CN104623649A - Vaccine for preventing streptococcus agalactiae disease of golden pomfret - Google Patents
Vaccine for preventing streptococcus agalactiae disease of golden pomfret Download PDFInfo
- Publication number
- CN104623649A CN104623649A CN201510091866.2A CN201510091866A CN104623649A CN 104623649 A CN104623649 A CN 104623649A CN 201510091866 A CN201510091866 A CN 201510091866A CN 104623649 A CN104623649 A CN 104623649A
- Authority
- CN
- China
- Prior art keywords
- streptococcus agalactiae
- vaccine
- golden
- disease
- stromateoides argenteus
- 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 invention provides an application of streptococcus agalactiae in preparation of medicines for preventing a streptococcus agalactiae disease of golden pomfret, and further provides a vaccine. The vaccine is prepared from the streptococcus agalactiae and pharmaceutically acceptable auxiliary materials or carriers. The invention further provides an application of the vaccine in preparation of medicines for preventing the streptococcus agalactiae disease of golden pomfret. The vaccine provided by the invention is capable of stimulating the golden pomfret to generate the immunity of resisting the streptococcus agalactiae disease of the golden pomfret. The streptococcus agalactiae disease of the golden pomfret can be effectively prevented.
Description
Technical field
The present invention relates to a kind of fish vaccine, particularly a kind of vaccine preventing golden Stromateoides argenteus streptococcus agalactiae disease.
Background technology
Gold Stromateoides argenteus formal name used at school golden pomfret, ground party name Cera Flava silvery pomfret, golden silvery pomfret, belong to Osteichthyes, Perciformes, Scad section, silvery pomfret Scad belongs to.The features such as it has delicious meat, nutritious, very popular.Therefore, golden Stromateoides argenteus as a main sea-farming kind, in China's Coastal Areas especially in Guangdong, Hainan obtains and develops rapidly, its cultivation scale and cultured output increase all year by year.
But along with cultivation amount and cultivation scale increase, the illness outbreak of gold Stromateoides argenteus also increases gradually, especially within nearly 2 years, there occurs a kind of new disease in gold Stromateoides argenteus culture zone, Hainan, spin in the main ill fish and water of this disease, fin ray fills hemorrhage, that anus is red and swollen disease, observe after dissecting and find that partial liver fills seriously hemorrhage in white, partial liver, a large amount of white slimy is had in intestinal, enteral wall congestive symptom obviously waits classical symptom, have the advantages that infectiousness is strong, morbidity is dead soon, mortality rate is high, bring larger economic loss to raiser.
Because the pathogenic factor of this disease it be unclear that, general fish disease prevention and cure medicine can only be adopted at present to carry out prophylactic treatment, but prevention effect is not obvious, also there is the potential risk of antibiotic remains simultaneously, and correspondingly increase the medicinal cost of cultivation, culture-cycle, what must bring is the increase of aquaculture cost thereupon.
Therefore, confirm the pathogenic factor of this disease, and prepare a species specificity prevention or the medicine for the treatment of this disease is a problem demanding prompt solution.
Summary of the invention
In order to solve the problem, the invention provides the vaccine that a species specificity prevents this disease.
Applicant is found by large quantity research, and aforementioned golden Stromateoides argenteus disease is caused by streptococcus agalactiae, can be described as golden Stromateoides argenteus streptococcus agalactiae sick.Before making the present invention, the report that streptococcus agalactiae causes golden Stromateoides argenteus disease is had no.
According to foregoing study results, the invention provides the purposes of streptococcus agalactiae (streptococcus agalactiae) in the medicine of the golden Stromateoides argenteus streptococcus agalactiae disease of preparation prevention.
Present invention also offers a kind of vaccine, it is prepared from by streptococcus agalactiae (streptococcus agalactiae) and pharmaceutically acceptable adjuvant or carrier.
Wherein, described vaccine comprises the streptococcus agalactiae of the deactivation causing immune effective dose and pharmaceutically acceptable adjuvant or carrier.
Preferably, the streptococcus agalactiae of described streptococcus agalactiae to be preserving number be ATCC 51487.
Preferably, the streptococcus agalactiae streptococcus agalactiae 14-48 of described streptococcus agalactiae to be preserving number be CGMCC NO.9974.
