CN105001276B

CN105001276B - A kind of less toxic Kdo2The preparation and its application of MPLA

Info

Publication number: CN105001276B
Application number: CN201510284792.4A
Authority: CN
Inventors: 王小元; 王碧雯; 韩亚宁; 李烨; 李颜颜
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2015-05-28
Filing date: 2015-05-28
Publication date: 2018-03-16
Anticipated expiration: 2035-05-28
Also published as: CN105001276A

Abstract

The invention discloses a kind of Kdo of novel low-toxicity₂Lipoid A preparation and its application, belongs to bioengineering field.Present invention transformation Escherichia coli, a kind of Kdo with special construction, low toxicity is synthesized₂MPLA, this Kdo₂Lipoid A is made up of without outer core polysaccharide long-chain, only two deoxidation of 2 ketone 3 octanoic acids, six fatty acid chains and C4 positions monophosphate.The Kdo₂Lipoid A, it is easy to separation and Extraction and detection, and maintains the biological immune activity of lipoid part A, 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, standard and step is simple and clear are easily operated.

Description

A kind of less toxic Kdo2The preparation and its application of-MPLA

Technical field

The present invention relates to a kind of Kdo of novel low-toxicity₂The preparation and its application of-MPLA, belong to bioengineering neck Domain.

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.The LPS, LPS on its top layer can be discharged after Gram-negative bacteria intrusion host It can be identified by the Toll-like receptor 4 (Toll Like Receptor 4, TLR-4) on host immune cell surface, then trigger Intracellular a series of biochemical reactions, produce a variety of inflammatory cytokines such as TNF-α, IL-6, IL-8.Wherein, Kdo₂- class Fat A is the major part combined with TLR-4 acceptors, and the fine structure of especially lipoid part A determines that LPS is released after stimulating cell The cell factor type and quantity put.

The lipoid part A of wild-type e. coli and salmonella generally comprises six or seven fatty acid chains and in C1 position And C4 ' positions are connected with two phosphate groups, there is higher cytotoxicity.And the lipid A derivatives of some special constructions, then show Go out relatively low toxicity, vaccine adjuvant can be used as, non-specifically strengthen the former immune response of body fight, improve vaccine effect Rate.But because lipoid A macromolecule water-solubilities are very low, effective bioactivity can not be showed in aqueous phase, so recent decades are extremely The present, basic scientific research or clinically or corresponding lipid A derivatives bioactivity is studied using LPS studies, but LPS is due to core sugar and O antigen part structure diversifications, and molecular weight is big and heterogeneity, can not pass through mass spectrum or nuclear magnetic resonance Method carries out detection and Rapid identification in real time, also the temporary method for extraction and purification without standardization.In addition, production lipoid A is adopted at present Salmonella is pathogenic bacteria and a variety of different structure lipoid A of synthesis, and the relatively difficult, product of operating is impure and in aqueous phase System bioactivity is bad, also need absorption some salt surfaces could with vaccine with reference to and preferably act on；Actual production process In be related to a variety of chemical treatments such as acid/base hydrolysis, step is various, cost is high, and can be with aliphatic acid in processing procedure Chain break, compound resulting structure is destroyed, influences product quality and yield.

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, only comprising two 2-keto-3-deoxyoctanoic acids, six fatty acid chains and C1, C4 ' Two phosphate groups in position, synthesized since intercellular membrane inner side, route of synthesis is very conservative, therefore Escherichia coli W3110 is excellent In the industrial production starting strain of salmonella.And Kdo₂- lipoid A with respect to lipoid A have preferably it is amphiphilic, be easy to directly carry Take and purify, and can also has preferable solubility and bioactivity by the direct Testing and appraisal of ESI mass spectrums in aqueous phase system.This Invention transformation Escherichia coli, a kind of Kdo with special construction, low toxicity is synthesized₂- MPLA, this Kdo₂- mono- phosphorus Acids fat A is free of core polysaccharide long-chain, only by two 2-keto-3-deoxyoctanoic acids, the single phosphate of six fatty acid chains and C4 ' positions Group is formed.This special Kdo₂- MPLA, it is easy to separation and Extraction and detection, industrial production efficiency is high, cost is low, And this special Kdo₂- MPLA compound maintains the effective immunostimulatory activity of lipoid part A, wilder Type W3110 LPS also has obvious attenuation, is extremely potential vaccine adjuvant.Meanwhile the present invention provides one kind and carried Take and purify Kdo₂- lipoid A standardized method, step is simple and clear, easily operated.

