CN102533669B - Duck byd virus inactivated vaccine and preparation method thereof - Google Patents
Duck byd virus inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention provides a vaccine for preventing duck egg reduction syndrome caused by BYD virus and a preparation method thereof. The invention provides a duck BYD virus (Family Flaviviridae, Genus Flavivirus, Ntaya virus group, Duck BYD virus) JXSP strain, and the preservation number is CGMCC No.5266. The virulent strain provided by the invention is duck BYD virus virulent strain with excellent immunogenicity. The virulent strain is vaccinated onto a sensitive cell to obtain cell sap, which is emulsified after being inactivated so as to obtain the safe, effective and controllable duck BYD virus inactivated vaccine, so that the prevention and the control of the duck egg reduction syndrome can be favored.
Description
Technical field
The present invention relates to the veterinary biological product technical field, particularly, relate to duck BYD virus vaccines and preparation method thereof.
Background technology
Since in April, 2010, the capable a kind of serious disease of the duck field flow in Southeast China part province, each mainly supports the heavy provinces and cities of duck to this disease rapid spread to China, comprises Zhejiang, Fujian, Jiangxi, Shandong, Hebei, Jiangsu and Beijing.Sickness influence comprises the multiple laying ducks of Beijing duck and sheldrake, it is exactly that food consumption descends suddenly and follows the rapid drawdown of laying rate that infected duck presents the most outstanding clinical symptom, laying rate can be down to 10% in 5 days, cut open that hemorrhage, atrophy takes place the visible ovary of inspection, serious pathology such as break.Initial stage visible part ovarian follicle has blutpunkte or blood spots, along with the development of the course of disease, and the hemorrhage and sex change that ovarian follicle is serious, follicular rupture and peritonitis, as seen some cases has the spleen enlargement.Lesion tissue shows as mainly that ovarian hemorrhage, follicular development stop, locking or disintegration, and visible circle or the red corpusculum that dyes of particulate state that differs in size in a large number is full of ovarian follicle or a matter of disintegration.The visible microglia of indivedual case brains is soaked into kitchen range, congested, inflammatory cell infiltration under the arachnoid of brain.Infect serious duck field kind egg at some and almost have no harvest, morbidity later stage kind duck mortality ratio is that 5%-30% does not wait, and the provisions duck has already been caused enormous economic loss.
Epidemiology survey and pathogen separation through system identify that the arch-criminal who studies confirm that this disease is a kind of new flavivirus, called after BYD virus.The duck field disease of this unknown cause has been explained in the discovery of BYD virus on the one hand, (most of member of Flavivirus is that important infecting both domestic animals and human venereal disease is former because the characteristics of flavivirus itself on the other hand, can give the people by media transmissions such as hematophagous buges, cause people's infection and morbidity), so the public hygienics meaning of duckling virus should cause the great attention of industry.
Owing in the duck group, do not find the infection of flavivirus before as yet; this duck BYD virus infection still belong to the first time the outburst and popular; and flaviviridae infections is still lacked medicine effectively both at home and abroad; still not having vaccine can protect it, therefore also not treatment and immunoprophylaxis measure effectively at present.
Summary of the invention
The object of the present invention is to provide a kind of BYD of prevention virus infection to cause inactivated vaccine of duck egg drop syndrome and preparation method thereof.
The invention provides duck BYD virus (Family Flaviviridae, GenusFlavivirus, Ntaya virus group, Duck BYD virus) JXSP strain, separate from duck field, Baiyang Lake, Chinese Hebei province morbidity duck cerebral tissue.The JXSP strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on September 22nd, 2011, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, the classification name: duck BYD virus, deposit number are CGMCC No.5266.
Duck BYD virus JXSP of the present invention strain belongs to flaviviridae (Flaviviridae) Flavivirus (flavivirus).Under the Electronic Speculum, virus particle is spherical in shape, and the about 45-60nm of size has the electro-dense nuclear core of the about 30nm of a diameter and the lipid cyst membrane of parcel nuclear core.Mainly in the kytoplasm of cells infected, copy.Viral genome is the sub-thread positive chain RNA, and size is 10990nt.Genome 5 ' and 3 ' end respectively have one section non-coding region (NCR), and the coded sequence of whole gene is 5 '-NCR-C-PrM-E-NS1-NS2A2B-NS3-NS4A4B-NS5-NCR-3 '.
