CN108904813A - A kind of preparation method of fowl vaccine diluent - Google Patents
A kind of preparation method of fowl vaccine diluent Download PDFInfo
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- CN108904813A CN108904813A CN201811165186.0A CN201811165186A CN108904813A CN 108904813 A CN108904813 A CN 108904813A CN 201811165186 A CN201811165186 A CN 201811165186A CN 108904813 A CN108904813 A CN 108904813A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
Abstract
The present invention provides a kind of preparation methods of fowl vaccine diluent, belong to biomedicine technical field, including：It is homogenized after chicken bursa, chicken intestines and water are mixed, the homogenate multigelation that will be obtained, it is successively stirred, is centrifuged after obtained freeze thawing liquid is mixed with acetic acid, it is inactivated after obtained centrifugal clear liquid is mixed with beta-propiolactone, 1~3h is hydrolyzed at 35~42 DEG C in obtained inactivation liquid, hydrolyzate will be obtained and carry out micro-filtration, adjust the pH value of obtained micro-filtrate, obtained adjusting liquid is subjected to ultrafiltration, the ultrafiltrate degerming that will be obtained obtains fowl vaccine diluent.The fowl that preparation method provided by the invention is prepared can kill the specific pathogen free bacterium of fowl vaccine with vaccine diluent completely, can guarantee vaccine using safe.
Description
Technical field
The present invention relates to biomedicine technical fields, and in particular to a kind of preparation method of fowl vaccine diluent.
Background technique
Vaccine refers to the biological products for immunization campaign made of all kinds of pathogenic microorganisms, and the purpose of immunity inoculation is
It induces body and generates the immune response powerful to antigen inoculation, to protect body from the invasion of corresponding pathogen.Current vaccine
Use be it is very universal, whether people or livestock and poultry require vaccine inoculation, to prevent certain diseases.Currently, on the market
Though vaccine have using transfer factor dilution carry out vaccine dilution, some fowl seedling transfer factor diluted
Afterwards, it cannot but accomplish integral asepsis.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation methods of fowl vaccine diluent, using preparation provided by the invention
The dilution that method is prepared, after diluting vaccine, so that integral asepsis.
The present invention provides a kind of preparation methods of fowl vaccine diluent, include the following steps：
1) it is homogenized after mixing chicken bursa, chicken intestines and water, obtains homogenate；
2) by the homogenate multigelation of the step 1) 6~8 times, freeze thawing liquid is obtained；
3) the freeze thawing liquid of the step 2) is successively stirred with the mixed mixed liquor of acetic acid, is centrifuged, by what is obtained
Centrifugal clear liquid mixes inactivation with beta-propiolactone, obtains inactivation liquid；
4) the inactivation liquid of the step 3) is obtained into hydrolyzate in 35~42 DEG C of 1~3h of hydrolysis；
5) hydrolyzate of the step 4) is subjected to micro-filtration, the pH value of the micro-filtrate adjusted is 6.5~7.5, is obtained
Adjust liquid；
6) the adjusting liquid of the step 5) is subjected to ultrafiltration, the ultrafiltrate degerming that will be obtained obtains fowl vaccine diluent.
Preferably, the quality of the step 1) chicken bursa and the quality of chicken intestines, the volume ratio of water are (0.5~1.5):
(0.1~0.5):(0.5~2.5).
Preferably, the speed of the step 1) homogenate is 15000~18000rpm, the time of the homogenate is 10~
20min。
Preferably, by volume percentage, the step 3) acetic acid in mixed liquor final concentration of 1~3%.
Preferably, the temperature of the step 3) stirring is 0~10 DEG C, and the time of the stirring is 10~14h, the stirring
Speed be 30~50rpm；The temperature of the centrifugation is 0~4 DEG C, and the time of the centrifugation is 10~20min, the centrifugation
Speed is 5000~10000rpm.
Preferably, by volume percentage, after the step 3) centrifugal clear liquid is mixed with beta-propiolactone, the end of beta-propiolactone
Concentration is 0.010~0.015%.
