CN101745106A - Porcine parvnvirus living vaccine and preparation method thereof - Google Patents
Porcine parvnvirus living vaccine and preparation method thereof Download PDFInfo
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- CN101745106A CN101745106A CN200810204266A CN200810204266A CN101745106A CN 101745106 A CN101745106 A CN 101745106A CN 200810204266 A CN200810204266 A CN 200810204266A CN 200810204266 A CN200810204266 A CN 200810204266A CN 101745106 A CN101745106 A CN 101745106A
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Abstract
The invention relates to a porcine parvnvirus living vaccine and a preparation method thereof. The living vaccine is prepared by adopting a PPVS-1A strain (the HA valence is not less than 29, and the TCID50 is not less than 107.0/ml) to obtain a cell culture through being inoculated with a swine testis (ST) subculture cell growing well, adding a suitable stabilizing agent and carrying out freezing vacuum drying. The virus content of each first part of the living vaccine is not less than 105.0TCID50. Compared with the prior art, the porcine parvnvirus living vaccine has good immunogenicity, rapid antibody generation after immunization, high titer and long maintenance time of the generated antibody, long retention period and small immunizing dose. When the porcine parvnvirus living vaccine is used to carry out vaccine injection a plurality of weeks before hybridization, the pregnant sow can maintain strong immunizing power in the easy infection period. The porcine parvnvirus living vaccine is the excellent choice for preventing the porcine parvnvirus. The adopted preparation method has reasonable process and lower cost and greatly reduces the cost burden of the livestock breeding.
Description
Technical field
The invention belongs to the biological new medicine technical field of veterinary, relate to the pig parvoviral disease vaccine, relate in particular to a kind of pig parvoviral disease live-vaccine and preparation method thereof.
Background technology
Porcine parvovirus is that (Porcine parvovirus PPV) infects and one of pig breeding dysfunction disease of causing by pig parvoviral.PPV infects and mainly causes the death of embryo and fetus, and stillborn fetus is produced in in-pig performance miscarriage, and mummy and weak son etc. especially see to produce the mummy tire more.Simultaneously, PPV can increase the weight of the pmws that pig circular ring virus causes, brings crushing blow to the pig farm often with other immunosuppressive disease accompanying infections.Primary disease is distributed widely in all over the world, causes enormous economic loss to pig industry.
Since Mayr separated this virus of report first in 1966, a lot of countries were separated to pig parvoviral and detect antibody from Europe, America and Asia etc. in succession.Just there is the report of primary disease in China in early 1980s, in the period of 1983～1985, Hou Shikuan, Pan Xuezhu, Wu Jie respectively in Heilungkiang, ground such as Shanghai, Sichuan is separated to PPV.In recent years, in Hubei, ground such as Henan, Shandong, Fujian, Guangxi, the Xinjiang report that all has PPV to infect.Investigation shows that the infection rate of PPV in swinery is in rising trend, at China multiparity sow and breeding boar infection rate up to 92%.The boar and the sow that infect are the main sources of infection, and virus is often infected by placental infection and copulation passes to fetus, also can pass to susceptible animal by respiratory tract, digestive tract by contaminated food, environment, and muroid is important communication media.
Popular often the betiding of primary disease farrowed season in spring and autumn, is common in first farrowing sow, and it is popular or distribute generally to be endemicity.In case PPV imports negative pig farm into, breed, propagate rapidly, almost 100% pig only all can be infected in 3 months, and after primary disease took place, the sow reproductive failure may constantly appear in pig farm for successive years.After sow infection period of pregnancy, its rate of embryonic death can reach 80%～100%.Because scale, intensive culture degree are more and more higher, PPV infects and presents more and more serious fashion trend in recent years, also causes serious harm for China's pig industry.
PPV infects does not also have effective Therapeutic Method at present, and the same with the anti-system of other many virosiss, this disease is also mainly puted prevention first with vaccine immunity.Because PPV serotype is single, immunogenicity is high, make vaccination become a kind of efficient ways that control PPV infects.At present existing more than 10 country developed the PPV vaccine, comprises inactivated vaccine and attenuated live vaccines.
1. inactivated vaccine
From beginning in 1976, external just report relevant for the PPV Inactivated Vaccine, and in the eighties in 20th century in national widespread usage such as the U.S., Australia, France.(1987) such as China Pan Xue pearls have at first successfully developed the PPV inactivated vaccine, and after this Wu Jie, Han Xiaocheng, Xiao Chi, leaf priority such as face south is successfully developed the PPV inactivated vaccine.
