CN108395458A - It is attenuated the preparation and application of lipoid A - Google Patents
It is attenuated the preparation and application of lipoid A Download PDFInfo
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Abstract
The invention discloses the preparation and application of attenuation lipoid A, belong to gene engineering technology field.The present invention carries out molecular modification in non-pathogenic bacteria expression in escherichia coli lpxD1 genetic fragments or lpxD2 genetic fragments to lipoid A, the lipoid A that fatty acid chain structure changes has been obtained, and the recombination bacillus coli for expressing lpxD1 genetic fragments or lpxD2 genetic fragments reduces the toxicity of cell.The present invention provides new method and thinking to prepare vaccine adjuvant.
Description
Technical field
The present invention relates to the preparation and application of attenuation lipoid A, belong to gene engineering technology field.
Background technology
Lipopolysaccharides (LPS), is commonly called as endotoxin, is located on the outside of the outer membrane of Gram-negative bacteria, and be the main of the position
Constituent.By lipoid A, core polysaccharide and O antigen three parts composition, lipoid A is the activated centre of lipopolysaccharides, is to cause to exempt from
The primary structure of epidemic disease reaction.LPS can excite the nospecific immunity of host.LPS binding protein (LBP) are by LPS
It is transported to cell surface, with the help of CD14 and MD-2, by the receptor Toll like receptor4 (TLR4) of cell surface
Identification then has occurred a series of signal transmission, eventually leads to a series of release of inflammatory factors.Mainly there is Mal- among these
MyD88 and two signal paths of TRAM-TRIF composition, can generate the different biological cell factors, Mal-MyD88 is mainly induced respectively
TNF-α is generated, IL-6 and IL-8, TRAM mainly generate RANTES and MCP-1 etc..
Vaccine adjuvant is exactly immunopotentiator, can play booster action in vaccine inoculation mechanism, contribute to more
Well nospecific immunity is more fully excited, vaccine is made preferably to play a role.LPS can cause a series of inflammatory factor
Release, and lipoid A is the activated centre of LPS, so being ground as the important of new vaccine adjuvant using lipoid A as vaccine adjuvant
Study carefully direction.The lipoid A vaccine adjuvants being widely used at present are monophosphate from salmonella, six fatty acid chain lipoid A
(MPL), this MPL by being generated from mutant salmonella strain, structure and the quasi-grease A structure of wild type E.coli are different,
C1 phosphate groups and C3 myristyl missings, hexadecane secondary fat acid chain is had more in C2 fatty acid chains.MPL is to access
The stimulation degree of TLR4-Mal-MyD88 is low, has effective stimulation to TLR4-TRAM-TRIF, to which MPL can be stimulated effectively
Nospecific immunity, and not will produce excessive inflammatory factor injury body.But MPL also has in industrial production and clinical application
Shortcoming has certain risk and answers if the production salmonella of MPL belongs to conditioned pathogen carrying out industrial mass production
Beasts vaccine is used, secondly the production technology of MPL is complicated, the problems such as being damaged to its structure during production purifies.
Invention content
The first purpose of the invention is to provide a kind of novel lipoid A, and structure is as shown in following formula I：
Second purpose of the invention is to provide the preparation method of the lipoid A, includes the following steps：
(1) recombination bacillus coli of structure expression lpxD1 genetic fragments or lpxD2 genetic fragments；
(2) recombination bacillus coli is cultivated, lipoid A is collected in extraction.Specifically, using mono- phase systems of Bligh-Dyer (chloroform/
Methanol/water solution, 1:2:0.8, v/v/v) lower phase is collected by centrifugation in suspension thalline；Under be added to sodium acetate (pH4.5) solution, boil
Water-bath cracks sugar chain；Then be added chloroform and methanol formed Bligh-Dyer two-phases system (chloroform/methanol/water, 2:2:1.8, v/
V/v), centrifuge, remove and mutually rotate, chloroform/methanol solution (4 is added:1, v/v) lipoid A is washed out.
In one embodiment of the invention, step (1) described Escherichia coli be E.coli RL25, W3110 or
HW002。
In one embodiment of the invention, step (1), by come from Francisella lpxD1 genetic fragments or
LpxD2 genetic fragment digestions connect plasmid pUC18, are then transferred to host cell.
In one embodiment of the invention, the nucleotide sequence of the lpxD1 genetic fragments is SEQ ID NO:1
Sequence, the nucleotide sequence of lpxD2 genetic fragments is SEQ ID NO:2 sequence.
In one embodiment of the invention, step (2) collects the recombination bacillus coli thalline that late log phase is arrived in culture,
ddH2After O washing thallines with mono- phase systems of Bligh-Dyer (chloroform/methanol/aqueous solution, 1:2:0.8, v/v/v) suspension thalline,
Magnetic agitation for a period of time, split-phase, use mono- phase systems of Bligh-Dyer washing cell fragment 2-3 times；Sodium acetate is added
(pH4.5) solution, ultrasonic vibration, boiling water bath crack sugar chain；Chloroform is added after being cooled to room temperature and methanol forms Bligh-Dyer bis-
Phase system (chloroform/methanol/water, 2:2:1.8, v/v/v) it, centrifuges, removes and mutually move into revolving bottle, rotary evaporation, addition chloroform/
Methanol solution (4:1, v/v) lipoid A is washed out.
