CN103421708B - One strain strangles strains of streptococcus XZ1 and the application in strangles vaccine thereof - Google Patents
One strain strangles strains of streptococcus XZ1 and the application in strangles vaccine thereof Download PDFInfo
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Abstract
The present invention relates to a strain strangles suis (also known as streptococcus equi horse subspecies, Streptococcus.equi? subsp? equi) XZ1, CGMCC? does No.7830, have the 16S in sequence table 1? rRNA gene order; This strains separation from the sick horse of strangles, gram positive bacterium, cell be spherical, and it is right to be arranged in, or in the reel chain bent, at ordinary broth and dull and stereotyped poor growth; β type haemolysis occurs 5% sheep blood flat board, and form completely transparent haemolysis band around dewdrop shape small colonies, width can reach 3mm.Amphimicrobian grow, growth temperature from 25 DEG C to 40 DEG C, optimum growth temperature 37 DEG C, the most suitable growth pH7.4-7.5.Glucose fermentation produces acid, unfermentable lactose, N.F,USP MANNITOL and sorbyl alcohol.This bacterial strain can be used for preparation strangles inactivated vaccine.The vaccine specific aim prepared by this bacterial strain is good, and cost is low, safety, and protected effect is good.
Description
Technical field
A strain provided by the invention is separated strangles suis (Streptococcus.equisubspequi) XZ1 bacterial strain CGMCCNo.7830 from Yili of Xinjiang, and this bacterial strain prepare in the control of strangles inactivated vaccine method and possible application.
Technical background
Along with economic globalization, modern horse industry has changed the huge enterprise of puting on display, driving the forms such as amusement with horse racing, performance into, and this has welcome unprecedented opportunity to develop for Chinese horse industry.The fast development of horse industry, the raising of horse mass-producing and intensive increasingly, makes the harm of strangles show become clear day by day.
Strangles (Stangles) is a kind of Acute exposure sexually transmitted disease being caused equus by strangles suis, mainly occurs purulent lymphangitis in Horse Neck portion and head.This disease is commonly called as spray larynx or groove knot, larynx bone is swollen, is by acute, the hot transmissible disease of the one of the microbial equus of strangles hammer.It is characterized in that acute, the suppurative inflammation of acute, Catarrhal, suppurative inflammation and neck inferior gluteal lymph node occur the mucous membrane such as nasopharynx.
This disease is high at horse keeping area sickness rate, in worldwide distribution, almost all wants once popular every year, not only directly affects growing of horses, also can cause the death of horses, cause serious financial consequences, still annoying the development of horse keeping industry at present.At present the control of this disease is in most cases controlled based on antibiotic therapy and environmental health, but antibiotic therapy often can cause unsatisfactory curative effect because of bacteriogenic resistance.
The streptococcic vaccine of application strangles can prevent and reduce the generation of this disease to a certain extent.At present domestic in the streptococcic commercial vaccine of strangles research and product seldom, the demand of horse industry fast development can not be adapted to.In addition because the bacterial strain that different areas are popular is not quite similar, between different epidemic strain, mutual immunological competence also has different, finds that this kind of bacterial strain has the feature of variation in addition, is only difficult to reach the generation effectively controlling this disease of different areas with a kind of vaccine strain.For adapting to the fast development of horse aquaculture, purify this disease as early as possible and control the generation of this bacterium and popular, need the pathological material of disease taking Yili of Xinjiang strangles morbidity horses, isolate local popular strangles suis, carry out the research such as biochemistry and Molecular Identification, this bacterial strain should be the epidemic link of Yili of Xinjiang, can be used for preparation strangles suis deactivation vaccine, realizes the effective control to local strangles.
Goal of the invention
For being badly in need of in the development of current horse industry and in horse disease prevention and control solving problem is prevented and treated to strangles, the present invention aims to provide a kind of strangles vaccine strains with strong points, and this bacterial strain can be utilized to preparation strangles suis deactivation vaccine, and this vaccine cost is low, safety, can be used for the prevention of strangles.
Summary of the invention
Strangles suis (Streptococcus.equisubspequi) XZ1 bacterial strain provided by the invention, on June 28th, 2013, be preserved in No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number is CGMCCNo.7830 in Beijing.
1. strangles suis (Streptococcus.equisubspequi) XZ1 bacterial strain CGMCCNo.7830 provided by the invention has following characteristics:
(1) colony characteristics: the tiny bacterium colony forming canescence, smooth surface, neat in edge on blood agar plate.
(2) cell morphological characteristic: cell circle or oval, general most in catenation, short person 4 ~ 8 bacteriums compositions, elder is made up of 20 ~ 30 bacteriums, Gram-positive.
