CN107537035A - Composite adjuvant and rabies vaccine containing composite adjuvant and its preparation method and application - Google Patents
Composite adjuvant and rabies vaccine containing composite adjuvant and its preparation method and application Download PDFInfo
- Publication number
- CN107537035A CN107537035A CN201710761727.5A CN201710761727A CN107537035A CN 107537035 A CN107537035 A CN 107537035A CN 201710761727 A CN201710761727 A CN 201710761727A CN 107537035 A CN107537035 A CN 107537035A
- Authority
- CN
- China
- Prior art keywords
- rabies vaccine
- adjuvant
- saponin
- cpgodn2006
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention relates to field of immunology, and in particular to composite adjuvant and the rabies vaccine containing composite adjuvant and its preparation method and application.The invention provides a kind of rabies vaccine composite adjuvant, and it includes Saponin and CpGODN2006, and the mass ratio of the Saponin and CpGODN2006 are 8:1‑1:10.Present invention also offers a kind of rabies vaccine composition, and it includes composite adjuvant and rabies vaccine, wherein, relative to 1/,100 1/10 parts of rabies vaccine, the Saponin is that the μ g of the 5 μ g 40 and CpGODN2006 are the μ g of 5 μ g 50.The rabies vaccine of the present invention can promote rabies vaccine to produce antibody and the antibody titer for improving anti-rabies vaccine as early as possible and can maintain the long period with composite adjuvant, in addition, the present invention rabies vaccine composition can inducing cellular immune reaction, it is possible to reduce rabies vaccine immune time.
Description
Technical field
The present invention relates to field of immunology, and in particular to composite adjuvant and containing the rabies vaccine of composite adjuvant and its preparation side
Method and application.
Background technology
Rabies (Rabies) are a kind of central nervous system acute infectious diseases of infecting both domestic animals and human as caused by rabies viruses,
It is the acute infectious disease for being only capable of preventing by vaccine and high rabbit anteserum, popularity is wide, case fatality rate 100%.The mad dog in the whole world 98%
Case concentrates on Asia, and China is rabic hotspot, and the eighties, average year number of the infected was 5530, and highest year is sent out
Patient's number is 7043.The dead major measure of human rabies morbidity is controlled to be treatment of wounds, be inoculated with rabies vacciness and injection
Anti-rabies immune serum or immunoglobulin.
Rabies vaccine (Rabies Vaccine (Vero Cell) for Human Use, Free-dried.) be earliest by
French microbiologist, chemist's Louis's Pasteur are prepared, and with the development of technology, Vaccines classes are increasingly
It is more.Rabies vaccine mainly after crazy animal bites are hurted sb.'s feelings implements immunoprophylaxis, i.e. virus infection formerly, is vaccinated rear, its
Effect is rescue the wounded's life, and virus was strangled within incubation period, blocks patient to enter the clinical onset phase.China makes now at present
There are refined VERO cell rabies and refined hamster kidney cell rabies vacciness.
Saponin (Saponin) is also known as saponin, is a kind of adjuvant, is one of a kind of component in immunostimulating complex, is
A kind of special glucoside of plant kingdom is widely present in, can even spend middle extraction to obtain from the crust of plant, leaf, stem, root.It
Lasting saponiform foam can be produced after aqueous solution shaking, there is bidirectional modulation immunization.Kensil separates from saponin
More than 10 kinds of individual saponins, wherein four kinds be saponin principal monomer, be respectively：Qs-7, Qs-17, Qs-18, Qs-21.Wherein
Numeral 7,17,18,21 represent using C4 posts reversed-phased high performace liquid chromatographic (RP-HPLC) elution time.
The non-CpG motifs to methylate are prevalent in DNA of bacteria, and have strong immunostimulation, CpGODN
Refer to the oligodeoxynucleotide containing cytosine-phosphate-guanine motif, the oligonucleotides of the artificial synthesized motif containing CpG
(CpGODN) immunostimulatory properties of CpG motifs in DNA of bacteria can be simulated, and the immune response based on Th1 types can be induced,
CpGODN can effectively induce body to produce immune response with the antigen combined application of protide, have potential immunologic adjuvant effect
Should.
Saponin or the artificial synthesized oligonucleotides containing CpG are injected or injected in advance simultaneously as adjuvant and antigen,
Can non-specifically strengthen or change the former immune response of body fight, but there is presently no on Saponin and
(sequence is CpGODN2006：TCGTCGTTTTGTCGTTTTGTCG) it is expelled to mouse simultaneously with rabies vaccine as composite adjuvant
In to induce the report of stronger humoral immune reaction and cell immune response.
The content of the invention
Therefore, the rabies vaccine the invention provides a kind of rabies vaccine composite adjuvant and containing the composite adjuvant,
When the rabies vaccine of the present invention carries out immune, stronger humoral immune reaction and cell immune response can be induced.
The invention provides a kind of rabies vaccine composite adjuvant, and it includes Saponin and CpGODN2006, described
Saponin and CpGODN2006 mass ratio is 8:1-1:10.
The invention provides a kind of preparation method of rabies vaccine composite adjuvant, its be by containing Saponin and
CpGODN2006 raw material is mixed.
The invention provides a kind of rabies vaccine composition, and it includes composite adjuvant and rabies vaccine, wherein, relative to mad
Canine vaccines 1/100-1/10 parts, the Saponin are that 5 μ g-40 μ g, CpGODN2006 dosage is 5 μ g-50 μ g.
The invention provides a kind of preparation method of rabies vaccine composition, and it is by containing Saponin, CpGODN2006
Mixed with the raw material of rabies vaccine.
Specifically, the present invention proposes following technical scheme.
A kind of rabies vaccine composite adjuvant, it includes Saponin and CpGODN2006, the Saponin with
CpGODN2006 mass ratio is 8:1-1:10.