The invention provides the application of duck BYD virus JXSP strain in the preparation vaccine.
The invention provides the vaccine that utilizes duck BYD virus JXSP strain preparation.
The invention provides a kind of method for preparing duck BYD virus JXSP strain virus liquid, comprise the steps:
1) cell of enlarged culturing is pressed 1% of nutrient solution and added JXSP strain virus seed, 37 ℃, adsorbed 1 hour;
2) add 1% foetal calf serum and keep liquid, cultivate down for 37 ℃, gather in the crops freeze thawing 3 times when cytopathy reaches 75% when above;
3) centrifugal or remove by filter cell debris, collect supernatant liquor, this supernatant liquor is duck BYD virus JXSP strain virus liquid.
Duck BYD virus JXSP strain can be cultivated propagation in DEF (DEFs), and generation CPE (cell rounding, shrinkage, pyknosis, being the kitchen range shape comes off), virus also can infect BHK-21, Vero cell and produce CPE, but does not all produce CPE on other cells such as PK-15, ST, MDCK.Therefore, above-mentioned cell may be selected to be BHK-21, Vero and/or DEF.
In one embodiment of the invention, select the BHK-21 cell to carry out the seed culture of viruses multiply test of duck BYD virus JXSP strain, be specially: BYDV-JXSP the 2nd generation duck embryo allantoic liquid poison is seeded in the BHK-21 cell cultures, in 5%CO after with 5 times of dilutions of serum-free DMEM substratum
2In the incubator, 37 ℃ leave standstill cultivation, and when treating that pathology appears in 75% cell, harvested cell liquid is got 100 times of dilutions of supernatant postoperative infection inoculation BHK-21 cell after the freeze thawing 2 times, so reached for the 8th generation, harvested cell liquid in-70 ℃ of preservations as kind of a poison, viral level 〉=10
5.5TCID
50/ mL.
During with BHK-21 cell proliferation virus, can carry out enlarged culturing before the inoculation: get the BHK-21 cell that liquid nitrogen is preserved, after placing 37 ℃ of water-baths to melt immediately, the centrifugal 5min of 800r/min, abandon supernatant, add the DMEM cell growth medium suspension cell precipitation that contains 10% foetal calf serum, change in the Tissue Culture Flask, in 5%CO
2In the incubator, 37 ℃ leave standstill cultivation, treat to carry out passage after cell monolayer forms.Discard cell culture fluid, add an amount of sterilization PBS and clean cell surface gently 2 times, abandon PBS, the trysinization liquid (0.25%) that adds 37 ℃ of preheatings leaves standstill 5～10min, the observation of cell gap obviously enlarges under the inverted microscope, cellular form becomes bowlder, and the tipping trypsin solution adds the DMEM piping and druming cell dispersion that contains 10% foetal calf serum gently, and install to new Tissue Culture Flask in 1: 3 ratio branch and cultivate, in 5%CO
2In the incubator, 37 ℃ leave standstill cultivation.
The present invention also provides the method for preparing duck BYD viral inactivation vaccine, is the duck BYD of above-mentioned preparation virus JXSP strain virus liquid is carried out deactivation and emulsification as seedling successively with viral liquid, obtains duck BYD viral inactivation vaccine.
Described viral liquid viral level 〉=10
5.5TCID
50/ mL.
Described deactivation is to be that 0.2% (V/V) ratio adds formaldehyde solution (formaldehyde content is 37%) with viral liquid in final concentration, and fully mixing is put 37 ℃ of effect 24h inactivation of viruses.
Described emulsification be with the viral liquid after the deactivation as water, after mixing according to water oil phase volume ratio 4: 6,10000～12000r/min emulsification 2min adds Thiomersalate solution before stopping stirring, and makes final concentration reach 0.01% volume ratio.
