CN102757911A - Salmonella typhimurium X3306lux and applications of salmonella typhimurium X3306lux in living body imaging - Google Patents

Salmonella typhimurium X3306lux and applications of salmonella typhimurium X3306lux in living body imaging Download PDF

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CN102757911A
CN102757911A CN2012102062465A CN201210206246A CN102757911A CN 102757911 A CN102757911 A CN 102757911A CN 2012102062465 A CN2012102062465 A CN 2012102062465A CN 201210206246 A CN201210206246 A CN 201210206246A CN 102757911 A CN102757911 A CN 102757911A
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x3306lux
salmonella typhimurium
salmonella
bacterium
bacteria
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黄瑞
李嫄渊
储元元
叶颖
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Suzhou University
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Suzhou University
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Abstract

The invention discloses a salmonella typhimurium X3306lux and applications of salmonella typhimurium X3306lux in living body imaging. The bacterial strain is preserved in depositary authority designated by the state intellectual property office, the preservation data is 13 March, 2012, the depositary authority is China general microbiological culture collection center, and the preservation number is CGMCC No.5895. The salmonella typhimurium X3306lux is a virulent strain carrying virulent plasmids pStSR100, can be taken as a standard strain, and is applied to the research of the pathogenic mechanism of salmonella typhimurium. X3306lux is the bacterial strain with stable bioluminescence property, which is constructed based on X3306. By the site-specific recombination, a housekeeping gene E.colifrr promoter and a Lux operon are inserted into chromosome of X3306 for stable expression (meanwhile, the insertion site does not damage the gene functions of a genome, and the bacteria can not suffer from the adverse influence). The Lux operon conducts constitutive expression on luciferase and a substrate under the regulation of the housekeeping gene E.colifrr promoter connected with the Lux operon at the upstream, the bacteria can light without additionally adding screening pressure and the substrate.

Description

Salmonella typhimurtum X3306lux and the application in living imaging thereof
 
Technical field
The invention belongs to microbial technology field, particularly Salmonella typhimurtum X3306lux and the application in living imaging thereof.
 
