CN106913867A - A kind of Vickers, Aeromonas hydrophila bivalent inactivated vaccine and prepare with scale technology - Google Patents
A kind of Vickers, Aeromonas hydrophila bivalent inactivated vaccine and prepare with scale technology Download PDFInfo
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- CN106913867A CN106913867A CN201710156501.2A CN201710156501A CN106913867A CN 106913867 A CN106913867 A CN 106913867A CN 201710156501 A CN201710156501 A CN 201710156501A CN 106913867 A CN106913867 A CN 106913867A
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- 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
- A61K39/0208—Specific bacteria not otherwise provided for
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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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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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
- A61K2039/70—Multivalent vaccine
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Abstract
The present invention is a kind of fresh water fish bacterial septicemia immune protection bivalent inactivated vaccine and large-scale production technology.The present invention is made inactivated vaccine using isolated velogen strain in morbidity fish body, large-scale production, and immunity inoculation is carried out in methods such as injection, immersions, and the cultured freshwater fishes such as crucian, grass carp are immunized, and can reach good protection effect；Overcome single inactivated vaccine just for a kind of shortcoming of pathogen simultaneously, there is provided a kind of bigeminy vaccine can applied once resist two kinds of pathogenic bacterial infections simultaneously, improve immune effect, vaccine by immersion, inject or mix and stir feedstuff feeding and can conveniently apply.
Description
Technical field
The invention belongs to aquiculture disease immune protection technical field, and in particular to a kind of fresh water fish bacterial septicemia is exempted from
Epidemic disease prevents and treats bivalent inactivated vaccine and prepare with scale technology.
Background technology
The bacillary hemorrhage of freshwater fish is that harm cultured freshwater fish is the most serious also known as bacterium hueppe's disease
One of infectious disease, the fish of harm mainly have the fish such as grass, silver carp, flathead, crucian carp, carp, triangular bream, dace, eel, mandarin fish, are caused to freshwater aquaculture industry
Heavy losses (reach more than 30%).The cause of disease is mainly Aeromonas Aeromonas veronii) and Aeromonas hydrophila, he
The bacterial septicemia disease that causes account for more than 80%, be the most common pathogenic bacteria of freshwater fish.It is general using disinfectant and
Chemical drug antibiotic etc. carries out anti-treatment, easy polluted-water, and causes aquatic products medicament residue problem.Vaccine can stimulate dynamic fish body to produce
The attack of raw immune defense cause of disease, can also strengthen the ability of the anti-distress of body, as one of disease prevention and control measure.It is domestic near
Aquatic products vaccine development makes fast progress over 10 years, but vaccine large-scale preparation method more lacks, essentially laboratory preparation techniques,
At present in the urgent need to can industrialization production aquatic products production of vaccine technology.
The content of the invention
In order to solve problems of the prior art, gone out the invention provides a kind of Vickers, Aeromonas hydrophila bigeminy
Live vaccine and prepare with scale technology.
The present invention is realized by the following technical solutions：
A kind of Vickers, Aeromonas hydrophila bivalent inactivated vaccine include following components：Aeromonas veronii culture is inactivated
Liquid and Aeromonas hydrophila culture inactivation liquid.
Preferably, in described Vickers, Aeromonas hydrophila bivalent inactivated vaccine, Aeromonas veronii culture inactivation liquid
50% is accounted for, wherein bacterial concentration is no less than 25 × 108cfu/ml；Aeromonas hydrophila culture inactivation liquid accounts for 50%, wherein carefully
Bacteria concentration is no less than 25 × 108cfu/ml。
Present invention also offers a kind of Vickers, the prepare with scale technology of Aeromonas hydrophila bivalent inactivated vaccine, including
Following steps：Aeromonas veronii culture is inactivated into liquid, Aeromonas hydrophila culture inactivation liquid presses 1:1 ratio is mixed
Uniformly, sterile filling, the Vickers of system, Aeromonas hydrophila bivalent inactivated vaccine finished product.
