CN102908625B - Chicken Newcastle disease and avian infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation process thereof - Google Patents
Chicken Newcastle disease and avian infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation process thereof Download PDFInfo
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Abstract
The invention discloses a chicken Newcastle disease and avian infectious bronchitis bivalent live vaccine heat-resistant lyophilization protective agent and a preparation process thereof. The heat-resistant lyophilization protective agent is composed of a liquid A and a liquid B according to a volume ratio of 1:1. The liquid A is composed of: 4-6g of L-sodium glutamate, 4-6g of sucrose, 1-3g of D-sorbitol, 2-4g of sodium thiosulfate, 0.1-0.3g of vitamin C, and injection water for complementing the liquid to 100mL. The liquid B is composed of: 4-6g of polyvinyl pyrrolidone, and injection water for complementing the liquid to 100mL. The protective agent provided by the invention has the advantages of simple preparation method, suitability for large-scale production, lower energy consumption, and lower cost. With the protective agent, a lyophilization time is reduced to 28h or shorter, such that application efficiency is greatly improved. The protective agent has good protection performance upon a chicken Newcastle disease and avian infectious bronchitis bivalent live vaccine. With the protective agent, the vaccine can be preserved for mother than 24 months under a temperature of 2-8 DEG C.
Description
Technical field
The present invention relates to a kind of heat resisting protective of animal virus live vaccine, refer to especially a kind of newcastle disease, infectious bronchitis of chicken bigeminal live vaccine heat resisting protective and preparation technology thereof.
Background technology
Freeze drying protectant and preparation technology thereof are key technologies during live vaccine is produced, and the preservation of vaccine, transportation and use are had a significant impact.At present, in China's live vaccine, the freeze dried vaccine of living accounts for sizable ratio, and domestic conventional protective agent is mainly milk, sucrose, gelatin etc., and its prescription is simple, poor to the defencive function of vaccine.Adopt the vaccine of these traditional protective agent lyophilizing; conventionally require goods to be kept at below-15 ℃; Zhe Gei manufacturer and user cause very large inconvenience; the long-term storage and transport that are vaccine are limited; and may preserve improperly due to vaccine, cause vaccine inactivation, effect reduces; do not reach epidemic prevention object, cause tremendous economic loss.
Heat-resisting lyophilized protecting agent is compared with conventional freeze drying protectant; freeze dried vaccine has the feature of high temperature resistant long shelf-life; thereby the performance of heat resisting protective is more better than traditional protection agent; live vaccine can be kept under 2～8 ℃ of conditions before the deadline; even also can preserve tens days under higher room temperature, therefore make storage, transportation and the use of live vaccine all more convenient, economical.
To the research starting of heat resisting protective early, their freeze-dried live vaccine is all used heat resisting protective substantially at present, but has no play-by-play in developed country.The protectant development of domestic veterinary biologics relatively lags behind; in recent years also there are some relevant research reports in China, mainly comprises the heat-resisting lyophilized protecting agent research of newcastle disease, chicken Marek's disease, chicken bursal disease, infectious bronchitis of chicken, fowlpox and swine fever freeze-dried live vaccine etc.At present existing Chinese patent CN 1772302 A heat resistant freeze drying protectant for chicken Newcastle disease live vaccine, its preparation method and application, used gelatin, lactose, arginine, sorbitol as protective agent.Chinese patent CN101612404A discloses the heat-resisting lyophilized protecting agent of newcastle disease and infectious bronchitis of chicken bigeminal live vaccine, at 2～8 ℃, can preserve 24 months.But in these patents, all deposit the in use long problem of freeze-drying time, affected the propagation and employment of heat resisting protective.Therefore develop a kind of compound method simple, cost is low, freeze-drying time is short, can large-scale production, the newcastle disease with good protection performance, infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent just seem particularly urgently important.
Summary of the invention
In view of this, main purpose of the present invention is to provide a kind of newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, A liquid and B liquid, according to the volume ratio of 1: 1, consists of; Wherein, described A liquid is comprised of following component: Pidolidone sodium 4～6g, and sucrose 4～6g, D-glucitol 1～3g, sodium thiosulfate 2～4g, vitamin C 0.1～0.3g, mends to 100mL with water for injection; Described B liquid is comprised of following component: polyvinylpyrrolidone 4～6g, mends to 100mL with water for injection.