Preferably, the temperature of the step 3) inactivation is 5~15 DEG C, and the time of the inactivation is 24~30h.
Preferably, the aperture for the filter membrane that the step 5) micro-filtration uses is 0.22 μm.
Preferably, the molecular cut off for the ultrafiltration membrane that the step 6) ultrafiltration uses is 4~8kD.
The present invention provides the fowl vaccine diluents that a kind of method described in above scheme is prepared.
Contain chicken intestines antibacterial peptide in the dilution that the preparation method of fowl provided by the invention vaccine diluent is prepared
And transfer factor, the chicken intestines antibacterial peptide binding transfer factor can kill the specific pathogen free bacterium in vaccine, to guarantee epidemic disease completely
Seedling use is more safe.
The embodiment of the present invention is as the result is shown：The fowl that preparation method provided by the invention is prepared vaccine diluent energy
Enough specific pathogen free bacterium for killing fowl vaccine completely, can guarantee vaccine using safe.
Specific embodiment
The present invention provides a kind of preparation methods of fowl vaccine diluent, include the following steps：
1) it is homogenized after mixing chicken bursa, chicken intestines and water, obtains homogenate；
2) by the homogenate multigelation of the step 1) 6~8 times, freeze thawing liquid is obtained；
3) the freeze thawing liquid of the step 2) is successively stirred with the mixed mixed liquor of acetic acid, is centrifuged, by what is obtained
Centrifugal clear liquid mixes inactivation with beta-propiolactone, obtains inactivation liquid；
4) 1~3h is hydrolyzed at 35~42 DEG C in the inactivation liquid of the step 3), obtains hydrolyzate；
5) hydrolyzate of the step 4) is subjected to micro-filtration, the pH value of the micro-filtrate adjusted is 6.5~7.5, is obtained
Adjust liquid；
6) the adjusting liquid of the step 5) is subjected to ultrafiltration, the ultrafiltrate degerming that will be obtained obtains fowl vaccine diluent.
The present invention is homogenized after mixing chicken bursa, chicken intestines and water, obtains homogenate.
Preferably chicken bursa and chicken intestines are eluted with water the present invention, remove the peel degreasing go after muscle after being rubbed again with meat grinder again with
Water mixing homogenate, the chicken bursa and chicken intestines are all from fresh healthy animal object.
In the present invention, the quality of chicken bursa and the quality of chicken intestines, the volume ratio of water are preferably (0.5~1.5):(0.1
~0.5):(0.5~2.5), more preferably (0.8~1.2):(0.15~0.3):(0.8~1.5), most preferably 1:0.2:
1.2.The present invention can from chicken bursa isolated transfer factor, the present invention can from chicken intestines isolated antibacterial peptide.
In the present invention, the water is preferably water for injection.
In the present invention, the speed of the homogenate is preferably 15000~18000rpm, more preferably 16000~
17000rpm, most preferably 16500rpm；The time of the homogenate is preferably 10~20min, more preferably 12~18min, most
Preferably 15min.
The present invention obtains freeze thawing liquid, preferably multigelation 7 times for the homogenate multigelation 6~8 times.In the present invention
In, when the multigelation, the temperature of freezing is independently preferably -22~-18 DEG C, more preferably -20 DEG C；The time of freezing is independent
Preferably 48~60h, more preferably 52h；The temperature of dissolution is independently preferably 20~30 DEG C, more preferably 25 DEG C.In the present invention
In, the multigelation is to make clasmatosis.
The freeze thawing liquid is successively stirred with the mixed mixed liquor of acetic acid, is centrifuged, the centrifugation that will be obtained by the present invention
Clear liquid inactivates after mixing with beta-propiolactone, obtains inactivation liquid.
In the present invention, by volume percentage, final concentration of the acetic acid in mixed liquor is preferably 1~3%, more excellent
It is selected as 2%.In the present invention, the acetic acid can be such that the antibacterial peptide in chicken intestines preferably separates.
