CN113755514B

CN113755514B - Construction method of escherichia coli mutant and preparation method of outer membrane vesicle

Info

Publication number: CN113755514B
Application number: CN202111048805.XA
Authority: CN
Inventors: 沈锡辉; 杨亚东; 王瑶; 孙宇; 唐亚楠; 肖志博
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2021-09-08
Filing date: 2021-09-08
Publication date: 2023-06-16
Anticipated expiration: 2041-09-08
Also published as: CN113755514A

Abstract

The invention discloses a construction method of an escherichia coli mutant and a preparation method of outer membrane vesicles. The method comprises the steps of constructing a high-yield mutant of the outer membrane vesicle of the escherichia coli and extracting the outer membrane vesicle, wherein the mutant is constructed by simultaneously knocking out an escherichia coli Tol-pal inner membrane system protein (shown as SEQ ID NO: 1) and an outer membrane phospholipid accumulation related protein (shown as SEQ ID NO: 2) through a CRISPR-Cas9 system. The extraction of outer membrane vesicles comprises: culturing the strain in LB liquid medium, filtering and sterilizing culture supernatant, ultrafiltering and concentrating, and ultracentrifugating to obtain outer membrane vesicle. Compared with the existing single-double gene mutant strain, the deletion of double genes plays a synergistic role, not only can remarkably improve the yield of outer membrane vesicles by about 180 times compared with the original strain, but also the obtained outer membrane vesicles have the characteristics of high purity, proper particle size and the like, are a low-cost and high-efficiency preparation method, solve the problem of insufficient yield of the outer membrane vesicles, and show good application prospects.

Description

Construction method of escherichia coli mutant and preparation method of outer membrane vesicle

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construction method of an escherichia coli mutant and a preparation method of outer membrane vesicles.

Background

Bacterial outer membrane vesicles (outer membrane vesicles, OMVs) are mainly vesicles produced by gram-negative bacteria during growth and have a particle size of 20-300nm. OMVs have a number of pathogen-associated molecular patterns, including lipopolysaccharide, immunomodulatory proteins, bacterial DNA, which bind to host pattern recognition receptors to induce immune responses, while these OMVs components can act as ligands for Toll-like receptors (TLRs) making OMVs a good immune adjuvant with endogenous biological functions that can be targeted into and interact with cells as a biofilm delivery system. Studies have shown that OMVs act as primary signaling vectors for bacteria, protecting and delivering effector molecules to target sites through surface ligands without error, and completing mass exchange of information between bacteria-bacteria, bacteria-hosts, so OMVs are also known as bacterial type 0 secretion system (T0 SS).

Compared with the classical bacterial I-VI type secretion system (T1 SS-T6 SS), the OMVs secretion system has a plurality of advantages which are not possessed by the classical secretion system, can be used as a nanoscale carrier under the condition of not depending on cell contact, can be provided for remote cells through long-distance targeted transportation, and can protect loaded cargoes such as proteins, nucleic acids and the like from being damaged and influenced by external environments. All gram-negative bacteria studied to date release OMVs. The OMVs are inactive, can effectively activate an immune system to generate a protective immune reaction, and simultaneously serve as a natural nano system, so that the OMVs have unique physiological characteristics which are not possessed by artificially synthesized nano material carriers, and can simultaneously present a plurality of exogenous proteins through genetic engineering operation, thereby becoming popular in vaccine research and development. For example, neisseria meningitidis type B (Neisseria meningitidis) OMVs vaccine (trade name Bexsero) has been marketed in europe and america, and vaccine research efforts based on OMVs to prevent other infectious diseases have also been underway.

A great deal of research work has been carried out on bacterial outer membrane vesicles as novel presentation vectors, mainly focusing on the secretion amount, safety, presentation efficiency and the like of OMVs, so that the bacterial outer membrane vesicles can become a better safe and efficient presentation vector system. The Bernadac and the like combine and delete different single gene mutants of the escherichia coli Tol-pal endomembrane system to screen out a double gene mutant JC8031, so that the yield of OMVs is obviously improved, and an application foundation of the OMVs is laid. In recent years, bacterial outer membrane vesicles are widely focused and studied as drug delivery systems, OMVs are used as nano carriers of biological sources, and the OMVs have biodegradability, can evade natural immune response of hosts, can carry different drugs to reach action targets by using the biocompatibility, and improve the permeability and action sensitivity of the drugs. Studies such as Gujrate show that OMVs secreted by escherichia coli can carry siRNA in an inner cavity through electrotransformation, and meanwhile, a specific affinity body of human epidermal growth factor receptor 2 (HER 2) is expressed on the surface as a tumor cell targeting ligand for treating cancer, so that good effect is shown, and good application prospect is shown.

