CN100516206C - Reovirus vaccine and its preparing method - Google Patents
Reovirus vaccine and its preparing method Download PDFInfo
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- CN100516206C CN100516206C CNB2006100127915A CN200610012791A CN100516206C CN 100516206 C CN100516206 C CN 100516206C CN B2006100127915 A CNB2006100127915 A CN B2006100127915A CN 200610012791 A CN200610012791 A CN 200610012791A CN 100516206 C CN100516206 C CN 100516206C
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Abstract
A reovirus vaccine and its method for preparation belong to the technical sphere of druggist sundries and are used to solve the indigenous fowl reovirus infection vaccine problem. The separated reovirus is of CGMCC No.1713 as its preserved number and has been preserved in China microbial bacterial preservation management committee common microorganism center on May 16th, 2006. The said vaccine is prepared by steps including virus isolation from organ of diseased chicken, serum check, virus breeding and generation and cryodesiccation. The invention separates the virus from the diseased chicken with ARV infection of indigenous fowls, applies them in immune chicken, and produces immune response within 1 week and produces strong immunity within 2 weeks. The immune time is 2-2.5 months, and the detoxicating protecting rate during immune period is 94.8%-98.8%, which is explicitly higher than import vaccine protecting rate and can make up for the domestic defect of vaccine vacancy in reovirus affection.
Description
Technical field
The present invention relates to a kind of virus vaccines and preparation method, specifically, is the preparation method of a kind of reovirus and vaccine, belongs to the medical product technical field.
Background technology
(Avianreovirus ARV) is one of the main pathogen of chicken viral sacroiliitis and tenosynovitis to Avianreovirus, belongs to Reoviridae, the member that reovirus belongs to.Since Fahey and Grawly (1954) were separated to (ARV) from the chicken respiratory of suffering from chronic respiratory tract disease first, many ARV strain isolateds were in the news in succession.Along with carrying out in a deep going way of research, the relation of ARV and other disease is also paid close attention to by people.Have been found that except that viral arthritis and tenosynovitis, ARV plays a part to a certain degree in the generation of diseases such as respiratory tract disease, enteron aisle disease, the sick damage of conscience, blue wing disease, the short and small syndromes of fryer, temporary Digestive tract disorder, all can be separated to ARV in the chicken body of suffering from these diseases.ARV worldwide exists, and is widely current in chicken, turkey, and other bird infects ARVS also report.Loss economically often is that feed conversion rate is low owing to sacroiliitis, tenosynovitis cause discarded rate height and growth-inhibiting.Flourish along with aviculture, the influence of ARV is increasing, and its disease incidence that causes rises, and complicacy increases.At present, the main means of control fowl group ARV morbidity are to use attenuated live vaccine or inactivated whole virus vaccines to control typical chicken reovirus infection by immunization.But because this virus is RNA viruses, a plurality of segmentations are arranged, ubiquity in commodity fowl group, between different strains in antigenic structure, pathogenic, cell cultures characteristic, have certain difference on to trypsinase susceptibility and host specificity.When genetic evolution, morph easily, the difference between two kinds of viruses of different zones is bigger.Use external vaccine for the native country should virus prevention and treatment be not very desirable, and price is very high.Therefore, at this viral serotype of the specific existence of China, the vaccine of the former unexistent special advantage of Development and Production can play a positive role to control native country fowl group ARV morbidity undoubtedly, and can produce favorable economic benefit.
Summary of the invention
Technical problem to be solved by this invention is: provide that a kind of immunity produces rapidly, longer duration, immunizing power are strong, at the reovirus vaccine and the preparation method of native country fowl group viral arthritis disease.
The alleged problem of the present invention is solved by following technical scheme:
A kind of reovirus, described virus were Avianreovirus (Avianreovirus, be called for short ARV), and strain is the RS13 strain, and preserving number is CGMCC No.1713, were deposited in Chinese microbial preservation management committee common micro-organisms center on May 16th, 2006.