Wherein, describedly immune effective dose is caused for being not less than 2 × 10
5cFU/ml.Preferably, the described immune effective dose that causes is 2 × 10
5-2 × 10
8cFU/ml.
Wherein, described adjuvant or carrier are water or adsorbent.Described water is normal saline or distilled water, and described adsorbent is zeolite or kieselguhr.
Present invention also offers a kind of combination medicine, it comprises aforesaid vaccine and Percutaneous absorption enhancer.
Described cutaneous permeable agent is azone or hyoscyamine.
Present invention also offers the purposes of aforementioned vaccine in the medicine of the golden Stromateoides argenteus streptococcus agalactiae disease of preparation prevention.The preparation of the medicine of described prevention golden Stromateoides argenteus streptococcus agalactiae disease is powder or water preparation.
Present invention also offers a kind of streptococcus agalactiae, it is by the preserving number of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation: the streptococcus agalactiae streptococcus agalactiae 14-48 of CGMCC NO.9974.
Streptococcus agalactiae streptococcus agalactiae 14-48 of the present invention, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) is deposited on November 15th, 2014, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCC NO.9974.
Vaccine provided by the invention can stimulate golden Stromateoides argenteus to produce immunity, golden Stromateoides argenteus streptococcus agalactiae effectively can be prevented sick, can avoid excessively using of pesticide, be that extensive aquaculture realizes safety, ecological a kind of effective ways, the quality of golden Stromateoides argenteus can be improved, realize good economic benefit.
Streptococcus agalactiae streptococcus agalactiae 14-48 of the present invention, the vaccine prepared with it, protected effect is better than streptococcus agalactiae reference culture (ATCC 51487), and application prospect is good.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The detailed description of the invention of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Detailed description of the invention
The isolation identification of embodiment 1 bacterial strain
1, experiment material and method
1.1 experiment material
Bacteria distribution to be identified is from the ill golden Stromateoides argenteus of Lingao, Hainan plant, and isolation medium is BHI, and cultivation temperature is 37 DEG C, is numbered TW2014-48.
Reagent consumptive material 16S rDNA universal amplification test kit (TAKARA), BHI culture medium, bacteria bio trace assessor (sky, Hangzhou and biological reagent company limited), Taq archaeal dna polymerase (TAKARA), agarose (TianGen), 100bp DNA maker (Tiangen).
1.2 experimental technique
1.2.1 Gram stain is observed
Get one, the fresh inclined-plane of incubated overnight, with a small amount of thalline of inoculating loop picking, being coated in drip has on the microscope slide of a small amount of physiological saline solution, carries out Gram’s staining, in basis of microscopic observation coloration result.
1.2.2 the amplification of 16S rDNA and analysis
Using the bacterial genomes DNA extracted as template, use the 16S rDNA total length of 16S rDNA universal amplification primer amplification antibacterial (PCR system and amplification program as follows), PCR primer carries out sequencing through DNA purification system purification Hou Song Sangon Biotech (Shanghai) Co., Ltd..Use NCBI Blast data base to carry out 16S rDNA sequence alignment analysis, obtain the kind of antibacterial.
PCR system:
Negative control uses the 16S-free H of 1 μ l
2o alternate template DNA.
Pcr amplification program:
1.2.3 bacterial biochemical assay
According to 16S rDNA sequence alignment analysis result, carry out biochemical identification to separated strain, authentication method carries out according to biochemical identification operating procedure.
2, qualification result
2.1 cultivate and dyeing observation
TW2014-48 is gram-positive cocci, single, Cheng Shuan, catenation, different in size.
