Monophosphate lipid A as well as synthesis method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to monophosphate lipid A as well as a synthesis method and application thereof. The monophosphate lipid A is shown as a formula I; the synthesis method comprises the following steps: constructing engineering bacteria for producing the monophosphate lipoid A, and carrying out metabolism extraction on the engineering bacteria to obtain the monophosphate lipoid A as shown in the formula 1. According to the method, the production cost of KMA is reduced by utilizing the constructed engineering strain, pathogenic bacteria are not involved in the production process, an inducer is not needed, and the method can be used for large-scale production of attenuated KMA. The attenuated KMA enhances the IgG antibody level of an influenza vaccine immunized mouse, and has better immunologic adjuvant activity on the influenza vaccine.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a monophospholipid A and a synthesis method and application thereof. Background Art

[0002] Since the 1990s, the outbreak of infectious diseases has posed a great threat to human life and property safety. Vaccination is an important measure to effectively prevent infectious diseases. However, some antigens in vaccines have weak immunogenicity, and vaccination alone often fails to achieve the desired effect. Immune adjuvants refer to substances that can stimulate the body by increasing the surface area of ​​antigens, slowly releasing antigens, and promoting inflammatory responses, thereby changing and enhancing the body's specific immune response to antigens. Aluminum salts can cause inflammation at the injection site and may lead to the formation of local erythema, granulomas, and subcutaneous nodules. At the same time, aluminum salt adjuvants may delay the release of some neutralizing antibodies, so they are subject to various restrictions when used. In summary, the development of new vaccine adjuvants is very necessary and meaningful.

[0003] Lipopolysaccharide derived from the cell membrane of Gram-negative bacteria, whose active unit lipid A can be recognized by Toll-like receptor 4 (TLR-4), which then stimulates the activation of the immune system and causes the body to produce a variety of inflammatory factors. However, wild-type lipid A can cause the human immune system to overreact, induce excessive inflammatory reactions and septic shock, and even lead to the production of sepsis. Therefore, it is not suitable for use as an adjuvant. Monophosphoryl lipid A (MPL) is a lipid A mixture with a monophosphate structure. Compared with wild-type lipid A, its adverse reaction rate is lower, but the currently available lipid A still has problems such as strong toxic reactions and high preparation costs, so it is difficult to popularize its use. The existing production methods of MPL are: one is to extract it from Salmonella, but Salmonella is a strong pathogenic bacterium and is more dangerous; the other is chemical synthesis, but there are more steps, longer routes, and expensive costs. Many problems restrict large-scale production and use. Summary of the invention

[0004] The purpose of the present invention is to solve the existing technical problems and further provide a monophospholipid A and a synthesis method and application thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solution:

[0006] A monophospholipid A, wherein the monophospholipid A is shown in Formula 1; its chemical formula is C 94 H 177 N2O 23P, molecular weight 1734.25, its structure is based on lipid A (with β-(1'-6) linked D-glucosamine disaccharide as the backbone, C1 and C4' positions are phosphorylated, C2 and C3 amino groups are respectively connected to tetradecyloxy, C3' position is connected to (R)-3-(tetradecyloxy)tetradecyl, and C2' amino group is connected to (R)-3-(dodecyloxy)tetradecyl,) but the phosphate group at C1 is missing, and a hydroxyl group is introduced into the C at position 2 of the secondary fatty acid chain at C3' to replace the original hydrogen atom;

[0007]

[0008] A method for synthesizing monophosphate lipid A comprises constructing an engineered bacterium for producing monophosphate lipid A, and utilizing the metabolic extraction of the engineered bacterium to obtain monophosphate lipid A as shown in formula 1.

[0009] The monophospholipid A producing engineering bacteria uses Escherichia coli as a host, causes deletion mutation in lacI in its genome, and knocks in expression genes lpxE and lpxO genes at the lacZ gene site, thus obtaining the monophospholipid A producing engineering bacteria E. coli W3110ΔlacI lacZ::Fn lpxE lpxO.

[0010] An application of the monophospholipid A, which is the application of the monophospholipid A as a vaccine adjuvant.

[0011] The invention discloses an engineered bacterium for producing monophospholipid A. The engineered bacterium uses Escherichia coli as a host, causes deletion mutation in lacI in its genome, and knocks in expression genes lpxE and lpxO genes at the lacZ gene site, thus obtaining the engineered bacterium E. coli W3110△lacI lacZ::Fn lpxE lpxO for producing monophospholipid A.

