CN101757619A - Veterinary rabies inactivated vaccine and preparation method thereof - Google Patents
Veterinary rabies inactivated vaccine and preparation method thereof Download PDFInfo
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- CN101757619A CN101757619A CN200910309555A CN200910309555A CN101757619A CN 101757619 A CN101757619 A CN 101757619A CN 200910309555 A CN200910309555 A CN 200910309555A CN 200910309555 A CN200910309555 A CN 200910309555A CN 101757619 A CN101757619 A CN 101757619A
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Abstract
The invention relates to a veterinary rabies inactivated vaccine and a preparation method thereof, the inactivated vaccine is rabies inactivated virus using liposomes as adjuvant, wherein the preparation method thereof includes the following steps of preparing rabies virus solution, concentrating and purifying the virus solution, inactivating the virus, preparing the vaccine, split-charging and packing the vaccine. The vaccine of the invention has the advantages of stable performance, good safety, quick and strong immunization function for immunized animals, and high protection rates and the like. The vaccine of the invention is veterinary vaccine, effectively prevents rabies virus spreading radically, thereby preventing and controlling rabies virus infection more effectively.
Description
Technical field
The present invention relates to a kind of veterinary rabies inactivated vaccine, also relate to the preparation method of this veterinary rabies inactivated vaccine simultaneously, belong to the live vaccine technical field.
Background technology
From 2001～2006 years, China's rabies year death toll was respectively 854,1159,1980,2651,2545,2649 examples.From above data as can be seen, run mad and death toll every year is all rising.These data show that rabies become the public health problem that Chinese society can not be ignored again.Take place and in the groove in people's rabies, wild animal is the main reservoir of primary disease, appearance is healthy and animal that carry virus also is the source of infection, but dog plays an important role carrying and propagate in the rabies, be human poultry infection's topmost source, the people runs mad and is propagated to the mankind by dog more than 93%.Along with the increase of raising pets amounts such as family dog, cat, house pet will more and more become for the people to infect rabic important source.
What at present infect that rabies mainly take for the people is that the people infects the immunity of back to the people; yet will fundamentally antirabic generation; control and elimination rabies; must carry out comprehensive comprehensive prevention measure to dog and wild animal and house pet; strengthen the monitoring management dynamics; improve their immune coverage rate, regularly detect its antibody titer and whether can reach immunoprotection level, booster immunization.Therefore developing veterinary rabies vaccine safely and effectively, is antirabic key, has become the task of top priority.
Summary of the invention
The object of the present invention is to provide a kind of veterinary rabies inactivated vaccine, with propagation from root blocking-up rabies virus.
The present invention also aims to provide a kind of preparation method of veterinary rabies inactivated vaccine.
To achieve these goals, technical program of the present invention lies in having adopted a kind of veterinary rabies inactivated vaccine, this vaccine is by the rabies virus liquid of deactivation and be aided with adjuvant and be prepared into, and the concentration of viral liquid is 1.0683 * 10 in the described vaccine
6TCID
50/ 0.03ml, adjuvant are liposome solutions, and wherein the concentration of phospholipid is 33mmol, and the concentration of cholesterol is 33mmol.
Wherein employed rabies virus be the SAD strain through former generation hamster kidney cell be the Vnukovo-32 Strain after generation cultivating the 40-107 that goes down to posterity under 32 ℃ of conditions, derive from Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences.The ablation method of Vnukovo-32 is HEPES1mol/LpH7.5 and the 0.14g/l NaHCO with 2%
3Virus stock solution used pH to 7.4～7.6 at 4 ℃ of downward modulation purification dropwise add 10%W/V BPL, and making its ultimate density is 0.025%.Behind the deactivation 24h, the viral liquid of deactivation was got final product through 37 ℃ of water-baths in 2 hours again.
Technical program of the present invention also lies in adopting a kind of preparation method of veterinary rabies inactivated vaccine, this preparation method may further comprise the steps:
(1) preparation rabies virus liquid: the Vnukovo-32 Strain that the inoculation rabies virus derives from SAD makes it infection cell in the BHK21 cell; The cell of virus inoculation at first behind 37 ℃ of cultivation 24h, is changed to 33 ℃ and cultivates 72h, then the cell culture multigelation is collected viral liquid 3 times.
(2) concentrated, the purification of rabies virus liquid: with zinc acetate sedimentation method concentrating virus liquid, with Sepharose 4FF gel chromatography column purified virus liquid.
(3) deactivation of rabies virus: the inactivator that uses is beta-propiolactone, and concrete ablation method is HEPES1mol/LpH7.5 and the 0.14g/lNaHCO with 2%
3Virus stock solution used pH to 7.4～7.6 at 4 ℃ of downward modulation purification dropwise add 10%W/VBPL, and making its ultimate density is 0.025%.Behind the deactivation 24h, the viral liquid of deactivation was got final product through 37 ℃ of water-baths in 2 hours again.