(3) physiological and biochemical property: nutritional requirement is higher, needing to be added with blood, serum, glucose etc. in ordinary culture medium could grow.Aerobic or amphimicrobian grows growth temperature from 25 DEG C to 40 DEG C, optimum temperuture 37 DEG C, pH growth scope 6-9, optimal pH 7.4 ~ 7.6.
(4) 16SrRNA gene sequence characteristic sees attached list 1.
With reference to " Bergey'sManualofSystematicBacteriology " (second edition), according to its morphological specificity and physiological and biochemical property, and according to the Search Results of its 16SrRNA gene order in GenBank, strangles strains of streptococcus XZ1 (CGMCCNo.7830) is accredited as streptococcus equi horse subspecies (Streptococcus.equisubspequi).
The substratum TM of CGMCCNo.7830 bacterial strain provided by the invention forms:
Be prepared from after PH=7.4112 DEG C of sterilizing in 30 minutes.
2. the preparation of inactivation antigen
Isolate is inoculated in respectively and is added with in the TM substratum of 2% horse serum, 37 DEG C, 180r/min cultivates 18 ~ 24h, adds 0.4% formalin-inactivated 36 ~ 48h.Centrifugally remove supernatant.Bacterial precipitation is diluted to 1 × 10 with sterilizing PBS (pH7.2,10mmol)
7cFU/mL.With immunizing rabbit after the emulsification of equivalent Fu Shi Freund's complete adjuvant, with the bacterium of same concentration and the emulsification of equivalent Fu Shi Freund's incomplete adjuvant after 3 weeks, preparation immunogen.
3. the inactivation antigen of preparation is inoculated in sheep blood agar plate by steriling test respectively, cultivates 24 ~ 72h, observes with or without bacterial growth for 37 DEG C;
4. safety examination
By the rabbit 5 of inactivation antigen subcutaneous injection 1.5 ~ 2.0kg body weight of preparation, 4mL/ only, separately gets 2 subcutaneous injection physiological saline and compares, and 4mL/ only, observes 10d.
5. mouse immuning test
Isolate is inoculated in respectively and is added with in the TM substratum of 2% horse serum, 37 DEG C, 180r/min cultivates 18 ~ 24h, adds 0.4% formalin-inactivated 36 ~ 48h.Centrifugally remove supernatant.Bacterial precipitation is diluted to 1 × 10 with sterilizing PBS (pH7.2,10mmol)
6cFU/mL.Immunogen is prepared in Fu Shi Freund's complete adjuvant and the emulsification of Fu Shi Freund's incomplete adjuvant of inactivated bacterial liquid qualified after steriling test and safety verification and equivalent.By the mouse 8 of the inactivation antigen subcutaneous injection 20g body weight of preparation, 0.5mL/ only, separately gets same mouse 8, skin subcutaneous injection physiological saline compares, and only, each group all carries out the 2nd immunity to 0.5mL/ after head exempts from after 3 weeks, 2 exempt from rear 15d live strain attacks, and challenge dose is 1 × 10
5cFU.
5. vaccine valence measures
Under identical rearing conditions, the rabbit of Stochastic choice 1.5 ~ 2.0kg body weight 5, dorsal sc multi-point injection vaccine 4mL/ only, separately get 5 subcutaneous injection physiological saline to compare, 4mL/ only, each group is all carried out the 2nd immunity after head exempts from after 3 weeks, 15,30,45d thalline makes envelope antigen, ELISA method detects antibody.
Invention effect
(1) the invention provides a strain to be separated from Yili of Xinjiang strangles suis advantage epidemic strain, have important value for the strangles that effectively the local epidemic link of prevention causes.The inactivated vaccine production cost prepared with it is low, and added value of product is high, and the market requirement is high, has good market outlook.
(2) this bacterial strain can be utilized to produce various dissimilar strangles vaccine comprise inactivated vaccine and subunit vaccine.This vaccine immunization is effective, with strong points, and characteristic is strong.
(3) the invention provides the strangles suis that a strain is separated the sick Malaysia and China of strangles from Yili of Xinjiang, this vaccine is because of with the thorough deactivation of formaldehyde, and the inactivated vaccine security prepared with it is good, dangerous without loose poison.
Accompanying drawing explanation
Fig. 1 is that the streptococcic genomic dna of strangles extracted carries out electrophoresis result figure in 1% sepharose.
Fig. 2 is the electrophoresis result figure of strangles suis 16SrRNAPCR amplified production.
Specific embodiments
In order to understand the present invention better, be further described by following embodiment, but be not limitation of the invention.
Embodiment 1: the cultivation of strangles strains of streptococcus XZ1CGMCCNo.7830 and biological property.
