CN113274489B - Chitin oligosaccharide vaccine for preventing fungal infection and preparation method thereof - Google Patents
Chitin oligosaccharide vaccine for preventing fungal infection and preparation method thereof Download PDFInfo
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Abstract
The application belongs to the field of biotechnology, and relates to a chitin oligosaccharide vaccine for preventing fungal infection and a preparation method thereof, wherein the vaccine is formed by coupling oligosaccharide with carrier protein, and is prepared by aldehyde (-CHO) on the oligosaccharide and amino (-NH) on the carrier protein 2 ) A coupling body which is condensed and formed by the action of a reducing agent. The carrier protein is non-human protein, and the oligosaccharide is a mixture of chitin oligosaccharides. The vaccine provided by the application has strong immunogenicity, can activate Th17 cell immunity, and can identify and protect infection caused by fungi.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a chitin oligosaccharide vaccine for preventing fungal infection and a preparation method thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the application and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
In recent years, due to the increasing number of patients suffering from immunodeficiency or injury (such as immunosuppressant use, tumor radiotherapy and chemotherapy, aids epidemic, bone marrow transplantation, etc.), the increasing number of patients suffering from gastrointestinal dysbacteriosis (such as gastrointestinal complex surgery, broad-spectrum antibiotics, etc.), the increasing number of patients suffering from invasive catheterization (such as mechanical respiration, deep vein nutrition, etc.), the incidence and mortality of systemic fungal infections have been on the rise all over the world. Common causative strains of systemic fungal infections are candida, aspergillus, cryptococcus neoformans, about 70% of which are fungal infections that involve internal organs of the human body or form eubacteremia causing death. The number of antifungal drugs used clinically at present is small, and the large amount of antifungal drugs is used, so that drug-resistant strains are continuously appeared, and serious drug resistance problems are generated. Because the systematic fungal infection has strong concealment and uncertainty of clinical specimen separation diagnosis, the clinical diagnosis is not obvious, the early infection is difficult to diagnose, and the optimal treatment time is easily delayed. The fungal vaccine is the most ideal preventive measure, but the research of the fungal vaccine starts late and progresses little, and no vaccine for preventing systemic fungal infection exists at home and abroad at present.
The fungal cell wall typically contains mannose, beta-glucan, and chitin polysaccharide. Early researches have found that the immunogenicity of sugar is low, the immune effect of organism is difficult to excite, and the research team couples beta-mannobiose, trisaccharose and tetrasaccharose with Keyhole Limpet Hemocyanin (KLH) to form glycoprotein compounds, and couples candida albicans virulence Als1 epitope short peptide, and glycoprotein compounds formed by coupling beta-glucan with KLH with different branches can all cause strong humoral immune response (IgG antibody reaction) in mice, but the number of varieties available for screening effective sugar antigens is limited due to the fact that tested antigens need to pass through complex chemical synthesis technology, and the protection effect of the current research vaccine still needs to be improved. There is a study to provide an oligosaccharide vaccine for preventing invasive fungal infection by amino (-NH) groups on chitosan oligosaccharides 2 ) With amino groups (-NH) on carrier proteins 2 ) By means of bridging agents (e.gCHO(CH 2 ) m CHO), a glycoprotein compound is formed, th17 and Th1 cell immunity can be effectively activated, but the coupling position with protein is undefined, the specificity is poor, the process is unstable and the quality is uncontrollable because the chitosan oligosaccharide contains a plurality of amino groups.
Chitin is widely present in the cell wall of fungi, and chitin oligosaccharide is a small molecule oligosaccharide obtained by enzymolysis of chitin, has the same monosaccharide composition as chitin, and can be used as a broad-spectrum antifungal target point. At present, series of chitooligosaccharides can be prepared in batches in vitro, and large-scale immunogen screening can be realized. The broad-spectrum fungus vaccine in the field is not reported yet, and the mechanism of recognition by immune system and the like is still unclear. Therefore, the research of antifungal vaccines based on fungal cell structures has very urgent practical significance.
Disclosure of Invention
In order to overcome the problems, the application aims to provide a chitin oligosaccharide vaccine for preventing fungal infection, which is used for preventing and treating fungal infection of immunosuppressed patients.
