CN109432416A - A kind of oligosaccharides vaccine preventing invasive infections with fungi - Google Patents
A kind of oligosaccharides vaccine preventing invasive infections with fungi Download PDFInfo
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Abstract
The present invention provides a kind of oligosaccharides vaccines for preventing invasive infections with fungi, chitosan oligosaccharide including conjugate immunogens carrier protein, the coupling mass ratio of the immunogenic carrier albumen and chitosan oligosaccharide is 0.7-210:1, the deacetylation of chitosan oligosaccharide is 0.05-98%, average relative molecular mass < 5000 Da, the immunogenic carrier albumen are inhuman source protein.Oligosaccharides vaccine provided by the invention activates Th17, Th1 cellular immunity effectively by deacetylation appropriate to prevent broad spectrum fungus infection.The oligosaccharides vaccine can treat or prevent invasive fungi, such as Candida albicans, aspergillus, cryptococcus and Ye Shi lung pityrosporion ovale.
Description
Technical field
The invention belongs to vaccine biomedicine fields, and in particular to a kind of oligosaccharides vaccine for preventing invasive infections with fungi.
Background technique
Rise with new immunosuppressive therapy method, organ transplant and AIDS disease incidence, it is aggressive drug resistance fungal infection morbidity, dead
Rate is died to significantly rise in the whole world.According to the current standard of international medical community, fungal infection is divided into shallow, subcutaneous tissue and invasion
3 seed type of (or systematicness) fungal infection, latter two are also known as deep fungal infection.Common causative strain is candida albicans, aspergillus, hidden ball
Bacterium and Ye Shi lung pityrosporion ovale account for about 70% of fungal infection or so, and infection involves human body viscera or forms fungemia.It is clinical deep
Portion's fungal infection has become the important cause of the death of nosocomial infection, and the whole world is every year because the number of fungal infection death is up to 1,500,000 people.Beauty
The white thought of state's studies have shown that occupies the 4th in nosocomial infection septicemia, and case fatality rate ranks first, and only the U.S. just reaches individual event treatment expense
Hundred million dollars/year of 20-40.Hospital, China different crowd studies of invasive fungal infections disease incidence 4.1-41.2%, death rate 9.8-60%.It is anti-
Fungi-medicine only more than ten, new drug progress are slow.The optional less varieties of clinical application, treatment cycle are long (> 1-6 the month), and curative effect is undesirable,
Make anti-invasion fungi at clinical difficult problems.
Fungi vaccine research starting evening, progress are small, at present still without listing vaccine.There are 2 to enter clinical vaccine needle in recent years
To the protein vaccine that the white adhesion factor for reading strain of certain specific kind and virulence factor design, plays to neutralize by humoral immunity and make
With, it is expected to infect applied to the white thought of gynaecology of specific pathogenic strain, and non-lethal invasive infections with fungi.
Fungal cell wall generally contains mannose, beta glucan and chitin polysaccharide.The immunogene of previous studies discovery sugar
Property it is lower, it is difficult to excite the immunological effect of body, and using beta glucan as the fungi vaccine research of representative, coupling can have been passed through and carried
Body protein improves immune response.The immunologic mechanism understanding for resisting fungal infection to body at present is gradually goed deep into, and is the discovery that
Cellular immunity and inherent immunity based on Th17 play a leading role.And the research of beta glucan-KLH vaccine is dripped with antibody
Degree, i.e. humoral immunity, to be leading evaluating effect, therefore the research of its efficacy of vaccines is also weak.Simultaneously as the tested sugar is
Chemistry is fully synthetic, be limited to radical protection one by one, the reaction that synthesizes limited degree of polymerization sugar chain mode one by one it is complicated, at high cost, one
As only synthesis the about 2-5 degree of polymerization several oligosaccharides tested, do not have screening effect.And using fungal cell wall chitin as target
The vaccine of point is not reported, still unclear by mechanism such as immune system identifications.Moreover, only having chitosan and albumen coupling at present
Afterwards as the report of immunologic adjuvant.
Summary of the invention
It is current lack prevention invasive infections with fungi vaccine aiming at the problem that, the present invention, which provides, a kind of prevents invasive fungi
The oligosaccharides vaccine of infection, at low cost, broad-spectrum antifungal.
It is a further object of the present invention to provide a kind of preparation method of the oligosaccharides vaccine of above-mentioned prevention invasive infections with fungi,
Raw material is easy to get, preparation process is simple.
To achieve the above object, the present invention adopts the following technical scheme that.
A kind of oligosaccharides vaccine preventing invasive infections with fungi, the chitosan oligosaccharide including conjugate immunogens carrier protein.Institute
The coupling mass ratio for stating immunogenic carrier albumen and chitosan oligosaccharide is 0.7-210:1, preferably 1-101:1.
The deacetylation of the chitosan oligosaccharide is 0. 05-98%, average relative molecular mass < 5000 Da.Preferably, de- second
Acyl degree is 0.1-20%.Preferably, the Relative average molecular weight 1000-3000Da of the chitosan oligosaccharide.Preferably, it is described not
The A of 2% aqueous solution of the chitosan oligosaccharide of coupling420<0.1；2% aqueous solution 13000rpm is centrifuged 2min, no precipitating.The A420Refer to molten
Absorbance value of the liquid to 420nm light.The chitosan oligosaccharide can be commercially available or voluntarily passes through degradation chitosan preparation.In general,
Referred to as chitinous oligomers when deacetylation degree is less than 15%, but it is also referred to as chitosan oligosaccharide in the present invention.
The immunogenic carrier albumen is inhuman source protein；Including but not limited to diphtheria toxin non-toxic variant, KLH
(keyhole limpet hemocyanin), BSA(bovine serum albumin(BSA)), OVA(chicken ovalbumin), Blue carrierTM(mollusk source property is high
Soluble hemocyanin), tetanus toxin/toxoid is isolated from the high-molecular-weight protein of non-acquisition type haemophilus influenzae
(HMP), the pseudomonal toxin A after detoxification, cholera toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens bud
Born of the same parents bacillus exotoxin/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP 7 albumen, diphtheria toxin mutation
CRM, CRM191, CRM3201 breathe syncystial virus F and G-protein.Preferably KLH, BSA, OVA.
The connection of above-mentioned chitosan oligosaccharide and the immunogenic carrier albumen can be such as coupled by conventional immunology means
Agent coupling.Preferably, the preparation method of above-mentioned oligosaccharides vaccine, comprising the following steps:
(1) immunogenic carrier albumen, chitosan oligosaccharide and coupling agent are dissolved in coupling liquid, are incubated for；
(2) terminator is added to be incubated for；
(3) purifying obtains oligosaccharides vaccine.
