CN102757912A - Salmonella typhimurium X3337lux and applications of salmonella typhimurium X3337lux in living body imaging - Google Patents

Abstract

The invention discloses a salmonella typhimurium X3337 and applications of the salmonella typhimurium X3337 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.5894. The salmonella typhimurium X3337 is the bacterial strain after the elimination of virulent plasmids, and can be taken as a reference to be used for researching the functions of the virulent plasmid of salmonella typhimurium. X3337lux is salmonella typhimurium bacterial strain with a stable biological luminescence property. The salmonella typhimurium X3337 complements have the defects that an excitation light source is needed by the detection of bioluminescence bacteria, the specificity of the salmonella typhimurium X3337 in a body is low, the background is high, the quantity is difficult to determine and the like. The salmonella typhimurium X3337 can be widely applied to establishing an in-vitro cell infection model and an in-vivo experiment model. A convenient tool is provided for the intensive study of the pathogenic mechanism of salmonella typhimurium, drug researching and screening and related disease preventing strategy researching and the like.

Description

Salmonella typhimurium X3337lux and its application in live imaging

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technical field

The invention belongs to the technical field of microorganisms, in particular to Salmonella typhimurium X3337lux and its application in live imaging.

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Background technique

Salmonella typhimurium ( Salmonella typhimurium ) has a wide range of hosts and can cause many diseases in humans and animals. It is of great practical significance to study its pathogenic mechanism. Plasmid is the genetic material outside the bacterial chromosome, which contains many important gene clusters, and is closely related to the symbiosis, metabolism and pathogenic mechanism of bacteria. Salmonella typhimurium X3337 is a strain after eliminating the virulence plasmid, which can be used as a control to study the function of the virulence plasmid of Salmonella typhimurium.

In recent years, in vivo optical imaging technology of living animals has been continuously developed in life science research due to its simple operation and intuitiveness. This imaging technology can directly observe the activities and responses of the marked genes and cells in living animals in real time. Bioluminescence technology (bioluminescence) is currently a hot spot in vivo imaging research, which is marked with luciferase (luciferase) gene. The bioluminescence using luciferase as the reporter source in the body emits light through the specific interaction between the enzyme and the substrate, and the specificity is extremely strong. Because the animal itself does not have any self-luminescence, the bioluminescence has extremely low background and high signal-to-noise Compare. Compared with traditional fluorescence imaging, the biggest feature of bioluminescent imaging is the high sensitivity of in vivo detection. When the fluorescence imaging method is excited by the excitation light, many substances in the organism (such as skin, hair, various tissues and food, etc.) will produce fluorescence. Especially when the marked target is hidden deep inside the tissue and requires higher energy excitation light, it also produces strong non-specific fluorescence. The background noise generated by these non-specific fluorescence makes the signal-to-noise ratio much lower than that of bioluminescence. In addition, the signal level of fluorescence depends on factors such as the intensity of excitation light, the number of luminescent cells, the depth of the target, the absorption and scattering of the tissue through which the light passes, making it difficult to quantify the fluorescence intensity. Bioluminescent technology can make up for its shortcomings.

Bacterial luciferase gene operon Lux CDABE is composed of luciferase gene and its substrate synthase gene, and the bacteria marked by it will continue to emit light without exogenous substrates. In recent years, researchers have successively cloned it into various vectors and introduced it into pathogenic bacteria, and applied bioluminescence technology and small animal imaging system to monitor the infection process of pathogenic bacteria. But plasmid-based bioluminescence is unstable without antibiotic selection and cannot be used for long-term studies in vivo. Injecting antibiotics into experimental mice to screen for Salmonella carrying intact luminescent plasmids will cause drug resistance in mice and interfere with experimental results to some extent. Due to the different copy numbers of the plasmids, the luminescence intensity of single cells is not stable, so it is impossible to quantitatively detect pathogenic bacteria by luminescence intensity. In 2010, Kevin Howe constructed the plasmid pBEN276, which can be used to clone the Lux operon into the bacterial chromosome (Figure 1), and its bioluminescent signal can be used for accurate quantification. Therefore, the present invention is constructed on the basis of Salmonella typhimurium X3337 Strain X3337lux with stable bioluminescent properties. Using the luminescent bacterium X3337lux as a control to establish an animal model, it is possible to dynamically track the dissemination process of bacteria in living animals in real time, in-depth study of the pathogenic mechanism of pathogenic bacteria, and drug research and screening.

