CN115181715B - A recombinant Escherichia coli capable of efficiently producing monophosphate lipid A vaccine adjuvant - Google Patents

Abstract

The invention discloses a recombinant Escherichia coli capable of efficiently producing monophosphoric acid lipid A vaccine adjuvant, belonging to the fields of genetic engineering and synthetic biology. The present invention knocks out the O-antigen gene cluster gnd-galF, the core sugar gene cluster rfaD-waaQ, the intestinal common antigen gene cluster rfe-rffM, the claric acid gene cluster wcaM-wza and the phospholipid transport systems mlaA, mlaC and pldA gene, and overexpress FnlpxE in Francisella chromosome and SepagP and SepagL gene in Salmonella chromosome to obtain recombinant strain MW012/pWEPL. Through simple fermentation, the MW012/pWEPL strain can efficiently synthesize monophosphate hexaacylated lipid A (MPL), and the proportion of MPL in all lipid A is as high as 75%.

Description

A recombinant Escherichia coli capable of efficiently producing monophosphate lipid A vaccine adjuvant

technical field

The invention relates to a recombinant Escherichia coli capable of efficiently producing monophosphoric acid lipid A vaccine adjuvant, belonging to the fields of genetic engineering and synthetic biology.

Lipopolysaccharide (LPS) is the main component of the outer membrane of Escherichia coli. Its hydrophilic sugar chain can prevent hydrophobic substances from entering the cell, maintain cell stability, and provide protection for the cell; its lipid A structure can be immune Pathogen recognition receptor TLR4 (Toll-like receptor4) recognition on cells (such as monocytes, macrophages, neutrophils and dendritic cells). When LPS is recognized by the receptor TLR4 on the surface of mammalian cells to activate the innate immune system, highly toxic LPS can cause excessive release of pro-inflammatory factors, causing heat shock and even death; while low-toxic LPS or its derivatives can effectively activate The innate immune system does not produce excess inflammatory molecules. Therefore, LPS or its derivatives with low toxicity can be used as immune system stimulators to be developed into vaccines or vaccine adjuvants.

Lipid A is the bioactive center of LPS and the recognition site of host immune cell TLR4. The number of phosphate groups, the number and length of fatty acid chains directly affect the types and quantities of pro-inflammatory cytokines released. Some structures of lipid A can induce excessive cytokine production, which can cause severe endotoxic shock, while some structures of lipid A can cause mild inflammatory responses, produce appropriate cytokines, induce Th1 type responses, attract and Activates macrophages and dendritic cells to help the host eliminate invading microorganisms. Therefore, different structures of lipid A have different immune functions, and some structures of lipid A can be used as vaccine adjuvants to enhance the intensity and duration of immune response. Vaccine adjuvants play an important role in the generation and enhancement of vaccine immune response. The most widely used vaccine adjuvant is aluminum adjuvant. It is an adsorbent that strongly adsorbs protein antigens from solution, forming a precipitate. When it is inoculated into the body, it can form an "antigen library", slowly release the antigen, fully prolong the action time of the antigen, and induce a high level of antibody response, but cannot induce strong T cell immunity. However, T cell immunity is urgently needed for the prevention and treatment of certain diseases, such as malaria, tuberculosis, and AIDS. Therefore, designing a vaccine adjuvant that can stimulate a strong Th1 cell immune response is a current research hotspot.

Monophosphate lipid A (MPL) is a molecular derivative of lipid A that can enhance the immune response, and is being developed into a new generation of vaccine adjuvant with broad application prospects. Commercial MPL can only be obtained by chemically processing the lipid A structure of Salmonella Minnesota R595, which is expensive and complex.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention knocks out four LPS-related gene clusters gnd-galF, rfaD-waaQ, rfe-rffM and wcaM-wza and phospholipid transport system genes mlaA, mlaC and pldA was obtained from the LPS streamlined strain MW012. Subsequently, the plasmid pWEPL carrying the lpxE in the Francisella chromosome and the pagP and pagL genes in the Salmonella chromosome was transformed into the LPS streamlined strain MW012 to obtain the recombinant strain MW012/pWEPL. The lipid A isolated from MW012/pWEPL was analyzed by thin layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS). The results showed that there were mainly two kinds of monophospholipid A in MW012/pWEPL, One is six acylation, the other is five acylation. More importantly, the proportion of hexaacylated monophosphate lipid A, the most effective component in the lipid A vaccine adjuvant, reached 75%. The Escherichia coli strain MW012/pWEPL constructed by this fermentation provides a good alternative for the production of lipid A vaccine adjuvant MPL.