In step (1), the coupling agent is selected from but not limited to CHO (CH2)mCHO, BS3(bis
[sulfosuccinimidyl] suberate, bis- (sulfo group) succinates), DSS(disuccinimidyl suberate, it is double
Succinimide suberate) and BS(PEG) n.Wherein, the value of m or n is bigger, and the coupling agent " arm " as bridge is longer,
More be conducive to immune stage reduction and combine steric hindrance, but too long coupling agent preparation purity decreases.Preferably, m=3-
10；n=2-15.
In step (1), the coupling liquid is selected from the 0.01-0.3 M buffer of water or pH 6.9-9 not amino-contained.It is preferred that
, it is coupled the pH 7.2-8 of liquid, concentration is 0.05-0.2 M.
In step (1), the concentration of the immunogenic carrier albumen is 0.5-100 mg/mL, preferably 10-90 mg/mL.
In step (1), the mass ratio of the immunogenic carrier albumen and chitosan oligosaccharide is 0.5-100:1, preferably 10-
90:1。
In step (1), the mass ratio of the coupling agent and chitosan oligosaccharide is 0.5-10:1, preferably 1-5:1.
In step (1), 0-35 DEG C of incubation temperature of the immunogenic carrier albumen, chitosan oligosaccharide and coupling agent, heat preservation
10-150 min, preferably, 4-25 DEG C, 20-120 min.
In step (1), coupling agent, chitosan oligosaccharide adding manner can be dissolved into solution appropriate after be added or with solid
Form is directly added into.
In step (2), the mass ratio of the terminator and coupling agent is 5-85:1, preferably 10-50:1.
In step (2), the terminator is selected from Tris-HCl, glycine, lysine or NaBH4.Preferably, terminator is
The Tris-HCl buffer of the 1-4 M of pH 7-8.It is furthermore preferred that terminator is the Tris-HCl buffering that 7.5 concentration of pH is 1 M
Liquid.
In step (2), 0-35 DEG C of incubation temperature after terminator is added keeps the temperature 5-75min, preferably, 4-25 DEG C,
10-60min。
In step (3), the purification process can be to be obtained by desalting column or dialysis with removing low molecule impurity
Immunogenic carrier albumen coupling chitosan oligosaccharide.Wherein, the desalination or dialysis buffer or water are lytic immunity immunogenic carrier
Solution used when albumen.
Preferably, the filler model of the desalting column has G10-G50, Bio-gel P2-P10, Bio-gel P2-
P6DG, Thermo Scientific Zeba Spin Desalting Columns, preferably G25 and Thermo
Scientific™ Zeba™ Spin Desalting Columns.The dialysis membrane aperture of the dialysis is 1-10kDa, choosing
From but be not limited to Thermo Scientific Slide-A-Lyzer Dialysis Cassettes；Preferably, aperture
For the Thermo Scientific Slide-A-Lyzer Dialysis Cassettes of 3kDa.
Preferably, further including the steps that oligosaccharides vaccine solution solid is made after step (3).Common operation can be used,
Such as vacuum freeze drying or spray drying.
A kind of above-mentioned oligosaccharides vaccine is as the purposes for treating or preventing invasive fungi drug；The fungi includes but unlimited
In Candida albicans, aspergillus, cryptococcus and Ye Shi lung pityrosporion ovale.
The invention has the following advantages:
Oligosaccharides vaccine provided by the invention exposes-NH using removing acetyl group using the chitosan oligosaccharide of deacetylation2, make
For chitosan oligosaccharide coupling protein is made with the site of carrier protein couplet, increase the immunogenicity of chitosan oligosaccharide.However, deacetylated spend
It is low cause can conjugation sites quantity it is very few, deacetylation is excessively high to make chitosan oligosaccharide molecule and pathogen cell wall chitin molecule difference
Excessive, the present invention activates Th17, Th1 cellular immunity effectively by deacetylation appropriate to prevent broad spectrum fungus and infect.
The oligosaccharides vaccine can treat or prevent invasive fungi, such as Candida albicans, aspergillus, cryptococcus and Ye Shi lung pityrosporion ovale.
Specific embodiment
Below with reference to embodiment, the present invention will be further described, but the present invention is not limited by the following examples.
The preparation of 1 chitosan oligosaccharide of embodiment
1.1
With the chitosan of 0.3M acetic acid 300mL dissolution 15g deacetylation 1%, pH 5.0 is adjusted after dissolving completely, water is added to be settled to
500mL.Chitosan enzyme 1.5g(130U/g is added), 25-35 DEG C of stirring water-bath 8h.Plate-frame filtering Enzymatic Hydrolysis of Chitosan liquid, then with not
Same aperture (400-5000Da) ultrafiltration through membranes, nanofiltration retention phase answer molecular weight section chitosan oligosaccharide (< 5000Da, < 4000 Da, <
3000 Da, < 2000 Da, < 1000Da, < 800Da, < 600Da, < 400Da).After rotary evaporation is concentrated 5 times, spray drying is collected
Chitosan oligosaccharide.The A of 2% aqueous solution of gained chitosan oligosaccharide420<0.1；2% aqueous solution 13000rpm is centrifuged 2min, no precipitating.
1.2
With the chitosan of 0.2 M hydrochloric acid 300mL dissolution 18g deacetylation 95%, pH 6.5 is adjusted after dissolving completely, adds water constant volume
To 500mL.Chitosan enzyme 1.5g(200U/g is added), 25-35 DEG C of stirring water-bath 16h.Plate-frame filtering Enzymatic Hydrolysis of Chitosan liquid, then use
Different pore size ultrafiltration through membranes retention phase answers the chitosan oligosaccharide (1000-3000Da, 3000-5000Da) of molecular weight section.Rotary evaporation
After 10 times of concentration, chitosan oligosaccharide is collected in spray drying.The A of 2% aqueous solution of gained chitosan oligosaccharide420<0.1；2% aqueous solution 13000rpm
It is centrifuged 2min, no precipitating.
1.3
With the chitosan of 0.3M acetic acid 300mL dissolution 10g deacetylation 10%, pH 5.5 is adjusted after dissolving completely, water is added to be settled to
500mL.Chitosan enzyme 1.5g(100U/g is added), 25-35 DEG C of stirring water-bath 8h.Plate-frame filtering Enzymatic Hydrolysis of Chitosan liquid, then with not
With aperture ultrafiltration through membranes retention phase answer molecular weight section chitosan oligosaccharide (1000-2000 Da, 1000-3000Da, 2000-3000Da,
3000-4000 Da, 4000-5000Da).After rotary evaporation is concentrated 15 times, chitosan oligosaccharide is collected in freeze-drying.Gained chitosan oligosaccharide
The A of 2% aqueous solution420<0.1；2% aqueous solution 13000rpm is centrifuged 2min, no precipitating.
