CN104844665B - A kind of Kdo of the low toxicity containing five fatty acid chains2The preparation and application of MPLA - Google Patents

Abstract

The invention discloses the preparation and application of Kdo ₂ monophosphoric acid lipid A containing five fatty acid chains with low toxicity, belonging to the field of bioengineering. The present invention transforms Escherichia coli, and synthesizes a five-chain Kdo ₂ -monophosphate lipid A with a special structure and low toxicity. This Kdo ₂ -lipid A molecule is not connected with a core polysaccharide long chain, and only consists of two 2-keto-3-deoxyoctanoic acid, five fatty acid chains and a single phosphate at C4'. The Kdo ₂ ‑lipid A has amphipathic properties, is convenient for separation, extraction and purification, can be detected and directly identified, and maintains the biological immune activity of the lipid A part, and has a significant attenuation effect compared with wild-type W3110 LPS, It is a vaccine adjuvant with great development potential. At the same time, the method for extracting and purifying the Kdo ₂ -lipid A provided by the present invention has simple steps and is easy to operate.

Description

Preparation and application of a low toxicity Kdo2-monophosphate lipid A containing five fatty acid chains

technical field

The invention relates to the preparation and application of Kdo ₂ -monophosphate lipid A containing five fatty acid chains with low toxicity, belonging to the field of bioengineering.

Background technique

Lipopolysaccharide (LPS), the bacterial endotoxin, is the main component of the outer membrane of most Gram-negative bacteria, mainly composed of Kdo ₂ -lipid A (Kdo ₂ -Lipid A), core polysaccharide (Core) and O-antigen (O-antigen) consists of three parts; among them, Kdo ₂ -lipid A is the hydrophobic end of LPS, and its structure is very conservative, connecting the hydrophilic core polysaccharide and O-antigen and assisting the two to adhere to the cell surface Constituting a complete cell wall, the core polysaccharides and O-antigens are heterogeneous, and there are various combinations of glycosyl types, numbers, and positions in different Gram-negative strains and even in the same strain. Gram-negative bacteria will release LPS on their surface after invading the host. LPS can be recognized by Toll Like Receptor 4 (TLR-4) on the surface of host immune cells, and then trigger a series of physiological and biochemical reactions in the cell. Produce TNF-α, IL-6, IL-8 and other inflammatory cytokines. Among them, Kdo ₂ -lipid A is the main part that binds to TLR-4 receptors, especially the fine structure of lipid A part determines the type and quantity of cytokines released after LPS stimulates cells.

In the LPS of wild-type E. coli and Salmonella, the lipid A part contains six or seven fatty acid chains, and has two phosphate groups at the C1 and C4' positions, which has high cytotoxicity. Certain lipid A with special structure and low toxicity can be used as a vaccine adjuvant to non-specifically enhance the body's immune response to antigens and improve vaccine efficiency. However, due to the very low water solubility of lipid A molecules, they cannot exhibit effective biological activity in the aqueous phase. Therefore, in recent decades, crude samples of LPS are still used in basic research or clinical practice to test the corresponding lipid A derivatives. activity research. However, due to the large molecular weight, structural diversity of core sugar and O antigen, LPS cannot be detected and quickly identified by mass spectrometry or nuclear magnetic resonance in real time, and there is no standardized extraction and purification method for the time being. In addition, in the actual production of lipid A vaccine adjuvant, Salmonella currently used is a pathogenic bacterium, which has certain safety hazards, and Salmonella simultaneously produces a variety of lipid A with different structures, and the extraction process is cumbersome and energy-intensive.

Escherichia coli W3110 is a non-pathogenic bacterium, its LPS part does not contain O-antigen, only Kdo ₂ -lipid A and core polysaccharide, and its Kdo ₂ -lipid A part has a single structure, which is superior to Salmonella as a starting strain for industrial production. The present invention transforms Escherichia coli and synthesizes a Kdo ₂ -lipid A with special structure and low toxicity. This Kdo ₂ -lipid A is not connected to the core polysaccharide long chain, and only consists of two 2-keto-3-deoxy Caprylic acid, five fatty acid chains and a single phosphate at the C4' position. This special Kdo ₂ - lipid A has amphipathic properties, is easy to separate, extract and purify, can be detected and directly identified, and maintains the biological activity of the lipid A part, and is significantly attenuated compared with wild-type W3110 LPS It is superior to lipid A and LPS molecules and has great development potential as a vaccine adjuvant. At the same time, the invention provides a standardized method for extracting and purifying Kdo ₂ -lipid A, with simple and clear steps and easy operation.

