CN105999284A - Heat-resisting cryoprotectant for classical swine fever live vaccines and preparation method and application thereof - Google Patents
Heat-resisting cryoprotectant for classical swine fever live vaccines and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides heat-resisting cryoprotectant for classical swine fever live vaccines and a preparation method and application thereof, and belongs to the technical field of veterinary biological product manufacturing. The cryoprotectant is prepared from, by mass, 0.2-2.5% of synanthrin, 0.5-12% of glucan, 1-6% of polyvinylpyrrolidone, 0.5-12% of lactose, 0.2-6% of saccharose, 0.05-2.5% of polyethylene glycol, 0.005-0.6% of histidine, 0.05-4% of cysteine and 0.2-4% of a DMEM culture medium, and the rest is water for injection. The invention further provides a preparation method and application of the heat-resisting cryoprotectant. The heat-resisting cryoprotectant does not contain foreign protein or gelatin, stress reactions and side effects on animals are avoided, the safety of the vaccines is greatly improved, the antigen activity can be efficiently protected in the freeze-drying and storing process, and the stability of the vaccines is improved.
Description
Technical field
The invention belongs to veterinary biologics manufacturing technology field, be especially for the heat-resisting of live vaccines of hog cholera
Freeze drying protectant, preparation method and applications.
Background technology
Swine fever (classical swine fever, CSF) is commonly called as " rinderpest ", in more American-European areas also known as pig
Cholera (hog cholera), pig calentura or classic swine fever, be by the swine fever virus of flaviviridae pestivirus
The one that (Classical Swine Fever Virus, CSFV) causes is acute, hot and high degree in contact infects
Disease, clinically anxious with morbidity, high heat is delaied, general property petechial hemorrhage of whole body and infarction of spleen be as principal character.Pig
The mortality rate of pestilence is high, can cause huge potential safety hazard and economic loss to pig industry.
1908, Hungary Hutyra was prepared for CSFV hyper-immune serum, and it can produce stronger protection,
But expensive due to hyper-immune serum, obtains more difficult, and has the danger disseminated, therefore scholars turns to
Inactivated vaccine research.Inactivated vaccine there is also that immunizing dose is big, duration of immunity is short, produces immunity more slowly, no
Local immunity can be caused, be only capable of subcutaneously or intramuscularly injecting and the shortcoming such as relatively costly, therefore gradually by the weak poison of swine fever
Vaccine is replaced.Nineteen fifty-five, the hog cholera lapinised virus vaccine (i.e. C strain) that China successfully develops, at world's model
Serve vital effect during enclosing interior hog cholera immune prevention and control and final elimination, be that prevention and control swine fever makes
With most vaccines.
Live vaccines of hog cholera the most common on market has 4 kinds, is all based on C strain, i.e. swine fever cell is lived
Vaccine, swine fever breast rabbit spleen drench tissue live vaccine, swine fever becomes rabbit spleen to drench tissue live vaccine and swine fever-pig erysipelas-Pulmonis Sus domestica
Epidemic disease trigeminy vaccine.Wherein immune effect preferably swine fever cell live vaccine becomes rabbit tissue spleen to drench Seedling with swine fever, is also
The special swine Fever Vaccine of government procurement.
The most conventional live vaccines of hog cholera freeze drying protectant prescription is simple, mostly contains sucrose, gelatin, Ox blood serum
The composition such as albumin, lactoalbumin hydrolysate, but by gelatin and foreign protein when having been found that injection freeze dried vaccine clinically
The side effect such as the allergy caused；Additionally, traditional protective agent heat resistance is poor, need under freezing conditions
-15 DEG C of ability preserve for a long time.
Summary of the invention
The present invention is directed to the heat-resisting lyophilized protecting agent being currently used for live vaccines of hog cholera because bringing containing foreign protein, gelatin
The problem of side reaction, it is provided that a kind of do not contain foreign protein and gelatin, heat resistant freeze drying for live vaccines of hog cholera is protected
Protecting agent, this heat-resisting lyophilized protecting agent can efficiently protect antigen active during lyophilization and high-temperature storage,
And to animal nonirritant and hazardness, to pig safety.