[0012] The regulatory gene lacI gene is deleted in the host strain, and the lpxE gene from Agrobacterium tumefaciens C58 and the lpxO gene from Salmonella typhimurium 14028S are inserted.

[0013] Methods and steps for constructing engineered bacteria

[0014] 1) The artificially constructed lacI knockout gene fragment was transformed into the competent cell of E. coli W3110 to obtain the mutant strain E. coli W3110△lacI::Fkan, and the kanamycin resistance gene was removed by site-specific recombination.

[0015] 2) Construction of the Fn-lpxE-lpxO knock-in gene fragment synthesis steps: ① Obtain the lpxE gene in the Agrobacterium tumefaciens C58 genome. ② Obtain the lpxO gene in the Salmonella typhimurium 14028S genome. ③ Obtain the Fn-lpxE-lpxO knock-in gene fragment by restriction endonuclease digestion and D4 ligase enzyme ligation.

[0016] 3) The artificially constructed knock-in gene fragment in step 2) was transferred into the E. coli W3110ΔlacI competent cells prepared in step 1) to obtain the mutant strain E. coli W3110ΔlacI lacZ::FnlpxE lpxO::Fkan, and the kanamycin resistance gene was removed by site-specific recombination.

[0017] The nucleotide sequence of the lacI knockout gene fragment is shown in SEQ ID NO.1.

[0018] The nucleotide sequence of the lpxE knock-in gene fragment is shown in SEQ ID NO.2.

[0019] The nucleotide sequence of the lpxO knock-in gene fragment is shown in SEQ ID NO.3.

[0020] The site-specific recombination in step 1) is loxp site-specific recombination mediated by Cre enzyme.

[0021] The site-specific recombination described in step 2) is FRT site-specific recombination mediated by FLP enzyme.

[0022] An application of the engineered bacteria for producing monophosphate lipid A, and an application of the engineered bacteria in producing monophosphate lipid A.

[0023] Furthermore, the engineered bacteria are propagated, centrifuged, crushed, hydrolyzed with sodium acetate, extracted, purified, etc.

[0024] The purification is performed by using a DEAE-52 cellulose anion exchange column, the mobile phase is chloroform / methanol / ammonium acetate solution, and the elution purification purpose is achieved by changing the concentration of ammonium acetate (0-480mM), and the elution flow rate is 0.03-12mL / min.

[0025] Furthermore, the revived engineered bacterial solution was inoculated into 0.2 L of fresh LB medium at a ratio of 1:1000 (v / v) and cultured at 37°C until OD 600=1, centrifuge at 4°C, 8000rpm for 20min, collect the cells, wash the cells once with ddH2O, suspend the cells with Bligh-Dyer one-phase system (chloroform / methanol / water, 1:2:0.8, volume ratio), stir magnetically for 1h, centrifuge at 3000rpm for 20min, wash the precipitate 2-3 times with the one-phase system; add 27mL of 1% sodium dodecyl sulfate (SDS) 12.5mM sodium acetate (pH4.5) solution, ultrasonically shake for 20min, and cleave the polysaccharide arm in a 100°C water bath for 30min. Cool to room temperature, add 30mL of chloroform and 30mL of methanol to prepare a Bligh-Dyer two-phase system (chloroform / methanol / water, 2:2:1.8, volume ratio), separate layers, extract multiple times, retain the chloroform layer and rotary evaporate, and finally add chloroform / methanol solution (4:1, volume ratio) to wash out KMA.

[0026] The above-obtained sample was purified by DEAE-52 cellulose anion exchange chromatography: the crude KMA sample was dissolved in chloroform / methanol / water (2:3:1, volume ratio) solution, and loaded onto a DEAE-52 cellulose anion exchange column equilibrated with 6 volumes of chloroform / methanol / water (2:3:1, volume ratio) solution in advance, and phospholipids and other impurities were eluted with chloroform / methanol / 120 mM ammonium acetate (2:3:1, volume ratio) solution, and then KMA was eluted and collected with chloroform / methanol / 480 mM ammonium acetate (2:3:1, volume ratio) solution, and the collected eluate was prepared into a chloroform / methanol / water two-phase system (2:2:1.8, volume ratio); the lower phase was removed by centrifugation, and the solvent was removed by rotary evaporation to obtain pure lipid A, which was stored at -20°C.

[0027] KMA detection and analysis

[0028] 1) Thin layer chromatography (TLC) detection:

[0029] The sample was spotted on a Gel 60 TLC thin layer plate, and the developing solvent was chloroform / methanol / water / ammonia water (40:25:4:2, volume ratio). After the chromatography, the residual developing solvent was blown dry, carbonized with 10% sulfuric acid ethanol, and placed on a heating plate for color development.

