CN102716475A - Preparation method of cow mammitis vaccines - Google Patents
Preparation method of cow mammitis vaccines Download PDFInfo
- Publication number
- CN102716475A CN102716475A CN2012102219777A CN201210221977A CN102716475A CN 102716475 A CN102716475 A CN 102716475A CN 2012102219777 A CN2012102219777 A CN 2012102219777A CN 201210221977 A CN201210221977 A CN 201210221977A CN 102716475 A CN102716475 A CN 102716475A
- Authority
- CN
- China
- Prior art keywords
- staphylococcus aureus
- mammitis
- vaccine
- incubators
- cow
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 96
- 208000004396 mastitis Diseases 0.000 title claims abstract description 68
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 104
- 241000193985 Streptococcus agalactiae Species 0.000 claims abstract description 49
- 238000000034 method Methods 0.000 claims abstract description 36
- 241000588724 Escherichia coli Species 0.000 claims abstract description 22
- 239000003053 toxin Substances 0.000 claims abstract description 16
- 231100000765 toxin Toxicity 0.000 claims abstract description 16
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 48
- 239000002609 medium Substances 0.000 claims description 33
- 239000006161 blood agar Substances 0.000 claims description 27
- 230000008021 deposition Effects 0.000 claims description 27
- 241000894006 Bacteria Species 0.000 claims description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 108010038854 colibacterin Proteins 0.000 claims description 21
- 235000015097 nutrients Nutrition 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 20
- 238000001556 precipitation Methods 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 18
- 239000011550 stock solution Substances 0.000 claims description 16
- 235000015278 beef Nutrition 0.000 claims description 15
- 239000002504 physiological saline solution Substances 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- 239000006916 nutrient agar Substances 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- 210000002249 digestive system Anatomy 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 9
- 229940033663 thimerosal Drugs 0.000 claims description 9
- 239000012531 culture fluid Substances 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 8
- 238000007747 plating Methods 0.000 claims description 8
- 239000011324 bead Substances 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 7
- 238000000576 coating method Methods 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 7
- 238000009631 Broth culture Methods 0.000 claims description 6
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 229960003067 cystine Drugs 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 239000004744 fabric Substances 0.000 claims description 3
- 238000001802 infusion Methods 0.000 claims description 3
- 239000012092 media component Substances 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- XINQFOMFQFGGCQ-UHFFFAOYSA-L (2-dodecoxy-2-oxoethyl)-[6-[(2-dodecoxy-2-oxoethyl)-dimethylazaniumyl]hexyl]-dimethylazanium;dichloride Chemical compound [Cl-].[Cl-].CCCCCCCCCCCCOC(=O)C[N+](C)(C)CCCCCC[N+](C)(C)CC(=O)OCCCCCCCCCCCC XINQFOMFQFGGCQ-UHFFFAOYSA-L 0.000 claims 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 claims 1
- 230000002265 prevention Effects 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 30
- 238000012360 testing method Methods 0.000 description 24
- 230000036039 immunity Effects 0.000 description 23
- 238000000151 deposition Methods 0.000 description 21
- 230000000694 effects Effects 0.000 description 19
- 230000001681 protective effect Effects 0.000 description 15
- 241000283973 Oryctolagus cuniculus Species 0.000 description 14
- 230000037396 body weight Effects 0.000 description 14
- 239000001963 growth medium Substances 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 9
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000000306 component Substances 0.000 description 7
- 239000003440 toxic substance Substances 0.000 description 7
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 231100000614 poison Toxicity 0.000 description 6
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 5
- 239000005864 Sulphur Substances 0.000 description 5
- 230000003115 biocidal effect Effects 0.000 description 5
- 230000001147 anti-toxic effect Effects 0.000 description 4
- 230000036760 body temperature Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000011046 pyrogen test Methods 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000013337 sub-cultivation Methods 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 229940031572 toxoid vaccine Drugs 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 230000000941 anti-staphylcoccal effect Effects 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000004448 titration Methods 0.000 description 2
- 231100000167 toxic agent Toxicity 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000004158 L-cystine Substances 0.000 description 1
- 235000019393 L-cystine Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000007711 Peperomia pellucida Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000011169 microbiological contamination Methods 0.000 description 1
- 231100000668 minimum lethal dose Toxicity 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 108010009004 proteose-peptone Proteins 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- GNBVPFITFYNRCN-UHFFFAOYSA-M sodium thioglycolate Chemical compound [Na+].[O-]C(=O)CS GNBVPFITFYNRCN-UHFFFAOYSA-M 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
A preparation method of cow mammitis vaccines relates to a preparation method of vaccines. The method comprises preparing staphylococcus aureus toxin with the titer of more than 1024, staphylococcus aureus similar toxin with the titer of more than 128, staphylococcus aureus mycoprotein, streptococcus agalactiae vaccines and escherichia coli vaccines, and obtaining the cow mammitis vaccines after compatibility of medicines. The cow mammitis vaccines are safe and free of toxin, and protection rate on a mouse in a toxin studying group achieves 91.7%. The preparation method is applied in the field of prevention and cure of cow mammitis.
Description
Technical field
The present invention relates to a kind of method for preparing of vaccine.
Background technology
Mammitis of cow (Mastitis) is the most general infectious disease of milch cow of growing up; Mainly be that the cow mammary gland tissue receives infected by microbes and a kind of inflammation of causing; Pilosity is born in age of sucking in puerperal; This disease extensively is present in all over the world, is milch cow commonly encountered diseases, frequently-occurring disease, is to cause milk product to help already to lose the most serious disease.Causing the nearly kind more than 150 of pathogenic microorganism of mammitis of cow, is main with staphylococcus aureus, escherichia coli, streptococcus agalactiae mainly, and these three kinds of bacterial mammitis of cow account for more than 90% of total incidence.
The Therapeutic Method of current mammitis of cow mainly is to adopt antibiotic therapy; Antibiotic has been used to treat the history in existing more than 50 year of mammitis of cow, and it has played certain function in the control of mammitis of cow, but because single, heavy dose of for a long time not science is used antibiotic; Caused the elimination of sensitivity antibacterial; Drug tolerant bacteria has accounted for main flow gradually, and especially the drug resistance problem of staphylococcus aureus is serious day by day, has caused antibiotic therapy to produce little effect.
Utilize vaccine to prevent and treat mammitis of cow and have good prospect, at first, vaccine can prevent the milch cow infection pathogen and cause mastitis; Secondly, vaccine helps to reduce the order of severity of intramammary infection, controls subclinical type mastitis; The 3rd, use the vaccine control mastitis can not have antibiotic remains problem in the milk; Be easy and simple to handle at last, cost.Current, the vaccine of succeeding in developing seldom, the overwhelming majority is the complete thalline vaccine of the first generation, DeGrain.Therefore, develop a kind of novel mammitis of cow vaccine, prevention and treatment mammitis of cow are present problem demanding prompt solutions.
Summary of the invention
The invention provides a kind of method for preparing of mammitis of cow vaccine.
