CN107281485B - A kind of immunopotentiator for improving haemophilus parasuis vaccine immunity effect - Google Patents
A kind of immunopotentiator for improving haemophilus parasuis vaccine immunity effect Download PDFInfo
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- CN107281485B CN107281485B CN201710453777.7A CN201710453777A CN107281485B CN 107281485 B CN107281485 B CN 107281485B CN 201710453777 A CN201710453777 A CN 201710453777A CN 107281485 B CN107281485 B CN 107281485B
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55522—Cytokines; Lymphokines; Interferons
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Abstract
The invention discloses a kind of immunopotentiators for improving haemophilus parasuis vaccine immunity effect.The immunopotentiator contains 6 PGD, IL 8, IL 6, glucose and gelatin, can significantly improve haemophilus parasuis immune effect of vaccine.It has also been found that 6 PGD, IL 6, IL 8 produce synergistic effect when using simultaneously to haemophilus parasuis immune effect of vaccine, the promoting immunity effect played is used separately the sum of the facilitation effect of generation higher than 6 PGD, IL 6, IL 8.What the immunopotentiator of the present invention may advantageously facilitate haemophilus parasuis vaccine is widely and effectively application, with answering prospect well.
Description
Technical field
The present invention relates to a kind of immunopotentiators for improving haemophilus parasuis vaccine immunity effect.
Background technology
Haemophilus parasuis is as caused by haemophilus parasuis, is the new hair bacterium for seriously endangering pig breeding industry in recent years
Sexually transmitted disease mainly causes polyserositis, arthritis and meningoencephalitis of pig etc., and incidence is generally 10-15%, dead
Rate has brought tremendous economic losses to pig breeding industry up to more than 50%, causes the pay attention to day by day of people.
The effective means for controlling the disease at present is vaccine inoculation.The use of document report commercial vaccine or oneself seedling can be controlled
The infection of haemophilus parasuis processed.But also having the example using vaccine prevention failure, the immune effect of vaccine is for effectively pre-
Prevent that the disease is particularly important.
The vaccine of high immune efficacy is in addition to the vaccine strain needed, and the antigen of high concentration, adjuvant is for improving vaccine
Immune effect is also quite important, and good immunologic adjuvant, which has, reduces vaccine antigen doses, reduces the advantages such as vaccine cost.The present invention
It is intended to develop a kind of immunopotentiator that can improve haemophilus parasuis immune effect of vaccine.
Invention content
The purpose of the present invention is to provide a kind of immunopotentiators for improving haemophilus parasuis vaccine immunity effect.
The technical solution used in the present invention is：
A kind of immunopotentiator for improving haemophilus parasuis vaccine immunity effect, which is characterized in that the immunopotentiator
By 10~50 parts of 6-PGD, 0.5~5 part of IL-8,0.3~5 part of IL-6,1~6 part of glucose, 2~8 parts of gelatin compositions, described part
Number is mass fraction.
Further, above-mentioned immunopotentiator is by 20~30 parts of 6-PGD, 2~4 parts of IL-8,1~3 part of IL-6,2~4 parts of Portugals
Grape sugar, 4~6 parts of gelatin compositions, the number is mass fraction.
A kind of immunopotentiator for improving haemophilus parasuis vaccine immunity effect, which contains 10~
50mg/mL6-PGD, 0.5~5mg/mL IL-8,0.3~5mg/mL IL-6,1~6mg/mL glucose, 2~8mg/mL gelatin,
Surplus is sterile saline.
Further, above-mentioned immunopotentiator contains 20~30mg/mL 6-PGD, 2~4mg/mL IL-8,1~3mg/mL
IL-6,2~4mg/mL glucose, 4~6mg/mL gelatin, surplus are sterile saline.
A kind of haemophilus parasuis vaccine mixture contains Immune-enhancing effect described in any one of the above embodiments in the vaccine mixture
Agent.
Further, the volume of above-mentioned immunopotentiator is the 1~15% of haemophilus parasuis vaccine.
6-PGD, IL-8 and IL-6 application as haemophilus parasuis vaccine immunopotentiator simultaneously.
