CN115181715A

CN115181715A - Recombinant escherichia coli capable of efficiently producing monophosphoryl lipid A vaccine adjuvant

Info

Publication number: CN115181715A
Application number: CN202210955587.6A
Authority: CN
Inventors: 王小元; 王震; 赵爱珍; 汪宸卉; 黄丹阳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-08-10
Filing date: 2022-08-10
Publication date: 2022-10-14
Anticipated expiration: 2042-08-10
Also published as: CN115181715B

Abstract

The invention discloses recombinant escherichia coli capable of efficiently producing a monophosphoryl lipid A vaccine adjuvant, belonging to the fields of genetic engineering and synthetic biology. The O-antigen gene cluster gnd-galF, the core sugar gene cluster rfaD-waaQ, the common intestinal antigen gene cluster rfe-rffM, the clavulanate gene cluster wcaM-wza and the genes mlaA, mlaC and pldA of a phospholipid transport system on the genome of Escherichia coli MG1655 are knocked out, and FnlpxE in a Francisella chromosome and SepagP and SepagL genes in a salmonella chromosome are overexpressed to obtain recombinant bacteria MW012/pWEPL. Through simple fermentation, the MW012/pWEPL strain can efficiently synthesize monophosphoryl hexaacyl lipid A (MPL), and the proportion of MPL in the whole lipid A is up to 75%.

Description

Recombinant escherichia coli capable of efficiently producing monophosphoryl lipid A vaccine adjuvant

Technical Field

The invention relates to recombinant escherichia coli capable of efficiently producing monophosphoryl lipid A vaccine adjuvant, and belongs to the field of genetic engineering and synthetic biology.

Lipopolysaccharide (LPS) is used as a main component of an outer membrane of an escherichia coli cell, and a hydrophilic sugar chain of the LPS can prevent a hydrophobic substance from entering the cell, maintain the stability of the cell and provide protection for the cell; its lipid a structure can be recognized by the pathogen recognition receptor TLR4 (Toll-like receptor 4) on immune cells (such as monocytes, macrophages, neutrophils and dendritic cells) in the host. When LPS is recognized by a receptor TLR4 on the surface of a mammalian cell to activate the innate immune system, the LPS with high toxicity can cause excessive release of proinflammatory factors and trigger heat shock and even death; while low toxicity LPS or its derivatives are effective in activating the innate immune system without producing excessive amounts of inflammatory molecules. Therefore, the LPS or the derivative thereof with low toxicity can be used as an immune system activator to be developed into a vaccine or a vaccine adjuvant.

Lipid A is a bioactive center of LPS and a recognition site of TLR4 of host immune cells, and the number of phosphate groups, the number of fatty acid chains and the length of the lipid A directly influence the release type and the release amount of proinflammatory cytokines. Some structures of lipid a induce the production of excessive cytokines, which in turn induce severe endotoxic shock, while some structures of lipid a induce mild inflammatory responses, produce moderate cytokines, induce Th 1-type responses, attract and activate macrophages and dendritic cells, and help the host to eliminate invading microorganisms. Thus, lipid A of different structures have different immunological functions, and some of the structures can be used as vaccine adjuvants to enhance the intensity and duration of the immune response. Vaccine adjuvants play an important role in the generation and enhancement of vaccine immune responses. The most widely used vaccine adjuvant is currently an aluminum adjuvant. It is an adsorbent that strongly adsorbs protein antigens from solution, forming a precipitate. When the antigen is inoculated into an organism, an antigen library can be formed, the antigen is slowly released, the action time of the antigen is fully prolonged, high-level antibody response can be induced, but stronger T cell immunity can not be induced. T cell immunity is urgently needed for the prevention and treatment of certain diseases, such as malaria, tuberculosis and AIDS. Therefore, designing vaccine adjuvants that can stimulate strong Th1 type cellular immune responses is the current focus of research.

Monophosphoryl lipid a (MPL) is a lipid a molecular derivative capable of enhancing immune response, and is being developed into a new generation vaccine adjuvant with wide application prospect. Commercial MPL is currently only available by chemical treatment of the lipid A structure of the Salmonella mutant Salmonella Minnesota R595, which is costly and complex.

Disclosure of Invention

In order to solve the technical problems, four LPS-related gene clusters gnd-galF, rfaD-waaQ, rfe-rffM and wcaM-wza and phospholipid transport system genes mlaA, mlaC and pldA on a genome are knocked out in Escherichia coli to obtain an LPS simplified strain MW012. Subsequently, plasmid pWEPL carrying lpxE genes in Francisella chromosome and pagP and pagL genes in Salmonella chromosome was transformed into LPS reduced strain MW012, to obtain recombinant strain MW012/pWEPL. Lipid A isolated from MW012/pWEPL was analyzed by Thin Layer Chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS) methods, which indicated that two monophosphoryl lipids A were produced predominantly in MW012/pWEPL, one hexaacylated and the other pentaacylated. More importantly, the proportion of the most effective component hexaacylated monophosphoryl lipid A in the lipid A vaccine adjuvant reaches 75 percent. The Escherichia coli strain MW012/pWEPL constructed by fermentation provides a good alternative for producing lipid A vaccine adjuvant MPL.

