Preparation method of escherichia coli extracellular vesicles and application thereof in anti-lung cancer drugs

Abstract

The application discloses a preparation method of E. coli extracellular vesicles and application of the E. coli extracellular vesicles in anti-lung cancer drugs. The preparation method of the E. coli extracellular vesicles comprises the following steps: knocking out a Braun lipoprotein gene of E. coli to obtain a recombinant bacterium; lysing the recombinant bacterium and performing low-speed centrifugation to obtain a supernatant and a lysis solution; performing high-speed centrifugation on the lysis solution to obtain a precipitate, which is the E. coli extracellular vesicles. The E. coli extracellular vesicles prepared by the method can be applied to the preparation of anti-tumor drugs. The Braun lipoprotein gene of E. coli is knocked out, a recombinant bacterium strain is constructed, and OMVs extracted from the bacterium strain can directly inhibit the proliferation of specific types of small cell lung cancer NCI-H69, but the inhibiting effect on the proliferation of non-small cell lung cancer and other small cell lung cancer strains is poor. The OMVs prepared by the application can provide a new path for the design and screening of anti-lung cancer drugs, and have a good drug-making prospect.

Description

Technical Field

[0001] This invention relates to an anti-lung cancer drug, and more particularly to a method for preparing Escherichia coli extracellular vesicles and their application in anti-lung cancer drugs. Background Technology

[0002] Lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lungs. The majority of patients have non-small cell lung cancer (NSCLC), while a smaller proportion have small cell lung cancer (SCLC). Surgery is the preferred treatment for early-stage NSCLC, aiming for radical resection; however, surgery is less effective for small cell lung cancer. Currently, chemotherapy is the main treatment for small cell lung cancer and is also important for advanced or metastatic NSCLC. Targeted therapy drugs are currently mainly used to treat NSCLC (especially adenocarcinoma), while there are fewer drugs available for targeted therapy in small cell lung cancer.

[0003] Bacterial outer-membranevesicles (OMVs) have garnered significant attention in preclinical studies as carriers for delivering antitumor drugs to target cells. OMVs are nanoscale protein-liposomes, small, spherical, bilayered vesicles (10-300 nm) released into the extracellular environment by Gram-negative bacteria. They are composed of lipids, proteins, lipopolysaccharides, phospholipids, DNA, RNA, an inner membrane, periplasm, and other molecules. These vesicles can transport proteins, virulence factors, lipopolysaccharides, DNA, enzymes, and toxins over long distances to their targets. Numerous studies have utilized biotechnology to design OMVs as carriers to deliver antitumor drugs to target cells, improving the targeting specificity of antitumor drugs and reducing systemic toxicity. However, reports on the direct inhibitory or killing effects of OMVs on tumor cells are limited, especially regarding the inhibitory effect of single-gene knockout OMVs on small cell lung cancer.

[0004] The Braun lipoprotein (Lpp) gene in Escherichia coli is mainly associated with the formation and trait regulation of OMVs, but no studies have yet shown that the Lpp gene is related to the direct anti-tumor effect of OMVs. Summary of the Invention

[0005] Objective of this invention: The objective of this invention is to provide a method for preparing Escherichia coli extracellular vesicles, solving the problem of how to prepare Escherichia coli extracellular vesicles with anti-lung cancer drug efficacy. Another objective of this invention is to propose an application of Escherichia coli extracellular vesicles in the preparation of anti-lung cancer drugs, solving the problem of how to prepare anti-lung cancer drugs.

[0006] Technical solution: The present invention provides a method for preparing extracellular vesicles of Escherichia coli, comprising the following steps: (1) The Braun lipoprotein gene of Escherichia coli was knocked out to obtain recombinant bacteria; (2) After lysing the recombinant bacterial cells, centrifuge at low speed and collect the supernatant to obtain the lysate; (3) Centrifuge the lysate at high speed and take the precipitate as the extracellular vesicles of Escherichia coli.

[0007] Preferably, in step (1), the targeted knockout method is CRISPR-Cas9.

[0008] Preferably, in CRISPR-Cas9, the sgRNA sequence is: 5'-GACGTTCAGGCTGCTAAAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (as shown in SEQ ID No. 1);

[0009] Preferably, in step (2), the method for preparing the recombinant bacterial cells is as follows: Inoculate a single colony of the recombinant bacteria into LB liquid medium and culture by shaking until the bacterial culture reaches OD500. 600 Value ≥ 1; Under aseptic conditions, after centrifuging the bacterial solution, discard the supernatant and take the precipitate, which is the recombinant bacterial cell.

[0010] Preferably, the method for lysing the bacterial cells is as follows: The recombinant bacterial cells were resuspended in lysis buffer to obtain a bacterial suspension, and the bacterial suspension was homogenized to obtain a lysed bacterial suspension.