In the present invention, the temperature of the stirring is preferably 0~10 DEG C, more preferably 2~8 DEG C, most preferably 4~6 DEG C；
The time of the stirring is preferably 10~14h, more preferably 11~13h, most preferably 12h；The speed of the stirring is preferably
30~50rpm, more preferably 35~45rpm, most preferably 40rpm.
In the present invention, the temperature of the centrifugation is preferably 0~4 DEG C；The time of the centrifugation is preferably 10~20min,
More preferably 12~18min, most preferably 15min；The speed of the centrifugation is preferably 5000~10000rpm, more preferably
6000~9000rpm, most preferably 8000rpm.
In the present invention, by volume percentage, the centrifugal clear liquid is mixed with beta-propiolactone, and beta-propiolactone is clear in centrifugation
Final concentration in liquid is preferably 0.010~0.015%, and more preferably 0.012%.In the present invention, the beta-propiolactone to from
Heart clear liquid is inactivated.
In the present invention, the time of the inactivation is preferably 24~30h, more preferably 26~28h, most preferably 27h；Institute
The temperature for stating inactivation is preferably 5~15 DEG C, more preferably 8~12 DEG C, most preferably 10 DEG C.The present invention hydrolyzes the inactivation liquid
Obtain hydrolyzate；The temperature of the hydrolysis is 35~42 DEG C, preferably 37 DEG C；The time of the hydrolysis is 1~3h, preferably
1.5~2.5h, more preferably 2h.In the present invention, the beta-propiolactone 37 DEG C can automatic hydrolysis be nontoxic β hydroxyl third
Acid.
Hydrolyzate is carried out micro-filtration by the present invention, and the pH value of the micro-filtrate adjusted is 6.5~7.5, obtains adjusting liquid.?
In the present invention, the aperture for the filter membrane that the micro-filtration uses is preferably 0.22 μm.In the present invention, the pH value for adjusting micro-filtrate
Preferably 6.8~7.2, more preferably 7.
The adjusting liquid is carried out ultrafiltration by the present invention, and the ultrafiltrate degerming that will be obtained obtains fowl vaccine diluent.
In the present invention, the molecular cut off for the ultrafiltration membrane that the ultrafiltration uses is preferably 4~8kD, more preferably 5~
7kD, most preferably 6kD.In the present invention, it is preferable to use sterilizing filters to carry out degerming, the sterilizing filter for the degerming
Filter sizes be preferably 0.1 μm.The present invention is not particularly limited the conditional parameter of the ultrafiltration, using the condition of conventional ultrafiltration
Parameter.
The present invention provides a kind of fowl vaccine diluents that above scheme the method is prepared.In the present invention,
The fowl is respectively provided with the effect that enhancing is immune and sterilizes containing transfer factor and chicken intestines antibacterial peptide in vaccine diluent.
The present invention is not particularly limited the application method and dosage of fowl vaccine diluent, according to being diluted
Vaccine carry out conventional selection.
A kind of preparation method of fowl vaccine diluent of the present invention is done further combined with specific embodiments below
Detailed introduction, technical solution of the present invention include but is not limited to following embodiment.
Embodiment 1
(1) chicken bursa and chicken intestines are cleaned up, peeling degreasing removes muscle, and spare, chicken bursa is cleaned with water for injection
Fresh healthy animal body is all from chicken intestines；
(2) by the chicken bursa 5Kg handled well and chicken intestines 1Kg be put into meat grinder rub, then by obtained rubbing object by
Volume ratio 1:1 adds water for injection, is placed in bruisher and is homogenized 20min in the case where revolving speed is 15000rpm, obtains homogenate；
(3) by homogenate multigelation 6 times, (temperature freezed when freeze thawing is -22 DEG C, time 48h, temperature when dissolution
It is 30 DEG C, time 1.5h), obtain freeze thawing liquid；
(4) after freeze thawing liquid to be added to final concentration of 1% acetic acid, 14h is stirred with the revolving speed of 30rpm in 10 DEG C, then set
In 4 DEG C, 20min is centrifuged with 5000rpm, takes centrifugal clear liquid in aseptic tank, it is final concentration of that centrifugal clear liquid is mixed addition
0.010% beta-propiolactone, inactivates for 24 hours within 10 DEG C, and obtained inactivation liquid is placed in 37 DEG C of hydrolysis 2h, obtains hydrolyzate；
(5) hydrolyzate is subjected to slipstream micro-filtration through 0.22 μm of filter membrane, collects filter transparent liquid, and adjust pH value to 7.5, obtains
Adjust liquid.