Third object of the present invention is to provide a kind of reduction coli somatics to TLR4 accesses stimulus intensity or cell
Immunocompetent method is in Escherichia coli is by pUC plasmid expressions lpxD1 genetic fragments, the Escherichia coli
W3110 or HW002.
Fourth object of the present invention is to provide the application of the recombination bacillus coli and lipoid A in terms of vaccine adjuvant.
Beneficial effects of the present invention：
(1) it is that host prepares lipoid A present invention employs non-pathogenic bacteria E.coli RL25, W3110 and HW002, widens
Adjuvant clinical application range.It expresses lpxD1 genetic fragments respectively in host or lpxD2 genetic fragments divides lipoid A
Son transformation, then uses chloroform/methanol/H2O solution mixed phase extractions, can fast and effectively extract lipoid A, opposite to reduce
Production procedure and the destruction to quasi-grease A structure.
(2) the lipoid A that the present invention is prepared by GC-MS and the method for basic hydrolysis mass spectral analysis is analyzed, phase
Than not expressing lpxD1 genetic fragments or lpxD2 genetic fragments, thalline class after lpxD1 genetic fragments or lpxD2 genetic fragments is expressed
The structure of fat A changes, and the structure of E.coli RL25/pUC-lpxD1 changes the fatty acid chain shown on 2 and 2 ' positions
By single C14:3-OH fatty acid chains become C14:3-OH、C16:3-OH and C18:3-OH is mixed.E.coli
The structure change of RL25/pUC-lpxD2 shows the fatty acid chain on 2 and 2 ' positions by single C14:3-OH fatty acid chains become
At C14:3-OH and C16:3-OH is mixed.Both bacterial strains are under 42 DEG C of condition of culture, to the signal path of TLR4
Stimulation ability reduces, and is reduced to the toxicity of cell.
(3) lpxD1 genetic fragments are expressed by pUC plasmids respectively in Escherichia coli W3110 and HW002, can dropped
Low W3110 and HW002 thalline are to TLR4 accesses stimulus intensity or Cell-mediated Immunity.
Generally speaking, novel lipoid A provided by the invention has safer property, is easy to extraction preparation as vaccine adjuvant
The advantages of.
Description of the drawings
Fig. 1 thalline E.coli RL25, E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2 lipoids A
Structure mass spectral analysis.
Fig. 2 thalline E.coli RL25, E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2 lipoids A
Structure mass spectral analysis after basic hydrolysis, A are 25% ammonium hydroxide results of hydrolysis, and B is the hydrolysis figure of the sodium hydroxide of 0.3M.
Fig. 3 thalline E.coli RL25, E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2 lipoids A
The GC-MS of middle fatty acid chain is analyzed.
Fig. 4 thalline E.coli RL25, E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2 self-solidifyings
The comparison of collection ability.
Fig. 5 thalline E.coli RL25, E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2 couple
TLR4 accesses stimulate the analysis of degree.
The comparison of Fig. 6 .RL25/pUC, RL25/pUC-lpxD1 and RL25/pUC-lpxD2 to Cell-mediated Immunity.A is not
The case where stimulating the cell factor of mouse RAW267.4 cells generation with thalline various concentration LPS, B are different thalline various concentrations
LPS stimulates the case where cell factor that Human THP-1 cells generate.
The quasi-grease A structure identification of Fig. 7 .LpxD1 expression bacterial strains.A is the lipoid A of W3110/pUC and W3110/pUC-lpxD1
ESI/MS analysis, B be HW002/pUC and HW002/pUC-lpxD1 lipoid A ESI/MS analysis.
Fig. 8 .LpxD1 expression bacterial strains stimulate cell TLR4 accesses with control strain the comparison of degree.
Comparisons of the LPS of Fig. 9 .LpxD1 expression bacterial strains to Cell-mediated Immunity.
Specific implementation mode
The structure of 1 genetic engineering bacterium of embodiment
Find out lpxD1 and lpxD2 gene orders.Enzyme is chosen according to the nucleotide sequence of lpxD1, lpxD2 and pUC plasmid
Enzyme site.Design primer carries out PCR, recycles lpxD1 and lpxD2 gene expression segments.Respectively digestion expressing gene fragment and
PUC18 plasmids.Expressing gene fragment after connection digestion and pUC18 plasmids.It will be connected by the method for chemical conversion
Plasmid imported into E.coli RL25, and (construction method of E.coli RL25 is referring to document：Bartling C M,Raetz C
R.Crystal structure and acyl chain selectivity of Escherichia coli LpxD,the
N-acyltransferase of lipid Abiosynthesis.Biochemistry,2009,48(36):8672-8683)、
(construction method of E.coli HW002 is referring to document by W3110 and HW002：Wang X,Zhang C,Shi F,Hu
X.Purification and characterization of lipopolysaccharides.Subcellular
Biochemistry.2010,53:27-51) in bacterial strain.Verification screening obtains the bacterial strain E.coli RL25/ containing correct plasmid
PUC-lpxD1, E.coli RL25/pUC-lpxD2, W3110/pUC-lpxD1 and HW002/pUC-lpxD1.
2 genetically engineered E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2 lipoids A's of embodiment
Structural analysis
1. the extracting method of lipoid A
The extracting method of lipoid A uses chloroform/methanol/H2O solution mixed phase extractions.By the bacterium solution being incubated overnight by first
Beginning OD600=0.02 is transferred in 200mL LB liquid mediums, when 37 DEG C of cultures are to the thalline logarithmic phase later stage, 4000rpm centrifugations
30min collects thalline, ddH2O washing thallines once afterwards use mono- phase systems of Bligh-Dyer (chloroform/methanol/aqueous solution, 1:2:
0.8, v/v/v) 76mL suspension thallines, magnetic agitation 1h, 2000rpm centrifuge 30min split-phases, broken using phase system washing cell
Piece 2-3 times；Sodium acetate (PH4.5) solution of 27mL 12.5mM, ultrasonic vibration 2min, 100 DEG C of water-bath 30min cracking sugar are added
Chain.Be cooled to after room temperature be added 30mL chloroforms and 30mL methanol be made into Bligh-Dyer two-phases system (chloroform/methanol/water, 2:2:
1.8, v/v/v), 2000rpm centrifuges 30min, removes and mutually moves into revolving bottle, rotary evaporation.Chloroform/methanol solution (4 is added:
1, v/v) lipidA is washed out.
The extracting method of 2.LPS
The extraction of LPS 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 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
Oscillation 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.
3. the mass spectrometric analysis method of lipoid A
Lipid samples are dissolved in chloroform/methanol solution (4:1, v/v) in, in WATERS SYNAPT Q-TOF Mass
Mass Spectrometer Method is carried out on Spectrometer mass spectrographs.Using ESI ion sources, Anionic recognition pattern, detection range is less than m/
z2500。
Mass spectral results show that the value of the m/z of E.coli RL25 is mainly 1796, is expressed when in E.coli RL25
When lpxD1, there are 1796,1824,1852,1880,1908 a series of values in the value of m/z, is expressed when in E.coli RL25
When lpxD2, there are 1796,1824,1852 a series of values in the value of m/z.Illustrate that the expression of lpxD1 and lpxD2 make
The quasi-grease A structure of E.coli RL25 is changed.
The GC-MS of 4.LPS is analyzed
The C15 (internal standard) and the 200 anhydrous acidified methanols of μ L2M of 50 μ L 0.2mg/mL will be added in the LPS samples of 200 μ g,
Lid is covered, 18 hours of effect in 90 DEG C of heating brick are placed in.It is cooled to room temperature, 200 μ L saturated sodium-chlorides are added.It is added
400 μ L hexanes, be vortexed concussion 30 seconds.It will be transferred in the sample injection bottle of GC by the supernatant of hexane extraction, sample introduction, GC analyses.
GC-MS analysis results are shown, in E.coli RL25 lipoids A, only exist C14:A kind of hydroxy fatty acids of 3-OH
Chain, and in E.coli RL25/pUC-lpxD1 lipoids A, there are C14:3-OH、C16:3-OH、C18:Tri- kinds of hydroxyl fat of 3-OH
Fat acid chain, in E.coli RL25/pUC-lpxD2 lipoids A, there are C14:3-OH and C16:Two kinds of fatty acid chains of 3-OH.GC-
MS results can show that lpxD1 is expressed as introducing C14 on 2 and 2 ' positions in quasi-grease A structure:3-OH、C16:3-OH、
C18:Tri- kinds of hydroxy fatty acid chains of 3-OH, lpxD2 are expressed as introducing C14 on 2 and 2 ' positions in quasi-grease A structure:3-OH and
C16:Two kinds of hydroxy fatty acid chains of 3-OH.
5. basic hydrolysis is analyzed
Lipoid A is dissolved in the sodium hydroxide (25% ammonium hydroxide) of 180 μ L 0.3M, room temperature is degraded 16 hours, with
The methanol of the chloroform and 200 μ L of 200 μ L is added afterwards, 3000rpm centrifuges 10 minutes, removes phase, is analyzed into MALDI-TOF-MS.
When lipoid A is by complete hydrolysis, the fatty acid chain of lipoid A is only left two fatty acid chains on 2 and 2 ' positions,
The value of the m/z of E.coli RL25 be 951, and the value of the m/z of E.coli RL25/pUC-lpxD1 be predominantly 979,1007,
1035, determine that quasi-grease A structure change is happened on 2 and 2 ' positions, so proving that the expression of lpxD1 makes E.coli RL25
Quasi-grease A structure changed on 2 and 2 ' positions, in conjunction with MS analysis and GC-MS analysis obtain, E.coli RL25/pUC-
There are C14 on 2 and 2 ' positions by lpxD1:3-OH、C16:3-OH、C18:Tri- kinds of hydroxy fatty acid chains of 3-OH.E.coli RL25/
The value of the m/z of pUC-lpxD2 is predominantly 979 and 1007, determines that quasi-grease A structure change is happened on 2 and 2 ' positions.So
The expression of lpxD2 is proved so that the quasi-grease A structure of E.coli RL25 is changed on 2 and 2 ' positions, in conjunction with MS analyses and
GC-MS analyses show that there are C14 on 2 and 2 ' positions by E.coli RL25/pUC-lpxD1:3-OH and C16:Two kinds of hydroxyls of 3-OH
Base fatty acid chain.
3 genetically engineered E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2 of embodiment is from agglutinability
The mutation analysis of power
By the bacterium solution being incubated overnight by initial OD600=0.02 is transferred in 50mLLB fluid nutrient mediums, 37 DEG C, 200r/
Min is cultivated to OD600=2；Thalline were collected by centrifugation, washed once and is resuspended with the phosphate buffer (PBS) of pH 7.4, and control is resuspended
OD processed600=2；It takes in 10mL to 15mL centrifuge tubes, rests in 4 DEG C of environment, after 24 hours, the top bacterium solution is taken to measure OD, and
By computational methods (2- bacterium solutions above OD600)/2 calculate thalline from agglutinability.
The results are shown in Figure 4, compares and obtains, E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2's
It is higher by 2 times or so from agglutinability ratio E.coli RL25.
4 genetically engineered E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2 of embodiment believes TLR4
The stimulation power analysis of number access
HEK-Blue hTLR4 cells because transfection expression mankind's TLR4 and NF-kB secretion inducing SEAP reporter genes,
It can specifically identify the LPS on bacterial body living surface in cultivating system and secrete blue SEAP kinases, monitor LPS in real time and lure
The power for the TLR4 immune pathways led indicates thalline to the stimulation ability of TLR4 and to cell by the SEAP depths to develop the color
Toxicity.Light absorption value is bigger, bigger to cytotoxicity.
The results are shown in Figure 5, E.coli RL25, E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-
Light absorption value situations of the lpxD2 at 650nm be：E.coli RL25 ＞ E.coli RL25/pUC-lpxD2 ＞ E.coli RL25/
PUC-lpxD1, when a concentration of the 10 of thalline4When CFU/mL, for the stimulation degree phase of the TLR4 of HEK-Blue hTLR4 cells
It is poor maximum.
The above results analytic explanation：E.coli RL25/pUC-lpxD1 and E.coli RL25/pUC-lpxD2 lipoids A knots
After structure changes, become smaller for the stimulation ability of the TLR4 accesses of cell, the toxicity of cell is reduced, and 2 and 2 ' positions
On fatty acid chain it is longer, thalline is smaller for the stimulation ability of the TLR4 accesses of cell.
The comparison of embodiment 5 RL25/pUC, RL25/pUC-lpxD1 and RL25/pUC-lpxD2 to Cell-mediated Immunity
The immunocompetence of LPS mainly by stimulating TLR4 accesses come what is played a role, after TLR4, there is Mal-MyD88
With two accesses of TRAM-TRIF, this two accesses correspond to the generation of different cell factors respectively.It is found by the research of front
The variation of fatty acid chain lengths of the lipoid A on 2 and 2 ' positions, results in immunocompetent difference, influences to TLR4 accesses
Stimulation ability, in order to deeper into immunocompetent influence of the research chain length for LPS, tested by ELISA, to different accesses
Antibiotic not of the same race be determined.Cell is stimulated with LPS after purification, then measures the amount of cell factor, LPS
It is a concentration of：0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1000ng/mL.In addition to this, lipoid A of the same race is for people
Immunocompetence with animal will not be completely the same, thus used respectively the cell THP-1 of mouse cell RAW267.4 and people into
Measurement is gone.
1, RL25/pUC, RL25/pUC-lpxD1 and RL25/pUC-lpxD2 are to the immunocompetent comparisons of RAW264.7
The corresponding different cell factor of two accesses under TLR4 is determined respectively, under Mal-MyD88 accesses
IL-6 and TNF-α, the RANTES under TRAM-TRIF accesses.
The result of measurement is as shown in Figure 6A, and as can be seen from Fig., the LPS stimulations RAW264.7 of different thalline various concentrations is thin
Born of the same parents generate the result of the amount of different cytokines, it is found that in general, the concentration of LPS is higher, and stimulation cell generates thin
The amount of intracellular cytokine is bigger, but the amount of cell factor is not increased with the increase of the concentration of LPS always, when LPS's
When concentration rises to 1000ng/mL by 100ng/mL, the amount ascendant trend of TNF-α is no longer apparent.Three kinds of thalline stimulate cell
Trend is consistent on the whole for the amount of the three kinds of cell factors generated, RL25/pUC ＞ RL25/pUC-lpxD2 ＞ RL25/pUC-
lpxD1.Moreover, the diversity ratio RL25/pUC-lpxD2 between RL25/pUC and RL25/pUC-lpxD2 and RL25/pUC-lpxD1
Difference between RL25/pUC-lpxD1 is more obvious.
So when the length of 2 and 2 ' the position fatty acid chains of lipoid A changes, lipoid A is for RAW264.7 cells
Immunocompetence change, all have a significant effect to two accesses of Mal-MyD88 and TRAM-TRIF, when fatty acid chain is elongated
When, the immunocompetence of cell is reduced, the corresponding cell factor of two accesses of generation is stimulated all to be substantially reduced.Three plants of bacteria thorns swash
The immunocompetence detection of RAW264.7 cells discloses immunocompetent influence of the fatty acid chain length on LPS on 2 and 2 ' positions,
Important foundation is provided for the exploitation of new vaccine adjuvant.