The oil phase preparation method adds stearic acid aluminium 2g for getting 94 parts of injection white oils (mL) among the present invention, and the limit edged stirs, and until transparent fully, adds 6 parts of (mL) Si Ben-80 again, abundant mixing, and autoclaving obtains the 100mL oil phase; The preparation method of water is that the cell culture and virus liquid 100 parts (mL) of getting deactivation adds the tween-80 that 4 parts (mL) sterilize, and fully shakes up to tween-80 and dissolves fully, obtains the 104mL water.
The duck BYD-JXSP viral inactivation vaccine outward appearance of the present invention's preparation is oyster white emulsion.
Can be used for of vaccine of the present invention, carried out the neck subcutaneous vaccination to the healthy duck of 5～7 ages in days, every 0.5mL vaccine.
In an embodiment of the present invention, safety examination to the BYD-JXSP viral inactivation vaccine that makes is found, the healthy susceptible duck (HI antibody titer≤1: 20) of 5～7 ages in days is through neck subcutaneous injection vaccine 1mL, and the part that occurs because of vaccinate and the untoward reaction of whole body did not take place in the observation period; In the efficacy test to the BYD-JXSP viral inactivation vaccine that makes, reach healthy susceptible duck (HI antibody titer≤1: 20) more than 60% with 5～7 ages in days or laying rate, every through neck subcutaneous injection vaccine 0.5mL, the strong poison of inoculation back 3 all intracranial inoculation JXSP (virus titer 〉=10
5.5TCID
50/ mL, 0.1mL/ are only), in the observation period, vaccine reaches more than 90% the protection ratio of immune duck.
Duck BYD virus JXSP provided by the invention strain has good immunogenicity, this strain is inoculated on the responsive host cell, and harvested cell liquid, emulsification after the deactivation obtains a kind of safe, effective, controlled duck BYD viral inactivation vaccine.Behind this vaccine inoculation duck, do not see untoward reaction, cut open the inspection inoculation position; not symptom such as swollen, the suppuration of show, and vaccine is absorbed fully, and security is good; good immune effect, the infection that can resist the strong poison of duck BYD is conducive to the prevention and control of duck egg drop syndrome in the large-scale cultivation.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, duck used among the embodiment is provided by Guangdong Dahuanong Animal Health Products Co., Ltd., used chemical reagent, raw material, equipment be for can buy the commercially available prod that obtains among the embodiment, the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Embodiment 1 virus is separated with pathogenic
1, isolation identification should virus be separated from duck field, Baiyang Lake, Chinese Hebei province morbidity duck cerebral tissue.10% homogenate is made with aseptic PBS by sick duck brain tissue sample, the centrifugal 20min of 3000r/min, after filtration sterilization has maternal antibody duck embryo by allantoic cavity approach inoculation 9～12 ages in days, 37 ℃ of hatchings 5 days, the results allantoic fluid also goes down to posterity in the duck embryo.With the 2nd generation the allantoic fluid poison be seeded on the BHK-21 cell monolayer, in 5% CO2gas incubator, leave standstill cultivation, observed 7 days, results culture when waiting cytopathy to occur ,-70 ℃ of preservations.
Observe under the ultrathin section(ing) transmission electron microscope of preparation cells infected, infection back more than 24 hours cell cytoplasm or vesicle in the visible a large amount of about 50nm of diameter, sphere have that the togavirus particle is poly-to be piled or be dispersed in, as seen most of virus particle has the electro-dense nuclear core of the about 30nm of diameter.The virus-culturing fluid that ultracentrifugation concentrates through the phospho-wolframic acid negative staining, is observed the about 50nm of visible diameter, the spherical virus particle of cyst membrane is arranged under the transmission electron microscope.
The whole genome sequence measurement result shows that viral genome is the sub-thread positive chain RNA, and size is 10990nt.Genome 5 ' and 3 ' end respectively have one section non-coding region (NCR), and the coded sequence of whole gene is 5 '-NCR-C-PrM-E-NS1-NS2A2B-NS3-NS4A4B-NS5-NCR-3 '.The kind system that has the full genome of flavivirus of sequence data to make in utilization takes place in the evolutionary tree, and BYD virus (JXSP strain) is nearest with bagaza virus (Bagaza Virus) evolutionary relationship, and homology is about 72%.The JXSP strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on September 22nd, 2011, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, classification name: duck BYD virus.Deposit number is CGMCC No.5266.