Further, the mass ratio of 6-PGD, IL-8 and IL-6 are (10~50)：(0.5~5)：(0.3~5).
The application as haemophilus parasuis vaccine immunopotentiator simultaneously of 6-PGD, IL-8, IL-6, glucose and gelatin.
Further, the mass ratio of 6-PGD, IL-8, IL-6, glucose and gelatin is (10~50)：(0.5~5)：
(0.3~5)：(1~6)：(2~8).
The beneficial effects of the invention are as follows：
Present invention discover that using haemophilus parasuis vaccine immunity can be significantly improved while 6-PGD, IL-8 and IL-6
Effect.It has also been found that association is produced to haemophilus parasuis immune effect of vaccine when 6-PGD, IL-6, IL-8 are used simultaneously
Same-action, the promoting immunity effect played are used separately the sum of the facilitation effect of generation higher than 6-PGD, IL-6, IL-8.
The present invention study containing 10~50mg/mL 6-PGD, 0.5~5mg/mL IL-8,0.3~5mg/mL IL-6,1
~6mg/mL glucose, the immunopotentiator of 2~8mg/mL gelatin have more preferably haemophilus parasuis immune effect of vaccine
Facilitation effect.What the immunopotentiator of the present invention may advantageously facilitate haemophilus parasuis vaccine is widely and effectively application, with very
Good answers prospect.
Description of the drawings
Haemophilus parasuis IgG antibody level, ordinate OD in piglet serum is immunized for each group in Fig. 1450nmValue；
Fig. 2 is horizontal for each group haemophilus parasuis IgG antibody, ordinate OD450nmValue.
Specific embodiment
With reference to specific embodiment, the present invention is further illustrated.
A kind of immunopotentiator for improving haemophilus parasuis vaccine immunity effect of embodiment 1
The present embodiment immunopotentiator is by 20 parts of 6-PGD albumen (6-Phosphogluconate dehydrogenase, 6-
Phosphogluconate dehydrogenase), 2 parts of IL-8 albumen, 3 parts of IL-6 albumen, 3 parts of glucose, 5 parts of gelatin composition, the number is equal
For mass fraction.
The preparation method of immunopotentiator：Protein 6-PGD, IL-8, IL-6, glucose, gelatin by said ratio prepare,
Mixing is dispensed into penicillin bottle, and freeze-drying saves backup, you can.
The use of immunopotentiator：
(1) sterile saline of corresponding amount is added in when using into immunopotentiator, makes each constituent concentration be respectively
20mg/mL6-PGD, 2mg/mL IL-8,3mg/mL IL-6,3mg/mL glucose, 5mg/mL gelatin, handy physiological saline solution
Immunopotentiator afterwards.
(2) it is vaccine volume 6% with immune after physiological saline solution that volume will be added in haemophilus parasuis vaccine
Reinforcing agent, you can.
A kind of immunopotentiator for improving haemophilus parasuis vaccine immunity effect of embodiment 2
Contain 10~50mg/mL 6-PGD albumen (6-Phosphogluconate in the present embodiment immunopotentiator
Dehydrogenase, 6-phosphogluconate dehydrogenase), 0.5~5mg/mL IL-8 albumen, 0.3~5mg/mL IL-6 eggs
In vain, 1~6mg/mL glucose, 2~8mg/mL gelatin, surplus are sterile saline.
The preparation method of immunopotentiator：Protein 6-PGD, IL-8, IL-6, glucose, gelatin are prepared by said ratio
It after good, dispenses into penicillin bottle, freeze-drying saves backup, the sterile saline of every bottle of addition corresponding amount during use.
The use of immunopotentiator：Physiological saline is used by vaccine volume 1~15% is added in haemophilus parasuis vaccine
Dissolved immunopotentiator.