In order to achieve the technical purpose, the application adopts the following technical scheme:
in a first aspect of the present application, there is provided a method for preparing a chitooligosaccharide vaccine for preventing fungal infection, comprising:
coupling the oligosaccharide mixture containing aldehyde groups with a carrier protein to form a conjugate vaccine.
The research finds that: the oligosaccharide used in the application is chitin oligosaccharide, which is a mixture, has different polymerization degrees, is not chemically modified, has strong structural similarity with chitin in fungal cell walls, and is easier to cause immune response of organism cells. At the same time, the chitin oligosaccharide contains aldehyde (-CHO) and amino (-NH) contained in carrier protein with immunity enhancing effect 2 ) The conjugate vaccine has stable structure, definite conjugate position and strong specificity, and can well prevent and treat the infection caused by fungi of immunosuppressed patients.
In a second aspect of the application, there is provided a chitin oligosaccharide vaccine prepared by any of the above methods for preventing fungal infection.
In a third aspect, the application provides an application of the chitin oligosaccharide vaccine for preventing fungal infection in preparing a medicament for preventing systemic fungal infection.
In a fourth aspect, the application provides an application of the chitin oligosaccharide vaccine for preventing fungal infection in preparation of a medicament for activating Th17 cellular immunity.
The application has the beneficial effects that:
(1) The oligosaccharide vaccine provided by the application utilizes aldehyde (-CHO) contained in chitin oligosaccharide and amino (-NH) contained in carrier protein with immune activation effect 2 ) Forming a coupling site, and adding a reducing agent to form the conjugate vaccine with stable structure. The coupling position of the conjugate vaccine is clear, the specificity is strong, wherein the chitin oligosaccharide used is a mixture with different polymerization degrees, has strong structural component similarity with chitin (i.e. polysaccharide) in the cell wall of fungi, improves the immunogenicity of the vaccine by increasing the coupling ratio of the oligosaccharide and carrier protein, effectively activates Th17 cell immune response, and identifies and protects infection caused by fungi (such as candida albicans, aspergillus, cryptococcus neoformans and the like).
(2) The method has the advantages of simplicity, low cost, universality and easiness in large-scale production.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
A chitin oligosaccharide vaccine for preventing fungal infection is prepared from aldehyde group (-CHO) on oligosaccharide mixture and amino group (-NH) on carrier protein 2 ) The condensation forms a compound with Schiff base (coupling intermediate), the Schiff base is formed as a relatively unstable reversible interaction, and the Schiff base is converted into a stable coupling body, namely an oligosaccharide vaccine under the action of a reducing agent, and the coupling mole ratio of the oligosaccharide and the carrier protein is more than (200-500): 1.
In some embodiments, the oligosaccharide is a chitin oligosaccharide, which is a mixture of different degrees of polymerization, a relative molecular mass of less than or equal to 5000Da, a degree of deacetylation of less than or equal to 15%, which is commercially available or prepared by degrading chitin.
In some embodiments, the carrier protein is a non-human protein selected from one of Concholepasconcholepashemocyanin (CCH), keyhole limpet hemocyanin (KLH, keyhole limpet hemocyanin), bovine Serum Albumin (BSA), tetanus toxin/toxoid, rotavirus VP7 protein, diphtheria toxin mutant CRM, and clostridium perfringens exotoxin/toxoid.
The application also provides a preparation method of the chitin oligosaccharide vaccine for preventing fungal infection, which comprises the following steps:
(1) The carrier protein is dissolved in phosphate buffer solution and mixed reversely.
(2) Adding chitin oligosaccharide, and mixing.
(3) Adding reducing agent, mixing, and standing for reacting for a certain time.
(4) Purifying to obtain the oligosaccharide vaccine.
In some embodiments, in step (1), the carrier protein is at a concentration of 1-100mg/ml, the phosphate buffer is 0.01-0.1M sodium phosphate, 0-0.15M sodium chloride, pH7.0-8.0.
In some embodiments, in step (2), the molar concentration of chitinase oligosaccharide is 100-1200 times the molar amount of carrier protein.