The first object of the present invention is to provide a recombinant Escherichia coli that efficiently produces MPL, and the recombinant Escherichia coli has knocked out the O-antigen gene cluster, the core sugar gene cluster, the intestinal common antigen gene cluster and claritic acid on the Escherichia coli genome gene cluster and mlaA, mlaC and pldA genes in the phospholipid transport system, and overexpression of dephosphatase (LpxE) from the Francisella genome and palmitoyltransferase (PagP) and deacylase (PagL ).

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

In one embodiment, the O-antigen gene cluster gnd-galF comprises 13 genes, respectively gnd, wbbL, wbbK, wbbJ, wbbI, rfc, glf, rfbX, rfbC, rfbA, rfbD, rfbB, galF, The NCBI accession numbers of its sequence are "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".

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

In one embodiment, the core sugar gene cluster rfaD-waaQ comprises 14 genes, which are rfaD, waaF, waaC, waaU, waaL, waaZ, waaY, waaJ, waaR, waaB, waaS, waaP, waaG, waaQ , the accession numbers of its sequence on NCBI are "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, respectively rfe, wzzE, wecB, wecC, rffG, rffH, rffC, wecE, wzxE, wecF, wzyE, rffM, the sequences of which are The accession numbers on NCBI are "NP_418231.1", "NP_418232.1", "YP_026253.1", "YP_026254.1", "YP_026255.1", "NP_418236.1", "YP_026256.1", " NP_418238.1", "NP_418239.1", "YP_026257.1", "NP_418241.1", "NP_418242.1".

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

In one embodiment, the clarified acid gene cluster wcaM-wza comprises 20 genes, which are respectively wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, fcl, gmm, wcaI, manC , manB, wcaJ, wzx, wcaK, wcaL, wcaM a total of 20 genes, the NCBI accession numbers of their sequences are "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", "NP_416547.1".

In one embodiment, the NCBI accession numbers of the phospholipid transport system mlaA, mlaC and pldA gene sequences are "NP_416848.1", "NP_417659.1", "NP_418265.1".

In one embodiment, the NCBI accession number of the sequence from the Francisella lpxE gene is "WP_159184080.1"; the NCBI accession numbers of the sequence from the Salmonella pagP and pagL genes are "NP_459620.1", " NP_416848.1".

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

The second object of the present invention provides a method for constructing the above-mentioned recombinant Escherichia coli, the method is to knock out the O-antigen gene cluster, the core sugar gene cluster, the intestinal common antigen gene cluster, and the claritic acid gene cluster on the Escherichia coli genome Genes in , as well as mlaA, mlaC and pldA genes in the phospholipid transport system, and overexpression of dephosphatase (LpxE) from the Francisella genome and palmitoyltransferase (PagP) and deacylase ( PagL).

In one embodiment, Francisella-derived lpxE and Salmonella-derived pagP and pagL are linked to the expression plasmid pWSK29.

The third object of the present invention is to provide a method for efficiently producing MPL. The method is to inoculate the above-mentioned recombinant Escherichia coli into a fermentation medium for 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 fermentation reaction conditions are a temperature of 37° C., and 180-220 rpm.

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

The fifth object of the present invention is to provide the application of said recombinant Escherichia coli in the production of lipid A vaccine adjuvant.

Beneficial effect:

(1) The present invention knocks out four LPS-related gene clusters on the E. coli genome and the mlaA, mlaC and pldA genes in the phospholipid transport system in Escherichia coli to obtain the reduced strain MW012 of LPS. The reduced strain MW012 has a good growth state and its LPS The structure is Kdo ₂ -lipid A, which is the simplest structure of LPS.

(2) The plasmid pWEPL carrying lpxE derived from Francisella and pagP and pagL derived from Salmonella was transformed into LPS streamlined strain MW012 to obtain recombinant strain MW012/pWEPL. The recombinant strain MW012/pWEPL mainly produces two kinds of monophosphate lipid A, one is hexaacylated and the other is pentaacylated, and more importantly, the most effective component of lipid A vaccine adjuvant is hexaacylated The proportion of monophospholipid A reaches 75%.

(3) The recombinant Escherichia coli MW012/pWEPL strain constructed by the present invention grows well, the lipid A has a simple structure, and the extraction method is simple and convenient. Compared with the existing MPL obtained from Salmonella Minnesota R595 through chemical treatment, the strain MW012/pWEPL of the present invention is an Escherichia coli that is easy to cultivate, grows rapidly, and reduces production risks.

(4) The microbial transformation method provided by the present invention has simple fermentation conditions, short fermentation time, and simple extraction process, and no chemical treatment is required to extract active ingredients.

Description of drawings

Figure 1 is a knockout flowchart; X is the target gene cluster gnd-galF, rfaD-waaQ, rfe-rffM, wcaM-wza, mlaA, mlaC and pldA.

Figure 2 is a flow chart of the construction of the LPS streamlined strain MW012; a: the knockout gene cluster involved in this discovery; b: the specific process of knockout.

