CN103417980A - Novel freeze-drying protective additive for duck virus hepatitis live vaccines - Google Patents
Novel freeze-drying protective additive for duck virus hepatitis live vaccines Download PDFInfo
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Abstract
The invention relates to a novel freeze-drying protective additive for duck virus hepatitis live vaccines. The novel freeze-drying protective additive has the advantages that the vaccine is low in loss of antigen activity during freeze drying, and the problem that the duck hepatitis virus is sensitive to the freeze drying is effectively solved; the duck virus hepatitis vaccines freeze-dried by the protective additive are good in heat resistance, and storage life thereof is longer than 24 months at the temperature of 2-8 DEG C, good activity is still maintained in ten days after a resistance aging test at the temperature of 37 DEG C, so that the problem of a strict low-temperature cold chain of the vaccines upon storage and transportation is solved, and meanwhile vaccine storage and transportation costs are greatly lowered.
Description
Technical field
The present invention relates to the novel freeze drying protectant of a kind of duck viral hepatitis live vaccine, belong to veterinary biologics manufacturing technology field.
Background technology
Duck viral hepatitis (duck viral hepatitis, DVH) is that the duckling that caused by DHV (duck hepatitis virus, DHV) a kind of be take acute, strong, the height lethal infectious disease that the liver hemorrhagic inflammation is feature.The duck liver inflammation mainly betides 3 week age with interior duckling, and morbidity suddenly, is propagated rapidly, and M & M can reach more than 90%.Since nineteen fifty Levine and Fabricant are separated to 1 type DHV from USA New York Long Island morbidity duckling first, at present knownly cause that the virus of duck liver inflammation has three kinds, be called 1 type, 2 types and 3 type DHVs.Wherein 1 type DHV belongs to the microRNA Viraceae, and 2 types and 3 type DHVs belong to Astroviridae.Three kinds of virus is pathogen independently each other, between the serum-free intersecting protective, and 2 types are detected in Britain, 3 types only occurred in the U.S..1 type duck liver is scorching popular the widest, and after last century, the fifties was found, duck country is supported in the world this disease report successively.China's Huang all build reported first in 1963 District of Shanghai duck field break out this disease, at first king's equality be separated to this virus in morbidity duck field, Beijing area in 1980, the generation that all there is primary disease in the duck area is supported by China afterwards.
The report such as Sandhu in 1992 is found the variant of a kind of 1 type DHV, with between Embryo Gallus domesticus cross neutralization test proof and 1 type, the part cross-protection being arranged.There are respectively the report of 1 type DHV variant and Novel duck hepatitis in China mainland, Taiwan and Korea S in recent years, though different researchers confirms that by molecular biology and serological test there is the existence of the scorching variant of duck liver or Novel duck hepatitis in China, duck liver inflammation popular still be take 1 type hepatitis of classics as main.
The year number of animals raised of China duck has reached 4,000,000,000, account for whole world duck and raise more than 70% of total amount, owing to there is no for a long time the scorching vaccine of legal commercialization duck liver for prophylactic immunization, this disease has become the No.1 epidemic disease that the duck industry is supported by harm China, and the annual direct economic loss caused because of the death of infected duck hepatitis reaches several hundred million units.Before this, China mainly adopts high immunity yolk antibody to the control of duck viral hepatitis, because the passive immunity persistent period is short, the prevention and therapeutic effect not good, usually need the multiple injection yolk antibody, cause expenses for prevention and control high, waste time and energy.Attenuated live vaccines is the most economical and effective method of prevention primary disease; we have cultivated by the effort of more than two decades, and 1 strain immunogenicity is good, virulence stable, safety is good, produce the fast vaccine strain of protection, develops duck viral hepatitis live vaccine (A66 strain).Immunity inoculation by vaccine is expected to control the popular of domestic duck viral hepatitis, significant to the mortality rate and the raising productivity effect that reduce duckling.