2, the comparison of RL25/pUC, RL25/pUC-lpxD1 and RL25/pUC-lpxD2 to THP-1 Cell-mediated Immunities
The LPS of three kinds of thalline stimulates THP-1 cells respectively, be then respectively compared Mal-MyD88 under TLR4 accesses and
The amount for the cell factor that two accesses of TRAM-TRIF generate furthers investigate three kinds of bacterium for the immunocompetent of THP-1 cells
Difference, the fatty acid chain length on 2 and 2 ' positions to react lipoid A is for its immunocompetent influence.So determining
Two kinds of cell factors of IL-8 and TNF-α under Mal-MyD88 accesses, the RANTES cell factors (figure under TRAM-TRIF accesses
6B)。
LPS has found that it is thin to THP-1 to affect LPS for the variation of the structure of lipoid A to the stimulus result of THP-1 cells in figure
The immunocompetence of born of the same parents.But the difference of the different cell factors of the LPS stimulation THP-1 cells generations of different thalline is different
, the difference of the amount of the IL-8 of the LPS stimulation THP-1 cells generations of three kinds of thalline is minimum, the TNF- that stimulation THP-1 cells generate
The difference of the amount of α becomes larger with the increase of the concentration of LPS, and the difference of the amount for the RANTES that stimulation THP-1 cells generate is in LPS
Each concentration is almost the same.So the length of the fatty acid chain on the 2 and 2 ' positions of lipoid A is longer, LPS is to THP-1 cells
Immunocompetence is lower, is all reduced to the immunocompetence of two accesses of Mal-MyD88 and TRAM-TRIF under TLR4 accesses.
Probe into application of 6 LpxD1 of embodiment in W3110 and HW002
1, W3110/pUC and W3110/pUC-lpxD1 quasi-grease A structures are identified
The mass spectrogram of the lipoid A of two kinds of thalline is as shown in Figure 7 A, m/ occurs in the lipoid A mass spectrograms of W3110/pUC
Two peaks z1716 and m/z1796, the structure of the lipoid A corresponding to wherein m/z1796 is as shown in the structure chart in figure, m/z1796
The structure of corresponding lipoid A is that the structure of lipoid A shown in figure falls off a phosphate in extraction process；W3110/
There are m/z1716, m/z1744, m/z1772, m/z1796, m/z1824, m/z1852 in the mass spectrogram of pUC-lpxD1 lipoids A,
M/z1880 corresponds to the quasi-grease A structure that 2 and 2 ' fatty acid chain lengths are increased on the basis of W3110 lipoid A, W3110/ respectively
Fatty acid chain type on 2 and 2 ' positions of pUC-lpxD1 quasi-grease A structures includes 3-OH-C14,3-OH-C16 and 3-OH-C18.
2, HW002/pUC and HW002/pUC-lpxD1 quasi-grease A structures are identified
The mass spectrogram of HW002/pUC and HW002/pUC-lpxD1 lipoids A is as shown in Figure 7 B, and m/ occurs in HW002/pUC
The peaks z1506, m/z1506 corresponding structures in peak are as shown, the structure of lipoid A is removed on the basis of HW001/pUC structures
Stimulation fatty acid chain on 3 ' positions；And there is m/z1506 in the mass spectrogram of HW002/pUC-lpxD1 lipoids A, m/
Z1534, m/z1562, m/z1590, m/z1618, the corresponding quasi-grease A structure in each peak are equivalent in HW002/pUC quasi-grease A structures
On the basis of increase fatty acid chain at 2 and 2 ' positions, be added on the basis of original 3-OH-C14 3-OH-C16 and
Two kinds of fatty acid chains of 3-OH-C18.
3, LpxD1, which expresses bacterial strain, stimulates cell TLR4 accesses with control strain the comparison of degree
Base of the quasi-grease A structure of W3110/pUC-lpxD1 and HW002/pUC-lpxD1 in W3110/pUC and HW002/pUC
The length of the fatty acid chain on 2 and 2 ' positions is increased on plinth, and compareing bacterium is changed on the basis of W3110
It makes, can further probe into the immunocompetent influence of the length and other structures combination of features of fatty acid chain on lipoid A in this way,
We compare stimulation ability of the thalline for TLR4 first.
Bacterial strain and control strain are expressed for the stimulation ability of TLR4 accesses as shown in figure 8, on the whole, with thalline
Concentration raising, thalline is increased for the stimulation ability of the TLR4 accesses of cell, but be not it is increased always, when
Cell concentration reaches 105When continuing growing the concentration of thalline when CFU/mL, thalline rises the stimulus intensity of TLR4 accesses
Be not it is obvious that the reason is that in the case where cell concentration is certain, the concentration of thalline reaches the thorn to cell TLR4 after saturation
Sharp ability just no longer rises.Each expression bacterial strain is substantially less than corresponding control for the stimulation ability of cell TLR4 accesses
Stimulation ability of the bacterial strain for cell TLR4 accesses is organized, and this species diversity is gradually apparent with the raising of cell concentration, arrives
Up to after maximum difference, with the raising of cell concentration, this species diversity is gradually reduced again.