It is a first object of the present invention to provide a recombinant E.coli for efficient MPL production, which knocks out the O-antigen gene cluster, core sugar gene cluster, intestinal common antigen gene cluster and clavulanic acid gene cluster on the E.coli genome and mlaA, mlaC and pldA genes in the phospholipid transport system, and overexpresses dephosphorylatase (LpxE) from Francisella genome and hexadecanoyl transferase (PagP) and deacylase (PagL) from Salmonella genome.

In one embodiment, the O-antigen gene cluster is gnd-galF.

In one embodiment, the O-antigen gene cluster gnd-galF comprises 13 genes, gnd, wbbL, wbbK, wbbjj, wbbI, rfc, glf, rfbX, rfbC, rfbA a, rfbD, rfbB b, galF, the sequences of which have the accession numbers "NP _416533.1", "NP _416534.1", "NP _416536.1", "NP _416537.1", "NP _416538.1", "NP _416539.1", "NP _416540.1", "NP _416541.1", "NP _416542.1", "NP _416543.1", "NP _416544.1", "NP _416545.1" and "NP _416546.1" in the order of NCBI.

In one embodiment, the core carbohydrate gene cluster is rfaD-waaQ.

In one embodiment, the core carbohydrate cluster rfaD-waaQ comprises 14 genes, rfaD, waaF, waaC, waaU, waaL, waaZ, waaY, waaJ, waaR, waaB, waaS, waaP, waaG, waaQ, and the like, which are sequenced in the order of "NP _418076.1", "NP _418077.1", "NP _418078.1", "NP _418079.1", "NP _418080.1", "NP _418081.1", "NP _418082.1", "NP _418083.1", "NP _418084.1", "NP _418085.1", "NP _418086.1", "NP _418087.1", "NP _418088.1", "NP _418089.1".

In one embodiment, the intestinal common antigen gene cluster is rfe-rffM.

In one embodiment, the intestinal common antigen gene cluster rfe-rffM comprises 12 genes, rfe, wzzE, wecB, wecC, rffG, rffH, rffC, wecE, wzxE, wecF, wzyE, rffM, and the sequences have accession numbers "NP _418231.1", "NP _418232.1", "YP _ 253.1", "YP _026254.1", "YP _026255.1", "NP _418236.1", "YP _026256.1", "NP _418238.1", "NP _418239.1", "YP _026257.1", "NP _418241.1", and "NP _418242.1" in the order of NCBI.

In one embodiment, the clavulanic acid gene cluster is wcaM-wza.

In one embodiment, the clavulanic acid gene cluster wcaM-wza comprises 20 genes, wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, fcl, gmm, wcaI, manC, manB, wcaJ, wzx, wcaK, wcaL, wcaM for a total of 20 genes, the NCBI of the sequence has accession numbers of NP _416566.1, NP _416565.1, NP _416564.1, NP _416563.1, NP _416562.1, NP _416561.1, NP _416560.1, NP _416559.1, NP _416558.1 "NP _416557.1", "NP _416556.1", "NP _416555.1", "NP _416554.1", "NP _416553.1", "NP _416552.1", "NP _416551.1", "NP _416550.1", "NP _416549.1", "NP _416548.1" and "NP _416547.1".

In one embodiment, the phospholipid transport system mlaA, mlaC and pldA gene sequences have accession numbers "NP _416848.1", "NP _417659.1", "NP _418265.1" on NCBI.

In one embodiment, the sequence from the francis lpxE gene has NCBI accession number "WP _159184080.1"; the NCBI accession numbers of the sequences from the salmonella pagP and pagL genes are "NP _459620.1", "NP _416848.1", respectively.

In one embodiment, the escherichia coli comprises escherichia coli MG1655.

The second object of the present invention is to provide a method for constructing the above recombinant E.coli by knocking out the genes in the O-antigen gene cluster, the core sugar gene cluster, the intestinal common antigen gene cluster, the clavulanic acid gene cluster on the genome of E.coli, and the mlaA, mlaC and pldA genes in the phospholipid transport system, and overexpressing dephosphorylatases (LpxE) from the Francisella genome and hexadecanoyl transferase (PagP) and deacylase (PagL) from the Salmonella genome.

In one embodiment, ipxe from franciscella and pagP, pagL from salmonella are ligated to expression plasmid ppsk 29.

The third object of the present invention is to provide a method for efficiently producing MPL by inoculating the recombinant escherichia coli into a fermentation medium and performing fermentation production.

In one embodiment, the fermentation medium contains 4-6 g/L yeast powder, 8-12 g/L peptone, and 8-12 g/L NaCl.

In one embodiment, the reaction conditions for the fermentation are a temperature of 37 ℃ and 180 to 220rpm.

The fourth purpose of the invention is to provide the application of the recombinant Escherichia coli in the field of biomedicine.

The fifth purpose of the invention is to provide the application of the recombinant Escherichia coli in the production of a lipoid A vaccine adjuvant.

Has the beneficial effects that:

(1) According to the invention, four LPS-related gene clusters on an Escherichia coli genome and mlaA, mlaC and pldA genes in a phospholipid transport system are knocked out in Escherichia coli to obtain an LPS simplified strain MW012, the simplified strain MW012 has a good growth state, and the LPS structure is Kdo ₂ Lipid A, the simplest structure of LPS.