In some embodiments, the steps areIn the step (3), the reducing agent is lithium aluminum hydride (LiAlH) 4 ) Potassium borohydride (KBH) 4 ) Sodium borohydride (NaBH) 4 ) Sodium cyanoborohydride (NaBH) 3 CN)、Na 2 SO 3 、FeSO 4 The concentration of the mixture is 1-100mg/ml, and the mixture is placed at 25-37 ℃ for reaction for 1-10 days.
In some embodiments, in step (4), the purification method is column chromatography or dialysis for removing small molecules of impurities, and the buffer used for column chromatography or dialysis is a solution used when dissolving the carrier protein. The packing types of the desalting column are Sephadex G10-G50, bio-gel P2-P10 and Thermo Scientific TM Zeba TM Spin Desalting Columns. The dialysis bag has a molecular weight cut-off of 1000-10000Da and a flattening width of 1-3.5cm.
The oligosaccharide vaccine is used for treating or preventing infection caused by systemic fungi including but not limited to candida albicans, aspergillus and cryptococcus neoformans.
The application will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
Example 1
Dissolving 80mg CCH in 1ml 0.01M sodium phosphate and 0.01M sodium chloride buffer solution with pH of 7.2, slightly oscillating to dissolve, adding 2mg chitin oligosaccharide with deacetylation degree of 5% and molecular weight less than or equal to 5000Da, dissolving, mixing, and mixing well to obtain 16mg KBH 4 Mixing the materials with light vibration, and standing at 25 ℃ for 2 days for reaction. The chitin oligosaccharide-carrier protein conjugate is obtained by removing small molecules of impurities by using 0.01M sodium phosphate and 0.01M sodium chloride (pH 7.2) as buffer solution for dialysis and by a dialysis bag with cutoff molecular weight of 10000Da, and the oligosaccharide vaccine is obtained by vacuum freeze drying.
Example 2
Dissolving 40mg of CCH in 1ml of 0.01M sodium phosphate and 0.01M sodium chloride (pH 7.2) buffer solution, slightly shaking for dissolving, adding 4mg of chitin oligosaccharide with deacetylation degree of 2% and molecular weight of less than or equal to 2000Da, dissolving, mixing, and mixing well to obtain 40mg KBH 4 Mixing the materials with light vibration, and standing at 28 ℃ for 3 days for reaction. With 0.01M sodium phosphate and 0.01M sodium chloride (pH 7.2)Removing small molecules of impurities from the buffer solution for dialysis through a dialysis bag with a cutoff molecular weight of 5000Da to obtain a chitin oligosaccharide-carrier protein coupling body, and performing vacuum freeze drying to obtain the oligosaccharide vaccine.
Example 3
Dissolving 10mg of CCH in 1ml of 0.1M sodium phosphate and 0.01M sodium chloride buffer solution with pH7.0, slightly oscillating for dissolving, adding 5mg of chitin oligosaccharide with deacetylation degree of 1% and molecular weight less than or equal to 1500Da, dissolving, mixing well, and 20mg of KBH 4 Mixing the materials with light vibration, and standing at 37 ℃ for 7 days for reaction. Taking buffer solution of 0.1M sodium phosphate and 0.01M sodium chloride (pH 7.0) as a mobile phase, passing through a Bio-gel P2 Desalting Columns column, starting to connect 10 drops (about 0.5 ml) of sample, continuously connecting 20 tubes, detecting to obtain a chitin oligosaccharide-carrier protein conjugate, and performing vacuum freeze drying to obtain the oligosaccharide vaccine.
Example 4
Dissolving 10mg BSA in 1ml 0.01M sodium phosphate buffer solution with pH7.0, dissolving with gentle shaking, adding 25mg chitin oligosaccharide with deacetylation degree of 15% and molecular weight of 800Da, dissolving, mixing, and mixing to obtain 40mg NaBH 3 CN, mixing with light vibration, standing at 25deg.C, and reacting for 1 day. Taking 0.01M sodium phosphate buffer solution with pH of 7.0 as a mobile phase, passing through Sephadex G10 Desalting Columns, starting to connect 10 drops (about 0.5 ml) of sample after the sample passes through a column, continuously connecting 20 tubes, detecting to obtain a chitin oligosaccharide-carrier protein conjugate, and performing vacuum freeze drying to obtain the oligosaccharide vaccine.
