CN101474401B - Method for producing concentrated inactivate vaccine for newcastle disease - Google Patents
Method for producing concentrated inactivate vaccine for newcastle disease Download PDFInfo
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Abstract
The invention provides a method for producing improved concentrated inactivated vaccine against Newcastle disease. The method comprises the steps of virus inoculation passage, ultrafiltration and concentration, mixing water phase and oil phase and the like, wherein, a 30KD membrane is used for ultra-filtrating and concentrating, and the concentrated water phase and oil phase are mixed and emulsified in the proportion of 1 to 1 in a high pressure emulsification pump. The invention uses ultrafilter membrane concentration and antigen ingredient instead of the content of live virus, so that the chicken can produce strong immunity after being immunized by the vaccine for 10 to 14 days; the immunity period is 10 months; the anti-virus protective rate is 100 percent within the immunity period; and the invention has wide application prospect.
Description
Technical field
What the present invention relates to is a kind of improvement of production technology of vaccine, specifically, is that a kind of newcastle disease concentrates improving one's methods of inactivated vaccine concentration technique.Belong to bioengineering field.
Background technology
(Newcastlediseasevirus NDV) claims philippine fowl disease virus, pseudo-fowl plague virus or avian pneumo-encephalitis virus again to Avian pneumo-encephalitis virus.Position in viral taxonomy belongs to paramyxovirus section (Paramyxoviridae), paramyxovirus belongs to a kind in (Paramyxovirus).This virus main harm chicken, galeeny and turkey, bamboo telegraph in stymied chicken group, virulent strain can make the full group of chicken group destroy.Low virulent strain only causes that chicken group respiratory tract infection and egg production descend, but rehabilitation rapidly.The mankind can cause conjunctivitis or lymphadenitis with the malicious vaccine of living because of contacting disease fowl, but very fast just rehabilitation.Avian pneumo-encephalitis virus is defined as the category-A eqpidemic disease by OIE, it is a kind of acute, height contagious disease, M ﹠ M is all more than 90%, and nineteen thirty-five, beam English, Ma Wentian etc. confirmed that first there is popular [1] of this disease in China, and primary disease is classified as I class eqpidemic disease by China now.
Avian pneumo-encephalitis virus is a ssRNA virus, and peplos is arranged.Virion tool pleomorphism has circle, ellipse and elongated rod shape etc.Sophisticated virion diameter 100～400mn.Peplos is the double-decker film, is combined with viral glycoprotein by the lipid of host cell adventitia and derives.There is the furcella of long 12～15nm on the peplos surface, has hemagglutinin, neuraminidase and hemolysin.The center of virus is ssRNA molecule and protein capsomere attached to it, is wound in the symmetric nucleocapsid of spiral, the about 18nm of diameter.Sophisticated virus is to be released into the extracellular in the mode of sprouting.
The Avian pneumo-encephalitis virus resistance of environment to external world is stronger, and 55 ℃ of effect 45min and direct sunlight effect 30min down just are inactivated.Virus is deposited several weeks in 4 ℃, deposits some months or deposited several years in-70 ℃ in-20 ℃, and its appeal is all unaffected.Break out within 8 weeks of back at newcastle, still can in hen house, egg nest, eggshell and feather, be separated to virus.
Virus is to the ether sensitivity.Most of detergents can be with its rapid deactivation.Alkaline matters such as sodium hydroxide are to its Disinfection Effect instability.Can be in 3%～5% lysol, phenol and the cresol 5min with exposed virion deactivation.In 37 ℃ cultivator, stifling 6h just can be its deactivation with 0.1% formalin.
Institute's toxic strain of Avian pneumo-encephalitis virus can both the multiple birds of coagulation and the erythrocyte of mammal.The erythrocyte of most of strains energy coagulation bulls and sheep.In the hemagglutination test of virus, the erythrocyte of chicken is the most commonly used.
This virus can be cultivated on the chick chorioallantoic membrane of 9～12 ages in days He in the allantoic cavity, and most of strains also can be cultivated in subcultures such as the nephrocyte of rabbit, pig, calf and monkey and chicken histiocyte or passage cell.The nephrocyte of the fibroblast of Embryo Gallus domesticus, Embryo Gallus domesticus and hamster is usually used in the cultivation of Avian pneumo-encephalitis virus.
