CN104971346B - It is a kind of using chitosan oligosaccharide as the Riemerellosis Anatipestifer bacterium shadow vaccine of adjuvant - Google Patents
It is a kind of using chitosan oligosaccharide as the Riemerellosis Anatipestifer bacterium shadow vaccine of adjuvant Download PDFInfo
- Publication number
- CN104971346B CN104971346B CN201510238788.4A CN201510238788A CN104971346B CN 104971346 B CN104971346 B CN 104971346B CN 201510238788 A CN201510238788 A CN 201510238788A CN 104971346 B CN104971346 B CN 104971346B
- Authority
- CN
- China
- Prior art keywords
- bacterium
- vaccine
- riemerellosis anatipestifer
- bacterium shadow
- shadow
- 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.)
- Expired - Fee Related
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The object of the present invention is to provide a kind of load to have interferon-induced active polyinosinic acid, and Riemerellosis Anatipestifer bacterium shadow vaccine of the chitosan oligosaccharide as adjuvant is added.The program constructs Human liver glutathione carrier first, and electrotransformation enters Riemerellosis Anatipestifer protoplast, 28 DEG C of Zengjing Granules after screening positive clone, the then expression of 42 DEG C of inducing lysis genes, to OD600When no longer reducing, polylysine is added and continues effect sufficiently to crack viable bacteria, is then centrifuged for separating thallus.Dry thallus is mixed with the poly IC solution equal proportion of 6mg/mL, enters poly IC embedding in bacterium shadow, 2% chitosan oligosaccharide aqueous solution is then added and stirs to form uniform mixture, be sub-packed in cillin bottle, be freeze-dried to get Riemerellosis Anatipestifer novel bacteria shadow vaccine is arrived.Bacterium shadow vaccine prepared by the present invention is immune through drinking water, and can both stimulate the local immunity of respiratory tract and alimentary canal mucous membrane, can also excite humoral immunity, to the protecting effect and injecting immune no significant difference of body.
Description
Technical field
The invention belongs to live vaccine production fields, are related to a kind of preparation method of duck bacterial vaccine, more particularly, to
It is a kind of using chitosan oligosaccharide as the preparation method of the Riemerellosis Anatipestifer bacterium shadow vaccine of adjuvant.
Background technique
Riemerellosis Anatipestifer (Riemerella Anatipestifer, RA) mainly encroaches on 2-7 week old duckling, with neurosis
Shape and the gentle capsulitis of fibrinous pericarditis, perihepatitis are characterized, and are the main bacteria sexually transmitted diseases for endangering duck culturing production at present
One of.The disease can be infected by respiratory tract, alimentary canal and skin trauma, and usually with the cause of diseases shape such as Escherichia coli, paramyxovirus
At mixed infection, cause large quantities of death of duck.
Although Riemerellosis Anatipestifer to some antibiotic sensitives, is difficult to fully erased in body；Simultaneously because antibiotic
Unreasonable use, drug resistance is more prevalent, on the one hand increases the difficulty of antibiotic treatment, on the other hand often leads to fowl
The medicament residue problem of product becomes increasingly conspicuous.The scientific method of the prevention and control disease is on the basis of strengthening feeding management, and inoculation is suitable
Suitable Riemerellosis Anatipestifer vaccine.
There are some researchs to report in terms of the vaccine of Riemerellosis Anatipestifer at present, for example, Riemerellosis Anatipestifer goes out
Live seedling, divalent composite adjuvant inactivated vaccine etc..Bacterium is often destroyed by inactivation, the structure of cell surface and influences antigen
Property, body cannot be stimulated to generate more comprehensive protection.In recent years, novel bacteria shadow (or ghost, ghost have been carried out in terms of vaccine preparation
Shadow) many researchs, solve the problems, such as keep bacterium appearance integrality.The research of this aspect also has one in Riemerellosis Anatipestifer
A little reports, it was demonstrated that the bacterium shadow vaccine protective rate with higher prepared using this method.The main infection approach of animal is breathing
Road and alimentary canal, respiratory tract, alimentary canal surface mucosa-immune barrier (using S-IgA SIgA as representative) be to
The first barrier of imperial pathogen infection.Energy is colonized in respiratory tract since Riemerellosis Anatipestifer is prepared into bacterium movie queen and loses substantially
Power can only be by injecting immune animal, thus the problem of still have following several respects so local immunity cannot be generated：①
By injecting immune, very big immunological stress is caused to animal, is often caused paramyxovirus, Escherichia coli to be taken advantage of a weak point, is caused
Serious secondary infection.2. inactivated vaccine can only cause humoral immunity, slight local mucosa-immune, Bu Nengti are not generated or only generated
For reliably supporting anti-infectious mucosa-immune barrier.