[0030] 2) Derivatization-HPLC detection:

[0031] Lipid A does not have a UV-chromogenic group, but the remaining hydroxyl group after removal of the phosphate group can be conjugated with 3,5-dinitrobenzyloxyamine hydrochloride (DNBA)-pyridine at 60°C to obtain O-dinitrobenzyl oxime with UV activity.

[0032] HPLC analysis was performed using a Thermo Fisher liquid chromatography system, column temperature 50°C, UV detector (254 nm). Thermoscientific Vanquish Chromeleon 7 software was used for system control and data collection / processing. This study used an Agilent ZORBAX C18, 5 μm particle size, 250 mm × 4.6 mm stainless steel column.

[0033] The mobile phases were (A) acetonitrile-5 mM tetrabutylammonium phosphate (TBAP) aqueous solution (95:5, volume ratio) and (B) isopropanol-5 mM tetrabutylammonium phosphate (TBAP) aqueous solution (95:5, volume ratio). The injection volume was 10 μL, the flow rate was 1.0 mL / min, and the mobile phase B was 10% to 80% linear gradient elution for 60 min, and UV detection was performed.

[0034] ESI / MS analysis: KMA samples were dissolved in chloroform / methanol (4:1, volume ratio) solution and mass spectrometry was performed on a WATERS SYNAPTQ-TOF Mass Spectrometer, using an ESI ion source, negative ion detection mode, and an M / z detection range of less than 1800.

[0035] An application of the monophosphate lipid A-producing engineered bacteria, and an application of the monophosphate lipid A obtained by culturing and metabolizing the engineered bacteria as a vaccine adjuvant.

[0036] A vaccine adjuvant, the adjuvant contains the monophospholipid A or the engineered strain.

[0037] Specifically

[0038] 1) Add monophospholipid A (KMA) to 12% lecithin / squalene (w / v) solution preheated at 65°C and place in a 65°C water bath for 60 min until completely dissolved.

[0039] 2) The above-obtained dissolved solution and 0.5% Tween 80 / water solution were preheated at 65° C., mixed at a ratio of 1:10 (v / v), and thoroughly emulsified using a homogenizer to obtain an adjuvant for standby use.

[0040] The vaccine is an influenza vaccine

[0041] The advantages of the present invention are:

[0042] The invention constructs a genetically engineered bacterium for producing monophospholipid A, and the production process does not involve pathogenic bacteria, and the bacterium can be used for large-scale production of KMA. The metabolite KMA obtained has a novel structure and immune adjuvant activity. In vivo experiments have proved that the KMA can enhance the immune response of the vaccine, has good immune adjuvant activity for influenza vaccine, and has good safety.

[0043] The KMA obtained by the present invention is a KMA with a special structure, attenuated toxicity, and six fatty acid chains. The compound is amphiphilic, can be directly extracted and identified by ESI mass spectrometry detection. On the basis of retaining its six fatty acid chains to have maximum immune activity, a hydroxyl group is introduced to improve its water solubility; the monophosphate structure reduces its toxicity, can activate the host's innate immune system, and can be used as an immune adjuvant and administered together with a vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 .Amplification program diagram, Note: Annealing temperature and extension time are set according to the requirements of each pair of primers and amplified fragments.

[0045] Figure 2 .KMA thin layer chromatogram; Note: Channel 1 is commercially available MPL, Channel 2 is KMA after preliminary purification, and Channel 3 is KMA.

[0046] Figure 3 .In the MS mass spectrum, the peak with an atomic mass of 1732.8671 is the atomic mass peak of KMA (M / Z is 1734.25).

[0047] Figure 4 .HPLC chromatogram of KMA. Note: 1 is the blank reagent chromatogram, 2 is the blank-derivatization chromatogram, and 3 is the KMA-derivatization chromatogram.

[0048] Figure 5 .Effect of KMA on IgG antibody content in vaccinated mice, Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Results: see Figure 4 As shown, compared with group C (negative administration group), group D (aluminum salt positive drug group) can significantly increase the IgG antibody level of mice immunized with influenza vaccine (P < 0.0001); compared with group D, group E (commercially available MPL adjuvant group) can significantly increase the IgG antibody level of mice immunized with influenza vaccine (P < 0.1), group G (KMA high-dose group) can significantly increase the IgG antibody level of mice immunized with influenza vaccine (P < 0.001), and has a dose-dependent relationship with group F (KMA low-dose group). Group A: blank group; Group B: water-in-oil blank carrier administration group. DETAILED DESCRIPTION

[0049] The following is a description of the exemplary embodiments of the present application, including various details of the embodiments of the present application to facilitate understanding, which should be considered as merely exemplary. Therefore, it should be recognized by those of ordinary skill in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. Similarly, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0050] The novel modified strain provided by the present invention is produced by modifying the genome of wild-type Escherichia coli W3110 through a new synthetic route to produce a novel monophospholipid A component (KMA), which is safe, low toxic, and has low production cost. It has good activity when used in combination with a vaccine and is suitable as a novel immune adjuvant.

