CN104984336A - Swine streptococcosis divalent propolis inactivated vaccine and preparation method thereof - Google Patents
Swine streptococcosis divalent propolis inactivated vaccine and preparation method thereof Download PDFInfo
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- CN104984336A CN104984336A CN201510478398.4A CN201510478398A CN104984336A CN 104984336 A CN104984336 A CN 104984336A CN 201510478398 A CN201510478398 A CN 201510478398A CN 104984336 A CN104984336 A CN 104984336A
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Abstract
The invention belongs to the technical field of animal vaccine preparation and particularly relates to swine streptococcosis divalent propolis inactivated vaccine. The vaccine is prepared with an antigen concentrated solution of swine streptococcosis type-two CVCC3313 bacterial strains, an antigen concentrated solution of swine streptococcosis type-two CVCC3307 bacterial strains, a propolis ethyl alcohol leach liquor, WONG's polysaccharide and water for injection according to the volume ratio of 13-25: 15-20: 4-6: 4-5: 34-61. A preparation method of the vaccine is further related to, and the preparation method comprises the procedures of production-used bacterial strain preparation, antigen solution preparation, viable counting, ultrafiltration and concentration, inactivation, emulsification, vaccine mixing and the like, thereby completing the preparation of the divalent propolis inactivated vaccine. By means of the divalent propolis inactivated vaccine, swine stress can be reduced, the inactivated vaccine is safe and reliable, and no potential danger of toxicity dispersing exists.
Description
Technical field
The invention belongs to animal vaccine preparing technical field, specifically, is about a kind of pig streptococcicosis bivalence inactivated propolis vaccines and preparation method thereof.
Background technology
Pig streptococcicosis is caused by hemolytic streptococcal infection, and clinical manifestation is acute hemorrhagic septicemia, meningitis, endocarditis, pneumonia, arthritis etc.Wherein streptococcus suis 2-type is pathogenic strong, extensively popular, and this type is also the serotype that clinical cross frequence is the highest.Streptococcus suis 2-type is a kind of important infecting both domestic animals and human encephalapthy agent, often causes ablactational baby pig or growing and fattening pigs sudden death; This bacterium of human infection, can cause meningitis, causes permanent deafness, septicemia, endocarditis, even dead.The pig streptococcicosis that streptococcus suis 2-type causes constitutes serious threat to the aquaculture of China and people ' s health.
Therefore, need to develop a kind of Streptococcus suis disease vaccine to solve the problem.
Summary of the invention
Object of the present invention provides a kind of pig streptococcicosis bivalence inactivated propolis vaccines, and this vaccine side effect is little, does not contain foreign protein, have good immunological safety containing in endotoxin and serum.
For achieving the above object, the present invention is by the following technical solutions:
Described antigen is that the antigen of streptococcus suis 2-type CVCC3313 bacterial strain and streptococcus suis 2-type CVCC3307 bacterial strain concentrates deactivation, and when joining Seedling, in every milliliter of vaccine, the bacterium number of each bacterial strain is 2.0 × 10
9cFU ~ 3.0 × 10
9cFU, described adjuvant is propolis alcohol leaching liquid and Huang Shi polysaccharide, when joining Seedling, in every milliliter of vaccine, propolis dry matter content is 8mg, and the antigen concentrated solution of described streptococcus suis 2-type CVCC3313 bacterial strain, the antigen concentrated solution of streptococcus suis 2-type CVCC3307 bacterial strain, propolis alcohol leaching liquid, volume ratio between Huang Shi polysaccharide and water for injection are 13 ~ 25:15 ~ 20:4 ~ 6:4 ~ 5:34 ~ 61.
According to the present invention, before described streptococcus suis 2-type CVCC3313 bacterial strain and the deactivation of streptococcus suis 2-type CVCC3307 bacterial strain strain antigens liquid, viable count is 3.0 × 10
9cFU/ml ~ 4.0 × 10
9between CFU/ml.
According to the present invention, the antigen liquid of described streptococcus suis 2-type CVCC3313 bacterial strain and streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain is through 4 times of ultrafiltration and concentration.
According to the present invention, adjuvant used is high-quality propolis, obtains through alcohol steep.
