CN103525771A - Goose parvovirus and applications thereof - Google Patents
Goose parvovirus and applications thereof Download PDFInfo
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Abstract
The invention provides goose parvovirus (GPV) and applications thereof, and belongs to the technical field of biology. The invention provides a goose parvovirus GPV-YZ strain as well as applications thereof in preparing medicaments for preventing and treating the gosling plague disease. The microorganism preservation number is CGMCC NO.8160. The invention also provides a gosling plague disease viral vaccine as well as an anti-gosling plague yolk antibody. The goose parvovirus YZ strain provided by the invention has favorable immunogenicity for the present epidemic goose parvovirus, can resist toxicity attacking of the GPV-AV240 strain, the GPV-GD strain as well as the toxic strain of the goose parvovirus, and has the protection capability against the GPV-AV240 strain and the GPV-GD strain. After breeding geese are immunized by the gosling plague disease viral vaccine provided by the invention, young geese can be protected by maternal antibodies, and can be prevented from being infected by gosling plague viruses. The anti-gosling plague yolk antibody provided by the invention can prevent and treat gosling disease.
Description
Technical field
The invention belongs to biological technical field, be specifically related to Goose Parvovirus and application thereof.
Background technology
Gosling plague is a kind of acute or subacute septic transmissible disease by the caused young goose of goose parvovirus (claim again Goose Parvovirus, Goose parvovirus, is abbreviated as GPV).Gosling plague is the deadly infectious disease of gosling, once gosling catches an illness, case fatality rate is up to 50～95%.The propagation such as the Goose Parvovirus water that mainly secretory product, movement, kind egg and the sick goose by sick goose drank, feed, apparatus, place.Clinical tired with spirit, peel off solely even, serosity nose liquid is flowed out in nostril, suffers from goose and again and again shakes the head, and draws lark or yellow-green colour loose stool, nervous disorders, the mucous membrane necrosis of small intestine posterior segment comes off and solidifies formation embolus with fibrinous exudate, shape as sausage shape be feature.Often be sepsis process, M & M is very high, very big to supporting goose industry disserve to produce.
Rounded or the sexangle of goose parvovirus (claiming again Goose Parvovirus) outward appearance, diameter 20～25nm, has the common morphological specificity of this genus virus.Nucleic acid is comprised of single-stranded dna.At present, Goose Parvovirus GD strain (being abbreviated as " GPV-GD strain ") is the low virulent strain of domestication, and the attenuated live vaccines of preparing with it and yolk antibody are one of these sick effective ways of current control, and all obtains the new veterinary drug certificate of country.Goose Parvovirus AV240(is abbreviated as GPV-AV240) Reference strains preserved of Zhu Shi China Veterinary Drugs Supervisory Inst., for the related science research of gosling plague disease.Above-mentioned two kinds of strains are all separated acquisitions in last century.Research data shows, through developing for many years, although the serum of Goose Parvovirus does not have significant variation, gene order has obvious difference.After vaccine prepared by Goose Parvovirus GPV-GD strain and GPV-AV240 strain or yolk antibody inoculation gosling, gosling plague epidemic disease still happens occasionally.
Summary of the invention
The object of this invention is to provide a kind of goose parvovirus GPV-YZ strain; current popular goose parvovirus is had to good immunogenicity; can resist GPV-AV240 strain, GPV-GD strain and self strain and attack poison, protective capability covers GPV-AV240 strain, GPV-GD strain.
Another object of the present invention is to provide Goose Parvovirus vaccine, and immunity is planted after goose, and young goose can obtain protection from maternal antibody, can resist Goose Parvovirus and infect.
A further object of the present invention is to provide anti gosling plague yolk antibody, can prevent and treat gosling plague.
Object of the present invention adopts following technical scheme to realize.
Goose parvovirus GPV-YZ strain, its microbial preservation number is: CGMCC NO.8160.
Goose parvovirus GPV-YZ strain is in preparation prevention and control the application in the medicine for the treatment of gosling plague disease, and the microbial preservation of described goose parvovirus GPV-YZ strain number is CGMCC NO.8160.
The medicine of described prevention gosling plague disease is Goose Parvovirus vaccine, described in control treating gosling plague disease medicine be anti gosling plague yolk antibody.
Goose Parvovirus vaccine, the goose parvovirus GPV-YZ strain virus liquid that contains deactivation, the microbial preservation of described goose parvovirus GPV-YZ strain number is CGMCC NO.8160.