1.4
With the chitosan of 0.3M acetic acid 300mL dissolution 15g deacetylation 20%, pH 5.5 is adjusted after dissolving completely, water is added to be settled to
500mL.Chitosan enzyme 1.5g(130U/g is added), 25-35 DEG C of stirring water-bath 8h.Plate-frame filtering Enzymatic Hydrolysis of Chitosan liquid, then with not
With aperture ultrafiltration through membranes, nanofiltration retention phase answer molecular weight section chitosan oligosaccharide (< 5000Da, < 4000 Da, < 3000 Da, < 2000
Da, < 1000Da, < 800Da, < 600Da, < 400Da).After rotary evaporation is concentrated 5 times, chitosan oligosaccharide is collected in spray drying.Gained shell
The A of 2% aqueous solution of oligosaccharides420<0.1；2% aqueous solution 13000rpm is centrifuged 2min, no precipitating.
1.5
With the chitosan of 0.3M acetic acid 300mL dissolution 15g deacetylation 50%, pH 6.0 is adjusted after dissolving completely, water is added to be settled to
500mL.Chitosan enzyme 1.5g(130U/g is added), 25-35 DEG C of stirring water-bath 8h.Plate-frame filtering Enzymatic Hydrolysis of Chitosan liquid, then with not
The chitosan oligosaccharide (2000-4000Da) of molecular weight section is answered with aperture ultrafiltration through membranes retention phase.After rotary evaporation is concentrated 5 times, freezing is dry
Dry collection chitosan oligosaccharide.The A of 2% aqueous solution of gained chitosan oligosaccharide420<0.1；2% aqueous solution 13000rpm is centrifuged 2min, no precipitating.
1.6
With the chitosan of 0.3M acetic acid 300mL dissolution 15g deacetylation 0.05%, pH 5.0 is adjusted after dissolving completely, adds water constant volume
To 500mL.Chitosan enzyme 1.5g(130U/g is added), 25-35 DEG C of stirring water-bath 8h.Plate-frame filtering Enzymatic Hydrolysis of Chitosan liquid, then use
Different pore size ultrafiltration through membranes, nanofiltration retention phase answer molecular weight section chitosan oligosaccharide (< 5000Da, < 4000 Da, < 3000 Da, <
2000 Da, < 1000Da, < 800Da, < 600Da, < 400Da).After rotary evaporation is concentrated 5 times, chitosan oligosaccharide is collected in spray drying.Institute
Obtain the A of 2% aqueous solution of 2 sugar of shell widow420<0.1；2% aqueous solution 13000rpm is centrifuged 2min, no precipitating.
1.7
With the chitosan of 0.3M acetic acid 300mL dissolution 15g deacetylation 0.1%, pH 5.0 is adjusted after dissolving completely, adds water constant volume
To 500mL.Chitosan enzyme 1.5g(130U/g is added), 25-35 DEG C of stirring water-bath 8h.Plate-frame filtering Enzymatic Hydrolysis of Chitosan liquid, then use
Different pore size ultrafiltration through membranes, nanofiltration retention phase answer molecular weight section chitosan oligosaccharide (< 5000 Da, < 4000 Da, < 3000 Da, <
2000 Da, < 1000 Da, < 800 Da, < 600 Da, < 400 Da).After rotary evaporation is concentrated 5 times, it is few that shell is collected in spray drying
Sugar.The A of 2% aqueous solution of gained chitosan oligosaccharide420<0.1；2% aqueous solution 13000rpm is centrifuged 2min, no precipitating.
1.8
With the chitosan of 0.3M acetic acid 300mL dissolution 15g deacetylation 98%, pH 5.0 is adjusted after dissolving completely, water is added to be settled to
500mL.Chitosan enzyme 1.5g(130U/g is added), 25-35 DEG C of stirring water-bath 8h.Plate-frame filtering Enzymatic Hydrolysis of Chitosan liquid, then with not
With aperture ultrafiltration through membranes, nanofiltration retention phase answer molecular weight section chitosan oligosaccharide (< 5000 Da, < 4000 Da, < 3000 Da, < 2000
Da, < 1000Da, < 800Da, < 600Da, < 400Da).After rotary evaporation is concentrated 5 times, chitosan oligosaccharide is collected in spray drying.Gained shell
The A of 2% aqueous solution of oligosaccharides420<0.1；2% aqueous solution 13000rpm is centrifuged 2min, no precipitating.
The preparation of 2 oligosaccharides vaccine of embodiment
50mgKLH is taken to be dissolved in the 1mL0.1M sodium phosphate buffer of pH7.2, NaCl containing 0.15M.1mg deacetylation, which is added, is
10%, the chitosan oligosaccharide of molecular weight 1000-3000Da, dissolution mix.Take (CHO (the CH containing glutaraldehyde2)3CHO) 50% aqueous solution, 2 μ L, adds
Enter substrate solution, mixes, keep 25 DEG C of slight vibration 60min.Glycine solid 50mg is added to mix, keeps 4 DEG C of 30min.With
0.1M sodium phosphate buffer, NaCl containing 0.15M are mobile phase, pass through Thermo Scientific Zeba Spin
Desalting Columns removes low molecule impurity, obtains the carrier protein solution of coupling chitosan oligosaccharide, is placed in 3000Da bag filter
Interior pure water dialysis for 24 hours, obtains oligosaccharides vaccine after freeze-drying.
The preparation of 3 oligosaccharides vaccine of embodiment
50mgKLH is taken to be dissolved in the 1mL0.1M sodium phosphate buffer of pH7.2, NaCl containing 0.15M.1mg deacetylation, which is added, is
95%, the chitosan oligosaccharide of molecular weight 1000-3000Da, dissolution mix.Take (CHO (the CH containing glutaraldehyde2)3CHO) 50% aqueous solution, 11 μ L,
Substrate solution is added, mixes, keeps 25 DEG C of slight vibration 60min.Glycine solid 250mg is added to mix, keeps 4 DEG C of 30min.
With 0.1M sodium phosphate buffer, NaCl containing 0.15M is mobile phase, passes through Thermo Scientific Zeba Spin
Desalting Columns removes low molecule impurity, obtains the carrier protein solution of coupling chitosan oligosaccharide, is placed in 2000Da bag filter
Interior pure water dialysis for 24 hours, obtains oligosaccharides vaccine after freeze-drying.
The preparation of 4 oligosaccharides vaccine of embodiment
50mgBSA is taken to be dissolved in the 1mL0.1M sodium phosphate buffer of pH7.2, NaCl containing 0.15M.1mg deacetylation, which is added, is
10%, the chitosan oligosaccharide of molecular weight 1000-3000Da, dissolution mix.Take (CHO (the CH containing glutaraldehyde2)3CHO) 50% aqueous solution, 2 μ L, adds
Enter substrate solution, mixes, keep 25 DEG C of slight vibration 60min.Glycine solid 50mg is added to mix, keeps 4 DEG C of 30min.With
0.1M sodium phosphate buffer, NaCl containing 0.15M are mobile phase, pass through Thermo Scientific Zeba Spin
Desalting Columns removes low molecule impurity, obtains the carrier protein solution of coupling chitosan oligosaccharide, is placed in 1000Da bag filter
Interior pure water dialysis for 24 hours, obtains oligosaccharides vaccine after freeze-drying.