Figure 3 is the LPS structure of strain MW012 analyzed by SDS-PAGE.

Figure 4 is the LPS structure of strain MW012 analyzed by LC-MS.

Fig. 5 is the construction of the expression plasmid; the construction of the plasmid pWEPL expressing lpxE derived from Francisella and pagP and pagL derived from Salmonella.

Figure 6 is the growth curves of strains MG1655, MW012 and MW012/pEPL.

Figure 7 is the TLC analysis of the lipid A structure of the MW012/pWEPL strain.

Fig. 8 is the structure of lipid A of MW012/pWEPL strain analyzed by LC-MS.

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

Figure 10 is the LC-MS analysis of lipid A in MG1655/pWEPL strain.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

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

The medium involved in the following examples is as follows:

The media were all prepared with ddH ₂ O, and sterilized at 121°C for 15-20 minutes after preparation.

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

LBA medium (g/L): yeast powder 5, peptone 10, NaCl 10, Amp 0.05.

Add corresponding antibiotics according to the resistance gene carried on the plasmid.

The primer sequences involved in the following examples of table 1 (the underlined part is the N20 sequence)

Bacterial strains and plasmids involved in the following examples of table 2

Embodiment 1: Construction of knockout plasmid

Using the CRISPR/Cas9 knockout system to knock out the four gene clusters related to LPS in Escherichia coli and the mlaA, mlaC and pldA genes in the phospholipid transport system, seven knockout plasmids need to be constructed: pT1, pT2, pT3, pT4, pTargetF -mlaA, pTargetF-mlaC and pTargetF-pldA, the construction process of these plasmids is as follows:

(1) Select a 20nt _N20 sequence complementary to the target gene target sequence, the specific _N20 sequences are the underlined sequences of the primers pT1, pT2, pT3, pT4, pTargetF-mlaA, pTargetF-mlaC and pTargetF-pldA, and modify these sequences To the 5' end of the plasmid pTargetF forward primer, forward primers pT1-F, pT2-F, pT3-F, pT4-F, pTargetF-mlaA-F, pTargetF-mlaC-F and pTargetF-pldA-F were obtained respectively.

Using the plasmid pTargetF as a template, the forward primers pT1-F, pT2-F, pT3-F, pT4-F, pTargetF-mlaA-F, pT argetF-mlaC-F and pTargetF-pldA-F are the same as the reverse primer pTargetF- R was used to amplify the open-circle plasmid with the _N20 sequence introduced by PCR. The PCR amplification products were verified by electrophoresis and purified and recovered.

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

(3) Use T4 polynucleotide kinase (T4 PNK) to phosphorylate the recovered product after digesting the template plasmid, react at 37°C for 30min, and then heat inactivate at 65°C for 10min.

(4) Add 1 μL of T4 DNA ligase to the phosphorylated reaction system, and react at 22° C. for 4 hours to obtain a ligation solution. After the reaction, the competent cells of Escherichia coli JM109 were taken out and thawed on ice, and the connection solution was added to the competent cells, mixed by blowing and aspiration, and kept in an ice bath for 30 minutes. Then the competent cells were heat-shocked in a water bath at 42°C for 90 s, and ice-bathed for 2 min, and 1 mL of LB medium was quickly added. Resuscitate at 37°C for 1 hour at 100 rpm, spread on LB plates supplemented with 50 mg/L spectinomycin (Spe), and culture upside down at 37°C to screen for knockout transformants. Using pTargetF as a negative control, colony PCR was performed on the transformants to verify whether the knockout plasmid was successfully constructed. Connect the correct transformants to LB liquid test tubes added with spectinomycin (Spe), extract the plasmids to obtain knockout plasmids pTargetF-gene (pT1 (knockout O-antigen gene cluster), pT2 (knockout core polysaccharide gene cluster) , pT3 (knockout of the claritic acid gene cluster), pT4 (knockout of the intestinal common antigen gene cluster), pTargetF-mlaA, pTargetF-mlaC and pTargetF-pldA).

Example 2: Construction of LPS streamlined strain MW012

The CRISPR/Cas9 knockout system was used to knock out the four gene clusters related to LPS and the mlaA, mlaC and pldA genes in the phospholipid transport system of wild-type Escherichia coli MG1655 to obtain the LPS streamlined strain MW012. The specific knockout process is as follows (Figure 1):

(1) Preparation of Escherichia coli electroporation knockout competent cells MG1655/pCas

The plasmid pCas is transformed into Escherichia coli MG1655 to obtain recombinant Escherichia coli MG1655/pCas containing the pCas plasmid, and the Escherichia coli MG1655/pCas is activated on the LB solid plate adding 30mg/L Kanamycin (Kan), and inoculated into LB (Kan ⁺ ) test tube was cultivated overnight to obtain seed liquid; transfer the seed liquid to 25mL LB (Kan+) medium at 1% (v/v), cultivate at 30°C and 200rpm until OD ₆₀₀ =0.2, add 500μL Induced by L-arabinose solution, continue to culture until OD ₆₀₀ = 0.5, ice-bath for 30 minutes; 4°C, 4000rpm centrifuge for 10 minutes to collect the bacteria, wash the bacteria three times with pre-cooled 10% glycerol solution; add 300 μL 10% glycerol solution to resuspend Bacteria were dispensed into sterile 1.5mL EP tubes, 80μL/tube.