The method for preparing of a kind of mammitis of cow vaccine of the present invention is carried out according to following steps:
One, staphylotoxin preparation
A, with inoculating loop picking staphylococcus aureus, be inoculated in the nutrient broth, place 37 ℃ of incubators to cultivate 18 ~ 24h, culture fluid; The culture fluid of drawing 10 μ L is coated with on the blood agar plating medium evenly; After in 37 ℃ of incubators, cultivating 18 ~ 24h; Picking one inoculating loop diameter is greater than single bacterium colony of 1mm; Be inoculated in nutrient agar slant medium, place 37 ℃ of incubators to cultivate 18 ~ 24h then, get staphylococcus aureus Nutrient agar slant culture; Picking one inoculating loop staphylococcus aureus Nutrient agar slant culture is inoculated in the toxin producing medium, and shaken cultivation 18 ~ 24h under 37 ℃ of conditions is seed liquor; B, seed liquor that step a is obtained by volume percentage composition are that 1% ~ 3% inoculum concentration is inoculated in the toxin producing medium; It is 37 ℃ in temperature then; Rotating speed is after cultivating 18 ~ 24h under the condition of 180 ~ 250r/min; Centrifugal 30min under the condition of 3000r/min collects supernatant and deposition simultaneously, and the supernatant of collection is staphylotoxin; The deposition of collecting, subsequent use;
Two, staphylococcus aureus toxoid preparation
C, get the staphylotoxin that step 1 makes; NaOH solution with 1N is regulated pH value to 6.5 ~ 8.5; Adding formaldehyde to formaldehyde volumetric concentration then is 0.2% ~ 0.5%, places 37 ℃ of incubators, the 3 ~ 7d that detoxifies, and promptly gets staphylococcus aureus toxoid stock solution; D, the staphylococcus aureus toxoid stock solution that step c is obtained are used filter paper filtering, and in filtrating, adding medical sodium chloride to sodium chloride mass concentration is 10% ~ 20%, regulate pH value to 2.0 ~ 4.0, hold over night with the HCl solution of 2N then; E, with the staphylococcus aureus toxoid stock solution centrifugal 10min under the 3000r/min rotating speed after the steps d overnight treatment; The aseptic redistilled water centrifuge washing deposition of collecting precipitation reuse 1 ~ 2 time; Collecting deposition after the washing, to be dissolved in pH be in 6.5 ~ 8.5 the phosphate buffer; Centrifugal 10min under the 3000r/min rotating speed once more, collecting precipitation and in deposition, to add mass concentration be that 1% thimerosal solution to thimerosal mass concentration is 0.01% promptly gets the staphylococcus aureus toxoid; Wherein, phosphate buffer and staphylococcus aureus toxoid stock solution volume ratio are 1:15 ~ 30;
Three, the preparation of staphylococcus aureus tropina
Get the deposition of collecting in the step 1,, get supernatant with 0 ~ 4 ℃ of physiological saline solution centrifuge washing 3 times; Collecting precipitation is that the ratio of 1:3 ~ 5 is put into aseptic homogenate cup and mixed with the deposition of collecting and sterile glass beads in mass ratio, adds 0 ~ 4 ℃ of physiological saline solution to cup more completely; Adopting homogenizer is 5 ℃ in temperature, and rotating speed is to vibrate 12min under the condition of 4000r/min, is 14000r/min at rotating speed then; Temperature is centrifugal 40min under 5 ℃ the condition, collects supernatant, with HCl solution adjusting supernatant pH value to 3.0 ~ 5.0 of 0.5N; Be 7000r/min at rotating speed once more, temperature is centrifugal 30min under 5 ℃ the condition, collecting precipitation; Pack in the bag filter,, promptly get the staphylococcus aureus tropina through the dialysis of 48 ~ 96h flowing water;
Four, the preparation of streptococcus agalactiae vaccine
Preparation bacterium amount is 1.5 ~ 3.0 * 10
9The streptococcus agalactiae vaccine, place 4 ℃ refrigerator to preserve subsequent use;
Five, the preparation of colibacterin
Preparation bacterium amount is 1.5 ~ 3.0 * 10
9Colibacterin, place 4 ℃ refrigerator to preserve subsequent use;
Six, the preparation of mammitis of cow vaccine
After the colibacterin that the staphylococcus aureus toxoid that step 2 is obtained, the staphylococcus aureus tropina that step 3 obtains, streptococcus agalactiae vaccine that step 4 obtains and step 5 obtain mixes, promptly get the mammitis of cow vaccine; Wherein, it is 10 ~ 30U that every milliliter of mammitis of cow vaccine contains anatoxic the tiring of staphylococcus aureus, and the protein content of staphylococcus aureus tropina is 200 ~ 400 μ g, and the bacterium number of streptococcus agalactiae is 1.5 ~ 3.0 * 10
9, colibacillary bacterium number is 1.5 ~ 3.0 * 10
9
Beneficial effect of the present invention:
The present invention adopts the main pathogenic bacterium staphylococcus aureus cause mammitis of cow, streptococcus agalactiae, escherichia coli as producing bacterial strain; With extracting, making with extra care the effective efficiency composition that forms: staphylococcus aureus toxoid and tropina, streptococcus agalactiae vaccine, colibacterin reasonable compatibility are built into a kind of novel mammitis of cow vaccine.Confirm through zoopery; The vaccine of the present invention's development has safe, nontoxic, higher characteristics such as immunity; Protective rate to staphylococcus aureus counteracting toxic substances group white mice has reached 93.3%; Also more than 80%, and the immunity effect of new generation vaccine all is better than the immunity effect of one-component vaccine, and (staphylococcus aureus toxoid vaccine protective rate is 73.3% to the protective rate of streptococcus agalactiae and escherichia coli counteracting toxic substances group white mice; Staphylococcus aureus tropina vaccine protective rate is 56.7%; Streptococcus agalactiae vaccine protective rate is 66.7%; The colibacterin protective rate is 70%).This has also fully confirmed staphylococcus aureus toxoid and tropina compatibility; Can significantly improve body and produce the level of anti-staphylococcus aureus antibody; And toxoid and tropina not only have specific immunity effect (to staphylococcus aureus); Also have the nonspecific immunity effect (through the quantity of leukocyte increasing, streptococcus agalactiae and escherichia coli are produced engulf, effect such as cleaning).
The specific embodiment
Technical scheme of the present invention is not limited to the following cited specific embodiment, also comprises the combination in any between each specific embodiment.
