CN106520623B - A kind of serum 7-type haemophilus parasuis low virulent strain and its application - Google Patents
A kind of serum 7-type haemophilus parasuis low virulent strain and its application Download PDFInfo
- Publication number
- CN106520623B CN106520623B CN201611091197.XA CN201611091197A CN106520623B CN 106520623 B CN106520623 B CN 106520623B CN 201611091197 A CN201611091197 A CN 201611091197A CN 106520623 B CN106520623 B CN 106520623B
- Authority
- CN
- China
- Prior art keywords
- vaccine
- haemophilus parasuis
- serum
- virulent strain
- low virulent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000606807 Glaesserella parasuis Species 0.000 title claims abstract description 67
- 210000002966 serum Anatomy 0.000 title claims abstract description 32
- 229960005486 vaccine Drugs 0.000 claims abstract description 43
- 241000894006 Bacteria Species 0.000 claims abstract description 38
- 239000007788 liquid Substances 0.000 claims abstract description 20
- 239000003814 drug Substances 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims description 42
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 13
- 239000002671 adjuvant Substances 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000003556 assay Methods 0.000 claims description 7
- 239000004411 aluminium Substances 0.000 claims description 6
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical group [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 6
- 229910052782 aluminium Inorganic materials 0.000 claims description 6
- 239000003292 glue Substances 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 235000015097 nutrients Nutrition 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 238000004945 emulsification Methods 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 230000007918 pathogenicity Effects 0.000 abstract description 7
- 238000012360 testing method Methods 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- 239000002574 poison Substances 0.000 description 6
- 231100000614 poison Toxicity 0.000 description 6
- 230000001681 protective effect Effects 0.000 description 5
- 229940031551 inactivated vaccine Drugs 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000606790 Haemophilus Species 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 229940031567 attenuated vaccine Drugs 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000002741 palatine tonsil Anatomy 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 229960001212 bacterial vaccine Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 201000011475 meningoencephalitis Diseases 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
Classifications
-
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/21—Haemophilus
-
- 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
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
-
- 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
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of serum 7-type haemophilus parasuis low virulent strain and its application, serum 7-type haemophilus parasuis low virulent strain is preserved in China typical culture collection center on October 13rd, 2016, and preserving number is CCTCC No:M2016560；The pathogenicity of the low virulent strain weakens significantly, is suitable for preparing the medicine for the treatment of Haemophilus parasuis；The application of the serum 7-type haemophilus parasuis low virulent strain, to prepare the medicine for the treatment of Haemophilus parasuis using serum 7-type haemophilus parasuis low virulent strain；The medicine is vaccine, and the preparation process of vaccine is prepared including 1) primary seed solution；2) prepared by secondary seed solution；3) prepared by original bacteria liquid；4) vaccine is prepared.
Description
Technical field
The present invention relates to a kind of serum 7-type haemophilus parasuis low virulent strain and its application, belong to haemophilus parasuis application
Technical field.
Background technology
Haemophilus parasuis is the new hair bacterial infectious disease for seriously endangering pig breeding industry in recent years, causes the multiple of pig
Scrositis, arthritis and meningoencephalitis etc., incidence are generally 10-15%, and the death rate is brought up to more than 50% to pig breeding industry
Huge economic loss, causes the pay attention to day by day of people.The serotype of the sick pathogenic bacteria haemophilus parasuis (Hps) is answered
It is miscellaneous various, can be at least 15 by Hps points by Kieleetin.Rapp-Gbarideosn (KRG) AGP test serotype method
A serotype, the separation strains serotype for separately having more than 20% can not determine.Existing data shows that Hps has obvious region
Feature.