Preferably, it is for described rabies vaccine composite adjuvant, the mass ratio of the Saponin and CpGODN2006
4:1-1:2, preferably 1:1.
A kind of preparation method for preparing rabies vaccine composite adjuvant, it is by containing Saponin and CpGODN2006
Raw material is mixed.
Preferably, for described preparation method, the mass ratio of the Saponin and CpGODN2006 are 8:1-1:10.
Preferably, for described preparation method, the mass ratio of the Saponin and CpGODN2006 are 4:1-1:2, it is excellent
Elect 1 as:1.
A kind of rabies vaccine composition, it includes composite adjuvant and rabies vaccine.
Preferably, it is described relative to rabies vaccine 1/100-1/10 parts for described rabies vaccine composition
Saponin be 5 μ g-40 μ g, the CpGODN2006 be 5 μ g-50 μ g, wherein, the rabies vaccine, Saponin and
CpGODN2006 dosage is the dosage of every mouse.
Preferably, for described rabies vaccine composition, relative to rabies vaccine 1/50-1/25 parts, the Saponin
It is 10 μ g-20 μ g for 10 μ g-40 μ g, the CpGODN2006, wherein, the rabies vaccine, Saponin and CpGODN2006
Dosage is every mouse more preferably dosage.It is furthermore preferred that the dosage of the Saponin is 10 μ g-20 μ g.
A kind of preparation method of rabies vaccine composition, it is by containing Saponin, CpGODN2006 and rabies vaccine
Raw material is mixed.
A kind of rabies vaccine parenteral solution, comprising above-mentioned rabies vaccine composition and dilution, the dilution is PBS,
The dosage of the PBS is 6.8-7.8ml.
Preferably, for described rabies vaccine parenteral solution, the rabies vaccine parenteral solution is intraperitoneal injection injection.
A kind of rabies vaccine composition, its application in mammalian immune response, preferably in people or in animal immune
Application in response.
Beneficial effect acquired by the present invention is：The present invention is immunized using the rabies vaccine containing composite adjuvant, is tied
Fruit shows that composite adjuvant can promote rabies vaccine to produce antibody and the antibody titer for improving anti-rabies vaccine as early as possible and can maintain
Long period, in addition, the present invention rabies vaccine composition can inducing cellular immune reaction, it is possible to reduce rabies vaccine is immunized
Number.
Brief description of the drawings
Fig. 1 is the anti-rabies vaccine of serum for implementing low dosage composite adjuvant, high dose composite adjuvant and control group PBS in seven
Antibody level detection OD450nm time history plots；
Fig. 2 is that the anti-rabies vaccine antibody titer of mice serum of all experiment packets in embodiment seven changes over time signal
Figure；
Fig. 3 is the schematic diagram that the IFN-γ of all dosage groups in embodiment seven changes over time.
Embodiment
Fig. 1 is the anti-rabies vaccine of serum for implementing low dosage composite adjuvant, high dose composite adjuvant and control group PBS in seven
Antibody level detection OD450nm time history plots, wherein low dosage composite adjuvant is 10 μ g Saponin+10
μg CpGODN2006；High dose composite adjuvant is 20 μ g Saponin+20 μ g CpGODN2006, it can be seen that single
Pure adjuvant immunity mouse does not induce the antibody of rabies vaccine antigen-specific to produce, because of same time point, adjuvant immunity mice serum
IgG antibody level and PBS immune group no difference of science of statistics.
Fig. 2 is that the anti-rabies vaccine antibody titer of mice serum of all experiment packets in embodiment seven changes over time signal
Figure, wherein, A:1/50 person-portion rabies vaccine；B：1/25 person-portion rabies vaccine；C:The 1/50 person-portion rabies vaccine+compound assistant of low dosage
Agent；D：1/50 person-portion rabies vaccine+high dose composite adjuvant；E：1/25 person-portion rabies vaccine+low dosage composite adjuvant；F：1/25
Person-portion rabies vaccine+high dose composite adjuvant.
From figure 2 it can be seen that exempting from 14d antibody titers at the beginning of A groups reaches highest, exempt from 12d antibody titers at the beginning of B groups and reach most
Height, exempt from 10d antibody titers at the beginning of C-F groups and reach higher level：Wherein exempt from 35d antibody titers at the beginning of C groups and reach highest, exempt from the beginning of D groups
14d antibody titers reach highest, exempt from 12d antibody titers at the beginning of E-F groups and reach highest.
Fig. 3 is the schematic diagram that the IFN-γ of all dosage groups in embodiment seven changes over time, wherein, A:PBS control；B：
1/50 person-portion rabies vaccine；C：1/25 person-portion rabies vaccine；D:Low dosage composite adjuvant (10 μ g Saponin+10 μ g
CpGODN2006)；E:High dose composite adjuvant (20 μ g Saponin+20 μ g CpGODN2006)；F:1/50 person-portion rabies vaccine
+ low dosage composite adjuvant；G：1/50 person-portion rabies vaccine+high dose composite adjuvant；H：1/25 person-portion rabies vaccine+low dosage is multiple
Combination adjuvant；I：1/25 person-portion rabies vaccine+high dose composite adjuvant.
As can be seen from the figure same immune different time detection secretion of gamma-IFN spot formation cell number (IFN-γ-
SFC change), the FN- γ-SFC that A-D groups, G-H groups are formed after just exempting from 8 days are more, what E groups were formed after just exempting from 4 days
IFN-γ-SFC is more, and the IFN-γ-SFC that F groups are formed after just exempting from 14 days is more, what I groups were formed after just exempting from 14 days
IFN-γ-SFC is more, and the IFN-γ-SFC that F-I groups are formed after just exempting from 35 days maintains higher level.
Term " 1 person-portion " refers to a people once dosage used.