(2) Construction of homology arm knockout fragments

The genome of Escherichia coli MG1655 was extracted, and the genome was used as a template to amplify the upstream homology arm and Downstream homology arm, gel recovery, and overlapping PCR with primers U1-F/D1-R to obtain homology arm knockout fragment 1; use homology arm primer U2-F/U2 to knock out the target gene cluster rfaD-waaQ -R, D2-F/D2-R were respectively amplified to obtain the upstream homology arm and the downstream homology arm, the gel was recovered, and primers were used to perform overlapping PCR on U2-F/D2-R to obtain the homology arm knockout fragment 2; Use the homology arm primers U3-F/U3-R and D3-F/D3-R to knock out the target gene cluster wza-wcaM to amplify respectively to obtain the upstream homology arm and the downstream homology arm, recover the gel, and use the primer pair U3 -F/D3-R carry out overlap PCR, obtain the homology arm knockout fragment 3; Use the homology arm primer U4-F/U4-R of knocking out target gene cluster rfe-rffM, D4-F/D4-R amplifies respectively Increase the upstream homology arm and downstream homology arm, recover the gel, and use the primers to perform overlapping PCR on U4-F/D4-R to obtain the homology arm knockout fragment 4; use the homology arm primer to knock out the target gene cluster mlaA U5-F/U5-R, D5-F/D5-R were amplified respectively to obtain the upstream homology arm and the downstream homology arm, recovered from the gel, and performed overlapping PCR on U5-F/D5-R with primers to obtain the homology arm Knock out fragment 5; use the homology arm primers U6-F/U6-R and D6-F/D6-R to knock out the target gene cluster mlaC to amplify respectively to obtain the upstream homology arm and the downstream homology arm, recover the gel, and use The primer pair U6-F/D6-R was overlapped by PCR to obtain the homology arm knockout fragment 6; the homology arm primers U7-F/U7-R and D7-F/D7-R were used to knock out the target gene cluster pldA respectively The upstream homology arm and the downstream homology arm were amplified, the gel was recovered, and the primer pair U7-F/D7-R was used for overlapping PCR to obtain the homology arm knockout fragment 7.

(3) Electroporation knockout of gene

Wash the electric shock cup three times with absolute ethanol, blow dry, and pre-cool for 20 minutes. The Escherichia coli MG1655/pCas competent cells of step (1) were placed on ice to thaw, and the 100ng knockout plasmid pTargetF-gene (pT1, pT2, pT3, pT4, pTargetF-mlaA, pTargetF-mlaC and pTargetF-pldA) and 500ng of the corresponding homology arm knockout fragment in step (2), gently pipette and mix well, and suck into the groove of the electric shock cup. Place the electric shock cup in an ice bath for 10 minutes, and then electric shock after drying. Then quickly add 1mL LB medium to the electric shock cup, suck out all the bacterial solution into a 1.5mL EP tube, recover at 30°C, 100rpm for 1.5h, spread on the LB plate containing 30mg/L Kan and 50mg/L Spe, and Incubate upside down in a 30°C incubator. Using MG1655 as a negative control, the forward primer of the upstream homology arm and the reverse primer of the downstream homology arm were used to screen the transformants for correct transformants.

(4) Removal of knockout plasmid pTargetF-gene and thermosensitive plasmid pCas

Connect the correct knockout transformant in step (3) to an LB test tube added with 30 mg/L Kan and 1 mM IPTG, induce the removal of the enzyme expression of the knockout plasmid pTargetF-gene by IPTG, culture at 30°C for 12 hours with shaking, and incubate Single colonies were isolated by streaking on LB (Kan+) plates. A single colony sensitive to spectinomycin is screened out to obtain a mutant strain that removes the pTargetF-gene knockout plasmid, which is then transferred to LB (Kan+) test tubes for preservation, and can be directly prepared for continuous knockout. Inoculate the mutated strain without the knockout plasmid into an LB test tube, culture it with shaking at 42°C, and isolate a single colony by streaking on an LB plate. A single colony sensitive to kanamycin was screened out, that is, a non-resistance mutant strain without pCas was obtained, and then transferred to LB test tubes for preservation.

