CN108220182A - One plant of horse source streptococcus zooepidemicus strain X JMSY16-1 and its application in streptococcus equi disease vaccine - Google Patents
One plant of horse source streptococcus zooepidemicus strain X JMSY16-1 and its application in streptococcus equi disease vaccine Download PDFInfo
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Abstract
The present invention relates to one plant of horse streptococcus zooepidemicus (also known as Malian drainage,Streptococcus equi subsp.zooepidemicus) XJMSY16 1, CGMCC No.12428, there is the 16S rRNA gene orders in sequence table 1；The strain isolation is from the sick horse of streptococcus equi disease, gram-positive bacterium, and cell is arranged in pair or the long-chain in bending in spherical, in ordinary broth and tablet undergrowth；β type haemolysis occurs on 5% sheep blood tablet, forms fully transparent haemolysis band around dewdrop shape petite, width is up to 2.5~3.2 mm.Growth is in amphimicrobian, and growth temperature is from 26 DEG C~40 DEG C, 37 DEG C of optimum growth temperature, the most suitable growth pH7.4~7.5.Glucose fermentation production acid, it is impossible to lactose fermenters, mannitol and sorbierite.The bacterial strain can be used for preparing streptococcus equi inactivated vaccine.The vaccine specific aim prepared by the bacterial strain is good, at low cost, safety, and protecting effect is good.
Description
Technical field
One plant provided by the invention be isolated from Yili of Xinjiang horse streptococcus zooepidemicus (Streptococcus equi
subsp. zooepidemicus) XJMSY16-1 bacterial strain CGMCC No.12428 and the bacterial strain be anti-streptococcus equi disease
Control the middle method for preparing inactivated vaccine and possible application.
Background technology
With economic globalization, modern horse industry has turned to the Pang in the form of horse racing, performance exhibition, riding amusement etc.
Big industry, this is that Chinese horse industry has welcome unprecedented opportunity to develop.The fast development of horse industry, the raising of horse are advised increasingly
Modelling and intensive, makes the harm of streptococcus equi disease show to become clear day by day.
Horse streptococcus zooepidemicus causes the streptococcosis of equus to be a kind of acute, hot contagious disease, main table
Now respiratory tract infection and purulent inflammation, and the symptoms such as septicemia, meningitis, arthritis can be caused.In addition bacterium or one
The important zoonosis cause of disease of kind.The microbial animal epidemic be in worldwide distribution, but different zones prevalence have it is respective
Feature.The ground such as the America and Europe disease mostly occurs in the animals such as horse, milk cow, sheep, and Nei Diduo provinces and cities of China are popular mainly in swinery.
The disease is high in horse keeping area incidence, almost every year will be popular primary in worldwide distribution, not only directly affects
The growth and development of horses also results in the death of horses, has resulted in serious financial consequences, still annoyings the hair of horse keeping industry at present
Exhibition.At present to the prevention of the disease in most cases based on antibiosis extract for treating and environmental sanitation control, but antibiosis extract for treating often can
Lead to unsatisfactory curative effect because of bacteriogenic drug resistance.
It can prevent and reduce the occurrence of this disease to a certain extent using the vaccine of horse streptococcus zooepidemicus.The country is in horse at present
Research and product are few in terms of the vaccine of the commercialization of source streptococcus zooepidemicus, it is impossible to adapt to the demand of horse industry fast development.This
Outside since the bacterial strain of different regions prevalence is not quite similar, mutual immunocompetence also has different between different epidemic strains,
Additionally, it is observed that this kind of bacterial strain has the characteristics that variation, only it is extremely difficult to effectively control the different regions disease with a kind of vaccine strain
Generation.To adapt to the fast development of horse aquaculture, purifying this disease as early as possible and controlling generation and the prevalence of the bacterium, need to take
The pathological material of disease of Yili of Xinjiang streptococcus equi disease morbidity horses, isolates the horse streptococcus zooepidemicus of local prevalence, carries out biochemical
The epidemic link of Yili of Xinjiang is should be with researchs, the bacterial strains such as Molecular Identifications, available for preparing the inactivation of streptococcus equi disease
Vaccine realizes effective prevention to local streptococcus equi disease.
Develop to neutralize for current horse industry and be badly in need of solving the problems, such as the prevention to streptococcus equi disease, purport of the present invention in horse disease prevention and control
A kind of streptococcus equi disease vaccine bacterial strain with strong points is being provided, which can be utilized to prepare streptococcus equi and inactivate
Seedling, the vaccine is at low cost, safety, the prevention available for streptococcus equi disease caused by horse streptococcus zooepidemicus.