In live vaccine, freeze-dried live vaccine accounts for sizable ratio.Freeze drying protectant and freeze-dry process are key technologies during live vaccine is produced, and lyophilizing, preservation, transportation and the use of vaccine had a significant impact.At present; domestic live vaccine freeze drying protectant composition for animals is mainly milk, sucrose, gelatin etc.; although the product shape prepared as protective agent with them is stable, ease of solubility is good, with low cost; but it is not strong to vaccine heat stability and antigen active protective capability; large to antigen active loss in some vaccine freeze-drying process; product need to preserved below-15 ℃ mostly, and storage life only has 1 year left and right time, and this brings very large difficulty to storage and transportation of vaccine.Live vaccine novel protective agent and freeze-dry process research abroad just start in the 1980s; external live vaccine for animals and domestic stranger generally adopt novel protected agent prescription and freeze-dry process with live vaccine now; vaccine storage life under 2～8 ℃ of conditions was reached more than 24 months, preserve for 37 ℃ and within 10 days, still keep active.
Virus is as the simplest life form, the kind difference is also different to the sensitivity of freezing sense, and this has reflected them to the tolerance feature of lyophilizing and has repaired the ability difference of damaging in dry run.Grieff and Rightsel are divided into different monoid [Grieff to the difference of lyophilizing sensitivity by virus according to virus, D.and Rightsel, W.A. (1965) Stabilities of virus after vacuum sublimation and storage.Cryobiology3, 435-443.], and 1 type DHV just in time belongs to the microRNA Viraceae member [Rightsel responsive especially to lyophilizing, W.A..and Grieff, D (1967) Freezing and freeze-drying of virus.Cryobiology3, 423-431.], we also verify this specific character when the scorching vaccine protective agent formula of screening duck liver, so protective agent formula involved in the present invention and freeze-drying curve are suitable for the microRNA Viraceae virus to the lyophilizing sensitivity, especially be suitable for the lyophilizing of DHV.
Summary of the invention
The object of the invention is to by the screening of frozen-dried protective agent prescription, optimally provide the novel freeze drying protectant of a kind of applicable duck viral hepatitis live vaccine and freeze-drying curve.Novel protective agent preparation of the present invention is simple, is applicable to large-scale production.
Technical scheme of the present invention
A kind of duck viral hepatitis heat-resisting lyophilized protecting agent of live vaccine, its prescription is:
The each component percent weight in volume is: trehalose 2%～5%, glucose 1%～3%, glycine 3%～8%, D-glucitol 1%～3%, polyvinylpyrrolidone 1%～3%, gelatin 0.8%～1.2%, and water for injection complements to 100%;
Compound method is:
Be dissolved in water for injection after the compositions such as the trehalose of freeze drying protectant component, glucose, glycine, D-glucitol, polyvinylpyrrolidone, gelatin are taken to mixing in the prescription ratio, put 116 ℃ of autoclaving 30min.
Novel protective agent involved in the present invention is suitable for the frozen-dried protective to the microRNA Viraceae virus of lyophilizing sensitivity, especially is suitable for the application in the duck viral hepatitis live vaccine.
The application of this novel protective agent in the duck viral hepatitis live vaccine, that this novel freeze drying protectant mixes with the volume ratio of 1:1 with the duck viral hepatitis antigen liquid, packing, adopt corresponding freeze-drying curve lyophilization to obtain the duck viral hepatitis live vaccine, and this corresponding freeze-drying curve is: the goods after packing are inserted in the body of freeze dryer that is chilled in advance 5 ℃, speed with 0.5 ℃～1 ℃/min cools the temperature to-45 ℃, maintaining 4～6 hours makes vaccine freeze reality, after being evacuated to 5Pa, speed with 1 ℃/min rises to 15 ℃ by temperature again, maintain 12～15 hours at this temperature, then with the speed of 0.5 ℃/min, be warming up to 25 ℃ and maintain 2 hours, speed with 0.5 ℃/min is warming up to 32 ℃ again, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is 26～30 hours, and the every plumage part of the duck viral hepatitis live vaccine viral level after lyophilizing answers>=10
4.3LD
50.
