Group B meningococcus low-endotoxin mutant strain and application thereof

Abstract

The invention relates to the technical field of microbial genetic engineering and vaccine development, and discloses a group B meningococcus low-endotoxin mutant strain, which is prepared by the following steps: providing a meningococcus mother strain; constructing a plasmid vector for gene replacement; replacing the target gene of the stock plant by using a gene editing technology to obtain a mutant strain; a group B meningococcal low-endotoxin mutant strain is obtained through screening, the invention further provides application of the group B meningococcal low-endotoxin mutant strain, the mutant strain is applied to preparation of outer membrane vesicles, and the preparation of the outer membrane vesicles comprises the following steps: regulating culture conditions of the mutant strain; optimizing lipopolysaccharide synthesis of the outer membrane vesicles by utilizing a dynamic metabolic flow model; the structural stability of the outer membrane vesicle is enhanced through chemical modification. Through gene editing, culture process optimization and an efficient extraction technology, the endotoxin activity is reduced, and the stability of the mutant strain and the extraction quality and immunogenicity of the outer membrane vesicles are improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial genetic engineering and vaccine development, in particular to a group B meningococcal low endotoxin mutant strain and application thereof. Background Art

[0002] Epidemic cerebrospinal meningitis is an acute central nervous system infection caused by Neisseria meningitidis, which is characterized by rapid onset, strong infectivity and high mortality. During the infection process, lipopolysaccharide (LPS) in the outer membrane of meningococci is the main toxic factor, and its endotoxin activity can trigger a strong inflammatory response, leading to multiple organ failure and even death in patients. At the same time, outer membrane vesicles (OMVs) are widely considered to be an important carrier for the development of meningococcal vaccines because they carry rich membrane proteins and immunogenic substances. Reducing the endotoxin activity of LPS while retaining the immunogenicity of OMVs is crucial for the development of safe and effective vaccines.

[0003] Existing technologies have made some progress in the development of outer membrane vesicles. For example, some studies have reduced the endotoxin activity of LPS through gene mutation, thereby reducing the inflammatory response caused by it in the body. At the same time, improved physical or chemical extraction methods can effectively release bacterial outer membrane vesicles and retain the structural integrity of the vesicles to a certain extent. In addition, the optimization of bacterial culture medium formula and production process has increased the extraction amount and consistency of vesicles. These technologies have laid a good foundation for the application of outer membrane vesicles in vaccine development.

[0004] However, the existing technology still has many shortcomings. First, the traditional gene mutation method is inefficient and lacks precision, making it difficult to achieve specific mutations in a complex genomic background. Some technologies will seriously affect the growth performance of the strain when reducing endotoxin activity, resulting in poor stability in the production process. Second, in the existing vesicle extraction methods, physical fragmentation methods can easily damage the vesicle structure, while chemical methods have the limitation of insufficient extraction purity. In addition, the culture process lacks dynamic regulation of osmotic pressure and pH, which can easily lead to bacterial lysis and affect vesicle yield. Finally, the existing technology fails to balance the contradiction between low endotoxin activity and high immunogenicity, weakening the potential of mutant strain outer membrane vesicles in vaccine applications. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a low endotoxin mutant strain of group B meningococcus and its application, which solves the problems in the prior art of low gene mutation efficiency, poor growth performance of mutant strains, easy structural damage during the outer membrane vesicle extraction process, insufficient purity, and difficulty in balancing low endotoxin activity and high immunogenicity.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a group B meningococcal low endotoxin mutant and its application, comprising the following steps: Provide meningococcal mother strains; Construction of plasmid vectors for gene replacement; Gene editing technology is used to replace the target gene of the mother strain to obtain a mutant strain; Screening and obtaining low endotoxin mutants of group B meningococci; The mutant strain was cultured; Extract outer membrane vesicles of mutant strains.

[0007] Preferably, the plasmid vector constructed for gene replacement comprises: Construct homologous sequences on both sides of lpxL1; Insertion of kanamycin resistance gene; The homologous sequences and resistance gene were ligated to the plasmid backbone.

[0008] Preferably, the target gene replacement is accomplished by the following steps: transforming the plasmid into a mother plant; Replace the lpxL1 gene in the mother strain by homologous recombination; Dynamic inhibition of lpxL2 gene expression.

[0009] Preferably, the mutant screening comprises: The mutants were screened in a medium containing kanamycin; Detect target gene replacement by PCR; HPLC was used to detect the modification degree of lipid A in LPS.

[0010] Preferably, the culturing of the mutant strain comprises: Initial culture stage: in modified GC liquid medium, control the temperature at 34℃~38℃ and CO2 concentration at 4%~6%; logarithmic growth stage: gradually increase the pH value to 7.6~7.8, and maintain the osmotic pressure at 280~320mOsmol / L; Late stage of culture: the osmotic pressure of the fermentation liquid is regulated to a stable level.

[0011] Preferably, the extraction of outer membrane vesicles comprises: Add 0.1M Tris-HCl buffer containing 5-10mM EDTA; Stir at room temperature for 20 to 30 minutes; The crude extract was separated at a centrifugal speed of 12,000×g to 15,000×g.

