CN111346057B - Aerosolisable plague F1 dry powder inhalant - Google Patents
Aerosolisable plague F1 dry powder inhalant Download PDFInfo
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Abstract
The invention discloses an aerosolizable plague F1 dry powder inhaler. The invention discloses a dry powder preparation, which comprises the following raw materials in parts by weight: f1 protein 0.08-0.12 g: CpG 0.08-0.12 g: 0.8-1.2g of mannitol: inositol 0.8-1.2 g: leucine 0.4-0.6 g: poloxamer 0.04-0.06 ml. The invention also provides a preparation method of the dry powder preparation, which comprises the following steps: carrying out spray freeze drying on the solution SY3 to obtain a dry powder preparation; the solution SY3 contains F1 protein 0.08-0.12g/100ml, CpG 0.08-0.12g/100ml, mannitol 0.8-1.2g/100ml, inositol 0.8-1.2g/100ml, leucine 0.4-0.6g/100ml, and poloxamer 0.04-0.06%. The dry powder preparation prepared by the invention has good stability, can be stored at normal temperature, does not depend on cold chain transportation, saves transportation cost compared with liquid vaccines, can resist plague bacteria from invading the spleen and lung of mice, and has immune protection effect on the mice.
Description
Technical Field
The invention relates to an aerosolizable plague F1 dry powder inhaler.
Background
According to the data provided by the world health organization, the epidemic situation of the world plague is on the rise, and the world health organization lists the plague as a new epidemic disease in 2000. Because Yersinia pestis can infect human body by aerosol approach, and plague as a kind of virulent infectious disease has the characteristics of fast transmission, high fatality rate and the like, Yersinia pestis has been listed as standard biological warfare agent and bioterrorism agent by the US army. The effective vaccine is the most effective mode for blocking disease transmission, and has great significance for better guaranteeing the life safety of people and strengthening the diagnosis, prevention and treatment of plague. Among them, the development of a safe and effective vaccine is the focus and direction of plague control.
The F1 antigen is encoded by the F1 operon on the pMT1 plasmid of Yersinia pestis, and forms a capsule on the cell surface in a polymer form, and the F1 antibody plays an immune protective role by blocking the anti-phagocytosis of the F1 protein. In order to effectively prevent the pulmonary plague, the vaccine is generally considered to be more effective only by immunization through a mucosal route, but the effect of simply inoculating the vaccine on a mucosal surface is not ideal.
At present, no effective lung inhalation immune vaccine aiming at plague exists at home and abroad.
Disclosure of Invention
The invention aims to provide an aerosolisable plague F1 dry powder inhalant.
The invention provides a protective dry powder preparation (dry powder preparation A), which comprises the following raw materials: f1 protein, CpG, mannitol, inositol, leucine, and poloxamer.
The invention also discloses a dry powder preparation (dry powder preparation B), which comprises the following raw materials in parts by weight:
f1 protein 0.08-0.12 g: CpG 0.08-0.12 g: 0.8-1.2g of mannitol: inositol 0.8-1.2 g: leucine 0.4-0.6 g: poloxamer 0.04-0.06 ml.
The invention also discloses a dry powder preparation (dry powder preparation C), which comprises the following raw materials in percentage by weight:
f1 protein 0.1 g: CpG 0.1 g: 1g of mannitol: 1g of inositol: leucine 0.5 g: poloxamer 0.05 ml.
The invention also provides a preparation method (method A) of the dry powder preparation, which comprises the following steps:
carrying out spray freeze drying on the solution SY3 to obtain a dry powder preparation;
solution SY3 contains F1 protein, CpG, mannitol, inositol, leucine and poloxamer.
The invention also provides a preparation method (method B) of the dry powder preparation, which comprises the following steps:
carrying out spray freeze drying on the solution SY3 to obtain a dry powder preparation;
the solution SY3 contains F1 protein 0.08-0.12g/100ml, CpG 0.08-0.12g/100ml, mannitol 0.8-1.2g/100ml, inositol 0.8-1.2g/100ml, leucine 0.4-0.6g/100ml, and poloxamer 0.04-0.06% (volume percentage content).
The invention also provides a preparation method (method C) of the dry powder preparation, which comprises the following steps:
carrying out spray freeze drying on the solution SY3 to obtain a dry powder preparation;
the SY3 solution contains F1 protein 0.1g/100ml, CpG 0.1g/100ml, mannitol 1g/100ml, inositol 1g/100ml, leucine 0.5g/100ml, and poloxamer 0.05% (by volume).