The content of the invention

The invention solves first technical problem be to provide it is a kind of with special construction, attenuation Kdo₂- monophosphate Lipoid A, its chemical formula are C₁₁₀H₂₀₁N₂O₃₆P, relative molecular mass 2157.37, it is the D-Glucose connected with β-(1 ' -6) Amine disaccharides is skeleton, and 6 ' positions connect two α -one base -3- deoxidation-D- sweet dew octanoic acids (Kdo₂), 4 ' positions be phosphorylated, 3 ' positions connection (R) -3- (tetradecyloxyaniline) myristyl, 2 ' bit aminos connection (R) -3- (dodecyloxy) myristyl, 3 and 2 ammonia Base connects tetradecyloxyaniline respectively and formed, and concrete structure formula is shown in formula I：

The invention solves second technical problem be to provide one kind and can directly produce the attenuation Kdo₂- monophosphate class Fat A Recombinant organism E.coli W3110 △ waaCF △ lacIlacZ::FnlpxE.Kdo₂- lipoid A and its spread out The various structures of biology now can not be by being chemically synthesized, and also the bacterial strain of no naturally occurring can directly synthesize Kdo₂- Lipoid A.Heretofore described Recombinant organism is by waaCF, lacI in E.coli W3110 DNA sequences Deletion mutation inactivation occurs for gene, is knocked in respectively obtained by the FnlpxE genes of expressing heterologous inside lacZ gene.The Escherichia coli Genetic engineering bacterium antibiotic-free mark, in growth course without derivant, can directly it is synthetically produced go out special construction low toxicity Kdo₂- MPLA, and product is single, purity is high, can be extracted directly and be easy to purify；Bacterial strain has higher hydrophobic It is property, higher from agglutinability and relatively low antibiotic resistance and Geng Gao safety in utilization.

Its construction method mainly includes the following steps that：First, the lacI on wild-type e. coli W3110 chromosomes is knocked out Gene, the regulatory protein in lacI gene code lac operators, knock out the gene and can release regulatory protein and the later stage is inserted The exogenous gene expression in lacZ sites checks effect, foreign gene is formed constitutive expression；Secondly, in W3110 △ lacI On the basis of bacterial strain, FnlpxE genes are knocked at chromosome lacZ sites, lpxE derives from Francisella, its egg encoded White enzyme can hydrolyze the phosphoric acid on lipoid A molecules 1, and structure obtains intermediate strains HW001；Again, struck on the basis of HW001 Except the waaC-waaF gene clusters on chromosome, bacterial strain BW001 is constructed, bacterial strain is lacked the second glycosyl of core polysaccharide the 1st Enzyme is transported, Kdo is directly produced so as to be not connected to outer core polysaccharide₂- MPLA.

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 waaCF of structure knocks out the nucleotide sequence such as SEQ ID NO.3 of fragment It is shown.

The present invention also provides a kind of conveniently application Recombinant organism production Kdo₂- lipoid A's Method, mainly include the following steps that：

(1) thalline is cultivated：Using general shake flask fermentation culture, seed bacterium solution is transferred and cultivated to initial stage stationary phase.

(2) extraction separation：Thalline is collected, Kdo is extracted using chloroform/methanol/water mixed phase extraction₂- lipoid A studies.

(3) DEAE fibre columns purify：Kdo₂- lipoid A studies mix membrane phospholipid, are passed through again by being splined on DEAE fibre columns Gradient elution, phosphatide impurity is removed, obtains the Kdo of high-purity₂- lipoid A.

It is not related to pathogenic bacteria in the production method, and without derivant, cracks sugar chain without being chemically treated, bacterial strain can Attenuation Kdo is obtained for directly extraction₂The large-scale production of-MPLA；And the Kdo of this strain gene engineering bacterium₂- monophosphate Lipoid A products are single, have it is amphiphilic, have certain solubility in organic phase and aqueous phase, be good vaccine adjuvant candidate.