Above-mentioned reovirus, the no cyst membrane of described virus is 20 body symmetries, and genome is made of 10 double-stranded RNA segmentations, can tolerate 60 ℃ of 8～10h, insensitive to ether, to the chloroform slight sensitive, to the alkali sensitivity, 3% sodium hydroxide effect 1h can kill virus, no blood clotting characteristic.
A kind of reovirus vaccine preparation method, it comprises the steps:
A. isolated viral: at position isolated virals such as the ight soil of the sick chicken that suffers from the viral arthritis disease, cloaca swab, tracheae, stndon sheaths, wash with the PBS damping fluid, filter, multigelation 3～5 times, discharge virus, go down to posterity breeding during virus is on chicken embryo or chick embryo fibroblast, make virus titer reach 10
9.5ELD
50/ 0.2ml uses viral seed as producing;
B. serum detects: with in the S1133 positive serum and after, inoculate in 10 age in days SPF chicken embryos or the chick embryo fibroblast, corresponding chicken idiosome and internal organ pathology or corresponding cytopathy do not appear, confirm that this viral serotype is identical with S1133;
C. viral proliferation: seed culture of viruses is gone down to posterity by chicken embryo or chick embryo fibroblast breeding, and virus titer detects and reaches 10
9.5ELD
50/ 0.2ml;
D. freeze-dried mixed: with viral dilution to 10
6.5ELD50/ml adds 5% milk powder protective material and viral liquid according to 1: 1.2 ratio, and the reovirus freeze-dried live vaccine is made in freeze-drying.
Above-mentioned reovirus vaccine preparation method, described viral proliferation went down to posterity for 50～70 generations.
The present invention isolates reovirus from native country fowl group infects the sick chicken of ARV; the reovirus that separation is obtained goes down to posterity on the chicken embryo; make freeze-dried live vaccine; be applied to immune chicken; produce immunne response in 1 week, can produce strong immunizing power in 2 weeks, duration of immunity is 2～2.5 months; attacking malicious protection ratio between duration of immunity is 94.8%～98.8%, apparently higher than external import vaccine protection ratio.This vaccine to China should virus prevention and treatment be even more ideal selection, be adapted to domestic each poultry-farm, safe and reliable, has very big actual application value, can remedy at present domestic aspect the reovirus infection defective of vaccine vacancy, for the control of this disease provide a kind of can practicable vaccine and preparation method.
Embodiment
Vaccine of the present invention is at native country reovirus RS13 strain, this virus does not have cyst membrane, is 20 body symmetries, and genome is made of 10 double-stranded RNA segmentations, heat there is resistibility, can tolerate 60 ℃ of 8～10h, insensitive to ether, to the chloroform slight sensitive, to the alkali sensitivity, 3% sodium hydroxide effect 1h can kill virus, and this virus does not have the blood clotting characteristic.
This virus is grown in the chicken embryo easily, can select yolk sac inoculation for use when separating for the first time, general 3～5 days chicken embryo deaths after inoculation, and the chicken embryo is purple because of extensive subcutaneous hemorrhage.Chorioallantoic membrane when inoculation, death in common 7～8 days, chorioallantoic membrane have protuberance slightly, dispersive white focus, chicken embryo mild dysplasia.
This virus also can be bred on chick embryo fibroblast, but needs to adapt to, and promptly adherent cell 1h often forms synplasm, comes across 24～48 hours the earliest, occurs the cavity after the monolayer cell sex change subsequently, and giant cells is suspended in the substratum.
Below provide the embodiment of preparation vaccine:
A. isolated viral: isolated viral from the stndon sheath of sick chicken, the stndon sheath (if other sick chicken is then extracted at other diseased region) of sick chicken is blended, use 5ml PBS (phosphate buffered saline buffer) dilution, add mycillin in the diluent, to eliminate other living contaminantses.Multigelation 5 times discharges virus from tissue.Centrifugal 20 minutes of 3000 commentaries on classics/min remove tissue block.Contain virus in the supernatant liquor, be the virus that is separated to.This virus was bred for 70 generations on the chicken embryo continuously, detect virus titer, reach 10
9.5ELD
50/ 0.2ml gets the 70th and withholds and obtain virus, uses viral seed as producing.