[0011] Preferably, in step (2), the conditions for low-speed centrifugation are 2-6℃, centrifugation at 1000-3000g for 5-30 minutes.

[0012] Preferably, in step (3), the high-speed centrifugation method is as follows: Centrifuge the lysis solution at 2-6℃ and 9000-11000g for 10-30 min, and collect the supernatant to obtain the first supernatant; Centrifuge the first supernatant at 12000-14000 g for 20-40 min, and then filter and sterilize the supernatant to obtain the second supernatant. Centrifuge the second supernatant at 2-6℃ and 150,000-200,000g for 30-90 minutes, discard the supernatant to obtain the first vesicle precipitate; The first vesicle precipitate was resuspended in buffer solution and centrifuged at 150,000-200,000g for 30-90 minutes at 2-6℃. The supernatant was discarded, and the precipitate was resuspended in buffer solution to obtain E. coli extracellular vesicles.

[0013] The second aspect of this invention discloses the application of the Escherichia coli extracellular vesicles prepared by the above-described method in the preparation of antitumor drugs.

[0014] Preferably, the tumor is lung cancer.

[0015] Furthermore, the lung cancer in question is small cell lung cancer.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: This invention constructs a recombinant bacterial strain by knocking out the Braun lipoprotein (Lpp) gene in *Escherichia coli*. OMVs extracted from this strain can directly inhibit the proliferation of a specific type of small cell lung cancer, NCI-H69, but its inhibitory effect on non-small cell lung cancer and other small cell lung cancer strains is poor. The OMVs obtained by this invention can provide a new pathway for the design and screening of anti-lung cancer drugs and have good prospects for drug development. Attached Figure Description

[0017] Figure 1 This is a morphological image of the extracellular vesicles of Escherichia coli prepared in Example 1. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0019] Example 1: A method for preparing extracellular vesicles of Escherichia coli is as follows: (1) Obtain the complete DNA sequence of the Escherichia coli Braun lipoprotein (Lpp) gene, Lpp [Escherichiacoli str. K-12 substr. MG1655]-Gene ID 946175. Within the coding region of the Lpp gene, select a highly specific sequence of about 20 bases: 5'-GACGTTCAGGCTGCTAAAGA-3'.

[0020] Based on this sequence, an sgRNA was designed, and its sequence is as follows: 5'-GACGTTCAGGCTGCTAAAGAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGGCUUUU-3'; Design Donor DNA (design repair template), including: Left homologous arm: a sequence approximately 500-1000 bp upstream of the Lpp gene; Right homologous arm: A sequence approximately 500-1000 bp downstream of the Lpp gene. The specific sequence is as follows: The donor DNA sequence is used to guide cells to completely delete or inactivate the Lpp gene after it has been cut.

[0021] (2) Constructing gene editing tools Constructing sgRNA expression plasmids: The designed sgRNA sequence was cloned into the expression vector pTargetF (with spectinomycin resistance) to obtain the recombinant plasmid pTargetF-sgRNA. After entering bacteria, this plasmid continuously transcribes sgRNA.

[0022] The Cas9 enzyme protein was expressed using the pCas plasmid (with kanamycin resistance).

[0023] (3) The gene editing tool from step (2) was transferred into E. coli to construct Lpp gene knockout recombinant bacteria. A final concentration of 75 ng / μL of sgRNA expression plasmid, a final concentration of 150 ng / μL of pCas plasmid, and a final concentration of 250 ng / μL of Donor DNA were added to competent Escherichia coli BL21. After incubation on ice for 20 min, heat shock at 42℃ for 60 s, and recovery at 37℃ for 45 min, transformed Escherichia coli were obtained.

[0024] The transformed BL21 bacteria were plated on double-antibiotic plates containing kanamycin and spectinomycin and incubated at 37°C. Single colonies were picked and added to liquid LB medium for shake culture. Colony PCR was performed using primers targeting the interior and flanking regions of the Lpp gene. The PCR products were sequenced, ultimately confirming that the Lpp gene sequence had been accurately deleted or destroyed.

[0025] The relevant primer sequences are as follows; (1) Internal validation primers (to detect whether the lpp gene has been knocked out) Forward primer: 5'-ATGAAAGCTACTAAACTGGTACTG-3' (target site sequence) Reverse primer: 5'-TTACTTGCGGTATTTAGTAGCCATG-3' (target site reverse complementary sequence) (2) External verification primers (to detect whether homologous recombination was successful) Left lateral primer (upstream of the left homologous arm): 5'-GATGCTGCTGCTGCTGCTGCTGCT-3' Right lateral primer (downstream of the right homologous arm): 5'-GCTGATGATGCTGCTGCTGCTGAT-3' By changing the temperature or adding an inducer (such as IPTG or arabinose), the pCas and pTargetF plasmids were removed from the identified recombinant bacteria to obtain Lpp gene knockout recombinant bacteria.

