CN103417980B - 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 a kind of with liver hemorrhagic inflammation acute, strong, the height lethal infectious diseases that is feature of the duckling caused by DHV (duck hepatitis virus, DHV).Duck liver inflammation mainly betides duckling within 3 week age, and suddenly, propagate rapidly, M & M can reach more than 90% in morbidity.Since nineteen fifty Levine and Fabricant is separated to 1 type DHV from USA New York Long Island morbidity duckling first, the known virus of duck liver inflammation that causes has three kinds at present, is called 1 type, 2 types and 3 type DHVs.Wherein 1 type DHV belongs to microRNA Viraceae, and 2 types and 3 type DHVs belong to Astroviridae.Three kinds of virus is independently pathogen each other, between serum-free intersecting protective, and 2 types are detected in Britain, and 3 types only occurred in the U.S..1 type duck hepatitis epidemic is the widest, and after last century, the fifties was found, duck country is supported in the world has this disease to report successively.China's Huang is all built reported first in 1963 duck field, District of Shanghai and is broken out that this is sick, and first king's equality be separated to this virus in morbidity duck field, Beijing area in 1980, and the regional generation all having primary disease of duck is supported by China afterwards.
The report such as Sandhu in 1992 finds the variant of a kind of 1 type DHV, proves there is partial intersection protective effect between 1 type with Embryo Gallus domesticus cross neutralization test.China mainland, Taiwan and Korea S have the report of 1 type DHV variant and Novel duck hepatitis respectively in recent years, though by molecular biology and serological test, different researchers confirms that there is the existence of the scorching variant of duck liver or Novel duck hepatitis in China, duck liver inflammation popular still based on 1 type hepatitis of classics.
The year number of animals raised of China duck has reached 4,000,000,000, account for more than 70% of whole world duck raising total amount, owing to there is no legal commercialization duck hepatitis vaccine for a long time for prophylactic immunization, this disease has become the No.1 epidemic disease that duck industry is supported by harm China, and the direct economic loss caused because of the death of infected duck hepatitis every year reaches several hundred million unit.Before this, the control of China to duck viral hepatitis mainly adopts high immunity yolk antibody, and because the passive immunity persistent period is short, prevention and therapy poor effect, usually needs multiple injection yolk antibody, cause expenses for prevention and control high, waste time and energy.Attenuated live vaccines is the most economical and most effective method of prevention primary disease; we have been cultivated by the effort of more than two decades, and 1 strain immunogenicity is good, virulence is stablized, safety good, produce the fast vaccine strain of protection, develops duck viral hepatitis live vaccine (A66 strain).Be expected to control the popular of domestic duck viral hepatitis by the immunity inoculation of vaccine, significant to the mortality rate and raising productivity effect reducing 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 have a significant impact the lyophilizing of vaccine, preservation, transport and use.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 to vaccine heat stability and antigen active protective capability not strong; large to antigen active loss in some vaccine freeze-drying process; product needs to preserve below-15 DEG C mostly, and storage life only has about 1 year time, and this brings very large difficulty to the storage of vaccine and transport.Live vaccine novel protective agent and freeze-dry process research abroad just start in the 1980s; present external live vaccine for animals and domestic stranger generally adopt novel protected agent prescription and freeze-dry process with live vaccine; make vaccine storage life under 2 ~ 8 DEG C of conditions reach more than 24 months, preserve for 37 DEG C and within 10 days, still keep active.
Virus is as the simplest life form, and kind is different, also different to the sensitivity of freezing sense, which reflects them to the ability difference damaged in the tolerance feature of lyophilizing and reparation dry run.Virus is divided into different monoid [Grieff according to virus to the difference of lyophilizing sensitivity by Grieff and Rightsel, 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 characteristic when screening duck liver inflammation vaccine protectant formula, so protective agent formula involved in the present invention and freeze-drying curve are suitable for the microRNA Flaviviridae virus to lyophilizing sensitivity, especially the lyophilizing of DHV is suitable for.
Summary of the invention
The object of the invention is to by the screening of frozen-dried protective agent prescription, optimally provide a kind of and be applicable to the novel freeze drying protectant of 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:
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 mixing is taken in prescription ratio to the composition such as trehalose, glucose, glycine, D-glucitol, polyvinylpyrrolidone, gelatin of freeze drying protectant component, put 116 DEG C of autoclaving 30min.