The preparation method of described goose parvovirus GPV-YZ strain virus liquid is: by goose parvovirus GPV-YZ strain inoculation goose embryo, cultivate 150-170h, collect the idiosome of allantoic fluid and removal gall-bladder, homogeneous after mixing, obtains described goose parvovirus GPV-YZ strain virus liquid.
Described Goose Parvovirus vaccine is oil-emulsion.
Described Goose Parvovirus vaccine is adopted preparation with the following method: by tween-80, be that 4:90-4:100 mixes with the goose parvovirus GPV-YZ strain virus liquid of deactivation according to volume ratio, obtain aqueous phase solution; By Si Ben-80, according to volume ratio, be that 6:90-6:100 mixes with white oil, obtain oil-phase solution; Described oil-phase solution and aqueous phase solution are that 2:1-4:1 mixes, obtain described Goose Parvovirus vaccine after emulsification according to volume ratio.
Anti gosling plague yolk antibody, adopts preparation with the following method: described Goose Parvovirus vaccine inoculation health is opened to laying hen, results high-immunity egg; The yolk of getting high-immunity egg, adds acidic aqueous solution, stirs, and standing rear centrifuging and taking supernatant liquor is crossed leaching filtrate, obtains described anti gosling plague yolk antibody.When the fine jade expansion of yolk, tire and reach 2
5time, results high-immunity egg.
Goose parvovirus GPV-YZ provided by the invention strain, has good immunogenicity to current popular Goose Parvovirus, can resist GPV-AV240 strain, GPV-GD strain and self strain and attack poison, and protective capability covers GPV-AV240 strain, GPV-GD strain.
Goose Parvovirus vaccine provided by the invention, immunity is planted after goose, plants goose and can obtain higher antibody horizontal, and the extended period is long.Due to goose parvovirus GPV-YZ strain, current popular Goose Parvovirus is had to good immunogenicity, so also can protect inoculation animal, avoid infection GPV-AV240 strain, GPV-GD strain and self strain after Goose Parvovirus vaccine immunity of the present invention.Young goose can obtain protection from maternal antibody, can resist Goose Parvovirus and infect.
Anti gosling plague yolk antibody provided by the invention, can prevent and treat gosling plague.
Accompanying drawing explanation
Fig. 1 is the electrophorogram of PCR product, the Maker that wherein swimming lane 1 is DL2000; The PCR product that swimming lane 2 and 3 is Virus Sample; Swimming lane 4 is the PCR product of healthy goose embryo allantoic liquid; Swimming lane 5 is the PCR product (positive control) of GPV-AV240 strain.
Fig. 2 is VP3 gene sequencing homology analysis result.
The preservation information of goose parvovirus GPV-YZ strain is as follows:
Classification And Nomenclature: goose parvovirus,
Latin name: Goose parvovirus,
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC),
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica,
Preservation date: on 09 05th, 2013,
Deposit number: CGMCC NO.8160.
Embodiment
Acquisition and the characteristic measurement of embodiment 1 goose parvovirus
1. virus is separated
The young foie gras tissue of doubtful infected goose parvovirus is ground to homogenate, obtain samples.Add physiological saline, the final concentration of 1/5 times of samples volume to be penicillin and the Streptomycin sulphate of 2000 units.Multigelation 3 times, 7000 r/min are centrifugal, and 20 min draw supernatant liquor.With filtering with microporous membrane, get filtered liquid through 10 pieces of allantoic cavity inoculation 12-14 age in days goose embryos, abandon dead embryo in 24 hours; Collect 24 ~ 96 hours dead embryos and get its allantoic fluid; After grinding to form homogenate, the hepatic tissue that gathers not dead goose embryo in 96 hours is mixed into suspension with above-mentioned allantoic fluid.By this suspension multigelation 3 times, get supernatant liquor after centrifugal, inoculation goose embryo, blind passage, after 3 generations, cuts open inspection to dead goose embryo under aseptic, observes goose embryo and has or not macroscopic pathological change, checks as aseptic with blood flat board simultaneously.Collection has the Virus Sample of blood clotting, and its pathology idiosome and allantoic fluid are placed in to-20 ℃ of preservations, in order to further identifying.