The rest of the SY3 solution is deionized water.
In any one of the solutions SY3, the balance is deionized water for inactivating nuclease.
The pH value of any one of the solutions SY3 is 7.2.
Any of the above solutions SY3 was adjusted to pH 7.2 with 1M sodium hydroxide solution.
The spray freeze-drying method can be specifically as follows: the solution SY3 after ice-bath precooling for 2 hours is directly sprayed into a cold medium (such as liquid nitrogen) through an inlet of a two-fluid nozzle, and the ice crystals are subjected to vacuum freeze drying.
The spray freeze-drying method can be specifically as follows: precooling the solution SY3 for 2 hours in an ice bath, then transferring 20ml into an injector, connecting the outlet of the injector with the inlet of a two-fluid nozzle, directly spraying the solution into liquid nitrogen (3/4 volume of liquid nitrogen is filled in a stainless steel basin and placed on a magnetic stirrer for stirring, if necessary, adding the liquid nitrogen in the middle of spraying, wherein the distance between the nozzle and the liquid level of the liquid nitrogen is approximately 10cm, the air pressure of an air pump is set to be 0.15 MPa), and rapidly freezing the sprayed atomized liquid drops into ice crystals under the action of the liquid nitrogen; and transferring the ice crystals and a small amount of residual liquid nitrogen into a stainless steel cup, sealing a layer of gauze at the opening, and drying in a vacuum freeze dryer for over 36 hours.
The invention also protects the dry powder preparation prepared by any one of the methods.
The invention also protects the application of any one of the dry powder preparations in preparing plague vaccines.
The invention also provides a plague vaccine, the active ingredient of which is any one of the dry powder preparations.
Any of the vaccines described above may be a therapeutic vaccine or a prophylactic vaccine.
Any one of the dry powder preparations is an aerosolizable plague F1 dry powder inhaler.
The dry powder preparation prepared by the invention has the median particle size (aerodynamics) of 2.72-2.88 mu m, spherical particles, loose and porous shape and uniform particle size distribution. The dry powder preparation is beneficial to the deposition of the vaccine in the deep part of the lung, can prolong the contact reaction time of the antigen and the mucosal tissue, and is beneficial to improving the immune protection effect.
The dry powder preparation prepared by the invention does not reduce the protection effect of the F1 protein, and has obvious protection effect on animals attacked by Yersinia pestis. The dry powder preparation prepared by the invention is used for immunizing animals, the antibody titer generation duration is up to three months, and the mucosal immune response of mice is successfully stimulated. The dry powder preparation prepared by the invention has good local adhesion of the lung, thereby delaying the clearance speed of the mucous membrane to the dry powder preparation and increasing the antigen presentation time. The dry powder preparation prepared by the invention has good stability, can be stored at normal temperature, does not depend on cold chain transportation, saves the transportation cost compared with a liquid vaccine, and can keep better activity. The dry powder preparation prepared by the invention can resist the plague bacteria from invading the spleen and lung of the mouse and has immune protection effect on the mouse.
The invention provides support for the research and development feasibility of the plague dry powder vaccine. The invention has great application value for preventing and controlling plague.
Drawings
Fig. 1 is an absorption curve of dry powder formulation SY 3.
Fig. 2 is an electron microscope image of dry powder preparation SY2 and dry powder preparation SY 3.
FIG. 3 is a survival curve for animal experiments.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
6-8 week old SPF grade BALB/c female mice: beijing Wittiulihua laboratory animal technology Co.
The F1 protein is shown as a sequence 1 in a sequence table. F1 protein dry powder is F1 protein in dry powder state.
CpG, which is called Class B CpG oligonucleotide, has immune activation function, is an immunostimulant for human or mouse TLR9, and is a typical mucosal immune adjuvant. The CpG used in the examples is a product with catalog number tlrl-2006 from Sigma, which has the website links: https:// www.invivogen.com/odn 2006.
Mannitol: sigma, cat # M1902. Inositol: sigma, cat # I7508. Leucine (L-leucine): sigma, cat # L8912. Poloxamers (Pluronic F-68 at a concentration of 10g/100 mL)
F-68 solution): sigma, cat # P5556.
HRP-labeled goat anti-mouse IgG, HRP-labeled goat anti-mouse IgG1, HRP-labeled goat anti-mouse Ig2a, HRP-labeled goat anti-mouse IgA, and HRP-labeled goat anti-mouse IgM are all products of abcam.