Background technology
Salmonella typhimurtum ( Salmonella typhimurium) be a kind of important infecting both domestic animals and human cause of disease bacterium, mainly through water and food transmission, can cause infectious intestinal diseases such as food poisoning, gastro-enteritis, its incidence of infection occupies the first place of Salmonella infection.X3306 is the Salmonella typhimurtum virulent strain that carries virulence plasmid pStSR100, can be used as standard strain, is applied to the research of Salmonella typhimurtum mechanism of causing a disease.
In recent years, in the living animal body optical image technology easy and simple to handle and intuitive is able to continuous development in life science with it.This imaging technique can real time direct be observed gene and the cell of mark in intravital activity of living animal and reaction.Utilize the bacterium of optical markings to set up animal model and can Real-time and Dynamic follow the trail of the course of infection of pathogenic bacteria in living animal, further investigation pathogenic bacteria mechanism of causing a disease is carried out drug research and screening etc.Main fluorescence (fluorescence) and two kinds of technology of noclilucence (bioluminescence) of adopting.
Fluorescence radiation is to produce emission light after arriving higher-energy state through the excitation fluorophor.Fluorescence technique is easy and simple to handle, and signal is stronger, but when being applied in vivo tests when receiving excitation a lot of materials of organism all can produce fluorescence, like skin, hair and various tissue and food etc.Particularly, the target spot that is labeled also produces very strong non-specific fluorescence when organization internal needs the exciting light of higher-energy when being hidden in deeply; And pathogenic bacteria has complicated location in animal body; The exciting light of fluorescence need pass tissue and arrive target spot, and emission light need come out in body, and the path is longer.Signal level depends on the degree of depth of the quantity that excites light intensity, photogenic cell, target spot, tissue that light the passes factors such as absorption and scattering to it, makes fluorescence intensity be difficult to quantitatively.
Noclilucence is with luciferase (luciferase) genetic marker.With luciferase as the noclilucence in report source in the body be specific action with enzyme and substrate and luminous, specificity is extremely strong, because of animal itself has no luminous, makes noclilucence have extremely low background, high SNR.The noclilucence imaging is with respect to fluorescence imaging, and its maximum characteristics are exactly the highly sensitive that detects in the body.Therefore, bioluminescence technique becomes the focus of present research.
Bacterium LuxOperon is made up of luciferase gene and its substrate synthase gene, and the bacterium that has this operon can continue the luminous exogenous substrate that do not need.Recent study persons are cloned into variety carrier successively and import in the pathogenic bacteria, applying biological luminescence technology and small animal imaging system monitoring pathogenic bacterial infection process.But with the plasmid is bioluminescence technique instability under antibiotic-free is selected on basis, can not be used for intravital studying for a long period of time.The experiment mice injection of antibiotics is screened the salmonella of carrying complete luminous plasmid, can cause the resistance of mouse, and interference experiment result to a certain extent.Different because of the copy number of plasmid again, unicellular luminous intensity is unstable, can't come the detection by quantitative pathogenic bacteria with luminous intensity.2010, Kevin Howe made up plasmid pBEN276, utilize this plasmid can with LuxOperon is cloned into bacterial chromosome (Fig. 1), and its noclilucence signal can be used for accurate quantification, and the present invention has made up the strain X 3306lux with stabilate luminescent properties thus on Salmonella typhimurtum X3306 basis.
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Summary of the invention
The technical problem that solves:
It is low to the present invention is directed to the interior detection specificity of fluorescently-labeled body; Background is high; Defectives such as difficulty is quantitative; And need add screening pressure and substrate with bioluminescent detection based on plasmid in the past, and and be difficult to quantitatively wait deficiency, a kind of Salmonella typhimurtum X3306lux and the application in living imaging thereof that can stabilized illumination be provided.
Technical scheme:
Salmonella typhimurtum ( Salmonella typhimurium) X3306lux; This bacterial strain is in the preservation of specified depositary institution of State Intellectual Property Office; Preservation date is on March 13rd, 2012, depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC No.5895.Salmonella typhimurtum ( Salmonella typhimurium) application of X3306lux in living imaging.
Salmonella typhimurtum ( Salmonella typhimurium) X3306 hereinafter referred X3306; Salmonella typhimurtum ( Salmonella typhimurium) X3306lux hereinafter referred X3306lux.
Beneficial effect:This Salmonella typhimurtum has stable noclilucence performance, and the Bacteria Detection that has remedied od-ray needs excitation light source and defective such as specificity is low in vivo, and background is high, and is difficult quantitative.Through the locus specificity reorganization, with house-keeping gene E. Coli frrPromotor with LuxOperon inserts stably express (insert the site simultaneously and do not destroy the genomic gene function, bacterium is had no adverse effect) in the host bacterium karyomit(e). LuxOperon is swum the house-keeping gene E. of connection above that Coli frrUnder the regulation and control of promotor, constructive expression's luciferase and substrate need not to add screening pressure and substrate is kept bacterial luminescence.And detect number of bacteria through experiment and be directly proportional, and in vivo can continue to detect the noclilucence of bacterium mouse with its luminous intensity.Remedied in the past that the noclilucence bacterium of plasmid mark need add screening pressure and substrate, influenced luminous detection, and weak point such as unit cell luminous quantity instability.Fluorescent enzyme gene inserts stably express in the karyomit(e), and the amount of luminescence of unit cell is very stable.Add specificity and SNR that bioluminescence technique is high,, also can directly draw the relative populations of photogenic cell from the signal level of animal body surface even if labeled cell has complicated location in animal body.Can be widely used in the foundation of experimental animal model in cell in vitro infection model and the body.But in conjunction with utilizing living imaging instrument real-time and dynamic to continue to monitor Salmonella typhimurtum in the intravital invasion of mouse, propagation and spread condition.Be the further investigation of Salmonella typhimurtum and Salmonella typhi mechanism of causing a disease, the instrument that the research of drug research and screening and relative disease control strategy etc. facilitates.
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Description of drawings
Fig. 1 is the pBEN276 plasmid map, TnsABCDGene for coding Tn7 transposon.Operon LuxCDABEPlain enzyme of coding fluorescence and substrate are introduced polyclone restriction enzyme site Xho1 and Not1, connect in Tn7 transposon flank. LuxOperon 3 ' end connects E .coliHouse-keeping gene FrrPromotor, start LuxThe constructive expression of operon.
Fig. 2 is bacterium colony bioluminescent detection result (1 and 2 are followed successively by the single bacterium colony of Χ 3306lux forms images under whitelight and luminescence condition, and 3 and 4 are followed successively by Χ 3306 single bacterium colonies forms images under whitelight and luminescence condition).
Fig. 3 be bacterium liquid bioluminescent detection result (1,2:white light mode imaging Χ 3306lux; 3:white light mode imaging Χ 3306; 4,5:luminescence mode imaging Χ 3306lux; 6:luminescence mode imaging Χ 3306).
Fig. 4 is Χ 3306lux stable luminescent property property detected result (relation of the light intensity and the fate that under no selective pressure condition, goes down to posterity)
Fig. 5 is a stabilized illumination bacterium signal detecting result (1,2,3 are followed successively by with the mouse that infects Χ 3306lux in white light mode imaging luminescence mode imaging and merge image) in vivo.
Fig. 6 is the luminous detection figure (1 ~ 5: doubling dilution Χ 3306lux bacterium liquid of number of photons and bacterial count relation; 6: Χ 3306 negative controls).
(1 ~ 5:10 doubly dilutes Χ 3306lux bacterium liquid to Fig. 7 for number of photons and bacterial count graph of a relation; 6: Χ 3306 negative controls).
 