The detection of tiring of reovirus, its detection method is TCID
50(cell median infective dose) and ELD
50(chicken embryo medium lethal dose).The chicken embryo goes down to posterity virus 10
-8With 10
-9The pathology hole occurs and be respectively 5 holes and 2 holes, i.e. TCID
50=10
-8.9/ 0.1ml, chicken embryo go down to posterity virus 10
-9Gradient is all dead, i.e. ELD50=10
-9.5/ 0.2ml.
B. serum detects: with in the S1133 positive serum and after, inoculate 10 age in days SPF (specific pathogen free microorganism) chicken embryo, corresponding chicken idiosome and internal organ pathology do not appear, confirm that this viral serotype is identical with S1133.
C. viral proliferation: with seed culture of viruses by the breeding of chicken embryo, get 20 pieces of well-developed SPF chicken embryos, 0.2ml/ piece injection, extract artificial air chamber near chicken embryo great vessels out, the virus of (part by weight, i.e. g/ml) is injected in the artificial air chamber will to be diluted to 1: 10000 with the PBS damping fluid, 37.5 ℃～38.5 ℃ of conditions cultivations, gather in the crops dead chicken embryo and the chorioallantoic membrane that the live chickens embryo of pathology is arranged after 24 hours, 1: 10 dilution proportion ,-20 ℃ are frozen standby.Continuous passage.
In the test that the present invention carries out, when virus through 70 generations of continuous passage, virulence weakens during obviously than separation just.The virus that goes down to posterity from the detection of tiring of the virus in continuous 20 generations in the 50th generation to 70 generations, all can be reached very high numerical value, promptly 10
9.5ELD
50About/0.2ml, illustrate that this virus has adapted in the chicken embryo to breed, and highly stable.From virulence attenuation of, stable, the safety, effective of virus multiplication, prove that this virus is adapted at producing large-scale application.Simultaneously, also conclusion unanimity therewith of the result of study of clinical trial.
D. freeze-dried mixed: with viral dilution to 10
6.5ELD50/ml adds 5% milk powder protective material and viral liquid according to 1: 1.2 ratio, and the reovirus freeze-dried live vaccine is made in freeze-drying.
Prepared freeze-dried live vaccine contains virus quantity 10 in every cillin bottle
6.5ELD50/1000 plumage part.
Safety check: 10 times of amounts of getting using dosage are 10
4.5ELD
50/ 0.2ml foot-pad immunization 1 age in days SPF chicken observed for 2 weeks, and situations such as footpad inoculation position NIP reaction occur.
Imitate inspection: getting using dosage is 10
3.5The subcutaneous immune 1 age in days chicken of ELD50/0.2ml nape portion after 2 weeks, is used strong virus attack, and protection ratio reaches 100%.Freeze dried vaccine immunity chicken can produce strong immunizing power in 1 week, and duration of immunity is 2.5 months, and attacking malicious protection ratio between duration of immunity is 94.8%～98.3%.
Vacuum tightness detects: get 10 bottles of freeze dried vaccines at random, measure the vacuum tightness of this sample with the high-frequency spark vacuum tester, all occur the purple aura in the vaccine bottle, illustrate that the vacuum tightness of this freeze dried vaccine is qualified.
Residual moisture is measured: adopt the vacuum drying method to measure, concrete operations are consulted " People's Republic of China's veterinary biologics rules " (version in 2000) and are carried out for 438 pages.The residual moisture that each stochastic sampling detects the freeze dried vaccine sample all is lower than 4.0%, illustrates that this freeze dried vaccine residual moisture is qualified, reaches standard.
Claims (3)
1. reovirus, it is characterized in that: described virus is Avianreovirus, Avianreovirus, be called for short ARV, strain was the RS13 strain, and preserving number is CGMCC No.1713, was deposited in Chinese microbial preservation management committee common micro-organisms center on May 16th, 2006.
