CN102836427B - Duck virus hepatitis (DVH) immunostimulating complex (ISCOM) and preparation method thereof - Google Patents
Duck virus hepatitis (DVH) immunostimulating complex (ISCOM) and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a duck virus hepatitis (DVH) immunostimulating complex (ISCOM). Particularly, chicken embryo is inoculated with viral hepatitis virus, allantoic fluid is collected, the chicken embryo is cultured in single-layer duck embryo fibroblasts so as to obtain virus liquid with virus content of DVH virus being no less than 10<7> EID50/0.1ml, then the virus liquid is dropped on a sucrose pad and centrifuged, a sample layer and a 10% sucrose layer are collected, saponin QuilA and lipid mixture are added, dialysis is carried out, and therefore the DVH ISCOM is manufactured. The ISCOM is high in immunological activity and little in toxic and side effect.
Description
Technical field
The present invention relates to a kind of duck viral hepatitis immunostimulating complex and preparation method thereof, belong to veterinary biologics technical field.
Background technology
Duck viral hepatitis (Duck virus Hepatitis is called for short DVH) is a kind of acute fatal infectious disease of duckling.This disease betide hatching duckling season, once occur, in duckling group propagate very fast, sickness rate can reach 100%.Its clinical manifestation is mainly opisthotonus, and pathological changes is mainly liver enlargement and has hemorrhage speckle.Primary disease betides the U.S. the earliest, popular successively in states such as Britain, Canada, Germany, Italy, India thereafter.China is from 1958 since Shanghai is separated to DHV DHV first, and the popular generation of many provinces and cities in the whole nation in succession, causes huge economic loss, has become impact and has supported the important threat that duck industry develops in a healthy way.
DHV has 3 serotypes, i.e. I, II, III, and DVH virus I-type belongs to Picornaviridae, spherical in shape or class is spherical, and diameter is at 20-40NM, without cyst membrane, without blood clotting, can be in duck, chicken, goose embryo allantoic cavity propagation.Viral resistance is strong, in natural environment, can survive the long period, and current ubiquitous duck viral hepatitis is caused by DHV I type.DVH virus II type belongs to Astrovirus, and DVH-III belongs to picornavirus.Between three kinds of serotypes of DVH virus without cross-protection.Duck viral hepatitis virus and DHB are without any dependency.
Duck viral hepatitis spreads in China in recent years, seriously hinder the sound development of the foster duck industry of China, the duck group who has occurred using I type duck viral hepatitis attenuated vaccine or high immunity yolk antibody prevention or fail to respond to any medical treatment is broken out the phenomenon of doubtful duck liver inflammation, suspects for the scorching variant of I type duck liver or Novel duck hepatitis.Use attenuated vaccine immunity duckling, be subject to the impact of the immune function of duckling own and maternal antibody, caused the part of duck viral hepatitis to break out, caused huge loss to aquaculture.
Summary of the invention
The present invention also aims to provide a kind of duck viral hepatitis immunostimulating complex.
The present invention also aims to provide a kind of preparation method of duck viral hepatitis immunostimulating complex.
In order to realize above object, the preparation process of duck viral hepatitis immunostimulating complex of the present invention is: be prepared as example with I type duck viral hepatitis,
(1) preparation of duck viral hepatitis virus allantoic fluid: will breed in the chick embryo allantoic cavity of duck viral hepatitis virus inoculation 9 ages in days, be placed in incubator and hatch to 5th～7 days, during if there is death, dysplasia, carry out at once the results of allantoic fluid, be placed in-80 ℃ of refrigerators and preserve;
(2) cultivate virus: by the duck viral hepatitis virus allantoic fluid inoculation monolayer DEF in step (1), 37 ℃ of cultivations, every 10～12h observation of cell, if when cytopathy reaches 80%, harvesting,-20 ℃ and room temperature multigelation 3 times, centrifugal 20～the 40min of 5000r/min, gets supernatant, abandons precipitation, supernatant is at the centrifugal 1.5～2.5h of 14000r/min, and viral level reaches 10
7eID
50when/0.1mL, collect duck viral hepatitis virus;
(3) preparation of lipid mixtures: it is in 20% mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes to dissolve completely, is diluted to 8～12mg/mL, subpackage, 2～8 ℃ save backup;
(4) preparation of saponin QuilA solution: 0.01g saponin QuilA is dissolved in to 10mL, in the PBS buffer that pH is 6.8, final concentration 1mg/mL;
(5) the PBS buffer virus dilution liquid that the preparation of duck viral hepatitis immunostimulating complex: pH is 7.2 is 10mg/mL to protein concentration, to add concentration be 20% mega-10 to its final concentration be 2%, room temperature effect 2～3h, virus liquid is taped against on sucrose pad, upper strata is 10% sucrose (containing 0.4%mega-10) of PBS preparation, bottom is the 30% sucrose pad with PBS preparation, centrifugal 2～the 3h of 14000r/min, collect the sucrose layer of sample layer and 10%, add saponin QuilA and lipid mixtures, wherein saponin mass content is 0.1～0.2%, the concentration of lipid mixtures in complex is 50～100uL/mL, this solution is added to the dialysis of carrying out 48～54h in bag filter, make duck viral hepatitis immunostimulating complex.