2, pathogenic virus inoculation laying ducks, clinical manifestation descends for searching for food and egg productivity sharply descends.Laying rate has dropped to below 12% from 60.9% in back 6 days of infection, until stopping production.Infect that to cut open the visible ovarian follicle of inspection in back 3 days hemorrhage, the degeneration that occurs ovary along with the development of the course of disease changes ovarian follicle sex change and atrophy.The visible sick duck ovarian hemorrhage of tissue examination, follicular development stops, locking or disintegration, and circle or the red corpusculum that dyes of particulate state that differs in size in a large number arranged, and is full of ovarian follicle or a matter of disintegration.The visible microglia of some cases brain is soaked into kitchen range, and little perivasculitis cell forms oversleeve spline structure (vascular cuffing), is apyetous encephalitis pathology.Histopathology changes and cuts open the inspection finding substantially and the duck clinical symptom of falling ill conforms to substantially, can detect and be recovered to virus with fluorescence RT-PCR and viral separation test.
Virus intracranial inoculation 1～7 duck in age in week can cause tangible nervous symptoms, shows as the decline of searching for food, weak and limp, circus movement, paralysis and death, and mortality ratio is 40%～60%.Histological examination shows as typical apyetous encephalitis pathology.
The preparation of embodiment 2 duck BYD virus JXSP strain virus liquid
1, seed culture of viruses is bred and will be seeded in the BHK-21 cell cultures after 5 times of dilutions of BYDV-JXSP the 2nd generation allantois duck blastochyle poison, in 5% CO2gas incubator, 37 ℃ leave standstill and cultivate when treating that pathology appears in 75% cell, harvested cell liquid, get 100 times of dilutions of supernatant postoperative infection inoculation BHK-21 cell after the freeze thawing 2 times, so reached for the 8th generation, harvested cell liquid in-70 ℃ of preservations as seed culture of viruses, viral level 〉=10
5.5TCID
50/ mL.
2, the BHK-21 cell that liquid nitrogen is preserved is got in cell seminal propagation, after placing 37 ℃ of water-baths to melt immediately, the centrifugal 5min of 800r/min, abandon supernatant, add the DMEM cell growth medium suspension cell precipitation that contains 10% foetal calf serum, change in the Tissue Culture Flask, in 5% CO2gas incubator, 37 ℃ leave standstill cultivation, treat to carry out passage after cell monolayer forms.Discard cell culture fluid, add an amount of sterilization PBS and clean cell surface gently 2 times, abandon PBS, the trysinization liquid (0.25%) that adds 37 ℃ of preheatings leaves standstill 5～10min, and the observation of cell gap obviously enlarges under the inverted microscope, cellular form becomes bowlder, tipping trypsin solution gently, add the DMEM piping and druming cell dispersion that contains 10% foetal calf serum, and install to new Tissue Culture Flask in 1: 3 ratio branch and cultivate, in 5% CO2gas incubator, 37 ℃ leave standstill cultivation.
3, after the viral liquid of preparation treats that the BHK-21 cell monolayer forms, discard cell culture fluid, add an amount of sterilization PBS and clean cell surface gently 2 times, abandon PBS; To plant poison and do 100 times of dilutions with the DMEM basal liquid, 10% (V/V) by the final volume of cell culture fluid is inoculated into cell monolayer, make liquid evenly cover cell surface, in 5% CO2gas incubator, 37 ℃ left standstill 1 hour, add the DMEM that contains 1% foetal calf serum then, put in 5% CO2gas incubator, cultivated 96 hours for 37 ℃, when pathology (showing as the cell circle contracts and come off) appears in the cell more than 75%, results, multigelation 3 times, through centrifugal or remove by filter cell debris, collecting cell nutrient solution (virus titer 〉=10
5.5TCID
50/ mL), be the viral liquid that the preparation vaccine is used, preserve below-20 ℃.，
The preparation of embodiment 3 duck BYD viral inactivation vaccines
1, the deactivation of viral liquid
Be that the ratio of 0.2% (volume ratio) adds formaldehyde solution with the viral liquid of preparation among the embodiment 2 in final concentration, mixing fully shakes up the back in 37 ℃ of effects 24 hours, inactivation of viruses.
