CN110438029A - A kind of novel ablation method of mycoplasma hyopneumoniae - Google Patents
A kind of novel ablation method of mycoplasma hyopneumoniae Download PDFInfo
- Publication number
- CN110438029A CN110438029A CN201910558888.3A CN201910558888A CN110438029A CN 110438029 A CN110438029 A CN 110438029A CN 201910558888 A CN201910558888 A CN 201910558888A CN 110438029 A CN110438029 A CN 110438029A
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- Prior art keywords
- mycoplasma hyopneumoniae
- mycoplasma
- inactivation
- group
- ablation method
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- 241000204045 Mycoplasma hyopneumoniae Species 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000002679 ablation Methods 0.000 title claims abstract description 10
- 230000002779 inactivation Effects 0.000 claims abstract description 22
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims abstract description 5
- 235000019345 sodium thiosulphate Nutrition 0.000 claims abstract description 5
- 241000204031 Mycoplasma Species 0.000 claims abstract description 4
- 229940031551 inactivated vaccine Drugs 0.000 claims description 12
- 241000204003 Mycoplasmatales Species 0.000 claims description 5
- 206010035664 Pneumonia Diseases 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 abstract description 8
- 230000005847 immunogenicity Effects 0.000 abstract description 6
- 241000700605 Viruses Species 0.000 abstract description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 14
- 239000000243 solution Substances 0.000 description 8
- 230000000937 inactivator Effects 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000002574 poison Substances 0.000 description 6
- 231100000614 poison Toxicity 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 6
- 229940033663 thimerosal Drugs 0.000 description 6
- 230000002633 protecting effect Effects 0.000 description 4
- 201000008827 tuberculosis Diseases 0.000 description 4
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 201000008235 Mycoplasma pneumoniae pneumonia Diseases 0.000 description 2
- 206010035724 Pneumonia mycoplasmal Diseases 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 206010022998 Irritability Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
Abstract
The present invention provides a kind of mycoplasma hyopneumoniae ablation methods, it is to mix the binary ethylenimine of final concentration 3mM with mycoplasma hyopneumoniae bacterium solution, is incubated at 37 DEG C for 24 hours, reuse in sodium thiosulfate and binary ethylenimine.The present invention can effectively realize inactivation of virus, and utmostly retain the immunogenicity of mycoplasma, have a good application prospect in vaccine preparation.
Description
Technical field
The present invention relates to a kind of mycoplasma hyopneumoniae NJ plants of novel ablation methods.
Background technique
In inactivated vaccine production, usually to use bacteria inactivation rate agent makes live vaccine lose infection ability, keeps simultaneously
Its antigenicity.
Common inactivator has formaldehyde and thimerosal.Formaldehyde is the traditional inactivator of comparison, and inactivation of viruses can reach very
Good effect, but formalin-inactivated has not only inactivated the immunogenicity that virus also destroys pathogen, and formalin-inactivated time
It is long, vulnerable to temperature, pH value, concentration, with the presence or absence of the factors such as organic matter, the type of pathogen and nitrogen content influence, residual
Free formaldehyde, if with vaccine inject body after, can generate irritability reaction.Thimerosal is commonly used for mycoplasma as preservative
Inactivation, thimerosal itself is more toxic, and should be noted the autoprotection to user of service in operating process.
Still without a kind of inactivating efficacy, mycoplasma hyopneumoniae good, harmless to immunogenicity and small to human toxicity goes out at present
Activating method.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of mycoplasma hyopneumoniae ablation methods.
Technical solution of the present invention includes:
A kind of mycoplasma hyopneumoniae ablation method, it is by the binary ethylenimine of final concentration 3mM and mycoplasma hyopneumoniae bacterium
Liquid mixing, is incubated for for 24 hours at 37 DEG C, reuses in sodium thiosulfate and binary ethylenimine.
A kind of mycoplasma hyopneumoniae ablation method, the mycoplasma are NJ plants.
A kind of preparation method of mycoplasma hyopneumoniae inactivated vaccine, it is to be inactivated using preceding method to mycoplasma hyopneumoniae
Afterwards, with Dao Daer A130 emulsify to get.
The obtained mycoplasma hyopneumoniae inactivated vaccine of aforementioned preparation process.