Because haemophilus parasuis easily produces drug resistance, so the effective means for controlling this sick is vaccine inoculation.Document report
The use of road commercial vaccine or oneself seedling can control the infection of haemophilus parasuis.But also have and prevent to lose using inactivated vaccine
The example lost, this is probably since pathogenic strain is different from vaccine strains serotype thus lacks cross protection.Protected although intersecting
Shield is the basic demand to commodity inactivated vaccine, but strain specificity inactivated vaccine may be because that there are more than one in swinery
Bacterial strain or serotype and lack effect, it is also possible to because later swinery in introduce new bacterial strain and lose effect.Due to secondary pig
The diversity of haemophilus serotype and account for significant proportion be unable to parting bacterial strain, the difference of Strain Virulence, and it is current right
Protective antigens and virulence factor lack heightened awareness, it has not been possible to have a kind of inactivated vaccine to be produced at the same time to all pathogenic strains
Raw cross-protection.It is prevention and the inevitable requirement and the general trend of events that control Haemophilus parasuis to develop the vaccine with cross protection
Become.
The exploitation of vaccine product is in addition to efficient technical requirements, and cost is immunized must be cheap, it is impossible to beyond holding for aquaculture
By ability.Subtract (weak) virus live vaccine because with immunizing potency high (antigen dose can be reduced), it is of low cost, broad-spectrum vaccine can be developed
The new technology advantage of (live bacterial vaccines have intersecting protective), is the research hotspot of Most current infectious disease.
The content of the invention
For overcome the deficiencies in the prior art, of the invention first purpose is to provide a kind of serum 7-type pair pig bloodthirsty
Bacillus HPKP-7 (Latin names：Haemophilus parasuis HPKP-7) natural low virulent strain, the pathogenicity of the low virulent strain
Weaken significantly, be suitable for preparing the medicine for the treatment of Haemophilus parasuis.
Achieving the object of the present invention can be reached by adopting the following technical scheme that：A kind of serum 7-type haemophilus parasuis
Low virulent strain, it is characterised in that：The serum 7-type haemophilus parasuis low virulent strain was preserved in Chinese Typical Representative on October 13rd, 2016
Culture collection, depositary institution address：In Wuhan, China Wuhan University；Preserving number is CCTCC No:M2016560；
The Classification And Nomenclature is haemophilus parasuis HPKP-7, Latin name：Haemophilus parasuis HPKP-7.
Second object of the present invention is to provide a kind of application of above-mentioned serum 7-type haemophilus parasuis low virulent strain, sharp
The medicine for the treatment of Haemophilus parasuis is prepared with serum 7-type haemophilus parasuis low virulent strain.
Third object of the present invention is to provide a kind of medicine for treating Haemophilus parasuis, to utilize serum 7-type
Haemophilus parasuis low virulent strain prepares the vaccine for the treatment of Haemophilus parasuis.
Achieving the object of the present invention can be reached by adopting the following technical scheme that：A kind of secondary pig for the treatment of as described above is thermophilic
The medicine of blood bacillosis, the medicine are vaccine.
Preferably, the vaccine is obtained by following preparation process：
1) prepared by primary seed solution：Take the serum 7-type haemophilus parasuis low virulent strain of lyophilized preservation to be inoculated in TSA solids to put down
Plate, 18~24h is cultivated under conditions of being 37 DEG C in temperature；Then picking single bacterium is fallen within TSB culture mediums, is 37 DEG C in temperature
Under the conditions of 16~18h of shaken cultivation, carry out purely after the assay was approved, as primary seed solution, put 2~8 DEG C and save backup；
2) prepared by secondary seed solution：Take the primary seed solution that step 1) obtains to be inoculated in TSB culture mediums, be 37 in temperature
16~18h of shaken cultivation under conditions of DEG C, purely after the assay was approved, as secondary seed solution, put 2~8 DEG C and save backup；
3) prepared by original bacteria liquid：The secondary seed nutrient solution that step 2) obtains is taken, is inoculated in TSB culture mediums, is in temperature
Under conditions of 37 DEG C after 24~28h of shaken cultivation, original bacteria liquid is obtained；
4) vaccine is prepared：Sampled in the original bacteria liquid that step 3) obtains, according to《Chinese veterinary pharmacopoeia》Annex is purely examined
Test and count plate；Then thalline original bacteria liquid centrifuging and taking precipitated, is washed after thalline is resuspended with sterile saline, washed
Thalline normal saline dilution into concentration be 3 × 107~3 × 1010The seedling bacterium solution of CFU/mL, seedling bacterium solution is mixed with adjuvant
Close, emulsification, the vaccine for the Haemophilus parasuis that obtains medical treatment, 2~8 DEG C save backup.