At present, though the use of vaccine makes this disease obtain effective control, but because immune effect of vaccine is undesirable, and the enhancing of the virulence of epidemic isolates and China's poultry husbandry level is uneven, make this disease remain one of disease of serious harm China aviculture, in China the generation of atypical newcastle disease even the outburst of typical newcastle are often arranged.
Current, the used vaccine of anti-this disease of system mainly contains live vaccine and inactivated vaccine, wherein live vaccine has attenuated vaccine II system, III system, IV system and mesogenic vaccine I system [2], the shortcoming of attenuated vaccine is to break through the interference of maternal antibody, though the mesogenic vaccine can be broken through the influence of maternal antibody, can produce antibody fast, can cause the chicken morbidity, exist the to loose danger of poison it is reported the atypical newcastle disease that can cause chicken.Inactivated vaccine is mainly oil emulsion inactivated vaccine, and it can avoid the influence of maternal antibody, does not have the danger of the poison that looses, and is suitable for the chicken immune of any age in days; But because the virulence of epidemic isolates strengthens at present, make immunizing dose continue to increase, this has brought inconvenience to immunity, and the side reaction of vaccine immunity strengthens, malabsorption or granuloma often appear in inoculation position, have influenced the quality of bird product, have caused certain loss to aviculture.Therefore, present domestic poultry husbandry urgent needs good immune effect, the newcastle disease inactivated vaccine that immunizing dose is little solve above problem.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, the preparation method that provides a kind of newcastle disease to concentrate inactivated vaccine, make its provide little, the immune generation time of side effect early, immunity is strong, duration of immunity is long vaccine.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
1) newcastle disease virus low virulent strain lyophilizing poison is redissolved, in 9 generations of continuous passage inoculated into chick embryo, lyophilizing is preserved respectively, as the basis kind poison of production newcastle disease concentrated type inactivated vaccine with produce with kind of a poison;
2), use 30KD film bag to 5 times of viral ultrafiltration and concentration with after the viral liquid deactivation;
3) water after will concentrating in the high-pressure emulsification pump and oil phase be by mixed emulsifying in 1: 1, newcastle disease concentrate inactivated vaccine, viral level＞10 before the deactivation
6.3EID
50/ 0.1ml.
In a specific embodiments, the newcastle disease virus low virulent strain is LaSota preferably, imitate and examine with the preferably strong malicious Beijing strain of newcastle disease virus (CVCCAV1611 strain) of strong poison, seed culture of viruses is used in the production and the check that are " newcastle disease inactivated vaccine " in existing " People's Republic of China's veterinary drug allusion quotation ", available from China Veterinary Drugs Supervisory Inst..
In a specific embodiments, described 30KD film is available from Millipore company.
In another embodiment, also aseptic 5% tween 80 being added in the deactivation venom before the implementation step 3, be water; Add 6% Si Ben-80 in No. 10 white oils, add 2% aluminium stearate again, the sterilization back is an oil phase.
Ultimate principle of the present invention is that the immune effect of inactivated vaccine depends primarily on the dosage form and the antigenic content of inactivated vaccine except that the influence that is subjected to virus or strain itself.With regard to the dosage form of viral inactivated vaccine, it seems that at present the effect of oil-emulsion inactivated vaccine is the most certain; With regard to antigenic content,, bring into use the antigen concentration technique for guaranteeing unit volume vaccine endoantigen content.General antigen concentration technique mainly contains and concentrates glue concentration method, precipitation concentration method, centrifugal concentration method, dialysis concentration method, freeze-drying, chromatography concentration method and filtration enrichment at present.Because it is low that filtration enrichment has a cost, filter membrane can use repeatedly, filtration yield is big, can carry out to a certain degree advantages such as purification to the antigen composition simultaneously, generally adopted by domestic and international research and Producer, wherein use a lot of abroad manufacturers of Milipore ultrafilter membrane concentration method (concentrated film and the accessory thereof of Miliporee ultrafiltration and concentration machine 30KD are available from U.S. Millipore company) to put into production use, domestic also have many producers to use [3] in research.The inventor introduces external advanced person's ultrafiltration and concentration technology consulting a large amount of pertinent literatures and analyzing from the basis of Avian pneumo-encephalitis virus molecular size, and adopting the molecular weight that dams is that the Milipore ultrafilter membrane of 30KD carries out the concentrated of NDV Embryo Gallus domesticus liquid.This technology can be handled number 10L antigen liquid in 1h, for large-scale production provides convenience.Remove the part foreign protein quilt in the vaccine simultaneously, increased effective antigenic content in the vaccine, made to produce efficient immune fast after the immunity, improved the efficient of immunne response.
