CN107970441A - A kind of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines and prepare with scale technology - Google Patents
A kind of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines and prepare with scale technology Download PDFInfo
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- CN107970441A CN107970441A CN201711256185.2A CN201711256185A CN107970441A CN 107970441 A CN107970441 A CN 107970441A CN 201711256185 A CN201711256185 A CN 201711256185A CN 107970441 A CN107970441 A CN 107970441A
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- streptococcusagalactiae
- tilapia mossambica
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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
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
- A61K39/092—Streptococcus
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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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
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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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
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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
- A61K2039/55588—Adjuvants of undefined constitution
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- Animal Behavior & Ethology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines, the vaccine includes following components：Inactivate Streptococcusagalactiae concentrate, physiological saline and propolis adjuvant.Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines batch production technology of preparing in the present invention, is produced on a large scale, and reduces vaccine manufacturing cost.Vaccine can be applied by injection system, and cultivating tilapia is immunized, and immune effect is good, can effectively prevent hemorrhage caused by Streptococcusagalactiae etc., reduce disease loss.
Description
Technical field
The invention belongs to aquiculture disease immune protection technical field, and in particular to a kind of Tilapia mossambica Streptococcusagalactiae propolis
Inactivated vaccine and scale technology of preparing.
Background technology
China is Rofe fish culture and big export country, and the Tilapia mossambica Streptococcusagalactiae disease incidence cultivated in recent years is in rising
Trend, the Tilapia mossambica of region break out and spread situation, pond culture and cage culture is supported in South China Tilapia mossambica master
Morbidity, for incidence 20%~60%, the death rate reaches as high as more than 90%.Tilapia mossambica Streptococcusagalactiae disease of the prior art
Rely primarily on antibiotic and chemicals is controlled, there is certain therapeutic effect, but medicine usage amount is big, aquaculture cost
Also restrict the development of culture fishery while higher.Thus research safety efficiently controls aquatic economic animal Major Diseases
The anti-measure processed of vaccine immunity should be used as one of main direction of studying from now on.Propolis is the treasure-house of flavone compound, so as to have
Have many natural drug characteristics, in propolis also containing alcohol, phenol, aldehyde, acid, ethers and alkene, hydrocarbon, terpene, steroid and
The bioactive substances such as a variety of amino acid, aliphatic acid, enzyme, multivitamin and trace element, though propolis does not have antigenic characteristic,
But there is immunological enhancement, can improve antibody-producing ability, improve haemocyanin and globulin content, improve leucocyte and huge
Phagocyte phagocytic activity, the immune enhancing function of propolis show as three immune defense, immunological homeostasis, immunosurveillance aspects, can
Strengthen organism immune response, activation and protection immune organ, be excellent immunologic adjuvant, have to body immune system extensive
Effect.China's aquatic products vaccine research is started late with exploitation, in the development and application of aquatic products vaccine with advanced developed country have compared with
Big gap, also uses, vaccine large-scale preparation method without industrialization Tilapia mossambica streptococcus vaccine product in produce reality so far
Lack, essentially laboratory preparation techniques.
The content of the invention
To achieve these goals, the present invention provides a kind of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines and scale
Technology of preparing.
The present invention realizes by the following technical solutions：A kind of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines, it is described
Vaccine includes following components：Inactivate Streptococcusagalactiae concentrate, physiological saline and propolis adjuvant.
Preferably, the bacterial concentration of the inactivation Streptococcusagalactiae concentrate is higher than 30 × 108cfu/ml。
Present invention also offers a kind of prepare with scale technology of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines, the technology
Include the following steps：Inactivation Streptococcusagalactiae concentrate uses normal saline dilution, then with propolis adjuvant, is mixed equal
Even, sterile filling, that is, obtain vaccine finished product.
Preferably, the prepare with scale technology of the Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines specifically includes following step
Suddenly：Inactivation Streptococcusagalactiae concentrate is diluted with sterile saline, seedling tank, mixing speed 50-100r/min, side are matched somebody with somebody in addition
The propolis solution that side adds 10% volume is stirred, after being stirred 30min, aseptically, filling obtained vaccine finished product.