Streptococcus equi horse subspecies XZ1CGMCCNo.7830 is separated the pus from the sick horse of strangles, and being inoculated in blood is in substrate cultivation base, in 37 DEG C of cultivations, and 12-15h.This bacterium has following characteristics: (1) colony characteristics: the colony diameter cultivated 1 day on blood agar plate is 0.2-1.0mm, and bacterium colony is rounded, smooth surface, neat in edge, protruding, canescence.(2) cell morphological characteristic: cell is circular or oval; Gram-positive; Cell dia 1.0-2.0 μm.(3) physiological and biochemical property: amphimicrobian grows; Growth temperature from 28 DEG C to 39 DEG C, optimum growth temperature 37 DEG C; PH growth scope 6-8, the most suitable growth pH7.4-7.6; Can glucose fermentation, sucrose, is not hydrolyzed Vitamin C2, does not grow in 6.5%NaC1, not liquefy gelatin, decomposes arginine, saligenin can be utilized as carbon source, can not utilize lactose, synanthrin, trehalose, N.F,USP MANNITOL, sorbyl alcohol.
With reference to " Bergey'sMannualofSystematicBacteriology " (second edition), according to its morphological specificity and physiological and biochemical property, and according to the Search Results of its 16SrRNA gene order in GenBank, strain X Z1CGMCCNo.7830 is accredited as strangles strains of streptococcus (streptococcus equi horse subspecies, Streptococcus.equisubspequi).
Embodiment 2: the pcr amplification of the 16SrRNA gene of strangles strains of streptococcus XZ1CGMCCNo.7830 and sequencing
Strangles strains of streptococcus XZ1CGMCCNo.7830 is inoculated in liquid nutrient medium, and centrifugal for the fermented liquid growing to late log phase (5000 revs/min, 5 minutes) are removed supernatant liquor, with TE (50mMTris, 50mMEDTA-Na
2) solution washes 2 times; Mixed by thalline with 0.5mLTES solution, add appropriate N,O-Diacetylmuramidase, 37 DEG C are incubated 2 hours; Add 0.2mL20%SDS, 60 DEG C are incubated 10 minutes; Add 0.3mL5MNaClO
4, mixing; Add equal-volume phenol-chloroform-primary isoamyl alcohol (25:24:1), shake up about 5 minutes gently, centrifugal (5000 revs/min, 5 minutes), Aspirate supernatant, then use phenol-chloroform-primary isoamyl alcohol (25:24:1) to process one time; Then chloroform-isoamyl alcohol (24:1, v/v) is used to process twice successively, until do not have protein film to occur; Supernatant liquor adds 20 μ l0.2%RNA enzyme 37 DEG C insulation 30 minutes, and chloroform-isoamyl alcohol (24:1, v/v) processes three times; Supernatant liquor 2 times of volume ice alcohol settling, 70% ice alcohol solution dipping 5 minutes.Be dissolved in after drying in TE solution as template.
The forward primer reacted for the PCR of 16SrRNA gene amplification is:
5 '-TGGAACGCACAGATGATAC-3 ', reverse primer is:
5’-GACTTCGGGTGTTACAAAC-3’。
PCR reaction system (50 μ l) is: 10 × buffer5 μ l, 25mmol/LMgCl
2each 1 μ l, the ddH of 4 μ l, 10mmol/LdNTPs1 μ l, 20pmol/L primer
2o36 μ l, TaqDNA enzyme 1 μ l, template 1 μ l.PCR reaction conditions is: 95 DEG C of 10min, 95 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1min, 30 circulations; 72 DEG C of 10min, 4 DEG C of preservations.16SrRNA gene order length is 1312bp.The nucleotide sequence of its 16SrRNA gene is as shown in sequence table 1.
Embodiment 3: the preparation of inactivation antigen
Isolate is inoculated in respectively and is added with in the TM substratum of 2% horse serum, 37 DEG C, 180r/min cultivates 18 ~ 24h, adds 0.4% formalin-inactivated 36 ~ 48h.After deactivation thoroughly, 7000rpm/min is centrifugal, and 15min removes supernatant.Bacterial precipitation is diluted to 1 × 10 with sterilizing PBS (pH7.2,10mmol)
7cFU/mL.With immunizing rabbit after the emulsification of equivalent Fu Shi Freund's complete adjuvant, with the bacterium of same concentration and the emulsification of equivalent Fu Shi Freund's incomplete adjuvant after 3 weeks, vaccine carries out steriling test and safety examination after fully vibrating and mixing.
Embodiment 4: steriling test
The inactivation antigen of preparation is inoculated in respectively sheep blood agar plate, cultivates 24 ~ 72h, observe with or without bacterial growth for 37 DEG C.After cultivating, result all loses bacteria growing, and show that inactivating efficacy is good, working specification is pollution-free.