Yersinia pestis 91001 is collectively referred to as yersinia pestis brucella abortus origin strain 91001, and is described in the following documents: sequence determination and preliminary analysis of the entire genome of Yersinia pestis strain 91001 in the Brucella pestis of Song Asia army 2003, doctor's academic thesis.
A handheld liquid aerosol lung delivery device: lung liquid quantitative atomizer of beijing huilong and technologies ltd. A handheld dry powder aerosol lung delivery device: lung dry powder quantitative atomizer of Beijing Huilong and science and technology Limited.
Scanning electron microscope: S-3400N, Hitachi, Japan. Small aerosol settlement evaluating cabin: beijing Huilong and science and technology Co. Laser particle size analyzer TSI 3321: APS-3321, TSI Inc., USA.
EXAMPLE 1 preparation of Dry powder formulation
Preparation of dry powder preparation SY1
1. Preparation of the solution
A solution SY1, a solution SY2 and a solution SY3 are prepared respectively. The pH of all three solutions was adjusted to 7.2 with 1M sodium hydroxide solution. CpG is an immunological adjuvant. Mannitol is used as bulking agent. Inositol is a freeze-drying protective agent. Leucine and poloxamer are used as dispersing agents. Deionized water used for preparing the solution is deionized water for inactivating nuclease.
The SY1 solution contains trypsinogen 0.05g/100ml, mannitol 1g/100ml, inositol 1g/100ml, leucine 0.5g/100ml, poloxamer 0.05% (volume percentage), and deionized water in balance. Filtering and sterilizing, and preparing for use.
The SY2 solution contains CpG 0.1g/100ml, mannitol 1g/100ml, inositol 1g/100ml, leucine 0.5g/100ml, poloxamer 0.05% (volume percentage), and deionized water in balance. Filtering and sterilizing, and preparing for use.
The SY3 solution contains F1 dry protein powder 0.1g/100ml, CpG 0.1g/100ml, mannitol 1g/100ml, inositol 1g/100ml, leucine 0.5g/100ml, poloxamer 0.05% (volume percentage), and deionized water in balance. Filtering and sterilizing, and preparing for use. The amount of dry matter contained in SY3 per 100ml of solution is equal to about 2.75 g.
2. Preparation of Dry powder
The vacuum freeze-drying machine is Bo Yi kang FD-1B-50 vacuum freeze-drying machine (http:// www.biocool.com.cn/mengbing-Products-30451529 /).
Precooling the sample liquid for 2 hours in an ice bath, then transferring 20ml into an injector, connecting the outlet of the injector with the inlet of a two-fluid spray head, directly spraying the sample liquid into liquid nitrogen (liquid nitrogen with the volume of 3/4 is filled in a stainless steel basin and placed on a magnetic stirrer for stirring, if necessary, adding the liquid nitrogen in the middle of spraying, enabling the spray head to be approximately 10cm away from the liquid level of the liquid nitrogen, setting the air pressure of an air pump to be 0.15 MPa), spraying atomized liquid drops, and rapidly freezing the atomized liquid drops into ice crystals under the action of the liquid nitrogen. And transferring the ice crystals and a small amount of residual liquid nitrogen into a stainless steel cup, sealing a layer of gauze at the opening, and drying in a vacuum freeze dryer for 36 hours (in actual operation, more than 36 hours can be used). The dry powder was collected into 5ml vials with rubber stoppers and stored sealed at-20 ℃.
The obtained dry powder is white powder by using the solution SY1 as a sample solution, and is named as dry powder preparation SY1, which is also called trypsinogen dry powder inhalant.
The obtained dry powder is white powder by using the solution SY2 as a sample solution, and is named as dry powder preparation SY2, which is also called CpG dry powder inhalant.
The solution SY3 is used as a sample liquid, and the obtained dry powder is white powder, named as dry powder preparation SY3, also named as F1 vaccine dry powder inhalant. 20ml of sample liquid is prepared to obtain 158mg-168mg of dry powder preparation SY 3. Yield ═ yield of dry powder formulation SY3 ÷ amount of dry matter contained in 20ml of sample liquid × 100%. The amount of dry matter contained in 20ml of the sample solution was 2.75 g/0.2 mg/550 mg. Therefore, the yield of the dry powder preparation SY3 prepared by using the solution SY3 as a sample liquid is 28.73-30.55%.