Preferably, the Vickers, the prepare with scale technology of Aeromonas hydrophila bivalent inactivated vaccine comprise the following steps：
1) prepared by first order seed：Aeromonas veronii, Aeromonas hydrophila bacterial strain are inoculated with nutrient agar panel, 28 DEG C respectively
Culture, 24 hours, chooses lawn inoculation nutrient broth medium, 28 DEG C, Shaking culture 18-20 hours, prepares primary seed solution each
3L；
2) prepared by secondary seed：Two primary seed solutions are each inoculated with 50L seeding tanks, culture medium is trained for 30L toxin producings
Base is supported, 28 DEG C, the throughput of 30L/min, stirring ventilation culture 12-14 hours prepares secondary seed solution；
3) seedling bacterium solution culture：Secondary seed solution is each inoculated with 500L fermentation tanks, culture medium is 300L toxin producing cultures
Base, 28 DEG C, the throughput of 175L/min, stirring ventilation culture 12-14 hours obtains preparing the bacterium solution of vaccine, viable count 50 ×
108More than cfu/ml is qualified；
4) bacterium solution inactivation：Culture bacterium solution moves to 500L inactivation tanks respectively, and two kinds of bacterium solutions are separately added into 0.30% formalin,
37 DEG C, inactivate 24 hours, carry out steriling test and residues of formaldehyde inspection it is qualified, obtain Aeromonas veronii culture inactivation liquid and
Aeromonas hydrophila culture inactivates liquid；
5) dispensed with seedling：Two kinds of bacterial cultures are inactivated into liquid respectively and presses 1：1 ratio input 1000L matches somebody with somebody seedling tank, in tank
Mixed liquor stirs.Dispensed under aseptic condition, roll lid, obtain Vickers, Aeromonas hydrophila bivalent inactivated vaccine finished product.
Vaccine preparation method is to be amplified to 500L fermentation tank cultures step by step by I and II seed liquor in the present invention, is gone to
500 inactivation tanks inactivation, then go to 1000L with seedling tank mix, with seedling, packing obtain vaccine finished product, be a kind of factorial praluction skill
Art.Technology maturation, can be mass-produced, vaccine finished product 700L can be produced per batch, be available at least 3,500,000 tail fish injecting immunes to make
With, stable and controllable for quality, low cost, better than small-scale laboratory's technology of preparing, can industrialization production.Vaccine can by injection,
The modes such as immersion are applied, and the cultured freshwater fish such as immune crucian, grass carp works well, injecting immune protective rate reach 80% with
On, immersion immunity protective rate can effectively prevent fresh water fish bacterial septicemia more than 50%, reduce making for the chemical drugs such as antibiotic
With, fish quality is improved, water pollution is reduced, increase fisherfolk income.Single inactivated vaccine is overcome just for one kind simultaneously
The shortcoming of pathogen, there is provided a kind of bigeminy vaccine can applied once resist two kinds of pathogenic bacterial infections simultaneously, improve immune effect,
Vaccine is by immersion, injecting or mix and stir feedstuff feeding can conveniently administration.
Specific 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.
The prepare with scale technology of a kind of Vickers, Aeromonas hydrophila bivalent inactivated vaccine is present embodiments provided, including
Following steps：
1st, prepared by primary seed solution
The strain (Aeromonas veronii strain, Aeromonas hydrophila strain) for being stored in refrigerator is taken out, inoculation nutrient agar (training
Foster based formulas are shown in annex), 28 DEG C are cultivated 24 hours, are chosen lawn and are connect nutrient agar, and 28 DEG C are cultivated 24 hours, are inoculated with nutrient broth
300ml/ bottles of (culture medium prescription is shown in annex), puts shaking table 200r/min by 10 bottles, 28 DEG C, cultivates 18 hours, obtains 3L first order seeds
Liquid.
The present embodiment selection strain be：Aeromonas veronii bacterial strain LYK090702 is isolated from ill silver carp kidney, thermophilic water
Aeromonas bacterial strain YYK090901 is isolated from ill bighead kidney, and current strain is stored in China Aquatic Science Research Institute pearl
River produces research institute.