(4) preparation of vaccine
The compound method of adjuvant is: the ratio by mole is 10: 2: 1, get hydrogenated soya phosphatide respectively, cholesterol and 18-amine., three kinds of materials after taking by weighing are dissolved in the 20ml chloroform, rotary evaporation is removed organic solvent, treat that lipid forms thin film at the bottle wall, added 60ml chloroform ether again (1: 1, v/v) mixed organic solvents dissolving, carry out water-bath, ultrasonic phosphate buffer (20mmol/LpH7.7) 20ml, the mixing of adding simultaneously, rotary evaporation is removed organic solvent, when emulsion formed jelly gradually, evacuation was removed remaining organic solvent, liposome solutions.Wherein contain the phospholipid of 33mmol and the cholesterol of 33mmol in the liposome solutions of 1mL.
The preparation of vaccine: the liposome solutions of getting the 1mL homogenize mixes with the rabies virus liquid of 1mL deactivation, and adds 0.02% Sodium Mercurothiolate, and wherein the concentration of viral liquid is 1.0683 * 10
6TCID
50/ 0.03ml.
(5) packing: every injection 1ml, can carry out packing by 1 part/bottle or 10 part/bottles, packing.
Strain used in the present invention can also be the Flury LEP strain of the source U.S., and the employed poison cell that connects can also be that the source African green monkey kidney cell is the vero cell
Strain used in the present invention and connect poison cell also can above-mentioned strain and connect in the poison cell any one, as meet the demands, also can be other Strain or connect poison cell, the strain that more than provides and to connect poison cell only be for example explanation the present invention.
Beneficial effect of the present invention: what the present invention adopted is veterinary rabies inactivated vaccine, has blocked the propagation of rabies virus from root.And the present invention uses liposome as adjuvant; because liposome (liposome) is the double-layer of lipoid microcapsule of synthetic; have and to merge with cell membrane; can be engulfed by phagocyte and can be by abundant biodegradable characteristics; can accelerate vaccine and produce immunization; improve its immunity, to the protective rate height of immunized animal.Therefore this vaccine can effectively prevent and control the infection of rabies virus, can produce remarkable economical and social benefit.
Specific embodiment
Embodiment 1
The preparation method of liposome
Ratio by mole is 10: 2: 1, get hydrogenated soya phosphatide, cholesterol and 18-amine. respectively, three kinds of materials after taking by weighing are dissolved in the 20ml chloroform, rotary evaporation is removed organic solvent, treat that lipid forms thin film at the bottle wall, added 60ml chloroform ether again (1: 1, v/v) mixed organic solvents dissolving, carry out water-bath, ultrasonic phosphate buffer (the 20mmol/L pH 7.7) 20ml that adds simultaneously, mixing, rotary evaporation is removed organic solvent, when emulsion formed jelly gradually, evacuation was prepared into liposome solutions after removing remaining organic solvent.
Embodiment 2
The preparation method of veterinary rabies inactivated vaccine
Preparation rabies virus liquid: the Vnukovo-32 strain is inoculated BHK-21 cell 2000mL square vase synchronously by 1% volume, 37 ℃ cultivate 24h after, be changed to 33 ℃ and the cell culture multigelation gathered in the crops viral liquid 3 times after cultivating 72h.
Concentrating of virus: 4 ℃ of centrifugal 30min of viral liquid, 3000r/min removes assorted sheet of cell and impurity, collects supernatant; Supernatant mixes in the zinc acetate (pH 5.0) of 1: 50 ratio adding 1mol/L, adjusts pH to 6.8, places 30-60min, 3000r/min, 4 ℃ of centrifugal 40min, collecting precipitation for 4 ℃; Again fully suspend with an amount of saturated EDTA, 3000r/min, 4 ℃ of centrifugal 20min draw supernatant, are spissated viral liquid.
The purification of virus liquid: the viral liquid after concentrating, carry out Sepharose 4FF chromatography purification.Medium Main form level 100 * 1.3
2* 3.14
3, be the PBS eluting of 0.04mol/L with pH7.2 concentration behind the last sample, elution speed is 3mL, UV-detector detects substep and collects first peak.
The deactivation of rabies virus: the inactivator that uses is beta-propiolactone, and concrete ablation method is HEPES1mol/LpH7.5 and the 0.14g/lNaHCO with 2%
3Virus stock solution used pH to 7.6 at 4 ℃ of downward modulation purification dropwise adds 10%W/V BPL, and making its ultimate density is 0.025%.Behind the deactivation 24h, the viral liquid of deactivation was got final product through 37 ℃ of water-baths in 2 hours again.