The present invention also provides a kind of preparation method of the pig streptococcicosis bivalence inactivated propolis vaccines as described in above-mentioned any one, comprises the steps:
1) propolis alcohol leaching liquid preparation: propolis is stored at low temperatures, first propolis is put-15 DEG C of freezing 24-48h during use, grind with freezing crusher again and sieve, 95% ethanol is added in the ratio of 1:4, at 37 DEG C of lixiviate 48-72 hour, cooling, filtration, obtain pure propolis alcohol leaching liquid (transparent maroon solution), the propolis dry matter content of leachate is not less than 32g/ml;
2) production bacterial strain is prepared: streptococcus suis 2-type CVCC3313 bacterial strain and streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain are carried out breeding preparation production bacterial strain, and this production bacterial strain is checked purely;
3) prepare antigen liquid: by step 2) production bacterial strain be inoculated into corresponding seedling culture medium culturing respectively, prepare antigen liquid; And purely check;
4) count plate: respectively from step 3) sample two kinds of antigen liquids obtaining, count plate is carried out to streptococcus suis 2-type CVCC3313 bacterial strain and streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain, to determine the bacterium number cultivated;
5) ultrafiltration and concentration: by concentrated 4 times of the antigen liquid ultrafiltration instrument of above-mentioned two kinds of deactivations, obtained antigen concentrated solution, obtained antigen concentrated solution contains bacterium number 1.2 × 10
10cFU/ml ~ 1.6 × 10
10cFU/ml.
6) deactivation: two kinds of antigen concentrated solutions obtain deactivation concentrated antigen liquid through formalin-inactivated, and carry out deactivation inspection;
7) vaccine finished product completes: measure 13000ml ~ 25000ml streptococcus suis 2-type CVCC3313 bacterial strain concentrated solution respectively, measure 15000ml ~ 20000ml 2 type BHZZ-L4 bacterial strain concentrated solution, measure 4000ml ~ 5000ml Huang Shi polysaccharide, measure 4000ml ~ 6000ml propolis alcohol leaching liquid, propolis alcohol leaching liquid is measured according to the standard of propolis-containing dry 8mg in every milliliter of vaccine, the propolis dry matter content of leachate must be not less than 32g/ml, is supplemented to 100,000 milliliters with water for injection; Low speed 2000rpm stirs 15-20min, obtained described pig streptococcicosis bivalence inactivated propolis vaccines.
According to the present invention, before described streptococcus suis 2-type CVCC3313 bacterial strain and the deactivation of streptococcus suis 2-type CVCC3307 bacterial strain strain antigens liquid, viable count is 3.0 × 10
9cFU/ml ~ 4.0 × 10
9between CFU/ml.
According to the present invention, step 7) adjuvant be propolis alcohol leaching liquid, selected propolis, through alcohol steep, prepares propolis alcohol leaching liquid dry matter content >=32g/ml.
Pig streptococcicosis bivalence inactivated propolis vaccines of the present invention, its beneficial effect is:
1, reduce the pig streptococcicosis that Streptococcus suis causes, improve the survival rate of piglet.
2, its immunity is high, and easy to use, reduce piglet pig stress, there is not the hidden danger of loose poison.
3, take propolis as adjuvant, be a kind of green, environmental protection, safe and effective novel adjuvant, the immunoprotection that body is quick, efficient, lasting can be stimulated.
4, these goods do not add the antiseptic such as thimerosal, reduce the introducing of exogenous chemical substances, high to foodsafety.
Detailed description of the invention
Below in conjunction with embodiment, pig streptococcicosis bivalence inactivated propolis vaccines of the present invention and preparation method thereof is described in further detail.
Strain is originated
(1) streptococcus suis 2-type CVCC3313 bacterial strain, purchased from China Veterinery Drug Inspection Office;
(2) streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain, purchased from China Veterinery Drug Inspection Office.
One, propolis alcohol leaching liquid preparation
Propolis is stored at low temperatures, first propolis is put-15 DEG C of freezing 24-48h during use, grind with freezing crusher again and sieve, 95% ethanol is added in the ratio of 1:4 (W/V), at 37 DEG C of lixiviate 48-72 hour, cooling, filtration, obtain pure propolis alcohol leaching liquid, alcohol leaching liquid is transparent maroon solution, and the propolis dry matter content of leachate is not less than 32g/ml.