As described above, the invention provides a kind of rabies vaccine composite adjuvant, rabies vaccine composite adjuvant bag
Containing Saponin and CpGODN2006, the mass ratio of the Saponin and CpGODN2006 are 8:1-1:10.
In another embodiment, the invention provides a kind of rabies vaccine composite adjuvant, the mad dog epidemic disease
Seedling includes Saponin and CpGODN2006 with composite adjuvant, and the mass ratio of the Saponin and CpGODN2006 are 4:1-1:2,
Preferably 1:1.
The invention provides a kind of preparation method of rabies vaccine composite adjuvant, its be by containing Saponin and
CpGODN2006 raw material is mixed.
Present invention also offers a kind of rabies vaccine composition, it includes composite adjuvant and rabies vaccine, and relative to
Rabies vaccine is 1/100-1/10 parts, and the Saponin is that 5 μ g-40 μ g, the CpGODN2006 are 5 μ g-50 μ g.Preferably,
It is 1/50-1/25 parts relative to rabies vaccine, the Saponin is that 10 μ g-40 μ g, the CpGODN2006 are 10 μ g-20 μ g,
More there is choosing, be 1/50 part or 1/25 part relative to rabies vaccine, the Saponin is 10 μ g or 20 μ g, described
CpGODN2006 is 10 μ g or 20 μ g.
The invention provides a kind of preparation method of rabies vaccine composition, and it is by containing Saponin, CpGODN2006
Solution is mixed to get with the raw material of rabies vaccine.
The present invention is by Saponin, CpGODN2006 and antigen wiring solution-forming, is then diluted, first adds dilution,
Then Saponin, CpGODN2006 and antigen are sequentially added, the solution diluted.
WO0009159 discloses CpG and Saponin composition and preparation method thereof, is used disclosed in specification
CpGODN1826 is immunized as the monomer QS-21 adjuvants in adjuvant, Saponin and OVA vaccines, but CpGODN1826
Sequence is TCCATGACGTTCCTGACGTT, and the effect of the CpGODN1826 is activation and I type of the inducing antigen in delivery cell
Activation [Ank MB.et al., the 2008.An important role for type III of IFN antiviral activities
interferon(IFN-lambda/IL-28)in TLR-induced antiviral activity.J Immunol 180:
2474-85.], show Th1 immune response；CpGODN2006 sequence is TCGTCGTTTTGTCGTTTTGTCG,
CpGODN2006 can significantly increase antigen-specific antibodies reaction and interferon mediation cellular immunity [Zhang Y.et al.,
2003.CpG ODN 2006and IL-12are comparable for priming Th1 lymphocyte and IgG
responses in cattle immunized with a rickettsial outer membrane protein in
alum.Vaccine21:3307-18.], therefore, CpGODN1826 is different from the CpGODN2006 of present invention sequence, rises
Effect is also different.
Below to the raw material used in the present embodiment and the manufacturer of equipment, and the equipment that uses of product analysis and analysis
Method is described as follows, wherein described chemical substance do not indicate be conventional reagent chemical pure rank.Wherein, it is real
Apply the raw material used in example information and experimental facilities information as shown in Table 1 and Table 2.
The information of raw material used in the present invention of table 1
The information of experimental facilities used in the present invention of table 2
| Equipment | Model | Producer |
| Centrifuge | TG16-WS table model high speed centrifuges | The ordinary instrument and meter Co., Ltd in Changsha |
| Centrifuge | L-530 | Hunan instrument centrifuge |
| ELIASA | Infinite M200 | Switzerland TECAN |
| ELISpot plate reader | S5-Verse | CTL companies of the U.S. |
Embodiment one
1. a kind of rabies vaccine composite adjuvant, comprising Saponin and CpGODN2006, be by Saponin with
CpGODN2006 mass ratio is 1:10 are obtained by mixing.
2. a kind of rabies vaccine composition, it includes Saponin, CpGODN2006 and rabies vaccine, in terms of every mouse,
5 μ g Saponin, 50 μ g CpGODN2006 and 1/10 person-portion rabies vaccine are obtained by mixing.
Embodiment two
1. a kind of rabies vaccine composite adjuvant, comprising Saponin and CpGODN2006, be by Saponin with
CpGODN2006 mass ratio is 8:1 is obtained by mixing.
2. a kind of rabies vaccine composition, it includes Saponin, CpGODN2006 and rabies vaccine, in terms of every mouse,
40 μ g Saponin, 5 μ g CpGODN2006 and 1/100 person-portion rabies vaccine are obtained by mixing.
Embodiment three
1. a kind of rabies vaccine composite adjuvant, comprising Saponin and CpGODN2006, be by Saponin with
CpGODN2006 mass ratio is 1:1 is obtained by mixing.
2. a kind of rabies vaccine composition, it includes Saponin, CpGODN2006 and rabies vaccine, in terms of every mouse,
10 μ g Saponin, 10 μ g CpGODN2006 and 1/25 person-portion rabies vaccine are obtained by mixing.
Example IV
1. a kind of rabies vaccine composite adjuvant, comprising Saponin and CpGODN2006, be by Saponin with
CpGODN2006 mass ratio is 1:1 is obtained by mixing.
2. a kind of rabies vaccine composition, it includes Saponin, CpGODN2006 and rabies vaccine, in terms of every mouse,
20 μ g Saponin, 20 μ g CpGODN2006 and 1/50 person-portion rabies vaccine are obtained by mixing.
Embodiment five
1. a kind of rabies vaccine composite adjuvant, comprising Saponin and CpGODN2006, be by Saponin with
CpGODN2006 mass ratio is 1:1 is obtained by mixing.