2.2 16S rDNA increase and analyze
Through 16S rDNA order-checking, TW2014-48 bacterial strain shows that this fragment has 1542bp, its sequence is:
GGAAATTTATCTTGGCTACGACGACGCTGGCGGCGTGCCTAATACATGCAAGTAGAACGCTGATGTTTGGTGTTTACACTAGACTGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTCATAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATAAGAGTGATTAACACATGTTAGTTATTTAAAAGGAGCAATTGCTTCACTGTGAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGGAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGAGAAGAACGTTGGTAGGAGTGGAAAATCTACCAAGTGACGGTAACTAACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTCTTTAAGTCTGAAGTTAAAGGCAGTGGCTTAACCATTGTACGCTTTGGAAACTGGAGGACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGCCCTTTCCGGGGCTTAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTCTGACCGGCCTAGAGATAGGCTTTCTCTTCGGAGCAGAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCTAAGGTGGGATAGATGATTGGGGTGAAGTCGTAACAAGGTAACCCGTAGTCGTGACTGGAAACACACTCGTGCGCAGA
The 16S rDNA sequence reported in the sequence of acquisition and Genbank is carried out Blast analysis, result shows that TW2014-48 gathers for cluster with streptococcus agalactiae streptococcus agalactiae on phylogenetic tree, its homology is 99%, combining form feature, is accredited as streptococcus agalactiae streptococcus agalactiae.
In conjunction with 16S rDNA and morphological feature, TW2014-48 is accredited as streptococcus agalactiae S.agalactiae, called after streptococcus agalactiae streptococcus agalactiae 14-48, and be preserved in China General Microbiological culture presevation administrative center, preserving number on November 15th, 2014: CGMCC NO.9974.
Preparation and the immune effect of embodiment 2 gold medal Stromateoides argenteus streptococcus agalactiae (TW2014-48) inactivated vaccine are studied
This experiment has carried out the preparation of the full vaccine of deactivation and the research of immune effect to the streptococcus agalactiae be separated in natural occurrence gold Stromateoides argenteus body, is intended to for the immune protection of golden Stromateoides argenteus streptococcus agalactiae disease provides theoretical and test basis.
1. materials and methods
1.1 material
Streptococcus agalactiae (TW2014-48) is separated from the ill golden Stromateoides argenteus of Lingao, Hainan plant, is preserved in China General Microbiological culture presevation administrative center, preserving number: CGMCC NO.9974.
Healthy golden Stromateoides argenteus is purchased from Hainan plant, and average weight 153.2 ± 3.5g, raises and train 10 days before experiment with in aquarium.
1.2 method
1.2.1 the preparation of vaccine
Aseptically, be inoculated into by the streptococcus agalactiae being stored in-70 DEG C in brain heart culture medium (BHI), 37 DEG C of constant temperature culture 24h, picking colony is inoculated in 37 DEG C of constant temperature culture 24h on BHI plating medium, use physiological saline solution eluting, adjustment bacterial concentration is 2 × 10
8cFU/ml, then the formalin adding 0.6% is at 37 DEG C of vibration deactivation 36h, be full bacterium inactivated vaccine, in 4 DEG C of environment, preservation is for subsequent use.
1.2.2 vaccine safety inspection
By the full bacterination of deactivation of preparation in sheep blood BHI agar plate, after 37 DEG C of constant temperature culture 36-48h, observe on flat board whether have bacterial growth to judge whether to contain in Bacteria vaccine the pathogen of non-deactivation; Adopt the method for lumbar injection simultaneously, the Bacteria vaccine of preparation is inoculated healthy golden Stromateoides argenteus by 5 times of normal inoculum concentration with 10 multiple doses and carries out artificial liver support, after inoculation, observe 21d, to check whether Bacteria vaccine causes a disease to the golden Stromateoides argenteus of health.
1.2.3 grouping is tested
Healthy for 360 tails golden Stromateoides argenteus stochastic averagina is divided into 12 groups, often organizes 30 tails.Adopt to soak and carry out immunity with injection two kinds of immunization routes to golden Stromateoides argenteus, the full vaccine of deactivation dilutes by test desired concn, and injecting normal saline is as test control group (table 1) simultaneously.
1.3 protective effect
Latter 3 weeks of immunity, adopts the experiment fish of golden Stromateoides argenteus streptococcus agalactiae (TW2014-48) to immune group and matched group to carry out artificial counteracting toxic substances infection, every tail experiment fish lumbar injection gold Stromateoides argenteus streptococcus agalactiae (TW2014-48) 8 × 10
7cFU/ml pathogen 0.2ml, observes the morbidity death condition of each experimental group, Computation immunity protective rate, the immune effect of evaluation vaccine.Calculate relative immunity protective rate (RPS) according to the following formula:
RPS (%)=(matched group mortality rate-experimental group mortality rate)/matched group mortality rate × 100%
2 results
The safety of 2.1 vaccines
The bacteria containing amount 2 × 10 in formalin-inactivated vaccine is drawn through colony counting method
8cFU/ml.Get vaccine 200ul and be inoculated in sheep blood BHI agar plate, at 37 DEG C, cultivate 24-48h, do not find bacterial growth.Preparation deactivation whole-bacterial-vaccine is carried out counteracting toxic substances infection to the golden Stromateoides argenteus of health, observes 21d and do not find that inoculation fish is abnormal.In body and Vitro Experimental Results show that concentration is the formalin of 0.6% (V/V) carries out 36h under 37 DEG C of conditions deactivation to pathogen, prepared vaccine is safe.
The protectiveness of 2.2 vaccines
The injection of gold Stromateoides argenteus and immersion, after 3 weeks, carry out artificial liver support with streptococcus agalactiae (TW2014-48) to the experiment fish after immunity.Found that: the full bacterium inactivated vaccine of streptococcus agalactiae (TW2014-48) of preparation has certain immune protection with injecting immune after golden Stromateoides argenteus through soaking, and the protective capability of injecting immune is better than immersion immunity, and during injection, its concentration is 2.0 × 10
8cFU/ml is then comparatively suitable.
Protection situation after the immunity of table 1 streptococcus agalactiae (TW2014-48) inactivated vaccine
3 conclusions
Full bacterium inactivated vaccine prepared by the streptococcus agalactiae (TW2014-48) be separated to from the golden Stromateoides argenteus of natural infection generation streptococcus agalactiae disease has good immunogenicity; through soaking and can produce after injection two kinds of mode Immuno gold Stromateoides argenteus the immunity of streptococcus agalactiae disease; its protective rate is between 53.3%-76.7%, and in immersion immunity, the cell concentration of inactivated vaccine is 2.0 × 10
8cFU/ml is comparatively suitable, and when injecting its concentration 2.0 × 10
8cFU/ml is then comparatively suitable.
Experimental result illustrates, the vaccine adopting streptococcus agalactiae streptococcus agalactiae 14-48 of the present invention to prepare to by it because golden Stromateoides argenteus streptococcus agalactiae sickly have good preventive effect.
The immune effect comparative study of deactivation whole-bacterial-vaccine prepared by embodiment 3 gold medal Stromateoides argenteus streptococcus agalactiae (TW2014-48) and streptococcus agalactiae reference culture (ATCC51487)
This experiment has carried out being separated inactivated vaccine prepared by streptococcus agalactiae and reference culture to the comparative study of golden Stromateoides argenteus streptococcus agalactiae disease immune effect, and better to carry out immune protection for golden Stromateoides argenteus streptococcus agalactiae disease provide reliable foundation.
1. materials and methods
1.1 strain
Gold Stromateoides argenteus streptococcus agalactiae disease (TW2014-48) is separated from ill golden Stromateoides argenteus, preserving number: CGMCC NO.9974.
Streptococcus agalactiae reference culture (ATCC 51487) is given by Sichuan Agricultural University's fish diseases research center.
1.2 experiment fishes
Be purchased from health gold Stromateoides argenteus (Trachinotus ovatus) of Haifeng county, Hainan Deep sea net cage plant, average weight 252.8 ± 3.5g, before experiment, in aquarium, raise and train 10 days.
The preparation of 1.3 vaccines
The streptococcus agalactiae (TW2014-48) and streptococcus agalactiae reference culture (ATCC 51487) of depositing in-70 DEG C are inoculated in respectively BHI dull and stereotyped in 37 DEG C of cultivation 24h recovery, the single bacterium colony of picking coats BHI solid medium again, colony counting method determination bacterial concentration is made under washing with normal saline, be 0.6% (V/V) formalin by concentration simultaneously, 37 DEG C of deactivation 36h, make full bacterium inactivated vaccine.