Efficient extraction of bacterial outer membrane vesicles is the basis of the above-mentioned research and application of OMVs, but the yield of wild-type escherichia coli OMVs is still limited in wide application, and how to obtain high-yield and high-purity OMVs is particularly important. Thus, the ability to obtain high yield E.coli strains that secrete OMVs in large amounts has become a critical issue in the art to be addressed as a viable extraction method.

Disclosure of Invention

The invention aims to solve the problem of insufficient yield of outer membrane vesicles in wide application, and provides a construction method of an escherichia coli mutant and a preparation method of the outer membrane vesicles, which not only can remarkably improve the yield of the outer membrane vesicles, but also can obtain the outer membrane vesicles with the characteristics of high purity, proper particle size and the like, and has good application prospects.

The invention solves the technical problems by adopting the technical scheme that:

a construction method of an escherichia coli mutant comprises the following steps: the Tol-pal inner membrane system protein of the escherichia coli is knocked out, and the outer membrane phospholipid accumulation related protein is constructed; the amino acid sequence of the Tol-pal endomembrane system protein is shown as SEQ ID NO. 1; the amino acid sequence of the outer membrane phospholipid accumulation related protein is shown as SEQ ID NO. 2.

Specifically, the Tol-pal inner membrane system protein and outer membrane phospholipid accumulation related protein of the escherichia coli are knocked out through a CRISPR-Cas9 system to construct.

Alternatively, the CRISPR-Cas9 system is characterized in that a pCas temperature-sensitive plasmid carrying Cas9 protein is introduced into escherichia coli, a recombinant knockout plasmid pTargetF carrying sg20 and upstream and downstream homology arms of a target gene is simultaneously introduced into the escherichia coli, and escherichia coli mutants for knocking out the target gene are obtained through cutting of the Cas9 protein and homologous recombination exchange of the upstream and downstream homology arms of the target gene.

Optionally, saidThe pCas temperature-sensitive plasmid expresses Cas9 protein under the induction of 50mM L-arabinose, 0.5mM Km ^r On LB solid medium, normally culturing at 30 ℃; the pCas temperature sensitive plasmid was eliminated by culturing at 37℃for 8-12 h.

Alternatively, the sgRNA transcribed from the recombinant knockout plasmid pTargetF directs Cas9 protein to cleave e.coli chromosome, replacing the fragmented DNA by homologous recombination; the cleavage disruption of the pMB1 replicon of pTargetF by the Cas9 protein under 0.25mM IPTG induction, abrogated the recombinant knockout plasmid pTargetF.

Alternatively, the nucleotide sequence for encoding the Tol-pal endomembrane system protein is shown as SEQ ID NO. 3; the nucleotide sequence of the outer membrane phospholipid accumulation related protein is shown as SEQ ID NO. 4.

The escherichia coli mutant is obtained by adopting the construction method of the escherichia coli mutant.

The invention relates to a preparation method of outer membrane vesicles, which takes an escherichia coli mutant as a culture object and extracts the outer membrane vesicles from culture supernatant; the culture conditions are as follows: LB liquid culture medium is cultivated for 20 to 24 hours at 37 ℃ and 200 r/min.

Optionally, the culture supernatant is centrifuged at 8000rpm for 5min and 6000rpm for 15min respectively to remove thalli; the supernatant was then subjected to molecular entrapment and centrifugation to obtain outer membrane vesicles.

A method of preparing outer membrane vesicles comprising: (1) construction of E.coli high-yield mutant: knocking out Tol-pal inner membrane system proteins and outer membrane phospholipid accumulation related proteins of escherichia coli through a CRISPR-Cas9 system; the amino acid sequence of the Tol-pal endomembrane system protein is shown as SEQ ID NO. 1; the amino acid sequence of the outer membrane phospholipid accumulation related protein is shown as SEQ ID NO. 2;

(2) Extracting outer membrane vesicles from culture supernatant of the escherichia coli high-yield mutant obtained in the step (1): the culture conditions are as follows: LB liquid culture medium is cultivated for 20 to 24 hours at 37 ℃ and 200 r/min.