[0012] Preferably, the purification of outer membrane vesicles comprises: Use a 100 kDa molecular cutoff membrane to ultrafilter and replace the solution until the EDTA concentration is less than 1 mM; Use DNase concentration of 2000-2500U / L in a solution containing 2-3mM MgCl2; The exosome extraction system is used to remove small molecule impurities below 30nm.

[0013] The present invention also provides the use of a low endotoxin mutant strain of group B meningococcus, including using the mutant strain for outer membrane vesicle preparation.

[0014] Preferably, the outer membrane vesicle preparation comprises: Controlling the culture conditions of mutant strains; Optimizing lipopolysaccharide synthesis in outer membrane vesicles using a dynamic metabolic flux model; Enhancing the structural stability of outer membrane vesicles by chemical modification.

[0015] Preferably, the outer membrane vesicle preparation further comprises chemical modification: A mixture of phosphatidylcholine and cholesterol was added to the extracted outer membrane vesicles; The weight ratio of phosphatidylcholine to cholesterol is 1:0.5 to 1:1; Treat at 37°C for 30 to 60 minutes.

[0016] The present invention provides a low endotoxin mutant of group B meningococcus and its application, which has the following beneficial effects: 1. The present invention combines gene editing technology and homologous recombination to accurately delete the lpxL1 gene and dynamically inhibit the expression of lpxL2 and lpxM genes, thereby effectively reducing the modification level of lipid A. Compared with the problems of low gene manipulation efficiency and unstable performance of mutant strains in the prior art, this technical solution improves the endotoxin control effect of mutant strains and solves the difficult problem of balancing stability and activity.

[0017] 2. In the process of extracting outer membrane vesicles, the present invention uses EDTA solution to chelate and destroy the outer membrane, combined with ultrafiltration concentration and enzyme degradation methods, which not only ensures the integrity of the vesicle structure, but also achieves high-purity extraction. The physical method in the prior art often causes damage to the vesicle structure, but the present invention avoids this problem and improves the vesicle extraction efficiency.

[0018] 3. By dynamically optimizing the culture conditions of the mutant strains, especially the regulation of osmotic pressure and pH value, the present invention ensures that the mutant strains have improved metabolic stability and vesicle release. Compared with the single scheme in the prior art that does not consider the regulation of the culture environment, this method is more suitable for industrial application needs.

[0019] 4. The present invention adopts a systematic metabolic optimization model to control the lipid A synthesis pathway, achieving the dual goals of low endotoxin activity and high immunogenicity. Compared with the shortcomings of the existing technology that simply reduces endotoxins but damages immune function, this solution effectively retains the core advantages of mutant strains in immune development and provides strong support for vaccine design. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a flow chart of the preparation method of the present invention; Figure 2 The schematic diagram of the present invention is a schematic diagram of constructing the group B meningococcal lpxL1 homologous recombination plasmid. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Please refer to the attached Figure 1 and attached Figure 2 The present invention provides a group B meningococcal low endotoxin mutant, comprising the following steps: S1. Provide meningococcal mother strains; The selection of the mother strain should meet certain genetic background requirements to ensure the high efficiency of gene editing and the stability of the strain. Generally, the selected mother strain needs to have a good outer membrane structure and maintain complete lipopolysaccharide (LPS) biosynthesis ability, thus laying the foundation for subsequent mutation operations.

[0023] In some embodiments, strains suitable as mother strains include, but are not limited to, group B meningococcal strains, which exhibit good operational stability during the extraction and purification of outer membrane vesicles. At the same time, the strain has high activity in modifying the lipid A part of lipopolysaccharide, providing a clear target for subsequent gene editing.

[0024] In this embodiment, the mother plant provided has the following characteristics: The genome of the mother plant contains a complete lpxL1 gene, which encodes a hydroxy fatty acyltransferase and plays an important role in the biosynthesis pathway of lipid A.

[0025] When the mother strain is cultured on modified GC solid medium (GCagarbase, supplemented with 1% IsoVitaleX), it can grow rapidly at 37°C and 5% CO2. The typical growth time is 12 to 15 hours, and the colonies are round, transparent and smooth.

[0026] The basic composition of the mother plant lipopolysaccharide can be expressed as the following general formula: LPS=LipidA+Core Polysaccharide+O-Antigen Among them, LipidA is the main determinant of endotoxin activity, and its molecular formula is: LipidA=C6H 10 O5N2+∑(R fa ) Where: C6H 10 O5N2+∑ represents the core disaccharide backbone of lipid A, which is composed of two molecules of D-glucosamine linked by β-1,6; ∑(R fa ) indicates modified fatty acid chains, the number and type of which are directly related to the activity of the lpxL1 gene; The core polysaccharide is the intermediate structure connecting LipidA and O-antigen, and its general formula is: Core Polysaccharide=∑(Sugar Units) Wherein, ∑ (Sugar Units): the sum of glycosyl units, usually including D-glucose (Glc), D-mannose (Man) and L-galactosamine (GalNAc).

[0027] The arrangement of the core polysaccharide is determined by the genes encoding the synthases (e.g., waaC and waaF); O-Antigen=n(Repeating Units) Wherein, n: the number of repeating units, usually 10 to 50; Repeating Units: the specific chemical formula of the repeating unit; Through the above formula, the molecular composition of LPS is broken down into three parts: LipidA, core polysaccharide, and O-antigen. Among them, LipidA is an important determinant of endotoxin activity, and its degree of modification directly affects the virulence of bacteria. The present invention precisely controls the fatty acid modification level of LipidA by deleting the lpxL1 gene and inhibiting the expression of lpxL2 / lpxM, thereby reducing its endotoxin activity while retaining the integrity of the core polysaccharide and O-antigen. This molecular-level regulation method effectively solves the problems of unstable growth of mutant strains and insufficient immunogenicity in traditional methods.

[0028] Supplementary calculation formula: Relationship between endotoxin activity and LipidA modification level Endotoxin activity (E) and the number of fatty acid chains in LipidA (n fa ) is positively correlated, which can be expressed as: E=k·n fa Where, E: endotoxin activity, unit is EU / mg; k: constant, reflecting the effect of environmental conditions on LipidA modification; n fa : Number of fatty acid chains, usually 4 to 6; gene editing can reduce n fa , which can achieve effective control of E. Experimental results show that when the number of fatty acid chains is reduced to 3 or less, the endotoxin activity decreases by more than 70%.

[0029] In a possible implementation, the modification of LipidA in the provided mother strain meets the following characteristics: The carbon number of the fatty acid chain is 12 to 16; Contains one or more phosphate groups, usually modified at the C1 and C4' positions of the disaccharide backbone.

[0030] Specifically, the preparation method of the mother plant in this embodiment is as follows: Generally, the mother strain stored in cryopreservation (-80℃) needs to be revived before use. Thaw the cryopreserved tube at room temperature for 3 to 5 minutes, take a small amount of bacterial solution with a sterilized inoculation loop, and streak it on the surface of the modified GC solid culture medium. Then, place the culture medium in a 37℃ incubator and culture it under 5% CO2 for 12 to 15 hours. During this process, it is necessary to avoid overgrowth of the strain to prevent the bacterial metabolites from interfering with the genome structure.

[0031] As an alternative, if the mother strain is used directly for gene editing operations, after solid culture, a single colony can be transferred to modified GC liquid medium (the formula contains 10g peptone, 1g starch and 1g glucose per liter, supplemented with 0.1% L-lysine and 0.2% L-glutamic acid) and cultured at 200rpm and 35℃ until OD 600 Reach 0.6-0.8. The cell state at this stage is optimal, which is conducive to the subsequent plasmid transformation or gene editing steps.

[0032] In some embodiments, to verify the genetic background of the mother plant, the following genomic analysis is performed: Whole genome sequencing (WGS) technology is used to analyze the gene sequence integrity of the mother plant. The specific operations are: Extract genomic DNA from the mother plant (using the traditional phenol-chloroform method or a commercial kit).

[0033] The Illumina NovaSeq platform was used to construct a 150 bp paired-end library.

[0034] BWA and Prokka software were used for data alignment and gene annotation, with a focus on confirming the integrity of the lpxL1, lpxL2, and lpxM genes.

[0035] The results showed that the lpxL1 gene sequence of the mother strain was 945bp long, located on the second genome fragment, and did not contain any frameshift mutation or deletion. The genes related to the synthesis of core polysaccharides (such as waaC and waaF) were intact, providing a guarantee for the normal biosynthesis of lipopolysaccharides.

[0036] In one possible implementation, the selection of the mother strain directly affects the yield and purity of subsequent outer membrane vesicles: Generally speaking, the selected mother plant should have good outer membrane stability. Specific evaluation methods include: The growth performance of the mother plant was tested under different osmotic pressure environments. The mother plant was inoculated into culture medium with osmotic pressures of 250, 300, and 350 mOsmol / L, and the growth curve was observed for 24 hours. The results showed that the appropriate osmotic pressure range was 280-320 mOsmol / L.

[0037] The outer membrane structure of the mother plant was observed by transmission electron microscopy (TEM). The image showed that the outer membrane was smooth, the distance between the inner and outer membranes was 3-5nm, and no obvious folding or rupture was observed.

[0038] This embodiment provides a reliable basis for the subsequent construction of mutant strains through the steps of providing and verifying the above-mentioned mother strains.

[0039] S2, construction of plasmid vector for gene replacement; The design of the plasmid vector needs to meet the basic requirements of gene replacement, including carrying homologous fragments upstream and downstream of the target gene to initiate homologous recombination, and having a selection marker gene to facilitate subsequent screening operations. In general, the vector backbone needs to have the ability to replicate and transform efficiently and be compatible with the genetic background of the meningococcal mother strain.

[0040] Specifically, the construction of the plasmid vector is achieved through multiple steps. First, the homologous sequence of the target gene is obtained, and the length of the upstream and downstream sequences must be long enough to ensure efficient homologous recombination. Then, a screening marker gene (such as a kanamycin resistance gene) is inserted, and the entire fragment is cloned into the plasmid backbone. The final design of the vector needs to be verified by enzyme digestion and sequencing to confirm its accuracy.

[0041] In one possible implementation, a plasmid vector is used to replace the lpxL1 gene in the parent strain. This gene encodes hydroxy fatty acyltransferase, an important enzyme for lipid A modification, and its deletion can reduce the endotoxin activity of the parent strain. The design of the plasmid vector must ensure that it does not affect the normal expression of other genes, while taking into account the growth stability of the mutant strain.