Embodiment
Embodiment 1
Bacterial strain is concrete to be made up and identifies:
1, material is prepared
1.1 plasmid and bacterial strain
Plasmid pBEN276 is so kind as to give by French Natural Resources Research Institute zoonosis and Pierre professor Germon of publilc health pathogenic bacteria research laboratory.【Kevin?Howe,?Attila?Karsi,?Pierre?Germon,?et.al.?Development?of?stable?reporter?system?cloning?luxCDABE?genes?into?chromosome?of?Salmonella?enterica?serotypes?using?Tn7?transposon?[J].?BMC?Microbiology?2010,?10:197】
Salmonella typhimurtum ( Salmonella typhimurium) X3306 is so kind as to give by biological scienology ROY CURTISS professor III of institute of the upright university of State of Arizona, US.【HIDENORI?MATSUI，?CHRISTOPHER?M.?BACOT，?WENDY?A.?Virulence?Plasmid-Borne?spvB?and?spvC?Genes?Can?Replace?the?90-Kilobase?Plasmid?in?Conferring?Virulence?to?Salmonella?enterica?Serovar?Typhimurium?in?Subcutaneously?Inoculated?Mice[J].?Journal?of?bacteriology，?2001，?15(183):?4652–4658.?】
1.2 instrument and reagent
Instrument: electroporation (Bio-RadGene Pulser II system) is a U.S. Bio-Rad product;
High speed freezing centrifuge Beckaman Company products;
The multi-mode animalcule living imaging DXS4000pro of system of Kodak;
Reagent: plasmid extraction kit QIAGEN Plasmid MaxKit is available from Qiagen company.
2 methods
2.1 the conversion of purpose bacterial strain
2.1.1 competent cell preparation: plate streak inoculation Salmonella typhimurtum X3306 to LB solid medium, be inverted overnight cultures for 37 ℃.The individual mellow and full glossiness single colony inoculation of picking is to 3mL LB liquid nutrient medium, 37 ℃, 200r/min overnight cultures.The ratio of culture with 1:100 is inoculated in the 100mL growth medium, and 37 ℃, it is 0.3 o'clock taking-up culture, ice bath 30min immediately that 250r/min is cultured to OD600.4 ℃, the centrifugal 10min of 4000r/min collects thalline.Give a baby a bath on the third day after its birth time with the ultrapure water of 4 ℃ of precoolings, resuspended with the ultrapure water of 50 μ L precoolings, place for use on ice.
2.1.2 electric conversion scheme: plasmid pBEN276 is joined in the 50 μ L competent cells, change the 1mm pole cup of precooling after mixing over to.Re-adjustment voltage, resistance and capacitance parameter are 2.5kV, 400 Ω, and 50 μ F, the electric shock back adds the SOC substratum 1mL of 30 ℃ of preheatings rapidly.Bacterium liquid after the electric shock is transferred to test tube, and 37 ℃, 100r/min cultivates 1 ~ 3h.Get 100 μ L and coat on the LB flat board that contains 40 μ g/mL penbritins 37 ℃ of overnight cultures.
2. 2 transformants are identified
Positive colony is chosen from the LB agar plate that contains 50 μ g/mL Amp and is seeded to 2mL and contains 30 ℃ of 50 μ g/mL Amp liquid LB substratum, and 200rpm cultivates 14 ~ 16h, and it is luminous that bacterium liquid is transferred to 24 orifice plate detection of biological.Extract plasmid simultaneously and carry out enzyme and cut and identify evaluation, and be inoculated in the SS solid medium and carry out the Salmonella typhimurtum biochemical identification with PCR.
2.3 induce the transposon reorganization
Be seeded to 30 ℃ of LB liquid nutrient mediums that contain the 0.1%wt pectinose with above-mentioned through the transformant of identifying, 200rpm cultivates 14 ~ 16h.Bacterium liquid after inducing is rule on antibiotic-free LB solid medium flat board, cultivates 14 ~ 16h for 42 ℃.Respectively select 6 mono-clonals and be inoculated in 42 ℃ of liquid LB substratum, 200rpm cultivates 14 ~ 16h, and it is luminous that bacterium liquid is transferred to 24 orifice plate detection of biological.
2.4 screening stably express LuxThe clone of operon
Can rule in containing on the 50 μ g/mL Amp solid LB substratum by luminous bacterium liquid after picking out reorganization, in the antibiotic-free substratum, continue to go down to posterity 37 ℃ simultaneously and cultivate detection by quantitative bacterial luminescence situation.Filter out the antibiotic-free resistance and the bacterium colony of the ability stabilized illumination that repeatedly goes down to posterity.
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The multiplication by culture method of bacterial strain:
Salmonella typhimurtum Χ 3306lux is a facultative anaerobe, in common LB substratum, can grow medium component: peptone 10g, and yeast extract 5g, NaCl 10g is dissolved in 1L water, and propagation can be stablized under 37 ℃ of conditions of culture temperature in pH=7 ± 0.2.
 