Invention content
Malian drainage (Streptococcus equi subsp.zooepidemicus) provided by the invention
XJMSY16-1 bacterial strains on May 10th, 2016, are preserved in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 " China Microbiological bacterium
Kind preservation administration committee common micro-organisms center " (CGMCC), preserving number are CGMCC No.12428.
1. horse streptococcus zooepidemicus provided by the invention (Streptococcus equi subsp. zooepidemicus)
XJMSY16-1 bacterial strain CGMCC No.12428 have following characteristics：
（1）Colony characteristics：Form that canescence, surface be smooth, tiny bacterium colony of neat in edge on blood agar plate.
（2）Bacterial morphological characteristic：Bacterium circle or oval, it is general most in catenation, short 4~8 bacterium groups of person
Into elder is made of 20~30 bacteriums, Gram-positive.
（3）Physiological and biochemical property：Nutritional requirement is higher, need to be added with the ability such as blood, serum, glucose in ordinary culture medium
Growth.Aerobic or amphimicrobian grows growth temperature from 26 DEG C to 39 DEG C, 37 DEG C of optimum temperature, pH growth scopes 6-9, most suitable
PH7.3~7.6.
（4）16S rRNA gene sequence characteristics see attached list 1.
Reference《Bergey’s Manual of Systematic Bacteriology》（The second edition）, according to its form spy
It seeks peace physiological and biochemical property and according to its search result of 16S rRNA gene orders in GenBank, horse streptococcus zooepidemicus
Strain X JMSY16-1 (CGMCC No.12428) be accredited as Malian drainage (Streptococcus equi
subsp. zooepidemicus)。
The culture medium TM compositions of CGMCC No.12428 bacterial strains provided by the invention：
Peptone 0.3%
NaCl 0.2%
Yeast extract 0.3%
Glucose 0.5%
Horse serum 5%
It is prepared after the sterilizing in 30 minutes of PH=7.4 112 DEG C.
2. the preparation of inactivation antigen
Separation bacterium is inoculated in respectively in the TM culture mediums added with 2% horse serum, 37 DEG C, 180 r/min cultures 18~for 24 hours, add
0.4% 36~48h of formalin-inactivated.Supernatant is removed in centrifugation.Bacterial precipitation is with the PBS that sterilizes（PH7.2,10mmol) it is diluted to 2 ×
107CFU/mL.Immunizing rabbit after being emulsified with equivalent Fu Shi Freund's complete adjuvants, it is endless with the bacterium of similary concentration and equivalent Fu Shi after 3 weeks
Full adjuvant emulsion prepares immunogene.
3. the inactivation antigen of preparation is inoculated in sheep blood agar plate by steriling test respectively, and 37 DEG C of cultures 24~
72 h, observation whether there is bacterial growth;
4. safety examination
The inactivation antigen of preparation is subcutaneously injected to the rabbit 6 of 1.5~2.5kg weight, 4mL/ only, separately takes 2 hypodermic injections
Physiological saline compares, and 4mL/ only, observes 10d.
5. mouse immuning test
Will separation bacterium be inoculated in respectively in the TM culture mediums added with 2% horse serum, 37 DEG C, 180 r/min culture 18~for 24 hours, add
0.4% 36~48h of formalin-inactivated.Supernatant is removed in centrifugation.Bacterial precipitation is with the PBS that sterilizes（PH7.2,10mmol) it is diluted to 1 ×
106CFU/mL.The Fu Shi Freund's complete adjuvants and Fu Shi of qualified inactivated bacterial liquid and equivalent be not after steriling test and safety verification
Freund's complete adjuvant emulsification prepares immunogene.The inactivation antigen of preparation is subcutaneously injected to the mouse 8 of 20g weight, 0.5mL/ only,
Another to take same mouse 8, skin is subcutaneously injected physiological saline and compares, and only, each group carries out 0.5 mL/ after head exempts from after 3 weeks
2nd time immune, and 2 exempt from rear 15d is attacked with live strain, and challenge dose is 1 × 105CFU。
5. vaccine valence measures
Under identical rearing conditions, the rabbit 5 of 1.5~2.0kg weight, dorsal sc multi-point injection vaccine are randomly choosed
4mL/ only, separately takes 5 physiological saline is subcutaneously injected and compares, only, each group after 3 weeks exempt from for the 2nd time 4mL/ after head exempts from
Epidemic disease, 15,30,45d thalline make envelope antigen, ELISA method detection antibody.
Invention effect
（1）The present invention provides one plant to be isolated from Yili of Xinjiang streptococcus equi disease advantage epidemic strain, for effectively preventing to work as
Streptococcus equi disease has important value caused by ground epidemic link.Inactivated vaccine production cost with its preparation is low, added value of product
Height, the market demand is high, there is good market prospects.
（2）The bacterial strain can be utilized to produce various types of streptococcus equi disease vaccine and include inactivated vaccine and Asia
Subunit vaccine.The vaccine immunization effect is good, and with strong points, characteristic is strong.
（3）The horse streptococcus zooepidemicus of streptococcus equi disease Malaysia and China for being isolated from Yili of Xinjiang the present invention provides one plant,
For the vaccine because thoroughly being inactivated with formaldehyde, the inactivated vaccine safety with its preparation is good, dangerous without poison is dissipated.
Description of the drawings
Fig. 1 is the result figure of genomic DNA electrophoresis in 1% Ago-Gel of the horse streptococcus zooepidemicus of extraction.M:
DNA Marker DL15000；1,2：The genomic DNA of streptococcus zooepidemicus bacterial strain for extraction.
Fig. 2 is the electrophoresis result figure of horse streptococcus zooepidemicus 16SrRNA pcr amplification products.M：DNA Marker
DL2000；1,2：The PCR amplification result of streptococcus zooepidemicus 16S rRNA.
Specific embodiment
For a better understanding of the present invention, it is further described by following embodiment, but not to the present invention's
It limits.
Embodiment 1：The culture of horse streptococcus zooepidemicus strain X JMSY16-1 CGMCC No.12428 and biological property.
Malian drainage XJMSY16-1 CGMCC No.12428 are isolated from the pus of streptococcus equi disease horse, inoculation
In blood in substrate cultivation base, to be cultivated in 37 DEG C, 12-15 h.The bacterium has following characteristics：（1）Colony characteristics：In blood
The colony diameter cultivated on agar plate 1 day is 0.3-1.2 mm, and bacterium colony is rounded, and surface is smooth, neat in edge, protrusion, ash
White.（2）Bacterial morphological characteristic：Bacterium is round or oval；Gram-positive；1.1-2.1 μm of bacterium diameter.（3）
Physiological and biochemical property：Amphimicrobian is grown；Growth temperature is from 27 DEG C to 39 DEG C, 37 DEG C of optimum growth temperature；PH growth scopes 6-
8, the most suitable growth pH7.3-7.6；It can not hydrolyze aesculin with glucose fermentation, sucrose, do not grow, do not liquefy in 6.5%NaCl
Gelatin decomposes arginine, can be by the use of salicin as carbon source, it is impossible to utilize lactose, synanthrin, trehalose, mannitol, sorbierite.
Reference《Bergey’s Mannual of Systematic Bacteriology》（The second edition）, according to its form
Feature and physiological and biochemical property and according to its search result of 16S rRNA gene orders in GenBank, bacterial strain
XJMSY16-1 CGMCC No.12428 are accredited as horse streptococcus zooepidemicus bacterial strain（Malian drainage,Streptococcus.equi subsp. zooepidemicus）.
Embodiment 2：The PCR of the 16S rRNA genes of horse streptococcus zooepidemicus strain X JMSY16-1 CGMCC No.12428 expands
Increasing and sequencing
Horse streptococcus zooepidemicus strain X JMSY16-1 CGMCC No.12428 are inoculated in fluid nutrient medium, will grow to logarithm evening
The zymotic fluid centrifugation of phase（4500 revs/min, 5 minutes）Supernatant is removed, with TE (50 mM Tris, 50 mM EDTA-Na2)
Solution is washed 2 times；With 0.5 mL TES solution by thalline mixing, appropriate lysozyme is added in, 37 DEG C keep the temperature 2 hours；Add in 0.2 mL
20 % SDS, 60 DEG C keep the temperature 10 minutes；Add in 0.3 mL 5M NaClO4, mixing；Add in isometric phenol-chloroform-isoamyl alcohol
(25: 24:1) it, gently shakes up 5 minutes or so, centrifuges（5000 revs/min, 5 minutes）, Aspirate supernatant, then with phenol-chloroform-
Isoamyl alcohol (25: 24:1) it handles one time；Then chloroform-isoamyl alcohol is used successively（24:1, v/v）Twice of processing, until not having egg
Tunica albuginea occurs；Supernatant adds in 37 DEG C of 20 μ l, 0.2 % RNA enzyme and keeps the temperature 30 minutes, chloroform-isoamyl alcohol（24:1, v/v）Place
Reason three times；2 times of volume ice ethanol precipitations of supernatant, 70% ice alcohol solution dipping 5 minutes.It is dissolved in TE solution and making after drying
For template.
