CN104844665B - A kind of Kdo of the low toxicity containing five fatty acid chains2The preparation and application of MPLA - Google Patents
A kind of Kdo of the low toxicity containing five fatty acid chains2The preparation and application of MPLA Download PDFInfo
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Abstract
The invention discloses a kind of Kdo of the low toxicity containing five fatty acid chains2The preparation and application of MPLA, belong to bioengineering field.The present invention transformation Escherichia coli, synthesized it is a kind of with special construction, low toxicity, five chain Kdo2MPLA, this Kdo2Lipoid A molecules are not connected with core polysaccharide long-chain, are only made up of two deoxidation of 2 ketone 3 octanoic acids, the single phosphoric acid of five fatty acid chains and C4 ' positions.The Kdo2Lipoid A has parents' property, is easy to separation and Extraction and purifying, can be carried out detection and Direct Identification, and maintains the biological immune activity of lipoid part A, and the LPS compared with wild type W3110 also has obvious attenuation, is extremely potential vaccine adjuvant.Meanwhile extraction provided by the invention and purify the Kdo2Lipoid A method, step is simple and clear, easily operated.
Description
Technical field
The present invention relates to a kind of Kdo of the low toxicity containing five fatty acid chains2The preparation and application of-MPLA, belong to raw
Thing engineering field.
Background technology
Lipopolysaccharides (LPS), i.e. bacterial endotoxin, be form most of gramnegative bacterium outer membrane outer layers it is main into
Point, mainly by Kdo2- lipoid A (Kdo2- Lipid A), core polysaccharide (Core) and O- antigens (O-antigen) three parts composition；
Wherein Kdo2- lipoid A is LPS hydrophobic side, and its structure is very conservative, is connected to hydrophilic core polysaccharide and O- antigens and assists
The two is adhered to cell surface and forms intact cell wall, and core polysaccharide and O- antigen parts have isomerism, different grams
There is multiple combinations form glycosyl species, number, position to negative strain even in same bacterial strain.Gram-negative bacteria invades host
The LPS on its top layer can be discharged afterwards, and LPS can be by (the Toll Like Receptor of Toll-like receptor 4 on host immune cell surface
4, TLR-4) identify, then trigger intracellular a series of biochemical reactions, produce a variety of inflammation such as TNF-α, IL-6, IL-8
Property cell factor.Wherein, Kdo2- lipoid A is the major part combined with TLR-4 acceptors, especially the fine knot of lipoid part A
Structure determines that LPS stimulates the cell factor type and quantity discharged after cell.
In wild-type e. coli and the LPS of salmonella, lipoid part A includes six or seven fatty acid chains, and
There are two phosphate groups C1 positions and C4 ' positions, have higher cytotoxicity.Some special constructions, the lipoid A of hypotoxicity can make
For vaccine adjuvant, non-specifically strengthen the former immune response of body fight, improve vaccine efficiency.But due to lipoid A
Macromolecule water-solubility is very low, and effective bioactivity can not be showed in aqueous phase, so recent decades are so far, basic scientific research or
Clinically or corresponding lipid A derivatives bioactivity is studied using LPS studies.However, LPS is due to molecular weight
Greatly, core sugar and O antigen part structure diversifications, can not carry out detecting in real time by the method for mass spectrum or nuclear magnetic resonance and quick
Identification, the also temporary method for extraction and purification without standardization.And in the production of actual lipoid A vaccine adjuvants, it is current to salmonella be
Pathogenic bacteria, have certain potential safety hazard, and salmonella produces the lipoid A of a variety of different structures simultaneously, extraction process is cumbersome,
High energy consumption.