Complex toxic and side effects of the present invention is little, the immune antibody height of tiring.
The specific embodiment:
Below in conjunction with some specific embodiment, the present invention is described in further detail.
Embodiment 1
(1) preparation of duck viral hepatitis virus allantoic fluid: will breed in the chick embryo allantoic cavity of duck viral hepatitis virus inoculation 9 ages in days, be placed on while hatching to the 5th day in incubator, observe Embryo Gallus domesticus and whether occur death or dysplasia or edema.During if there is death or dysplasia and edema, carry out at once the results of allantoic fluid.The allantoic fluid of collecting is placed on-80 ℃ of refrigerators and preserves.If not there is observing Embryo Gallus domesticus and whether occur death or dysplasia for the 7th day, can not regather, discard whole Embryo Gallus domesticus;
(2) cultivate virus: by ready duck viral hepatitis virus allantoic fluid inoculation monolayer DEF, 37 ℃ of cultivations, carry out observation of cell every 12h, if when cytopathy reaches 80%, harvesting,-20 ℃ and room temperature multigelation 3 times, the centrifugal 30min of 5000r/min, gets supernatant, abandons precipitation, supernatant is at the centrifugal 2h of 14000r/min, and the sample that takes a morsel carries out negative staining electron microscopic observation.Viral level reaches 10
7eID
50/ 0.1mL just can be used as lower step experiment;
(3) preparation of lipid mixtures stock solution: it is in 20% mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes to dissolve completely, and being diluted to final concentration is 8mg/mL, subpackage, 8 ℃ save backup;
(4) preparation of saponin QuilA solution: it is in 6.8 PBS buffer that 0.01g saponin QuilA is dissolved in to 10mL pH, final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: it is 10mg/mL that virus liquid is diluted to protein concentration by the PBS buffer that is 7.2 with pH, to add concentration be 20 %mega-10 to its final concentration be 2%, room temperature effect 3h, virus liquid is taped against on sucrose pad, upper strata is 10% sucrose (containing 0.4%mega-10) of PBS preparation, bottom is the 30% sucrose pad with PBS preparation, the centrifugal 2h of 14000r/min, collect the sucrose layer of sample layer and 10%, add saponin QuilA and the lipid mixtures 50uL/mL of 0.1% amount, this solution is added to the dialysis of carrying out 48h in bag filter, make duck viral hepatitis immunostimulating complex.
Embodiment 2
(1) preparation of duck viral hepatitis virus allantoic fluid: will breed in the chick embryo allantoic cavity of duck viral hepatitis virus inoculation 9 ages in days, be placed on while hatching to the 6th day in incubator, observe Embryo Gallus domesticus and whether occur death or dysplasia or edema.During if there is death or dysplasia and edema, carry out at once the results of allantoic fluid.The allantoic fluid of collecting is placed on-80 ℃ of refrigerators and preserves;
(2) cultivate virus: by ready duck viral hepatitis virus allantoic fluid inoculation monolayer DEF, 37 ℃ of cultivations, carry out observation of cell every 10h, if when cytopathy reaches 80%, harvesting,-20 ℃ and room temperature multigelation 3 times, the centrifugal 40min of 5000r/min, gets supernatant, abandons precipitation, supernatant is at the centrifugal 1.5h of 14000r/min, and the sample that takes a morsel carries out negative staining electron microscopic observation.Viral level is used.Viral level reaches 10
7eID
50/ 0.1mL just can be used as lower step experiment;
(3) preparation of lipid mixtures stock solution: it is in 20% mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes to dissolve completely, and being diluted to final concentration is 10mg/mL subpackage, and 5 ℃ save backup;
(4) preparation of saponin QuilA solution: it is in 6.8 PBS buffer that 0.01g saponin QuilA is dissolved in to 10mL pH, final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: it is 10mg/mL that virus liquid is diluted to protein concentration by the PBS buffer that is 7.2 with pH, add 20%mega-10 to final concentration be 2%, room temperature effect 2.5h, virus liquid is taped against on sucrose pad, upper strata is 10% sucrose (containing 0.5%mega-10) of PBS preparation, bottom is the 30% sucrose pad with PBS preparation, the centrifugal 2.5h of 14000r/min, collect the sucrose layer of sample layer and 10%, add saponin QuilA and the lipid mixtures 100uL/mL of 0.2% amount, this solution is added to the dialysis of carrying out 50h in bag filter, make duck viral hepatitis immunostimulating complex.
Embodiment 3
(1) preparation of duck viral hepatitis virus allantoic fluid: will breed in the chick embryo allantoic cavity of duck viral hepatitis virus inoculation 9 ages in days, be placed on while hatching to the 7th day in incubator, observe Embryo Gallus domesticus and whether occur death or dysplasia or edema.During if there is death or dysplasia and edema, carry out at once the results of allantoic fluid.The allantoic fluid of collecting is placed on-80 ℃ of refrigerators and preserves;
(2) cultivate virus: by ready duck viral hepatitis virus allantoic fluid inoculation monolayer DEF, 37 ℃ of cultivations, carry out observation of cell every 11h, if when cytopathy reaches 80%, harvesting,-20 ℃ and room temperature multigelation 3 times, the centrifugal 20min of 5000r/min, gets supernatant, abandons precipitation, supernatant is at the centrifugal 2.5h of 14000r/min, and the sample that takes a morsel carries out negative staining electron microscopic observation.Viral level reaches 10
7eID
50/ 0.1mL just can be used as lower step experiment;
(3) preparation of lipid mixtures stock solution: it is in 20% mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes to dissolve completely, and being diluted to final concentration is 12mg/mL, subpackage, 2 ℃ save backup;
(4) preparation of saponin QuilA solution: it is in 6.8 PBS buffer that 0.01g saponin QuilA is dissolved in to 10mL pH, final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: it is 10mg/mL that virus liquid is diluted to protein concentration by the PBS buffer that is 7.2 with pH, add 20%mega-10 to final concentration be 2%, room temperature effect 2h, virus liquid is taped against on sucrose pad, upper strata is 10% sucrose (containing 0.4%mega-10) of PBS preparation, bottom is the 30% sucrose pad with PBS preparation, the centrifugal 3h of 14000r/min, collect the sucrose layer of sample layer and 10%, add saponin QuilA and the lipid mixtures 80uL/mL of 0.15% amount, this solution is added to the dialysis of carrying out 54h in bag filter, make duck viral hepatitis immunostimulating complex.
Claims (4)
1. a duck viral hepatitis immunostimulating complex, is characterized in that: this complex is prepared from saponin QuilA, lipid mixtures by duck viral hepatitis virus on sucrose pad; Preparation process comprises:
(1) preparation of duck viral hepatitis virus allantoic fluid: will breed in the chick embryo allantoic cavity of duck viral hepatitis virus inoculation 9 ages in days, be placed in incubator and hatch to 5th～7 days, during if there is death or dysplasia, carry out at once the results of allantoic fluid, be placed in-80 ℃ of refrigerators and preserve; If exceeding the 7th day, Embryo Gallus domesticus do not occur that death or dysplasia should discard Embryo Gallus domesticus at once;
(2) cultivate virus: by the duck viral hepatitis virus allantoic fluid inoculation monolayer DEF in step (1), 37 ℃ of cultivations, every 10～12h observation of cell, if when cytopathy reaches 80%, harvesting,-20 ℃ and room temperature multigelation 3 times, centrifugal 20～the 40min of 5000r/min, gets supernatant, abandons precipitation, supernatant, at the centrifugal 1.5～2.5h of 14000r/min, obtains duck viral hepatitis virus;
(3) preparation of lipid mixtures: it is in 20% mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes to dissolve completely, is diluted to 8～12mg/mL, subpackage, 2～8 ℃ save backup;
(4) preparation of saponin QuilA solution: 0.01g saponin QuilA is dissolved in 10mL PBS buffer to final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: phosphate buffer PBS virus dilution liquid is 10mg/mL to protein concentration, to add concentration be 20% mega-10 to its final concentration be 2%, room temperature effect 2～3h, virus liquid is taped against on sucrose pad, upper strata is 10% sucrose of PBS preparation, wherein contain 0.4%mega-10, bottom is the 30% sucrose pad with PBS preparation, centrifugal 2～the 3h of 14000r/min, collect the sucrose layer of sample layer and 10%, add saponin QuilA and lipid mixtures, wherein the mass content of saponin in complex is 0.1～0.2%, the concentration of lipid mixtures in complex is 50～100uL/mL, this solution is added to the dialysis of carrying out 48～54h in bag filter, make duck viral hepatitis immunostimulating complex.