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Contents of the invention

Technical issues solved:

The present invention aims at the defects of low specificity, high background, and difficult quantification of fluorescent markers in vivo detection, and the shortcomings of conventional plasmid-based bioluminescence detection that require additional screening pressure and substrates, and are difficult to quantify, etc., and provides a stable luminescence Salmonella typhimurium X3337lux and its use in intravital imaging.

Technical solutions:

Salmonella typhimurium ( Salmonella typhimurium ) X3337lux, the strain has been preserved in the preservation unit designated by the State Intellectual Property Office, the preservation date is March 13, 2012, the name of the preservation unit: China Microorganism Culture Collection Management Committee General Microbiology Center, deposit Number: CGMCC No.5894. Application of Salmonella typhimurium ( Salmonella typhimurium ) X3337lux in live imaging.

Salmonella typhimurium ( Salmonella typhimurium ) X3337 is hereinafter referred to as X3337; Salmonella typhimurium ( Salmonella typhimurium ) X3337lux is hereinafter referred to as X3337lux.

Beneficial effects: the Salmonella typhimurium has stable bioluminescent performance, which makes up for the defects that the detection of bioluminescent bacteria requires an excitation light source, low specificity in vivo, high background, and difficult quantification. Through site-specific recombination, the housekeeping gene E. coli frr promoter and Lux operon are inserted into the host bacterial chromosome for stable expression (at the same time, the insertion site does not destroy the genome gene function and has no adverse effects on the bacteria). Under the regulation of the housekeeping gene E. coli frr promoter connected upstream, the Lux operon constitutively expresses luciferase and substrates, without additional screening pressure and substrates to maintain bacterial luminescence. And through the experiment, it is detected that the number of bacteria is proportional to its luminous intensity, and the bioluminescence of bacteria can be continuously detected in the living mice. It makes up for the shortcomings of previous plasmid-labeled bioluminescent bacteria that require additional screening pressure and substrates, which affect luminescence detection, and the amount of luminescence per unit cell is unstable. The luciferase gene is inserted into the chromosome and expressed stably, and the luminescent quantity per unit cell is very stable. Coupled with the extremely high specificity and signal-to-noise ratio of bioluminescent technology, even if the labeled cells have complex positioning in the animal body, the relative number of luminescent cells can be directly obtained from the signal level on the animal body surface. It can be widely used in the establishment of in vitro cell infection models and in vivo experimental animal models. Combining the use of in vivo imaging equipment can continuously monitor the invasion, proliferation and spread of Salmonella typhimurium in vivo in real time and dynamically. It provides a convenient tool for in-depth research on the pathogenic mechanism of Salmonella typhimurium and Salmonella typhimurium, drug research and screening, and related disease prevention and control strategies.

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Description of drawings

Figure 1 is a map of the pBEN276 plasmid, and tnsABCD is the gene encoding the Tn7 transposon. The operon luxCDABE , which encodes luciferase and substrate, introduces multiple cloning restriction sites Xho1 and Not1, which are flanked by the Tn7 transposon. The 3' end of the Lux operon is linked to the promoter of the housekeeping gene frr of E. coli to initiate the constitutive expression of the Lux operon.

Figure 2 shows the results of colony bioluminescence detection (1 and 2 are images of X3337lux single colonies under whitelight and luminescence conditions, and 3 and 4 are images of X3337 single colonies under whitelight and luminescence conditions).

Figure 3 shows the bioluminescence detection results of the bacterial liquid (1~5: luminescence mode imaging Χ3337lux; 6: luminescence mode imaging Χ3337).

Fig. 4 is the graph of the relationship between the amount of Χ3337lux photons and the number of passage days under the condition of no selection pressure.

Figure 5 shows the signal detection results of stable luminescent bacteria in mice (1, 2, and 3 are imaging of mice infected with X3337lux in white light mode, luminescence mode imaging and merge image).

Figure 6 is a luminescence detection diagram of the relationship between the number of photons and the number of bacteria (1~5: 10-fold dilution of X3337lux bacterial solution; 6: X3337 negative control).