Example 5
Dissolving 50mg BSA in 1ml 0.01M sodium phosphate buffer solution and 0.15M sodium chloride buffer solution with pH8.0, slightly oscillating to dissolve, adding 75mg chitosan oligosaccharide with deacetylation degree of 15% and molecular weight less than or equal to 500Da, dissolving, mixing well, and 50mg LiAlH 4 Mixing with light vibration, standing at 37deg.C for 1 day. And (3) taking 0.01M sodium phosphate and 0.15M sodium chloride with pH of 8.0 as a buffer solution for dialysis, removing small foreign molecules by a dialysis bag with a cutoff molecular weight of 5000Da to obtain a chitin oligosaccharide-carrier protein conjugate, and performing vacuum freeze drying to obtain the oligosaccharide vaccine.
Example 6
50mg of tetanus toxin was dissolved in 1ml of 0.01M sodium phosphate, pH8.0, and 0.15M chlorideDissolving in sodium buffer solution with gentle shaking, adding 45mg chitosan oligosaccharide with deacetylation degree of 15% and molecular weight less than or equal to 1000-2000Da, dissolving, mixing, and mixing to obtain 10mg LiAlH 4 Mixing with light vibration, standing at 37deg.C for 1 day. And (3) taking 0.01M sodium phosphate and 0.15M sodium chloride with pH of 8.0 as a buffer solution for dialysis, removing small foreign molecules by a dialysis bag with a cutoff molecular weight of 5000Da to obtain a chitin oligosaccharide-carrier protein conjugate, and performing vacuum freeze drying to obtain the oligosaccharide vaccine.
Example 7 determination of the degree of chitin oligosaccharide-carrier protein coupling Using example 3 as an example, the degree of coupling is expressed as the coupling ratio, which is the number of moles of chitin oligosaccharide attached per mole of carrier protein.
(1) Determination of the content of reducing sugars (free chitooligosaccharides)
0.1g (accurate to 0.001 g) of N-acetyl-D-glucosamine is weighed, the N-acetyl-D-glucosamine is dissolved in a 10ml volumetric flask by purified water, the solution is uniformly shaken after the volume is fixed to the scale, 10mg/ml standard solution mother solution is obtained, and the solution is stored in a refrigerator at the temperature of 4 ℃ in a dark place for two weeks and is effective. Respectively sucking 0, 20, 40, 80, 120, 160 and 240 mu LN-acetyl-D-glucosamine standard solution into a 1.5ml centrifuge tube, and supplementing 1ml with purified water to obtain standard solutions with different concentrations. Meanwhile, purified water is used for preparing 2, 1 and 0.5mg/ml chitin oligosaccharide solution.
Sucking 30 μl of standard or solution to be detected, adding 30 μl of LDNS, mixing, boiling water bath for 5min, tap water cooling, adding 180 μl of purified water, mixing, adding 200 μl of purified water into 96-well plate, and measuring absorbance OD with enzyme-labeled instrument 540 . The molar concentration of N-acetyl-D-glucosamine is taken as an abscissa, the absorbance is taken as an ordinate, a standard curve is established, and the standard curve y= 148.18x-0.0585 and R are obtained 2 = 0.9991. The concentration of reducing sugars in the sample and chitooligosaccharide solution was determined by standard curves.
(2) Determination of total sugar content
1g (accurate to 0.001 g) of N-acetyl-D-glucosamine is weighed, dissolved in a 10ml volumetric flask by purified water, and shaken uniformly after the volume is fixed to the scale, 100mg/ml standard solution mother solution is obtained, and the standard solution mother solution is stored in a refrigerator at 4 ℃ in a dark place for two weeks and is effective. The standard solutions of 0, 20, 40, 80, 120, 160, 200, 240 and 280 mu LN-acetyl-D-glucosamine were respectively aspirated into 1.5ml centrifuge tubes, and 1ml were filled with purified water to obtain standard solutions of different concentrations. Simultaneously, purified water is used for preparing 20, 10 and 5mg/ml chitin oligosaccharide solution.