Wherein, more preferably, step 1)~3) in, the rotating speed of the shaken cultivation is 120~180rpm.
More preferably, the holding time of the primary seed solution and secondary seed solution is no more than 3 days.
More preferably, in step 2), by seed liquor and the volume ratio of culture medium 1:20, primary seed solution is inoculated in TSB
In culture medium.
More preferably, in step 3), by seed liquor and the volume ratio of culture medium 1:100, secondary seed solution is inoculated in TSB
In culture medium.
More preferably, in step 4), original bacteria liquid is centrifuged into 10min under conditions of rotating speed is 8000~10000rpm, is taken
The thalline of precipitation.
The vaccine of the present invention includes but not limited to oil in water vaccine, water-in-oil type vaccine, water-in-oil-in water vaccine, can
Selection adjuvant as needed；As the preferred embodiment of the present invention, in step 4), the adjuvant is aluminium glue adjuvant, and by seedling bacterium
Liquid and aluminium glue adjuvant are with volume ratio 4：1 mixes, and 10~20min is emulsified under conditions of 5000~8000rpm of rotating speed.
Compared with prior art, the beneficial effects of the present invention are：
1st, serum 7-type haemophilus parasuis low virulent strain in the present invention, the pathogenicity of the low virulent strain weaken, are suitable for significantly
Prepare the medicine for the treatment of Haemophilus parasuis；
2nd, need not when preparing medicine in terms of serum 7-type haemophilus parasuis low virulent strain is applied to medicine preparation by the present invention
Inactivation, without carrying out inactivation inspection, condition is simple, the production efficiency higher of medicine.
Brief description of the drawings
Fig. 1 is the growthform figure on tablet of 1 bacterium colony of embodiment；
Fig. 2 is the colonial morphology figure under 1 microscope of embodiment；
Fig. 3 is the gel electrophoresis figure of embodiment 1PCR amplifications；
It is described that a kind of serum 7-type haemophilus parasuis natural low virulent strain is provided, it is preserved in China on October 13rd, 2016
Type Tissue Collection, depositary institution address：In Wuhan, China Wuhan University；Preserving number is CCTCC No:
M2016560；The Classification And Nomenclature is haemophilus parasuis HPKP-7, Latin name：Haemophilus parasuis HPKP-
7。
Embodiment
In the following, with reference to attached drawing and embodiment, the present invention is described further：
A kind of serum 7-type haemophilus parasuis low virulent strain, the serum 7-type haemophilus parasuis low virulent strain was in 2016 10
The moon is preserved in China typical culture collection center on the 13rd, and preserving number is CCTCC No:M2016560.
Above serum 7-type haemophilus parasuis low virulent strain is prepared to the vaccine for the treatment of Haemophilus parasuis.
The preparation method of vaccine comprises the following steps：
1) prepared by primary seed solution：Take the serum 7-type haemophilus parasuis low virulent strain of lyophilized preservation to be inoculated in TSA solids to put down
Plate, 18~24h is cultivated under conditions of being 37 DEG C in temperature；Then picking single bacterium is fallen within TSB culture mediums, is 37 DEG C in temperature
Under the conditions of, with rotating speed 120~180rpm, 16~18h of shaken cultivation, carry out purely after the assay was approved, as primary seed solution, putting 2
~8 DEG C save backup, and the holding time is no more than 3 days；
2) prepared by secondary seed solution：The primary seed solution that step 1) obtains is taken, by seed liquor and the volume ratio of culture medium 1:
20 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, with rotating speed 120~180rpm, 16~18h of shaken cultivation, into
Row purely after the assay was approved, as secondary seed solution, is put 2~8 DEG C and is saved backup, and the holding time is no more than 3 days；
3) prepared by original bacteria liquid：The secondary seed nutrient solution that step 2) obtains is taken, by seed liquor and the volume ratio of culture medium 1:
100 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, after 24~28h of rotating speed 120~180rpm shaken cultivations,
Obtain original bacteria liquid；
4) vaccine is prepared：Sampled in the original bacteria liquid that step 3) obtains, according to《Chinese veterinary pharmacopoeia》Annex is purely examined
Test and count plate；By original bacteria liquid, centrifugation 10min takes the thalline of precipitation under conditions of rotating speed is 8000~10000rpm, with nothing
Bacterium physiological saline be resuspended thalline after wash, washed thalline normal saline dilution into concentration be 3 × 107~3 × 1010CFU/
The seedling bacterium solution of mL, seedling bacterium solution is mixed with aluminium glue adjuvant, and 10~20min is emulsified under conditions of 5000~8000rpm of rotating speed,
Obtain medical treatment the vaccine of Haemophilus parasuis, and 2~8 DEG C save backup.
