CN105854011A - Preparation and application of efficient cell inactivation vaccine against new duck reovirus disease - Google Patents
Preparation and application of efficient cell inactivation vaccine against new duck reovirus disease Download PDFInfo
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- CN105854011A CN105854011A CN201610265577.4A CN201610265577A CN105854011A CN 105854011 A CN105854011 A CN 105854011A CN 201610265577 A CN201610265577 A CN 201610265577A CN 105854011 A CN105854011 A CN 105854011A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention relates to preparation and application of an efficient cell inactivation vaccine against new duck reovirus disease. The preparation method comprises the following steps: an antigen preparation step: inoculating a virus NDRV-TH11 strain with immortalized duck embryo fibroblast to obtain cytotoxicity with 80% of cytopathic effect, and performing cryopreservation at -20 DEG C; and a vaccine preparation step: inactivating the obtained cytotoxicity and mixing with a homemade nano adjuvant and stirring uniformly to obtain the efficient cell inactivation vaccine against new duck reovirus disease. After the duck is immunized by the efficient cell inactivation vaccine against new duck reovirus disease prepared in the invention, the duck can effectively resist the attack of a virulent strain of the new duck reovirus.
Description
Technical field
The present invention relates to preparation and the application of a kind of new duck reovirus disease high efficiency cell inactivated vaccine, be specifically related to one
Plant new duck reovirus disease high efficiency cell inactivated vaccine and the application on new duck reovirus disease of prevention thereof, belong to
In Preventive Veterinary Medicine field.
Background technology
Avianreovirus infects the report of aquatic bird and is initially detected in Muscovy duck, and referred to as muscovy duck reovirus is sick, its pathogenic because of
Son is referred to as muscovy duck reovirus (Muscovy duck reovirus, MDRV).Research shows that MDRV is to China
Beijing duck and other local varieties duck do not have infectivity.Along with the development of duck culturing industry, reovirus disease infects
Multi items duck also appears in the newspapers, but due to epidemic regions, morbidity population and the difference of disease time, reovirus infection
Duck shows difference in various degree.Cheng Anchun etc. report ground, Sichuan, Yunnan etc. in 1998 " the duck viral head that swells goes out
Mass formed by blood stasis " epidemic situation, follow-up study is shown to be by caused by duck reovirus.2008, Liu Hong etc. was to duck field, Guangdong Province one
Planting with swollen head, weak foot, shed tears, it is main special for drawing yellow green loose stool, hepatorrhagia and downright bad and esophagus cloaca ulcer and incrustation
When the epidemic situation levied carries out Study on Etiology, be separated to many strain virus, including 1 strain duck reovirus (Duck reovirus,
DRV-GZ strain).2009, it is a kind of hemorrhage and downright bad for major lesions feature with liver that old few Oriolus chinensis diffusus etc. reports Fujian Province
Mule duck and sheldrake reovirus disease, afterwards by studying the infectivity of this strain and immunogenic attributes, find that this is
There is the novel reovirus of notable difference in one strain and muscovy duck reovirus.
2011, Liu etc. was also isolated to a strain reovirus (HC strain) in Beijing duck body, and its animal returns real
Testing and show, DRV-HC strain causes the death failing to cause duck in 15d, but can cause the death of SPF chicken, and this strain is described
Pathogenicity the strongest.
In the same year, some duckeries of China East China there occurs that one is main with weak foot and liver, spleen necrosis successively
The infectious disease of feature, sickness rate about 20%～50%, case fatality rate, more than 80%, causes the most serious warp to duck culturing industry
Ji loss.China Agriculture Academe Shanghai Veterinary Institute is from Pathogen Biology, animal Orthogonal Rotational Regressive Tests, genome spy
Property and molecular level on confirm that this cause of disease is that a kind of in the past the duck reovirus of separation, Muscovy duck of being different from exhales intestinal first
Lonely virus, Avianreovirus, test proves that this epidemic situation is to be caused by reovirus, and the cause of disease being separated to claims
For DRV-TH11 strain.It is worthy of note, exhale with previously reported duck reovirus such as NDRV-JM85 strain, Muscovy duck
Intestinal orphan virus, Avianreovirus are compared, and the host range of new duck reovirus is wider, and pathogenic higher, it can
Infect the duck of multiple kinds including including sheldrake, Beijing duck, cherry valley duck etc., and the case fatality rate of infected duck is
50-80%, prompting TH11 strain is new duck reovirus (the Novel duck that a strain pathogenicity has morphed
reovirus,NDRV).Up to now, this disease there is no effectively preventing method.