Absorbing 100 μl of standard or solution to be detected, adding 300 μl of anthrone-sulfuric acid solution, boiling water for 10min, immediately placing into ice water for 15min, taking 200 μl, placing into 96-well plate, and measuring absorbance OD with enzyme-labeling instrument 620 . The molar concentration of N-acetyl-D-glucosamine is taken as an abscissa, the absorbance is taken as an ordinate, and a standard curve is established to obtain a standard curve y=8.9869x+0.0232, R 2 = 0.9829. The concentration of total sugars in the sample and chitooligosaccharide solution was determined by standard curve.
(3) Protein content determination by Bradford method
Preparing 5mg/ml BSA standard solution mother liquor and preparing 0, 0.02, 0.05, 0.1, 0.3, 0.5 and 0.7mg/ml standard solution.
Adding 20 μl of standard solution or 10-fold diluted sample into 96-well plate, adding 200 μl of coomassie brilliant blue 1×G250 staining solution into each well, standing at room temperature for 3-5min, and measuring absorbance OD with enzyme marker 595 . Taking BSA concentration as an abscissa and absorbance as an ordinate, and making a standard curve to obtain a standard curve y=1.6331x+0.0761, R 2 =0.9884. The concentration of protein in the sample was determined by a standard curve.
(4) Calculation of coupling ratio
The degree of polymerization of chitooligosaccharides = moles of total chitooligosaccharides/moles of chitooligosaccharide reducing sugars;
the molar number of chitooligosaccharides attached to the protein = (total sugar-reducing sugar x degree of polymerization)/degree of polymerization;
coupling ratio = moles of chitin oligosaccharides attached to protein/(10 x moles of protein).
(5) Analysis of results
The 20-tube chromatographic sample is not subjected to color reaction through the determination of the reducing sugar, which indicates that no reducing sugar (free chitooligosaccharide) exists, namely, aldehyde (-CHO) on the free chitooligosaccharide is completely reacted; the total sugar measurement shows that the 3 rd, 4 th and 5 th tubes have color reaction, brown color development, larger absorbance value and other non-color development; the Bradford method is used for measuring proteins, the 3 rd, 4 th and 5 th tubes have obvious color reaction, light blue color and larger light absorption value, the result shows that the chromatography parts of the coupling of the chitin oligosaccharides and the carrier protein are concentrated in the 3 rd, 4 th and 5 th tubes, the polymerization degree calculation of the chitin oligosaccharides is shown in table 1, the coupling ratios of the 3 rd, 4 th and 5 th tube samples are 321, 3395 and 2302 respectively, the data are shown in table 2, and the 4 th tube sample has the highest coupling ratio and can be used as a preparation for mouse immunization.
TABLE 1 degree of polymerization of chitin oligosaccharides
TABLE 2 coupling data of chitin oligosaccharides to carrier proteins
Example 8 immunization experiments with oligosaccharide vaccine
The effectiveness of the vaccine was studied using a mouse model of immunocompromised (neutropenia), which is considered a suitable model that can be used to simulate patients suffering from such defects due to chemotherapy for the treatment of various malignant tumors, etc. Immunocompromised mice resulting from cyclophosphamide injection will have increased susceptibility to fungi, similar to increased susceptibility to neutropenia in patients. Examining T cell mediated immune response, and evaluating immunogenicity of the chitin oligosaccharide-carrier protein vaccine by detecting changes in cytokine IL-17F level in Th17 cells with mouse blood.
1. Establishment of model of immunocompromised mice
Female mice of 6-8 weeks old Balb/c were selected and fed adaptively for 3-7 days. Cyclophosphamide 20g/L was prepared with physiological saline, and was injected intraperitoneally at 200mg/kg body weight, and 3 days later, was measured with SYSMEX XT-2000i fully automatic blood analyzerNumber of neutrophils in blood of mice, number of neutrophils<500 pieces/mm 3 The experiment can be continued, after which cyclophosphamide is injected every 10 days at a dose of 150mg/kg and maintained throughout the experiment.
2. Mouse immunization protocol
Saline placebo, CCH, chitooligosaccharide-CCH of example 3 (tube 4 sample) and fluconazole (as positive control for challenge test) were set up, 10 per group. The mice were injected subcutaneously at the neck and back at the beginning of the experiment at multiple points of 2.5mg/kg, the saline blank was injected with saline, the fluconazole was not injected, the mice were intragastrically at 50 mg/kg.d at week 6, and the other groups were injected with the adjuvanted samples for 7 days.