[0051] The KMA of the present invention is derived from genetically engineered Escherichia coli. As long as it conforms to the structure provided in this application, it is within the protection scope of this application. In a preferred embodiment, the KMA can be extracted from Escherichia coli.

[0052] The present invention further provides a composition, comprising any one of the KMAs provided in the present application and a pharmaceutically acceptable carrier and / or excipient.

[0053] The present invention also provides use of any one of the KMAs or the above-mentioned compositions as vaccine adjuvants.

[0054] Through the experimental research of the present application, it was found that the KMA of the present invention can non-specifically change or enhance the immune response of the vaccine and can significantly increase the IgG antibody level of vaccine-immunized mice.

[0055] Therefore, the KMA or composition of the present invention can be used as a vaccine adjuvant in vaccines.

[0056] As used herein, the term "vaccine" refers to any preparation of antigenic or immunogenic material suitable for stimulating immune activity in animals or humans.

[0057] As used herein, the term "adjuvant" refers to any substance or mixture of substances that enhances, strengthens, upregulates, alters or otherwise promotes an animal's immune response (eg, humoral or cellular immune response) to an antigen.

[0058] As used herein, the term "antigen" refers to any substance that, when introduced into an immunocompetent human or animal, stimulates a humoral and / or cell-mediated immune response. The antigen may be a pure substance, a mixture of substances, or particulate matter (including cells, cell fragments, or cell-derived fragments) or a live (usually attenuated) organism or virus. Examples of suitable antigens include, but are not limited to, proteins, glycoproteins, lipoproteins, peptides, carbohydrates / polysaccharides, lipopolysaccharides, toxins, viruses, bacteria, fungi, and parasites. Antigens may be natural (naturally expressed or produced), synthetic, or derived by recombinant DNA methodology familiar to those skilled in the art.

[0059] As used herein, the term "pharmaceutical excipient" refers to a general term for all medicinal materials other than the main drug added to the prescription to solve the formability, effectiveness, stability and safety of the preparation when producing medicines and preparing prescriptions. It has been reasonably evaluated in terms of safety and is contained in the pharmaceutical preparation. In addition to excipients, serving as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, solubilization, and sustained release. They are important ingredients that may affect the quality, safety and effectiveness of drugs. The pharmaceutical excipients described in this application can be appropriate carriers or excipients, emulsifiers, wetting agents, preservatives, stabilizers, antioxidants, adjuvants (such as aluminum hydroxide adjuvants, oil adjuvants, Freund's complete adjuvants and Freund's incomplete adjuvants), etc.

[0060] Example 1: Construction of E. coli W3110ΔlacI mutant strain

[0061] 1. Obtaining lacI gene knockout fragment

[0062] Using the genome of Escherichia coli W3110 as a template, the lacI gene knockout fragment was obtained through chemical synthesis and PCR step-by-step amplification, enzyme digestion, enzyme ligation and other technologies. Its two ends are the upstream homology arm lacI-U and the downstream homology arm lacI-D, and the middle is the loxp-kan-loxp resistance gene fragment. The amplification primers are shown in Table 1, and the amplification conditions are shown in Figure 1 The constructed lacI gene knockout fragment was digested and ligated into the plasmid pBlue ScriptII SK(+) (purchased) to obtain the recombinant knockout plasmid pBS-I-kan, which was introduced into DH5α Escherichia coli competent cells (purchased), the plasmid was amplified, and a sufficient amount of the LacI gene knockout fragment was obtained by PCR amplification.