2nd, prepared by secondary seed solution
By 30L toxin producings culture medium (culture medium prescription is shown in annex) that formula is prepared, 50L seed fermentation tanks, 121 are added to
DEG C, 20 minutes sterilize, culture medium cooling after, by 3L primary seed solutions be inoculated with seed fermentation tank, 28 DEG C, throughput 30L/min,
250r/min, stir culture 12 hours, obtains secondary liquid seed liquor.
3rd, the bacterium solution culture of vaccine is prepared
By the 300L toxin producing culture mediums that formula is prepared, 500L fermentation tanks are added to, 121 DEG C, sterilized within 20 minutes, culture medium
After cooling, pure and qualified secondary seed solution is accessed into fermentation tank, culture-liquid temp is 28 DEG C, filtrated air stream in fermentation tank
Amount 175L/min, 200r/min culture bacterium solution 12 hours, culture terminates rear aseptic sampling, carries out purely and viable count detects (ginseng
According to《Republic of China Veterinary Pharmacopoeia》2005 version the 3rd), viable count need to reach 50 × 108More than CFU/mL is qualified
Bacterium solution.
2.4th, inactivate
Qualified bacterium solution is pipelined to inactivate tank, sterile working adds formalin, and formalin ultimate density is
0.30%, with 37 DEG C of inactivations, inactivate 24 hours, period, every 4 hours stirring at low speed once the bacterium solution for having inactivated was cooled to 1~5
Between DEG C, steriling test and residues of formaldehyde inspection (reference are carried out《Republic of China Veterinary Pharmacopoeia》In 2010 version).
2.5th, with seedling and packing
Two kinds of qualified inactivation liquid of bacterium are pressed 1 respectively：1 ratio input 1000L matches somebody with somebody seedling tank, and the stirring of in-tank mixing liquid is equal
It is even.Dispensed under aseptic condition, rolled lid, obtained Vickers, Aeromonas hydrophila bivalent inactivated vaccine finished product.
After measured, culture bacterium solution purely checks qualified, and Aeromonas veronii nutrient solution viable count is 106 × 108Cfu/ml,
Aeromonas hydrophila nutrient solution viable count is 117 × 108Cfu/ml, is above 50 × 108cfu/ml；Inactivated bacterial liquid steriling test
Qualified, in the fresh-water fishes Aeromonas bivalent inactivated vaccine of gained, Aeromonas hydrophila culture inactivation liquid accounts for 50%, Vickers gas
Pseudomonas culture inactivates liquid, accounts for 50%, and vaccine finished product formalin residual (average out to 0.182%) meets national standard.
The Vickers of present invention preparation, the immune effect of Aeromonas hydrophila bivalent inactivated vaccine are as described below：
Healthy crucian (20-30g/ tails) is randomly divided into three groups, every group of 100 tails.First group is injecting immune group, per tail abdomen
Bivalent inactivated vaccine 0.2ml prepared by chamber injection embodiment one；Second group is immersion immunity group, will first test fish 1%
3-5min is soaked in NaCL solution, immersion 10-15min is then inflated in 10 times of bigeminy vaccine liquid of dilution；3rd group is right
According to group.Water temperature is 22-26 DEG C.After immune 28d, respectively with 2 times of Aeromonas veronii strain LYK090702 of LD50 dosage (6 ×
108Cfu/ml) and Aeromonas hydrophila strain YYK090901 (3 × 107Cfu/ml every group of each 50 tail test fish is infected in) intraperitoneal injection,
Often tail 0.2ml, and breeding observing 7 days, records morbidity and the death condition of each group test fish.