The specific embodiment one: the method for preparing of a kind of mammitis of cow vaccine of this embodiment is carried out according to following steps:
One, staphylotoxin preparation
A, with inoculating loop picking staphylococcus aureus, be inoculated in the nutrient broth, place 37 ℃ of incubators to cultivate 18 ~ 24h, culture fluid; The culture fluid of drawing 10 μ L is coated with on the blood agar plating medium evenly; After in 37 ℃ of incubators, cultivating 18 ~ 24h; Picking one inoculating loop diameter is greater than single bacterium colony of 1mm; Be inoculated in nutrient agar slant medium, place 37 ℃ of incubators to cultivate 18 ~ 24h then, get staphylococcus aureus Nutrient agar slant culture; Picking one inoculating loop staphylococcus aureus Nutrient agar slant culture is inoculated in the toxin producing medium, and shaken cultivation 18 ~ 24h under 37 ℃ of conditions is seed liquor; B, seed liquor that step a is obtained by volume percentage composition are that 1% ~ 3% inoculum concentration is inoculated in the toxin producing medium; It is 37 ℃ in temperature then; Rotating speed is after cultivating 18 ~ 24h under the condition of 180 ~ 250r/min; Centrifugal 30min under the condition of 3000r/min collects supernatant and deposition simultaneously, and the supernatant of collection is staphylotoxin; The deposition of collecting, subsequent use;
Two, staphylococcus aureus toxoid preparation
C, get the staphylotoxin that step 1 makes; NaOH solution with 1N is regulated pH value to 6.5 ~ 8.5; Adding formaldehyde to formaldehyde volumetric concentration then is 0.2% ~ 0.5%, places 37 ℃ of incubators, the 3 ~ 7d that detoxifies, and promptly gets staphylococcus aureus toxoid stock solution; D, the staphylococcus aureus toxoid stock solution that step c is obtained are used filter paper filtering, and in filtrating, adding medical sodium chloride to sodium chloride mass concentration is 10% ~ 20%, regulate pH value to 2.0 ~ 4.0, hold over night with the HCl solution of 2N then; E, with the staphylococcus aureus toxoid stock solution centrifugal 10min under the 3000r/min rotating speed after the steps d overnight treatment; The aseptic redistilled water centrifuge washing deposition of collecting precipitation reuse 1 ~ 2 time; Collecting deposition after the washing, to be dissolved in pH be in 6.5 ~ 8.5 the phosphate buffer; Centrifugal 10min under the 3000r/min rotating speed once more, collecting precipitation and in deposition, to add mass concentration be that 1% thimerosal solution to thimerosal mass concentration is 0.01% promptly gets the staphylococcus aureus toxoid; Wherein, phosphate buffer and staphylococcus aureus toxoid stock solution volume ratio are 1:15 ~ 30;
Three, the preparation of staphylococcus aureus tropina
Get the deposition of collecting in the step 1,, get supernatant with 0 ~ 4 ℃ of physiological saline solution centrifuge washing 3 times; Collecting precipitation is that the ratio of 1:3 ~ 5 is put into aseptic homogenate cup and mixed with the deposition of collecting and sterile glass beads in mass ratio, adds 0 ~ 4 ℃ of physiological saline solution to cup more completely; Adopting homogenizer is 5 ℃ in temperature, and rotating speed is to vibrate 12min under the condition of 4000r/min, is 14000r/min at rotating speed then; Temperature is centrifugal 40min under 5 ℃ the condition, collects supernatant, with HCl solution adjusting supernatant pH value to 3.0 ~ 5.0 of 0.5N; Be 7000r/min at rotating speed once more, temperature is centrifugal 30min under 5 ℃ the condition, collecting precipitation; Pack in the bag filter,, promptly get the staphylococcus aureus tropina through the dialysis of 48 ~ 96h flowing water;
Four, the preparation of streptococcus agalactiae vaccine
Preparation bacterium amount is 1.5 ~ 3.0 * 10
9The streptococcus agalactiae vaccine, place 4 ℃ refrigerator to preserve subsequent use;
Five, the preparation of colibacterin
Preparation bacterium amount is 1.5 ~ 3.0 * 10
9Colibacterin, place 4 ℃ refrigerator to preserve subsequent use;
Six, the preparation of mammitis of cow vaccine
After the colibacterin that the staphylococcus aureus toxoid that step 2 is obtained, the staphylococcus aureus tropina that step 3 obtains, streptococcus agalactiae vaccine that step 4 obtains and step 5 obtain mixes, promptly get the mammitis of cow vaccine; Wherein, it is 10 ~ 30U that every milliliter of mammitis of cow vaccine contains anatoxic the tiring of staphylococcus aureus, and the protein content of staphylococcus aureus tropina is 200 ~ 400 μ g, and the bacterium number of streptococcus agalactiae is 1.5 ~ 3.0 * 10
9, colibacillary bacterium number is 1.5 ~ 3.0 * 10
9
The main pathogenic bacterium staphylococcus aureus of this embodiment employing causing mammitis of cow, streptococcus agalactiae, escherichia coli are as producing bacterial strain; With extracting, making with extra care the effective efficiency composition that forms: staphylococcus aureus toxoid and tropina, streptococcus agalactiae vaccine, colibacterin reasonable compatibility are built into a kind of novel mammitis of cow vaccine.Confirm through zoopery; The vaccine of the present invention's development has safe, nontoxic, higher characteristics such as immunity; Protective rate to staphylococcus aureus counteracting toxic substances group white mice has reached 93.3%; Also more than 80%, and the immunity effect of new generation vaccine all is better than the immunity effect of one-component vaccine, and (staphylococcus aureus toxoid vaccine protective rate is 73.3% to the protective rate of streptococcus agalactiae and escherichia coli counteracting toxic substances group white mice; Staphylococcus aureus tropina vaccine protective rate is 56.7%; Streptococcus agalactiae vaccine protective rate is 66.7%; The colibacterin protective rate is 70%).This has also fully confirmed staphylococcus aureus toxoid and tropina compatibility; Can significantly improve body and produce the level of anti-staphylococcus aureus antibody; And toxoid and tropina not only have specific immunity effect (to staphylococcus aureus); Also have the nonspecific immunity effect (through the quantity of leukocyte increasing, streptococcus agalactiae and escherichia coli are produced engulf, effect such as cleaning).
The specific embodiment two: what this embodiment and the specific embodiment one were different is: the operation that step 1 to step 6 is carried out is all carried out under aseptic condition.Other is identical with the specific embodiment one.
The specific embodiment three: what this embodiment and the specific embodiment one to two were different is: it is 1024 ~ 4096 that the staphylotoxin described in the step 1 is tired.Other is identical with the specific embodiment one to two.
The specific embodiment four: what this embodiment was different with one of specific embodiment one to three is: it is 128 ~ 256 that the staphylococcus aureus toxoid described in the step 2 is tired.Other is identical with one of specific embodiment one to three.
The specific embodiment five: what this embodiment was different with one of specific embodiment one to four is: protein content is 10 ~ 50mg/mL in the staphylococcus aureus tropina described in the step 3.Other is identical with one of specific embodiment one to four.
The specific embodiment six: what this embodiment was different with one of specific embodiment one to five is: the sterile glass beads described in the step 3 is identical for one of
other and specific embodiment one to five.
The specific embodiment seven: what this embodiment was different with one of specific embodiment one to six is: the product acid nutrient media components described in the step 1 is: the MgSO of 0.3g
47H
2The KCl of O, 0.2g, the K of 3.0g
2HPO
43H
2The glucose of O, 2.0g, 2mL mass concentration are 10% cystine, the beef infusion broth of 700mL and the beef Digestive system of 300mL; Wherein, beef Digestive system method for preparing is: beef is rubbed, take by weighing 1000g, add the Na of distilled water 1500mL, 1.5g
2CO
3, the pancreatin of 6g and the chloroform of 30mL, place 37 ℃ of incubators, using mass concentration is that 4% NaOH regulates pH to 8.0; Digestion 36h; The HCL of reuse 2N regulates pH to 4.5, is heated to 90 ℃, filters with double-layer cloth then; In filtrating, adding volumetric concentration is 2% chloroform mixing, promptly gets the beef Digestive system.Other is identical with one of specific embodiment one to six.
The specific embodiment eight: what this embodiment was different with one of specific embodiment one to seven is: the method for preparing of the streptococcus agalactiae vaccine described in the step 4 is following:: a, get an inoculating loop streptococcus agalactiae; Be inoculated in the blood agar plating medium with the sectional streak inocalation method; Place 37 ℃ of incubators to cultivate 18 ~ 24h then; Picking one inoculating loop diameter is inoculated in the nutrient broth medium greater than single bacterium colony of 1mm, places 37 ℃ of incubators to cultivate 18 ~ 24h; Get streptococcus agalactiae nutrient broth culture 2mL; Be inoculated on the blood agar slant medium, coating evenly is placed in 37 ℃ of incubators cultivates 24 ~ 48h, promptly gets streptococcus agalactiae blood agar slant culture; B, use concentration are that the sterile phosphate normal saline of 0.02 ~ 0.2mol/L washes the streptococcus agalactiae blood agar slant culture that step a obtains in the aseptic vial; Filter with aseptic silk then; Adding formalin to formaldehyde volumetric concentration is 1.0% ~ 1.2%; After placing 37 ℃ of incubators to cultivate 1 ~ 7d, adding phenol to final concentration is 5.0g/L, places 4 ℃ refrigerator to preserve at last.Other is identical with one of specific embodiment one to seven.