[0063] 2. Preparation of Chemically Competent E. coli W3110

[0064] Initial bacterial concentration of E. coli W3110 OD 600 =0.02, 37°C, 200 rpm, culture until OD 600The concentration of the culture medium is about 0.4. The cultured bacterial liquid is placed in an ice bath for 30 minutes, transferred to a pre-cooled sterile centrifuge tube, centrifuged at 8000rpm, 4℃, for 10 minutes, the bacterial cells are collected, and the bacterial cells are washed with pre-cooled 15% glycerol. The centrifuge tube is inverted to allow the liquid to flow out; the bacterial precipitate is resuspended with 0.6mL of pre-cooled 100mM CaCL2-MgCL2 solution (80mM MgCL2-20 mM CaCl2) per 1mL of cultured bacterial liquid, centrifuged at 8000rpm, 4℃, for 10 minutes, and the bacterial cells are collected. The treated liquid is discarded, and the centrifuge tube is inverted to allow the liquid to flow out. The chemically competent state of Escherichia coli W3110 is prepared by resuspending the bacterial precipitate with 0.04mL of pre-cooled 100mM CaCL2 (containing 15% glycerol) solution per 1mL of cultured bacterial liquid.

[0065] 3. Construction of E. coli W3110△lacI mutant strain

[0066] The Red enzyme recombinant plasmid pKD46 (purchased) was transformed into chemically competent E. coli W3110 to obtain the strain E. coli W3110 / plasmid pKD46. 600 =0.02, 30°C, 200rpm, culture for 1.5h (OD600≈0.1) Add 30mM L-arabinose to induce the expression of Red homologous recombinase, continue to culture for 2h, OD600 is about 0.4; ice bath the cultured bacterial solution for 30min, transfer to a pre-cooled sterile centrifuge tube, centrifuge at 8000rpm, 4°C for 10min, collect the bacteria, wash the bacteria with pre-cooled 15% glycerol, invert the centrifuge tube to drain the liquid; resuspend the bacterial precipitate with 0.6mL pre-cooled 100mM CaCL2-MgCL2 solution (80mM MgCL2-20 mM CaCl2) per 1mL of cultured bacterial solution, centrifuge at 8000rpm, 4°C for 10min, collect the bacteria. Discard the treated solution and invert the centrifuge tube to drain the liquid. The bacterial pellet was resuspended with 0.04 mL of pre-cooled 100 mM CaCL2 (containing 15% glycerol) solution per 1 mL of culture solution to prepare E. coli W3110 / plasmid pKD46 chemically competent cells.

[0067] Add 1000ng knockout gene LacI fragment (step 1) to the competent strain of E. coli W3110 / plasmid pKD46, mix gently, place on ice for 30min, heat shock in a 42℃ water bath for 45s, quickly put back on ice for 2min, add 700μL LB sterile liquid culture medium without antibiotics, mix evenly, culture at 30℃, 200rpm for 2h, draw an appropriate amount and spread evenly on LB solid plate culture medium containing corresponding kanamycin and ampicillin, invert and culture overnight, and screen the target engineering strain E. coli W3110△lacI / pKD46.

[0068] 4. Construction of temperature-sensitive expression plasmid and removal of resistance genes

[0069] The genome of plasmid pSH47 (purchased) was used as a template, and the Cre gene fragment was amplified by PCR, and SacI and NcoI restriction sites were introduced at both ends. The Cre gene fragment was double-digested with a non-restriction endonuclease and then ligated into the temperature-sensitive plasmid pKD46 treated with the same double restriction enzymes to obtain the temperature-sensitive Cre enzyme expression vector plasmid pKD46-Cre.

[0070] The target engineering strain obtained in step 3 was cultured and subcultured at 42°C to remove the temperature-sensitive plasmid pKD46, and then the competent cells were transformed into the temperature-sensitive plasmid pKD46-Cre to promote site-specific recombination of the LoxP sites at both ends of the kan fragment on the transformant chromosome, thereby removing the kan marker gene fragment. Finally, the pKD46-cre plasmid was removed by culture at 42°C to obtain the engineered strain mutant E. coli W3110△lacI.

[0071] Example 2: Construction of E. coli W3110ΔlacI lacZ::FnlpxE lpxO mutant strain

[0072] 1. Obtaining the knock-in gene fragment lpxE-lpxO-Fkan

[0073] Using the purchased genomes of Salmonella enterica serovar Typhimurium 14028S, Agrobacterium tumefaciens C58, and pKD13 plasmid as templates, lpxO, lpxE, and the kan fragment with FRT site were amplified by PCR, and restriction sites were introduced respectively. The amplification primers are shown in Table 1. The amplification conditions are shown in Table 1. Figure 1.; After the three gene fragments were digested and ligated with the pBlue Script IISK(+) plasmid fragments digested with the same enzymes, the ligation products were transformed into DH5α competent cells, coated with LB solid plates containing kanamycin, and the recombinant plasmid pBS-lpxE-lpxO-Fkan was screened. Using lacZ-F / R as primers, PCR amplified the lpxE-lpxO-Fkan knock-in gene fragment, which had lacZ-α fragments at both ends and could be used as natural homology arms. After 1% agarose gel electrophoresis, the gel was cut and recovered for purification.