Escherichia coli W3110 is non-pathogenic bacteria, and its LPS parts do not include O- antigens, only Kdo2- lipoid A and core are more
Sugar, and its Kdo2- lipoid part A structure is single, is an advantage over the industrial production starting strain of salmonella.Present invention transformation large intestine
Bacillus, a kind of Kdo with special construction, low toxicity is synthesized2- lipoid A, this Kdo2- lipoid A be not connected to core polysaccharide long-chain,
Only it is made up of two 2-keto-3-deoxyoctanoic acids, the single phosphoric acid of five fatty acid chains and C4 ' positions.This special Kdo2- lipoid A
With parents' property, it is easy to separation and Extraction and purifying, detection and Direct Identification can be carried out, and maintain lipoid A parts
Bioactivity, the LPS compared with wild type W3110 also have obvious attenuation, are an advantage over lipoid A and LPS molecule, great development
The vaccine adjuvant of potentiality.Meanwhile the present invention provides a kind of extraction and purifying Kdo2- lipoid A standardized method, step are simply bright
, it is easily operated.
The content of the invention
The invention solves first technical problem be to provide it is a kind of with special construction, low toxicity, five fatty acid chains
Kdo2- MPLA, its chemical formula are C96H175N2O35P, molecular mass 1947.17, its structure include 2 α -one bases-
3- deoxidation-D- sweet dews octanoic acid (Kdo), 2 Glucosamines, 1 phosphate group and 5 fatty acid chains, connected with β-(1 ' -6)
The D-Glucose amine disaccharides connect is skeleton, and 6 ' positions connect two α -one base -3- deoxidation-D- sweet dew octanoic acids (Kdo2), 4 ' positions are by phosphoric acid
Change, 2 ' bit aminos connection (R) -3- (dodecyloxy) myristyl, 3 ' positions, 3 and 2 bit aminos connect tetradecane oxygen respectively
Base and form, concrete structure formula is shown in formula I.Compound tool is amphiphilic, can directly be extracted by organic system while can quilt
The direct Testing and appraisal of ESI mass spectrums；There is preferable solubility in aqueous phase system, can be specifically congenital by TLR4/MD2 receptor activations
Property immune system, but show low cytotoxicity simultaneously, there are the potentiality as immunologic adjuvant.
The invention solves second technical problem be to provide one kind and can directly produce the attenuation Kdo2- lipoid A's
Recombinant organism E.coli W3110 △ waaCF △ lacI △ lpxMlacZ::FnlpxE.The genetic engineering bacterium strikes
Except the regulatory protein in lacI gene code lac operators；FnlpxE genes are knocked at chromosome lacZ sites, it is compiled
The protease of code can hydrolyze the phosphoric acid on lipoid A molecules 1；The protein for having knocked out coding can be in the position of quasi-grease A structure 3 '
The lpxM genes of 14 carbon hydroxy fatty acid chains are added, knock out waaCF gene clusters, obtain the purpose thing without core polysaccharide.
The construction method of the Recombinant organism be by waaCF in E.coli W3110 DNA sequences,
Deletion mutation inactivation occurs for lacI, lpxM gene, and the FnlpxE genes of expressing heterologous are knocked in inside lacZ gene.
Methods described mainly includes the following steps that：First, the lacI bases on wild-type e. coli W3110 chromosomes are knocked out
Cause, the regulatory protein in lacI gene code lac operators, knock out the gene and can release regulatory protein and lacZ is inserted to the later stage
The exogenous gene expression in site checks effect；Secondly, on the basis of W3110 △ lacI bacterial strains, in chromosome lacZ sites
Place knocks in FnlpxE genes, and (lpxE derives from Francisella, and its protease encoded can be hydrolyzed on lipoid A molecules 1
Phosphoric acid), it is allowed to form constitutive expression, structure intermediate strains HW001；Again, knocked out on the basis of HW001 on chromosome
LpxM genes, its protein encoded can add 14 carbon hydroxy fatty acid chains in the position of quasi-grease A structure 3 ', build intermediate strains
HW002；Finally, on the basis of HW002, waaCF gene clusters are knocked out, construct five fat of directly production synthesis special construction
Sour chain Kdo2- MPLA target gene engineering bacteria BW002.
In one embodiment of the invention, the lacI of structure knocks out the nucleotide sequence such as SEQ ID NO.1 of fragment
It is shown.
In one embodiment of the invention, the nucleotide sequence such as SEQ of the FnlpxE-Fkan Insert Fragments of structure
Shown in ID NO.2.