2, inactivating efficacy check
The BHK-21 cell is cultured to individual layer at 24 orifice plates, the viral liquid of the BYDV-JXSP of 10 times of dilutions of formalin-inactivated processing in the step 1 is inoculated on the BHK-21 cell monolayer by the 0.2mL/ hole, each sample is inoculated 3 holes, hatch after 1 hour and discard sample liquid, after keeping the liquid washing, continue to keep cultivation, establish the contrast of positive control and blank well simultaneously; Observe positive control hole visible cell pathology after 48 hours, sample and blank hole are negative; 2 generations of blind passage, the same observation, CPE appears in positive hole if CPE does not appear in the cell in experimental port and blank hole, illustrates that then inactivating efficacy is good.
3, the preparation of vaccine
(1) injection white oil 94mL is got in the preparation of 100mL oil phase, adds stearic acid aluminium 2g, and the limit edged stirs, and until transparent fully, adds 6mL Si Ben-80 again, abundant mixing, and autoclaving is standby;
(2) tween-80 of 4 parts of (mL) sterilizations of 100 parts of (mL) addings of cell culture and virus liquid of deactivation is got in the water preparation, fully shakes up to tween-80 and dissolves fully;
(3) join seedling and get 6 parts of oil phases and put into colloidal mill or emulsion cylinder, stir at a slow speed, slowly add 4 parts of waters simultaneously, add behind the water with 10000～12000r/min emulsification 2min; Add 1% Thiomersalate solution before stopping stirring, final concentration reaches 0.01%.After the emulsification, sampling 5～10mL is with the centrifugal 15min of 3000r/min, if demixing phenomenon, emulsification are again arranged.
(4) the quantitative packing of the vaccine that packing is good with emulsification seals.
The safety examination of embodiment 4 vaccines
1, vaccine and laboratory animal
Get the duck BYD viral inactivation vaccine of three crowdes of embodiment, 3 preparations, lot number is respectively 2011001,2011002,2011003.Healthy duck is provided by Guangdong Dahuanong Animal Health Products Co., Ltd..
2, vaccine uses a single dose inoculation to minimum days old animal
Every batch of vaccine is distinguished 10 of the healthy ducks of immunity 5～7 ages in days, every neck subcutaneous injection vaccine 0.5mL; Injection back is continued to observe and was raised 14 days, observes the reaction of immunity back animal, every group the part duck is dissected, and observes the absorbing state of injection site vaccine.
3, vaccine is to target animals single dose repeated inoculation
Every batch of vaccine is distinguished 10 of the healthy ducks of immunity 5～7 ages in days, every neck subcutaneous injection vaccine 0.5mL; In 2 weeks of back of immunity for the first time, carry out two and exempt from, every neck subcutaneous injection vaccine 0.5mL again; Injection back is continued to observe and was raised 14 days, observes the reaction of immunity back animal, every group the part duck is dissected, and observes the absorbing state of injection site vaccine.
4, vaccine is to an overdose inoculation of target animals
Every batch of vaccine is distinguished 10 of the healthy ducks of immunity 5～7 ages in days, every neck subcutaneous injection vaccine 1mL; Injection back is continued to observe and was raised 14 days, observes the reaction of immunity back animal, every group the part duck is dissected, and observes the absorbing state of injection site vaccine.
5, test-results
More than each group test duck part and systemic adverse reactions all do not appear, the mental status of animal and appetite are good after the vaccination, the part duck is carried out anatomic observation show; The vaccination position is symptom such as swollen, the suppuration of show not, and vaccine absorbs fully, the results are shown in Table 1.Therefore, three batches of vaccines of the embodiment of the invention 3 preparation under the situation of single dose, overdose, repeatedly inoculation all to duck safety.