2. a kind of rabies vaccine composition, it includes Saponin, CpGODN2006 and rabies vaccine, in terms of every mouse,
20 μ g Saponin, 20 μ gCpGODN2006 and 1/25 person-portion rabies vaccine are obtained by mixing.
Embodiment six
1. a kind of rabies vaccine composite adjuvant, comprising Saponin and CpGODN2006, be by Saponin with
CpGODN2006 mass ratio is 1:1 is obtained by mixing.
2. a kind of rabies vaccine composition, it includes Saponin, CpGODN2006 and rabies vaccine, in terms of every mouse,
10 μ g Saponin, 10 μ g CpGODN2006 and 1/50 person-portion rabies vaccine are obtained by mixing.
Implement seven compliance test results
1. the humoral immune reaction of adjuvant rabies vaccine induction
(1) experimental animal and packet
BALB/c mouse, female, every mouse 18-20g.It is divided into 12 groups, every group of 30 mouse.
(2) antigen and adjuvant
Antigen：Human lyophilized rabies vaccine (Vero cells)
Adjuvant：Saponin, CpGODN2006
(3) it is grouped
Rabies vaccine by specification dissolves rabies vaccine, and 0.5ml waters for injection dissolve 1 person-portion rabies vaccine,
CpGODN2006 concentration is 2mg/ml, and Saponin concentration is 10mg/ml.This is the concentrated solution of three kinds of solution, then by it
It is diluted, that is, adds dilution PBS, then sequentially add Saponin, CpGODN2006 and antigen, the solution diluted.
Low dosage antigen group：0.4ml rabies vaccines+7.6mlPBS；(1/50 person-portion rabies vaccine/mouse)
High dose antigen group：0.8ml rabies vaccines+7.2mlPBS；(1/25 person-portion rabies vaccine/mouse)
Low dosage composite adjuvant group：0.2mlCpGODN2006+0.04mlSaponin+7.8mlPBS；
High dose composite adjuvant group：0.4mlCpGODN2006+0.08mlSaponin+7.6mlPBS；
Low dosage antigen+low dosage composite adjuvant group：
0.4ml rabies vaccines+0.2mlCpGODN2006+0.04mlSaponin+7.4mlPBS；
Low dosage antigen+high dose composite adjuvant group：
0.4ml rabies vaccines+0.4mlCpGODN2006+0.08mlSaponin+7.2mlPBS；
High dose antigen+low dosage composite adjuvant group：
0.8ml rabies vaccines+0.2mlCpGODN2006+0.04mlSaponin+7.0mlPBS；
High dose antigen+high dose composite adjuvant group：
0.8ml rabies vaccines+0.4mlCpGODN2006+0.08mlSaponin+6.8mlPBS；
PBS control group：8mlPBS.
(4) mixture of corresponding antigen and antigen and adjuvant is immunized respectively, is immunized 2 times in 0 day, intraperitoneal injection in 7 days.
(5) mouse dissection and serum obtain
4 days, 8 days, 10 days, 12 days, 14 days, 35 days after just exempting from, every group is dissected 5 mouse respectively, is plucked eyeball and is taken blood, room temperature
Place 3 hours, 3000 revs/min of centrifugation 10min, take supernatant, 12000 revs/min of centrifugation 10min of supernatant, take -30 DEG C of preservations of supernatant.
(6) IgG antibody in indirect elisa method (enzyme-linked immunosorbent assay) measure serum
Dissecting every time for every group selects 1 different dilution factor of mouse dilution to be detected, and determines that different anatomic time difference is exempted from
The antibody titer scope of mouse is immunized in epidemic focus, and every group of each time point dilutes 2-3 concentration side serum OD value, and its step is as follows：
A. antigen coat：Every lyophilized rabies vaccine is dissolved using 0.5ml waters for injection, coating buffer 1:300 times of dilutions
Rabies vaccine, 100 μ l are added per hole, 4 DEG C of coatings are overnight；
B. board-washing：3min/ times, wash altogether three times, washing lotion PBST, the PBS as containing 0.05%TWEEN20 (uses hand
Wash)；
C. close：200 μ l confining liquids (PBST for containing 1% bovine serum albumin (BSA)), 37 DEG C of closing 1h are added per hole；
D. board-washing：Board-washing is carried out using with step (b) identical method；
E. serum to be checked is diluted with confining liquid, and dilution ratio is：PBS groups, low dosage adjuvant group and high dose adjuvant group, often
The serum that individual time point dissection mouse is collected is 1:100 dilutions；Just exempt from each group mice serum of latter 4 days also 1:100 dilutions；Just
8 days after exempting from：Low, high dose mice immunized with antigen serum 1:100、1:500 and 1:Mouse 1 is immunized in 1000 dilutions, antigen adjuvant:
100、1:100 and 1:2000 dilutions；Collect serum within 10 days after just exempting from：Low, high dose mice immunized with antigen serum 1:100、1:
4000 and 1:8000 dilutions, antigen adjuvant immune serum 1:8000、1:16000 and 1:20000 dilutions；12 days after just exempting from,
14 days, 35 days：Low, high dose mice immunized with antigen serum 1:8000 and 1:10000 dilutions, antigen adjuvant immune serum 1:
20000 and 1:30000 dilutions；100 μ l serum dilutions are added per hole, set two multiple holes, 37 DEG C of incubations per dilution factor per sample
2h；
F. board-washing：Board-washing is carried out using with step (b) identical method；
G. the rabbit antigen mouse IgG secondary antibodies (1 of confining liquid dilution HRP marks:30000) 100 μ l, 37 DEG C of incubations, are added per hole
1h；
H. board-washing：Board-washing is carried out using with step (b) identical method；
I. the μ l TMB nitrite ions of nitrite ion 100 are added per hole, lucifuge room temperature places 20-30min, adds 50 μ l terminate liquids
(2N H2SO4) color development stopping, survey OD450nm light absorption values.