[0026] (4) The recombinant bacteria with the Lpp gene knocked out were inoculated into LB liquid medium and shaken at 37°C and 160 rpm for 12 h until the bacterial culture reached OD. 600 The value is 1; after centrifuging the bacterial culture at 6000 rpm for 5 minutes, discard the supernatant, take 20 μL of bacterial cells and spot them onto an empty nematode growth medium (NGM empty plate). Check for contamination after 12 hours. If no contamination is found, the experiment can continue, and the recombinant bacterial cells should be temporarily stored in a refrigerator at 4℃.

[0027] (5) After resuspending the recombinant bacterial cells in lysis buffer, obtain a bacterial suspension. Pipette 3-4 mL of the bacterial suspension into a dedicated disruption tube, add 1 mL of 0.1 mm magnetic beads, and homogenize the bacterial suspension using a biological homogenizer. The parameters of the biological homogenizer are 6 m / s. Repeat twice for 30 seconds, and place the sample on ice during the process. Then centrifuge the bacterial suspension at 2000 g for 10 min at 4°C, and collect the supernatant to obtain the lysis buffer. (6) Centrifuge the lysate at 4°C and 10,000g for 20 min, and take the supernatant to obtain the first supernatant; The first supernatant was centrifuged at 13000 g for 30 min, and the supernatant was filtered through a 0.22 μm filter to remove bacteria, thus obtaining the second supernatant. The second supernatant was centrifuged at 170,000 g for 60 min at 4 °C, and the supernatant was discarded to obtain the first vesicle precipitate. The first vesicle precipitate was resuspended in 1×PBS at a ratio of 1g:20mL, centrifuged at 170000g for 60min at 4℃, the supernatant was discarded, and the precipitate was resuspended in 200μL of 1×PBS to obtain Escherichia coli extracellular vesicles.

[0028] Example 2: Everything else is the same as in Example 1, except that: In step (6), the lysis solution is centrifuged at 2°C and 9000g for 30 min, and the supernatant is taken to obtain the first supernatant. The first supernatant was centrifuged at 12000 g for 40 min, and the supernatant was filtered through a 0.22 μm filter to remove bacteria, thus obtaining the second supernatant. The second supernatant was centrifuged at 2°C and 150,000g for 90 minutes, and the supernatant was discarded to obtain the first vesicle precipitate. The first vesicle precipitate was resuspended in 1×PBS at a ratio of 1g:20mL, centrifuged at 150000g for 90min at 2℃, the supernatant was discarded, and the precipitate was resuspended in 200μL of 1×PBS to obtain Escherichia coli extracellular vesicles.

[0029] Example 3: Everything else is the same as in Example 1, except that: In step (6), the lysis solution is centrifuged at 11000g for 10 min at 6°C, and the supernatant is taken to obtain the first supernatant. The first supernatant was centrifuged at 14000 g for 20 min, and the supernatant was filtered through a 0.22 μm filter to remove bacteria, thus obtaining the second supernatant. The second supernatant was centrifuged at 200,000g for 30 minutes at 6°C, and the supernatant was discarded to obtain the first vesicle precipitate. The first vesicle precipitate was resuspended in 1×PBS at a ratio of 1g:20mL, centrifuged at 200000g for 30min at 6℃, the supernatant was discarded, and the precipitate was resuspended in 200μL of 1×PBS to obtain Escherichia coli extracellular vesicles.

[0030] Comparative Example 1: Everything else is the same as in Example 1, except that: In step (4), the recombinant bacteria with the Lpp gene knocked out were replaced with wild-type K-12 Escherichia coli. Wild-type K-12 extracellular vesicles were finally obtained.

[0031] The microstructure of the Lpp single-gene knockout mutant OMVs prepared in Example 1 was examined (TEM), and the results are as follows: Figure 1 As shown.

[0032] The protein concentration of bacterial extracellular vesicles (OMVs) obtained in Examples 1-3 and Comparative Example 1 was determined by the BCA method. OMVs were then added to nematode liquid culture medium at the same final protein concentration (specifically 0.126 μg / μL) for nematode lifespan testing, as follows: After synchronizing GC833 nematodes (tumor model), once the nematodes reached the L4 stage in NGM, 10 nematodes per well were selected and placed into a 96-well plate. The liquid system per well consisted of 120 μL S culture + 30 pL FUDR (5-fluorouridine-2'-deoxynucleoside). Each group was fed OMVs every other day, and the number of dead nematodes was counted daily. Nematodes were considered dead when they were upright and unresponsive to platinum needle touch. The blank control group received an equal volume of 1×PBS with OMVs. The blank control group consisted of at least 30 nematodes, and all experiments were repeated at least three times. The results are as follows: Table 1. Effects of different OMVs on the lifespan of nematodes in a tumor model.