Embodiment 1
The separation identification of haemophilus parasuis low virulent strain：
Level land area slaughterhouse collection pig tonsil is opened from Guangdong within 2016, under sterile working, wiped with cotton ball soaked in alcohol whole
Tonsil, cuts a fritter, and the TSA containing 10% calf serum of percent by volume and 0.1%NAD is first inoculated in section and is put down
Plate, 37 DEG C of overnight incubations.
The growthform on tablet of bacterium colony as shown in Figure 1, picking well-grown on tablet, colonial morphology is smaller,
Canescence, translucent, neat in edge, slightly slime mould fall, through Gram's staining, micro- Microscopic observation, and the colonial morphology under microscope
As shown in Fig. 2, in pale red, in long-chain or the bacterium colony of short chain, to expand the mirror that 16S rRNA are carried out after cultivating to suspicious bacterium colony
It is fixed.The primer sequence used is：
Above-mentioned bacterial strains 16S rRNA PCR products serve Hai Shenggong bioengineering Co., Ltd sequencing, sequencing result with
Known array carries out Blast comparisons in GenBank, and comparison result shows that the genetic fragment that PCR is obtained is 822bp, genetic fragment
Sequence as shown in sequence table SEQ ID No.1, with the homology of known haemophilus parasuis 98.4~100%, determine point
From to bacterial strain be haemophilus parasuis.
The haemophilus parasuis of above-mentioned identification is subjected to serotype PCR identifications, primer sequence：
As shown in figure 3, obtaining the purpose fragment that size is about 600bp through PCR amplification, amplified production is through sequencing, sequencing knot
For fruit as shown in sequence table SEQ ID No.2, it is 100% to compare with the homology of the funQ genes of haemophilus parasuis serum 7-type,
It is thus determined that separated bacterial strain is serum 7-type, most at last strain was named HPKP-7.
Embodiment 2
The serum 7-type haemophilus parasuis low virulent strain HPKP-7 that embodiment 1 is obtained carries out pathogenicity detection：
1) pathogenicity of cavy is detected
Virulence determination is carried out to the HPKP-7 that separation obtains using cavy as model animal.
Cavy is observed three days after buying, and after situation without exception, is randomly divided into 12 groups, every group 5,12 groups of isolation are raised
Support, keep same experimental conditions.
5 groups of cavys of artificial challenge are carried out by being injected intraperitoneally with the HPKP-7 that embodiment 1 obtains during experiment, in addition 6
Group cavy is infected using haemophilus parasuis with reference to velogen strain Serotype 5 Nagasaki as control strain, and 1 group of cavy is as cloudy
Property control, injecting normal saline, continuous observation 10 days observes and records cavy morbidity and death condition, as shown in table 1.
1 cavy of table attacks malicious result
Result of the test shows that Nagasaki plants of inoculum concentrations of strong poison control group are 1.5 × 108、1.5×109、1.5×1010、
1.5×1011、1.5×1012During CFU, the death of test group whole cavy is responsible for, and separated strain HPKP-7 is being inoculated with
Dosage is 1.5 × 108、1.5×109、1.5×1010、1.5×1011During CFU, cavy does not show any clinical symptoms, growth
In good condition, dosage of inoculation is 1.5 × 1012During CFU, when cavy to the off-test for having 2/5, which dissects, visible intraperitoneal has fiber
Disposition is oozed out, but does not cause death.