The specific embodiment of the invention
Duck viral hepatitis live vaccine (A66 strain) antigen preparation: (inventor causes weak acquisition by the DHV be separated to by China Anhui in 1986 through the continuous passage of SPF Embryo Gallus domesticus to get DHV A66 strain for producing, and preserved by the inventor, this strain DHV (duck hepatitis virus) was delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 on 08 13rd, 2013, Institute of Microorganism, Academia Sinica, the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCCNo.8020) 100 times of dilutions of sterile saline do for seed culture of viruses, inoculate well-developed 9～10 age in days SPF chick embryo allantoic cavities, every embryo 0.2ml, after sealing of hole, putting 36～37 ℃ continues to hatch, abandon Embryo Gallus domesticus dead before 36 hours.After 36 hours, every 4～8 hours photograph eggs once, take out at any time 72 hours Embryo Gallus domesticus with interior death, put 4～8 ℃ cooling.The Embryo Gallus domesticus of cooling 4～24 hours are taken out, with iodine tincture disinfection air chamber section, remove shell breaking with aseptic operation, collect pathological changes chick chorioallantoic membrane, blastochyle (allantoic fluid and amniotic fluid), fetus, it is one group that every several Embryo Gallus domesticus mix, and high-speed homogenization is made suspension, is placed in sterilization container, add appropriate antibiotic room temperature effect 1～2 hour, the postposition that the keeps sample-15 ℃ preservation of freezing.Should>=10 through checking aseptic and viral level to measure every 1ml
7.5ELD
50, can be for joining Seedling.
Join Seedling and packing: take out the Embryo Gallus domesticus tissue antigen be up to the standards and thaw, weigh, first add appropriate protective agent to mix in same container, filter, the deduction level of residue, add the surplus protective agent according to the actual tissue mass that filters.Add suitable antibiotic again, fully mix, quantitative separating, then insert rapidly in the body of freeze dryer of pre-cooling, by the preferred freeze-drying curve of the present invention, carries out lyophilisation.
Test product important technological parameters and the method for inspection:
The physical behavior of goods, residual moisture are measured and vacuum mensuration is undertaken by method and the standard of existing " People's Republic of China's veterinary drug allusion quotation " (in 2010 version) appendix.
Viral level assay method and standard:
Viral level is measured and is pressed the dated plumage part of label, and vaccine is diluted to 1 plumage part/0.1ml with sterile saline, remakes 10 times of serial dilutions, gets 10
-3, 10
-4, 10
-53 dilution factors, inoculate 5 of 8～9 age in days SPF Embryo Gallus domesticus in allantoic cavity respectively, and every embryo 0.1ml, put 37 ℃ and continue to hatch, and before 24 hours, dead Embryo Gallus domesticus discards and disregards, and at 24～120 hours dead Embryo Gallus domesticus, takes out at any time calculating ELD
50.Viral level in every plumage part vaccine should be not less than 10
4.3ELD
50.
5% sucrose defatted milk contrasts as conventional freeze drying protectant.
Test product is placed under-15 ℃, 2～8 ℃, 37 ℃ conditions and preserves, different time sampling and measuring viral level.
Novel freeze drying protectant of the present invention is comprised of the following component of weight percent meter: trehalose 2%～5%, glucose 1%～3%, glycine 3%～8%, D-glucitol 1%～3%, polyvinylpyrrolidone (PVP-k30) 1%～3%, gelatin 0.8%～1.2%, and water for injection complements to 100%; .
The compositions such as the trehalose of above-mentioned novel freeze drying protectant component, glucose, glycine, D-glucitol, polyvinylpyrrolidone (PVP-k30), gelatin are dissolved in to water for injection in ratio separately; can adopt the water-bath alr mode of heating for dissolving above-mentioned substance fully; until solution is transparent; divide again and be filled to the rearmounted 116 ℃ of autoclaving 30min of appropriate containers, standby.
Novel freeze drying protectant after above-mentioned sterilizing is mixed with the volume ratio of 1:1 with the duck viral hepatitis antigen liquid, divide and be filled in the 7ml cillin bottle, every bottle of 2～4ml, by the butyl rubber trident plug postposition of partly jumping a queue, enter in the body of freeze dryer that is chilled in advance 5 ℃, speed with 0.5 ℃～1 ℃/min cools the temperature to-45 ℃, maintaining 4～6 hours makes vaccine freeze reality, after being evacuated to 5Pa, speed with 1 ℃/min rises to 15 ℃ by temperature again, maintain 12～15 hours at this temperature, then with the speed of 0.5 ℃/min, be warming up to 25 ℃ and maintain 2 hours, speed with 0.5 ℃/min is warming up to 32 ℃ again, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is 26～30 hours, and the every plumage part of the duck viral hepatitis live vaccine viral level after lyophilizing answers>=10
4.3LD
50.
