CN104667269B - A kind of Porcine epidemic diarrhea virus nasal cavity immunity vaccine and preparation method thereof - Google Patents
A kind of Porcine epidemic diarrhea virus nasal cavity immunity vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN104667269B CN104667269B CN201510053897.9A CN201510053897A CN104667269B CN 104667269 B CN104667269 B CN 104667269B CN 201510053897 A CN201510053897 A CN 201510053897A CN 104667269 B CN104667269 B CN 104667269B
- Authority
- CN
- China
- Prior art keywords
- vaccine
- epidemic diarrhea
- activator
- nasal cavity
- virus
- 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.)
- Active
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of Porcine epidemic diarrhea virus PEDV nasal cavity immunity vaccines and preparation method thereof, vaccine of the present invention is mixed to prepare according to a specific ratio by the vaccinogen liquid, assist agent solution and sodium azide solution of Porcine epidemic diarrhea virus, wherein described assist agent solution contains TLR activator, activator, carbomer and the PVPK of NOD sample acceptors.PEDV nasal cavity immunities vaccine prepared by the present invention compares existing related vaccines, and its potency is high, good immune effect.The vaccine of the present invention carries out vaccine immunity by bronchia mucosal tissue; can simultaneously excitating organism mucosa-immune and systemic immunity; substantial amounts of sIgA is produced in sow milk; maternal antibody is passed to by piglet by milk; piglet is obtained effective immunoprotection, prevent the harm of piglet epidemic diarrhea.
Description
Technical field
The invention belongs to technical field of veterinary biology, and in particular to epidemic disease is immunized in a kind of Porcine epidemic diarrhea virus bronchia mucosal
Seedling and preparation method thereof.
Background technology
Porcine epidemic diarrhea virus（Porcine epidemic diarrhea virus, PEDV）Each age can be caused
Pig vomiting, diarrhoea and dehydration occurs, each age pig is susceptible, especially the newborn piglet in 1 week old is endangered maximum.It is newborn
For the incidence of disease of piglet up to 60%~100%, the death rate is up to 100%.Cause large quantities of death of piglet, slaughter rate of hogs declines, made
Into market cut of pork deficiency, it is stable to influence pork price.China is successive in main pig province since in the December, 2010
Large-scale outbreak pig epidemic diarrhea, due to the high incidence in susceptible piglet group and high fatal rate, to whole pig industry band
To give a heavy blow to, serious economic loss is caused.In May, 2013, the U.S. confirm pig epidemic diarrhea occurs first within the border
（Porcine epidemic diarrhea, PED), epidemic situation diffuses to rapidly 23 states in the U.S. afterwards, in some plants
Interior, the disease has been even up to 100% to pig age less population fatal rate.Report, the sow in the U.S. about 10% infects
Therefore this virus, piglet can be affected, cause 5,000,000 piglet death.Up to the present, pig epidemic diarrhea does not have also
Highly effective vaccine can use.
Most infectious cause of diseases are all to invade body by mucous membrane surface.Mucous membrane is the first of body defenses infection
Road defence line, it is highly important that increasing research shows that mucosal immune response serves in prophylaxis against infection diseases.Specifically
Mucosal immune response induction caused by secretory IgA (sIgA) antibody prevent to include it is viral including infectious cause of disease enter
Key effect, and the Main Factors of mucosa-immune protective effect are played in invading.
Mucosal immune system is different from systemic immune system, and mucosal immune system, which can stimulate, produces mucosal immune response and complete
Body immune response, and general immunity induction body fluid immune response, it is impossible to activate mucosal immune system.Mucosal immune response produces
Secretory IgA（sIgA）Antibody, invasion of the pathogen to mucous membrane surface is prevented, and general immunity response can not then produce.In addition,
Mucosa-immune is the local maximally efficient approach for producing long-term immunological memory of induction.
The major way of the pig epidemic diarrhea of China's prevention at present is vaccine inoculation, existing pig epidemic diarrhea commodity epidemic disease
The immunization wayses of seedling are injected for Houhai acupoint, main to induce humoral immunity of organism reaction, produce IgG antibody, great Liang Lin in blood
Bed experiment confirms that its immune effect is unsatisfactory.PEDV mainly encroaches on the intestinal epithelial cell of piglet, and IgG antibody does not reach
The reason for this position is probably vaccine immunity failure.At home and abroad to the pathogen Preventive strategy of infringement mucous membrane tissue
In, mucosal vaccine has been widely applied, and is made a breakthrough.Due to intramuscular injection systemic vaccination can only activating system exempt from
Epidemic disease, and mucosal vaccine technology can excite mucosa-immune and systemic immunity simultaneously, therefore mucosal vaccine is maximally effective prevention mucous membrane
The immunization wayses of infection.The country is simultaneously had no on the effective mucosa-immune vaccines of PEDV.