Bacterial classification form and biochemical characteristic detect:
37 ℃ of overnight cultures bacteriums of liquid LB substratum are evenly muddy.Get a ring bacterium liquid and suitably dilute back gram's staining microscopy, this bacterium is a gram negative bacilli.Cultivate 18h for 37 ℃ with solid LB substratum, form the oyster white bacterium colony, smooth surface, neat in edge, diameter 1 ~ 3mm.It is median size, circle, smooth, oyster white, bacterium colony central black that 37 ℃ of SS substratum are cultivated the last colonial morphology of 18 ~ 24h, the hydrogen sulfide positive.Picking list colony inoculation detects biochemical characteristic in 37 ℃ of overnight cultures of biochemistry pipe, this bacterium nonfermented lactose and sucrose, ability glucose fermentation, N.F,USP MANNITOL, SANMALT-S.Picking list colony inoculation is cultivated 18 ~ 24h for 37 ℃ in semi-solid agar, and bacterium is plumose growth, and power is positive.
 
Single bacterium colony bioluminescent detection:
The sectional streak method is inoculated in the LB flat board with Salmonella typhimurtum Χ 3306lux, 37 ℃, cultivates 14 ~ 16h.After waiting to grow single bacterium colony, place multi-mode living imaging appearance to carry out bioluminescent detection flat board.Under the bioluminescent detection pattern, the single bacterium colony (Salmonella typhimurtum Χ 3306lux) of Visible Luminescence bacterium, and common bacteria list bacterium colony (Salmonella typhimurtum Χ 3306) invisible (Fig. 2).
 