Two, production bacterial strain is prepared
1, streptococcus suis 2-type CVCC3313 bacterial strain
(1) first order seed breeding: by the streptococcus suis 2-type CVCC3313 inoculation of lyophilizing in TSA solid medium, put 37 DEG C to cultivate 24 hours, then choose well-grown bacterium colony, inoculation TSA solid medium, put 37 DEG C to cultivate 12 ~ 16 hours, as first order seed.
(2) secondary seed breeding: the first order seed getting streptococcus suis 2-type CVCC3313 bacterial strain is inoculated in TSB fluid medium, puts 37 DEG C and cultivates 12 ~ 16 hours, purely check by " Chinese veterinary pharmacopoeia " annex, qualified rear as secondary seed.
2, streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain
(1) first order seed breeding: by the streptococcus suis 2-type CVCC3307 bacterial strain inoculation of lyophilizing in TSA solid medium, put 37 DEG C to cultivate 24 hours, then choose well-grown bacterium colony, inoculation TSA solid medium, put 37 DEG C to cultivate 12 ~ 16 hours, as first order seed.
(2) secondary seed breeding: the first order seed getting streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain is inoculated in TSB fluid medium, puts 37 DEG C and cultivates 12 ~ 16 hours, purely check by " Chinese veterinary pharmacopoeia " annex, qualified rear as secondary seed.
Three, antigen liquid is prepared
1, the secondary seed solution of streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain is inoculated in TSB fluid medium by 1%, puts 37 DEG C and cultivates 12 hours;
2, the secondary seed solution of streptococcus suis 2-type CVCC3313 bacterial strain is inoculated in TSB fluid medium by 1%, puts 37 DEG C and cultivates 12 hours.
Four, purely check
Purely check by existing " Chinese veterinary pharmacopoeia " annex.
Five, count plate
Sample from obtained two kinds of antigen liquids respectively, carry out count plate with blood agar plate, to determine the bacterium number cultivated, obtained bacterium number is diluted to 3.0 × 10
9cFU/ml ~ 4.0 × 10
9between CFU/ml.
Six, ultrafiltration and concentration
Concentrated with 4 times, the MODELNo.XX814V230 ultrafiltration instrument of Milliproe company respectively by the antigen liquid of above-mentioned two kinds of deactivations, obtained antigen concentrated solution contains bacterium number 1.2 × 10
10cFU/ml ~ 1.6 × 10
10cFU/ml.
Seven, deactivation
Respectively to add in two kinds of antigen concentrated solutions each antigen liquid total amount of liquid 0.4% formalin, 37 DEG C of deactivations 48 hours, period stirred 1 time every 6 hours, and then sampling carries out deactivation inspection by " Chinese veterinary pharmacopoeia ", should without bacterial growth.
Eight, vaccine finished product
Embodiment 1
(1) the streptococcus suis 2-type CVCC3313 bacterial strain measured needed for 13000ml concentrates deactivation liquid, and after concentrated, bacterium number is 1.6 × 10
10cFU/ml, measures 17000ml streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain and concentrates deactivation liquid, and after concentrated, bacterium number is 1.2 × 10
10cFU/ml;
(2) 4000ml Huang Shi polysaccharide is measured;
(3) 5000ml propolis alcohol leaching liquid is measured, propolis alcohol leaching liquid is measured according to the standard of propolis-containing dry 8mg in every milliliter of vaccine, the propolis dry matter content of leachate must be not less than 32g/ml, slowly adds in emulsion tank, and 2000rpm stirs 5min;
(4) be supplemented to 100000ml with water for injection, be placed in and join Seedling emulsion tank, 2000rpm stirs 15-20min, fully emulsified, obtains described pig streptococcicosis bivalence inactivated propolis vaccines.
After testing, in vaccine, the bacterium number of streptococcus suis 2-type CVCC3313 bacterial strain is 2.0 × 10
9the bacterium number of CFU/ml, streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain is 2.0 × 10
9cFU/ml, propolis-containing dry 8mg in every milliliter of vaccine.