The CRISP/Cas9 knockout method successfully knocked out four LPS-related gene clusters gnd-galF, rfaD-waaQ, wza-wcaM, rfe-rffM and three phospholipid transport-related mlaA, mlaC and pldA genes from the large intestine. The genome of Bacillus MG1655 was sequentially knocked out, and the LPS-stripped strain MW012 was obtained (Fig. 2).

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

The gene cluster rfaD-waaQ contains rfaD, waaF, waaC, waaU, waaL, waaZ, waaY, waaJ, waaR, waaB, waaS, waaP, waaG, waaQ a total of 14 genes;

The gene cluster wza-wcaM contains wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, fcl, gmm, wcaI, manC, manB, wcaJ, wzx, wcaK, wcaL, wcaM a total of 20 Gene;

The gene cluster rfe-rffM comprises rfe, wzzE, wecB, wecC, rffG, rffH, rffC, wecE, wzxE, wecF, wzyE, rffM total 12 genes.

Example 3: Extraction and structure verification of strain MW012 lipopolysaccharide (LPS)

(1) LPS extraction and purification method: the strain was inoculated into 500 mL of LB medium with an initial OD ₆₀₀ of 0.02, cultured at 37°C for 18 hours, centrifuged for 20 minutes, the supernatant was discarded, and the bacterial precipitate was collected. Add 20 mL of water to the bacterial cell pellet to fully suspend, then add 20 mL of 90% phenol preheated at 68°C, and shake in a constant temperature water bath at 68°C for 1 hour. After shaking, cool to room temperature, centrifuge for 20 minutes to separate the phases, absorb the upper phase into a centrifuge tube, let stand at 4°C for 12 hours, transfer the supernatant to a dialysis bag, and dialyze for 24 hours. Vacuum freeze-dried to obtain a crude sample of LOS. Add 9 mL of water, 1 mL of reaction buffer (100 mM Tris-HCl, 25 mM MgCl ₂ , 1 mM CaCl ₂ , pH 7.5), appropriate amount of DNase I and RNase A to the crude LOS sample, and let stand at 37°C for 4 h (weigh the crude LOS sample with a precision balance , 1mg lipopolysaccharide added 1μg enzyme). Add an appropriate amount of proteinase K to the reacted solution, and let stand at 37° C. for 12 hours (1 μg of enzyme is added to 1 mg of lipopolysaccharide). To remove residual protein, add 5 mL of water-saturated phenol to the centrifuge tube and mix well. Centrifuge for 30 minutes to separate the phases, absorb the upper phase into a dialysis bag, dialyze in water for 24 hours, change the water every 4 hours, and remove the residual phenol in the solution. The liquid in the dialysis bag was poured into a centrifuge tube, and vacuum freeze-dried to obtain semi-pure LOS. Redissolve the semi-pure LOS in a mixture of chloroform and methanol (2:1, v/v), centrifuge at 12,000 rpm for 20 min, and discard the supernatant. Repeat the above operations, blow dry, redissolve in water, and vacuum freeze-dry to obtain pure LOS.

(2) SDS-PAGE analysis of LPS: prepare 20g/L LOS solution. Add 5 μL of 4×SDS loading buffer (50M Tris-HCL, 2% SDS, 10% sucrose and 0.01% bromophenol blue, pH 6.8) to 15 μL of LOS solution, heat in a boiling water bath for 10 min, and wait for the sample to cool to room temperature Afterwards, polyacrylamide gel electrophoresis was performed, and 20 μL of the solution was added to the wells of the gel. The stacking gel current was set to 15mA, and when the band ran to the interface between the separating gel and the stacking gel, the current was adjusted to 25mA. When the sample band runs to about 5mm from the bottom of the gel, turn off the power and stop the electrophoresis. At room temperature, use fixative solution (30% ethanol and 10% acetic acid) to fix the gel for 20 min; use oxidizing solution (30% ethanol, 10% acetic acid and 0.7% periodic acid) to treat for 20 min; shake the water to wash the gel for 1 h, every Change the water every 20 minutes and wash thoroughly. Use silver ammonia solution (56mL, 0.1M NaOH, add 4mL concentrated ammonia water, add water to 230mL, then add 10mL 20% AgNO ₃ solution drop by drop, the solution should be clear and transparent, if precipitation occurs, it needs to be re-prepared, silver ammonia solution needs to be fresh Treat the gel with freshly prepared) for 10 minutes; wash the gel with shaking water for 30 minutes, and change the water every 10 minutes. Add chromogenic solution (0.05g/L citric acid and 0.02% formaldehyde) to treat until the LPS band appears. To prevent over-staining, immediately add 7% glacial acetic acid to terminate the reaction when obvious LPS bands appear.

