CN101185757B - Vaccine containing composite adjuvant and preparation method thereof - Google Patents
Vaccine containing composite adjuvant and preparation method thereof Download PDFInfo
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- CN101185757B CN101185757B CN2007101560157A CN200710156015A CN101185757B CN 101185757 B CN101185757 B CN 101185757B CN 2007101560157 A CN2007101560157 A CN 2007101560157A CN 200710156015 A CN200710156015 A CN 200710156015A CN 101185757 B CN101185757 B CN 101185757B
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Abstract
The invention discloses a vaccine containing compound adjuvant and a preparation technology thereof, which pertains to the filed of biotechnology and relates to the prevention of animal diseases. The vaccine contains the compound adjuvant which is combined by oil adjuvant and ginsenosides adjuvant, and the content of ginsenosides adjuvant per milliliter vaccine is 20 to 1000 micrograms, and the oil adjuvant accounts for one half of the total volume of the vaccine. The preparation method of the vaccine containing compound adjuvant has the following steps: the ginsenosides is dissolved in a small amount of physiological saline; the ginsenosides liquid is mixed with antigen, and then the mixture is fully emulsified and mixed with the oil adjuvant under the function of emulsifier; or the oil adjuvant and the ginsenosides solution are firstly emulsified and mixed, and then the mixture is mixed with the antigen for emulsion; or the ginsenosides solution is added in conventional adjuvant vaccine and then is emulsified and mixed. The compound adjuvant vaccine of the invention is proved that the invention can induce the higher intensity of immune response compared with the usage of a single adjuvant by the antigen animal immune experiments which take the inactivated food and mouth disease virus as the module.
Description
Technical field
The invention belongs to biological technical field, relate to the prevention technique of Animal diseases, particularly a kind of vaccine that uses composite adjuvant and preparation method thereof adopts the vaccine of this composite adjuvant preparation can increase substantially vaccine-induced specific immune response intensity.
Background technology
Infectious disease is a class important diseases of harm animal health, and the economic loss that causes to animal husbandry is very serious.To animal vaccine is the pathogenetic conventional methods of many national infection preventions.Adjuvant can improve the immune effect of vaccine, and oily adjuvant is an important component forming many animal infection disease vaccines (as foot-and-mouth disease vaccine).But the immune effect of some tank oil Adjuvanted vaccines is not very good at present.Investigation such as people such as Xie Qin shows, having injected only has behind the foot-and-mouth disease vaccine of oil-containing adjuvant 20.9% piglet to produce enough strong immunoreation, and still there is the danger of infection in remaining piglet.People such as Hao Dali find after having analyzed 91 parts of foot-and-mouth disease vaccine piglet serum of having injected the oil-containing adjuvant, only have 31.9% serum contained antibody to reach the protectiveness level.
Summary of the invention
The objective of the invention is to protect the problem of immunized animal effectively, the vaccine of more effective immunological adjuvant processing and the preparation method of this vaccine are provided at a little less than the inductive immunoreation behind more existing oil-adjuvant vaccine injection animals.
In order to overcome the shortcoming of some oil-adjuvant vaccine immune effect differences, the invention provides a kind of new vaccine formulation, adopt the vaccine of this formulation can increase substantially vaccine-induced immunoreation intensity, the ability that enhance immunity animal opposing infectious disease infects.
Technical scheme of the present invention is to make to contain the ginsenoside in the oil-adjuvant vaccine.Ginsenoside's content is 20～1000 μ g/ml, promptly contains ginsenoside 20～1000 micrograms in every milliliter of vaccine.
A kind of feature that contains the composite adjuvant vaccine of the present invention is: contain oily adjuvant and ginsenoside's adjuvant in the described vaccine, the content of ginsenoside's adjuvant is 20～1000 micrograms in every milliliter of vaccine, and oily adjuvant accounts for 1/2nd of vaccine volume.
A kind of preparation method that contains the composite adjuvant vaccine of the present invention is following steps:
(1) ginsenoside is dissolved in a small amount of normal saline;
(2) with ginsenoside's solution and antigen mix homogeneously, the fully emulsified mixing under the effect of emulsifying agent with this mixture and oily adjuvant then; Or, mix with antigen emulsifying then oily adjuvant and the mixing of ginsenoside's emulsifying soln; Or emulsifying mixes add ginsenoside's solution in conventional oil-adjuvant vaccine after.
Beneficial effect of the present invention is: owing to while oil-containing adjuvant and ginsenoside in a kind of vaccine, vaccine-induced immunoreation intensity is increased substantially.Its reason may be that two kinds of different adjuvants of model of action have produced synergism together: oily adjuvant can make antigen slowly discharge in the injection site, make the immunocyte of the persistent stimulation of antigen animal, the ginsenoside is a kind of immunologic stimulant, can improve the animal immune cell to antigenic respond.
Description of drawings
Fig. 1 is: the composite adjuvant (every milliliter 20 microgram of content of ginsenoside) and the immune effect of vaccine figure that uses a kind of adjuvant that contain ginsenoside and white oil.Give mice (n=6/ group) muscle inoculation: 1) FMDV (O type) antigen (matched group), 2) FMDV (O type) antigen+ginsenoside (4 μ g), 3) FMDV (O type) antigen+white oil (100 μ l), 4) FMDV (O type) antigen+ginsenoside (4 μ g)+white oil (100 μ l).Use after 3 weeks with quadrat method mediating recipe amount booster immunization 1 time.The 2nd week blood sampling behind booster immunization, preparation serum detects serum FMDV specific IgG with indirect sandwich ELISA.With mean+SD (SD) expression testing result.The different persons of subscript letter represent that significant difference is arranged (P＜0.05).
Fig. 2 is: every milliliter of composite adjuvant foot and mouth disease vaccine immune effect figure that contains 500 μ g ginsenosides.The foot and mouth disease vaccine (O type) and the ginsenoside (1000 μ g) of 1 dosage (2ml) are mixed the immune piglet in back (10/group).3 weeks back blood sampling before immunity and after the immunity, preparation serum.Measure serum I HA titre with indirect hemagglutination method.The different persons of subscript letter represent that significant difference is arranged (P＜0.05) in same detection time. go up asterisk (*) person and represent to be significantly higher than same treated animal immunity level (P＜0.05) before.
Fig. 3 is: every milliliter of composite adjuvant foot and mouth disease vaccine immune effect figure that contains 1000 μ g ginsenosides.The foot and mouth disease vaccine (O type) and the ginsenoside (2000 μ g) of 1 dosage (2ml) are mixed the immune piglet in back (11/group).3 weeks back blood sampling before immunity and after the immunity, preparation serum.Measure serum I HA titre with indirect hemagglutination method.The different persons of subscript letter represent that significant difference is arranged (P＜0.05) in same detection time. go up asterisk (*) person and represent to be significantly higher than same treated animal immunity level (P＜0.05) before.
The specific embodiment
For verifying the result of use of composite adjuvant vaccine of the present invention, carry out animal experiment for embodiment with foot and mouth disease (FMD) vaccine.
Example 1: ginsenoside and white oil are in conjunction with the potentiation to foot and mouth disease (FMD) vaccine immunity.
One material and method
1. laboratory animal: 24 of female ICR mices, available from the Shanghai Experimental Animal Center.
2. antigen and adjuvant: antigen is deactivation O type foot and mouth disease virus (FMDV), is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences (LVRI).White oil is available from Hangzhou industrial chemicals company limited.The ginsenoside is HongJiu Co., Ltd's product.
3. vaccine production: at first will be with a small amount of physiological saline solution ginsenoside, O type foot and mouth disease virus (FMDV) antigenic solution with deactivation mixes again, fully mixes with oily adjuvant then, promptly.
4. immunization method: 24 mices are divided into 4 groups at random, 6 every group.Every the each intramuscular injection vaccine of mice 0.2ml injects 2 times, at interval 3 weeks.Adjuvant and antigen dose see Table 1.
