CN107684621A - Application of the corynebacterium diphtheroides as immunologic adjuvant in fowl oil emulsion inactivated vaccine - Google Patents
Application of the corynebacterium diphtheroides as immunologic adjuvant in fowl oil emulsion inactivated vaccine Download PDFInfo
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Abstract
The invention discloses application of the corynebacterium diphtheroides as immunologic adjuvant in fowl oil emulsion inactivated vaccine, described corynebacterium diphtheroides refers to similar with corynebacterium diphtheriae corynebacteria in terms of form and biological characteristics, i.e., the low pathogenic or non-pathogenic corynebacteria in addition to highly pathogenic Bacterium diphtheriae.Corynebacterium diphtheroides active adjuvant provided by the invention has high immunostimulatory activity, can nonspecific stimulation T, bone-marrow-derived lymphocyte immunologic function, promote cytokine profiles secretion, it is added to the generation that vaccine-induced animal's antibody can be obviously promoted in fowl oil emulsion inactivated vaccine.
Description
Technical field
The present invention relates to microbial technology field, more particularly to a kind of corynebacterium diphtheroides is used as immunologic adjuvant in fowl
Application in oil emulsion inactivated vaccine.
Background technology
After being particularly Avian Influenza in recent years, fowl turns into most important fowl Vaccines classes with oil emulsion inactivated vaccine.
But fowl is highly prone to the influence of inactivation antigen quality, particularly high quality vaccine with oil emulsion inactivated vaccine and typically led to
The realization of high power concentrated antigen technology is crossed, the fowl of high-quality is higher with oil emulsion inactivated vaccine production cost and production capacity is limited, it is impossible to
Effectively meet the market demand.Therefore, safe efficient, economic fowl oil emulsion inactivated vaccine special immune adjuvant is researched and developed
As research emphasis.
Immunologic adjuvant is also known as nonspecific stimulation agent, and body can be strengthened to vaccine product by referring to be used cooperatively with vaccine antigen
The material of immune response.Conventional immunologic adjuvant has the mycobacterium tuberculosis (BCG vaccine) of inactivation, thymic peptide, double-strand poly-nuclear
Thuja acid, aluminium hydroxide aluminium glue adjuvant and mineral oil adjuvant etc..Immunologic adjuvant generally existing problems with present：(1) knot of inactivation
Core mycobacterial adjuvants, because natural mycobacterium tuberculosis has high virulence, there is pathogenicity to humans and animals, therefore at present all
It is that adjuvant is prepared using the BCG vaccine of attenuation, but requires high there are still subsequent treatment, easily causing inactivation does not cause thoroughly
Trigger the animal health harmfulness of height when being applied as adjuvant, additionally cause serious animal adverse reaction or local granulation
Swollen lesion etc., along with bacterial fermentation condition is strict, fermentation period is long, slow-growing, high to nutritional requirement, therefore it forbids to answer
For commercially available vaccine.(2) thymosin extrasin has been applied to human tumor treatment, but price is costly, it is impossible to for animal vaccine.
(3) double stranded polynucleotide (poly IC) has become conventional anticancer drugs, but in view of its half-life short, in vivo in transient
Reaction, it is still indefinite with the effectiveness of oil emulsion inactivated vaccine immunologic adjuvant as fowl.(4) aluminium hydroxide aluminium glue adjuvant is current
Animal vaccine often uses adjuvant, and it is mainly by making antigen adjuvant compound form slowly releasing effect, and certain journey in injection site
Immune response stimulating on degree and play adjuvant effect, but it is better than viral vaccine in bacterial vaccine positive effect, and helps
Agent stimulation is limited.
In summary, animal vaccine adjuvant really efficiently, economic, therefore new immune assistant are lacked in the market
Agent is used to improve animal immune effect, turns into livestock and poultry breeding industry major issue urgently to be resolved hurrily.
The content of the invention
It is an object of the invention to overcome shortcoming present in prior art, there is provided a kind of bioactivity is high, inanimate object poison
Application of the corynebacterium diphtheroides of property as immunologic adjuvant in fowl oil emulsion inactivated vaccine, the corynebacterium diphtheroides
With the adjuvant effect similar to Bacterium diphtheriae.
The purpose of the present invention is achieved through the following technical solutions：
Application of the corynebacterium diphtheroides as immunologic adjuvant in fowl oil emulsion inactivated vaccine.
Described corynebacterium diphtheroides refers to similar with corynebacterium diphtheriae bar-shaped in terms of form and biological characteristics
Bacillus, i.e., the low pathogenic or non-pathogenic corynebacteria in addition to highly pathogenic Bacterium diphtheriae are main to include but is not limited to such as
Lower bacterium：Such as Corynebacterium pseudodi phtheriae, corynebacterium ulcerrans, Corynebacterium pseudotuberculosis, propionibacterium acnes (i.e. short and small rod-like stem
Bacterium) etc..
