CN109833474A - Temperature sensitive type adjuvant and preparation method thereof - Google Patents
Temperature sensitive type adjuvant and preparation method thereof Download PDFInfo
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- CN109833474A CN109833474A CN201910104090.1A CN201910104090A CN109833474A CN 109833474 A CN109833474 A CN 109833474A CN 201910104090 A CN201910104090 A CN 201910104090A CN 109833474 A CN109833474 A CN 109833474A
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Abstract
The present invention provides a kind of temperature sensitive type adjuvant, is used to prepare animal disease vaccine and can change with ambient temperature and phase transition occurs；The temperature sensitive type adjuvant includes: the chitosan or chitosan derivatives of 20wt% to 60wt%；The crosslinking agent of 10wt% to 40wt%；The colloid extract of 1.0wt% to 5.0wt%；The plasticizer of 1.0wt% to 5wt%；And remaining is diluent.The immunogenicity of viral antigen in vivo can be improved in the temperature sensitive type adjuvant, increase the time stimulated immune system, and then the memory immune effect of animal can be increased, and the production method of the temperature sensitive type adjuvant is simple, low in cost, can be widely used for the vaccine of Animal diseases.
Description
Technical field
The present invention relates to a kind of vaccine adjuvants, can change and undergoing phase transition with temperature especially with regard to a kind of
Temperature sensitive type adjuvant.
Background technique
It is way for preventing the most effective of virus-infected animal and most efficiency, Hen Duochuan that vaccine inoculation, which is recognized,
The catch an illness basis of control plan is vaccine to be made and to animal inoculation pvaccination with microorganism living or through the microorganism inactivated or its product
To induce specific immunity.Effective vaccination program can form the immunological memory energy for single-minded antigen in animal
Power, thus in the future with cause quick and strong immune response when antigen contact in animal.However, so far still there are many
Viral or bacteriosis there is no available vaccine, or not have the vaccine that can reach enough immunizations also.In addition, being permitted
Multi-vaccine because its antigen efficiency it is too low, using adjuvant inferior, to generate serious side effects, stability low or expensive
And it is not suitable for.Therefore, it needs to develop more effective vaccine or related adjuvant products.
By after the material mixing of antigen and referred to as adjuvant, immune system can be enhanced to antigen by being injected in animal body together
Immune resistance, it is described be known as adjuvant substance by directly act on immune system or by modify antigen pharmacokinetics
Learn feature come enhance animal body to field virus resist reaction, and whereby increase antigen and immune system interaction when
Between.For example, it is not enough to generate that immunogenicity, especially immunogenicity be low, molecule when animal is individually immunized in inactivation antigen
Measure small antigen, it is therefore desirable to the immune response induced with different types of adjuvant come enhancement antigen.However, domestic and international beast
Contain a large amount of mineral oil with the oily vaccine that production of vaccine quotient promotes and applies, while escorting for aviculture
Many food-safety problems are brought, for example, the vaccine residue problem of broiler chicken carcass has caused the load of more and more people
Sorrow.
Water-In-Oil (W/O) type oil vaccine is after being mixed by antigen liquid, interfacial agent and mineral oil, in high shear
It is prepared under power effect.Its immune mechanism is after inoculation position stores a period of time, and part generates granuloma and inflammation is anti-
It answers, attracts the aggregation such as macrophage, lymphocyte to identify antigen, generate antibody.Meanwhile the oily adjuvant slowly spread makes
The time that antigen is transported to secondary lymphoid organ extends, and constantly stimulates the immune system of body, renews long, antibody to generate
The high immune efficacy of level.Although the production technology of domestic and international production of vaccine producer is continuously improved, also constantly mentioned with oil quality
Height, but due to the oil-continuous phase of this preparation have the tendency that in injection site it is resident, in many situations, this w/o type oil
Matter vaccine generates a large amount of granulomas near inoculation position, and protection antibody generates speed and makes slowly, easily to animal carcass quality
At adverse effect, and it may cause violent pain during inoculation.
In addition, there is oil-in-water (O/W) type Adjuvanted vaccines injection site to show that local reaction is small and that stability is high is excellent
Gesture, however the antigen for including or being exposed in continuous phase disperses in injection site quickly and is decomposed by body fluid enzyme, it is difficult to it provides
Long-term protection antibody, it is often necessary to which special immunostimulant and multiple booster immunization, preparation cost is high, not extensive
It uses.
Therefore, occurred a kind of sustained release vaccine on the market in recent years, be to utilize phase can occur as temperature changes
Adjuvant of the temperature sensitive type in-situ gel of variation as vaccine.The stream that situ-gel is tended to appear as outside animal body for injection
Dynamic property solution state, antigen or adjuvant are uniformly distributed wherein；After injecting vaccine in animal body, solution state will receive animal
Temperature influence and injection site occur phase transition become poor fluidity gel state or solid state, Antigen distribution exists at this time
The drug-reservoir of slow release is formed in semi-solid gel, Antigen distribution forms slow release in semi-solid gel at this time
Drug-reservoir, such temperature sensitive type adjuvant have Water-In-Oil (W/O) type oil vaccine and oil-in-water (O/W) type Adjuvanted vaccines
Advantage, in addition to that can reduce administration number of times, improve patient compliance, enhancing and extend humoral immune response, raising cellular immunity
Response intensity and be conducive to immunological memory reaction generation other than, will not more cause serious inflammatory reaction.