The preparation of 5 oligosaccharides vaccine of embodiment
50mgBSA is taken to be dissolved in the 1mL0.1M sodium phosphate buffer of pH7.2, NaCl containing 0.15M.1mg deacetylation, which is added, is
95%, the chitosan oligosaccharide of molecular weight 1000-3000Da, dissolution mix.Take (CHO (the CH containing glutaraldehyde2)3CHO) 50% aqueous solution, 11 μ L,
Substrate solution is added, mixes, keeps 25 DEG C of slight vibration 60min.Glycine solid 250mg is added to mix, keeps 4 DEG C of 30min.
With 0.1M sodium phosphate buffer, NaCl containing 0.15M is mobile phase, passes through Thermo Scientific Zeba Spin
Desalting Columns removes low molecule impurity, obtains the carrier protein solution of coupling chitosan oligosaccharide, is placed in 3000Da bag filter
Interior pure water dialysis for 24 hours, obtains oligosaccharides vaccine after freeze-drying.
The preparation of 6 oligosaccharides vaccine of embodiment
50mgOVA is taken to be dissolved in the 1mL0.01M sodium phosphate buffer of pH7.2.It is 10%, molecular weight that 1mg deacetylation, which is added,
The chitosan oligosaccharide of 1000-3000Da, dissolution mix.Take (CHO (the CH containing glutaraldehyde2)3CHO it is molten that substrate is added in) 50% aqueous solution, 2 μ L
Liquid mixes, keeps 25 DEG C of slight vibration 60min.Glycine solid 50mg is added to mix, keeps 4 DEG C of 30min.With pH7.2's
0.01M sodium phosphate buffer is mobile phase, passes through Thermo Scientific Zeba Spin Desalting
Columns removes low molecule impurity, obtains the carrier protein solution of coupling chitosan oligosaccharide, is placed in pure water in 3000Da bag filter and dialyses
For 24 hours, oligosaccharides vaccine is obtained after freeze-drying.
The preparation of 7 oligosaccharides vaccine of embodiment
50mgOVA is taken to be dissolved in the 1mL0.01M sodium phosphate buffer of pH7.2.It is 95%, molecular weight that 1mg deacetylation, which is added,
The chitosan oligosaccharide of 1000-3000Da, dissolution mix.Take (CHO (the CH containing glutaraldehyde2)3CHO it is molten that substrate is added in) 50% aqueous solution, 11 μ L
Liquid mixes, keeps 25 DEG C of slight vibration 60min.Glycine solid 250mg is added to mix, keeps 4 DEG C of 30min.With pH7.2's
0.01M sodium phosphate buffer is mobile phase, passes through Thermo Scientific Zeba Spin Desalting
Columns removes low molecule impurity, obtains the carrier protein solution of coupling chitosan oligosaccharide, is placed in pure water in 3000Da bag filter and dialyses
For 24 hours, oligosaccharides vaccine is obtained after freeze-drying.
The preparation of 8 oligosaccharides vaccine of embodiment
100mgBSA is taken to be dissolved in the 1mL0.1M sodium phosphate buffer (NaCl containing 0.15M) of pH6.9.1mg deacetylation, which is added, is
0.05%, molecular weight < 400Da chitosan oligosaccharide, dissolution mix.Take 50% glutaraldehyde (CHO (CH2)3CHO) 1 μ L of aqueous solution is added mixed
It is even, keep 0 DEG C of slight vibration 150min.NaBH is added4Solid 42.5mg is mixed, and keeps 0 DEG C of 75min.It is slow with 0.1M sodium phosphate
Fliud flushing (NaCl containing 0.15M) is mobile phase, removes low molecule impurity by desalination prepacked column G10, obtains the load of coupling chitosan oligosaccharide
Body protein solution is placed in pure water dialysis in 3000Da bag filter and for 24 hours, oligosaccharides vaccine is obtained after freeze-drying.
The preparation of 9 oligosaccharides vaccine of embodiment
90mgKLH is taken to be dissolved in the 1mL0.1M sodium phosphate buffer of pH7.It is 0.05%, molecular weight < 600Da that 1mg deacetylation, which is added,
Chitosan oligosaccharide, dissolution mix.Take 50% hexandial (CHO (CH2)4CHO) 1.4 μ L of aqueous solution, which is added, mixes, and keeps 2 DEG C of slight vibrations
140min.The 463 μ Lmg of 1M Tris-HCl buffer that pH7.4 is added is mixed, and keeps 2 DEG C of 70min.With 0.1M sodium phosphate buffer
Liquid is mobile phase, removes low molecule impurity by desalination prepacked column G15, obtains the carrier protein solution of coupling chitosan oligosaccharide, be placed in
Pure water dialysis for 24 hours, obtains oligosaccharides vaccine in 3000Da bag filter after freeze-drying.
The preparation of 10 oligosaccharides vaccine of embodiment
80mgOVA is taken to be dissolved in the 1mL0.01M sodium phosphate buffer of pH7.2.Be added 1mg deacetylation be 0.1%, molecular weight <
The chitosan oligosaccharide of 800Da, dissolution mix.Take 50% heptan dialdehyde (CHO (CH2)5CHO) 1.8 μ L of aqueous solution, which is added, mixes, and keeps 4 DEG C light
Micro-vibration 120min.Glycine 67.5mg is added to mix, keeps 4 DEG C of 60min.It is stream with 0.01M sodium phosphate buffer (pH7.2)
Dynamic phase removes low molecule impurity by desalination prepacked column G25, obtains the carrier protein solution of coupling chitosan oligosaccharide, be placed in 3000Da
Pure water dialysis for 24 hours, obtains oligosaccharides vaccine in bag filter after freeze-drying.
The preparation of 11 oligosaccharides vaccine of embodiment
Take 70mgBlue carrierTMIt is dissolved in the 1mL0.1M carbonate buffer solution of pH7.1.Be added 1mg deacetylation be 0.1%,
Molecular weight < 1000Da chitosan oligosaccharide, dissolution mix.Take 50% suberic aldehyde (CHO (CH2)6CHO) 2 μ L of aqueous solution, which is added, mixes, and keeps
8 DEG C of slight vibration 110min.Lysine 70mg is added to mix, keeps 8 DEG C of 55min.Using 0.1M carbonate buffer solution as mobile phase,
Low molecule impurity is removed by desalination prepacked column G50, obtains the carrier protein solution of coupling chitosan oligosaccharide.