1.4 vaccine safety inspections
The full vaccine of deactivation (TW2014-48) of preparation and streptococcus agalactiae reference culture (ATCC51487) are seeded on sheep blood BHI agar plate respectively, after 37 DEG C of constant temperature culture 24-48h, observe on flat board whether have bacterial growth to judge to prepare the non-complete inactivation of vaccine; Adopt the method for lumbar injection simultaneously, the vaccine of preparation is inoculated healthy golden Stromateoides argenteus by 5 times of normal inoculum concentration with 10 multiple doses and carries out artificial liver support, after inoculation, observe 21d, check whether Bacteria vaccine causes a disease to the golden Stromateoides argenteus of health.
1.5 experiment groupings
Healthy for 450 tails golden Stromateoides argenteus stochastic averagina is divided into 6 groups, and often organize 90 tails, often group establishes 3 repetitions.Adopt respectively to soak with deactivation whole-bacterial-vaccine prepared by golden Stromateoides argenteus streptococcus agalactiae (TW2014-48) and streptococcus agalactiae reference culture (ATCC 51487), with injection two kinds of immunization routes, immunity is carried out to golden Stromateoides argenteus, saline control group is set simultaneously.
1.6 protective effect
Latter 3 weeks of immunity, adopts the test fish of golden Stromateoides argenteus streptococcus agalactiae (TW2014-48) to immune group and matched group to carry out artificial counteracting toxic substances infection, lumbar injection experiment fish 8 × 10
7the streptococcus agalactiae (TW2014-48) of CFU/ml, every tail 0.2ml, observes the morbidity death condition of each experimental group, draws protective rate.
1.7 date processing
Data represent with " average ± SD ", and adopt SPSS19.0 software to carry out variance analysis to data, and carry out multiple comparisons by LSD and Duncan ' s method, P<0.05 represents significant difference.
2. result
The safety of 2.1 vaccines
Show that the streptococcus agalactiae TW2014-48 strain vaccine of preparation and the cell concentration of streptococcus agalactiae reference culture (ATCC 51487) are 2 × 10 by colony counting method
8cFU/ml.This vaccine 200ul is inoculated on sheep blood TSA agar plate, cultivate 24 ~ 48h at 37 DEG C after, does not find bacterial growth.Carry out safety testing to show, at 21d viewing duration, there is not exception in test fish.Result of the test shows that concentration is the formalin of 0.5% (V/V) carries out 36h under 28 DEG C of conditions deactivation to pathogen, can by thorough for pathogen deactivation, and the vaccine of preparation is safe.
The protectiveness of 2.2 vaccines
By two kinds of full bacterium inactivated vaccines, after adopting injection and immersion two kinds of immunization routes to carry out immune 3 weeks to the golden Stromateoides argenteus of health, with golden Stromateoides argenteus streptococcus agalactiae (TW2014-48), artificial liver support is carried out to the experiment fish after immunity.Simultaneously according to following formulae discovery relative immunity protective rate (RPS).
RPS (%)=(matched group mortality rate-experimental group mortality rate)/matched group mortality rate × 100%
Found that as following table: in immersion or injecting immune group; gold Stromateoides argenteus streptococcus agalactiae (TW2014-48) inactivated vaccine protective rate apparently higher than streptococcus agalactiae reference culture inactivated vaccine, and with reference culture inactivated vaccine significant difference (P<0.05) (as table 2, table 3).
Table 2 gold Stromateoides argenteus streptococcus agalactiae (TW2014-48) is compared (injection group) with the protectiveness of vaccine prepared by streptococcus agalactiae reference culture (ATCC 51487)
Note: different lowercase alphabet shows significant difference (P<0.05), lower same.
Table 3 gold Stromateoides argenteus streptococcus agalactiae (TW2014-48) is compared (immersion group) with the protectiveness of vaccine prepared by streptococcus agalactiae reference culture (ATCC 51487)
3. conclusion
This experiment finds; from natural infection occur full bacterium inactivated vaccine prepared by streptococcus agalactiae (TW2014-48) that golden Stromateoides argenteus is separated to through soak with injection two kinds of mode Immuno gold Stromateoides argenteus after can produce certain immunity, and its protective rate is all apparently higher than vaccine protective rate prepared by streptococcus agalactiae reference culture (ATCC 51487).
Result shows, when preparing the vaccine of immunoprophylaxis gold Stromateoides argenteus streptococcus agalactiae, streptococcus agalactiae streptococcus agalactiae 14-48 of the present invention has more excellent clinical value than streptococcus agalactiae reference culture (ATCC 51487).