Described corynebacterium diphtheroides has following characteristics：Aerobic or amphimicrobian, aerobic growth form skin covering of the surface；Connect
Catalase-positive；Gram's staining is strong positive, is not easy to decolourize；Microscopy is in single, paired, V-shaped or arranged in parallel, and is occurred
The bacterium of significant proportion coccobacillus state, its microscopy state is observed has high similarity with corynebacterium diphtheriae；Bacteria cell wall peptide
The racemization diaminopimelic acid of being characterized property of glycan is main component；It is low to humans and animals pathogenicity, not pathogenic or opportunistic.
The preferably aerobic culture of described corynebacterium diphtheroides；Contact enzyme positive；Gram's staining is strong positive, and dyeing is solid
Put forth effort to be not easy to decolourize by force；Large scale coccobacillus state is presented in microscopy；Bacteria cell wall peptide glycan is racemization diaminopimelic acid knot
Structure；It is not pathogenic to humans and animals normal condition；The whole cell peptidoglycan of Bacterium diphtheriae sample with adjuvanticity and bar-shaped
Bacillus acid.
Current research it has been proved that Bacterium diphtheriae due to whole cell peptidoglycan and rod with special chemical structure
Shape bacillus acid and there is strong adjuvant effect, but it has highly pathogenic, thus its biological safety is very low, limits its as helping
The application of agent.Corynebacterium diphtheroides provided by the invention has the cell membrane peptide similar in Bacterium diphtheriae chemical constitution
Glycan and corynebacteria acid, thus there is the adjuvant effect similar to Bacterium diphtheriae, but biological safety is very high simultaneously, generation
Table strain is Corynebacterium pseudodi phtheriae (Corynebacterium pseudodiphtheriticum) and Corynebacterium pseudotuberculosis
(Corynebacterium pseudotuberculosis)；It is preferred that there is high biological security, to reality under laboratory condition
Test animal without or extremely low pathogenicity Corynebacterium pseudodi phtheriae.
The feature of described Corynebacterium pseudodi phtheriae is as follows：Acid is not produced in aerobic culture, common carbohydrates fermentation；Catalase sun
Property；Gram's staining is strong positive, and dyeing pull-out capacity is not easy to decolourize by force；Large scale coccobacillus state is presented in microscopy；Bacterial cell
Wall peptide glycan is racemization diaminopimelic acid structure；It is not pathogenic to humans and animals normal condition；Diphtheria rod with adjuvanticity
Whole cell peptidoglycan and the corynebacteria acid of shape bacillus sample.
Corynebacterium pseudodi phtheriae has the following advantages that as adjuvant：(1) there is the Bacterium diphtheriae sample of high expression quantity
Whole cell peptidoglycan and corynebacteria acid, it is strong positive to show as Gram's staining, and dyeing pull-out capacity is not easy to decolourize by force；(2) certainly
It right avirulence, will not cause researcher and producers that inadvertent contamination occurs, reduce the leakage of laboratory biological material and cause
Environmental pollution possibility；(3) aerobic fermentation, fermentation condition require low；(4) growth rate is fast, and fermentation period is short；(5) nutrition
Less demanding, fermentation costs are low；(6) post processing is simple, and active component loss is small.
A kind of preparation method of fowl oil emulsion inactivated vaccine, corynebacterium diphtheroides is employed as immunologic adjuvant, bag
Include following step：
(1) preparation of adjuvant liquid：The mass parts of corynebacterium diphtheroides adjuvant powder 1~10 are taken, is dissolved in physiological saline and making
The standby adjuvant liquid into 10~100mg/mL concentration, preferably 25mg/mL concentration；By 4 DEG C of guarantors after the conventional bacteria removing of adjuvant liquid progress
Deposit standby；
(2) preparation of antigen aqueous phase：The inactivation antigen of 75~96 parts by volume, preferably 94 parts by volume is taken, is added under aseptic condition
Add 1~22 parts by volume, preferably 2 parts by volume adjuvant liquid, add the Tween 80 (TWEEN80) of 3~6 parts by volume sterilizing, preferably add
Enter the Tween 80 (TWEEN80) of 4 parts by volume sterilizing, vibration or stirring obtain antigen aqueous phase after being completely dissolved to TWEEN80；
(3) preparation of vaccine：In sterilizing oil phase this 80 (SPAN80) and 1% (volume hundred are taken charge of containing 6% (percent by volume)
Point ratio) aluminum stearate, antigen aqueous phase with sterilizing oil phase volume ratio be 1:1.5~1:3, preferred antigens aqueous phases and sterilizing oil phase
Volume ratio is 1:2, antigen aqueous phase is slowly added into sterilizing oil phase, emulsified, fowl oil emulsion inactivated vaccine is prepared.