The preparation of vaccine: liposome is pressed the method preparation of embodiment 1, and the liposome solutions of getting the homogenize of 1mL mixes with the rabies virus liquid of deactivation, and adds 0.02% Sodium Mercurothiolate, and wherein the concentration of viral liquid is 1.0683 * 10
6TCID
50/ 0.03ml, the concentration of phospholipid is 33mmol, the concentration of cholesterol is 33mmol.
Every injection 1ml can carry out packing by 1 part/bottle or 10 part/bottles, packing.3 monthly ages above dog, cat, subcutaneous or intramuscular injection, every 1ml.
Embodiment 3
The infectivity check of rabies virus
With the deactivation purification with there is not the rabies virus of deactivation to inoculate white mice respectively, observed 14 days, the former white mice of virus inoculation all survives as a result, but nervous symptoms occurs at the 6th day for the mice of the not viral liquid inoculation of fire extinguishing, and the 8th day begins to occur dead.As seen has good inactivating efficacy with the beta-propiolactone inactivation of viruses.
Embodiment 4
The inactivation of viruses efficacy test
Made vaccine is preserved 4,6 and 12 months respectively at 4 ℃ of general refrigerators, select 9 dogs to carry out inoculation by the holding time, every 1mL, and before immunity and the antibody in its serum of back about 30 days blood sampling mensuration of immunity.The effectiveness of different time sections inactivation of viruses sees Table 1.
The effectiveness of table 1 different time sections inactivation of viruses
Embodiment 5
The mensuration of standard strain virulence
Standard strain CVS-11 with the dilution that PBS makes 10 times of series, is got 10
-4, 10
-5, 10
-6, 10
-7, 10
-8The mice of 5 dilution factor difference intracranial inoculation 10-12g, 10 of each dilution factor inoculations, every 0.03mL observed 14 days, and mice dead in 3 days is disregarded, and calculates LD
50, the measurement result statistics is as shown in table 2.
The mensuration of table 2 rabies virus CVS-11 median lethal dose(LD 50)
Titre with Reed-Muench method basis of calculation strain:
Distance is than=(being higher than 50% percent-50)/(be higher than 50% percent-be lower than 50% percent)
＝(100-50)/(100-30)
＝0.715
IgLD
50=Ig (dilution factor that is higher than 50% mortality rate)+(distance is than the logarithm of * extension rate)
＝-6+(0.715×-1)
＝-6.715
Promptly the CVS-11 that surveys be 10 to the median lethal dose(LD 50) of white mice
-6.715/ mL.
Embodiment 6
The immune efficacy challenge test
Make 5 times of serial dilutions with the vaccine of deactivation purification with reference to vaccine respectively with the PBS of 0.04mol/L pH7.6, got 4 dilution factors (1: 5; 1: 25; 125,1: the 625) mice of abdominal cavity inoculation 11-12g, 10 of each dilution factor inoculations, 0.5mL/ only does negative control simultaneously.In 2 weeks after the vaccination, whole test mices are with 100LD
50Only attack standard strain CVS-11 rabies suspension 0.03mL/ in the brain, observed for 2 weeks again, 5-14 days the mice of record behind the counteracting toxic substances presents the mice (behind the counteracting toxic substances in 4 days each dilution factor can allow dead 2 at most) of rabies, and mice in control group is death all.Calculate vaccine to be checked and with reference to vaccine half protective number (PD with the logarithm method
50), its computing formula is calculated according to the computational methods of routine.
The liposome Adjuvanted vaccines of beta-propiolactone deactivation shows that through NIH efficacy determinations experimental result this vaccine valence has reached 7.00IU, and higher immune efficacy is arranged, and can reach the effect of opposing rabies virus protection mice fully.
Claims (7)
1. veterinary rabies inactivated vaccine, it is characterized in that: described vaccine comprises the rabies virus liquid and the adjuvant of deactivation.
2. a kind of veterinary rabies inactivated vaccine according to claim 1 is characterized in that: the concentration of viral liquid is 1.0683 * 10 in the described vaccine
6TCID
50/ 0.03ml, adjuvant are liposome solutions, and wherein the concentration of phospholipid is 33mmol, and the concentration of cholesterol is 33mmol.
3. a kind of veterinary rabies inactivated vaccine according to claim 1, it is characterized in that: described deactivation is meant the process of using inactivator beta-propiolactone (being BPL) inactivation of viruses, and concrete ablation method is HEPES 1mol/L pH7.5 and the 0.14 g/l NaHCO with 2%
3Virus stock solution used pH to 7.4～7.6 at 4 ℃ of downward modulation purification dropwise add 10%W/V BPL, and making its ultimate density is 0.025%, behind the deactivation 24h, the viral liquid of deactivation are got final product through 37 ℃ of water-baths in 2 hours again.
4. the preparation method of a veterinary rabies inactivated vaccine as claimed in claim 1, it is characterized in that: described preparation method may further comprise the steps
(1) preparation rabies virus liquid; (2) concentrated, the purification of viral liquid; (3) deactivation of viral liquid; (4) preparation of vaccine; (5) packing of vaccine, packing.
5. the preparation method of a kind of veterinary rabies inactivated vaccine according to claim 4 is characterized in that: concentrated being meant with zinc acetate sedimentation method concentrating virus liquid in the step (2), purification is meant with Sepharose 4FF gel chromatography column purified virus liquid.
6. the preparation method of a kind of veterinary rabies inactivated vaccine according to claim 4, it is characterized in that: described deactivation is meant the process of using inactivator beta-propiolactone (being BPL) inactivation of viruses, and concrete ablation method is HEPES 1mol/L pH7.5 and the 0.14 g/l NaHCO with 2%
3Virus stock solution used pH to 7.4～7.6 at 4 ℃ of downward modulation purification dropwise add 10%W/V BPL, and making its ultimate density is 0.025%.Behind the deactivation 24h, the viral liquid of deactivation was got final product through 37 ℃ of water-baths in 2 hours again.
7. the preparation method of a kind of veterinary rabies inactivated vaccine according to claim 4, it is characterized in that: the compound method of vaccine is that the liposome solutions of getting the homogenize of 1ml mixes with the rabies virus liquid of 1ml deactivation in the step (4), and add 0.02% Sodium Mercurothiolate, wherein contain the phospholipid of 33mmol and the cholesterol of 33mmol in the liposome solutions of 1mL, the concentration of viral liquid is 1.0683 * 10
6TCID
50/ 0.03ml.
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| CN200910309555A CN101757619A (en) | 2009-11-11 | 2009-11-11 | Veterinary rabies inactivated vaccine and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EA015458B1 (en) * | 2010-11-03 | 2011-08-30 | Открытое Акционерное Общество "Институт Биотехнологий Ветеринарной Медицины" | Virus vaccine against rabies for oral immunization in wild carnivores and method for its production |
| CN102166354A (en) * | 2011-04-12 | 2011-08-31 | 广州市华南农大生物药品有限公司 | Rabies inactivated vaccine and preparation method thereof |
| CN102228686A (en) * | 2011-06-30 | 2011-11-02 | 金宇保灵生物药品有限公司 | Process for preparing veterinary rabies inactivated and freeze-dried vaccine through suspension culture cell |
| RU2563542C1 (en) * | 2014-10-30 | 2015-09-20 | Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский и технологический институт биологической промышленности" | Rabies vaccine for oral immunisation of wild and stray carnivorous animals and method for producing same |
| CN105505887A (en) * | 2015-12-29 | 2016-04-20 | 浙江美保龙生物技术有限公司 | Porcine reproductive and respiratory syndrome virus diluent and preparation method thereof |
| CN108524929A (en) * | 2017-03-06 | 2018-09-14 | 广州瑞贝斯药业有限公司 | A kind of production method of rabies vacciness |
-
2009
- 2009-11-11 CN CN200910309555A patent/CN101757619A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EA015458B1 (en) * | 2010-11-03 | 2011-08-30 | Открытое Акционерное Общество "Институт Биотехнологий Ветеринарной Медицины" | Virus vaccine against rabies for oral immunization in wild carnivores and method for its production |
| CN102166354A (en) * | 2011-04-12 | 2011-08-31 | 广州市华南农大生物药品有限公司 | Rabies inactivated vaccine and preparation method thereof |
| CN102228686A (en) * | 2011-06-30 | 2011-11-02 | 金宇保灵生物药品有限公司 | Process for preparing veterinary rabies inactivated and freeze-dried vaccine through suspension culture cell |
| CN102228686B (en) * | 2011-06-30 | 2013-04-24 | 金宇保灵生物药品有限公司 | Process for preparing veterinary rabies inactivated and freeze-dried vaccine through suspension culture cell |
| RU2563542C1 (en) * | 2014-10-30 | 2015-09-20 | Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский и технологический институт биологической промышленности" | Rabies vaccine for oral immunisation of wild and stray carnivorous animals and method for producing same |
| CN105505887A (en) * | 2015-12-29 | 2016-04-20 | 浙江美保龙生物技术有限公司 | Porcine reproductive and respiratory syndrome virus diluent and preparation method thereof |
| CN108524929A (en) * | 2017-03-06 | 2018-09-14 | 广州瑞贝斯药业有限公司 | A kind of production method of rabies vacciness |
| CN108524929B (en) * | 2017-03-06 | 2019-05-07 | 广州瑞贝斯药业有限公司 | A kind of production method of rabies vacciness |
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Application publication date: 20100630 |