(6) liquid will be adjusted and carries out cross-flow ultrafiltration through 8kD ultrafiltration membrane, and collect ultrafiltration permeate, then with 0.1 μm of aseptic filtration
Filtrate is collected in device degerming, and aseptic subpackaged is fowl vaccine diluent finished product.
Embodiment 2
(1) chicken bursa and chicken intestines are cleaned up, peeling degreasing removes muscle, and spare, chicken bursa is cleaned with water for injection
Fresh healthy animal body is all from chicken intestines；
(2) by the chicken bursa 5Kg handled well and chicken intestines 1Kg be put into meat grinder rub, then by obtained rubbing object by
Volume ratio 1:2 add water for injection, are placed in bruisher and are homogenized 15min in the case where revolving speed is 16500rpm, obtain homogenate；
(3) by homogenate multigelation 7 times, (temperature freezed when freeze thawing is -20 DEG C, time 52h, temperature when dissolution
It is 25 DEG C, time 1.5h), obtain freeze thawing liquid；
(4) freeze thawing liquid is added to final concentration of 2% acetic acid, 10h is stirred with the revolving speed of 50rpm in 10 DEG C, then be placed in 0
DEG C, 10min is centrifuged with 10000rpm, takes centrifugal clear liquid in aseptic tank, centrifugal clear liquid is mixed and is added final concentration of 0.015%
Beta-propiolactone, inactivate 30h within 10 DEG C, obtained inactivation liquid be placed in 37 DEG C of hydrolysis 2h, obtains hydrolyzate；
(5) hydrolyzate is subjected to slipstream micro-filtration through 0.22 μm of filter membrane, collects filter transparent liquid, and adjust pH value to 7.5, obtains
Adjust liquid.
(6) liquid will be adjusted and carries out cross-flow ultrafiltration through 4kD ultrafiltration membrane, and collect ultrafiltration permeate, then with 0.1 μm of aseptic filtration
Filtrate is collected in device degerming, and aseptic subpackaged is fowl vaccine diluent finished product.
Embodiment 3
(1) chicken bursa and chicken intestines are cleaned up, peeling degreasing removes muscle, and spare, chicken bursa is cleaned with water for injection
Fresh healthy animal body is all from chicken intestines；
(2) by the chicken bursa 5Kg handled well and chicken intestines 1Kg be put into meat grinder rub, then by obtained rubbing object by
Volume ratio 1:3 add water for injection, are placed in bruisher and are homogenized 10min at 18000rpm, obtain homogenate；
(3) by homogenate multigelation 8 times, (temperature freezed when freeze thawing is -18 DEG C, time 48h, temperature when dissolution
It is 20 DEG C, time 1.5h), obtain freeze thawing liquid；
(4) freeze thawing liquid is added to final concentration of 3% acetic acid, 12h is stirred with the revolving speed of 40rpm in 10 DEG C, then be placed in 0
DEG C, 15min is centrifuged with 8000rpm, takes centrifugal clear liquid in aseptic tank, centrifugal clear liquid is mixed and is added final concentration of 0.012%
Beta-propiolactone, inactivate 27h within 10 DEG C, obtained inactivation liquid be placed in 37 DEG C of hydrolysis 2h, obtains hydrolyzate；
(5) hydrolyzate is subjected to slipstream micro-filtration through 0.22 μm of filter membrane, collects filter transparent liquid, and adjust pH value to 7.5, obtains
Adjust liquid；
(6) liquid will be adjusted and carries out cross-flow ultrafiltration through 6kD ultrafiltration membrane, and collect ultrafiltration permeate, then with 0.1 μm of aseptic filtration
Filtrate is collected in device degerming, and aseptic subpackaged is fowl vaccine diluent finished product.