Contents of the invention

The first technical problem to be solved by the present invention is to provide a special structure, low toxicity, five fatty acid chain Kdo ₂ - monophosphate lipid A, its chemical formula is C ₉₆ H ₁₇₅ N ₂ O ₃₅ P, and its molecular weight is 1947.17, its structure contains 2 α-keto-3-deoxy-D-mannooctanoic acid (Kdo), 2 glucosamine, 1 phosphate group and 5 fatty acid chains, based on β-(1'-6) The linked D-glucosamine disaccharide is the backbone, the 6'-position is connected with diα-keto-3-deoxy-D-mannooctanoic acid (Kdo ₂ ), the 4'-position is phosphorylated, and the 2'-position is amino-linked (R)- 3-(dodecyloxy)tetradecyl, the 3'-position, 3-position and 2-position amino groups are respectively connected with tetradecyloxy groups, and the specific structural formula is shown in formula I. The compound is amphipathic and can be directly extracted by organic systems and directly detected and identified by ESI mass spectrometry; it has good solubility in aqueous systems and can specifically activate the innate immune system through TLR4/MD2 receptors, but at the same time exhibits Low cytotoxicity, has potential as an immune adjuvant.

The second technical problem to be solved by the present invention is to provide a genetically engineered Escherichia coli E. coli W3110ΔwaaCFΔlacIΔlpxMlacZ::FnlpxE capable of directly producing the attenuated Kdo ₂ -lipid A. The genetically engineered bacteria knocked out the regulatory protein in the lac operon encoded by the lacI gene; knocked in the FnlpxE gene at the lacZ site of the chromosome, and the protease encoded by it can hydrolyze the phosphate on the 1-position of the lipid A molecule; knocked out the encoded protein can The lpxM gene of the tetradecyl hydroxy fatty acid chain is added to the 3' position of the lipid A structure, and the waaCF gene cluster is knocked out to obtain the target object without the core polysaccharide.

The construction method of the Escherichia coli genetically engineered bacteria is to inactivate the waaCF, lacI, and lpxM genes in the E. coli W3110 chromosome genome by deletion mutations, and knock in the FnlpxE gene expressing heterologously in the lacZ gene.

The method mainly includes the following steps: first, knock out the lacI gene on the chromosome of wild-type Escherichia coli W3110, the lacI gene encodes a regulatory protein in the lac operon, and knocking out this gene can release the regulatory protein's effect on the late insertion of the lacZ site. The repression of source gene expression; secondly, on the basis of W3110△lacI strain, the FnlpxE gene was knocked in at the lacZ site of chromosome (lpxE is derived from Francisella, and the protease encoded by it can hydrolyze the phosphate at position 1 of lipid A molecule ), so that it forms a constitutive expression, and constructs the intermediate strain HW001; again, on the basis of HW001, the lpxM gene on the chromosome is knocked out, and the protein encoded by it can add a tetradecyl hydroxy fatty acid chain at the 3' position of the lipid A structure, The intermediate strain HW002 was constructed; finally, on the basis of HW002, the waaCF gene cluster was knocked out, and the genetically engineered strain BW002 for the purpose of directly producing and synthesizing five fatty acid chain Kdo ₂ -monophosphate lipid A with a special structure was constructed.

In one embodiment of the present invention, the nucleotide sequence of the constructed lacI knockout fragment is shown in SEQ ID NO.1.

In one embodiment of the present invention, the nucleotide sequence of the constructed FnlpxE-Fkan insert is shown in SEQ ID NO.2.

In one embodiment of the present invention, the nucleotide sequence of the constructed lpxM knockout fragment is shown in SEQ ID NO.3.

In one embodiment of the present invention, the nucleotide sequence of the constructed waaCF knockout fragment is shown in SEQ ID NO.4.

The present invention also provides a kind of convenient and rapid application of said Escherichia coli genetically engineered bacteria to produce the method for attenuated Kdo ₂ -lipid A, mainly comprising the following steps:

(1) Cultivation of bacteria: general shake flask fermentation culture is used, and the seed bacteria liquid is transferred to the initial stage of the stable period.

(2) Extraction and separation: the bacteria are collected, and the attenuated Kdo ₂ -lipid A is extracted by a chloroform/methanol/water mixed phase extraction method.

(3) DEAE fiber column purification: the attenuated Kdo ₂ -lipid A crude sample is mixed with membrane-coated phospholipids, and the phospholipid impurities can be washed out by loading the sample on the DEAE fiber column and then gradient elution to obtain high-purity Kdo ₂ - Lipid A.

