CN111607550A - High-yield Klebsiella alcoholic beverage expressing luciferase and application thereof - Google Patents
High-yield Klebsiella alcoholic beverage expressing luciferase and application thereof Download PDFInfo
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Abstract
The invention constructs high-yield Klebsiella alcoholic beverage containing luciferase gene cluster (LUX) by utilizing plasmid construction and electric transformation technology, and finally obtains the high-yield Klebsiella alcoholic beverage (HiAlc Kpn-LUX) capable of stably expressing luciferase through autofluorescence identification, passage stability test of strains and observation of culture characteristics. Provides an important visual research tool for researching the progress of relevant diseases of the Klebsiella alcohol production and screening a proper sterilization method, antibiotics and the like.
Description
Technical Field
The invention relates to the field of microbial medicine, in particular to a construction method of genetically modified high-yield klebsiella wine containing a pan-host plasmid of a luciferase gene and stably expressing luciferase, a strain constructed by the method, and application of the strain in the aspects of calculating bacterial concentration, screening and sterilizing methods, screening medicines, preparing infected animal models and the like.
Background
Research shows that about 60% of non-alcoholic fatty liver diseases in China are related to high-alcohol-producing Klebsiella pneumoniae (high-alc Kpn) in intestinal tracts of patients. HiAlc Kpn isolated from the intestine of a patient with nonalcoholic fatty liver disease was transplanted into mice, and the results showed that HiAlc Kpn induced the production of nonalcoholic fatty liver disease in mice. Feeding glucose to the above mice with HiAlc Kpn implanted in the intestine enables detection of alcohol in the blood of the mice. Therefore, HiAlc Kpn can be used as a new detection index of the nonalcoholic fatty liver disease.
At present, research on pathogenesis and the like of HiAlc Kpn induced non-alcoholic fatty liver disease is just started, so a powerful tool is urgently needed to research the infection way of HiAlc Kpn and the characteristics of the HiAlc Kpn in vivo location and diffusion, evaluate the curative effect of the existing antibiotics and the effect of suitable medicines, such as antibiotics or other sterilization methods.
Bioluminescence is a phenomenon in which light is emitted by an enzyme within an organism. Bioluminescence is widely found in organisms such as bacteria and insects. The bioluminescence technology of using luciferase gene (LUX) gene labeled cells has the advantages of simple and convenient operation, intuitive result, no dependence on substrates, small damage to organisms and the like. The technology is widely applied to a plurality of fields of tumor growth monitoring and metastasis tracing, gene therapy, target gene expression detection and the like.
We constructed a pan-host plasmid pBBR1-LUX containing the luciferase gene LUX A/B/C/D/E. The plasmid can stably express luciferase in carbapenem drug-resistant Klebsiella and Klebsiella oxytoca. The plasmid was able to stably exist at HiAlcKpn and gave chloramphenicol resistance, but failed to express luciferase. Therefore, there is a need for an improved method for stably expressing luciferase from high-yielding klebsiella pneumoniae.
Disclosure of Invention
To overcome the above-mentioned drawbacks, pBBR1-LUX was modified by adding a constitutive promoter before LUX A/B/C/D/E. The invention constructs a plasmid containing a constitutive promoter and a luciferase gene LUX A/B/C/D/E gene cluster, converts the plasmid into high-yield Klebsiella alcoholic beverage through electric transformation, screens the genetically modified high-yield Klebsiella alcoholic beverage capable of stably expressing luciferase through passage, and uses the genetically modified high-yield Klebsiella alcoholic beverage in the aspects of calculating the bacterial concentration, screening a proper sterilization method, screening medicaments, preparing an infected animal model and the like. Therefore, the temperature of the molten metal is controlled,
the present invention provides a plasmid comprising a constitutive promoter and a luciferase gene LUX a/B/C/D/E gene cluster.
Preferably, the present invention constructs a plasmid containing a constitutive promoter and LUX A/B/C/D/E. The plasmid is a pan-host plasmid, the plasmid can complete the construction process in Escherichia coli, and a promoter of the plasmid can be recognized by Klebsiella pneumoniae of high-yield wine.