The accompanying drawing explanation
Fig. 1 vacuum lyophilization curve synoptic diagram.As shown in the figure, vaccine after packing enters in the body of freeze dryer that is chilled in advance 5 ℃, speed with 0.5 ℃～1 ℃/min cools the temperature to-45 ℃, maintain 4～6 hours and make vaccine freeze reality, after being evacuated to 5Pa, then with the speed of 1 ℃/min, temperature is risen to 15 ℃, maintain 12～15 hours at this temperature, then with the speed of 0.5 ℃/min, be warming up to 25 ℃ and maintain 2 hours, then be warming up to 32 ℃ with the speed of 0.5 ℃/min, maintain 6 hours outlets of jumping a queue.
The microbial resources information the present invention relates to
The microbial resources that the present invention relates to are DHV (duck hepatitis virus) A66 strain [DHV that the inventor was separated to by China Anhui in 1986 (duck hepatitis virus) A strain causes weak acquisition through the continuous passage of SPF Embryo Gallus domesticus], this strain DHV (duck hepatitis virus) was delivered Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 on 08 13rd, 2013, Institute of Microorganism, Academia Sinica, the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.8020.
Positive effect of the present invention
The present invention relates to the novel freeze drying protectant of a kind of duck viral hepatitis live vaccine.This novel protective agent great advantage is to make vaccine antigen active loss in freeze-drying process little, efficiently solves the responsive difficult problem of DHV to lyophilizing.Secondly; good with the scorching live-vaccine heat-proof of the duck liver of this protective agent lyophilizing; vaccine storage life under 2～8 ℃ of conditions reached more than 24 months; still keep excellent activity after 10 days through 37 ℃ of anti-aging tests; solved vaccine to storing, transporting strict low temperature cold chain problem, also reduced greatly vaccine simultaneously and stored and cost of transportation.
Embodiment
Below in conjunction with concrete embodiment, the present invention is described in further detail, the present embodiment is not construed as limiting the present invention.
Embodiment 1
---the preparation of duck viral hepatitis live vaccine (A66 strain) antigen
Get to produce with DHV A66 strain seed culture of viruses and do 100 times of dilutions with sterile saline, inoculate well-developed 9～10 age in days SPF chick embryo allantoic cavities, every embryo 0.2ml, after sealing of hole, put 36～37 ℃ and continue to hatch, and abandons Embryo Gallus domesticus dead before 36 hours.After 36 hours, every 4～8 hours photograph eggs once, take out at any time 72 hours Embryo Gallus domesticus with interior death, put 4～8 ℃ cooling.The Embryo Gallus domesticus of cooling 4～24 hours are taken out, with iodine tincture disinfection air chamber section, remove shell breaking with aseptic operation, collect pathological changes chick chorioallantoic membrane, blastochyle (allantoic fluid and amniotic fluid), fetus, it is one group that every several Embryo Gallus domesticus mix, and high-speed homogenization is made suspension, is placed in sterilization container, add appropriate antibiotic room temperature effect 1～2 hour, the postposition that the keeps sample-15 ℃ preservation of freezing.Should>=10 through checking aseptic and viral level to measure every 1ml
7.5ELD
50.
Embodiment 2
---protectant preparation and vaccine preparation (1)
The following component of novel freeze drying protectant weight percent meter forms: trehalose 2, glucose 1%, glycine 3%, D-glucitol 1%, polyvinylpyrrolidone (PVP-k30) 1%, gelatin 0.8%, surplus is water for injection.Dissolve above-mentioned substance fully, through 116 ℃ of autoclaving 30min.
Get novel freeze drying protectant and the 5% sucrose skim milk protective agent for contrasting, with the duck viral hepatitis antigen liquid be up to the standards, with the volume ratio of 1:1, mix respectively, divide and be filled in the 7ml cillin bottle, every bottle of 2ml, by the butyl rubber trident plug postposition of partly jumping a queue, enter in the body of freeze dryer that is chilled in advance 5 ℃, speed with 0.5 ℃/min cools the temperature to-45 ℃, maintaining 4 hours makes vaccine freeze reality, after being evacuated to 5Pa, speed with 1 ℃/min rises to 15 ℃ by temperature again, maintain 12 hours at this temperature, then with the speed of 0.5 ℃/min, be warming up to 25 ℃ and maintain 2 hours, speed with 0.5 ℃/min is warming up to 32 ℃ again, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is about 26 hours.
Embodiment 3
---protectant preparation and vaccine preparation (2)
The following component of novel freeze drying protectant weight percent meter forms: trehalose 3.5%, glucose 2%, glycine 5.5%, D-glucitol 2%, polyvinylpyrrolidone (PVP-k30) 2%, gelatin 1% inject water to 100%.Dissolve above-mentioned substance fully, through 116 ℃ of autoclaving 30min.