(7) result
A. the level of serum is determined
Measurement result is shown in Table 3, wherein, it is the flat of every group of 5 mice serum antibody test OD450nm that data in table, which represent,
The data of mean value ± error amount, low dosage adjuvant and high dose adjuvant are as shown in Figure 1.
Table 3 is exempted from 4 days at the beginning of being shown and the level (serum 1 of 8 days antibody serums:100 dilutions)
| Group | 4d | 8d | 4d is compared with 8d |
| PBS control group | 0.094±0.014 | 0.099±0.043 | T=-0.29, P=0.78 |
| Low dosage antigen group | 0.096±0.007 | 0.360±0.199 | T=-2.96, P=0.042 |
| High dose antigen group | 0.116±0.013 | 0.665±0.148 | T=-8.28, P=0.004 |
| Low dosage antigen+low dosage adjuvant group | 0.132±0.015 | 1.076±0.369 | T=-5.71, P=0.005 |
| Low dosage antigen+high dose adjuvant group | 0.146±0.045 | 1.058±0.431 | T=-4.71, P=0.009 |
| High dose antigen+low dosage adjuvant group | 0.161±0.019 | 1.476±0.523 | T=-5.60, P=0.005 |
| High dose antigen+high dose adjuvant group | 0.146±0.047 | 1.039±0.799 | T=-3.35, P=0.044 |
Compared with control group PBS：4 days after just exempting from, low dosage antigen+high dose adjuvant group, high dose antigen+low dosage
The equal ＜ 0.01 of P values of adjuvant group and high dose antigen+high dose adjuvant group, shows extremely significant difference, low dosage antigen+low
The P values ＜ 0.05 of dosage adjuvant group, assumes a marked difference；It is low dosage antigen+low dosage adjuvant group, low 8 days after just exempting from
The P values of doses of antigen group+high dose adjuvant group, high dose antigen+low dosage adjuvant group and high dose antigen+high dose adjuvant group
Equal ＜ 0.01, shows extremely significant difference.
Compared with low dosage antigen group：4 days after just exempting from, low dosage antigen+high dose adjuvant group, high dose antigen+
The equal ＜ 0.01 of P values of low dosage adjuvant group and high dose antigen+high dose adjuvant group, shows extremely significant difference；Exempt from just
8 days afterwards, the P ＜ 0.01 of high dose antigen+low dosage adjuvant group, show extremely significant difference, low dosage antigen+low dosage assistant
The equal ＜ 0.05 of P values of agent group, low dosage antigen+high dose adjuvant and high dose antigen+high dose adjuvant, it is significant poor to show
It is different.
Compared with high dose antigen group：4 days, the P ＜ 0.05 of high dose antigen+low dosage adjuvant group after just exempting from, table
Reveal significant difference；8 days, the P ＜ 0.01 of high dose antigen+low dosage adjuvant group after just exempting from, it is extremely significant poor to show
It is different.
Table 3 shows the immune mouse 4d antibody levels of rabies vaccine, and statistically there was no significant difference with control group, and helps
The antibody level of serum that mouse 4d is immunized in agent rabies vaccine is all remarkably higher than PBS control group, but aggregate level is relatively low；The mad dog of adjuvant
Vaccine immune mouse, 8d antibody level of serum are significantly higher than PBS control group, at the same rabies vaccine and adjuvant rabies vaccine be immunized it is small
Mouse antibody level of serum is significantly higher than the mice serum level for just exempting from 4d.Therefore, adjuvant promotes rabies vaccine to produce antibody as early as possible,
And enhancing antibody level.
From figure 1 it appears that simple adjuvant immunity mouse does not induce the antibody of rabies vaccine antigen-specific to produce, because of phase
Same time point, adjuvant immunity mice serum IgG antibody level and PBS immune group no difference of science of statistics.
B. adjuvant improves rabies vaccine antibody titer and maintains the long period
Detection at twice, just exempts to collect Virus monitory 1 time in 4 days, 8 days, 10 days, just exempts to collect serum in 12 days, 14 days, 35 days
Detection 1 time；With PBS groups (the totally 15 mouse data) immune serum 1 detected every time:100 dilution survey OD average values ±
2SD values are standard, determine Titer of serum IgG antibody.Wherein, A is 1/50 person-portion rabies vaccine (low dosage antigen group), B 1/
25 person-portion rabies vaccines (high dose antigen group), C are the μ g Saponin/10 μ g CpGODN2006 of 1/50 person-portion rabies vaccine+10
(low dosage antigen+low dosage composite adjuvant group), D are the μ g Saponin/20 μ g of 1/50 person-portion rabies vaccine+20
CpGODN2006 (low dosage antigen+high dose composite adjuvant group), E are the μ g Saponin/10 μ g of 1/25 person-portion rabies vaccine+10
CpGODN2006 (high dose antigen+low dosage composite adjuvant group), F are the μ g Saponin/20 μ g of 1/25 person-portion rabies vaccine+20
CpGODN2006 (high dose antigen+high dose composite adjuvant group).
As can be seen from Figure 2：Exempt from 14d antibody titers at the beginning of A groups and reach highest, exempt from 12d antibody titers at the beginning of B groups and reach highest,
Exempt from 10d antibody titers at the beginning of C-F groups and reach higher level：Wherein exempt from 35d antibody titers at the beginning of C groups and reach highest, exempt from 14d at the beginning of D groups
Antibody titer reaches highest, exempts from 12d antibody titers at the beginning of E-F groups and reaches highest.