The preparation of 3 haemophilus parasuis vaccine of embodiment
Haemophilus parasuis vaccine is prepared by existing conventional techniques, concrete operation method is as follows：
1) prepared by primary seed solution：The haemophilus parasuis deposited of going bail for is inoculated in TSA solid plates, is 37 DEG C in temperature
Under the conditions of cultivate 18h；Then picking single bacterium is fallen in TSB culture mediums, under conditions of temperature is 37 DEG C, is shaken with rotating speed 120rpm
Culture 18h is swung, purely after the assay was approved, as primary seed solution, put 2~8 DEG C and save backup, the holding time is no more than
3 days；
2) prepared by secondary seed solution：The primary seed solution that step 1) obtains is taken, by seed liquor and the volume ratio of culture medium 1:
20 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, with rotating speed 120rpm shaken cultivation 16h, are purely examined
It after qualification, as secondary seed solution, puts 2~8 DEG C and saves backup, the holding time is no more than 3 days；
3) prepared by original bacteria liquid：The secondary seed culture solution that step 2) obtains is taken, by seed liquor and the volume ratio of culture medium 1:
100 are inoculated in TSB culture mediums, in temperature under conditions of 37 DEG C, with rotating speed 120rpm shaken cultivations for 24 hours after, obtain opportunistic pathogen
Liquid；
4) count plate：The bacterium solution obtained in step 3) is subjected to count plate, step is as follows：
Haemophilus parasuis is inoculated on TSA solid mediums, 37 DEG C are cultivated 12h or so, and lower bacterium is washed with pH 7.4PBS
Piping and druming is uniformly mixed repeatedly after tongue, then carries out plate count：Each sample takes 1mL, and it is dilute to do 10 times of series with pH 7.4PBS
It releases, each dilution changes 1 branched pipette.Specific method is：It is accurate to draw 100 μ L of bacterium solution, it is put into and fills the first of 900 μ L of dilution
(liquid level is not contacted in branch 1.5mL centrifuge tubes), separately takes 1 1mL pipette tips that the first pipe liquid after mixing, is drawn 100 μ L and put
In the 2nd 1.5mL centrifuge tube for entering to fill 900 μ L of dilution, it is diluted to last 1 pipe successively.Estimate according to sample containing bacterium number
Meter selects acceptable diluent degree, and the bacterium solution for drawing final dilution is inoculated with suitable culture medium flat plate 3, each to drip 100 μ L, makes
Bacterium solution is scattered, and after putting 37 DEG C of airings, is overturn tablet, is cultivated 48~72h, counts the bacterium colony in each plate in following manner
Number is calculated per the viable count (CFU/mL) in mL bacterium solutions.
5) it inactivates：Final concentration of 0.2~0.4% formalin is added in above-mentioned bacterium solution, 37 DEG C of oscillation inactivations are for 24 hours.
6) it emulsifies：It is sampled in the inactivated bacterial liquid obtained in step 5), according to《Republic of China Veterinary Pharmacopoeia》Annex into
Row is purely examined；By original bacteria liquid, centrifugation 10min takes the thalline of precipitation under conditions of rotating speed is 8000rpm, with sterile physiological salt
Water washs after thalline is resuspended, and washed thalline normal saline dilution is into a concentration of 3 × 109The seedling bacterium solution of CFU/mL, will
Seedling bacterium solution is with aluminium glue adjuvant with volume ratio 4：1~9：1 mixes, and emulsifies 20min under conditions of rotating speed 8000rpm, obtains secondary pig
Influenzae vaccine, 2~8 DEG C save backup.
The immunoenhancement result comparative studies of 4 immune adjuvants of embodiment
In research process of the present invention, a large amount of cell factor is had studied as immunologic adjuvant to haemophilus parasuis vaccine
The influence of immune effect, is set forth below part of test results.
30 10 age in days piglets are taken, 5 groups is randomly divided into, every group 5, adds respectively in the vaccine of the preparation of above-described embodiment 3
Enter following A~E groups immunologic adjuvant, F groups are control group (being shown in Table 1), and the above-mentioned secondary pig of A~E group immunologic adjuvant direct immunizations is bloodthirsty
Coli vaccine, every piglet immunological 2mL, two exempt from 2mL/ heads after 15 days, two exempt from 20 days after carry out attacking poison, while the secondary pig of measure of taking a blood sample
Haemophilus antibody titer.
Table 1 tests grouping situation
Note：Concentration in table is final concentration of the cell factor in vaccine.
Each group is immunized that the incidence after piglet is attacked poison is as shown in table 2, there it can be seen that the middle IL-2 of A, B group,
During immunologic adjuvant as haemophilus parasuis vaccine of IL-4, IFN-α, IFN-β, not playing the role of enhancing to vaccine,
It is not conducive to the generation of haemophilus parasuis immune effect of vaccine even, has aggravated the incidence of piglet.It can from table 2
Go out, C~E groups illustrate that immunologic adjuvant is conducive to improve haemophilus parasuis immune effect of vaccine without finding piglet morbidity.
The incidence after piglet is attacked poison is immunized in 2 each group of table
Further detection each group is immunized haemophilus parasuis IgG antibody level in piglet serum and finds, such as 3 institute of Fig. 1 and table
Show, A (0.91), B (0.95) organize IgG antibody level and vaccine control group F groups (0.91) quite, without significant difference；C
(1.18), the IgG antibody level of E (1.29) groups is higher than F (0.91) group, but does not have significant difference, and the immune effect of D groups is notable
Higher than F groups, antibody level illustrates that 6-PGD, IL-6, IL-8 are used simultaneously up to 1.69, and higher than the sum of 2 groups of facilitation effects of C, E group
When, the effect that collaboration promotes is produced, significantly improves haemophilus parasuis immune effect of vaccine.
Haemophilus parasuis IgG antibody level in piglet serum is immunized for each group in table 3
| Group | IgG antibody level (OD450nmValue) |
| A | 0.91 |
| B | 0.95 |
| C | 1.18 |
| D | 1.69 |
| E | 1.29 |
| F | 0.91 |
The immune effect detection of 5 immunopotentiator of the present invention of embodiment
In the haemophilus parasuis vaccine of above-mentioned preparation the embodiment of the present invention is added in the ratio of vaccine volume 1~15%
The immunopotentiator of physiological saline solution is used in 1, can be used after fully vibrating mixing.10 age in days piglet 25 is taken, random point
Into 5 groups, A~E groups, every group 5, the volume additive amount as described in table 4 (0%, 1%, 5%, 10%, 15%) is by immune increasing respectively
Strong agent is added in haemophilus parasuis vaccine, then piglet is immunized, 2mL/ heads, and two are carried out after 15 days and is exempted from, 2mL/ heads,
Two exempt to carry out within 20 days attacking poison or take blood measuring antibody level afterwards, relative immunity effect.
The incidence after piglet is attacked poison is immunized in 4 each group of table
5 each group haemophilus parasuis IgG antibody of table is horizontal
| Group | IgG antibody level (OD450nmValue) |
| A | 0.91 |
| B | 1.12 |
| C | 1.56 |
| D | 1.69 |
| E | 1.59 |
As can be seen that adding Immune-enhancing effect of the present invention by vaccine volume 1~15% from the testing result of 4,5 and Fig. 2 of table
Agent can effectively improve the immune effect of haemophilus parasuis vaccine, wherein when additive amount be 10% when best results.
A kind of immunopotentiator for improving haemophilus parasuis vaccine immunity effect of embodiment 6
The present embodiment immunopotentiator is by 10 parts of 6-PGD albumen, 0.6 part of IL-8 albumen, 5 parts of IL-6 albumen, 6 portions of grapes
Sugar, 2 parts of gelatin compositions, the number is mass fraction.
The preparation method of immunopotentiator：Protein 6-PGD, IL-8, IL-6, glucose, gelatin by said ratio prepare,
Mixing is dispensed into penicillin bottle, and freeze-drying saves backup, you can.
The use of immunopotentiator：
(1) sterile saline of corresponding amount is added in when using into immunopotentiator, makes each constituent concentration be respectively
10mg/mL6-PGD, 0.6mg/mL IL-8,5mg/mL IL-6,6mg/mL glucose, 2mg/mL gelatin, handy physiology salt are water-soluble
Immunopotentiator after solution.
(2) it is vaccine volume 15% with immune after physiological saline solution that volume will be added in haemophilus parasuis vaccine
Reinforcing agent, you can.