Get novel freeze drying protectant and the 5% sucrose skim milk protective agent for contrasting, with the duck viral hepatitis antigen liquid be up to the standards, with the volume ratio of 1:1, mix respectively, divide and be filled in the 7ml cillin bottle, every bottle of 3ml, by the butyl rubber trident plug postposition of partly jumping a queue, enter in the body of freeze dryer that is chilled in advance 5 ℃, speed with 0.75 ℃/min cools the temperature to-45 ℃, maintaining 5 hours makes vaccine freeze reality, after being evacuated to 5Pa, speed with 1 ℃/min rises to 15 ℃ by temperature again, maintain 13.5 hours at this temperature, then with the speed of 0.5 ℃/min, be warming up to 25 ℃ and maintain 2 hours, speed with 0.5 ℃/min is warming up to 32 ℃ again, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is about 28 hours.
---protectant preparation and vaccine preparation (3)
The following component of novel freeze drying protectant weight percent meter forms: trehalose 5%, glucose 3%, glycine 8%, D-glucitol 3%, polyvinylpyrrolidone (PVP-k30) 3%, gelatin 1.2%, surplus is water for injection.Dissolve above-mentioned substance fully, through 116 ℃ of autoclaving 30min.
Get novel freeze drying protectant and the 5% sucrose skim milk protective agent for contrasting, with the duck viral hepatitis antigen liquid be up to the standards, with the volume ratio of 1:1, mix respectively, divide and be filled in the 7ml cillin bottle, every bottle of 4ml, by the butyl rubber trident plug postposition of partly jumping a queue, enter in the body of freeze dryer that is chilled in advance 5 ℃, speed with 1 ℃/min cools the temperature to-45 ℃, maintaining 6 hours makes vaccine freeze reality, after being evacuated to 5Pa, speed with 1 ℃/min rises to 15 ℃ by temperature again, maintain 15 hours at this temperature, then with the speed of 0.5 ℃/min, be warming up to 25 ℃ and maintain 2 hours, speed with 0.5 ℃/min is warming up to 32 ℃ again, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is about 30 hours.
Embodiment 5
---the detection of freeze-dried products
The physical behavior of table 1 freeze-dried products, residual moisture are measured and the vacuum measurement result
* measure product with the vacuum determinator and all present the milky aura, meet People's Republic of China's veterinary drug allusion quotation relevant regulations (Chinese veterinary pharmacopoeia committee compiles. three ones of in 2010 versions of People's Republic of China's veterinary drug allusion quotation. Chinese agriculture publishing house 2011).
Table 2 freeze-dried products is preserved under-15 ℃ of conditions, different time sampling and measuring viral level measurement result
Table 3 freeze-dried products is preserved under 2～8 ℃ of conditions, different time sampling and measuring viral level measurement result
Table 4 freeze-dried products is preserved under 37 ℃ of conditions, different time sampling and measuring viral level measurement result
From result of the test, show; the forward and backward malicious valency loss of 5% sucrose defatted milk protective agent vaccine freeze-drying is larger; surpass 2 titres; and the forward and backward malicious valency loss of novel freeze drying protectant vaccine freeze-drying of the present invention is less; be no more than 1 titre, show that novel protective agent of the present invention has good protective effect in freeze-drying process to DHV.
Result of the test also shows, the scorching vaccine of the duck liver of 5% sucrose defatted milk protective agent lyophilizing can only be preserved 12 months under-15 ℃ of conditions, under 2～8 ℃ of conditions, preserves 12 months, preserves viral titer loss in 3 days under 37 ℃ of conditions and surpasses 1 titre; And the preservation under-15 ℃ and 2～8 ℃ of conditions of the scorching vaccine of the duck liver of novel protective agent lyophilizing of the present invention all reaches more than 24 months; preserve 10 days viral titers under 37 ℃ of conditions and be lost in 1 titre scope, show to use duck viral hepatitis live vaccine prepared by novel freeze drying protectant of the present invention to there is stability preferably under 2～8 ℃ of conditions.
Embodiment 6
---the correlation test of vaccine
(batch piece number: 200844), we carry out the work according to the clinical trial protocol of approval in time to obtain Ministry of Agriculture's veterinary biologics clinical trial approval in duck viral hepatitis live vaccine (A66) clinical trial application.To June, at 5 duck field 5 batches of vaccine products that prepare by novel protective agent of the present invention of application, carry out clinical trial from February, 2009, test amounts to immune duckling 5.9 ten thousand plumages.Through the statistics of clinical observation, challenge test, antibody test and fertility performance, result shows: duck viral hepatitis live vaccine (A66) to immune duckling safety, effectively, have no adverse reaction.
Lot number, the quantity of vaccine is used in 3 clinical trials, sees the following form 5.
Scorching (A66) live vaccine of the duck liver that table 5 test is used
| The vaccine lot number | Specification | Use plumage part | Use the duck field | The kind of duck |
| 070901 | 1000 plumages/bottle | 12000 | 1 | Cherry Village Ducks (SM3) |
| 070902 | 1000 plumages/bottle | 8000 | 2 | Gaoyou duck (meat egg dual-purpose) |
| 70903 | 1000 plumages/bottle | 12000 | 3 | Cherry Village Ducks (SM3) |
| 071004 | 1000 plumages/bottle | 15000 | 4 | Cherry Village Ducks (SM3) |
| 071005 | 1000 plumages/bottle | 12000 | 5 | Chaohu sheldrake (meat egg dual-purpose) |
Five test duck field test arrangement basic conditions of table 6
Annotate: every batch of vaccine separately arranges respectively 100 ducklings to use 1/10 plumage part and 10 plumage doses injecting immunes.
1. clinical observation situation
At five test sites, no matter be the test duck of taking injecting immune or drinking water immune, in immune 5 days, duck group mental status is normal, without assembling, roll up, beat the heap phenomenon, searches for food, drinks water and be normal, and dead, naughty situation is normal; To every batch of vaccine injection immunity and the immune duck group of drinking-water, get at random respectively 10 ducklings in the time of latter 5 days is dissected in immunity, inoculation position regulating liver-QI, spleen, lung, the heart, digestive tract and the brain of injecting immune is showed no pathological changes and abnormal, gets at random 5 ducks of heavy dose (10 times of immunizing doses) injecting immune group and cuts open the inspection observation also without abnormal.
During experimental observation (to 42 ages in days), morbidity and dead increase situation appear when No. 1 duck group and contrast duck group 15 age in days are tested in the duck field, be colibacillosis through clinical and laboratory diagnosis, select enrofloxacin drinking-water drug treatment 5 days after drug sensitive test, rehabilitation; Occurring the sudden death case during No. 3 nonimmune contrast in duck field duck group 8 ages in days, is duck viral hepatitis through clinical and laboratory diagnosis, and mine massively and get 4 times of immunizing dose subcutaneous injection methods and carry out urgent prophylactic immunization that afternoon to this duck, and after 3 days, death stops, rehabilitation; 2,4 and No. 5 San Ge test site immune duck groups are with nonimmune while contrasting duck group to 42 age in days, duck group mental status, search for food, drink water, extremely wash in a pan and the growth promoter situation all normal.Duck liver inflammation and other untoward reaction, do not occur in every batch of 100 ducklings low doses immunity (1/10 recommends immunizing dose) and heavy dose of (recommending immunizing dose for 10 times) injecting immune that vaccine arranges respectively during to 42 age in days.
The Clinical observation result shows, duck viral hepatitis live vaccine (A66) adopts injection (subcutaneous or muscle) and drinking-water is immune that duckling safety is had no adverse reaction to growth promoter, and the urgent prevention of the duck viral hepatitis of clinical generation is had to good result.
2. challenge test result
Test duck group to each batch selects 10 in 5 days after vaccine immunity at random, randomly draws 10 with batch nonimmune contrast ducks simultaneously, takes back laboratory and carries out challenge test.With the strong malicious W strain of DHV, by every duck 1000LD
50Dosage carry out challenge test, immunity test group (injecting immune and drinking-water immunity) counteracting toxic substances protective rate all reaches more than 80% as a result, nonimmune matched group counteracting toxic substances mortality rate, more than 80%, shows that duck viral hepatitis live vaccine (A66) immune effect is certain.Each batch of clinical Immunization result of the test of vaccine is in Table 7.