W3110/pUC has found that on the whole, W3110/pUC is logical to the TLR4 of cell compared with W3110/pUC-lpxD1
The stimulation ability on road is above W3110/pUC-lpxD1, and HW002/pUC has found compared with HW002/pUC-lpxD1, two kinds
Bacterium is all relatively low to the overall stimulation level of TLR4 accesses, when cell concentration is 102CFU/mL、103CFU/mL、104CFU/mL、
105When CFU/mL, absorbance of the stimulation liquid at 650nm is below 0.1.When cell concentration is 106When CFU/mL, thalline is to thin
The irritation level of born of the same parents TLR4 rises, at this point, HW002/pUC to the irritation level and HW002/pUC-lpxD1 of TLR4 to TLR4's
Irritation level is variant, and HW002/pUC-lpxD1 is about irritation levels of the HW002/pUC to TLR4 to the irritation level of TLR4
70%.When cell concentration is 107When CFU/mL, thalline continues to rise to the irritation level of cell TLR4, at this point, HW002/
PUC is still variant to the irritation level of TLR4 to the irritation level and HW002/pUC-lpxD1 of TLR4, HW002/pUC-lpxD1
HW002/pUC is about to the 84% of the irritation level of TLR4 to the irritation level of TLR4.Two plants of bacterium pierce cell TLR4 accesses
Sharp degree discloses immunocompetent influence of the fatty acid chain length on LPS on 2 and 2 ' positions, is the exploitation of new vaccine adjuvant
Important foundation is provided.
4, LpxD1 expresses comparisons of the LPS of bacterial strain to Cell-mediated Immunity
LPS is mainly by stimulating TLR4 accesses to trigger an immune response, and there are Mal-MyD88 and TRAM- below TLR4
Two accesses of TRIF, so in order to which the length for further studying 2 and 2 ' fatty acid chains on lipoid location A is lived for LPS is immune
Property influence determine the cell factors of the different accesses of correspondence that different thalline LPS stimulate different cells to generate respectively, survey respectively
RANTES the and Mal-MyD88 accesses under the TRAM-TRIF accesses of the lower RAW264.7 cells of LPS stimulations and THP-1 cells are determined
Under two kinds of cell factors of TNF-α.
The results are shown in Figure 9, and generally speaking, the LPS stimulation THP-1 cells and RAW264.7 of the expression bacterial strain of LpxD1 are thin
The cell factor that born of the same parents generate is less than control strain.
The LPS of W3110/pUC and W3110/pUC-lpxD1 stimulates the amount of the different cytokines of different cells generations as schemed
It is shown.The stimulus result of THP-1 cells is shown, the TNF-α difference that the LPS stimulation cells of two kinds of bacterium generate is apparent, when LPS is dense
When degree is 1ng/mL, the amount of TNF-α is very low, and there is no apparent differences, when LPS concentration rises to 10ng/mL, stimulates cell
There is notable difference in the TNF-α amount of generation, and the TNF-α amount that the LPS stimulation cells of W3110/pUC-lpxD1 generate at this time is only
The 25% of the TNF-α amount that the LPS stimulation cells of W3110/pUC generate.When LPS concentration rises to 100ng/mL, cell is stimulated
The difference that the TNF-α amount of generation occurs is still apparent, the TNF-α amount that the LPS stimulation cells of W3110/pUC-lpxD1 generate at this time
The 50% of the TNF-α amount that the LPS stimulation cells of only W3110/pUC generate.When LPS concentration rises to 1000ng/mL, stimulation
The difference that the TNF-α amount that cell generates occurs is no longer apparent, and the LPS stimulation cells of W3110/pUC-lpxD1 generate at this time
TNF-α amount is slightly above the TNF-α amount that the LPS stimulation cells of W3110/pUC generate.And two kinds of thalline stimulation THP-1 cells generate
RANTES amount but without so apparent difference, the RANTES's that stimulation generates when the LPS of two kinds of bacterium is 10ng/mL
The LPS that the amount for measuring the RANTES that difference is maximum, and the LPS stimulation cells of W3110/pUC-lpxD1 generate at this time is W3110/pUC is pierced
Swash the 66% of the RANTES amounts that cell generates, under other concentration of LPS, two kinds of bacteria thorns swash the RANTES of cell generation not
Apparent difference.So on the basis of the quasi-grease A structure of W3110, LpxD1 is expressed, increases the fatty acid chain on 2 and 2 ' positions,
The stimulation ability that LPS is less than TLR4 accesses is reduced, integrally reduces immunocompetences of the LPS for THP-1 cells so that TNF-
The production quantity of α and RANTES reduces.And TNF-α amount decline it is more obvious, it was demonstrated that the growth of fatty acid chain length for
The influence of Mal-MyD88 accesses is more obvious.The stimulus result of RAW267.4 cells is shown, W3110/pUC-lpxD1 stimulations
The amount for the cell factor that cell generates integrally is less than the amount for the cell factor that W3110/pUC stimulation cells generate.Two kinds of thalline thorns
Swash the difference maximum that the amount for the TNF-α that cell generates is shown at a concentration of 10ng/mL of LPS, W3110/pUC-lpxD1's
The LPS that LPS stimulates the amount of the TNF-α of cell generation to be W3110/pUC stimulates the 65% of the amount of the TNF-α of cell generation.And two
The difference that the amount of the RANTES of the LPS stimulation cell bad students of kind bacterium is shown at a concentration of 10ng/mL of LPS is maximum, W3110/
The RANTES that the LPS that the amount for the RANTES that the LPS stimulation cells of pUC-lpxD1 generate is W3110/pUC stimulates cell to generate
The 36% of amount.But under other LPS concentration, there is no bright for the amount of the RANTES of the LPS stimulation cell generations of two kinds of thalline
Aobvious difference.