(2) Plasmid pWEPL carrying lpxE from Francisella and pagP and pagL from salmonella is transformed into LPS simplified strain MW012 to obtain recombinant strain MW012/pWEPL. The recombinant bacterium MW012/pWEPL mainly produces two monophosphoryl lipid A, one is hexaacyl, the other is pentaacyl, and more importantly, the proportion of the most effective component hexaacyl monophosphoryl lipid A in the lipid A vaccine adjuvant reaches 75%.

(3) The recombinant Escherichia coli MW012/pWEPL strain constructed by the invention has good growth condition, the lipoid A has simple structure and the extraction method is simple and convenient. Compared with the prior MPL obtained by chemical treatment from Salmonella Minnesota R595, the strain MW012/pWEPL of the invention is Escherichia coli which is easy to culture, grows rapidly and reduces production risk.

(4) The microbial conversion method provided by the invention has the advantages of simple fermentation condition, short fermentation time and simple extraction process, and can extract effective components without chemical treatment.

Drawings

FIG. 1 is a knockout flow chart; x is the target gene cluster gnd-galF, rfAD-waaQ, rfe-rffM, wcaM-wza, mlaA, mlaC and pldA.

FIG. 2 is a flow chart of the construction of LPS reduced strain MW 012; a: the knockout gene cluster involved in the present discovery; b: and (3) knocking out.

FIG. 3 shows the LPS structure of strain MW012 analyzed by SDS-PAGE.

FIG. 4 shows the LPS structure of strain MW012 analyzed by LC-MS.

FIG. 5 shows the construction of an expression plasmid; construction of plasmid pWEPL expressing IpxE derived from Francisella and pagP and pagL derived from Salmonella.

FIG. 6 shows growth curves for strains MG1655, MW012 and MW 012/pEPL.

FIG. 7 shows TLC analysis of lipid A structure of MW012/pWEPL strain.

FIG. 8 shows LC-MS analysis of lipid A structure of MW012/pWEPL strain.

FIG. 9 is a TLC analysis of lipid A in MG1655 and MG1655/pWEPL strains.

FIG. 10 is an LC-MS analysis of lipid A in MG1655/pWEPL strain.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, the reagents and materials used in the following examples are all commercially available or may be prepared by known methods.

The media involved in the following examples are as follows:

all the culture media use ddH ₂ And O, sterilizing at 121 ℃ for 15-20 min after preparation.

LB medium (g/L): 5 parts of yeast powder, 10 parts of peptone and 10 parts of NaCl.

LBA Medium (g/L): yeast powder 5, peptone 10, naCl 10, amp 0.05.

The corresponding antibiotic is selectively added according to the resistance gene carried on the plasmid.

TABLE 1 primer sequences (N20 sequence underlined) referred to in the following examples

TABLE 2 strains and plasmids referred to in the examples below

Example 1: construction of knock-out plasmids

Four gene clusters related to LPS in escherichia coli and mlaA, mlaC and pldA genes in a phospholipid transport system are knocked out by adopting a CRISPR/Cas9 knocking-out system, and 7 knocking-out plasmids are required to be constructed: pT1, pT2, pT3, pT4, pTargetF-mlaA, pTargetF-mlaC and pTargetF-pldA, and the construction process of these plasmids is specifically as follows:

(1) Selecting 20nt of N complementary to the target sequence of the target gene ₂₀ Sequence, in particular N ₂₀ The sequences are underlined for primers pT1, pT2, pT3, pT4, pTargetF-mlaA, pTargetF-mlaC and pTargetF-pldA, respectively, and these sequences were modified to the 5' -end of the forward primer of plasmid pTargetF to give forward primers pT1-F, pT2-F, pT3-F, pT4-F, pTar getF-mlaA-F, pTargetF-mlaC-F and pTargetF-pldA-F, respectively.

The N-introduced plasmid pTargetF was PCR-amplified using the plasmid pTargetF as a template and the forward primers pT1-F, pT2-F, pT3-F, pT4-F, pTargetF-mlaA-F, pT argetF-mlaC-F and pTargetF-pldA-F as the reverse primers pTargetF-R ₂₀ Open circular plasmid of sequence. And (3) carrying out electrophoretic verification on the PCR amplification product and purifying and recovering.

(2) Since the recovered product may contain the template plasmid pTargetF, which may affect the subsequent experiments, dpn I was added to the recovered product, and the recovered product was reacted at 37 ℃ for 2 hours to digest the template plasmid.

(3) The recovered product after digesting the template plasmid was phosphorylated using T4 polynucleotide kinase (T4 PNK), reacted at 37 ℃ for 30min, and then heat inactivated at 65 ℃ for 10min.

(4) mu.L of T4 DNA ligase was added to the phosphorylated reaction system and reacted at 22 ℃ for 4 hours to obtain a ligation solution. After the reaction, escherichia coli JM109 competent cells were taken out and thawed on ice, and the ligation solution was added to the competent cells and gently aspirated and mixed, and ice-cooled for 30min. The competent cells were then heat shocked in a water bath at 42 ℃ for 90s, ice-cooled for 2min, and added rapidly to 1mL of LB medium. After 1 hour at 37 ℃ and 100rpm, the cells were plated on LB plates supplemented with 50mg/L spectinomycin (Spe), and inverted at 37 ℃ to select knockout transformants. And (3) carrying out colony PCR on the transformant by taking pTargetF as a negative control, and verifying whether the knockout plasmid is successfully constructed. The correct transformants were ligated to LB liquid tubes to which spectinomycin (Spe) was added, and plasmids were extracted to obtain knock-out plasmids pTargetF-gene (pT 1 (O-antigen gene cluster knock-out), pT2 (core polysaccharide gene cluster knock-out), pT3 (clavulanate gene cluster knock-out), pT4 (intestinal common antigen gene cluster knock-out), pTargetF-mlaA, pTargetF-mlaC and pTargetF-pldA).