2. virus is identified
The DNA that extracts the Virus Sample that obtains in the present embodiment title 1, concrete steps are as follows: get virus liquid 400 μ L, add the SDS solution that 12.5 μ L Proteinase Ks and 50 μ L, concentration are 10%, mix, 56 ℃ of effect 30min; Add successively 200 μ L chloroforms and 200 μ L phenol, thermal agitation 15s, room temperature is placed 5min; 4 ℃, the centrifugal 10min of 12000r/min; Get supernatant, add isopyknic chloroform to mix, room temperature is placed 5min; 4 ℃, the centrifugal 10min of 12000r/min; Get supernatant and add the sodium acetate solution that the dehydrated alcohol of 2 times of volumes and 1/10 volume, concentration are 3M, place 30min for-20 ℃; 4 ℃, the centrifugal 10min of 12000r/min; Abandon supernatant, add 75% ethanol 1000 μ L washings; 4 ℃, the centrifugal 5min of 7500r/min; Abandon supernatant, dry 10min; Add 15 μ L distilled water dissolution precipitations and carry out pcr amplification as template.
Design pair of primers, carries out PCR evaluation to viral VP3 gene,
Upstream primer (SEQ ID NO:1): 5 '-ATGGCAGAGGGAGGAGGCGG-3 ';
Downstream primer (SEQ ID NO:2): 5 '-TGTGCATTGTGCAATACCCG-3 '.
The reaction system of pcr amplification (50ul): template 5 μ L, 10 * Buffer, 5 μ L, Mg
2+3 μ L, dNTPs 2 μ L, upstream primer 1 μ L, downstream primer 1 μ L, Taq archaeal dna polymerase 0.5 μ L, distilled water is supplied volume to 50 μ L.
PCR response procedures: 94 ℃ of denaturation 5min; Enter circulation: 94 ℃ of effect 60s, 62 ℃ of effect 45s, 72 ℃ of effect 45s, 35 circulations; 72 ℃ are extended 10min.
Pcr amplification product is detected with 1% agarose gel electrophoresis, and result shows that clip size is about 538bp(Fig. 1).Amplified fragments is checked order, and concrete sequence is as shown in SEQ ID NO:3.
Homology analysis result (Fig. 2) shows, the goose parvovirus sequence homology comparison of announcing in above-mentioned fragment (SEQ ID NO:3) and NCBI website GENEBANK, homology is more than 94%, illustrate that the virus that the present invention obtains is goose parvovirus, called after goose parvovirus GPV-YZ strain (being abbreviated as GPV-YZ strain).Bing Song China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, its microbial preservation number is: CGMCC NO.8160.
3. virus culture
The sample that is accredited as goose parvovirus in this title 2 is done to 100 times of dilutions with physiological saline, inoculate 11 ~ 13 age in days goose embryos, every embryo 0.2mL, discards 48 hours dead goose embryos.Cultivate after 168 hours for 37 ℃, collect dead goose embryo allantoic liquid and idiosome, idiosome is removed after gall-bladder, through homogeneous, adds in allantoic fluid, and through 5000r/min, 10min, collects supernatant as seed culture of viruses ,-20 ℃ of preservations.
4. virus characteristic
Detect the seed culture of viruses characteristic in the present embodiment title 3.
4.1 viral level
Seed culture of viruses is made to 10 times of doubling dilutions with physiological saline, inoculate respectively the fine hair allantoic cavity of 11 ~ 13 age in days goose embryos, every goose embryonic breeding kind 0.2mL, and make blank.5 goose embryos of each extent of dilution inoculation, with paraffin wax sealing, are placed in 37 ℃ of incubators and carry out constant temperature culture.Cultivate 24 hours photograph embryos once, discard embryo dead in 24 hours, with Reed-Mueeh method, calculate the medium lethal dose (ELD of GPV-YZ strain to goose embryo
50).By calculating, obtain ELD
50be not less than 10
-5.0/ 0.2mL.
4.2 virulence
Seed culture of viruses is made 10 times of doubling dilutions with physiological saline, and neck subcutaneous injection 0.2mL inoculates the 3 young geese in age in days left and right, observes the death condition of observing young goose 10.With Reed-Mueeh method, calculate the medium lethal dose (LD of GHV to young goose
50).By calculating, obtain LD
50be not less than 10
-4.50/ 0.2mL.