2) pathogenicity of piglet is detected
25 age in days weanling pigs 30, are randomly divided into 6 groups, every group 5, wherein 5 are used as negative control, inject physiology salt
Water, 1 group of Nagasaki plants of intraperitoneal injection haemophilus parasuis, 4 groups of HPKP-7 plants of injections, continuous observation 10 days, observes and records examination
Piglet morbidity and death condition are tested, the results are shown in Table 2.
2 piglet of table attacks malicious result
Result of the test is shown, is injected intraperitoneally 3 × 109After Nagasaki plants of CFU, 5 experiment piglets fall ill, experiment knot
Dead 3 during beam, and inject 3 × 109、3×1010、3×1011During HPKP-7 plants of CFU, piglet does not cause morbidity, and injection 3 ×
1012There are slight clinical symptoms in 1 piglet during CFU, does not occur the phenomenon of piglet death.Cavy and piglet challenge test result
Confirm, haemophilus parasuis HPKP-7 pathogenicities weaken significantly.
Embodiment 3
The immune efficacy detection of haemophilus parasuis low virulent strain HPKP-7：
Vaccine is prepared by following steps：
1) prepared by primary seed solution：Take the serum 7-type haemophilus parasuis low virulent strain of lyophilized preservation to be inoculated in TSA solids to put down
Plate, 18h is cultivated under conditions of being 37 DEG C in temperature；Then picking single bacterium is fallen within TSB culture mediums, in the condition that temperature is 37 DEG C
Under, with rotating speed 120rpm shaken cultivation 18h, carry out purely after the assay was approved, as primary seed solution, it is standby putting 2~8 DEG C of preservations
With the holding time is no more than 3 days；
2) prepared by secondary seed solution：The primary seed solution that step 1) obtains is taken, by seed liquor and the volume ratio of culture medium 1:
20 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, with rotating speed 120rpm shaken cultivation 16h, are purely examined
After qualification, as secondary seed solution, put 2~8 DEG C and save backup, the holding time is no more than 3 days；
3) prepared by original bacteria liquid：The secondary seed nutrient solution that step 2) obtains is taken, by seed liquor and the volume ratio of culture medium 1:
100 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, after rotating speed 120rpm shaken cultivations 24h, obtain opportunistic pathogen
Liquid；
4) vaccine is prepared：Sampled in the original bacteria liquid that step 3) obtains, according to《Chinese veterinary pharmacopoeia》Annex is purely examined
Test and count plate；By original bacteria liquid, centrifugation 10min takes the thalline of precipitation under conditions of rotating speed is 8000rpm, uses sterile physiological
Brine be resuspended thalline after wash, washed thalline normal saline dilution into concentration be 3 × 1010The seedling bacterium solution of CFU/mL,
By seedling bacterium solution and aluminium glue adjuvant with volume ratio 4：1 mixes, and emulsifies 20min under conditions of rotating speed 8000rpm, obtain medical treatment secondary pig
The vaccine of haemophilus disease, 2~8 DEG C save backup.
The vaccine of obtained treatment Haemophilus parasuis is subjected to Immunoprotection test：
A. to the Immunoprotection test of cavy
Cavy 15 is taken, is randomly divided into 3 groups, every group 5, the haemophilus parasuis vaccine muscle of the above-mentioned preparation of test group
Cavy is injected, only, two exempt from 2mL/ after 14 days, and 2mL/ only, after two exempt from 14 days, attacks poison control and physiological saline is immunized in blank control,
To immune group and attack malicious control group cavy and carry out attacking poison for Nagasaki plant with Serotype 5 velogen strain, intraperitoneal injection, 1.5 ×
109Only, continuous observation 10 days, observes and records cavy morbidity and death condition, calculates every group of protective rate, as shown in table 3 CFU/.
3 vaccine immunity protection test result of table
Result of the test shows, vaccine immunity group, which is attacked after poison, not to be occurred falling ill and dead, and 5 cavys of control group fall ill, 4
It is only dead, it was demonstrated that vaccine has certain protective efficacy to velogen strain.
B. to the cross-protection test of different serotypes
Because of 4,5,12,13 type haemophilus parasuis of Major Epidemic serum in China's clinic, thus we to vaccine to this 4
The cross-protection test of serotype is verified.40 cavys are taken, are randomly divided into 8 groups, wherein 4 groups of intramuscular injection pair pigs are bloodthirsty
Bacillus attenuated vaccine, only, two exempt from 2mL/ after 14 days, and 2mL/ only, attacks malicious control group injecting normal saline, two exempt from 14 days after use respectively
4,5,12,13 type haemophilus parasuis of serum attack poison, and 2 × 109Only, continuous observation 10 days, observes and records cavy hair to CFU/
Disease and death condition, calculate every group of protective rate, as shown in table 4.
Table 4 attacks different serotypes bacterial strain the immunoprotection result of poison
Result of the test shows that the haemophilus parasuis attenuated vaccine of preparation is capable of providing different serotypes preferable friendship
Fork protection.
For those skilled in the art, technical solution that can be as described above and design, make other each
Kind is corresponding to be changed and deforms, and all these change and deform the protection model that should all belong to the claims in the present invention
Within enclosing.
SEQUENCE LISTING
<110>Guangdong Haida Husbandry and Veterinary Institute Co., Ltd. of Agricultural University Of South China
<120>A kind of serum 7-type haemophilus parasuis low virulent strain and its application
<130> 2016
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 822
<212> DNA
<213>Haemophilus parasuis HPKP-7
<400> 1
tgaccagcca cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga 60
atattgcrca atggggggaa ccctgatgca gccatgccgc gtgaatgaag aaggccttcg 120
ggttgtaaag ttctttcggt gangaggaag ggtggtgttt taatagaaca ctacattgac 180
gttagtcaca gaagaagcac cggctaactc cgtgccagca gccgcggtaa tacggagggt 240
gcgagcgtta atcggaatga ctgggcgtaa agggcacgca ggcggtgact taagtgagat 300
gtgaaagccc cgagcttaac ttgggaattg catttcatac tgggttgcta gagtatttna 360
gggagggnta gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaataccgaa 420
ggcgaaggca gccccttggg aaaatactga cgctcatgtg cgaaagcgtg gggagcaaac 480
aggattagat accctggtag tccacgctgt aaacgctgtc gattnnggga ttgggctnag 540
agcttggtgc ccgtagctaa cgtgataaat cgaccgcctg gggagtacgg ccgcaaggtt 600
aaaactcaaa tnaattgacg ggggccgcac aagcggtgga gcatgtggtt taattcgatg 660
naacgcgaag aaccttacct actcttgaca tcctaagaag aactcagaga tgagtttgtg 720
ccttcgggaa cttagagaca ggtgctgcat ggctgtcgtc agctcgtgtt gtgaaatgtt 780
gggttnagtc ccgcaacgag cgcaaccctt atcctttgtt gc 822
<210> 2
<211> 600
<212> DNA
<213>Haemophilus parasuis HPKP-7
<400> 2
tagttggtat attattttct aaacgttatc ctataaaatg gaataatgtt aatttagaat 60
ttaaaattcc gtactgtata agtataataa taattttatt ttcaatattt attagttttc 120
ttgcatttga ttttaatgag ttactttatc gtcagaagta tgttactaat gaaactcata 180
gggttttagt tattttattt aaagtgttat ctccgatttc atcttttcta tgtggcttta 240
ttcgaaataa atatttgaag tataactttc tatttcttat tctaagtgtt ccttttagcc 300
tcaattctag aataatatcc ttagatttaa tgatgtttta tattgggagt gttttgtgtt 360
ttaaaagagg ttttttgtat aaatccataa ttatttttag tgtgattttt tctatatcct 420
ctcttatttt aagggggatg tcagagcaag gttttatata taacatatta aatttatctt 480
acatttttga tattagctta gatgtttttt ttagaggggt gaattatgtg accagttttt 540
cagtatatga aatggaatat gctttagaac agaaacttag tggtacagat gcattctaat 600
Claims (9)
- A kind of 1. serum 7-type haemophilus parasuis low virulent strain, it is characterised in that：The serum 7-type haemophilus parasuis （Haemophilus parasuis）Low virulent strain was preserved in China typical culture collection center, preservation on October 13rd, 2016 Number it is CCTCC No: M2016560.