Example 2: construction of LPS simplified strain MW012

Four gene clusters related to LPS of wild type Escherichia coli MG1655 and mlaA, mlaC and pldA genes in a phospholipid transport system are knocked out by using a CRISPR/Cas9 knocking-out system, and an LPS simplified strain MW012 is obtained. The specific knockout procedure is as follows (FIG. 1):

(1) Preparation of E.coli electroporation knockout competent cell MG1655/pCas

The plasmid pCas was transformed into E.coli MG1655 to obtain recombinant E.coli MG1655/pCas containing the pCas plasmid, and E.coli MG1655/pCas was activated on an LB solid plate to which 30MG/L kanamycin (Kan) was added, and inoculated to LB (Kan) ⁺ ) Performing test tube overnight culture to obtain seed solution; transferring the seed solution into 25mL LB (Kan +) culture medium according to 1% (v/v), and culturing at 30 deg.C and 200rpm to OD ₆₀₀ =0.2, add 500 μ L L-arabinose solution to induce, continue culturing to OD ₆₀₀ =0.5, ice bath for 30min; centrifuging at 4 deg.C and 4000rpm for 10min, collecting thallus, and washing thallus with precooled 10% glycerol solution for three times; 300. Mu.L of 10% glycerol solution was added to resuspend the cells, and the cells were aliquoted into sterile 1.5mL EP tubes (80. Mu.L/tube).

(2) Construction of homologous arm knockout fragments

Extracting a genome of escherichia coli MG1655, using the genome as a template, respectively amplifying by using homologous arm primers U1-F/U1-R and D1-F/D1-R for knocking out a target gene cluster gnd-galF to obtain an upstream homologous arm and a downstream homologous arm, recovering glue, and performing overlapping PCR on U1-F/D1-R by using primers to obtain a homologous arm knock-out fragment 1; respectively amplifying the homologous arm primers U2-F/U2-R and D2-F/D2-R for knocking out the target gene cluster rfaD-waaQ to obtain an upstream homologous arm and a downstream homologous arm, recovering glue, and performing overlapping PCR on the U2-F/D2-R by using the primers to obtain a homologous arm knock-out fragment 2; respectively amplifying by using homology arm primers U3-F/U3-R and D3-F/D3-R for knocking out a target gene cluster wza-wcAM to obtain an upstream homology arm and a downstream homology arm, recovering glue, and performing overlapping PCR on the U3-F/D3-R by using the primers to obtain a homology arm knock-out fragment 3; respectively amplifying the homology arm primers U4-F/U4-R and D4-F/D4-R for knocking out the rfe-rffM of the target gene cluster to obtain an upstream homology arm and a downstream homology arm, recovering glue, and performing overlapping PCR on the U4-F/D4-R by using the primers to obtain a homology arm knock-out fragment 4; respectively amplifying by using homology arm primers U5-F/U5-R, D5-F/D5-R for knocking out the target gene cluster mlaA to obtain an upstream homology arm and a downstream homology arm, recycling glue, and performing overlapping PCR on the U5-F/D5-R by using the primers to obtain a homology arm knock-out fragment 5; respectively amplifying by using homology arm primers U6-F/U6-R, D6-F/D6-R for knocking out the target gene cluster mlAC to obtain an upstream homology arm and a downstream homology arm, recycling glue, and performing overlapping PCR on the U6-F/D6-R by using the primers to obtain a homology arm knock-out fragment 6; respectively amplifying the upstream homology arm and the downstream homology arm by using homology arm primers U7-F/U7-R and D7-F/D7-R for knocking out the target gene cluster pldA, recovering glue, and performing overlapping PCR on the U7-F/D7-R by using the primers to obtain a homology arm knock-out fragment 7.

(3) Electrotransfer knockout of genes

Washing the electric shock cup with anhydrous ethanol for three times, drying, and precooling for 20min. Coli MG1655/pCas competent cells of step (1) were thawed on ice, 100ng of the knockout plasmid pTargetF-gene (pT 1, pT2, pT3, pT4, pTargetF-mlaA, pTargetF-mlaC and pTargetF-pldA) of example 1 and 500ng of the corresponding homology arm knockout fragment of step (2) were added, gently pipetted, mixed well and pipetted into the wells of an electric cuvette. The electric shock cup is subjected to ice bath for 10min, and is electrically shocked after being wiped dry. Then, 1mL of LB medium was quickly added to the cuvette, the whole amount of the culture was aspirated into a 1.5mL EP tube, resuscitated at 30 ℃ and 100rpm for 1.5 hours, spread on an LB plate containing 30mg/L of Kan and 50mg/L of Spe, and cultured in an inverted state in an incubator at 30 ℃. Correct transformants were selected with forward primer for upstream homology arm and reverse primer for downstream homology arm using MG1655 as negative control.

(4) Removal of knock-out plasmid pTargetF-gene and temperature sensitive plasmid pCas

Transferring the correct knockout transformant in the step (3) to an LB test tube added with 30mg/L Kan and 1mM IPTG, removing the enzyme expression of the knockout plasmid pTargetF-gene by IPTG induction, carrying out shake culture at 30 ℃ for 12h, and streaking out a single colony on an LB (Kan +) plate. Screening out a single colony sensitive to spectinomycin to obtain a mutant strain with pTargetF-gene knockout plasmid removed, inoculating to an LB (Kan +) test tube for seed protection, and directly preparing competence for continuous knockout. The mutant strain from which the knockout plasmid was removed was inoculated into an LB tube, cultured with shaking at 42 ℃ and streaked on an LB plate to isolate a single colony. Screening out single bacterial colony sensitive to kanamycin to obtain non-anti-mutation bacterial strain without pCas, and connecting to LB test tube for conservation.

Four LPS-related gene clusters gnd-galF, rfaD-waaQ, w za-wcaM, rfe-rffM and 3 phospholipid transport-related mlaA, mlaC and pldA genes are successively knocked out from the genome of Escherichia coli MG1655 by using a CRISP/Cas9 knocking-out method, and an LPS simplified strain MW012 (figure 2) is obtained.

The gene cluster gnd-galF comprises 13 genes of gnd, wbbL, wbbK, wbbJ, wbbI, rfc, glf, rfbX, rfbC, rfbA, rfbD, rfbB and galF;

the gene cluster rfaD-waaQ comprises 14 genes of rfaD, waaF, waaC, waaU, waaL, waaZ, waaY, waaJ, waaR, waaB, waaS, waaP, waaG and waaQ;

the gene cluster wza-wcAM comprises 20 genes of wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, fcl, gmm, wcaI, manC, manB, wcaJ, wzx, wcaK, wcaL and wcaM;

the gene cluster rfe-rffM comprises 12 genes including rfe, wzzzE, wecB, wecC, rffG, rffH, rffC, wecE, wzxE, wec F, wzyE and rffM.

Example 3: extraction and structure verification of bacterial strain MW012 Lipopolysaccharide (LPS)

(1) And (3) an LPS extraction and purification method: the strains were tested for initial OD ₆₀₀ 0.02 of the strain was inoculated into 500mL of LB medium, cultured at 37 ℃ for 18 hours, centrifuged for 20 minutes, the supernatant was removed, and the pellet was collected. 20mL of water is added into the bacterial sediment for full suspension, then 20mL of 90% phenol preheated at 68 ℃ is added, and the mixture is shaken for 1h in a water bath kettle with a constant temperature of 68 ℃. Cooling to room temperature after shaking, centrifuging for 20min for phase separation, sucking the upper phase into a centrifuge tube, standing at 4 ℃ for 12h, transferring the supernatant into a dialysis bag, and dialyzing for 24h. Vacuum freeze-drying to obtain crude LOS. To the LOS crude was added 9mL of water, 1mL of reaction buffer (100 mM Tris-HCl,25mM MgCl) ₂ ，1mM CaCl ₂ pH 7.5), appropriate amounts of DNase I and RNase A, and standing at 37 ℃ for 4h (crude LOS sample weighed on a precision balance, 1. Mu.g of enzyme added to 1mg of lipopolysaccharide). To the reacted solution was added an appropriate amount of proteinase K, and the mixture was allowed to stand at 37 ℃ for 12 hours (1 mg of lipopolysaccharide to 1. Mu.g of enzyme). To remove residual protein, 5mL of water saturated phenol was added to the centrifuge tube and mixed well. Centrifuging for 30min, separating phase, sucking the upper phase into dialysis bag, dialyzing in water for 24 hr, and replacing water every 4 hr to remove residual phenol in the solution. Pouring the liquid in the dialysis bag into a centrifuge tube, and carrying out vacuum freeze drying to obtain the LOS semi-pure product. The semi-pure LOS was redissolved in a mixture of chloroform and methanol (2, 1,v/v), centrifuged at 12000rpm for 20min, and the supernatant was decanted off. Repeating the above operations, blow-drying, re-dissolving in water, and vacuum freeze-drying to obtain pure LOS product.

(2) Analysis of LPS by SDS-PAGE: prepare 20g/L LOS solution. After adding 5. Mu.L of 4 XSDS loading buffer (50M Tris-HCl, 2% SDS, 10% sucrose and 0.01% bromophenol blue, pH 6.8) to 15. Mu.L of LOS solution, heating in a boiling water bath for 10min, after the sample had cooled to room temperature, performing polyacrylamide gel electrophoresis, and adding 20. Mu.L of the solution to the gel wells in its entirety. The current of the concentrated glue is set to be 15mA, and when the strip runs to the interface of the concentrated glue, the current is adjusted to be 25mA. When the sample strip runs to a distance of about 5mm from the bottommost part of the adhesive, the power supply is turned off and stoppedStopping electrophoresis. Fixing the gel with fixing solution (30% ethanol and 10% acetic acid) at room temperature for 20min; treating with oxidizing solution (30% ethanol, 10% acetic acid and 0.7% periodic acid) for 20min; the gel is washed for 1h by shaking water, and the water is changed every 20min and is fully washed. Using a silver ammonia solution (56 mL, 4mL of concentrated aqueous ammonia added to 0.1M NaOH, water to 230mL, then dropwise adding 10mL of 20% ₃ The solution is clear and transparent, and needs to be prepared again if precipitation occurs, and the silver ammonia solution needs to be prepared at present) for 10min; the gel was washed with shaking water for 30min and the water was changed every 10min. Color developing solution (0.05 g/L citric acid and 0.02% formaldehyde) was added until LPS bands appeared. To prevent excessive staining, when a clear LPS band appeared, the reaction was stopped by immediately adding 7% glacial acetic acid.