However, temperature sensitive type adjuvant on the market is using polyvinyl alcohol as main component mostly at present, manufacturing process is numerous
Multiple and production cost is high, therefore is only applied to applicable object and is the vaccine of the mankind, and can not be widely used in Animal diseases use
Vaccine；In addition also having as chitosan is temperature sensitive type adjuvant obtained by principal component, but the class adjuvant is mostly with chitosan/sweet
Based on oleophosphoric acid sodium salt formula, it is formed by that gel mechanical performance is poor, and gel shaped state labile causes for disease
The clad ratio of malicious antigen liquid is bad, while it is unstable to also result in subsequent drug rate of release in vivo.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of that present invention to provide a kind of temperature sensitive type adjuvants, and the temperature sensitive type adjuvant is with shell
Glycan is main component and improves conventional chitosan/phosphoglycerol sodium salt formula, with good biocompatibility and in vivo
Degradability, and the production method of the temperature sensitive type adjuvant is simple and low in cost, can be widely used in Animal diseases
Vaccine.
The purpose of the present invention is achieved through the following technical solutions:
A kind of temperature sensitive type adjuvant comprising: the chitosan or chitosan derivatives of 20wt% to 60wt%；10wt% is extremely
The crosslinking agent of 40wt%；The colloid extract of 1.0wt% to 5.0wt%；The plasticizer of 1.0wt% to 5wt%；And remaining
For diluent；The immunogenicity of viral antigen in vivo can be improved in the temperature sensitive type adjuvant, to immune system stimulation
Time increases, and then can increase the memory immune effect of animal.
A viewpoint according to the present invention, in temperature sensitive type adjuvant of the invention, in order to improve the infection or increasing that prevent virus
Add vaccine stability and efficiency and achieve the effect that epidemic prevention, product can be made to reach good effect, in actually application, institute
The Blend proportion (Adg:Vat) for stating temperature sensitive type adjuvant (Adj) and viral antigen liquid (Vat) is 1:4 (v/v)~1:1 (v/v).Example
Such as, it can be 1:4 (v/ in the ratio of the temperature sensitive type adjuvant (Adj) of the invention and the Blend proportion of viral antigen liquid (Vat)
V)~1:1 (v/v)；In certain specific embodiments, preferably 1:4 (v/v)~2:3 (v/v)；More preferably 1:4 (v/v)~3:7
(v/v)；Most preferably 1:4 (v/v).
Viewpoint according to the present invention, the chitosan or chitosan derivatives can relative to the weight ratio of the crosslinking agent
To be 1:10~10:1, preferably 1:10~6:1 is more 1:10~2:1；Most preferably 1:10~1:3.
A viewpoint according to the present invention can be used and not add especially in the buffer of temperature sensitive type adjuvant of the invention
To limit, for example, the deacetylated degree of the chitosan is generally 50% to 100%；Preferably 60% to 100%；More
Good is 70% to 100%；Most preferably 80% to 100%.In addition, the molecular weight of the chitosan is generally 50,000~2,000,000；Compared with
Good is 100,000~2,000,000；More preferably 500,000~2,000,000；Most preferably 1,000,000~2,000,000.
A viewpoint according to the present invention, can be used in temperature sensitive type adjuvant of the invention the chitosan derivatives not
Especially it is limited；For example, the chitosan derivatives are selected from carboxymethyl chitosan, chitosan hydrochloride, chitosan cream
Hydrochlorate, chitosan acetate, chitosan sulfate rouge, chitosan quaternary amine, Chitosan poly oligosaccharide or chitin.
A viewpoint according to the present invention can be used and not add especially in the crosslinking agent of temperature sensitive type adjuvant of the invention
To limit；For example, the crosslinking agent in cellulose, polyacrylic acid, polymethylacrylic acid and polyacrylamide extremely
Few one kind；Preferably selected from least one of polyacrylic acid, polymethylacrylic acid and polyacrylamide；Most preferably polypropylene
Acid.
A viewpoint according to the present invention can be used and not add especially in the plasticizer of temperature sensitive type adjuvant of the invention
To limit；For example, the plasticizer can be glycerol, propylene glycol, ethylene glycol, diethylene glycol monoethyl ether, glyceride and
At least one of cithrol；Preferably at least one of glycerol, propylene glycol, ethylene glycol and glyceride；More
Good is at least one of glycerol, ethylene glycol and glyceride；Most preferably glycerol.
A viewpoint according to the present invention, can be used in the colloid extract of temperature sensitive shape adjuvant of the invention includes β-Portugal
Glycan.
A viewpoint according to the present invention, the viscosity of above-mentioned beta glucan are generally 4.0 to 7.0 mm2/s；It is preferably 4.5
To 7.0mm2The range of/s；More preferably 4.5 to 6.0mm2The range of/s；Most preferably 5.0 to 6.0mm2The range of/s.
A viewpoint according to the present invention, the average molecular weight of above-mentioned beta glucan are generally the model 16000 to 20000
It encloses；Preferably 17000 to 20000；More preferably 17500 to 20000；Most preferably 18000 to 20000.
A viewpoint according to the present invention, the pH value of above-mentioned beta glucan are generally 5 to 8；Preferably 5.5 to 8；More preferably
5.5 to 7；Most preferably 5.5 to 6.
A viewpoint according to the present invention, can be used can be into the colloid extract of temperature sensitive type adjuvant of the invention
One step includes aloe extract, cortex acanthopanacis extract, purslane extract, grapefruit extract, persimmon leaf extract, mulberry
At least one of yellow mushroom extract, cordyceps sinensis extract and its derivative；It the use of source is preferably aloe extract, five
Add skin extract, purslane extract, grapefruit extract, persimmon leaf extract, Phellinus mushroom extract and cordyceps sinensis extraction
Take at least one of object；It the use of source is more preferably aloe extract, cortex acanthopanacis extract, purslane extract and grape fruit
At least one of extract；Optimal use source is at least one of aloe extract and cortex acanthopanacis extract.