[0042] Generally, homologous sequences upstream and downstream of the target gene lpxL1 are amplified from the genome of the meningococcal mother strain. Each homologous sequence is 800 to 1200 bp long and is obtained by PCR amplification. The formula of the PCR reaction system is as follows: PCR reaction system = dNTPs + primer pair + Taq enzyme + template DNA + buffer, where dNTPs: deoxynucleotide mixture, final concentration is 200 μM; primer pair: upstream and downstream primers, concentration is 0.5 μM; Taq enzyme: DNA polymerase, concentration is 2U / 50 μL; template DNA: parent plant genomic DNA, concentration is 50 ng / μL; buffer: reaction buffer, containing Mg 2+ ions, concentration is 1.5 mM.

[0043] The amplified product was verified by 1.0% agarose gel electrophoresis to verify whether the fragment size was consistent with the expected value. If the fragment size deviation exceeded 50 bp, the PCR conditions should be optimized, such as lowering the annealing temperature or increasing the extension time.

[0044] As an option, the amplified homologous sequence fragment can be double-digested with restriction endonucleases to facilitate subsequent cloning into a plasmid vector. Recommended restriction sites include EcoRI and HindIII.

[0045] During the plasmid construction process, it is necessary to select a suitable backbone vector. Specifically, the vector backbone used in this embodiment is pUC18, which has high copy replication ability and a multiple cloning site (MCS) for easy insertion of exogenous fragments. The pUC18 vector also has an AmpR resistance gene, which provides the selective pressure of ampicillin for preliminary screening of transformation efficiency. The lpxL1 upstream homologous sequence, kanamycin resistance gene and downstream homologous sequence are sequentially inserted into the multiple cloning site (MCS) region of the vector. The basic structure of the plasmid backbone can be expressed as: Plasmid=Promoter+MCS+Selectable Marker+Replication Origin Among them, Promoter: promoter sequence, used to drive the expression of the selectable marker gene. It is recommended to use a medium-strong promoter; MCS: multiple cloning site, providing a variety of restriction sites for inserting exogenous fragments; Selectable Marker: selectable marker gene, such as kanamycin resistance gene, used for resistance screening; Replication Origin: replication initiation sequence, determines the number of copies of the plasmid in the mother plant.

[0046] In a possible implementation, the sequence of the kanamycin resistance gene is inserted between the upstream and downstream homologous sequences. The specific sequence of the kanamycin resistance gene must be ensured to be complete and functionally expressed to avoid screening failure.

[0047] The specific plasmid verification method is: After the plasmid is constructed, the accuracy of the vector structure needs to be confirmed by double enzyme digestion experiment. Taking EcoRI and HindIII as an example, the cutting result can produce three fragments, the sizes of which correspond to the upstream and downstream homologous sequences and the kanamycin resistance gene respectively. The enzyme digestion reaction system is as follows: Enzyme digestion system = DNA template + restriction enzyme + buffer + ddH2O Among them, DNA template: constructed plasmid vector, concentration is 100 ng / μL; restriction enzymes: EcoRI and HindIII, usage amount is 2U / 20μL; buffer: 10× reaction buffer, final concentration is 1×; ddH2O: deionized water, used to make up the total reaction volume.

[0048] The digestion products were analyzed by agarose gel electrophoresis. The size of the target band should be consistent with the expected value, otherwise the primers need to be redesigned or the vector structure needs to be repaired.

[0049] In some embodiments, the optimization of the plasmid vector may also include the addition of additional regulatory elements. For example, a ribosome binding site (RBS) is added to both ends of the upstream and downstream homologous sequences to improve the initiation efficiency of gene transcription. The sequence of the RBS may be AGGAGG or a similar sequence, and its length is 6 to 10 bp.

[0050] In the process of plasmid expression, in order to avoid non-specific recombination, the target sequence of the Cas9 system can also be introduced as a negative screening method. The specific operation is to insert a 20bp Cas9 recognition sequence in the non-target gene region to ensure the specific cutting of the plasmid that has not been successfully replaced in the mother strain.

[0051] The above-mentioned plasmid vector construction method ensures the efficient expression and homologous recombination ability of the vector in the meningococcal mother strain through reasonable design and verification steps, providing technical guarantee for subsequent gene replacement operations.

[0052] S3, using gene editing technology to replace the target gene of the mother strain to obtain a mutant strain; In order to achieve the construction of low-endotoxin mutant strains of group B meningococci, on the basis of providing the mother strain and constructing the plasmid vector, it is necessary to use gene editing technology to replace the target gene in the mother strain. In general, the core steps of target gene replacement include plasmid transformation, homologous recombination and gene editing regulation. Through homologous recombination, the lpxL1 gene is deleted, and the expression of lpxL2 and lpxM genes is further dynamically regulated to optimize the modification level of lipopolysaccharide (LPS).

[0053] Specifically, gene editing technology needs to be combined with the genetic background of the mother plant and the design characteristics of the plasmid vector. Condition optimization, selective screening and verification methods during the transformation process are the key to ensuring the successful construction of mutant strains. In one possible implementation method, the CRISPR-dCas9 system can be used to further inhibit the expression of non-target genes, reduce the modification level of fatty acid chains in lipopolysaccharides, and ensure the efficiency of target gene replacement and the stability of mutant strains.

[0054] In this embodiment, the specific steps of the gene editing operation are as follows: After plasmid construction is completed, the plasmid vector is introduced into the mother plant by autonomous uptake. Generally, the mother plant needs to be revived before transformation and cultured on GC agar plates overnight (~16 hours).

[0055] Use a marker to draw a 1x1cm square on the back of a GC agar plate and streak a small amount of overnight grown bacteria onto this square: the streak should be barely visible.

[0056] ~5 ug of plasmid was dropped onto the square of bacteria and the plate was incubated at 37°C and 5% (v / v) CO2 for 6-8 hours.

[0057] After this period, bacterial growth should be seen within the squares. All of this growth was plated onto fresh GC agar plates containing the appropriate antibiotic (50 ug / mL Kan).

[0058] Individual colonies were picked within the next 48 hours and streaked onto fresh GC agar plates and grown to logarithmic growth phase.

[0059] In some embodiments, homologous recombination is accomplished by: After the plasmid is introduced into the mother strain, the lpxL1 gene in the mother strain is replaced by homologous recombination mediated by upstream and downstream homologous sequences. This process mainly relies on the endogenous recombinase system of meningococci. To improve the recombination efficiency, it is necessary to ensure that the length of the homologous sequence reaches 800 to 1200 bp.

[0060] The basic principle of homologous recombination can be expressed as the following formula: Among them, P HR : Homologous recombination probability; L HS : Homologous sequence length (800-1200 bp); L TS : total length of target sequence; E RS : Recombinase activity index.

[0061] In general, P HR With L HS Positively correlated, when L HS When it is greater than 0.8 times the length of the target gene sequence, the recombination probability can reach more than 90%.

[0062] Alternatively, the CRISPR-dCas9 system can be used to dynamically regulate the expression of non-target genes: Specifically, dCas9 binds to specific sgRNAs, guiding the dCas9 protein to bind to the promoter regions of the lpxL2 and lpxM genes, thereby inhibiting their transcription. The efficiency of this transcriptional inhibition depends on the design of the sgRNA and the binding properties of the Cas9 protein.

[0063] The core sequence of sgRNA must meet the following requirements: Length: 20 bp; GC content: 40% to 60%; The terminal three nucleotides must match the PAM sequence of the parent strain.

[0064] Utilizing the above system, the modification of fatty acid chains in lipopolysaccharide can be effectively reduced, further reducing endotoxin activity.

[0065] Screening methods for targeted gene replacement: After gene editing is completed, it is necessary to verify whether the replacement of the target gene is successful through various means. In some embodiments, the following steps are used: The transformed bacteria were inoculated into a modified GC solid medium containing kanamycin at a concentration of 60-90 μg / mL to screen for resistant strains; Use PCR to amplify upstream and downstream homologous sequence regions, and verify the products by agarose gel electrophoresis. The target fragment length should be about 2000-3000 bp; High performance liquid chromatography (HPLC) was used to detect the composition of LPS, focusing on the fatty acid modification level of Lipid A. The experiment showed that when the number of fatty acid chains was reduced from 6 to 3, the endotoxin activity was reduced by more than 70%.

[0066] In one possible implementation, the genetic stability of the mutant strain is verified by: The mutant strain was subcultured for 20 generations, and the integrity of the target gene was tested by PCR in each generation. The results showed that the lpxL1 gene replacement region in the mutant strain did not mutate, and the inhibition levels of lpxL2 and lpxM remained stable.

[0067] In the growth performance test of mutant strains, the mutant strains were cultured to the logarithmic growth phase (12 to 24 hours) and their OD 600 The values ​​were not significantly different from those of the non-mutated parent strain, indicating that the gene replacement operation had no adverse effects on the growth performance of the strain.

[0068] Through the above method, this example successfully completed the replacement of the target gene and obtained a low endotoxin mutant strain of group B meningococcus.

[0069] S4. Screening and obtaining low endotoxin mutant strains of group B meningococci; This step involves key links such as selective culture, gene verification and endotoxin activity detection. Through screening, it can be ensured that the mutant strain not only successfully deletes the target gene, but also retains good growth performance and outer membrane structure.

[0070] Specifically, the screening work should combine resistance screening, genomic verification and biochemical testing to ensure that the selected mutants have low endotoxin characteristics and meet the needs of subsequent research and application. In one possible implementation, PCR technology is used to verify the replacement of the target gene, and the modification level of the lipid A part in lipopolysaccharide (LPS) is analyzed by high-performance liquid chromatography (HPLC) to further confirm the low endotoxin activity of the mutant.

[0071] In this embodiment, the steps for screening mutants are as follows: Generally, the transformed bacteria are inoculated into a modified GC solid medium containing kanamycin to screen for resistant single colonies. The antibiotic concentration of the medium ranges from 60 to 90 μg / mL, and the culture conditions are 37°C and 5% CO2 for 12 to 16 hours.

[0072] For the single colony obtained in the initial screening, it is necessary to further verify whether the target gene in its genome has been correctly replaced. Specifically, PCR amplification is performed by extracting the genomic DNA of the single colony. The PCR reaction conditions are as follows: PCR reaction system = template DNA + primer pair + dNTPs + Taq enzyme + 10× buffer. When the size of the amplified product is consistent with the expected value (about 2000-3000 bp), it indicates that the target gene has been successfully replaced.