Table 1 safety testing result
The efficacy test of embodiment 5 vaccines
1, vaccine and laboratory animal
Get three batches of duck BYD viruses (JXSP strain) inactivated vaccine that embodiment 3 makes, lot number is 2011001,2011002,2011003.Healthy duck is provided by Guangdong Dahuanong Animal Health Products Co., Ltd..
2, vaccine immunization
Every batch of vaccine is distinguished 10 of the healthy ducks of immunity 5～7 ages in days, every neck subcutaneous injection vaccine 0.5mL; Establish 10 of non-immune control group ducks simultaneously.
3, Serum Antibody Detection
Immunity blood sampling in back 21 days separation of serum adopts indirect ELISA method to detect antibody horizontal.Concrete operation method is as follows:
1. the virus antigen breeding is done BYD virus (JXSP strain) to be inoculated on the DEF that grows up to individual layer after 100 times of dilutions with the DMEM basal liquid with purifying, inhale behind 37 ℃ of absorption 1h and abandon viral liquid, add and keep liquid continuation cultivation, observe every day, treat that pathology appears in 75%～90% cell, collect viral liquid, in 4 ℃, get supernatant behind 10, the 000r/min high speed centrifugation 45min, supernatant is in 4 ℃, 40,000r/min ultracentrifugation 2.5h abandons supernatant, the 1/100 usefulness sterilization PBS that presses original volume suspends and precipitates, afterwards suspension is placed on the sucrose layer of 20% (W/V) and 50% (W/V) in 4 ℃ 40,000r/min ultracentrifugation 3h, get two concentrating virus bands between the sucrose layer in-80 ℃ of preservations, used antigen as the bag that ELISA detects.
2. above-mentioned purifying antigen is diluted to 6 μ g/mL with carbonate buffer solution (pH9.6), joins in (100 μ L/ hole) in the 96 hole enzyme-linked reaction plates, 4 ℃ of bags that spend the night are by enzyme-linked reaction plate;
3. get bag by good enzyme-linked reaction plate, the tipping coating buffer was with PBS (pH7.4) washings (PBST) washing that contains 0.05% tween 20 3 times, each 3 minutes;
4. every hole add 150 μ L confining liquids (5% skimmed milk, PBST), 37 ℃ the effect 2 hours, 3. each hole liquid of tipping wash 3 times with reference to step;
5. every hole adds 100 μ L duck serum to be checked (1: 200) dilution, and 37 ℃ act on 1 hour.Each Sptting plate is respectively set up 2 positive and negative serum control wells simultaneously.3. each hole liquid of tipping wash 3 times with reference to step;
6. every hole adds the goat-anti duck IgG (H+L) (dilution in 1: 500, U.S. KPL company produces) of 100 μ L horseradish peroxidase-labeled, and 37 ℃ act on 1 hour.3. each hole liquid of tipping wash 3 times with reference to step;
7. every hole adds tmb substrate liquid 100 μ L, and 15min is made in 37 ℃ of lucifuge senses, adds 50 μ L 2M sulphuric acid soln termination reactions, measures OD with microplate reader
450nmValue, the record result.
4, Serum Antibody Detection result
The antibody test result all reaches more than 0.85, and control group serum is negative (the results are shown in Table 2) all.Serum Antibody Detection result shows, can induce behind this vaccine of duck immunization to produce good immune response.
Adopt fixed virus-dilute serum neutralization test serum to be carried out parallel detection, duck serum ELISA antibody titer OD
450nm〉=0.85 o'clock, virus neutralization tires 〉=and 1: 100.The protection ratio of strong poison being attacked poison can reach more than 90%.