The specific embodiment nine: what this embodiment was different with one of specific embodiment one to eight is: the method for preparing of the colibacterin described in the step 5 is following: a, get escherichia coli one inoculating loop; Be inoculated in the blood agar flat board with the sectional streak inocalation method, place 37 ℃ of incubators to cultivate 18 ~ 24h, picking one inoculating loop diameter is greater than single bacterium colony of 1mm; Be inoculated in the nutrient broth medium; Place 37 ℃ of incubators to cultivate 18 ~ 24h, get an inoculating loop escherichia coli nutrient broth culture 2mL, be inoculated on the blood agar slant medium; Coating evenly is placed in 37 ℃ of incubators cultivates 24 ~ 48h, promptly gets colibacterin; B, concentration are that the colibacterin that the sterile phosphate normal saline of 0.02 ~ 0.2mol/L obtains step a washes in the aseptic vial; Filter with aseptic silk then; Adding formalin to formaldehyde volumetric concentration is 1.0% ~ 1.2%; After placing 37 ℃ of incubator 1 ~ 7d, adding phenol to final concentration is 5.0g/L, places 4 ℃ refrigerator to preserve at last.Other is identical with one of specific embodiment one to eight.
Through following verification experimental verification effect of the present invention:
The method for preparing of a kind of mammitis of cow vaccine of this test is carried out according to following steps:
One, staphylotoxin preparation
A, with inoculating loop picking staphylococcus aureus, be inoculated in the nutrient broth, place 37 ℃ of incubators to cultivate 24h, culture fluid; The culture fluid of drawing 10 μ L is even in the coating of blood agar plating medium; After in 37 ℃ of incubators, cultivating 24h; Picking one inoculating loop diameter is greater than single bacterium colony of 1mm; Be inoculated in nutrient agar slant medium, place 37 ℃ of incubators to cultivate 18 ~ 24h then after, staphylococcus aureus Nutrient agar slant culture; Get an inoculating loop staphylococcus aureus Nutrient agar slant culture, be inoculated in the toxin producing medium, shaken cultivation 24h under 37 ℃ of conditions is seed liquor; B, seed liquor that step a is obtained by volume percentage composition are that 1% inoculum concentration is inoculated in the toxin producing medium; It is 37 ℃ in temperature then; Rotating speed is after cultivating 24h under the condition of 200r/min; Centrifugal 30min under the condition of 3000r/min collects supernatant and deposition simultaneously, and the supernatant of collection promptly gets staphylotoxin; The deposition of collecting, subsequent use;
Two, the staphylotoxin mensuration of tiring
Get 96 hole ELISA Plates, add the 0.1mL physiological saline solution, draw the staphylotoxin 0.1mL that step 1 obtains again and add the 1st hole at every Kong Zhongjun of first row; With the application of sample rifle inhale repeatedly beat to mix after, draw 0.1mL and add in the 2nd hole, inhale once more and beat mix homogeneously; With the method doubling dilution to the 12 holes, in every hole, adding the concentration of using physiological saline solution to wash 3 times respectively then is 1% fresh rabbit erythrocyte 0.1mL, puts 1h in 37 ℃ of incubators; Take out and observe, the maximum dilution multiple of complete hemolysis is toxin and tires; Get the staphylococcus aureus poison of tiring more than or equal to 1024 units, subsequent use;
Three, staphylococcus aureus toxoid preparation
C, get the staphylotoxin that step 2 makes, regulate pH value to 8.0 with the NaOH solution of 1N, adding formaldehyde to formaldehyde volumetric concentration then is 0.4%, places 37 ℃ of incubators 7d that detoxifies, and promptly gets staphylococcus aureus toxoid stock solution; D, the staphylococcus aureus toxoid stock solution that step c is obtained are used filter paper filtering, and in filtrating, adding medical sodium chloride to sodium chloride mass concentration is 15%, regulate pH value to 3.0, hold over night with the HCl solution of 2N then; E, with the staphylococcus aureus toxoid stock solution centrifugal 10min under the 3000r/min rotating speed after the steps d overnight treatment; The aseptic redistilled water centrifuge washing deposition of collecting precipitation reuse 1 time; Collecting deposition after the washing, to be dissolved in pH be in 8.0 the phosphate buffer; Centrifugal 10min under the 3000r/min rotating speed once more, collecting precipitation and in deposition, to add mass concentration be that 1% thimerosal solution to thimerosal mass concentration is 0.01% promptly gets the staphylococcus aureus toxoid; Wherein, phosphate buffer and staphylococcus aureus toxoid stock solution volume ratio are 1:20;
Four, the staphylococcus aureus toxoid mensuration of tiring
⑴ antitoxic serum preparation: select 3 ~ 5 of the healthy rabbits of body weight more than 2 kilograms, from ear vein blood sampling 3mL, separation of serum; Survey basic antibody and carry out following test,, whenever later on injected 1 time at a distance from 3 days at the staphylococcus aureus toxoid 1mL (10U tires) that rabbit back leg intramuscular injection step 3 obtains less than the rabbit of 1:8; Inject altogether 3 times; Behind the 7d from ear vein blood sampling 3mL, separation of serum, subsequent use;
⑵ antitoxic serum titration: get 96 hole ELISA Plates, the physiological saline solution to every Kong Zhongjun of first row adds 0.1mL adds the isolating serum 0.1mL of step ⑴ in the 1st hole; With the application of sample rifle inhale repeatedly beat to mix after, draw 0.1mL and add in the 2nd hole, inhale once more and beat mix homogeneously; With the method doubling dilution to the 12 holes, add 0.1mL staphylotoxin (2U/mL) to every Kong Zhongzai then, slightly shake mixing; Place 37 ℃ of incubators to cultivate 30min, add in every hole then that to use the volumetric concentration of physiological saline solution washing 3 times be 1% fresh rabbit erythrocyte 0.1mL, observe after placing 37 ℃ of incubators to cultivate 60min; Complete anhemolytic maximum dilution multiple is serum titer, and replication is once got the immunizing rabbit carotid artery blood-letting of tiring to more than the 1:128; Separation of serum; Be antitoxic serum, add mass concentration and be 0.01% thimerosal, it is subsequent use to place-20 ℃ of refrigerator-freezers to preserve;
⑶ toxoid titration: get 96 hole ELISA Plates, add the 0.1mL physiological saline solution, in the 1st hole, add the staphylococcus aureus toxoid 0.1mL that step 3 obtains again to every Kong Zhongjun of first row; With the application of sample rifle inhale repeatedly beat to mix after, draw 0.1mL and add in the 2nd hole, inhale once more and beat mix homogeneously; With the method doubling dilution to the 12 holes, in every hole, add antitoxic serum (2U/mL) 0.1mL that step ⑵ obtains respectively, place 37 ℃ of incubators to cultivate 30min; In every hole, add 0.1mL staphylotoxin (2U/mL) again; Slight concussion mixing places 37 ℃ of incubators to cultivate 30min, and in every hole, adding the volumetric concentration of using physiological saline solution to wash 3 times again is 1% fresh rabbit erythrocyte 0.1mL; Observe after placing 37 ℃ of incubators to cultivate 60min; The maximum dilution multiple of complete hemolysis is the staphylococcus aureus toxoid and tires, and getting tires is the staphylococcus aureus toxoid more than 128, subsequent use;
Five, the preparation of staphylococcus aureus tropina