The invention also provides genetically modified high-yield Klebsiella pneumoniae which can stably express luciferase. Preferably, the genetically modified high-yield Klebsiella pneumoniae can stably express luciferase under the action of a constitutive promoter.
The invention also provides a construction method of the genetically modified high-yield Klebsiella alcoholic beverage, which comprises the following steps:
(1) preparing a plasmid comprising the LUX A/B/C/D/E gene cluster; preferably, the plasmid is a pan-host plasmid, and more preferably, the plasmid comprises a constitutive promoter that is recognized by klebsiella pneumoniae, so that klebsiella pneumoniae stably expresses luciferase and fluorescence can be detected.
(2) Preparing the electrotransformation competence of the high-yield Klebsiella alcoholic strain;
(3) and (3) electrically transforming the plasmid obtained in the step (1) into the competent high-yield klebsiella alcoholic beverage obtained in the step (2), and selecting a positive bacterial colony to obtain the genetically modified high-yield klebsiella alcoholic beverage.
Preferably, the constitutive promoter is a constitutive strong promoter, including but not limited to PJ23119, CaMV35S, P32 and the like. Preferably, the constitutive strong promoter is PJ 23119.
Preferably, the step (1) includes: (a) before LUX A/B/C/D/E amplification, a sequence of a constitutive promoter is added during primer design, and a LUX A/B/C/D/E gene cluster obtained by primer amplification is cloned to a pan-host plasmid pBBR 1; (b) electrically transforming the plasmid into an escherichia coli competent cell; (c) screening chloramphenicol positive escherichia coli; (d) a pan-host plasmid containing a constitutive promoter and LUX A/B/C/D/E gene cluster was extracted from positive E.coli.
Preferably, the constitutive promoter has the sequence: TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC (SEQ ID NO. 1).
Further, the step (a) comprises amplifying the LUXA/B/C/D/E gene cluster containing a promoter sequence; ② pBBR1MCS-1 double enzyme digestion; thirdly, recovering the enzyme digestion fragment gel of the second step, and connecting the enzyme digestion fragment gel with the LUXA/B/C/D/E gene cluster containing the promoter sequence amplified in the first step by using a Gibson connection method to obtain circular plasmids.
Furthermore, in the step (i), the LUX A/B/C/D/E gene cluster is amplified by using a high-fidelity enzyme, wherein the annealing temperature is 1-3 ℃ lower than the recommended temperature, such as 59-61 ℃.
The invention also provides application of the genetically modified high-yield Klebsiella alcoholic beverage in an evaluation method for calculating the concentration of bacteria.
The invention also provides an application of the genetically modified high-yield Klebsiella alcoholic beverage in a screening and sterilizing method.
The invention also provides an application of the genetically modified high-yield Klebsiella alcoholic beverage in drug screening.
The invention also provides application of the genetically modified high-yield Klebsiella alcoholic beverage in preparation of animal models.
Preferably, the animal model can be a bacterial localization research model, an antibiotic sterilization effect verification model, a bacterial localization and wine production efficiency analysis model, an influence model on liver injury and the like.
The invention also provides an evaluation method for calculating the concentration of bacteria, which comprises the steps of (1) culturing the genetically modified high-yield Klebsiella inebrians, (2) adjusting the concentration of strains by a dilution method, and calculating the concentration of the high-yield Klebsiella inebrians by detecting fluorescence values, and (3) drawing a standard curve of the fluorescence values of the genetically modified high-yield Klebsiella inebrians with different concentrations.
The invention also provides an evaluation method of the sterilization method, which comprises the step of evaluating the sterilization method by using the genetically modified high-yield Klebsiella alcoholic beverage.
Preferably, the sterilization method comprises sterilizing the articles carrying the high-yield klebsiella vinifera by using ultraviolet irradiation, alcohol, NaOH or 84 disinfectant, and more preferably, the sterilization method comprises irradiating the articles for more than 30min by using ultraviolet irradiation, or sterilizing the articles by using 10% of 84 disinfectant.