Preparation and the application thereof of embodiment 2 Goose Parvovirus vaccines
1 virus culture
Seed culture of viruses (embodiment 1) is done to 100 times of dilutions with physiological saline, inoculate 11 ~ 13 age in days goose embryos, every embryo 0.2mL, 37 ℃ of cultivations.Discard 48 hours dead goose embryos.Incubation time reaches 168 hours, collects allantoic fluid and the idiosome of dead goose embryo, and idiosome is removed after gall-bladder, through homogeneous, adds in allantoic fluid, through the centrifugal 10min of 5000r/min, collects supernatant as GPV-YZ strain virus liquid ,-20 ℃ of preservations.
2 viral levels
GPV-YZ strain virus liquid is made to 10 times of doubling dilutions with physiological saline, inoculate respectively 11 ~ 13 age in days goose embryo fine hair allantoic cavities, every goose embryonic breeding kind 0.2mL, and make blank.5 goose embryos of each extent of dilution inoculation, with paraffin wax sealing, are placed in 37 ℃ of incubators and carry out constant temperature culture.Every 24 hours photograph embryos once, observe 168 hours.With Reed-Mueeh method, calculate the medium lethal dose (ELD of GPV-YZ strain virus liquid to goose embryo
50), ELD
50should be not less than 10
-5.0/ 0.2mL.
The deactivation of 3 virus liquids
In GPV-YZ strain virus liquid, add formaldehyde, make the final concentration of formaldehyde reach 0.1%(volumetric concentration), in 37 ℃ of insulations 24 hours, every 1 hour, stir therebetween or jolting once, obtain inactivation of viruses liquid.Deactivation finishes rearmounted 2-8 ℃, can preserve 1 month.
4 deactivation checks
After inactivation of viruses liquid is 1:10 dilution with physiological saline according to extent of dilution, inoculate 5 pieces of 12 age in days susceptible goose embryos, inoculum size is every goose embryo 0.2ml.At 37 ℃, cultivate, observe 120 hours.In 2 generations of blind passage,, if goose embryo without death, shows that deactivation is complete.
5 prepare Goose Parvovirus vaccine
Prepare water: by tween-80 and inactivation of viruses liquid by volume 4:96 mix, obtain water.
Oil phase: be that 6:94 mix with injection white oil according to volume ratio by Si Ben-80, obtain oil phase.
Emulsification: be that 3:1 mixes also emulsification by oil phase and water according to volume ratio, obtain Goose Parvovirus vaccine.
6 Goose Parvovirus vaccine safety checks
Every batch of Goose Parvovirus vaccine produces each 5 of kind of geese with the young goose of 3 ages in days with opening, every incidence subcutaneous injection 4 plumage part (2ml) Goose Parvovirus vaccines, and the not vaccinated similar goose of another setting compares group.
After inoculation, continue to observe 14 days, the young goose of all tests is without clinical symptom, and health condition is good.Therefore this Goose Parvovirus vaccine is safe.
Goose Parvovirus vaccine is used for inoculating kind of a goose, the gosling plague that prevents Goose Parvovirus to cause by maternal antibody.The route of inoculation of this vaccine is neck subcutaneous injection.
7 Goose Parvovirus vaccine immunity dose determinations and protectiveness thereof are measured
To open product kind of a goose and be divided into 4 groups, 10 every group.1 group every neck subcutaneous injection 0.3ml Goose Parvovirus vaccine; 2 groups every neck subcutaneous injection 0.5ml Goose Parvovirus vaccine; 3 groups every neck subcutaneous injection 1.0ml Goose Parvovirus vaccine; 4 groups every neck subcutaneous injection 1.0ml physiological saline.Test adopts single immunization, and the goose egg that produces is collected in latter 21 days to 28 days of immunity, and 20 pieces of every group of random chooses are hatched young goose.After young goose hatching 3 ~ 4 days, choose 10 young gooseneck subcutaneous injection GPV-YZ of portion strain for every group, attacking toxic agent amount is every young goose 100ELD
50, attack the rear Continuous Observation of poison 10 days, record young goose death condition.