Traditionally, lipid A is obtained by first extracting lipopolysaccharide with phenol, and then performing a series of processes such as hydrolysis, chromatography, and purification. The present invention directly synthesizes the Kdo ₂ -lipid A molecule containing only five fatty acid chains by knocking out the lpxM gene without trace, inserts and expresses the FnlpxE gene, and the protease encoded by it modifies the five fatty acid chain Kdo ₂ -lipid A molecules into five Fatty acid chain Kdo ₂ -monophosphate lipid A molecule, followed by seamless knockout of the waaCF gene, makes the strain unable to convert the first and second heptoses of the core polysaccharide in the inner membrane due to the lack of outer membrane heptose transferase Linked with the structurally modified Kdo ₂ -monophosphate lipid A molecule containing five fatty acid chains, the five fatty acid chain Kdo ₂ -monophosphate lipid A is directly turned out of the outer membrane during growth and produced. Kdo ₂ -lipid A with this special structure can be directly extracted without chemical hydrolysis or chromatography. The low-toxic five-fatty acid chain Kdo ₂ -monophosphate lipid A obtained by conventional shake flask fermentation can be directly extracted by the chloroform-methanol system, which is similar to the extraction of membrane phospholipids, without adding phenol and without acid/alkali hydrolysis. Compared with lipid A, the extraction process is simpler. And this novel Kdo ₂ -lipid A can be detected and analyzed directly by thin-layer chromatography (TLC) and electrospray mass spectrometry (ESI/MS), and usually lipopolysaccharide is because it contains a large amount of hydrophilic sugar chains, the molecular weight is too large, The fat solubility is so low that it cannot be directly analyzed by TLC and EIS/MS. In general, the Escherichia coli genetically engineered bacteria constructed by the present invention do not need inducers in the production process, and the product does not need further chemical treatment to crack sugar _chains . Large-scale production of phospholipid A; and the special structure Kdo ₂ -lipid A product produced by this strain of engineering bacteria is single, has amphipathicity, and has certain solubility in both organic phase and aqueous phase, which is more conducive to mixing with different vaccines with multiple The combination of these two forms has good vaccine adjuvant potential.

Description of drawings

Figure 1 TLC analysis of five-chain Kdo ₂ -monophosphate lipid A produced by genetically engineered bacteria BW002

1: WBB06Kdo ₂ - Lipid A sample; 2: Genetically engineered bacteria BW002Kdo ₂ - Lipid A sample

Figure 2 ESI/MS analysis of five-chain Kdo ₂ -monophosphate lipid A produced by genetically engineered bacteria BW002

A: WBB06Kdo ₂ - lipid A sample; B: genetically engineered bacteria BW002Kdo ₂ - lipid A sample

Figure 3 Toxicity analysis of mouse macrophage RAW264.7 stimulated by wild-type Escherichia coli W3110LPS and genetically engineered bacteria BW002 five-chain Kdo ₂ -monophosphate lipid A

Figure 4 Toxicity analysis of human macrophage THP-1 stimulated by wild-type Escherichia coli W3110LPS and genetically engineered bacteria BW002 five-chain Kdo ₂ -monophospholipid A

Figure 5 Analysis of hydrophobicity and self-aggregation characteristics of wild-type Escherichia coli W3110 and genetically engineered bacteria BW002

Figure 6 Antibiotic resistance analysis of wild-type Escherichia coli W3110 and genetically engineered bacteria BW002

Detailed ways

Example 1 Extraction and structural analysis of Kdo ₂ -lipid A produced by genetically engineered bacteria BW002

LPS produced by W3110 and containing long sugar chains cannot be directly extracted, and its structure cannot be directly analyzed by thin layer chromatography (TLC) and ESI/MS analysis due to its large mass and low solubility in the organic phase. Therefore, we selected Escherichia coli WBB06 (W3110waaCF::tet6), the heptose-deficient mutant of W3110 as a control for structural analysis. WBB06 was confirmed to be able to directly synthesize Kdo ₂ -lipid A with lipid A partially composed of six fatty acid chains and bisphosphates.