Antibody titer compares between just exempting from 8d each groups：C-F group mice serum antibody titers are all remarkably higher than A and B groups (P<
0.01), A groups and B group mice serum antibody titers and C-F group mice serums antibody titer two-by-two compared with, difference is without statistics
Learn meaning (P>0.05).Antibody titer compares between just exempting from 10d each groups：C-F group mice serum antibody titers are all remarkably higher than A
Group, B groups (P<0.01).Antibody titer compares between just exempting from 12d each groups：D-F group mice serum antibody titers are all remarkably higher than A groups
(P<0.05 or P<0.01), E-F groups mice serum antibody titer is significantly higher than B groups and C groups (P<0.05 or P<0.01), D groups are small
Mouse serum antibody titer is substantially less than F groups (P=0.004).Antibody titer compares between just exempting from 14d each groups：D-F group mice serums
Antibody titer is all remarkably higher than A groups and B groups (P<0.05 or P<0.01), C groups mice serum antibody titer is substantially less than D groups (P
=0.004) and F groups (P=0.039).Antibody titer compares between just exempting from 35d each groups：C-F group mice serums antibody titer is aobvious
Work is higher than A groups and B groups (P<0.01).
2. the cell immune response of adjuvant rabies vaccine induction
(1) experimental animal and packet
BALB/c mouse, female, every mouse 18-20g.It is divided into 12 groups, every group of 20 mouse.
(2) antigen and adjuvant
Antigen：Human lyophilized rabies vaccine (Vero cells)
Adjuvant：Saponin, CpGODN2006
(3) it is grouped
Rabies vaccine by specification dissolves rabies vaccine, and 0.5ml waters for injection dissolve 1 person-portion rabies vaccine,
CpGODN2006 concentration is 2mg/ml, and Saponin concentration is 10mg/ml.
High dose antigen group：0.8ml rabies vaccines+7.2mlPBS；
Low dosage antigen group：0.4ml rabies vaccines+7.6mlPBS；
Low dosage adjuvant group：0.2mlCpGODN2006+0.04mlSaponin+7.8mlPBS；
High dose adjuvant group：0.4mlCpGODN2006+0.08mlSaponin+7.6mlPBS；
Low dosage antigen+low dosage adjuvant group：
0.4ml rabies vaccines+0.2mlCpGODN2006+0.04mlSaponin+7.4mlPBS；
Low dosage antigen+high dose adjuvant group：
0.4ml rabies vaccines+0.4mlCpGODN2006+0.08mlSaponin+7.2mlPBS；
High dose antigen+low dosage adjuvant group：
0.8ml rabies vaccines+0.2mlCpGODN2006+0.04mlSaponin+7.0mlPBS；
High dose antigen+high dose adjuvant group：
0.8ml rabies vaccines+0.4mlCpGODN2006+0.08mlSaponin+6.8mlPBS；
PBS control group：8mlPBS.
(4) mixture of corresponding antigen and antigen and adjuvant is immunized respectively, in 0 day, 7 days peritoneal immunities 2 times.
(5) mouse is dissected
4 days, 8 days, 14 days, 35 days after just exempting from, every group is dissected 5 mouse respectively, is plucked eyeball and is taken blood, and disconnected neck execution is small
Mouse, 2-3min in 75% ethanol is soaked in, the sterile excision spleen in super-clean bench.
(6) ELISpot detects
1) splenic lymphocytes are prepared：Added in 35mm culture dishes 4-5ml mouse lymphocytes separating liquids (Mouse
Lymph separating liquid, it is biological Engineering Co., Ltd up to section, to room temperature and is shaken up using preceding recovery), 200 mesh nylon wires are placed one
Secondary property culture dish, spleen are placed on nylon wire, and scissors shreds, and spleen is ground with 5ml syringe pistons, thin after nylon net filter
Born of the same parents' suspension is immediately transferred in 15ml centrifuge tubes, covers 500 μ l-1ml RPMI-1640 culture mediums；Room temperature, 800g (L350 from
Scheming, about Hunan Xiang Yi Instrument Ltd., 2140 turns) centrifugation 30min (reduction of speed is 2 grades), buffy coat is suctioned out, then add
Enter 10-12ml RPMI-1640 culture mediums, overturn and mix.(1200 turns) centrifugation 10min of room temperature 250g, topple over supernatant, with nothing
Blood serum medium (being biological Engineering Co., Ltd up to section) 0.5ml suspension cells, diluting cells, counting, adjustment cell concentration is extremely
2.5×106/ml。
2) ELISpot (ELISPOT) detects：According to pre-coated Mouse IFN-γ ELISpot PLUS kit
(ALP) the spot SFC of kit (Mabtech, Sweden) specification detection splenic lymphocytes secretion of gamma-IFN, concrete operations：(1)
The PBS board-washings of 0.2 μm of membrane filtration 5 times, the RPMI-1640 culture mediums that 200 μ l contain 10% hyclone are added per hole, and room temperature is incubated
Educate the plate after 30min or PBS washings and directly add cell culture.(2) culture medium is discarded, adds 100 μ l lymphocytes suspensions and 50 μ
The rabies vaccine or CoA (final concentration of 5 μ g/ml) of the people's dosage of l 1/50, negative control adds 50 μ l serum free mediums.5%
CO2, 37 DEG C be incubated 48h.(3) develop the color：Microwell plate is taken out, abandons liquid, 200 μ l PBS (0.22 μm of filtering) board-washing is added per hole 5 times,
100 μ l detection antibody is sequentially added per hole, is incubated at room temperature 2h.Solution in hole is discarded, ibid board-washing 5 times, 100 are sequentially added per hole
μ l Streptavidin-ALP, it is incubated at room temperature 1h.Solution in hole is discarded, ibid board-washing 5 times, add 100 μ l BCIP/NBT bottoms
Thing solution (0.45 μm filtering), room temperature place 10-30min, and after spot to appear, with each hole of distillation water washing, microwell plate is put
Ventilation lucifuge is dried.(4) SFC is detected：ELISpot plate reader (CTL, the U.S.) read plate, automatically analyzes IFN-γ
SFC.Measurement result is shown in Table 4.