The comparative result of the different immunizing doses of table 1 to the acquired protection of young goose source of parents
As can be seen from Table 1, every kind goose immunity 0.3ml Goose Parvovirus vaccine, it is 50% that young goose is attacked malicious protection ratio; Every kind goose immunity 0.5ml Goose Parvovirus vaccine, it is 70% that young goose is attacked malicious protection ratio; Every kind goose immunity 1.0ml Goose Parvovirus vaccine, it is 100% that young goose is attacked malicious protection ratio.The young goose of hatching is attacked malicious result and shows, more than every kind goose inoculation Goose Parvovirus vaccine 1.0ml, can protect young goose.This test shows that Goose Parvovirus vaccine has good immunogenicity.
Young goose challenge test, has shown that Goose Parvovirus vaccine can effectively protect young goose to avoid being subject to GPV-YZ strain and attack, so this vaccine does not need to carry out antibody horizontal test to the protection effect of young goose.
8. immune duration is measured
Take away and produce 10 of kind of geese, every neck subcutaneous injection 1.5ml Goose Parvovirus vaccine, respectively at immunity latter 30 days, 60 days, 90 days, 120 days and blood sampling in 150 days, separation of serum, detects and tires with agar diffusion test; Respectively at 30-35 after immunity days, 60-65 days, 90-95 days, 120-125 days with within 150-155 days, collect kind of an egg and hatch, after hatching, poison is attacked in the 4th day every neck subcutaneous injection GPV-YZ strain, and dosage is 100ELD
50/ plumage.Attack the rear Continuous Observation of poison 10 days, record young goose death condition.
Table 2 immune duration is measured
| 30 days | 60 days | 90 days | 120 days | 150 days | |
| Female goose antibody titer (Log2) | 7.2 | 6.7 | 6.0 | 5.1 | 3.6 |
| Young goose protection ratio (%) | 100 | 100 | 100 | 100 | 66.7 |
As can be seen from Table 2, in the monitoring phase, from latter 30 days of immunity, female goose antibody titer was continuous decrease trend.Latter 120 days of immunity, the antibody titer of female goose is from 2
7.2drop to 2
5.1, but the protection ratio of young goose is still 100%.Latter 150 days of immunity, the antibody titer of female goose is down to 2
3.6, the protection ratio of young goose is only 66.7%(4/6).Above-mentioned test explanation, effective immune duration of Goose Parvovirus vaccine is 120 days.In order to make young goose obtain sustainable protection, can be chosen in female goose immunity and carry out booster immunization in latter about 100 days.
9. intersect the mensuration of passive protection power
With method, press the vaccine preparation method of the present embodiment, containing the vaccine of GPV-AV240 strain (purchased from China Veterinery Drug Inspection Office) with containing GPV-GD strain, (Guangdong Province province Foshan Science &. Technology College provides in preparation respectively, Chen Bailun, Ye Benheng, Li Jiehong. the research [J] of gosling plague duck embryo GD attenuated vaccine. journal of animal science and veterinary medicine, the fourth phase in 1985) vaccine.Vaccine containing GPV-AV240 strain is denoted as control vaccine 1, the ELD of the GPV-AV240 strain virus liquid using in preparation process<sub TranNum="202">50</sub>content is 10<sup TranNum="203">-5.0</sup>/ 0.2mL.Vaccine containing GPV-GD strain is denoted as control vaccine 2, the ELD of the GPV-GD strain virus liquid using in preparation process<sub TranNum="204">50</sub>content is 10<sup TranNum="205">-5.0</sup>/ 0.2mL.
Get 9 healthy non-young geese of exempting from of 7 ages in days, be divided into 3 groups, 3 every group.By the immune one group of young goose of Goose Parvovirus vaccine (according to the preparation of the present embodiment title 1-5 method), control vaccine 1 and control vaccine 2 difference, immunizing dose is 1.0ml, presses the dosage booster immunization of 1.5ml/ plumage part during 21 age in days.Each is organized to young goose and in 49 ages in days, gets blood, separation of serum, measure agar diffusion test tire (be that fine jade expands and tires, with reference to " 2000 editions Chinese veterinary pharmacopoeias ", lower with).Adjustment is for the serum titer to 2 of each strain
4, obtain respectively the hyper-immune serum of GPV-YZ strain, GPV-AV240 strain and GPV-AV240 strain ,-20 ℃ save backup.
Young 90 of the geese of 3 ages in days are divided into 3 groups, 30 every group at random.Wherein, first group of young goose, neck subcutaneous vaccination GPV-AV240 strain hyper-immune serum, 0.5ml/ is only.Second group of young goose, neck subcutaneous vaccination GPV-GD strain hyper-immune serum, 0.5ml/ is only.The 3rd group of young goose, neck subcutaneous vaccination GPV-YZ strain hyper-immune serum, 0.5ml/ is only.Hyper-immune serum is inoculated latter 24 hours, to every group of young goose: get at random 10 young geese, neck subcutaneous vaccination GPV-AV240 strain virus liquid; Separately get 10 young geese, neck subcutaneous vaccination GPV-YZ strain virus liquid; 10 remaining young geese, neck subcutaneous vaccination GPV-GD strain virus liquid.The toxic agent amount of attacking of each strain is every 100ELD
50/ 0.2ml.Observe 10 days, the morbidity of young goose and death condition respectively organized in record, intersects protest test.
Table 3 intersection passive protection test result
As can be seen from Table 3, GPV-AV240 strain hyper-immune serum is 100% to the protection ratio of self strain, and the protection ratio of GPV-GD strain and GPV-YZ strain is respectively to 70% and 60%; GPV-GD strain hyper-immune serum is 90% to the protection ratio of self strain, and the protection ratio of GPV-AV240 strain and GPV-YZ strain is respectively to 80% and 70%.GPV-YZ strain not only can be protected the poison of attacking of self strain, and other strain is also had to good cross protection, and protection effect is better than other 2 strain virus.
Adopt the Goose Parvovirus vaccine (GPV-YZ strain) of embodiment 2 preparations to prepare anti gosling plague yolk antibody.
1. laying hen immunity
Adopt health to open laying hen, chest muscle multi-point injection Goose Parvovirus vaccine successively, 0.5ml/ only, exempts from headed by this.Head exempts from latter 10 days, every laying hen immunity Goose Parvovirus vaccine 1.0ml.Head exempts from latter 20 days, every laying hen inoculation Goose Parvovirus vaccine 2.0ml.After three immunity, make regular check on yolk agar diffusion test and tire (detection method is with reference to " 2000 editions Chinese veterinary pharmacopoeias "), when fine jade expands to tire, reach 2
5, start to collect the high egg of exempting from.
2. prepare anti gosling plague yolk antibody
The height that sterilization is collected is exempted from egg, takes the separated egg white of craft or mechanical system and yolk.In 2 ℃ ~ 30 ℃ environment, in yolk, add 7 times of its volumes acidic aqueous solution (with hydrochloric acid regulate water to pH be 4 ~ 5), stir, 4 ℃ standing 8 hours, 8000rpm gets supernatant liquor after centrifugal 30 minutes.Supernatant liquor, through the membrane filtration in 0.22 μ m aperture, is got filtrate, and adjusting that its agar diffusion test tires is 2
4, obtain anti gosling plague yolk antibody.-20 ℃ save backup.
3. the young goose gosling plague infection ability of prevention is measured
Get 50 of the young geese of the 3 nonimmune health in age in days left and right, be divided at random 5 groups, 10 every group.The 1st group is blank group, and every neck subcutaneous injection anti gosling plague yolk antibody 1.0ml, does not attack poison, isolated rearing separately.The 2nd group (test group) every neck subcutaneous injection anti gosling plague yolk antibody 0.3ml, the 3rd group (test group) every neck subcutaneous injection anti gosling plague yolk antibody 0.5ml, the 4th group (test group) every neck subcutaneous injection anti gosling plague yolk antibody 1.0ml, the 5th group (attacking malicious control group) every neck subcutaneous injection physiological saline 0.5ml.Inoculate latter 24 hours, 2nd ~ 5 groups of neck subcutaneous injection GPV-YZ strain, every 100ELD
50, Continuous Observation 10 days.
Table 4 gosling plague yolk antibody prophylactic tria result
As can be seen from Table 4, every young goose inoculation 0.3ml anti gosling plague yolk antibody, attacked poison after 24 hours, and protection ratio only has 50%; Every young goose inoculation 0.5ml anti gosling plague yolk antibody, attacked poison after 24 hours, and protection ratio reaches 100%; Every young goose inoculation 1.0ml anti gosling plague yolk antibody, attacked poison after 24 hours, and protection ratio reaches 100%.The demonstration of prophylactic tria result, young goose injection 0.5ml and above anti gosling plague yolk antibody, can protect young goose completely.