Figure 7 is a diagram of the relationship between the number of photons and the number of bacteria (1~5: Χ3337lux bacterial count doubling; 6: Χ3337 negative control).

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Detailed ways

Example 1

Specific construction and identification of strains:

1. Material preparation

1.1 Plasmids and strains

Plasmid pBEN276 was kindly donated by Prof. Pierre Germon, Laboratory of Animal Infectious Diseases and Public Health Pathogen Research Laboratory, French Natural Resources Institute. 【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 typhimurium ( Salmonella typhimurium ) X3337 was kindly provided by Professor ROY CURTISS III, College of Biological Sciences, Arizona State University, USA. [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 bacteriology1, 15001 (183): 4652–4658.]

1.2 Instruments and reagents

Instrument: Electrotransfer instrument (Bio-RadGene Pulser II system) is a product of American Bio-Rad;

High-speed refrigerated centrifuge Beckaman products;

Kodak multi-mode small animal live imaging system DXS4000pro;

Reagents: Plasmid extraction kit QIAGEN Plasmid MaxKit was purchased from Qiagen.

2 methods

2.1 Transformation of the target strain

2.1.1 Competent cell preparation: Inoculate Salmonella typhimurium X3337 into LB solid medium by streaking method on a plate, and incubate overnight at 37°C. Pick individual round and glossy single colonies and inoculate them into 3mL LB liquid medium, and culture overnight at 37°C and 200r/min. The culture was inoculated into 100 mL of growth medium at a ratio of 1:100, cultured at 37°C and 250 r/min until the OD600 was 0.3, the culture was taken out, and immediately placed in an ice bath for 30 min. Centrifuge at 4000r/min for 10min at 4°C to collect the cells. Wash three times with 4°C pre-cooled ultrapure water, resuspend in 50 μL pre-cooled ultra-pure water, and place on ice until use.

2.1.2 Electroporation scheme: Add the plasmid pBEN276 into 50 μL of competent cells, mix well and transfer to a pre-cooled 1 mm electrode cup. Then adjust the voltage, resistance and capacitance parameters to 2.5kV, 400Ω, 50μF, and quickly add 1mL of SOC medium preheated at 30°C after the electric shock. Transfer the electroshocked bacterial solution to a test tube, and incubate at 37°C, 100r/min for 1-3h. 100 μL was spread on LB plates containing 40 μg/mL ampicillin, and incubated overnight at 37°C.

2.2 Identification of transformants

Positive clones were picked from the LB agar plate containing 50 μg/mL Amp and inoculated into 2 mL liquid LB medium containing 50 μg/mL Amp at 30°C and 200 rpm for 14-16 hours, and the bacterial liquid was transferred to a 24-well plate to detect bioluminescence. At the same time, the plasmid was extracted for enzyme digestion and PCR identification, and inoculated on SS solid medium for biochemical identification of Salmonella typhimurium.

2.3 Induction of transposon recombination

The above-identified transformants were inoculated into LB liquid medium containing 0.1%wt arabinose at 30°C and cultured at 200rpm for 14-16h. The induced bacterial solution was streaked on the LB solid medium plate without antibiotics, and cultured at 42°C for 14-16h. Each selected 6 single clones were inoculated in liquid LB medium at 42°C, cultured at 200rpm for 14-16h, and the bacterial solution was transferred to a 24-well plate to detect bioluminescence.

2.4 Screening for clones stably expressing the Lux operon

The bacterial solution capable of luminescence after recombination was selected and streaked on the solid LB medium containing 50 μg/mL Amp, and at the same time continued to subculture in the antibiotic-free medium and cultured at 37°C to quantitatively detect the luminescence of the bacteria. Colonies without antibiotic resistance and stable luminescence after multiple passages were screened.

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Bacterial strain culture propagation method:

Χ3337lux is a facultative anaerobic bacterium that can grow in ordinary LB medium. Medium ingredients: peptone 10g, yeast extract 5g, NaCl 10g, dissolved in 1L water, pH=7±0.2, culture temperature 37 ℃ conditions can be stable proliferation. The difference between the new strain Χ3337lux and Χ3337 is that it has stable bioluminescent properties. The Lux operon site-specific insertion into the chromosome has no effect on other physiological characteristics of bacteria.