Traditional lipoid A acquisition is first to use phenol extraction lipopolysaccharides, then carries out a series of processes such as hydrolysis, chromatography, purifying. The special Kdo of the present invention₂- MPLA has good parents' property, can directly be extracted with chloroform methanol mixed system Obtain, method is similar to the extraction of membrane phospholipid, without adding phenol, is hydrolyzed without by acid/base, compared with lipoid A extraction Cheng Gengwei is simple.The Kdo extracted₂- MPLA sample can directly pass through thin-layer chromatography (TLC) and electrospray ionization mass spectrum (ESI/MS) detect, analyze, and generally lipopolysaccharides is because it contains a large amount of hydrophily sugar chains, molecular weight is excessive, fat-soluble extremely low nothing Method is directly analyzed by TLC and EIS/MS.

To cytotoxicity, immunostimulatory activity assessment in, MPLA water solubility is extremely low, and immune effect is bad, Typically also need to be combined or be adsorbed in aluminium salt surface with other adjuvants in actual clinical and could preferably play a role.The present invention's Special construction Kdo₂Higher solubility is also presented in-MPLA sample in aqueous phase cell culture system, maintains lipoid The bioactivity of part A, the LPS compared with wild type W3110 also have obvious attenuation, the parents assigned along with special construction Property is more favorable to it and combined in a variety of forms with vaccine composition, is extremely potential vaccine adjuvant candidate.

Brief description of the drawings

Fig. 1 genetic engineering bacteriums BW001 produces Kdo₂The TLC analyses of-MPLA

1:WBB06Kdo₂- lipoid A samples； 2:BW001Kdo₂- MPLA sample

Fig. 2 genetic engineering bacteriums BW001 produces Kdo₂- MPLA ESI/MS is analyzed

A:WBB06Kdo₂- lipoid A samples；B:BW001Kdo₂- MPLA sample

Fig. 3 wild-type e. colis W3110LPS and genetic engineering bacterium BW001Kdo₂- MPLA stimulates mouse huge Phagocyte RAW264.7 oxicity analysis

Fig. 4 wild-type e. colis W3110LPS and genetic engineering bacterium BW001Kdo₂- MPLA stimulates people's macrophage Cell THP-1 oxicity analysis

Fig. 5 wild-type e. colis W3110 and genetic engineering bacterium BW001 hydrophobicity with from aggegation specificity analysis

Fig. 6 wild-type e. colis W3110 and genetic engineering bacterium BW001 antibiotic resistance is analyzed

Embodiment

The Kdo that the genetic engineering bacterium BW001 of embodiment 1 is produced₂The extraction and structural analysis of-MPLA

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 can It is six fatty acid chains, the Kdo of bis phosphoric acid directly to synthesize lipoid part A₂- 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 molten to be eventually adding chloroform/methanol Liquid (2:1, v/v) by Kdo₂- lipoid A is washed out.

2nd, the Kdo that genetic engineering bacterium BW001 is produced₂Thin-layer chromatography (TLC) analysis of-MPLA

The Kdo that the WBB06 of wash-off and BW001 is produced₂- 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, because containing bis phosphoric acid, hydrophobicity is weaker and migration velocity is slower；Mutant strain BW001 Kdo₂- lipoid A (swimming lane 2) structure It is very single, high hydrophobicity is showed, migration velocity is substantially speeded compared with WBB06, infers its lipoid part A for one polarity of missing The monophosphate form of six fatty acid chains of phosphate group.TLC structures illustrate that, by directional transformation, genetic engineering bacterium BW001 is true Surely produce single, special construction Kdo₂- MPLA, and can be extracted directly, Rapid identification.

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 BW001 is produced₂- MPLA, its lipoid part A C1 positions lack a phosphate group (m/z values are 80), are Six fatty acid chain forms of monophosphate, main [M-H]^-Peak m/z values are 2156.0, illustrate the special construction Kdo of BW001 productions₂- Lipoid A definite molecular weight is 2157；M/z values 2177.9 be add sodium ion after caused by [M+Na-2H]^-Peak, actually Same substance.MS results further identify the Kdo that genetic engineering bacterium BW001 is produced₂- lipoid A precision architecture, says again Bright genetic engineering bacterium BW001 can directly produce the very single Kdo of structure really₂- MPLA, and this compound exists High sensitivity is shown in the detection of ESI/MS mass spectrographs, is very beneficial for implementing monitoring in later phase clinical development process and administration is controlled System.