4. the young goose gosling plague infection ability for the treatment of is measured
Get 50 of the young geese of the 3 nonimmune health in age in days left and right, be divided at random 5 groups, 10 every group.The 1st group is blank group, and every injection anti gosling plague yolk antibody 1.0ml, does not attack poison, isolated rearing separately.2nd ~ 5 groups of neck subcutaneous injection GPV-YZ strain, every 100ELD
50.After 12 hours, the 2nd group (test group) every neck subcutaneous injection anti gosling plague yolk antibody 0.3ml, the 3rd group (test group) every neck subcutaneous injection anti gosling plague yolk antibody 0.5ml, the 4th group (test group) every neck subcutaneous injection anti gosling plague yolk antibody 1.0ml, the 5th group (attacking malicious control group) every neck subcutaneous injection physiological saline 0.5ml.After injection anti gosling plague yolk antibody, Continuous Observation 10 days.
Table 5 gosling plague yolk antibody therapeutic test result
| Grouping | Attack toxic agent amount | Dosage | Quantity | Attack poison protection number | Attack |
| 1 group | 0 | 1.0ml | 10 | 10/10 | 100% |
| 2 groups | 100ELD 50 | 0.3ml | 10 | 3/10 | 30% |
| 3 groups | 100ELD 50 | 0.5ml | 10 | 5/10 | 50% |
| 4 groups | 100ELD 50 | 1.0ml | 10 | 10/10 | 100% |
| 5 groups | 100ELD 50 | 0 | 10 | 0/10 | 0% |
As shown in table 5, adopt GPV-YZ strain to attack poison latter 12 hours to young goose, every inoculation anti gosling plague yolk antibody 0.3ml, young goose protection ratio is only 30%; Young goose is attacked poison latter 12 hours, every injection 0.5ml anti gosling plague yolk antibody, and young goose protection ratio is only 50%; Young goose is attacked poison latter 12 hours, every injection 1.0ml anti gosling plague yolk antibody, and young goose protection ratio reaches 100%.The demonstration of emergency treatment test-results, GPV-YZ strain is attacked after poison, and every young goose injection 1.0ml and above anti gosling plague yolk antibody, can protect young goose completely.
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>goose parvovirus and application thereof
<130> 20131017
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> artificial
<220>
<223>upstream primer
<400> 1
atggcagagg gaggaggcgg 20
<210> 2
<211> 20
<212> DNA
<213> artificial
<220>
<223>downstream primer
<400> 2
tgtgcattgt gcaatacccg 20
<210> 3
<211> 538
<212> DNA
<213>goose parvovirus GPV-YZ strain
<400> 3
atggcagagg gaggaggcgg agctttgggc gacgcttcag ggggtgccga tggagtgggt 60
aatgcctcgg gaaattggca ttgcgattcc caatggatgg gaaacacagt catcacaaaa 120
actacccgaa cttgggtctt gccgagctac aataaccaca tctacaaagc aattaccagc 180
ggaacctctc aggacgcaac tgtccagtat gctggataca gtactccctg ggggtacttc 240
gatttcaacc gcttccactg ccacttctcc ccgagagact ggcaaagact tatcaacaac 300
cattggggaa tcagacccaa gtcccttaaa tttaagatct tcaatgtcca agtcaaagaa 360
gtcacaacgc aggatcagac gaagaccatt gcaaacaatc tcacgtcaac gattcaagtg 420
ttcacggatg atgagcatca actcccgtat gtgctgggct cggctacgga aggcaccatg 480
ccgccgttcc cgtcggatgt gtatgccctg ccgcagtacg ggtattgcac aatgcaca 538
Claims (9)
1. goose parvovirus GPV-YZ strain, its microbial preservation number is: CGMCC NO.8160.
2. described in claim 1, goose parvovirus GPV-YZ strain is in preparation prevention with control the application in the medicine for the treatment of gosling plague disease, and the microbial preservation of described goose parvovirus GPV-YZ strain number is CGMCC NO.8160.
3. application according to claim 2, the medicine that it is characterized in that described prevention gosling plague disease is Goose Parvovirus vaccine, described in control treating gosling plague disease medicine be anti gosling plague yolk antibody.
4. Goose Parvovirus vaccine, is characterized in that the goose parvovirus GPV-YZ strain virus liquid that contains deactivation, and the microbial preservation of described goose parvovirus GPV-YZ strain number is CGMCC NO.8160.