The preparation of 12 oligosaccharides vaccine of embodiment
65mg tetanus toxin is taken to be dissolved in the HEPES buffer solution of the 1mL0.02M of pH7.3.It is 1%, molecule that 1mg deacetylation, which is added,
The chitosan oligosaccharide of amount < 2000Da, dissolution mix.Take 50% azel aldehyde (CHO (CH2)7CHO) 3 μ L of aqueous solution, which is added, mixes, and is kept for 10 DEG C
Slight vibration 90min.The 403 μ Lmg of 2M Tris-HCl buffer that pH8.0 is added is mixed, and keeps 10 DEG C of 45min.With 0.02M's
HEPES buffer solution is mobile phase, removes low molecule impurity by desalting column Bio-gel P2, obtains the carrier egg of coupling chitosan oligosaccharide
White solution.
The preparation of 13 oligosaccharides vaccine of embodiment
60mgHMP is taken to be dissolved in the 1mL0.05M borate buffer solution of pH7.4.Be added 1mg deacetylation be 1%, molecular weight <
The chitosan oligosaccharide of 3000Da, dissolution mix.Take 50% decanedial (CHO (CH2)8CHO) 4 μ L of aqueous solution, which is added, mixes, and keeps 14 DEG C light
Micro-vibration 80min.Glycine 120mg is added to mix, keeps 14 DEG C of 40min.Using 0.05M borate buffer solution as mobile phase, lead to
It crosses desalting column Bio-gel P4 and removes low molecule impurity, obtain the carrier protein solution of coupling chitosan oligosaccharide.
The preparation of 14 oligosaccharides vaccine of embodiment
55mg pseudomonal toxin A is taken to be dissolved in the 1mL pure water of pH7.5.It is 10%, molecular weight 1000- that 1mg deacetylation, which is added,
The chitosan oligosaccharide of 2000 Da, dissolution mix.Take 50%CHO (CH2)105 μ L of CHO aqueous solution, which is added, to be mixed, and 17 DEG C of slight vibrations are kept
70min.Lysine 137.5mg is added to mix, keeps 17 DEG C of 35min.Using pure water as mobile phase, pass through desalting column Bio-gel P6
Low molecule impurity is removed, the carrier protein solution of coupling chitosan oligosaccharide is obtained.
The preparation of 15 oligosaccharides vaccine of embodiment
50mg cholera toxin is taken to be dissolved in the 1mL0.01M sodium phosphate buffer of pH7.6.It is 10%, molecule that 1mg deacetylation, which is added,
The chitosan oligosaccharide of 2000-3000 Da is measured, dissolution mixes.80 μ L of 4mgBS3 originate sodium phosphate buffer dissolution, take 60 μ L that bottom is added
Object solution mixes, keeps 20 DEG C of slight vibration 60min.The 413 μ Lmg of 3MTris-HCl buffer that pH7.0 is added is mixed, and is kept
20℃30min.Using 0.01M sodium phosphate buffer as mobile phase, low molecule impurity is removed by desalting column Bio-gel P10, is obtained
To the carrier protein solution of coupling chitosan oligosaccharide.
The preparation of 16 oligosaccharides vaccine of embodiment
40mg pertussis toxin is taken to be dissolved in the 1mL0.1M sodium phosphate buffer of pH7.7.It is 10%, molecule that 1mg deacetylation, which is added,
The chitosan oligosaccharide of 3000-4000 Da is measured, dissolution mixes.The 4mgDSS DMSO of 120 μ L dissolves, and takes 105 μ L that substrate solution is added mixed
It is even, keep 22 DEG C of slight vibration 50min.Glycine 157.5mg is added to mix, keeps 22 DEG C of 25min.With 0.1M sodium phosphate buffer
Liquid is mobile phase, removes low molecule impurity by desalination prepacked column Bio-gel P6DG, obtains the carrier protein of coupling chitosan oligosaccharide
Solution.
The preparation of 17 oligosaccharides vaccine of embodiment
30mg clostridium perfringens exotoxin is taken to be dissolved in the 1mL0.1M sodium phosphate buffer of pH7.8 (containing 0.15M
NaCl).It is 20%, molecular weight < 4000Da chitosan oligosaccharide that 1mg deacetylation, which is added, and dissolution mixes.By 100mg(100 μ L) BS
(PEG)2The DMSO that 0.9mL is added is configured to 250mM reagent, takes 40 μ L that substrate solution is added and mixes, keeps 24 DEG C of slight vibrations
40min.Lysine 160mg is added to mix, keeps 24 DEG C of 20min.It is stream with 0.1M sodium phosphate buffer (NaCl containing 0.15M)
Dynamic phase removes low molecule impurity by Thermo Scientific Zeba Spin Desalting Columns, obtains
It is coupled the carrier protein solution of chitosan oligosaccharide.
The preparation of 18 oligosaccharides vaccine of embodiment
20mg hepatitis B surface antigen is taken to be dissolved in the 1mL0.1M carbonate buffer solution of pH7.9.1mg deacetylation is added to be 20%, divide
The chitosan oligosaccharide of son amount < 5000Da, dissolution mix.By 100mg(100 μ L) BS(PEG)4The DMSO for being dissolved in 720 μ L is configured to 252mM
Reagent takes 36.9 μ L that substrate solution is added and mixes, keeps room temperature DEG C slight vibration 20min.The 3MTris-HCl that pH7.5 is added is slow
434 μ Lmg of fliud flushing is mixed, and keeps room temperature DEG C 10min.Using 0.1M carbonate buffer solution as mobile phase, pass through Thermo
Scientific Slide-A-Lyzer Dialysis Cassettes removes low molecule impurity, obtains coupling chitosan oligosaccharide
Carrier protein solution.
The preparation of 19 oligosaccharides vaccine of embodiment
15mg hepatitis B core antigen is taken to be dissolved in the HEPES buffer solution of the 1mL0.02M of pH8.1mg deacetylation is added to be 20%, divide
The chitosan oligosaccharide of son amount < 3000Da, dissolution mix.By 100mg(100 μ L) BS(PEG)5The DMSO that 650 μ L are added is configured to 250mM
Reagent takes 37.5 μ L that substrate solution is added and mixes, room temperature slight vibration 30min.Glycine 150mg is added to mix, room temperature
15min.Using the HEPES buffer solution of 0.02M as mobile phase, low molecule impurity is removed by the bag filter of aperture 10kDa, obtains idol
Join the carrier protein solution of chitosan oligosaccharide.
The preparation of 20 oligosaccharides vaccine of embodiment
10mg rotavirus VP 7 albumen is taken to be dissolved in the 1mL0.05M borate buffer solution of pH8.1.1mg deacetylation, which is added, is
50%, the chitosan oligosaccharide of molecular weight 2000-4000Da, dissolution mix.By 100mg(100 μ L) BS(PEG)6The DMSO of 594 μ L is added
It is configured to 251mM reagent, takes 38.17 μ L that substrate solution is added and mixes, keep room temperature slight vibration 40min.Lysine is added
137.5mg is mixed, and is stored at room temperature 20min.Using 0.05M borate buffer solution as mobile phase, removed by the bag filter of aperture 8kDa
Low molecule impurity is removed, the carrier protein solution of coupling chitosan oligosaccharide is obtained.