Table 2 Serum Antibody Detection result
| The test grouping | The vaccine lot number | Detected result |
| One group of immunity | 2011001 | OD 450＞1.05 |
| Two groups of immunity | 2011002 | OD 450＞0.89 |
| Three groups of immunity | 2011003 | OD 450＞0.95 |
| Non-immune control group | / | OD 450＜0.2 |
5, challenge test
The strong poison of 3 all intracranial inoculation checks (BYD-JXSP strain BHK-21 cell cultures the 3rd generation virus liquid that embodiment 2 makes, its virus titer 〉=10 after the immunity of experiment duck
5.5TCID
50/ mL, 0.1mL/ are only), continue to observe 14 days, the reaction of recording each treated animal comprises appetite, clinical symptom, cuts open the variation of inspection general pathology.To dying of illness duck and attack the test duck that slaughtered on the 14th day poison back, detect brain and the in-house BYD-JXSP virus of spleen with the fluorescence RT-PCR method.
6, challenge test result
(1) attacks poison contrast duck and attack back 9/10 duck morbidity of poison, 5/10 duck death.
Sick duck shows as: 1. clinical observation is seen the minimizing of searching for food, contracting neck, is trembled, walking step state is undesired, ataxia and paralysis etc.; 2. cut open inspection natural death duck splenomegaly, extravasated blood substantially, observation was slaughtered the duck eye to 14 days and is not seen obvious pathology; 3. apyetous encephalitis such as the little blood vessel oversleeve of the visible brain of pathological study sample glial cell invasion, glial cell invasion kitchen range change.
To dying of illness duck and attack the contrast duck slaughtered in back 14 days of poison, getting brain and spleen, to detect BYD virus (JXSP strain) with the fluorescence RT-PCR method all positive.See table 3 for details.
Table 3 contrast duck challenge test is table as a result
(2) vaccine immunity duck
Three batches of inactivated vaccine immune group, clinical manifestation is normal after most of immune duck immunity, every group has the part duck can occur one in 4～8 days and cross property and subtract food, contracting neck, clinical symptom such as rock, immune three groups of 1 duck death in the 8th day after attacking poison, the visible ataxia of duck individually attacking poison back.Cut open the inspection no abnormality seen substantially, the accidental spongiocyte of pathological study is little focal hyperplasia.Brain and spleen detect BYD virus (JXSP strain) with the fluorescence RT-PCR method, and three groups only 4 samples are positive.
The results are shown in Table 4.
By experimental result as seen, behind three batches of vaccine immunity ducks that the embodiment of the invention 3 makes, can make the infection of duck opposing duck BYD virus, not cause untoward reaction, have good protection and render a service.
Table 4 vaccine immunity group is attacked poison back summary sheet as a result
The comprehensive test of embodiment 6 immune effect of vaccine
1, vaccine and laboratory animal
Get 1 batch of the duck BYD viral inactivation vaccine that embodiment 3 makes, lot number is 2011001.Healthy duck is provided by Guangdong Dahuanong Animal Health Products Co., Ltd..
2, the safety examination of vaccine
10 of the healthy ducks of 5～7 ages in days, every neck subcutaneous injection vaccine 1mL; The injection back continues to observe raising 14 days, does not see part and systemic adverse reactions, and the inoculation duck searches for food normally.
3, efficacy test
1. vaccine immunization is with 10 of the healthy ducks of vaccine inoculation 5 ages in days, every neck subcutaneous injection vaccine 0.5mL; Establish 10 of non-immune control group ducks simultaneously.
2. strong malicious (BYD-JXSP strain BHK-21 cell cultures the 3rd generation virus liquid that embodiment 2 makes, its virus titer 〉=10 are used in 3 all intracranial inoculation checks after attacking malicious protection test immunity
5.5TCID
50/ mL, 0.1mL/ are only), continue to observe 14 days.4/10 duck occurred one in 3～8 days and crosses property and subtract food, contracting neck, clinical symptom such as rock after immunity, to attack poison do not have in back 14 days dead, the whole survival.Cuing open inspection after slaughtering substantially shows no obvious abnormalities.It is positive that brain and spleen detect 0/10 in BYD virus (JXSP strain) and 1/10 with the fluorescence RT-PCR method.
Non-immune contrast duck is attacked back 9/10 duck morbidity of poison, 5/10 duck death.