The pig parvoviral disease vaccine that present China has obtained new veterinary drug certificate has: the pig parvoviral oil emulsion inactivated vaccine of academy of agricultural sciences, Shanghai animal and veterinary Research Institute (No. the 04th, (94) new veterinary drug card word), No. 08, the new veterinary drug card of Wuhan Zhongbo Biochemical Co., Ltd's porcine parvovirus inactivated vaccines (CP-99 strain) (2006) word, Hua Zhong Agriculture University, Wuhan Keqian Animal Biological Products Co., Ltd., the porcine parvovirus inactivated vaccines of Zhongmu Industry Co.,Ltd's joint research and development (wH-1 strain) (No. 71, (2006) new veterinary drug card word).The producer of selling the pig parvoviral inactivated vaccine on market mainly contains: Shanghai Jiamu Biological Products Co., Ltd.'s (technical backstopping is Shanghai Academy of Agricultural Sciences's animal and veterinary institute), Qilu Animal Health Products Co., Ltd., Wuhan Keqian Animal Biological Products Co., Ltd., the sharp biologics company limited in sea, Shanghai, Songjiang, Shanghai biologics company limited etc.
The using dosage of inactivated vaccine is big, and side reaction is also bigger, has promoted the research and development of live vaccine.
2. live vaccine
The existing abroad commercial prod of PPV attenuated live vaccines, finding the earliest and being applied to clinical is the PPV NADL-2 low virulent strain of the U.S., it is weak that this strain utilizes the cell culture continuous passage to cause more than 50 times at laboratory the strong poison of PPV.Japan scholar Fujisaki etc. passed for 54 generations continuously with the wild malicious PPV 90HS of PPV low temperature (30 ℃) on porcine kidney cell, produce the HT variant, on this basis, Akihiro etc. go down to posterity the HT strain in cultivation on the porcine kidney cell and after with ultraviolet radiation, obtained the better HT-SK-C strain of safety, the attenuated live vaccines that utilizes this strain production is in Japanese goodsization.
Because a large amount of existence of PPV virulent strain, people return strong worry to virus reorganization and weak poison makes the application of attenuated live vaccines be subjected to certain limitation, but above-mentioned live vaccine effect for many years clinically confirms that it is safe and reliable.
Summary of the invention
Purpose of the present invention is exactly for a kind of have good safety and immunogenic pig parvoviral disease live-vaccine and preparation method thereof are provided.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of pig parvoviral disease live-vaccine is characterized in that, this live vaccine adopts the weak malicious PPV of pig parvoviral
S-1The A strain, pig testis (ST) passage cell that inoculation is grown fine, the harvesting culture adds suitable stabilizing agent, makes every part viral level 〉=10 of described live vaccine through lyophilisation
5.0TCID
50
The weak malicious PPV of described pig parvoviral
S-1The A strain is PPV
S-1Strain causes the weak low virulent strain that obtains in continuous passage to the on the pig testis cell line more than 40 generations, and its HA tires 〉=and 2
9, TCID
50〉=10
7.0/ ml.
Described stabilizing agent comprises 5% sucrose defatted milk freeze drying protectant.
A kind of preparation method of pig parvoviral disease live-vaccine is characterized in that, this method may further comprise the steps:
(1) prepare the pig testis cell according to conventional method, growth-promoting media is the MEM that contains the 5%-8% new-born calf serum, connects poison when cell grows up to 2/3 above monolayer;
(2) get the weak malicious PPV of pig parvoviral
S-1The A strain is diluted by 0.1%, the pig testis cell monolayer that grows fine that inoculation step (1) obtains, and 37 ℃ adsorbed 1.5 hours, added cell maintenance medium, put 37 ℃ of static or rotating and culturing;
(3) when cytopathy appears in 75% above cell, harvesting culture, multigelation 3 times;
(4) get that supernatant is measured malicious valency and HA tires;
(5) the cell venom that is up to the standards is placed in the sterilization container, add stabilizing agent, fully shake up, quantitatively packing, every part viral level 〉=10 according to 1: 1 volume ratio
5.5TCID
50
(6) carry out lyophilisation rapidly, make the pig parvoviral disease live-vaccine, every part viral level 〉=10
5.0TCID
50
The weak malicious PPV of described pig parvoviral
S-1The A strain is PPV
S-1Strain causes the weak low virulent strain that obtains in continuous passage to the on the pig testis cell line more than 40 generations, and its HA tires 〉=and 2
9, TCID
50〉=10
7.0/ ml.
Cell maintenance medium in the described step (2) is the cell maintenance medium that contains the MEM of 2% new-born calf serum.
In preserving below-20 ℃, the holding time is no more than 30 behind the middle multigelation of described step (3).