The SFC of the secretion of gamma-IFN of table 4
As can be seen from the above table, 8 days, 14 days and 35 days after immune, compared with control group PBS, low dosage antigen+high agent
Adjuvant, the equal ＜ 0.01 of the P values of high dose antigen+low dosage adjuvant and high dose antigen+high dose adjuvant are measured, is shown extremely notable
Difference；After immune in 8 days, the P values of low dosage antigen group, high dose plateau group and low dosage antigen+low dosage adjuvant are equal
＜ 0.01, extremely significant difference is shown, after immune in 35 days, the P values ＜ 0.05 of low dosage antigen+low dosage adjuvant, table
Reveal significant difference.
Compared with low dosage antigen group, after immune in 14 days, 35 days, the equal ＜ of P values of high dose antigen+high dose adjuvant
0.01, extremely significant difference is shown, after immune in 14 days, low dosage antigen+low dosage adjuvant, high dose antigen+low dose
The equal ＜ 0.01 of P values of adjuvant is measured, shows extremely significant difference, after immune in 35 days, low dosage antigen+high dose adjuvant,
The equal ＜ 0.05 of P values of high dose antigen+low dosage adjuvant, assumes a marked difference.
Compared with high dose antigen, after immune in 14 days, 35 days, high dose antigen+low dosage adjuvant, high dose antigen
The equal ＜ 0.01 of P values of+high dose adjuvant, shows extremely significant difference, after immune in 14 days, low dosage antigen+low dosage
The P values ＜ 0.01 of adjuvant, shows extremely significant difference, after immune in 35 days, the P values of low dosage antigen+high dose adjuvant
＜ 0.01, show extremely significant difference.
Compared with low dosage adjuvant, after immune in 8 days, 14 days, 35 days, low dosage antigen+high dose adjuvant, high dose
The equal ＜ 0.01 of P values of antigen+low dosage adjuvant, shows extremely significant difference, after immune in 8 days, low dosage antigen group, height
Doses of antigen group, the equal ＜ 0.01 of the P values of low dosage antigen+low dosage adjuvant, show extremely significant difference, and high dose antigen+
The P values ＜ 0.05 of high dose adjuvant, assumes a marked difference, after immune in 14 days, low dosage antigen+low dosage adjuvant,
The equal ＜ 0.01 of P values of high dose antigen+high dose adjuvant, shows extremely significant difference, and after immune in 35 days, low dosage resists
The P values ＜ 0.05 of original+low dosage adjuvant, assumes a marked difference, the P values ＜ 0.01 of high dose antigen+high dose adjuvant, table
Reveal extremely significant difference.
Compared with high dose adjuvant, after immune in 8 days, 14 days, 35 days, low dosage antigen+high dose adjuvant, high dose
Antigen+low dosage adjuvant, the equal ＜ 0.01 of the P values of high dose antigen+high dose adjuvant, show extremely significant difference, exempt from just
In 8 days afterwards, 14 days, the equal ＜ 0.01 of P values of low dosage antigen+low dosage adjuvant, extremely significant difference is shown, 8 after just exempting from
In it, low dosage antigen, the equal ＜ 0.01 of the P values of high dose antigen, extremely significant difference is shown, it is low after just exempting from 35 days
The P values ＜ 0.05 of doses of antigen+low dosage adjuvant, assumes a marked difference.
Illustrate that the rabies vaccine containing composite adjuvant composition can promote the maculiform of mouse boosting cell secretion of gamma-IFN
Into cell number, i.e., the cell immune response that the rabies vaccine containing composite adjuvant composition of the invention can be stronger.
It is described above, only it is the preferred embodiment that the present invention is implemented, any formal limitation not is done to the present invention, it is all
Modifications, equivalent substitutions and improvements done within the spirit and principles in the present invention etc., it is required to the protection included in the present invention
Within the scope of.
Claims (13)
1. a kind of rabies vaccine composite adjuvant, it is characterised in that it includes Saponin and CpGODN2006, the Saponin
Mass ratio with CpGODN2006 is 8:1-1:10.
2. rabies vaccine composite adjuvant according to claim 1, wherein, the matter of the Saponin and CpGODN2006
Amount is than being 4:1-1:2, preferably 1:1.
A kind of 3. preparation method of the rabies vaccine composite adjuvant described in claim 1 or 2, it is characterised in that its be by containing
The raw material for having Saponin and CpGODN2006 is mixed.
4. preparation method according to claim 3, wherein, the mass ratio of the Saponin and CpGODN2006 are 8:1-
1:10。
5. preparation method according to claim 4, wherein, the mass ratio of the Saponin and CpGODN2006 are 4:1-
1:2, preferably 1:1.
6. a kind of rabies vaccine composition, it is characterised in that contain any one of the composite adjuvant described in claim 1 or 2 or 3-5
Composite adjuvant and rabies vaccine prepared by methods described.
7. rabies vaccine composition according to claim 6, wherein, it is described relative to rabies vaccine 1/100-1/10 parts
Saponin is that 5 μ g-40 μ g and the CpGODN2006 are 5 μ g-50 μ g.
8. rabies vaccine composition according to claim 7, wherein, it is described relative to rabies vaccine 1/50-1/25 parts
Saponin is that 10 μ g-40 μ g, the CpGODN2006 are 10 μ g-20 μ g；Preferably, the Saponin is 10 μ g-20 μ g.
A kind of 9. preparation method of the rabies vaccine composition described in any one of claim 6-8, it is characterised in that its be by containing
The raw material for having Saponin, CpGODN2006 and rabies vaccine is mixed.