Novel protective agent involved in the present invention is suitable for the frozen-dried protective of the microRNA Flaviviridae virus to lyophilizing sensitivity, is especially suitable for the application in duck viral hepatitis live vaccine.
The application of this novel protective agent in duck viral hepatitis live vaccine, that this novel freeze drying protectant mixes with the volume ratio of 1:1 with duck viral hepatitis antigen liquid, subpackage, corresponding freeze-drying curve lyophilization is adopted namely to obtain duck viral hepatitis live vaccine, and this corresponding freeze-drying curve is: the goods after subpackage insert be chilled to 5 DEG C in advance body of freeze dryer in,-45 DEG C are cooled the temperature to the speed of 0.5 DEG C ~ 1 DEG C/min, maintaining makes vaccine freeze reality in 4 ~ 6 hours, after being evacuated to 5Pa, with the speed of 1 DEG C/min, temperature is risen to 15 DEG C again, maintain 12 ~ 15 hours at this temperature, then be warming up to 25 DEG C with the speed of 0.5 DEG C/min and maintain 2 hours, 32 DEG C are warming up to again with the speed of 0.5 DEG C/min, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is 26 ~ 30 hours, and the duck viral hepatitis live vaccine every plumage part viral level after lyophilizing answers>=10
4.3lD
50.
The specific embodiment of the invention
Prepared by duck viral hepatitis live vaccine (A66 strain) antigen: (the present inventor causes weak acquisition by the DHV that 1986 are separated to by China Anhui through the continuous passage of SPF Embryo Gallus domesticus to get production DHV A66 strain, and preserved by the present inventor, this strain DHV (duck hepatitis virus) delivered No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 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) seed culture of viruses sterile saline does 100 times of dilutions, inoculate well-developed 9 ~ 10 age in days SPF chick embryo allantoic cavities, every embryo 0.2ml, after sealing of hole, put 36 ~ 37 DEG C to continue to hatch, abandon Embryo Gallus domesticus dead before 36 hours.After 36 hours, every 4 ~ 8 hours photograph eggs once, take out Embryo Gallus domesticus dead within 72 hours at any time, put 4 ~ 8 DEG C of coolings.The cooling Embryo Gallus domesticus of 4 ~ 24 hours is taken out, with iodine tincture disinfection air chamber portion, with aseptic operation except shell breaking, collect pathological changes chick chorioallantoic membrane, blastochyle (allantoic fluid and amniotic fluid), fetus, it is one group that several Embryo Gallus domesticus every mix, and high-speed homogenization makes suspension, is placed in sterilization container, add appropriate antibiotic room temperature effect 1 ~ 2 hour, keep sample rearmounted-15 DEG C of preservations of freezing.Should>=10 through checking aseptic and viral level to measure every 1ml
7.5eLD
50, can be used for joining Seedling.
Join Seedling and subpackage: take out the chicken embryo tissue antigen be up to the standards and thaw, weigh, first add appropriate protective agent and mix in same container, filter, deduction level of residue, add surplus protective agent according to the actual tissue mass that filters.Add appropriate antibiotics again, fully mix, quantitative separating, then insert rapidly in the body of freeze dryer of pre-cooling, carry out lyophilisation by the preferred freeze-drying curve of the present invention.
Test product important technological parameters and the method for inspection:
The physical behavior of goods, residual moisture measure and vacuum measures and undertaken by the method for existing " People's Republic of China's veterinary drug allusion quotation " (in 2010 version) annex and standard.
Viral level assay method and standard:
Viral level measures the plumage part of pressing label and indicating, and vaccine sterile saline is diluted to 1 plumage part/0.1ml, remakes 10 times of serial dilutions, get 10
-3, 10
-4, 10
-53 dilution factors, respectively inoculation 8 ~ 9 age in days SPF Embryo Gallus domesticus 5 in allantoic cavity, every embryo 0.1ml, puts 37 DEG C and continues to hatch, and Embryo Gallus domesticus dead before 24 hours discards to be disregarded, and the Embryo Gallus domesticus dead at 24 ~ 120 hours, takes out at any time, calculates 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.