Get the precipitation of collecting in the step 1; With 4 ℃ of SPSS centrifuge washings 3 times; Get supernatant; Collecting precipitation; Is that the ratio of 1:5 is put into aseptic homogenate cup and mixed with the precipitation of collecting and sterile glass beads in mass ratio; It is full to add 4 ℃ of SPSSs to cup again; Adopting homogenizer is 5 ℃ in temperature; Rotating speed is to vibrate 12min under the condition of 4000r/min, is 14000r/min at rotating speed then, and temperature is centrifugal 40min under 5 ℃ the condition; Collect supernatant; Regulating supernatant pH value to 4.5 with the HCl solution of 0.5N, is 7000r/min at rotating speed once more, and temperature is centrifugal 30min under 5 ℃ the condition; Collecting precipitation; Pack in the bag filter,, promptly get the staphylococcus aureus mycoprotein through the dialysis of 72h flowing water;
Six, staphylococcus aureus tropina Determination on content
With Kjeldahl tropina is carried out the mensuration of nitrogen content, the protein content of depletion Staphylococcus aureus tropina is that the above staphylococcus aureus tropina of 30mg/mL is subsequent use;
Seven, the preparation of streptococcus agalactiae vaccine
F, get an inoculating loop streptococcus agalactiae, be inoculated in the blood agar plating medium, place 37 ℃ of incubators to cultivate 24h then with the sectional streak inocalation method; Picking one inoculating loop diameter is inoculated in the nutrient broth medium greater than single bacterium colony of 1mm, places 37 ℃ of incubators to cultivate 24h; Get streptococcus agalactiae nutrient broth culture 2mL; Be inoculated on the blood agar slant medium, coating evenly is placed in 37 ℃ of incubators cultivates 48h, promptly gets streptococcus agalactiae blood agar slant culture; G, use concentration are that the sterile phosphate normal saline of 0.02 ~ 0.2mol/L washes the streptococcus agalactiae blood agar slant culture that step f obtains in the aseptic vial; Filter with aseptic silk then; Adding formalin to volumetric concentration is 1.0%; After placing 37 ℃ of incubators to cultivate 7d, adding phenol to concentration is 5.0g/L, places 4 ℃ refrigerator to preserve at last;
Eight, the preparation of colibacterin
H, get escherichia coli one inoculating loop, be inoculated in the blood agar flat board, place 37 ℃ of incubators to cultivate 24h with the sectional streak inocalation method; Picking one inoculating loop diameter is inoculated in the nutrient broth medium greater than single bacterium colony of 1mm, places 37 ℃ of incubators to cultivate 24h; Get an inoculating loop escherichia coli nutrient broth culture 2mL; Be inoculated on the blood agar slant medium, coating evenly is placed in 37 ℃ of incubators cultivates 48h, promptly gets colibacterin; I, use concentration are that the colibacterin that the sterile phosphate normal saline of 0.02 ~ 0.2mol/L obtains step h washes in the aseptic vial; Filter with aseptic silk then; Adding formalin to volumetric concentration is 1.0%; After placing 37 ℃ of incubator 7d, adding phenol to concentration is 5.0g/L, places 4 ℃ refrigerator to preserve at last;
Nine, the preparation of mammitis of cow vaccine
After the colibacterin that the staphylococcus aureus toxoid that step 2 is obtained, the staphylococcus aureus tropina that step 3 obtains, streptococcus agalactiae vaccine that step 4 obtains and step 5 obtain mixes, promptly get the mammitis of cow vaccine; Wherein, every milliliter of mammitis of cow vaccine contains the staphylococcus aureus toxoid tires and is that 15U, the protein content of staphylococcus aureus tropina are 375 μ g, and it is 2.7 * 10 that the streptococcus agalactiae bacterium is counted content
9, it is 2.7 * 10 that the escherichia coli bacterium is counted content
9
This test is all carried out under aseptic condition from step 1 to step 9.
Toxin producing medium component described in this test procedure one is: the MgSO of 0.3g
47H
2The KCl of O, 0.2g, the K of 3.0g
2HPO
43H
2The glucose of O, 2.0g, 2mL quality percentage composition are 10% cystine, the beef infusion broth of 700mL and the beef Digestive system of 300mL; Wherein, beef Digestive system method for preparing is: beef is rubbed, take by weighing 1000g, add the Na of distilled water 1500mL, 1.5g
2CO
3, the pancreatin of 6g and the chloroform of 30mL, place 37 ℃ of incubators, using mass concentration is that 4% NaOH regulates pH to 8.0; Digestion 36h; The HCL of reuse 2N regulates pH to 4.5, is heated to 90 ℃, filters with double-layer cloth then; In filtrating, adding volumetric concentration is 2% chloroform mixing, promptly gets the beef Digestive system.
Toxin producing medium method for making: mentioned component is mixed, be heated to fusing fully, regulate pH value to 7.6; Boil filtration, add active carbon 2.0g, be sub-packed in after being mixed in the 250mL conical flask; The 60mL/ bottle places in the high pressure steam sterilizer, and 20min sterilizes under 115 ℃ of temperature.
Aseptic vial in this test procedure seven and the step 8 and aseptic silk all use at 180 ℃ of high temperature sterilize 2h, and purpose is in order to remove pyrogen.
Adding 4 ℃ of physiological saline solution to cup in this test procedure five is full of in order to make deposition and the abundant mixing of sterile glass beads, to the physiological saline solution amount of adding and the volume no requirement (NR) of cup.
The nutrient broth of this test is bought the extensive and profound in meaning star Bioisystech Co., Ltd from Beijing; The blood agar plating medium is available from Zhengzhou Bosai Biotechnology Co., Ltd.
The staphylococcus aureus of this test is bought from Institute of Microorganism, Academia Sinica, and deposit number is: ATCC25923; Streptococcus agalactiae is bought from Institute of Microorganism, Academia Sinica, and deposit number is: ATCC12386; Escherichia coli are bought from Institute of Microorganism, Academia Sinica, and deposit number is: ATCC25922.
The staphylotoxin of this test is produced 8 batches altogether; Tiring of 8 batches of toxin; The soprano has reached 4096, and the lowest 2048 also far surpasses 1024 standard, has proved absolutely that this project is very ripe, stable in the technology aspect the preparation staphylotoxin.
The staphylococcus aureus toxoid of this test is produced 8 batches altogether, and 8 batches of toxoids are tired and are 128 or 256 through mensuration, has proved absolutely that this project is very ripe, stable in the technology aspect the preparation staphylococcus aureus toxoid.
Mammitis of cow vaccine to this test makes is examined and determine
1) sterility test
Under gnotobasis; In 3 groups of sulphur glycollate culture mediums, insert each 1mL of mammitis of cow vaccine respectively; In 20 ℃ ~ 25 ℃ incubators, cultivate 3 ~ 4d; Subcultivation to sulphur glycollate culture medium, blood agar culture-medium and improvement Martin culture medium then, every kind of each subcultivation 2 pipe of culture medium, every pipe 0.5mL; Sulphur glycollate culture medium, each 1 pipe of blood agar culture-medium of getting behind the above-mentioned subcultivation place 30 ℃ ~ 35 ℃ incubator to cultivate, and all the other each pipes place 20 ℃ ~ 25 ℃ incubator to cultivate, and cultivate 14d, observe the microbiological contamination situation that has or not;
Wherein, the sulphur glycollate culture medium component is: pancreas casein peptone 15g, yeast extract powder 5g, glucose 5g, sodium chloride 2.5g, L-cystine 0.5g, sodium thioglycollate 0.5g, agar 0.7g, 0.2% methylene blue solution 1.0mL, distilled water 1000mL.With the said components heating for dissolving, regulate pH value to 7.1, packing places in the high pressure steam sterilizer, is using behind the sterilization 20min under 115 ℃ of conditions;
Improvement Martin nutrient media components is: glucose 20g, peptone 5g, yeast extract powder 2g, K
2HPO
43H
2O1g, MgSO
47H
2O 0.5g, distilled water 1000mL.Method for making: with the said components heating for dissolving, regulate pH value to 6.4, packing places in the high pressure steam sterilizer, is using behind the sterilization 20min under 115 ℃ of conditions;
Blood agar culture-medium is available from Zhengzhou Bosai Biotechnology Co., Ltd.