[0074] 2. Preparation of knock-in competent strains and chemical transformation

[0075] First, the pKD46 plasmid was transferred into the E. coli W3110ΔlacI chemically competent strain obtained and prepared in Example 1 to obtain the E. coli W3110ΔlacI / pKD46 strain. Then, the E. coli W3110ΔlacI / pKD46 chemically competent strain was prepared.

[0076] Add 1000ng of lpxE-lpxO-Fkan exogenous gene fragment to the competent bacterial solution of E. coli W3110△lacI / pKD46, mix gently, place on ice for 30 min, heat shock in a 42°C water bath for 45 s, quickly put back on ice for 2 min, add 700μL of LB sterile liquid culture medium without antibiotics, mix evenly, culture at 30°C and 200 rpm for 2 h, draw an appropriate amount and evenly spread it on an LB solid plate containing kanamycin, screen for mutants with the lpxE-lpxO-Fkan gene fragment inserted in the lacZ site, and then use the heat shock method to remove the pKD46 plasmid.

[0077] Finally, the pCP20 plasmid (purchased) was transferred into the E. coli W3110△lacI lacZ::FnlpxE lpxO chemically competent strain to induce the expression of FLP enzyme. The kan resistance marker gene was removed by the recognition of the FRT site by the FLP enzyme to obtain the mutant strain E. coli W3110△lacI lacZ::FnlpxElpxO, and the lpxE-lpxO gene was integrated and expressed on the E. coli chromosome.

[0078] Nucleotide sequence of the lacI knockout gene fragment.

[0079] 2846 bp

[0080] DNA

[0081] Artificial sequence

[0082] SEQ ID NO.1.

[0083]

[0084]

[0085] The nucleotide sequence of the lpxE knock-in gene fragment is 730 bp

[0086] DNA

[0087] Artificial sequence

[0088] SEQ ID NO.2

[0089]

[0090] Nucleotide sequence of pxO knock-in gene fragment SEQ ID NO. 3909 bp

[0091] DNA

[0092] Artificial sequence

[0093] SEQ ID NO.3

[0094]

[0095] Table 1. Primer sequence list

[0096]

[0097] Example 3: Extraction and purification of genetically engineered bacterial lipid KMA

[0098] 1. Extraction of Lipid KMA

[0099] The E. coli W3110△lacI lacZ::FnlpxE lpxO mutant strain was inoculated into 0.2 L fresh LB medium at a ratio of 1:1000 (v / v) and cultured at 37°C until OD 600=1, centrifuge at 4°C, 8000rpm for 20min, collect the cells, wash the cells once with ddH2O, and use Bligh-Dyer extraction. Centrifuge 0.2L overnight culture to collect the cells, wash once with double distilled water, resuspend the cells with 38mL chloroform / methanol / water one-phase system (1:2:0.8, volume ratio), break them with magnetic stirring for 1h, collect the solid fragments by centrifugation, and wash them three times with one-phase system to remove lipid compounds. Add 27 mL of 12.5 mM sodium acetate solution (pH 4.5) containing 1% sodium dodecyl sulfate (SDS) to the lower phase solid, and ultrasonically disrupt it for 20 min. Then heat it in a water bath at 100°C for 30 min. After cooling, add 30 mL of chloroform and 30 mL of water to form a chloroform / methanol / water two-phase system (2:2:1.8, volume ratio). Centrifuge again to separate the phases, transfer the lower phase liquid to a rotary evaporator and evaporate it to dryness. The crude sample is stored at -20°C.

[0100] 2. Purification of lipid KMA

[0101] The purification preparation of lipid KMA samples uses DEAE-52 cellulose anion exchange chromatography: the crude sample is dissolved in 5 mL of chloroform / methanol / water (2:3:1, volume ratio) solution, and loaded onto a 10 mL DEAE-52 cellulose anion exchange column that has been equilibrated with 6 volumes of chloroform / methanol / water (2:3:1, volume ratio) solution in advance; phospholipids and other impurities are eluted with chloroform / methanol / 120 mM ammonium acetate (2:3:1, volume ratio) solution, and lipid A is eluted and collected with chloroform / methanol / 480 mM ammonium acetate (2:3:1, volume ratio) solution; the eluate is collected and prepared into a chloroform / methanol / water two-phase system (2:2:1.8, volume ratio); the lower phase is removed by centrifugation, and the solvent is removed by rotary evaporation to obtain pure lipid A, which is stored at -20°C.

[0102] Example 4: Detection and analysis of lipid structure of genetically engineered bacteria

[0103] 1. Thin layer chromatography (TLC) analysis

[0104] The sample KMA was dissolved in chloroform / methanol (4:1, volume ratio), and the sample was spotted on a silica gel 60TLC plate and chromatographed in a developing agent of chloroform / methanol / water / ammonia water (40:25:4:2, volume ratio). After the chromatography, the developing agent was blown dry, and 10% sulfuric acid ethanol color developing solution was evenly sprayed for carbonization, and heated at high temperature for color development (see Figure 2 ).

[0105] 2. Derivatization-High Performance Liquid Chromatography (HPLC) Analysis

[0106] Lipid A does not have a UV-chromogenic group, but the hydroxyl group after dephosphorylation can be conjugated with DNBA-pyridine at 60°C to obtain O-dinitrobenzyl oxime with UV activity.

[0107] HPLC analysis was performed using a Thermo Fisher liquid chromatography system, column temperature 50°C, UV detector (254 nm). Thermoscientific Vanquish Chromeleon 7 software was used for system control and data collection / processing. This study used an Agilent ZORBAX C18, 5 μm particle size, 250 mm × 4.6 mm stainless steel column.

[0108] The mobile phases were (A) acetonitrile-5 mM TBAP aqueous solution (95:5, volume ratio) and (B) isopropanol-5 mM TBAP aqueous solution (95:5, volume ratio). The flow rate was 1.0 mL / min and the injection volume was 10 μL. The detection was performed with a linear gradient elution of 10% to 80% B for 60 min (see Figure 4 ).

[0109] 3. Electrospray ionization mass spectrometry (ESI / MS) analysis

[0110] KMA samples were dissolved in chloroform / methanol solution (4:1, volume ratio) and directly injected into a Waters quadrupole time-of-flight mass spectrometer. ESI / MS was performed in negative ion detection mode with the cone voltage set to 120 V and the detection range of m / z 1700-1800 (see Figure 3 ).

[0111] Example 5: Preparation of KMA-Oil-in-Water Emulsion

[0112] 1. Prepare a 12% lecithin / squalene (w / v) stock solution.

[0113] Weigh 600 mg of lecithin and add 5 mL of squalene, place in a 65°C water bath to dissolve, and sterilize the solution through a 0.22 μm filter.

[0114] 2. Prepare a stock solution of 0.5% Tween 80 / water (v / v).

[0115] Take 250 μL of Tween 80 and add 49.75 mL of dH2O, and dissolve in a 65°C water bath. Sterilize the solution by passing it through a 0.22 μm filter.

[0116] 3. Dissolve KMA in 12% lecithin / squalene stock solution.

[0117] 10 mg of the purified KMA was added to 1 mL of 12% lecithin / squalene solution preheated at 65°C and placed in a 65°C water bath for 60 min until MPL was completely dissolved.

[0118] 4. Preparation of Emulsion

[0119] The KMA / lecithin / squalene solution and the 0.5% Tween 80 / water solution were preheated simultaneously, mixed at a ratio of 1:10, and emulsified evenly using a homogenizer.

[0120] Example 6: Study on the adjuvant activity of KMA on influenza vaccine

[0121] Influenza vaccine: produced by Liaoning Chengda Biological Co., Ltd.

[0122] Animals: 56 female ICR mice, 6-8 weeks old, purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0123] Preparation of influenza vaccine preparations containing KMA: Weigh and prepare oil-in-water preparations respectively, and configure the preparation concentrations according to the dosage of each group.

[0124] Experimental groups and dosages:

[0125] A: Blank saline group

[0126] B: Blank oil-in-water preparation group

[0127] C: Influenza vaccine group: 3 μg influenza vaccine / rat.

[0128] D: Aluminum salt adjuvant group: 3 μg influenza vaccine + 250 μg aluminum salt adjuvant / rat;

[0129] E: Commercially available lipid A (MPL)-water-in-oil group: 3 μg influenza vaccine + 5 μg MPL adjuvant / rat

[0130] F: KMA-O / W low-dose group: 3μg influenza vaccine + 5μg KMA adjuvant / rat

[0131] G: KMA-O / W high-dose group: 3μg influenza vaccine + 15μg KMA adjuvant / rat

[0132] Immunization scheme: 56 mice were randomly divided into 7 groups, 8 mice in each group. 0.1 mL / mouse was injected intramuscularly.