Embodiment 5: safety examination
By the rabbit 5 of inactivation antigen subcutaneous injection 1.5 ~ 2.0kg body weight of preparation, 4mL/ only, separately gets 2 subcutaneous injection physiological saline and compares, and 4mL/ only, observes 10d.Inoculation rabbit is dead without 1 example, illustrates that vaccine safety is better; Check the reaction of inoculation local and whole body, rabbit of result all the other inoculations except 1 inoculation position has light inflammation reaction are without significantly local and systemic reaction, and spirit, body temperature, appetite, breathing and pulse are normal.
Embodiment 6: mouse immuning test
Isolate is inoculated in and is added with in the TM substratum of 5% horse serum, 37 DEG C, 180rpm/min cultivates 18 ~ 24h, adds 0.4% formalin-inactivated 36 ~ 48h.Centrifugally remove supernatant.Bacterial precipitation is diluted to 1 × 10 with sterilizing PBS (pH7.2,10mmol)
6cFU/mL.Immunogen is prepared in Fu Shi Freund's complete adjuvant and the emulsification of Fu Shi Freund's incomplete adjuvant of inactivated bacterial liquid qualified after steriling test and safety verification and equivalent.By the mouse 20 of the inactivation antigen subcutaneous injection 20g body weight of preparation, 0.5mL/ only, separately get same mouse 20, subcutaneous injection physiological saline compares, 0.5mL/ only, each group is all carried out the 2nd immunity after head exempts from after 3 weeks, the 2 viable bacteria cultures of exempting from rear 15d lethal dose are attacked, and the dosage often organizing intraperitoneal inoculation attack bacterium is 5 × 10
5cFU, observes one week.Result control group is all dead, dead 4 of immune group, and the immune protective efficiency of immune group can reach 80%.
Embodiment 7: vaccine valence measures
Under identical rearing conditions, the rabbit of Stochastic choice 1.5 ~ 2.0kg body weight 5, dorsal sc multi-point injection vaccine 4mL/ only, separately gets 5 subcutaneous injection physiological saline and compares, and only, each group all carries out the 2nd immunity to 4mL/ after head exempts from after 3 weeks, 45d is with 10
8thalline makes envelope antigen, and ELISA method detects antibody.Detected result shows that the antibody titer of rabbit 45d after twice immunity is 7.3 × 10
5.
Test proves: the inactivated vaccine prepared by this bacterium is to laboratory animal safety, and toxicity is little, non-evident effect, can produce higher immunizing potency after immunizing rabbit 2 times, 2 exempt from after ELISA tire and can reach 7.3 × 10
5, can 80% be reached to the protection ratio of laboratory animal mouse.
Claims (3)
1. strangles suis (Streptococcus.equisubspequi) XZ1 of strangles vaccine is prepared in a strain, and its preserving number is CGMCCNo.7830.
2. strangles suis XZ1 according to claim 1, is characterised in that its 16SrDNA is the nucleotide sequence shown in sequence table 1.
3. the method for the inactivated vaccine of strangles is prepared with strangles suis XZ1 according to claim 1, comprise and strangles suis XZ1 according to claim 1 is inoculated in TM substratum, 37 DEG C, 180r/min cultivates 18 ~ 24h, add 0.4% formalin-inactivated, the precipitation after centrifugal is diluted to 1 × 10 with sterilizing PBS
8cFU/mL, the adjuvant emulsion through steriling test and the qualified inactivated bacterial liquid of safety verification and equivalent is prepared into inactivated vaccine, the substratum TM that strangles suis XZ1 uses,
After 121 DEG C of sterilizings in 20 minutes, add 5% horse serum, pH=7.4.
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| CN104789587B (en) * | 2015-04-10 | 2018-04-27 | 新疆农业大学 | Eukaryotic expression recombinant plasmid compound is preparing the application of strangles hammer bacteria vaccine |
| CN108220182B (en) * | 2016-12-22 | 2021-02-26 | 新疆农业大学 | Streptococcum zooepidemicus strain XJMSY16-1 and application thereof in streptococcus equi vaccine |
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| CN1833723A (en) * | 2006-04-25 | 2006-09-20 | 范红结 | Chain coccus recombination subunit vaccine and prepn. thereof |
| WO2010069335A2 (en) * | 2008-12-19 | 2010-06-24 | Københavns Universitet | Diagnosis of endometritis |
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| CN1833723A (en) * | 2006-04-25 | 2006-09-20 | 范红结 | Chain coccus recombination subunit vaccine and prepn. thereof |
| WO2010069335A2 (en) * | 2008-12-19 | 2010-06-24 | Københavns Universitet | Diagnosis of endometritis |
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| 马腺疫链球菌的分离鉴定与疫苗的研制;康立超，等;《黑龙江畜牧兽医》;20130531;第109-111页，具体参见摘要和讨论 * |
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