The outer membrane vesicle prepared by the preparation method of the outer membrane vesicle is applied to the preparation of vaccines, immunopotentiators or nanometer presentation vectors.

Compared with the prior art, the invention has the following beneficial effects:

the preparation of engineering escherichia coli can be easily realized by CRISPR-Cas9 genetic engineering technology, and the escherichia coli mutant of the high-yield outer membrane vesicle is obtained, so that the operation is simple and easy to implement; the escherichia coli mutant constructed by the invention does not need antibiotics to maintain, has genetic stability, and can be cultured in a large amount in industrial production;

compared with the existing Bernadac and other reported double-gene mutants JC8031 with the deletion of different single-gene mutants, the yield of the outer membrane vesicle is obviously increased by about 180 times compared with that of the original strain, the defect of low yield of the outer membrane vesicle in clinical application can be well overcome, and the production cost is obviously reduced. The invention can realize continuous concentration of a large amount of culture supernatant filtrate by a tangential flow membrane ladle instrument with molecular retention of 100kDa, can be widely applied to industrial production, and can obtain outer membrane vesicles with high purity and proper particle size.

Drawings

The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:

FIG. 1 is a schematic diagram showing the construction process of a double-gene mutant of Escherichia coli Δb0783 Δb3149 in the present invention;

FIG. 2 is a schematic diagram showing the construction results of a double-gene mutant of Escherichia coli Δb0783 Δb3149 in the present invention;

FIG. 3 is a schematic diagram showing growth trends of wild-type E.coli and other mutants;

FIG. 4 is a graph showing particle size of each strain by particle size analysis (Nanosight);

FIG. 5 is a view of extracted outer membrane vesicles by Transmission Electron Microscopy (TEM);

FIG. 6 detection of wild type strain and mutant strain outer membrane vesicle production by SDS-PAGE of major outer membrane proteins.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to the specific drawings and the embodiments.

In the invention, the escherichia coli mutant is constructed by knocking out escherichia coli Tol-pal inner membrane system protein (shown as SEQ ID NO: 1) and outer membrane phospholipid accumulation related protein (shown as SEQ ID NO: 2) through a CRISPR-Cas9 system.

In the invention, the nucleotide sequence for encoding the Tol-pal endomembrane system protein is shown as SEQ ID NO. 3.

In the invention, the nucleotide sequence of the outer membrane phospholipid accumulation related protein is shown as SEQ ID NO. 4.

In the CRISPR-Cas9 system, pCas temperature-sensitive plasmid carrying Cas9 protein is introduced into escherichia coli, recombinant knockout plasmid pTargetF carrying sg20 and upstream and downstream homology arms of target genes is simultaneously introduced into escherichia coli, and escherichia coli outer membrane vesicle high-yield mutant for knocking out target genes is obtained through cutting of Cas9 protein and homologous recombination exchange of upstream and downstream homology arms of target genes.

In the invention, the pCas temperature-sensitive plasmid expresses Cas9 protein under the induction of 50mM L-arabinose, and 0.5mM Km ^r On LB solid medium, normal culture is carried out at 30 ℃. The pCas temperature sensitive plasmid was eliminated by culturing at 37℃for 8-12 h.

In the invention, the sgRNA transcribed by the recombinant knockout plasmid pTargetF guides Cas9 protein to cut E.coli chromosome, and the broken DNA is replaced by homologous recombination. The cleavage disruption of the pMB1 replicon of pTargetF by the Cas9 protein under 0.25mM IPTG induction, abrogated the recombinant knockout plasmid pTargetF.

In the invention, the extraction of the outer membrane vesicles in the bacterial culture supernatant comprises the following steps:

(1) E.coli was cultured in LB liquid medium at 37℃for 20-24 hours at 200 r/min. Centrifuging at 8000rpm for 5min and at 6000rpm for 15min to remove thallus, filtering culture supernatant with 0.45 μm filter membrane, and concentrating;

(2) The filtrate was concentrated by a tangential flow membrane packer with a molecular cutoff of 100kDa, and the concentrate was ultracentrifuged at 150000g for 2h by a Beckman Coulter ultracentrifuge Type70Ti rotor to obtain bacterial outer membrane vesicles.

The invention relates to a preparation method of the yield of outer membrane vesicles of escherichia coli, which comprises the following steps: (1) constructing a high-yield mutant of the outer membrane vesicle of the escherichia coli; (2) extraction of outer membrane vesicles from bacterial culture supernatants.