1. Extraction of Kdo ₂ - Lipid A

The Kdo ₂ -lipid A produced by the control bacteria WBB06 and the genetically engineered bacteria BW001 was extracted using the chloroform/methanol/water mixed phase extraction method. The specific operation is as follows: Transfer the overnight cultured seed bacteria solution to 400mL LB liquid medium according to the initial OD ₆₀₀ = 0.02, cultivate at 37°C until OD ₆₀₀ = 1, collect the bacteria by centrifugation at 8000rpm for 10 minutes, wash the bacteria once with ddH ₂ O Use Bligh-Dyer one-phase system (chloroform/methanol/water, 1:2:0.8, v/v/v) to suspend the bacteria, magnetically stir for 1 hour, and centrifuge at 2000rpm for 20 minutes to separate the phases, take the upper phase, and then add appropriate amount of chloroform and water Make a Bligh-Dyer two-phase system (chloroform/methanol/water, 2:2:1.8, v/v/v), centrifuge at 2000rpm for 10min, remove the phase and transfer it to a rotary evaporator, and rotate to evaporate; finally add chloroform/methanol solution (2:1, v/v) Kdo ₂ -lipid A was washed out.

2. Thin-layer chromatography (TLC) analysis of Kdo ₂ -lipid A produced by genetically engineered bacteria BW002

The washed out WBB06 and the Kdo ₂ -lipid A sample produced by BW002 were spotted on the Gel 60TLC plate with a glass capillary, and the developing agent was chloroform/methanol/glacial acetic acid/water (25:15:4:4, 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, and place it on a heating plate for color development (Figure 1). According to the properties of the spreading agent, the higher the hydrophobicity of the sample, the faster the migration speed, so the sample with fewer phosphate groups and more fatty acid chains should have a higher migration speed. TLC results showed that the structure of Kdo ₂ -lipid A (swimming lane 1) produced by WBB06 without modification of lipid A part was relatively simple and the migration speed was slow; the structure of Kdo ₂ -lipid A (swimming lane 2) of genetically engineered bacteria BW002 was the same Very homogeneous, with slightly faster sample migration than WBB06, its lipid A moiety was deduced to be the Kdo ₂ -monophospholipid A form containing five fatty acid chains. The TLC structure shows that after directional transformation, the genetically engineered bacteria BW002 can produce a single Kdo ₂ -lipid A with a special structure.

3. ESI/MS analysis of Kdo ₂ - lipid A structure

ESI/MS analysis: Lipid samples were dissolved in chloroform/methanol solution (2:1, v/v), and mass spectrometry was performed on a WATERS SYNAPT Q-TOFMass Spectrometer mass spectrometer. Using ESI ion source, negative ion detection mode, the detection range is less than m/z 2500 (Figure 2). The main [MH]-peak m ^/ z value of the Kdo ₂ -lipid A produced by the control bacteria WBB06 is 2236.0, indicating that the exact molecular weight of the Kdo ₂ -lipid A produced by WBB06 is 2237; the m/z values are at 2258.0 and 2279.9 The mass spectrum peaks are the [M+Na-2H] ^- and [M+2Na-3H] ^- peaks generated by the ionization process of the sample plus the sodium ions in the instrument, which are actually the same substance. The Kdo ₂ -lipid A produced by the genetically engineered bacteria BW002, the Kdo ₂ -monophosphate lipid A form of five fatty acid chains in the lipid A part, has lost the C1 phosphate group in the lipid A part (m/z value 80) and C3' secondary fatty acid chain (m/z value 210), the main [MH] -peak m ^/ z value is 1946.2, indicating that the exact molecular weight of the special structure Kdo ₂ - lipid A produced by BW002 is 1947.2 ; Similarly, the m/z value at 1968.2 is the [M+Na ^- 2H]-peak generated by adding sodium ions, which is actually the same substance. MS results identified the exact structure of Kdo ₂ -lipid A produced by the genetically engineered strain BW002, further indicating that the genetically engineered BW002 can directly produce Kdo ₂ -monophosphate lipid A with a very simple structure containing five fatty acid chains.

Example ₂ Toxicity analysis of five-chain Kdo2-monophosphate lipid A produced by genetically engineered bacteria BW002

The directly extracted Kdo ₂ -lipid A sample is mixed with various membrane phospholipids, so it needs to be purified to remove phospholipid interference before performing cytotoxicity analysis. The purification of Kdo ₂ -lipid A is mainly accomplished by gradient elution on a DEAE fiber column. In the cell stimulation experiment, the purified sample of lipopolysaccharide (LPS) from the wild-type strain W3110 was used as a positive control, and PBS was used as a hard control; an attenuated strain produced by genetically engineered bacteria BW001 (E.coli W3110△waaCF△lacIlacZ::FnlpxE) was also introduced. , monophosphate six fatty acid chains Kdo ₂ -lipid A for comparison.