A kind of immunopotentiator for improving haemophilus parasuis vaccine immunity effect of embodiment 7
The present embodiment immunopotentiator is by 50 parts of 6-PGD albumen (6-Phosphogluconate dehydrogenase, 6-
Phosphogluconate dehydrogenase), 5 parts of IL-8 albumen, 0.3 part of IL-6 albumen, 1 part of glucose, 8 parts of gelatin compositions, the number
It is mass fraction.
The preparation method of immunopotentiator：Protein 6-PGD, IL-8, IL-6, glucose, gelatin by said ratio prepare,
Mixing is dispensed into penicillin bottle, and freeze-drying saves backup, you can.
The use of immunopotentiator：
(1) sterile saline of corresponding amount is added in when using into immunopotentiator, makes each constituent concentration be respectively
50mg/mL6-PGD, 5mg/mL IL-8,0.3mg/mL IL-6,1mg/mL glucose, 8mg/mL gelatin, handy physiology salt are water-soluble
Immunopotentiator after solution.
(2) it is vaccine volume 1% with immune after physiological saline solution that volume will be added in haemophilus parasuis vaccine
Reinforcing agent, you can.
A kind of immunopotentiator for improving haemophilus parasuis vaccine immunity effect of embodiment 8
Contain 30mg/mL 6-PGD albumen (6-Phosphogluconate in the present embodiment immunopotentiator
Dehydrogenase, 6-phosphogluconate dehydrogenase), 4mg/mL IL-8 albumen, 1mg/mL IL-6 albumen, 2mg/mL Portugals
Grape sugar, 4mg/mL gelatin, surplus is sterile saline.
The preparation method of immunopotentiator：Protein 6-PGD, IL-8, IL-6, glucose, gelatin are prepared by said ratio
It after good, dispenses into penicillin bottle, freeze-drying saves backup, the sterile saline of every bottle of addition corresponding amount during use.
The use of immunopotentiator：Physiological saline solution is used by vaccine volume 8% is added in haemophilus parasuis vaccine
Immunopotentiator afterwards.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. it is a kind of improve haemophilus parasuis vaccine immunity effect immunopotentiator, which is characterized in that the immunopotentiator by
10~50 parts of 6-PGD, 0.5~5 part of IL-8,0.3~5 part of IL-6,1~6 part of glucose, 2~8 parts of gelatin compositions, the number
It is mass fraction.
2. immunopotentiator according to claim 1, which is characterized in that the immunopotentiator is by 20~30 parts of 6-PGD, and 2
~4 parts of IL-8,1~3 part of IL-6,2~4 parts of glucose, 4~6 parts of gelatin compositions, the number is mass fraction.
3. it is a kind of improve haemophilus parasuis vaccine immunity effect immunopotentiator, which is characterized in that the immunopotentiator by
Following component forms：10~50mg/mL 6-PGD, 0.5~5mg/mL IL-8,0.3~5mg/mL IL-6,1~6mg/mL Portugals
Grape sugar, 2~8mg/mL gelatin, surplus is sterile saline.
4. immunopotentiator according to claim 3, which is characterized in that the immunopotentiator consists of the following compositions：20
~30mg/mL 6-PGD, 2~4mg/mL IL-8,1~3mg/mL IL-6,2~4mg/mL glucose, 4~6mg/mL gelatin,
Surplus is sterile saline.
5. a kind of haemophilus parasuis vaccine mixture, which is characterized in that appoint in the vaccine mixture containing Claims 1 to 4
Immunopotentiator described in one.
A kind of 6. haemophilus parasuis vaccine mixture according to claim 5, which is characterized in that the immunopotentiator
Volume be haemophilus parasuis vaccine 1~15%.
7.6-PGD, IL-8 and IL-6 application as haemophilus parasuis vaccine immunopotentiator simultaneously.
8. application according to claim 7, it is characterised in that：The mass ratio of 6-PGD, IL-8 and IL-6 for (10~
50)：(0.5~5)：(0.3~5).
The application as haemophilus parasuis vaccine immunopotentiator simultaneously of 9.6-PGD, IL-8, IL-6, glucose and gelatin.
10. application according to claim 9, it is characterised in that：6-PGD, IL-8, IL-6, glucose and gelatin quality
Ratio is (10~50)：(0.5~5)：(0.3~5)：(1~6)：(2~8).
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