The preparation of 21 oligosaccharides vaccine of embodiment
5mg diphtheria toxin mutation CRM is taken to be dissolved in the 1mL pure water of pH8.2.It is 50%, molecular weight 2000- that 1mg deacetylation, which is added,
The chitosan oligosaccharide of 4000Da, dissolution mix.Take the BS(PEG of 38.7 μ L)7, substrate solution is added, mixes, keeps 25 DEG C of slight vibrations
20min.The 331 μ Lmg of 3MTris-HCl buffer that pH7.2 is added is mixed, and keeps 25 DEG C of 10min.Using pure water as mobile phase, lead to
The bag filter for crossing aperture 6kDa removes low molecule impurity, obtains the carrier protein solution of coupling chitosan oligosaccharide.
The preparation of 22 oligosaccharides vaccine of embodiment
3mg diphtheria toxin mutation CRM191 is taken to be dissolved in the 1mL 0.01M sodium phosphate buffer of pH8.3.1mg deacetylation is added
For the chitosan oligosaccharide of 95%, molecular weight 1000-3000Da, dissolution is mixed.Take the BS(PEG of 42.14 μ L)8, it is mixed that substrate solution is added
It is even, keep 28 DEG C of slight vibration 18min.Glycine 105mg is added to mix, keeps 28 DEG C of 9min.With 0.01M sodium phosphate buffer
Liquid is mobile phase, removes low molecule impurity by the bag filter of aperture 5kDa, obtains the carrier protein solution of coupling chitosan oligosaccharide.
The preparation of 23 oligosaccharides vaccine of embodiment
1mg diphtheria toxin mutation CRM3201 is taken to be dissolved in the 1mL0.2M sodium phosphate buffer of pH8.5.1mg deacetylation is added
For the chitosan oligosaccharide of 95%, molecular weight 3000-500Da, dissolution is mixed.Take the BS(PEG of 45.2 μ L)9, substrate solution is added, mixes,
Keep 30 DEG C of slight vibration 15min.Lysine 80mg is added to mix, keeps 30 DEG C of 7.5min.It is with 0.2M sodium phosphate buffer
Mobile phase removes low molecule impurity by the bag filter of aperture 4kDa, obtains the carrier protein solution of coupling chitosan oligosaccharide.
The preparation of 24 oligosaccharides vaccine of embodiment
0.8mg breathing syncystial virus F protein is taken to be dissolved in the 1mL0.1M sodium phosphate buffer (NaCl containing 0.15M) of pH8.8.It is added
1mg deacetylation is the chitosan oligosaccharide of 98%, molecular weight 4000-5000Da, and dissolution mixes.Take the BS(PEG of 42.84 μ L)12, it is added
Substrate solution mixes, keeps 33 DEG C of slight vibration 12min.The 149 μ Lmg of 4MTris-HCl buffer that pH7.8 is added is mixed, and is protected
Hold 33 DEG C of 6min.With 0.1M sodium phosphate buffer (NaCl containing 0.15M) for mobile phase, removed by the bag filter of aperture 3kDa
Low molecule impurity obtains the carrier protein solution of coupling chitosan oligosaccharide, is lyophilized to obtain the final product.
The preparation of 25 oligosaccharides vaccine of embodiment
0.5mg breathing syncytial virus G protein is taken to be dissolved in the 0.5mL 0.3M carbonate buffer solution of pH9.With the 0.5mL 0.1M of pH9
It is 98%, molecular weight < 1000Da chitosan oligosaccharide that carbonate buffer solution, which dissolves 1mg deacetylation, is mixed with carrier protein solution.Directly
Meet the BS(PEG that 41.2 μ L are added)15, substrate solution is added, mixes, keeps 35 DEG C of standing 10min.It is mixed that glycine 50mg is added
It is even, keep 35 DEG C of 5min.Using 0.3M carbonate buffer solution as mobile phase, it is miscellaneous that low molecule is removed by the bag filter of aperture 1kDa
Matter obtains the carrier protein solution of coupling chitosan oligosaccharide, is spray-dried to obtain the final product.
The detection of 26 coupling ratio of embodiment
(1) standard curve precision weighs DEXTROSE ANHYDROUS 0.1030g(water content 1.22%), 50mL, which is settled to, with pure water mixes.
Take 5mL in 50mL measuring bottle, moisturizing to 50mL obtains 0.2035 mg/mL standard solution.Take 0,10,20,40,60,80,100,
120, then 140,160 μ L add 200,190,180,160,140,120,100,80,60,40 in 1.5 mL centrifuge tubes respectively
μ L water mixes, and obtains 0-0.1628 mg/mL series of concentrations.
(2) sample solution weighs oligosaccharides vaccine formulation into the aqueous solution of 1 mg/mL of mass concentration, is not coupled with same concentration
The carrier protein of chitosan oligosaccharide is blank control.It takes 400 μ L oligosaccharides vaccine solutions to hydrolyze in 2 mL to manage, the 80 μ L concentrated sulfuric acids is added,
Lid is screwed, in 105 DEG C of 1.5 h of heat preservation.Moisturizing dilute hydrolyzate to 0.5 mL of scale place, with pipettor blow and beat mix to
Sample solution.Same treatment carrier protein blank solution.Numerical value over range is such as detected, is diluted with appropriate amount of water.
(3) sample measurement takes series standard solution, 200 μ L of testing sample solution in 1.5 mL centrifuge tubes, is added 0.2%
(w/v) 600 μ L of anthrone sulfuric acid solution, buckle closure after mixing, boiling water bath heat 15min, and ice-water bath cools down 15min after taking-up.It is mixed
It takes 200 μ L that 96 orifice plates are added after even, absorbance at 625nm is detected by microplate reader.Standard curve tested concentration range y=
4.840x-0 R2=0.991.Sample solution is read in 625nm absorbance, reduces the blank absorption value of respective carrier albumen, according to
Standard curve calculates sample sugar concentration.And carrier protein and chitosan oligosaccharide coupling mass ratio are calculated according to the following formula:
1 carrier protein of table and chitosan oligosaccharide are coupled mass ratio
。
The antigenicity and immunity of 27 oligosaccharides vaccine of embodiment
It is immune by animal inoculation pvaccination, take key IL-17F cytokine levels in peripheral blood detection Th17 cell to change to evaluate
The antigenicity and immunity of the chitosan oligosaccharide of conjugate immunogens carrier protein, the meaning of the parameter are equivalent to the body of B cell mediation
IgG or IgM variation during liquid is immune.