Preferably; in newcastle disease of the present invention and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent; described A liquid is comprised of following component: the constituent of A liquid is: Pidolidone sodium 4g; sucrose 5g; D-glucitol 2g; sodium thiosulfate 2g, vitamin C 0.2g, mends to 100mL with water for injection.
Preferably, in newcastle disease of the present invention and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, described A liquid is comprised of following component: Pidolidone sodium 6g; sucrose 6g, D-glucitol 3g, sodium thiosulfate 3g; vitamin C 0.3g, mends to 100mL with water for injection.
In newcastle disease of the present invention and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, described B liquid is comprised of following component: polyvinylpyrrolidone 4g, mends to 100mL with water for injection.
Preferably, in newcastle disease of the present invention and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, described B liquid is comprised of following component: polyvinylpyrrolidone 6g, mends to 100mL with water for injection.
The compound method that another object of the present invention is to provide a kind of newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, comprises the following steps:
(1) preparation of A liquid: press described quality proportioning by Pidolidone sodium, sucrose, D-glucitol, sodium thiosulfate, filtration sterilization after vitamin C is fully dissolved in water for injection;
(2) preparation of B liquid: autoclaving after described quality proportioning is fully dissolved in water for injection by polyvinylpyrrolidone;
(3) after the A liquid preparing and B liquid are mixed according to the volume ratio of 1: 1, obtain.
Preferably, the aseptic filtration in the preparation method of newcastle disease of the present invention and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, step 1) adopts 0.22 μ m filtering with microporous membrane degerming.
Preferably, the autoclaving in the preparation method of newcastle disease of the present invention and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, step 2) is sterilizing 30 minutes at the temperature of 116 ℃.
The present invention also provides a kind of newcastle disease and the application of infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent in preparation newcastle disease and the heat-resisting dosage form of infectious bronchitis of chicken bigeminal live vaccine.
Preferably; the application process of newcastle disease of the present invention and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent is: newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent and newcastle disease and infectious bronchitis of chicken bigeminal live vaccine are joined to Seedling with the volume ratio of 1: 1, carry out lyophilization after subpackage.
More preferably, in the application process of newcastle disease of the present invention and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, the described cryodesiccated time is less than 28 hours.
As seen from the above, applicant adjusts the formula of heat-resisting lyophilized protecting agent by great many of experiments, solved freeze drying protectant oversize problem consuming time in step of freeze drying, under the prerequisite of heat-resisting protective effect that guarantees freeze drying protectant, obtained a kind of compound method simple, energy consumption is lower, cost still less can large-scale production, the newcastle disease with good protection performance, infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, particularly freeze-drying time is reduced in 28 hours, that has greatly improved heat-resisting lyophilized protecting agent is protecting newcastle disease, service efficiency in infectious bronchitis of chicken bigeminal live vaccine.
The specific embodiment
With specific embodiment, be described in further detail technical scheme of the present invention below.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall in the protection domain of Ben Fanming.
embodiment 1the preparation of newcastle disease, infectious bronchitis of chicken heat-resisting lyophilized protecting agent
Formula 1:
The constituent of A liquid is: Pidolidone sodium 4g, and sucrose 5g, D-glucitol 2g, sodium thiosulfate 2g, vitamin C 0.2g, mends to 100mL with water for injection after dissolving completely.Through 0.22 μ m membrane filtration degerming, standby.
The constituent of B liquid is: polyvinylpyrrolidone 4g, mends to 100mL with water for injection after dissolving completely.116 ℃ of autoclavings 30 minutes, standby.
A liquid and B liquid mix according to the volume proportion of 1: 1, are the heat-resisting lyophilized protecting agent of formula 1.
Formula 2:
The constituent of A liquid is: Pidolidone sodium 5g, and sucrose 4g, D-glucitol 1g, sodium thiosulfate 4g, vitamin C 0.1g, mends to 100mL with water for injection after dissolving completely.Through 0.22 μ m membrane filtration degerming, standby.
The constituent of B liquid is:: polyvinylpyrrolidone 5g, mends to 100mL with water for injection after dissolving completely.116 ℃ of autoclavings 30 minutes, standby.
A liquid and B liquid mix according to the volume proportion of 1: 1, are the heat-resisting lyophilized protecting agent of formula 2.
Formula 3:
The constituent of A liquid is: Pidolidone sodium 6g, sucrose 6g, D-glucitol 3g, sodium thiosulfate 3g, vitamin C 0.3g, be finished CL after water for injection mend to 100mL.Through 0.22 μ m membrane filtration degerming, standby.