Preserve under test product being placed in-15 DEG C, 2 ~ 8 DEG C, 37 DEG C conditions, different time sampling and measuring viral level.
Novel freeze drying protectant of the present invention is made up 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 water for injection in respective ratio; water-bath can be adopted to heat alr mode for dissolving above-mentioned substance completely; until solution is transparent; divide again and be filled to the rearmounted 116 DEG C of autoclaving 30min of appropriate containers, for subsequent use.
Novel freeze drying protectant after above-mentioned sterilizing is mixed with the volume ratio of 1:1 with duck viral hepatitis antigen liquid, dividing is filled in 7ml cillin bottle, every bottle of 2 ~ 4ml, partly jump a queue rearmountedly to enter to be chilled in advance in the body of freeze dryer of 5 DEG C with butyl rubber trident plug,-45 DEG C are cooled the temperature to the speed of 0.5 DEG C ~ 1 DEG C/min, maintaining makes vaccine freeze reality in 4 ~ 6 hours, after being evacuated to 5Pa, with the speed of 1 DEG C/min, temperature is risen to 15 DEG C again, maintain 12 ~ 15 hours at this temperature, then be warming up to 25 DEG C with the speed of 0.5 DEG C/min and maintain 2 hours, 32 DEG C are warming up to again with the speed of 0.5 DEG C/min, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is 26 ~ 30 hours, and the duck viral hepatitis live vaccine every plumage part viral level after lyophilizing answers>=10
4.3lD
50.
Accompanying drawing explanation
Fig. 1 vacuum lyophilization curve synoptic diagram.As shown in the figure, vaccine after subpackage enter be chilled to 5 DEG C in advance body of freeze dryer in,-45 DEG C are cooled the temperature to the speed of 0.5 DEG C ~ 1 DEG C/min, maintaining makes vaccine freeze reality in 4 ~ 6 hours, after being evacuated to 5Pa, then with the speed of 1 DEG C/min, temperature is risen to 15 DEG C, maintain 12 ~ 15 hours at this temperature, then be warming up to 25 DEG C with the speed of 0.5 DEG C/min and maintain 2 hours, then be warming up to 32 DEG C with the speed of 0.5 DEG C/min, maintain 6 hours outlets of jumping a queue.
The microbial resources information that the present invention relates to
The microbial resources that the present invention relates to are DHV (duck hepatitis virus) A66 strain [DHV (duck hepatitis virus) the A strain that the present inventor was separated in 1986 by China Anhui causes weak acquisition through the continuous passage of SPF Embryo Gallus domesticus], this strain DHV (duck hepatitis virus) delivered No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 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 makes 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 DEG C of conditions reaches more than 24 months; still excellent activity is kept after 10 days through 37 DEG C of anti-aging tests; solve vaccine to storing, transporting strict low temperature cold chain problem, also greatly reduce vaccine storage and cost of transportation simultaneously.
Embodiment
Be described in further detail the present invention below in conjunction with concrete embodiment, the present embodiment is not construed as limiting the present invention.
Embodiment 1
---prepared by duck viral hepatitis live vaccine (A66 strain) antigen
Get production DHV A66 strain seed culture of viruses sterile saline and do 100 times of dilutions, inoculate well-developed 9 ~ 10 age in days SPF chick embryo allantoic cavities, every embryo 0.2ml, after sealing of hole, put 36 ~ 37 DEG C and continue to hatch, abandon Embryo Gallus domesticus dead before 36 hours.After 36 hours, every 4 ~ 8 hours photograph eggs once, take out Embryo Gallus domesticus dead within 72 hours at any time, put 4 ~ 8 DEG C of coolings.The cooling Embryo Gallus domesticus of 4 ~ 24 hours is taken out, with iodine tincture disinfection air chamber portion, with aseptic operation except shell breaking, collect pathological changes chick chorioallantoic membrane, blastochyle (allantoic fluid and amniotic fluid), fetus, it is one group that several Embryo Gallus domesticus every mix, and high-speed homogenization makes suspension, is placed in sterilization container, add appropriate antibiotic room temperature effect 1 ~ 2 hour, keep sample rearmounted-15 DEG C of preservations 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 composition of novel freeze drying protectant weight percent meter: 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 completely, through 116 DEG C of autoclaving 30min.