(1) immunization protocol
By 4-6 week old Kun Ming mice (SPF grades) female mice, every group 6, physiological saline blank group, KLH adjuvant group, reality are set
Apply the chitosan oligosaccharide-KLH group of 10 % of deacetylation in example 2, in embodiment 3 95 % of deacetylation chitosan oligosaccharide-KLH group.Exist respectively
On-test 0,2,4,6 week, to physiological saline group mouse neck, dorsal sc multi-point injection, dosage was 0.2 mL/；Remaining
Given the test agent 0.2 mL/ of each group mouse same procedure injection containing adjuvant is only.
To activate immune response to reduce uncontrollability again as far as possible, select conventional Freund completely and Freund's incomplete adjuvant.Exempt within 0th week
Freund's complete adjuvant is selected in epidemic disease injection, and incomplete Freund's adjuvant is selected in inoculation in the 2nd, 4,6 week.With the PBS(pH of 0.02 M
7.2-7.4) buffer by KLH, the deacetylated-KLH of 95 % of chitosan oligosaccharide, that the deacetylated-KLH of 1 % of chitosan oligosaccharide is configured to 0.1 mg/mL is molten
Liquid, then mixed in equal volume with Freund's complete adjuvant or incomplete Freund's adjuvant, it is uniform by ultrasonic emulsification.
(2) intracellular cytokine detects
Lymphocyte extracts and stimulation: before inoculation in the 0th week and after inoculation in the 6th week, respectively to vein under mouse jaw
It takes 0.3 mL of blood to mix in 1.5 mL anticoagulant tubes, is added 3 mL1 × erythrocyte cracked liquid splitting erythrocyte, 4 min, 4 DEG C 500
G is centrifuged 10 min and inhales abandoning supernatant, and 500 g centrifugation, 10 min, which inhale, after the PBS of addition 10 mL, 0.01 M abandons supernatant, with 200 μ L thorn
Swash liquid (stimulation formula of liquid: PMA 1/1000, inonycin 1/1000, BFA(brefeldin) 1/1000, monensin1/
1000 fit in 1640 complete medium) lymphocyte of precipitating is suspended in 96 orifice plate of round bottom, it is incubated in cell incubator
Educate 4 h.
Extracellular dyeing: 96 orifice plates are centrifuged 10 min(centrifugal conditions similarly hereinafter in 4 DEG C of 1600 rpm), supernatant is abandoned, by 70
Add FACS buffer solution (CD3e containing fluorescent staining, CD4, CCR6,7-AAD antibody, additive amount containing extracellular staining antibodies in the hole μ L/
Respectively 1:100,1:200,1:100,1:100) piping and druming, 4 DEG C of 40 min of incubation are protected from light, extracellular dyeing is carried out.
Rupture of membranes: being added 180 hole μ L/ of FACS buffer solution without antibody and blow and beat, and supernatant is abandoned in centrifugation, is added by 100 holes μ L/
Rupture of membranes liquid is protected from light 4 DEG C of 40 min of incubation, carries out rupture of membranes processing.
Dyeing intracellular: with 1 × 180 hole μ L/ of rupture of membranes buffer blow and beat, supernatant is abandoned in centrifugation, is added by 70 holes μ L/ containing born of the same parents
The rupture of membranes buffer (antibody of IL-17F containing fluorescent staining, additive amount are respectively 1:100) of interior staining antibodies, room temperature is protected from light incubation 40
Min carries out intracellular cytokine dyeing.
Flow cytomery: by 180 holes μ L/ addition FACS buffer solution piping and druming, supernatant is abandoned in centrifugation, then presses 100 holes μ L/
FACS buffer solution piping and druming is added, upper machine testing Th17 IL-17F intracellular is horizontal, and reflection given the test agent swashs to what mouse cell was immunized
Effect living.
Chitosan oligosaccharide-BSA the group of 10 % of deacetylation, 5 deacetylation of embodiment, 95 % in same operation detection embodiment 4
Chitosan oligosaccharide-BSA group and embodiment 6 in the chitosan oligosaccharide-OVA group of 10 % of deacetylation, in embodiment 7 95 % of deacetylation shell
Oligosaccharides-OVA group.Th17 IL intracellular accounts for Th17 ratio as shown in table 2- table 4 before and after each oligosaccharides vaccine immune mouse.
Th17 IL-17F accounting intracellular before and after mouse is immunized in 2 KLH coupled chitosan of table
* compared with physiological saline blank group and KLH adjuvant group, there is statistical difference, α=0.05.
Th17 IL-17% accounting intracellular before and after mouse is immunized in 3 BSA coupled chitosan of table
# has statistical difference, α=0.05 compared with physiological saline blank group and BSA adjuvant group.
Th17 IL-17 accounting intracellular before and after mouse is immunized in 4 OVA coupled chitosan of table
+ compared with physiological saline blank group and BSA adjuvant group, there is statistical difference, α=0.05.
It can be seen that compared with " physiological saline blank group " and " carrier adjuvant " group of only empty vectors and adjuvant,
Rising in immune front and back IL-17F level has statistical significance, shows that the chitosan oligosaccharide for being coupled KLH, BSA, OVA can be effective
The IL-17F for activating Th17 cell secretion antifungal efficacy, has antigenicity and immunity, the low ratio deacetylation of deacetylation
High has higher rush IL-17F effect.
The challenge viral dosage of 28 oligosaccharides vaccine of embodiment
4-6 week old Kun Ming mice (SPF grades) female mice is taken, every group 6, physiological saline blank group, KLH adjuvant group, reality are set
Apply the chitosan oligosaccharide-KLH group of 10 % of deacetylation in example 2, the chitosan oligosaccharide-KLH group of 95 % of deacetylation, Fluconazole in embodiment 3
Totally 5 groups of control group.
Respectively at on-test 0,2,4,6 week, to physiological saline group mouse neck, dorsal sc multi-point injection, dosage is
0.2 mL/ is only；KLH adjuvant group, the chitosan oligosaccharide-KLH group of 10 % of deacetylation, 95 % of deacetylation chitosan oligosaccharide-KLH organize mouse
Given the test agent 0.2 mL/ of the same procedure injection containing adjuvant is only；Fluconazole group is not injected, and is given at the 6th week by 50 mg/kgd
Intragastric administration on mice, continuous gavage 7 days, for the positive controls for attacking preventative antimycotic administration before poison.
Freund's complete adjuvant is selected in inoculation in 0th week, and incomplete Freund's adjuvant is selected in inoculation in the 2nd, 4,6 week.With
The PBS(pH 7.2-7.4 of 0.02 M) buffer is by KLH, the chitosan oligosaccharide-KLH group of 10 % of deacetylation, 95 % of deacetylation
Chitosan oligosaccharide-KLH group is configured to 0.5 mg/mL solution, then mixes in equal volume with Freund's complete adjuvant or incomplete Freund's adjuvant,
It is uniform by ultrasonic emulsification.