Result of the test shows all do not have bacteria growing in sulphur glycollate culture medium, blood agar culture-medium, the improvement Martin culture medium, and hence one can see that, and mammitis of cow vaccine sterility test of the present invention is qualified.
2) safety test
Choose body weight and be 5 of the healthy mices of 18 ~ 20g, under gnotobasis,, observe day by day, should have no way of toxicant and the symptom that causes or dead for qualified to 7d to every mouse subcutaneous injection mammitis of cow vaccine 0.5mL.
Result of the test shows, the Total Test white mice is all had no way of toxicant and the symptom that causes, all strong depositing, and hence one can see that, and mammitis of cow vaccine of the present invention has security reliability.
3) abnormal toxicity test
Choosing body weight is 5 of 18 ~ 20g white mice, takes by weighing every white mice body weight before the injection, under gnotobasis, to every mouse peritoneal injection 0.5mL mammitis of cow vaccine, observes 7d, and every white mice weight increase behind the 7d is qualified mammitis of cow vaccine.
Result of the test shows that in the observation period, the test white mice is all strong deposits, no abnormal reaction, and every white mice body weight all increases behind the 7d, and the increase scope is between 8 ~ 15g, and hence one can see that, and mammitis of cow vaccine abnormal toxicity test of the present invention is qualified.
4) pyrogen test
Choose 3 healthy do not have hinder, female no pregnant and body weight is the rabbit of 1.7 ~ 2.5kg, it is clean after 180 ℃ of high temperature sterilize 2h remove pyrogen test used syringe, syringe needle and other vessel that contacts with test liquid, tests more than the preceding rabbit fasting 2h; Then rabbit is fixed; Begin to measure normal body temperature behind 30 ~ 60min, continuous measurement twice, each 60min at interval; Twice temperature difference must not be greater than 0.2 ℃, is the normal body temperature of this rabbit with the meansigma methods of this twice body temperature; Get the mammitis of cow vaccine, before injection, be preheated to 38 ℃, survey rabbit and reach in the normal body temperature 15min, slowly inject the mammitis of cow vaccine of 3mL to tame rabbit ear vein, every at a distance from the 30min take temperature once, tie-in 6 times.
Result of the test shows, 3 rabbit of pyrogen test are heated up and all are lower than 0.6 ℃, and 3 rabbit intensification summations are no more than 1.4 ℃, and hence one can see that, and mammitis of cow vaccine pyrogen test of the present invention is qualified.
5) immunity test of mammitis of cow vaccine
Immune group 1: through mammitis of cow vaccine immunity, body weight is 30 of 14 ~ 16g white mice;
Immune group 2: through the immunity of staphylococcus aureus toxoid vaccine, body weight is 30 of 14 ~ 16g white mice;
Immune group 3: through staphylococcus aureus tropina vaccine immunity, body weight is 30 of 14 ~ 16g white mice;
Immune group 4: through streptococcus agalactiae vaccine immunization, body weight is 30 of 14 ~ 16g white mice;
Immune group 5: through colibacterin immunity, body weight is 30 of 14 ~ 16g white mice;
Immune group 6: through mammitis of cow vaccine immunity, body weight is 30 of 14 ~ 16g white mice;
Immune group 7: through mammitis of cow vaccine immunity, body weight is 30 of 14 ~ 16g white mice;
Matched group: same batch is 9 groups of 14 ~ 16g white mice (5 every group) without vaccine immunity, body weight.
Under gnotobasis; With above-mentioned vaccine immunity correspondence respectively group white mice; Every subcutaneous injection 0.5mL injects 2 times continuously, each 7d at interval; The last immunity was carried out challenge test after 10 days: to the staphylococcus aureus (being contained among the 0.5mL) of immune group 1, immune group 2, every lumbar injection 1MLD of immune group 3 white mice (MLD is a minimum lethal dose, promptly is enough to make the minimum of animal dead under certain condition); Streptococcus agalactiae (being contained among the 0.5mL) to every lumbar injection 1MLD of immune group 4 white mice; Escherichia coli (being contained among the 0.5mL) to every lumbar injection 1MLD of immune group 5 white mice; Streptococcus agalactiae (being contained among the 0.5mL) to every lumbar injection 1MLD of immune group 6 white mice; Escherichia coli (being contained among the 0.5mL) to every lumbar injection 1MLD of immune group 7 white mice; Inject the toadstool (staphylococcus aureus, streptococcus agalactiae, escherichia coli) of 2MLD, 1MLD and 1/2MLD respectively to 9 groups of control group mice abdominal cavities respectively, observe 3d.
Table 1 immunity test result
Result of the test is as shown in table 1, can be known by table 1:
1) matched group mice infection 2MLD and 1MLD toadstool person are all dead, and infecting 1/2MLD person has part dead, and the used counteracting toxic substances bacterium liquid of this test of susceptible of proof meets the requirement of " Chinese biological goods rules " defined;
2) protective rate of immune group 1 white mice is 93.3%; Much larger than immune group 2 73.3% with 56.7% of immune group 3, the immunity effect that prove the novel mammitis of cow vaccine of the present invention structure is better than the simple vaccine that uses staphylococcus aureus toxoid or tropina as main component;
3) protective rate of immune group 6 white mice is 83.8%; Be better than 66.7% of immune group 4; The mammitis of cow immune effect of vaccine that proof the present invention makes up is better than the independent vaccine of streptococcus agalactiae; Explain that staphylococcus aureus toxoid and tropina unite use and not only infection of staphylococcus aureus is had the specificity prevention effect; But also streptococcus agalactiae is had non-specific prevention effect, predict that its mechanism possibly be that toxoid and tropina are united to use and improved the body leucocyte level, thereby streptococcus agalactiae has been played the effect of engulfing, clearing up;
4) protective rate of immune group 7 white mice is 86.7%; Be better than 70.0% of immune group 5; The mammitis of cow immune effect of vaccine that proof the present invention makes up is better than the independent vaccine of escherichia coli; Explain that staphylococcus aureus toxoid and tropina unite use and not only infection of staphylococcus aureus is had the specificity prevention effect; But also escherichia coli are had non-specific prevention effect, predict that its mechanism possibly be that toxoid and tropina are united to use and improved the body leucocyte level, thereby escherichia coli have been played the effect of engulfing, clearing up.
Above result of the test shows, the mammitis of cow vaccine that the present invention makes up has characteristics such as safety, reliable, immunity height, and its immune effect will be better than merely the single vaccine that is the main body with a certain composition wherein far away.
Claims (9)
1. the method for preparing of a mammitis of cow vaccine is characterized in that the method for preparing of mammitis of cow vaccine is carried out according to following steps:
One, staphylotoxin preparation
A, with inoculating loop picking staphylococcus aureus, be inoculated in the nutrient broth, place 37 ℃ of incubators to cultivate 18 ~ 24h, culture fluid; The culture fluid of drawing 10 μ L is coated with on the blood agar plating medium evenly; After in 37 ℃ of incubators, cultivating 18 ~ 24h; Picking one inoculating loop diameter is greater than single bacterium colony of 1mm; Be inoculated in nutrient agar slant medium, place 37 ℃ of incubators to cultivate 18 ~ 24h then, get staphylococcus aureus Nutrient agar slant culture; Picking one inoculating loop staphylococcus aureus Nutrient agar slant culture is inoculated in the toxin producing medium, and shaken cultivation 18 ~ 24h under 37 ℃ of conditions is seed liquor; B, seed liquor that step a is obtained by volume percentage composition are that 1% ~ 3% inoculum concentration is inoculated in the toxin producing medium; It is 37 ℃ in temperature then; Rotating speed is after cultivating 18 ~ 24h under the condition of 180 ~ 250r/min; Centrifugal 30min under the condition of 3000r/min collects supernatant and deposition simultaneously, and the supernatant of collection is staphylotoxin; The deposition of collecting, subsequent use;
Two, staphylococcus aureus toxoid preparation
C, get the staphylotoxin that step 1 makes; NaOH solution with 1N is regulated pH value to 6.5 ~ 8.5; Adding formaldehyde to formaldehyde volumetric concentration then is 0.2% ~ 0.5%, places 37 ℃ of incubators, the 3 ~ 7d that detoxifies, and promptly gets staphylococcus aureus toxoid stock solution; D, the staphylococcus aureus toxoid stock solution that step c is obtained are used filter paper filtering, and in filtrating, adding medical sodium chloride to sodium chloride mass concentration is 10% ~ 20%, regulate pH value to 2.0 ~ 4.0, hold over night with the HCl solution of 2N then; E, with the staphylococcus aureus toxoid stock solution centrifugal 10min under the 3000r/min rotating speed after the steps d overnight treatment; The aseptic redistilled water centrifuge washing deposition of collecting precipitation reuse 1 ~ 2 time; Collecting deposition after the washing, to be dissolved in pH be in 6.5 ~ 8.5 the phosphate buffer; Centrifugal 10min under the 3000r/min rotating speed once more, collecting precipitation and in deposition, to add mass concentration be that 1% thimerosal solution to thimerosal mass concentration is 0.01% promptly gets the staphylococcus aureus toxoid; Wherein, phosphate buffer and staphylococcus aureus toxoid stock solution volume ratio are 1:15 ~ 30;
Three, the preparation of staphylococcus aureus tropina
Get the deposition of collecting in the step 1,, get supernatant with 0 ~ 4 ℃ of physiological saline solution centrifuge washing 3 times; Collecting precipitation is that the ratio of 1:3 ~ 5 is put into aseptic homogenate cup and mixed with the deposition of collecting and sterile glass beads in mass ratio, adds 0 ~ 4 ℃ of physiological saline solution to cup more completely; Adopting homogenizer is 5 ℃ in temperature, and rotating speed is to vibrate 12min under the condition of 4000r/min, is 14000r/min at rotating speed then; Temperature is centrifugal 40min under 5 ℃ the condition, collects supernatant, with HCl solution adjusting supernatant pH value to 3.0 ~ 5.0 of 0.5N; Be 7000r/min at rotating speed once more, temperature is centrifugal 30min under 5 ℃ the condition, collecting precipitation; Pack in the bag filter,, promptly get the staphylococcus aureus tropina through the dialysis of 48 ~ 96h flowing water;
Four, the preparation of streptococcus agalactiae vaccine
Preparation bacterium amount is 1.5 ~ 3.0 * 10
9The streptococcus agalactiae vaccine, place 4 ℃ refrigerator to preserve subsequent use;
Five, the preparation of colibacterin
Preparation bacterium amount is 1.5 ~ 3.0 * 10
9Colibacterin, place 4 ℃ refrigerator to preserve subsequent use;
Six, the preparation of mammitis of cow vaccine
After the colibacterin that the staphylococcus aureus toxoid that step 2 is obtained, the staphylococcus aureus tropina that step 3 obtains, streptococcus agalactiae vaccine that step 4 obtains and step 5 obtain mixes, promptly get the mammitis of cow vaccine; Wherein, it is 10 ~ 30U that every milliliter of mammitis of cow vaccine contains anatoxic the tiring of staphylococcus aureus, and the protein content of staphylococcus aureus tropina is 200 ~ 400 μ g, and the bacterium number of streptococcus agalactiae is 1.5 ~ 3.0 * 10
9, colibacillary bacterium number is 1.5 ~ 3.0 * 10
9
2. according to the method for preparing of the said a kind of mammitis of cow vaccine of claim 1, it is characterized in that the operation that step 1 to step 6 is carried out all carries out under aseptic condition.
3. according to the method for preparing of claim 1 or 2 said a kind of mammitis of cow vaccines, it is characterized in that it is 1024 ~ 4096 that the staphylotoxin described in the step 1 is tired.
4. according to the method for preparing of the said a kind of mammitis of cow vaccine of claim 3, it is characterized in that it is 128 ~ 256 that the staphylococcus aureus toxoid described in the step 2 is tired.
5. according to the method for preparing of the said a kind of mammitis of cow vaccine of claim 4, it is characterized in that protein content is 10 ~ 50mg/mL in the staphylococcus aureus tropina described in the step 3.
6. according to the method for preparing of the said a kind of mammitis of cow vaccine of claim 5, it is characterized in that the sterile glass beads described in the step 3 is
7. according to the method for preparing of the said a kind of mammitis of cow vaccine of claim 6, it is characterized in that the product acid nutrient media components described in the step 1 is: the MgSO of 0.3g
47H
2The KCl of O, 0.2g, the K of 3.0g
2HPO
43H
2The glucose of O, 2.0g, 2mL mass concentration are 10% cystine, the beef infusion broth of 700mL and the beef Digestive system of 300mL; Wherein, beef Digestive system method for preparing is: beef is rubbed, take by weighing 1000g, add the Na of distilled water 1500mL, 1.5g
2CO
3, the pancreatin of 6g and the chloroform of 30mL, place 37 ℃ of incubators, using mass concentration is that 4% NaOH regulates pH to 8.0; Digestion 36h; The HCL of reuse 2N regulates pH to 4.5, is heated to 90 ℃, filters with double-layer cloth then; In filtrating, adding volumetric concentration is 2% chloroform mixing, promptly gets the beef Digestive system.
8. according to the method for preparing of the said a kind of mammitis of cow vaccine of claim 7; The method for preparing that it is characterized in that the streptococcus agalactiae vaccine described in the step 4 is following: a, get an inoculating loop streptococcus agalactiae; Be inoculated in the blood agar plating medium with the sectional streak inocalation method, place 37 ℃ of incubators to cultivate 18 ~ 24h then, picking one inoculating loop diameter is greater than single bacterium colony of 1mm; Be inoculated in the nutrient broth medium; Place 37 ℃ of incubators to cultivate 18 ~ 24h, get streptococcus agalactiae nutrient broth culture 2mL, be inoculated on the blood agar slant medium; Coating evenly is placed in 37 ℃ of incubators cultivates 24 ~ 48h, promptly gets streptococcus agalactiae blood agar slant culture; B, use concentration are that the sterile phosphate normal saline of 0.02 ~ 0.2mol/L washes the streptococcus agalactiae blood agar slant culture that step a obtains in the aseptic vial; Filter with aseptic silk then; Adding formalin to formaldehyde volumetric concentration is 1.0% ~ 1.2%; After placing 37 ℃ of incubators to cultivate 1 ~ 7d, adding phenol to final concentration is 5.0g/L, places 4 ℃ refrigerator to preserve at last.