Freund's adjuvant is used to stimulate the body to produce strong immune response. Freund's complete adjuvant is selected for week 0, and Freund's incomplete adjuvant is selected for week 2, 4 and 6. Preparing 1mg/mL solution of CCH, chitooligosaccharide and chitooligosaccharide-CCH with 0.02M PBS (pH 7.2-7.4) buffer solution, mixing with Freund adjuvant, and emulsifying by ultrasonic wave.
3. Cytokine detection
Cytokine measurement step:
(1) Extraction and stimulation of lymphocytes: before and after week 0 and 6 immunization, the mice were subjected to submaxillary venous blood collection and mixing in an anticoagulant tube of 1.5mL, 3mL of 1 Xerythrocyte lysate was added, mixing was performed for 4min, 500g was centrifuged at 4℃for 10min, the supernatant was aspirated off, 10mL of 0.01MPBS was added, 500g was centrifuged for 10min, the supernatant was aspirated off, 200. Mu.L of stimulation solution was used, the precipitated lymphocytes were suspended in a round bottom 96-well plate, and incubated in a cell incubator for 4h.
(2) Extracellular staining: the 96-well plate was centrifuged at 1600rpm for 10min (same centrifugation conditions), the supernatant was discarded, and FACS buffer containing extracellular staining antibodies (containing fluorescent staining CD3e, CCR6, CD4, 7-AAD antibodies in amounts of 1:100, 1:200, 1:100, respectively) was added at 70 μl/well and was blown up and incubated for 40min under dark conditions to stain extracellular.
(3) Rupture of membranes: 180. Mu.L/well of FACS buffer without antibody was added and blown, the supernatant was centrifuged off, and the membrane-disrupting solution was added at 100. Mu.L/well, incubated at 4℃for 40min in the absence of light, and membrane-disrupting treatment was performed.
(4) Intracellular staining: the supernatant was centrifuged with 180. Mu.L/Kong Chuida of 1 Xof membrane-disrupting buffer, and 70. Mu.L/well of membrane-disrupting buffer containing intracellular staining antibody was added and incubated at room temperature for 40min in the absence of light to stain intracellular cytokines.
(5) Cytokine detection: the FACS buffer was added at 180. Mu.L/well and blown, the supernatant was centrifuged off, and the FACS buffer was added at 100. Mu.L/well and blown, and the flow cytometer detected Th17 intracellular IL-17F levels, reflecting the activation of the mouse cellular immunity by the test sample. The ratio of intracellular IL-17F to Th17 before and after immunization of each group of mice is shown in Table 3, for example.
TABLE 3 Th17 intracellular IL-17F duty cycle before and after mouse immunization
Note that: ＊＊ P<before the immunization at 0.01vs, ## P<blank group of 0.01vs
As can be seen from table 3, the ratio of intracellular IL-17F in Th17 of mice in CCH group and chitooligosaccharide group was improved after immunization compared with that before immunization, but the effect was not significant, and the ratio of intracellular IL-17F in mice in chitooligosaccharide-CCH group in example 3 was significantly improved (P < 0.01); compared with the blank control group, the intracellular IL-17F ratio of the CCH group and the chitooligosaccharide group is improved to a certain extent, and the intracellular IL-17F ratio of the chitooligosaccharide-CCH group mice in the embodiment 3 is remarkably improved (P < 0.01), which shows that the oligosaccharide coupled with the carrier protein can excite Th17 cell immunity, promote the oligosaccharide coupled with the carrier protein to secrete cell factor IL-17F, and play an antifungal role.
EXAMPLE 9 toxicity test of immunized mice
Candida albicans is used as a virus-attacking strain, and the protection effect of the oligosaccharide vaccine on fungi after virus attack is studied.
1. Determination of lethal doses of Candida albicans infection
Suspending cultured Candida albicans with 0.01M PBS to adjust concentration to 0.5X10 6 、1.0×10 6 、0.5×10 7 、1.0×10 7 、0.5×10 8 、1.0×10 8 CFU/ml, 0.1 ml/mouse i.v. to combat toxicity, mice were selected from immunocompromised mice (neutrophil count) injected with cyclophosphamide in step 1 of example 8<500 pieces/mm 3 ) At the same time, a physiological saline control group was set, the mortality of the mice within 5 days was counted, and the minimum lethal dose was determined, and the results are shown in table 4.