The LPS of HW002/pUC and HW002/pUC-lpxD1 stimulates the amount of the different cytokines of different cells generations as schemed
It is shown.The stimulus result of THP-1 cells is shown, the TNF-α of generation and the amount of RANTES are all relatively low levels, so simultaneously
Apparent difference is not shown.The stimulus result of RAW264.7 cells is shown, for cytokine TNF-α, two kinds of thalline
The amount that generates of LPS stimulation cells it is with the biggest gap in a concentration of 10ng/mL of LPS, the LPS thorns of HW002/pUC-lpxD1 at this time
Swash the 33% of the amount that the LPS that the amount that cell generates is HW002/pUC stimulates cell to generate.For cell factor RANTES, two kinds
The amount that the LPS stimulation cells of thalline generate is with the biggest gap in a concentration of 10ng/mL of LPS, at this time HW002/pUC-lpxD1
The LPS that the amount that LPS stimulates cell to generate is HW002/pUC stimulates the 27% of the amount of cell generation.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>It is attenuated the preparation and application of lipoid A
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1014
<212> DNA
<213>Francisella
<400> 1
atgtatagtt tagattttct agcgtcaaag cttgatggtg aggttaaggg cgataagaat 60
gtcgagataa aaaaaatagc tacactatca caagctggtg aaggtgatat ttctttttgt 120
actaacccta aatatttaaa agctttgtct gaaacaaaag cttctgcagt tttaataaca 180
gaagaggcac tagagttttg taatacaaac gcggtggtac tatcaaatcc ttatatggct 240
ttagcaaagg ttatggagct atttgataaa tcgccgcgtc ctgatggtaa aattcatagt 300
aaagctgtga tagccgcaag tgctataatt ggtgaaaatg tcactatagg cgctaatgcg 360
gtagttggtg aaaatgtagt aattggtgac aatgtttata taggtgcctg tgcaactatt 420
gataatggta ccaagatagg taatgacacg ctgataaaga gcaatgtatc tatagctcac 480
gatgttgaga ttggtactgg gtgtattatt catcaaaatg cagtaattgg ctgtgatggt 540
tttggtaatg caagagatga agatggtagt tggacaaaaa ttcctcagct aggaagagta 600
attatcgaag atgatgttga gattggttct ggtacaactg ttgatagagg agctattgac 660
gatacaataa ttaaaaaggg cgcacgtatt gataatttag tacagatagc tcataatgta 720
gttatcggta gaaacactgc tttagccggt gttacagctg ttgcaggtag tacaacgatt 780
ggcgataact gccttattgg aggtcagtca gcaataactg gccatattag catttgtgat 840
aatactatta taggtggcgc atctaatatt gggaagtcaa tcactaagcc aggaatgtac 900
tatgctgcgt ttgaagctaa acctaggatt caatggggta ggttcgtagc taaattagct 960
aagattgata ctctaattac aaaagtaaag caactagaag aaaaaattaa ataa 1014
<210> 2
<211> 1044
<212> DNA
<213>Francisella
<400> 2
ttgcaataca ctctaaaaca aatatccgag catctaaatg caaaagtaat taatgaacca 60
agtggtgaag ttattatatc ggggcttaac tacgctgaac aagcaaaaga aaatgatcta 120
actcttattg ataaacaaga acatattaag ctttggcaaa attcaaaagc tgcagctgct 180
attgtaagta aaaaaattag taaagaatta gcacaggtaa atgacaagcc tctaattgta 240
gtcaataatg ctgatttagc tatggctaaa atattagaac ttttttctgt gccataccca 300
gaacaaaatg gtattcatga aaaagctgta atagatccaa ctgctaaaat tggtaaaaat 360
gtctcaattg gacccggcgc atatatcggt aaaaatgtcg agattggtga taatacgatt 420
atctatgcta acgtatgtat ctataacgat gcaaaagttg gaactaactg tattatctgg 480
ccaagtgtga caattcgtga tagaactatt ataggtcatt tttgtaggtt atactcaaac 540
tgttcaatcg gatcagatgg ttttggctat agaccgtctg aagatggtag aacaattgtt 600
aggattccac acattggtaa tgttgttatc ggtagctttg tcgatattgg ttcaaatact 660
tgtattgata atgctaaata tggctcaact ataattggag attataccaa aattgataat 720
cttgttcaga taggtcacaa tgtaattatt ggtaaaggct gcatgatttg tggacaagct 780
ggtatcagtg gttcagtaac aattggtgac ggcgttataa ttgccggtaa tgctggtatc 840
aaagatcata caaatattgg tagtggtgca cgtatcggtg gtaaagctgg agttatgtgg 900
gatgtacctg ctggtgaaag ccatatgggc tatcccgctt acaaagacag tgagcttgcc 960
aaacaatgga ttgctatcag gaagctccct gagactatga aaaaacttaa ggctattgct 1020
aaaagcctaa acatagatct ttaa 1044
Claims (10)
1. lipoid A, which is characterized in that its structure is as shown in following formula I or formula II：
2. a kind of method preparing lipoid A described in claim 1, which is characterized in that include the following steps：
(1) recombination bacillus coli of structure expression lpxD1 genetic fragments or lpxD2 genetic fragments；
(2) recombination bacillus coli is cultivated, lipoid A is collected in extraction.