The invention also provides a drug screening method, which comprises screening drugs, such as antibiotics, by using the genetically modified high-yield klebsiella alcoholic beverage to search for sensitive and effective antibiotics for the high-yield klebsiella alcoholic beverage.
The assessment method may be an in vivo or in vitro assessment method. The assessment method is not a treatment method. The evaluation method is used for evaluating candidate sterilization methods, and detecting and comparing the sterilization effects of the candidate sterilization methods to determine which sterilization methods can be used for killing high-yield Klebsiella alcoholic beverage and which sterilization methods cannot be used for killing high-yield Klebsiella alcoholic beverage, or comparing the sterilization degrees of different sterilization methods, namely, the sterilization effects are not necessary and are only a possibility.
The drug screening method may be an in vivo or in vitro screening method. The drug screening method is not a therapeutic method. The method is used for screening drugs, detecting and comparing the drug effects of candidate drugs to determine which candidate drugs can be used as drugs and which can not be used as drugs, or comparing the drug effect sensitivity degrees of different drugs, namely, the treatment effect is not necessary and is only a possibility.
The invention also provides a preparation method of the animal model, which comprises the step of injecting the genetically modified high-yield Klebsiella into an animal body. Preferably, the animal model may be a burn model, a surgical model, an infection model, or the like. The in vivo injection mode can be nasal feeding, intragastric perfusion, intraperitoneal injection, tail vein injection and the like.
Preferably, the enrichment concentration of the strain in the animal body is not less than 105Cfu/mL。
The invention also provides a sterilization method of the high-yield klebsiella vinifera, which comprises the step of sterilizing articles carrying the high-yield klebsiella vinifera by utilizing ultraviolet irradiation, alcohol, NaOH or 84 disinfectant.
Preferably, the article carrying klebsiella vinifera comprises medical instruments, medical materials and other medical articles, such as operation beds, surgical instruments, medical beds, medical bedsheets and the like, or food or soil carrying klebsiella vinifera and the like.
Preferably, the sterilization method comprises ultraviolet irradiation for more than 30min, or sterilizing the articles with 10% of 84 sterilizing liquid.
The invention has the following good technical effects:
1. the invention utilizes the constitutive promoter to ensure that the high-yield Klebsiella alcohol can stably express luciferase.
2. The invention uses the electrotransformation method to prepare the competent cell, has simple and convenient operation and easy obtaining, and has high transformation efficiency. Preferably, the LUX A/B/C/D/E gene cluster is amplified by using high-fidelity enzyme, and the amplification efficiency is highest when the annealing temperature is 1-3 ℃ lower than the recommended temperature. And the Gibson connection method is used, so that the connection efficiency is improved.
3. Luciferase is converted into the high-yield Klebsiella alcoholic beverage by a pan-host plasmid for the first time by an electric conversion method, so that the bioluminescent genetically modified high-yield Klebsiella alcoholic beverage is obtained, and various performances of the high-yield Klebsiella alcoholic beverage, including various performances such as growth, in vivo distribution and the like, can be conveniently observed;
4. the high-yield Klebsiella alcoholic beverage transformed by the luciferase-expressing gene can conveniently, quickly and intuitively screen appropriate drugs, such as various antibiotics, so that the patient can quickly find the appropriate antibiotics or other therapeutic drugs to recover the patient early.
5. The high-yield Klebsiella alcoholic beverage modified by the luciferase-expressing gene is screened to a proper sterilization method, such as ultraviolet rays, 84 disinfectant and the like, so that medical articles can be effectively sterilized, and nosocomial infection is prevented;
6. the sterilization effect of the high-yield Klebsiella alcohol is visually observed, the environment is clinically and reasonably sterilized, a basis is provided for adopting more intervention measures such as contact prevention, patient isolation, environment cleaning and the like, and the method has important significance for preventing and controlling nosocomial infection;
7. the method has the advantages that the distribution condition of the high-yield Klebsiella in the experimental animal body can be visually tracked, and the method has important guiding significance for optimizing the use scheme of the antibacterial drugs.
The foregoing is merely a summary of aspects of the invention and is not, and should not be taken as, limiting the invention in any way. All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention. The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.
Drawings
FIG. 1: schematic diagram of plasmid construction of pBBR1-PJ-LUX A/B/C/D/E, wherein A is schematic diagram of plasmid construction of pBBR1-LUX A/B/C/D/E, above: PCR linear amplification of the LUX A/B/C/D/E gene group, as follows: the pBBR1 plasmid was digested twice with KpnI/HindIII. B is a schematic diagram of the transformation of pBBR1-LUX A/B/C/D/E plasmid into Escherichia coli.
FIG. 2: fluorescence intensity of different passaged strains, P3-P10 sequentially represents the third generation to the tenth generation.
FIG. 3: the Luminescence fluorescence intensity of the genetically modified high-yield Klebsiella pneumoniae with stably expressed luciferase at different concentrations.
Detailed Description
The present invention will be described in detail below by way of preparation examples and experimental examples to make the features and advantages of the present invention clearer. It should be noted, however, that the preparation and test examples are for understanding the concept of the present invention, and the scope of the present invention is not limited to only the preparation examples and the test examples listed herein.
The experimental procedures used in the following preparations and experimental examples were all conventional ones unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.
1. Test materials:
coli (e.coli) DH5a competent cells, a plasmid mini-cartridge was purchased from tiangen biochemistry technologies, ltd. The gel recovery kit was purchased from QIAGEN, Germany. High fidelity DNA polymerase Q5, high fidelity DNA ligase, KpnI, HindIII restriction enzymes were purchased from NEB Inc. of USA. The pBBR1MCS-LUX plasmid was constructed by the applicant.
2. A detection instrument:
veritas microplate photometers (Turner BioSystems), NanoDropTM One ultramicro UV spectrophotometers (Thermo), IVIS Lumina LT small animal Living imager (Perkin Elmer).
3. High-yield wine klebsiella collection and preparation
The high-yield Klebsiella alcoholic beverage is provided for research and collection of the capital department. The high-yield klebsiella wine is identified as the high-yield klebsiella wine sensitive to chloramphenicol by performing bacteria identification and drug sensitivity test by a full-automatic microbial analyzer (American BD) (the judgment of drug sensitivity result refers to the recommended standard of the American clinical laboratory standardization research institute).
4. Statistical analysis:
statistical analysis was performed using GraphPad prism7.0 software, and fluorescence data were examined for normality using Kolmogorov-Simrnov. The fluorescence signal detection data of the genetically modified high-yield Klebsiella alcoholic beverage expressing luciferase is in a skewed distribution, the fluorescence value is expressed by a median, and Wilcoxon rank sum test is adopted between the two groups. The detection data of the light signals of the genetically modified high-yield Klebsiella alcoholic beverage with different concentrations of luciferase are in a skewed distribution, the fluorescence value is expressed by a mean value, and the OneWay anova test is adopted. The difference is statistically significant when P is less than 0.05.
EXAMPLE 1 construction of a Pan-host plasmid containing the luciferase Gene
Amplification of the LUXA/B/C/D/E gene cluster:
(1) primer pair for designing LUX gene cluster containing strong promoter
LUX F：
agggaacaaaagctgggtacTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGCGGATAACAATTACTAGAG (SEQ ID NO.2, underlined sequence in italics is PJ23119 promoter sequence);
LUX R: gctgcaggaattcgatatcaATTTGTCCTACTCAGGAGAG (SEQ ID NO.3) the lower case bold sequence of the primer is the sequence complementary to pBBR 1.
The LUX A/B/C/D/E gene cluster in the pBBR1-LUX plasmid is controlled by a T5 promoter, and experimental verification results show that the LUX A/B/C/D/E gene cluster is not expressed under the control of the T5 promoter, so that a PJ23119 Promoter (PJ)) is inserted. We amplified the LUXA/B/C/D/E group of pBBR1-LUX plasmid using Q5 high fidelity DNA polymerase and a primer pair containing the LUX gene cluster, with the addition of the same sequence as pBBR1 during primer design (shown in FIG. 1A).
(2) LUX A/B/C/D/E gene cluster PCR amplification
Table 1: LUX A/B/C/D/E gene cluster amplification PCR reaction system
After the reaction system is prepared, PCR reaction is carried out, and the PCR program is set as shown in Table 2:
table 2: PCR program set-up
When the program is carried out to 4 ℃, the reaction product is taken out, and the subsequent PCR product identification is carried out after the PCR instrument is closed.
The LUX A/B/C/D/E gene cluster is amplified by using high-fidelity enzyme, the annealing temperature (62 ℃) of the high-fidelity enzyme can effectively amplify the LUX A/B/C/D/E gene cluster at the recommended temperature, and further, the applicant finds that the annealing temperature is lower than the recommended temperature by 1-3 ℃ (namely 59-61 ℃) through gradient PCR detection, and the amplification efficiency can be further improved by 20%. This example uses a 60 ℃ anneal.
(3) Agarose gel electrophoresis:
1) preparation of an appropriate concentration (1.5%) agarose gel: 1.5g of agarose (Gene Star) was weighed and dissolved in 100ml of TAE, and the mixture was heated in a microwave oven until the agarose was completely dissolved, and the solution was clear and transparent. Cooling is carried out at 40-50 ℃ and 10. mu.l of nucleic acid dye (Gene star) are added. And pouring the mixture into an assembled glue groove after uniformly mixing, and standing the mixture for 30 minutes at room temperature.
2) Loading after assembling the electrophoresis device: DNA Ladder (Gene Star) and PCR products (0.66. mu.l of 10 × loading buffer, if necessary) were added to each well, followed by electrophoresis at 120V for 10 minutes at constant pressure. And (3) observing whether the PCR strip is positive under an ultraviolet gel imager, comparing the position of the positive strip with the DNA Ladder, and identifying the strip as positive when the molecular weight of the strip is consistent with that of the target DNA (about 5000 bp).
(4) PCR product sequencing and analysis:
gel recovery of PCR products: collection of DNA of the correct size: the DNA fragment of interest was cut with a clean blade under an ultraviolet lamp and weighed, and the cut gel was collected in a 1.5ml centrifuge tube. DNA gel recovery and purification kit is used. Add 300. mu.l sol buffer to 100. mu.g gel, incubate with shaking at 50 ℃ and add isopropanol (100. mu.l per 100. mu.g gel) after the gel is observed to be completely molten. Transferring 600 μ l of the above liquid to a centrifugal column, centrifuging at 12000rpm for 1 min, discarding waste liquid in the casing, inserting the centrifugal column into the casing, and adding the residual liquid to the same centrifugal column if the liquid is residual, and repeating the steps. Adding 500 mul of cleaning solution into the centrifugal column, centrifuging at 12000rpm for 1 minute, and discarding the waste liquid in the sleeve. The spin column was carefully removed, a new tube was inserted, centrifuged at 12000rpm for 1 minute, then the lid was opened and left to stand at room temperature for two minutes to remove residual isopropanol H2O. The column was placed in a new 1.5ml centrifuge tube, 50. mu.l of nuclease-free water was added thereto, and after standing at room temperature for 2 minutes, it was centrifuged at 12000rpm for 1 minute, and the recovered DNA was collected. The recovered DNA was labeled and sent to sequencing company (Huada Gene) for sequencing for one generation. And comparing the sequencing result with the pBAV1k-T5-LUX plasmid, and analyzing, wherein the sequencing result shows that the amplified fragment is a LUX gene group, and carrying out subsequent experiments.
pBBR1MCS-1 double digestion:
pBBR1 is a 4707bp gram-negative ubiquitous host plasmid carrying the Chloramphenicol resistance gene (cmR), pBBR1 replication origin (pBBR1ori), pBBR1(pBBR1rep) regulatory factors. 3321/3281bp of the plasmid has single restriction sites of HindIII and KpnI restriction enzymes, and a linearized PBBR1 plasmid is obtained by adopting a HindIII/KpnI restriction enzyme digestion method. The schematic diagram is shown in fig. 1. a. The HindIII/KpnI cleavage system of the pBBR1MCS-1 plasmid is shown in Table 3.