As a result, as shown in FIG. 3, lane 1 is an electrophoretogram of LOS of wild-type E.coli strain MG1655, and LPS of strain MG1655 is only core-kdo ₂ -a lipid a structure; lane 2 shows the LPS structure of LPS-reduced strain MW012, in which only Kdo is present in the LPS structure of strain MW012 ₂ A lipid A structure, and thus its migration speed is fast. The structure of LPS extracted from the MW012 strain was further analyzed by LC-MS. There are three main peaks in LC-MS spectra, 1797.2, 2157.5 and 2395.7, corresponding to lipid A and kdo respectively ₂ -1-dephosphorylated A and kdo 2-1-dephosphorylated-2-palmityl-lipidated A. This indicates that a significant portion of lipid a is palmitoylated in the LPS structure of the MW012 strain, which further indicates that MW012 is a very suitable efficient producer of MPL.

Example 4: construction of expression plasmid pWEPL

Firstly, taking a Francis genome as a template, performing PCR amplification on a 720bp gene fragment by using primers FnlpxE-F and FnlpxE-R, and performing electrophoresis verification, purification and recovery; carrying out enzyme digestion reaction on the vector pWSK29 by using SacI endonuclease respectively, wherein the reaction temperature is 37 ℃ and the reaction time is 30min, and then recovering an enzyme digestion product and carrying out electrophoresis verification; mixing the recovered enzyme digestion product and the gene fragment, and reacting for 30min at 37 ℃ according to the instruction of the one-step cloning kit to obtain a reaction solution; after the reaction is finished, taking out the competent cells of the Escherichia coli JM109, melting the competent cells on ice, adding the reaction solution into the competence, slightly blowing, sucking and uniformly mixing, and carrying out ice bath for 30min; then the competence is thermally shocked for 90s in a water bath at 42 ℃, ice-washed for 2min, and rapidly added into 1mL of LB culture medium; resuscitated at 37 ℃ for 1h at 100rpm, then spread on LB plates supplemented with 50mg/L ampicillin (Amp), and cultured in an inverted manner at 37 ℃ to screen knockout transformants; carrying out colony PCR on the transformant by taking pWSK29 as a negative control, and verifying whether the plasmid is successfully constructed; the correct transformant was transferred to an LB liquid tube to which ampicillin (Amp) was added, and the plasmid was extracted to obtain plasmid pWE.

Secondly, taking salmonella genome as a template, performing PCR amplification on a 570bp gene fragment by using primers SepagP-F and SepagP-R, and performing electrophoresis verification, purification and recovery; carrying out enzyme digestion reaction on plasmid pWE by utilizing HindIII endonuclease, wherein the reaction temperature is 37 ℃ and the reaction time is 30min, and then respectively recovering enzyme digestion products and carrying out electrophoresis verification; mixing the recovered enzyme digestion product and the gene fragment, and reacting for 30min at 37 ℃ according to the instruction of the one-step cloning kit to obtain a reaction solution; the reaction solution was transformed into competent cells of Escherichia coli JM109, spread on an LB plate to which 50mg/L of ampicillin (Amp) was added, and subjected to inverted culture at 37 ℃ to screen knockout transformants; carrying out colony PCR on the transformant by taking pWE as a negative control to verify whether the plasmid is successfully constructed; the correct transformant was inoculated into an LB liquid tube to which ampicillin (Amp) was added, and the plasmid was extracted to obtain plasmid pWEP.

Thirdly, taking salmonella genome as a template, performing PCR amplification on a 705bp gene fragment by using primers SepagL-F and SepagL-R, and performing electrophoresis verification, purification and recovery; carrying out enzyme digestion reaction on plasmid pWEP by using XhoI endonuclease at 37 ℃ for 30min, and then respectively recovering enzyme digestion products and carrying out electrophoresis verification; mixing the recovered enzyme digestion product and the gene fragment, and reacting for 30min at 37 ℃ according to the instruction of the one-step cloning kit to obtain a reaction solution; the reaction solution was transformed into competent cells of Escherichia coli JM109, spread on an LB plate to which 50mg/L of ampicillin (Amp) was added, and subjected to inverted culture at 37 ℃ to select a knockout transformant; carrying out colony PCR on the transformant by taking pWEP as a negative control to verify whether the plasmid is successfully constructed; the correct transformant was transferred to an LB liquid tube to which ampicillin (Amp) was added, and the plasmid was extracted to obtain plasmid pWEPL. The plasmid map is shown in FIG. 4.