1. DEAE fiber column purification of Kdo ₂ -lipid A

The genetically engineered bacteria BW002 and the control engineered bacteria BW001 were inoculated into 800 mL systems respectively, cultured overnight, and the produced Kdo ₂ -lipid A was extracted according to the method described in Example 1. Add DEAE-cellulose stored in chloroform/methanol/2.4M ammonium acetate (v/v/v 2:3:1) into a glass tube to make an adsorption separation column, and control the column volume to 20ml; use 6 times the volume Equilibrate the column with chloroform/methanol/water (v/v/v 2:3:1); wash out Kdo ₂ -lipid with one volume of chloroform/methanol/water (v/v/v 2:3:1) A crude sample, load the sample into the column; when the sample flows to the upper end of the filler, use chloroform/methanol/ammonium acetate (v/v/v2:3:1) solution for stage elution, in which the concentration gradient of ammonium acetate in the aqueous phase Take 60mM, 120mM, 240mM, 360mM, and 480mM respectively; collect the eluate and add appropriate amount of chloroform and water to make the eluate into a Bligh-Dyer two-phase system, let it stand for about 30mins, remove the phase, and blow with nitrogen. The phospholipid impurities were mainly washed out when the concentration of ammonium acetate was 60mM, 120mM and 240mM, and the Kdo ₂ - lipid A samples produced by the two engineering bacteria were mainly washed out when the concentration of ammonium acetate was 360mM and 480mM.

2. Extraction and purification of wild-type Escherichia coli W3110LPS

LPS was extracted by hot phenol method. Transfer the overnight cultured W3110 bacteria solution to 800mL LB liquid medium according to the initial OD ₆₀₀ = 0.02, cultivate at 37°C for 12 hours, then centrifuge at 8000 rpm for 10 minutes to collect the bacteria, wash the bacteria once with ddH ₂ O, and weigh the wet weight of the bacteria. Dissolve every 3g of wet bacteria in 10mL ddH ₂ O and preheat at 68°C. Add an equal volume of preheated 90% phenol, shake vigorously at 68°C for 1 hour. After low-temperature cooling, centrifuge at 4000 rpm for 20 minutes at 4° C., dialyze the supernatant against ddH ₂ O for three days to remove phenol, and vacuum freeze-dry to obtain crude LPS. After resuspending the crude product with an appropriate amount of ddH ₂ O, add RNase A (final concentration 50 μg/ml) and DNase I (final concentration 100 μg/ml) to treat at 37°C for 2 hours, then add proteinase K (final concentration 100 μg/ml) to treat overnight at 37°C . 5 mL of water-saturated phenol was added to the enzyme-treated sample, mixed evenly, centrifuged at 4000 rpm for 30 minutes, the supernatant was dialyzed against ddH ₂ O for one day, and vacuum freeze-dried to obtain LPS. Redissolve LPS in chloroform/methanol solution (2:1, v/v), vortex for 30 seconds, centrifuge at 12,000 rpm for 10 minutes, remove the supernatant, redissolve the precipitate in water, and vacuum freeze-dry to obtain pure LPS.

3. Analysis of the production of inflammatory cytokines after Kdo ₂ -lipid A stimulated mouse macrophage leukemia cells RAW264.7