Bacterium liquid bioluminescent detection:
Single colony inoculation is to 37 ℃ of liquid LB substratum, and 200rpm cultivates 14 ~ 16h, and it is luminous that bacterium liquid is transferred to 24 orifice plate detection of biological.Following visible luminescent bacteria (Salmonella typhimurtum Χ 3306lux) of noclilucence pattern, common bacteria (Salmonella typhimurtum Χ 3306) invisible (Fig. 3).
The noclilucence Detection of Stability
With single colony inoculation to the antibiotic-free liquid of Χ 3306lux LB substratum, going down to posterity every day is cultured to 12 days, collects bacterium liquid in per 3 days, with OD 600For standard is diluted to the equal volume same concentrations, in the living imaging appearance, carry out bioluminescent detection, the record luminous intensity.Χ 3306lux has stable noclilucence performance (like Fig. 4).
 
Embodiment 2
Salmonella typhimurtum ( Salmonella typhimurium) Χ 3306lux living imaging
1. microbial culture
Select mono-clonal from solid LB agar plate and be seeded to the LB liquid nutrient medium, 37 ℃, 200rpm cultivates 14 ~ 16h to logarithmic phase.4000rpm, 4 ℃, the centrifugal collection thalline of 10min, through saline water wash twice (4000rpm, 4 ℃, 10min) after, resuspended with an amount of saline water, survey OD600 and calculate bacterial concentration.For use with the saline water gradient dilution.
2. mouse infection method (abdominal injection or mouth raise infection)
2.1 abdominal injection infects
Choose 8-12 week BALB/C or C57BL mouse, with 50 μ L concentration 10 5The bacterium liquid abdominal injection infecting mouse of CFU/mL.
2.2. mouth is raised infection
After water 6h is prohibited in 8-12 week BALB/C or C57BL mouse fasting, with the NaHCO of 50 μ L 10%wt 3Irritate stomach, in and hydrochloric acid in gastric juice, with volume 0.2 mL concentration 10 7The bacterium liquid of CFU/mL is irritated the stomach infecting mouse, recovers feeding after half a hour.
3. living imaging method
, treat that anesthesia comes into force to utilize after (about 30min) the small animal imaging appearance to form images through the intraperitoneal injection of anesthesia mouse with 10%wt Chloral Hydrate 0.1mL.At first under white light (white light) condition, form images only visible mouse.Under noclilucence (luminescence) condition, form images only visible luminescent bacteria then.With twice imaging results merger (merge), the position (Fig. 5) that the Visible Luminescence bacterium is sent out after infecting.Can calculate luminescent bacteria propagation situation (Fig. 6,7) in vivo through the photon component analysis.

Claims (2)

  1. Salmonella typhimurtum ( Salmonella typhimurium) X3306lux; This bacterial strain is in the preservation of specified depositary institution of State Intellectual Property Office; Preservation date is on March 13rd, 2012, depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC No.5895.
  2. Salmonella typhimurtum ( Salmonella typhimurium) application of X3306lux in living imaging.
CN2012102062465A 2012-06-21 2012-06-21 Salmonella typhimurium X3306lux and applications of salmonella typhimurium X3306lux in living body imaging Pending CN102757911A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103352012A (en) * 2013-05-31 2013-10-16 苏州大学 Salmonella typhimurium X3337-pRST98lux and its application in living imaging
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

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001077160A1 (en) * 2000-04-10 2001-10-18 University College London A modified h-ns peptide of salmonella typhimurium, capable of modulating the oligomerization of h-ns

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001077160A1 (en) * 2000-04-10 2001-10-18 University College London A modified h-ns peptide of salmonella typhimurium, capable of modulating the oligomerization of h-ns

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HIDENORI MATSUI ET AL.: "Virulence Plasmid-Borne spvB and spvC Genes Can Replace the 90-Kilobase Plasmid in Conferring Virulence to Salmonella enterica Serovar Typhimurium in Subcutaneously Inoculated Mice", 《JOURNAL OF BACTERIOLOGY》, vol. 183, no. 15, 31 August 2001 (2001-08-31), pages 4652 - 4658 *
KEVIN HOWE ET AL.: "Development of stable reporter system cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7 transposon", 《BMC MICROBIOLOGY》, vol. 10, no. 197, 23 July 2010 (2010-07-23), pages 1 - 8 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103352012A (en) * 2013-05-31 2013-10-16 苏州大学 Salmonella typhimurium X3337-pRST98lux and its application in living imaging
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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Application publication date: 20121031