9. the method for preparing of said according to Claim 8 a kind of mammitis of cow vaccine is characterized in that the method for preparing of the colibacterin described in the step 5 is following: a, get escherichia coli one inoculating loop, be inoculated in the blood agar flat board with the sectional streak inocalation method; Place 37 ℃ of incubators to cultivate 18 ~ 24h; Picking one inoculating loop diameter is inoculated in the nutrient broth medium greater than single bacterium colony of 1mm, places 37 ℃ of incubators to cultivate 18 ~ 24h; Get an inoculating loop escherichia coli nutrient broth culture 2mL; Be inoculated on the blood agar slant medium, coating evenly is placed in 37 ℃ of incubators cultivates 24 ~ 48h, promptly gets colibacterin; B, concentration are that the colibacterin that the sterile phosphate normal saline of 0.02 ~ 0.2mol/L obtains step a washes in the aseptic vial; Filter with aseptic silk then; Adding formalin to formaldehyde volumetric concentration is 1.0% ~ 1.2%; After placing 37 ℃ of incubator 1 ~ 7d, adding phenol to final concentration is 5.0g/L, places 4 ℃ refrigerator to preserve at last.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012102219777A CN102716475A (en) | 2012-06-29 | 2012-06-29 | Preparation method of cow mammitis vaccines |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012102219777A CN102716475A (en) | 2012-06-29 | 2012-06-29 | Preparation method of cow mammitis vaccines |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102716475A true CN102716475A (en) | 2012-10-10 |
Family
ID=46942430
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012102219777A Pending CN102716475A (en) | 2012-06-29 | 2012-06-29 | Preparation method of cow mammitis vaccines |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102716475A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103800899A (en) * | 2014-01-27 | 2014-05-21 | 内蒙古华希生物科技有限公司 | Milk cattle mastitis vaccine |
| CN108717125A (en) * | 2018-06-22 | 2018-10-30 | 北京农学院 | Milk cow early stage mammitis monitoring method |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1141000A (en) * | 1993-11-05 | 1997-01-22 | 伊莱利利公司 | Vaccine Design and Production |
| CN1310022A (en) * | 2000-02-22 | 2001-08-29 | 哈尔滨高科技集团天健药业有限责任公司 | Method of making medicine from extracting metabolic product of staphylococcus aureus and thallus protein |
| CN1451437A (en) * | 2002-04-18 | 2003-10-29 | 黑龙江省科学院应用微生物研究所 | Staphlococcus aureus preparation and producting method thereof |
| WO2004004759A1 (en) * | 2002-07-05 | 2004-01-15 | The United States Of America, Represented By The Secretary Of Agriculture | Vaccine for the prevention of bacterial infection of the bovine mammary gland |
| WO2007148229A2 (en) * | 2006-02-22 | 2007-12-27 | Stefan Knight | Immunogenic multivalent adhesin particles |
| CN101177451A (en) * | 2007-11-02 | 2008-05-14 | 山东省农业科学院奶牛研究中心 | Micrococcus pyogenes adhesion functional peptide and coded sequence thereof |
| CN101730543A (en) * | 2007-07-23 | 2010-06-09 | 疫苗研究国际有限公司 | The staphylococcal whole-cell vaccine of deactivation |
| CN102120761A (en) * | 2010-12-24 | 2011-07-13 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Acapsular type staphylococcus aureus extracellular polysaccharide-protein conjugate and preparation method thereof |
| CN102205117A (en) * | 2011-05-19 | 2011-10-05 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof |
| CN102294026A (en) * | 2010-11-17 | 2011-12-28 | 赤峰博恩药业有限公司 | Milk cow streptococcal mastitis inactivated vaccine and preparation method thereof |
-
2012
- 2012-06-29 CN CN2012102219777A patent/CN102716475A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1141000A (en) * | 1993-11-05 | 1997-01-22 | 伊莱利利公司 | Vaccine Design and Production |
| CN1310022A (en) * | 2000-02-22 | 2001-08-29 | 哈尔滨高科技集团天健药业有限责任公司 | Method of making medicine from extracting metabolic product of staphylococcus aureus and thallus protein |
| CN1451437A (en) * | 2002-04-18 | 2003-10-29 | 黑龙江省科学院应用微生物研究所 | Staphlococcus aureus preparation and producting method thereof |
| WO2004004759A1 (en) * | 2002-07-05 | 2004-01-15 | The United States Of America, Represented By The Secretary Of Agriculture | Vaccine for the prevention of bacterial infection of the bovine mammary gland |
| WO2007148229A2 (en) * | 2006-02-22 | 2007-12-27 | Stefan Knight | Immunogenic multivalent adhesin particles |
| CN101730543A (en) * | 2007-07-23 | 2010-06-09 | 疫苗研究国际有限公司 | The staphylococcal whole-cell vaccine of deactivation |
| CN101177451A (en) * | 2007-11-02 | 2008-05-14 | 山东省农业科学院奶牛研究中心 | Micrococcus pyogenes adhesion functional peptide and coded sequence thereof |
| CN102294026A (en) * | 2010-11-17 | 2011-12-28 | 赤峰博恩药业有限公司 | Milk cow streptococcal mastitis inactivated vaccine and preparation method thereof |
| CN102120761A (en) * | 2010-12-24 | 2011-07-13 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Acapsular type staphylococcus aureus extracellular polysaccharide-protein conjugate and preparation method thereof |
| CN102205117A (en) * | 2011-05-19 | 2011-10-05 | 新疆维吾尔自治区畜牧科学院兽医研究所 | Dairy cow mammitis staphylococcus aureus-Escherichia coli bigeminy polyvalent vaccine and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 曲晓军等: "新型奶牛乳房炎多联苗的研制及免疫力试验", 《生物技术》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103800899A (en) * | 2014-01-27 | 2014-05-21 | 内蒙古华希生物科技有限公司 | Milk cattle mastitis vaccine |
| CN108717125A (en) * | 2018-06-22 | 2018-10-30 | 北京农学院 | Milk cow early stage mammitis monitoring method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104189898B (en) | P. aeruginosa bacteria vaccine and preparation method thereof | |
| CN105963692B (en) | Combined vaccine for preventing hand-foot-and-mouth disease | |
| CN102813917B (en) | Acellular pertussis/Haemophilus influenzae type b - group A and C meningococcus combined vaccine | |
| CN111500482B (en) | Clostridium perfringens type A strain of sheep, inactivated vaccine and vaccine preparation method | |
| CN107267466A (en) | A kind of method for mass producing swine pseudorabies vaccine | |
| CN102181457B (en) | Clostridium difficile exotoxin B amino-terminal gene sequence with optimized codon and nucleic vaccine of clostridium difficile exotoxin B | |
| CN102125687B (en) | Production method for bivalent inactivated vaccine for infectious serositis of ducks | |
| CN102805864B (en) | Newcastle disease and H9N2 subtype avian influenza dual inactivated vaccine and preparation method thereof | |
| CN105999256B (en) | Combined vaccine for preventing hand-foot-and-mouth disease | |
| CN103160555A (en) | Culture medium, culture method and application of high-yield exotoxin of clostridium perfringens | |
| CN102716475A (en) | Preparation method of cow mammitis vaccines | |
| CN113957007A (en) | Inactivated vaccine for mycoplasma synoviae | |
| Marecki et al. | Cellular and humoral aspects of host resistance in murine salmonellosis | |
| CN106075423B (en) | Combined vaccine for preventing hand-foot-and-mouth disease | |
| CN102276720A (en) | High-biological-activity egg yolk antibody and preparation method thereof | |
| CN103800900B (en) | Staphylococcus aureus and the mammitis of cow vaccine that its deactivation is obtained | |
| CN113069539B (en) | Rabbit A-type and D-type pasteurella multocida and staphylococcus aureus triple inactivated vaccine and preparation method thereof | |
| CN113046271B (en) | A rabbit strain of Pasteurella multocida type F and its application in the preparation of inactivated vaccines | |
| CN106389475A (en) | Applications of Bacteroides fragilis in prevention and/or treatment of meningitis | |
| CN101406697B (en) | Preparation method of bacillus pyocyaneus vaccine | |
| JPS6234725B2 (en) | ||
| CN108295253A (en) | A kind of A, C group meningitis cocci-b types haemophilus influenzae/encephalitis B combined vaccine | |
| CN103800901B (en) | Streptococcus and the mammitis of cow vaccine that its deactivation is obtained | |
| CN103007265B (en) | Duck infection serositis trivalent inactivated vaccine and preparation method thereof | |
| CN103800899B (en) | A kind of mammitis of cow vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121010 |