Summary of the invention
For defect of the prior art, it is an object of the invention to provide a kind of new duck reovirus disease high-efficiency fine
The preparation of born of the same parents' inactivated vaccine and application, provide one safely and effectively method for the new duck reovirus disease of sending out of preventing and treating.
It is an object of the invention to be achieved through the following technical solutions:
First aspect, the invention provides the preparation method of a kind of new duck reovirus disease high efficiency cell inactivated vaccine,
Comprise the following steps:
Antigen preparation process: by the DEF of virus N DRV-TH11 strain inoculation immortalization, harvesting is sick
Becoming the cell toxicant reaching 80% ,-20 DEG C frozen；
Vaccine preparation process: mix after the cell toxicant inactivation of results with self-control nanometer adjuvant, stir, i.e. get Xin Fa
Duck reovirus disease high efficiency cell inactivated vaccine.
Preferably, described duck reovirus NDRV-TH11 strain is deposited in China Committee for Culture Collection of Microorganisms
(China General Microbiological Culture Collection Center is called for short at common micro-organisms center
CGMCC), depositary institution CGMCC.Address is in North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, preservation day 2013
On November 28, in, deposit number: CGMCC NO:8554.
Preferably, the DEF of described immortalization is prepared by this laboratory, (its preparation sees Yuzhi Fu,
Zongyan Chen,Chuanfeng Li,Guangqing Liu.Establishment of a duck cell line
susceptible to duck hepatitis virus type 1.J Virol Methods.2012
Sep；184(1-2):41-5.doi:10.1016/j.jviromet.2012.05.004.Epub 2012May 23.)
Can not be compared with continuous passage or SPF duck embryo cost height cause Virus culture difficulty with conventional primary cell, this high efficiency cell
Cell used by inactivated vaccine development is the cell of this laboratory immortalization, and cell can continuous passage, it is possible to suspension culture,
Growth conditions is good, and virus adapts to this DEF cell, and can cause cytopathy, is beneficial to breeding and the production of vaccine of virus.
Preferably, described self-control nanometer adjuvant includes each component of following weight/mass percentage composition: Nano chitosan 25%, breast
Agent 25%, mineral oil 50%.Compared with existing white-oil adjuvant, side reaction is less.
Preferably, in antigen preparation process, described virus N DRV-TH11 strain inoculation DEF monolayer.
Preferably, in vaccine preparation process, described inactivation specifically uses following steps: by the cell toxicant of results, by total
0.001% addition beta-propiolactone (BPL) of volume, 37 DEG C of inactivation 24h.The present invention uses BPL to inactivate, and can efficiently go out
Live virus.Compared with the inactivation mode of conventional inactivated vaccine, inactivation is more thorough and safe and efficient.And avoid because adding
When entering formalin-inactivated virus, formaldehyde amount too much causes immunity side reaction.
Preferably, in vaccine preparation process, the cell toxicant after described inactivation is 1:1 with the volume ratio of self-control nanometer adjuvant.
Second aspect, the invention provides a kind of new duck reovirus disease high efficiency cell of sending out prepared based on described method and goes out
The application of live vaccine, described application includes for preventing Novel duck reovirus disease.
Preferably, described prevention is newly sent out the method for duck reovirus disease and is specifically included: take inactivated vaccine immunity kind duck one
Secondary, 1ml/ plumage；After 4 weeks, booster immunization is once, 1ml/ plumage.
Prior art is compared, and the present invention has a following beneficial effect:
1. the present invention plants duck raising filial generation maternal antibody level by immunity, makes duckling can resist Novel duck at Sensitivity age and exhales
Intestinal orphan's virus infects.