2. according to the vaccine of the described reovirus of claim 1 preparation, it is characterized in that: described reovirus was bred for 70 generations, get the 70th and withhold and obtain virus, detect its virus titer and reach 10
9.5Behind the ELD50/0.2ml, through serum detect, viral proliferation went down to posterity for 50～70 generations, virus titer detects and reaches 10
9.5ELD
50/ 0.2ml, make vaccine after freeze-dried mixed; Described reovirus does not have cyst membrane, is 20 body symmetries, and genome is made of 10 double-stranded RNA segmentations, can tolerate 60 ℃ of 8～10h, and is insensitive to ether, and to the chloroform slight sensitive, to the alkali sensitivity, 3% sodium hydroxide effect 1h can kill virus, no blood clotting characteristic.
3. method for preparing as reovirus vaccine as described in the claim 2 is characterized in that it carries out as follows:
A. the virus of preservation in the described claim 1 is bred on the chicken embryo and went down to posterity for 70 generations, got for the 70th generation, results virus detects its virus titer and reaches 10
9.5ELD50/0.2ml uses viral seed as producing;
B. serum detects: with in the S1133 positive serum and after, inoculate in 10 age in days specific pathogen free microorganism chicken embryos or the chick embryo fibroblast, corresponding chicken idiosome and internal organ pathology or corresponding cytopathy do not appear, confirm that this viral serotype is identical with S1133;
C. viral proliferation: viral seed was gone down to posterity for 50～70 generations by chicken embryo or chick embryo fibroblast breeding, and virus titer detects and reaches 10
9.5ELD
50/ 0.2ml;
D. freeze-dried mixed: with viral dilution to 10
6.5ELD
50/ ml adds 5% milk powder protective material and viral liquid according to 1: 1.2 ratio, and the reovirus freeze-dried live vaccine is made in freeze-drying.
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|---|---|---|---|
| CNB2006100127915A CN100516206C (en) | 2006-06-06 | 2006-06-06 | Reovirus vaccine and its preparing method |
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|---|---|---|---|
| CNB2006100127915A CN100516206C (en) | 2006-06-06 | 2006-06-06 | Reovirus vaccine and its preparing method |
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| CN100516206C true CN100516206C (en) | 2009-07-22 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1895667B (en) * | 2006-06-06 | 2010-11-03 | 瑞普（保定）生物药业有限公司 | Reovirus inactivated vaccine and its preparation |
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| CN102352346B (en) * | 2011-10-24 | 2013-03-13 | 福建省农业科学院畜牧兽医研究所 | Novel-pathotype duck reovirus and attenuated vaccine thereof |
| CN104711240B (en) * | 2015-03-18 | 2017-10-10 | 广西壮族自治区兽医研究所 | The application of Avianreovirus σ A albumen and its relevant biological material |
| CN105925541A (en) * | 2016-05-13 | 2016-09-07 | 云南省畜牧兽医科学院 | Blue tongue virus cryoprotective agent and cryopreservation method |
| CN115184344A (en) * | 2022-01-30 | 2022-10-14 | 北京市疾病预防控制中心 | Novel coronavirus IgG antibody detection method suitable for novel biological sample oral fluid and application thereof |
| CN116478937B (en) * | 2023-06-12 | 2023-08-18 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Gene III avian reovirus variant and application thereof in preparing vaccine |
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2006
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1895667B (en) * | 2006-06-06 | 2010-11-03 | 瑞普（保定）生物药业有限公司 | Reovirus inactivated vaccine and its preparation |
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| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20061227 Assignee: Tianjin Ringpu Bio-Technology Co., Ltd. Assignor: Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. Contract record no.: 2010120000014 Denomination of invention: Reovirus vaccine and its preparing method Granted publication date: 20090722 License type: Exclusive License Record date: 20100308 |
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| EC01 | Cancellation of recordation of patent licensing contract |
Assignee: Tianjin Ringpu Bio-Technology Co., Ltd. Assignor: Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. Contract record no.: 2010120000014 Date of cancellation: 20141023 |
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