The constituent of B liquid is: polyvinylpyrrolidone 6g, mends to 100mL with water for injection after dissolving completely.116 ℃ of autoclavings 30 minutes, standby.
A liquid and B liquid mix according to the volume proportion of 1: 1, are the heat-resisting lyophilized protecting agent of formula 3.
embodiment 2the heat-resisting lyophilized protecting agent of newcastle disease, infectious bronchitis of chicken bigeminal live vaccine join Seedling and protection potency test
1. test material and reagent
1.1 strain
Newcastle disease virus Lasota strain is purchased from China Veterinery Drug Inspection Office.
Avian infectious bronchitis virus H120 strain, H52 strain are purchased from China Veterinery Drug Inspection Office.
1.2SPF egg is purchased from Beijing Cimmeria laboratory animal technology company limited.
The all reagent of 1.3 reagent the present invention is purchased from Sigma company.
2. the setting of joining Seedling and lyophilization program of test group bigeminal live vaccine and freeze drying protectant
According to the freeze drying protectant of three formulas of embodiment 1, join after Seedling by 1: 1 volume ratio with newcastle disease, infectious bronchitis of chicken live vaccine respectively; aseptic subpackaged in cillin bottle; (1 the lyophilized preparation of filling a prescription is A to be respectively tri-groups of A, B, C; the lyophilized preparation of formula 2 is B; the lyophilized preparation of formula 3 is C), test respectively 01,02,03 3 batch for every group.Cartonning under-40 ℃ of conditions, goods this temperature pre-freeze after 3 hours, start vacuum pump, start heater after reaching max vacuum, and primary drying Temperature Setting is-10～-5 ℃, and the used time is in 18 hours; Redrying Temperature Setting is 25 ℃, and the used time is in 5 hours.Therefore, secondary freeze-drying time is only 23 hours.If adjust temperature-time in the middle of counting in, whole lyophilization cycle is also no more than 28h.
3. test group goods technical parameter and the method for inspection
The physical behavior of 3.1 products, steriling test, mycoplasma check, exogenous virus check, specificity check, safety verification, residual moisture are measured and vacuum mensuration is undertaken by method and the standard of the regulations > > of the < < People's Republic of China (PRC) (2005 editions).
3.2 viral level assay method and standards: using Embryo Gallus domesticus median infective dose (EID50) as the standard of weighing vaccine valence.Titration method is carried out according to the regulations > > of the < < People's Republic of China (PRC) (2005 editions) newcastle disease, infectious bronchitis of chicken bigeminal live vaccine titer detection method.
(1) standard of the weak malicious freeze-dried vaccine of newcastle disease:
A. after lyophilizing, virus loss is less than 0.3 titre.
B. every plumage part viral level>=10 after lyophilizing
7.0eID
50.
C. after lyophilizing, goods are put in 37 ℃ and preserve 10d and carry out viral level mensuration, and viral level declines in 1 titre.
D. after lyophilizing, goods are put in 2～8 ℃ of preservations 24 months, every plumage part viral level>=10
6.2eID
50.
(2) standard of the weak malicious freeze-dried vaccine of infectious bronchitis of chicken:
A. after lyophilizing, virus loss is less than 0.3 titre.
B. every plumage part viral level>=10 after lyophilizing
4.5eID
50.
C. after lyophilizing, goods are put in 37 ℃ and preserve 10d and carry out viral level mensuration, and viral level declines in 1 titre.
D. after lyophilizing, goods are put in 2～8 ℃ of preservations 24 months, every plumage part viral level>=10
3.6eID
50.
4. result of the test and comparison
Table 1.37 ℃ is preserved titration (EID50/ plumage part) newcastle part
Table 237 ℃ is preserved titration (EID50/ plumage part) infectious bronchitis part
Titration (EID50/ plumage part) newcastle part is preserved in table 32～8 ℃
Titration (EID50/ plumage) infectious bronchitis of chicken part is preserved in table 52～8 ℃
From result of the test, show; under 2～8 ℃ of preservation conditions; heat-resisting lyophilized protecting agent of the present invention is suitable to the protection effect of the protection effect of newcastle disease, infectious bronchitis of chicken live vaccine and existing heat-resisting lyophilized protecting agent (patent CN101612404A); but use this protective agent greatly to shorten freeze-drying time; lyophilization cycle, in 28 hours, has improved production efficiency.
Result of the test explanation, adopts heat-resisting lyophilized protecting agent of the present invention to have good protection effect to newcastle, infectious bronchitis of chicken live vaccine.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (11)
1. newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, is characterized in that: A liquid and B liquid, according to the volume ratio of 1: 1, consist of; Wherein, described A liquid is comprised of following component: Pidolidone sodium 4～6g, and sucrose 4～6g, D-glucitol 1～3g, sodium thiosulfate 2～4g, vitamin C 0.1～0.3g, mends to 100mL with water for injection; Described B liquid is comprised of following component: polyvinylpyrrolidone 4～6g, mends to 100mL with water for injection.
2. newcastle disease according to claim 1 and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent; it is characterized in that: described A liquid is comprised of following component: Pidolidone sodium 4g; sucrose 5g; D-glucitol 2g; sodium thiosulfate 2g; vitamin C 0.2g, mends to 100mL with water for injection.
3. newcastle disease according to claim 1 and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent; it is characterized in that: described A liquid is comprised of following component: Pidolidone sodium 6g; sucrose 6g; D-glucitol 3g; sodium thiosulfate 3g; vitamin C 0.3g, mends to 100mL with water for injection.
4. newcastle disease according to claim 1 and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, is characterized in that: described B liquid is comprised of following component: polyvinylpyrrolidone 4g, mends to 100mL with water for injection.
5. newcastle disease according to claim 1 and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent, is characterized in that: described B liquid is comprised of following component: polyvinylpyrrolidone 6g, mends to 100mL with water for injection.
6. a method of preparing claim 1-5 newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent described in any one, comprising:
(1) preparation of A liquid: press described quality proportioning by Pidolidone sodium, sucrose, D-glucitol, sodium thiosulfate, filtration sterilization after vitamin C is fully dissolved in water for injection;
(2) preparation of B liquid: autoclaving after polyvinylpyrrolidone being fully dissolved in water for injection by described quality proportioning;
(3) after the A liquid preparing and B liquid are mixed according to the volume ratio of 1: 1, obtain.
7. method according to claim 6, is characterized in that: the filtration sterilization described in step (1) adopts 0.22 μ m microporous filter membrane.
8. method according to claim 6, is characterized in that: the autoclaving described in step (2) is sterilizing at the temperature of 116 ℃ 30 minutes.
9. the newcastle disease described in claim 1-5 any one and the infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent application in preparation newcastle disease and the heat-resisting dosage form of infectious bronchitis of chicken bigeminal live vaccine.
10. application according to claim 9; it is characterized in that; newcastle disease described in claim 1-5 any one and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent and newcastle disease and infectious bronchitis of chicken bigeminal live vaccine are joined to Seedling with the volume ratio of 1: 1, after subpackage, carry out lyophilization.
11. application according to claim 10, is characterized in that, the described cryodesiccated time is less than 28 hours.
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| CN104367996B (en) * | 2013-08-15 | 2017-09-05 | 普莱柯生物工程股份有限公司 | A kind of method and its product that pseudorabies disease live-vaccine is produced with subculture cell source |
| CN111184871A (en) * | 2020-03-23 | 2020-05-22 | 辽宁益康生物股份有限公司 | Heat-resistant protective agent for newcastle disease and bronchitis combined live vaccine and preparation process thereof |
| CN114042167B (en) * | 2021-12-08 | 2022-10-21 | 山东滨州博莱威生物技术有限公司 | Newcastle disease and infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation method and application thereof |
| CN116440282B (en) * | 2023-05-09 | 2023-10-20 | 中海生物制药(泰州)有限公司 | Freeze-drying protective agent for live vaccine of infectious bronchitis and preparation method thereof |
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| CN1528458A (en) * | 2001-12-21 | 2004-09-15 | 卫广森 | Heat-resisting lyophilized protectant for chicken newcastle disease, chicken infectious bronchitis (H120, H52) dual lyophilized vaccine and preparing process thereof |
| CN101612404A (en) * | 2009-07-29 | 2009-12-30 | 哈药集团生物疫苗有限公司 | Newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent |
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| CN1528458A (en) * | 2001-12-21 | 2004-09-15 | 卫广森 | Heat-resisting lyophilized protectant for chicken newcastle disease, chicken infectious bronchitis (H120, H52) dual lyophilized vaccine and preparing process thereof |
| CN101612404A (en) * | 2009-07-29 | 2009-12-30 | 哈药集团生物疫苗有限公司 | Newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent |
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