The bacterial outer membrane vesicle obtained in the invention can be applied to vaccine preparation and immunopotentiator, and can be applied to medicines and other small molecules as nano presentation carriers.

Example 1: construction of E.coli Δb0783 Δb3149 double-gene mutant

The embodiment provides a method for knocking out target genes in escherichia coli by using a CRISPR-Cas9 system, wherein the target genes are respectively named as b0783 and b3149, and the nucleotide sequences of the target genes are shown as SEQ ID NO. 3 and SEQ ID NO. 4 of a sequence table. The method is characterized in that an escherichia coli delta b0783 single gene mutant, an escherichia coli delta b3149 single gene mutant and an escherichia coli delta b0783 delta b3149 double gene mutant are respectively constructed, wherein the delta b0783 delta b3149 double gene mutant is obtained by further knocking out b3149 on the basis of the delta b0783 single gene mutant, and the method is consistent.

In the embodiment, the specific process of mutant construction is described by taking the Δb0783 single gene mutant as an example, and the construction process is shown in fig. 1, and the steps are as follows:

(1) Material

Coli DH 5. Alpha. Was purchased from Beijing Co., ltd. Of Tiangen biotechnology, and E.coli Nissle 1917 was purchased from China center for type culture Collection with a collection number of CCTCC NO: m2017345, pTargetF plasmid and pCas plasmid and various restriction enzymes were purchased from Takara Bio Inc.;

LB liquid medium: 0.01g/mLNaCl,0.01g/mL tryptone, 0.005g/mL yeast extract; LB solid medium: 0.01g/mLNaCl,0.01g/mL tryptone, 0.005g/mL yeast extract, 0.015g/mL agar; each medium was supplemented with kanamycin at a final concentration of 50. Mu.g/mL, 25. Mu.g/mL, and chloramphenicol as needed.

(2) Primer design

The target gene b078 was designed based on the published gene sequence of E.coli K-12MG1655 strain (GenBank No. U00096.3) on GenBank3 upstream and downstream homology arm primer, primer P _A /P _B Amplification of upstream homology arm AB of b0783 Gene with primer P _C /P _D Downstream homology arm CD was amplified. P (P) _E /P _F To knock out the upstream and downstream primers of sg20 in plasmid pTargetF.

a) The primer sequences were as follows:

P _A :5'-CCGAGTCGGTGCTTTTTTTGAACCGGAAGCCGTAGTGGAA-3'；

P _B :5'-GCAAGGGAAACGCAGATGTTGGACTTGAGATCGCGACGAC-3'；

P _C :5'-GTCGTCGCGATCTCAAGTCCAACATCTGCGTTTCCCTTGC-3'；

P _D :5'-GGTAATAGATCTAAGCTTCTGCAGGTCGACCCTCTTCCGCTTGCTTCTG-3'；

P _E :5'-AGCTAGCTCAGTCCTAGGTATAATACTAGTCCAGTGATTGTTGAAGTGTCGTTTTAGAGCTAGAAATAGC-3'；

P _F :5'-TTCCACTACGGCTTCCGGTTCAAAAAAAGCACCGACTCGG-3'。

b) The amplification system is shown in Table 1:

TABLE 1 PCR amplification System for Gene fragments

c) The reaction procedure is:

pre-denaturation at 95℃for 5min; (denaturation at 95 ℃,30s; annealing at 50 ℃,30s; extension at 72 ℃,1 min/kb). Times.10 cycles; (denaturation at 95 ℃,30s; annealing at 52 ℃,30s; extension at 72 ℃,1 min/kb). Times.20 cycles; finally, the mixture is extended for 10min at 72 ℃; preserving heat at 16 ℃. And after the completion, carrying out electrophoresis detection on the PCR product.

(3) Preparation of recombinant knockout plasmid

Referring to the instruction book of a Tiangen plasmid recovery kit, recovering each gene fragment product, and constructing a fusion gene fragment AF of sg20 and upstream and downstream homology arms of a target gene b0783 by overlapping PCR; with reference to the root plasmid extraction kit, the knockout plasmid pTargetF was extracted.

a) The cleavage reaction system and conditions are shown in Table 2:

TABLE 2 double cleavage reaction System

After vortex mixing, placing the mixture in a 37 ℃ incubator for enzyme digestion for 8-12 hours; DNA recovery was performed after completion of cleavage.

b) The ligation reaction system and conditions are shown in Table 3:

TABLE 3 ligation reaction System

Placing in a 50 ℃ connector for 1h.

c) Screening and identification of clones: the ligation product was transformed into E.coli DH 5. Alpha. And plated on LB solid plates containing chloramphenicol, and positive clone pTargetF-AF was obtained by colony PCR verification and sequencing.