It is a further object of the present invention to provide the preparation method of heat-resisting lyophilized protecting agent, the method is easy to operate.
Another object of the present invention is to provide above-mentioned heat-resisting lyophilized protecting agent answering in terms of preparing live vaccines of hog cholera
With, the live vaccines of hog cholera of preparation, it is possible to tolerance higher temperature stores.
The purpose of the present invention adopts the following technical scheme that realization.
A kind of heat-resisting lyophilized protecting agent for live vaccines of hog cholera, is made up of the component of following weight/mass percentage composition:
Inulin 0.2%～2.5%, glucosan 0.5%～12%, polyvinylpyrrolidone 1%～6%, lactose 0.5%～
12%, sucrose 0.2%～6%, Polyethylene Glycol 0.05%～2.5%, histidine 0.005%～0.6%, half Guang ammonia
Acid 0.05%～4%, DMEM culture medium 0.2%～4%, remaining is water for injection.
In the present invention, the degree of polymerization of inulin is 2～10, and glucosan is Dextran-40 or Dextran-70,
Polyvinylpyrrolidone is PVP K30 or PVP K90, and Polyethylene Glycol is PEG 200～400.
The present invention also provides for the preparation method of described heat-resisting lyophilized protecting agent, comprises the steps:
(1) inulin, glucosan, polyvinylpyrrolidone, lactose, sucrose, Polyethylene Glycol are dissolved in water,
Autoclaving is used to carry out sterilizing；
(2) histidine, cysteine and DMEM culture medium are dissolved in water, use Filtration to carry out degerming；
(3) step (1) and (2) gained solution are mixed, supply water, obtain the protection of described heat resistant freeze drying
Agent.
The present invention also provides for the application in terms of preparing live vaccines of hog cholera of the described heat-resisting lyophilized protecting agent and employing
Live vaccines of hog cholera prepared by described heat-resisting lyophilized protecting agent.
The present invention also provides for the preparation method of live vaccines of hog cholera, comprise the steps: heat-resisting lyophilized protecting agent and
Hog cholera venom 1:0.8 by volume～1.2 mixing, obtains described live vaccines of hog cholera after lyophilization.
In the present invention, described freeze-drying method is as follows: mixed by heat-resisting lyophilized protecting agent and hog cholera venom
Compound is placed on the dividing plate in body of freeze dryer, in normal pressure, pre-cooling 20～40min under the conditions of 2～5 DEG C, then
It is cooled to-38～-42 DEG C and maintain 3～5h according to the speed of 0.5～1 DEG C/min；The cold-trap of freeze dryer is down to
-48～-52 DEG C, it is evacuated to 300～400mTorr in casing；Then by described dividing plate with 0.2～0.8 DEG C/min
Speed be warming up to-22～-28 DEG C and maintain 14～18h, be warming up to the speed of 0.1～0.4 DEG C/min afterwards
-12～-17 DEG C and maintain 8～12h, finally it is warming up to 20～24 DEG C with 0.2～0.8 DEG C/min and maintains 5～7h.
Compared with prior art, the present invention for live vaccines of hog cholera heat-resisting lyophilized protecting agent beneficial effect such as
Under: heat-resisting lyophilized protecting agent of the present invention does not contains foreign protein and gelatin, it is to avoid to the stress of animal and
Side effect, substantially increases the safety of vaccine.Heat-resisting lyophilized protecting agent of the present invention is in lyophilization and stores
Journey can efficiently be protected antigen active, efficiently solve vaccine low temperature cold chain problem in preserving, transporting,
Add the stability of vaccine.Use live vaccines of hog cholera prepared by heat-resisting lyophilized protecting agent of the present invention, at 2-8 DEG C
Under the conditions of at least can store 36 months, at least can store 15 days under the conditions of 37 DEG C.Therefore, use
Live vaccines of hog cholera prepared by heat-resisting lyophilized protecting agent of the present invention will be substantially reduced inactivation risk, improve vaccine safety
Property.