Get novel freeze drying protectant and the 5% sucrose skim milk protective agent for contrasting, mix with the volume ratio of 1:1 with the duck viral hepatitis antigen liquid be up to the standards respectively, dividing is filled in 7ml cillin bottle, every bottle of 2ml, partly jump a queue rearmountedly to enter to be chilled in advance in the body of freeze dryer of 5 DEG C with butyl rubber trident plug,-45 DEG C are cooled the temperature to the speed of 0.5 DEG C/min, maintaining makes vaccine freeze reality in 4 hours, after being evacuated to 5Pa, with the speed of 1 DEG C/min, temperature is risen to 15 DEG C again, maintain 12 hours at this temperature, then be warming up to 25 DEG C with the speed of 0.5 DEG C/min and maintain 2 hours, 32 DEG C are warming up to again with the speed of 0.5 DEG C/min, 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 composition of novel freeze drying protectant weight percent meter: 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 completely, through 116 DEG C of autoclaving 30min.
Get novel freeze drying protectant and the 5% sucrose skim milk protective agent for contrasting, mix with the volume ratio of 1:1 with the duck viral hepatitis antigen liquid be up to the standards respectively, dividing is filled in 7ml cillin bottle, every bottle of 3ml, partly jump a queue rearmountedly to enter to be chilled in advance in the body of freeze dryer of 5 DEG C with butyl rubber trident plug,-45 DEG C are cooled the temperature to the speed of 0.75 DEG C/min, maintaining makes vaccine freeze reality in 5 hours, after being evacuated to 5Pa, with the speed of 1 DEG C/min, temperature is risen to 15 DEG C again, maintain 13.5 hours at this temperature, then be warming up to 25 DEG C with the speed of 0.5 DEG C/min and maintain 2 hours, 32 DEG C are warming up to again with the speed of 0.5 DEG C/min, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is about 28 hours.
Embodiment 4
---protectant preparation and vaccine preparation (3)
The following component composition of novel freeze drying protectant weight percent meter: 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 completely, through 116 DEG C of autoclaving 30min.
Get novel freeze drying protectant and the 5% sucrose skim milk protective agent for contrasting, mix with the volume ratio of 1:1 with the duck viral hepatitis antigen liquid be up to the standards respectively, dividing is filled in 7ml cillin bottle, every bottle of 4ml, partly jump a queue rearmountedly to enter to be chilled in advance in the body of freeze dryer of 5 DEG C with butyl rubber trident plug,-45 DEG C are cooled the temperature to the speed of 1 DEG C/min, maintaining makes vaccine freeze reality in 6 hours, after being evacuated to 5Pa, with the speed of 1 DEG C/min, temperature is risen to 15 DEG C again, maintain 15 hours at this temperature, then be warming up to 25 DEG C with the speed of 0.5 DEG C/min and maintain 2 hours, 32 DEG C are warming up to again with the speed of 0.5 DEG C/min, maintain 6 hours outlets of jumping a queue.Lyophilizing whole process is about 30 hours.
Embodiment 5
---the detection of freeze-dried products
Physical behavior, the residual moisture of table 1 freeze-dried products measure and vacuum measurement result
* measure product with vacuum determinator and all present milky aura, meet People's Republic of China's veterinary drug allusion quotation relevant regulations (Chinese veterinary pharmacopoeia committee compiles. People's Republic of China's veterinary drug allusion quotation in 2010 version three. Chinese agriculture publishing house 2011).
Table 2 freeze-dried products is preserved under-15 DEG C of conditions, different time sampling and measuring viral level measurement result
Table 3 freeze-dried products is preserved under 2 ~ 8 DEG C of conditions, different time sampling and measuring viral level measurement result
Table 4 freeze-dried products is preserved under 37 DEG C of conditions, different time sampling and measuring viral level measurement result
Show from result of the test; the forward and backward malicious valency loss of 5% sucrose defatted milk protective agent vaccine freeze-drying is larger; more than 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 to DHV in freeze-drying process.
