CN104971346A - riemerella anatipestifer bacterial ghost vaccine adopting chitosan oligosaccharide as adjuvant - Google Patents
riemerella anatipestifer bacterial ghost vaccine adopting chitosan oligosaccharide as adjuvant Download PDFInfo
- Publication number
- CN104971346A CN104971346A CN201510238788.4A CN201510238788A CN104971346A CN 104971346 A CN104971346 A CN 104971346A CN 201510238788 A CN201510238788 A CN 201510238788A CN 104971346 A CN104971346 A CN 104971346A
- Authority
- CN
- China
- Prior art keywords
- bacterium
- riemerellosis anatipestifer
- vaccine
- bacterium shadow
- riemerella anatipestifer
- 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.)
- Granted
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The purpose of the present invention is to provide a riemerella anatipestifer bacterial ghost vaccine loaded with polyinosinic acid-polycytidylic acid having interferon inducing activity and added with chitosan oligosaccharide as an adjuvant. According to the technical scheme, a temperature control expression vector is constructed and is electrotransformed into riemerella anatipestifer protoplast, positive clones are screened and then are subjected to bacterial amplification culture at a temperature of 28 DEG C, lysis gene expression is induced at a temperature of 42 DEG C, polylysine is added to continuously act when the OD600 is no longer be reduced so as to completely lyze the live bacteria, centrifugation is performed to separate the bacteria, the dried bacteria and a 6 mg/mL Poly I:C solution according to an equal ratio so as to make the Poly I:C be embedded into the bacterial ghosts, a 2% chitosan oligosaccharide aqueous solution is added, stirring is performed to form a uniform mixture, sub-packaging is performed into penicillin bottles, and freeze-drying is performed so as to obtain the novel riemerella anatipestifer bacterial ghost vaccine. According to the present invention, the prepared riemerella anatipestifer bacterial ghost vaccine is immunized through drinking water, such that the local immunity on the respiratory tract mucous membrane and the digestive tract mucous membrane can be irritated, the humoral immunity can be stimulated, and the protection effect on the human body is not different from the injection immunity.
Description
Technical field
The invention belongs to live vaccine production field, relate to a kind of preparation method of duck bacterial vaccine, especially relating to a kind of take oligochitosan as the preparation method of the Riemerellosis Anatipestifer bacterium shadow vaccine of adjuvant.
Background technology
Riemerellosis Anatipestifer (
riemerella Anatipestifer, RA) and main infringement 2-7 duckling in age in week, with nervous symptoms and fibrinous pericarditis, perihepatitis and airsacculitis for feature, is that one of main bacteria sexually transmitted disease that duck produces is supported in harm at present.This disease infects by respiratory tract, digestive tract and skin trauma, and usually forms mixed infection with the cause of disease such as escherichia coli, paramyxovirus, causes large quantities of death of duck.
Although Riemerellosis Anatipestifer is to some antibiotic sensitive, be difficult in body remove completely; Simultaneously due to antibiotic unreasonable use, its drug resistance is more prevalent, increases the difficulty of antibiotic therapy on the one hand, often causes the drug residue problem of poultry product to become increasingly conspicuous on the other hand.The scientific method of this disease of prevention and control is on the basis of strengthening feeding and management, the Riemerellosis Anatipestifer vaccine that inoculation is suitable.
