The processing of endonuclease reaction system:

(1) once with the saturated phenol/chloroform of Tris/primary isoamyl alcohol extracting, 4 ℃, 12000rpm, centrifugal 10min shifts supernatant to another new centrifuge tube;

(2) add the 3M NaAC (pH4.8) of 1/10 volume, the dehydrated alcohol of 2 times of volume precoolings, mixed evenly after, in-20 ℃, place 30min;

(3) 4 ℃, the centrifugal 10min of 12000rpm, DNA precipitates with 70% washing with alcohol twice, vacuum-drying.(10mM Tris.HCl, 1mM EDTA is pH8.0) in damping fluid or the sterilized water to be dissolved in an amount of TE.

For preventing carrier self ligation, reduce the background when transforming, improve joint efficiency, remove the pUC18 molecule 5 ' terminal phosphate of linearization with small intestine alkaline phosphomonoesterase (CIAP).Concrete grammar carries out to specifications.

Connect, transform and screening

1. ligation system (cumulative volume is 20 μ l):

Being connected of genomic dna and plasmid pUC18 (15 μ l system):

DNA 1μL

ddH ₂O 10μL

pUC18 1μL

Mixing, 45 ℃ of water-baths are after 5 minutes, and ice bath adds T4 ligase enzyme 1.5 μ L more rapidly, 10 * buffer1.5 μ L, and 14-16 ℃ of water-bath spent the night.

2. connect the processing of product:

(1) TE complements to 200 μ L.

(2) add the dehydrated alcohol of 2 times of volumes and the 3mol/LNaAc (pH4.8) of 1/10 volume, place 20min deposit D NA for-20 ℃.

(3) 4 ℃ of centrifugal 10min of 12000rpm abandon supernatant.

(4) the cold ethanol that adds 1mL70% is washed DNA precipitation twice, vacuum-drying.

(5) precipitation is dissolved in 5 μ L ddH ₂Among the O.14 μ l ddH ₂O, 1 μ l enzyme is cut the genomic dna after the processing, and 1 μ l enzyme is cut the carrier pUC18 fragment after the processing, and is evenly mixed, 45 ℃ of water bath heat preservation 5min.

(6) place immediately on ice after the taking-up, add 2 μ l, 10 * T4 DNALigase Buffer, 2 μ l T4 DNALigase, mixing, 16 ℃ of reaction 16-24h;

3. the preparation of competent escherichia coli cell

(1) the fresh streak culture single colony inoculation of picking is in 5mL LB liquid nutrient medium, and 37 ℃, 180rpm, overnight shaking is cultivated;

(2) be forwarded in the 200mL LB liquid nutrient medium in 1: 100 ratio, 37 ℃, 180rpm, shaking culture is to OD ₆₀₀=0.5-0.6, about 3-5h;

(3) bacterium liquid is transferred in the aseptic 250mL centrifuge tube, 4 ℃, 6000rpm, centrifugal 10min collects thalline;

(4) supernatant discarded, the aseptic deionized water (precooling) of adding 200mL, resuspended precipitation, 4 ℃, 6000rpm, centrifugal 10min;

(5) supernatant discarded, the aseptic deionized water (precooling) of adding 100mL, the operation of repeating step (4);

(6) aseptic 10% glycerine (precooling) of adding 100mL, the operation of repeating step (4);

(7) add aseptic 10% glycerine of 2mL (precooling), resuspended precipitation divides to be filled in the aseptic centrifuge tube of 1.5ml, 95 μ L/ pipe ,-70 ℃ of preservations.

4. the electric method for transformation of bacillus coli DH 5 alpha:

(1) 5 μ LDNA connects product and 95 μ L competent escherichia coli cell mixings, ice bath 5-10min.

(2) mixture is transferred to electric the conversion in the cup (negative control does not add mixture, only adds competent cell) of 0.2cm of precooling, on Bio-Rad electricity conversion instrument 2.50kv is set, and shocks by electricity.

(3) transform sucking-off electricity transformation system the cup from electricity, and after SOC liquid nutrient medium (20g/L Tryptones, 5g/L yeast powder, 0.5g/L NaCl 20mM glucose, the 10mM magnesium chloride) dilution with 1mL37 ℃ of preheating, 37 ℃, cultivate 1h for 150 rev/mins.

(4) bacterium liquid is all coated and is contained 20mmol/L MgSO ₄On the LB solid medium of 50 μ g/mL kantlex and 100 μ g/mL penbritins, be inverted incubated overnight for 37 ℃.

5. screening, the evaluation of clone's:

(1) from two anti-dull and stereotyped picking transformants of going up of incubated overnight, extract plasmid in a small amount, cut, and to cut product with pUC18BamH I enzyme is contrast with restriction enzyme BamH I enzyme.

(2) be the DNA standard with DL15000, electrophoresis detection is identified in 0.8% sepharose, selects clone's (Fig. 3) that has pUC18 carrier band and external source goal gene band simultaneously.

(3) be template simultaneously, carry out PCR (concrete grammar is with described in the embodiment 1) with the Mu transposon detection primer Mu1 and the Mu2 of Mu transposon dna fragmentation with the plasmid that from transformant, extracts.Electrophoresis detection PCR product is the DNA standard with DL2000, is over against photograph with plasmid pHTH2, if can amplify the specificity band of 700bp, then illustrates and contains artificial Mu transposon dna fragmentation in the transformant plasmid, clones successfully (Fig. 2).

(4) the positive transformant plasmid is checked order.

6. the dna sequencing of positive transformant plasmid:

Reverse sequence with Mu transposon two ends serves as to check order as primer according to the AM42P1 that designs.Entrust the handsome company in Shanghai to carry out dna sequencing, measure the nucleotide sequence of transposon insertion site flank 500bp.

7. the assignment of genes gene mapping:

Sequencing result the genome database (http//www.pseudomonas.com) of Pseudomonas aeruginosa or GenBank ( Http:// www.ncbi.nlm.nih.gov/) in BLAST carry out sequence alignment and find the position of gene that Mu inserts in the pseudomonas aeruginosa gene group.

Sequence table

＜110〉Nankai University

＜120〉Patina pseudomonas migration ability correlated gene PA 5001 and application

<160>1

<210>1

<211>957

<212>DNA

＜213〉Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA)

<400>1

Claims (2)

1. Patina pseudomonas migration ability correlated gene PA 5001, its nucleotide sequence is shown in sequence table sequence 1.

2. the application of the described Patina pseudomonas migration ability correlated gene PA 5001 of claim 1, being used for this gene is the medicine that design of new drug target spot and preparation suppress thalline swimming ability, to reduce its virulence and pathogenic, strengthens antibiotic drug effect.

Images