The results show that, as shown in Figure 3, lane 1 is the electropherogram of the wild-type E. coli MG1655 strain LOS, and the LPS of the MG1655 strain has only a structure of core-kdo ₂ -lipid A; lane 2 is the LPS structure of the LPS streamlined strain MW012, the strain Only Kdo ₂ -lipid A structure exists in the LPS structure of MW012, so its migration speed is relatively fast. The structure of LPS extracted from MW012 strain was further analyzed by LC-MS. There are three main peaks in the LC-MS spectrum, which are 1797.2, 2157.5 and 2395.7, corresponding to lipid A, kdo ₂ -1-dephospho-lipid A and kdo2-1-dephospho-2-palmitoyl-lipid A, respectively. This shows that in the LPS structure of the MW012 strain, a considerable part of lipid A has undergone palmitoylation, which further indicates that MW012 is a very suitable strain for high-efficiency production of MPL.

Embodiment 4: Construction of expression plasmid pWEPL

In the first step, using the Francisella genome as a template, a 720bp gene fragment was amplified by PCR using primers FnlpxE-F and FnlpxE-R, and verified by electrophoresis and purified; the vector pWSK29 was digested with SacI endonuclease respectively Reaction, the reaction temperature is 37°C, the time is 30min, and then the digested product is recovered and verified by electrophoresis; the recovered digested product and gene fragment are mixed and reacted at 37°C for 30min according to the instruction manual of the one-step cloning kit to obtain the reaction solution After the reaction, take out the competent cells of Escherichia coli JM109 and thaw on ice, add the reaction solution into the competent cells and mix them by blowing gently, and put them in an ice bath for 30 minutes; Bath for 2 minutes, quickly add 1mL LB medium; recover at 100rpm for 1h at 37°C, then spread on LB plates supplemented with 50mg/L ampicillin (Amp), and culture upside down at 37°C to screen for knockout transformants; use pWSK29 As a negative control, colony PCR was performed on the transformant to verify whether the plasmid was successfully constructed; the correct transformant was connected to an LB liquid test tube added with ampicillin (Amp), and the plasmid was extracted to obtain the plasmid pWE.

The second step is to use the Salmonella genome as a template, use primers SepagP-F and SepagP-R to perform PCR amplification of a 570bp gene fragment, and perform electrophoresis verification and purification recovery; use HindIII endonuclease to carry out enzyme digestion reaction on plasmid pWE, The reaction temperature was 37°C, and the reaction time was 30 minutes. Then the digested products were recovered separately and verified by electrophoresis; the recovered digested products and gene fragments were mixed and reacted at 37°C for 30 minutes according to the instruction manual of the one-step cloning kit to obtain a reaction solution; Transform the reaction solution into Escherichia coli JM109 competent cells, smear it on an LB plate supplemented with 50 mg/L ampicillin (Amp), and culture it upside down at 37°C to screen knockout transformants; Colony PCR was carried out to verify whether the plasmid was successfully constructed; the correct transformant was connected to an LB liquid test tube added with ampicillin (Amp), and the plasmid was extracted to obtain plasmid pWEP.

The third step is to use the Salmonella genome as a template, use primers SepagL-F and SepagL-R to perform PCR amplification of a 705bp gene fragment, and perform electrophoresis verification and purification recovery; use XhoI endonuclease to carry out enzyme digestion reaction on plasmid pWEP, The reaction temperature was 37°C, and the reaction time was 30 minutes. Then the digested products were recovered separately and verified by electrophoresis; the recovered digested products and gene fragments were mixed and reacted at 37°C for 30 minutes according to the instruction manual of the one-step cloning kit to obtain a reaction solution; Transform the reaction solution into Escherichia coli JM109 competent cells, smear it on an LB plate supplemented with 50 mg/L ampicillin (Amp), and culture it upside down at 37°C to screen knockout transformants; Colony PCR was carried out to verify whether the plasmid was successfully constructed; the correct transformant was connected to an LB liquid test tube added with ampicillin (Amp), and the plasmid was extracted to obtain plasmid pWEPL. The plasmid map is shown in Figure 4.

Embodiment 5: Construction of recombinant bacterial strain MW012/pWEPL

(1) Preparation of Escherichia coli MW012 Competent Cells

Escherichia coli MW012 in the inoculation embodiment 2 is in LB liquid medium, 37 ℃, 200rpm cultivates overnight, seed liquid is transferred to 50mL LB liquid medium by 2% (v/v) inoculum amount, 37 ℃, 200rpm cultivates To OD ₆₀₀ =0.4-0.6, put the culture medium in an ice bath for half an hour and then transfer it to a pre-cooled 50mL centrifuge tube, centrifuge at 8000rpm for 10min at 4°C to collect the bacteria, and wash the precipitate three times with pre-cooled 0.01M _CaCl2 , Finally, suspend with 1 mL of 0.01M CaCl ₂ , add 1 mL of 30% glycerol to mix, and distribute 200 μL of each tube into pre-cooled sterile EP tubes.