A viewpoint according to the present invention can be used and not add especially in the buffer of temperature sensitive type adjuvant of the invention
To limit, for example, the buffer source can be phosphate buffer, Tris-HCl buffer, sodium citrate-phosphorus
At least one of hydrochlorate buffer, Hepes, Hepes buffer, maleate buffer and its derivative；
Being suitable for the invention buffer and preferably using source is phosphate buffer, Tris-HCl buffer, sodium citrate-phosphoric acid
Salt buffer agent, Hepes, Hepes buffer or maleate buffer；It the use of source is more preferably phosphate-buffered
Agent, Tris-HCl buffer, sodium citrate-phosphate buffer or Hepes；Optimal use source is phosphate
Buffer.
A viewpoint according to the present invention can be used and resist in what the adjuvant of temperature sensitive type adjuvant of the invention was used cooperatively
Stoste is not limited especially, for example, it is anti-for example to can be malicious antigen of living, inactivation antigen, dead malicious antigen, secondary unit
Former or antigen nucleic acid；Being suitable for the invention antigen liquid preferably uses source for malicious antigen living or inactivation antigen；More preferably using next
Source is inactivation antigen.
In addition, a viewpoint according to the present invention can also provide a kind of preparation method of temperature sensitive type adjuvant, be include with
Lower step: by the chitosan of 20wt% to 60wt% or chitosan derivatives and 1.0wt% parts by weight to 5wt% parts by weight
Plasticizer is sufficiently mixed with the first operating condition, and being subsequently added into the crosslinking agent of 10wt% to 40wt%, persistently to stir 7 small
When more than and form constituent A；Then the colloid extract of 1.0wt% to 5.0wt% and diluent are operated into item in second
It is sufficiently mixed under part and obtains constituent B；And above-mentioned (b) resulting constituent B is slowly dropped into resulting group of above-mentioned (a)
At in object A, and it is sufficiently mixed obtains temperature sensitive type adjuvant in a third operating condition.
A viewpoint according to the present invention, the viscosity of the constituent B are 102MPas~108MPas, if constituent B
Viscosity be lower than 102MPas can make temperature sensitive shape adjuvant structural instability when mutually conversion forms gel, can not be effective
Antigen liquid is coated, if the viscosity of constituent B is higher than 108MPas can be too high to be released effectively in vivo because of viscosity
Antigen.Also, the viscosity of the constituent B is preferably 103MPas~108mPa·s；More preferably 104MPas~
108mPa·s；More preferably 105MPas~108mPa·s。
A viewpoint according to the present invention, wherein (a) first operating condition is included in 40~80 DEG C of temperature environment, stirring
Revolving speed 1200rpm~2400rpm and pH value are reacted 1~5 hour under conditions of being 5~8.
A viewpoint according to the present invention, wherein (b) the second operating condition be included in room temperature environment with 600rpm~
The speed of agitator of 1200rpm persistently stirs 2~3 hours.
A viewpoint according to the present invention, wherein (c) third operating condition is included at a temperature of 40~80 DEG C with 800rpm
The speed of agitator of~1200rpm persistently stirs 1~3 hour.
Then, the present invention will be further described in conjunction with specific embodiments.
Detailed description of the invention
Fig. 1 is partial size-temperature variation of temperature sensitive type adjuvant S1 to S3 of the invention.
Fig. 2 is that the data result for carrying out biphase equilibrium system test using temperature sensitive type adjuvant S1 to S3 of the invention compares
Figure.
Fig. 3 A is that comparative example 1 of the invention, embodiment 1, embodiment 2 and embodiment 3 are carrying out percutaneous absorbtion attachment effect
When fruit is tested, the pigskin M1 of mixed liquor P1 to P4 is not yet sprayed to the schematic picture on the surface M4.
Fig. 3 B is that comparative example 1 of the invention, embodiment 1, embodiment 2 and embodiment 3 are carrying out percutaneous absorbtion attachment effect
When fruit is tested, the pigskin M1 of mixed liquor P1 to P4 is sprayed to the result schematic picture on the surface M4.
Fig. 3 C is that comparative example 1 of the invention, embodiment 1, embodiment 2 and embodiment 3 are carrying out percutaneous absorbtion attachment effect
When fruit is tested, result schematic picture of the pigskin M1 of mixed liquor P1 to P4 to the surface M4 after 2 hours is sprayed.
Fig. 4 compares figure for comparative example 3-4 and embodiment 7-9 of the invention the power valence verification result for carrying out malicious vaccine living
Fig. 5 is that the power valence verification result of comparative example 5-6 of the invention and embodiment 10-12 progress inactivated vaccine compares figure
Specific embodiment
Hereinafter, enumerate different specific embodiments for embodiments of the present invention and more fully hereinafter describe and illustrate,
It is apparent to keep spirit of the invention more complete with content；However, those skilled in the art are it will be clear that the present invention
Certainly these examples are not only restricted to, also can reach the present invention using other same or impartial function and sequence of steps.
Firstly, for particular terms as used in this specification or the explanation of being described property of noun；However, following
Illustrate to be only to be illustrated, it is non-as limitation description of the invention and claim.Unless this specification is defined otherwise
In addition, the meaning of science and technology vocabulary used herein understands and usual meaning phase with those skilled in the art
Together.