Some research reports have been had at present in the vaccine of Riemerellosis Anatipestifer, such as, Riemerellosis Anatipestifer inactivated vaccine, bivalence composite adjuvant inactivated vaccine etc.Antibacterial is through deactivation, and the structure of its cell surface is often destroyed and affect antigenicity, body can not be stimulated to produce and comparatively comprehensively protect.In recent years, many researchs that Novel bacteria shadow (or ghost, ghost) has been carried out in aspect prepared by vaccine, solve the problem keeping antibacterial appearance integrity.The research of this aspect also has some to report at Riemerellosis Anatipestifer, confirms that the bacterium shadow vaccine utilizing the method to prepare has higher protective rate.The main infection approach of animal is respiratory tract and digestive tract, and the mucosa-immune barrier (with S-IgA SIgA for representative) on respiratory tract, digestive tract surface is the first barrier resisting pathogen infection.Substantially colonization ability at respiratory tract is lost because Riemerellosis Anatipestifer is prepared into bacterium movie queen, so can not local immunity be produced, injecting immune animal can only be passed through, thus still there is the problem of following several respects: 1. pass through injecting immune, very large immunological stress is caused to animal, often cause paramyxovirus, escherichia coli take advantage of a weak point, cause serious secondary infection.2. inactivated vaccine can only cause humoral immunization, does not produce or only produces slight local mucosa-immune, can not provide and support anti-infective mucosa-immune barrier reliably.
Summary of the invention
In view of the deficiencies in the prior art, the object of the present invention is to provide a kind of loading to have the polyinosinic acid of interferon-induced activity, and add the Riemerellosis Anatipestifer bacterium shadow vaccine of oligochitosan as adjuvant.Technical scheme provided by the present invention is to effectively control Riemerellosis Anatipestifer infection, and the use, minimizing drug residue etc. that reduce antibacterials in poultry farming process are significant.
In order to solve the problem existing for background technology, the present invention is by the following technical solutions:
Bacterial strain based on the 2 type Riemerellosis Anatipestifer clinical separation strains (being numbered WF1) selecting this laboratory qualification to preserve.After the strain recovery of freezen protective, to be prepared into protoplast for subsequent use for test method routinely.
Utilize conventional molecular biological technology, the E gene (being called for short E) of Phage PhiX174 is connected as E-SN is dual-gene with Staphylococcal Nuclease A gene order (SN) by 15 flexible amino acid, then be connected with Human liver glutathione carrier pBV220, build Human liver glutathione carrier pBV-E-SN.By this expression vector electric Riemerellosis Anatipestifer protoplast transforming aforementioned preparation of test method routinely, screening positive clone obtains Riemerellosis Anatipestifer engineering bacteria.
By the Riemerellosis Anatipestifer engineering bacteria conventional method 28 DEG C of Zengjing Granule obtained, grow to OD
600to 0.6 ~ 0.7 time rapid temperature increases to 42 DEG C inducing lysis gene expression.Work as OD
600when no longer reducing, be cooled to 37 DEG C of polylysines adding 10 μ g/mL in the ratio of 9:1 in bacterium shadow and act on 24h under 37 DEG C of conditions, the viable bacteria remaining with abundant deactivation and do not destroy its surface texture.Thalline is sampled, and is coated with dull and stereotyped detection after dilution, and the judgement without viable bacteria growth is qualified.Then centrifugalize thalline, lyophilization.
Dry thalline is in 1:1(w/v) ratio to join the concentration prepared according to a conventional method be in the polyinosini solution of 6mg/mL, stir into the preliminary swelling 6h of bacterium mud, polyinosini embedding entered in bacterium shadow.
Oligochitosan is mixed with 2%(w/v) aqueous solution, add above-mentioned in the ratio of 2:8 through swelling bacterium mud, fully stir and form homogeneous mixture, be sub-packed in cillin bottle, lyophilization, namely obtain Riemerellosis Anatipestifer bacterium shadow oligochitosan Adjuvanted vaccines.
Before vaccine uses, add normal saline and suitably dilute, drink water immune duckling.Due to bacterium shadow wrap up by positively charged oligochitosan, can more closely be adsorbed in alimentary canal mucous membrane surface, be convenient to be caught by dendritic cell, antigen presentation and irritates nucous membrane immunoreation occur, and chitosan has protection antigen as carrier and exempts degraded and slow releasing function; On the other hand, in antigen processing pathways, the polyinosinic acid that bacterium shadow comprises is released, and stimulates body endogenous interferon level to raise, improves immunity of organisms, especially enhance the defensive ability/resistance ability to viral cause of disease.