Ultimate density of the adjuvant in antigen aqueous phase is 0.2~2mg/mL, preferably 0.5mg/mL.
The emulsification, it is holding 4000~6000rpm/min, preferably 5000rpm/min in antigen aqueous phase adition process
Stirring at low speed, after antigen aqueous phase is completely added to sterilizing oil phase, regulation rotating speed is 12000~20000rpm/min, preferably
16000rpm/min, carry out 2~5min of high speed shearing emulsification, preferably 3min.
The corynebacterium diphtheroides active component of the present invention has the following advantages that as adjuvant compared with existing adjuvant products
And effect：
(1) corynebacterium diphtheroides active adjuvant provided by the invention has height oxygen resistence, does not need anaerobic fermentation, hair
The features such as ferment technique is simple and with short production cycle, yield is high, and there is high immunostimulatory activity, can nonspecific stimulation T, B
Lymphocyte immunologic function, promote cytokine profiles secretion, it is added in fowl oil emulsion inactivated vaccine and can substantially promoted
Enter the generation of vaccine-induced animal's antibody, and testing proves induction of antibodies level in coccidia positive field without being decreased obviously.
(2) corynebacterium diphtheroides active adjuvant provided by the invention possesses tight security, and its active component is compared to normal
The BCG vaccine adjuvant (mycobacterium tuberculosis of inactivation) of rule possesses obviously security, without any obvious adverse reaction, moves
After thing application, vaccine injection is local to be remained without obvious, and conventional BCG vaccine adjuvant part granuloma strong reaction.
(3) corynebacterium diphtheroides active adjuvant provided by the invention research and production has a tight security, product with
Accessory substance does not pollute the environment.
(4) tests prove that, fowl prepared by the present invention is used to the antibody induced after animal be immunized with oil emulsion inactivated vaccine
Level is significantly higher than the control group vaccine of no adjuvant addition, therefore corynebacterium diphtheroides provided by the invention can be used as effectively
Adjuvant is used for fowl oil emulsion inactivated vaccine.
Embodiment
Further detailed description is done to the present invention with reference to embodiment, but the implementation of the present invention is not limited to this.
Embodiment 1：The preparation of ewcastle disease+H9 subtype avian influenzas Combined Oil inactivated Vaccine (abbreviation new stream bigeminy vaccine)
(1) adjuvant sample sterilization treatment and quantitative
Corynebacterium diphtheroides adjuvant formulation Powder samples 1g is taken, is dissolved in after 40mL physiological saline that to be prepared into 25mg/mL dense
Adjuvant liquid is spent, 4 DEG C after the conventional bacteria removing of adjuvant liquid progress are saved backup.
(2) preparation of antigen aqueous phase：
1. without vehicle control group antigen aqueous phase：Take the ewcastle disease+H9 hybrid antigens (ewcastle disease antigen (abbreviation that 94mL is inactivated
ND antigens)：H9 subtype avian influenzas antigen=1:1 ratio mixes, and ewcastle disease antigen and H9 subtype avian influenza antigens are chicken embryo system
Standby antigen stock), 2mL sterile salines are added under aseptic condition, add the TWEEN80 of 4mL sterilizings, vibration or stirring are extremely
After TWEEN80 is completely dissolved, as without vehicle control group antigen aqueous phase (total amount 100mL).
2. adjuvant group antigen aqueous phase (the final concentration of 0.5mg/mL of adjuvant)：Ewcastle disease+H9 the hybrid antigens for taking 94mL to inactivate
(ewcastle disease antigen：H9 subtype avian influenzas antigen=1:1 ratio mixes, and ewcastle disease antigen and H9 subtype avian influenza antigens are chicken
Embryo prepares antigen stock), 2mL adjuvants are added under aseptic condition, add the TWEEN80 of 4mL sterilizings, vibration or stirring are extremely
After TWEEN80 is completely dissolved, as adjuvant group antigen aqueous phase (total amount 100mL).
(3) prepared by vaccine：
1. prepared without vehicle control group vaccine：Take above-mentioned preparation without vehicle control group antigen aqueous phase 100mL, be slowly added to
200mL sterilizings oil phase (containing 6%SPAN80 and 1% aluminum stearate), is emulsified, it is desirable to which mulser is anti-using IKA T25 mulsers
5000rpm/min stirring at low speed is kept in former adition process, after antigen is completely added to oil phase, regulation mulser rotating speed is
16000rpm/min high speed shearing emulsifications 3min.After the vaccine packing prepared after emulsification, it is standby to be placed in 4 DEG C of holdings.