As used herein, " vaccine " or " vaccine mixed liquor " is that can be used for inducing protective immunity in recipient
Composition.Therefore, after subject antigen inoculation, vaccine, which can prevent, delays or mitigate, is exposed to identical or related antigen
Subject disease development severity (subject relative to non-vaccine inoculation).By being protected provided by vaccine
Property it is immune can be (antibody-mediated) the immune and/or cellular immunity of body fluid.For example, vaccine inoculation can eliminate or reduce pathogen or
The load of infected cell, or mitigate any other measurable infection.Vaccine inoculation, which also can reduce to be immunized, (has been inoculated with epidemic disease
Seedling) subject tumor load.
As used herein, term " protective immunity " refers to when subject has been exposed to antigen, thus in subject
In cause to resist antigen immunity obtained when immune response (active/day after tomorrow and/or passive/congenital), so that antigen be made to lose
Living and/or reduction antigen load and formation immunological memory (such as memory T cell or B cell).
By protective immunity provided by vaccine inoculation may part or only a part of vaccine inoculation by
It is provided in inspection person.Therefore, vaccine can induce protective immunity in a part of immune population, because some individuals may be powerless
Establish strong immune response or protective immunological reaction or any immune response (in some cases).This ability may not enough return
Cause is in individual genetic background or is attributed to immunodeficiency symptom (day after tomorrow or congenital) or immunosupress (for example, due to warp
Regimen chemotherapy or using immunosuppressive drug with for example prevent organ rejection response or inhibit auto-immune pathologies).Xiang Yi
The vaccine that partial immunity group provides protection stands good and is considered as effectively.
As used herein, term " adjuvant " refers to when together with antigen administration, and antigen is immunized in enhancing subject
The compound of reaction.
" animal " of this paper term means all non-human animals, including mammal.
Herein, for the numerical value and parameter to define the scope of the invention, substantially inevitably containing because a
Standard deviation caused by other test method, thus indicated with rough quantitative value mostly, however in specific embodiment then
The correlation values of presentation reported as precisely as possible.Herein, " about " usually depending on the considering of those skilled in the art, usually
Reference table actual numerical value is fallen within the acceptable standard error of average value, for example, the actual numerical value is in a certain number
Within ± 10%, ± 5%, ± 1% or ± the 0.5% of value or range.
" preparation example 1 "
Firstly, by the chitosan of 2.0kg (deacetylated degree is 80%, molecular weight 80 ten thousand) and the glycerol one of 0.4kg
It rises and is added in allotment bottle, using blender (model: UHM-10, label: NIHONSEIKI Japan) with temperature in gnotobasis
45 DEG C, speed of agitator 1200rpm, pH value be 6.0 under conditions of, carry out reaction 2 hours；Confirmation adds after mixing
The polyacrylic acid of 4.0kg simultaneously continues stirring 8 hours, and then obtains constituent A1.
Then, as shown in table 1, by the aloe colloid extract of 0.1kg (1000g aloe is extracted using 70% alcohol,
The effective component that aloe colloid extract may further be extracted, the form that tincture is made use, concentration 18.8g/
Ml, and contain 20% or more barbaloin), the cortex acanthopanacis colloid extract of 0.2kg (1000g five is extracted using 70% alcohol
Add skin, the effective component of cortex acanthopanacis colloid extract may further be extracted, the form that tincture is made uses, and concentration is
18.8g/ml, and contain 20% or more eleutheroside) and 0.2kg β-glucan (viscosity 6.0mm2/ s, average mark
Son amount about 1.9 × 104And pH value is 5.5) to be added together in the phosphate buffer of 3.45kg, in room in gnotobasis
Constituent B1 was obtained with speed of agitator 800rpm uniform stirring 2 hours under temperature, and the viscosity of the constituent B 1 is 7.1
×106mPa·s。
Then, constituent B1 is slowly instilled in constituent A1 with dropper, and continue with ultrahigh speed homogenizer with
800rpm, 60 DEG C of condition homogeneous stir 1 hour, then obtain temperature sensitive type adjuvant S1 of the invention via condensation cooling.It connects
, using the particle diameter distribution of dynamic light scattering detection temperature sensitive type adjuvant S1 at 25 DEG C, 37 DEG C, 50 DEG C and 60 DEG C, calculate
It average grain diameter and is reported in Table 1 below out.
" preparation example 2 "
Firstly, by the chitosan of 4.0kg (deacetylated degree is 80%, molecular weight 80 ten thousand) and the glycerol one of 0.4kg
It rises and is added in allotment bottle, using blender (model: UHM-10, label: NIHONSEIKI Japan) with temperature in gnotobasis
45 DEG C, speed of agitator 1500rpm, pH value be 6.5 under conditions of, carry out reaction 2.5 hours；Confirmation adds after mixing
The polyacrylic acid of 2.5kg simultaneously continues stirring 7 hours, and then obtains constituent A2.
Then, as shown in table 1, by the aloe colloid extract of 0.1kg (1000g aloe is extracted using 70% alcohol,
The effective component that aloe colloid extract may further be extracted, the form that tincture is made use, concentration 18.8g/
Ml, and contain 20% or more barbaloin) and 0.3kg beta glucan (viscosity 4.5mm2/ s, average molecular weight are about
1.85 ×104And pH value be 6.5) together be added 2.3kg phosphate buffer in, in gnotobasis at room temperature with
Speed of agitator 600rpm uniform stirring 1.5 hours and obtain constituent B2, and the viscosity of the constituent B2 be 1.2 ×
107mPa·s。
Then, constituent B2 is slowly instilled in constituent A2 with dropper, and continue with ultrahigh speed homogenizer with
900rpm, 65 DEG C of condition homogeneous stir 1.5 hours, then obtain temperature sensitive type adjuvant S2 of the invention via condensation cooling.It connects
, using the particle diameter distribution of dynamic light scattering detection temperature sensitive type adjuvant S2 at 25 DEG C, 37 DEG C, 50 DEG C and 60 DEG C, calculate
It average grain diameter and is reported in Table 1 below out.