Beneficial effect
The present invention adopts the newcastle disease strain of standard, through the Embryo Gallus domesticus cultivation of going down to posterity, makes oil emulsion vaccine after adopting Milipore ultrafilter membrane concentration method to concentrate deactivation, the film of evidence 30KD can with in the viral blastochyle all the antigen compositions hold back;
The concentrated effect of ultrafiltration and concentration film has good stability, and antigenic HA tires and becomes positive correlation with cycles of concentration after concentrating;
Can tire by the method raising that strengthens cycles of concentration to the blastochyle on the low side of tiring in the production.Can require to determine cycles of concentration according to goods in the actual production by simple and rapid HA test;
To inactivated vaccine, what the inventor used mainly is its antigen composition, rather than the content of live virus, therefore should tire to detect HA when antigenic content after concentrating is measured.And easy, quick, the easy row of HA titration also is convenient to carry out aborning;
Use Millipore ultrafiltration and concentration membrance concentration antigen, effective, stability is strong, and the production that is suitable for certain scale is used;
With producing strong immunity in 10～14 days behind the vaccine immune chicken, counteracting toxic substances protective rate 100% in the duration of immunity 10 months, duration of immunity.
The specific embodiment
The present invention will be described further in conjunction with specific embodiments, and these examples only are used for illustration purpose, and are not used in the restriction scope of the invention.
Embodiment 1
The weak malicious LaSota strain of newcastle disease virus is as seed culture of viruses, inoculate 10 age in days SPF Embryo Gallus domesticus, death in 72-120 hour of results inoculation back and the tangible Embryo Gallus domesticus liquid of lesion, the employing molecular weight that dams is that the Milipore ultrafilter membrane of 30KD carries out concentrating of NDV Embryo Gallus domesticus liquid, concentrates the back and adds formalin solution and makes content reach 0.2%.Shake up back 37 ℃ of deactivations 16 hours.Before deactivation, carry out the mensuration of viral level, viral level＞10 by ministry standard
8.8EID
50/ 0.1ml.Get inactivation of viruses liquid and be inoculated in 6 of SPF Embryo Gallus domesticus, observed 5, the non-specific death of Embryo Gallus domesticus is no more than 1.All blastochyles are measured the blood clotting valency respectively, standby when all blood clotting not occurring.In the deactivation venom, add aseptic 5% tween 80, be water; Add 6% Si Ben-80 in No. 10 white oils, add 2% aluminium stearate again, the sterilization back is an oil phase.In the high-pressure emulsification pump with water and oil phase by mixed emulsifying in 1: 1, newcastle disease concentrate inactivated vaccine, viral level＞10
8.8EID
50/ 0.1ml.
This vaccine shows through property determination and safety testing, for the Water-In-Oil type, has good stability, and modest viscosity, safe and reliable.This vaccine is preserved at 4 ℃ and was not occurred layering in 12 months.Before measuring it simultaneously and concentrating and the EID50 after concentrating from 10
6.31EID
50/ 0.1ml is elevated to 10
8.84EID
50/ 0.1ml; The blood clotting valency is from 2
9Rise to 2
11Test chicken is inoculated behind this vaccine 10～14 days and is produced strong immunity, and the counteracting toxic substances protective rate in the duration of immunity 10 months, duration of immunity is 100%.