Result of the test also shows, and the duck hepatitis vaccine of 5% sucrose defatted milk protective agent lyophilizing can only be preserved 12 months under-15 DEG C of conditions, preserves 12 months under 2 ~ 8 DEG C of conditions, preserves viral titer loss in 3 days more than 1 titre under 37 DEG C of conditions; And the duck hepatitis vaccine of novel protective agent lyophilizing of the present invention is preserved and is all reached more than 24 months under-15 DEG C and 2 ~ 8 DEG C of conditions; preserve 10 days viral titers under 37 DEG C of conditions to be lost within the scope of 1 titre, show that the duck viral hepatitis live vaccine using novel freeze drying protectant of the present invention to prepare has good stability under 2 ~ 8 DEG C of conditions.
Embodiment 6
---the correlation test of vaccine
(official written reply 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.From in February, 2009 to June, apply 5 batches of vaccine products prepared by novel protective agent of the present invention carry out clinical trial 5 duck fields, test amounts to immune duckling 5.9 ten thousand plumage.Through the statistics of clinical observation, challenge test, antibody test and fertility performance, result shows: duck viral hepatitis live vaccine (A66) is to immune duckling safety, effectively, have no adverse reaction.
3 clinical trials use lot number, the quantity of vaccine, see the following form 5.
Duck liver inflammation (A66) live vaccine used tested by table 5
| Vaccine lot number | Specification | Use plumage part | Use 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, table 6 test duck field test arrangement basic condition
Note: often criticize vaccine and separately arrange 100 ducklings to use 1/10 plumage part and 10 plumage doses injecting immunes respectively.
1. clinical observation situation
At five test sites, be no matter the test duck of taking injecting immune or drinking-water immunity, in immunity 5 days, duck group mental status is normal, and without assembling, rolling up, beat heap phenomenon, search for food, drink water normal, extremely, naughty situation is normal; To the duck group often criticizing vaccine injection immunity and drinking-water immunity, get 10 ducklings respectively at random when latter 5 days of immunity to dissect, the inoculation position regulating liver-QI of injecting immune, spleen, lung, the heart, digestive tract and brain are showed no pathological changes and exception, get heavy dose (10 times of immunizing doses) injecting immune group 5 ducks at random and cut open inspection and observe also without exception.
During experimental observation (to 42 ages in days), morbidity and dead increase situation is there is when No. 1 duck field test duck group and contrast duck group's 15 age in days, be colibacillosis through clinical and laboratory diagnosis, after drug sensitive test, select enrofloxacin drinking water administration to treat 5 days, rehabilitation; Occurring sudden death case during No. 3 nonimmune contrast in duck field duck group's 8 ages in days, is duck viral hepatitis through clinical and laboratory diagnosis, and that afternoon mines massively to this duck and gets 4 times of immunizing dose subcutaneous injection methods and carry out urgent prophylactic immunization, deadly after 3 days stops, rehabilitation; 2,4 and No. 5 three test site immune duck groups with nonimmune contrast duck group to 42 age in days time, duck group mental status, search for food, drink water, extremely wash in a pan and growth promoter situation all normal.Often criticize the low dose of immunity of 100 ducklings (1/10 recommends immunizing dose) and heavy dose of (recommending immunizing dose for 10 times) injecting immune that vaccine arranges respectively, to duck liver scorching and other untoward reaction do not occur during 42 age in days.
Clinical observation result shows, duck viral hepatitis live vaccine (A66) adopts injection (subcutaneous or muscle) and drinking-water immunity to duckling safety, growth promoter is had no adverse reaction, has good result to the urgent prevention of the duck viral hepatitis of clinical generation.
2. challenge test result
To the test duck group of each batch 5 days Stochastic choice 10 after vaccine immunity, randomly draw 10 with batch nonimmune contrast ducks simultaneously, take back laboratory and carry out challenge test.With the strong malicious W strain of DHV, by every duck 1000LD
50dosage carry out challenge test, result immunity test group (injecting immune and drinking-water immunity) counteracting toxic substances protective rate all reaches more than 80%, 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 vaccine clinical Immunization result of the test is in table 7.