Table 3: HindIII/KpnI enzyme digestion system of pBBR1MCS-1 plasmid
3. And (3) recovering the enzyme digestion fragment gel, and connecting the enzyme digestion fragment gel with the amplified LUXA/B/C/D/E gene cluster by using a Gibson connection method:
after the two linearized fragments were identified and gel recovered, they were ligated using 2 XNEBuilder HiFi DNAassembly ligase, and the successfully ligated circular plasmid product was named pBBR1-lux, as shown in FIG. 1. B. pBBR1MCS-1HindIII/KpnI restriction sites are connected with LUXA/B/C/D/E gene cluster system as shown in Table 4:
table 4: pBBR1MCS-1HindIII/KpnI restriction site connected with LUXA/B/C/D/E gene cluster system (20 μ l)
4. The product was transformed into E.coli competent cells:
and electrically transforming the connected product to escherichia coli DH5a competent cells, screening positive bacteria, amplifying the bacteria, extracting plasmids, performing first-generation sequencing identification, and identifying the correctly sequenced plasmid to be named as pBBR 1-PJ-LUX. Coli containing pBBR1-PJ-LUX was collected and stored in a freezer at-70 ℃.
Example 2 detection of luciferin Gene expression and fluorescence stability in genetically engineered high yielding Klebsiella vinifera containing luciferase.
1. High-yield wine Klebsiella electrotransformation competence preparation
And (3) selecting a single colony of the high-yield Klebsiella alcohol to inoculate the single colony in a liquid BHI culture medium, and standing and culturing the colony overnight at 37 ℃. Inoculating overnight-cultured high-yield Klebsiella alcoholic strain liquid into fresh BHI culture medium at a ratio of 1:10, and standing at 37 deg.C for culture to OD6000.3 to 0.4. Taking out the logarithmic phase bacterial liquid, carrying out ice bath for 30min, centrifuging at 4 ℃ and 4000rcf, collecting thalli, and washing the thalli with precooled sterile double distilled water and precooled 1% sucrose solution respectively to obtain the microbial inoculumHigh-yield Klebsiella alcohol sensitive cells.
Electrotransformation of pBBR1-LUX and pBBR1-PJ-LUX plasmids:
5 ul of plasmid pBBR1-LUX and pBBR1-PJ-LUX were added to 100 ul of high-yielding Klebsiella alcoholic competent cells, mixed well and ice-cooled for 10 minutes. The mixture was transferred to a pre-cooled 0.1cm electric shock cup (berle, usa) at electric field strength: 22.5 kV/mm; resistance: 200 omega; capacitance: electrotransformation was carried out at 25. mu.F. Immediately after the completion of the electric shock, 950. mu.l of BHI medium preheated at 37 ℃ was added to the cuvette, and the cuvette was subjected to static culture at 37 ℃ for 1 hour and then spread on a Columbia blood plate medium containing 25. mu.g/ml chloramphenicol. Culturing at 37 deg.C for 24h, and selecting positive colony for identification.
The results showed that pBBR1-LUX transformed high yielding Klebsiella alcoholic had chloramphenicol resistance but no luminence signal. Whereas chloramphenicol resistant pBBR1-PJ-LUX strains have a Luminence signal.
After the pBBR 1-PJ-LUX-transformed positive colony with a Luminescence signal is cultured for the third generation, the Luminescence value is detected by using a Veritas microplate photometer, and the result shows that the genetically modified high-yield Klebsiella inescense after the third generation culture still has the Luminescence signal, so the colony is named as the genetically modified high-yield Klebsiella inescense stably expressing the luciferase gene (HiAlc Kpn-pBBR1-PJ-LUX A/B/C/D/E).
3. The stability detection of the genetically modified high-yield Klebsiella alcoholic beverage with stably expressed luciferase gene comprises the following steps:
positive colonies were frozen at-70 ℃. After 7 days, a single colony obtained by streaking recovery was a third generation strain (P3), and P3 was transformed into a strain of 1: 100 dilution and incubation to log phase (OD)6000.5-0.8) was obtained as a fourth generation strain (P4). And the like to obtain strains P5, P6, P7, P8, P9 and P10. The fluorescence intensity of the above-mentioned strain was measured after dilution to OD600 of 1, and the results are shown in FIG. 2, and the colonies of the present invention were stably passaged until the tenth passage, and the fluorescence value was maintained at about 10000/1 OD.