Second, analysis of pharmaceutical Properties
1. Moisture absorption curve
The constant temperature and humidity chamber is set at 60% RH/25 deg.C, the electronic balance is placed therein, zero setting is performed, the dry powder preparation SY3 is placed on the balance, the dry powder weight is recorded every 30min for a total time of 6 h. The percentage (%) of weight increase relative to 0 time point at each time point was calculated as the water uptake capacity, and a moisture absorption curve was plotted.
The results are shown in FIG. 1. The moisture absorption capacity of the dry powder preparation SY3 is between 20-25% at 25 deg.C and 60% RH, and reaches equilibrium about 30 min.
2. Particle morphology and particle size distribution
The dry powder preparation SY2 and the dry powder preparation SY3 are respectively observed in particle morphology through a plurality of visual fields by a scanning electron microscope, and the single particle size distribution (median and mean) is estimated.
An electron micrograph of dry powder formulation SY2 is shown in fig. 2A. The median of the single particle sizes was 10.44 μm, and the mean was 11.55. mu.m. The dry powder particles are spherical, loose and porous.
An electron micrograph of the dry powder formulation SY3 is shown in fig. 2B. The median of the single particle sizes was 10.58 μm, and the mean was 11.58. mu.m. The dry powder particles are spherical, loose and porous.
3. Aerodynamic property analysis
Opening a small aerosol settlement evaluating cabin (the diameter of the bottom in the cabin is 0.76m, the height is 0.73m), and adjusting to a circulating air mode.
Secondly, starting a mass concentration detector TSI 8530, monitoring the mass concentration of the aerosol particles in the cabin in real time, and sampling the flow at 3L/min until the mass concentration of the aerosol particles in the cabin is reduced to 0.005mg/m3。
And thirdly, closing the circulating air in the cabin and opening a low-power fan arranged at the bottom in the cabin.
Opening a sampling port of the evaluation cabin, and generating dry powder aerosol in the test preparation into the cabin by using the handheld dry powder aerosol lung delivery device, wherein the generation amount is 5 mg/time and only 1 time.
Fifthly, after the dry powder aerosol is generated, uniformly mixing for 30S by a fan, and closing the fan.
Sixthly, a laser particle size analyzer TSI3321 is used, a Huilong and production diluter is connected to the front section of the sampling port, the sampling flow is 5L/min, and the sampling time is 5 min. The test index is the aerodynamic number median diameter (median particle diameter) of the total particles.
The test preparation is dry powder preparation SY1, dry powder preparation SY2 or dry powder preparation SY 3.
The dry powder preparation SY1 has a median particle size of 2.29-2.52 μm. The median particle size of the dry powder preparation SY2 was 3.09. mu.m. The dry powder preparation SY3 has a median particle size of 2.72-2.88 μm.
Example 2 Effect test of Dry powder preparation
Preparation of plague bacillus liquid
1. Activation of
Yersinia pestis 91001 preserved at-80 deg.C is thawed, then 20 μ l is pipetted and inoculated into 20ml of liquid BHI medium, and cultured at 26 deg.C under shaking at 200rpm for 36 h.
2. Preculture
Finish the stepAfter the step 1, 1ml of the bacterial liquid is taken and inoculated into 19ml of liquid BHI culture medium, and the liquid BHI culture medium is subjected to shaking culture at 26 ℃ and 200rpm until the system OD is reached600nmThe value was 1.0.
3. Formal culture
After the step 2 is completed, 200 mul of bacterial liquid is taken and inoculated to 20ml of liquid BHI culture medium, and the bacterial liquid is subjected to shaking culture at 26 ℃ and 200rpm until the system OD is reached600nmThe value was 1.0, and the cells were cultured at 37 ℃ for 3 hours with shaking at 200 rpm.
4. After the step 3 is finished, centrifuging for 10min at 3000g, collecting the thalli, and resuspending the thalli by using physiological saline containing 0.05% (volume ratio) poloxamer to obtain bacterial liquid.
5. And (4) taking the bacterial liquid obtained in the step (4), diluting the bacterial liquid by 5 times by using physiological saline containing 0.05 percent (volume ratio) of poloxamer, then inoculating the diluted bacterial liquid to a BHI culture medium plate, carrying out inverted culture at 26 ℃ for 3 days, and then counting the number of colonies.