Table 75 batch vaccine clinical trial Immunization protection result
3. immune antibody assay
Test duck group to five duck fields, within 7,14,28,42 days, random select to gather 10 Sanguis Anas domestica samples by different immunization routes after respectively at vaccine immunity, with batch 5 Sanguis Anas domestica samples of nonimmune matched group, get after separation of serum in the equivalent group and mix, adopt Embryo Gallus domesticus neutralization test method to measure antibody.Test adopts the method for fixed virus dilute serum, and DHV A66 is mixed with to 200ELD with normal saline
50/ 0.1ml, normal saline for serum (containing each 1000 units/ml of penicillin and streptomycin) is done with the equivalent virus liquid, to mix after doubling dilution, puts 37 ℃ of effect 1h.Each dilution factor is inoculated respectively 5 of 9 age in days SPF Embryo Gallus domesticus, and every embryonic breeding kind 0.2ml, put 37 ℃ and continue to hatch 120h, calculates the duck hepatitis antibody neutralization by the Reed-Muench method and tires.Result shows: duckling is accepted the scorching vaccine immunity of duck liver (no matter injecting immune or drinking-water immunity); latter 7 days antibody horizontals of immunity are apparently higher than nonimmune duck; after immunity, 4～6 weeks immune duck group antibody titers maintain the peak level, can protect duckling to spend the scorching age bracket that infects of duck liver.Therefore, from the angle of immune neutralizing antibody, confirm that duck viral hepatitis live vaccine (A66) has good immunogenicity, each batch of clinical immune duck group of vaccine antibody horizontal detection of dynamic the results are shown in Table 8.
Table 85 batch vaccine immunity duckling antibody horizontal dynamic measurement result
4. fertility performance is added up
Statistical summaries is carried out to fertility performance such as the death rate in test duck group clinical trial process, feedstuff-meat ratio, weightening finish etc. in 5 clinical trial duck fields, from statistical result, can find out, duck viral hepatitis live vaccine (A66) on fertility performance such as death rate, feedstuff-meat ratio, weightening finishes without impact.Each is tested duck field test duck all living creatures and produces performance statistics and the results are shown in Table 9.
Fertility performance statistical summaries result during table 95 batch vaccine clinical trial duck group to 42 age in days
Brief summary: 5 duck field immunity 1～3 age in days duckling 5.9 ten thousand plumages have carried out clinical test results and have shown: injecting immune or the duck group who drinks water immune has no adverse reaction no matter, duck group mental status, search for food, drink water all normal, duck group's death rate, meat feed ratio, weightening finish and growth promoter situation are all normal, and in process of the test, duck viral hepatitis does not occur immunity test duck group.By the statistics of clinical observation, laboratory TPPA and challenge test and fertility performance, result proof duck viral hepatitis (A66) live vaccine safety, effective.
Claims (4)
1. a duck viral hepatitis heat-resisting lyophilized protecting agent of live vaccine, it is characterized in that, prescription is: the each component percent weight in volume is: trehalose 2%～5%, glucose 1%～3%, glycine 3%～8%, D-glucitol 1%～3%, polyvinylpyrrolidone 1%～3%, gelatin 0.8%～1.2%, and water for injection complements to 100%;
Compound method is: be dissolved in water for injection after the compositions such as the trehalose of freeze drying protectant component, glucose, glycine, D-glucitol, polyvinylpyrrolidone, gelatin are taken to mixing in the prescription ratio, put 116 ℃ of autoclaving 30min.
2. the application of novel freeze drying protectant according to claim 1, is characterized in that this novel protective agent is suitable for the frozen-dried protective to the microRNA Viraceae virus of lyophilizing sensitivity, especially is suitable for the application in the duck viral hepatitis live vaccine.
3. the application of novel freeze drying protectant as claimed in claim 2; it is characterized in that the application of described this novel protective agent in the duck viral hepatitis live vaccine; this novel freeze drying protectant with the duck viral hepatitis antigen liquid with the volume ratio of 1:1 mix, packing, adopt corresponding freeze-drying curve to carry out vacuum lyophilization and obtain the duck viral hepatitis live vaccine.