It is for the PCR of the 16S rRNA gene magnifications forward primers reacted：
5 '-TGGAACGCACAGATGATAC -3 ', reverse primer are：
5’- GACTTCGGGTGTTACAAAC -3’ 。
PCR reaction systems（50 μL）For：10×buffer 5μL、25 mmol/L MgCl2 4μL、10 mmol/L
1 μ L of dNTPs, each 1 μ L of 20 pmol/L primers, ddH236 μ l of O, 1 μ L of Taq DNA enzymatics, 1 μ L of template.PCR reaction conditions are：
95 DEG C of 10 min, 95 DEG C of 1 min, 55 DEG C of 1 min, 72 DEG C of 1 min, 30 cycles；72 DEG C of 10 min, 4 DEG C of guarantors
It deposits.16S rRNA gene orders length is 1312 bp.The nucleotide sequence of its 16S rRNA gene is as shown in sequence table 1.
Embodiment 3：The preparation of inactivation antigen
Will separation bacterium be inoculated in respectively in the TM culture mediums added with 2% horse serum, 37 DEG C, 180r/min culture 18~for 24 hours, add
0.4% 36~48h of formalin-inactivated.After inactivation thoroughly, 7000rpm/min centrifugations 15min removes supernatant.Bacterial precipitation is to sterilize
PBS（PH7.2,10mmol) it is diluted to 1 × 107CFU/mL.With equivalent Fu Shi Freund's complete adjuvants emulsify after immunizing rabbit, after 3 weeks with
The bacterium of similary concentration emulsifies with equivalent Fu Shi Freund's incomplete adjuvants, and vaccine carries out steriling test and safety inspection after fully vibrating mixing
It tests.
Embodiment 4：Steriling test
The inactivation antigen of preparation is inoculated in sheep blood agar plate, 37 DEG C of 24~72 h of culture respectively, observation whether there is bacterium
Growth.Result, which loses, after culture bacterium growth, shows that inactivating efficacy is good, working specification is pollution-free.
Embodiment 5：Safety examination
The inactivation antigen of preparation is subcutaneously injected to the rabbit 6 of 1.5~2.5kg weight, 4mL/ only, separately takes 2 hypodermic injections
Physiological saline compares, and 4mL/ only, observes 10d.Rabbit is inoculated with without 1 death, illustrates that vaccine safety is preferable;Inspection connects
The reaction of kind locally and systemically, the rabbit that as a result remaining is inoculated in addition to 1 inoculation position has light inflammation reaction is without apparent office
Portion and general reaction, spirit, body temperature, appetite, breathing and pulse are normal.
Embodiment 6：Mouse immuning test
Will separation bacterium be inoculated in the TM culture mediums added with 5% horse serum, 37 DEG C, 180rpm/min culture 18~for 24 hours, add 0.4%
36~48h of formalin-inactivated.Supernatant is removed in centrifugation.Bacterial precipitation is with the PBS that sterilizes（PH7.2,10mmol) it is diluted to 1 × 106CFU/
mL.The Fu Shi Freund's complete adjuvants and Fu Shi of qualified inactivated bacterial liquid and equivalent are not exclusively helped after steriling test and safety verification
Agent emulsification prepares immunogene.The inactivation antigen of preparation is subcutaneously injected to the mouse 20 of 20g weight, 0.5mL/ only, separately takes phase
With mouse 20, physiological saline is subcutaneously injected and compares, only, each group after 3 weeks exempt from for the 2nd time 0.5mL/ after head exempts from
Epidemic disease, 2 exempt from the viable bacteria culture attack of rear 15d lethal doses, and the dosage of every group of intraperitoneal inoculation attack bacterium is 5 × 105CFU is seen
It examines one week.As a result control group is all dead, and dead 4 of immune group, the immune protective efficiency of immune group is up to 80%.
Embodiment 7：Vaccine valence measures
Under identical rearing conditions, the Kun ming white mouse 12 of 15~20kg weight, subcutaneous multi-point injection vaccine are randomly choosed
0.5 mL/ only, separately takes 12 physiological saline is subcutaneously injected and compares, only, each group carries out 0.5 mL/ after head exempts from after 3 weeks
2nd time immune, and 45d is with 108Thalline makees envelope antigen, ELISA method detection antibody.Testing result shows mouse through being immunized twice
The antibody titer of 45d is 8.1 × 10 afterwards3。
ATACGTAGCTTGCTACAATTATCTGTGAGTCGCGAACGGGTGAGTAACGCGTAGGTAACCTAGCTTATAGCGG
GGGATAACTATTGGAAACGATAGCTAATACCGCATAAAAGTGGTTGACCCATGTTAACCATTTAAAAGGAGCAACAG
CTCCACTATGAGATGGACCTGCGTTGTATTAGCTAGTTGGTAGGGTAAAGGCCTACCAAGGCGACGATACATAGCCG
ACCTGAGAGGGTGAACGGCCACACTGGGACTAAGACACAGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTC
GGCAATGGGGGGAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGA
GAAGAACAGTGATGGGAGTGGAAAGTCCATCATGTGACGGTAACTAACCAGAAAGGGACGGCTAACTACGTGCCAGC
AGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTGATAAGTC
TGAAGTTAAAGGCAGTGGCTTAACCATTGTATGCTTTGGAAACTGTTAAACTTGGGTGCAGAAGGGGAGAGTGGAAT
TCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGAC
GCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGCTGAGTGCTAGGT
GTTAGGCCCTTTCCGGGGCTTAGTGCCGTAGCTAACGCATTAAGCACTCCGCCTGGGGAGTATGACCGCAAGGTTGA
AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAGCCTTAC
CAGGTCTTGACATCCCGATGCTATTCTTAGAGATAAGAAGTTACTTCGGTACATTGGAGACAGGTGGTGCATGGTTG
TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTAAG
TTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGA
CCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTGAAAGCCA
ATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAACCGCTAGTAATCGCGGATCAGCACGCCGCG
GTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAA
CCGTTAA
Claims (3)
1. the horse streptococcus zooepidemicus of one plant of preparation horse streptococcus zooepidemicus disease vaccine（Streptococcus equi subsp.zooepidemicus) XJMSY16-1, preserving number is CGMCC No.12428.
2. horse streptococcus zooepidemicus XJMSY16-1 according to claim 1, it is characterised in that its 16S rDNA has such as sequence
Nucleotide sequence shown in table 1.
3. horse streptococcus zooepidemicus XJMSY16-1 according to claim 1 prepares the side of horse streptococcus zooepidemicus inactivated vaccine
Method is inoculated in TM culture mediums, 37 DEG C, 180 r/min trainings including horse streptococcus zooepidemicus XJMSY16-1 described in claim 1
Foster 18~for 24 hours, add 0.4% formalin-inactivated, centrifugation is with the PBS that sterilizes（PH7.2,10mmol) it is diluted to 1 × 108CFU/mL,
Fu Shi Freund's complete adjuvants and Fu Shi the Freund's incomplete adjuvants emulsification of inactivated bacterial liquid and equivalent by steriling test and safety verification qualification
It is prepared into inactivated vaccine, the TM culture mediums that horse streptococcus zooepidemicus XJMSY16-1 is used,
Peptone 0.3%
NaCl 0.2%
Yeast extract 0.3%
Glucose 0.5%
After 121 DEG C of sterilizings in 20 minutes, 5% horse serum, PH=7.4 are added in.
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| CN112011479A (en) * | 2020-08-06 | 2020-12-01 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Streptococcus equi subsp equi HLJ2018D-LX strain and application thereof in preparation of streptococcus equi subsp equi inactivated vaccine |
| CN114591841A (en) * | 2022-03-02 | 2022-06-07 | 吉林大学 | Rhodococcus equi strain and application thereof in preparation of inactivated vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011479A (en) * | 2020-08-06 | 2020-12-01 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Streptococcus equi subsp equi HLJ2018D-LX strain and application thereof in preparation of streptococcus equi subsp equi inactivated vaccine |
| CN112011479B (en) * | 2020-08-06 | 2022-04-22 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Streptococcus equi subsp equi HLJ2018D-LX strain and application thereof in preparation of streptococcus equi subsp equi inactivated vaccine |
| CN114591841A (en) * | 2022-03-02 | 2022-06-07 | 吉林大学 | Rhodococcus equi strain and application thereof in preparation of inactivated vaccine |
| CN114591841B (en) * | 2022-03-02 | 2024-01-30 | 吉林大学 | Rhodococcus equi and application thereof in preparation of inactivated vaccine |
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