" preparation example 3 "
Firstly, by the chitosan of 6.0kg (deacetylated degree is 80%, molecular weight 80 ten thousand) and the glycerol one of 0.1kg
It rises and is added in allotment bottle, using blender (model: UHM-10, label: NIHONSEIKI Japan) with temperature in gnotobasis
50 DEG C, 1500 rpm of speed of agitator, pH value be 6.5 under conditions of, carry out reaction 3 hours；Confirmation adds after mixing
The polyacrylic acid of 1.0kg simultaneously continues stirring 10 hours, and then obtains constituent A3.
Then, as shown in table 1, by the aloe colloid extract of 0.2kg (1000g aloe is extracted using 70% alcohol,
The effective component that aloe colloid extract may further be extracted, the form that tincture is made use, concentration 18.8g/
Ml, and contain 20% or more barbaloin) and 0.05kg beta glucan (viscosity 4.0mm2/ s, average molecular weight are about
It is 1.69 × 104And pH value is 5.5) to be added together in the phosphate buffer of 2.65kg, in gnotobasis at room temperature
Obtain constituent B3 with speed of agitator 700rpm uniform stirring 2 hours, and the viscosity of the constituent B2 be 2.1 ×
106mPa·s。
Then, constituent B3 is slowly instilled in constituent A3 with dropper, and continue with ultrahigh speed homogenizer with
900rpm, 60 DEG C of condition homogeneous stir 2 hours, then obtain temperature sensitive type adjuvant S3 of the invention via condensation cooling.It connects
, using the particle diameter distribution of dynamic light scattering detection temperature sensitive type adjuvant S3 at 25 DEG C, 37 DEG C, 50 DEG C and 60 DEG C, calculate
It average grain diameter and is reported in Table 1 below out.
Table 1
Fig. 1 is the tendency chart that the average grain diameter of temperature sensitive type adjuvant S1, S2 and S3 vary with temperature.As shown in Figure 2, work as temperature
When degree increases, the particle size of temperature sensitive type adjuvant S1, S2 and S3 also can with increase, indicate temperature sensitive type adjuvant S1 of the invention,
The kenel of S2 and S3 can change with temperature change.
" biphase equilibrium system test "
By the obtained temperature sensitive type adjuvant S1 to S3 of above-mentioned preparation example 1 to 3 respectively with phosphate buffer (PBS) with such as
Volume ratio shown in table 2 is mixed and is heated, and the mixed liquor for observing different proportion is changed into the process of gel by liquid,
And phase transition temperature is reported in Table 2 below.
Table 2
Then, by the data information drafting pattern 2 of above-mentioned table 2.By the result of table 2 and Fig. 2 it is found that working as temperature sensitive type adjuvant
Weight ratio of the S1 to S3 in mixed liquor be 15% when, by liquid be changed into colloid phase transition temperature be respectively 43 DEG C,
42.5℃,45℃；When temperature sensitive type adjuvant is when weight ratio of the S1 to S3 in mixed liquor is 20%, glue is changed by liquid
The phase transition temperature of body is respectively at 37 DEG C, 36 DEG C, 37.5 DEG C；When weight ratio of the temperature sensitive type adjuvant in S1 to S3 in mixed liquor
It is respectively at 30 DEG C, 32.5 DEG C, 31 DEG C by the phase transition temperature that liquid is changed into colloid when being 25%；When temperature sensitive type adjuvant exists
It is respectively in 26 DEG C, 27 by the phase transition temperature that liquid is changed into colloid when weight ratio of the S1 to S3 in mixed liquor is 30%
℃, 26℃；When temperature sensitive type adjuvant is when weight ratio of the S1 to S3 in mixed liquor is 35%, colloid is changed by liquid
Phase transition temperature is respectively at 23 DEG C, 22 DEG C, 24 DEG C；When temperature sensitive type adjuvant is 40% in weight ratio of the S1 to S3 in mixed liquor
When, it is respectively at 21 DEG C, 20 DEG C, 22.5 DEG C by the phase transition temperature that liquid is changed into colloid；When temperature sensitive type adjuvant is in S1 to S3
It is respectively in 23 DEG C, 22 DEG C, 24 by the phase transition temperature that liquid is changed into colloid when weight ratio in mixed liquor is 35%
℃；When temperature sensitive type adjuvant is when weight ratio of the S1 to S3 in mixed liquor is 40%, it is changed into the phase alternating temperature of colloid by liquid
Degree is respectively at 21 DEG C, 20 DEG C, 22.5 DEG C；When temperature sensitive type adjuvant is when weight ratio of the S1 to S3 in mixed liquor is 45%,
It is respectively at 19 DEG C, 20 DEG C, 19 DEG C by the phase transition temperature that liquid is changed into colloid；When temperature sensitive type adjuvant is being mixed in S1 to S3
It is respectively at 17 DEG C, 18 DEG C, 16 DEG C by the phase transition temperature that liquid is changed into colloid when weight ratio in liquid is 50%.
Since the body temperature of general poultry is about at 37~40 DEG C or so, it is contemplated that the preserving type of vaccine and poultry are just
Chang Tiwen, therefore the ratio of the temperature sensitive type adjuvant (Adj) and the Blend proportion of viral antigen liquid (Vat) of the invention can be
About 1:4 (v/v)~1:1 (v/v)；In certain specific embodiments, preferably about 1:4 (v/v)~2:3 (v/v)；More preferably about
1:4 (v/v)~3:7 (v/v)；Most preferably about 1:4 (v/v).