2. the kind duck before laying eggs with above-mentioned high efficiency cell inactivated vaccine immunity, 1ml/ plumage, it is spaced 28 days booster immunizations, makes
Immunity is planted the filial generation of duck and is contained higher maternal antibody, and the filial generation duckling that immunity kind duck is produced can be resisted in 15 ages in days
The infection of Novel duck reovirus.
3. the present invention is simple and feasible, practical, provides one safely and effectively for the new duck reovirus disease of sending out of preventing and treating
Method.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.Following example will assist in those skilled in the art
Member is further appreciated by the present invention, but limits the present invention the most in any form.It should be pointed out that, the common skill to this area
For art personnel, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement.These broadly fall into
Protection scope of the present invention.
Embodiment 1
The DEF list of this laboratory immortalization is inoculated in the present embodiment application duck reovirus NDRV-TH11 strain
Layer, the harvesting poison when cytopathy reaches 80%；Above-mentioned virus is suitably concentrated and after beta-propiolactone (BPL) inactivates,
By proper proportion and self-control nanometer adjuvant mixing, make new duck reovirus disease high efficiency cell inactivated vaccine of sending out, then will
Above-mentioned inactivated vaccine is applied to prevent Novel duck reovirus disease；Plant duck immunity and neutralizing antibody: take inactivated vaccine immunity
Plant duck once, 10ml/ plumage；After 4 weeks, booster immunization is once, 10ml/ plumage.Described duck reovirus NDRV-TH11 strain
It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (China General
Microbiological Culture Collection Center, is called for short CGMCC), depositary institution CGMCC.Address exists
North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, November 28 2013 preservation day, deposit number: CGMCC NO:
8554。
Described vaccine preparation method specifically comprises the following steps that
(1) prepared by antigen: virus N DRV-TH11 strain is inoculated the DEF of this laboratory immortalization, results
Cytopathy reaches the cell toxicant of 80%, and-20 DEG C frozen.As the cell toxicant TCID to DEF50≥
10-67.5During/0.1ml, can be used for inactivation of virus.By the cell toxicant of results, by 0.001% addition β-the third of cumulative volume
Ester (BPL), 4 DEG C of inactivation 4h.Take the virus liquid after inactivation and inoculate 9 age in days SPF Embryo Gallus domesticus or 11 age in days duck embryos, Embryo Gallus domesticus and duck
Embryo should all be survived.
(2) prepared by vaccine: adds self-control nanometer adjuvant and the virus liquid of inactivation in the ratio of 1:1, stirs and make
Water-in-oil emulsion vaccine.This vaccine steriling test result is asepsis growth.
Experiment proves: the healthy susceptible duck of 3 ages in days, and after the vaccine of every plumage cervical region subcutaneous injection 1.0ml, duck should all be deposited in inoculation
Live, search for food, drink water, growth promoter etc. the most normal；Cut open at random and kill duck, the internal organs no abnormality seen such as the heart, liver, spleen.
In two exempt from rear different time (i.e. PI 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, 56
My god, 63 days, 70 days, 77 days, 84 days, 91 days) together with comparison duck blood sampling, measure NAT.Knot
Fruit shows, planting duck just has certain antibody for latter 7 days in second time immunity, and two exempt from latter 14 days antibody titers rises substantially, in
Two reach 2 when exempting from latter 28-49 days50, it is 2 until antibody horizontal when 91 days425。
Duckling (immunity plant duck generation) passive protection: take two exempt from after plant the hatching egg that duck was produced in 60 days, select with for the moment
Between to hatch an age in days of shell duck 20 plumage containing NDRV maternal antibody be maternal antibody group (A group)；Set nonimmune kind simultaneously
1 age in days duck 20 plumage of duck generation (without NDRV maternal antibody) is non-maternal antibody group (B group)；By A group and B group duckling
Penetrate NDRV poison 05ml/ plumage by force through lower limb intramuscular injection, observe 21 days.And after counteracting toxic substances the 7th day, cut open at random and kill 5 plumages, observe each device
Official's pathological changes situation.Result: A group duckling is injected after strong poison and all survives, search for food, drink water, the aspect such as spirit normal, with
It is all normal that machine cuts open internal organs such as killing the liver of 5 plumage ducks, spleen, has no the changes such as hemorrhagic necrosis.And after B group duckling injects strong poison,
Search for food, minimizing of drinking water, spirit is depressed, and comparison of whole observation period duck weight ratio maternal antibody group is light and obvious difference, at random
Cut open and kill 5 plumage duck liver, spleen all observes serious hemorrhagic necrosis speckle.Result shows that kind of duck is through twice immunity NDRV high efficiency cell
After inactivated vaccine, its filial generation duckling is highly resistant to NDRV poison by force and infects.