Example 5: construction of recombinant Strain MW012/pWEPL

(1) Preparation of Escherichia coli MW012 competent cells

Escherichia coli MW012 obtained in example 2 was inoculated into LB liquid medium, cultured overnight at 37 ℃ and 200rpm, and the seed solution was inoculated at 2% (v/v) to 50mL of LB liquid medium, cultured at 37 ℃ and 200rpm to OD ₆₀₀ =0.4-0.6, transferring culture solution into pre-cooled 50mL centrifuge tube after ice bath for half an hour, centrifuging at 4 deg.C and 8000rpm for 10min, collecting thallus, precipitating with pre-cooled 0.01M CaCl ₂ Washing 3 times, and finally using 1mL of 0.01M CaCl ₂ Suspending, adding 1mL of 30% glycerol, mixing, and subpackaging 200 μ L each tube into a pre-cooled sterile EP tube.

(2) Transformation of

100-200ng of plasmid pWEPL obtained in example 4 was added to the competent cells of Escherichia coli MW012 prepared in step (1), mixed well, subjected to heat shock at 42 ℃ for 90s in an ice bath for 2-3 min, resuscitated in 1mL of LB medium, incubated at 37 ℃ for 2h, spread on a 50. Mu.g/mL LB solid plate for ampicillin, cultured at 37 ℃, and transformants were selected for seed culture in LB liquid medium containing 100. Mu.g/mL ampicillin. The recombinant strain MW012/pWEPL was obtained.

Example 6: growth Performance assay for E.coli MG1655, MW012 and MW012/pEPL

The strain to be tested is streaked on a plate (wild type escherichia coli is streaked on an LB plate, and recombinant bacteria is streaked on an LBA plate), and a single colony is selected and inoculated into 5mL of seed culture medium for overnight culture. The following day the seed medium was used as the initial OD ₆₀₀ =0.02 inoculations in 50mL LB medium, three replicates per strain. Samples were taken every 2 hours and the absorbance at 600nm of the samples was measured, and two time points were measured after the strain had grown to a plateau. And drawing a line graph by taking the time as an abscissa and the light absorption value as an ordinate to obtain a growth curve.

The results showed that, as shown in FIG. 5, the strain MG1655 grew in LB medium for 12h to reach stationary phase, and its highest OD ₆₀₀ The value of (b) is 4.29; the strain MW012 grows in LB culture medium for 12h to reach the stationary phase, and the highest OD thereof ₆₀₀ The value of (b) is 2.99; the strain MW012/pWEPL grows for 12h in LB culture medium to reach the stationary phase, and the highest OD of the strain ₆₀₀ The value of (A) is 2.56. The result shows that the recombinant bacteria and the wild bacteria have the same growth tendency in the LB culture medium, but the growth performance is reduced.

Example 7: extraction and structure verification of recombinant strain MW012/pWEPL lipoid A structure

The extraction of the lipid A structure of E.coli in example 6 was performed by chloroform/methanol/water mixed phase extraction. The overnight cultured broth was expressed as the initial OD ₆₀₀ =0.02 transfer to 200mL LB liquid medium, 37 ℃ culture to OD ₆₀₀ When the concentration is 1, the cells are centrifuged at 8000r/min for 10min to collect the cells. 25Mm EDTA is added before the fermentation of the strain MW012/pWEPL is finished. ddH ₂ O washing the thalli once, suspending the thalli by a Bligh-Dyer one-phase system (chloroform/methanol/water, 1:2:0.8, v/v/v), magnetically stirring for 1h, centrifuging at 2000r/min for 20min, separating phases, and washing cell fragments for 2-3 times by using the one-phase system; 27mL of a 12.5mmol/L sodium acetate (pH 4.5) solution was added, followed by ultrasonic shaking for 10min and water bath at 100 ℃ for 30min to cleave the sugar chains. Cooling to room temperature, adding 30mL of chloroform and 30mL of methanol to prepare a Bligh-Dyer two-phase system (chloroform/methanol/water, 2; finally lipid A was washed out by adding chloroform/methanol solution (4; blowing the organic solvent with a nitrogen blower, and storing the lipoid A at-20 deg.C for use.

Lipid a was dissolved in chloroform/methanol solution (4, 1,v/v) and the sample was spotted on a gel 60TLC plate with chloroform/methanol/water/ammonia (40. After the chromatography is finished, the residual spreading agent on the plate is dried by blowing, carbonized by 10 percent sulfuric acid dissolved in ethanol, placed on a heating plate and developed at 180 ℃.

Lipid A was dissolved in chloroform/methanol solution (4, 1,v/v) and Mass spectrometric detection was performed on a WATERS SYNAPT Q-TOF Mass Spectrometer. And the detection range is less than m/z 2500 by adopting an anion detection mode. Data were acquired and analyzed using MassLynx v4.1software software.

The results show that: as shown in FIG. 6, lane 1, a control group, is lipid A extracted from the strain MG1655/pWEPL, and four bands represent from top to bottom: 1-dephosphoryl-2-palmityl-lipid A, MPL, 1-dephosphoryl-lipid A and 1-dephosphoryl-3-Deacyl-lipid A; lane 2 is lipid A extracted from the strain MW012/pEPL, with only two structures, MPL and 1-dephosphoryl-3-Deacyl-lipid A, respectively, from top to bottom, wherein the proportion of MPL is up to 75%. Lipid A extracted from strain MW012/pWEPL was analyzed by LC-MS. There are two major peaks in the LC-MS spectra, 1490.1 and 1728.3, corresponding to 1-dephosphoryl-3-deacyl-lipid A and MPL, respectively. The secondary mass spectral analysis of the 1728.3 peak showed that the 1500.1 peak in the primary mass spectrum was the fragment peak of the 1728.3 peak, which was generated during LC-MS. These results indicate that MPL can be efficiently synthesized in recombinant bacteria MW012/pWEPL.