2. prepared by adjuvant group vaccine：The adjuvant group antigen aqueous phase 100mL of above-mentioned preparation is taken, is slowly added to 200mL sterilizing oil
Phase (containing 6%SPAN80 and 1% aluminum stearate), is emulsified, it is desirable to which mulser is in antigen adition process using IKA T25 mulsers
5000rpm/min stirring at low speed is kept, after antigen is completely added to oil phase, regulation mulser rotating speed is that 16000rpm/min is high
Fast emulsification pretreatment 3min.After the vaccine packing prepared after emulsification, it is standby to be placed in 4 DEG C of holdings.
Pay attention to, then vaccine preparation order prepares adjuvant group vaccine, to most again first to prepare no vehicle control group vaccine
Big possible reduction test error.
(4) oil emulsion inactivated vaccine physical behavior detects
Above-mentioned preparation and the vaccine dispensed are taken, rises again to after room temperature, detects following physical behavior respectively：
A. every group of vaccine takes 10mL samples rotation viscometer to detect vaccine viscosity；
B. every group of vaccine is slowly dropped to respectively and stood on the water surface, observe vaccine spread condition, in addition to first drips, subsequently
Be added drop-wise to the water surface vaccine be not separated into it is qualified；
C. every group takes 30mL vaccines, is distributed into 3 10mL cone bottom centrifuge tubes respectively, and 3000rpm/min centrifuges 20min,
Observation whether there is layering demulsifying phenomenon；It is layered if any demulsification, illustrating that vaccine is unqualified need to prepare again；
D. it is above-mentioned to pass through the vaccine centrifuged, 37 DEG C are respectively placed in after mark, room temperature, 4 DEG C preserve 1 month, see whether brokenly
Breast, it is qualified that ttom of pipe, which aqueous phase occurs less than 0.5mL,.
Physical behavior test result is：
A. test group vaccine viscosity 36.4cp, control group vaccine 41.6cp.
B. test group vaccine and control group vaccine are qualified.
C. for test group vaccine with control group vaccine without demulsification, the two is qualified.
D. for test group vaccine with control group vaccine without demulsification, the two is qualified.
(5) oil emulsion inactivated vaccine safety testing：21 age in days SPF chickens are immunized respectively using 2 kinds of vaccines of above-mentioned preparation,
1mL/, every group of vaccine immunity 10, the daily health status for observing chicken, Continuous Observation 14 days.
Testing result：Animal is in a good state of health without death, qualified.
(6) oil emulsion inactivated vaccine effect is tested：Using the age in days SPF chickens of vaccine immunity 21 of above-mentioned preparation, 0.3mL/
Only, every group of vaccine immunity 10, every chicken marks pin number, and after vaccine injection from 1 week, blood sampling separation serum carries out ND and H9 weekly
The HI detections of subtype avian influenza, continuous detection 3 weeks, it is poor with experimental group immune effect of vaccine that adjuvant adds to compare control group vaccine
It is different.Testing result is as shown in table 1, table 2.
The H9 TPPA results of the new stream bigeminy vaccine of table 1.
The ND TPPA results of the new stream bigeminy vaccine of table 2.
Conclusion：In the experiment of new stream Combined Oil inactivated Vaccine, 15 days after being immunized, adjuvant group vaccine ratio is without vehicle control group
Vaccine shows the trend of more High antibody level, to 21 days after immune, adjuvant group than control group have more High antibody level and
Antibody level difference increases between the two.This experiment is as can be seen that adjuvant can significantly increase new stream bigeminy vaccine antibody level.
Embodiment 2：The preparation of H5 subtype avian influenzas (D7 strains) oil emulsion inactivated vaccine
(1) adjuvant sample sterilization treatment and quantitative
Corynebacterium diphtheroides adjuvant formulation Powder samples 1g is taken, is dissolved in after 40mL physiological saline that to be prepared into 25mg/mL dense
The adjuvant liquid of degree, 4 DEG C after the conventional bacteria removing of adjuvant liquid progress are saved backup.
(2) preparation of antigen aqueous phase：
1. without vehicle control group antigen aqueous phase：94mL H5 subtype avian influenza D7 strain inactivation antigens are taken, are added under aseptic condition
2mL sterile salines, the TWEEN80 of 4mL sterilizings is added, after vibration or stirring are completely dissolved to TWEEN80, as without assistant
Agent control group antigen aqueous phase (total amount 100mL).
2. adjuvant group antigen aqueous phase (the final concentration of 0.5mg/mL of adjuvant)：Take 94mL H5 subtype avian influenza D7 strains inactivation anti-
Original, 2mL adjuvants are added under aseptic condition, add the TWEEN80 of 4mL sterilizings, vibration or stir to TWEEN80 is completely dissolved
Afterwards, as adjuvant group antigen aqueous phase (total amount 100mL).
(3) prepared by vaccine：
1. prepared without vehicle control group vaccine：Take above-mentioned preparation without vehicle control group antigen aqueous phase 100mL, be slowly added to
200mL sterilizings oil phase (containing 6%SPAN80 and 1% aluminum stearate), is emulsified, it is desirable to which mulser is anti-using IKA T25 mulsers
5000rpm/min stirring at low speed is kept in former adition process, after antigen is completely added to oil phase, regulation mulser rotating speed is
16000rpm/min high speed shearing emulsifications 3min.After the vaccine packing prepared after emulsification, it is standby to be placed in 4 DEG C of holdings.
2. prepared by adjuvant group vaccine：The adjuvant group antigen aqueous phase 100mL of above-mentioned preparation is taken, is slowly added to 200mL sterilizing oil
Phase (containing 6%SPAN80 and 1% aluminum stearate), is emulsified, it is desirable to which mulser is in antigen adition process using IKA T25 mulsers
5000rpm/min stirring at low speed is kept, after antigen is completely added to oil phase, regulation mulser rotating speed is that 16000rpm/min is high
Fast emulsification pretreatment 3min.After the vaccine packing prepared after emulsification, it is standby to be placed in 4 DEG C of holdings.
Pay attention to, then vaccine preparation order prepares adjuvant group vaccine, to most again first to prepare no vehicle control group vaccine
Big possible reduction test error.
(4) oil emulsion inactivated vaccine physical behavior detects：Above-mentioned preparation and the vaccine dispensed are taken, is risen again to after room temperature, point
Following physical behavior is not detected：
A. every group of vaccine takes 10mL samples rotation viscometer to detect vaccine viscosity.
B. every group of vaccine is slowly dropped to respectively and stood on the water surface, observe vaccine spread condition, in addition to first drips, subsequently
Be added drop-wise to the water surface vaccine be not separated into it is qualified.
C. every group takes 30mL vaccines, is distributed into 3 10mL cone bottom centrifuge tubes respectively, and 3000rpm/min centrifuges 20min,
Observation whether there is layering demulsifying phenomenon；It is layered if any demulsification, illustrating that vaccine is unqualified need to prepare again.
D. it is above-mentioned to pass through the vaccine centrifuged, 37 DEG C are respectively placed in after mark, room temperature, 4 DEG C preserve 1 month, see whether brokenly
Breast, it is qualified that ttom of pipe, which aqueous phase occurs less than 0.5mL,.
Physical behavior test result：
A. test group vaccine viscosity 40.1cp, control group vaccine 38.7cp.
B. test group vaccine and control group vaccine are qualified.
C. for test group vaccine with control group vaccine without demulsification, the two is qualified.
D. for test group vaccine with control group vaccine without demulsification, the two is qualified.
(5) oil emulsion inactivated vaccine safety testing：21 age in days SPF chickens are immunized respectively using 2 kinds of vaccines of above-mentioned preparation,
Only, every group of vaccine immunity 10 is daily to observe chicken health status, Continuous Observation 14 days by 1mL/.
Testing result：Animal is in a good state of health without death, qualified.
(6) oil emulsion inactivated vaccine effect is tested：Using the age in days SPF chickens of vaccine immunity 21 of above-mentioned preparation, 0.3mL/
Only, every group of vaccine immunity 10, every chicken marks pin number, and after vaccine injection from 1 week, blood sampling separation serum carries out D7 strains weekly
HI is detected, and continuous detection 3 weeks, compares control group vaccine and the experimental group immune effect of vaccine difference of adjuvant addition.
Table 3.H5 subtype avian influenzas (D7 strains) oil emulsion inactivated vaccine SPF chicken effect testing inspection results
Conclusion：In the experiment of H5 subtype avian influenzas (D7 strains) oil emulsion inactivated vaccine, 21 days after being immunized, adjuvant group vaccine compares
The trend of more High antibody level is shown according to group vaccine, to after being immunized 28 days, adjuvant group increases than control group antibody level gap,
I.e. adjuvant group is higher 1log2 than control group antibody.This experiment is as can be seen that adjuvant immunity enhancing is notable.
(7) Commercial meat-type duck immunity test：Using Commercial meat-type duck purchased in market, vaccine immunity, 0.3mL/ are carried out in 8 ages in days or so
Only, every group of vaccine immunity 10, every duck marks pin number, and after vaccine injection from 1 week, blood sampling separation serum carries out D7 strains weekly
HI is detected, and continuous detection 4 weeks, compares control group vaccine and the experimental group immune effect of vaccine difference of adjuvant addition.
Table 4.H5 subtype avian influenzas (D7 strains) oil emulsion inactivated vaccine Commercial meat-type duck immunity test result
Conclusion：In the experiment of H5 subtype avian influenzas (D7 strains) oil emulsion inactivated vaccine, 21,28,42 days after being immunized, adjuvant group epidemic disease
Seedling shows more High antibody level than control group vaccine, and particularly to after being immunized 42 days, control group antibody is in be decreased obviously
Gesture and adjuvant group downward trend unobvious.As can be seen that for immune duck, adjuvant can not only significantly increase vaccine for this experiment
Antibody level, and can be more lasting maintenance High antibody level.
Embodiment 3：The preparation of H5 subtype avian influenzas (D7+rD8 strains) Infectious coryza
(1) adjuvant sample sterilization treatment and quantitative
Corynebacterium diphtheroides adjuvant formulation Powder samples 1g is taken, is dissolved in after 40mL physiological saline that to be prepared into 25mg/mL dense
Adjuvant liquid is spent, 4 DEG C after the conventional bacteria removing of adjuvant liquid progress are saved backup.
(2) preparation of antigen aqueous phase：
1. without vehicle control group antigen aqueous phase：The D7 strains of subtype avian influenza containing H5 and rD8 strain hybrid antigens that 94mL is inactivated are taken,
2mL sterile salines are added under aseptic condition, add the TWEEN80 of 4mL sterilizings, vibration or stirring are complete to TWEEN80
After dissolving, as without vehicle control group antigen aqueous phase (total amount 100mL).
2. adjuvant group antigen aqueous phase (the final concentration of 0.5mg/mL of adjuvant)：The D7 strains of subtype avian influenza containing H5 for taking 94mL to inactivate
With rD8 strain hybrid antigens, 2mL adjuvants are added under aseptic condition, add the TWEEN80 of 4mL sterilizings, vibration or stirring are extremely
After TWEEN80 is completely dissolved, as adjuvant group antigen aqueous phase (total amount 100mL).
(3) emulsification prepares vaccine：
1. prepared without vehicle control group vaccine：Take above-mentioned preparation without vehicle control group antigen aqueous phase 100mL, be slowly added to
200mL sterilizings oil phase (containing 6%SPAN80 and 1% aluminum stearate), is emulsified, it is desirable to which mulser is anti-using IKA T25 mulsers
5000rpm/min stirring at low speed is kept in former adition process, after antigen is completely added to oil phase, regulation mulser rotating speed is
16000rpm/min high speed shearing emulsifications 3min.After the vaccine packing prepared after emulsification, it is standby to be placed in 4 DEG C of holdings.
2. prepared by adjuvant group vaccine：The adjuvant group antigen aqueous phase 100mL of above-mentioned preparation is taken, is slowly added to 200mL sterilizing oil
Phase (containing 6%SPAN80 and 1% aluminum stearate), is emulsified, it is desirable to which mulser is in antigen adition process using IKA T25 mulsers
5000rpm/min stirring at low speed is kept, after antigen is completely added to oil phase, regulation mulser rotating speed is that 16000rpm/min is high
Fast emulsification pretreatment 3min.After the vaccine packing prepared after emulsification, it is standby to be placed in 4 DEG C of holdings.
Pay attention to, then vaccine preparation order prepares adjuvant group vaccine, to most again first to prepare no vehicle control group vaccine
Big possible reduction test error.
(4) oil emulsion inactivated vaccine physical behavior detects
Above-mentioned preparation and the vaccine dispensed are taken, rises again to after room temperature, detects following physical behavior respectively：
A. every group of vaccine takes 10mL samples rotation viscometer to detect vaccine viscosity.
B. every group of vaccine is slowly dropped to respectively and stood on the water surface, observe vaccine spread condition, in addition to first drips, subsequently
Be added drop-wise to the water surface vaccine be not separated into it is qualified.
C. every group takes 30mL vaccines, is distributed into 3 10mL cone bottom centrifuge tubes respectively, and 3000rpm/min centrifuges 20min,
Observation whether there is layering demulsifying phenomenon；It is layered if any demulsification, illustrating that vaccine is unqualified need to prepare again.
D. it is above-mentioned to pass through the vaccine centrifuged, 37 DEG C are respectively placed in after mark, room temperature, 4 DEG C preserve 1 month, see whether brokenly
Breast, it is qualified that ttom of pipe, which aqueous phase occurs less than 0.5mL,.