Viral level 〉=10 of the cell venom that is up to the standards in the described step (5)
7.0TCID
50/ ml, red cell agglutination valency HA 〉=2
9
Stabilizing agent in the described step (5) comprises 5% sucrose defatted milk freeze drying protectant.
I Pan Xuezhu equal nineteen eighty-two and at first be separated to pig parvoviral on the morbidity pig farm of suburb of Shanghai, and the PPV inactivated vaccine has been developed in the success that took the lead at home in 1987, obtained new veterinary drug certificate in 1994, produce by Shanghai Jiamu Biological Products Co., Ltd..On this basis, the cultivation and the research of pig parvoviral low virulent strain have been carried out, with PPV
S-1Strain is gone up 40 generations of continuous passage to the in pig testis cell line (ST) and is caused weak low virulent strain, the called after PPV of obtaining
S-1The A strain.PPV
S-1The pathological changes rule appears in the A low virulent strain on the ST cell, after measured HA tire 〉=2
11, TCID
50Be stabilized in 10
7.5～10
8.0/ 0.1ml.With 4ml dosage intramuscular injection PPV HI negative antibody pig, there is not obvious clinical symptoms, all be not separated to virus from inoculation porcine blood plasma, internal organs and nose anus secretions.Test confirms PPV
S-1The A strain has good safety and immunogenicity, does not cause viremia, toxin expelling not.
The present invention adopts PPV
S-1The A low virulent strain is inoculated well-grown ST cell, results freeze thawing when treating that pathological changes appears in 75% above cell, after measured HA tire 〉=2
9, TCID
50〉=10
7.0/ ml.
Compared with prior art, it is fast that pig parvoviral disease live-vaccine immunogenicity of the present invention is good, the immunity back produces antibody, high and the length of holding time of the antibody titer that produces, long shelf-life, immunizing dose is little, can make farrowing sow keep strong immunity in vulnerable period in the former Zhou Jinhang vaccine injections of breeding, it is the splendid selection of prevention porcine parvovirus, the preparation method technology that adopts is reasonable, and price is lower, greatly reduces the cost burden of aquaculture.
The specific embodiment
The invention will be further described below in conjunction with specific embodiment.
Embodiment 1
A kind of preparation method of pig parvoviral disease live-vaccine, this method may further comprise the steps:
(1) prepare the ST cell according to conventional method, growth-promoting media is the MEM that contains 8% new-born calf serum, connects poison when cell grows up to 2/3 above monolayer;
(2) get the production weak malicious PPV of pig parvoviral
S-1A strain seed culture of viruses (PPV
S-1Strain causes the weak low virulent strain that obtains in continuous passage to the on the pig testis cell line more than 40 generations, and its HA tires 〉=and 2
9, TCID
50〉=10
7.0/ ml) by 0.1% dilution, the ST cell monolayer that inoculation is grown fine, 37 ℃ adsorbed 1.5 hours, added the MEM cell maintenance medium that contains 2% new-born calf serum, put 37 ℃ of static or rotating and culturing;
(3) observe 2 every day, record cytopathy situation, when cytopathy appears in 75% above cell, the harvesting culture, multigelation 3 times is preserved below-20 ℃, is no more than 30;
(4) get that supernatant is measured malicious valency and HA tires, viral level 〉=10
7.0TCID
50/ ml, and red cell agglutination valency HA 〉=2
9
(5) the cell venom that is up to the standards is mixed in the same sterilization container, adds 5% sucrose defatted milk freeze drying protectant, fully shake up according to 1: 1 volume ratio, quantitatively packing, every part viral level answers 〉=10
5.5TCID
50
(6) carry out lyophilisation after the packing rapidly, make the pig parvoviral disease live-vaccine, every part viral level answers 〉=10
5.0TCID
50
Use the pig parvoviral disease live-vaccine of the present invention's development, every part viral level answers 〉=10
5.0TCID
50Antibody response all appears in 1 week behind the immune swine, and back 7 months counteracting toxic substances of immunity still can be resisted the strong malicious attack of PPV, can keep 6 months at least the immune duration of pig, and-20 ℃ of storage lives are 18 months.
After the vaccination of pig body no bad clinical should, appetite, body temperature are normal, the mental status is good, does not cause viremia and the toxin expelling of inoculation pig, not diaplacental infection, safety is good.The fertility performance of vaccination of sows is improved, and has produced good society and economic benefit, and the sow breeding difficulty that causes for the prevention parvovirus has played positive effect.
The use of the vaccine that the present invention makes and points for attention:
1. act on and purposes
Be used to prevent porcine parvovirus.