(4) Preparation of b0783 Gene-deleted Strain

(1) Referring to the root plasmid extraction kit, PCas plasmid expressing Cas9 protein is extracted, and the plasmid is transformed into corresponding E.coli Nissle 1917 by a heat shock method.

(2) Preparation of competent cells of E.coli containing PCas plasmid overnight cultured E.coli was transferred to LB liquid medium containing 50mM L-ArG, shaken to logarithmic phase at 30℃and sterilized ddH ₂ O was washed 3 times, centrifuged at 5000rpm each time at 4℃for 5min, and finally the appropriate ddH was added ₂ O is mixed evenly and split charging is carried out at 100 mu L/tube for electric rotation.

(3) Screening of mutants the recombinant knock-out plasmid pTargetF-AF, which was confirmed to be correct, was electrotransferred to competent cells as described above, resuscitated at 30℃and plated on LB solid plates containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL chloramphenicol, cultured overnight and then using the homologous upper arm upstream primer P _A And a downstream primer P of the homology lower arm _D The mutants were identified to obtain mutant strains containing pTargetF and PCas plasmids.

(4) Plasmid elimination the mutant strains containing pTargetF and PCas plasmids obtained above were cultured in LB liquid medium containing 0.25mM IPTG, under induction, cas9 protein was destroyed to the pMB1 replicon cleavage of pTargetF, eliminating the recombinant knockout plasmid pTargetF. Then the mutant strain only containing PCas plasmid is cultivated for 8-12h at 37 ℃ to eliminate the pCas temperature sensitive plasmid, and finally the Escherichia coli delta b0783 single gene mutant is obtained.

(5) Construction of E.coli Δb3149 Single Gene mutant and Δb0783 Δb3149 double Gene mutant

The construction of the E.coli delta b3149 single gene mutant is consistent with the construction method of the b0783 gene deletion strain, wherein the delta b0783 delta b3149 double gene mutant is obtained by further knocking out the b3149 on the basis of the delta b0783 single gene mutant, the construction method is consistent with the construction method of the b0783 gene deletion strain, and the construction and identification results are shown in figure 2, so that the E.coli delta b3149 and delta b0783 single gene mutant and delta b0783 delta b3149 double gene mutant are successfully obtained.

Example 2: culture of strains and preparation of outer membrane vesicles

(1) The obtained escherichia coli delta b0783 single gene mutant, delta b3149 single gene mutant and delta b0783 delta b3149 double gene mutant are streaked and inoculated on an LB solid culture medium, cultured overnight at 37 ℃, and single colony is selected and inoculated on an LB liquid culture medium as mother liquor for expansion culture. The mother solution was transferred to a conical flask containing 1L of LB liquid medium at a volume ratio of 1:100, and cultured with shaking at 37℃and 200rpm/min for 20 to 24 hours.

(2) Centrifuging the cultured bacterial liquid at 8000rpm for 5min and at 6000rpm for 15min to remove bacterial body, filtering culture supernatant with 0.45 μm filter membrane, and concentrating; the above filtrate was concentrated by a tangential flow membrane wrapper having a molecular cutoff of 100kDa, the concentrate was ultracentrifuged at 150000g for 2h by a Beckman Coulter ultracentrifuge Type70Ti rotor to collect outer membrane vesicles, then suspended again with 50mM HEPES (pH 8.0), washed twice with 50mM HEPES (pH 8.0) in a Millipore 100-kDa ultrafiltration tube, OMVs suspended with 50mM HEPES (pH 8.0), and then filtered through a 0.22 μm filter to sterilize to obtain bacterial outer membrane vesicles, which were stored at-80 ℃.