Embodiment 2
(1) the streptococcus suis 2-type CVCC3313 bacterial strain measured needed for 18000ml concentrates deactivation liquid, and after concentrated, bacterium number is 1.4 × 10
10cFU/ml, measures 15000ml streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain and concentrates deactivation liquid, and after concentrated, bacterium number is 1.6 × 10
10cFU/ml;
(2) 4500ml Huang Shi polysaccharide is measured;
(3) 4000ml propolis alcohol leaching liquid is measured, propolis alcohol leaching liquid is measured according to the standard of propolis-containing dry 8mg in every milliliter of vaccine, the propolis dry matter content of leachate must be not less than 32g/ml, slowly adds in emulsion tank, and 2000rpm stirs 5min;
(4) be supplemented to 100000ml with water for injection, be placed in and join Seedling emulsion tank, 2000rpm stirs 15-20min, fully emulsified, obtains described pig streptococcicosis bivalence inactivated propolis vaccines.
After testing, in vaccine, the bacterium number of streptococcus suis 2-type CVCC3313 bacterial strain is 2.5 × 10
9the bacterium number of CFU/ml, streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain is 2.4 × 10
9cFU/ml, propolis-containing dry 8mg in every milliliter of vaccine.
Embodiment 3
(1) the streptococcus suis 2-type CVCC3313 bacterial strain measured needed for 25000ml concentrates deactivation liquid, and after concentrated, bacterium number is 1.2 × 10
10cFU/ml, measures 20000ml streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain and concentrates deactivation liquid, and after concentrated, bacterium number is 1.5 × 10
10cFU/ml;
(2) 5000ml Huang Shi polysaccharide is measured;
(3) 6000ml propolis alcohol leaching liquid is measured, propolis alcohol leaching liquid is measured according to the standard of propolis-containing dry 8mg in every milliliter of vaccine, the propolis dry matter content of leachate must be not less than 32g/ml, slowly adds in emulsion tank, and 2000rpm stirs 5min;
(4) be supplemented to 100000ml with water for injection, be placed in and join Seedling emulsion tank, 2000rpm stirs 15-20min, fully emulsified, obtains described pig streptococcicosis bivalence inactivated propolis vaccines.
After testing, in vaccine, the bacterium number of streptococcus suis 2-type CVCC3313 bacterial strain is 3.0 × 10
9the bacterium number of CFU/ml, streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain is 3.0 × 10
9cFU/ml, propolis-containing dry 8mg in every milliliter of vaccine.
Nine, the safety experiment of the vaccine finished product of embodiment 1, embodiment 2 and embodiment 3
Above-mentioned obtained bivalence inactivated propolis vaccines is seeded to 10 piglets, every intramuscular injection 4ml, matched group intramuscular injection 4mlTSB fluid medium, Continuous Observation 2 weeks.Experimental group and the matched group of embodiment 1, embodiment 2 and embodiment 3 are all healthy.
Ten, the potency test of the vaccine finished product of embodiment 1, embodiment 2 and embodiment 3
With 28 ~ 35 age in days piglet 20, bivalence inactivated propolis vaccines obtained by every intramuscular injection 2mL, immunity is after 21 days, be divided into 2 groups at random, often organize each 10, carry out lumbar injection 1ml with the streptococcus suis 2-type CVCC3313 bacterial strain of a minimum lethal dose and streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain respectively, 10 pigs are set in addition respectively, not immunely compare, observe 2 weeks, assay is in table 2.
The potency test result of table 1 piglet
As can be seen from above-described embodiment, pig streptococcicosis bivalence inactivated propolis vaccines has good protection to pig streptococcicosis.
Claims (7)
1. a pig streptococcicosis bivalence inactivated propolis vaccines, comprise antigen and adjuvant, it is characterized in that, described antigen is that the antigen of streptococcus suis 2-type CVCC3313 bacterial strain and streptococcus suis 2-type CVCC3307 bacterial strain concentrates deactivation, and when joining Seedling, in every milliliter of vaccine, the bacterium number of each bacterial strain is 2.0 × 10
9cFU ~ 3.0 × 10
9cFU, described adjuvant is propolis alcohol leaching liquid and Huang Shi polysaccharide, when joining Seedling, in every milliliter of vaccine, propolis dry matter content is 8mg, and the antigen concentrated solution of described streptococcus suis 2-type CVCC3313 bacterial strain, the antigen concentrated solution of streptococcus suis 2-type CVCC3307 bacterial strain, propolis alcohol leaching liquid, volume ratio between Huang Shi polysaccharide and water for injection are 13 ~ 25:15 ~ 20:4 ~ 6:4 ~ 5:34 ~ 61.