" test of percutaneous absorbtion adhesion effect "
Firstly, take fresh porcine skin totally four of 4cmx4cm size, respectively pigskin M1 to M4, then by the pig hair on pigskin
It shaves, and is rinsed with clear water and remove subcutaneous tissue；Then it is irradiated with ultraviolet light flashlight and record of taking pictures, gained photo
As shown in Figure 3A.
Then, twice of temperature sensitive type adjuvant S1 is diluted by phosphate buffer (PBS), red fluorescent protein, with PBS
(2X), the stoste of five times of temperature sensitive type adjuvant S1 (5X) and temperature sensitive type adjuvant S1 is diluted with ratio as shown in table 3 with PBS
It is uniformly mixed, obtains mixed liquor P1 to P4, and mixed liquor P1 to P4 is uniformly sprayed to pigskin M1 respectively to be fitted into aerosol can
To the surface of M4；Then observation red fluorescent protein is irradiated with ultraviolet light flashlight and is attached to pigskin M1 to the surface M4
Situation and record of taking pictures, gained photo it is as shown in Figure 3B.
After pigskin M1 to M4 is statically placed in 2 hours of room temperature, then observation red fluorescence is irradiated with ultraviolet light flashlight
Albumen be attached to pigskin M1 to the situation on the surface M4 and record of taking pictures, gained photo it is as shown in Figure 3 C.
Table 3
It can confirm that mixed liquor P1 to P4 is all sprayed on the surface of pigskin M1 to M4 via spray bottle by the photo of Fig. 3 B,
But after two hours, the reaction of redfree fluoroprotein on the surface of pigskin M1 can be observed by the photo of Fig. 3 C, show
Show that red fluorescent protein is absorbed by pigskin M1 completely；And pigskin M2 still all is able to observe red fluorescent protein to M4
Reaction, and as the concentration of temperature sensitive type adjuvant is higher, the reaction of red fluorescent protein is stronger, shows of the invention temperature sensitive
Type adjuvant can effectively extend the time of drug release, have the function of sustained release.
The temperature sensitive type adjuvant S1 to S3 of 20ml is respectively put into beaker, and using homogenizer with the revolving speed of 3000rpm into
Then row stirring is slowly added into the 1mg/ml bovine serum albumen solution (bovine serum albumin weight is 80mg) of 80ml, when whole adds
Revolving speed 5000rpm is tuned into again after complete persistently to stir 10 minutes.
Then, the mixed liquor that stirring is completed is heated to 38 DEG C, so that it is frozen into colloid, and colloid is put into 10ml's
Slight wobble in PBS (7.4,10mM, 38 DEG C of pH) makes to remain in the bovine serum albumin that colloid surface does not coat into complete
It is dissolved in PBS, and is placed in UV spectrophotometer after clarified solution is separated, measure the absorption value at 280nm, and
The concentration that bovine serum albumin is conversed with the standard curve of bovine serum albumen solution will because the total volume of clarified solution is 10ml
Total volume is bovine serum albumin weight contained in clarified solution at upper concentration, and the calculation formula of clad ratio is as follows:
Calculated clad ratio is as shown in table 4.
In addition, remaining operating condition is in aforementioned phase using composition A1 obtained in preparation example 2 in comparative example 1
Together, and the clad ratio of calculation composition A1.
Table 4
By the result of above-mentioned table 4 it is found that for the cladding of bovine serum albumin when composition A1 being used only in comparative example 2
Rate only has 85.1%, and temperature sensitive type adjuvant S1, S2 and S3 used in embodiment 4 to 6 is for the packet of bovine serum albumin
The rate of covering is respectively 99.5%, 98.1% and 97.4%, all better than comparative example 2 as a result, be shown in addition colloid extract after
Temperature sensitive type adjuvant of the invention have good clad ratio, can will effectively by antigen cladding wherein, reduce material loss.
" the power valence verifying of poison vaccine living "
Firstly, by temperature sensitive type adjuvant S1 to S3 obtained in phosphate buffer (PBS), preparation example 1 to 3 with such as 4 institute of table
The ratio shown is respectively put into 2000 milliliters of beaker, and homogenizer is put into beaker, and the solution in beaker is allowed to be covered in
The stirring rod of matter machine up to after 2/3 (about 15 centimeters), rotation homogenizer (model: T.T-S.M-S, label: the big ancient cooking vessel in Taiwan is mechanical)
High shear is to 3000rpm, then slowly by the VII genotype Newcastle of ratio as shown in table 4 malicious antigen liquid living
(106.5TCID50/Ml it) is added in the solution in rotation；Finally, revolving speed can be tuned into 5000rpm when antigen liquid all adds, then
It carries out last mixing time five to ten minutes, poison vaccine V1 to V5 living is made.
Then, by above-mentioned obtained work poison vaccine V1 to V5 respectively with injected s.c. injection in immune 1 age in days chicken
Only, each embodiment injection 6, the vaccine dose of every chicken injection are 0.2ml, and separation of taking a blood sample after 1 week, 2 weeks and 3 weeks
Test serum.
Secondly, carrying out hemagglutination (hemagglutination；HA it) tests, i.e., each row on the U-shaped disk in 96 holes adds
The test serum of 25 μ l is added then at the 1st row for 0.85% physiological saline for entering 25 μ l.