Grouping of table 1 animal and processing
| Group | The ginsenoside | Oil | FMDV(O) | Normal saline |
| Ginsenoside ginsenoside+oily normal saline | 4μg4μg | 100μl 100μl | 100μl 100μl 100μl 100μl | 100μl 100μl |
5. blood specimen collection: two exempt from back 2,4,6 week blood sampling.Blood sample is statically placed in 4 ℃ of refrigerator overnight, and 600g is centrifugal, draws serum, is sub-packed in the 0.2ml plastic centrifuge tube-20 ℃ of preservations.
6. specific IgG detects
(1) every hole adds the cattle resisting O-type foot and mouth disease virus antibody (1: 1000) (Lanzhou Veterinary Inst., Chinese Academy of Agricultural Science) of 50 μ l through carbonate buffer solution (pH9.6) dilution on elisa plate, shrouding, and 4 ℃ are spent the night.
(2) with cleaning mixture washing 5 times, each 300 μ l pat dry.Every hole adds 300 μ l phosphate buffers (containing 5% defatted milk+0.05% tween 20) sealing, hatches 2 hours for 37 ℃.
(3) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds the O type foot-and-mouth disease virus antigen of 50 μ l through dilution in 1: 3, and the vibration mixing was hatched 2 hours for 4 ℃.
(4) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds the to be checked serum of 50 μ l through phosphate buffer (containing 0.05% tween 20) 1: 50 dilution, hatches 1 hour for 37 ℃.
(5) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds goat anti-mouse IgG (h+l) antibody (U.S. Betheyl Laboratory company) through dilution in 1: 1000.Hatched 1 hour for 37 ℃.
(6) with phosphate buffer washing 5 times, each 300 μ l pat dry.Every hole adds through 1: 10000 anti-goat IgG FC-HRP of rabbit, hatches 1 hour for 37 ℃.With cleaning mixture washing 5 times, each 300 μ l pat dry.
(7) every hole adds 100 μ l tmb substrate solution.Incubated at room 15 minutes, every hole add 50 μ l, 2 M H2SO4 cessation reactions, and the mixing that vibrates gently.In 15 minutes, be determined at the OD value of 450nm wavelength with microplate reader.
Two results
The vaccine that contains ginsenoside and the two adjuvants of oil vaccine-inducedly goes out higher anti-FMDV antibody (see figure 1) (P＜0.05) than what only contain a kind of adjuvant.
Example 2: contain the test of ginsenoside's's (500 mcg/ml) the immune piglet of oily adjuvant foot-and-mouth disease vaccine (O type)
One material and method
1. laboratory animal: 20 of 40 age in days piglets.
2. vaccine and adjuvant: O type foot-and-mouth disease vaccine is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences (LVRI).Ginsenoside (GS) is provided by HongJiu Co., Ltd.
3. vaccine production: at first will in vaccine, add ginsenoside's solution then, and fully mix, and make the content of ginsenoside in every milliliter of vaccine be respectively 0 (comparing) and 500 micrograms with a small amount of physiological saline solution ginsenoside.
4. immunization method: 20 piglets are divided into 2 groups at random, 10 every group.An intramuscular injection vaccine of every pig 2ml, blood sampling before and after inoculation, separation of serum is sub-packed in the plastic centrifuge tube ,-20 ℃ of preservations.
5. indirect hemagglutination test (IHA): tested serum is put 56 ℃ of deactivations in 30 minutes.Every hole adds 50 μ l diluents on the hemagglutination test plate, adds tested serum 50 μ l at the 1st row again, does 2 times of dilutions then from left to right.Every hole adds the sheep red blood cell (SRBC) of 25 μ l sensitization, 37 ℃, hatches result of determination after 2 hours.
Two results
The composite adjuvant vaccine that contains ECMS and oil induces higher anti-IHA antibody (P＜0.05) than oil-adjuvant vaccine, sees Fig. 2.
Example 3: contain the test of ginsenoside's's (1000 mcg/ml) the immune piglet of oily adjuvant foot-and-mouth disease vaccine (O type)
One material and method
1. laboratory animal: 22 of 40 age in days piglets.
2. vaccine and adjuvant: O type foot-and-mouth disease vaccine is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences (LVRI).Ginsenoside (GS) is provided by HongJiu Co., Ltd.
3. vaccine production: at first will in vaccine, add ginsenoside's solution then, and fully mix, and make the content of ginsenoside in every milliliter of vaccine be respectively 0 (comparing) and 1000 micrograms with a small amount of physiological saline solution ginsenoside.
4. immunization method: 22 piglets are divided into 2 groups at random, 11 every group.An intramuscular injection vaccine of every pig 2ml, blood sampling before and after inoculation, separation of serum is sub-packed in the plastic centrifuge tube ,-20 ℃ of preservations.
5. indirect hemagglutination test (IHA): tested serum is put 56 ℃ of deactivations in 30 minutes.Every hole adds 50 μ l diluents on the hemagglutination test plate, adds tested serum 50 μ l at the 1st row again, does 2 times of dilutions then from left to right.Every hole adds the sheep red blood cell (SRBC) of 25 μ l sensitization, 37 ℃, hatches result of determination after 2 hours.
Two results
The two Adjuvanted vaccines that contain ECMS and oil induce higher anti-IHA antibody (P＜0.05) than oil-adjuvant vaccine, see Fig. 3.
Claims (2)
1. vaccine that contains composite adjuvant is characterized in that: contain white-oil adjuvant and ginsenoside's adjuvant in the described vaccine, the content of ginsenoside's adjuvant is 20～1000 micrograms in every milliliter of vaccine, and white-oil adjuvant accounts for 1/2nd of vaccine volume.
2. a kind of according to claim 1 composite adjuvant vaccine that contains is characterized in that it is prepared from by following steps:
(1) ginsenoside is dissolved in a small amount of normal saline;
(2) ginsenoside's solution and antigen are mixed the fully emulsified mixing under the effect of emulsifying agent with this mixture and white-oil adjuvant then; Or earlier white-oil adjuvant and ginsenoside's emulsifying soln are mixed, mix with antigen emulsifying then; Or emulsifying mixes add ginsenoside's liquid in conventional white-oil adjuvant vaccine after.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103751775A (en) * | 2013-12-31 | 2014-04-30 | 浙江大学 | Ginsenoside-containing vegetable oil adjuvant and preparation method and application thereof |
| CN104857511B (en) * | 2015-02-13 | 2018-03-30 | 浙江大学 | Thinner for vaccine containing panaxoside |
| CN104922667B (en) * | 2015-07-09 | 2018-07-27 | 宁波荣安生物药业有限公司 | A kind of Antirabic Vaccine and preparation method thereof |
| CN113797328A (en) * | 2020-06-16 | 2021-12-17 | 浙江洪晟生物科技股份有限公司 | Compound adjuvant for animal vaccine, preparation method and vaccine |
| CN112220922A (en) * | 2020-10-19 | 2021-01-15 | 浙江美保龙生物技术有限公司 | Porcine pseudorabies live vaccine immunity-enhancing diluent and use method thereof |
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| CN1367022A (en) * | 2002-01-16 | 2002-09-04 | 浙江大学 | Ginseng total saponin or monomer saponin RbI vaccine immunological adjuvant application |
| CN1136010C (en) * | 1999-12-23 | 2004-01-28 | 中国兽药监察所 | Making process of subunit antigen vaccine cream |
| US6861410B1 (en) * | 2002-03-21 | 2005-03-01 | Chiron Corporation | Immunological adjuvant compositions |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1136010C (en) * | 1999-12-23 | 2004-01-28 | 中国兽药监察所 | Making process of subunit antigen vaccine cream |
| CN1367022A (en) * | 2002-01-16 | 2002-09-04 | 浙江大学 | Ginseng total saponin or monomer saponin RbI vaccine immunological adjuvant application |
| US6861410B1 (en) * | 2002-03-21 | 2005-03-01 | Chiron Corporation | Immunological adjuvant compositions |
Non-Patent Citations (1)
| Title |
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| 胡松华,林锋强.人参皂甙Rb1的免疫佐剂作用.《中国兽医学报》.2003,第23卷(第5期),480-482. * |
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