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Species morphology and biochemical characteristics detection:

The liquid LB medium was cultivated overnight at 37°C and the bacteria were uniformly turbid. Take a ring of bacterial solution and dilute it properly, then check it with Gram staining microscope, the bacteria are Gram-negative bacilli. Cultivate with solid LB medium at 37°C for 18 hours to form a milky white colony with a smooth surface, neat edges, and a diameter of 1-3mm. The SS medium was cultured at 37°C for 18-24 hours, and the colony morphology was medium-sized, round, smooth, milky white, with a black center of the colony, and positive for hydrogen sulfide. Pick a single colony and inoculate it in a biochemical tube for overnight cultivation at 37°C to test its biochemical characteristics. The bacteria do not ferment lactose and sucrose, but can ferment glucose, mannitol, and maltose. Pick a single colony and inoculate it on semi-solid agar, culture at 37°C for 18-24 hours, the bacteria grow in a feathery shape, and the motility is positive.

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Colony bioluminescence detection:

Inoculate Χ3337lux on LB plates by the method of partitioning and streaking, culture at 37°C for 14-16 hours. After a single colony grows, the plate is placed in a multi-mode in vivo imager for bioluminescence detection. In the bioluminescence detection mode, a single colony of luminescent bacteria (Χ3337lux) was visible, but a single colony of common bacteria (Χ3337) was invisible (Figure 2).

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Bacterial liquid bioluminescence detection and stability detection:

A single colony of Χ3337lux was inoculated into liquid LB medium at 37°C, cultured at 200rpm for 14-16h, and the bacterial solution was transferred to a 24-well plate to detect bioluminescence. In the bioluminescence mode, only luminescent bacteria (Χ3337lux) were visible, and common bacteria (Χ3337) were invisible (Figure 3). Subculture under the condition of no selection pressure, collect the OD ₆₀₀ of the bacterial liquid every day and dilute it to an equal volume and concentration of the bacterial liquid for bioluminescence detection, record the amount of photons, and the luminescence performance of Χ3337lux is stable (Figure 4).

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Example 2

Salmonella typhimurium ( Salmonella typhimurium ) Χ3337lux live imaging

1. Bacterial culture

Select a single clone from the solid LB agar plate and inoculate it into LB liquid medium, and culture at 37°C, 200rpm for 14-16h to logarithmic growth phase. Collect bacteria by centrifugation at 4000rpm, 4°C, 10min, wash twice with normal saline (4000rpm, 4°C, 10min), resuspend with normal saline, measure OD600 to calculate the bacterial concentration. Gradiently diluted with normal saline for use.

2. Mice infection method (intraperitoneal injection or oral feeding infection)

2.1 Intraperitoneal injection infection

BALB/C or C57BL mice of 8-12 weeks were selected, and 50 μL of bacterial solution with a concentration of 10 ⁵ CFU/mL was injected intraperitoneally to infect the mice.

2.2. Oral feeding infection

After 8-12 weeks of fasting, BALB/C or C57BL mice were fasted for 6 hours, and were given 50 μL of ¹⁰ % wt NaHCO ₃ to neutralize gastric acid. Rats, resume feeding after half an hour.

3. In vivo imaging method

The mice were anesthetized by intraperitoneal injection of 0.1 mL of 10%wt chloral hydrate, and imaged with a small animal imager after the anesthesia took effect (about 30 min). First imaged under white light conditions, only mice were visible. Then imaged under luminescence conditions, only the luminescent bacteria were visible. The two imaging results were merged, and the site where the luminescent bacteria spread after infection could be seen (Figure 5). The proliferation of luminescent bacteria in vivo can be calculated by photon analysis (Figure 6, 7).

Claims (2)

1. Salmonella typhimurium ( Salmonella typhimurium ) X3337lux, the strain has been preserved in the preservation unit designated by the State Intellectual Property Office, and the preservation date is March 13, 2012. The name of the preservation unit: General Microbiology Center of China Committee for the Preservation of Microbial Cultures , deposit number: CGMCC No.5894. 2. Application of Salmonella typhimurium ( Salmonella typhimurium ) X3337lux in live imaging.

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