The content of the invention
In order to solve above-mentioned problem, the present invention by research, made a kind of potency height, good immune effect,
The PEDV mucosa-immune vaccines almost having no side effect.The vaccine of the present invention carries out vaccine immunity by bronchia mucosal tissue, can be with
Excitating organism mucosa-immune and systemic immunity simultaneously, produce substantial amounts of sIgA in sow milk, by milk by maternal antibody
Piglet is passed to, piglet is obtained effective immunoprotection, prevents the harm of piglet epidemic diarrhea.
It is an object of the invention to provide a kind of preparation method of pig epidemic diarrhea PEDV nasal cavity immunity vaccines.
The technical solution used in the present invention is：
A kind of vaccine adjuvant solution, the activator of the activator containing TLR and NOD sample acceptors in the assist agent solution.
Further, above-mentioned assist agent solution contains 0.05~0.5 μ g/mL TLR activator, 0.01~0.1 μ g/mL
Activator, 0.1~10mg/mL carbomers and the 0.5~5mg/mL polyvinylpyrrolidones of NOD sample acceptors, surplus is physiology salt
Water.
Further, above-mentioned TLR activator is that can activate TLR2, TLR3, TLR4, TLR7 activator, or energy simultaneously
TLR2, TLR4, TLR8, TLR9 activator are activated simultaneously, or can be activated simultaneously in TLR2, TLR4, TLR3, TLR9 activator
At least one.
Further, above-mentioned NOD samples receptor stimulating agent be NOD1 samples receptor stimulating agent, in NOD2 sample receptor stimulating agents extremely
Few one kind.
A kind of Porcine epidemic diarrhea virus nasal cavity immunity vaccine, its preparation method comprise the following steps：
1）Prepare the cell inactivation seedling of Porcine epidemic diarrhea virus, i.e. vaccinogen liquid；
2）By vaccinogen liquid, any described assist agent solution of claim 1 ~ 4 and sodium azide solution by volume（94~
88）：（5~10）：（1~2）Mix, produce Porcine epidemic diarrhea virus nasal cavity immunity vaccine.
Further, above-mentioned steps 1）Concrete operations be：By Porcine epidemic diarrhea virus vero cells infection, work as cell
When lesion is up to more than 80%, virus liquid is harvested, virus liquid is inactivated, the virus concentration after inactivation is adjusted to 4.5 × 107~
5.5×107TCID50/ mL, produce cell inactivation seedling, i.e. vaccinogen liquid.
Further, the concrete operations of above-mentioned inactivation are：By in virus liquid add volume for virus liquid 1%~2% 8 ~
12g/L beta-propiolactones solution inactivates 24~48h, then adds sodium thiosulfate and is neutralized, inactivation process is blocked, i.e.,
Can.
Further, above-mentioned steps 2）Described in sodium azide solution concentration be 8 ~ 12g/L.
The beneficial effects of the invention are as follows：
PEDV nasal cavity immunities vaccine prepared by the present invention compares existing related vaccines, and its potency is high, good immune effect.
The vaccine of the present invention carries out vaccine immunity by bronchia mucosal tissue, can excitating organism mucosa-immune and be simultaneously
System is immune, and substantial amounts of sIgA is produced in sow milk, maternal antibody is passed into piglet by milk, obtains piglet effective
Immunoprotection, prevent piglet epidemic diarrhea harm.
Brief description of the drawings
Fig. 1 is the testing result of sIgA antibody.
Embodiment
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
The preparation of the vaccine of embodiment 1
Porcine epidemic diarrhea virus CHYJ130330 passes through vero cells infection（African green monkey kidney cell）, culture, harvest
Virus liquid, measure TCID50, obtain vaccinogen liquid after inactivation, for further preparing vaccine.Concrete operation step is as follows：
1）The preparation of vaccinogen liquid
Virus culture：Recovery Vero cells, through amplification, after growing up to thin individual layer to cell, by PEDV viruses CHYJ130330
By volume（1:10）~（1:100）It is seeded in cell bottle, in 37 DEG C, 5% CO2Cultivated in incubator, observe cell daily
Lesion, when cytopathy is up to more than 80%, virus liquid is harvested, -20 DEG C save backup.