[0073] In some embodiments, to further confirm the integrity of the replacement region, the PCR amplification product can be sent to a commercial sequencing agency for Sanger sequencing. The sequencing results should show that the kanamycin resistance gene (kanR) is accurately inserted into the target gene replacement position.

[0074] As an option, the composition of lipopolysaccharide can be determined by HPLC: Lipopolysaccharide was extracted from the screened single colony and the modification level of lipid A was analyzed. The basic method for lipopolysaccharide extraction is the hot phenol water method, which includes the following steps: Take 5 mL of bacterial solution in the logarithmic growth phase, centrifuge at 4°C and 5000×g for 10 min to collect the bacteria; Add 1 mL of water-saturated phenol solution to the bacteria and incubate in a 95°C water bath for 10 minutes; After cooling, the mixture was centrifuged at 12,000 × g for 15 min, the aqueous phase was collected, and the extraction was repeated twice.

[0075] The extracted lipopolysaccharide samples were detected by high performance liquid chromatography. The fatty acid modification level of the lipid A part can be characterized by the following formula: Among them, R fa : fatty acid modification ratio; ∑ fa-mod : The total amount of modified fatty acids (such as hydroxylated and acylated fatty acids); fa-total : The total amount of all fatty acids in lipopolysaccharide.

[0076] Generally, when the fatty acid modification ratio of the mutant strain is lower than 0.5, it can be judged as having low endotoxin activity.

[0077] In a possible implementation, the growth performance of the mutant strain needs to be tested: The selected mutant strains were inoculated into the improved GC liquid medium and the OD 600 The growth performance of the mutant strain can be quantified by the following formula: Among them, G rate : Growth rate, in OD 600 / h; ΔOD 600 : OD per unit time 600 ; Δt: time interval, in hours.

[0078] The growth rate of the mutant strain should not be significantly different from that of the parent strain to ensure its suitability for subsequent applications.

[0079] Screening mutants for genetic stability: To verify the genetic stability of the mutant strain, it can be continuously propagated for 20 generations. At the end of each generation, genomic DNA is extracted and PCR is performed to detect the replacement of the target gene. If the target gene replacement does not undergo reverse mutation after 20 generations and the expression of the resistance gene is stable, the mutant strain is considered to be genetically stable.

[0080] In addition, the outer membrane vesicles of the mutant strains need to be morphologically observed. The vesicle images were taken using a transmission electron microscope (TEM). The average diameter of the outer membrane vesicles of the mutant strains ranged from 60 to 90 nm, with uniform morphology and clear boundaries.

[0081] Through the above method, the screened mutant strains not only successfully deleted the target gene, but also showed excellent characteristics in lipopolysaccharide modification, endotoxin activity and growth performance.

[0082] S5, culturing the mutant strain; After successfully screening out the expected low-endotoxin mutants, they need to be properly cultured to ensure that the mutants have stable growth performance and provide sufficient bacterial volume for the subsequent extraction of outer membrane vesicles. In general, the culture process needs to include initial culture, amplification during the logarithmic growth phase, and stable regulation in the later stage of culture. By optimizing the culture medium composition, culture conditions, and process parameters, the outer membrane integrity of the mutants can be maintained to the maximum extent, while avoiding the effect of bacterial lysis on the extraction efficiency of the target product.

[0083] Specifically, the culture of mutant strains should be combined with their metabolic characteristics to dynamically adjust the osmotic pressure and pH value to meet growth requirements. In one possible implementation, the OD of the culture medium can be monitored. 600 Changes in the growth rate of mutant strains were quantified and combined with biochemical analysis to assess the fitness of culture conditions.

[0084] In this embodiment, the culture process of the mutant strain is as follows: Generally, after the mutant strain is revived from the frozen state (-80°C), it needs to be streaked on a solid culture medium. The culture medium is a modified GC solid medium supplemented with 1% IsoVitaleX, and the culture conditions are 37°C, 5% CO2, and the culture time is 12 to 15 hours.

[0085] Pick a small amount of bacteria from a single colony, inoculate it into the modified GC liquid medium, and culture it with shaking. The basic formula of the liquid medium is as follows: Culture medium components = peptone + starch + glucose + NaCl + L-lysine + L-glutamic acid The culture conditions are: temperature: 35-37°C; rotation speed: 200-250 rpm; CO2 concentration: 4-6%.

[0086] In some embodiments, to further improve the culture effect, an osmotic pressure regulator (such as glycerol or sucrose) may be added in a concentration range of 0.5-1% (w / v).

[0087] In one possible implementation, dynamic regulation of the data growth period includes the following: By real-time monitoring of the pH value and osmotic pressure of the culture medium, the culture conditions can be dynamically adjusted. The osmotic pressure adjustment formula is as follows: P osm =R·T·∑c i Among them, P osm : osmotic pressure of culture medium, unit is mOsmol / L; R: gas constant, value is 0.0821L·atm / (K·mol); T: temperature of culture medium, unit is K; ∑c i : The total concentration of solutes in the culture medium, in mol / L.

[0088] Generally, the suitable osmotic pressure range of mutant strains is 280-320mOsmol / L. When the osmotic pressure is lower than 280mOsmol / L, NaCl is added to increase the solute concentration; when the osmotic pressure is higher than 320mOsmol / L, it can be adjusted by diluting the culture medium.

