CN101880651B - Preparation method of Muscovy duck parvo novel vaccines - Google Patents
Preparation method of Muscovy duck parvo novel vaccines Download PDFInfo
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- CN101880651B CN101880651B CN 201010215818 CN201010215818A CN101880651B CN 101880651 B CN101880651 B CN 101880651B CN 201010215818 CN201010215818 CN 201010215818 CN 201010215818 A CN201010215818 A CN 201010215818A CN 101880651 B CN101880651 B CN 101880651B
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Abstract
The invention relates to a preparation method of Muscovy duck parvo novel vaccines, which comprises the following steps of: using Muscovy duck parvovirus attenuated virus P1 strain (MPV-P1 strain with a preservation number of CCTCC NO:V201013) and Muscovy duck origin goose parvovirus attenuated virus D strain ( GPV-D strain with a preservation number of CCTCC NO:V201014) as seed viruses; proliferating viruses by applying the muscovy duck embryone fibroblast spinner culture technology to obtain cell toxic liquids; mixing the cell toxic liquids according to the proper proportion and freezing a dry protective agent to freeze and dry to research safe and effective Muscovy duck parvo novel vaccines. The young Muscovy ducks are inoculated once so as to prevent and control the Muscovy duck parvovirus infection and the Muscovy duck gosling blast dieases at the same time, thereby the stress reaction of the Muscovy ducks caused by immunization for many times is solved. The invention is suitable for scale production under the GMP (Good Manufacturing Practices) condition, and saves the production cost of the vaccines.
Description
Technical field
The present invention relates to kind preparation method of the tiny vaccine of duck.
Background technology
Kind duck parvovirus (being commonly called as " three weeks are sick ") is the 1-3 kind duck in age in week at first found at our province in 1987, and to rush down be a kind of new eqpidemic disease of clinical symptom to breathe heavily.This disease is mainly encroached on the young bird kind duck of 7-21 age in days, sickness rate 27-62% and case fatality rate 22-43%, and the duck major part of recovering becomes stiff duck, already causes very large economy loss for kind duck.Aspect this disease pathogen, diagnosis and study on prevention, I all occupy the top standard of international similar research.We at first separate, have identified a kind duck parvovirus (MPV) at home and abroad, confirm that MPV is kind cause of disease of duck parvovirus, are the newcomers that the Parvoviridae parvovirus belongs to, and have enriched the animal virology content.Set up on this basis and prepared 5 strain monoclonal antibodies, use a wherein strain monoclonal antibody sensitization polystyrene latex, take the lead in having set up latex agglutination (LPA) and agglutination inhibition test (LPAI) detection virus and antibody at home and abroad; Utilize biotechnology to select simultaneously and meet the low virulent strain that the manufacturing living vaccine is used, the vaccine of preparation can effectively prevent a kind duck parvovirus." kind duck parvovirus disease live-vaccine " and " kind duck parvovirus diagnostic reagent " obtained one type of new veterinary drug certificate of country respectively in 2000.
Kind duck gosling blast is to have raised district's outbreak of epidemic kind duck on ground such as Putian, Fujian Province, Fuqing since 1997, and all there is generation in each foster duck area, the whole nation at present.Young clinically kind duck takes place to come off with diarrhoea, intestines mucosa, and to form embolism be characteristic, sickness rate 50-70%, case fatality rate 40-65%.Use various microbiotic, kind duck parvovirus vaccine and the high immunity yolk antibody etc. all can not disease controlling, the provisions duck be already caused than large economy and loses.For effectively anti-system should disease; We have carried out cause of disease, diagnosis and study on prevention: from 1997 should disease Fujian Province kind duck raise the district popular since, we successively are separated to 8 strains kind duck source goose parvovirus from morbidity kind duck internal organs, and have carried out comprehensive and systematic evaluation; Affirmation should virus be a kind duck gosling blast virus; Clinically should disease normal and kind duck parvovirus polyinfection, very harmful, artificial challenge or find all that clinically its M & M all is higher than a kind duck parvovirus.On this basis, our seed selection becomes kind duck gosling blast virocyte to adapt to low virulent strain and is developed into a kind duck gosling blast less toxic vaccine, and can effectively prevent should disease.
Kind duck parvovirus and kind duck gosling blast all are important transmissible diseases of kind duck; Use vaccine and can effectively prevent the generation of these two diseases; The distress that the duck crowd causes is reacted with reducing because of immunization repeatedly in order to reduce manpower and materials; We develop the tiny vaccine of kind duck again, and young kind of duck immunity of 1-2 age in days can prevent kind duck parvovirus and kind duck gosling blast 1 time throughout one's life.
Summary of the invention
The purpose of this invention is to provide kind preparation method of the tiny vaccine of duck, the present invention realizes through following technical scheme:
A kind ofly derive from malicious P1 strain (MPV-P1 strain) a little less than the exquisite weak kind duck parvovirus of people, be deposited in CCTCC, June 2 2010 preservation day, deposit number: CCTCC NO:V201013.
A kind ofly derive from malicious D strain (GPV-D strain) a little less than the exquisite weak kind duck source goose parvovirus of people, be deposited in CCTCC, June 2 2010 preservation day, deposit number: CCTCC NO:V201014.
Malicious D strain a little less than malicious P1 strain a little less than the above-mentioned kind duck parvovirus and kind duck source goose parvovirus is applied to the tiny vaccine of preparation kind duck.
The present invention with kind duck parvovirus a little less than a little less than malicious P1 strain (MPV-P1 strain) and kind duck source goose parvovirus malicious D strain (GPV-D strain) serve as kind of a poison; Use kind DEF rolling bottle culture technique virus of proliferation; The harvested cell venom; Add the lyophilized vaccine lyophilize after mixing by suitable proportion, be developed into genetic stability, security is good, immunogenicity is strong and cost the is low tiny vaccine of kind duck, reach the purpose that once inoculation could prevent and control two kinds of eqpidemic diseases simultaneously.The present invention is suitable for the tiny vaccine of scale operation kind duck under the GMP condition.
Embodiment
Below in conjunction with specific embodiment the present invention is further specified.(but not being limitation of the present invention).
Derive from malicious P1 strain (MPV-P1 strain) a little less than the exquisite weak kind duck parvovirus of people; Be to utilize kind duck embryo and kind DEF alternately to go down to posterity etc. to form a little less than the biotechnology artificially breeding causes; Lost pathogenic to the young kind duck of an age in days; And have following characteristic (1) virus titer: the MPV-P1 strain is inoculated 12 ages in days kind duck embryo, its ELD through allantoic cavity
50Answer>=10
-4.5/ 0.1ml; To a kind duck embryo fibroblast monolayer TCID
50>=10
-5.0/ 0.1ml; (2) security: with young kind duck 10 plumages of the healthy susceptible of 1 age in days, 0.2 milliliter of every plumage leg muscle injection seed culture of viruses stoste was observed 20 days, should all survive; (3) immunogenicity: the healthy responsive young kind duck of an age in days, 0.2 milliliter of leg muscle injection, its minimum immunity amount is 100TCID
50/ 0.2ml; (4) virus multiplication: select MDEF multiplication culture MPV-P1 for use, inoculum size is 4～5 days for the time that 1%, 37 ℃ of rotating and culturing to cytopathy of cultivating liquid measure reaches more than 75% synchronously.The MPV-P1 strain be deposited in Chinese typical culture collection center (or be called for short: CCTCC), June 2 2010 preservation day, deposit number CCTCCNO:V201013.
Derive from malicious D strain (GPV-D strain) a little less than the exquisite weak kind duck source goose parvovirus of people; Be to utilize a kind DEF variable to go down to posterity alternately to form a little less than seed selection causes; Lost pathogenic to the young kind duck of an age in days, and had following characteristic (1) virus titer: the GPV-D strain is to kind duck embryo fibroblast monolayer TCID50>=10
-4.5/ 0.1ml; LPA tires and reaches 2
3(2) security: with young kind duck 10 plumages of the healthy susceptible of 1 age in days, 0.2 milliliter of every plumage leg muscle injection seed culture of viruses stoste was observed 20 days, should all survive; (3) immunogenicity: the healthy responsive young kind duck of an age in days, 0.2 milliliter of leg muscle injection, its minimum immunity amount is 300TCID
50/ 0.2ml; (4) virus multiplication: select MDEF multiplication culture GPV-D for use; Inoculum size is 4～5 days for the time that 1%, 37 ℃ of rotating and culturing to cytopathy of cultivating liquid measure reaches more than 75% synchronously.The GPV-D strain is deposited in CCTCC, June 2 2010 preservation day, deposit number CCTCC NO:V201014.
Malicious D strain a little less than malicious P1 strain a little less than the above-mentioned kind duck parvovirus and kind duck source goose parvovirus is applied to kind preparation method of the tiny vaccine of duck:
(1) MPV-P1 strain propagation, by the 1% synchronous inoculation kind DEF (MDEF) of cultivating liquid measure, 37 ℃ of rotating and culturing to cytopathies reach more than 75%, results MPV-P1 cell toxicant with MPV-P1 strain kind poison.
(2) GPV-D strain propagation, by the 1% synchronous inoculation kind DEF (MDEF) of cultivating liquid measure, 37 ℃ of rotating and culturing to cytopathies reach more than 75%, results GPV-D cell toxicant with GPV-D strain kind poison.
(3) viral level TCID
50Measure, MPV-P1 and GPV-D are to a kind duck embryo fibroblast monolayer TCID50 difference>=10
-4.5/ 0.1ml and>=10
-4.0When/0.1ml is above, can be used for preparing vaccine.(4) vaccine preparation adds the lyophilized vaccine lyophilize with MPV-P1 and GPV-D after by 1: 1～1.5 mixed and forms.
How reducing production costs and improving immune effect is the gordian technique that the present invention will solve, and for this reason, the present invention further takes following measure:
(1) cell culture fluid: get 0.5% newborn Chinese liquid and add equivalent DMEM liquid, include each 100 units/ml of 3% calf serum, penicillium mould and Streptomycin sulphate, and with 7.5%NaHCO
3Accent pH is 7.4-7.6.
(2) kind DEF preparation: get and hatch well-developed kind of duck embryo of 17 ages in days, on the ovoscopy lamp, use the stroke discharge chamber.Place on the incubator tray, use 75% alcohol disinfecting eggshell air chamber, aseptic taking-up embryo again with 2% tincture of iodine earlier; Decaptitate, four limbs and internal organ, put into the glassware of sterilization, use Hank ' s liquid washing idiosome; Be cut into 2～3mm with the scissors of sterilizing and organize fritter, pour in the sterilization triangle digestion bottle, use Hank ' s liquid to wash again 2～3 times; (each embryo 8～10ml) is placed on that stirring at low speed left standstill 5 minutes after 30 minutes on constant temperature (37 ℃) magnetic stirring apparatus to add 37 ℃ 0.25% pancreatin; Tissue block is avaled, and the upper strata cell suspension is poured in the 500ml sterilization vial, and vial places ice bath.Tissue block in the digestion bottle adds 0.25% an amount of pancreatin again, the samely repeats to digest 3～4 times, when tissue block is flocculence, promptly stops digestion.Cell suspension in packing 250～500ml sterilization centrifugal bottle (pipe), in centrifugal 15～20 minutes kinds of 4 ℃～8 ℃ low speed (800～1000 rev/mins), is abandoned supernatant after filtering with 4 layers of sterile gauze.The cell precipitation piece of each centrifugal bottle; The nutrient solution that contains 5% calf serum with 3～5ml dispels; Collect in each centrifugal bottle (pipe) spissated cell suspension in the 100ml vial; Count every milliliter and contain cell count, with cell culture fluid the concentrating cells suspension is diluted to every milliliter and contains 150～1,800,000 viable cell.Subsequent use.
(3) virus multiplication: kind DEF is inoculated MPV-P1 and GPV-D kind poison respectively synchronously, and inoculum size is to cultivate 1% of liquid measure, the rearmounted 37 ℃ of rotating and culturing of mixing, and rotating speed is per hour 9～11 commentaries on classics.Every day, observation of cell was 2 times, when pathology (cytopathy is that the cell refractivity strengthens, and the cell circle contracts) appears in 75% above cell, put culturing bottle-20 ℃ frozen.
(4) vaccine immunity is learned index determining
4.1 steriling test: undertaken by existing " the People's Republic of China's veterinary drug allusion quotation " appendix, answer asepsis growth.
4.2 viral level is measured: on MDEF individual layer 96 orifice plates, carry out respectively, every extent of dilution connects 4 holes, and the 0.1ML/ hole is put under 6.0,37 ℃ of conditions of CO2 and cultivated, and observes 7 days, calculates TCID
50MPV-P1 answers>=10
5.5TCID
50/ ml and GPV-D answer>=10
5.0TCID
50/ ml.
4.3 safety verification: with young kind duck 10 plumages of the healthy susceptible of 1 age in days, the vaccine of 10 times of amounts of every plumage leg muscle injection was observed 20 days, and the inoculation duck does not all see diet desire and psychological problem, and it is good to grow, all survivals.
4.4 efficacy test: can know that from table 1 the young kind duck leg flesh of an age in days susceptible is inoculated 1 plumage part vaccine, and, supply the LPAI antibody titer to measure in inoculating back 7 days together with not immune control group blood sampling separation of serum; Attack MPV and GPV poison by force simultaneously respectively, every group 10 plumage.The result shows: immune duck all can be resisted the attack with the source strength poison, and protection ratio reaches 100% and 87.5% respectively; Attack simultaneously before the poison that MPV-LPAI antibody and GPV-LPAI antibody reach 2 respectively in the immune duck serum
-2.2With 2
-2.3, and the contrast duck is 2
0.6Below.
After the young kind pest of duck seedling immunity of table 1 age in days to measuring with the protection ratio of source strength poison
4.5 immune generation phase and immune duration are measured: can know that from table 2 back 7 days LPAI antibody horizontals of immunity are all 2
2.0More than, and can resist strong virus attack, and continue to i.e. 87 ages in days of back 80 days of immunity always, met or exceeded a kind duck and delivered age in days for sale this moment.The result shows that young kind of duck immunity of an age in days can protect for 1 time throughout one's life, means that also 1 immunization can prevent kind a duck parvovirus and a gosling blast simultaneously, not only reduces manpower and materials, also reduces the stress response that causes because of immunization repeatedly.
Antibody horizontal dynamic change in the serum after the young kind pest of duck seedling immunity of table 2 age in days
4.6 behind the vaccine immunity anti-MPV LPAI antibody horizontal with attack poison protection dependency: can know from table 3, measure anti-MPV LPAI antibody titer in 7 days behind the young kind duck immunization vaccine of 1 age in days, attack MPV poison by force simultaneously.Antibody titer is 2 as a result
-1More than, it is 100% that MPV is attacked malicious protection ratio, antibody titer＜2
-1, its sickness rate 62.5%, mortality ratio 33.3% proves that LPAI tires and protection is proportionate.Simultaneously, measure the MPV antibody titer with the LPAI method and can replace the method for vaccine potency check with strong virus attack, both easy, can prevent strong poison diffusion again.
Table 3 vaccine immunity is injected the relation with protection of tiring of anti-MPV LPAI in back 7 days kinds of duck serum
4.7 behind the vaccine immunity anti-GPV LPAI antibody horizontal with attack poison protection dependency: can know from table 4, measure anti-GPV LPAI antibody titer in 7 days behind the young kind duck immunization vaccine of 1 age in days, attack GPV poison by force simultaneously.Antibody titer is 2 as a result
-2More than, it is 100% that GPV is attacked malicious protection ratio; Antibody titer is 2
-1, its sickness rate 13%, mortality ratio 0; Antibody titer＜2
-1, its sickness rate 70%, mortality ratio 35%; Proof LPAI tires and protection is proportionate.Simultaneously, measure the GPV antibody titer with the LPAI method and can replace the method for vaccine potency check with strong virus attack, both easy, can prevent strong poison diffusion again.
Table 4 vaccine immunity is injected the relation with protection of tiring of anti-GPV LPAI in back 7 days kinds of duck serum
4.8 clinical application effect evaluation: be total to the young kind duck of immunization plumage more than 5000 part one age in days in test duck field, tracing observation 20d does not see that untoward reaction appears in immune duck; And the immune group surviving rate reaches more than 95%, apparently higher than control group (surviving rate 68%).Further this vaccine safety of proof, effective can be used for preventing clinically kind a duck parvovirus and a gosling blast.
Claims (3)
1. derive from malicious P1 strain a little less than the exquisite weak kind duck parvovirus of people, be deposited in CCTCC, June 2 2010 preservation day, deposit number: CCTCC NO:V201013.
2. derive from malicious D strain a little less than the exquisite weak kind duck source goose parvovirus of people, be deposited in CCTCC, June 2 2010 preservation day, deposit number: CCTCC NO:V201014.
3. with the application of malicious D strain a little less than malicious P1 strain a little less than described kind of duck parvovirus of claim 1 and described kind of duck source of claim 2 goose parvovirus on the tiny vaccine of preparation kind duck.
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Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102346191B (en) * | 2011-06-17 | 2013-09-11 | 福建省农业科学院畜牧兽医研究所 | Muscovy duck gosling plague latex particle agglutination reagent and preparation method thereof |
| CN102426238B (en) * | 2011-09-07 | 2013-09-04 | 福建省农业科学院畜牧兽医研究所 | Immunofluorescent reagent used for detecting Muscovy duck gosling blast diseases |
| CN102847147B (en) * | 2012-02-24 | 2014-10-01 | 福建省农业科学院畜牧兽医研究所 | Combined live vaccine for muscovy duck parvovirus disease and muscovy duck reovirus disease |
| CN103272230B (en) * | 2012-02-24 | 2015-01-28 | 福建省农业科学院畜牧兽医研究所 | Triple vaccine special for Muscovy duck |
| CN103820397B (en) * | 2014-03-07 | 2016-05-11 | 青岛易邦生物工程有限公司 | A kind of kind Duck parvovirus and application thereof |
| CN105154410B (en) * | 2015-09-21 | 2018-04-03 | 山东农业大学 | A kind of Duck parvovirus strain and its live vaccine |
| CN105920596B (en) * | 2016-06-18 | 2021-02-05 | 青岛易邦生物工程有限公司 | Muscovy duck parvovirus disease and gosling plague bivalent vaccine |
| CN105907728B (en) * | 2016-06-18 | 2019-03-12 | 青岛易邦生物工程有限公司 | A kind of kind duck source Goose Parvovirus |
| CN106075426B (en) * | 2016-06-18 | 2021-02-05 | 青岛易邦生物工程有限公司 | Gosling pestivirus vaccine |
| CN106620687A (en) * | 2016-11-10 | 2017-05-10 | 刘同明 | Immunization method for gosling plague |
| CN108707588B (en) * | 2018-06-05 | 2021-11-16 | 山东信得科技股份有限公司 | Muscovy duck parvovirus strain and application thereof |
| CN108465107B (en) * | 2018-06-05 | 2020-02-21 | 山东信得科技股份有限公司 | Duck type 2 adenovirus and Muscovy duck parvovirus disease combined inactivated vaccine |
| CN112501132A (en) * | 2020-08-28 | 2021-03-16 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Duck source goose parvovirus artificial attenuated strain and application thereof |
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| CN1218748C (en) * | 2001-12-19 | 2005-09-14 | 福建省农业科学院畜牧兽医研究所 | Foreign duck liver ichthyophthirius active vaccine and preparation method thereof |
| CN1179751C (en) * | 2001-12-19 | 2004-12-15 | 福建省农业科学院畜牧兽医研究所 | Foreign duck liver ichthyophthirius active vaccine and its preparation method |
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