CN106520623A - Serum 7 type haemophilus parasuis low virulent strain and application thereof - Google Patents

Serum 7 type haemophilus parasuis low virulent strain and application thereof Download PDF

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Publication number
CN106520623A
CN106520623A CN201611091197.XA CN201611091197A CN106520623A CN 106520623 A CN106520623 A CN 106520623A CN 201611091197 A CN201611091197 A CN 201611091197A CN 106520623 A CN106520623 A CN 106520623A
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haemophilus parasuis
medicine
serum
virulent strain
low virulent
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CN106520623B (en
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贾爱卿
廖明
王贵平
樊惠英
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GUANGDONG HAID ANIMAL HUSBANDRY VETERINARY RESEARCH INSTITUTE Co Ltd
South China Agricultural University
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GUANGDONG HAID ANIMAL HUSBANDRY VETERINARY RESEARCH INSTITUTE Co Ltd
South China Agricultural University
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    • 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

Abstract

The invention discloses a serum 7 type haemophilus parasuis low virulent strain and application of the serum 7 type haemophilus parasuis low virulent strain. The serum 7 type haemophilus parasuis low virulent strain was preserved in China Center for Type Culture Collection on October 13th, 2016, and the preservation number is CCTCC No: M2016560. The virulence of the low virulent strain is greatly weakened, and the low virulent strain is suitable for being used for preparing the medicine for treating haemophilus parasuis. According to application of the serum 7 type haemophilus parasuis low virulent strain, the serum 7 type haemophilus parasuis low virulent strain is utilized for preparing the medicine for treating haemophilus parasuis. The medicine is a vaccine. A preparation method of the vaccine comprises the first step of preparation of a primary seed solution, the second step of preparation of a secondary seed solution, the third step of original bacteria liquid and the fourth step of preparation of the vaccine.

Description

A kind of serum 7-type haemophilus parasuis low virulent strain and its application
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 are newly bacterial infectious diseases of serious harm pig industry in recent years, cause the multiple of pig Scrositis, arthritis and meningoencephalitis etc., the incidence of disease are generally 10-15%, and the death rate is brought to pig industry up to more than 50% Huge economic loss, causes the pay attention to day by day of people.The serotype of the sick pathogenic bacteria haemophilus parasuis (Hps) is multiple It is miscellaneous various, by Kieleetin.Rapp-Gbarideosn (KRG) AGP test serotype method, can be at least 15 by Hps point Individual serotype, the separation strains serotype for separately having more than 20% can not be determined.Existing data shows that Hps has obvious region Feature.
Because haemophilus parasuis easily produces drug resistance, so it is vaccine inoculation to control the sick effective means.Document report The use of road commercial vaccine or oneself seedling can control the infection of haemophilus parasuis.But also have and lost using inactivated vaccine prevention The example for losing, this be likely due to pathogenic strain it is different from vaccine strains serotype thus lack cross protection.Although intersect protecting Shield is the basic demand to commodity inactivated vaccine, but strain specificity inactivated vaccine is probably due to have more than one in swinery Bacterial strain or serotype and lack effect, it is also possible to because introducing new bacterial strain and losing effect in swinery later.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 current right Protective antigens and virulence factor lack heightened awareness, it has not been possible to have a kind of inactivated vaccine while producing to all of pathogenic strain Raw cross-protection.It is the inevitable requirement and the general trend of events for preventing and controlling Haemophilus parasuis to develop the vaccine with cross protection Become.
In addition to efficient technical requirements, immune cost must be cheap, it is impossible to holding beyond aquaculture for the exploitation of vaccine product By ability.Subtract (weak) virus live vaccine because with immunizing potency height (antigen dose can be reduced), it is with low cost, broad-spectrum vaccine can be developed The new technology advantage of (live bacterial vaccines have intersecting protective), is the study hotspot of Most current infectious disease.
The content of the invention
In order to overcome the deficiencies in the prior art, first purpose of the present invention is that a kind of serum 7-type pair pig of offer is 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.
Realize that the purpose 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 is 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 be 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, profit 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, is to utilize serum 7-type Haemophilus parasuis low virulent strain prepares the vaccine for the treatment of Haemophilus parasuis.
Realize that the purpose of the present invention can be reached by adopting the following technical scheme that：A kind for the treatment of pair pig as 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：The serum 7-type haemophilus parasuis low virulent strain for taking lyophilized preservation is inoculated in TSA solids and puts down Plate, cultivates 18～24h under conditions of temperature is for 37 DEG C；Then picking single bacterium colony, 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 primary seed solution, putting 2～8 DEG C and saving backup；
2) prepared by secondary seed solution：Taking step 1) primary seed solution that obtains is inoculated in TSB culture mediums, is 37 in temperature 16～18h of shaken cultivation under conditions of DEG C, carries out purely after the assay was approved, as secondary seed solution, putting 2～8 DEG C and saving backup；
3) prepared by original bacteria liquid：Take step 2) the secondary seed nutrient solution that obtains, it is inoculated in TSB culture mediums, in temperature is Under conditions of 37 DEG C after 24～28h of shaken cultivation, original bacteria liquid is obtained；
4) prepare vaccine：In step 3) sample in the original bacteria liquid that obtains, according to《Chinese veterinary pharmacopoeia》Annex is purely examined Test and count plate；Then the thalline for original bacteria liquid centrifuging and taking being precipitated, is washed with after the resuspended thalline of SPSS, scrubbed Thalline normal saline dilution into concentration be 3 × 107～3 × 1010The seedling bacterium solution of CFU/mL, seedling bacterium solution and adjuvant are mixed Close, emulsification, the vaccine of the Haemophilus parasuis that obtain 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 less than 3 days.
More preferably, step 2) in, by seed liquor and the volume ratio 1 of culture medium:20, primary seed solution is inoculated in into TSB In culture medium.
More preferably, step 3) in, by seed liquor and the volume ratio 1 of culture medium:100, secondary seed solution is inoculated in into TSB In culture medium.
More preferably, step 4) in, 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 is not limited to oil in water vaccine, water-in-oil type vaccine, water-in-oil-in water vaccine, can Adjuvant is selected as needed；As the preferred embodiment of the present invention, step 4) in, the adjuvant is aluminium glue adjuvant, and by seedling bacterium Liquid and aluminium glue adjuvant are with volume ratio 4：1 mixing, emulsifies 10～20min under conditions of 5000～8000rpm of rotating speed.
Compared to existing technology, the beneficial effects of the present invention is：
1st, serum 7-type haemophilus parasuis low virulent strain in the present invention, the pathogenicity of the low virulent strain weaken significantly, are suitable for Prepare the medicine for the treatment of Haemophilus parasuis；
2nd, in terms of serum 7-type haemophilus parasuis low virulent strain is applied to medicine preparation by the present invention, need not when preparing medicine Inactivation, need not carry out inactivation inspection, and condition is simple, and the production efficiency of medicine is higher.
Description of the drawings
Growthform figures on flat board of the Fig. 1 for 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 amplification；
It is described that a kind of serum 7-type haemophilus parasuis natural low virulent strain is provided, China is preserved 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 be haemophilus parasuis HPKP-7, Latin name：Haemophilus parasuis HPKP- 7。
Specific embodiment
Below, with reference to accompanying drawing and specific 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 It is preserved within 13rd China typical culture collection center the moon, preserving number is CCTCC No:M2016560.
Above serum 7-type haemophilus parasuis low virulent strain is prepared into the vaccine for the treatment of Haemophilus parasuis.
The preparation method of vaccine is comprised the following steps：
1) prepared by primary seed solution：The serum 7-type haemophilus parasuis low virulent strain for taking lyophilized preservation is inoculated in TSA solids and puts down Plate, cultivates 18～24h under conditions of temperature is for 37 DEG C；Then picking single bacterium colony, in 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 less than 3 days；
2) prepared by secondary seed solution：Take step 1) primary seed solution that obtains, by the volume ratio 1 of seed liquor and culture medium: 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, enter 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 less than 3 days；
3) prepared by original bacteria liquid：Take step 2) the secondary seed nutrient solution that obtains, by the volume ratio 1 of seed liquor and culture medium: 100 are inoculated in TSB culture mediums, temperature be 37 DEG C under conditions of, with rotating speed 120～180rpm, 24～28h of shaken cultivation after, Obtain original bacteria liquid；
4) prepare vaccine：In step 3) sample in the original bacteria liquid that obtains, according to《Chinese veterinary pharmacopoeia》Annex is purely examined Test and count plate；Original bacteria liquid is centrifuged into the thalline that 10min takes precipitation under conditions of rotating speed is 8000～10000rpm, with nothing Wash after the resuspended thalline of bacterium physiological saline, scrubbed thalline normal saline dilution is 3 × 10 into concentration7～3 × 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, is first inoculated in the TSA containing 10% calf serum of percent by volume and 0.1%NAD with tangent plane and puts down Plate, 37 DEG C of overnight incubations.
As shown in figure 1, picking well-grown on flat board, colonial morphology is less for the growthform on flat board of bacterium colony, Canescence, translucent, neat in edge, slightly slime mould fall, Jing Gram's staining, basis of microscopic observation, the colonial morphology under microscope As shown in Fig. 2 be in pale red, in long-chain or short chain bacterium colony, the mirror to 16S rRNA are carried out after suspicious bacterium colony Amplification Culture It is fixed.The primer sequence for using is：
Above-mentioned bacterial strains 16S rRNA PCR primers serve Hai Shenggong bioengineering Co., Ltd sequencing, sequencing result with In GenBank, known array carries out Blast comparisons, 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%, it is determined that point It is haemophilus parasuis from the bacterial strain for arriving.
The haemophilus parasuis of above-mentioned identification is carried out into the identification of serotype PCR, primer sequence：
As shown in figure 3, Jing PCR amplifications obtain the purpose fragment that size is about 600bp, amplified production Jing sequencings, sequencing knot As shown in sequence table SEQ ID No.2, comparison is 100% with the homology of the funQ genes of haemophilus parasuis serum 7-type to fruit, It is thus determined that separated bacterial strain is serum 7-type, most the Strain Designation is HPKP-7 at last.
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 separating the HPKP-7 for obtaining as model animal with cavy.
Cavy is observed three days after buying, and after situation without exception, is randomly divided into 12 groups, 5 per group, and 12 groups of isolation are raised Support, keep same experimental conditions.
The HPKP-7 obtained with embodiment 1 in process of the test carries out 5 groups of cavys of artificial challenge by lumbar injection, and in addition 6 Cavy is infected as control strain with reference to velogen strain Serotype 5 Nagasaki group with haemophilus parasuis, and 1 group of cavy is used as the moon Property control, injecting normal saline, Continuous Observation 10 days, observed and recorded cavy morbidity and death condition, as shown in table 1.
Table 1 cavy attacks malicious result
Result of the test shows that strong poison control group Nagasaki strains inoculum concentration is 1.5 × 108、1.5×109、1.5×1010、 1.5×1011、1.5×1012During CFU, it is dead to be responsible for test group whole cavy, and detached strain HPKP-7, in inoculation 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, dissecting when having 2/5 cavy to off-test 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, 5 per group, wherein 5 used as negative control, inject physiology salt Water, 1 group of lumbar injection haemophilus parasuis Nagasaki strain, 4 groups of injection HPKP-7 strains, Continuous Observation 10 days, observed and recorded examination Piglet morbidity and death condition are tested, as a result as shown in table 2.
2 piglet of table attacks malicious result
Result of the test shows, lumbar injection 3 × 109After CFU Nagasaki strains, 5 test piglets fall ill, test knot Dead 3 during beam, and inject 3 × 109、3×1010、3×1011During CFU HPKP-7 strains, piglet does not cause morbidity, and injection 3 × 1012During CFU, there are slight clinical symptoms in 1 piglet, the dead phenomenon of piglet does not occur.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：The serum 7-type haemophilus parasuis low virulent strain for taking lyophilized preservation is inoculated in TSA solids and puts down Plate, cultivates 18h under conditions of temperature is for 37 DEG C；Then picking single bacterium colony is in TSB culture mediums, in the condition that temperature is 37 DEG C Under, with rotating speed 120rpm shaken cultivations 18h, carry out purely after the assay was approved, as primary seed solution, putting 2～8 DEG C of preservations standby With the holding time is less than 3 days；
2) prepared by secondary seed solution：Take step 1) primary seed solution that obtains, by the volume ratio 1 of seed liquor and culture medium: 20 are inoculated in TSB culture mediums, under conditions of temperature is 37 DEG C, with rotating speed 120rpm shaken cultivations 16h, are purely checked After qualified, as secondary seed solution, put 2～8 DEG C and save backup, the holding time is less than 3 days；
3) prepared by original bacteria liquid：Take step 2) the secondary seed nutrient solution that obtains, by the volume ratio 1 of seed liquor and culture medium: 100 are inoculated in TSB culture mediums, temperature be 37 DEG C under conditions of, with rotating speed 120rpm shaken cultivations 24h after, obtain opportunistic pathogen Liquid；
4) prepare vaccine：In step 3) sample in the original bacteria liquid that obtains, according to《Chinese veterinary pharmacopoeia》Annex is purely examined Test and count plate；Original bacteria liquid is centrifuged into the thalline that 10min takes precipitation under conditions of rotating speed is 8000rpm, sterile physiological is used Wash after the resuspended thalline of salt solution, scrubbed thalline normal saline dilution is 3 × 10 into concentration10The seedling bacterium solution of CFU/mL, By seedling bacterium solution with aluminium glue adjuvant with volume ratio 4：1 mixing, emulsifies 20min under conditions of rotating speed 8000rpm, and obtain medical treatment secondary pig The vaccine of haemophilus disease, 2～8 DEG C save backup.
The vaccine of the treatment Haemophilus parasuis for obtaining is carried out into Immunoprotection test：
A. the Immunoprotection test to cavy
Cavy 15 is taken, 3 groups are randomly divided into, 5 per group, haemophilus parasuis vaccine muscle of the test group with above-mentioned preparation Injection cavy, only, after 14 days, two exempt from 2mL/, and 2mL/ only, after two exempt from 14 days, attacks poison control and blank immunity physiological saline, To immune group and attack malicious control group cavy Serotype 5 velogen strain Nagasaki strains and carry out attacking poison, lumbar injection, 1.5 × 109CFU/ only, fall ill and death condition by Continuous Observation 10 days, observed and recorded cavy, calculates every group of protective rate, as shown in table 3.
3 vaccine immunity protection test result of table
Result of the test shows that morbidity and death does not occur in vaccine immunity group after attacking poison, 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. the cross-protection test to different serotypes
Because China clinic in 4,5,12,13 type haemophilus parasuis of Major Epidemic serum, so we to vaccine to this 4 The cross-protection test of serotype is verified.40 cavys are taken, 8 groups are randomly divided into, wherein 4 groups of intramuscular injection pair pigs are bloodthirsty Bacillus attenuated vaccine, only, after 14 days, two exempt from 2mL/, 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 carries out attacking poison, and 2 × 109CFU/ only, send out by Continuous Observation 10 days, observed and recorded cavy Disease and death condition, calculate every group of protective rate, as shown in table 4.
Table 4 attacks the immunoprotection result of poison to different serotypes bacterial strain
Result of the test shows that the haemophilus parasuis attenuated vaccine of preparation can provide preferably friendship to different serotypes Fork protection.
For a person skilled in the art, technical scheme that can be as described above and design, make other each Plant corresponding change and deform, and all these changes and deforms 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 (10)

1. 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 China typical culture collection center is preserved on October 13rd, 2016, preserving number is CCTCC No:M2016560.
2. a kind of application of serum 7-type haemophilus parasuis low virulent strain as claimed in claim 1, it is characterised in that：Using blood Clear 7 type haemophilus parasuis low virulent strain prepares the medicine for the treatment of Haemophilus parasuis.
3. one the medicine of Haemophilus parasuis is treated as claimed in claim 2, it is characterised in that：The medicine is vaccine.
4. the medicine of Haemophilus parasuis is treated as claimed in claim 3, it is characterised in that：The vaccine is by following system Standby step is obtained：
1) prepared by primary seed solution：The serum 7-type haemophilus parasuis low virulent strain for taking lyophilized preservation is inoculated in TSA solid plates, 18～24h is cultivated under conditions of temperature is for 37 DEG C；Then picking single bacterium colony is in TSB culture mediums, in the bar that temperature is 37 DEG C 16～18h of shaken cultivation under part, carries out purely after the assay was approved, as primary seed solution, putting 2～8 DEG C and saving backup；
2) prepared by secondary seed solution：Taking step 1) primary seed solution that obtains 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, putting 2～8 DEG C and saving backup；
3) prepared by original bacteria liquid：Take step 2) the secondary seed nutrient solution that obtains, it 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) sample in the original bacteria liquid that obtains, according to《Chinese veterinary pharmacopoeia》Annex carry out purely inspection and Count plate；Then the thalline for original bacteria liquid centrifuging and taking being precipitated, is washed with after the resuspended thalline of SPSS, scrubbed bacterium Body normal saline dilution is 3 × 10 into concentration7～3 × 1010The seedling bacterium solution of CFU/mL, seedling bacterium solution is mixed with adjuvant, Emulsification, the vaccine of the Haemophilus parasuis that obtain medical treatment, 2～8 DEG C save backup.
5. the medicine of Haemophilus parasuis is treated as claimed in claim 4, it is characterised in that：Step 1)～3) in, it is described The rotating speed of shaken cultivation is 120～180rpm.
6. the medicine of Haemophilus parasuis is treated as claimed in claim 4, it is characterised in that：The primary seed solution and two The holding time of level seed liquor is less than 3 days.
7. the medicine of Haemophilus parasuis is treated as claimed in claim 4, it is characterised in that：Step 2) in, by seed liquor With the volume ratio 1 of culture medium:20, primary seed solution is inoculated in TSB culture mediums.
8. the medicine of Haemophilus parasuis is treated as claimed in claim 4, it is characterised in that：Step 3) in, by seed liquor With the volume ratio 1 of culture medium:100, secondary seed solution is inoculated in TSB culture mediums.
9. the medicine of Haemophilus parasuis is treated as claimed in claim 4, it is characterised in that：Step 4) in, by original bacteria liquid 10min is centrifuged under conditions of rotating speed is 8000～10000rpm, the thalline of precipitation is taken.
10. the medicine of Haemophilus parasuis is treated as claimed in claim 4, it is characterised in that：Step 4) in, the adjuvant For aluminium glue adjuvant, and by seedling bacterium solution and aluminium glue adjuvant with volume ratio 4：1 mixing, breast under conditions of 5000～8000rpm of rotating speed Change 10～20min.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108018230A (en) * 2017-11-30 2018-05-11 华中农业大学 A kind of natural less-virulent strain of serum 7-type haemophilus parasuis and its application
CN109806389A (en) * 2019-02-22 2019-05-28 河南省农业科学院畜牧兽医研究所 A kind of trivalent inactivated vaccine against Haemophilus parasuis infection and its application

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CN102399724A (en) * 2011-11-08 2012-04-04 山东省农业科学院畜牧兽医研究所 Haemophilus parasuis LC strain and application thereof
CN105524857A (en) * 2015-09-17 2016-04-27 龙岩学院 Haemophilus parasuis strain

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CN102399724A (en) * 2011-11-08 2012-04-04 山东省农业科学院畜牧兽医研究所 Haemophilus parasuis LC strain and application thereof
CN105524857A (en) * 2015-09-17 2016-04-27 龙岩学院 Haemophilus parasuis strain

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108018230A (en) * 2017-11-30 2018-05-11 华中农业大学 A kind of natural less-virulent strain of serum 7-type haemophilus parasuis and its application
CN109806389A (en) * 2019-02-22 2019-05-28 河南省农业科学院畜牧兽医研究所 A kind of trivalent inactivated vaccine against Haemophilus parasuis infection and its application
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

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