In one embodiment of the invention, the lpxM of structure knocks out the nucleotide sequence such as SEQ ID NO.3 of fragment
It is shown.
In one embodiment of the invention, the waaCF of structure knocks out the nucleotide sequence such as SEQ ID NO.4 of fragment
It is shown.
The present invention also provides a kind of conveniently application Recombinant organism production attenuation Kdo2- lipoid
A method, is mainly included the following steps that：
(1) thalline is cultivated：Using general shake flask fermentation culture, by seed bacterium solution switching culture to initial stage stationary phase.
(2) extraction separation：Thalline is collected, the attenuation Kdo is extracted using chloroform/methanol/water mixed phase extraction2- class
Fat A.
(3) DEAE fibre columns purify：The attenuation Kdo2- lipoid A studies mix membrane phospholipid, by being splined on DEAE
Fibre columns can remove phosphatide impurity through gradient elution again, obtain the Kdo of high-purity2- lipoid A.
Traditional lipoid A acquisition is first to use phenol extraction lipopolysaccharides, then carries out a series of processes such as hydrolysis, chromatography, purifying.
The present invention comprises only the Kdo of five fatty acid chains by traceless knockout lpxM genes, directly synthesis2- lipoid A molecules, by inserting
Enter to express FnlpxE genes, protease that it is encoded is by five fatty acid chain Kdo2- lipoid A molecular modifications are into five fatty acid chain Kdo2-
MPLA molecule, then pass through traceless knockout waaCF genes so that in bacterial strain due to lack outer membrane heptose transferase and
In inner membrance can not by first, second heptose of core polysaccharide with complete structural modification, it is containing five fatty acid chains
Kdo2- MPLA molecule is connected, the five fatty acid chain Kdo in growth course2- MPLA is directly tilted out outer
Produced on the outside of film.The Kdo of this special construction2- lipoid A can directly extract acquisition without chemical hydrolysis, chromatography.Pass through
The five less toxic fatty acid chain Kdo that conventional shake flask fermentation obtains2- MPLA can directly be extracted by chloroform-methanol system,
Method is similar to the extraction of membrane phospholipid, without adding phenol, is hydrolyzed without by acid/base, the extraction process compared with lipoid A is more
Simply.And this new Kdo2- lipoid A can directly be detected by thin-layer chromatography (TLC) and electrospray ionization mass spectrum (ESI/MS), divided
Analysis, and generally lipopolysaccharides is because it contain a large amount of hydrophily sugar chains, molecular weight is excessive, it is fat-soluble it is extremely low can not directly by TLC with
EIS/MS is analyzed.Generally speaking, the Recombinant organism that the present invention is built, in process of production without derivant, production
Thing can be used for directly extraction to obtain low toxicity, five fatty acid chain Kdo without further chemical treatment cracking sugar chain, bacterial strain2-
The large-scale production of MPLA；And the special construction Kdo of this plant of engineering bacteria production2- lipoid A products are single, have parents
Property, there is certain solubility in organic phase and aqueous phase, be more beneficial for combining in a variety of forms from different vaccines, there is good epidemic disease
Seedling adjuvant potentiality.
Brief description of the drawings
Fig. 1 genetic engineering bacteriums BW002 produces five chain Kdo2The TLC analyses of-MPLA
1:WBB06Kdo2- lipoid A samples；2:Genetic engineering bacterium BW002Kdo2- lipoid A samples
Fig. 2 genetic engineering bacteriums BW002 produces five chain Kdo2The ESI/MS analyses of-MPLA
A:WBB06Kdo2- lipoid A samples；B:Genetic engineering bacterium BW002Kdo2- lipoid A samples
Fig. 3 wild-type e. colis W3110LPS and chain Kdo of genetic engineering bacterium BW002 five2- MPLA stimulates small
Mouse macrophage RAW264.7 oxicity analysis
Fig. 4 wild-type e. colis W3110LPS and chain Kdo of genetic engineering bacterium BW002 five2- MPLA stimulates people
Macrophage THP-1 oxicity analysis
Fig. 5 wild-type e. colis W3110 and genetic engineering bacterium BW002 hydrophobicity with from aggegation specificity analysis
Fig. 6 wild-type e. colis W3110 and genetic engineering bacterium BW002 antibiotic resistance is analyzed
Embodiment
The Kdo that the genetic engineering bacterium BW002 of embodiment 1 is produced2- lipoid A extraction and structural analysis
Produced by W3110, the LPS containing long sugar chain can not directly extract, and due to quality is too big, in the dissolving of organic phase
Property low thin-layer chromatography (TLC) and the ESI/MS of can not also passing through analyze Direct Analysis structure.Therefore, we choose Escherichia coli
WBB06 (W3110waaCF::Tet6), control of the W3110 heptose deletion mutation strain as structural analysis.WBB06 is proved
Lipoid part A can directly be synthesized as six fatty acid chains, the Kdo of bis phosphoric acid2- lipoid A.
1、Kdo2- lipoid A extraction
The Kdo that control bacterium WBB06 and genetic engineering bacterium BW001 is produced2- lipoid A extraction using chloroform/methanol/
Water mixed phase extraction.Concrete operations are as follows：By the seed bacterium solution being incubated overnight by initial OD600=0.02 is transferred to 400mL
In LB fluid nutrient mediums, 37 DEG C of cultures to OD6008000rpm centrifuges 10min and collects thalline, ddH when=12O washing thallines are once
Afterwards with the phase systems of Bligh-Dyer mono- (chloroform/methanol/water, 1:2:0.8, v/v/v) suspension thalline, magnetic agitation 1h, 2000rpm
Centrifuge 20min split-phases, take phase, add appropriate chloroform and water be made into Bligh-Dyer two-phases system (chloroform/methanol/water,
2:2:1.8, v/v/v), 2000rpm centrifuges 10min, removes and mutually moves into revolving bottle, rotary evaporation；It is eventually adding chloroform/methanol
Solution (2:1, v/v) by Kdo2- lipoid A is washed out.
2nd, the Kdo that genetic engineering bacterium BW002 is produced2- lipoid A thin-layer chromatography (TLC) analysis
By the Kdo of the WBB06 of wash-off and BW002 productions2- lipoid A samples are with capillary glass tube point in Gel 60TLC plates
On, developing agent is chloroform/methanol/glacial acetic acid/water (25：15：4：4, v/v/v/v).Chromatography terminates the exhibition remained on after-blow dry plate
Layer agent, is carbonized with 10% sulfuric acid for being dissolved in ethanol, is placed in colour developing (Fig. 1) in heating plate.It is hydrophobic according to the property of developing agent
Property higher sample migration speed it is faster, therefore phosphate group less, the more sample of fatty acid chain quantity should have compared with Gao Qian
Move speed.TLC results show the Kdo of the unmodified WBB06 productions of lipoid part A2- lipoid A (swimming lane 1) structure is more single
One, migration velocity is slower；Genetic engineering bacterium BW002 Kdo2- lipoid A (swimming lane 2) structure is equally very single, sample migration speed
Degree is slightly faster than WBB06, and it is the Kdo containing five fatty acid chains to infer its lipoid part A2- MPLA form.TLC is tied
Structure illustrates that, by directional transformation, genetic engineering bacterium BW002 can produce single, special construction Kdo2- lipoid A.
3、Kdo2The ESI/MS analyses of-quasi-grease A structure
ESI/MS is analyzed：Lipid samples are dissolved in chloroform/methanol solution (2:1, v/v) in, in WATERS SYNAPT Q-TOF
Mass Spectrometer Method is carried out on Mass Spectrometer mass spectrographs.It is small using ESI ion guns, Anionic recognition pattern, detection range
In m/z 2500 (Fig. 2).Compare the Kdo of bacterium WBB06 productions2- lipoid A is main [M-H]-Peak m/z values are 2236.0, illustrate WBB06
The Kdo of production2- lipoid A definite molecular weight is 2237；Mass spectra peak of the m/z values at 2258.0 and 2279.9 is sample ions
Change process is added in instrument caused by sodium ion [M+Na-2H]-[M+2Na-3H]-Peak, actually same substance.Genetic engineering
The Kdo that bacterium BW002 is produced2- lipoid A, the Kdo of its five fatty acid chain of lipoid part A2- MPLA form, in class
Fat part A has lacked C1 positions phosphate group (m/z values are 80) and C3 ' precedence levels fatty acid chain (m/z values are 210), mainly
[M-H]-Peak m/z values are 1946.2, illustrate the special construction Kdo of BW002 productions2- lipoid A definite molecular weight is 1947.2；Together
Sample, m/z values 1968.2 be add sodium ion after caused by [M+Na-2H]-Peak, actually same substance.MS results identify
Genetic engineering bacterium BW002 produces Kdo2- lipoid A precise structure, further illustrate that genetic engineering BW002 can be produced directly
The very single Kdo containing five fatty acid chains of structure2- MPLA.
The five chain Kdo that the genetic engineering bacterium BW002 of embodiment 2 is produced2The oxicity analysis of-MPLA
The Kdo directly extracted2- lipoid A samples are carried out again because being mixed with a variety of membrane phospholipids, it is necessary to purify and remove after phosphatide disturbs
Cytotoxicity analysis.Kdo2- lipoid A purifying is mainly completed by DEAE fibre columns gradient elution.It is wild in cell Stimulation Assay
Raw type goes out bacterium germination W3110 lipopolysaccharides (LPS) purification of samples and compareed as positive control, PBS as hardness；Separately introduce by gene work
Journey bacterium BW001 (E.coli W3110 △ waaCF △ lacIlacZ::FnlpxE) six attenuation, monophosphate aliphatic acid of production
Chain Kdo2- lipoid A is compared.
1、Kdo2- lipoid A DEAE fibre columns purifying
Genetic engineering bacterium BW002 and control engineering bacteria BW001 expand inoculation 800mL systems respectively, are incubated overnight, according to reality
Apply method described in example 1 and extract produced Kdo2- lipoid A.Chloroform/methanol/2.4M ammonium acetates (v/v/v 2 will be stored in:3:
1) DEAE-cellulose in, which is added in glass tube, is made adsorbing separation post, controls column volume to be acted on for 20ml；With 6 times of bodies
Long-pending chloroform/methanol/water (v/v/v 2:3:1) pillar is balanced；With monoploid product chloroform/methanol/water (v/v/v 2:3:1) solution
Wash out Kdo2- lipoid A studies, are loaded in pillar；When sample flow to filler upper end, with chloroform/methanol/ammonium acetate (v/v/v
2:3:1) solution carry out stepwise elution, wherein the concentration gradient of aqueous phase ammonium acetate take respectively 60mM, 120mM, 240mM, 360mM,
480mM；Collect eluent and add appropriate chloroform and water, eluent is become Bligh-Dyer two-phase systems, stand
30mins or so, removes phase, and nitrogen blows.Phosphatide impurity mainly washes out when acetic acid ammonium concentration is 60mM, 120mM, 240mM, two plants
The Kdo of engineering bacteria production2- lipoid A samples mainly wash out when acetic acid ammonium concentration is 360mM, 480mM.
2nd, wild-type e. coli W3110LPS extraction and purifying
LPS extraction uses hot phenol method.By the W3110 bacterium solutions being incubated overnight by initial OD600=0.02 is transferred to 800mL
In LB fluid nutrient mediums, 37 DEG C culture 12 hours after 8000rpm centrifugation 10min collect thalline, ddH2O washing thallines once after
Thalline weight in wet base is weighed, is dissolved in 10mL ddH per 3g wet thallus2In O, 68 DEG C of preheatings.90% phenol that addition preheats in equal volume, 68
DEG C acutely vibration 1 hour.After sub-cooled, 4 DEG C, 4000rpm is centrifuged 20 minutes, supernatant ddH2O dialyses three days with removal
Phenol, vacuum freeze drying obtain LPS crude products.The appropriate ddH of crude product2After O is resuspended, RNaseA (the μ g/ml of final concentration 50) is added
37 DEG C are handled 2 hours with DNase I (the μ g/ml of final concentration 100), add 37 DEG C of processing of Proteinase K (the μ g/ml of final concentration 100)
Overnight.5mL water saturation phenol is added in sample after ferment treatment, is mixed, 4000rpm is centrifuged 30 minutes, supernatant ddH2O is saturating
Analysis one day, vacuum freeze drying obtains LPS.LPS is redissolved and arrives chloroform/methanol solution (2:1, v/v), vortex oscillation 30 seconds,
12000rpm is centrifuged 10 minutes, removes supernatant, and precipitation is redissolved in water, and vacuum freeze drying obtains LPS sterlings.
3、Kdo2Point of inflammatory cytokine is produced after-lipoid A Stimulated Macrophages leukaemias RAW264.7
Analysis
Mouse cell RAW264.7 is inoculated in 96 orifice plates, per hole 105Individual cell, 37 DEG C, 5%CO2After culture 12 hours, carefully
Born of the same parents are adherent, change fresh medium, add various concentrations LPS (final concentration is respectively 0.1,1,10,100ng/ml), stimulate 24 small
When after take supernatant, detect interleukin-6 (IL-6) respectively using ELISA kit (R＆D Systems, DY406, DY410)
With tumor necrosis factor α (TNF-α) content (Fig. 3 A, B).So the results of stimulation of sample has carried out difference using minimum significant poor method
Different analysis, * * * P<0.001,**P<0.01, * P<0.1.Under each irritaiting concentration, the work of genetic engineering bacterium BW002 and attenuation
Kdo caused by journey bacterium BW0012- lipoid A samples, have in varying degrees to the effect of stimulation of cell compared with wild type W3110LPS
Decrease, caused pre- inflammatory cytokine IL-6 and TNF-α are also reduced in varying degrees after inducing cell；Wherein genetic engineering
Kdo caused by bacterium BW0022- lipoid A sample lipoid part As decrease C3 ' precedences while C1 positions phosphate group has been lacked
Level fatty acid chain, its cytotoxicity weaken clearly.For example, when sample concentration is 100ng/ml, wild type W3110LPS
Kdo comprising six fatty acid chains and two phosphate groups2- lipoid A, it stimulates IL-6 caused by cell and TNF-α high respectively
Up to 3871.5pg/ml and 14164.7pg/ml, six fatty acid chain Kdo of monophosphate of BW001 productions2- lipoid A stimulates cell
Caused IL-6 and TNF-α are relatively low, respectively 1533.2pg/ml and 12067.1pg/ml.And caused by genetic engineering bacterium BW002
The Kdo of five fatty acid chains of monophosphate2- lipoid A sample lipoid part As have lacked C1 positions phosphate group and C3 ' precedences at the same time
Cytotoxicity weakens more obviously after level fatty acid chain, cell caused pre- inflammatory cytokine IL-6 and TNF- after being stimulated
α is much lower compared with the yield that wild type W3110LPS is stimulated, also below six fatty acid chains of monophosphate of C1 positions phosphate group
Sample inducing amount.When sample concentration is 100ng/ml, Kdo caused by BW0022- lipoid A samples induction IL-6 yield be
The 21% of 814.0pg/ml, only wild type W3110LPS induction, it induces the secretory volume of TNF-α also there was only 9059.9pg/ml,
Account for the 66% of wild type W3110LPS inductions.Therefore, Kdo caused by genetic engineering bacterium BW0022- lipoid A has to mouse cell
There is the effect being significantly attenuated, be properly applied to the exploitation of follow-up animal vaccine adjuvant.
4、Kdo2- lipoid A stimulates the analysis of generation inflammatory cytokine after people's acute leukemia monocyte THP-1
People's cell THP-1 is pressed per hole 105Individual cell is inoculated in 96 orifice plates, adds PMA to 20ng/ml, 37 DEG C, 5%CO2Lure
Lead 48 hours and be manipulated into differentiating to adherent macrophage, then change fresh medium, while it is (dense eventually to add various concentrations LPS
Degree be respectively 0.1,1,10,100ng/ml), stimulate 24 hours after take supernatant, using ELISA kit (R＆D Systems,
DY208, DY210) interleukin-8 (IL-8) and tumor necrosis factor α (TNF-α) content (Fig. 4 A, B) are detected respectively.So sample
Results of stimulation carried out variance analysis, * * * P using minimum significant poor method<0.001, * * P<0.01, * P<0.1.It is thin with mouse
Born of the same parents' moderate stimulation result is similar, Kdo caused by genetic engineering bacterium BW0022IL- caused by-lipoid A samples induction people's cell THP-1
8 with TNF-α and wild type W3110LPS, BW001Kdo2- lipoid A induction yield compares, and also significantly decreases.In sample
When product concentration is 100ng/ml, special construction Kdo caused by BW0022IL-8 caused by-lipoid A inductions is only 7379.1pg/
Ml, and wild type W3110LPS inducing amount is 19347.3pg/ml, the BW001 Kdo of six fatty acid chains2- lipoid A samples lure
The amount of leading is 14782.5pg/ml.Kdo caused by genetic engineering bacterium BW0022- lipoid A and wild type W3110LPS, BW001Kdo2-
Lipoid A stimulates after people's cell THP-1 caused TNF-α compared with mouse cell RAW264.7 order of magnitude lower, caused by BW002
Special construction Kdo2TNF-α caused by-lipoid A inductions is also significantly lower than W3110 LPS, BW001Kdo2- lipoid A inducing amount,
The 46% of only wild type W3110LPS induction yield.It can be seen that Kdo caused by genetic engineering bacterium BW0022- lipoid A is to people's cell
Also there are obvious attenuating effects, be also suitable for the exploitation of the vaccine adjuvant applied to follow-up people.
Five chain Kdo of the low toxicity of embodiment 32- MPLA producer gene engineering bacteria BW002 hydrophobicitys and from agglutinability
Power is analyzed
1st, genetic engineering bacterium BW002 and the hydrophobic comparisons of wild-type e. coli W3110
Bacterial strain hydrophobicity assay method is：It is incubated overnight bacterium solution centrifugation (7000g, 10min) and collects thalline；Thalline is used
PBS7.4 is washed 2 times；Then OD is made with PBS7.4 suspension thallines600=1.0 (are designated as A0, retain 3 decimals)；Finally by 2mL bacterium
Suspension mixes with 800 μ L dimethylbenzene, DL 2min, is stored at room temperature the OD of measure aqueous phase after 1h600(being designated as A, retain 3 decimals),
Calculate cell surface hydrophobicity:H%=(A0-A)/A0*100。
Because LPS is the main macromolecular of Escherichia coli outer membrane face, after waaCF is knocked out, LPS hydrophily core polysaccharide chains
Missing can make cell surface hydrophobicity strengthen (Fig. 5 A).After measured, wild-type e. coli W3110 hydrophobicity is
39.6%, and genetic engineering bacterium BW002 hydrophobicity rises to 78.7%, with the engineering bacteria BW001 compareed hydrophobicity
(82.8%) relatively, it is 2 times of wild type or so.Existing document proves that bacterial strain is hydrophobic with cell surface from compendency
Property positive correlation (r=0.71), stronger cell surface hydrophobicity is advantageous to strengthen the hydrophobic interaction between cell, makes thin
Born of the same parents are easier to aggegation in aqueous and settled down.
2nd, comparisons of the genetic engineering bacterium BW002 and wild-type e. coli W3110 from agglutinability
Bacterial strain is from agglutinability assay method：Centrifugation (7000g, 5min) is incubated overnight bacterium solution to collect thalline,
PBS7.4 washes thalline 2 times；Then OD is made with PBS7.4 suspension thallines600=2.0 (are designated as A0, retain 3 decimals)；Take 12mL should
Bacterium solution is added in test tube or graded tube, in 22 DEG C of standings；0h, 1.5h, 3h, 6h, 9h, 12h, 15h, 18h, 24h are determined respectively
OD600Value (is designated as Ai, retain 3 decimals)；Calculate from aggegation percentage AAg%=[(A0-Ai)/A0]×100.AAg% is got over
Show the stronger from agglutinability of thalline greatly.Draw curve and show dynamic change of the self-solidifying collection capacity of water with the time.
Such as Fig. 5 B, BW002 is from aggegation percentage apparently higher than W3110 after standing 6 hours, after standing 12 hours, W3110,
BW001, BW002 from aggegation percentage be respectively 17.7%, 72.6%, 39.8%, BW002 from agglutinability compared with compared with wild type
Being higher by one times has more, but is less than BW001.Thalline from the strong bacterial strain of aggegation in the industrial production advantageously in being collected rapidly,
Increase production efficiency.
Five chain Kdo of the low toxicity of embodiment 42- MPLA producer gene engineering bacteria BW002 antibiotic resistance analysis
Bacterial outer membrane is resisting antibiotic into playing an important role in cell as the natural cover for defense.LPS structure changes
Afterwards, it is contemplated that security when engineered strain actual production uses, genetic engineering bacterium is determined using micro broth dilution method respectively
Strain and wild-type e. coli are to the minimal inhibitory concentration of erythromycin and ovobiocin (MIC).Erythromycin (Erythromycin)
Be macrolide antibiotics, can be combined with 23SrRNA specific regions, produce structure damage effect, make peptidyl tRNA from
Dissociated earlier on ribosomes, so as to influence the generation of peptide chain and extension；Ovobiocin (Novobiocin) is that Coumarins resists
Raw element, can suppress archaeal dna polymerase.Influence nucleic acid metabolism.
MIC assay methods are：Antibiotic is diluted with LB fluid nutrient mediums, and the erythromycin of doubling dilution and ovobiocin are added
Into 96 orifice plates, add 100 μ L per hole, wherein erythromycin concentration gradient be 500,250,125,62.5,31.3,15.6,7.8,
3.9 μ g/mL, novobiocin concentration gradient are 1000,500,250,125,62.5,31.3,15.6,7.8 μ g/mL, last hole
Not dosing is used as growth control.
Measurement result shows that engineering strain BW002 bacterial strains are to erythromycin (Erythromycin) and ovobiocin
(Novobiocin) sensitiveness is basically identical, and resistance is very low.MIC of the erythromycin to W3110, BW001 and BW002 bacterial strain
Respectively 125 μ g/mL, 7.8 μ g/mL and 3.9 μ g/mL；Ovobiocin is respectively to W3110, BW001 and BW002 bacterial strain MIC
500μg/mL、31.2μg/mL、7.8μg/mL.BW002 bacterial strains are remarkably reinforced to the sensitiveness of erythromycin and ovobiocin, are dropped
It is low to the 1/32 of about wild type, also significantly lower than BW001, after illustrating while lacking C1 positions phosphate group and core polysaccharide,
Epicyte permeability is greatly improved so as to which antibiotic is easier to enter cell, and removes C2 ' precedence level fat again on this basis
Fat acid chain, then be further increased epicyte permeability, makes genetic engineering bacterium BW002 all highly quick to various antibiotic
Sense.Sum it up, the hypersensitivity and low resistance to antibiotic show that engineering strain BW002 bacterial strains are that very safety can
The industrial producing strain of control.
The engineering strain that the present invention is built can directly produce special construction, low toxicity, containing five fatty acid chain chains
Kdo2- MPLA, avoid and produce LPS or lipoid A, while the use of the phenol avoided and cumbersome using pathogenic bacteria
Chemical hydrolysis, chromatography, bacterial strain itself antibiotic-free selection markers simultaneously show higher antibiotics sensitivity and higher from agglutinability
Power, it is more beneficial for safe, green, large-scale industrial production.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by 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.
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| Escherichia coli Mutants That Synthesize Dephosphorylated Lipid A Molecules;Brian O. Ingram，等;《Biochemistry》;20100826;第49卷(第38期);8325–8337 * |
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