Detailed description of the invention
The present invention is only described in further detail by following embodiment, but does not constitute any limitation of the invention.
Embodiment 1 heat-resisting lyophilized protecting agent 1
Heat-resisting lyophilized protecting agent 1, is made up of the component of following weight/mass percentage composition: (degree of polymerization (DP) is inulin
9) 0.5%, glucosan (Dextran-70) 5%, polyvinylpyrrolidone (PVP K30) 3%, lactose 5%, sugarcane
Sugar 2%, Polyethylene Glycol (PEG 200) 1%, histidine 0.3%, cysteine 1.5%, DMEM cultivate
Base (Gibco company) 3%, remaining is water for injection.
The concrete preparation process of heat-resisting lyophilized protecting agent 1 is as follows:
(1) weigh inulin (DP is 9), glucosan (Dextran-70), polyvinylpyrrolidone (PVP K30),
Lactose, sucrose, Polyethylene Glycol (PEG200) are dissolved in a small amount of water for injection, autoclaving sterilizing
30min, controlling temperature in sterilization process is 110 DEG C, standby；
(2) weigh histidine, cysteine and DMEM culture medium to be dissolved in a small amount of water for injection, use hole
Footpath is that the membrane filtration of 0.22 μm is degerming rear standby；
(3) solution mixing step (1) and (2) prepared, supplies water for injection, obtains heat resistant freeze drying and protects
Protect agent 1.
Prepare live vaccines of hog cholera 1: by heat-resisting lyophilized protecting agent 1 and hog cholera venom (C strain, viral level 40
×104RID/mL) mix with volume ratio 1:1, in subpackage to 7ml cillin bottle, 2ml/ bottle, pitch with butyl four
After plug is partly jumped a queue, carry out lyophilization in inserting body of freeze dryer, obtain live vaccines of hog cholera 1.
Cryodesiccated concretely comprise the following steps: the mixture of heat-resisting lyophilized protecting agent 1 with hog cholera venom is placed on and freezes
On dividing plate in dry cabinet bulk, at normal pressure, pre-cooling 30min under the conditions of 4 DEG C, then according to the speed of 1 DEG C/min
Degree is cooled to-40 DEG C, maintains 4h at-40 DEG C；The cold-trap of freeze dryer is down to-50 DEG C, takes out in body of freeze dryer
Vacuum is to 350mTorr；Then dividing plate is warming up to-25 DEG C with the speed of 0.5 DEG C/min, maintains 16h at-25 DEG C,
It is warming up to-15 DEG C with the speed of 0.25 DEG C/min afterwards, maintains 10h at-15 DEG C, finally with 0.5 DEG C/min liter
Temperature, to 20 DEG C, maintains 5h at 20 DEG C.
Embodiment 2 heat-resisting lyophilized protecting agent 2
Heat-resisting lyophilized protecting agent 2, is made up of the component of following weight/mass percentage composition: inulin (DP is 5) 2%,
Glucosan (Dextran-70) 10%, polyvinylpyrrolidone (PVP K90) 2%, lactose 1%, sucrose 0.5%,
Polyethylene Glycol (PEG400) 2%, histidine 0.01%, cysteine 3%, DMEM culture medium 0.5%,
Remaining is water for injection.
The concrete preparation process of heat-resisting lyophilized protecting agent 2 is as follows:
(1) weigh inulin (DP is 5), glucosan (Dextran-70), polyvinylpyrrolidone (PVP K90),
Lactose, sucrose, Polyethylene Glycol (PEG400) are dissolved in a small amount of water for injection, autoclaving sterilizing
30min, controlling temperature in sterilization process is 110 DEG C, standby；
(2) weigh histidine, cysteine and DMEM to be dissolved in a small amount of water for injection, be 0.22 with aperture
The membrane filtration of μm is degerming rear standby；
(3) solution mixing step (1) and (2) prepared, supplies water for injection, obtains heat resistant freeze drying and protects
Protect agent 2.