4. the application of novel freeze drying protectant as claimed in claim 2, it is characterized in that the freeze-drying curve that described vacuum lyophilization process is used is: the goods after packing are inserted in the body of freeze dryer that is chilled in advance 5 ℃, speed with 0.5 ℃～1 ℃/min cools the temperature to-45 ℃, maintaining 4～6 hours makes vaccine freeze reality, after being evacuated to 5Pa, speed with 1 ℃/min rises to 15 ℃ by temperature again, maintain 12～15 hours at this temperature, then with the speed of 0.5 ℃/min, be warming up to 25 ℃ and maintain 2 hours, speed with 0.5 ℃/min is warming up to 32 ℃ again, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is 26～30 hours, and the every plumage part of the duck viral hepatitis live vaccine viral level after lyophilizing answers>=10
4.3LD
50.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310354836.7A CN103417980B (en) | 2013-08-14 | 2013-08-14 | Novel freeze-drying protective additive for duck virus hepatitis live vaccines |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201310354836.7A CN103417980B (en) | 2013-08-14 | 2013-08-14 | Novel freeze-drying protective additive for duck virus hepatitis live vaccines |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103893776A (en) * | 2014-03-18 | 2014-07-02 | 中国农业科学院兰州兽医研究所 | Heat-resistant freezing and drying protecting agent for contagious ovine ecthyma virus cell attenuated vaccine as well as preparation method and application of heat-resistant freezing and drying protecting agent |
| CN104127870A (en) * | 2014-08-13 | 2014-11-05 | 四川农业大学 | Method for preparing duck hepatitis A virus freeze-drying preparation and prepared freeze-drying preparation |
| CN104726414A (en) * | 2015-01-07 | 2015-06-24 | 南京天邦生物科技有限公司 | Serum type 3 duck hepatitis A virus live vaccine and preparation method thereof |
| CN104127870B (en) * | 2014-08-13 | 2017-01-04 | 四川农业大学 | Preparation method of duck hepatitis A virus freeze-dried preparation and freeze-dried preparation thereof |
| CN106310250A (en) * | 2015-06-19 | 2017-01-11 | 上海市农业科学院 | Swine fever oral attenuated freezing-dry vaccine and preparation method thereof and freeze-drying protective agent |
| CN112807423A (en) * | 2020-12-29 | 2021-05-18 | 肇庆大华农生物药品有限公司 | Freeze-drying process of chick embryo culture vaccine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103007289A (en) * | 2012-11-09 | 2013-04-03 | 上海创宏生物科技有限公司 | Heat-resisting protective agent for duck virus hepatitis live vaccines and preparation method and application of protective agent |
-
2013
- 2013-08-14 CN CN201310354836.7A patent/CN103417980B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103007289A (en) * | 2012-11-09 | 2013-04-03 | 上海创宏生物科技有限公司 | Heat-resisting protective agent for duck virus hepatitis live vaccines and preparation method and application of protective agent |
Non-Patent Citations (2)
| Title |
|---|
| 张小飞: ""鸭病毒性肝炎弱毒疫苗（A66株）的研制"", 《中国博士学位论文全文数据库》 * |
| 李正丰等: ""保护剂及其在口蹄疫疫苗中的研究进展"", 《生物技术通报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103893776A (en) * | 2014-03-18 | 2014-07-02 | 中国农业科学院兰州兽医研究所 | Heat-resistant freezing and drying protecting agent for contagious ovine ecthyma virus cell attenuated vaccine as well as preparation method and application of heat-resistant freezing and drying protecting agent |
| CN103893776B (en) * | 2014-03-18 | 2016-04-06 | 中国农业科学院兰州兽医研究所 | Sheep infective pustule virus cell weak-toxic vaccine heat-resisting lyophilized protecting agent and its preparation method and application |
| CN104127870A (en) * | 2014-08-13 | 2014-11-05 | 四川农业大学 | Method for preparing duck hepatitis A virus freeze-drying preparation and prepared freeze-drying preparation |
| CN104127870B (en) * | 2014-08-13 | 2017-01-04 | 四川农业大学 | Preparation method of duck hepatitis A virus freeze-dried preparation and freeze-dried preparation thereof |
| CN104726414A (en) * | 2015-01-07 | 2015-06-24 | 南京天邦生物科技有限公司 | Serum type 3 duck hepatitis A virus live vaccine and preparation method thereof |
| CN106310250A (en) * | 2015-06-19 | 2017-01-11 | 上海市农业科学院 | Swine fever oral attenuated freezing-dry vaccine and preparation method thereof and freeze-drying protective agent |
| CN112807423A (en) * | 2020-12-29 | 2021-05-18 | 肇庆大华农生物药品有限公司 | Freeze-drying process of chick embryo culture vaccine |
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| CN103417980B (en) | 2015-05-13 |
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