3. a kind of method preparing lipoid A described in claim 1 according to claim 2, which is characterized in that step (1)
The Escherichia coli are E.coli RL25, W3110 or HW002.
4. a kind of method preparing lipoid A described in claim 1 according to claim 2 or 3, which is characterized in that step
(1), the lpxD1 genetic fragments for coming from Francisella or lpxD2 genetic fragment digestions are connected into plasmid pUC18, then turned
Enter host cell.
5. a kind of method preparing lipoid A described in claim 1 according to claim 2, which is characterized in that described
The nucleotide sequence of lpxD1 genetic fragments is SEQ ID NO:The nucleotide sequence of 1 sequence, lpxD2 genetic fragments is SEQ
ID NO:2 sequence.
6. a kind of method preparing lipoid A described in claim 1 according to claim 2, which is characterized in that step (2)
Culture is collected to the recombination bacillus coli thalline of late log phase, ddH2Mono- phase system (chlorine of Bligh-Dyer is used after O washing thallines
Imitative/methanol/water solution, 1:2:0.8, v/v/v) suspension thalline, magnetic agitation for a period of time, split-phase, use Bligh-Dyer mono-
Phase system washs cell fragment 2-3 times；Sodium acetate (pH4.5) solution, ultrasonic vibration is added, boiling water bath cracks sugar chain；It is cooled to room
Be added after temperature chloroform and methanol formed Bligh-Dyer two-phases system (chloroform/methanol/water, 2:2:1.8, v/v/v) it, centrifuges, takes
Lower phase moves into revolving bottle, rotary evaporation, and chloroform/methanol solution (4 is added:1, v/v) lipoid A is washed out.
7. a kind of recombination bacillus coli, which is characterized in that expression lpxD1 genetic fragments or lpxD2 genetic fragments.
8. a kind of recombination bacillus coli according to claim 7, which is characterized in that with Escherichia coli RL25, W3110 or
HW002 is host.
9. a kind of reducing method of the coli somatic to TLR4 accesses stimulus intensity or Cell-mediated Immunity, which is characterized in that
It is by pUC plasmid expressions lpxD1 genetic fragments in Escherichia coli, the Escherichia coli are W3110 or HW002.
10. recombination bacillus coli described in lipoid A or claim 7 described in claim 1 and in preparing vaccine or vaccine adjuvant
Application.
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| JP2020510726A (en) * | 2017-03-13 | 2020-04-09 | デ スタート デル ネーデルランデン， ヴェルト． ドール デ ミニステル ヴァン ヴイダブリューエス ミニステリー ヴァン ボルクスゲツォントヘイト， ベルジーン エン シュポルトＤｅ Ｓｔａａｔ Ｄｅｒ Ｎｅｄｅｒｌａｎｄｅｎ， Ｖｅｒｔ． Ｄｏｏｒ Ｄｅ Ｍｉｎｉｓｔｅｒ Ｖａｎ Ｖｗｓ Ｍｉｎｉｓｔｅｒｉｅ Ｖａｎ Ｖｏｌｋｓｇｅｚｏｎｄｈｅｉｄ， Ｗｅｌｚｉｊｎ Ｅｎ Ｓｐｏｒｔ | Bordetella vaccine containing LPS with reduced reactogenicity |
| CN111729074A (en) * | 2020-06-02 | 2020-10-02 | 遵义医科大学珠海校区 | Application of polypeptide RL25 in preparation of antitumor drugs |
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| JP2020510726A (en) * | 2017-03-13 | 2020-04-09 | デ スタート デル ネーデルランデン， ヴェルト． ドール デ ミニステル ヴァン ヴイダブリューエス ミニステリー ヴァン ボルクスゲツォントヘイト， ベルジーン エン シュポルトＤｅ Ｓｔａａｔ Ｄｅｒ Ｎｅｄｅｒｌａｎｄｅｎ， Ｖｅｒｔ． Ｄｏｏｒ Ｄｅ Ｍｉｎｉｓｔｅｒ Ｖａｎ Ｖｗｓ Ｍｉｎｉｓｔｅｒｉｅ Ｖａｎ Ｖｏｌｋｓｇｅｚｏｎｄｈｅｉｄ， Ｗｅｌｚｉｊｎ Ｅｎ Ｓｐｏｒｔ | Bordetella vaccine containing LPS with reduced reactogenicity |
| JP7311227B2 (en) | 2017-03-13 | 2023-07-19 | イントラヴァック ビー．ブイ． | Bordetella vaccines containing LPS with reduced reactogenicity |
| CN111729074A (en) * | 2020-06-02 | 2020-10-02 | 遵义医科大学珠海校区 | Application of polypeptide RL25 in preparation of antitumor drugs |
| CN111729074B (en) * | 2020-06-02 | 2021-03-26 | 遵义医科大学珠海校区 | Application of polypeptide RL25 in preparation of antitumor drugs |
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