[0089] Specifically, the cultivation process needs to be regulated in stages: In the initial culture stage, the pH value of the culture medium is controlled at 7.2 to 7.4. The growth rate of the mutant strain is slow at this stage, and the influence of rapid changes in pH on the cell outer membrane should be avoided as much as possible.

[0090] Entering the logarithmic growth phase (12 to 24 hours), gradually increase the pH value to 7.6 to 7.8 and keep the osmotic pressure stable. 600 The OD value was used to evaluate the proliferation of bacteria. 600 The changing law of can be expressed by the following formula: N t =N0·e μ·t Among them, N t : bacterial concentration at time t (measured by OD 600 N0: initial bacterial concentration; μ: bacterial specific growth rate, unit is h -1 ; t: cultivation time, in hours.

[0091] In the later stage of culture (24-48 hours), the rotation speed needs to be appropriately reduced to 150-200 rpm and the pH value should be maintained at 7.6-7.7 to slow down the metabolic activity of the bacteria and avoid excessive lysis interfering with the extraction of outer membrane vesicles.

[0092] In some embodiments, monitoring and optimization of the culture process includes the following: Use online sensors to monitor changes in DO (dissolved oxygen) and pH of the culture solution. DO levels should be maintained at 30-40%, as levels below this range may inhibit bacterial metabolism.

[0093] At the end of the culture, the culture fluid needs to be collected and the wet weight of the bacteria needs to be recorded.

[0094] During the culture process, the osmotic pressure, pH value and DO level were dynamically regulated to ensure the growth performance and outer membrane integrity of the mutant strain, which laid the foundation for the subsequent efficient extraction of outer membrane vesicles.

[0095] S6. Extract outer membrane vesicles of mutant strains; After the mutant strain is cultured, the outer membrane vesicles need to be extracted from the bacteria. Generally, the extraction of outer membrane vesicles is completed through steps such as chemical dissolution, centrifugation and filtration purification. This process not only requires the destruction of the outer membrane structure of the mutant strain to release the vesicles, but also the avoidance of excessive loss or structural damage of the outer membrane vesicles. The extracted OMVs will serve as the core component of vaccine development or other immune-related applications, and their purity and stability directly determine the quality of the final product.

[0096] Specifically, key parameters need to be controlled during the extraction of outer membrane vesicles, such as the concentration of the dissolving solution, stirring time, and centrifugation speed. In one possible implementation, EDTA solution is used to chelate divalent cations in the outer membrane, destroying the outer membrane structure, thereby effectively releasing the vesicles, and the purity is improved by ultrafiltration and DNA enzyme treatment.

[0097] In this embodiment, the specific steps of extracting outer membrane vesicles are as follows: Generally, after collecting the bacteria from the culture medium, the bacteria need to be pretreated. Take 500mL of culture medium, centrifuge at 4℃, 8000×g for 20 minutes, collect the bacterial precipitate, and the calculation formula of the wet weight of the bacteria is as follows: W wet =V·C OD ·F Among them, W wet : wet weight of bacteria, in g; V: volume of culture medium, in L; C OD :OD 600 Conversion factor to wet weight, usually 0.5g / L / OD 600 ; F: adjustment factor, depends on the strain and culture conditions, and ranges from 0.8 to 1.2.

[0098] Add 5 mL of 0.1 M Tris-HCl buffer (pH 8.6) to the collected bacteria, and add 10 mM EDTA solution, and stir at room temperature for 20 to 30 minutes. EDTA destroys the Mg in the bacterial outer membrane by chelation.2+ and Ca 2+ bridge, thereby releasing the vesicles.

[0099] As an option, the stirring speed can be adjusted according to the amount of bacteria and the viscosity of the solution.

[0100] In some embodiments, the stirring speed is 100-150 rpm to avoid excessive shear force causing damage to the vesicle structure.

[0101] Isolate OMVs by high-speed centrifugation at 4°C, 12,500 × g to 15,000 × g for 60 minutes. The supernatant after centrifugation contains crude outer membrane vesicles that need to be further purified.

[0102] In one possible implementation, purifying outer membrane vesicles comprises the following steps: Use a 100kDa molecular cutoff ultrafiltration membrane for concentration and liquid exchange. The buffer for liquid exchange is 10mMTris-HCl (pH7.4), and the EDTA concentration is controlled below 1mM. During the ultrafiltration process, the integrity of the vesicles is ensured by adjusting the pressure and flow rate. The typical operating pressure is 0.2-0.4MPa and the flow rate is 20-40mL / min.

[0103] To remove DNA contamination, DNase solution was added to the sample with a final concentration of 2000-2500 U / L, and 2-3 mM MgCl2 was added to increase DNase activity. The reaction conditions were 37°C, 30-60 minutes, and then the degradation products were removed by filtration.

[0104] The filtration system uses a 0.22μm pore size filter membrane to further remove fine impurities. For high-purity applications (such as vaccine preparation), an exosome extraction system (such as EXODUS) can be used for final purification.

[0105] Quantification and characterization of outer membrane vesicles: The concentration and quality of extracted outer membrane vesicles need to be determined by specific methods. Quantitative methods for OMVs concentration include protein content determination and lipopolysaccharide content determination.

