CN101612404B - Heat-resisting lyophilized protectant for chicken newcastle disease and chicken infectious bronchitis dual living vaccine - Google Patents
Heat-resisting lyophilized protectant for chicken newcastle disease and chicken infectious bronchitis dual living vaccine Download PDFInfo
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- CN101612404B CN101612404B CN2009101606592A CN200910160659A CN101612404B CN 101612404 B CN101612404 B CN 101612404B CN 2009101606592 A CN2009101606592 A CN 2009101606592A CN 200910160659 A CN200910160659 A CN 200910160659A CN 101612404 B CN101612404 B CN 101612404B
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Abstract
The invention discloses a heat-resisting lyophilized protectant for chicken newcastle disease and chicken infectious bronchitis dual living vaccine and a preparation method thereof. The heat-resisting protectant consists of solution A and solution B in a volume ratio of 1:1, wherein the solution A consists of the following compositions: 4 to 6g of sucrose, 3 to 5g of polypeptone, 1 to 3 g of D-sorbitol and the balance of water for injection in each 100 milliliters of the solution A; and the solution B consists of the following compositions: 3 to 5g of polyvinylpyrrolidone, 0.5 to 1.5g of L-arginine and the balance of water for injection in each 100 milliliters of the solution B. The heat-resisting lyophilized protectant has the advantages of good heat resistance, capacity of effectively protecting the living vaccine, simple formula components and preparation method, low production cost, and mass production and the like. The heat-resisting lyophilized protectant can ensure that the chicken newcastle disease and chicken infectious bronchitis dual living vaccine is preserved for more than 24 months at a temperature of between 2 and 8 DEG C.
Description
Technical field
The present invention relates to a kind of protective agent of viral lived vaccine, relate in particular to heat-resisting lyophilized protecting agent of newcastle disease and infectious bronchitis of chicken bigeminal live vaccine and preparation method thereof, belong to animal and make the field with viral lived vaccine.
Background technology
Freeze drying protectant and preparation technology thereof are key technologies during live vaccine is produced, and preservation, transportation and the use of vaccine had a significant impact.At present, in China's live vaccine, the freeze dried vaccine of living accounts for sizable ratio, and domestic protective agent commonly used is mainly milk, sucrose, gelatin etc., and its prescription is simple, and is poor to the defencive function of vaccine.Adopt the freeze dried vaccine of these traditional protective agents; owing to need be kept at the low temperature below-15 ℃; this puts to no little inconvenience to manufacturer and user; make the long preservation and the transportation of finished product vaccine limited; and may be because vaccine is preserved the improper vaccine inactivation that causes; render a service and reduce, do not reach the epidemic prevention purpose, cause the tremendous economic loss.
Heat-resisting lyophilized protecting agent is compared with the freeze drying protectant of routine; its freeze dried vaccine has the characteristics 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 under higher room temperature, also can preserve tens days, therefore make storage, transportation and the use of live vaccine all more convenient, economical.
The defencive function of freeze drying protectant mainly has very big relation with the factor of two aspects.The one, microbiological factor, the kind of microorganism has determined that with aspects such as itself architectural difference (having or not cyst membrane as virus), cultural method and vaccine kinds protectant prescription of variety classes vaccine also should be different; The 2nd, protectant prescription factor is as protective agent raw material and character thereof, proportioning, freeze-drying curve.The prescription of heat-resisting lyophilized protecting agent will be selected prescription according to above the two principal element, and is reasonably combined, and checking repeatedly, formulates rational freeze-drying curve.After the product lyophilizing, its physical behavior is carried out storage life test, and sample is placed on contrast tests such as carrying out storage life length and titration, safety examination under the different temperatures.According to result of the test, constantly adjust prescription and freeze-drying curve, to reach ideal effect.
The eighties in 20th century, developed country just begins to carry out the research of heat resisting protective, and their freeze-dried live vaccine uses heat resisting protective basically at present, but does not see play-by-play.The protectant development of domestic veterinary biologics relatively lags behind; in recent years also there is the research report of some this respects in China; mainly comprise the heat-resisting lyophilized protecting agent research of newcastle (ND), IBD, MD, IBV, fowlpox and swine fever freeze-dried live vaccine etc.; but do not reach the level of applying yet, at present the domestic report of not seeing newcastle disease, infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent as yet.
