CN102210857A - Method for improving antigen amount of inactivated porcine circovirus 2 (PCV2) vaccine - Google Patents
Method for improving antigen amount of inactivated porcine circovirus 2 (PCV2) vaccine Download PDFInfo
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- CN102210857A CN102210857A CN201010138882XA CN201010138882A CN102210857A CN 102210857 A CN102210857 A CN 102210857A CN 201010138882X A CN201010138882X A CN 201010138882XA CN 201010138882 A CN201010138882 A CN 201010138882A CN 102210857 A CN102210857 A CN 102210857A
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Abstract
The invention relates to a preparation method for improving the antigen amount of an inactivated porcine circovirus 2 (PCV2) vaccine. The technical process of the method mainly comprises the following steps of: acquiring a cell proliferation inactivated PCV2 culture solution, primarily filtering by 4 layers of sterile gauze, medially filtering by a 100KD hollow fiber ultrafiltration membrane component, and high efficiently filtering by a 6KD hollow fiber ultrafiltration membrane component. Shown by experimental results, the antigen amount of the inactivated PCV2 vaccine is improved by 10 times by concentration through the method, and the concentrated PCV2 antigens are not influenced in characteristics, thus enhancing the immune efficacy of the vaccine. Therefore, the method is simple in preparation and easy in production, and has the advantages of low cost, controllable quality, high concentration ratio, good actual effect, no three wastes and the like.
Description
Technical field
The present invention relates to a kind of method that improves porcine circovirus 2 type totivirus killed vaccine antigen amount, specifically with porcine circovirus 2 type cell culture deactivation thing through primarily efficient filter, medium air filtration and high efficiency filter three phases, reach the manufacture method that concentrate to improve totivirus killed vaccine antigen amount.
Background technology
Porcine circovirus 2 type (PCV2) is the cause of disease that causes pig circular ring virus 2 viral disease (PCVD), comprises pig wean back multisystemic exhaustion syndrome (PMWS), Corii Sus domestica inflammation-nephrotic syndrome (PDNS), pig hypertrophy necrotizing pneumonia (PNP), the congenital brain of pig the tremble miscarriage of (CT), pig and breeding difficulty etc.At present, PCVD is the trend that rises year by year at the positive rate of China, has that popular scope is wide, each growth stage pig infects characteristics such as serious, that the nursery pig sickness rate is the highest, mixed infection is outstanding, serious threat the development of China's pig industry.
The immunity of PCV2 vaccine can provide good protection, and effect is obvious, gains universal acceptance, as obvious reduction mortality rate, minimizing death rate, raising production achievement, reduction Drug therapy expense etc.Yet in the development process of PCV2 vaccine, the subject matter that runs into is: the outer multiplication capacity of this virion is poor, the culture viral level is low, and the virus inoculation cell does not produce pathological changes, and viral level is difficult for measuring, viral infection animal clinical response differs, and immunity is estimated difficulty.The antigen amount that wherein improves vaccine is the bottleneck of PCV2 production of vaccine and application, so the insider proposes, by domestication and screening adapt to the PCV2 growth cell line, set up High Density Cultivation technology etc. and be expected to solve this type of problem.But up to now, do not see relevant successful report as yet.
At present, existing two PCV2 totivirus inactivated vaccines obtain Chinese patent (CN200810024423.1 and CN200810079560.5).The former has adopted PCV2 Shanghai separated strain SH, breeds in a large number in the PK-15 cell, adds white-oil adjuvant through formalin-inactivated and carries out emulsifying, the white-oil adjuvant vaccine that preparation is conventional; So improving, its vaccine antigen amount is restricted.Latter system isolates organs and tissues in the sick pig body of typical PCV2, obtain the organs and tissues extract through grinding, filtering, add formalin-inactivated, make original inactivated vaccine with the normal saline dilution, this vaccination after interior short its morbidity of health pig body or death, is gathered organs and tissues, through grinding, filtering, add formalin-inactivated, make the PCV2 inactivated vaccine with the normal saline dilution; So its vaccine antigen amount is determined difficulty, preparation cost and is increased.
Summary of the invention
In order to address the above problem, the invention provides a kind of manufacture method of concentrated raising PCV2 totivirus killed vaccine antigen amount.
Realize that the concrete technical solution of above-mentioned purpose is as follows:
1, a kind of manufacture method that improves PCV2 totivirus killed vaccine antigen amount comprises that PK-15 cell proliferation PCV2 cultivates that deactivation liquid is obtained, primarily efficient filter, medium air filtration, high efficiency filter.