Example 3: gene modified Klebsiella alcohol-producing luciferase strength detection method for stably expressing luciferase with different concentrations
A genetically modified Klebsiella clavulans single colony strain stably expressing luciferase is picked, cultured at 37 ℃ for 4 hours, and then detected by a NanoDropTM One ultramicro ultraviolet spectrophotometer to determine the OD value of the bacterium. Adjusting the concentration OD of the strain by dilution 6002. The obtained OD600Strain 2 was as follows 1: the luminence values were measured on a microplate reader at 2 dilutions on 9 gradients, with the results shown in figure 3, where series 1 is: the high-yield wine pneumonia Klebsiella with different concentrations transformed into pBBR1-LUX plasmid, the high-yield wine pneumonia Klebsiella with gene modification transformed into pBBR1-PJ-LUX plasmid in series 2, the fluorescence value is logarithm of series 2. And (3) displaying data: the luminence value decreased with decreasing colony concentration, with the trend of decreasing in a logarithmic manner. The fitting curve is as follows: y ═ 1E +05ln (x) +235135R2 ═ 0.96. Meanwhile, it can be observed that the luminence value decreases with the decrease of the colony concentration when the strain concentration is 106Cfu and 104Cfu, the fluorescence intensities are 2353 and 65, respectively, which are less than 105Cfu, the fluorescence of the strain cannot be detected in a small animal imager, so that the enrichment concentration of the strain in animals cannot be less than 10 when the strain is recommended to be used for animal experiments5Cfu/mL。
When the OD value of the strain is between 0.125 and 1 (the bacteria are in the logarithmic growth phase), the Luminescence value decreases with the decrease of the colony concentration, and the decrease trend conforms to the linear decrease form. The fitting curve is as follows: y-28009 x +155158R 2-0.9558.
Based on the above results and the lowest value of fluorescence detected in the small animal imager, the optimal concentration of inoculated bacteria for the preparation of animal infection models can be calculated.
Example 4: verification of sterilization effect of high-yield Klebsiella vinifera stably expressing luciferase by different treatment modes
Different physical and chemical methods are adopted for treatment. The physical method comprises the following steps: the gene modified high-yield Klebsiella alcoholic beverage for stably expressing luciferase is streaked at three regions by using an inoculating loop (1 μ l), and is applied in a box at 37 ℃ for culturing for 2h, and is irradiated by ultraviolet for 30 min. The chemical method comprises the following steps: alcohol (75%), 84 disinfectant (10%), H2O2(30%) and NaOH (0.4%, 4%) were soaked separatelyAnd inoculating the brevibacterium (1ul) for 30min, and respectively picking 1 mu l of genetically modified high-yield klebsiella wine stably expressing luciferase for three-region lineation. After the treated genetically modified high-yield klebsiella wine is cultured for 8 hours at 37 ℃ on the plate, the fluorescence of the genetically modified high-yield klebsiella wine for stably expressing luciferase is observed by using a small animal imager.
The results show that the ultraviolet irradiation is more than 30min, or the sterilizing effect of 84% disinfectant on the articles is best.
Example 5: gene modified high-yield Klebsiella alcoholic beverage for stably expressing luciferase and used for screening antibiotics
We have devised different antibiotic sterilization protocols. Firstly, the pH value and the oxygen content of the infection environment are simulated, and whether the infection environment contains cell factors and the like can simulate the environments of sputum, blood, urine, excrement and the like. Different antibiotic concentrations were tested at specific bactericidal effects on the bacteria. Different antibiotic use concentrations are preferred.
For multi-drug resistant bacteria, different antibiotic ratios are preferably used in combination under specific conditions according to clinical drug resistant bacteria treatment schemes.
Example 6: hialc Kpn-pBBR1-PJ-LUX A/B/C/D/E for preparing infection animal model
The genetically modified high-yielding klebsiella alcoholic were diluted to the corresponding concentrations according to the concentrations recommended for establishing animal infection models in example 3. The model animals are infected by modes of gavage, nasal feeding, tail vein injection or intraperitoneal injection and the like. And observing the bacterial spreading path or the colonization position on a small animal imager.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Sequence listing
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Claims (10)
1.A plasmid comprising a constitutive promoter capable of promoting expression of the LUX a/B/C/D/E gene in klebsiella vinifera and the luciferase gene cluster LUX a/B/C/D/E.
2. A genetically modified high-yield Klebsiella alcoholic beverage is characterized in that the genetically modified high-yield Klebsiella alcoholic beverage can stably express luciferase.
3. A construction method of genetically modified high-yield Klebsiella alcoholic beverage is characterized by comprising the following steps:
(1) preparing a plasmid comprising the LUX A/B/C/D/E gene cluster, preferably said plasmid further comprising a constitutive promoter;
(2) preparing electrotransformation competent high-yield Klebsiella alcoholic beverage;
(3) and (3) electrically transforming the plasmid obtained in the step (1) into the competent high-yield klebsiella alcoholic beverage obtained in the step (2), and selecting a positive bacterial colony to obtain the genetically modified high-yield klebsiella alcoholic beverage.
4. The method of claim 3, wherein the plasmid is a pan-host plasmid comprising a constitutive promoter, preferably the constitutive promoter is PJ23119 or CaMV35S, P32 promoter.
5. Use of the genetically engineered klebsiella vinifera of claim 2 in an evaluation method for calculating bacterial concentration, in a method for screening for sterilization, in a method for screening for drugs, or in the preparation of animal models.
6. An evaluation method for calculating a concentration of bacteria, the evaluation method comprising: (1) culturing the genetically modified high-yielding klebsiella alcoholic beverage of claim 2, (2) adjusting the concentration of the strain by a dilution method, and calculating the concentration of the genetically modified high-yielding klebsiella alcoholic beverage by detecting a fluorescence value, and (3) drawing a standard curve of the fluorescence values of the genetically modified high-yielding klebsiella alcoholic beverage with different concentrations.
7. An evaluation method of a sterilization method, wherein the evaluation method comprises evaluating the sterilization method by using the genetically modified high-yielding klebsiella alcoholic beverage of claim 2, preferably, the sterilization method comprises sterilizing an article carrying the high-yielding klebsiella alcoholic beverage by using ultraviolet irradiation, alcohol, NaOH or 84 sterilizing solution, more preferably, the sterilization method comprises ultraviolet irradiation for more than 30min, or sterilizing the article by using 10% of 84 sterilizing solution.
8. A drug screening method comprising screening a drug using the genetically modified high-yielding Klebsiella alcoholic beverage of claim 2.
9. A method for preparing an animal model, comprising injecting the genetically modified Klebsiella into an animal by nasal feeding, gavage, intraperitoneal injection, tail vein injection, etc.
10. The sterilization method of the high-yield Klebsiella alcoholic beverage is characterized by comprising the step of sterilizing an article carrying the high-yield Klebsiella alcoholic beverage by using ultraviolet irradiation, alcohol, NaOH or 84 disinfectant, preferably, the sterilization method comprises the step of irradiating the article for more than 30min by using ultraviolet irradiation, or sterilizing the article by using 10% of 84 disinfectant.
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| CN202010475653.0A Pending CN111607550A (en) | 2020-05-29 | 2020-05-29 | High-yield Klebsiella alcoholic beverage expressing luciferase and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112481182A (en) * | 2020-12-07 | 2021-03-12 | 首都医科大学附属北京地坛医院 | VRE expressing luciferase and application thereof |
| EP3988652A1 (en) * | 2020-10-23 | 2022-04-27 | Aden Hygiene, Ent. | Biological indicator for rapid verification of disinfection or sterilization |
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2020
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3988652A1 (en) * | 2020-10-23 | 2022-04-27 | Aden Hygiene, Ent. | Biological indicator for rapid verification of disinfection or sterilization |
| CN112481182A (en) * | 2020-12-07 | 2021-03-12 | 首都医科大学附属北京地坛医院 | VRE expressing luciferase and application thereof |
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