Compared with prior art, what the present invention relates to utilizes temperature control lysis genes to prepare Riemerellosis Anatipestifer bacterium shadow vaccine tool to have the following advantages and marked improvement:
1. Riemerellosis Anatipestifer bacterium shadow vaccine provided by the present invention, by immunity of drinking water, what avoid injecting immune stress; Immunity not only can stimulate the antibody of body generation to Riemerellosis Anatipestifer, body endogenous interferon level also can be caused to raise, prevent the generation of viral infection.
2. traditional inactivated vaccine and current reported relevant Riemerellosis Anatipestifer bacterium shadow vaccine, by injecting immune, can only cause humoral immune reaction, protected effect has certain limitation.Technical scheme provided by the present invention, stimulating the mucosa-immune reaction of respiratory tract and digestive tract local on the one hand, providing first immune defence barrier for resisting infecting of Riemerellosis Anatipestifer; On the other hand, oligochitosan can promote that bacterium shadow enters in body through mucosa, stimulates the humoral immunization of body, thus provides more comprehensive immunoprotection.
Detailed description of the invention
Be below specific embodiments of the invention, technical scheme of the present invention is further described, but protection scope of the present invention is not limited to these embodiments.Every do not deviate from the present invention's design change or equivalent substituting include within protection scope of the present invention.
The preparation of embodiment one Riemerellosis Anatipestifer bacterium shadow
the preparation of 1.1 protoplasts:after being activated by the RA2 type bacterial strain being numbered WF1, be cultured to OD
600when being 0.7, bacterium liquid is placed in ice-water bath 30min, gets 10mL bacterium liquid, 4 DEG C, the centrifugal 10min of 5000rpm, abandons supernatant, with 10mL ice-water bath aquesterilisa washing bacterium mud, and recentrifuge, repeated washing like this three times.Suspend with ice-water bath high osmotic buffer, add the lysozyme soln of preheating to 5mg/mL, after 37 DEG C of water-bath 30min, then the EDTA solution adding preheating is to 0.01mol/L, 37 DEG C of water-bath 20min.In mechanism, get the bacterium liquid of process on microscope slide every 5min, azaleine dyes, the generation situation of oily Microscopic observation protoplast.When the formation rate of protoplast reaches more than 95%, the centrifugal 20min of 5000rpm, abandons supernatant, then adds improvement TSB liquid regeneration culture medium centrifuge washing 3 times, abandons supernatant.Finally protoplast improvement TSB liquid regeneration culture medium is suspended.
the structure of 1.2 Human liver glutathione carriers:by Phage PhiX174 crack protein E gene order (E, GI:9626372) and Staphylococcal Nuclease A gene order (SN, GI:21281729) by 15 flexible amino acid linker (G
4s)
3series connection is that E-SN is dual-gene, proceeds in cloning vehicle PMD19T-simple, builds recombinant cloning vector pMD-E-SN.Then with PST I with ECOR I respectively Human liver glutathione carrier pBV220 is carried out double digestion with recombinant cloning vector pMD-E-SN and is connected, build restructuring Human liver glutathione carrier pBV-E-SN.
the screening of 1.3 electricity conversions and positive colony:get 10 μ L bacteriolyze plasmids to add in the protoplasm somatocyte that 40 μ L prepare, carefully mix, move in the electric shock cup of 0.1cm after placing 5min on ice.Electricity ginseng is set to: 15Kv/cm, 50 μ Fd, 150 Ω, 5ms, carry out electroporated.After electric shock, in electric shock cup, add 960 μ L TSB regeneration culture mediums immediately, be transferred to by bacterium liquid in sterilizing 1.5mL centrifuge tube, 28 DEG C, 150r/min shakes bacterium 2h.Centrifugal bacterium liquid, discard 800 μ L supernatants, with the resuspended thalline of remaining liq, regenerate soft agar medium (35 μ g/mL Amp+) with the TSB being chilled to 45 DEG C and mix, be poured into above TSB regenerated solids culture medium (35 μ g/mL Amp+), 28 DEG C of cultivations.Single bacterium colony that picking flat board grows, be inoculated in the test tube containing the TSB fluid medium of Amp+ (100 μ L/mL), 28 DEG C of jolting overnight incubation, are accredited as the positive by bacterium liquid PCR.