2, according to the manufacture method of above-mentioned 1 described raising PCV2 totivirus killed vaccine antigen amount, concrete steps and condition are as follows:
(1) PK-15 cell proliferation PCV2 cultivation deactivation liquid is obtained
After utilizing indirect immunofluorescence detection PK-15 cell proliferation PCV2 culture fluid TCID50, through 37 ℃ of deactivation 30h of final concentration 0.1% formaldehyde.
(2) primarily efficient filter
Utilize 4 layers of sterile gauze tentatively to filter PCV2 cell culture deactivation liquid.Its purpose is to remove granule foreign big in the cell culture.
(3) medium air filtration
With the polysulfone hollow fiber ultrafiltration membrane system of primarily efficient filter liquid by 100KD.Its purpose is to remove the protein of macromolecule.
(4) high efficiency filter
The polysulfone hollow fiber ultrafiltration membrane system by 6KD again with medium air filtration liquid.Its purpose is to concentrate totivirus antigen, can reach 10 times and concentrate.
Processing technology of the present invention is simple, process stabilizing, easy-regulating, success rate height.The production small investment of the inventive method, and do not have three wastes problem.Manufacture method of the present invention is fit to suitability for industrialized production, is not subjected to the limitation in time, place, can be big-and-middle-sized veterinary biological product manufacturing enterprise and adopts.
The present invention passes through PCV2 cell culture deactivation thing through primarily efficient filter → medium air filtration → high efficiency filter three phases, reach the antigen amount of 10 times of concentrated totivirus inactivated vaccines, this current as yet not acclimation and screening go out to adapt to PCV2 growth cell line, realize setting up under the situation of High Density Cultivation technology, manufacture method of the present invention provides condition for the raising of PCV2 totivirus killed vaccine antigen amount undoubtedly, thereby lays a good foundation for the high-quality production of PCV2 totivirus inactivated vaccine.
The inventive method concentrates the antigen measurer that improves PCV2 totivirus inactivated vaccine unique advance: one, material therefor is easy to get, and required facility is easy.Two, production process is simple, low production cost.Three, concentrate the ratio height, and to having no effect through spissated PCV2 antigenic characteristic.Four, strengthen the immune efficacy of vaccine, and do not had three wastes problem, had society and economical effects.
The specific embodiment
In conjunction with the embodiments the present invention is done to describe further.
Embodiment:
Should concentrate the manufacture method that improves PCV2 totivirus killed vaccine antigen amount, comprise following manufacturing process:
1, PK-15 cell proliferation PCV2 cultivation deactivation liquid is obtained
Is that 1: 5 ratio is inoculated in and grows up to 70% monolayer PK-15 cell with PCV2 in viral liquid: DMEM, and 37 ℃ of absorption 2h abandon Incubating Solution and add an amount of DMEM that contains 2% hyclone (FBS), put 37 ℃, 5%CO
2After cultivating 18～24h under the condition, handle cell 30min, continue to use the DMEM that contains 2%FBS in 37 ℃, 5%CO with the D-glucosamine hydrochlorate of 300mmoL
2Receive poison after cultivating 48h under the condition, multigelation three sub-samplings carry out PCR and detect back results virocyte culture.After utilizing indirect immunofluorescence to detect the TCID50 of final virocyte culture, get PCV2 cell culture (6000mL for example) and, get final product through 37 ℃ of deactivation 30h of final concentration 0.1% formaldehyde.
2, primarily efficient filter
Utilization is through 4 layers of sterile gauze of 115 ℃ of autoclaving 15min, and (6000mL for example) filters under aseptic condition to PCV2 cell culture deactivation liquid, collects filtrate, promptly gets the primarily efficient filter liquid that does not contain large granular impurities such as cell debris.
3, medium air filtration
Earlier the ultrafiltration apparatus pressure regulator valve is opened before the medium air filtration, making pressure is 0MPa, then 6000mL primarily efficient filter liquid being pumped into molecular cut off by biological peristaltic pump is that the polysulfone hollow fiber ultrafiltration membrane system of 100KD is to being full of membrane module, stabilate peristaltic pump rotating speed, regulate pressure regulator valve gradually to operating pressure 0.05MPa, the beginning ultrafiltration, suspend ultrafiltration during for 500mL to concentrating return-flow liquid, with isopyknic 0.01mol/L pH is that ultrafiltration is continued in 7.6 aseptic PBS dilution back, collect ultrafiltrate 6000mL, promptly get the PCV2 totivirus inactivation antigen (medium air filtration liquid) that does not contain greater than 100KD molecular weight protein matter.