2. usage and consumption
Press head part that label indicates, with sterile saline or special-purpose diluted vaccine, deep intramuscular injection, 1 part/head.Preceding 2～3 weeks immunity of insemination of sows, boar head when 6 monthly ages exempts from, later immunity every half a year 1 time.
3. points for attention
3.1 vaccine should prevent high temperature, disinfectant and solar radiation in transportation, preservation, use.
3.2 vaccine dilution back limit used up in 4 hours.
3.3 the reply injection site carries out strict sterilization.
3.4 remaining vaccine and apparatus should be discarded after harmless treatment.
3.5 this vaccine is only effective to the sow breeding difficulty that is caused by PPV, and the sow breeding difficulty that is caused by other infectious disease is not had preventive effect, when using this vaccine, should note preventing other infectious disease.
4. storage
Below-15 ℃, effect duration is 18 months.
Embodiment 2
A kind of preparation method of pig parvoviral disease live-vaccine, this method may further comprise the steps:
(1) prepare the ST cell according to conventional method, growth-promoting media is the MEM that contains 5% new-born calf serum, connects poison when cell grows up to 2/3 above monolayer;
(2) get the production weak malicious PPV of pig parvoviral
S-1A strain seed culture of viruses (PPV
S-1Strain causes the weak low virulent strain that obtains in continuous passage to the on the pig testis cell line more than 40 generations, and its HA tires 〉=and 2
9, TCID
50〉=10
7.0/ ml) by 0.1% dilution, the ST cell monolayer that inoculation is grown fine, 37 ℃ adsorbed 1.5 hours, added the MEM cell maintenance medium that contains 2% new-born calf serum, put 37 ℃ of static or rotating and culturing;
(3) observe 2 every day, record cytopathy situation, when cytopathy appears in 75% above cell, the harvesting culture, multigelation 3 times is preserved below-20 ℃, is no more than 30;
(4) get that supernatant is measured malicious valency and HA tires, viral level 〉=10
7.0TCID
50/ ml, and red cell agglutination valency HA 〉=2
9
(5) the cell venom that is up to the standards is mixed in the same sterilization container, adds 5% sucrose defatted milk freeze drying protectant, fully shake up according to 1: 1 volume ratio, quantitatively packing, every part viral level answers 〉=10
5.5TCID
50
(6) carry out lyophilisation after the packing rapidly, make the pig parvoviral disease live-vaccine, every part viral level answers 〉=10
5.0TCID
50
Use the pig parvoviral disease live-vaccine of the present invention's development, every part viral level answers 〉=10
5.0TCID
50Antibody response all appears in 1 week behind the immune swine, and back 7 months counteracting toxic substances of immunity still can be resisted the strong malicious attack of PPV, can keep 6 months at least the immune duration of pig, and-20 ℃ of storage lives are 18 months.
Claims (9)
1. a pig parvoviral disease live-vaccine is characterized in that, this live vaccine adopts the weak malicious PPV of pig parvoviral
S-1The A strain, pig testis (ST) passage cell that inoculation is grown fine, the harvesting culture adds suitable stabilizing agent, makes every part viral level 〉=10 of described live vaccine through lyophilisation
5.0TCID
50
2. pig parvoviral disease live-vaccine according to claim 1 is characterized in that, the weak malicious PPV of described pig parvoviral
S-1The A strain is PPV
S-1Strain causes the weak low virulent strain that obtains in continuous passage to the on the pig testis cell line more than 40 generations, and its HA tires 〉=and 2
9, TCID
50〉=10
7.0/ ml.
3. pig parvoviral disease live-vaccine according to claim 1 is characterized in that, described stabilizing agent comprises 5% sucrose defatted milk freeze drying protectant.
4. the preparation method of a pig parvoviral disease live-vaccine as claimed in claim 1 is characterized in that, this method may further comprise the steps:
(1) prepare the pig testis cell according to conventional method, growth-promoting media is the MEM that contains the 5%-8% new-born calf serum, connects poison when cell grows up to 2/3 above monolayer;
(2) get the weak malicious PPV of pig parvoviral
S-1The A strain is diluted by 0.1%, the pig testis cell monolayer that grows fine that inoculation step (1) obtains, and 37 ℃ adsorbed 1.5 hours, added cell maintenance medium, put 37 ℃ of static or rotating and culturing;
(3) when cytopathy appears in 75% above cell, harvesting culture, multigelation 3 times;
(4) get that supernatant is measured malicious valency and HA tires;
(5) the cell venom that is up to the standards is placed in the sterilization container, add stabilizing agent, fully shake up, quantitatively packing, every part viral level 〉=10 according to 1: 1 volume ratio
5.5TCID
50
(6) carry out lyophilisation rapidly, make the pig parvoviral disease live-vaccine, every part viral level 〉=10
5.0TCID
50
5. the preparation method of pig parvoviral disease live-vaccine according to claim 4 is characterized in that, the weak malicious PPV of described pig parvoviral
S-1The A strain is PPV
S-1Strain causes the weak low virulent strain that obtains in continuous passage to the on the pig testis cell line more than 40 generations, and its HA tires 〉=and 2
9, TCID
50〉=10
7.0/ ml.