Table 75 batch vaccine clinical test Immunization protection result
3. immune antibody assay
To the test duck group of five duck fields, 10 Sanguis Anas domestica samples are gathered respectively at 7,14,28,42 days Stochastic choice after vaccine immunity by different immunization route, with batch nonimmune matched group 5 Sanguis Anas domestica samples, after separation of serum, get mixing in equivalent group, adopt Embryo Gallus domesticus neutralization test method to measure antibody.Test adopts the method for fixed virus dilute serum, and DHV A66 normal saline is become 200ELD
50/ 0.1ml, mixes with equivalent virus liquid after serum normal saline (containing penicillin and streptomycin each 1000 units/ml) makes doubling dilution, puts 37 DEG C of effect 1h.Each dilution factor inoculates 9 age in days SPF Embryo Gallus domesticus 5 respectively, and every embryonic breeding kind 0.2ml puts 37 DEG C and continues to hatch 120h, calculates duck hepatitis antibody Neutralizing titer by Reed-Muench method.Result shows: duckling accepts the scorching vaccine immunity of duck liver (no matter injecting immune or drinking-water immunity); immunity latter 7 days antibody horizontals are apparently higher than nonimmune duck; after immunity, 4 ~ 6 weeks immune duck group antibody titers maintain plateau level, and duckling can be protected to spend duck virus infection age bracket.Therefore, confirm that duck viral hepatitis live vaccine (A66) has good immunogenicity from the angle of immune neutralizing antibody, each batch of vaccine clinical immune duck group 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 statistics
Statistical summaries is carried out to the fertility performance etc. such as death rate, feedstuff-meat ratio, weightening finish in test duck group clinical trial process in 5 clinical trial duck fields, as can be seen from statistical result, duck viral hepatitis live vaccine (A66) on fertility performance such as death rate, feedstuff-meat ratio, weightening finishes without impact.Each test duck field test duck all living creatures produces performance statistics and the results are shown in Table 9.
Fertility performance statistical summaries result during table 95 batch vaccine clinical test duck group's 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 shown: the duck group of no matter injecting immune or drinking-water immunity has no adverse reaction, duck group mental status, search for food, drink water all normal, the death rate of duck group, 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 proves duck viral hepatitis (A66) live vaccine safety, effectively.
Claims (5)
1. a duck viral hepatitis heat-resisting lyophilized protecting agent of live vaccine, it is characterized in that, prescription is: each composition weight percent by 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: trehalose, glucose, glycine, D-glucitol, polyvinylpyrrolidone, gelatin are dissolved in water for injection after taking mixing in prescription ratio, put 116 DEG C of autoclaving 30min.
2. the application of freeze drying protectant according to claim 1, is characterized in that this protective agent is suitable for the frozen-dried protective of the microRNA Flaviviridae virus to lyophilizing sensitivity.
3. the application of freeze drying protectant according to claim 1, is characterized in that this protective agent is suitable for the application in duck viral hepatitis live vaccine.
4. the application of freeze drying protectant as claimed in claim 3; it is characterized in that the application of described protective agent in duck viral hepatitis live vaccine; be that this freeze drying protectant mixes with the volume ratio of 1:1 with duck viral hepatitis antigen liquid, subpackage, adopt corresponding freeze-drying curve to carry out vacuum lyophilization and namely obtain duck viral hepatitis live vaccine.
5. the application of freeze drying protectant as claimed in claim 4, it is characterized in that adopt freeze-drying curve be: the goods after subpackage insert be chilled to 5 DEG C in advance body of freeze dryer in,-45 DEG C are cooled the temperature to the speed of 0.5 DEG C ~ 1 DEG C/min, maintaining makes vaccine freeze reality in 4 ~ 6 hours, after being evacuated to 5Pa, with the speed of 1 DEG C/min, temperature is risen to 15 DEG C again, maintain 12 ~ 15 hours at this temperature, then be warming up to 25 DEG C with the speed of 0.5 DEG C/min and maintain 2 hours, 32 DEG C are warming up to again with the speed of 0.5 DEG C/min, maintain 6 hours outlets of jumping a queue, lyophilizing whole process is 26 ~ 30 hours, duck viral hepatitis live vaccine every plumage part viral level after lyophilizing answers>=10
4.3lD
50.
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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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| 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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| CN103417980B true CN103417980B (en) | 2015-05-13 |
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| 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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| "保护剂及其在口蹄疫疫苗中的研究进展";李正丰等;《生物技术通报》;20091231(第3期);第6-10页 * |
| 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》.2010,第91-98页. * |
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