Prepare live vaccines of hog cholera 2: (C strain, virus contains with swine fever virus antigen liquid by heat-resisting lyophilized protecting agent 2
Amount 40 × 104RID/mL) mix with volume ratio 1:1, in subpackage to 7ml cillin bottle, 2ml/ bottle, use fourth
After base four fork plug is partly jumped a queue, carry out lyophilization in inserting body of freeze dryer, obtain live vaccines of hog cholera 2.
Cryodesiccated concretely comprise the following steps: the mixture of heat-resisting lyophilized protecting agent 2 with hog cholera venom is placed on and freezes
On dividing plate in dry cabinet bulk, at normal pressure, pre-cooling 25min under the conditions of 3 DEG C, then according to the speed of 0.5 DEG C/min
Degree is cooled to-38 DEG C, maintains 5h at-38 DEG C；The cold-trap of freeze dryer is down to-48 DEG C, takes out in body of freeze dryer
Vacuum is to 400mTorr；Then dividing plate is warming up to-28 DEG C with the speed of 0.2 DEG C/min, maintains 14h at-28 DEG C,
It is warming up to-17 DEG C with the speed of 0.1 DEG C/min afterwards, maintains 8h at-17 DEG C, finally heat up with 0.4 DEG C/min
To 24 DEG C, maintain 7h at 24 DEG C.
Embodiment 3 swine fever heat resistance freeze drying protectant 3
Swine fever heat resistance freeze drying protectant 3, is made up of the component of following weight/mass percentage composition: inulin (DP
Be 2) 0.2%, glucosan (Dextran-40) 1%, polyvinylpyrrolidone (PVP K30) 5%, lactose 10%,
Sucrose 5%, Polyethylene Glycol (PEG 250) 0.1%, histidine 0.5%, cysteine 0.1%, DMEM
Culture medium 1.5%, remaining is water for injection.
The concrete preparation process of heat-resisting lyophilized protecting agent 3 is as follows:
(1) weigh inulin, glucosan (Dextran-40), polyvinylpyrrolidone (PVP K30), lactose,
Sucrose, Polyethylene Glycol are dissolved in a small amount of water for injection, and autoclaving sterilizing 30min, in sterilization process
Controlling temperature is 110 DEG C, standby；
(2) weigh histidine, cysteine and DMEM to be dissolved in a small amount of water for injection, be 0.22 with aperture
The membrane filtration of μm is degerming rear standby；
(3) solution mixing step (1) and (2) prepared, supplies and injects with water, obtain heat resistant freeze drying
Protective agent 3.
Prepare live vaccines of hog cholera 3: by heat-resisting lyophilized protecting agent 3 of the present invention and hog cholera venom (C strain, virus
Content 40 × 104RID/mL) with volume 1:1 than mixing, in subpackage to 7ml cillin bottle, 2ml/ bottle, use
After butyl four fork plug is partly jumped a queue, carry out lyophilization in inserting body of freeze dryer, obtain live vaccines of hog cholera 3.
Cryodesiccated concretely comprise the following steps: the mixture of heat-resisting lyophilized protecting agent 3 with hog cholera venom is placed on and freezes
On dividing plate in dry cabinet bulk, at normal pressure, pre-cooling 20min under the conditions of 2 DEG C, then according to the speed of 1 DEG C/min
Degree is cooled to-42 DEG C, maintains 3h at-42 DEG C；The cold-trap of freeze dryer is down to-52 DEG C, takes out in body of freeze dryer
Dividing plate, to 300mTorr, is then warming up to-22 DEG C with the speed of 0.8 DEG C/min by vacuum, maintains 18h at-22 DEG C,
It is warming up to-12 DEG C with the speed of 0.4 DEG C/min afterwards, maintains 12h at-12 DEG C, finally heat up with 0.7 DEG C/min
To 22 DEG C, maintain 6h at 22 DEG C.