The Kdo that the genetic engineering bacterium BW001 of embodiment 2 is produced₂The oxicity analysis of-MPLA

The Kdo that the genetic engineering bacterium BW001 directly extracted is produced₂- MPLA sample is because being mixed with multiple film phosphorus Fat carries out cytotoxicity analysis again, it is necessary to purify and remove after phosphatide disturbs.Kdo₂The purifying of-MPLA mainly passes through DEAE fibre columns gradient elution is completed.In cell Stimulation Assay, wild type goes out bacterium germination W3110 lipopolysaccharides (LPS) purification of samples work For positive control, PBS is as negative control.

1、Kdo₂The DEAE fibre columns purifying of-MPLA

BW001 bacterial strains expand inoculation 1L systems, are incubated overnight, and are produced according to the extraction of method described in embodiment 1 Kdo₂- MPLA.Chloroform/methanol/2.4M ammonium acetates (v/v/v 2 will be stored in:3:1) DEAE-cellulose in It is added in glass tube and adsorbing separation post is made, controls column volume to be acted on for 20ml；With chloroform/methanol/water (v/v/v of 6 times of volumes 2:3:1) pillar is balanced；With monoploid product chloroform/methanol/water (v/v/v 2:3:1) solution washes out Kdo₂- MPLA is thick Sample, it is loaded in pillar；When sample flow to filler upper end, with chloroform/methanol/ammonium acetate (v/v/v 2:3:1) solution is carried out Stepwise elution, the concentration gradient of wherein aqueous phase ammonium acetate take 60mM, 120mM, 240mM, 360mM, 480mM respectively；Collect elution Liquid simultaneously adds appropriate chloroform and water, eluent is become Bligh-Dyer two-phase systems, stands 30mins or so, removes phase, Nitrogen blows.Phosphatide impurity mainly washes out when acetic acid ammonium concentration is 60mM, 120mM, 240mM, the Kdo of purifying₂- MPLA Sample mainly washes 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 bacterium solution being incubated overnight by initial OD₆₀₀=0.02 is transferred to 800mL LB liquid In body culture medium, 37 DEG C culture 12 hours after 8000rpm centrifugation 10min collect thalline, ddH₂O washing thallines once weigh bacterium afterwards Body weight in wet base, it is dissolved in 10mL ddH per 3g wet thallus₂In O, 68 DEG C of preheatings.90% phenol preheated in equal volume is added, 68 DEG C acutely Vibration 1 hour.After sub-cooled, 4 DEG C, 4000rpm is centrifuged 20 minutes, supernatant ddH₂O dialyses three days to remove phenol, very Vacuum freecing-dry obtains LPS crude products.The appropriate ddH of crude product₂After O is resuspended, RNaseA (the μ g/ml of final concentration 50) and DNase is added 37 DEG C of I (the μ g/ml of final concentration 100) is handled 2 hours, adds 37 DEG C of processing of Proteinase K (the μ g/ml of final concentration 100) overnight.At enzyme 5mL water saturation phenol is added in sample after reason, is mixed, 4000rpm is centrifuged 30 minutes, supernatant ddH₂O dialyses one day, very Vacuum freecing-dry obtains LPS.LPS is redissolved and arrives chloroform/methanol solution (2:1, v/v), vortex oscillation 30 seconds, 12000rpm centrifugations 10 minutes, supernatant is removed, precipitation is redissolved in water, and vacuum freeze drying obtains LPS sterlings.