Table 4 mortality of candida albicans by i.v. mouse tail
As can be seen from table 4, the control mice did not die. 0.5×10 5 CFU and 1.0X10 5 Mice did not die within 5 days of CFU dose group; 0.5×10 6 Mortality rate 30% within 5 days of CFU dose group; 0.5×10 7 The death rate of the CFU dose group reaches 100% after 5 days; 1.0X10 7 Mortality within 12h of CFU dose group mortality was 100%, and it can be seen that the minimum lethal dose of candida albicans to mice was 0.5 x 10 7 CFU。
2. Toxicity test of immunized mice
Following the immunization protocol of example 8, a lethal dose of candida albicans was intravenously injected at week 8 to combat the death of each group of mice. Mice survival was calculated after 7 days and shown in table 5.
TABLE 5 survival rate of immunized mice after challenge
From the table above, the survival rate of mice in the chitin oligosaccharide-CCH group reaches 70% and is obviously higher than that of mice in other groups, which shows that the prepared oligosaccharide vaccine has an immunoprotection effect on infection caused by candida albicans.
Finally, it should be noted that the above-mentioned embodiments are only preferred embodiments of the present application, and the present application is not limited to the above-mentioned embodiments, but may be modified or substituted for some of them by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application. While the foregoing describes the embodiments of the present application, it should be understood that the present application is not limited to the embodiments, and that various modifications and changes can be made by those skilled in the art without any inventive effort.
Claims (8)
1. A method for preparing a chitin oligosaccharide vaccine for preventing fungal infection, comprising:
coupling the oligosaccharide mixture containing aldehyde groups with an amino-containing carrier protein to form an oligosaccharide mixture conjugate vaccine;
the coupling comprises condensation of aldehyde groups on the oligosaccharide mixture and amino groups on the carrier protein and treatment with a reducing agent;
the coupling mole ratio of the chitin oligosaccharide and the carrier protein is up to 500: more than 1;
the standing reaction time after adding the reducing agent is 1-7 days;
the oligosaccharide is a chitin oligosaccharide mixture with different polymerization degrees, the relative molecular mass is less than or equal to 5000Da, and the deacetylation degree is less than or equal to 15%;
the carrier protein is non-human protein, and the non-human protein is one of Concholepasconcholepas hemocyanin, keyhole limpet hemocyanin, bovine serum albumin, tetanus toxin/toxoid, rotavirus VP7 protein, diphtheria toxin mutant CRM and clostridium perfringens exotoxin/toxoid.
2. The method for preparing a chitin oligosaccharide vaccine for preventing fungal infection according to claim 1, wherein the reducing agent is lithium aluminum hydride, potassium borohydride, sodium cyanoborohydride, na 2 SO 3 、FeSO 4 One of them.
3. The method for preparing a chitin oligosaccharide vaccine for preventing fungal infection according to claim 2, wherein the concentration of the reducing agent is 1-100 mg/ml.
4. The method for preparing a chitooligosaccharide vaccine for preventing fungal infection according to claim 1, further comprising: removing small molecules of impurities and freeze-drying; the method for removing the small molecules of the impurities is dialysis or column chromatography.
5. The method for preparing a chitin oligosaccharide vaccine for preventing fungal infection according to claim 1, wherein the concentration of carrier protein is 1-100mg/ml;
or, the molar concentration of the chitin oligosaccharide is 100-1200 times of the molar amount of the carrier protein.
6. A chitin oligosaccharide vaccine for preventing fungal infection prepared by the method of any one of claims 1-5.
7. Use of the chitooligosaccharide vaccine of claim 6 for preventing fungal infection in the manufacture of a medicament for preventing systemic fungal infection.
8. Use of the chitooligosaccharide vaccine of claim 7 for the prevention of fungal infection in the manufacture of a medicament for activating Th17 cell immunity.
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| PCT/CN2021/000154 WO2022226672A1 (en) | 2021-04-30 | 2021-07-13 | Chitin oligosaccharide vaccine for preventing fungal infection and preparation method therefor |
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