[0106] Protein content determination: The protein concentration of the sample was determined using the Bradford method, and the formula is as follows: C protein =A 595 ·k Among them, C protein : Sample protein concentration, in μg / mL; A 595 : absorbance at 595 nm; k: slope of the standard curve, in μg / mL / OD.

[0107] Lipopolysaccharide content determination: The LAL test was used to determine the lipopolysaccharide content. The following formula is used: Among them, C LPS : LPS concentration, in EU / mL; T: gelation time generated in the reaction tube, in seconds; V: sample volume, in mL.

[0108] In some embodiments, the morphology of the vesicles is observed using a transmission electron microscope (TEM). Typical OMVs have a diameter of 60 to 90 nm, a smooth surface, and are evenly distributed.

[0109] The integrity of the vesicles was ensured by using steps such as EDTA, centrifugation and ultrafiltration during the extraction process, and the purity was further improved by means of DNA degradation and molecular retention.

[0110] The use of the serogroup B meningococcal low endotoxin mutant described below can be referenced to the serogroup B meningococcal low endotoxin mutant described above.

[0111] The present invention also provides the use of a low endotoxin mutant strain of group B meningococcus, including using the mutant strain for outer membrane vesicle preparation.

[0112] In a preferred embodiment of the present invention, the outer membrane vesicle preparation comprises: Controlling the culture conditions of mutant strains; Optimizing lipopolysaccharide synthesis in outer membrane vesicles using a dynamic metabolic flux model; Enhancing the structural stability of outer membrane vesicles by chemical modification.

[0113] In a preferred embodiment of the present invention, the outer membrane vesicle preparation further comprises chemical modification: A mixture of phosphatidylcholine and cholesterol was added to the extracted outer membrane vesicles; The weight ratio of phosphatidylcholine to cholesterol is 1:0.5 to 1:1; Treat at 37°C for 30 to 60 minutes.

[0114] This embodiment can be used to execute the above-mentioned preparation method embodiment, and its principle and technical effect are similar, which will not be described in detail here.

[0115] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A low endotoxin mutant of group B meningococcus, characterized in that: The following steps are involved: Provide meningococcal mother strains; Construction of plasmid vectors for gene replacement; Gene editing technology is used to replace the target gene of the mother strain to obtain a mutant strain; Screening and obtaining low endotoxin mutants of group B meningococci; The mutant strain was cultured; Extract outer membrane vesicles of mutant strains.

2. The low endotoxin mutant of group B meningococcus according to claim 1, characterized in that: The plasmid vector constructed for gene replacement includes: Construct homologous sequences on both sides of lpxL1; Insertion of kanamycin resistance gene; The homologous sequences and resistance gene were ligated to the plasmid backbone.

3. The low endotoxin mutant of group B meningococcus according to claim 1, characterized in that: The target gene replacement is accomplished by the following steps: transforming the plasmid into a mother plant; Replace the lpxL1 gene in the mother strain by homologous recombination; Dynamic inhibition of lpxL2 gene expression.

4. The low endotoxin mutant of group B meningococcus according to claim 1, characterized in that: The mutant screening comprises: The mutants were screened in a medium containing kanamycin; Detect target gene replacement by PCR; HPLC was used to detect the modification degree of lipid A in LPS.

5. The low endotoxin mutant of group B meningococcus according to claim 1, characterized in that: The cultivation of the mutant strain comprises: Initial culture stage: in modified GC liquid medium, control the temperature at 34℃~38℃ and CO2 concentration at 4%~6%; Logarithmic growth phase: gradually increase the pH value to 7.6-7.8, and maintain the osmotic pressure at 280-320mOsmol / L; Late stage of culture: the osmotic pressure of the fermentation liquid is regulated to a stable level.

6. The low endotoxin mutant of group B meningococcus according to claim 1, characterized in that: The extraction of outer membrane vesicles comprises: Add 0.1M Tris-HCl buffer containing 5-10mM EDTA; Stir at room temperature for 20 to 30 minutes; The crude extract was separated at a centrifugal speed of 12,000×g to 15,000×g.

7. The low endotoxin mutant of group B meningococcus according to claim 1, characterized in that: The purification of outer membrane vesicles comprises: Use a 100 kDa molecular cutoff membrane to ultrafilter and replace the solution until the EDTA concentration is less than 1 mM; Use DNase concentration of 2000-2500U / L in a solution containing 2-3mM MgCl2; The exosome extraction system is used to remove small molecule impurities below 30nm.

8. Use of a low endotoxin mutant of group B meningococcus, characterized in that: Use of the group B meningococcal low endotoxin mutant strain according to any one of claims 1 to 7 comprises using the mutant strain for outer membrane vesicle preparation.

9. The use of the low endotoxin mutant of group B meningococcus according to claim 8, characterized in that: The outer membrane vesicle preparation comprises: Controlling the culture conditions of mutant strains; Optimizing lipopolysaccharide synthesis in outer membrane vesicles using a dynamic metabolic flux model; Enhancing the structural stability of outer membrane vesicles by chemical modification.

10. The use of the low endotoxin mutant of group B meningococcus according to claim 8, characterized in that: The outer membrane vesicle preparation also includes chemical modification: A mixture of phosphatidylcholine and cholesterol was added to the extracted outer membrane vesicles; The weight ratio of phosphatidylcholine to cholesterol is 1:0.5 to 1:1; Treat at 37°C for 30 to 60 minutes.

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