Binary ethylenimine of the invention can play effective deactivation to mycoplasma hyopneumoniae, and can maintain higher
Immunogenicity is produced particularly suitable for inactivated vaccine.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Above content of the invention is described in further detail again below by way of specific embodiment.But it should not be by this
The range for being interpreted as the above-mentioned theme of the present invention is only limitted to example below.All technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
Specific embodiment
The preparation of 1 binary ethylenimine of embodiment (BEI)
It is pre-configured with 100ml 0.2M NaOH solution, weighs 2.05g BEA, 100ml 0.2M is added in BEA powder
In NaOH solution, then closed container, cavity volume cannot be excessive (it is recommended that not exceeding 1/2).37 DEG C of stirrings are cyclized 1h, and pH is answered
8.5 should be reduced to by 12.5, cyclisation is completed.It adjusts pH to 7.5~7.6 (as close as possible to 7.6), 0.22 μm of aseptic filtration saves.
The concentration of BEI is 0.1M at this time.
The inactivation of 2 mycoplasma hyopneumoniae of embodiment
(1) method
1. bacterium solution inactivates
Three crowdes of each 50ml of mycoplasma hyopneumoniae bacterium solution are taken, first is former according to final concentration of 3mM BEI inactivation pig pneumonia branch
Body adds inactivator mother liquor 1.5ml；Second batch inactivates mycoplasma hyopneumoniae, addition according to final concentration of 0.15% formalin
Inactivator mother liquor 75ul.Third batch inactivates mycoplasma hyopneumoniae according to final concentration of 0.005% thimerosal, adds inactivator
2.5ug.Three batches are placed in 37 DEG C of constant-temperature tables simultaneously, sample afterwards for 24 hours, are neutralized after the completion of first inactivation using sodium thiosulfate
BEI, the concentration of sodium thiosulfate are 1M, and additive amount is 10% (volume ratio) of BEI additive amount.It is separately sampled after sample neutralizes
Carry out inactivation inspection.
2. the preparation of inactivated vaccine and effect detection
Three batches of mycoplasma hyopneumoniae bacterium solutions emulsify three batches of vaccines by examining inactivation thoroughly, using Dao Daer A130.Epidemic disease
Seedling detection is qualified, carries out potency test, 25 susceptible piglets of health are randomly divided into 5 groups, every group 5.1st~3 group is vaccine
Immune group, the 4th group is to attack malicious control group, and the 5th group is blank control group.The grouping and design of test are as follows:
Head exempt from after 35 days, together with poison control pig 5 is attacked, by force with Js the plant of mycoplasma hyopneumoniae of normal saline dilution tissues
Poison, every 5ml are attacked in poison, the interior injection of transtracheal.Blank control group does not attack poison, attacks poison after 28 days, and all test pigs of dissect observe lung
Lesion is determined by porcine mycoplasmal pneumonia tuberculosis varying index scoring criteria.It is not small to attack malicious control group tuberculosis varying index arithmetic mean of instantaneous value
In 10, it is judged to test and sets up；It is calculated by porcine mycoplasmal pneumonia vaccine immunity group thumps varying index slip, vaccine immunity group pig
Tuberculosis varying index slip is not less than 60%, is judged to vaccine qualification.
(2) result
1. mycoplasma hyopneumoniae inactivation is examined
Three batches of inactivation liquid take 1ml to be inoculated in the T75 cell bottle equipped with 50ml DB-124 culture solution respectively, observe 5,
Color change is not found；It is continuous to see simultaneously from the DB-124 culture solution for taking 0.5ml to be inoculated in 4.5ml in two batches T75 cell bottle
10 are examined, three groups of mycoplasma inactivation liquid colors have not been changed, and inactivation is thorough.Specifically it see the table below:
| Group | Ablation method | Inactivator concentration | Inactivation temperature | Inactivation time/for 24 hours |
| First group | BEI | 3mmol/L final concentration | 37℃ | - |
| Second group | Formaldehyde | 0.15% | 37℃ | - |
| Third group | Thimerosal | 0.005% | 37℃ | - |
*+: for inactivation failure-: to inactivate successfully
2. mycoplasma hyopneumoniae efficacy test result
28 days all test pigs of dissect after poison are attacked, observation tuberculosis becomes, and scores, the result is as follows:
Attack malicious control group thumps varying index arithmetic mean of instantaneous value=22
First group of Immunization group arithmetic average=6.8
Second group of Immunization group arithmetic average=14.2
Third group Immunization group arithmetic average=12.2
First immune group thumps varying index slip=69.1% is greater than 60%, therefore the pig pneumonia branch of BEI inactivation is former
Body inactivated vaccine reaches protecting effect.Second immune group thumps varying index slip=35.5%, less than 60%, therefore formaldehyde
The mycoplasma hyopneumoniae inactivated vaccine of inactivation fails to reach protecting effect.Third immune group thumps varying index slip=
44.5%, less than 60%, therefore the mycoplasma hyopneumoniae inactivated vaccine of thimerosal inactivation fails to reach protecting effect.
(3) conclusion
Three kinds of methods can achieve the purpose that inactivation, but the mycoplasma hyopneumoniae inactivated vaccine of BEI inactivation can be utmostly
Immunogenicity is maintained, optimal protecting effect is reached.
To sum up, inactivator of the invention can effectively inactivate mycoplasma hyopneumoniae, and maintain its immunogenicity, can be used for phase
The preparation for closing inactivated vaccine, has a good application prospect.
Claims (4)
1. a kind of mycoplasma hyopneumoniae ablation method, which is characterized in that it is by the binary ethylenimine and pig pneumonia of final concentration 3mM
The mixing of mycoplasma bacterium solution, is incubated for for 24 hours at 37 DEG C, reuses in sodium thiosulfate and binary ethylenimine.
2. mycoplasma hyopneumoniae ablation method as described in claim 1, which is characterized in that the mycoplasma is NJ plants.
3. a kind of preparation method of mycoplasma hyopneumoniae inactivated vaccine, which is characterized in that it is using such as claims 1 or 2 institute
State method to mycoplasma hyopneumoniae inactivation after, with Dao Daer A130 emulsify to get.
4. the obtained mycoplasma hyopneumoniae inactivated vaccine of preparation method described in claim 3.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910558888.3A CN110438029A (en) | 2019-06-25 | 2019-06-25 | A kind of novel ablation method of mycoplasma hyopneumoniae |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910558888.3A CN110438029A (en) | 2019-06-25 | 2019-06-25 | A kind of novel ablation method of mycoplasma hyopneumoniae |
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| CN110438029A true CN110438029A (en) | 2019-11-12 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114134098A (en) * | 2021-12-15 | 2022-03-04 | 成都史纪生物制药有限公司 | Inactivation method of mycoplasma hyopneumoniae |
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| US5565205A (en) * | 1990-08-16 | 1996-10-15 | Solvay Animal Health, Inc. | Inactivated Mycoplasma hypopneumoniae bacterin and method of use thereof |
| WO2006056841A1 (en) * | 2004-11-24 | 2006-06-01 | Pharmacia & Upjohn Company Llc | Multiple-strain mycoplasma hyopneumoniae vaccines |
| CN102580082A (en) * | 2012-02-28 | 2012-07-18 | 肇庆大华农生物药品有限公司 | Inactived vaccine for poultry and preparation method thereof |
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-
2019
- 2019-06-25 CN CN201910558888.3A patent/CN110438029A/en active Pending
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| CN114134098A (en) * | 2021-12-15 | 2022-03-04 | 成都史纪生物制药有限公司 | Inactivation method of mycoplasma hyopneumoniae |
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Address after: No.358, lingchi street, Chengdu Economic and Technological Development Zone, Sichuan 610000 Applicant after: Chengdu Shiji biopharmaceutical Co.,Ltd. Applicant after: MA'ANSHAN SHIJI ANIMAL HEALTH MANAGEMENT Co.,Ltd. Address before: No.358, lingchi street, Chengdu Economic and Technological Development Zone, Sichuan 610000 Applicant before: CHENGDU TECBOND BIOLOGICAL PRODUCTS CO.,LTD. Applicant before: MA'ANSHAN SHIJI ANIMAL HEALTH MANAGEMENT Co.,Ltd. |
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Effective date of registration: 20221208 Address after: No.358, lingchi street, Chengdu Economic and Technological Development Zone, Sichuan 610000 Applicant after: Chengdu Shiji biopharmaceutical Co.,Ltd. Address before: No.358, lingchi street, Chengdu Economic and Technological Development Zone, Sichuan 610000 Applicant before: Chengdu Shiji biopharmaceutical Co.,Ltd. Applicant before: MA'ANSHAN SHIJI ANIMAL HEALTH MANAGEMENT Co.,Ltd. |
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Application publication date: 20191112 |