Comparative example 1

One of the conditions that the MPL-producing strains have to meet is that the strains must have the ability to highly palmitoylate the lipid A structure. While palmitoylation of the lipid a structure needs to satisfy two conditions: the PagP protein (which is contained in plasmid pWEPL) is required to disrupt the lipid asymmetry of the bacterial outer membrane in order to allow efficient localization of phospholipids to the outer membrane. The strain MW012 knocks out the O-antigen, core polysaccharide, clavulanic acid and common intestinal antigen gene cluster, then the lipid asymmetry of the outer membrane is interfered, and on the basis, the mlaA, mlaC and pldA genes related to a phospholipid transport system are knocked out, so that the phospholipid can be stably positioned on the outer layer of the outer membrane.

Such as: pWEPL was directly expressed in wild-type strain MG1655, the data are as follows: direct expression of pWEPL in wild-type MG1655 produced MPL but at very low yields (FIGS. 9 and 10).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A recombinant Escherichia coli, wherein an O-antigen gene cluster, a core sugar gene cluster, an intestinal common antigen gene cluster, a clavulanic acid gene cluster and a phospholipid transport system-associated gene on the genome of Escherichia coli are knocked out, and dephosphorylatases from the genome of Francisella and hexadecanoyl transferases and deacylases from the genome of Salmonella are overexpressed.

2. The recombinant Escherichia coli of claim 1, wherein the O-antigen gene cluster wbBL-galF comprises 13 genes, which are gnd, wbBL, wbbK, wbBJ, wbBI, rfc, glf, rfbX, rfbC, rfbA, rfbD, rfbB, galF, the NCBI sequence has accession numbers of NP-416533.1, NP-416534.1, NP-416536.1, NP-416537.1, NP-416538.1, NP-416539.1 "NP _416540.1", "NP _416541.1", "NP _416542.1", "NP _416543.1", "NP _416544.1", "NP _416545.1", "NP _416546.1";

the core carbohydrate cluster rfaD-waaQ comprises 14 genes, which are rfaD, waaF, waaC, waaU, waaL, waaZ, waaY, waaJ, waaR, waaB, waaS, waaP, waaG, waaQ, and the sequence of which has the accession numbers "NP _418076.1", "NP _418077.1", "NP _418078.1", "NP _418079.1", "NP _418080.1", "NP _418081.1", "NP _418082.1", "NP _418083.1", "NP _418084.1", "NP _418085.1", "NP _418086.1", "NP _418087.1", "NP _418088.1", "NP _418089.1".

3. The recombinant Escherichia coli according to claim 1, wherein the intestinal common antigen gene cluster rfe-rffM comprises 12 genes rfe, wzzE, wecB, wecC, rffG, rffH, rffC, wecE, wzxE, wecF, wzyE, rffM, and the sequences thereof are sequentially "NP _418231.1", "NP _418232.1", "YP _026253.1", "YP _026254.1", "YP _026255.1", "NP _418236.1", "YP _ 02682256.1", "NP _418238.1", "NP _418239.1", "YP _026257.1", "NP _418241.1", "NP 418242.1";

the clavulanic acid gene cluster wcaM-wza contains 20 genes, which are respectively 20 genes of wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, fcl, gmm, wcaI, manC, manB, wcaJ, wzx, wcaK, wcaL, wcaM, and the sequences of the genes are numbered as NP _416566.1"," NP _416565.1"," NP _416564.1"," NP _416563.1"," NP _416562.1"," NP _416561.1"," NP _416560.1"," NP _416559.1"," NP _416558.1"," NP _416557.1"," NP _416556.1"," NP _416551.1"," NP _416552.1"," NP _416551 "," NP _416551 _ 41651 "," NP _416552.1"," NP _416551 _ 416551.41651 "," NP _416551 "," NP _416552.1"," 41651 ";

the sequences of the genes related to the phosphate transport system, mlaA, mlaC and pldA, have accession number "NP _416848.1" at the NCBI. "NP-417659.1". "NP _418265.1".

4. The recombinant Escherichia coli according to claim 1, wherein the sequence derived from the IpxE gene of Francisella has NCBI accession number "WP _159184080.1";

the NCBI accession numbers for the sequences from the salmonella pagP and pagL genes are "NP _459620.1", "NP _416848.1", respectively.

5. The recombinant Escherichia coli of any one of claims 1 to 4, wherein the Escherichia coli is Escherichia coli MG1655.

6. A method for efficiently producing MPL, which comprises fermenting MPL using the recombinant Escherichia coli according to any one of claims 1 to 5.

7. The method according to claim 6, wherein the recombinant Escherichia coli is inoculated into a culture medium for culture.

8. The method according to claim 7, wherein MPL is extracted from the recombinant Escherichia coli after 2 to 18 hours of culture.

9. The method according to claim 7 or 8, wherein the culture medium contains 4-6 g/L yeast powder, 8-12 g/L peptone and 8-12 g/L NaCl.

10. Use of a recombinant E.coli as claimed in any one of claims 1 to 5 in the production of a lipid A vaccine adjuvant.