4, high efficiency filter
Earlier the ultrafiltration apparatus pressure regulator valve is opened before the high efficiency filter, making pressure is 0MPa, then 6000mL medium air filtration liquid being pumped into molecular cut off by biological peristaltic pump is that the polysulfone hollow fiber ultrafiltration membrane system of 6KD is to being full of membrane module, stabilate peristaltic pump rotating speed, regulate pressure regulator valve gradually to operating pressure 0.05MPa, the beginning ultrafiltration and concentration stops ultrafiltration and concentration during for 500mL to concentrating return-flow liquid, collect ultrafiltration and concentration liquid, promptly get and be not less than 10 times of spissated PCV2 totivirus inactivation antigens.
Claims (4)
1. manufacture method that improves porcine circovirus 2 type (PCV2) totivirus killed vaccine antigen amount comprises that PK-15 cell proliferation PCV2 cultivates that deactivation liquid is obtained, primarily efficient filter, medium air filtration, high efficiency filter.
2. the manufacture method of raising PCV2 totivirus killed vaccine antigen amount according to claim 2, it is characterized in that: primarily efficient filter system utilizes 4 layers of sterile gauze through 115 ℃ of autoclaving 15min, PCV2 cell culture deactivation liquid is filtered under aseptic condition, obtain the primarily efficient filter liquid that does not contain large granular impurities such as cell debris.
3. the manufacture method of raising PCV2 totivirus killed vaccine antigen amount according to claim 2, it is characterized in that: earlier the ultrafiltration apparatus pressure regulator valve is opened before the medium air filtration, making pressure is 0MPa, then primarily efficient filter liquid being pumped into molecular cut off by biological peristaltic pump is that the polysulfone hollow fiber ultrafiltration membrane system of 100KD is to being full of membrane module, stabilate peristaltic pump rotating speed, regulate pressure regulator valve gradually to operating pressure 0.05MPa, the beginning ultrafiltration, suspend ultrafiltration when being a certain amount of to concentrating return-flow liquid, with isopyknic 0.01mol/L pH is that ultrafiltration is continued in 7.6 aseptic PBS dilution back, collect ultrafiltrate, be the medium air filtration liquid that does not contain greater than the PCV2 totivirus inactivation antigen of 100KD molecular weight protein matter.
4. the manufacture method of raising PCV2 totivirus killed vaccine antigen amount according to claim 2, it is characterized in that: earlier the ultrafiltration apparatus pressure regulator valve is opened before the high efficiency filter, making pressure is 0MPa, then medium air filtration liquid being pumped into molecular cut off by biological peristaltic pump is that the polysulfone hollow fiber ultrafiltration membrane system of 6KD is to being full of membrane module, stabilate peristaltic pump rotating speed, regulate pressure regulator valve gradually to operating pressure 0.05MPa, the beginning ultrafiltration and concentration, stop ultrafiltration and concentration when being a certain amount of to concentrating return-flow liquid, collect ultrafiltration and concentration liquid, be and be not less than 10 times of spissated PCV2 totivirus inactivation antigens.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443572A (en) * | 2011-10-21 | 2012-05-09 | 武汉生物制品研究所有限责任公司 | Method for purifying detoxified pertussis vaccine antigen solution |
| CN102716476A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Cell inactivated vaccine, egg yolk antibody injection, and preparation method of cell inactivated vaccine |
| CN107308446A (en) * | 2017-07-14 | 2017-11-03 | 瑞普（保定）生物药业有限公司 | A kind of production method of porcine circovirus 2 type inactivated vaccine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101108248A (en) * | 2006-07-18 | 2008-01-23 | 洛阳普莱柯生物工程有限公司 | Method of preparing newcastle disease, infectiousness bronchitis bigeminy killed vaccine |
-
2010
- 2010-04-02 CN CN201010138882XA patent/CN102210857A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101108248A (en) * | 2006-07-18 | 2008-01-23 | 洛阳普莱柯生物工程有限公司 | Method of preparing newcastle disease, infectiousness bronchitis bigeminy killed vaccine |
Non-Patent Citations (2)
| Title |
|---|
| 姜北宇等: "禽用疫苗病毒浓缩技术的研究", 《华北农学报》 * |
| 王宏伟 等: "不同分子量超滤浓缩Vero 细胞狂犬病疫苗的效果研究", 《中国公共卫生》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102443572A (en) * | 2011-10-21 | 2012-05-09 | 武汉生物制品研究所有限责任公司 | Method for purifying detoxified pertussis vaccine antigen solution |
| CN102716476A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Cell inactivated vaccine, egg yolk antibody injection, and preparation method of cell inactivated vaccine |
| CN107308446A (en) * | 2017-07-14 | 2017-11-03 | 瑞普（保定）生物药业有限公司 | A kind of production method of porcine circovirus 2 type inactivated vaccine |
| CN107308446B (en) * | 2017-07-14 | 2021-03-23 | 瑞普（保定）生物药业有限公司 | Production method of porcine circovirus type 2 inactivated vaccine |
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Application publication date: 20111012 |