5. Goose Parvovirus vaccine according to claim 4, the preparation method who it is characterized in that described goose parvovirus GPV-YZ strain virus liquid is: by goose parvovirus GPV-YZ strain inoculation goose embryo, collect dead goose embryo in 48-180h, get the allantoic fluid of described dead goose embryo and the idiosome of removal gall-bladder; By mixing with described allantoic fluid after the idiosome homogeneous of described removal gall-bladder, centrifuging and taking supernatant liquor, obtain described goose parvovirus GPV-YZ strain virus liquid.
6. Goose Parvovirus vaccine according to claim 5, is characterized in that described Goose Parvovirus vaccine is oil-emulsion.
7. Goose Parvovirus vaccine according to claim 6, is characterized in that described Goose Parvovirus vaccine adopts preparation with the following method: by tween-80, be that 4:90-4:100 mixes with the goose parvovirus GPV-YZ strain virus liquid of deactivation according to volume ratio, obtain water; By Si Ben-80, according to volume ratio, be that 6:90-6:100 mixes with white oil, obtain oil phase; Described oil phase and water are that 2:1-4:1 mixes, obtain described Goose Parvovirus vaccine after emulsification according to volume ratio.
8. anti gosling plague yolk antibody, is characterized in that adopting preparation with the following method: Goose Parvovirus vaccine inoculation health described in claim 5 is opened to laying hen, gather in the crops the high egg of exempting from; Get height and exempt from the yolk of egg, add acidic aqueous solution, stir, standing rear centrifuging and taking supernatant liquor, crosses leaching filtrate, obtains described anti gosling plague yolk antibody.
9. anti gosling plague yolk antibody according to claim 8, is characterized in that fine jade when yolk expands to tire and reaches 2
5time, gather in the crops the high egg of exempting from.
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| CN103866048A (en) * | 2014-02-27 | 2014-06-18 | 广东省农业科学院动物卫生研究所 | HRM (High Resolution Melt) identification method, kit and primer group for muscovy duck parvovirus and goose parvovirus |
| CN104232672A (en) * | 2014-08-22 | 2014-12-24 | 福建省农业科学院畜牧兽医研究所 | Method for cloning complete sequence of muscovy duck origin goose parvovirus genome encoding area |
| CN106310247A (en) * | 2015-07-03 | 2017-01-11 | 扬州优邦生物制药有限公司 | Preparation method of gosling plague inactivated vaccines as well as prepared inactivated vaccines |
| CN106310247B (en) * | 2015-07-03 | 2019-09-10 | 扬州优邦生物药品有限公司 | The preparation method of gosling plague inactivated vaccine and its inactivated vaccine of preparation |
| CN105861449A (en) * | 2016-03-31 | 2016-08-17 | 福建省农业科学院畜牧兽医研究所 | Preparation method of short-billed dwarf syndrome gosling plague inactivated vaccine |
| CN105861449B (en) * | 2016-03-31 | 2019-07-12 | 福建省农业科学院畜牧兽医研究所 | The preparation method of the short short and small syndrome gosling plague inactivated vaccine of mouth |
| CN106893732A (en) * | 2017-04-18 | 2017-06-27 | 扬州大学 | A kind of rescue method of Goose Parvovirus clone |
| CN108607095A (en) * | 2018-06-05 | 2018-10-02 | 山东信得科技股份有限公司 | A kind of strain of goose parvovirus and its application |
| CN111704655A (en) * | 2020-04-24 | 2020-09-25 | 吉林农业大学 | Goose parvovirus protein capable of inducing goose embryo fibroblast apoptosis and preparation method thereof |
| 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 |
| CN113355293A (en) * | 2021-07-02 | 2021-09-07 | 辽宁益康生物股份有限公司 | Gosling plague virus vaccine strain, vaccine and egg yolk antibody based on vaccine |
| CN113355293B (en) * | 2021-07-02 | 2022-08-12 | 辽宁益康生物股份有限公司 | Gosling plague virus vaccine strain, vaccine and egg yolk antibody based on vaccine |
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Application publication date: 20140122 Assignee: Chongqing Health Forever Biotechnology Co., Ltd. Assignor: Jiangsu Academy of Agricultural Sciences Contract record no.: 2017320000199 Denomination of invention: Goose parvovirus and application thereof Granted publication date: 20150408 License type: Common License Record date: 20171031 |