- A kind of 2. medicine for treating Haemophilus parasuis, it is characterised in that：The medicine is to utilize blood as claimed in claim 1 Vaccine prepared by clear 7 type haemophilus parasuis low virulent strain.
- 3. a kind of preparation method of vaccine as claimed in claim 2, it is characterised in that the preparation process of the vaccine is：1）It is prepared by primary seed solution：The serum 7-type haemophilus parasuis low virulent strain of lyophilized preservation is taken to be inoculated in TSA solid plates, 18~24h is cultivated under conditions of being 37 DEG C in temperature；Then picking single bacterium is fallen within TSB culture mediums, in the bar that temperature is 37 DEG C 16~18h of shaken cultivation under part, purely after the assay was approved, as primary seed solution, put 2~8 DEG C and save backup；2）It is prepared by secondary seed solution：Take step 1）Obtained primary seed solution is inoculated in TSB culture mediums, is 37 DEG C in temperature Under the conditions of 16~18h of shaken cultivation, carry out purely after the assay was approved, as secondary seed solution, put 2~8 DEG C and save backup；3）It is prepared by original bacteria liquid：Take step 2）Obtained secondary seed nutrient solution, is inoculated in TSB culture mediums, is 37 DEG C in temperature Under conditions of after 24~28h of shaken cultivation, obtain original bacteria liquid；4）Prepare vaccine：In step 3）Sampled in obtained original bacteria liquid, according to《Chinese veterinary pharmacopoeia》Annex carry out purely examine and Count plate；Then thalline original bacteria liquid centrifuging and taking precipitated, is washed, washed bacterium after thalline is resuspended with sterile saline Body normal saline dilution into concentration be 3 × 107~3 × 1010The seedling bacterium solution of CFU/mL, seedling bacterium solution is mixed with adjuvant, Emulsification, the vaccine for the Haemophilus parasuis that obtains medical treatment, 2~8 DEG C save backup.
- 4. the preparation method of vaccine as claimed in claim 3, it is characterised in that：Step 1）~3）In, the shaken cultivation turns Speed is 120~180rpm.
- 5. the preparation method of vaccine as claimed in claim 3, it is characterised in that：The primary seed solution and secondary seed solution Holding time is no more than 3 days.
- 6. the preparation method of vaccine as claimed in claim 3, it is characterised in that：Step 2）In, by seed liquor and culture medium Volume ratio 1:20, primary seed solution is inoculated in TSB culture mediums.
- 7. the preparation method of vaccine as claimed in claim 3, it is characterised in that：Step 3）In, by seed liquor and culture medium Volume ratio 1:100, secondary seed solution is inoculated in TSB culture mediums.
- 8. the preparation method of vaccine as claimed in claim 3, it is characterised in that：Step 4）In, it is in rotating speed by original bacteria liquid 10min is centrifuged under conditions of 8000~10000rpm, takes the thalline of precipitation.