Summary of the invention
In view of the deficiencies in the prior art, load the object of the present invention is to provide a kind of with interferon-induced active
Polyinosinic acid, and Riemerellosis Anatipestifer bacterium shadow vaccine of the chitosan oligosaccharide as adjuvant is added.Technical solution provided by the present invention
It infects Riemerellosis Anatipestifer is effectively prevented, and the use of antibacterials, reduction medicament residue during reduction poultry farming
Etc. being of great significance.
In order to solve the problems existing in background technology, the present invention adopts the following technical solutions：
The 2 type Riemerellosis Anatipestifer clinical separation strains (number WF1) for selecting this laboratory qualification to save are as basic bacterium
Strain.After the strain recovery of freezen protective, routinely it is spare to be prepared into protoplast for test method.
Using conventional molecular biological technology, by the E gene (abbreviation E) of Phage PhiX174 and staphylococcus aureus
Nuclease A gene order (SN) is that E-SN is dual-gene by the series connection of 15 flexible amino acids, then with Human liver glutathione carrier
PBV220 connection constructs Human liver glutathione carrier pBV-E-SN.By the expression vector routinely aforementioned preparation of test method electrotransformation
Riemerellosis Anatipestifer protoplast, screening positive clone obtains Riemerellosis Anatipestifer engineering bacteria.
The 28 DEG C of Zengjing Granules of Riemerellosis Anatipestifer engineering bacteria conventional method that will be obtained, grow to OD600When to 0.6~0.7
It is brought rapidly up to the expression of 42 DEG C of inducing lysis genes.Work as OD600When no longer reducing, 37 DEG C are cooled in bacterium shadow by 9:1 ratio
The polylysine that 10 μ g/mL are added in example acts on for 24 hours under the conditions of 37 DEG C, sufficiently to inactivate the viable bacteria of remaining without destroying its surface
Structure.Thallus sampling applies plate detection after dilution, the judgement of no viable bacteria growth is qualification.It is then centrifuged for separating thallus, freezing is dry
It is dry.
Dry thallus presses 1：It is molten that the ratio of 1 (w/v) is added to the poly IC that the concentration prepared according to a conventional method is 6mg/mL
It in liquid, stirs into bacterium mud and is tentatively swollen 6h, enter poly IC embedding in bacterium shadow.
Chitosan oligosaccharide is configured to the aqueous solution of 2% (w/v), and the above-mentioned bacterium mud by swelling is pressed 2：8 ratio is added, sufficiently
Stirring forms homogeneous mixture, is sub-packed in cillin bottle, is freeze-dried to get Riemerellosis Anatipestifer bacterium shadow chitosan oligosaccharide adjuvant epidemic disease is arrived
Seedling.
Vaccine before use, plus physiological saline suitably dilute, drink water immune duckling.Since bacterium shadow is by positively charged chitosan oligosaccharide
It is wrapped up, can relatively closely be adsorbed in alimentary canal mucous membrane surface, convenient for being captured by Dendritic Cells, antigen submission occurred and pierces
Swash mucosa-immune reaction, and chitosan oligosaccharide as carrier there is protection antigen to exempt degradation and slow releasing function；On the other hand, antigen
In process, the polyinosinic acid that bacterium shadow includes is released, and stimulation body endogenous interferon level increases, and improves body
Immunity especially enhances the resilience to viral cause of disease.