6. the preparation method of pig parvoviral disease live-vaccine according to claim 4 is characterized in that, the cell maintenance medium in the described step (2) is the cell maintenance medium that contains the MEM of 2% new-born calf serum.
7. the preparation method of pig parvoviral disease live-vaccine according to claim 4 is characterized in that, in preserving below-20 ℃, the holding time is no more than 30 behind the middle multigelation of described step (3).
8. the preparation method of pig parvoviral disease live-vaccine according to claim 4 is characterized in that, viral level 〉=10 of the cell venom that is up to the standards in the described step (5)
7.0TCID
50/ ml, red cell agglutination valency HA 〉=2
9
9. the preparation method of pig parvoviral disease live-vaccine according to claim 4 is characterized in that, the stabilizing agent in the described step (5) comprises 5% sucrose defatted milk freeze drying protectant.
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|---|---|---|---|
| CN200810204266A CN101745106A (en) | 2008-12-09 | 2008-12-09 | Porcine parvnvirus living vaccine and preparation method thereof |
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|---|---|---|---|
| CN200810204266A CN101745106A (en) | 2008-12-09 | 2008-12-09 | Porcine parvnvirus living vaccine and preparation method thereof |
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| CN101745106A true CN101745106A (en) | 2010-06-23 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101947318A (en) * | 2010-09-09 | 2011-01-19 | 扬州优邦生物制药有限公司 | Method for preparing porcine parvovirus inactivated vaccines |
| CN102038945A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing swine parvovirus vaccine by utilizing bioreactor |
| CN102091329A (en) * | 2011-01-30 | 2011-06-15 | 哈药集团生物疫苗有限公司 | Preparation method of inactivated porcine parvovirus vaccine and product thereof |
| CN101851609B (en) * | 2010-02-02 | 2012-10-03 | 哈药集团生物疫苗有限公司 | Porcine parvovirus L strain and use thereof in preparation of porcine parvovirus inactivated vaccines |
| CN102727881A (en) * | 2012-07-04 | 2012-10-17 | 广东大华农动物保健品股份有限公司 | Highly pathogenic porcine reproductive and respiratory syndrome JXAl-R strain- porcine parvovirus disease bigeminal live vaccine and preparation method and application thereof |
| CN108795884A (en) * | 2018-07-02 | 2018-11-13 | 苏州良辰生物医药科技有限公司 | A kind of enrichment procedure of pig parvoviral |
-
2008
- 2008-12-09 CN CN200810204266A patent/CN101745106A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851609B (en) * | 2010-02-02 | 2012-10-03 | 哈药集团生物疫苗有限公司 | Porcine parvovirus L strain and use thereof in preparation of porcine parvovirus inactivated vaccines |
| CN101947318A (en) * | 2010-09-09 | 2011-01-19 | 扬州优邦生物制药有限公司 | Method for preparing porcine parvovirus inactivated vaccines |
| CN101947318B (en) * | 2010-09-09 | 2012-09-05 | 扬州优邦生物制药有限公司 | Method for preparing porcine parvovirus inactivated vaccines |
| CN102038945A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing swine parvovirus vaccine by utilizing bioreactor |
| CN102091329A (en) * | 2011-01-30 | 2011-06-15 | 哈药集团生物疫苗有限公司 | Preparation method of inactivated porcine parvovirus vaccine and product thereof |
| CN102091329B (en) * | 2011-01-30 | 2013-08-07 | 哈药集团生物疫苗有限公司 | Preparation method of inactivated porcine parvovirus vaccine and product thereof |
| CN102727881A (en) * | 2012-07-04 | 2012-10-17 | 广东大华农动物保健品股份有限公司 | Highly pathogenic porcine reproductive and respiratory syndrome JXAl-R strain- porcine parvovirus disease bigeminal live vaccine and preparation method and application thereof |
| CN108795884A (en) * | 2018-07-02 | 2018-11-13 | 苏州良辰生物医药科技有限公司 | A kind of enrichment procedure of pig parvoviral |
| CN108795884B (en) * | 2018-07-02 | 2021-11-30 | 苏州良辰生物医药科技有限公司 | Porcine parvovirus propagation method |
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Open date: 20100623 |