3、Kdo₂After-MPLA Stimulated Macrophages leukaemia RAW264.7 produce inflammatory cell because The analysis of son

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 hours After take supernatant, detected respectively using ELISA kit (R＆D Systems, DY406, DY410) interleukin-6 (IL-6) with it is swollen Tumor necrosis factor α (TNF-α) content (Fig. 3 A, B).So the results of stimulation of sample has carried out difference point using minimum significant poor method Analysis, * * P<0.01, * P<0.1.Under each irritaiting concentration, Kdo caused by genetic engineering bacterium BW001₂- MPLA sample Lipoid part A C1 positions phosphate group is removed, and its toxicity to mouse cell RAW264.7 substantially weakens.It is in sample concentration During 100ng/ml, Kdo caused by BW001₂- MPLA sample can substantially lower the inducing amount to IL-6, and it stimulates cell Caused IL-6 is only the 39.6% of wild type W3110LPS inductions, and it induces the secretory volume of TNF-α also to compare wild type The low 2097.6pg/ml of W3110LPS inductions.It can be seen that Kdo caused by genetic engineering bacterium BW001₂- MPLA is to mouse Cytositimulation toxicity substantially reduces, and is properly applied to the exploitation of follow-up animal vaccine adjuvant.

4、Kdo₂- MPLA produces dividing for inflammatory cytokine after stimulating people's acute leukemia monocyte THP-1 Analysis

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<0.01, * P<0.1.With mouse cell moderate stimulation result It is similar, Kdo caused by genetic engineering bacterium BW001₂IL-8 and TNF- caused by-MPLA sample induction people's cell THP-1 α also significantly decreases compared with wild type W3110LPS induction yield.When sample concentration is 100ng/ml, BW001 productions Raw special construction Kdo₂IL-8 caused by the induction of-MPLA is 14782.5pg/ml, and wild type W3110LPS is induced Generate 19347.3pg/ml.Kdo caused by genetic engineering bacterium BW001₂- MPLA stimulates with wild type W3110LPS Caused TNF-α is compared with mouse cell RAW264.7 order of magnitude lower after people's cell THP-1, but trend and IL-8 are basically identical. This result shows, Kdo caused by genetic engineering bacterium BW001₂The stimulation toxicity of-lipoid A to people's cell also significantly weakens, and also fits Preferably it is applied to the exploitation of the vaccine adjuvant of follow-up people.

The Kdo of the low toxicity of embodiment 3₂- MPLA producer gene engineering bacteria BW001 hydrophobicitys and from agglutinability point Analysis

1st, genetic engineering bacterium BW001 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 2mL bacterium are hanged Liquid 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), meter 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, the outer core polysaccharide of LPS hydrophilies The missing of chain can make cell surface hydrophobicity strengthen (Fig. 5 A).After measured, wild-type e. coli W3110 hydrophobicity is 39.6%, and engineering bacteria BW001 hydrophobicity substantially rises to 82.8%, about 2 of wild type times or so.Existing document card Bright, bacterial strain is advantageous to strengthen from compendency and cell surface hydrophobicity positive correlation (r=0.71), stronger cell surface hydrophobicity Hydrophobic interaction between cell, makes cell be easier to aggegation in aqueous and settle down.

2nd, comparisons of the genetic engineering bacterium BW001 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 A_i, retain 3 decimals)；Calculate from aggegation percentage AAg%=[(A₀-A_i)/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, BW001 is from aggegation percentage apparently higher than W3110 after standing 6 hours, after standing 12 hours, BW001 with W3110 is respectively 72.6% and 17.7% from aggegation percentage, and BW001 is higher by three times from agglutinability compared with wild type.Thalline The bacterial strain strong from aggegation advantageously in being collected rapidly, increases production efficiency in the industrial production.

The Kdo of the low toxicity of embodiment 4₂- MPLA producer gene engineering bacteria BW001 antibiotic resistances are analyzed

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, 100 μ L are added per hole, wherein erythromycin concentration gradient is 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, and last hole is not added with Medicine is as growth control.

Measurement result shows that engineering strain BW001 is to erythromycin (Erythromycin) and ovobiocin (Novobiocin) sensitiveness is basically identical, and resistance is very low.Erythromycin is respectively to the MIC of W3110 and BW001 bacterial strains 125μg/mL、7.8μg/mL；Ovobiocin is respectively 500 μ g/mL, 31.3 μ g/mL to W3110 and BW001 bacterial strains MIC.BW001 Bacterial strain is remarkably reinforced to the sensitiveness of erythromycin and ovobiocin, is reduced to about the 1/16 of wild type, illustrates that missing is core After heart polysaccharide and C1 positions phosphate group, epicyte permeability is improved to some extent so as to which antibiotic is easier to enter carefully Born of the same parents.Sum it up, the hypersensitivity and low resistance to antibiotic show that engineering strain BW001 is very safely controllable work Industry produces bacterial strain.