Problems such as in a word, it is complicated how existing heat-resisting lyophilized protecting agent exists prescription to some extent, is not easy to operation, and protective value is undesirable.Develop that a kind of compound method is simple, cost is low, 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
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provide that a kind of compound method is simple, cost is low, can large-scale production, the newcastle disease with good protection performance and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent.
Technical problem to be solved by this invention realizes by following technological approaches:
A kind of newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent are made up of according to 1: 1 volume proportion A liquid and B liquid.
Wherein, each constituent of described A liquid is: contain sucrose 4-6g in per 100 ml volumes, and polyprotein peptone 3-5g, D-sorbitol 1-3g, all the other components are water for injection; Preferably: contain sucrose 5g in per 100 ml volumes, polyprotein peptone (Polypeptone) 4g, D-sorbitol 3g, all the other components are water for injection;
Each constituent of described B liquid is: contain polyvinylpyrrolidone (PVPK90) 3-5g in per 100 ml volumes, and L-arginine 0.5-1.5g, all the other components are water for injection; Preferably, the consumption of each component is: contain polyvinylpyrrolidone (PVP K90) 4g in per 100 ml volumes, and L-arginine 1g, all the other components are water for injection.
Another technical problem to be solved by this invention provides the preparation method of above-mentioned newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent.
Another technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of method for preparing above-mentioned newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent comprises:
(1) preparation of A liquid: press described quality proportioning with sucrose, the polyprotein peptone, the D-sorbitol fully is dissolved in back autoclaving in the water for injection, and is standby;
(2) preparation of B liquid: by described quality proportioning polyvinylpyrrolidone (PVP K90) and L-arginine fully are dissolved in after-filtration degerming in the water for injection, standby;
(3) the A liquid for preparing and B liquid are mixed according to 1: 1 volume ratio after, promptly.
Wherein, the autoclaving described in the step 1) is preferably under 116 ℃ temperature and sterilized 20 minutes;
Step 2) aseptic filtration described in preferably adopts the mode of microporous filter membrane to carry out aseptic filtration; Further preferred, described microporous filter membrane is preferably the microporous filter membrane of aperture 0.22 μ m.
The using method of heat-resisting lyophilized protecting agent of the present invention: heat-resisting lyophilized protecting agent of the present invention and newcastle disease and infectious bronchitis of chicken bigeminal live vaccine are joined Seedling with 1: 1 volume ratio, get final product.
Biggest advantage of the present invention is to have solved defectives such as existing heat-resisting lyophilized protecting agent ubiquitous complicated component of institute and cost height; have heat resistance good, can effectively protect newcastle disease and infectious bronchitis of chicken bigeminal live vaccine; formula components and compound method are simple; production cost is low, all can realize advantages such as mass production fully at any biological factory.
The invention solves newcastle disease and infectious bronchitis of chicken bigeminal live vaccine and need a series of problems such as freezing transportation, the inconvenience that stores and cost height.Heat-resisting lyophilized protecting agent of the present invention can make newcastle disease and infectious bronchitis of chicken bigeminal live vaccine under 2～8 ℃ of conditions, preserves more than 24 months.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
The preparation of embodiment 1 heat-resisting lyophilized protecting agent
The preparation of A liquid: sucrose 5g, polyprotein peptone 4g, D-sorbitol 3g adds the injection water to 100ml; 116 ℃ of sterilizations of solution dissolving back high pressure 20 minutes;
The preparation of B liquid: PVP K90 4g, L-arginine 1g adds the injection water to 100ml; Solution dissolving back aperture 0.22 μ m filter aseptic filtration.
With A liquid and B liquid volume ratio mix homogeneously, promptly get heat-resisting lyophilized protecting agent of the present invention according to 1: 1.
The preparation of embodiment 2 heat-resisting lyophilized protecting agents
The preparation of A liquid: sucrose 6g, polyprotein peptone 5g, D-sorbitol 3g adds the injection water to 100ml; 116 ℃ of sterilizations of solution dissolving back high pressure 20 minutes;
The preparation of B liquid: PVP K90 5g, L-arginine 1.5g adds the injection water to 100ml; Solution dissolving back aperture 0.22 μ m filter aseptic filtration.
With A liquid and B liquid volume ratio mix homogeneously, promptly get heat-resisting lyophilized protecting agent of the present invention according to 1: 1.
The preparation of embodiment 3 heat-resisting lyophilized protecting agents
The preparation of A liquid: sucrose 4g, polyprotein peptone 3g, D-sorbitol 1g adds the injection water to 100ml; 116 ℃ of sterilizations of solution dissolving back high pressure 20 minutes;
The preparation of B liquid: PVP K90 3g, L-arginine 0.5g adds the injection water to 100ml; Solution dissolving back aperture 0.22 μ m filter aseptic filtration.
With A liquid and B liquid volume ratio mix homogeneously, promptly get heat-resisting lyophilized protecting agent of the present invention according to 1: 1.
Test example 1 heat-resisting lyophilized protecting agent of the present invention is to the protection potency test of newcastle disease and infectious bronchitis of chicken bigeminal live vaccine
1, test material
(1), newcastle disease poison
The infectious bronchitis of chicken poison is available from China Veterinery Drug Inspection Office
(2), the SPF Embryo Gallus domesticus is available from the Harbin veterinary institute
2, the preparation of experimental vaccine
Experimental vaccine is divided into A, B, C, D, five groups of E.The preparation method of wherein respectively organizing experimental vaccine is as follows:
A, B, C organize experimental vaccine: join Seedling with newcastle disease and infectious bronchitis of chicken bigeminal live vaccine stock solution (being prepared by " veterinary biologics rules " by Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. animal live vaccine workshop) by 1: 1 volume ratio respectively with the heat-resisting lyophilized protecting agent that embodiment of the invention 1-3 is prepared; by rules test preparation vaccine, every component is 01,02,03,04 4 batch.Product is-30 ℃ of cartonnings, and pre-freeze was heated after 4 hours, and the curve overall process amounts to 45 hours, and vacuum remains on 10Pa in sublimation process, and condenser temperature remains on below-60 ℃.
D organizes experimental vaccine: with protective agent (the sucrose 5g of conventional 5% sucrose skimmed milk preparation; defatted milk powder 11g; add the injection water to 100ml) and newcastle disease and infectious bronchitis of chicken bigeminal live vaccine stock solution; in 1.5: 1 ratio test preparations of rules vaccine, be divided into 01,02,03,04 4 batch.Product is-30 ℃ of cartonnings, and pre-freeze was heated after 4 hours, and the curve overall process amounts to 45 hours, and vacuum remains on 10Pa in sublimation process, and condenser temperature remains on below-60 ℃.
E organizes experimental vaccine: join Seedling available from the heat-resisting lyophilized protecting agent of legal person The Chemo-Sero-Therapeutic Research Institute of Japanese interest group and newcastle disease and infectious bronchitis of chicken bigeminal live vaccine stock solution according to 1: 1 ratio, manufacture experimently 1 batch.Product is-30 ℃ of cartonnings, and pre-freeze was heated after 4 hours, and the curve overall process amounts to 45 hours, and vacuum remains on 10Pa in sublimation process, and condenser temperature remains on below-60 ℃.
3, the preservation of experimental vaccine and titration
More than each batch experimental vaccine place respectively under 2～8 ℃ ,-15 ℃ conditions and preserve, detect the experimental vaccine situation of change of tiring in different time.
(1) titration of experimental vaccine: this test is with Embryo Gallus domesticus median infective dose (EID
50) as the standard of weighing vaccine valence.
(2) titration method:, carry out according to " veterinary biologics rules " newcastle disease, the infectious bronchitis of chicken bigeminal live vaccine detection method of tiring according to the real toxic amount of experimental vaccine.
(3) titration standard is Embryo Gallus domesticus median infective dose (EID with viral level
50) weigh vaccine tire every plumage part viral level newcastle disease poison＞10
6.0EID
50The infectious bronchitis of chicken poison answers＞10
3.5EID
50
4, result of the test
(1) experimental vaccine is preserved 12 months, 24 months titration results under 2～8 ℃ of conditions, sees Table 1, table 2.
Table 1 experimental vaccine is preserved 12 months, 24 months (newcastle disease part) titration results under 2～8 ℃ of conditions
Table 2 experimental vaccine is preserved 12 months, 24 months (infectious bronchitis of chicken part) titration results under 2～8 ℃ of conditions
(2) experimental vaccine is preserved 12 months, 24 months titration results under-15 ℃ of conditions, sees Table 3, table 4.
Table 3 experimental vaccine is preserved 12 months, 24 months (newcastle disease part) titration results under-15 ℃ of conditions
Table 4 experimental vaccine is preserved 12 months, 24 months (infectious bronchitis of chicken part) titration results under-15 ℃ of conditions
From above result of the test as can be seen; under condition of different temperatures; heat-resisting lyophilized protecting agent of the present invention is to the protection effect of newcastle disease and infectious bronchitis of chicken bigeminal live vaccine; protective agent (the sucrose 5g that obviously is better than conventional 5% sucrose skimmed milk preparation; defatted milk powder 11g; add the injection water to 100ml) the protection effect, compare with import heat-resisting lyophilized protecting agent vaccine, its protection effect approaching with it.
The result of the test explanation; the newcastle disease, the infectious bronchitis of chicken bigeminal live vaccine heat resistance that adopt heat-resisting lyophilized protecting agent of the present invention to prepare are good, and heat-resisting lyophilized protecting agent of the present invention has the excellent protection effect to newcastle disease and infectious bronchitis of chicken bigeminal live vaccine.
Claims (9)
1. newcastle disease and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent is characterized in that: be made up of according to 1: 1 volume ratio A liquid and B liquid; Wherein, described A liquid is made up of following each component: contain sucrose 4-6g in per 100 ml volumes, and polyprotein peptone 3-5g, D-sorbitol 1-3g, all the other components are water for injection; Described B liquid is made up of following each component: contain polyvinylpyrrolidone 3-5g in per 100 ml volumes, and L-arginine 0.5-1.5g, all the other components are water for injection.
2. according to described newcastle disease of claim 1 and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent; it is characterized in that: described A liquid is made up of following each component: contain sucrose 5g in per 100 ml volumes; polyprotein peptone 4g; D-sorbitol 3g, all the other components are water for injection.
3. according to described newcastle disease of claim 1 and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent; it is characterized in that: described B liquid is made up of following each component: contain polyvinylpyrrolidone 4g in per 100 ml volumes; L-arginine 1.0g, all the other components are water for injection.
4. method for preparing any one described newcastle disease of claim 1-3 and infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent comprises:
(1) preparation of A liquid: press described quality proportioning with sucrose, the polyprotein peptone, the D-sorbitol fully is dissolved in back autoclaving in the water for injection, and is standby;
(2) preparation of B liquid: described quality proportioning fully is dissolved in after-filtration degerming in the water for injection with polyvinylpyrrolidone and L-arginine, standby;
(3) the A liquid for preparing and B liquid are mixed according to 1: 1 volume ratio after, promptly.
5. it is characterized in that in accordance with the method for claim 4: the autoclaving described in the step 1) is sterilization under 116 ℃ temperature 20 minutes.
6. it is characterized in that: step 2 in accordance with the method for claim 4) described in aseptic filtration adopt the mode of microporous filter membrane to carry out aseptic filtration.
7. it is characterized in that in accordance with the method for claim 6: described microporous filter membrane is that the aperture is the microporous filter membrane of 0.22 μ m.
8. any one described newcastle disease of claim 1-3 and the infectious bronchitis of chicken bigeminal live vaccine heat-resisting lyophilized protecting agent application in protection newcastle disease and infectious bronchitis of chicken bigeminal live vaccine.
9. according to the described application of claim 8; it is characterized in that, comprising: any one described newcastle disease of claim 1-3 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 Seedling with 1: 1 volume ratio.
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| CN102908625B (en) * | 2011-08-01 | 2014-04-16 | 普莱柯生物工程股份有限公司 | Chicken Newcastle disease and avian infectious bronchitis bivalent live vaccine heat-resistant protective agent and preparation process thereof |
| CN102309759B (en) * | 2011-09-09 | 2013-02-20 | 乾元浩生物股份有限公司 | Poultry freeze-drying live vaccine heat-resisting protective agent and preparation method thereof |
| CN103550780B (en) * | 2013-10-30 | 2015-03-18 | 郑州邦和生物药业有限公司 | Protein protective agent for Pasteur inactivating human intravenous immunoglobulin and inactivation method of protein protective agent |
| 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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