2. a preparation method for duck viral hepatitis immunostimulating complex, is characterized in that: preparation process comprises:
(1) preparation of duck viral hepatitis virus allantoic fluid: will breed in the chick embryo allantoic cavity of duck viral hepatitis virus inoculation 9 ages in days, be placed in incubator and hatch to 5th～7 days, during if there is death or dysplasia, carry out at once the results of allantoic fluid, be placed in-80 ℃ of refrigerators and preserve; If exceeding the 7th day, Embryo Gallus domesticus do not occur that death or dysplasia should discard Embryo Gallus domesticus at once;
(2) cultivate virus: by the duck viral hepatitis virus allantoic fluid inoculation monolayer DEF in step (1), 37 ℃ of cultivations, every 10～12h observation of cell, if when cytopathy reaches 80%, harvesting,-20 ℃ and room temperature multigelation 3 times, centrifugal 20～the 40min of 5000r/min, gets supernatant, abandons precipitation, supernatant, at the centrifugal 1.5～2.5h of 14000r/min, obtains duck viral hepatitis virus;
(3) preparation of lipid mixtures: it is in 20% mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes to dissolve completely, is diluted to 8～12mg/mL, subpackage, 2～8 ℃ save backup;
(4) preparation of saponin QuilA solution: 0.01g saponin QuilA is dissolved in 10mL PBS buffer to final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: phosphate buffer PBS virus dilution liquid is 10mg/mL to protein concentration, to add concentration be 20% mega-10 to its final concentration be 2%, room temperature effect 2～3h, virus liquid is taped against on sucrose pad, upper strata is 10% sucrose of PBS preparation, wherein contain 0.4%mega-10, bottom is the 30% sucrose pad with PBS preparation, centrifugal 2～the 3h of 14000r/min, collect the sucrose layer of sample layer and 10%, add saponin QuilA and lipid mixtures, wherein the mass content of saponin in complex is 0.1～0.2%, the concentration of lipid mixtures in complex is 50～100uL/mL, this solution is added to the dialysis of carrying out 48～54h in bag filter, make duck viral hepatitis immunostimulating complex.
3. the preparation method of duck viral hepatitis immunostimulating complex as claimed in claim 3, is characterized in that: duck viral hepatitis viral level>=10 that step (2) obtains
7eID
50/ 0.1mL.
4. the preparation method of duck viral hepatitis immunostimulating complex as claimed in claim 3, is characterized in that: in step (4) and step (5), phosphate buffer PBS pH is respectively 6.8 and 7.2.
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| CN101475627A (en) * | 2008-12-11 | 2009-07-08 | 天津瑞普生物技术股份有限公司 | Preparation of transfer factor against swine fever |
| CN101954079A (en) * | 2010-09-21 | 2011-01-26 | 江苏省农业科学院 | Vaccine adjuvant of swine mycoplasmal pneumonia live vaccine, and preparation method and application thereof |
| CN102517331A (en) * | 2011-12-26 | 2012-06-27 | 武汉中博生物股份有限公司 | 2 type subunit vaccine for porcine circovirus as well as preparation method and application thereof |
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| CN101475627A (en) * | 2008-12-11 | 2009-07-08 | 天津瑞普生物技术股份有限公司 | Preparation of transfer factor against swine fever |
| CN101954079A (en) * | 2010-09-21 | 2011-01-26 | 江苏省农业科学院 | Vaccine adjuvant of swine mycoplasmal pneumonia live vaccine, and preparation method and application thereof |
| CN102517331A (en) * | 2011-12-26 | 2012-06-27 | 武汉中博生物股份有限公司 | 2 type subunit vaccine for porcine circovirus as well as preparation method and application thereof |
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| 梅兴国.微载体药物递送系统.《微载体药物递送系统》.2009,第385-386页. * |
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