Preferably, the preparation of the inactivation Streptococcusagalactiae concentrate includes the steps：
(1) prepared by Streptococcusagalactiae first order seed；
(2) prepared by Streptococcusagalactiae secondary seed；
(3) Streptococcusagalactiae zymotic fluid ferments；
(4) Streptococcusagalactiae zymotic fluid inactivates；
(5) centrifugation of Streptococcusagalactiae liquid, concentration are inactivated.
Preferably, the Streptococcusagalactiae first order seed is prepared and included the following steps：Streptococcusagalactiae is inoculated with brain heart infusion
Agar, when culture 24 is small under the conditions of 37 DEG C, chooses lawn inoculation 300ml brain-heart infusion mediums, at 37 DEG C, 180r/min conditions
Under, when culture 24 is small, obtain primary seed solution.
Preferably, the Streptococcusagalactiae secondary seed is prepared and included the following steps：Primary seed solution, inoculation 30L fermentations
Culture medium, air agitation culture 12h, obtains secondary seed under the conditions of 37 DEG C.
Preferably, the Streptococcusagalactiae zymotic fluid fermentation, includes the following steps：Secondary seed is inoculated with 300L fermented and cultureds
Base, 37 DEG C, 200r/min, throughput 250L/min cultures, when culture 12-14 is small, sampling, measures bacterium solution A600 values, until bacterium
Liquid A600 is begun to decline, and stops fermentation.
Preferably, the Streptococcusagalactiae zymotic fluid inactivation includes the following steps：Streptococcusagalactiae zymocyte liquid is transferred to
Inactivation tank, addition 0.40% formaldehyde of final concentration, 37 DEG C, when 50-100r/min inactivations 24 are small.
Preferably, the inactivation Streptococcusagalactiae liquid centrifugation, concentration include the following steps：Streptococcusagalactiae liquid is inactivated to utilize
Thalline is collected by centrifugation in tube centrifuge, is resuspended with sterile saline and inactivation Streptococcusagalactiae concentrate is made.
Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines batch production technology of preparing in the present invention, is produced on a large scale, drop
Low vaccine manufacturing cost.Vaccine can be applied by injection system, cultivating tilapia be immunized, immune effect is good, can effectively prevent
Hemorrhage caused by Streptococcusagalactiae etc., reduces disease loss.
Embodiment
Technical scheme is described in detail below by specific embodiment, but the scope of the present invention
It is not restricted by the embodiments.
Embodiment
Embodiment provides a kind of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines modelling production technology, specifically includes as follows
Step：
(1) prepared by first order seed：Streptococcusagalactiae is inoculated with brain heart infusion agar, 37 DEG C, when culture 24 is small, chooses lawn inoculation
9 bottles of 300ml brain heart infusions/bottles, when culture 24 is small, obtain 2.7L shake-flask seed liquids by 37 DEG C, 180r/min；
(2) prepared by secondary seed：2.7L shake-flask seed liquids, inoculation 40L fermentation mediums, 37 DEG C, 250r/min, throughput
40L/min cultivates 12h, obtains secondary seed solution；
(3) secondary seed solution is inoculated with：300L fermentation mediums (500L fermentation tanks), 37 DEG C, 200r/min, throughput
250L/min is cultivated, and when 12-14 is small, sampling, measures bacterium solution A600 values, and until bacterium solution A600 is begun to decline, stopping is fermented；
(4) Streptococcusagalactiae zymocyte liquid is transferred to inactivation tank, adds 0.40% formaldehyde of final concentration, 37 DEG C, 50-100r/
When min inactivations 24 are small.
(5) inactivate Streptococcusagalactiae liquid and collect thalline using tube centrifuge centrifugation (13500r/min), use sterile physiological
Brine, which is resuspended, is made inactivation Streptococcusagalactiae concentrate.
(6) aseptically, 10L is inactivated into Streptococcusagalactiae concentrate, adds 80L sterile salines, addition is matched somebody with somebody
Seedling tank, mixing speed 50-100r/min, adds 10% volume propolis solution, after being stirred 30min, sterile while stirring
Under the conditions of, filling obtained vaccine finished product；Filling to preserve (100mL plastic bottles), sealing, puts 2~8 DEG C of preservations.