Embodiment 2
10 age in days SPF Embryo Gallus domesticus are inoculated in the weak malicious LaSota strain of newcastle disease virus, death in 72-120 hour of results inoculation back and the tangible Embryo Gallus domesticus liquid of lesion, the employing molecular weight that dams is that the Milipore ultrafilter membrane of 30KD carries out concentrating of NDV Embryo Gallus domesticus liquid, concentrates the back and adds formalin solution and makes content reach 0.2%.Shake up back 37 ℃ of deactivations 16 hours.Viral liquid after the deactivation is done the deactivation check, and deactivation venom completely is standby.In the deactivation venom, add aseptic 5% tween 80, be water; Add 6% Si Ben-80 in No. 10 white oils, add 2% aluminium stearate again, the sterilization back is an oil phase.In the high-pressure emulsification pump with water and oil phase by mixed emulsifying in 1: 1, newcastle disease concentrate inactivated vaccine, viral level＞10
8.8EID
50/ 0.1ml.
This Seedling is applicable to the immunity inoculation of each poultry-farm to birds, and is safe and reliable.Produced strong immunity in 10～14 days behind the vaccine immune chicken, the counteracting toxic substances protective rate in the duration of immunity 10 months, duration of immunity is 100%.
Claims (2)
1. a newcastle disease concentrates the production method of inactivated vaccine, may further comprise the steps:
1) newcastle disease virus low virulent strain LaSota lyophilizing poison is redissolved, in 9 generations of continuous passage inoculated into chick embryo, lyophilizing is preserved respectively, as the basis kind poison of production newcastle disease concentrated type inactivated vaccine with produce with kind of a poison;
2), use 30KD film bag to 5 times of viral ultrafiltration and concentration with after the viral liquid deactivation;
3) add aseptic 5% tween 80 in the venom after deactivation, be water, in No. 10 white oils, add 6% Si Ben-80, add 2% aluminium stearate again, the sterilization back is an oil phase, water after will concentrating in the high-pressure emulsification pump then and oil phase be by mixed emulsifying in 1: 1, newcastle disease concentrate inactivated vaccine, viral level＞10 before the deactivation
6.3EID
50/ 0.1ml.
2. the described method of claim 1, wherein to imitate inspection be the strong malicious Beijing strain CVCCAV1611 of newcastle disease virus strain with strong poison to vaccine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834620A (en) * | 2013-07-19 | 2014-06-04 | 北京中联康生物科技有限公司 | Newcastle disease virus, newcastle disease inactivated vaccine and preparation method of newcastle disease inactivated vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103013928A (en) * | 2010-06-04 | 2013-04-03 | 上海市动物疫病预防控制中心 | H9N2 avian influenza virus and application thereof |
| CN102600465A (en) * | 2010-12-28 | 2012-07-25 | 华威特（北京）生物科技有限公司 | Newcastle disease (ND) vaccine, and its production method |
| CN102352345B (en) * | 2011-08-19 | 2013-05-22 | 北京市兽医生物药品厂 | Preparation method of newcastle disease HI antigen |
| CN102978166A (en) * | 2011-11-16 | 2013-03-20 | 普莱柯生物工程股份有限公司 | Method for preparing newcastle disease viruses and vaccines by using serial passage cells, and products thereof |
| CN103599532A (en) * | 2013-09-23 | 2014-02-26 | 天津瑞普生物技术股份有限公司 | Chicken infectious bronchitis divalent live vaccine and preparation method thereof |
| CN103611156A (en) * | 2013-09-23 | 2014-03-05 | 天津瑞普生物技术股份有限公司 | Avian pentavalent vaccine and preparation method thereof |
| CN104031888B (en) * | 2014-03-26 | 2016-04-06 | 东北林业大学 | Newcastle disease virus low virulent strain, inactivated vaccine and application thereof |
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2008
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Non-Patent Citations (1)
| Title |
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| 孙德君 姜力 潘传毅 毛春玲 邬立权.鸡新城疫（LaSota株）浓缩油乳剂灭活疫苗的研究.《黑龙江畜牧兽医》.2007,(第4期),93. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834620A (en) * | 2013-07-19 | 2014-06-04 | 北京中联康生物科技有限公司 | Newcastle disease virus, newcastle disease inactivated vaccine and preparation method of newcastle disease inactivated vaccine |
| CN103834620B (en) * | 2013-07-19 | 2016-05-18 | 北京中联康生物科技股份有限公司 | NDV, ewcastle disease inactivated vaccine and preparation method thereof |
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