Inactivation of virus：Measure TCID before virus liquid inactivation50（Organize median infective dose）, by above-mentioned virus 10g/L BPL
（Beta-propiolactone）Solution inactivates 24~48h（Volume is virus liquid 1%~2%,）, then add 10g/L sodium thiosulfate（With
Measure as the 0.5%~2% of cumulative volume）Neutralized, inactivation process is blocked；Virus concentration is finally adjusted to 5 × 107TCID50/
ML, produce cell inactivation seedling, i.e. vaccinogen liquid.
2）The preparation of assist agent solution
By TLR（Toll-like receptor）Activator, activator, carbomer, the PVPK of NOD sample acceptors（Polyvinylpyrrolidine
Ketone）It is slowly added in physiological saline, is placed on magnetic stirring apparatus stirring to all substances with 100~500 rpm and is completely dissolved,
Then assist agent solution can be obtained by being sterile filtered, and 4 DEG C save backup.
In the assist agent solution obtained, final concentration of 0.05~0.5 μ g/mL, the NOD samples receptor stimulating agent of TLR activators is whole
Concentration is 0.01~0.1 μ g/mL, final concentration of 0.5~5 mg/mL of final concentration of 0.1~10mg/mL of carbomer, PVPK.
Wherein above-mentioned TLR activator is the activator that can activate TLR2, TLR3, TLR4, TLR7 simultaneously, or can be swashed simultaneously
TLR2, TLR4, TLR8, TLR9 living activator, or can activate simultaneously in TLR2, TLR4, TLR3, TLR9 activator at least
It is a kind of.
Above-mentioned NOD samples receptor stimulating agent is at least one of NOD1 samples receptor stimulating agent, NOD2 sample receptor stimulating agents.
3）The preparation of PEDV nasal cavity immunity vaccines
By the vaccinogen liquid of above-mentioned preparation, assist agent solution and 10g/L sodium azide solutions by volume（94~88）：（5~
10）：（1~2）PEDV virus CHYJ130330 vaccine semi-finished product can be obtained after mixing, vaccine semi-finished product are distributed into 100mL/
Bottle, it is PEDV nasal cavity immunity vaccines after steriling test.
The PEDV nasal cavity immunity vaccines prepared below to above-described embodiment make further effect detection.
First, safety testing
By above-mentioned vaccine injection mouse, only, for the observation mouse state of mind to 48 hours, mouse diet was normal, spirit by 1mL/
In good condition, body temperature is normal, it was demonstrated that the vaccine safety of above-mentioned preparation.
It is another to take vaccine to give farrowing sow collunarium, 4mL/ heads, totally 5, observe 7 days.Sow growth conditions are normal, and body temperature is just
Often, have no to the irritant effect of nasal cavity, it was demonstrated that the vaccine safety.
2nd, Study On Immunogenicity
PEDV nasal cavity immunities vaccine prepared by embodiment 1 is subjected to Study On Immunogenicity to piglet.
Method：By PEDV nasal cavity immunities vaccine in farrowing sow it is antenatal 40 days, 20 days carry out Nasal immunization, 4mL/ heads/time,
Per side nostril 2mL, the health condition and farrowing situation of sow are observed and recorded during whole experiment, and is adopted when Farrowing
Collect the blood of 5 sows, collect serum, carry out antibody neutralizing mensuration.Colostrum is gathered simultaneously surveys sIgA antibody.Set collunarium not simultaneously
Vaccine group containing adjuvant, injection group, oral group, commercial goods vaccinate group.
1）Antibody neutralizing mensuration
Neutralizing antibody assay method is as follows：The sow serum that separation obtains is added in 96 porocyte culture plates, uses cell
After growth-promoting media doubling dilution to finite concentration（4 repetitions of each dilution holes）, add and be diluted to 200 TCID in advance50/ mL disease
Venom, 37 DEG C are put into after well mixed, 5% CO21~2h is neutralized in incubator, then adds cell suspension, cell concentration 2
×105~3 × 105Individual/mL, 37 DEG C are placed in, 5% CO2Cultivated in incubator.Cytopathy situation is observed after 5~7 days, its
In using half cell there is the highest extension rate of the serum of lesion as the neutralization titer of serum, as a result see the table below 1.