Measured through examining, Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines steriling test, residual formaldehyde, which are examined, to be met《In
Magnificent people's republic's veterinary drug allusion quotation》Version requirement in 2010.
Immune effect
The immune effect of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines prepared by the present invention is as described below：By healthy Rofe
Fish (10-20g/ tails) is randomly divided into group, every group of 50 tails.Immune group 1 is vaccinated, per tail prepared by intraperitoneal injection embodiment one
Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines 0.2ml；Immune group 2 is vaccinated, intraperitoneal injection is free of propolis adjuvant per tail
Vaccine 0.2ml；Inactivation Streptococcusagalactiae concentrate, saline control group do not vaccinate.Water temperature is 24-28 DEG C.Immune
After 28d, respectively with 6 × 107NSH1 plants of intraperitoneal injection every group of test fish of infection of Streptococcusagalactiae of cfu/mL, and breeding observing 7
My god, the morbidity of record each group test fish and death condition.Three batches of vaccines attack malicious immune protective effect test result indicates that, injection is exempted from
Epidemic disease group is to the relative immunity protective rate of NSH1 plants of Streptococcusagalactiae more than 60%.Wherein propolis adjuvant inactivated vaccine can improve
16% protective rate is horizontal.
Culture medium has following component in the present embodiment：BHI culture mediums (g/L)：Ox brain heart infusion 250, peptone 10, chlorine
Change sodium 5, glucose 2, disodium hydrogen phosphate 2.5；Physiological saline (g/L)：6.5, effect is as shown in the table.
| Group | Attack malicious quantity (tail) | Survival number (tail) | Survival rate (%) | Immune protective rate (%) |
| Vaccine group 1 | 50 | 45 | 90 | 88.6 |
| Vaccine group 2 | 50 | 38 | 76 | 72.7 |
| Control group | 50 | 6 | 12 | / |
The present embodiment is merely illustrative of the technical solution of the present invention, rather than its limitations；It is although right with reference to the foregoing embodiments
The present invention is described in detail, it will be understood by those of ordinary skill in the art that：It still can be to foregoing embodiments
Described technical solution is modified, or carries out equivalent substitution to which part technical characteristic；And these are changed or replace
Change, the essence of appropriate technical solution is departed from the spirit and scope of various embodiments of the present invention technical solution.
Claims (9)
1. a kind of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines, the vaccine includes following components：It is dense to inactivate Streptococcusagalactiae
Contracting liquid, physiological saline and propolis adjuvant.
2. Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines according to claim 1, it is characterised in that：The inactivation agalasisa
The bacterial concentration of hammer bacterium concentrate is higher than 30 × 108cfu/ml。
A kind of 3. method for preparing the Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines of claim 1 or 2, it is characterised in that
Including the steps：Inactivation Streptococcusagalactiae concentrate is diluted with sterile saline, seedling tank, mixing speed are matched somebody with somebody in addition
50-100r/min, adds the propolis solution of 10% volume while stirring, aseptically, filling after being stirred 30min
Vaccine finished product is made.
4. the method for Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines according to claim 3, it is characterised in that：It is described to go out
Streptococcusagalactiae concentrate living is prepared with the following method：
(1) prepared by Streptococcusagalactiae first order seed；
(2) prepared by Streptococcusagalactiae secondary seed；
(3) Streptococcusagalactiae zymotic fluid ferments；
(4) Streptococcusagalactiae zymotic fluid inactivates；
(5) centrifugation of Streptococcusagalactiae liquid, concentration are inactivated.
5. the method for Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines according to claim 4, it is characterised in that the nothing
Prepared by streptococcus lactis first order seed includes the following steps：Streptococcusagalactiae is inoculated with brain heart infusion agar, and 24 are cultivated under the conditions of 37 DEG C
Hour, lawn inoculation 300ml brain-heart infusion mediums are chosen, at 37 DEG C, under the conditions of 180r/min, when culture 24 is small, obtain level-one
Seed liquor.
6. the method for Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines according to claim 5, it is characterised in that the nothing
Prepared by streptococcus lactis secondary seed includes the following steps：Primary seed solution, is inoculated with 30L fermentation mediums, leads under the conditions of 37 DEG C
Gas agitating culture 12h, obtains secondary seed.