Next after being sufficiently mixed using micropipet, take the 25 μ l of test serum of the 2nd row that the second hole is added, to take again
The 3rd row is added in 25 μ l, and with this serial dilution to the 10th row, 2 times to 1024 times dilutions are presented in antigen, and 25 μ l in the 11st row are mixed
Liquid is closed not use.Every row simultaneously repeats to test with the identical sample in 3 holes.
Then, contain 8HAU's into every 25 μ l dilution with HI buffering dilution agent VII genotype Newcastle antigen liquid
VII genotype Newcastle antigen liquid, and 25 μ l dilutions are added in each hole and stand 15 minutes.
Then, 50 μ L, 0.96% Red blood cell suspensions are added then at every hole, are stood interpretation blood after sixty minutes at room temperature
Whether ball has agglutination, and the still highest antigen diluent multiple with hamegglution is antibody power valence, then by each reality
The average value for applying the antibody power valence of every group of 6 chicken test results of example and comparative example is recorded in table 4 and drafting pattern 4 respectively.
Table 4
By the result of above-mentioned table 3 and Fig. 4 it is found that in comparative example 3, only do not generated in the chicken body of injection PBS anti-
Body, and in comparative example 4, injection using PBS as adjuvant made by the chicken of malicious vaccine V2 living intracorporal at the 1st week resist
Physical strength valence only rises to 64, be snapped down to 40 and 20 by week within latter 2nd week and the 3rd week.Relatively, in 7~embodiment of embodiment 9
In, the chicken of vaccine V3~V4 living malicious through injection added with temperature sensitive type adjuvant of the invention, intracorporal antibody power valence is the
128 are risen to rapidly within 1 week, though having downslide at the 2nd week and 3 weeks, its intracorporal antibody power valence is still compared with using PBS as assistant
Poison vaccine V2 high living made by agent.
" the power valence of inactivated vaccine is verified "
Firstly, by temperature sensitive type adjuvant S1 to S3 obtained in phosphate buffer (PBS), preparation example 1 to 3 with such as 4 institute of table
The ratio shown is respectively put into 2000 milliliters of beaker, and homogenizer is put into beaker, and the solution in beaker is allowed to be covered in
The stirring rod of matter machine up to after 2/3 (about 15 centimeters), rotation homogenizer (model: T.T-S.M-S, label: the big ancient cooking vessel in Taiwan is mechanical)
High shear is to 3000rpm, then slowly by ratio VII genotype Newcastle inactivation antigen liquid as shown in table 4
(108.5TCID50/ ml) it is added in the solution in rotation；Finally, revolving speed can be tuned into 5000rpm when antigen liquid all adds,
It carries out last mixing time five to ten minutes, inactivated vaccine X1 to X5 is made.
Then, by above-mentioned obtained inactivated vaccine X1 to X5 respectively with injected s.c. injection in an immune week old
Chicken, each Examples and Comparative Examples injection 6, then, second of injection in two week old.The vaccine dose of every chicken injection
Amount is 0.2ml, and separates test serum in blood sampling in 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks and 6 weeks.
Secondly, carrying out hemagglutination (hemagglutination；HA it) tests, i.e., each row on the U-shaped disk in 96 holes adds
Enter 0.85% physiological saline of 25 μ l, then carrys out the test serum that 25 μ l are added in the 1st row.
Next after being sufficiently mixed using micropipet, take the 25 μ l of test serum of the 2nd row that the second hole is added, to take again
The 3rd row is added in 25 μ l, and with this serial dilution to the 10th row, 2 times to 1024 times dilutions are presented in antigen, and 25 μ l in the 11st row are mixed
Liquid is closed not use.Every row simultaneously repeats to test with the identical sample in 3 holes.
Then, contain 8HAU's into every 25 μ l dilution with HI buffering dilution agent VII genotype Newcastle antigen liquid
VII genotype Newcastle antigen liquid, and 25 μ l dilutions are added in each hole and stand 15 minutes.
Then, 50 μ L, 0.96% Red blood cell suspensions are added then at every hole, are stood interpretation blood after sixty minutes at room temperature
Whether ball has agglutination, and the still highest antigen diluent multiple with hamegglution is antibody power valence, then by each reality
The average value for applying the antibody power valence of every group of 6 chicken test results of example and comparative example is recorded in table 5 and drafting pattern 5 respectively.
Table 5
By the result of above-mentioned table 5 and Fig. 5 it is found that in comparative example 5, only do not generated in the chicken body of injection PBS anti-
Body, and in comparative example 6, injection using PBS as adjuvant made by inactivated vaccine X2 chicken it is intracorporal anti-at the 1st week
Physical strength valence only rises to 64, persistently rise within latter 2nd week and start within the 400, the 3rd week to be snapped down to the 200, the 4th week to the 6th Zhou Zewei
It holds 180.Relatively, the malicious vaccine living in 3~embodiment of embodiment 5, through injection added with temperature sensitive type adjuvant of the invention
The chicken of X3~X4, intracorporal antibody power valence rose to rapidly 120 or more at the 1st week, had reached 371 in the 2nd Zhou Shigeng
More than, although starting slightly to glide after the 3rd week, at the 4th week to the 6th week, the intracorporal antibody power valence of chicken can still be maintained
500 or more.
It is thus possible to which immune guarantor can be increased using the animal of the vaccine of temperature sensitive type adjuvant of the invention through injection by confirming
Shield ability, and then have the effect of to the excellent of viral infection resisting.So temperature sensitive type adjuvant of the invention can be mentioned significantly
The immunity and immune cell propagation of high animal, enhancing vaccine induce protective immunological reaction on infecting disease to animal.