Sick duck shows as: 1. clinical observation is seen the minimizing of searching for food, contracting neck, is trembled, walking step state is undesired, ataxia and paralysis etc.; 2. cut open inspection natural death duck splenomegaly, extravasated blood substantially, observation was slaughtered the duck eye to 14 days and is not seen obvious pathology; 3. apyetous encephalitis such as pathological study visible vessels oversleeve sample glial cell invasion, glial cell invasion kitchen range change.
To the contrast duck that dies of illness duck and slaughtered in 14 days, it is positive that brain and spleen detect 8/10 in BYD virus (JXSP strain) and 9/10 with the fluorescence RT-PCR method.See table 5 for details.
Table 5 efficacy test result
4, determine that duck morbidity standard is
Following 3 indexs meet 2, can be judged to be the morbidity duck:
1. dead;
2. the minimizing of searching for food is seen in clinical observation; The contracting neck occurs, tremble, nervous symptoms such as walking step state is undesired, ataxia and paralysis; The laying ducks egg number obviously descends, even is 0;
3. 8/10 above brain and spleen fluorescence RT-PCR detect the BYD virus-positive.
According to experimental result, as seen BYD-JXSP virus strain provided by the invention has good immunogenicity, utilize the duck BYD viral inactivation vaccine of its preparation to be applied to duck, can effectively help the infection of host duck opposing BYD virus, and security is good, untoward reaction can be do not caused, the prevention and control of the duck egg drop syndrome that the infection of duck BYD virus causes can be widely used in.
Claims (7)
1. an inactivated vaccine is characterized in that, contains duck BYD virus JXSP strain, and the deposit number of described duck BYD virus JXSP strain is CGMCC No.5266.
2. a method for preparing the described inactivated vaccine of claim 1 comprises the steps:
1) cell of enlarged culturing is pressed 1% of nutrient solution volume and added JXSP strain virus seed in the claim 1,37 ℃, adsorbed 1 hour;
2) add 1% foetal calf serum and keep liquid, cultivate down for 37 ℃, gather in the crops freeze thawing 3 times when cytopathy reaches 75% when above;
3) centrifugal or remove by filter cell debris, collect supernatant liquor, this supernatant liquor is duck BYD virus JXSP strain virus liquid;
4) step 3) is prepared duck BYD virus JXSP strain virus liquid once carries out deactivation and emulsification as seedling with viral liquid, obtains duck BYD viral inactivation vaccine.
3. method as claimed in claim 2 is characterized in that, described cell is BHK-21, Vero and/or DEF.
4. as claim 2 or 3 described methods, it is characterized in that step 3) gained virus liquid viral level 〉=10
5.5TCID
50/ mL.
5. as claim 2 or 3 arbitrary described preparation methods, it is characterized in that described deactivation is to be that 0.2% volume ratio adds formaldehyde solution, mixing, 37 ° of C effect 24h with viral liquid by final concentration.
6. as claim 2 or 3 arbitrary described preparation methods, described emulsification be with the viral liquid after the deactivation as water, after mixing according to water oil phase volume ratio 4:6,10000～12000r/min emulsification 2min, add Thiomersalate solution before stopping stirring, make final concentration reach 0.01% volume ratio.
7. preparation method as claimed in claim 6 is characterized in that, gets injection white oil 94mL, adds stearic acid aluminium 2g, and the limit edged stirs, and until transparent fully, adds 6mL Si Ben-80 again, abundant mixing, and autoclaving obtains the 100mL oil phase; The cell culture and virus liquid 100mL that gets deactivation adds the tween-80 of 4mL sterilization, fully shakes up to tween-80 and dissolves fully, obtains the 104mL water.
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| CN104109206B (en) * | 2014-07-03 | 2017-06-20 | 北京市动物疫病预防控制中心 | Duck tembusu virus monoclonal antibody, antigen detection kit and application |
| CN111484984B (en) * | 2019-01-28 | 2021-10-29 | 福建省农业科学院畜牧兽医研究所 | Duck circovirus strain and preparation method of inactivated vaccine 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 |
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| CN102220291A (en) * | 2011-05-18 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Duck flavivirus and inactivated vaccine thereof |
| CN102220290A (en) * | 2011-05-25 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Cell culture method for duck flavivirus |
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