10. a kind of rabies vaccine parenteral solution, containing any one of the claim 6-8 rabies vaccine compositions and dilution,
The dilution is PBS, and the dosage of the PBS is 6.8-7.8ml.
11. rabies vaccine parenteral solution according to claim 10, wherein, the rabies vaccine parenteral solution is intraperitoneal injection pin
Agent.
12. a kind of rabies vaccine composition described in any one of claim 6-8 is prepared into the method described in claim 9
The rabies vaccine composition arrived, its application in mammalian immune response.
13. application according to claim 12, it is in people or the application in animal immune response.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710761727.5A CN107537035A (en) | 2017-08-30 | 2017-08-30 | Composite adjuvant and rabies vaccine containing composite adjuvant and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710761727.5A CN107537035A (en) | 2017-08-30 | 2017-08-30 | Composite adjuvant and rabies vaccine containing composite adjuvant and its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107537035A true CN107537035A (en) | 2018-01-05 |
Family
ID=60957960
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710761727.5A Pending CN107537035A (en) | 2017-08-30 | 2017-08-30 | Composite adjuvant and rabies vaccine containing composite adjuvant and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107537035A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000009159A1 (en) * | 1998-08-10 | 2000-02-24 | Aquila Biopharmaceuticals, Inc. | Compositions of cpg and saponin adjuvants and methods thereof |
| CN1330553A (en) * | 1998-10-16 | 2002-01-09 | 史密丝克莱恩比彻姆生物有限公司 | Adjuvant systems and vaccines |
| CN101212983A (en) * | 2005-07-13 | 2008-07-02 | 财团法人首尔大学校产学协力财团 | A vaccine composition comprising alpha-galactosylceramide as an adjuvatnt for intranasal administration |
| US20110070575A1 (en) * | 2004-12-08 | 2011-03-24 | Coley Pharmaceutical Group, Inc. | Immunomodulatory Compositions, Combinations and Methods |
-
2017
- 2017-08-30 CN CN201710761727.5A patent/CN107537035A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000009159A1 (en) * | 1998-08-10 | 2000-02-24 | Aquila Biopharmaceuticals, Inc. | Compositions of cpg and saponin adjuvants and methods thereof |
| CN1330553A (en) * | 1998-10-16 | 2002-01-09 | 史密丝克莱恩比彻姆生物有限公司 | Adjuvant systems and vaccines |
| US20110070575A1 (en) * | 2004-12-08 | 2011-03-24 | Coley Pharmaceutical Group, Inc. | Immunomodulatory Compositions, Combinations and Methods |
| CN101212983A (en) * | 2005-07-13 | 2008-07-02 | 财团法人首尔大学校产学协力财团 | A vaccine composition comprising alpha-galactosylceramide as an adjuvatnt for intranasal administration |
Non-Patent Citations (2)
| Title |
|---|
| ANNA CAROLINA A等: "A rabies vaccine adjuvanted with saponins from leaves of the soap tree (Quillaja brasiliensis) induces specifific immune responses and protects against lethal challenge", 《VACCINE》 * |
| 刘丽等: "新型佐剂CpG ODN 的研究进展", 《CHINESE JOURNAL OF NEW DRUGS 》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ROBERTSON et al. | TICK-BORNE RICKETTSIAE OF THE SPOTTED FEVER GROUP IN WEST PAKISTAN: II. SEROLOGICAL CLASSIFICATION OF ISOLATES FROM WEST PAKISTANAN AND THAILAND: EVIDENCE FOR TWO NEW SPECIES | |
| CN106497890B (en) | A kind of XF-1 plants of porcine pseudorabies virus variant and preparation method and application | |
| CN102636644B (en) | Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen | |
| CN111018995A (en) | B, T cell epitope tandem fusion vaccine for African swine fever | |
| CN116059337B (en) | Novel adjuvant recombinant herpes zoster vaccine and preparation and application thereof | |
| CN103665152B (en) | Canine parvovirus single domain antibody and its preparation method and application | |
| CN107267466A (en) | A kind of method for mass producing swine pseudorabies vaccine | |
| Morefield et al. | Synthetic Toll-like receptor 4 agonist enhances vaccine efficacy in an experimental model of toxic shock syndrome | |
| CN116064548B (en) | Novel CpG vaccine adjuvant and application thereof | |
| CN101475639B (en) | Mixed yolk antibody for preventing and treating duck infectious serositis and preparation thereof | |
| CN104560890A (en) | H9 subtype avian influenza virus and application thereof | |
| CN104829690A (en) | Fabricius bursa undecapeptide capable of promoting immunity | |
| CN102772794A (en) | Application of brucellosis A19 molecular marking vaccine and immunological identification thereof | |
| CN102805864A (en) | Newcastle disease and H9N2 subtype avian influenza bivalent inactivated vaccine and preparation method thereof | |
| CN102580074B (en) | Riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine and preparation method thereof | |
| Liang et al. | Effects of Taishan Robinia pseudoacacia Polysaccharides on immune function in chickens | |
| CN107537035A (en) | Composite adjuvant and rabies vaccine containing composite adjuvant and its preparation method and application | |
| CN104667273A (en) | Vaccine adjuvant and application thereof in preparation of Newcastle disease inactivating vaccine | |
| CN104672302A (en) | Polypeptin, and preparation method as well as application thereof | |
| CN102268090B (en) | Siamese crocodile blood extract and extraction method and application thereof | |
| CN104208666A (en) | Vaccine composition, and preparation method and application thereof | |
| CN101332297B (en) | Preparation method of novel subunit vaccine of pig pseudorabies | |
| CN102988970A (en) | Foot-and-mouth disease purification vaccine, preparation method and applications thereof | |
| JP2019527191A (en) | Immune enhancer, foot-and-mouth disease inactivated vaccine, and method for producing the same | |
| CN102813920A (en) | Vaccine adjuvant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |