Plasmid for fluorescent labeling of bacterial outer membrane vesicle, and preparation method therefor

Abstract

A plasmid capable of automatically inserting a membrane-localized luciferase reporter gene into a chromosome of a bacterium / recipient bacterium. After the plasmid inserts a luciferase reporter gene into a chromosome of a bacterium / recipient bacterium, the plasmid can be lost. Since the plasmid can be lost, the bacterium / recipient bacterium into whose chromosome the luciferase reporter gene is inserted does not require the addition of antibiotics during subsequent use, providing convenience and reducing interference.

Description

A plasmid for fluorescent labeling bacterial outer membrane vesicles and its preparation method Technical Field

[0001] This invention belongs to the fields of medicine and biotechnology. It utilizes the characteristic of bacterial outer membrane vesicles carrying high abundance of bacterial outer membrane protein A (OmpA) to construct an OmpA-NanoLuc luciferase fusion protein and integrate the gene expressing the OmpA-NanoLuc luciferase fusion protein into the host bacterial genome. Background Technology

[0002] Bacterial outer membrane vesicles (OMVs) are small, spherical membrane structures released from the bacterial outer membrane. They are generally believed to be formed by the shedding of the bacterial outer membrane and typically range in diameter from 20 to 400 nanometers. Their contents include lipids, proteins, and nucleic acids.

[0003] Bacterial outer membrane vesicles serve multiple functions in bacterial biology. First, they act as a tool for interbacterial signaling, regulating bacterial growth, metabolism, and biological behavior. Second, they can be used as a means of molecular transfer between bacteria and host cells. Third, they are believed to activate the host immune system and participate in the host's immune response to bacterial infection. Furthermore, as nanoscale particles, they possess numerous potential applications in medicine and biotechnology, including drug delivery systems, vaccine candidates, and biomarkers, contributing to the development of new treatments, diagnostic tools, and biotechnological applications. Therefore, research on bacterial outer membrane vesicles is crucial for a deeper understanding of bacterial biology, immunology, antibiotic resistance, and drug delivery, attracting the attention of numerous scientists and researchers. Technical issues

[0004] Luciferase proteins catalyze the emission of light from substrates, allowing them to be recognized by imaging systems. They are commonly used in in vivo animal imaging experiments of bacteria. However, the original luciferase protein is often located inside the bacteria, causing the entire bacterium to glow. This doesn't guarantee that it's also located inside the bacterial outer membrane vesicles, thus limiting its application in in vivo animal localization experiments of bacterial outer membrane vesicles. Furthermore, when using bacterial outer membrane vesicles in in vivo experiments, the most common method to observe their targeting in animal organs is to label them with fluorescent dyes such as Cy5, followed by imaging to observe their target location. However, labeling bacterial outer membrane vesicles with fluorescent dyes presents several problems: 1. The added dye undoubtedly affects the membrane structure of the outer membrane vesicles, impairing their permeability and cell membrane integrity. 2. If handled improperly, such as using high concentrations of dye or prolonged exposure, Cy5 dye may have adverse effects on cell surface proteins. This could include non-specific binding of the dye to proteins or toxic effects of the dye, ultimately leading to changes in the surface structure of the bacterial outer membrane vesicle membrane. 3. When using fluorescent dyes in animals, it is necessary to consider the dye's chemical properties, dosage, concentration, route of entry, and timing to minimize toxicity to the experimental animals. 4. Cy5 dyes are relatively expensive, especially for large-scale applications or experiments requiring large quantities of dye; cost is a significant factor.

[0005] The previous article, "Establishment of a Bacterial Outer Membrane Vesicle Labeling Method Based on Luciferase Reporter Gene" (Journal of Clinical Laboratory Medicine, October 2019, Vol. 37, No. 10), disclosed a bacterial outer membrane vesicle labeling method based on luciferase reporter gene. However, this method can only be successfully expressed in protein-expressing strains such as BL21(DE3) and Rosetta(DE3), which has certain limitations. Furthermore, the applicant found in subsequent studies that the artificially designed plasmid containing the luciferase reporter gene must be present in this method. In order to prevent plasmid loss, antibiotics need to be used continuously, which will interfere with subsequent experiments and is inconvenient.

[0006] In view of this, the present invention is hereby proposed. Technical solutions

[0007] The purpose of this invention is to enable the luciferase protein to be localized on the outer membrane vesicles. Bacterial outer membrane vesicles all carry luminescent proteins, which can then be imaged and observed. Furthermore, the luciferase protein gene is integrated into the bacterial chromosome, allowing bacteria to continuously express the OmpA-NanoLuc luciferase fusion protein. Subsequent experiments do not require antibiotics and will not interfere with subsequent experiments.

[0008] The technical solution adopted by the present invention to solve its technical problem is: to provide a plasmid for fluorescent labeling of bacterial outer membrane vesicles, wherein the plasmid contains a recombinant protein DNA sequence as shown in SEQ ID NO: 1.

[0009] In an optional embodiment of the present invention, the plasmid comprises a Tn7 transposon and a tnsABCD transposase.

[0010] In an optional embodiment of the present invention, the plasmid is a pGRG25 vector, and the MCS region contains a recombinant protein DNA sequence as shown in SEQ ID NO: 1.

[0011] The present invention further provides a method for preparing bacterial outer membrane vesicles labeled with luciferase reporter gene, comprising the following steps:

[0012] A) Constructing the plasmids of this invention;

[0013] B) Transform the plasmid into the S17-1λpir strain;

[0014] C) Plasmids are transferred to recipient bacteria via conjugation transfer, the recipient bacteria are cultured, the plasmids are lost, and bacterial outer membrane vesicles are extracted.

[0015] In this invention, the recipient bacteria may be bacteria containing the glmS gene, preferably Escherichia coli prokaryotic expression strain BL21DE3.

[0016] As described above, step A is constructed by enzyme digestion and ligation, wherein the enzyme digestion reaction system consists of: 1 µg restriction endonuclease, 1 µl DNA fragment, 5 µl 10*NE Buffer, and 43 µl H2O, and the reaction is carried out at 37°C for 1 hour.

[0017] As described above, the reaction system for the ligation consists of: 1.0 μl Blunt Enzyme Mix, 2.5 μl 1 mM dNTP Mix, 2.5 μl 10x Blunting Buffer, and 19 μl Purified DNA, reacted at room temperature for 15 minutes.

[0018] The method described above, wherein the conjugation transfer plasmid to the recipient bacterium includes the following steps:

[0019] (1) After the S17-1λpir strain and the recipient strain were revived, they were cultured overnight at 37°C in 5 ml LB liquid medium until they reached the logarithmic phase;

[0020] (2) Centrifuge and collect the bacterial cells. Wash the donor bacteria twice with 2 ml of physiological saline.

[0021] (3) Resuspend both in 200 μl LB liquid medium and mix them into one EP tube, then incubate at 37°C for 3 hours;

[0022] (4) Dilute the LB liquid medium to 1 ml again, and take 20 μl to spread on a selective solid medium designed based on the recipient bacteria;

[0023] (5) Incubate overnight at 37°C to obtain a single clone, and incubate at 42°C to lose the plasmid.

[0024] In this invention, the plasmid loss method is as follows: bacteria are plated at 42°C, and single colonies grow the next day. A single colony is picked and cultured in LB broth with shaking. After 6-8 hours of incubation, a small amount of the bacterial solution is taken for PCR amplification verification. If the plasmid is still present, a band will be produced after PCR, indicating that plasmid removal was unsuccessful. If no band is produced after PCR, it indicates that plasmid loss was successful.

[0025] The method described above, wherein the extraction of bacterial outer membrane vesicles includes the following steps:

[0026] (a) After centrifugation at 11000g for 5 min, the supernatant was collected, then filtered through a 0.45μm filter membrane and the flow-through liquid was collected;

[0027] (b) Centrifuge at 130,000g for 70 min, discard the supernatant, add 500 μL PBS and resuspend by pipetting to complete the extraction of bacterial outer membrane vesicles.

[0028] The present invention further provides a recipient bacterium, wherein the recipient bacterium is a recipient bacterium that has undergone plasmid transfer via conjugation transfer as described above.

[0029] The present invention further provides a recipient bacterium, wherein a membrane-localized luciferase reporter gene is inserted into the chromosome of the recipient bacterium; in the present invention, the membrane-localized luciferase reporter gene may be the recombinant protein DNA sequence shown in SEQ ID NO: 1.

[0030] The present invention further provides a bacterial outer membrane vesicle, which is prepared by the preparation method described above.

[0031] The present invention further provides an application of the extracellular membrane vesicles described above in diagnosis or detection. Beneficial effects

[0032] The advantages and positive effects of this invention are as follows:

[0033] 1. Fluorescent labeling of bacterial outer membrane vesicles can be easily achieved without any dyes.

[0034] 2. Because the bacteria emit their own light rather than through the action of dyes, it has excellent biocompatibility, preserves the original state of the bacterial outer membrane vesicles to the greatest extent, and avoids cytotoxicity in animal experiments.

[0035] 3. Since the glmS gene is widely present in bacteria, most bacteria can use this plasmid to label their outer membrane vesicles and conduct subsequent experiments.

[0036] 4. Conventionally, inserting a DNA segment into bacteria to perform its function often requires the use of plasmids. To prevent plasmid loss after entry into bacteria, or to ensure that all bacteria carry the plasmid, a specific antibiotic resistance gene needs to be encoded on the plasmid. This gives the bacteria that acquire the plasmid a resistance to a previously undeveloped antibiotic. When the corresponding antibiotic is added during bacterial culture, if the bacteria survive, it indicates that they carry the plasmid, allowing for subsequent experiments. Of course, all subsequent experiments also require the addition of antibiotics to ensure that all bacteria carry the plasmid. However, the plasmid based on Tn7 transposon technology in this invention can insert a membrane-localized luciferase reporter gene into the bacterial chromosome. After insertion, the plasmid is no longer needed, preventing plasmid loss. Subsequent experiments do not require the addition of antibiotics, resulting in less experimental interference and greater convenience. Attached Figure Description

[0037] Figure 1: pGRG25 temperature-sensitive Tn7 transposon vector. pGRG25 is available on GenBank (accession number DQ460223). The best embodiment of the present invention

[0038] To better understand the present invention, the present invention will be further described in detail below with reference to the embodiments. However, the scope of protection of the present invention is not limited to the scope represented by the embodiments.

[0039] Unless otherwise specified, all raw materials used in this invention are conventional commercially available products. Unless otherwise specified, all methods used in this invention are conventional methods in the field. All substances used in this invention are of conventional usage quality.

[0040] Example 1: Cloning of recombinant plasmids

[0041] The pGRG25 plasmid, preserved in the laboratory, was transformed into DH5α chemocompetent cells and amplified by low-temperature culture at 30℃. The plasmid was then extracted using a plasmid miniprep kit to obtain sufficient pGRG25 plasmid for the cloning step. After designing the recombinant protein DNA sequence, NotI and XhoI restriction sites were added to the 5' and 3' ends of the recombinant protein DNA sequence, respectively, based on the MCS site information of pGRG25, for subsequent cloning experiments. The recombinant protein DNA synthesis was performed by GenScript Biotech Co., Ltd. Cloning was performed using an enzyme digestion-ligation method to construct the recombinant plasmid. Restriction endonucleases were purchased from NEB. The reaction system consisted of 1 µg restriction endonuclease, 1 µl DNA fragment, 5 µl 10*NEBuffer, and 43 µl H2O, and was incubated at 37℃ for 1 h. After digestion, the digested products were purified using a PCR product purification kit (Omega).

[0042] The ligation of the recombinant protein DNA sequence to the plasmid was performed using the Quick Ligation™ Kit (NEB). The reaction mixture consisted of 1.0 μl Blunt Enzyme Mix, 2.5 μl 1 mM dNTP Mix, 2.5 μl 10X Blunting Buffer, and 19 μl Purified DNA. The mixture was reacted at room temperature for 15 minutes to complete the ligation.

[0043] The recombinant protein DNA sequence (SEQ ID NO: 1) is as follows:

[0044] Embodiments of the present invention

[0045] Example 2: Amplification and Enzyme Digestion Identification of Transformed DH5α Strains

[0046] After the DNA fragment was ligated to the recombinant plasmid, the ligation product was immediately added to DH5α chemocompetent cells, placed on ice for 30 minutes, heat-shocked at 42°C for 45 seconds, and then incubated in LB liquid medium containing 0.1% glucose at 37°C for 1 hour to recover. The cells were then plated on LB solid medium containing 100 µg / mL ampicillin and incubated overnight at 35°C. The next day, after single clones grew, clones were selected for PCR amplification and agarose gel electrophoresis. The size of the electrophoretic bands was consistent with expectations.

[0047] The clone quality was verified by sequencing. After verification, the DH5α strain containing the recombinant plasmid was amplified again using solid culture medium and stored at -80℃ for later use.

[0048] Example 3: Conjugation transfer delivery of recombinant plasmids

[0049] Following the method in Example 2, the DNA fragment and recombinant plasmid were ligated beforehand and then transferred into the donor bacteria (S17-1λpir). After recovery, both the donor bacteria (S17-1λpir) and the recipient bacteria (Escherichia coli prokaryotic expression strain BL21DE3) were cultured overnight at 37°C in 5 mL LB liquid medium. Antibiotics and 0.1% glucose were added to the donor bacteria according to the plasmid characteristics, and the culture was allowed to reach the logarithmic growth phase. The bacteria were centrifuged and collected. The donor bacteria were washed twice with 2 mL of physiological saline to remove antibiotics and glucose. Both bacteria were resuspended in 200 μL LB liquid medium and mixed into an EP tube, incubated at 37°C for 3 hours. The culture was then diluted again to 1 mL with LB liquid medium, and 20 μL was spread onto a selective solid medium designed based on the recipient bacteria. The culture was incubated overnight at 37°C to obtain a single clone. Plasmid loss was achieved at least once at 42°C. pGRG25 is a temperature-sensitive plasmid, and the plasmid loss was confirmed by PCR amplification.

[0050] The plasmid loss method is as follows: Bacteria are plated at 42°C, and single colonies grow the following day. A single colony is picked and cultured in LB broth with shaking. After 6-8 hours of incubation, a small amount of the bacterial solution is taken for PCR amplification verification. If the plasmid is still present, a band will be produced after PCR, indicating that plasmid removal was unsuccessful. If no band is produced after PCR, the plasmid loss was successful.

[0051] Example 4: Extraction of bacterial outer membrane vesicles

[0052] The recipient bacteria (Escherichia coli prokaryotic expression strain BL21DE3) were cultured in LB liquid medium at 37°C. The bacterial culture was collected the next day, centrifuged at 11,000g for 5 min, and the supernatant was collected. The supernatant was then filtered through a 0.45 μm filter membrane and the flow-through was collected. Subsequently, the culture was ultracentrifuged at 130,000g for 70 min, the supernatant was discarded, and the bacteria were resuspended in 500 μL of PBS by pipetting to complete the extraction of bacterial outer membrane vesicles.

[0053] The Nano Glo Luciferase Assay kit (N1110) was used to detect NanoLuc luciferase activity in bacterial outer membrane vesicles. Before the assay, the chromogenic substrate and reaction buffer were diluted 1:50 and 100 μL was added to each well of a white 96-well plate. During the assay, 5 μL of the sample was added, and the bioluminescence intensity was measured using the "Luminescence" channel of a multi-mode microplate reader. The luciferase activity in the sample was then recorded.

[0054] The fluorescence intensity of the supernatant from the bacterial culture was 0.4*10. 5RLU / μL. The luciferase in the sample accumulated at the bottom of the centrifuge tube after ultracentrifugation, and the fluorescence intensity of the precipitate at the bottom after ultracentrifugation was 6*10. 5 RLU / μL, the fluorescence intensity of the supernatant after ultracentrifugation was 0.04*10. 5 RLU / μL, consistent with the enrichment process of bacterial outer membrane vesicles, indicates that the recombinant protein has been successfully localized on the surface of the outer membrane vesicles, which can effectively label bacterial outer membrane vesicles. Furthermore, the amount and proportion of the outer membrane vesicles remaining in the supernatant after centrifugation of the present invention are lower, and more are enriched in the precipitate.

[0055] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A plasmid for fluorescent labeling of bacterial outer membrane vesicles, characterized in that, The plasmid comprises a recombinant protein DNA sequence as shown in SEQ ID NO:

1.

2. The plasmid according to claim 1, characterized in that, The plasmid comprises a Tn7 transposon and tnsABCD transposase.

3. The plasmid according to claim 1 or 2, characterized in that, The plasmid is a pGRG25 vector, and the MCS region comprises a recombinant protein DNA sequence as shown in SEQ ID NO:

1.

4. A method of preparing a luciferase reporter labeled bacterial outer membrane vesicle, characterized in that, The method comprises the following steps: A) constructing the plasmid as claimed in claims 1-3; B) transforming the plasmid into a S17-1 lambda pir strain; C) transferring the plasmid to a recipient bacterium by conjugation, culturing the recipient bacterium, losing the plasmid, and extracting bacterial outer membrane vesicles.

5. The method of claim 4, wherein, The step A is constructed by enzyme digestion and ligation, wherein the enzyme digestion reaction system is: 1 μg restriction enzyme, 1 μl DNA fragment, 5 μl 10*NEBuffer, 43 μl H2O, and the reaction is carried out at 37°C for 1 hour.

6. The method of claim 5, wherein, The ligation reaction system is: 1.0 μl Blunt Enzyme Mix, 2.5 μl 1 mM dNTP Mix, 2.5 μl 10x Blunting Buffer, 19 μl Purified DNA, and the reaction is carried out at room temperature for 15 minutes.

7. The method of claim 4, wherein, The step C of transferring the plasmid to a recipient bacterium by conjugation comprises the following steps: (1) after recovering the S17-1 lambda pir strain and the recipient bacterium, respectively, using LB liquid medium to culture overnight at 37°C, reaching the logarithmic phase; (2) centrifuging and collecting the bacterial cells, and using physiological saline to wash the donor bacteria; (3) respectively resuspending them using LB liquid medium, and conjugating at 37°C for 3 hours; (4) diluting again using LB liquid medium, and coating on a selective solid medium based on the design of the recipient bacterium; (5) culturing overnight at 37°C to obtain single clones, and culturing at high temperature at 42°C to lose the plasmid.

8. The method of claim 4, wherein, The step C of extracting bacterial outer membrane vesicles comprises the following steps: (a) after centrifugation at 11000g, taking the supernatant, and then filtering using a filter and collecting the flow-through liquid; (b) ultracentrifugation at 130000g, discarding the supernatant, adding PBS to resuspend by blowing, and completing the extraction of bacterial outer membrane vesicles.

9. A bacterial outer membrane vesicle, characterized in that, Prepared by the preparation method of any one of claims 4 to 8.

10. The use of the extracellular membrane vesicles of claim 9 in diagnosis or detection.

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