6. According to the result of the step 5, the bacterial liquid for counteracting the toxin is prepared by using the physiological saline containing 0.05 percent (volume ratio) of poloxamer and the bacterial liquid obtained in the step 4.
Second, animal test
SPF-grade BALB/c female mice 6-8 weeks old were divided into four groups of 40 mice each.
The test process comprises the following steps: the first immunization was performed on day 1 of the experiment, the second immunization was performed on day 22 of the experiment, the third immunization was performed on day 43 of the experiment, and the challenge was performed on day 64 of the experiment.
Grouping condition: group one (F1 dry powder group), 0.5mg of dry powder formulation SY3 per mouse immunized at a time; a second group ((F1 liquid group)), each mouse being immunized with 50. mu.l of solution (containing 0.5mg of SY3 as dry powder formulation in a physiological saline solution containing 0.05% poloxamer); a third group (CpG dry powder group), 0.5mg of dry powder preparation SY2 is immunized for each mouse; in the fourth group (CpG liquid group), each mouse was immunized with 50. mu.l of solution (containing 0.5mg of SY2 as dry powder formulation in a physiological saline solution containing 0.05% poloxamer).
The dry powder formulation is administered using a hand-held dry powder aerosol lung delivery device.
The solution is administered using a handheld liquid aerosol lung delivery device.
Counteracting toxic substances: and (3) administering the challenge bacteria solution prepared in the step one by adopting a handheld liquid aerosol lung delivery device, wherein the challenge dose of each mouse is 2000 CFU.
1. Survival curve drawing
After challenge, 10 mice are taken from each group, and the number of dead mice is recorded at the time points of 0d, 1d, 2d, 2.5d, 3d, 3.5d, 4d, 5d, 6d, 7d, 8d, 9d, 10d, 11d, 12d, 13d and 14d after challenge, and a survival curve is drawn.
The results are shown in FIG. 3. After 14 days of challenge, the survival rates of mice in both the F1 dry powder group and the F1 liquid group were 90%. After 4 days of challenge, all CpG fluid group mice died. After 6 days of challenge, all mice in the CpG dry powder group died.
2. Observation of clinical symptoms
CpG dry powder group/CpG liquid group: the mice all have shrugging phenomenon in the second day after the attack of toxin, the mice with serious symptoms in the third day have the symptoms of secretion in canthus, slow response after external stimulation and the like, and then the symptoms of the mice gradually get worse, so that forced abdominal respiration is seen.
F1 dry powder group/F1 liquid group: mice show only slight shrugging after challenge.
3. Specific antibody detection
At time point 1 (2 days before the second immunization), time point 2 (2 days before the third immunization), time point 3 (2 days before challenge), and time point 4 (two weeks after challenge), mouse serum and lung homogenate were taken, respectively, and titers of 2 specific antibodies (IgG, IgA) were detected. At time point 4, no detection was performed if all mice in the group died.
The lung homogenate was obtained by homogenizing lung with 800. mu.l of physiological saline.
Detecting specific antibody by enzyme linked immunosorbent assay. The coating is F1 dry protein powder with the coating concentration of 0.2 mug/ml. The primary antibody was a gradient dilution of serum or lung homogenate (gradient dilution with sterile saline). The secondary antibody is goat anti-mouse IgG marked by HRP or goat anti-mouse IgA marked by HRP. The color development liquid is TMB color development liquid.
CpG dry powder group/CpG liquid group: at each time point, the 2 antibody titers in serum and lung homogenates were zero.
F1 dry powder group/F1 liquid group: the IgA titer in serum and lung homogenates continued to increase at time 1, time 2, with time 3 reaching a maximum (time 3, dry F1 powder group, serum IgA titer 1920, lung homogenate IgA titer 1920; time 3, F1 liquid group, serum IgA titer 23360, lung homogenate IgA titer 7360), and slightly decreased at time 4.
F1 dry powder group/F1 liquid group: IgG titers in serum and lung homogenates continued to increase at time 1, time 2, with time 3 reaching the highest (time 3, F1 dry powder group, serum IgG titer 307200, lung homogenate IgG titer 17920; time 3, F1 liquid group, serum IgG titer 665600, lung homogenate IgG titer 33280), and slightly decreased at time 4.
4. Detection of amount of bacteria carried
At time 1 (2 days before primary immunization), time 2 (2 days before challenge), time 3 (2 days after challenge), and time 4 (2 weeks after challenge), spleen homogenate and lung homogenate of mice were each taken, and gradient-diluted with sterile physiological saline, and then the amount of the carrier was measured by plate culture (BHI medium plate, 26 ℃, 3 days). At time point 4, no test was performed if all mice in the group died (indicated with a-in table 1).
The lung homogenate was obtained by homogenizing lung with 800. mu.l of physiological saline.
Spleen homogenate was obtained by homogenizing spleen with 800. mu.l of physiological saline.
The bacterial load in the spleen and lungs of each group of mice was 0 at time point 1 and time point 2.
The results of splenic and pulmonary bacterial loads for each group of mice at time points 3 and 4 are shown in table 1.
TABLE 1
2 days after the challenge, a large amount of yersinia pestis was detected in the spleens of the mice in the CpG dry powder group and the CpG liquid group, yersinia pestis was not detected in the spleens of the mice in the F1 dry powder group, and a small amount of yersinia pestis was detected in the spleens of the mice in the F1 liquid group.
A large amount of Yersinia pestis is detected in the lungs of the mice in the CpG dry powder group and the CpG liquid group 2 days after the challenge, and the Yersinia pestis number in the lungs of the mice in the F1 dry powder group and the F1 liquid group is extremely obviously lower than that in the CpG dry powder group and the CpG liquid group.
SEQUENCE LISTING
<110> military medical research institute of military science institute of people's liberation force of China
<120> an aerosolizable plague F1 dry powder inhaler
<130> GNCYX182416
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 149
<212> PRT
<213> Artificial sequence
<400> 1
Ala Asp Leu Thr Ala Ser Thr Thr Ala Thr Ala Thr Leu Val Glu Pro
1 5 10 15
Ala Arg Ile Thr Leu Thr Tyr Lys Glu Gly Ala Pro Ile Thr Ile Met
20 25 30
Asp Asn Gly Asn Ile Asp Thr Glu Leu Leu Val Gly Thr Leu Thr Leu
35 40 45
Gly Gly Tyr Lys Thr Gly Thr Thr Ser Thr Ser Val Asn Phe Thr Asp
50 55 60
Ala Ala Gly Asp Pro Met Tyr Leu Thr Phe Thr Ser Gln Asp Gly Asn
65 70 75 80
Asn His Gln Phe Thr Thr Lys Val Ile Gly Lys Asp Ser Arg Asp Phe
85 90 95
Asp Ile Ser Pro Lys Val Asn Gly Glu Asn Leu Val Gly Asp Asp Val
100 105 110
Val Leu Ala Thr Gly Ser Gln Asp Phe Phe Val Arg Ser Ile Gly Ser
115 120 125
Lys Gly Gly Lys Leu Ala Ala Gly Lys Tyr Thr Asp Ala Val Thr Val
130 135 140
Thr Val Ser Asn Gln
145
Claims (7)
1. A dry powder preparation comprises the following raw materials in parts by weight:
f1 protein 0.08-0.12 g: CpG 0.08-0.12 g: 0.8-1.2g of mannitol: inositol 0.8-1.2 g: leucine 0.4-0.6 g: poloxamer 0.04-0.06 ml.
2. A dry powder preparation comprises the following raw materials in parts by weight:
f1 protein 0.1 g: CpG 0.1 g: 1g of mannitol: 1g of inositol: leucine 0.5 g: poloxamer 0.05 ml.
3. A method for preparing a dry powder formulation comprising the steps of:
carrying out spray freeze drying on the solution SY3 to obtain a dry powder preparation;
the solution SY3 contains F1 protein 0.08-0.12g/100ml, CpG 0.08-0.12g/100ml, mannitol 0.8-1.2g/100ml, inositol 0.8-1.2g/100ml, leucine 0.4-0.6g/100ml, and poloxamer 0.04-0.06%.
4. A method for preparing a dry powder formulation comprising the steps of:
carrying out spray freeze drying on the solution SY3 to obtain a dry powder preparation;
the SY3 solution contains F1 protein 0.1g/100ml, CpG 0.1g/100ml, mannitol 1g/100ml, inositol 1g/100ml, leucine 0.5g/100ml, and poloxamer 0.05%.
5. A dry powder formulation prepared by the method of claim 3 or 4.
6. Use of the dry powder formulation of claim 1 or 2 or 5 in the preparation of a plague vaccine.
7. A plague vaccine, the active ingredient of which is the dry powder formulation of claim 1 or 2 or 5.
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