2. vaccine as claimed in claim 1, is characterized in that, before described streptococcus suis 2-type CVCC3313 bacterial strain and the deactivation of streptococcus suis 2-type CVCC3307 bacterial strain strain antigens liquid, viable count is 3.0 × 10
9cFU/ml ~ 4.0 × 10
9between CFU/ml.
3. vaccine as claimed in claim 1, it is characterized in that, the antigen liquid of described streptococcus suis 2-type CVCC3313 bacterial strain and streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain is through 4 times of ultrafiltration and concentration.
4. vaccine as claimed in claim 1, it is characterized in that, adjuvant used is high-quality propolis, obtains through alcohol steep.
5. a preparation method for pig streptococcicosis bivalence inactivated propolis vaccines, is characterized in that, comprises the steps:
1) propolis alcohol leaching liquid preparation: propolis is stored at low temperatures, first propolis is put-15 DEG C of freezing 24-48h during use, grind with freezing crusher again and sieve, 95% ethanol is added in the ratio of 1:4, at 37 DEG C of lixiviate 48-72 hour, cooling, filtration, obtain pure propolis alcohol leaching liquid (transparent maroon solution), the propolis dry matter content of leachate is not less than 32g/ml;
2) production bacterial strain is prepared: streptococcus suis 2-type CVCC3313 bacterial strain and streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain are carried out breeding preparation production bacterial strain, and this production bacterial strain is checked purely;
3) prepare antigen liquid: by step 2) production bacterial strain be inoculated into corresponding seedling culture medium culturing respectively, prepare antigen liquid; And purely check;
4) count plate: respectively from step 3) sample two kinds of antigen liquids obtaining, count plate is carried out to streptococcus suis 2-type CVCC3313 bacterial strain and streptococcus suis 2-type CVCC3307 bacterial strain bacterial strain, to determine the bacterium number cultivated;
5) ultrafiltration and concentration: by concentrated 4 times of the antigen liquid ultrafiltration instrument of above-mentioned two kinds of deactivations, obtained antigen concentrated solution, obtained antigen concentrated solution contains bacterium number 1.2 × 10
10cFU/ml ~ 1.6 × 10
10cFU/ml.
6) deactivation: two kinds of antigen concentrated solutions obtain deactivation concentrated antigen liquid through formalin-inactivated, and carry out deactivation inspection;
7) vaccine finished product completes: measure 13000ml ~ 25000ml streptococcus suis 2-type CVCC3313 bacterial strain concentrated solution respectively, measure 15000ml ~ 20000ml 2 type BHZZ-L4 bacterial strain concentrated solution, measure 4000ml ~ 5000ml Huang Shi polysaccharide, measure 4000ml ~ 6000ml propolis alcohol leaching liquid, propolis alcohol leaching liquid is measured according to the standard of propolis-containing dry 8mg in every milliliter of vaccine, the propolis dry matter content of leachate must be not less than 32g/ml, is supplemented to 100,000 milliliters with water for injection; Low speed 2000rpm stirs 15-20min, obtained described pig streptococcicosis bivalence inactivated propolis vaccines.
6. the preparation method of a kind of pig streptococcicosis bivalence inactivated propolis vaccines as claimed in claim 5, is characterized in that, before described streptococcus suis 2-type CVCC3313 bacterial strain and the deactivation of streptococcus suis 2-type CVCC3307 bacterial strain strain antigens liquid, viable count is 3.0 × 10
9cFU/ml ~ 4.0 × 10
9between CFU/ml.
7. the preparation method of a kind of pig streptococcicosis bivalence inactivated propolis vaccines as claimed in claim 5, it is characterized in that, step 7) adjuvant be propolis alcohol leaching liquid, selected propolis, through alcohol steep, prepares propolis alcohol leaching liquid dry matter content >=32g/ml.
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Application publication date: 20151021 |