CN102286391B - Mink hemorrhagic pneumonia divalent inactivated vaccine and preparation method threof - Google Patents
Mink hemorrhagic pneumonia divalent inactivated vaccine and preparation method threof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, disclosing two pseudomonas aeruginosa virulent strains and a mink hemorrhagic pneumonia divalent inactivated vaccine prepared from the two strains. The pseudomonas aeruginosa virulent strains are inoculated in a culture medium to be amplified and cultured by ventilation; after the obtained cultured object is inactivated, the products are mixed at the ratio of 1-2:1; and adjuvant is added to prepare the mink hemorrhagic pneumonia divalent inactivated vaccine. The divalent inactivated vaccine disclosed by the invention has good immunity effect on mink hemorrhagic pneumonia, at least five-month protection periods can be obtained, the protection ratio is above 80%, the mink hemorrhagic pneumonia can be prevented, and the vaccine has good application prospect.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of vaccine and preparation method thereof.
Background technology
The mink hemorrhagic pneumonia claims again the mink pseudomonas pneumonia, mainly to occur in annual a kind of acute infectious disease that 9～November, mink moulted season, take hemorrhagic pneumonia, acute death is feature, occur the symptoms such as expiratory dyspnea, nostril outflow red zone foam liquid before dead, after death cut open the visible whole lobe of the lung diffuse hemorrhage of inspection and septicemia and change.This disease all has generation all over the world, and can cause the mortality ratio of plant 10%～50% every year, is the significant bacterial sexually transmitted disease of harm mink farming industry.
This disease pathogen is the Pseudomonas aeruginosa (Pseudomonas aeruginolsa) in pseudomonadaceae (Pseudomonadaceae) Rhodopseudomonas (Pseud-omonas), also be called Pseudomonas aeruginosa (Bacterium pyocyaneum), for gram negative bacillus, long 1.5～3.0 μ m, wide 0.5～0.7 μ m, the blunt circle in two ends, an end has a flagellum, can move.Can not form brood cell and pod membrane.Well-grown on the plain agar substratum, form bacterium colony not of uniform size.Majority can produce the blue-greenish colour water colo(u)r, makes the periphery of bacterial colonies nutrient agar painted.
Pseudomonas aeruginosa is distributed widely in soil, water and air, on normal people and animal intestinal and skin, existence is also arranged, also easily from discoveries such as wound, burn and urinary tract infections, can cause people's burn infection and trouble cystic fibrosis patient's pulmonary infection, therefore there is multiple virulence factor and strong invasiveness is arranged, think that this bacterium is not potentially pathogenic organism but pathogenic bacterium fully.
The morbidity of mink hemorrhagic pneumonia is anxious, dead fast, often have little time treatment during morbidity, common drug tilmicosin, Azythromycin, kantlex result for the treatment of are not remarkable, and antibiotic abuse causes Pseudomonas aeruginosa to produce extremely strong resistance, therefore need effective vaccine to prevent, domesticly there is no commercial vaccine and prevent the mink hemorrhagic pneumonia, report is arranged by bacterial cell wall extracts (JAMES E.PENNINGTON abroad, 1979), with the common protective antigen OEP extracted in thalline (common protective antigen) (ABEC, 1975; E.HONDA; 1976) add toxoid composition elastoser and make immunogen; certain immune effect is arranged, but the preparation process complexity, cost is high; be not suitable for a large amount of production and applications; and immune time is many, duration of immunity is short, is not suitable for clinical application; the Pseudomonas aeruginosa antigenic type is various, and the cell extract of single component often is difficult to reach good protection effect.
Summary of the invention
The mink hemorrhagic pneumonia bivalent inactivated vaccine that the purpose of this invention is to provide two kinds of serotype Pseudomonas aeruginosa virulent strains and prepared by these two kinds of virulent strains, described mink hemorrhagic pneumonia bivalent inactivated vaccine prevention mink hemorrhagic pneumonia, the epidemic prevention effect is good, and security is good.
A kind of pseudomonas aeruginosa strains DL15 provided by the invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.3811.
Another kind of pseudomonas aeruginosa strains JL08 provided by the invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.3812.