In conclusion the contents of the present invention are had been illustrated with embodiment as above, however the present invention not only limits
Due to these embodiments.Those skilled in the art without departing from the spirit and scope of the present invention, can carry out various again
Change and modification；For example, each technology contents illustrated by previous embodiment are combined or are changed and become new reality
Mode is applied, these embodiments are also considered as content belonging to the present invention certainly.Therefore, the range to be protected of this case also includes power
Sharp claim and its range defined.
Claims (10)
1. a kind of temperature sensitive type adjuvant, which is characterized in that be used to prepare animal disease vaccine and can change with ambient temperature and send out
Raw phase transition, the temperature sensitive type adjuvant include:
The chitosan or chitosan derivatives of 20wt% to 60wt%；
The crosslinking agent of 10wt% to 40wt%；
The colloid extract of 1.0wt% to 5.0wt%；
The plasticizer of 1.0wt% to 5wt%；And
Remaining is diluent；Wherein
The deacetylated degree of the chitosan is 50% to 100%, and molecular weight is 50,000~2,000,000；
The colloid extract includes beta glucan, and the viscosity of the beta glucan is 4.0 to 7.0mm2/ s, average molecular weight
It is 5 to 8 for 16000 to 20000 and pH value；And
In actually application, the Blend proportion (Adg:Vat) of the temperature sensitive type adjuvant (Adj) and viral antigen liquid (Vat) are 1:4
(v/v)~1:1 (v/v).
2. temperature sensitive type adjuvant according to claim 1, which is characterized in that the chitosan derivatives are carboxymethyl chitosan
Sugar, chitosan hydrochloride, chitosan lactate, chitosan acetate, chitosan sulfate rouge, chitosan quaternary amine, Chitosan poly oligosaccharide
Or chitin.
3. temperature sensitive type adjuvant according to claim 1, which is characterized in that the crosslinking agent be selected from cellulose, polyacrylic acid,
At least one of polymethylacrylic acid and polyacrylamide.
4. temperature sensitive type adjuvant according to claim 1, which is characterized in that the plasticizer is selected from glycerol, propylene glycol, second two
At least one of alcohol, diethylene glycol monoethyl ether, glyceride and cithrol.
5. temperature sensitive type adjuvant according to claim 1, which is characterized in that the colloid extract further includes aloe extraction
Take object, cortex acanthopanacis extract, purslane extract, grapefruit extract, persimmon leaf extract, Phellinus mushroom extract and worm summer in winter
At least one of careless extract.
6. temperature sensitive type adjuvant according to claim 1, which is characterized in that the diluent is selected from phosphate buffer
(PBS), Tris-HCl buffer, sodium citrate-phosphate buffer, Hepes, Hepes buffer and maleic acid
At least one of salt buffer agent.
7. a kind of preparation method of temperature sensitive type adjuvant, which comprises the following steps:
(a) by the increasing of the chitosan of 20wt% to 60wt% or chitosan derivatives and 1.0wt% parts by weight to 5wt% parts by weight
Mould agent, be sufficiently mixed with the first operating condition, be subsequently added into 10wt% to 40wt% crosslinking agent persistently stir 7 hours with
Above form constituent A；
(b) the colloid extract of 1.0wt% to 5.0wt% and diluent are sufficiently mixed under the second operating condition and obtain group
At object B, and the viscosity of the constituent B is 102MPas~108mPa·s；And
(c) above-mentioned (b) resulting constituent B is slowly dropped into above-mentioned (a) resulting constituent A, and in third operating condition
Under be sufficiently mixed and obtain temperature sensitive type adjuvant；Wherein the colloid extract includes beta glucan, and the beta glucan is glutinous
Degree is 4.0 to 7.0mm2/ s, average molecular weight are 16000 to 20000 and pH value is 5 to 8.
8. the preparation method of temperature sensitive type adjuvant according to claim 7, which is characterized in that the first operation in step (a)
Condition, which is included under conditions of 40~80 DEG C of temperature environment, speed of agitator 1200rpm~2400rpm and pH value is 5~8, reacts
1~5 hour.
9. the preparation method of temperature sensitive type adjuvant according to claim 7, which is characterized in that the second operation in step (b)
Condition includes persistently being stirred 2~3 hours in room temperature environment with the speed of agitator of 600rpm~1200rpm.
10. the preparation method of temperature sensitive type adjuvant according to claim 7, which is characterized in that the third operation in step (c)
Condition is included at a temperature of 40~80 DEG C, is persistently stirred 1~3 hour with the speed of agitator of 800rpm~1200rpm.
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| CN112402603A (en) * | 2020-12-02 | 2021-02-26 | 林建宏 | Temperature-sensitive adjuvant for aerosol immunization and preparation method thereof |
| TWI750929B (en) * | 2020-12-02 | 2021-12-21 | 林建宏 | Temperature-sensitive adjuvant for aerosol immunity and its preparation method |
| TWI772097B (en) * | 2021-07-08 | 2022-07-21 | 宏有投資有限公司 | Human thermosensitive adjuvant and preparation method thereof |
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| CN107551268A (en) * | 2016-06-30 | 2018-01-09 | 国年实业有限公司 | Birds Newcastle Disease vaccine adjuvant and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112402603A (en) * | 2020-12-02 | 2021-02-26 | 林建宏 | Temperature-sensitive adjuvant for aerosol immunization and preparation method thereof |
| TWI750929B (en) * | 2020-12-02 | 2021-12-21 | 林建宏 | Temperature-sensitive adjuvant for aerosol immunity and its preparation method |
| TWI772097B (en) * | 2021-07-08 | 2022-07-21 | 宏有投資有限公司 | Human thermosensitive adjuvant and preparation method thereof |
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