7. the method for Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines according to claim 6, it is characterised in that the nothing
The fermentation of streptococcus lactis zymotic fluid includes the following steps：Secondary seed is inoculated with 300L fermentation mediums, 37 DEG C, 200r/min, ventilates
Amount 250L/min is cultivated, and when culture 12-14 is small, sampling, measures bacterium solution A600 values, and until bacterium solution A600 is begun to decline, stopping is sent out
Ferment.
8. the method for Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines according to claim 7, it is characterised in that the nothing
The inactivation of streptococcus lactis zymotic fluid includes the following steps：Streptococcusagalactiae zymocyte liquid is transferred to inactivation tank, adds final concentration
0.40% formaldehyde, 37 DEG C, when 50-100r/min inactivations 24 are small.
9. the method for Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines according to claim 8, it is characterised in that described to go out
Streptococcusagalactiae liquid living centrifuges, concentration includes the following steps：Bacterium is collected by centrifugation using tube centrifuge in inactivation Streptococcusagalactiae liquid
Body, is resuspended with sterile saline and inactivation Streptococcusagalactiae concentrate is made.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711256185.2A CN107970441A (en) | 2017-12-04 | 2017-12-04 | A kind of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines and prepare with scale technology |
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| CN201711256185.2A CN107970441A (en) | 2017-12-04 | 2017-12-04 | A kind of Tilapia mossambica Streptococcusagalactiae inactivated propolis vaccines and prepare with scale technology |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108524927A (en) * | 2018-05-17 | 2018-09-14 | 广州普麟生物制品有限公司 | A kind of grass carp hemorrhage disease inactivated vaccine and preparation method thereof |
| CN113549572A (en) * | 2021-06-21 | 2021-10-26 | 广东海洋大学 | Streptococcus combined vaccine for marine fishes and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101890159A (en) * | 2010-06-23 | 2010-11-24 | 广西壮族自治区水产研究所 | Method for preparing bivalent inactivated vaccine of tilapia streptococcus |
| CN102847146A (en) * | 2011-09-15 | 2013-01-02 | 通威股份有限公司 | Vaccine used for preventing tilapia streptococcal disease |
| CN103834669A (en) * | 2014-02-21 | 2014-06-04 | 通威股份有限公司 | Oral vaccine of streptococcus agalactiae and preparation method thereof |
| CN105797149A (en) * | 2016-04-21 | 2016-07-27 | 中国水产科学研究院珠江水产研究所 | Grass carp bacterial septicemia and grass carp bacterial red skin disease bigeminy propolis inactivated vaccine and preparing technology |
-
2017
- 2017-12-04 CN CN201711256185.2A patent/CN107970441A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101890159A (en) * | 2010-06-23 | 2010-11-24 | 广西壮族自治区水产研究所 | Method for preparing bivalent inactivated vaccine of tilapia streptococcus |
| CN102847146A (en) * | 2011-09-15 | 2013-01-02 | 通威股份有限公司 | Vaccine used for preventing tilapia streptococcal disease |
| CN103834669A (en) * | 2014-02-21 | 2014-06-04 | 通威股份有限公司 | Oral vaccine of streptococcus agalactiae and preparation method thereof |
| CN105797149A (en) * | 2016-04-21 | 2016-07-27 | 中国水产科学研究院珠江水产研究所 | Grass carp bacterial septicemia and grass carp bacterial red skin disease bigeminy propolis inactivated vaccine and preparing technology |
Non-Patent Citations (2)
| Title |
|---|
| 朱洁莲 等: "罗非鱼源无乳链球菌灭活疫苗免疫效果的研究", 《中国兽医科学》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108524927A (en) * | 2018-05-17 | 2018-09-14 | 广州普麟生物制品有限公司 | A kind of grass carp hemorrhage disease inactivated vaccine and preparation method thereof |
| CN113549572A (en) * | 2021-06-21 | 2021-10-26 | 广东海洋大学 | Streptococcus combined vaccine for marine fishes and preparation method thereof |
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Application publication date: 20180501 |