The candida albicans of culture is made to the PBS suspension of 0.01 M, by 2 × 10 on the 8th week4A/0.1 mL dosage warp
Mouse tail vein injection attacks poison.After 10 days, mouse is put to death, double kidneys are cut in length and breadth respectively, takes half to fix through glutaraldehyde, passes through HE
Dyeing observation inflammatory conditions；Remaining half renal tissue is taken to weigh, homogenate is improveing Sabouraud culture medium flat plate inoculated and cultured,
Calculate fungi kidney carrying capacity CFU.
HE dyes main detection multicore granulocyte, lymphocyte, thick liquid cell, macrophage, megacaryocyte infiltration and fiber
Change degree etc., for evaluating inflammatory reaction degree.HE dyeing is divided into 0-4 grades, (1) 0 point: without infiltration；(2) 1 points: few leaching
Profit (10-50,400 times of microscopic observations)；(3) 2 points: mild infiltration (50-200,400 times of microscopic observations)；(4) 3 points: severe
Infiltration (200-500,400 times of microscopic observations)；(5) 4 points: severe infiltration (> 500,400 times of microscopic observations).
Deacetylation 95 in the chitosan oligosaccharide-BSA group, embodiment 5 of 10 % of deacetylation in same operation detection embodiment 4
Renal inflammation reaction influences after the chitosan oligosaccharide-BSA group of % attacks poison to Brazil's aspergillus, and the shell of 10 % of deacetylation is few in embodiment 6
Chitosan oligosaccharide-OVA the group of 95 % of deacetylation attacks the influence of renal inflammation reaction after poison to cryptococcus in sugar-OVA group, embodiment 7.
Mouse kidney inflammatory effect is as shown in table 5 after each oligosaccharides vaccine attacks poison to fungi.
Kidney HE dyeing scoring influences after the different vaccines of table 5 attack poison to fungi
* compared with physiological saline blank group, KLH adjuvant group, Fluconazole group, there are statistical difference, α=0.05；
# has statistical difference, α=0.05 compared with physiological saline blank group；
+ compared with physiological saline blank group, KLH adjuvant group, Fluconazole group, there are statistical difference, α=0.05.
By table 5 as it can be seen that the chitosan oligosaccharide of coupling carrier albumen, which can effectively mitigate fungi, attacks inflammatory reaction of the poison to mouse kidney,
The high inflammation of the low ratio deacetylation of deacetylation is lighter.
Claims (10)
1. a kind of oligosaccharides vaccine for preventing invasive infections with fungi, which is characterized in that including conjugate immunogens carrier protein
Chitosan oligosaccharide.
2. oligosaccharides vaccine according to claim 1, which is characterized in that the idol of the immunogenic carrier albumen and chitosan oligosaccharide
Connection mass ratio is 0.7-210:1, preferably 1-101:1.
3. oligosaccharides vaccine according to claim 1, which is characterized in that the deacetylation of the chitosan oligosaccharide is 0.05-98%,
Average relative molecular mass < 5000 Da；Preferably, deacetylation 0.1-20%, Relative average molecular weight 1000-3000Da.
4. oligosaccharides vaccine according to claim 1, which is characterized in that the immunogenic carrier albumen is non-source of people egg
It is white；Preferably, immunogenic carrier albumen is selected from diphtheria toxin non-toxic variant, KLH, BSA, OVA, Blue carrierTM, break
Cold toxin/toxoid, is isolated from the high-molecular-weight protein of non-acquisition type haemophilus influenzae, the pseudomonal toxin after detoxification
A, cholera toxin/toxoid, pertussis toxin/toxoid, clostridium perfringens exotoxin/toxoid, hepatitis B table
Born of the same parents are closed in face antigen, hepatitis B core antigen, rotavirus VP 7 albumen, diphtheria toxin mutation CRM, CRM191, CRM3201, breathing
Any one in viral F and G-protein.
5. a kind of preparation method of the oligosaccharides vaccine as described in claim 1-4 is any, which comprises the following steps:
(1) immunogenic carrier albumen, chitosan oligosaccharide and coupling agent are dissolved in coupling liquid, are incubated for；
(2) terminator is added to be incubated for；
(3) purifying obtains oligosaccharides vaccine.
6. preparation method according to claim 5, which is characterized in that in step (1), the coupling agent is selected from CHO (CH2)mCHO, BS3, DSS or BS(PEG)nIn it is any；Preferably, CHO (CH2)mM=3-10 in CHO；BS(PEG)nMiddle n=2-15；
In step (2), the terminator is selected from Tris-HCl, glycine, lysine or NaBH4。
7. preparation method according to claim 5, which is characterized in that in step (1), the immunogenic carrier albumen with
The mass ratio of chitosan oligosaccharide is 0.5-100:1；The mass ratio of the coupling agent and chitosan oligosaccharide is 0.5-10:1；It is described in step (2)
The mass ratio of terminator and coupling agent is 5-85:1.
8. preparation method according to claim 5, which is characterized in that in step (1), the coupling liquid is selected from water or pH
The 0.01-0.3 M buffer of 6.9-9 not amino-contained；Preferably, it is coupled the pH 7.2-8 of liquid, concentration is 0.05-0.2 M；
The concentration of the immunogenic carrier albumen is 0.5-100 mg/mL；It is preferred that 10-90 mg/mL.
9. preparation method according to claim 5, which is characterized in that further include by oligosaccharides vaccine solution system after step (3)
The step of at solid.
10. a kind of oligosaccharides vaccine as described in right 1-3 is any is as the purposes for treating or preventing invasive fungi drug.
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| CN113274488A (en) * | 2021-04-30 | 2021-08-20 | 山东省药学科学院 | Oligosaccharide vaccine for specifically preventing fungal infection and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113274489A (en) * | 2021-04-30 | 2021-08-20 | 山东省药学科学院 | Chitin oligosaccharide vaccine for preventing fungal infection and preparation method thereof |
| CN113274488A (en) * | 2021-04-30 | 2021-08-20 | 山东省药学科学院 | Oligosaccharide vaccine for specifically preventing fungal infection and preparation method thereof |
| WO2022226672A1 (en) * | 2021-04-30 | 2022-11-03 | 山东省药学科学院 | Chitin oligosaccharide vaccine for preventing fungal infection and preparation method therefor |
| CN113274489B (en) * | 2021-04-30 | 2023-08-29 | 山东省药学科学院 | Chitin oligosaccharide vaccine for preventing fungal infection and preparation method thereof |
| CN113274488B (en) * | 2021-04-30 | 2023-08-29 | 山东省药学科学院 | Oligosaccharide vaccine for specifically preventing fungal infection and preparation method thereof |
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