Measure the medium lethal dose (LD50) of this two strains strain to small white mouse by the Reed-Muench method, the LD50 of bacterial strain DL15 is 2,500,000 CFU, and the LD50 of bacterial strain JL08 is 2,000 ten thousand CFU; Bacterial strain DL15 is 1,000,000 CFU to the medium lethal dose (LD50) of mink, and the LD50 of bacterial strain JL08 is 2,500,000 CFU.
The present invention also provides the mink hemorrhagic pneumonia prepared by above-mentioned pseudomonas aeruginosa strains vaccine.
Described mink hemorrhagic pneumonia vaccine, as preferably, is bivalent inactivated vaccine.
The present invention also provides a kind of preparation method of mink hemorrhagic pneumonia bivalent inactivated vaccine, comprises the following steps:
Step 1: pseudomonas aeruginosa strains DL15 and pseudomonas aeruginosa strains JL08 are inoculated in respectively to the PYG culture medium culturing, and adjusting the bacterium number is 80-100 hundred million CFU/mL 1-2 in proportion: 1 mixes;
Step 2: inactivation step 1 gained mixed culture;
Step 3: add sanitas and adjuvant in step 2 gained culture, obtain described mink hemorrhagic pneumonia bivalent inactivated vaccine.
As preferably, the described inoculation of step 1 is for pressing the 3-4% inoculating strain of PYG culture volume.
As preferably, the described cultivation of step 1 is at 37 ℃ of aerated culture 12-15h, controls the incubation time of Pseudomonas aeruginosa in 15h, prevents the long generation that causes Pseudomonas Exotoxin of incubation time, ensures better vaccine safety.
As preferably, the described deactivation of step 2 is the formalin-inactivated with final concentration 0.15%.More preferably, in 37 ℃ of deactivation 24h, during jolting 2～3 times.
As preferably, the described sanitas of step 3 is sodium azide, and preferred whole mass concentration is 0.01%.
As preferably, the described adjuvant of step 3 is aluminium hydroxide solution, and preferably the aluminium hydroxide liquor capacity is nutrient solution 4%.
The mink hemorrhagic pneumonia bivalent inactivated vaccine that pseudomonas aeruginosa strains DL15 of the present invention and bacterial strain JL08 are mixed; once can reach 80% above protection ratio; duration of immunity can reach five months; can effectively prevent domestic popular mink hemorrhagic pneumonia at present; its preparation technology is simple; be applicable to commercialization production, have a good application prospect.
Biological preservation information:
Bacterial strain DL15: Classification And Nomenclature: Pseudomonas aeruginosa, Pseudomonas aeruginosa. is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 5th, 2010, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.3811.
Bacterial strain JL08: Classification And Nomenclature: Pseudomonas aeruginosa, Pseudomonas aeruginosa. is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 5th, 2010, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.3812.
The accompanying drawing explanation
Fig. 1 is shown form under the microscope of pseudomonas aeruginosa strains JL08 of the present invention, bacterial strain DL15;
Fig. 2 shows the protection efficiency of bivalent inactivated vaccine immunity mink of the present invention.
Embodiment:
Mink hemorrhagic pneumonia bivalent inactivated vaccine that the invention discloses pseudomonas aeruginosa strains JL08, bacterial strain DL15 and prepare with described two kinds of bacterial strains and preparation method thereof, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they all are deemed to be included in the present invention.Product of the present invention, method and application are described by preferred embodiment, the related personnel obviously can be changed methods and applications as herein described or suitably change and combination within not breaking away from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
Test materials
The PYG Media Components: peptone 2%, glucose 1%, sodium-chlor 0.5%, yeast powder 0.3%, with the deionized water preparation, joined adjust pH 7.2-7.4,116 ℃ of 30min.
The Pseudomonas aeruginosa somatotype is given birth to and is ground Co., Ltd. purchased from Japan with standard serum; Bacteria Identification micro biochemical reaction tubes is purchased from sky, Hangzhou and microorganism reagent company; 18～22g small white mouse is purchased from Jilin decent job biological products company limited; Formaldehyde, sodium azide is purchased from Beijing chemical reagent factory.
Embodiment 1: the screening of powerful strain
Isolate 67 strain Pseudomonas aeruginosas from the mink lung of hemorrhagic pneumonia is suffered from each ermine field on the ground such as Shandong, Hebei, Dalian, the Inner Mongol, Jilin, through Observation of biological characteristics, biochemical test, be accredited as Pseudomonas aeruginosa.Adopt Japan to give birth to the standard serum that grinds Co., Ltd. and carry out somatotype by slide agglutination test, determine the serotype of strain isolated.
Attack poison experiment by mouse and determine virulent isolates, by inoculation in the PYG liquid nutrient medium, 37 ℃ of jolting 12h, the uniform liquid muddiness, be yellow-green colour or white, forms mycoderm.After doing the bacterium counting, the bacterium number is adjusted to 2,000,000,000 CFU/mL, bacterium liquid is diluted respectively to 10 times, 100 times, be that the bacterium amount is respectively 2,000 ten thousand CFU/mL and 2,000,000 CFU/mL, and attack mouse by these two concentration, every each concentration of strain bacterium is attacked three mouse, every mouse is attacked 0.2ml, and One serotype is attacked bacterial strain totally one strain of the dead mouse of 100 times of extent of dilution, and another kind of serotype is attacked bacterial strain totally one strain of the dead mouse of 10 times of extent of dilution.The bacterial strain that this two strains virulence is stronger, as alternative vaccine strain, is distinguished called after bacterial strain JL08, bacterial strain DL15.Bacterial strain JL08, bacterial strain DL15 are gram negative bacillus, and single or one-tenth is two to be existed, and the blunt circle in two ends, do not form brood cell and pod membrane.
Measure the medium lethal dose (LD50) of this two strains virulent strain to small white mouse by the Reed-Muench method, the LD50 that is respectively bacterial strain DL15 is 2,500,000 CFU, and the LD50 of bacterial strain JL08 is 2,000 ten thousand CFU; To the medium lethal dose (LD50) of mink, the LD50 of bacterial strain DL15 is 1,000,000 CFU, and the LD50 of bacterial strain JL08 is 2,500,000 CFU.
Above-mentioned two kinds of powerful bacterial strain bacterial strain JL08, bacterial strain DL15 are carried out to biological preservation, and its relevant information is as follows:
Bacterial strain DL15: Classification And Nomenclature: Pseudomonas aeruginosa, Pseudomonas aeruginosa. is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 5th, 2010, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.3811.
Bacterial strain JL08: Classification And Nomenclature: Pseudomonas aeruginosa, Pseudomonas aeruginosa. is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 5th, 2010, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.3812.
Embodiment 2: the preparation of bivalent inactivated vaccine of the present invention
(1) vaccine preparation: by bacterial strain DL15, bacterial strain JL08 streak inoculation is cultivated 18h in 37 ℃ in the PYG nutrient agar, choose afterwards single colony inoculation in the 5mlPYG broth culture 37 ℃ of jolting 12h as generation seed liquor, again in 3% ratio be inoculated in 300mlPYG meat soup 37 ℃ of jolting 12h as two generation seed liquor, by two generation seed liquor be inoculated in 37 ℃ of ventilation 12h in 10 liter bottles of 6000mLPYG liquid in 4% ratio, be cultured vaccine bacterium liquid, by bacterial plate counts, bacterium liquid is counted, and by count results, the bacterium number of this two strain all is adjusted into to 8,000,000,000 CFU/mL, afterwards bacterium liquid is pressed to bacterial strain DL15, bacterial strain JL08 ratio is to mix at 1: 1, add formalin-inactivated after mixing, by the formaldehyde final concentration, it is 0.15% deactivation, 37 ℃ of deactivation 24h, shake 2-3 time during this time.
(2) deactivation of vaccine check: the bacterium liquid after formalin-inactivated is got to 200 microlitres and be connected in the 5mL liquid nutrient medium, once cultivate and get sample three times, each sample is inoculated in three test tubes to observe and within five days, carries out the deactivation safety verification, five days afterwards the liquid nutrient medium of inoculation still clarify that safety verification is qualified without muddiness.
(3) finished product vaccine preparation: the bacterium liquid that deactivation is upchecked adds 20% aluminium hydroxide sol solution in bacterium liquid and the aluminium glue ratio of 4: 1, add the sodium azide of 0.01% concentration after fully mixing, and mixes.
Embodiment 3: the safety verification of bivalent inactivated vaccine of the present invention
Three batches of vaccine single doses that embodiment 2 is prepared, single dose repeat and overdose is injected healthy mink, the inboard intramuscular inoculation of hind leg.By 14d, observe, inoculation mink body temperature and appetite are normal, and inoculation is local without swelling and inflammation, part and systemic reaction do not occur; Every batch of vaccine is selected 3, cut open and kill the Skin observing injection site pathological change of rear incision injection site, the subcutaneous nothing of mink inoculation position changes extremely, the inboard muscle inoculation position of hind leg muscle tissue is normal, only see injection site and be Vandyke brown, the corn grain size is arranged, distinguish with sorrel muscle on every side, injection site has no inflammatory reaction.The young ermine of 60-70 age in days continues to observe to the rear 30d of inoculation, and young ermine physically well develops.Illustrate that vaccine has security preferably to the mink of different ages.
Embodiment 4: bivalent inactivated vaccine of the present invention is measured the potency test of mink
The three batches of mink hemorrhagic pneumonia bivalent inactivated vaccines are 20 of immunity 16～18g small white mouses respectively, each 10 of experimental group and control groups, and only, immunity is used respectively 10LD together with 20 of control group small white mouses in latter 21 days to 0.2ml/
50the strong poison of DL15, JL08 attack poison, observe 7, the three batches of vaccine immunity group small white mouses equal 100% obtain protection, equal 100% morbidity of control group small white mouse.
The three batches of mink hemorrhagic pneumonia bivalent inactivated vaccines are the healthy mink at immune 2-10 monthly ages respectively, 10 of the every batch of vaccine intramuscular inoculation minks, 1ml/ only, latter 21 days of immunity together with 10 of control group minks respectively with containing 10LD
50the strong poison of DL15, JL08 strain attack poison (5 of each groups) by splashing in tracheae, observe 7, three batches of vaccine immunity group minks equal 100% obtain protection, equal 100% morbidity of control group mink.
Embodiment 5: bivalent inactivated vaccine of the present invention is measured the immune period of mink
Adopt three batches of bivalent inactivated vaccines to carry out immune period mensuration.Be divided into experimental group and control group, experimental group is divided into six groups, every group of 30 minks, and with three batches of bivalent inactivated vaccine immunity of the present invention, 10 minks of every batch of vaccine immunity, every inboard intramuscular injection 1mL of hind leg; Control group is divided into six groups; every group of 10 minks; the inboard muscle injecting normal saline contrast of hind leg 1mL; within 1st～6 months after immunity, attack respectively poison (5 minks of every batch of every strain inoculation) with bacterial strain JL08 and the bacterial strain DL15 of 10 times of amount LD50 per month, measure respectively the protection of vaccine to these two kinds of strains.Vaccine to the immune protective broken line graph of mink as shown in Figure 2; experimental result shows; bivalent inactivated vaccine of the present invention is 6 months to the protection period of mink anti Bacillus pyocyaneu Flugge virulent strain DL15; immune protective rate is more than 80%; protection period to mink anti Bacillus pyocyaneu Flugge virulent strain JL08 is 5 months, and immune protective rate is more than 80%.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. a Pseudomonas aeruginosa (Pseudomonas aeruginosa) DL-15, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.3811.
2. mink hemorrhagic pneumonia vaccine prepared by Pseudomonas aeruginosa (Pseudomonas aeruginosa) JL-08 that is CGMCC No.3812 by the described Pseudomonas aeruginosa DL-15 of claim 1 and deposit number.
3. mink hemorrhagic pneumonia vaccine as claimed in claim 2, is characterized in that, described vaccine is bivalent inactivated vaccine.
4. the preparation method of a mink hemorrhagic pneumonia bivalent inactivated vaccine comprises the following steps:
Step 1: the Pseudomonas aeruginosa JL-08 that the Pseudomonas aeruginosa DL-15 that is CGMCC No.3811 by deposit number and deposit number are CGMCC No.3812 is inoculated in respectively the PYG culture medium culturing, adjusting the bacterium number is 80-100 hundred million CFU/mL, and 1-2:1 mixes in proportion;
Step 2: inactivation step 1 gained mixed culture;
Step 3: add sanitas and adjuvant in step 2 gained culture, obtain described mink hemorrhagic pneumonia bivalent inactivated vaccine.
5. preparation method as claimed in claim 4, is characterized in that, the described inoculation of step 1 is for pressing the 3%-4% inoculating strain of PYG culture volume.
6. preparation method as claimed in claim 4, is characterized in that, the described cultivation of step 1 is at 37 ℃ of aerated culture 12-15h.
7. preparation method as claimed in claim 4, is characterized in that, the described deactivation of step 2 is the formalin-inactivated with final concentration 0.15%.
8. preparation method as claimed in claim 4, is characterized in that, the described sanitas of step 3 is sodium azide.
9. preparation method as claimed in claim 4, is characterized in that, the described adjuvant of step 3 is the aluminium hydroxide sol solution.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102626516A (en) * | 2012-04-05 | 2012-08-08 | 青岛农业大学 | Pseudomonas aeruginosa and propolis inactivated vaccine for minks and preparation process |
| CN103948918A (en) * | 2014-03-21 | 2014-07-30 | 母连志 | Method for preparing trivalent inactivated vaccine for preventing mink hemorrhagic pneumonia |
| CN104892768A (en) * | 2015-05-12 | 2015-09-09 | 青岛农业大学 | Mink pseudomonas aeruginosa ExoA-FliC chimeric protein vaccine |
| CN105420153B (en) * | 2015-12-16 | 2019-01-01 | 中国农业科学院特产研究所 | A kind of Pseudomonas aeruginosa fermentation medium and its fermentation culture method, vaccine preparation method |
| CN105543127B (en) * | 2015-12-26 | 2019-08-23 | 哈药集团生物疫苗有限公司 | Mink Pseudomonas aeruginosa serum Type B bacterial strain and its inactivated vaccine and application |
| CN109652344A (en) * | 2016-02-17 | 2019-04-19 | 中国农业科学院特产研究所 | Bacterial strain and its application and vaccine and preparation method thereof |
| CN105670972B (en) * | 2016-03-11 | 2019-08-09 | 哈药集团生物疫苗有限公司 | Mink Pseudomonas aeruginosa serum G type bacterial strain and its inactivated vaccine and application |
| CN105754905A (en) * | 2016-04-19 | 2016-07-13 | 青岛农业大学 | Pseudomonas aeruginosa of minks and application of pseudomonas aeruginosa |
| CN105749266B (en) * | 2016-04-26 | 2021-02-02 | 齐鲁动物保健品有限公司 | Mink hemorrhagic pneumonia and clostridium botulinum poisoning bivalent inactivated vaccine and preparation method thereof |
| CN106390111A (en) * | 2016-11-25 | 2017-02-15 | 于彦强 | Preparation method of mink hemorrhagic pneumonia inactivated vaccine and application thereof |
| CN108441457B (en) * | 2018-06-20 | 2021-04-13 | 中国农业科学院特产研究所 | Mink source D-type pseudomonas aeruginosa, application thereof, inactivated vaccine thereof and preparation method of inactivated vaccine |
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| RU1066074C (en) * | 1982-07-23 | 1995-12-10 | Всесоюзный государственный научно-контрольный институт ветеринарных препаратов | Pseudomonose vaccine for fur-bearing animals, mainly, minks and method of producing the same and method of its prophylaxis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| RU1066074C (en) * | 1982-07-23 | 1995-12-10 | Всесоюзный государственный научно-контрольный институт ветеринарных препаратов | Pseudomonose vaccine for fur-bearing animals, mainly, minks and method of producing the same and method of its prophylaxis |
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