By healthy grass carp (15-20g/ tails) 200 tails, two groups, each group of 100 tails are randomly divided into.First group vaccinates and exempts from
Epidemic disease group, per the bivalent inactivated vaccine 0.2ml prepared by tail intraperitoneal injection embodiment one；Second group of control group.Water temperature is 22-26
℃.After immune 28d, respectively with 2 times of Aeromonas veronii strain LYK090702 (9 × 10 of LD50 dosage8) and thermophilic water cfu/ml
Aeromonas strain YYK090901 (3 × 108Cfu/ml every group of each 50 tail test fish is infected in) intraperitoneal injection, per tail 0.2ml, and raises
Observation 7 days is supported, morbidity and the death condition of each group test fish is recorded.
Crucian attack malicious immune protective effect test result indicate that, injecting immune group is to bacterial strain LYK090702, YYK090901
Relative immunity protective rate be respectively 89%, 85%, immersion group is respectively 59%, 57% (being shown in Table 1)；Injecting immune grass carp, note
Penetrate immune group and 90%, 50% (being shown in Table 2) is respectively to the relative immunity protective rate of bacterial strain LYK090702, YYK090901.
The bivalent inactivated vaccine immune effect (crucian) of table 1
Immune protective rate=(the control group death rate-inoculation group the death rate)/control group death rate × 100%.
The bivalent inactivated vaccine immune effect (grass carp) of table 2
Nutrient broth medium：(1 liter of distilled water) 10 grams of peptone, 5 grams of sodium chloride (NaCl), 3 grams of beef extract, phosphoric acid hydrogen
1 gram of dipotassium (K2HOP4), heating for dissolving, with 10N NaOH pH to 7.5,121 DEG C sterilize 20 minutes.
Nutrient agar：(1 liter of distilled water) 10 grams of peptone, 5 grams of sodium chloride (NaCl), 3 grams of beef extract, phosphoric acid hydrogen
1 gram of dipotassium (K2HPO4), 15 grams of agar, heating for dissolving, with 10N NaOH pH to 7.5,121 DEG C sterilize 20 minutes.
Toxin producing culture medium：(1 liter of distilled water) 10 grams of peptone, 5 grams of sodium chloride (NaCl), 3 grams of beef extract, phosphoric acid hydrogen two
1.5 grams of potassium (K2HPO4), 5 grams of glucose, 5 grams of ammonium sulfate ((NH4) 2SO4), 8.6 milligrams of zinc sulfate (ZnSO4), magnesium sulfate
(MgSO4) 200 milligrams, heating for dissolving, with 10N NaOH pH to 7.5,121 DEG C sterilize 20 minutes.
Inactivator：40% formaldehyde.
The present embodiment is merely illustrative of the technical solution of the present invention, rather than its limitations；Although right with reference to the foregoing embodiments
The present invention has been described in detail, it will be understood by those within the art that：It still can be to foregoing embodiments
Described technical scheme is modified, or carries out equivalent to which part technical characteristic；And these are changed or replace
Change, do not make the spirit and scope of the essence disengaging various embodiments of the present invention technical scheme of appropriate technical solution.
Claims (4)
1. a kind of Vickers, Aeromonas hydrophila bivalent inactivated vaccine, it is characterised in that including following components：Aeromonas veronii
Culture inactivates liquid and Aeromonas hydrophila culture inactivation liquid.
2. Kazakhstan according to claim 1, vibrio alginolyticus bivalent inactivated vaccine, it is characterised in that：Described Vickers, thermophilic water
In Aeromonas bivalent inactivated vaccine, Aeromonas veronii culture inactivation liquid accounts for 50%, wherein bacterial concentration no less than 25 ×
108cfu/ml；Aeromonas hydrophila culture inactivation liquid accounts for 50%, and wherein bacterial concentration is no less than 25 × 108cfu/ml。
3. the prepare with scale skill of a kind of Vickers prepared described in claim 1 or 2, Aeromonas hydrophila bivalent inactivated vaccine
Art, comprises the following steps：Aeromonas veronii culture is inactivated into liquid, Aeromonas hydrophila culture inactivation liquid presses 1:1 ratio
Mixing and stirring, sterile filling, the Vickers of system, Aeromonas hydrophila bivalent inactivated vaccine finished product.
