CN108721616B - A kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines and preparation method thereof - Google Patents
A kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines and preparation method thereof.Avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines provided by the invention; it is formed by connecting with diphtheria toxoid carrier protein by nanosphere by avian pasteurella multocida capsular polysaccharide; using pasteurella multocida capsular polysaccharide in conjunction with carrier protein diphtheria toxoid; vaccine immunity effect is enhanced, and plays the role of cross protection to the infection of homologous strain and heterologous strain.The preparation method of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines provided by the invention, easy to operate, save the cost has a good application prospect.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines and
Preparation method.
Background technique
Pasteurella multocida abbreviation Pasteurella is a kind of encapsulated Gram-negative brevibacterium, can infect house
The many animals such as poultry, poultry and wild fowl.Clinical research shows that avian cholera is mainly by serotypes A: 1, A: 3 or A: 4 Pasteurella
A kind of caused contagious disease causes serious economic loss to aviculture.
Chinese patent application CN105288607A discloses a kind of preparation method of avian pasteurella multocida inactivated vaccine, the party
The avian pasteurella multocida inactivated vaccine immunizing rabbit of method preparation, plays the role of good immunizing rabbit, still, was using
Cheng Zhong, the action time of this inactivated vaccine in vivo is shorter, needs repeatedly to be inoculated with, take time and effort to install.
Chinese patent CN102139116B discloses a kind of preparation method of envelope protein A gene vaccine for fowl cholera, the party
For the vaccine of method preparation by introducing eukaryotic vector, the ability with efficient high-speed transfection enhances immunocompetence.But the preparation side
Method is complicated for operation, needs strict operating process, and preparation cost is higher.
Capsular polysaccharide is main virulence factor, is colloidal state or mucous substance that bacterium generates in certain environment,
The substance is mostly polysaccharide, polypeptide or polysaccharide protein complex etc., and accumulation forms stable, thicker fine and close guarantor outside cell wall
Sheath.A large amount of scholars studied and found that pasteurella multocida can be divided into five kinds of pod membrane serum of A, B, D, E and F the 1990s
Group.Research finds more stronger than acapsular correlation variation Strain Virulence containing encapsulated pasteurella multocida separation strains, it was demonstrated that
Pod membrane plays an important role to the pathogenic of pasteurella multocida.Research shows that A type without pod membrane mutant strain to test chicken without
It is pathogenic, and the mutant strain cannot be proliferated on chicken muscle cell, however the acapsular mutant strain of high dose makees vaccine
It but can the relevant immunity protection of irritant test chicken generation.Show that pod membrane is reasonable effective Pasteurella candidate subunit epidemic disease
Seedling.
Currently, preventing avian cholera with inactivated vaccine and live bacterial vaccines, but inactivated vaccine only has the infection of homologous strain
There is protective effect, and live bacterial vaccines have certain cross-protection to the infection of homologous strain and heterologous strain, therefore have
Have wide practical use.
Summary of the invention
Against the above technical problems, the object of the present invention is to provide a kind of avian pasteurella multocida capsular polysaccharide-protein binding epidemic diseases
Seedling and preparation method thereof.Avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines provided by the invention use pasteurella multocida
Capsular polysaccharide, and avian cholera is prevented using attenuated live vaccine, vaccine virulence is enhanced, and to homologous strain and heterologous strain
Infection play the role of cross protection.The preparation of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines provided by the invention
Method, easy to operate, save the cost.
Technical scheme is as follows:
The present invention provides a kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines, by avian pasteurella multocida capsular polysaccharide
It is formed by connecting with diphtheria toxoid carrier protein by nanosphere.
Further, the nanosphere is prepared by bioabsorbable polymer material.
Further, the bioabsorbable polymer material is chitosan, polyethylene glycol, PLA-PEG copolymer, sea
One or more of mosanom.
Further, the avian pasteurella multocida capsular polysaccharide, nanosphere and diphtheria toxoid carrier protein weight ratio
For 6-25:7-30:10.
Further, the avian pasteurella multocida capsular polysaccharide is extracted from Pasteurella obtains.
The present invention also provides a kind of preparation method of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines, including it is following
Step:
S1, to weigh weight be that bioabsorbable polymer material used in 1.2-1.5 times of capsular polysaccharide of preparation nanosphere is dissolved in
In the methylene chloride of 3-5 times of weight, then it is added in the polyvinyl alcohol water solution for being 1-10% containing mass concentration, stirs molten
Solution, obtains the mixed liquor of the final concentration of 0.1-5mg/mL of bioabsorbable polymer material；
S2, by mixed liquor made from step S1 pressure be 20-50MPa condition it is 5-8 times emulsified, each 2-3min, so
10-30min is stirred with 1000-3000 revs/min of speed afterwards, obtains emulsion, the organic solvent being evaporated in emulsion obtains glue
Liquid solution；
S3, the colloidal solution made from centrifuge centrifugation step S2, revolving speed 8000-12000r/min, obtaining diameter is
The nanosphere of 50-150nm washes nanosphere, is placed in freeze dryer and preservation is lyophilized；
S4, membrane filter method and ammonium sulfate precipitation method separating-purifying avian pasteurella multocida capsular polysaccharide are utilized；
S5, by nanosphere made from avian pasteurella multocida capsular polysaccharide made from step S4 and step S3 with weight ratio 1:
The ratio of 1.2-1.5 is coupled, and obtains polysaccharide nanosphere couplet, is then carried out aldehyde glycosylation reaction and is obtained the more of surface aldehydes
Sugared nanosphere couplet；
S6, the formalin for being 0.3%-0.4% with mass fraction carry out detoxification treatment preparation to diphtheria toxin and divide
Diphtheria toxoid carrier protein is obtained from purification；
S7, by diphtheria toxoid made from surface aldehydes polysaccharide nanosphere couplet made from step S5 and step S6
Carrier protein combine to get.
Further, aldehyde glycosylation reaction concrete operations in the step S5 are as follows:
In phosphate buffer, temperature is 30-37 DEG C, and under conditions of pH=5-7, polysaccharide nanosphere couplet is passed through
The glutaraldehyde water solution that mass fraction is 25% is modified, obtains the polysaccharide nanosphere couplet of surface aldehydes.
Further, avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines provided by the invention can be with any existing
Approach is immunized, including the forms such as skin corium or percutaneous drug delivery, intramuscular delivery.Wherein, amount to be administered is art technology
Personnel are confirmable according to common sense.
Compared with prior art, present invention has the advantage that
(1) avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines provided by the invention use pasteurella multocida pod membrane
Polysaccharide, and avian cholera is prevented using attenuated live vaccine, enhance vaccine virulence, and the sense to homologous strain and heterologous strain
Dye plays the role of cross protection.
(2) preparation method of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines provided by the invention, easy to operate, section
About cost.
Specific embodiment
The present invention is made further to illustrate in detail, completely below with reference to embodiment.Reagent used below or equipment are
Commercial product operates to specifications unless otherwise specified, and this will not be repeated here.
Embodiment 1, a kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines
Avian pasteurella multocida capsular polysaccharide-the protein conjugate vaccines are carried by avian pasteurella multocida capsular polysaccharide and diphtheria toxoid
Body protein is formed by connecting by nanosphere.The avian pasteurella multocida capsular polysaccharide, nanosphere and diphtheria toxoid carrier egg
White mass ratio is 15:18:25.
The preparation method is as follows:
S1, to weigh weight be that bioabsorbable polymer material used in 1.2 times of capsular polysaccharide of preparation nanosphere is dissolved in 3 times of weights
In the methylene chloride of amount, then it is added in the polyvinyl alcohol water solution for being 1% containing mass concentration, stirring and dissolving obtains biology
The mixed liquor of the final concentration of 0.1mg/mL of high molecular material；
S2, by mixed liquor made from step S1 pressure be 20MPa condition emulsified 5 times, each 2min, then with 1000
Rev/min speed stirs 30min, obtains emulsion, the organic solvent being evaporated in emulsion obtains colloidal solution；
S3, the colloidal solution made from centrifuge centrifugation step S2, revolving speed 8000r/min, obtaining diameter is 150nm's
Nanosphere washes nanosphere, is placed in freeze dryer and preservation is lyophilized；
S4, membrane filter method and ammonium sulfate precipitation method separating-purifying avian pasteurella multocida capsular polysaccharide are utilized；
S5, by nanosphere made from avian pasteurella multocida capsular polysaccharide made from step S4 and step S3 with weight ratio 1:
1.2 ratio coupling, obtains polysaccharide nanosphere couplet, and then in phosphate buffer, temperature is 30 DEG C, the item of pH=5
Under part, polysaccharide nanosphere couplet is modified by the glutaraldehyde water solution that mass fraction is 25%, obtains surface aldehydes
Polysaccharide nanosphere couplet；
S6, the formalin for being 0.3% with mass fraction carry out detoxification treatment preparation and separating-purifying to diphtheria toxin
Obtain diphtheria toxoid carrier protein；
S7, by diphtheria toxoid made from surface aldehydes polysaccharide nanosphere couplet made from step S5 and step S6
Carrier protein combine to get.
Embodiment 2, a kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines
Avian pasteurella multocida capsular polysaccharide-the protein conjugate vaccines are carried by avian pasteurella multocida capsular polysaccharide and diphtheria toxoid
Body protein is formed by connecting by nanosphere.
The mass ratio of the avian pasteurella multocida capsular polysaccharide, nanosphere and diphtheria toxoid carrier protein is 10:15:4.
The preparation method is as follows:
S1, to weigh weight be that bioabsorbable polymer material used in 1.5 times of capsular polysaccharide of preparation nanosphere is dissolved in 3-5 times
In the methylene chloride of weight, then it is added in the polyvinyl alcohol water solution for being 10% containing mass concentration, stirring and dissolving is given birth to
The mixed liquor of the final concentration of 5mg/mL of object high molecular material；
S2, by mixed liquor made from step S1 pressure be 50MPa condition emulsified 8 times, each 3min, then with 3000
Rev/min speed stirs 10min, obtains emulsion, the organic solvent being evaporated in emulsion obtains colloidal solution；
S3, the colloidal solution made from centrifuge centrifugation step S2, revolving speed 12000r/min, obtaining diameter is 50-
The nanosphere of 150nm washes nanosphere, is placed in freeze dryer and preservation is lyophilized；
S4, membrane filter method and ammonium sulfate precipitation method separating-purifying avian pasteurella multocida capsular polysaccharide are utilized；
S5, by nanosphere made from avian pasteurella multocida capsular polysaccharide made from step S4 and step S3 with weight ratio 1:
The ratio of 1.2-1.5 is coupled, and obtains polysaccharide nanosphere couplet, and then in phosphate buffer, temperature is 37 DEG C, pH=7
Under conditions of, polysaccharide nanosphere couplet is modified by the glutaraldehyde water solution that mass fraction is 25%, obtains surface aldehyde
The polysaccharide nanosphere couplet of base；
S6, the formalin for being 0.3%-0.4% with mass fraction carry out detoxification treatment preparation to diphtheria toxin and divide
Diphtheria toxoid carrier protein is obtained from purification；
S7, by diphtheria toxoid made from surface aldehydes polysaccharide nanosphere couplet made from step S5 and step S6
Carrier protein combine to get.
Embodiment 3, a kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines
A kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines, the avian pasteurella multocida capsular polysaccharide-protein binding epidemic disease
Seedling is formed by connecting with diphtheria toxoid carrier protein by nanosphere by avian pasteurella multocida capsular polysaccharide.The avian pasteurella multocida
The mass ratio of capsular polysaccharide, nanosphere and diphtheria toxoid carrier protein is 15:21:10.
The preparation method is as follows:
S1, to weigh weight be that bioabsorbable polymer material used in 1.4 times of capsular polysaccharide of preparation nanosphere is dissolved in 4 times of weights
In the methylene chloride of amount, then it is added in the polyvinyl alcohol water solution for being 7% containing mass concentration, stirring and dissolving obtains biology
The mixed liquor of the final concentration of 3mg/mL of high molecular material；
S2, by mixed liquor made from step S1 pressure be 38MPa condition emulsified 7 times, each 3min, then with 2000
Rev/min speed stirs 15min, obtains emulsion, the organic solvent being evaporated in emulsion obtains colloidal solution；
S3, the colloidal solution made from centrifuge centrifugation step S2, revolving speed 11000r/min, obtaining diameter is 80nm's
Nanosphere washes nanosphere, is placed in freeze dryer and preservation is lyophilized；
S4, membrane filter method and ammonium sulfate precipitation method separating-purifying avian pasteurella multocida capsular polysaccharide are utilized；
S5, by nanosphere made from avian pasteurella multocida capsular polysaccharide made from step S4 and step S3 with weight ratio 1:
1.4 ratio coupling, obtains polysaccharide nanosphere couplet, and then in phosphate buffer, temperature is 32 DEG C, the item of pH=6
Under part, polysaccharide nanosphere couplet is modified by the glutaraldehyde water solution that mass fraction is 25%, obtains surface aldehydes
Polysaccharide nanosphere couplet；
S6, the formalin for being 0.3% with mass fraction carry out detoxification treatment preparation and separating-purifying to diphtheria toxin
Obtain diphtheria toxoid carrier protein；
S7, by diphtheria toxoid made from surface aldehydes polysaccharide nanosphere couplet made from step S5 and step S6
Carrier protein combine to get.
Comparative example 1, a kind of avian pasteurella multocida vaccine
(1) the production preparation of strain
Avian pasteurella multocida standard strain A type NCTC1502 is inoculated in Tryptose soy Solid agar culture plate,
Recovery is cultivated in 37 DEG C of insulating boxs；
(2) preparation of seedling bacterium solution
Picking colonies typical is inoculated in Tryptose soy meat soup fluid nutrient medium, cultivates under the conditions of 37 DEG C, 200rpm more
Killing property Pasteurella takes 5mL culture bacterium solution in the Tryptose soy meat soup liquid training in 1000mL limit iron hoop border to logarithmic growth phase
It supports in base, continues to cultivate 12h in 37 DEG C, 200rpm, culture bacterium in limit iron hoop border harvests bacterium solution as seedling when reaching plateau
Use bacterium solution；Bacterium solution culture needed for limit iron culture reaches 0.6 to logarithmic growth phase to OD630nm, limits iron in practice to determine to produce
Environmental bacteria culture logarithmic phase provides technical parameter, eliminates every batch of and does the complexity of count of bacteria, time-consuming process；Limit iron hoop border
Are as follows: the D (2,2-dipyridyl2,2 '-of final concentration of 220 μm of ol/L is added in common Tryptose soy meat soup fluid nutrient medium
Bipyridyl), the iron ion in culture environment can be effectively chelated, the iron deficiency ring for breeding its extreme in host close to pathogen
Border；Culture PM in limit iron hoop border reaches time plateau for 12h；
(3) inactivation of seedling bacterium solution
The bacterium solution that above-mentioned culture obtains is adjusted to the sodium hydroxide solution of 1mol/L to pH=7.2, formaldehyde is then added
Solution makes, and is inactivated so that formaldehyde final mass score reaches 0.4%, mixing is sufficiently stirred, inactivate 72h at 37 DEG C, point
It does not take each inactivated bacterial liquid to be inoculated on a small quantity on plate to be cultivated, carries out steriling test, the requirement of no bacterial growth should be reached；
(4) preparation of vaccine
Inactivation is examined to qualified bacterium solution concentration, is adjusted to its concentration more than or equal to 3.25 × 1010CFU/mL is white with sterilizing
Oil adjuvant is sufficiently mixed emulsification in the ratio of 1:1 volume ratio, and 0.01g thimerosal is added according to every milliliter of emulsifying mixt and mixes
It is even, it can be obtained target inactivated vaccine.
Comparative example 2, a kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines
A kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines, the avian pasteurella multocida capsular polysaccharide-protein binding epidemic disease
Seedling is formed by connecting with diphtheria toxoid carrier protein by nanosphere by avian pasteurella multocida capsular polysaccharide.The avian pasteurella multocida
The mass ratio of capsular polysaccharide, nanosphere and diphtheria toxoid carrier protein is 5:7:5.
The preparation method is as follows:
S1, to weigh weight be that bioabsorbable polymer material used in 1.4 times of capsular polysaccharide of preparation nanosphere is dissolved in 4 times of weights
In the methylene chloride of amount, then it is added in the polyvinyl alcohol water solution for being 7% containing mass concentration, stirring and dissolving obtains biology
The mixed liquor of the final concentration of 3mg/mL of high molecular material；
S2, by mixed liquor made from step S1 pressure be 38MPa condition emulsified 7 times, each 3min, then with 2000
Rev/min speed stirs 15min, obtains emulsion, the organic solvent being evaporated in emulsion obtains colloidal solution；
S3, the colloidal solution made from centrifuge centrifugation step S2, revolving speed 11000r/min, obtaining diameter is 80nm's
Nanosphere washes nanosphere, is placed in freeze dryer and preservation is lyophilized；
S4, membrane filter method and ammonium sulfate precipitation method separating-purifying avian pasteurella multocida capsular polysaccharide are utilized；
S5, by nanosphere made from avian pasteurella multocida capsular polysaccharide made from step S4 and step S3 with weight ratio 1:
1.4 ratio coupling, obtains polysaccharide nanosphere couplet, and then in phosphate buffer, temperature is 32 DEG C, the item of pH=6
Under part, polysaccharide nanosphere couplet is modified by the glutaraldehyde water solution that mass fraction is 25%, obtains surface aldehydes
Polysaccharide nanosphere couplet；
S6, the formalin for being 0.3% with mass fraction carry out detoxification treatment preparation and separating-purifying to diphtheria toxin
Obtain diphtheria toxoid carrier protein；
S7, by diphtheria toxoid made from surface aldehydes polysaccharide nanosphere couplet made from step S5 and step S6
Carrier protein combine to get.
Comparative example 2 is substantially the same manner as Example 3, and difference is, avian pasteurella multocida capsular polysaccharide and diphtheria class in comparative example 2
The mass ratio of toxin vector albumen is 1:1.
Test example one, safety testing
1. test material: avian pasteurella multocida capsular polysaccharide-protein binding prepared by embodiment 1, embodiment 2 and embodiment 3
Vaccine.
2. subjects: the specific pathogen free chicken (SPF chicken) 60 of 80 ages in days.
3. test method: the SPF chicken of 60 80 ages in days is randomly divided into 3 groups, every group 20, respectively in each group SPF chicken
Avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines of 2mL embodiment 1-3 preparation are subcutaneously injected in neck, are denoted as embodiment 1 respectively
Group, 2 groups of embodiment, 3 groups of embodiment, are all injected intravenously the avian pasteurella multocida of lethal dose after 28 days, after 14 days, observe SPF chicken
Local inflammation reaction and strong living situation.Table 1 is safety testing result.
1 safety testing result of table
| Group | It observes and records | Dissect record | As a result |
| Example 1 group | It is asymptomatic | Without local inflammation reaction | It is strong to live |
| 2 groups of embodiment | It is asymptomatic | Without local inflammation reaction | It is strong to live |
| 3 groups of embodiment | It is asymptomatic | Without local inflammation reaction | It is strong to live |
As shown in Table 1, the SPF chicken of embodiment 1-3 group is after vaccinating 14, do not occur the state of mind it is bad,
Local inflammation and dead situation show that avian pasteurella multocida capsular polysaccharide provided by the invention-protein conjugate vaccines are non-toxic, tool
There is higher safety.
Test example two, Vaccine potency test
1. test material: avian pasteurella multocida capsular polysaccharide-egg prepared by embodiment 1, embodiment 2, embodiment 3, comparative example 2
Avian pasteurella multocida vaccine prepared by white combined vaccine and comparative example 1.
2. subjects: the SPF chicken at 3-6 monthly age 100.
3. test method: the SPF chicken at 120 3-6 monthly ages is divided into 6 groups, every group 20, respectively by embodiment 1, embodiment
2, fowl Pasteur prepared by the avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines and comparative example 1 that prepared by embodiment 3, comparative example 2
Coli vaccine is administered to the neck subcutaneous injection vaccine 0.5mL of 1-5 group SPF chicken, and is denoted as example 1 group, 2 groups of embodiment, reality
Apply 3 groups of example, 2 groups of comparative example and 1 group of comparative example, one group compares and do not vaccinate.After 21 days, every chicken muscle injects velogen strain
Bacterium solution 1mL, injection dosage are 3.1 × 103CFU/mL.Observation 14 days, whether there is or not clinical response and death states.Table 2 is vaccine effect
Power test data record sheet.
2 Vaccine potency test data record sheet of table
As shown in Table 2,2 groups of SPF chickens of example 1 group and embodiment occur arranging green loose stool situation, but spirit is good, observation
Restore simultaneously strong in phase to live, for 3 groups of embodiment of SPF chicken without any clinical symptoms in 14 days, effect is best, is of the invention best
Embodiment, 1 group of comparative example occurred arranging green loose stool, the bad situation of the state of mind within the observation period on the 14th, and final SPF chicken is complete
Portion is dead, and 2 groups of comparative example are arranged green loose stool, listless situation in appearance in 15 hours, and SPF chicken was all dead at 30 hours
It dies, control group is all dead at 22 hours, shows that avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines prepared by the present invention are micro
Injection, protective rate reach 100%.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.Any label in claim should not be construed as limiting the claims involved.
Claims (3)
1. a kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines, which is characterized in that by avian pasteurella multocida capsular polysaccharide with it is white
Larynx toxoid carrier protein is formed by connecting by nanosphere；The avian pasteurella multocida capsular polysaccharide, nanosphere and diphtheria class
The weight ratio of toxin vector albumen is 15:21:10；The nanosphere is prepared by bioabsorbable polymer material；The biology
High molecular material is one or more of chitosan, polyethylene glycol, PLA-PEG copolymer, sodium alginate.
2. the preparation method of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines as described in claim 1, which is characterized in that
The following steps are included:
S1, to weigh weight be that bioabsorbable polymer material used in 1.4 times of capsular polysaccharide of preparation nanosphere is dissolved in 4 times of weight
In methylene chloride, then it is added in the polyvinyl alcohol water solution for being 7% containing mass concentration, stirring and dissolving obtains biological high score
The mixed liquor of the final concentration of 3mg/mL of sub- material；
S2, by mixed liquor made from step S1 pressure be 38MPa condition emulsified 7 times, each 3min, then with 2000
Rev/min speed stirs 15min, obtains emulsion, the organic solvent being evaporated in emulsion obtains colloidal solution；
S3, the colloidal solution made from centrifuge centrifugation step S2, revolving speed 11000r/min obtain the nanometer that diameter is 80nm
Microballoon washes nanosphere, is placed in freeze dryer and preservation is lyophilized；
S4, membrane filter method and ammonium sulfate precipitation method separating-purifying avian pasteurella multocida capsular polysaccharide are utilized；
S5, by nanosphere made from avian pasteurella multocida capsular polysaccharide made from step S4 and step S3 with weight ratio 1:1.4's
Ratio coupling, obtain polysaccharide nanosphere couplet, then carry out aldehyde glycosylation reaction obtain surface aldehydes polysaccharide nanometer it is micro-
Ball couplet；
S6, the formalin for being 0.3% with mass fraction carry out detoxification treatment preparation and separating-purifying to diphtheria toxin
Obtain diphtheria toxoid carrier protein；
S7, by diphtheria toxoid made from surface aldehydes polysaccharide nanosphere couplet made from step S5 and step S6
Carrier protein combine to get.
3. the preparation method of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines as claimed in claim 2, which is characterized in that
Aldehyde glycosylation reaction concrete operations in the step S5 are as follows:
In phosphate buffer, temperature is 32 DEG C, and under conditions of pH=6, polysaccharide nanosphere couplet is passed through mass fraction
It is modified for 25% glutaraldehyde water solution, obtain the polysaccharide nanosphere couplet of surface aldehydes.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811032530.9A CN108721616B (en) | 2018-09-05 | 2018-09-05 | A kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines and preparation method thereof |
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| CN201811032530.9A CN108721616B (en) | 2018-09-05 | 2018-09-05 | A kind of avian pasteurella multocida capsular polysaccharide-protein conjugate vaccines and preparation method thereof |
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