Duckling (filial generation of immunity kind duck) maternal antibody Fluctuation: take kind of duck two and exempt from produced hatching egg in latter 60 days, hatching
After going out shell, blood sampling measures duckling NDRV maternal antibody growth and decline situation.1 age in days duckling NDRV maternal antibody is higher, neutralizing antibody
Titer reaches 23；Being gradually reduced afterwards, also having certain maternal antibody, NAT when 15 age in days is 215, 21
During age in days, maternal antibody disappears substantially.
Clinical application effect is evaluated: selects 3 Ge Zhong duck plants in the popular epidemic-stricken area of NDRV and carries out the control test of the method.
Planting duck and opening antenatal twice immunity NDRV inactivated vaccine, the duckling hatched is followed the tracks of and observes 30 days, and duck is planted in test and duckling does not all have
Occur NDRV to infect.And periphery does not takes above measure duckery to occur that duckling is dead because there is this disease in various degree.
Prove the method safely, effectively further, can be used for preventing clinically newly to send out duck reovirus disease.
Vaccine safety is tested: with 100 times of minimum immune dosage, muscle or 10 1 age in days test young birds of cervical region subcutaneous vaccination
Duck, observes the reaction of inoculation duckling, observation period 15d.As a result without morbidity and death after 1 age in days Muscovy duck immune vaccine, cut open
Inspection has no macroscopic substantially pathological changes.
Storage life is tested: goes bail for and is stored in the inactivated vaccine of 6,12,15,18,24,28,30 months under the conditions of 2-8 DEG C,
By the optimum immuning dose immunity 1 age in days duckling determined, concurrently set nonimmune matched group, 14 age in days counteracting toxic substances.Result shows
Show that 2-8 DEG C preserves the vaccine after 6,12,15 months duckling opposing all can be protected completely with the attack of source strength poison；Preserve 24
Vaccine protective rate after Yue is only 80%, and the vaccine protective rate preserving 28,30 months is respectively 25%, 20%.This vaccine is
Long shelf life is 24 months.
The concrete application approach of the present invention is a lot, and the above is only the preferred embodiment of the present invention.It should be pointed out that, above
Embodiment is merely to illustrate the present invention, and is not limited to protection scope of the present invention.Common for the art
For technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement, these improvement also should regard
For protection scope of the present invention.
Claims (6)
1. the preparation method newly sending out duck reovirus disease high efficiency cell inactivated vaccine, it is characterised in that include with
Lower step:
Antigen preparation process: by DEF or the duck of 11 ages in days of virus N DRV-TH11 strain inoculation immortalization
Embryo, harvesting pathological changes reaches the cell toxicant of 80%, and-20 DEG C frozen；
Vaccine preparation process: mix after the cell toxicant inactivation of results with self-control nanometer adjuvant, stir, i.e. get Xin Fa
Duck reovirus disease high efficiency cell inactivated vaccine.
2. the new preparation method sending out duck reovirus disease high efficiency cell inactivated vaccine as claimed in claim 1, it is special
Levying and be, described self-control nanometer adjuvant includes each component of following weight/mass percentage composition: Nano chitosan 25%, emulsifying agent
25%, mineral oil 50%.
3. the new preparation method sending out duck reovirus disease high efficiency cell inactivated vaccine as claimed in claim 1, it is special
Levying and be, in vaccine preparation process, described inactivation specifically uses following steps: by the cell toxicant of results, by cumulative volume
0.001% adds beta-propiolactone, 37 DEG C of inactivation 24h.
4. the new preparation method sending out duck reovirus disease high efficiency cell inactivated vaccine as claimed in claim 1, it is special
Levying and be, in vaccine preparation process, the cell toxicant after described inactivation is 1:1 with the volume ratio of self-control nanometer adjuvant.
5. newly send out duck reovirus disease high efficiency cell inactivated vaccine based on prepared by method described in claim 1 for one kind
Application, it is characterised in that described application includes newly sending out duck reovirus disease for preventing.
6. the new application sending out duck reovirus disease high efficiency cell inactivated vaccine as claimed in claim 5, its feature exists
In, described prevention is newly sent out the method for duck reovirus disease and is specifically included: take inactivated vaccine immunity kind duck once, 1ml/ plumage；
After 4 weeks, booster immunization is once, 1ml/ plumage.
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| CN201610265577.4A CN105854011A (en) | 2016-04-26 | 2016-04-26 | Preparation and application of efficient cell inactivation vaccine against new duck reovirus disease |
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| CN201610265577.4A CN105854011A (en) | 2016-04-26 | 2016-04-26 | Preparation and application of efficient cell inactivation vaccine against new duck reovirus disease |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108498794A (en) * | 2018-05-22 | 2018-09-07 | 山东农业大学 | A kind of inactivated vaccine and preparation method thereof of prevention novel duck reovirus |
| CN108660118A (en) * | 2018-05-22 | 2018-10-16 | 山东农业大学 | One kind leading to the arthritic novel duck reovirus of duck and its application |
| CN108743932A (en) * | 2018-07-03 | 2018-11-06 | 重庆永健生物技术有限责任公司 | A kind of inactivated vaccine preparation method for preventing duck reovirus |
| CN108977579A (en) * | 2018-08-10 | 2018-12-11 | 山东农业大学 | It is a kind of to detect the PCR kit for fluorescence quantitative for leading to the novel duck reovirus of duck spleen necrosis |
| CN109718370A (en) * | 2019-02-27 | 2019-05-07 | 广东渔跃生物技术有限公司 | A kind of duck reovirus vaccine and preparation method thereof |
| CN111000993A (en) * | 2020-01-07 | 2020-04-14 | 齐鲁动物保健品有限公司 | Bivalent inactivated vaccine for duck viral hepatitis and duck reovirus disease and preparation method thereof |
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| CN102631674A (en) * | 2012-03-02 | 2012-08-15 | 福建省农业科学院畜牧兽医研究所 | Novel muscovy duck reovirus disease vaccine and application thereof |
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| YUZHI FU, ET AL.: "Establishment of a duck cell line susceptible to duck hepatitis virus type 1", 《 JOURNAL OF VIROLOGICAL METHODS》 * |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108498794A (en) * | 2018-05-22 | 2018-09-07 | 山东农业大学 | A kind of inactivated vaccine and preparation method thereof of prevention novel duck reovirus |
| CN108660118A (en) * | 2018-05-22 | 2018-10-16 | 山东农业大学 | One kind leading to the arthritic novel duck reovirus of duck and its application |
| CN108498794B (en) * | 2018-05-22 | 2020-07-07 | 山东农业大学 | Inactivated vaccine for preventing and treating novel duck reovirus and preparation method thereof |
| CN108743932A (en) * | 2018-07-03 | 2018-11-06 | 重庆永健生物技术有限责任公司 | A kind of inactivated vaccine preparation method for preventing duck reovirus |
| CN108977579A (en) * | 2018-08-10 | 2018-12-11 | 山东农业大学 | It is a kind of to detect the PCR kit for fluorescence quantitative for leading to the novel duck reovirus of duck spleen necrosis |
| CN109718370A (en) * | 2019-02-27 | 2019-05-07 | 广东渔跃生物技术有限公司 | A kind of duck reovirus vaccine and preparation method thereof |
| CN109718370B (en) * | 2019-02-27 | 2019-11-29 | 广东渔跃生物技术有限公司 | A kind of duck reovirus vaccine and preparation method thereof |
| CN111000993A (en) * | 2020-01-07 | 2020-04-14 | 齐鲁动物保健品有限公司 | Bivalent inactivated vaccine for duck viral hepatitis and duck reovirus disease and preparation method thereof |
| CN111000993B (en) * | 2020-01-07 | 2022-08-23 | 齐鲁动物保健品有限公司 | Bivalent inactivated vaccine for duck viral hepatitis and duck reovirus disease and preparation method thereof |
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