[0133] One week after the first immunization, the second immunization was performed. 14 days after the second immunization, the IgG antibody level in the mouse serum was detected by ELISA experiment (see Figure 4 ).

[0134] Depend on Figure 4It can be seen that compared with group C (adjuvant negative control group of vaccination), group D (aluminum salt adjuvant group) had a better antibody level than group C, with a P value of <0.0001, which was a very significant difference, indicating that the mouse body produced a higher level of IgG antibodies and the model was successfully established. After the administration of two doses of KMA (groups F and G), the antibody level of mice was significantly increased, and the effect was better than that of aluminum salt adjuvant (P <0.001), and its adjuvant activity was slightly better than that of commercially available MPL (group E).

[0135] Example 7: KMA safety evaluation study

[0136] According to the general pyrogen test method provisions of Chapter 1142 of the 2020 edition of the "Chinese Pharmacopoeia", the safety of bioengineering KMA was tested through rabbit pyrogen experiments.

[0137] The specific experimental process is as follows: healthy female rabbits weighing 1.7-3.0 kg are fed for 3 days and their body temperature is measured every 30 minutes for a total of 8 times. Rabbits with 8 body temperatures within the range of 38.0-39.7℃ and a temperature difference of no more than 0.4℃ between the highest and lowest temperatures meet the experimental requirements and are used for pyrogen testing.

[0138] The syringe needles and experimental utensils used in the experiment were placed in a 250°C oven and heated for 30 minutes to remove pyrogens and avoid interference with the experimental results.

[0139] The bioengineering KMA was accurately prepared to a concentration of 162.9 μg / mL (the dosage was 16.29 μg, which was 5 times the clinical dosage) and administered by intravenous injection at the ear margin. The body temperature of the rabbits was measured and recorded every 30 minutes after administration for a total of 6 times.

[0140] Table 2. Rabbit pyrogen test results

[0141]

[0142] Note: The corresponding dose of MPLA in the human adjuvant AS04 is 50μg. By conversion to rabbits, 50μg / 60kg*2.3*1.7kg*5 is 16.29μg.

[0143] The results are shown in Table 2. According to the provisions of the general pyrogen test method in Chapter 1142 of the 2020 edition of the "Chinese Pharmacopoeia", it can be seen from Table 2 that in the rabbits in the initial test, the temperature increase was all below 0.6°C, which determined that the pyrogen test of the test sample met the regulations and had good safety.

Claims

1. A monophospholipid A, characterized in that: Monophospholipid A is shown in Formula 1; 2. A method for synthesizing monophospholipid A according to claim 1, characterized in that: An engineering bacterium producing monophosphate lipid A is constructed, and monophosphate lipid A shown in formula 1 is obtained by metabolic extraction of the engineering bacterium.

3. The method for synthesizing monophospholipid A according to claim 1, characterized in that: The monophospholipid A-producing engineering bacteria uses Escherichia coli as a host, causes a deletion mutation in lacI in its genome, and knocks in expression genes lpxE and lpxO genes at the lacZ gene site, thereby obtaining the monophospholipid A-producing engineering bacteria E. coli W3110ΔlacI lacZ::Fn lpxElpxO.

4. A use of the monophospholipid A according to claim 1, characterized in that: The monophospholipid A is used as a vaccine adjuvant.

5. An engineered bacterium for producing monophospholipid A, characterized in that: The engineered bacteria uses Escherichia coli as a host, causes a deletion mutation in lacI in its genome, and knocks in expression genes lpxE and lpxO genes at the lacZ gene site, thereby obtaining the engineered bacteria E. coli W3110△lacI lacZ::Fn lpxE lpxO that produces monophospholipid A.

6. The monophospholipid A engineered bacterium according to claim 5, characterized in that: The regulatory gene lacI gene is deleted in the host strain, and the lpxE gene from Agrobacterium tumefaciens C58 and the lpxO gene from Salmonella typhimurium 14028S are inserted.

7. Use of the engineered bacteria for producing monophospholipid A according to claim 5, characterized in that: The application of the engineered bacteria in producing monophospholipid A.

8. Use of the engineered bacteria producing monophospholipid A according to claim 5, characterized in that: The monophospholipid A obtained by culturing and metabolizing the engineered bacteria is used as a vaccine adjuvant.

9. A vaccine adjuvant, characterized in that: The adjuvant contains the monophospholipid A described in claim 1 or the engineered strain described in claim 5.

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