The sow immunity test neutralizing antibody testing result of table 1
As it can be seen from table 1 the PEDV nasal cavity immunities vaccine prepared using the present invention can be induced through Nasal immunization in sow
Higher neutralizing antibody is produced, compared to other groups, the highest extension rate highest of lesion occurs in its half cell, it is seen then that it is anti-
Body potency is compared with collunarium group（Without adjuvant）, injection group, the height of oral group and commercially available vaccine control group.
2）The detection of sIgA antibody in colostrum
In each group, the testing result of sIgA antibody is as shown in figure 1, it can be seen that collunarium vaccine in sow colostrum（Contain
Adjuvant）The content of sIgA in the sow milk of group is apparently higher than other each groups, i.e., the PEDV nasal cavity immunity epidemic diseases that prepared by the present invention
The potency of seedling is high, good immune effect.
It is readily appreciated that for those skilled in the art, the foregoing is only the preferred embodiment of patent of the present invention, and
Not to limit the present invention, all any modification, equivalent and improvement made within all the spirit and principles in the present invention etc., fall
Within the protection domain of application claims.
Claims (5)
1. a kind of application of vaccine adjuvant solution in pig epidemic diarrhea inactivation of viruses nasal cavity immunity vaccine adjuvant is prepared, the epidemic disease
Seedling assist agent solution contains 0.05~0.5 μ g/mL TLR activator, the activator of 0.01~0.1 μ g/mL NOD sample acceptors, 0.1
~10mg/mL carbomers and 0.5~5mg/mL polyvinylpyrrolidones, surplus are physiological saline；
The activator of the TLR is that can activate TLR2, TLR3, TLR4, TLR7 activator simultaneously, or can activate simultaneously TLR2,
TLR4, TLR8, TLR9 activator, or at least one of TLR2, TLR4, TLR3, TLR9 activator can be activated simultaneously；
The NOD samples receptor stimulating agent is at least one of NOD1 samples receptor stimulating agent, NOD2 sample receptor stimulating agents.
2. a kind of application of vaccine adjuvant solution in pig epidemic diarrhea inactivation of viruses nasal cavity immunity vaccine is prepared, its feature exist
In：
The vaccine preparation method comprises the following steps：
1) the cell inactivation seedling of Porcine epidemic diarrhea virus, i.e. vaccinogen liquid are prepared；
2) by the vaccine adjuvant solution described in vaccinogen liquid, claim 1 and sodium azide solution by volume (94~88)：(5~
10)：(1~2) mix, produce Porcine epidemic diarrhea virus nasal cavity immunity vaccine.
3. application according to claim 2, it is characterised in that：The concrete operations of step 1) are：By Porcine Epidemic Diarrhea
Malicious vero cells infection, when cytopathy is up to more than 80%, virus liquid is harvested, virus liquid is inactivated, after inactivation
Virus concentration is adjusted to 4.5 × 107~5.5 × 107TCID50/ mL, produce cell inactivation seedling, i.e. vaccinogen liquid.
4. application according to claim 3, it is characterised in that：The concrete operations of the inactivation are：It will be added in virus liquid
Volume inactivates 24~48h for 8~12g/L beta-propiolactones solution of virus liquid 1%~2%, then adds sodium thiosulfate and enters
Row is neutralized, and inactivation process is blocked, you can.