Embodiment 4
Before and after fowl cooperates the use of infectious laryngotracheitis of chicken heat resisting protective live vaccine with vaccine diluent, specific pathogen free
Bacterium number amount comparative test
The chicken infection that 6 bottles are numbered as H331-1, H332-1, H333-1, H331-2, H332-2, H333-2 different batches
Property laryngotracheitis heat resisting protective live vaccine, wherein H331-1 and H331-2 are same batch, and H332-1 and H332-2 is same
A batch, H333-1 and H333-2 are same batch, and P881-1, P882-1 and P883-1 is dilute with 2mL bursopoietin dilution
It releasing, H331-2, H332-2, H333-2 are diluted with the fowl that the 2mL embodiment of the present invention 1 is prepared with vaccine diluent, point
It does not take 100 μ L to be connected to 6 TSA plates and carries out bed board, whole process answers sterile working, the plate after bed board is placed in 37 DEG C of constant temperature
Case is incubated overnight, next day observation, as a result such as table 1.
1 steriling test result of table
By table 1 it can be concluded that, using 3 plates of fowl prepared by the present invention vaccine diluent group all without long bacterium, and
Bacterium has been grown using diluted 3 plate of bursopoietin diluted.It can be seen that fowl prepared by the present invention is diluted with vaccine
Liquid can kill pathogen in fowl vaccine completely really, to ensure the safety that vaccine uses, improve animal immune level, value
It must promote.
Embodiment 5
Before and after fowl cooperates the use of infectious bronchitis of chicken heat resisting protective live vaccine with vaccine diluent, specific pathogen free
Bacterium number amount comparative test
The avian infectious branch that 6 bottles are numbered as Q01-1, Q02-1, Q03-1, Q0H331-2, Q02-2, Q03-2 different batches
Tracheitis heat resisting protective live vaccine, wherein Q01-1 and Q01-2 is same batch, and Q02-1 and Q02-2 are the same batch,
Q03-1 and Q03-2 is same batch, by Q01-1, Q02-1 and Q03-1 2mL bursopoietin diluted, Q01-2, Q02-
2, Q03-2 is diluted with the fowl that the 2mL embodiment of the present invention 2 is prepared with vaccine diluent, and 100 μ L is taken to be connected to 6 respectively
TSA plate carries out bed board, and whole process answers sterile working, the plate after bed board is placed in 37 DEG C of insulating boxs and is incubated overnight, next day
Observation, the results are shown in Table 2.
2 steriling test result of table
| Number | Clump count |
| Q01-1 | 1 |
| Q02-1 | 0 |
| Q03-1 | 1 |
| Q01-2 | 0 |
| Q02-2 | 0 |
| Q03-2 | 0 |
By table 2 it can be concluded that, using 3 plates of fowl prepared by the present invention vaccine diluent group all without long bacterium, and
Using having grown bacterium in diluted 1 plate of bursopoietin diluted.It can be seen that fowl vaccine prepared by the present invention
Dilution can kill pathogen in fowl vaccine completely really, to ensure the safety that vaccine uses.
Embodiment 6
Fowl vaccine diluent cooperates in Bursal Disease before and after the use of heat resisting protective live vaccine, no feature disease
Opportunistic pathogen quantitative comparison test
The chicken infection that 6 bottles are numbered as F001-1, F002-1, F003-1, F001-2, F002-2, F003-2 different batches
Property bursal disease in heat resisting protective live vaccine, wherein F001-1 and F001-2 are same batch, and F002-1 and F002-2 are same
One batch, F003-1 and F003-2 are same batch, by F001-1, F002-1 and F003-1 2mL bursopoietin dilution
Dilution, F001-2, F002-2, F003-2 are diluted with the fowl that the 2mL embodiment of the present invention 3 is prepared with vaccine diluent,
It takes 100 μ L to be connected to 6 TSA plates respectively and carries out bed board, whole process answers sterile working, the plate after bed board is placed in 37 DEG C of perseverances
Incubator is incubated overnight, next day observation, and concrete outcome is as shown in table 3.
3 steriling test result of table
| Number | Clump count |
| F001-1 | 1 |
| F002-1 | 1 |
| F003-1 | 1 |
| F001-2 | 0 |
| F002-2 | 0 |
| F003-2 | 0 |
By table 3 it can be concluded that, using 3 plates of fowl prepared by the present invention vaccine diluent group all without long bacterium, and
Bacterium has been grown using diluted 3 plate of bursopoietin diluted.It can be seen that fowl prepared by the present invention is diluted with vaccine
Liquid can kill pathogen in fowl vaccine completely really, to ensure the safety that vaccine uses.
By above embodiments, it can be concluded that, the fowl that preparation method provided by the invention is prepared can with vaccine diluent
Completely kill fowl vaccine specific pathogen free bacterium, can guarantee vaccine using safe.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of fowl vaccine diluent, includes the following steps：
1) it is homogenized after mixing chicken bursa, chicken intestines and water, obtains homogenate；
2) by the homogenate multigelation of the step 1) 6~8 times, freeze thawing liquid is obtained；
3) the freeze thawing liquid of the step 2) is successively stirred with the mixed mixed liquor of acetic acid, is centrifuged, the centrifugation that will be obtained
Clear liquid mixes inactivation with beta-propiolactone, obtains inactivation liquid；
4) the inactivation liquid of the step 3) is obtained into hydrolyzate in 35~42 DEG C of 1~3h of hydrolysis；
5) hydrolyzate of the step 4) is subjected to micro-filtration, the pH value of the micro-filtrate adjusted is 6.5~7.5, is adjusted
Liquid；
6) the adjusting liquid of the step 5) is subjected to ultrafiltration, the ultrafiltrate degerming that will be obtained obtains fowl vaccine diluent.
2. preparation method according to claim 1, which is characterized in that the quality of the step 1) chicken bursa and chicken intestines
Quality, the volume ratio of water are (0.5~1.5):(0.1~0.5):(0.5~2.5).
3. preparation method according to claim 1, which is characterized in that the speed of the step 1) homogenate is 15000~
18000rpm, the time of the homogenate are 10~20min.
4. preparation method according to claim 1, which is characterized in that based on volumetric concentration, the step 3) acetic acid is mixed
Close final concentration of 1~3% in liquid.
5. preparation method according to claim 1, which is characterized in that the temperature of the step 3) stirring is 0~10 DEG C, institute
The time for stating stirring is 10~14h, and the speed of the stirring is 30~50rpm；The temperature of the centrifugation be 0~4 DEG C, it is described from
The time of the heart is 10~20min, and the speed of the centrifugation is 5000~10000rpm.
6. preparation method according to claim 1, which is characterized in that based on volumetric concentration, the step 3) centrifugal clear liquid
After being mixed with beta-propiolactone, final concentration of the 0.010~0.015% of beta-propiolactone.
7. preparation method according to claim 1, which is characterized in that the temperature of the step 3) inactivation is 5~15 DEG C, institute
The time for stating inactivation is 24~30h.
8. preparation method according to claim 1, which is characterized in that the aperture for the filter membrane that the step 5) micro-filtration uses is
0.22μm。
9. preparation method according to claim 1, which is characterized in that the ultrafiltration membrane that the step 6) ultrafiltration uses is cut
Staying molecular weight is 4~8kD.
10. the fowl vaccine diluent that method described in any one of claim 1 to 9 is prepared.
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Application publication date: 20181130 |