Embodiment 4 compares heat-resisting lyophilized protecting agent 1
Comparison heat-resisting lyophilized protecting agent 1 (being disclosed in CN102793927A), by following weight percentage
Component forms: trehalose 5%, polyvinylpyrrolidone (PVP K30) 2.5%, gelatin 2%, sodium glutamate
2%, D-glucitol 3.5%, polyprotein peptone 4.5%, L-arginine 0.75%, vitamin C 0.75%,
Surplus is the water for injection after sterilizing.
The preparation method of comparison heat-resisting lyophilized protecting agent 1 is as follows: (1) weighs polyethylene pyrrole by described weight proportion
Pyrrolidone (PVP K30), gelatin, D-glucitol, polyprotein peptone are dissolved in a small amount of water for injection, and 110 DEG C go out
Bacterium 30min is standby；(2) trehalose, sodium glutamate, L-arginine, dimension life are weighed by described weight proportion
Element C is dissolved in a small amount of water for injection, degerming rear standby with the membrane filtration that aperture is 0.22 μm；(3) will step
Suddenly the solution mixing that (1), (2) prepare, adds to the full amount of water for injection and get final product.
Comparison live vaccines of hog cholera 1: take comparison heat-resisting lyophilized protecting agent 1 and swine fever virus antigen liquid (C strain, disease
Poison content 40 × 104RID/mL) according to volume ratio be 1:1.2 mixing, in subpackage to 7ml cillin bottle, 2ml/
Bottle, after partly jumping a queue with butyl rubber plug, in body of freeze dryer.
Concretely comprising the following steps of lyophilizing: mixed liquor is inserted in the freeze drying box of 2 DEG C, with the rate of temperature fall of 1 DEG C/min
Being down to-35 DEG C, maintain 3.5h at this temperature, open vacuum pump, to case, vacuum is opened when reaching 0.01mbar
Beginning to heat up, 20h rises to-6 DEG C, then rises to 25 DEG C with the heating rate of 6 DEG C/h, maintains 3h at this temperature.
Embodiment 5 compares heat-resisting lyophilized protecting agent 2
Comparison heat-resisting lyophilized protecting agent 2 (being disclosed in CN103341176A), concrete preparation method is as follows: (1) A
Liquid is prepared: take NZ-amine 3g, monopotassium glutamate salt 0.375g, sucrose 25g, lactoalbumin hydrolysate 1.875g,
Above composition is dissolved in 100mL water for injection in order, shakes up, with the filter of 0.22 μm after fully dissolving
Membrane filtration degerming after 2～8 DEG C standby；(2) B liquid preparation: take gelatin hydrolysate 20g, with the note of 70～80 DEG C
Penetrate after fully dissolving with water 100mL, 121 DEG C, 15 pounds of autoclavings 30 minutes, it is saved in 37 DEG C after sterilizing；
(3) in the ratio of 4:1, A liquid and B liquid being carried out proportioning, mix homogeneously is heat-resisting lyophilized protecting agent,
The quality concentration of volume percent of the most each component is as follows: 2.4%NZ-amine, 0.3% monopotassium glutamate salt, 20%
Sucrose, 1.5% lactoalbumin hydrolysate, 4% gelatin hydrolysate.
Comparison live vaccines of hog cholera 2: take comparison heat-resisting lyophilized protecting agent 2 and swine fever virus antigen liquid (C strain, disease
Poison content 40 × 104RID/mL) according to volume ratio be 1:4 mixing, in subpackage to 7ml cillin bottle, 2ml/ bottle,
After partly jumping a queue with butyl rubber plug, in body of freeze dryer.
Concrete step of freeze drying is: is inserted by mixed liquor in freeze drying box, chooses pre-freezing temperature and be-50 DEG C, during pre-freeze
Cryogenic hold-time is 2h, pre-freeze beforehand control at-15 DEG C, resolve quick freezing after heat until product release: rise
China work-in-process temperature for-33 DEG C, flaggy temperature be set as-8 DEG C, sublimation time be 11h；Resolution temperature is
26 DEG C, the parsing time be 4h, lyophilizing whole process is 24h, outlet of jumping a queue, obtain compare live vaccines of hog cholera 2.
The detection of embodiment 6 live vaccines of hog cholera
Measure the physical behavior of each live vaccines of hog cholera, residual moisture in embodiment 1～5, store up under the conditions of 37 DEG C
Deposit 0d, 10d impact on virus of live vaccine content, under the conditions of 2～8 DEG C, store different time to live vaccine
The impact of viral level.
The physical behavior of freeze-dried products, residual moisture measure attached according to existing " People's Republic of China's veterinary drug allusion quotation "
Method and the standard of record are carried out.Concrete outcome is shown in Table 1.As it can be seen from table 1 all meet, " the China people are altogether
With state's veterinary drug allusion quotation " regulation of annex, and live vaccines of hog cholera 1,2 and 3 is at the aspect such as physical behavior, vacuum
All meeting regulation, residual moisture is more lower slightly than comparison live vaccines of hog cholera 1 and 2.Live vaccines of hog cholera freeze-dried products is sick
Poison detection method of content is carried out according to phenomenon " People's Republic of China's regulations ".
The physical behavior of table 1 live vaccines of hog cholera, residual moisture measurement result
| Group | Physical behavior | Residual moisture (4%, n=4) | Vacuum |
| Live vaccines of hog cholera 1 | Loose spongy | 1.01±0.09 | Meet regulation |
| Live vaccines of hog cholera 2 | Loose spongy | 1.25±0.03 | Meet regulation |
| Live vaccines of hog cholera 3 | Loose spongy | 1.09±0.12 | Meet regulation |
| Comparison live vaccines of hog cholera 1 | Loose spongy | 1.88±0.22 | Meet regulation |
| Comparison live vaccines of hog cholera 2 | Loose spongy | 1.69±0.19 | Meet regulation |
The measurement result of 37 DEG C of 10d and 15d restrovirus content of table 2 live vaccines of hog cholera
In table 2: lyophilizing loss refers to the difference of 0d vaccine virus content after noxious dampness and lyophilizing.Resistance to heat loss refers to vaccine freeze-drying
The difference of vaccine virus content after preserving 10 days or 15 days with 37 DEG C after 0 day.
From table 2 it can be seen that live vaccines of hog cholera 1,2 and 3 viral level in freeze-drying process be lost in 0.3～
Below 0.43Lg RID/1mL；After storing 10 days under the conditions of 37 DEG C, viral level loses all≤1.0Lg
RID/1mL, after storing 15 days under the conditions of 37 DEG C, viral level loss is 1.3Lg RID/1mL.Swine fever is novel resistance to
Hot live vaccine 1,2 and 3 lyophilizing and heat-resisting during viral level loss be respectively less than or equal to comparison swine fever
Heat-resisting live vaccine.
Table 3 live vaccines of hog cholera viral level measurement result after 2～8 DEG C of preservation different times
From table 3 it can be seen that the novel live vaccine of swine fever 1,2 and 3 preserves 36 at 2～8 DEG C after freeze drying
In month, viral level loss is 0.87～1.0LgRID/1mL, and lyophilizing loss is 1.3Lg with resistance to heat loss sum
RID/1mL, in the case of without foreign protein, gelatin, the resistance to heat loss of heat-resisting lyophilized protecting agent of the present invention
Less than matched group；Reduce the side effect to animal simultaneously, add the safety of goods.
The safety of embodiment 7 live vaccines of hog cholera
With normal saline, live vaccines of hog cholera 1, live vaccines of hog cholera 2, live vaccines of hog cholera 3 are redissolved into solution respectively,
Every part content is 7500RID, according to 1 part, 5 parts and 10 doses immunity.
Animal experiment is divided into 4 groups (tables 4), is 1 part, 5 parts and 10 parts respectively, immunity way
Footpath is intramuscular injection, and blank group (the most immune).The piglet of 20 ages in days, often group 10 are chosen in test.