The engineering strain that the present invention is built can directly produce special construction, less toxic Kdo₂- MPLA, is avoided Using pathogenic bacteria production LPS or lipoid A, at the same the use of the phenol avoided and cumbersome chemical hydrolysis, chromatography, bacterial strain itself Antibiotic-free selection markers simultaneously show higher antibiotics sensitivity and higher from agglutinability, are 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

1. one kind produces less toxic Kdo₂The method of-MPLA, it is characterised in that the Kdo of application production novel low-toxicity₂- mono- phosphorus Acids fat A Recombinant organism is produced, including three key steps, (1) shake flask fermentation culture thalline, (2) Collect thalline, Bligh-Dyer mixed systems extraction separation attenuation Kdo₂- lipoid, (3) DEAE fibre columns purifying Kdo₂- lipoid；

The genotype of the genetic engineering bacterium is E.coli W3110 △ waaCF △ lacIlacZ::FnlpxE；It is described less toxic Kdo₂- MPLA chemical formula is C₁₁₀H₂₀₁N₂O₃₆P, structure include 2 α -one base -3- deoxidation-D- sweet dews octanoic acids, 2 ammonia Base glucose, 1 phosphate group and 6 fatty acid chains, be using β-(1 ' -6) connection D-Glucose amine disaccharides as skeleton, 6 ' positions Connect two α -one base -3- deoxidation-D- sweet dews octanoic acid, 4 ' positions are phosphorylated, 3 ' positions connection (R) -3- (tetradecyloxyaniline) tetradecane Base, 2 ' bit aminos connection (R) -3- (dodecyloxy) myristyl, 3 and 2 bit aminos connect tetradecyloxyaniline and structure respectively Into；Its structural formula is as shown in Equation 1,

The construction method of described genetic engineering bacterium comprises the following steps：1) artificial constructed lacI is knocked out into fragment to be transformed into In E.coli W3110/pKD46 competent cells, mutant strain E.coli W3110lacI are obtained::Pkan, mediated by Cre enzymes LoxP locus specificities restructuring remove kalamycin resistance gene；2) FnlpxE fragments electricity is transferred to the E.coli of step 1) preparation In W3110 △ 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 respectively, It is built into intermediate strains HW001；3) artificial constructed waaCF is knocked out into fragment on the basis of HW001 and is transformed into HW001/ In pKD46 competent cells, recombinant bacterium E.coli HW001waaCF are obtained:Pkan, the FRT sites mediated again by FLP enzymes Specificity restructuring remove kalamycin resistance gene therein respectively, final genotype is respectively E.coli W3110 △ waaCF△lacIlacZ::FnlpxE, it is built into engineering strain BW001；The lacI of structure knocks out the nucleotides sequence of fragment Row are as shown in SEQ ID NO.1；The nucleotide sequence of the FnlpxE-Fkan Insert Fragments of structure is as shown in SEQ ID NO.2；Structure The waaCF built knocks out the nucleotide sequence of fragment as shown in SEQ ID NO.3；

The DEAE fibre columns purifying, which is, to be 2 with the volume ratio of 6 times of DEAE fibre columns volumes:3:1 chloroform/methanol/level Weigh pillar；The volume ratio accumulated with monoploid is 2:3:1 chloroform/methanol/aqueous solution washes out Kdo₂- MPLA study, on Sample is into pillar；It is 2 with volume ratio when sample flow to filler upper end:3:1 chloroform/methanol/ammonium acetate solution carries out the stage Elution, the concentration gradient of wherein aqueous phase ammonium acetate take 60mM, 120mM, 240mM, 360mM, 480mM respectively；Collect eluent simultaneously Appropriate chloroform and water are added, eluent is become Bligh-Dyer two-phase systems, 30mins is stood, removes phase, nitrogen blows.

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