Physical behavior testing result：
A. test group vaccine viscosity 38.2cp, control group vaccine 40.0cp.
B. test group vaccine and control group vaccine are qualified.
C. for test group vaccine with control group vaccine without demulsification, the two is qualified.
D. for test group vaccine with control group vaccine without demulsification, the two is qualified.
(5) oil emulsion inactivated vaccine safety testing：21 age in days SPF chickens are immunized respectively using 2 kinds of vaccines of above-mentioned preparation,
Only, every group of vaccine immunity 10 is daily to observe chicken health status, Continuous Observation 14 days by 1mL/.
Testing result：Animal is in a good state of health without death, qualified.
(6) oil emulsion inactivated vaccine effect is tested：Using the age in days SPF chickens of vaccine immunity 21 of above-mentioned preparation, 0.3mL/
Only, every group of vaccine immunity 10, every chicken marks pin number, after vaccine injection 1 week from, weekly blood sampling separation serum progress D7 with
RD8 HI detections, continuous detection 3 weeks, compare control group vaccine and the experimental group immune effect of vaccine difference of adjuvant addition.
Table 5.H5 subtype avian influenzas (D7+rD8 strains) divalence oil emu contrast test rD8 TPPA results
Table 6.H5 subtype avian influenzas (D7+rD8 strains) divalence oil emu contrast test D7 TPPA results
Conclusion：In the experiment of H5 subtype avian influenzas (D7+rD8 strains) Infectious coryza, 21,28,35,42 after being immunized
My god, adjuvant group vaccine shows more High antibody level than control group vaccine.This experiment is as can be seen that adjuvant can significantly increase two
Valency vaccine antibody is horizontal.
(7) commercial broiler immunity test
Using commodity Chinese ephedra broiler chicken purchased in market, vaccine immunity is carried out in 10 ages in days or so, 0.3mL/ only, every group of vaccine immunity
10, every chicken marks pin number, and after vaccine injection from 1 week, blood sampling separation serum carries out D7 and rD8 HI detections weekly, continuously
Detection 5 weeks, compare control group vaccine and the experimental group immune effect of vaccine difference of adjuvant addition.
Table 7.H5 subtype avian influenzas (D7+rD8 strains) divalence oil emu market broiler chicken inactivated vaccine effect contrast test D7 resists
Body measurement result
Table 8.H5 subtype avian influenzas (D7+rD8 strains) divalence oil emu market broiler chicken inactivated vaccine effect contrast test rD8 resists
Body is horizontal relatively
Conclusion：H5 subtype avian influenzas (D7+rD8 strains) Infectious coryza is immunized in Chinese ephedra broiler trial, is immunized
21,28,35 days afterwards, adjuvant group vaccine showed more High antibody level than control group vaccine.This experiment is as can be seen that adjuvant energy
Significantly increase bivalent vaccine antibody level.
Proved by zoopery, add the oil emulsion inactivated vaccine immune efficacy of adjuvant with it is un-added have it is significant poor
It is different；And the oil emulsion vaccine for adding adjuvant possesses tight security, vaccine injection is local without obvious residual.
Claims (10)
- A kind of 1. purposes of corynebacterium diphtheroides, it is characterised in that：Corynebacterium diphtheroides is as immunologic adjuvant in fowl oil Application in emulsion inactivated vaccine.
- 2. the purposes of corynebacterium diphtheroides according to claim 1, it is characterised in that：Described corynebacterium diphtheroides Refer to similar with corynebacterium diphtheriae corynebacteria in terms of form and biological characteristics, i.e., except highly pathogenic Bacterium diphtheriae Outer low pathogenic or non-pathogenic corynebacteria.
- 3. the purposes of corynebacterium diphtheroides according to claim 2, it is characterised in that：Described corynebacterium diphtheroides With following characteristics：Aerobic or amphimicrobian, aerobic growth form skin covering of the surface；Contact enzyme positive；Gram's staining is strong positive, It is not easy to decolourize；Microscopy is in single, paired, V-shaped or arranged in parallel, and the bacterium of significant proportion coccobacillus state occurs, its Microscopy state is observed has high similarity with corynebacterium diphtheriae；Racemization diaminourea heptan two of being characterized property of bacteria cell wall peptide glycan Acid is main component；It is low to humans and animals pathogenicity, not pathogenic or opportunistic.
- 4. the purposes of corynebacterium diphtheroides according to claim 2, it is characterised in that：Described corynebacterium diphtheroides It is Corynebacterium pseudodi phtheriae, corynebacterium ulcerrans, Corynebacterium pseudotuberculosis or propionibacterium acnes.
- 5. the purposes of corynebacterium diphtheroides according to claim 4, it is characterised in that：Described corynebacterium diphtheroides It is Corynebacterium pseudodi phtheriae.
- 6. the purposes of corynebacterium diphtheroides according to claim 5, it is characterised in that：Described Corynebacterium pseudodi phtheriae It is characterized in：Acid is not produced in aerobic culture, common carbohydrates fermentation；Contact enzyme positive；Gram's staining is strong positive, dyes pull-out capacity It is not easy to decolourize by force；Large scale coccobacillus state is presented in microscopy；Bacteria cell wall peptide glycan is racemization diaminopimelic acid structure；It is right Humans and animals normal condition is not pathogenic；The whole cell peptidoglycan and corynebacteria of Bacterium diphtheriae sample with adjuvanticity Acid.
- A kind of 7. preparation method of fowl oil emulsion inactivated vaccine, it is characterised in that：Employ any one of claim 1~6 Described corynebacterium diphtheroides comprises the steps as immunologic adjuvant：(1) preparation of adjuvant liquid：The mass parts of corynebacterium diphtheroides adjuvant powder 1~10 are taken, is dissolved in physiological saline and being prepared into The adjuvant liquid of 10~100mg/mL concentration；4 DEG C after the conventional bacteria removing of adjuvant liquid progress are saved backup；(2) preparation of antigen aqueous phase：The inactivation antigen of 75~96 parts by volume is taken, the assistant of 1~22 parts by volume is added under aseptic condition Agent liquid, adds the Tween 80 of 3~6 parts by volume sterilizing, and vibration or stirring obtain antigen aqueous phase after being completely dissolved to Tween 80；(3) preparation of vaccine：Contain 6% (percent by volume) department sheet 80 and 1% (percent by volume) stearic acid in sterilizing oil phase The volume ratio of aluminium, antigen aqueous phase and sterilizing oil phase is 1:1.5~1:3, antigen aqueous phase is slowly added into sterilizing oil phase, carries out breast Change, fowl oil emulsion inactivated vaccine is prepared.
- 8. the preparation method of fowl oil emulsion inactivated vaccine according to claim 7, it is characterised in that：In step (1), assistant The concentration of agent liquid is 25mg/mL；In step (2), the dosage of inactivation antigen is 94 parts by volume, and the dosage of adjuvant liquid is 2 parts by volume, The dosage of the Tween 80 of sterilizing is 4 parts by volume；In step (3), the volume ratio of antigen aqueous phase and sterilizing oil phase is 1:2.
- 9. the preparation method of fowl oil emulsion inactivated vaccine according to claim 7, it is characterised in that：Adjuvant is in antigen water Ultimate density in phase is 0.2~2mg/mL.
- 10. the preparation method of fowl oil emulsion inactivated vaccine according to claim 7, it is characterised in that：The emulsification, it is 4000~6000rpm/min stirring at low speed is kept in antigen aqueous phase adition process, treats that antigen aqueous phase is completely added to sterilizing oil Xiang Hou, regulation rotating speed are 12000~20000rpm/min, carry out 2~5min of high speed shearing emulsification.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114028558A (en) * | 2021-12-20 | 2022-02-11 | 漳州片仔癀药业股份有限公司 | Preparation method of inactivated vaccine adjuvant using bacterial lysate |
| CN114028554A (en) * | 2021-10-14 | 2022-02-11 | 广东渔跃生物技术有限公司 | Rhodococcus ruber immunopotentiator and application thereof in poultry vaccine |
| CN114288403A (en) * | 2021-12-20 | 2022-04-08 | 浙江洪晟生物科技股份有限公司 | Newcastle disease and H9 subtype avian influenza bivalent inactivated vaccine containing immunopotentiator and preparation method and application thereof |
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| CN102579339A (en) * | 2012-03-12 | 2012-07-18 | 天津瑞普高科生物药业有限公司 | Oil emulsion vaccine for broilers and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102579339A (en) * | 2012-03-12 | 2012-07-18 | 天津瑞普高科生物药业有限公司 | Oil emulsion vaccine for broilers and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114028554A (en) * | 2021-10-14 | 2022-02-11 | 广东渔跃生物技术有限公司 | Rhodococcus ruber immunopotentiator and application thereof in poultry vaccine |
| CN114028554B (en) * | 2021-10-14 | 2023-12-12 | 广东渔跃生物技术有限公司 | Rhodococcus erythropolis immunopotentiator and application thereof in poultry vaccine |
| CN114028558A (en) * | 2021-12-20 | 2022-02-11 | 漳州片仔癀药业股份有限公司 | Preparation method of inactivated vaccine adjuvant using bacterial lysate |
| CN114288403A (en) * | 2021-12-20 | 2022-04-08 | 浙江洪晟生物科技股份有限公司 | Newcastle disease and H9 subtype avian influenza bivalent inactivated vaccine containing immunopotentiator and preparation method and application thereof |
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