(2) conversion

Add 100-200ng of the plasmid pWEPL in Example 4 to the Escherichia coli MW012 competent cells prepared in step (1), mix well, bathe in ice for 30min, heat shock at 42°C for 90s, bathe in ice for 2-3min, add 1mL LB for culture The base was revived, incubated at 37°C for 2 hours, coated with 50 μg/mL ampicillin on an LB solid plate, cultured at 37°C, and the transformants were picked and cultured in LB liquid medium containing 100 μg/mL ampicillin. The recombinant strain MW012/pWEPL was obtained.

Embodiment 6: Escherichia coli MG1655, MW012 and MW012/pEPL growth performance assay

Streak the strains to be tested on the plate (the wild-type Escherichia coli is streaked on the LB plate, and the recombinant bacteria are streaked on the LBA plate), and a single colony is picked and inoculated into 5 mL of seed medium for overnight culture. The next day, the seed medium was inoculated in 50 mL of LB medium with an initial OD ₆₀₀ =0.02, and each strain was replicated in triplicate. Samples were taken every 2 hours and the absorbance value of the sample at 600nm was measured, and two time points were measured after the strain grew to a stationary phase. Take time as the abscissa and the absorbance value as the ordinate to make a line graph, which is the growth curve.

The results showed that, as shown in Figure 5, the strain MG1655 grew in LB medium until 12h and reached the stationary phase, and its highest OD ₆₀₀ value was 4.29; the strain MW012 grew in LB medium until 12h and reached the stationary phase, and its highest OD ₆₀₀ The value of 2.99; strain MW012/pWEPL grown in LB medium to 12h reached the stationary phase, the highest OD ₆₀₀ value of 2.56. The results showed that the recombinant bacteria and wild bacteria had the same growth tendency in LB medium, but the growth performance was reduced.

Example 7: Extraction and structural verification of the lipid A structure of the recombinant strain MW012/pWEPL

The extraction of Escherichia coli lipid A structure in Example 6 adopts the chloroform/methanol/water mixed phase extraction method. The overnight cultured bacterial solution was transferred to 200 mL of LB liquid medium at an initial OD ₆₀₀ =0.02, cultivated at 37°C until OD ₆₀₀ =1, and centrifuged at 8000 r/min for 10 min to collect the bacterial cells. 25Mm EDTA should be added to the strain MW012/pWEPL before the end of fermentation. After washing the cells with ddH ₂ O once, suspend the cells in a Bligh-Dyer one-phase system (chloroform/methanol/water, 1:2:0.8, v/v/v), stir them magnetically for 1 h, and centrifuge at 2000 r/min for 20 min to separate the phases. Use a one-phase system to wash the cell debris 2-3 times; add 27mL of 12.5mmol/L sodium acetate (pH 4.5) solution, ultrasonically shake for 10min, and cleavage the sugar chains in a water bath at 100°C for 30min. After cooling to room temperature, add 30mL chloroform and 30mL methanol to form a Bligh-Dyer two-phase system (chloroform/methanol/water, 2:2:1.8, v/v/v), centrifuge at 2000r/min for 10min, remove the phase and transfer it to a rotary evaporator Rotary evaporation in the bottle; finally add chloroform/methanol solution (4:1, v/v) to wash out lipid A; use a nitrogen blower to dry the organic solvent, and store lipid A at -20°C for future use.

Lipid A was dissolved in chloroform/methanol solution (4:1, v/v), and the sample was spotted on a gel 60TLC plate, and the developing agent was chloroform/methanol/water/ammonia (40:25:4:2, v /v/v/v). After the end of the chromatography, dry the developing agent remaining on the plate, carbonize it with 10% sulfuric acid dissolved in ethanol, place it on a heating plate, and develop the color at 180°C.

Lipid A was dissolved in chloroform/methanol solution (4:1, v/v) and detected by mass spectrometry on a WATERS SYNAPT Q-TOF MassSpectrometer. Using negative ion detection mode, the detection range is less than m/z 2500. Data were acquired and analyzed using MassLynx V4.1 software.