Example 3: identification of E.coli mutant high-yield outer membrane vesicles

(1) Measurement of growth conditions of each strain, each strain was cultured overnight at 37℃in LB liquid medium, diluted 10-fold continuously with sterile physiological saline, 100. Mu.L of a proper dilution of the bacterial liquid was uniformly spread on LB solid plates, 3 replicates were made for each dilution, cultured overnight at 37℃and counted, and the CFU of the stock solution was calculated as an average value. Then at a final concentration of about 10 ⁶ CFU/mL was transferred to the same liquid medium, cultured at 200r/min and 37 ℃, and the colony growth was measured by OD600 by sampling at 24h, and the results are shown in FIG. 3, wherein compared with the original strain, the single gene knockout of Δb3149 or Δb0783 can affect the growth state of the strain, and the double gene knockout of Δb3149 and Δb0783 is most obvious.

(2) And detecting the particle size of outer membrane vesicles of each strain, taking an outer membrane vesicle sample, diluting the outer membrane vesicle sample with filtered PBS according to the ratio of 1:10, 1:100 and 1:1000, detecting the particle size of vesicles produced by each strain by using a nanometer laser particle sizer until the value of the instrument detection result Pdl is not more than 0.5 and the result quality is evaluated as good. The working volume of the detection sample is 1-1.5 mL, the detection sample is placed in a cuvette for uniform mixing, and the detection sample is placed in an instrument for detection, and the instrument can automatically perform repeated detection for 3 times. As shown in FIG. 4, the outer membrane vesicles produced by each strain were not significantly different in particle size and were concentrated around 100 nm.

(3) And (3) carrying out transmission electron microscope detection on the outer membrane vesicles extracted from each strain, adopting a negative staining method, taking a proper amount of outer membrane vesicle samples, dripping the outer membrane vesicle samples on a copper mesh with a supporting film, standing for 5min, sucking redundant suspension by filter paper, and placing the filter paper in the transmission electron microscope for observation. As a result, as shown in FIG. 5, the obtained vesicles have a typical bilayer membrane structure and have the characteristics of high purity, uniform particle size, and the like.

(4) Detection of outer membrane vesicle yield of each strain, based on the content of main outer membrane proteins of outer membrane vesicles, the main protein band brightness after SDS-PAGE was evaluated by Image J software, and the results are shown in FIG. 6, and compared with the delta b3149 or delta b0783 single gene mutant and the existing Bernadac et al reported double gene mutant JC8031 in which other different single gene mutants are deleted in combination, the delta b3149 and delta b0783 double gene mutants in the invention remarkably improve the yield of outer membrane vesicles.

The preparation of engineering escherichia coli can be easily realized by CRISPR-Cas9 genetic engineering technology, and the escherichia coli mutant of the high-yield outer membrane vesicle is obtained, so that the operation is simple and easy to implement; the escherichia coli mutant constructed by the invention does not need antibiotics to maintain, has genetic stability, and can be cultured in a large amount in industrial production; compared with the existing Bernadac and other reported double-gene mutants JC8031 with the deletion of different single-gene mutants, the yield of the outer membrane vesicle is obviously increased by about 180 times compared with that of the original strain, the defect of low yield of the outer membrane vesicle in clinical application can be well overcome, and the production cost is obviously reduced. The invention can realize continuous concentration of a large amount of culture supernatant filtrate by a tangential flow membrane ladle instrument with molecular retention of 100kDa, can be widely applied to industrial production, and can obtain outer membrane vesicles with high purity and proper particle size.

The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.

In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.

Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Nucleotide sequence Listing electronic document

<110> university of agriculture and forestry science and technology in northwest

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Asn Pro Pro Val Ile Val Glu Val Ser Gly Ile Gly Gln Tyr Thr Val Val Val Glu Lys 80

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Val Ser Gly Val Phe Ile Gly Met Val Leu Gly Leu Gln Gly Tyr Leu Val Leu Thr Thr 80

Tyr Ser Ala Glu Thr Ser Leu Gly Met Leu Val Ala Leu Ser Leu Leu Arg Glu Leu Gly 100

Pro Val Val Ala Ala Leu Leu Phe Ala Gly Arg Ala Gly Ser Ala Leu Thr Ala Glu Ile 120

Gly Leu Met Arg Ala Thr Glu Gln Leu Ser Ser Met Glu Met Met Ala Val Asp Pro Leu 140

Arg Arg Val Ile Ser Pro Arg Phe Trp Ala Gly Val Ile Ser Leu Pro Leu Leu Thr Val 160

Ile Phe Val Ala Val Gly Ile Trp Gly Gly Ser Leu Val Gly Val Ser Trp Lys Gly Ile 180