Table 4 is the safety animal experiment packet of heat-resisting lyophilized protecting agent
| Group | Pig quantity (only) | Immunizing dose | Immunity volume |
| A | 10 | 1 part | 0.5ml |
| B | 10 | 5 parts | 2.5ml |
| C | 10 | 10 parts | 5ml |
| D | 10 | The most immune | / |
The safety animal test method of heat-resisting lyophilized protecting agent and the index of research:
Clinical observation: in 24h after inoculation, observes a piglet every 2h and has no adverse reaction；48h～
72h, observes a piglet every 8h and has no adverse reaction；After inoculation in 3～14d, every 24h observes to be had
Without abnormal response.If any nonspecific death, immune group all should be less than 1 with matched group.
Result: use live vaccines of hog cholera 1, live vaccines of hog cholera 2, live vaccines of hog cholera 3 respectively according to 1 part,
After 5 parts and 10 doses immunity 20 age in days piglets, pig mental status, search for food, drink water, grow
Developmental state is all normal, does not all find untoward reaction.Heat-resisting lyophilized protecting agent the most of the present invention is peace to pig
Complete.
Claims (7)
1. the heat-resisting lyophilized protecting agent for live vaccines of hog cholera; it is characterized in that being made up of the component of following weight/mass percentage composition: inulin 0.2%～2.5%, glucosan 0.5%～12%, polyvinylpyrrolidone 1%～6%, lactose 0.5%～12%, sucrose 0.2%～6%, Polyethylene Glycol 0.05%～2.5%, histidine 0.005%～0.6%, cysteine 0.05%～4%, DMEM culture medium 0.2%～4%, remaining is water for injection.
The most according to claim 1 for the heat-resisting lyophilized protecting agent of live vaccines of hog cholera; the degree of polymerization that it is characterized in that inulin is 2～10; glucosan is Dextran-40 or Dextran-70, and polyvinylpyrrolidone is PVP K30 or PVP K90, and Polyethylene Glycol is PEG 200～400.
3. the preparation method of heat-resisting lyophilized protecting agent described in claim 1 or 2, it is characterised in that comprise the steps:
(1) inulin, glucosan, polyvinylpyrrolidone, lactose, sucrose, Polyethylene Glycol are dissolved in water, use autoclaving to carry out sterilizing；
(2) histidine, cysteine and DMEM culture medium are dissolved in water, use Filtration to carry out degerming；
(3) step (1) and (2) gained solution are mixed, supply water, obtain described heat-resisting lyophilized protecting agent.
4. the application in terms of preparing live vaccines of hog cholera of the heat-resisting lyophilized protecting agent described in claim 1 or 2.
5. use live vaccines of hog cholera prepared by one of claim 1-2 described heat-resisting lyophilized protecting agent.
6. the preparation method of live vaccines of hog cholera described in claim 5, it is characterised in that comprise the steps:, by heat-resisting lyophilized protecting agent and hog cholera venom 1:0.8 by volume～1.2 mixing, to obtain described live vaccines of hog cholera after lyophilization.
The preparation method of live vaccines of hog cholera the most according to claim 6; it is characterized in that described freeze-drying method is as follows: be placed on the dividing plate in body of freeze dryer by the mixture of heat-resisting lyophilized protecting agent and hog cholera venom; in normal pressure, pre-cooling 20～40min under the conditions of 2～5 DEG C, then according to the speed of 0.5～1 DEG C/min is cooled to-38～-42 DEG C and maintain 3～5h；The cold-trap of freeze dryer is down to-48～-52 DEG C, in casing, is evacuated to 300～400mTorr；Then described dividing plate is warming up to-22～-28 DEG C and maintain 14～18h with the speed of 0.2～0.8 DEG C/min, it is warming up to-12～-17 DEG C and maintain 8～12h with the speed of 0.1～0.4 DEG C/min afterwards, is finally warming up to 20～24 DEG C with 0.2～0.8 DEG C/min and maintains 5～7h.
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