the production of 1.4 bacterium shadow vaccines:with 1:100 ratio, the little bacterium liquid inoculation 200mL that shakes is contained in the fresh TSB culture medium of Amp+ resistance, mixing, get appropriate bacterium liquid and survey the OD before shaking bacterium cultivation
600, then 28 DEG C of joltings are cultivated, and survey OD every 1h
600, treat OD
600when being 0.6,42 DEG C of inductions that heat up.After 42 DEG C of intensifications, still survey OD every 1h
600, and get appropriate bacterium liquid carry out time mark preserve, until OD
600value no longer declines, and then adds polylysine continuation effect 24h under 37 DEG C of conditions of 10 μ g/mL in the ratio of 9:1.Then get bacterium liquid 100 μ L spread plate, cultivate 16-20h, observe with or without bacterial growth for 37 DEG C.By qualified for sterility test bacterium shadow according to OD
600value adjustment concentration, makes bacterium shadow content reach 5 × 10
10cf μ/mL, dry thalline is in 1:1(w/v) ratio to join the concentration prepared according to a conventional method be in the polyinosini solution of 6mg/mL, stir into the preliminary swelling 6h of bacterium mud, polyinosini embedded and enters in bacterium shadow.Oligochitosan is mixed with 2%(w/v) aqueous solution, add above-mentioned in the ratio of 2:8 through swelling bacterium mud, fully stir and form homogeneous mixture, be sub-packed in cillin bottle, lyophilization, namely obtain Riemerellosis Anatipestifer bacterium shadow oligochitosan Adjuvanted vaccines.
The protection test of embodiment two vaccine immunity
In order to verify that bacterium shadow vaccine prepared by the present invention is to the clinical protection effect of duck; select Growth of Cherry Valley Commercial meat-type duck as laboratory animal; by bacterium shadow vaccine respectively by adding different adjuvants; inoculate with different immunization routes, carry out the controlled trial of homology strain oil adjuvant Seedling prepared by traditional formalin-inactivated method simultaneously.Its immune protective effect is evaluated by the contrast test of antibody horizontal detection and counteracting toxic substances protective rate.
1. materials and methods
1.1 test material
Bacterium shadow+chitosan adjuvant: by method preparation described in embodiment 1.4.
Bacterium shadow+oily adjuvant: mixed according to 1:1 ratio with mineral oil adjuvant by the RA2 type bacterial strain (WF2) of aforementioned preparation, makes bacterium number content reach 5 × 10
10cf μ/mL.
Formalin-inactivated Seedling+oily adjuvant: the RA2 type bacterial strain (WF2) of formalin-inactivated mixes according to 1:1 ratio with mineral oil adjuvant, makes bacterium number content reach 5 × 10
9cf μ/mL.
Laboratory animal: 1 age in days Growth of Cherry Valley Commercial meat-type duck.
1.2 test method
1.2.1 testing program
Be health 1 age in days Growth of Cherry Valley Commercial meat-type duck 50 plumage of RA feminine gender after testing, raise to 5 ages in days, be divided into 5 groups to carry out immunity respectively at random.Within 2 weeks, carry out homotype RA (WF1) leg muscle counteracting toxic substances and observe one week after immunity, counteracting toxic substances dosage is determined according to trial test result.Grouping immunity and counteracting toxic substances situation are in table 1.
Table 1 vaccine immunity protected effect
1.2.2 antibody horizontal detects
Using 2 type RA ultrasonic degradation supernatants as envelope antigen coated elisa plate, detect the Fluctuation of the serum antibody be separated of taking a blood sample weekly.
2. result of the test
2.1 challenge test results
Counteracting toxic substances result shows: 48h after counteracting toxic substances, and non-Immunization group starts to occur death, all has nervous symptoms before the death of morbidity duck, and after death in opisthotonus posture, cut open the visible hepatosplenomegaly of inspection, meninges is congested, and can be separated to counteracting toxic substances bacterial strain from above-mentioned tissue.Within one week observation period, non-Immunization group 10 ducks have 8 to fall ill dead, remaining 2 duck poor growths; Formalin-inactivated Seedling immune group has 2 ducks to occur turning round the nervous symptoms of neck but dead; There is 1 duck morbidity but death by the bacterium shadow vaccine group of immunization route of drinking water, in feeding process subsequently, form stiff duck; And all there is not falling ill dead situation in the bacterium shadow vaccine immunity group of injecting pathway; The all healthy survival of non-immunity non-counteracting toxic substances group duck.No matter show that the RA2 type bacterium shadow vaccine prepared passes through injection or drinking-water approach all has good protected effect to susceptible duck.
2.2 antibody dynamic regularity
After vaccine immunity in 4 weeks, antibody horizontal continues to rise, and within 4 weeks, peaks, after immunity, within 5 weeks, still maintain higher level.Wherein bacterium shadow+oily adjuvant immunity group is suitable with the antibody horizontal that formalin-inactivated Seedling+oily adjuvant immunity group induces body to produce.And bacterium shadow+chitosan adjuvant immunity group at the antibody horizontal in each stage all lower than the antibody horizontal (Fig. 1) of injecting immune group, trace it to its cause, may be the antibody mainly mucosal antibodies that drinking-water immunization route produces, and the mainly circulating antibody adopting ELISA method to detect.But the counteracting toxic substances result of 2 weeks after immunity, the protected effect that drinking-water immunization route provides and injecting pathway there is no significant difference.
Accompanying drawing explanation
Figure of description 1 shows Riemerellosis Anatipestifer inactivated vaccine and the Antibody dynamics change of the different immunization route of bacterium shadow vaccine after immune Growth of Cherry Valley Commercial meat-type duck in 5 weeks.
Claims (7)
1. prepare the method for Riemerellosis Anatipestifer bacterium shadow vaccine for one kind, it is characterized in that: utilize the E gene of Phage PhiX174 (being called for short E) and Staphylococcus nuclease A gene (being called for short SN) to build the two lysis genes pBV-E-SN of temperature control, its electricity is transformed Riemerellosis Anatipestifer protoplast, and screening obtains positive colony.
2. the method preparing Riemerellosis Anatipestifer bacterium shadow vaccine according to claim 1, it is characterized in that: the positive bacterium colony that screening obtains is through 28 DEG C of amplification cultivation, then in the expression of 42 DEG C of two lysis genes of the induction that heats up, finally add polylysine to continue to do in order to abundant cracking viable bacteria, then centrifugalize thalline carry out living stems.
3. the method preparing Riemerellosis Anatipestifer bacterium shadow vaccine according to claim 2, is characterized in that: by the whole concentration of qualified for sterility test bacterium tone, makes bacterium shadow content reach 5 × 10
10cf μ/mL.
4. dry thalline joins in 1:1 ratio the concentration prepared according to a conventional method is in the polyinosini solution of 6mg/mL, stirs into the preliminary swelling 6h of bacterium mud, polyinosini is embedded and enters in bacterium shadow.
5. oligochitosan is mixed with the aqueous solution of 2%, adds above-mentioned through swelling bacterium mud in the ratio of 2:8, stirs and makes it form homogeneous mixture, be sub-packed in cillin bottle, lyophilization, namely obtain Riemerellosis Anatipestifer bacterium shadow oligochitosan Adjuvanted vaccines.
6. the method preparing Riemerellosis Anatipestifer bacterium shadow vaccine according to any one of claim 1-3, is characterized in that: this vaccine is by drinking-water immune animal.
7. the method preparing Riemerellosis Anatipestifer bacterium shadow vaccine according to claim 1-6, is characterized in that: described animal is commodity laying ducks, 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 true CN104971346A (en) | 2015-10-14 |
| CN104971346B 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) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2017080098A1 (en) * | 2015-11-10 | 2017-05-18 | 林海祥 | Polyinosinic cell-amino compound-calcium chloride adjuvant and vaccine containing polyinosinic cell-amino compound-calcium chloride adjuvant |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101934072A (en) * | 2010-03-24 | 2011-01-05 | 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 |
| CN103146732A (en) * | 2013-01-28 | 2013-06-12 | 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
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101934072A (en) * | 2010-03-24 | 2011-01-05 | 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 |
| CN103146732A (en) * | 2013-01-28 | 2013-06-12 | 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 |
Non-Patent Citations (1)
| Title |
|---|
| 王勇: "鸭疫里默氏杆菌菌蜕技术的初步研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017080098A1 (en) * | 2015-11-10 | 2017-05-18 | 林海祥 | Polyinosinic cell-amino compound-calcium chloride adjuvant and vaccine containing polyinosinic cell-amino compound-calcium chloride adjuvant |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104971346B (en) | 2018-11-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102847146B (en) | Vaccine used for preventing tilapia streptococcal disease | |
| CN102086447B (en) | Duck virus hepatitis strains and inactivated vaccine | |
| CN102430120B (en) | Adjuvant for enhancing fish vaccine immunization effect and application thereof | |
| CN101144062B (en) | Lactobacillus casei strain and application for products thereof in bird immunity | |
| CN103182076B (en) | Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof | |
| CN106497890B (en) | A kind of XF-1 plants of porcine pseudorabies virus variant and preparation method and application | |
| CN105031639A (en) | Mink distemper-canine parvovirus enteritis bivalent vaccine and its preparation method and use | |
| CN105647841A (en) | Construction method and application of pseudomonas aeruginosa mutant strain | |
| CN108486067A (en) | The inactivated vaccine and application of Porcine epidemic diarrhea virus variant and its preparation | |
| CN106754594A (en) | A kind of Salmonella choleraesuls attenuated carrier bacterium and its construction method | |
| CN117467581B (en) | Pediococcus acidilactici King73 capable of improving canine immunity and application thereof | |
| CN106146626B (en) | A kind of erysipelothrix ruhsiopathiae subunit vaccine and preparation method and application | |
| CN104971346A (en) | riemerella anatipestifer bacterial ghost vaccine adopting chitosan oligosaccharide as adjuvant | |
| CN103060250A (en) | Genetically engineered bacteria strain expressing porcine transmissible gastroenteritis virus | |
| CN101638661B (en) | Construction of recombinant lactic acid bacteria with HN gene and F gene of Newcastle disease virus | |
| CN101829321B (en) | Vaccine for preventing red head disease of Pseudobagrus fulvidraco | |
| CN107158370B (en) | Raccoon dog canine distemper freeze-dried live vaccine and preparation method and application thereof | |
| CN100443502C (en) | Vitellus immune globulin for preventing prawn virus, tis preparing method and use thereof | |
| CN110862938B (en) | Porcine pathogenic escherichia coli strain and inactivated vaccine thereof | |
| CN104975038A (en) | Riemerella anatipestifer pre-bacterial ghost vaccine preparation method | |
| KR101622554B1 (en) | Method for producing a composition for the treatment of diseases of dogs with immune antibody | |
| CN109223716B (en) | Quadruple yolk antibody soluble powder for resisting porcine epidemic diarrhea, swine fever, pseudorabies and transmissible gastroenteritis and preparation method thereof | |
| CN107236715B (en) | Raccoon dog distemper virus attenuated vaccine strain and application thereof in preparation of raccoon dog distemper live vaccine | |
| CN104623649A (en) | Vaccine for preventing streptococcus agalactiae disease of golden pomfret | |
| CN105126092A (en) | Preparation method for riemerella anatipestifer ghost vaccine with chitosan oligosaccharide as adjuvant load escherichia coli pili adhesion 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 |
Granted publication date: 20181127 Termination date: 20210513 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |