CN102836427A - 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 the 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 betides the season of hatching duckling, in case take place, in the duckling crowd, propagates very soon, and sickness rate can reach 100%.Its clinical manifestation mainly is an opisthotonus, and pathological changes is mainly the liver enlargement and hemorrhage speckle is arranged.Primary disease betides the U.S. the earliest, and is popular successively in states such as Britain, Canada, Germany, Italy, India thereafter.China from 1958 since Shanghai is separated to DHV DHV first, the popular generation of many provinces and cities in the whole nation in succession causes enormous economic loss, has become influence and has supported the important threat that duck is already developed in a healthy way.
DHV has 3 serotypes, i.e. I, II, III, and the DVH virus I-type belongs to Picornaviridae, and is spherical in shape or type spherical, and diameter is at 20-40NM, no cyst membrane, no blood clotting property can be bred at duck, chicken, goose embryo allantoic cavity.Viral resistance is strong, but long period survival in natural environment, and present ubiquitous duck viral hepatitis is caused by DHV I type.DVH virus II type belongs to Astrovirus, and DVH-III belongs to picornavirus.There is not cross-protection between three kinds of serotypes of DVH virus.Duck viral hepatitis virus does not have any dependency with DHB.
Duck viral hepatitis spreads in China in recent years; Seriously hinder China and supported duck sound development already; The duck crowd who has occurred using I type duck viral hepatitis attenuated vaccine or high immunity yolk antibody to prevent or failed to respond to any medical treatment is broken out the scorching phenomenon of doubtful duck liver, suspects to be that scorching variant of I type duck liver or novel duck liver are scorching.Use the attenuated vaccine immunity duckling, receive the influence of immune function of duckling own and maternal antibody, caused the part of duck viral hepatitis to break out, caused tremendous 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 method for preparing of duck viral hepatitis immunostimulating complex.
In order to realize above purpose, 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: breed in the chick embryo allantoic cavity with duck viral hepatitis virus inoculation 9 ages in days; Be placed on and hatch in the incubator to the 5th～7 day; If during dead, dysplasia; Carry out the results of allantoic fluid at once, place-80 ℃ of refrigerators to preserve;
(2) cultivate virus: with the virus of the duck viral hepatitis in the step (1) allantoic fluid inoculation monolayer DEF, 37 ℃ of cultivations, every at a distance from 10～12h observation of cell; If cytopathy reaches at 80% o'clock, harvesting ,-20 ℃ with room temperature multigelation 3 times; Centrifugal 20～the 40min of 5000r/min gets supernatant, abandons deposition; Supernatant is at the centrifugal 1.5～2.5h of 14000r/min, and viral level reaches 10
7EID
50During/0.1mL, collect duck viral hepatitis virus;
(3) preparation of lipid mixtures: it is among 20% the mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes dissolving fully, is diluted to 8～12mg/mL, packing, and 2～8 ℃ of preservations are subsequent use;
(4) preparation of saponin QuilA solution: QuilA is dissolved in 10mL with the 0.01g saponin, and pH is in 6.8 the PBS buffer, final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: pH is that 7.2 PBS buffer virus dilution liquid is 10mg/mL to protein concentration, and adding concentration and be 20% mega-10 is 2% to its final concentration, room temperature effect 2～3h; Viral liquid is taped against on the sucrose pad; The upper strata is 10% sucrose (containing 0.4%mega-10) of PBS preparation, and the bottom is 30% a sucrose pad with the PBS preparation, the centrifugal 2～3h of 14000r/min; Collect the sucrose layer of sample layer and 10%; Add saponin QuilA and lipid mixtures, wherein the saponin mass content is 0.1～0.2%, and the concentration of lipid mixtures in complex is 50～100uL/mL; This solution is added the dialysis of carrying out 48～54h in the bag filter, promptly make the 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 further introduced in detail.
Embodiment 1
(1) preparation of duck viral hepatitis virus allantoic fluid: breed in the chick embryo allantoic cavity with duck viral hepatitis virus inoculation 9 ages in days, be placed on when hatching in the incubator, observe Embryo Gallus domesticus and dead or dysplasia or edema whether occur to the 5th day.If when dead or dysplasia and edema, carry out the results of allantoic fluid at once.The allantoic fluid of collecting is placed on-80 ℃ of refrigerators and preserves.Whether occur dead or dysplasia then can not regather if occur to observe Embryo Gallus domesticus on the 7th day, discard whole Embryo Gallus domesticus;
(2) cultivate virus: with ready duck viral hepatitis virus allantoic fluid inoculation monolayer DEF, 37 ℃ of cultivations are whenever carried out observation of cell at a distance from 12h; If cytopathy reaches at 80% o'clock, harvesting ,-20 ℃ with room temperature multigelation 3 times; The centrifugal 30min of 5000r/min gets supernatant, abandons deposition; Supernatant is at the centrifugal 2h of 14000r/min, and the sample that takes a morsel carries out the negative staining electron microscopic observation.Viral level reaches 10
7EID
50/ 0.1mL could be used as down the step experiment;
(3) preparation of lipid mixtures stock solution: it is among 20% the mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes dissolving fully, and being diluted to final concentration is 8mg/mL, packing, and 8 ℃ of preservations are subsequent use;
(4) preparation of saponin QuilA solution: it is in 6.8 the PBS buffer that 0.01g saponin QuilA is dissolved in 10mL pH, final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: using pH is that 7.2 PBS buffer is diluted to protein concentration with viral liquid and is 10mg/mL, and adding concentration and be 20 %mega-10 is 2% to its final concentration, room temperature effect 3h; Viral liquid is taped against on the sucrose pad; The upper strata is 10% sucrose (containing 0.4%mega-10) of PBS preparation, and the bottom is 30% a sucrose pad with the PBS preparation, the centrifugal 2h of 14000r/min; Collect the sucrose layer of sample layer and 10%; The saponin QuilA and the lipid mixtures 50uL/mL that add 0.1% amount add the dialysis of carrying out 48h in the bag filter with this solution, promptly make the duck viral hepatitis immunostimulating complex.
Embodiment 2
(1) preparation of duck viral hepatitis virus allantoic fluid: breed in the chick embryo allantoic cavity with duck viral hepatitis virus inoculation 9 ages in days, be placed on when hatching in the incubator, observe Embryo Gallus domesticus and dead or dysplasia or edema whether occur to the 6th day.If when dead or dysplasia and edema, carry out the results of allantoic fluid at once.The allantoic fluid of collecting is placed on-80 ℃ of refrigerators and preserves;
(2) cultivate virus: with ready duck viral hepatitis virus allantoic fluid inoculation monolayer DEF, 37 ℃ of cultivations are whenever carried out observation of cell at a distance from 10h; If cytopathy reaches at 80% o'clock, harvesting ,-20 ℃ with room temperature multigelation 3 times; The centrifugal 40min of 5000r/min gets supernatant, abandons deposition; Supernatant is at the centrifugal 1.5h of 14000r/min, and the sample that takes a morsel carries out the negative staining electron microscopic observation.Viral level is used.Viral level reaches 10
7EID
50/ 0.1mL could be used as down the step experiment;
(3) preparation of lipid mixtures stock solution: it is among 20% the mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes dissolving fully, and being diluted to final concentration is the 10mg/mL packing, and 5 ℃ of preservations are subsequent use;
(4) preparation of saponin QuilA solution: it is in 6.8 the PBS buffer that 0.01g saponin QuilA is dissolved in 10mL pH, final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: using pH is that 7.2 PBS buffer is diluted to protein concentration with viral liquid and is 10mg/mL, and adding 20%mega-10 is 2% to final concentration, room temperature effect 2.5h; Viral liquid is taped against on the sucrose pad; The upper strata is 10% sucrose (containing 0.5%mega-10) of PBS preparation, and the bottom is 30% a sucrose pad with the PBS preparation, the centrifugal 2.5h of 14000r/min; Collect the sucrose layer of sample layer and 10%; The saponin QuilA and the lipid mixtures 100uL/mL that add 0.2% amount add the dialysis of carrying out 50h in the bag filter with this solution, promptly make the duck viral hepatitis immunostimulating complex.
Embodiment 3
(1) preparation of duck viral hepatitis virus allantoic fluid: breed in the chick embryo allantoic cavity with duck viral hepatitis virus inoculation 9 ages in days, be placed on when hatching in the incubator, observe Embryo Gallus domesticus and dead or dysplasia or edema whether occur to the 7th day.If when dead or dysplasia and edema, carry out the results of allantoic fluid at once.The allantoic fluid of collecting is placed on-80 ℃ of refrigerators and preserves;
(2) cultivate virus: with ready duck viral hepatitis virus allantoic fluid inoculation monolayer DEF, 37 ℃ of cultivations are whenever carried out observation of cell at a distance from 11h; If cytopathy reaches at 80% o'clock, harvesting ,-20 ℃ with room temperature multigelation 3 times; The centrifugal 20min of 5000r/min gets supernatant, abandons deposition; Supernatant is at the centrifugal 2.5h of 14000r/min, and the sample that takes a morsel carries out the negative staining electron microscopic observation.Viral level reaches 10
7EID
50/ 0.1mL could be used as down the step experiment;
(3) preparation of lipid mixtures stock solution: it is among 20% the mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes dissolving fully, and being diluted to final concentration is 12mg/mL, packing, and 2 ℃ of preservations are subsequent use;
(4) preparation of saponin QuilA solution: it is in 6.8 the PBS buffer that 0.01g saponin QuilA is dissolved in 10mL pH, final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: using pH is that 7.2 PBS buffer is diluted to protein concentration with viral liquid and is 10mg/mL, and adding 20%mega-10 is 2% to final concentration, room temperature effect 2h; Viral liquid is taped against on the sucrose pad; The upper strata is 10% sucrose (containing 0.4%mega-10) of PBS preparation, and the bottom is 30% a sucrose pad with the PBS preparation, the centrifugal 3h of 14000r/min; Collect the sucrose layer of sample layer and 10%; The saponin QuilA and the lipid mixtures 80uL/mL that add 0.15% amount add the dialysis of carrying out 54h in the bag filter with this solution, promptly make the duck viral hepatitis immunostimulating complex.
Claims (7)
1. duck viral hepatitis immunostimulating complex is characterized in that: this complex is prepared from the sucrose pad with saponin QuilA, lipid mixtures in duck viral hepatitis virus.
2. duck viral hepatitis immunostimulating complex as claimed in claim 1 is characterized in that: the mass content of saponin QuilA is 0.1～0.2%.
3. duck viral hepatitis immunostimulating complex as claimed in claim 1 is characterized in that: lipid mixtures is that 20% mega-10 mixes by PHOSPHATIDYL ETHANOLAMINE, cholesterol and concentration.
4. duck viral hepatitis immunostimulating complex as claimed in claim 3 is characterized in that: the concentration of lipid mixtures is 50～100uL/mL.
5. the method for preparing of a duck viral hepatitis immunostimulating complex as claimed in claim 1, it is characterized in that: preparation process comprises:
(1) preparation of duck viral hepatitis virus allantoic fluid: with the chick embryo allantois of duck viral hepatitis virus inoculation 9 ages in days
Breed in the chamber, be placed on and hatch in the incubator,, place-80 ℃ of refrigerators to preserve if when dead or dysplasia, carry out the results of allantoic fluid at once to the 5th～7 day; If appearance that Embryo Gallus domesticus surpasses the 7th day is dead or dysplasia should discard Embryo Gallus domesticus at once;
(2) cultivate virus: with the virus of the duck viral hepatitis in the step (1) allantoic fluid inoculation monolayer DEF, 37 ℃ of cultivations, every at a distance from 10～12h observation of cell; If cytopathy reaches at 80% o'clock, harvesting ,-20 ℃ with room temperature multigelation 3 times; Centrifugal 20～the 40min of 5000r/min gets supernatant, abandons deposition; Supernatant obtains duck viral hepatitis virus at the centrifugal 1.5～2.5h of 14000r/min;
(3) preparation of lipid mixtures: it is among 20% the mega-10 that PHOSPHATIDYL ETHANOLAMINE and cholesterol are added to concentration, makes dissolving fully, is diluted to 8～12mg/mL, packing, and 2～8 ℃ of preservations are subsequent use;
(4) preparation of saponin QuilA solution: 0.01g saponin QuilA is dissolved in the 10mL PBS buffer final concentration 1mg/mL;
(5) preparation of duck viral hepatitis immunostimulating complex: phosphate buffer PBS virus dilution liquid is 10mg/mL to protein concentration, and adding concentration and be 20% mega-10 is 2% to its final concentration, room temperature effect 2～3h; Viral liquid is taped against on the sucrose pad; The upper strata is 10% sucrose (containing 0.4%mega-10) of PBS preparation, and the bottom is 30% a sucrose pad with the PBS preparation, the centrifugal 2～3h of 14000r/min; Collect the sucrose layer of sample layer and 10%; Add saponin QuilA and lipid mixtures, wherein the saponin mass content is 0.1～0.2%, and the concentration of lipid mixtures in complex is 50～100uL/mL; This solution is added the dialysis of carrying out 48～54h in the bag filter, promptly make the duck viral hepatitis immunostimulating complex.
6. the method for preparing of duck viral hepatitis immunostimulating complex as claimed in claim 5 is characterized in that: duck viral hepatitis viral level>=10 that step (2) obtains
7EID
50/ 0.1mL.
7. the method for preparing of duck viral hepatitis immunostimulating complex as claimed in claim 5 is characterized in that: phosphate buffer PBS pH is respectively 6.8 and 7.2 in step (4) and the step (5).
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 梅兴国: "《微载体药物递送系统》", 30 November 2009, article "微载体药物递送系统", pages: 385-386 * |
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Inventor after: Wu Hongyun Inventor after: Han Gaihui Inventor after: Xu Jin Inventor after: Liang Jun Inventor after: Zhang Yuli Inventor before: Wu Hongyun Inventor before: Han Gaihui Inventor before: Xu Jin |
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Effective date of registration: 20160302 Address after: 450061 Xingang Avenue, Xinzheng port, Henan, Zhengzhou Patentee after: Henan Hou Yi bioengineering Limited by Share Ltd Address before: 451162 Xingang, Henan, Zhengzhou, Hong Kong airport on the eastern side of the road on the eastern side of Zhengzhou Patentee before: Zhengzhou Houyi Pharmaceutical Co., Ltd. |
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