Compared with prior art, of the present invention to prepare Riemerellosis Anatipestifer bacterium shadow vaccine tool using temperature control lysis genes
It has the following advantages and marked improvement：
1. Riemerellosis Anatipestifer bacterium shadow vaccine provided by the present invention, immune by drinking water, answering for injecting immune is avoided
Swash；It is immune that body can not only be stimulated to generate the antibody to Riemerellosis Anatipestifer, it can also cause body endogenous interferon horizontal
It increases, prevents the generation of viral infection.
2. traditional inactivated vaccine and the related Riemerellosis Anatipestifer bacterium shadow vaccine reported at present, by injecting immune,
It can only cause humoral immune reaction, protecting effect has certain limitation.Technical solution provided by the present invention, on the one hand stimulation is exhaled
The mucosa-immune reaction for inhaling road and alimentary canal part, provides first of immune defense screen to resist infecting for Riemerellosis Anatipestifer
Barrier；On the other hand, chitosan oligosaccharide can promote bacterium shadow through mucous membrane enter in vivo, stimulate the humoral immunity of body, thus provide compared with
Comprehensive immunoprotection.
Specific embodiment
The following is specific embodiments of the present invention, is further described to technical solution of the present invention, but of the invention
Protection scope be not limited to these examples.It is all to be included in this hair without departing substantially from the change of present inventive concept or equivalent substitute
Within bright protection scope.
The preparation of one Riemerellosis Anatipestifer bacterium shadow of embodiment
The preparation of 1.1 protoplasts：After numbering the RA2 type bacterial strain activation for WF1, culture to OD600When being 0.7, by bacterium
Liquid is placed in 30min in ice-water bath, takes 10mL bacterium solution, and 4 DEG C, 5000rpm is centrifuged 10min, supernatant is abandoned, with 10mL ice-water bath aqua sterilisa
Bacterium mud is washed, is centrifuged again, is so washed repeatedly three times.It is suspended again with ice-water bath high osmotic buffer, the lysozyme of preheating is added
Solution is to 5mg/mL, after 37 DEG C of water-bath 30min, adds the EDTA solution of preheating to 0.01mol/L, 37 DEG C of water-bath 20min.Make
With in the process, take the bacterium solution of processing on glass slide every 5min, azaleine dyes, the generation feelings of oily microscopic observation protoplast
Condition.When the formation rate of protoplast reaches 95% or more, 5000rpm is centrifuged 20min, abandons supernatant, adds improvement TSB liquid
Regeneration culture medium centrifuge washing 3 times, abandon supernatant.Finally protoplast improvement TSB liquid regeneration culture medium is suspended.
The building of 1.2 Human liver glutathione carriers：By Phage PhiX174 crack protein E gene order (E, GI:9626372)
With Staphylococcal Nuclease A gene order (SN, GI:21281729) pass through 15 flexible amino acid linker (G4S)3
Series connection is that E-SN is dual-gene, is transferred in cloning vector PMD19T-simple, and recombinant cloning vector pMD-E-SN is constructed.Then it uses
ECOR I and PST I carries out double digestion and connection, structure to Human liver glutathione carrier pBV220 and recombinant cloning vector pMD-E-SN respectively
Build recombination Human liver glutathione carrier pBV-E-SN.
The screening of 1.3 electrotransformations and positive colony：Take 10 μ L bacteriolyze plasmids that the protoplasm somatocyte that 40 μ L are prepared is added
In, it is careful to mix, it is moved into the electric shock cup of 0.1cm after placing 5min on ice.Electricity ginseng is set as:15Kv/cm, 50 μ Fd, 150 Ω,
5ms is carried out electroporated.After electric shock, 960 μ L TSB regeneration culture mediums are added into electric shock cup immediately, bacterium solution is transferred to and is gone out
In bacterium 1.5mL centrifuge tube, 28 DEG C, 150r/min shakes bacterium 2h.It is centrifuged bacterium solution, discards 800 μ L supernatants, bacterium is resuspended with remaining liq
Body regenerates soft agar medium (35 μ g/mL Amp+) with the TSB for being cooled to 45 DEG C and mixes, is poured into TSB regenerated solids culture medium
(35 μ g/mL Amp+) above, 28 DEG C are cultivated.The single colonie grown on picking plate is inoculated into containing Amp+'s (100 μ L/mL)
In the test tube of TSB fluid nutrient medium, 28 DEG C of shaking overnight incubations are accredited as the positive by bacterium solution PCR.
The production of 1.4 bacterium shadow vaccines：With 1:100 ratios train the small fresh TSB for shaking bacterium solution inoculation 200mL resistance containing Amp+
It supports in base, mixes, appropriate bacterium solution is taken to survey the OD before shaking bacterium culture600, then 28 DEG C of shaking cultures, every 1h survey OD600, to OD600
When being 0.6,42 DEG C of heating inductions.After 42 DEG C of heatings, OD still is surveyed every 1h600, and take appropriate bacterium solution to carry out time label and protect
It deposits, until OD600Value no longer declines, and then presses 9:The polylysine that 10 μ g/mL are added in 1 ratio continues to make under the conditions of 37 DEG C
With for 24 hours.Then 100 μ L spread plate of bacterium solution is taken, 37 DEG C of culture 16-20h, whether there is or not bacterial growths for observation.By sterility test qualification
Bacterium shadow is according to OD600Value adjustment concentration, makes bacterium shadow content reach 5 × 1010Cf μ/mL, dry thallus press 1:The ratio of 1 (w/v) adds
Enter in the poly IC solution for being 6mg/mL to the concentration prepared according to a conventional method, stirs into bacterium mud and be tentatively swollen 6h, make poly IC
Embedding enters in bacterium shadow.Chitosan oligosaccharide is configured to the aqueous solution of 2% (w/v), and the above-mentioned bacterium mud by swelling is pressed 2:8 ratio adds
Enter, be sufficiently stirred to form uniform mixture, is sub-packed in cillin bottle, freeze-drying is to get few to Riemerellosis Anatipestifer bacterium shadow shell
Sugared Adjuvanted vaccines.
Two vaccine immunity protection test of embodiment
In order to verify bacterium shadow vaccine prepared by the present invention to the clinical protection effect of duck, select Growth of Cherry Valley Commercial meat-type duck as
Bacterium shadow vaccine is passed through respectively and different adjuvants is added, is inoculated with different immunization routes, while being passed by experimental animal
The check experiment of the congenic stock oil adjuvant seedling of formalin-inactivated method of uniting preparation.Pair of malicious protective rate is detected and attacked by antibody level
Than its immune protective effect of test evaluation.
1. materials and methods
1.1 test material
Bacterium shadow+chitosan oligosaccharide adjuvant：It is prepared by 1.4 the method for embodiment.
Bacterium shadow+oil adjuvant：By the RA2 type bacterial strain (WF2) and mineral oil adjuvant of aforementioned preparation according to 1:The mixing of 1 ratio, makes
Bacterium number content reaches 5 × 1010cfμ/mL。
Formalin-inactivated seedling+oil adjuvant：The RA2 type bacterial strain (WF2) and mineral oil adjuvant of formalin-inactivated are according to 1:1 ratio is mixed
It closes, bacterium number content is made to reach 5 × 109cfμ/mL。
Experimental animal：1 age in days Growth of Cherry Valley Commercial meat-type duck.
1.2 test method
1.2.1 testing program
It is detected as 1 age in days Growth of Cherry Valley Commercial meat-type duck of health, 50 plumage of RA feminine gender, raises to 5 ages in days, is randomly divided into 5 components
It is not immunized.2 weeks progress homotype RA (WF1) leg muscles are attacked poison and are observed one week after immune, attack toxic dose according to trial test
As a result it determines.Grouping, which is immunized and attacks malicious situation, is shown in Table 1.
1 vaccine immunity protecting effect of table
1.2.2 antibody level detects
Using 2 type RA ultrasounds cracking supernatant as envelope antigen coated elisa plate, the serum antibody for separation of taking a blood sample weekly is detected
Fluctuation.
2. test result
2.1 challenge test results
Attack poison as the result is shown：The 48h after attacking poison, non-Immunization group start death occur, have mind before morbidity duck is dead
It is after death in opisthotonos posture, the visible hepatosplenomegaly of dissect, meninx is congested, and can be separated to and attack from above-mentioned tissue through symptom
Toxic bacterial strain.Within one week observation period, 10 ducks of non-Immunization group have 8 morbidities dead, are left 2 duck slow growths；Formaldehyde
Inactivated vaccine immune group has 2 ducks to occur turning round the nervous symptoms of neck but not death；Gone out by the bacterium shadow vaccine group for immunization route of drinking water
Existing 1 duck morbidity but not dead, the stiff duck of formation in subsequent breeding process；And the bacterium shadow vaccine immunity group of injecting pathway does not go out
It now falls ill dead situation；It is not immune not attack the health survival of poison group duck.No matter showing that the RA2 type bacterium shadow vaccine of preparation passes through note
It penetrates or drinking-water approach all has good protecting effect to susceptible duck.
2.2 antibody dynamic regularity
After vaccine immunity in 4 weeks, antibody level persistently rises, and peaks within 4 weeks, still remains within 5 weeks higher to after being immunized
It is horizontal.The wherein antibody level phase that bacterium shadow+oil adjuvant immune group is generated with formalin-inactivated seedling+oil adjuvant immune group induction body
When.And bacterium shadow+antibody level of the chitosan oligosaccharide adjuvant immunity group in each stage is below the antibody level (result of injecting immune group
See Figure of description 1), it traces it to its cause, it may be possible to which the antibody that immunization route of drinking water generates is mainly mucosal antibodies, and is used
The mainly circulating antibody of ELISA method detection.But after immune 2 weeks attack malicious result from the point of view of, drinking-water immunization route offer
Protecting effect and injecting pathway have no significant difference.
Detailed description of the invention
Figure of description 1 shows Riemerellosis Anatipestifer inactivated vaccine and bacterium shadow vaccine difference immunization route in immune Growth of Cherry Valley
Antibody dynamics variation after Commercial meat-type duck in 5 weeks.
Claims (3)
1. a kind of method for preparing Riemerellosis Anatipestifer bacterium shadow vaccine, it is characterised in that：Utilize the E gene of Phage PhiX174
With the double lysis genes pBV-E-SN of Staphylococcus nuclease A gene constructed temperature control, by its electrotransformation Riemerellosis Anatipestifer plasm
Body, screening obtain positive colony；
Obtained positive bacterium colony is screened through 28 DEG C of amplification cultivations, the expression of double lysis genes is then induced in 42 DEG C of heatings, finally
Polylysine is added and continues effect sufficiently to crack viable bacteria, is then centrifuged for separating thallus and carries out living stems；
By the whole concentration of sterility test qualification bacterium tone, bacterium shadow content is made to reach 5 × 1010cfμ/mL；It is centrifugated thallus, freezing is dry
It is dry；
Dry thallus presses 1:It is to stir into the poly IC solution of 6mg/mL that 1 ratio, which is added to the concentration prepared according to a conventional method,
Bacterium mud is tentatively swollen 6h, enters poly IC embedding in bacterium shadow；
Chitosan oligosaccharide is configured to 2% aqueous solution, the above-mentioned bacterium mud by swelling is pressed 2:8 ratio is added, and stirring makes its shape
At homogeneous mixture, it is sub-packed in cillin bottle, is freeze-dried to get Riemerellosis Anatipestifer bacterium shadow chitosan oligosaccharide Adjuvanted vaccines are arrived.
2. the method according to claim 1 for preparing Riemerellosis Anatipestifer bacterium shadow vaccine, it is characterised in that：The vaccine is logical
Cross the immune animal of drinking-water.
3. the method according to claim 2 for preparing Riemerellosis Anatipestifer bacterium shadow vaccine, it is characterised in that：The animal
For commodity laying duck, Commercial meat-type duck, kind duck, goose and turkey.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510238788.4A CN104971346B (en) | 2015-05-13 | 2015-05-13 | It is a kind of using chitosan oligosaccharide as the Riemerellosis Anatipestifer bacterium shadow vaccine of adjuvant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510238788.4A CN104971346B (en) | 2015-05-13 | 2015-05-13 | It is a kind of using chitosan oligosaccharide as the Riemerellosis Anatipestifer bacterium shadow vaccine of adjuvant |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104971346A CN104971346A (en) | 2015-10-14 |
| CN104971346B true CN104971346B (en) | 2018-11-27 |
Family
ID=54268665
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510238788.4A Expired - Fee Related CN104971346B (en) | 2015-05-13 | 2015-05-13 | It is a kind of using chitosan oligosaccharide as the Riemerellosis Anatipestifer bacterium shadow vaccine of adjuvant |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104971346B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105396130A (en) * | 2015-11-10 | 2016-03-16 | 林海祥 | Polyriboinosinic polyribo-cytoidylic acid (PIC), ammonia and calcium adjuvant and vaccine containing same |
| CN105797153A (en) * | 2016-05-02 | 2016-07-27 | 浙江农林大学 | Veterinary vaccine immunologic adjuvant as well as preparation and application method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101934072B (en) * | 2010-03-24 | 2012-05-23 | 天津农学院 | Method for preparing actinobacillus pleuropneumoniae (App) bacterial ghost and method for preparing subunit vaccine by loading pasteurella antigen with App bacterial ghost |
| CN103146732B (en) * | 2013-01-28 | 2014-11-12 | 山东省农业科学院畜牧兽医研究所 | Efficient splitting tandem gene, efficient splitting plasmid and construction method and appliance |
-
2015
- 2015-05-13 CN CN201510238788.4A patent/CN104971346B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN104971346A (en) | 2015-10-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102086447B (en) | Duck virus hepatitis strains and inactivated vaccine | |
| CN106497890B (en) | A kind of XF-1 plants of porcine pseudorabies virus variant and preparation method and application | |
| CN102847146B (en) | Vaccine used for preventing tilapia streptococcal disease | |
| CN105031639A (en) | Mink distemper-canine parvovirus enteritis bivalent vaccine and its preparation method and use | |
| CN106754594A (en) | A kind of Salmonella choleraesuls attenuated carrier bacterium and its construction method | |
| CN113198011A (en) | Duck adenovirus type-3 strain inactivated vaccine and application thereof | |
| CN113491767A (en) | Triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and preparation method thereof | |
| CN104971346B (en) | It is a kind of using chitosan oligosaccharide as the Riemerellosis Anatipestifer bacterium shadow vaccine of adjuvant | |
| CN104587460A (en) | Mink viral enteritis and canine distemper binary living vaccine as well as preparation method and application thereof | |
| CN106729692B (en) | Triple inactivated vaccine for bovine viral diarrhea, bovine infectious rhinotracheitis and bovine parainfluenza and preparation method thereof | |
| CN102010876A (en) | Recombinant bacillus subtilis EV71-VP1 expression vector and preparation method and application thereof | |
| CN107823639B (en) | Inactivated vaccine for bovine viral diarrhea virus and preparation method thereof | |
| CN108066758B (en) | Raccoon dog canine distemper-parvovirus enteritis bigeminal live vaccine and preparation method and application thereof | |
| CN106146626B (en) | A kind of erysipelothrix ruhsiopathiae subunit vaccine and preparation method and application | |
| CN104087559B (en) | A kind of infectious bursa of Fabricius virus, inactivated vaccine and preparation method thereof | |
| CN103937817B (en) | Newcastle disease virus YT strain, its whole genome sequence and application thereof | |
| CN103060250A (en) | Genetically engineered bacteria strain expressing porcine transmissible gastroenteritis virus | |
| CN105733987A (en) | Mycoplasma synoviae | |
| CN101638661B (en) | Construction of recombinant lactic acid bacteria with HN gene and F gene of Newcastle disease virus | |
| CN108285885A (en) | A kind of comma bacillus bacterium shadow preparation method and its application in poultry vaccine | |
| CN110302374B (en) | Canine vaccine and preparation method and application thereof | |
| CN104667269B (en) | A kind of Porcine epidemic diarrhea virus nasal cavity immunity vaccine and preparation method thereof | |
| CN100443502C (en) | Vitellus immune globulin for preventing prawn virus, tis preparing method and use thereof | |
| CN103865884A (en) | Duck viral hepatitis bivalent yolk antibody, preparation method and application thereof | |
| CN107158370B (en) | Raccoon dog canine distemper freeze-dried live vaccine and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20181127 Termination date: 20210513 |