The results show that: as shown in Figure 6, lane 1 is the control group, which is lipid A extracted from bacterial strain MG1655/pWEPL, and the four bands represent from top to bottom: 1-Dephospho-2-palmitoyl-lipid A, MPL , 1-Dephospho-lipid A and 1-Dephospho-3-Deacyl-lipid A; Lane 2 is the lipid A extracted from the strain MW012/pEPL, there are only two structures, from top to bottom are MPL and 1-Dephospho- 3-Deacyl-lipid A, in which the proportion of MPL is as high as 75%. Lipid A extracted from strain MW012/pWEPL was analyzed by LC-MS. There are two main peaks in the LC-MS spectrum, respectively 1490.1 and 1728.3, corresponding to 1-dephospho-3-deacyl-lipid A and MPL respectively. The secondary mass spectrometry analysis of the 1728.3 peak shows that the 1500.1 peak in the primary mass spectrum is a fragment peak of the 1728.3 peak, which is generated in the process of LC-MS. These results indicated that the recombinant strain MW012/pWEPL could efficiently synthesize MPL.

Comparative example 1

One of the conditions that MPL high-yielding strains need to meet is that the strain must have the ability to highly palmitoylate the lipid A structure. The palmitoylation of the lipid A structure needs to meet two conditions: the need for PagP protein (the plasmid pWEPL contains the PagP protein), and the need to destroy the lipid asymmetry structure of the bacterial outer membrane so that the phospholipids can be effectively positioned on the outer membrane. However, the lipid asymmetry of the outer membrane of the strain MW012 has been disturbed after knocking out the O-antigen, core polysaccharide, claric acid and intestinal common antigen gene clusters, and on this basis, the mlaA, mlaC and pldA gene, so that phospholipids can be stably located in the outer layer of the outer membrane.

For example: pWEPL was directly expressed in the wild-type strain MG1655, and the data were as follows: Although the direct expression of pWEPL in the wild-type MG1655 could produce MPL, the yield was extremely low (Figure 9 and Figure 10).

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (7)

1. A recombinant escherichia coli, it is characterized in that, described recombinant escherichia coli has knocked out O-antigen gene cluster, core sugar gene cluster, intestinal common antigen gene cluster, clarified acid gene cluster and phospholipid transport system on the escherichia coli genome gene, and overexpressed dephosphatase from the Francisella genome and hexadecyltransferase and deacylase from the Salmonella genome; The O-antigen gene cluster wbbL - galF contains 13 genes, respectively gnd , wbbL , wbbK , wbbJ , wbbI , rfc , glf , rfbX , rfbC , rfbA , rfbD , rfbB , galF , and the accession numbers of their sequences on NCBI "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 sugar gene cluster rfaD - waaQ contains 14 genes, respectively rfaD , waaF , waaC , waaU , waaL , waaZ , waaY , waaJ , waaR , waaB , waaS , waaP, waaG , waaQ , the sequences of which are registered on NCBI The numbers are "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"; The intestinal common antigen gene cluster rfe - rffM contains 12 genes, which are respectively rfe , wzzE , wecB , wecC , rffG , rffH , rffC , wecE , wzxE , wecF , wzyE , rffM , and the accession numbers on NCBI of their sequences are "NP_418231.1", "NP_418232.1", "YP_026253.1", "YP_026254.1", "YP_026255.1", "NP_418236.1", "YP_026256.1", "NP_418238.1", "NP_418239 .1", "YP_026257.1", "NP_418241.1", "NP_418242.1"; The clarified acid gene cluster wcaM - wza contains 20 genes, respectively wza , wzb , wzc , wcaA , wcaB , wcaC , wcaD , wcaE , wcaF , gmd , fcl , gmm , wcaI , manC , manB , wcaJ , wzx , wcaK , wcaL , wcaM have a total of 20 genes, and the accession numbers of their sequences on NCBI are "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","NP_416547.1"; The genes related to the phosphate transport system are mlaA , mlaC and pldA , and the NCBI accession numbers of the sequences are "NP_416848.1", "NP_417659.1", "NP_418265.1"; The NCBI accession number of the sequence from Francisella lpxE gene is "WP_159184080.1"; The NCBI accession numbers of the sequences of the pagP and pagL genes from Salmonella are "NP_459620.1" and "NP_416848.1", respectively. 2. The recombinant Escherichia coli according to claim 1, wherein the Escherichia coli is Escherichia coli MG1655. 3. A method for efficiently producing monophosphoric acid lipid A, characterized in that, the method is to use the recombinant Escherichia coli according to claim 1 to ferment and produce monophosphoric acid lipid A. 4. The method according to claim 3, characterized in that, the recombinant Escherichia coli is inoculated into the culture medium for culturing. 5. The method according to claim 4, characterized in that the monophosphate lipid A is extracted from the recombinant Escherichia coli after culturing for 2 to 18 hours. 6. according to the described method of claim 4 or 5, it is characterized in that, described substratum contains 4～6 g/L yeast powder, 8～12g/L peptone, 8～12 g/L NaCl. 7. The application of the recombinant escherichia coli described in claim 1 in the production of monophosphate lipid A vaccine adjuvant.