The preparation of embodiment 4 inactivated vaccine water preparation
1. the preparation of streptococcus agalactiae inactivated vaccine
1.1 strain recoveries
Aseptically, it is dull and stereotyped in 37 DEG C of cultivation 24h recovery that the streptococcus agalactiae strain (TW2014-48) preserved under getting-800 DEG C of conditions is inoculated in BHI, with inoculating loop from the single colony inoculation of picking flat board in 10mlBHI meat soup, postvaccinal meat soup is put into water bath chader, 37 DEG C, 150r/min, shaken cultivation 14h.
1.2 bacterium liquid enrichment cultures
1. elementary cultivation
Aseptically, the bacterium liquid 10ml that recovery is cultivated is inoculated in 100ml BHI meat soup, postvaccinal meat soup is put into water bath chader, 37 DEG C, 150r/min, shaken cultivation 16h.
2. the secondary cultivation of bacterium liquid
Gradation is got BHI meat soup that 2ml level cultivate and is coated on uniformly on the BHI massive plate for preparing, coated BHI flat board is put into 37 DEG C of constant incubators, cultivates 24h (noticing that incubation time covers with whole flat board for degree with antibacterial tongue); In aseptic operating room, with the lawn of the normal saline flushing grow on plates of sterilizing, the bacteria suspension under washing is placed in the infusion bottle of sterilizing.
3. eluting bacterium liquid amplification culture
Bacterium liquid from eluting on flat board is inoculated 200ml BHI meat soup by every 5ml, carries out the amplification culture of bacterium liquid, 37 DEG C, 150r/min, shaken cultivation 24h.Obtain the bacterial suspension of high concentration.
(1) counting of bacterium liquid
Plating dilutions bacteria technology method is adopted to count to the bacterium liquid after enrichment culture
1., after choosing flat board cleaning autoclaving of the same size, preparation BHI flat board is for subsequent use;
2. the bacterium liquid got in vaccine preparation process is carried out multiple proportions (10n) diluted for use by sterile working;
3. the bacterium liquid of dilution is got 0.1ml and be coated with BHI flat board, 37 DEG C, cultivate 16 hours in calorstat, counting bacterium colony number;
4. select average colony number dilution flat board between 30-300, being multiplied by extension rate with this dilution average colony number, is then the total number of bacteria of this bacterium.
5. the concentration dilution of antibacterial is defined as 2.0 × 10
8cFU/ml.
(2) deactivation of bacterium liquid
To the bacterium liquid of amplification culture, by volume ratio adds the formalin of 60.6%, puts into water bath chader, 37 DEG C, 150r/min, vibration 36h deactivation.
(3) inactivating efficacy inspection
Bacteria suspension after deactivation need carry out the inspection of inactivating efficacy at every turn, the vaccine prepared of 300ul is drawn on sheep blood BHI agar plate with liquid-transfering gun under sterile working, observe after 37 DEG C of constant temperature culture 24-48h on flat board and whether have bacterial growth to judge to prepare the non-complete inactivation of vaccine, check with or without bacterial growth, if there is bacterial growth need carry out inactivation treatment again, if without bacterial growth, carry out operation sequence below.
(4) subpackage of vaccine
Deactivation is vaccine completely, and sterile working carries out being sub-packed in 500ml sterilizing high temperature bottle, and has the paraffin that should adopt of hole to close to rubber stopper.Divide the inactivated vaccine installed should be placed in 4 DEG C of preservations, for subsequent use.
When being prepared into water preparation, when immersion immunity uses can with the azone penetrating agent use in conjunction of 10mg/L, can be simultaneously used in combination, also can be used alone.
The preparation of embodiment 5 inactivated vaccine powder
1 freeze-drying
The Formalin inactivation whole-bacterial-vaccine aqueous solution that lower point of aseptic technique embodiment 4 prepared installs, puts into vacuum freeze drier and carries out pre-freeze, and when pre-freeze reaches uniform temperature, evacuation is dry.After lyophilizing terminates, be filled with dry aseptic air and enter drying baker, then carry out sealing of jumping a queue as soon as possible, in case again suck the moisture in air.