4. the prepare with scale technology of Vickers according to claim 3, Aeromonas hydrophila bivalent inactivated vaccine, its feature
It is to comprise the following steps：
1) prepared by first order seed：Aeromonas veronii, Aeromonas hydrophila bacterial strain are inoculated with nutrient agar panel, 28 DEG C of trainings respectively
Support, 24 hours, choose lawn inoculation nutrient broth medium, 28 DEG C, Shaking culture 18-20 hours, prepare each 3L of primary seed solution；
2) prepared by secondary seed：Two primary seed solutions are each inoculated with 50L seeding tanks, culture medium is 30L toxin producing culture mediums,
28 DEG C, the throughput of 30L/min, stirring ventilation culture 12-14 hours prepares secondary seed solution；
3) seedling bacterium solution culture：Secondary seed solution is each inoculated with 500L fermentation tanks, culture medium is 300L toxin producing culture mediums, 28
DEG C, the throughput of 175L/min, stirring ventilation culture 12-14 hours obtains preparing the bacterium solution of vaccine, and viable count 50 ×
108More than cfu/ml is qualified；
4) bacterium solution inactivation：Culture bacterium solution moves to 500L inactivation tanks respectively, and two kinds of bacterium solutions are separately added into 0.30% formalin, 37
DEG C, inactivate 24 hours, carry out steriling test and residues of formaldehyde inspection is qualified, obtain Aeromonas veronii culture inactivation liquid and thermophilic
Hydrophila culture inactivates liquid；
5) dispensed with seedling：Two kinds of bacterial cultures are inactivated into liquid respectively and presses 1：1 ratio input 1000L matches somebody with somebody seedling tank, in-tank mixing
Liquid stirs.Dispensed under aseptic condition, roll lid, obtain Vickers, Aeromonas hydrophila bivalent inactivated vaccine finished product.
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| CN201710156501.2A CN106913867A (en) | 2017-03-16 | 2017-03-16 | A kind of Vickers, Aeromonas hydrophila bivalent inactivated vaccine and prepare with scale technology |
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| CN201710156501.2A CN106913867A (en) | 2017-03-16 | 2017-03-16 | A kind of Vickers, Aeromonas hydrophila bivalent inactivated vaccine and prepare with scale technology |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109568572A (en) * | 2018-12-02 | 2019-04-05 | 河南师范大学 | A kind of preparation method and applications of Aeromonas Multivalent DNA Vaccine |
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| CN102366629A (en) * | 2011-01-25 | 2012-03-07 | 中国水产科学研究院珠江水产研究所 | Freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine and industrialized preparation method thereof |
| CN104689310A (en) * | 2014-09-15 | 2015-06-10 | 新乡医学院 | Aeromonas hydrophila and aeromonas veronii duplex oral sustained-release microsphere vaccine and preparation method thereof |
| CN104784683A (en) * | 2015-03-16 | 2015-07-22 | 西南大学 | Preparation and use method of Aeromonas veronii fishery injection vaccine |
| CN105031636A (en) * | 2014-09-15 | 2015-11-11 | 新乡医学院 | Bi-combined inactivate vaccine of aeromonas hydrophila and aeromonas veronii and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102366629A (en) * | 2011-01-25 | 2012-03-07 | 中国水产科学研究院珠江水产研究所 | Freshwater fish aeromonas hydrophila-aeromonas sobria bivalent inactivated vaccine and industrialized preparation method thereof |
| CN104689310A (en) * | 2014-09-15 | 2015-06-10 | 新乡医学院 | Aeromonas hydrophila and aeromonas veronii duplex oral sustained-release microsphere vaccine and preparation method thereof |
| CN105031636A (en) * | 2014-09-15 | 2015-11-11 | 新乡医学院 | Bi-combined inactivate vaccine of aeromonas hydrophila and aeromonas veronii and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109568572A (en) * | 2018-12-02 | 2019-04-05 | 河南师范大学 | A kind of preparation method and applications of Aeromonas Multivalent DNA Vaccine |
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Application publication date: 20170704 |