5. application according to claim 2, it is characterised in that：Sodium azide solution concentration described in step 2) for 8~
12g/L。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510053897.9A CN104667269B (en) | 2015-02-02 | 2015-02-02 | A kind of Porcine epidemic diarrhea virus nasal cavity immunity vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510053897.9A CN104667269B (en) | 2015-02-02 | 2015-02-02 | A kind of Porcine epidemic diarrhea virus nasal cavity immunity vaccine and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104667269A CN104667269A (en) | 2015-06-03 |
| CN104667269B true CN104667269B (en) | 2018-01-09 |
Family
ID=53303204
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510053897.9A Active CN104667269B (en) | 2015-02-02 | 2015-02-02 | A kind of Porcine epidemic diarrhea virus nasal cavity immunity vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104667269B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107177001B (en) * | 2016-03-09 | 2021-01-01 | 北京大伟嘉生物技术股份有限公司 | Egg yolk antibody for preventing and treating porcine epidemic diarrhea and preparation method thereof |
| JP6253210B1 (en) | 2016-08-17 | 2017-12-27 | 一般財団法人日本生物科学研究所 | Method for preventing or treating swine epidemic diarrhea, vaccine, and vaccine kit |
| CN106729691A (en) * | 2017-01-09 | 2017-05-31 | 北京大伟嘉生物技术股份有限公司 | A kind of Porcine epidemic diarrhea virus variant inactivated vaccine and its application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101117627B (en) * | 2007-02-01 | 2010-07-14 | 中国农业科学院哈尔滨兽医研究所 | Pig epidemic diarrhea virus attenuated vaccine strain and uses thereof |
| BR112013032675A2 (en) * | 2011-06-19 | 2017-08-08 | Vaxine Pty Ltd | adjuvant vaccine composition comprising inulin particles |
| CN103705918B (en) * | 2013-12-24 | 2019-08-06 | 北京大北农科技集团股份有限公司动物医学研究中心 | Porcine epidemic diarrhea resisting virus hyper-immune serum and preparation method thereof |
-
2015
- 2015-02-02 CN CN201510053897.9A patent/CN104667269B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104667269A (en) | 2015-06-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106267195B (en) | Triple antigen-antibody complex for porcine viral diarrhea and preparation method thereof | |
| CN106554944B (en) | Porcine epidemic diarrhea virus low-virulent strain, vaccine composition prepared from porcine epidemic diarrhea virus low-virulent strain and application of vaccine composition | |
| CA3170184A1 (en) | Adjuvant and vaccine compositions | |
| CN105153303A (en) | Egg yolk antibody for preventing and controlling duck parvovirus and preparation method thereof | |
| CN104667269B (en) | A kind of Porcine epidemic diarrhea virus nasal cavity immunity vaccine and preparation method thereof | |
| US9783788B2 (en) | Porcine pseudorabies virus (PRV)-YF strain and its application | |
| CN110893235B (en) | Inactivated vaccine for crucian hematopoietic necrosis and preparation method thereof | |
| CN105112349B (en) | A kind of brucella melitensis molecular marker vaccine strain and its application | |
| KR101654023B1 (en) | Live vaccine composition, inactivated vaccine composition and oral vaccine comprising the same from currently isolated attenuated porcine epidemic diarrhea virus | |
| CN108969492B (en) | Oral attenuated freeze-dried vaccine for swine fever and preparation method thereof | |
| CN111808826B (en) | Porcine type-A seneca virus SVA/CH-Fuj strain and application thereof | |
| US10064930B2 (en) | Immunogenic compositions and methods for pigeon fever | |
| CN116813718B (en) | Recombinant porcine circovirus 2 type Cap protein trimer, expression system and application thereof | |
| CN111729091B (en) | Method for checking efficacy of porcine Seika virus inactivated vaccine by using rabbit | |
| CN101745106A (en) | Porcine parvnvirus living vaccine and preparation method thereof | |
| CN104511015B (en) | A kind of vaccine combination and preparation method and application | |
| CN108517318A (en) | A kind of variation strain of Porcine epidemic diarrhea virus and its application | |
| CN110302374B (en) | Canine vaccine and preparation method and application thereof | |
| CN104758928A (en) | Goatpox virus-orf virus combined cell attenuated vaccine and its preparation method and use | |
| EP2034012A1 (en) | Vaccine against viral rabbit hemorrhagic disease | |
| CN104800842B (en) | A kind of goatpox, sheep pox divalence cell weak-toxic vaccine and its preparation method and application | |
| CN104250623B (en) | One plant of mycoplasma hyorhinis bacterial strain, vaccine combination and its preparation method and application | |
| CN104288762B (en) | A kind of vaccine combination and its preparation method and application | |
| CN111686246B (en) | Antigen-antibody complex vaccine for porcine epidemic diarrhea virus and preparation method thereof | |
| CN101380470B (en) | Pig parvovirus live vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Jia Aiqing Inventor after: Feng Xiaosheng Inventor after: Wang Guiping Inventor after: Liu Qi Inventor after: Zhou Ruyue Inventor before: Jia Aiqing Inventor before: Feng Xiaosheng Inventor before: Wang Guiping Inventor before: Liu Qi Inventor before: Wang Yueyun Inventor before: Zhou Ruyue Inventor before: An Xinyu |
|
| COR | Change of bibliographic data | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |