CN102286391A - 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 the mink pseudomonas pneumonia again, be mainly to occur in annual a kind of acute infectious disease that 9～November, mink moulted season, with hemorrhagic pneumonia, acute death is feature, occur symptoms such as expiratory dyspnea, nostril outflow red zone foam liquid before dead, after death cut open 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 important bacteria 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), be 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 have a flagellum, can move.Can not form brood cell and pod membrane.Well-grown on the plain agar substratum forms 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, the 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 have 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, the common drug tilmicosin, Azythromycin, the kantlex result of treatment is not remarkable, and antibiotic abuse causes Pseudomonas aeruginosa to produce extremely strong resistance, therefore need effective vaccine to prevent, domestic still do not have commercial vaccine and prevent the mink hemorrhagic pneumonia, report is arranged with bacterial cell wall extracts (JAMES E.PENNINGTON abroad, 1979), with the common protective antigen OEP that in thalline, extracts (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, the cost height; being not suitable for mass production uses; and immune time is many, and 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 prepare 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 is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.3811.
Another kind of pseudomonas aeruginosa strains JL08 provided by the invention is deposited in China Committee for Culture Collection of Microorganisms 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 with 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 vaccine by above-mentioned pseudomonas aeruginosa strains preparation.
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, may further comprise the steps:
Step 1: pseudomonas aeruginosa strains DL15 and pseudomonas aeruginosa strains JL08 are inoculated in the PYG culture medium culturing respectively, 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: in step 2 gained culture, add sanitas and adjuvant, promptly get 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, and the incubation time of control Pseudomonas aeruginosa prevents that incubation time is long and causes the ectotoxic generation of Pseudomonas aeruginosa in 15h, ensures vaccine safety better.
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 a sodium azide, and preferred whole mass concentration is 0.01%.
As preferably, the described adjuvant of step 3 is an aluminium hydroxide solution, and preferred aluminium hydroxide liquor capacity is 4% of a nutrient solution.
The mink hemorrhagic pneumonia bivalent inactivated vaccine that pseudomonas aeruginosa strains DL15 of the present invention and bacterial strain JL08 are mixed and made into; once can reach 80% above protection ratio; duration of immunity can reach five months; can effectively prevent present domestic popular mink hemorrhagic pneumonia; its preparation technology is simple; be fit to commercialization production, have a good application prospect.
Biological preservation information:
Bacterial strain DL15: classification name: Pseudomonas aeruginosa, Pseudomonas aeruginosa. is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 5th, 2010, the 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 name: Pseudomonas aeruginosa, Pseudomonas aeruginosa. is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 5th, 2010, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.3812.
Description of drawings
Fig. 1 is shown the microscopically form of pseudomonas aeruginosa strains JL08 of the present invention, bacterial strain DL15;
Fig. 2 shows the protection efficient of bivalent inactivated vaccine immunity mink of the present invention.
Embodiment:
The invention discloses pseudomonas aeruginosa strains JL08, bacterial strain DL15 and with mink hemorrhagic pneumonia bivalent inactivated vaccine of described two kinds of bacterial strains preparation 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 that all similarly replace and change apparent to those skilled in the art, they all are regarded as being included in the present invention.Product of the present invention, method and application are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
Test materials
PYG substratum composition: peptone 2%, glucose 1%, sodium-chlor 0.5%, yeast powder 0.3% with the deionized water preparation, has been joined adjust pH 7.2-7.4,116 ℃ of 30min.
The Pseudomonas aeruginosa somatotype is given birth to available from Japan with standard serum and is ground Co., Ltd.; Bacteria Identification trace biochemical reaction pipe is available from sky, Hangzhou and microorganism reagent company; 18～22g small white mouse is available from Jilin decent job biological products company limited; Formaldehyde, sodium azide is available from Beijing chemical reagent factory.
Embodiment 1: the screening of powerful strain
From the mink lung of hemorrhagic pneumonia is suffered from each ermine field on ground such as Shandong, Hebei, Dalian, the Inner Mongol, Jilin, isolate 67 strain Pseudomonas aeruginosas, through biological characteristics observe, biochemical test is accredited as Pseudomonas aeruginosa.Adopt Japan to give birth to the standard serum that grinds Co., Ltd. and carry out somatotype, determine the serotype of strain isolated by slide agglutination test.
Attack the poison experiment by mouse and determine the strong virus force bacterial strain, in the PYG liquid nutrient medium, 37 ℃ of jolting 12h, liquid are evenly muddy, are yellow-green colour or white, form mycoderm with inoculation.After doing the bacterium counting bacterium number is adjusted to 2,000,000,000 CFU/mL, bacterium liquid is diluted 10 times, 100 times respectively, be that the bacterium amount is respectively 2,000 ten thousand CFU/mL and 2,000,000 CFU/mL, and attack mouse with these two concentration, every each concentration of strain bacterium is attacked three mouse, every mouse is attacked 0.2ml, and a kind of 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 is distinguished called after bacterial strain JL08, bacterial strain DL15 as alternative vaccine strain.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 does not form brood cell and pod membrane.
Measure the medium lethal dose (LD50) of this two strains virulent strain to small white mouse with 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 biological preservation, and its relevant information is as follows:
Bacterial strain DL15: classification name: Pseudomonas aeruginosa, Pseudomonas aeruginosa. is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 5th, 2010, the 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 name: Pseudomonas aeruginosa, Pseudomonas aeruginosa. is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 5th, 2010, the 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 production: with 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 the 300mlPYG meat soup 37 ℃ of jolting 12h as two generation seed liquor, with two generation seed liquor be inoculated in 37 ℃ of ventilation 12h in the 10 liter bottles of 6000mLPYG liquid in 4% ratio, be cultured vaccine bacterium liquid, by bacterial plate counts bacterium liquid is counted, and the bacterium number average of this two strain is adjusted into 8,000,000,000 CFU/mL by count results, afterwards bacterium liquid is pressed bacterial strain DL15, bacterial strain JL08 ratio is mixing in 1: 1, add formalin-inactivated after the mixing, by the formaldehyde final concentration is 0.15% deactivation, 37 ℃ of deactivation 24h, during the concussion 2-3 time.
(2) deactivation of vaccine check: the bacterium liquid behind the formalin-inactivated is got 200 microlitres be connected in the 5mL liquid nutrient medium, once cultivate and get sample three times, each sample is inoculated in to observe in three test tubes and carried out the deactivation safety verification in five days, and the liquid nutrient medium of inoculating after five days is still clarified no muddiness, and then safety verification is qualified.
(3) finished product vaccine production: the bacterium liquid that deactivation is upchecked adds 20% aluminium hydroxide sol solution in bacterium liquid and 4: 1 ratio of aluminium glue, fully adds the sodium azide of 0.01% concentration behind the mixing, mixing.
Embodiment 3: the safety verification of bivalent inactivated vaccine of the present invention
Three batches of vaccine single doses, single dose repetition and overdose that embodiment 2 prepares are injected healthy mink, the inboard intramuscular inoculation of hind leg.Observe by 14d, inoculation mink body temperature and appetite are normal, and part and systemic reaction do not appear in local no swelling of inoculation and inflammation; Every batch of vaccine is selected 3, cut open and kill back incision injection site Skin observing injection site pathological change, the subcutaneous no abnormal variation of mink inoculation position, the inboard muscle inoculation position of hind leg muscle tissue is normal, only see the injection site and be Vandyke brown, the corn grain size is arranged, distinguish mutually with sorrel muscle on every side, inflammatory reaction is not seen in the injection site.60-70 age in days children ermine continues to observe to inoculation back 30d, 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, 0.2ml/ only, back 21 days of immunity is used 10LD respectively together with 20 of control group small white mouses
50The strong poison of DL15, JL08 attack poison, observed 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, back 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 the tracheae, observed 7, three batches of vaccine immunity group minks equal 100% obtain protection, control group mink equal 100% falls ill.
Embodiment 5: bivalent inactivated vaccine of the present invention was measured the immunoprotection phase of mink
Adopting three batches of bivalent inactivated vaccines to carry out the immunoprotection phase measures.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, the 1st～6 month every month bacterial strain JL08 and bacterial strain DL15 with 10 times of amount LD50 after the immunity attack poison (5 minks of every batch of every strain inoculation) respectively, measure the protection of vaccine to these two kinds of strains respectively.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 only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle 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 (10)
1. a pseudomonas aeruginosa strains DL15 is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.3811.
2. a pseudomonas aeruginosa strains JL08 is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.3812.
3. by the mink hemorrhagic pneumonia vaccine of the described pseudomonas aeruginosa strains preparation of claim 1 and claim 2.
4. mink hemorrhagic pneumonia vaccine as claimed in claim 3 is characterized in that described vaccine is a bivalent inactivated vaccine.
5. the preparation method of a mink hemorrhagic pneumonia bivalent inactivated vaccine may further comprise the steps:
Step 1: pseudomonas aeruginosa strains DL15 and pseudomonas aeruginosa strains JL08 are inoculated in the PYG culture medium culturing respectively, 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: in step 2 gained culture, add sanitas and adjuvant, promptly get described mink hemorrhagic pneumonia bivalent inactivated vaccine.
6. preparation method as claimed in claim 5 is characterized in that, the described inoculation of step 1 is for pressing the 3%-4% inoculating strain of PYG culture volume.
7. preparation method as claimed in claim 5 is characterized in that, the described cultivation of step 1 is at 37 ℃ of aerated culture 12-15h.
8. preparation method as claimed in claim 5 is characterized in that, the described deactivation of step 2 is the formalin-inactivated with final concentration 0.15%.
9. preparation method as claimed in claim 5 is characterized in that, the described sanitas of step 3 is a sodium azide.
10. preparation method as claimed in claim 5 is characterized in that, the described adjuvant of step 3 is the aluminium hydroxide sol solution.
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Cited By (11)
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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 |
| CN105420153A (en) * | 2015-12-16 | 2016-03-23 | 中国农业科学院特产研究所 | Pseudomonas aeruginosa fermentation medium, fermentation culture method thereof and vaccine preparation method |
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| CN105754905A (en) * | 2016-04-19 | 2016-07-13 | 青岛农业大学 | Pseudomonas aeruginosa of minks and application of pseudomonas aeruginosa |
| CN105749266A (en) * | 2016-04-26 | 2016-07-13 | 齐鲁动物保健品有限公司 | Mink hemorrhagic pneumonia and botulism combined inactivate vaccine and preparing method thereof |
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