[0033] As shown in Table 1, Lpp single-gene knockout mutant OMVs significantly prolonged the lifespan of the tumor model nematode GC833. Compared with Comparative Example 1, the lifespan of Example 1 group was significantly prolonged by 33.57% (p<0.05). There was no significant difference between Comparative Example 1 and the blank control group, indicating that wild-type E. coli OMVs do not have a lifespan-prolonging function. The significant lifespan extension of the tumor model nematodes in the experimental group may be due to the inhibition of tumor progression, which can serve as indirect evidence that Lpp single-gene knockout mutant OMVs have anti-tumor activity.

[0034] The OMVs prepared in Examples 1-3 and Comparative Example 1 were used in anti-tumor proliferation experiments on non-small cell lung cancer (A549, NCI-H226, NCI-H460 cell lines) and small cell lung cancer (NCI-H69, DMS114). The tumor cell proliferation inhibition rate was determined using the CCK-8 assay. Add 1×10 tumor cells to each well. 5 The final concentration of OMVs (based on protein concentration) added to cells in each experimental group was 1 μg / μL. Cells in the blank control group were added with 1×PBS. After 48 h of routine culture, the absorbance of each well was measured using the CCK-8 assay.

[0035] The formula for calculating the tumor cell proliferation inhibition rate is: Proliferation inhibition rate = (mean OD value of blank control group - mean OD value of experimental group) / (mean OD value of blank control group - background value of blank well) × 100%.

[0036] The results are as follows: Table 2. Inhibitory effects of different OMVs on the proliferation of different types of lung cancer

[0037] As shown in Table 2, wild-type BL21 OMVs in Comparative Example 1 had no significant inhibitory effect on various lung cancer cells. In contrast, Lpp single-gene knockout mutant OMVs in Examples 1-3 only showed a high inhibitory effect on the proliferation of typical small cell lung cancer NCI-H69. However, they had a poor inhibitory effect on the proliferation of non-small cell lung cancers such as A549 (adenocarcinoma), NCI-H226 (squamous cell carcinoma), and large cell carcinoma (NCI-H460), as well as DMS114 (which has characteristics of both small cell lung cancer and some non-small cell lung cancers). This indicates that Lpp single-gene knockout mutant OMVs specifically inhibit the proliferation of typical small cell lung cancer.

Claims

1. A method for preparing extracellular vesicles of *Escherichia coli*, characterized in that, Includes the following steps: (1) The Braun lipoprotein gene of Escherichia coli was knocked out to obtain recombinant bacteria; (2) After lysing the recombinant bacterial cells, centrifuge at low speed and collect the supernatant to obtain the lysate; (3) Centrifuge the lysate at high speed and take the precipitate as the extracellular vesicles of Escherichia coli.

2. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, In step (1), the targeted knockout method is CRISPR-Cas9.

3. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 2, characterized in that, In CRISPR-Cas9, the sgRNA sequence is shown in SEQ ID No. 1, and the repair template DNA sequence is shown in SEQ ID No.

2.

4. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, In step (2), the method for preparing the recombinant bacterial cells is as follows: Inoculate a single colony of the recombinant bacteria into LB liquid medium and culture by shaking until the bacterial culture reaches OD500. 600 Value ≥ 1; Under aseptic conditions, after centrifuging the bacterial solution, discard the supernatant and take the precipitate, which is the recombinant bacterial cell.

5. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, The method for lysing the bacterial cells is as follows: The recombinant bacterial cells were resuspended in lysis buffer to obtain a bacterial suspension, and the bacterial suspension was homogenized to obtain a lysed bacterial suspension.

6. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, In step (2), the conditions for low-speed centrifugation are 2-6℃, 1000-3000g for 5-30min.

7. The method for preparing extracellular vesicles of *Escherichia coli* according to claim 1, characterized in that, In step (3), the high-speed centrifugation method is as follows: Centrifuge the lysis solution at 2-6℃ and 9000-11000g for 10-30 min, and collect the supernatant to obtain the first supernatant; Centrifuge the first supernatant at 12000-14000 g for 20-40 min, and then filter and sterilize the supernatant to obtain the second supernatant. Centrifuge the second supernatant at 2-6℃ and 150,000-200,000g for 30-90 minutes, discard the supernatant to obtain the first vesicle precipitate; The first vesicle precipitate was resuspended in buffer solution and centrifuged at 150,000-200,000g for 30-90 minutes at 2-6℃. The supernatant was discarded, and the precipitate was resuspended in buffer solution to obtain E. coli extracellular vesicles.

8. The use of Escherichia coli extracellular vesicles prepared by any one of claims 1-7 in the preparation of antitumor drugs.

9. The application according to claim 8, characterized in that, The tumor is lung cancer.

10. The application according to claim 9, characterized in that, The lung cancer in question is small cell lung cancer.

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