- 9. the preparation method of vaccine as claimed in claim 3, it is characterised in that：Step 4）In, the adjuvant is aluminium glue adjuvant, And by seedling bacterium solution and aluminium glue adjuvant with volume ratio 4：1 mixes, and 10~20min is emulsified under conditions of 5000~8000rpm of rotating speed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611091197.XA CN106520623B (en) | 2016-12-01 | 2016-12-01 | A kind of serum 7-type haemophilus parasuis low virulent strain and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611091197.XA CN106520623B (en) | 2016-12-01 | 2016-12-01 | A kind of serum 7-type haemophilus parasuis low virulent strain and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106520623A CN106520623A (en) | 2017-03-22 |
| CN106520623B true CN106520623B (en) | 2018-04-24 |
Family
ID=58354923
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611091197.XA Expired - Fee Related CN106520623B (en) | 2016-12-01 | 2016-12-01 | A kind of serum 7-type haemophilus parasuis low virulent strain and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106520623B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018230B (en) * | 2017-11-30 | 2020-09-11 | 华中农业大学 | Serum 7 type haemophilus parasuis natural attenuated strain and application thereof |
| CN109806389B (en) * | 2019-02-22 | 2022-03-22 | 河南省农业科学院畜牧兽医研究所 | Haemophilus parasuis trivalent inactivated vaccine and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399724B (en) * | 2011-11-08 | 2013-01-02 | 山东省农业科学院畜牧兽医研究所 | Haemophilus parasuis LC strain and application thereof |
| CN105112342B (en) * | 2015-09-17 | 2019-03-15 | 龙岩学院 | A kind of preparation method of haemophilus parasuis bacterial strain pentavalent vaccine |
-
2016
- 2016-12-01 CN CN201611091197.XA patent/CN106520623B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN106520623A (en) | 2017-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103263666B (en) | Porcine circovirus 2 type, porcine mycoplasmal pneumonia bivalent inactivated vaccine and preparation method thereof | |
| CN105327344B (en) | Vaccine composition and the preparation method and application thereof containing porcine circovirus 2 type antigen Yu haemophilus parasuis antigen | |
| CN104498441B (en) | Duck hepatitis A virus (HAV) type III low virulent strain and live vaccine prepared therefrom and application | |
| CN102847146B (en) | Vaccine used for preventing tilapia streptococcal disease | |
| CN108441446A (en) | A kind of trivalent inactivated vaccine against Haemophilus parasuis infection and its production method and application | |
| CN108486067A (en) | The inactivated vaccine and application of Porcine epidemic diarrhea virus variant and its preparation | |
| CN107513510A (en) | Riemerella anatipestifer disease attenuated live vaccines and preparation method thereof | |
| CN104232535B (en) | Nocardia seriolae induced low virulent strain and application thereof | |
| CN107513506A (en) | Mycoplasma hyopneumoniae, vaccine combination and its application | |
| CN108018230A (en) | A kind of natural less-virulent strain of serum 7-type haemophilus parasuis and its application | |
| CN103908665B (en) | A kind of vaccine combination and its preparation method and application | |
| CN106520623B (en) | A kind of serum 7-type haemophilus parasuis low virulent strain and its application | |
| CN105154410B (en) | A kind of Duck parvovirus strain and its live vaccine | |
| CN104087559B (en) | A kind of infectious bursa of Fabricius virus, inactivated vaccine and preparation method thereof | |
| CN107164273B (en) | A kind of strong 2 type Streptococcus suis of serum of immunogenicity and its application | |
| CN104096222B (en) | A kind of vaccine combination and its preparation method and application | |
| CN103705918B (en) | Porcine epidemic diarrhea resisting virus hyper-immune serum and preparation method thereof | |
| CN103784951B (en) | Prevent and treat antigen composition of respiratory disease of scabies secondary infection of pig and its preparation method and application | |
| CN107365720B (en) | It is a kind of with the Serotype 5 haemophilus parasuis of cross-protection and its application | |
| CN101829321B (en) | Vaccine for preventing red head disease of Pseudobagrus fulvidraco | |
| CN104388396B (en) | Porcine pseudorabies poison strain and inactivated vaccine prepared therefrom and application | |
| CN104288762B (en) | A kind of vaccine combination and its preparation method and application | |
| CN104069489B (en) | Newcastle disease and infectious bursa of Fabricius bivalent inactivated vaccine and preparation method thereof | |
| CN103656634B (en) | Resisting porcine circovirus and porcine contagious pleuropneumonia infect vaccine combination and preparation | |
| CN103157101B (en) | Combined inactivate vaccine for haemophilus parasuis disease and streptococcus suis disease and preparation method for same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180424 |