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Asn Cys Leu Ile Lys Ser Val Val Phe Ala Ile Thr Val Thr Trp Ile Ser Leu Phe Asn 220

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gcaccgctgc tggtgcgcca gctctataat gtcggcgtcc tgtcgatgct gattattgtg 180

gtttctggcg tgttcatcgg aatggtgttg gggctgcaag gttatctggt tctgaccact 240

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gatagtgggt tcttctggtc ggcaatgcaa aatgccgtcg actggcgtat ggatctcgtc 600

aactgtctga ttaagagcgt ggtgttcgct atcacggtga cgtggatttc gttgtttaac 660

ggctacgacg ccatcccgac gtctgccggg attagccggg caaccactcg caccgttgtc 720

cactcgtctc tggctgttct ggggctggat tttgtgctga ccgcattgat gtttgggaat 780

Claims

1. A construction method of an escherichia coli mutant is characterized in that Tol-pal inner membrane system proteins and outer membrane phospholipid accumulation related proteins of escherichia coli are knocked out;

the amino acid sequence of the Tol-pal endomembrane system protein is shown as SEQ ID NO. 1;

the amino acid sequence of the outer membrane phospholipid accumulation related protein is shown as SEQ ID NO. 2.

2. The method for constructing a mutant of E.coli according to claim 1, wherein the Tol-pal endomembrane system protein and the outer membrane phospholipid accumulation-associated protein of E.coli are knocked out by CRISPR-Cas9 system.

3. The method for constructing a mutant of escherichia coli according to claim 2, wherein the CRISPR-Cas9 system is characterized in that a pCas temperature-sensitive plasmid carrying Cas9 protein is introduced into escherichia coli, a recombinant knockout plasmid pTargetF carrying sg20 and upstream and downstream homology arms of a target gene is simultaneously introduced into escherichia coli, and the escherichia coli mutant for knocking out the target gene is obtained through cleavage of Cas9 protein and homologous recombination exchange of the upstream and downstream homology arms of the target gene.

4. The method for constructing a mutant E.coli according to claim 3, wherein the pCas temperature-sensitive plasmid expresses Cas9 protein under the induction of 50mM L-arabinose, 0.5mM Km ^r On LB solid medium, normally culturing at 30 ℃;

the pCas-temperature sensitive plasmid was deleted by culturing 8-12h at 37 ℃.

5. The method for constructing a mutant E.coli according to claim 3, wherein the sgRNA transcribed from the recombinant knock-out plasmid pTargetF directs the Cas9 protein to cleave E.coli chromosome and replace the cleaved DNA by homologous recombination;

cas9 protein vs pTargetF under 0.25mM IPTG inductionpMB1Replicon cleavage disruption, elimination of recombinant knockout plasmid pTargetF.

6. The method for constructing a mutant of E.coli according to any one of claims 1 to 5, wherein the nucleotide sequence encoding the Tol-pal endomembrane system protein is shown in SEQ ID NO. 3;

the nucleotide sequence of the outer membrane phospholipid accumulation related protein is shown as SEQ ID NO. 4.

7. An E.coli mutant obtained by the method for constructing an E.coli mutant according to any one of claims 1 to 5.

8. A method for preparing outer membrane vesicles, comprising extracting outer membrane vesicles from a culture supernatant using the escherichia coli mutant of claim 7 as a culture object;

the culture conditions are as follows: LB liquid culture medium is cultured at 37 ℃ and 200r/min for 20-24h.

9. The method for preparing outer membrane vesicles according to claim 8, wherein the culture supernatant is centrifuged at 8000rpm for 5min and 6000rpm for 15min to remove cells, respectively; the supernatant was then subjected to molecular entrapment and centrifugation to obtain outer membrane vesicles.

10. A method of preparing outer membrane vesicles, comprising: (1) construction of E.coli mutant: knocking out the Tol-pal inner membrane system protein and outer membrane phospholipid accumulation related protein of the escherichia coli through a CRISPR-Cas9 system; the amino acid sequence of the Tol-pal endomembrane system protein is shown as SEQ ID NO. 1; the amino acid sequence of the outer membrane phospholipid accumulation related protein is shown as SEQ ID NO. 2;

(2) Extracting outer membrane vesicles from a culture supernatant of the E.coli mutant obtained in (1): the culture conditions are as follows: LB liquid culture medium is cultured at 37 ℃ and 200r/min for 20-24h.