Mouse cells RAW264.7 were inoculated in a 96-well plate, with 105 cells per well, cultured at 37°C, ⁵ % CO ₂ for 12 hours, after the cells adhered to the wall, fresh culture medium was added, and different concentrations of LPS were added (final concentrations were 0.1 , 1, 10, 100ng/ml), the supernatant was taken after 24 hours of stimulation, and the interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α ) content (Fig. 3A,B). Therefore, the stimulation results of the samples were analyzed using the least significant difference method, ***P<0.001, **P<0.01, *P<0.1. At each stimulation concentration, the Kdo ₂ -lipid A samples produced by the genetically engineered bacteria BW002 and the attenuated engineered bacteria BW001 were all weakened in varying degrees compared with the wild type W3110LPS on the stimulation of the cells, and the predicted effect of the cells was induced. The inflammatory cytokines IL-6 and TNF-α were also reduced to varying degrees; the lipid A part of the Kdo ₂ -lipid A sample produced by the genetically engineered bacteria BW002 also reduced the C3' position while missing the C1 phosphate group Secondary fatty acid chain, its cytotoxicity weakened very obviously. For example, when the sample concentration is 100ng/ml, the wild-type W3110LPS contains six fatty acid chains and two phosphate groups Kdo ₂ -lipid A, which stimulates cells to produce IL-6 and TNF-α up to 3871.5pg/ml and 14164.7pg/ml, the IL-6 and TNF-α produced by BW001-produced monophosphate six fatty acid chains Kdo ₂ -lipid A stimulated cells were lower, 1533.2pg/ml and 12067.1pg/ml, respectively. However, the lipid A part of the Kdo ₂ -lipid A sample of the five fatty acid chains of monophosphate produced by the genetically engineered bacteria BW002 was more obviously weakened after the C1 phosphate group and the C3' secondary fatty acid chain were simultaneously deleted. The pro-inflammatory cytokines IL-6 and TNF-α produced after being stimulated were much lower than those stimulated by wild-type W3110LPS, and were also lower than the sample-induced amount of six fatty acid chains of monophosphate at the C1 phosphate group. When the sample concentration was 100ng/ml, the production of Kdo ₂ -lipid A sample induced by BW002 was 814.0pg/ml, which was only 21% of that induced by wild type W3110LPS, and the secretion of TNF-α was also induced Only 9059.9 pg/ml accounted for 66% of wild-type W3110LPS induction. Therefore, the Kdo ₂ -lipid A produced by the genetically engineered bacteria BW002 has an obvious attenuating effect on mouse cells, and is suitable for the development of subsequent animal vaccine adjuvants.

4. Analysis of the production of inflammatory cytokines after Kdo ₂ -lipid A stimulates human acute leukemia mononuclear cells THP-1

Human cell THP- ¹ was seeded in a 96-well plate at 105 cells per well, added PMA to 20ng/ml, induced for 48 hours at 37°C, 5% CO ₂ to differentiate into adherent macrophages, and then replaced with fresh culture At the same time, different concentrations of LPS were added (final concentrations were 0.1, 1, 10, and 100 ng/ml), and the supernatant was taken after 24 hours of stimulation, and interleukin-8 ( IL-8) and tumor necrosis factor α (TNF-α) content (Fig. 4A, B). Therefore, the stimulation results of the samples were analyzed using the least significant difference method, ***P<0.001, **P<0.01, *P<0.1. Similar to the stimulation results in mouse cells, Kdo ₂ -lipid A samples produced by genetically engineered bacteria BW002 induced IL-8 and TNF-α produced by human cells THP-1 and wild-type W3110LPS, BW001 Kdo ₂ -lipid A Compared with the output, there is also a significant decline. When the sample concentration was 100ng/ml, the IL-8 induced by the special structure Kdo ₂ - lipid A produced by BW002 was only 7379.1pg/ml, while the induction amount of wild-type W3110LPS was 19347.3pg/ml, and BW001 with six fatty acid chains The induced amount of Kdo ₂ -lipid A sample was 14782.5 pg/ml. The TNF-α produced by the Kdo ₂ -lipid A produced by the genetically engineered bacteria BW002 and the wild type W3110LPS and BW001 Kdo ₂ -lipid A stimulated by human cell THP-1 was an order of magnitude lower than that of the mouse cell RAW264.7, and the special protein produced by BW002 The TNF-α induced by the structure Kdo ₂ -lipid A was also significantly lower than that induced by W3110 LPS and BW001 Kdo ₂ -lipid A, only 46% of that induced by wild type W3110LPS. It can be seen that the Kdo ₂ -lipid A produced by the genetically engineered bacteria BW002 also has an obvious attenuating effect on human cells, and is also suitable for the development of subsequent human vaccine adjuvants.

Example 3 Analysis of hydrophobicity and self-aggregation ability of genetically engineered bacteria BW002 producing low-toxic five-chain Kdo ₂ -monophosphate lipid A

1. Comparison of hydrophobicity between genetically engineered bacteria BW002 and wild-type Escherichia coli W3110

The method for measuring the hydrophobicity of the bacterial strain is as follows: overnight cultured bacterial liquid was centrifuged (7000g, 10min) to collect the bacterial cells; the bacterial cells were washed twice with _{PBS7.4 ;} 3 decimal places); Finally, 2 mL of bacterial suspension was mixed with 800 μL xylene, vortexed for 2 minutes, and left at room temperature for 1 hour to measure the OD ₆₀₀ of the aqueous phase (denoted as A, 3 decimal places), and calculate the cell surface hydrophobicity: H%=(A ₀ −A)/A ₀ *100.

Since LPS is the major macromolecule on the surface of the outer membrane of E. coli, the absence of the hydrophilic core polysaccharide chains of LPS after waaCF knockout would enhance the hydrophobicity of the cell surface (Fig. 5A). After determination, the hydrophobicity of wild-type Escherichia coli W3110 is 39.6%, while the hydrophobicity of genetically engineered bacteria BW002 rises to 78.7%, which is relatively close to the hydrophobicity (82.8%) of the engineered bacteria BW001 of the control, both of which are 2 of the wild type. about times. It has been proved in the literature that the self-agglutination of the strain is positively correlated with the hydrophobicity of the cell surface (r=0.71), and the strong hydrophobicity of the cell surface is conducive to enhancing the hydrophobic interaction between cells, making it easier for cells to aggregate and settle in aqueous solution down.

2. Comparison of self-agglutination ability of genetically engineered bacteria BW002 and wild-type Escherichia coli W3110

The method for measuring the self-agglutination ability of the bacterial strain is: centrifuge (7000g, 5min) to culture the bacterial liquid overnight to collect the bacterial cells, wash the bacterial cells twice with _PBS7.4 ; then suspend the bacterial cells with _PBS7 . , keep 3 decimal places); take 12mL of the bacterial solution into a test tube or a graduated tube, and let it stand at 22°C; measure the OD ₆₀₀ values of 0h, 1.5h, 3h, 6h, 9h, 12h, 15h, 18h, and 24h respectively ( Denote as A _i , keep 3 decimal places); calculate the autoagglutination percentage AAg%=[(A ₀ -A _i )/A ₀ ]×100. The greater the AAg%, the stronger the self-agglutination ability of the bacteria. Draw a curve to show the dynamic change of self-agglutination ability with time.

As shown in Figure 5B, the self-agglutination percentage of BW002 was significantly higher than that of W3110 after standing for 6 hours. After standing for 12 hours, the self-agglutination percentages of W3110, BW001, and BW002 were 17.7%, 72.6%, and 39.8%, respectively. The wild type is more than double, but lower than BW001. Strains with strong bacterial self-agglutination are more conducive to rapid collection in industrial production and increase production efficiency.

Example 4 Antibiotic resistance analysis of the low-toxic five-chain Kdo ₂ -monophosphate lipid A production genetically engineered bacterium BW002

The bacterial outer membrane acts as a natural barrier and plays an important role in preventing antibiotics from entering the cell. After the structure change of LPS, considering the safety of engineering strains in actual production and use, the minimum inhibitory concentration (MIC) of genetically engineered strains and wild-type Escherichia coli to erythromycin and novobiocin were respectively determined by micro broth dilution method. Erythromycin is a macrolide antibiotic, which can bind to the specific region of 23SrRNA, causing structural damage, causing peptidyl tRNA to dissociate from ribosomes earlier, thereby affecting the formation and elongation of peptide chains ; Novobiocin (Novobiocin) is a coumarin antibiotics, can inhibit DNA polymerase. Affect nucleic acid metabolism.

The method of MIC determination is as follows: the antibiotics are diluted with LB liquid medium, and the double-diluted erythromycin and novobiocin are added to a 96-well plate, and 100 μL is added to each well, where the concentration gradient of erythromycin is 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9 μg/mL, the concentration gradient of novobiocin was 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8 μg/mL, and no drug was added to the last well as a growth control.

The measurement results showed that the sensitivity of the genetically engineered strain BW002 to erythromycin and novobiocin was basically the same, and the resistance was very low. The MICs of erythromycin against W3110, BW001 and BW002 strains were 125 μg/mL, 7.8 μg/mL and 3.9 μg/mL, respectively; 7.8 μg/mL. The sensitivity of the BW002 strain to erythromycin and novobiocin was significantly enhanced, and both were reduced to about 1/32 of the wild type, which was also significantly lower than that of BW001, indicating that after the simultaneous deletion of the C1 phosphate group and the core polysaccharide, the outer membrane The permeability is greatly improved so that antibiotics can enter the cells more easily, and on this basis, the secondary fatty acid chain at the C2' position is removed, which further increases the permeability of the outer membrane of the cell, making the genetically engineered bacteria BW002 highly sensitive to various antibiotics. All in all, the high sensitivity and low resistance to antibiotics indicated that the genetically engineered strain BW002 was a very safe and controllable strain for industrial production.

The genetically engineered bacterial strain constructed by the present invention can directly produce Kdo ₂ -monophosphate lipid A with special structure, low toxicity, and five fatty acid chain chains, avoiding the use of pathogenic bacteria to produce LPS or lipid A, and avoiding the use of phenol And cumbersome chemical hydrolysis and chromatography, the strain itself has no antibiotic selection markers and shows high antibiotic sensitivity and high self-agglutination ability, which is more conducive to safe, green and large-scale industrial production.

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 (2)

1. a kind of produce less toxic, containing five fatty acid chains Kdo₂The method of-MPLA, it is characterised in that including Following steps：(1) thalline, chloroform/methanol/water mixing phase system extraction separation monophosphate are collected in shake flask fermentation culture thalline, (2) The attenuation Kdo of five fatty acid chains₂- lipoid, (3) DEAE fibre columns purify the attenuation Kdo of five fatty acid chains of monophosphate₂- mono- phosphorus Acids fat A；Step (1) carries out fermented and cultured using genetic engineering bacterium, and the genotype of the genetic engineering bacterium is E.coli W3110△waaCF△lacI△lpxMlacZ::FnlpxE；Less toxic, containing five fatty acid chains the Kdo₂- monophosphate Lipoid A chemical formulas are C₉₆H₁₇₅N₂O₃₅P, its structure include 2 α -one base -3- deoxidation-D- sweet dews octanoic acids, 2 Glucosamines, 1 Individual phosphate group and 5 fatty acid chains, be using β-(1 ' -6) connection D-Glucose amine disaccharides as skeleton, 6 ' positions connection two α -one Base -3- deoxidation-D- sweet dews octanoic acid, 4 ' positions are phosphorylated, 2 ' bit aminos connection (R) -3- (dodecyloxy) myristyl, and 3 ' Position, 3 and 2 bit aminos connect tetradecyloxyaniline respectively and formed, and its structural formula is as shown in Equation 1；

The structure of the genetic engineering bacterium comprises the following steps：1) artificial constructed lacI is knocked out into fragment and is transformed into E.coli In W3110/pKD46 competent cells, mutant strain E.coli W3110lacI are obtained::Pkan, the loxP mediated by Cre enzymes Locus specificity restructuring removes kalamycin resistance gene；2) FnlpxE fragments electricity is transferred to the E.coli W3110 of step 1) preparation In △ lacI/pKD46 competent cells, recombinant bacterium is obtained, its genotype is E.coli W3110 △ lacIlacZ::FnlpxE- Fkan, the specificity restructuring in the FRT sites mediated by FLP enzymes remove kalamycin resistance gene therein, are built into respectively Intermediate strains HW001；3) artificial constructed lpxM knockout fragments are transferred on the basis of HW001 mutant strains and are transformed into HW001/ In pKD46 competent cells, recombinant bacterium E.coli W3110 △ lacIlacZ are obtained::FnlpxE lpxM::Pkan, lead to again The specificity restructuring for crossing the loxP sites of Cre enzymes mediation removes kalamycin resistance gene therein respectively, is built into middle bacterium Strain HW002；4) artificial constructed waaCF is knocked out into fragment on the basis of HW002 mutant strains and is transformed into HW002/pKD46 impressions In state cell, recombinant bacterium E.coli HW002waaCF are obtained:Pkan, again by the specificity in the FRT sites of FLP enzymes mediation Restructuring removes kalamycin resistance gene therein respectively, and final genotype is E.coli W3110 △ waaCF △ lpxM △ lacIlacZ::FnlpxE, it is built into engineering strain BW002；LacI knocks out the nucleotide sequence such as SEQ ID of fragment Shown in NO.1；The nucleotide sequence of the FnlpxE-Fkan Insert Fragments of structure is as shown in SEQ ID NO.2；The lpxM of structure strikes Except the nucleotide sequence of fragment is as shown in SEQ ID NO.3；The waaCF of structure knocks out the nucleotide sequence such as SEQ ID of fragment Shown in NO.4；

It with the volume ratios of 6 times of volumes is 2 that the DEAE fibre columns purifying of the step (3), which is,:3:1 chloroform/methanol/water balance DEAE adsorption columns；The volume ratio accumulated with monoploid is 2:3:1 chloroform/methanol/aqueous solution washes out Kdo₂- lipoid A studies, loading Into pillar；It is 2 with volume ratio when sample flow to filler upper end:3:1 chloroform/methanol/ammonium acetate solution progress stage washes De-, the concentration gradient of wherein aqueous phase ammonium acetate takes 60mM, 120mM, 240mM, 360mM, 480mM respectively；Collect eluent and add Add appropriate chloroform and water, eluent is become Bligh-Dyer two-phase systems, stand 30mins, remove phase, nitrogen blows.

2. application of claim 1 methods described in vaccine adjuvant is prepared.