2 oven drying at low temperature methods
The Formalin inactivation whole-bacterial-vaccine aqueous solution that lower point of aseptic technique embodiment 4 prepared installs carries out moisture evaporation under cryogenic dries, and add adsorbents zeolite or kieselguhr oven drying at low temperature, the powder after oven dry is deposited in aseptic vial.Whole operating process need aseptically be carried out, bake out temperature at 40 DEG C-50 DEG C, in order to avoid affect antigenicity.
Add adjuvant soluble starch after drying, obtain full vaccine powder, specification: 1.1 × 10
8cFU/g.
To sum up, vaccine provided by the invention can stimulate golden Stromateoides argenteus to produce immunity, can effectively prevent golden Stromateoides argenteus streptococcus agalactiae sick, excessively using of pesticide can be avoided, that extensive aquaculture realizes safety, ecological a kind of effective ways, the quality of golden Stromateoides argenteus can be improved, realize good economic benefit.
Claims (14)
1. the purposes of streptococcus agalactiae (streptococcus agalactiae) in the medicine of the golden Stromateoides argenteus streptococcus agalactiae disease of preparation prevention.
2. a vaccine, is characterized in that: it is prepared from by streptococcus agalactiae (streptococcus agalactiae) and pharmaceutically acceptable adjuvant or carrier.
3. vaccine according to claim 2, is characterized in that: described vaccine comprises the streptococcus agalactiae of the deactivation causing immune effective dose and pharmaceutically acceptable adjuvant or carrier.
4. vaccine according to claim 3, is characterized in that: the streptococcus agalactiae of described streptococcus agalactiae to be preserving number be ATCC 51487.
5. vaccine according to claim 3, is characterized in that: the streptococcus agalactiae streptococcus agalactiae 14-48 of described streptococcus agalactiae to be preserving number be CGMCC NO.9974.
6. vaccine according to claim 5, is characterized in that: described causes immune effective dose for being not less than 2 × 10
5cFU/ml.
7. vaccine according to claim 6, is characterized in that: the described immune effective dose that causes is 2 × 10
5-2 × 10
8cFU/ml.
8. vaccine according to claim 3, is characterized in that: described adjuvant or carrier are water or adsorbent.
9. vaccine according to claim 8, is characterized in that: described water is normal saline or distilled water, and described adsorbent is zeolite or kieselguhr.
10. a combination medicine, is characterized in that: it comprises vaccine described in claim 2-9 any one and Percutaneous absorption enhancer.
11. combination medicines according to claim 10, is characterized in that: described cutaneous permeable agent is azone or hyoscyamine.
The purposes of vaccine described in 12. claim 2-9 any one in the medicine of the golden Stromateoides argenteus streptococcus agalactiae disease of preparation prevention.
13. purposes according to claim 12, is characterized in that: the preparation of the medicine of described prevention golden Stromateoides argenteus streptococcus agalactiae disease is powder or water preparation.
14. 1 kinds of streptococcus agalactiaes, is characterized in that: it is by the preserving number of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation: the streptococcus agalactiae streptococcus agalactiae 14-48 of CGMCC NO.9974.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510091866.2A CN104623649A (en) | 2015-02-28 | 2015-02-28 | Vaccine for preventing streptococcus agalactiae disease of golden pomfret |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510091866.2A CN104623649A (en) | 2015-02-28 | 2015-02-28 | Vaccine for preventing streptococcus agalactiae disease of golden pomfret |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104623649A true CN104623649A (en) | 2015-05-20 |
Family
ID=53203204
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510091866.2A Pending CN104623649A (en) | 2015-02-28 | 2015-02-28 | Vaccine for preventing streptococcus agalactiae disease of golden pomfret |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104623649A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112773891A (en) * | 2021-02-01 | 2021-05-11 | 北部湾大学 | Trachinotus ovatus source streptococcus agalactiae DNA vaccine as well as preparation method and application 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 (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101317904A (en) * | 2007-06-07 | 2008-12-10 | 何诚 | Uses of smoked plum extract against virus, bacteria, mycoplasma or chlamydia of livestock and poultry |
| CN101492739A (en) * | 2009-01-09 | 2009-07-29 | 华南农业大学 | Detection gene chip and method for main pathogen of bovine mastitis |
| CN102676683A (en) * | 2012-05-31 | 2012-09-19 | 广西壮族自治区水产研究所 | Method for screening candidate bacterial strain from fish streptococcus agalactiae vaccine |
-
2015
- 2015-02-28 CN CN201510091866.2A patent/CN104623649A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101317904A (en) * | 2007-06-07 | 2008-12-10 | 何诚 | Uses of smoked plum extract against virus, bacteria, mycoplasma or chlamydia of livestock and poultry |
| CN101492739A (en) * | 2009-01-09 | 2009-07-29 | 华南农业大学 | Detection gene chip and method for main pathogen of bovine mastitis |
| CN102676683A (en) * | 2012-05-31 | 2012-09-19 | 广西壮族自治区水产研究所 | Method for screening candidate bacterial strain from fish streptococcus agalactiae vaccine |
Non-Patent Citations (4)
| Title |
|---|
| AMAL MN ET AL: "An outbreak of Streptococcus agalactiae infection in cage-cultured golden pompano, Trachinotus blochii, in Malaysia", 《JOURNAL OF FISH DISEASE》 * |
| AMAL MN ET AL: "Molecular characterization of Streptococcus agalactiae strains isolated from fishes in Malaysia", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 陈贺: "罗非鱼无乳链球菌灭活疫苗的研制及其免疫效果的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑 E059-136》 * |
| 黄婷 等: "卵形鲳鲹感染无乳链球菌与海豚链球菌的研究", 《大连海洋大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112773891A (en) * | 2021-02-01 | 2021-05-11 | 北部湾大学 | Trachinotus ovatus source streptococcus agalactiae DNA vaccine as well as preparation method and application thereof |
| CN112773891B (en) * | 2021-02-01 | 2022-03-25 | 北部湾大学 | Trachinotus ovatus source streptococcus agalactiae DNA vaccine as well as preparation method and application 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102847146B (en) | Vaccine used for preventing tilapia streptococcal disease | |
| CN103215209B (en) | Bacillus amyloliquefaciens and application thereof | |
| CN108486012A (en) | Duck infectious serositis live vaccine and preparation method thereof | |
| CN106479938B (en) | A kind of Brevibacillus brevis bacterial strain and its application | |
| CN109234193B (en) | Rahnella aquatilis ZF7 and application thereof in plant growth promotion and disease prevention | |
| CN104450556A (en) | Serum-12 type haemophilus lus paradis vaccine strain and application thereof | |
| CN102839131A (en) | Trichoderma viride and application thereof | |
| CN102399724A (en) | Haemophilus parasuis LC strain and application thereof | |
| CN104232535B (en) | Nocardia seriolae induced low virulent strain and application thereof | |
| CN103109870B (en) | Application of fermentation supernatant liquid of pseudomonas | |
| CN107245459A (en) | One plant of haemophilus parasuis and its application | |
| CN104450559B (en) | New mycoplasma hyopneumoniae bacterial strain and its vaccine combination | |
| CN105441368A (en) | Mycoplasma bovis and application thereof | |
| CN105779346B (en) | A kind of enterococcus faecium and its application of bacteriocinogeny | |
| CN103908665B (en) | A kind of vaccine combination and its preparation method and application | |
| CN105274064B (en) | A kind of duck tembusu virus attenuated vaccine strain and its application | |
| CN104450555A (en) | Serum-13 type haemophilus lus paradis vaccine strain and application thereof | |
| CN110172423A (en) | One plant of Bei Laisi bacillus and its application in prevention and treatment root-knot nematode | |
| CN104087559B (en) | A kind of infectious bursa of Fabricius virus, inactivated vaccine and preparation method thereof | |
| CN101565686A (en) | Fusant bacterial strain and applications thereof | |
| CN107164273B (en) | A kind of strong 2 type Streptococcus suis of serum of immunogenicity and its application | |
| CN104623649A (en) | Vaccine for preventing streptococcus agalactiae disease of golden pomfret | |
| CN105018393A (en) | Bacillus megaterium strain and application thereof | |
| CN101829321B (en) | Vaccine for preventing red head disease of Pseudobagrus fulvidraco | |
| CN105112315A (en) | Endophyte strain Alcaligenes faecalis used for biocontrol of tobacco mosaic virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150520 |