CN105420141A - Haemophilus parasuis strain - Google Patents
Haemophilus parasuis strain Download PDFInfo
- Publication number
- CN105420141A CN105420141A CN201510620815.4A CN201510620815A CN105420141A CN 105420141 A CN105420141 A CN 105420141A CN 201510620815 A CN201510620815 A CN 201510620815A CN 105420141 A CN105420141 A CN 105420141A
- Authority
- CN
- China
- Prior art keywords
- bacterial strain
- haemophilus parasuis
- strain
- vaccine
- lyw1
- 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.)
- Granted
Links
Abstract
The invention provides a haemophilus parasuis strain. The strain is Haemophilus parasuis strain LYW1 and is preserved in China General Microbiological Culture Collection Center on June 21st, 2015. The address of the China General Microbiological Culture Collection Center is No.1 yard No.3, Beichen west road, Chaoyang district, Beijing, and the preservation number of the strain is CGMCC No.11146. The strain LYW1 can be used for participating in preparing a vaccine for preventing and curing diseases caused by various serotype haemophilus parasuis, and the finally prepared vaccine is good in vaccine safety and preventing and curing capacity.
Description
[technical field]
The invention belongs to biological field, be specifically related to a kind of haemophilus parasuis bacterial strain.
[background technology]
Haemophilus parasuis had once once been considered to by piglet conditionality, the sporadic disease that stress cause, and along with the raising of pig-rearing industry, intensive degree, nowadays it has become harm piglet and the most serious bacteriosis of growing swine.
The piglet in 2 ~ 28 week age of haemophilus parasuis main harm and growing swine, the most serious with 5-8 child care piglet harm of weaning age in week, cause the symptom such as polyserositis, sacroiliitis, sickness rate is up to 15% ~ 90%, and mortality ratio sometimes can up to 90%.Originally haemophilus parasuis is called as haemophilus suis or haemophilus influenzae suis, confirms that its growth does not need the X factor (materials of protoheme and other porphyrins) afterwards, is thus renamed as haemophilus parasuis.Haemophilus parasuis has multiform state property, strictly needs NAD or the V factor during growth, and serotype complexity is various, according to the difference of homotype Bacterial outer membrane proteins antigen, at least can be divided into 15 kinds of serotypes, separately has the strain isolated of more than 20% can not somatotype; And the popular serotype in country variant or area is different, according to Cai Xuwang etc., thinks that the serotype of China's current popular mainly contains 4,5,12,13 types, but different local predominant serotype is different, so increase difficulty to the prevention and control of disease.According to the qualification of China's seroepidemiological survey and isolated strains, the most popular with 4,5 types, be secondly 12 types, 13 types.In recent years, between each haemophilus parasuis serogroups, Immunogenicity power is strong, and the cause of disease serogroups of the Haemophilus parasuis that the vaccine strains serogroups of inoculation is popular with there and then does not conform to, and brings certain difficulty to the prevention and therapy of haemophilus parasuis.
The domestic vaccine for the Haemophilus parasuis immunoprophylaxis Haemophilus parasuis inactivated vaccine (Z-1517 strain) that has Boehringer Ingelheim animal health (U.S.) company limited to produce at present, pig Haemophilus parasuis 1 type that the biological large pharmaceutical factory of Spain Hai Bolai produces and 6 type inactivated vaccines, Haemophilus parasuis 4 type that Hua Zhong Agriculture University, Wuhan Keqian Animal Biological Products Co., Ltd., Zhongmu Industry Co., Ltd produce and 5 type inactivated vaccines; The application of these vaccines serves certain prophylactic effect, and the cause of disease epidemic strain of this disease and various serotype infection have made existing vaccine be difficult to reach the immune effect of expection, therefore, be practitioner's problem in the urgent need to address for participating in preparing the proper strain of the control various serotype Haemophilus parasuis former Haemophilus parasuis vaccine caused.
[summary of the invention]
Technical problem to be solved by this invention is to provide a kind of haemophilus parasuis bacterial strain LYW1 (Haemophilusparasuis), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on 07 21st, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNo.11146.
The present invention solves the problems of the technologies described above by the following technical programs:
2011, applicant aseptically gathers lungs, hydrarthrosis, the tracheae of doubtful Hps morbidity piglet in new Luo pig farm, Quanzhou City of Fujian Province, and difference streak inoculation is in TSA substratum, be placed in 37 DEG C of constant incubators, candle cylinder Anaerobic culturel 24-48h, carrying out physicochemical property and molecules is identified by cultivating the bacterial strain obtained, finally determining that it is a bacterial strain of haemophilus parasuis Pseudomonas.
Beneficial effect of the present invention is: provide a kind of haemophilus parasuis bacterial strain LYW1 (Haemophilusparasuis), the vaccine of disease that the haemophilus parasuis that this bacterial strain can be used in participating in preparation control various serotype causes, final obtained vaccine safety and prevention and control capability all good.
[accompanying drawing explanation]
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is 1.0% agarose gel electrophoresis figure of PCR primer in embodiment of the present invention 1-5.
[embodiment]
When in the present invention, haemophilus parasuis bacterial strain LYW1 (Haemophilusparasuis) is 2011, the lungs of collection doubtful Hps morbidity piglet in new Luo pig farm, Quanzhou City of Fujian Province, hydrarthrosis are carried out cultivating the bacterial strain obtained.And it should be noted that, also relate to other several bacterial strain in the present invention, be respectively haemophilus parasuis bacterial strain LYD1, haemophilus parasuis bacterial strain LYH5, haemophilus parasuis bacterial strain LY02 and haemophilus parasuis bacterial strain LYC2.
Haemophilus parasuis bacterial strain LYD1 (Haemophilusparasuis) was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 07 21st, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNo.11143; Haemophilus parasuis bacterial strain LYH5 (Haemophilusparasuis) was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 07 21st, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNo.11144; Haemophilus parasuis bacterial strain LY02 (Haemophilusparasuis) was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 07 21st, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNo.11145; Haemophilus parasuis bacterial strain LYC2 (Haemophilusparasuis) was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 07 21st, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNo.11147.
Illustrate in order to the present invention will be described in detail, applicant gives following specific embodiment and illustrates in order to the present invention will be described in detail, and applicant gives following specific embodiment.
The Isolation and ldentification of embodiment 1 bacterial strain LYW1
1, the separation of bacterial strain LYW1:
2011, applicant aseptically gathers lungs, hydrarthrosis, the tracheae of doubtful Hps morbidity piglet in new Luo pig farm, Quanzhou City of Fujian Province, and difference streak inoculation is in TSA substratum, be placed in 37 DEG C of constant incubators, candle cylinder Anaerobic culturel 24-48h is bacterial strain LYW1 by cultivating the Strain Designation obtained.
2, the qualification of bacterial strain LYW1:
Preliminary evaluation:
Obtained bacterial strain LYW1 is carried out the mensuration of morphology and biochemical characteristic, result is as follows: in Gram-negative, tiny bacillus, has multiple different form, from single coccobacillus to long, elongated so that thread thalline; Without haemolysis, strain isolated edwardsiella hoshinae, energy glucose fermentation, fructose, sucrose, nonfermented wood sugar, L-arabinose, lactose, raffinose, catalase, urease test feminine gender, unstable to seminose biochemical reaction, growth all needs the V factor and does not need the X factor; Therefore, tentatively can assert that bacterial strain LYW1 belongs to haemophilus parasuis Pseudomonas.
Further qualification:
Extract template: single bacterium colony of picking bacterial strain LYW1 carries out subclone cultivation, carefully scrapes lawn and be placed in 1.5mL centrifuge tube with the distilled water of sterilizing, and centrifugal 30s under 12000rpm, abandons supernatant afterwards, retain cell precipitation; Then add 567 μ LTE suspension precipitations, with equal-volume first extract (volume ratio is the phenol of 25: 24: 1: chloroform: primary isoamyl alcohol) extracting, two-phase is mixed completely, ice bath 10 minutes; Then centrifugal 10 minutes of 12000rpm, Aspirate supernatant, equal-volume second extract (volume ratio is the chloroform of 24: 1: primary isoamyl alcohol) extracting 1 time again, has cotton-shaped DNA precipitation to occur to solution; DNA precipitation is transferred in 1mL70% ethanol and washed, then in 65 DEG C of loft drier dry 2 minutes; Finally use 50-100 μ LTE damping fluid (containing 20 μ g/mLRNase) dissolving DNA to precipitate, be required template, 4 DEG C save backup.
PCR identifies: entrust Shanghai Sheng Gong biotechnology company limited synthesis upstream primer P1 (as shown in SEQIDNO:1): 5'-GTGATGAGGAAGGGTGGTGT-3'; Downstream primer P2 (as shown in SEQIDNO:2): 5'-GGCTTCGTCACCCTCTGT-3'.
With the present embodiment extract acquisition template for template, and with above-mentioned primer (P1/P2) for primer pair carries out PCR reaction, and with existing generally acknowledged haemophilus parasuis reference culture for negative control:
PCR reaction system (50 μ L): 10 × PCRBuffer (Mg
2+) 5 μ L, 2.5mmol/LdNTPs4 μ L, 5U/ μ LTaqDNA polysaccharase 0.5 μ L, H
2o36.5 μ L, 10 μm of ol/L upstream primer 1 μ L, 10 μm of ol/L downstream primer 1 μ L, template 2 μ L;
PCR response procedures is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of renaturation 1min, repeat 30 circulations; Last 72 DEG C of downward-extension 7min.
The detection of PCR primer: by PCR primer with 1% agarose gel electrophoresis PCR primer is identified, using DNAMarkerDL2000 as standard molecular weight, electrophoresis 20min in 80V voltage, 1 × TAE damping fluid.
Namely carry out gel electrophoresis and be separated inspection, (in Fig. 1, M is DNAladder to electrophoretic separation assay such as Fig. 1; 1 is the present embodiment test strains; 2 is negative control; 3 is distilled water blank) shown in, as shown in Figure 1, the present embodiment test strains and bacterial strain LYW1 the same with haemophilus parasuis reference culture, all amplify single band in 821bp position, namely confirm that this bacterial strain LYW1 and haemophilus parasuis reference culture belong to same Pseudomonas.
According to above-mentioned qualification, final confirmation bacterial strain LYW1 belongs to a bacterial strain of haemophilus parasuis (Haemophilusparasuis) Pseudomonas.
Serotype Identification: bacterial strain LYW1 is inoculated in TSA substratum, after 37 DEG C of a large amount of propagation of cultivation, centrifuging and taking thalline under 7000r/min; 9 volumes are added doubly to the pH7.4PBS buffered soln of thalline in thalline, 121 DEG C of steam treatment 2h, the centrifugal 10min of 7000r/min gets supernatant liquor and carries out agar diffusion test again, the agar diffusion test method specifically set up according to Keilstein and Rapp-Gabrielson identifies its Hps serotype, and qualification result is Hps12 type.
The Isolation and ldentification of embodiment 2 bacterial strain LYD1
1, the separation of bacterial strain LYD1:
2009, applicant aseptically gathers lungs, hydrarthrosis, the tracheae of doubtful Hps morbidity piglet in new Luo pig farm, Quanzhou City of Fujian Province, and difference streak inoculation is in TSA substratum, be placed in 37 DEG C of constant incubators, candle cylinder Anaerobic culturel 24-48h is bacterial strain LYD1 by cultivating the Strain Designation obtained.
2, the qualification of bacterial strain LYD1:
Preliminary evaluation:
Obtained bacterial strain LYD1 is carried out the mensuration of morphology and biochemical characteristic, its result is consistent with the preliminary evaluation result in embodiment 1; Therefore, tentatively can assert that bacterial strain LYD1 belongs to haemophilus parasuis Pseudomonas equally.
Further qualification:
Extract template: with reference to the extraction template operation in embodiment 1 using the DNA extracting bacterial strain LYD1 as required template, 4 DEG C save backup.
PCR identifies: with the present embodiment extract acquisition template for template, and with primer described in embodiment 1 (P1/P2) for primer pair carries out PCR reaction, and with existing generally acknowledged haemophilus parasuis reference culture for negative control:
PCR reaction system (50 μ L): 10 × PCRBuffer (Mg
2+) 5 μ L, 2.5mmol/LdNTPs4 μ L, 5U/ μ LTaqDNA polysaccharase 0.5 μ L, H
2o36.5 μ L, 10 μm of ol/L upstream primer 1 μ L, 10 μm of ol/L downstream primer 1 μ L, template 2 μ L;
PCR response procedures is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of renaturation 1min, repeat 30 circulations; Last 72 DEG C of downward-extension 7min.
The detection of PCR primer: by PCR primer with 1% agarose gel electrophoresis PCR primer is identified, using DNAMarkerDL2000 as standard molecular weight, electrophoresis 20min in 80V voltage, 1 × TAE damping fluid.Namely carry out gel electrophoresis and be separated inspection, electrophoretic separation assay is consistent with embodiment 1, namely as shown in Figure 1, then confirm this bacterial strain LYD1 equally and haemophilus parasuis reference culture belong to same Pseudomonas.
According to above-mentioned qualification, final confirmation bacterial strain LYD1 belongs to a bacterial strain of haemophilus parasuis (Haemophilusparasuis) Pseudomonas.
Serotype Identification: carry out Serotype Identification with reference to Serotype Identification operation in embodiment 1 to bacterial strain LYD1, qualification result is Hps1 type.
The Isolation and ldentification of embodiment 3 bacterial strain LYH5
1, the separation of bacterial strain LYH5:
2011, applicant aseptically gathers lungs, hydrarthrosis, the tracheae of doubtful Hps morbidity piglet in pig farm, Yongding, Longyan, and difference streak inoculation is in TSA substratum, be placed in 37 DEG C of constant incubators, candle cylinder Anaerobic culturel 24-48h is bacterial strain LYH5 by cultivating the Strain Designation obtained
2, the qualification of bacterial strain LYH5:
Preliminary evaluation:
Obtained bacterial strain LYH5 is carried out the mensuration of morphology and biochemical characteristic, its result is consistent with the preliminary evaluation result in embodiment 1; Therefore, tentatively can assert that bacterial strain LYH5 belongs to haemophilus parasuis Pseudomonas equally.
Further qualification:
Extract template: with reference to the extraction template operation in embodiment 1 using the DNA extracting bacterial strain LYH5 as required template, 4 DEG C save backup.
PCR identifies: with the present embodiment extract acquisition template for template, and with primer described in embodiment 1 (P1/P2) for primer pair carries out PCR reaction, and with existing generally acknowledged haemophilus parasuis reference culture for negative control:
PCR reaction system (50 μ L): 10 × PCRBuffer (Mg
2+) 5 μ L, 2.5mmol/LdNTPs4 μ L, 5U/ μ LTaqDNA polysaccharase 0.5 μ L, H
2o36.5 μ L, 10 μm of ol/L upstream primer 1 μ L, 10 μm of ol/L downstream primer 1 μ L, template 2 μ L;
PCR response procedures is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of renaturation 1min, repeat 30 circulations; Last 72 DEG C of downward-extension 7min.
The detection of PCR primer: by PCR primer with 1% agarose gel electrophoresis PCR primer is identified, using DNAMarkerDL2000 as standard molecular weight, electrophoresis 20min in 80V voltage, 1 × TAE damping fluid.Namely carry out gel electrophoresis and be separated inspection, electrophoretic separation assay is consistent with embodiment 1, namely as shown in Figure 1, then confirm this bacterial strain LYH5 equally and haemophilus parasuis reference culture belong to same Pseudomonas.
According to above-mentioned qualification, final confirmation bacterial strain LYH5 belongs to a bacterial strain of haemophilus parasuis (Haemophilusparasuis) Pseudomonas.
Serotype Identification: carry out Serotype Identification with reference to Serotype Identification operation in embodiment 1 to bacterial strain LY02, qualification result is Hps4 type.
The Isolation and ldentification of embodiment 4 bacterial strain LY02
1, the separation of bacterial strain LY02:
2012, applicant aseptically gathers lungs, hydrarthrosis, the tracheae of doubtful Hps morbidity piglet in pig farm, Yongding, Longyan, and difference streak inoculation is in TSA substratum, be placed in 37 DEG C of constant incubators, candle cylinder Anaerobic culturel 24-48h is bacterial strain LY02 by cultivating the Strain Designation obtained.
2, the qualification of bacterial strain LY02:
Preliminary evaluation:
Obtained bacterial strain LY02 is carried out the mensuration of morphology and biochemical characteristic, its result is consistent with the preliminary evaluation result in embodiment 1; Therefore, tentatively can assert that bacterial strain LY02 belongs to haemophilus parasuis Pseudomonas equally.
Further qualification:
Extract template: with reference to the extraction template operation in embodiment 1 using the DNA extracting bacterial strain LY02 as required template, 4 DEG C save backup.
PCR identifies: with the present embodiment extract acquisition template for template, and with primer described in embodiment 1 (P1/P2) for primer pair carries out PCR reaction, and with existing generally acknowledged haemophilus parasuis reference culture for negative control:
PCR reaction system (50 μ L): 10 × PCRBuffer (Mg
2+) 5 μ L, 2.5mmol/LdNTPs4 μ L, 5U/ μ LTaqDNA polysaccharase 0.5 μ L, H
2o36.5 μ L, 10 μm of ol/L upstream primer 1 μ L, 10 μm of ol/L downstream primer 1 μ L, template 2 μ L;
PCR response procedures is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of renaturation 1min, repeat 30 circulations; Last 72 DEG C of downward-extension 7min.
The detection of PCR primer: by PCR primer with 1% agarose gel electrophoresis PCR primer is identified, using DNAMarkerDL2000 as standard molecular weight, electrophoresis 20min in 80V voltage, 1 × TAE damping fluid.Namely carry out gel electrophoresis and be separated inspection, electrophoretic separation assay is consistent with embodiment 1, namely as shown in Figure 1, then confirm this bacterial strain LY02 equally and haemophilus parasuis reference culture belong to same Pseudomonas.
According to above-mentioned qualification, final confirmation bacterial strain LY02 belongs to a bacterial strain of haemophilus parasuis (Haemophilusparasuis) Pseudomonas.
Serotype Identification: carry out Serotype Identification with reference to Serotype Identification operation in embodiment 1 to bacterial strain LY02, qualification result is Hps5 type.
The Isolation and ldentification of embodiment 5 bacterial strain LYC2
1, the separation of bacterial strain LYC2:
2012, applicant aseptically gathers lungs, hydrarthrosis, the tracheae of doubtful Hps morbidity piglet in new Luo pig farm, Quanzhou City of Fujian Province, and difference streak inoculation is in TSA substratum, be placed in 37 DEG C of constant incubators, candle cylinder Anaerobic culturel 24-48h is bacterial strain LYC2 by cultivating the Strain Designation obtained.
2, the qualification of bacterial strain LYC2:
Preliminary evaluation:
Obtained bacterial strain LYC2 is carried out the mensuration of morphology and biochemical characteristic, its result is consistent with the preliminary evaluation result in embodiment 1; Therefore, tentatively can assert that bacterial strain LYC2 belongs to haemophilus parasuis Pseudomonas equally.
Further qualification:
Extract template: with reference to the extraction template operation in embodiment 1 using the DNA extracting bacterial strain LYC2 as required template, 4 DEG C save backup.
PCR identifies: with the present embodiment extract acquisition template for template, and with primer described in embodiment 1 (P1/P2) for primer pair carries out PCR reaction, and with existing generally acknowledged haemophilus parasuis reference culture for negative control:
PCR reaction system (50 μ L): 10 × PCRBuffer (Mg
2+) 5 μ L, 2.5mmol/LdNTPs4 μ L, 5U/ μ LTaqDNA polysaccharase 0.5 μ L, H
2o36.5 μ L, 10 μm of ol/L upstream primer 1 μ L, 10 μm of ol/L downstream primer 1 μ L, template 2 μ L;
PCR response procedures is: 94 DEG C of denaturation 5min; Then 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C of renaturation 1min, repeat 30 circulations; Last 72 DEG C of downward-extension 7min.
The detection of PCR primer: by PCR primer with 1% agarose gel electrophoresis PCR primer is identified, using DNAMarkerDL2000 as standard molecular weight, electrophoresis 20min in 80V voltage, 1 × TAE damping fluid.Namely carry out gel electrophoresis and be separated inspection, electrophoretic separation assay is consistent with embodiment 1, namely as shown in Figure 1, then confirm this bacterial strain LYC2 equally and haemophilus parasuis reference culture belong to same Pseudomonas.
According to above-mentioned qualification, final confirmation bacterial strain LYC2 belongs to a bacterial strain of haemophilus parasuis (Haemophilusparasuis) Pseudomonas.
Serotype Identification: carry out Serotype Identification with reference to Serotype Identification operation in embodiment 1 to bacterial strain LYC2, qualification result is Hps13 type.
The preparation of embodiment 6 haemophilus parasuis pentavalent inactivated vaccine
Breeding is cultivated: get above-mentioned bacterial strain LYD1, bacterial strain LYH5, bacterial strain LY02, bacterial strain LYW1 and bacterial strain LYC2 and distinguish streak inoculation on TSA agar plate, and in 5% ~ 10%CO
2(the CO namely containing 5% ~ 10% mass percent in culture environment
2), cultivate 24 ~ 48h at 37 DEG C, picking colonies typical is also inoculated on TSB substratum, be placed in 37 DEG C of shaking culture 24 ~ 48h, obtain the primary seed solution of the primary seed solution of bacterial strain LYD1, the primary seed solution of bacterial strain LYH5, the primary seed solution of bacterial strain LY02, the primary seed solution of bacterial strain LYW1 and bacterial strain LYC2; Get the primary seed solution of the primary seed solution of obtained strains LYD1, the primary seed solution of bacterial strain LYH5, the primary seed solution of bacterial strain LY02, the primary seed solution of bacterial strain LYW1 and bacterial strain LYC2 and line respectively on TSA agar plate, and in 5% ~ 10%CO
2(the CO namely containing 5% ~ 10% mass percent in culture environment
2), cultivate 24 ~ 48h at 37 DEG C, picking colonies typical is also inoculated on TSB substratum, be placed in 37 DEG C of shaking culture 24 ~ 48h, obtain the secondary seed solution of the secondary seed solution of bacterial strain LYD1, the secondary seed solution of bacterial strain LYH5, the secondary seed solution of bacterial strain LY02, the secondary seed solution of bacterial strain LYW1 and bacterial strain LYC2; The secondary seed solution of the secondary seed solution of the secondary seed solution of the secondary seed solution of bacterial strain LYD1, bacterial strain LYH5, bacterial strain LY02, the secondary seed solution of bacterial strain LYW1 and bacterial strain LYC2 is seeded to fermention medium respectively, and in 37 DEG C, cultivate 22 ~ 24h under 180r/min, obtain bacterial strain LYD1 bacterium liquid, bacterial strain LYH5 bacterium liquid, bacterial strain LY02 bacterium liquid, bacterial strain LYW1 bacterium liquid and bacterial strain LYC2 bacterium liquid;
Wherein, fermention medium is semisynthetic medium, the compound method of this semisynthetic medium is: get TSB30g and be dissolved in deionized water, and be settled to 1000mL, fully to shake up after dissolving in 121 DEG C of steam sterilizing 15min, after cooling, add the 0.02%NAD of the new-born calf serum of 50ml filtration sterilization, 200 μ L filtration sterilizations;
Concentrate and deactivation: breeding is cultivated each bacterium liquid of gained and concentrate respectively, and bacterial strain LYD1 bacterium liquid, bacterial strain LYW1 bacterium liquid, the bacterial strain LYC2 bacterium liquid antigenic content be all concentrated in bacterium liquid are 2.5 × 10
9cFU/mL, bacterial strain LYH5 bacterium liquid, the bacterial strain LY02 bacterium liquid antigenic content be then all concentrated in bacterium liquid are 2.0 × 10
9cFU/mL; Then each bacterium liquid after concentrated is carried out deactivation respectively;
The preparation of total mixed bacteria liquid: get bacterial strain LYD1 bacterium liquid, bacterial strain LYW1 bacterium liquid and the bacterial strain LYC2 bacterium liquid after concentrated, deactivation and mix by the volume ratio of 1:1:1, obtaining the first mixed bacteria liquid; Get the bacterial strain LYH5 bacterium liquid after concentrated, deactivation, bacterial strain LY02 bacterium liquid mixing by the volume ratio of 1:1, obtain the second mixed bacteria liquid; Afterwards by gained first mixed bacteria liquid and the second mixed bacteria liquid by the volume ratio of 1:1.5 mix then total mixed bacteria liquid;
Aqueous phase is prepared: by volume part gets sterilized tween-80 4 parts, total mixed bacteria liquid 96 parts that steriling test is qualified, and imports in Agitation Tank, opens agitator motor, is at the uniform velocity stirred to tween-80 and dissolves completely, obtains aqueous phase;
Oil phase is prepared: by volume part gets injection white oil 94 parts, Si Ben-806 parts add in oil phase preparation tank, opens agitator motor, at the uniform velocity stirs, and opens thermal oil switch simultaneously, and 125 DEG C of heating 30min, namely obtain oil phase after cooling;
Emulsification: get oil phase 1.5 parts by volume and be placed in emulsion tank, starts motor 1500-2000r/min and stirs, and slowly adds aqueous phase 1 parts by volume simultaneously, then with 4000r/min emulsification 30min; After emulsification, sampling 10ml with the centrifugal 15min of 3000r/min, if not stratified, emulsification gained emulsion is described haemophilus parasuis pentavalent inactivated vaccine; If there is demixing phenomenon, gained emulsion repeated emulsification until not stratified, finally namely obtain described haemophilus parasuis pentavalent inactivated vaccine.
In the present invention, the compound method of TSA agar plate and TSB substratum is: get Tryptose soy agar 40g, add deionized water and be settled to 1000mL, fully shake up dissolving, 121 DEG C of high pressure steam sterilization 15min, add the 0.2g/mLNAD of the new-born calf serum of 50mL filtration sterilization, 200 μ L filtration sterilizations after cooling.Fermention medium is semisynthetic medium, the compound method of this semisynthetic medium is: get TSB30g and be dissolved in deionized water, and be settled to 1000mL, fully to shake up after dissolving in 121 DEG C of high pressure steam sterilization 15min, after cooling, add the 0.02%NAD of the new-born calf serum of 50ml filtration sterilization, 200 μ L filtration sterilizations.The compound method of TSB substratum is: get Tryptose soy meat soup and TSB30g is dissolved in deionized water, be settled to 1000mL, fully shake up and dissolve rear 121 DEG C of steam sterilizing 15min, add the 0.02%NAD of the new-born calf serum of 50ml filtration sterilization, 200ul filtration sterilization.
The proof test of embodiment 7 haemophilus parasuis pentavalent inactivated vaccine
1, haemophilus parasuis vaccine safety testing that target animals single dose is inoculated
Preparation method of the present invention and the obtained haemophilus parasuis pentavalent inactivated vaccine of embodiment 6 preparation method 01 crowd is adopted to carry out musculi colli injection respectively to 5 10 ~ 15 age in days piglets (purchased from breeding pig farm, Fujian Longyan city), injection volume is 2ml/ head, separately establishes physiological saline immunized controls group.With in after the vaccination of viewing test pig 2 weeks, test pig is with or without allergy, and spirit, diet have without exception, and injection site is with or without red and swollen phenomenon and weight gain of piglets situation.Test-results is as shown in table 1 below.
The once inoculation safety testing result of table 1 pair non-usage age in days target animals
As shown in Table 1, after injection haemophilus parasuis pentavalent inactivated vaccine, piglet spirit is good, and after inoculation, red and swollen phenomenon does not all appear in injection site, and spirit, search for food all normal; Namely basically identical with the piglet state of saline control group; Thus show that the haemophilus parasuis vaccine obtained by the present invention is safe to target animals single dose inoculation.
2, haemophilus parasuis vaccine is to the safety testing of a target animals overdose inoculation
Preparation method of the present invention is adopted to obtain the haemophilus parasuis pentavalent inactivated vaccine of 3 batches, 01 batch, 02 batch, 03 batch respectively; Adopt this vaccine of 3 batches to carry out musculi colli injection to the healthy susceptible piglet of 10 ~ 15 ages in days respectively, inject 5, injection volume 4ml/ head for each batch, and each batch is all established physiological saline immunized controls group; Security after the vaccination of viewing test pig.Result shows to refer to table 2.
The safety testing of table 2 haemophilus parasuis vaccine overdose inoculation
As shown in Table 2, inject respectively 3 batches of haemophilus parasuis pentavalent inactivated vaccines 10 ~ 15 weeks age piglet injection site all there is not red and swollen reaction, and spirit, search for food all normally, with the piglet no significant difference of control group; Thus illustrate that the haemophilus parasuis pentavalent inactivated vaccine overdose inoculation that the present invention obtains is safe.
3, single dose repeated inoculation proof test
The 01 batch of haemophilus parasuis pentavalent inactivated vaccine adopting preparation method of the present invention to obtain carries out vaccine inoculation to the healthy susceptible piglet of 10 ~ 15 ages in days, and after 14 days, repeated inoculation is once, and to inoculate physiological saline for control group; Observe vaccine single dose repeated inoculation to the security of Pigs Inoculated.The results detailed in Table 3.
Table 3 is tested piglet single dose and is repeated safety testing result
Shown by table 3, this vaccine has no adverse effects to Pigs Inoculated, and without allergic phenomena, inoculation position is without untoward reactions such as rednesses, and spirit, the food consumption of Pigs Inoculated are all normal, the no significant difference of weight gain of piglets and control group; Thus prove that haemophilus parasuis pentavalent inactivated vaccine single dose repeated inoculation of the present invention is safe.
4, the safety testing of farrowing sow
Test group, the haemophilus parasuis pentavalent inactivated vaccine adopting preparation method of the present invention to obtain carries out musculi colli injection to antenatal 15 days farrowing sow (carrying out on pig farm, Longyan) each 5, and injection volume is 2ml/ head; Separately establish physiological saline immunized controls group.With in after the vaccination of viewing test pig 2 weeks, test pig is with or without allergy, and spirit, diet have without exception, and injection site is with or without red and swollen phenomenon, and whether sow produces subcase normal.Result shows, and after the inoculation of test group antenatal 15 days farrowing sows, red and swollen phenomenon does not all appear in injection site, inoculates 2-3 days, and body temperature slightly raises, the 4th day, and temperature recovery is normal, spirit, searches for food all normal, and institute farrows pig all normally, and farrowing rate reaches 96.8; Controlled trial pig is all normal, and farrowing rate is 97.2%; Thus illustrate that haemophilus parasuis pentavalent inactivated vaccine of the present invention is safe to farrowing sow inoculation.
The test of pesticide effectiveness of embodiment 8 haemophilus parasuis vaccine
Test group and immune group, the healthy susceptible pig 25 of the haemophilus parasuis pentavalent inactivated vaccine adopting preparation method of the present invention to obtain to 10 ~ 15 ages in days carries out musculi colli injection, injection volume is 2ml/ head, after 21 days, second immunisation, dosage is constant, after second immunisation 14 days, the immunity test pig of injecting described haemophilus parasuis pentavalent inactivated vaccine is divided into 5 groups at random, attacks poison with haemophilus parasuis serum 1 type, 4 types, 5 types, 12 types and 13 type bacterial strains respectively; Physiological saline immunized controls group is set simultaneously.Result is as shown in table 4.
Table 4 piglet attacks poison and protectiveness test
As shown in Table 4, the attack malicious immunization rate of the haemophilus parasuis pentavalent inactivated vaccine that preparation method of the present invention obtains to haemophilus parasuis serum 1,5,12 type bacterial strain reaches 100%, is 60% to the malicious immunization rate of attacking of haemophilus parasuis serum 4,13 type; Therefore, the disease that the haemophilus parasuis pentavalent inactivated vaccine that preparation method of the present invention obtains can not only cause for the haemophilus parasuis of various serotype simultaneously carries out immune protection, and has good immune protection ability.
To sum up, the invention provides a kind of haemophilus parasuis bacterial strain LYW1 (Haemophilusparasuis), the vaccine of disease that the haemophilus parasuis that this bacterial strain can be used in participating in preparation control various serotype causes, final obtained vaccine safety and prevention and control capability all good; In addition, also there is the comparatively cheap feature of cost.
Claims (1)
1. a haemophilus parasuis bacterial strain, it is characterized in that: described bacterial strain is haemophilus parasuis bacterial strain LYW1 (Haemophilusparasuis), China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on 07 21st, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNo.11146.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510620815.4A CN105420141B (en) | 2015-09-17 | 2015-09-25 | A kind of preparation method of five bivalent inactivated vaccine of haemophilus parasuis bacterial strain |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2015105918902 | 2015-09-17 | ||
| CN201510591890 | 2015-09-17 | ||
| CN201510620815.4A CN105420141B (en) | 2015-09-17 | 2015-09-25 | A kind of preparation method of five bivalent inactivated vaccine of haemophilus parasuis bacterial strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105420141A true CN105420141A (en) | 2016-03-23 |
| CN105420141B CN105420141B (en) | 2019-05-14 |
Family
ID=54660460
Family Applications (6)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510618836.2A Pending CN105524857A (en) | 2015-09-17 | 2015-09-25 | Haemophilus parasuis strain |
| CN201510616476.2A Active CN105112342B (en) | 2015-09-17 | 2015-09-25 | A kind of preparation method of haemophilus parasuis bacterial strain pentavalent vaccine |
| CN201510616477.7A Pending CN105505811A (en) | 2015-09-17 | 2015-09-25 | Haemophilus parasuis strain |
| CN201510617867.6A Active CN105543120B (en) | 2015-09-17 | 2015-09-25 | Haemophilus parasuis pentavalent inactivated vaccine |
| CN201510620815.4A Active CN105420141B (en) | 2015-09-17 | 2015-09-25 | A kind of preparation method of five bivalent inactivated vaccine of haemophilus parasuis bacterial strain |
| CN201510620812.0A Active CN105148262B (en) | 2015-09-17 | 2015-09-25 | A kind of preparation method of five bivalent inactivated vaccine of haemophilus parasuis |
Family Applications Before (4)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510618836.2A Pending CN105524857A (en) | 2015-09-17 | 2015-09-25 | Haemophilus parasuis strain |
| CN201510616476.2A Active CN105112342B (en) | 2015-09-17 | 2015-09-25 | A kind of preparation method of haemophilus parasuis bacterial strain pentavalent vaccine |
| CN201510616477.7A Pending CN105505811A (en) | 2015-09-17 | 2015-09-25 | Haemophilus parasuis strain |
| CN201510617867.6A Active CN105543120B (en) | 2015-09-17 | 2015-09-25 | Haemophilus parasuis pentavalent inactivated vaccine |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510620812.0A Active CN105148262B (en) | 2015-09-17 | 2015-09-25 | A kind of preparation method of five bivalent inactivated vaccine of haemophilus parasuis |
Country Status (1)
| Country | Link |
|---|---|
| CN (6) | CN105524857A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520623B (en) * | 2016-12-01 | 2018-04-24 | 华南农业大学 | A kind of serum 7-type haemophilus parasuis low virulent strain and its application |
| CN107365720B (en) * | 2017-06-12 | 2018-10-12 | 广东海大畜牧兽医研究院有限公司 | It is a kind of with the Serotype 5 haemophilus parasuis of cross-protection 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102908615A (en) * | 2011-08-01 | 2013-02-06 | 普莱柯生物工程股份有限公司 | Novel haemophilus parasuis disease trivalent inactivated vaccine and preparation method thereof |
| US20130129682A1 (en) * | 2009-11-04 | 2013-05-23 | Regents Of The University Of Minnesota | Haemophilus parasuis polypeptides and methods of use |
| CN104248755A (en) * | 2013-10-30 | 2014-12-31 | 普莱柯生物工程股份有限公司 | Haemophilus parasuis disease vaccine composition, preparation method and application thereof |
| CN104452556A (en) * | 2014-12-02 | 2015-03-25 | 吉林大学 | Verification system for vehicle-mounted pavement primary crack acquisition system based on line structured light reference |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875944A (en) * | 2009-11-18 | 2010-11-03 | 北京市农林科学院 | Haemophilus parasuis aroA gene suicide vector and construction method thereof |
| CN101721696A (en) * | 2009-12-18 | 2010-06-09 | 西南民族大学 | Trivalent oil emulsion inactivated vaccine for 4 type, 5 type and 13 type serum of haemophilus parasuis |
| US20130039941A1 (en) * | 2010-04-23 | 2013-02-14 | Intervet International B.V. | Vaccine comprising inactivated cells of haemophilus parasuis bacteria of serotype 5 |
| CN101968490A (en) * | 2010-08-26 | 2011-02-09 | 广东省农业科学院兽医研究所 | Indirect ELISA (Enzyme-Linked Immunosorbent Assay) method and kit for detecting haemophilus parasuis antibodies |
| CN102329746B (en) * | 2011-08-16 | 2013-01-09 | 武汉科前动物生物制品有限责任公司 | Porcine streptococcus disease and haemophilus parasuis disease combined inactivated vaccine and preparation method thereof |
| 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 |
| CN102499982B (en) * | 2011-12-21 | 2013-07-17 | 青岛易邦生物工程有限公司 | Method for producing trivalent inactivated vaccine against Haemophilus parasuis infection |
| CN102851249B (en) * | 2012-04-01 | 2013-11-13 | 山东省农业科学院畜牧兽医研究所 | Haemophilus parasuis LZ-20100109 strain and application thereof |
| CA2926190C (en) * | 2013-10-04 | 2023-05-09 | Merial, Inc. | Haemophilus parasuis vaccine serovar type four |
| CN104450556A (en) * | 2014-10-09 | 2015-03-25 | 扬州优邦生物制药有限公司 | Serum-12 type haemophilus lus paradis vaccine strain and application thereof |
| CN104450555A (en) * | 2014-10-09 | 2015-03-25 | 扬州优邦生物制药有限公司 | Serum-13 type haemophilus lus paradis vaccine strain and application thereof |
| CN104498384B (en) * | 2014-10-28 | 2017-05-24 | 中国动物卫生与流行病学中心 | Serum 13 type haemophilus parasuis and use thereof |
| CN104611274A (en) * | 2015-02-11 | 2015-05-13 | 青岛农业大学 | Haemophilus parasuis and application thereof |
-
2015
- 2015-09-25 CN CN201510618836.2A patent/CN105524857A/en active Pending
- 2015-09-25 CN CN201510616476.2A patent/CN105112342B/en active Active
- 2015-09-25 CN CN201510616477.7A patent/CN105505811A/en active Pending
- 2015-09-25 CN CN201510617867.6A patent/CN105543120B/en active Active
- 2015-09-25 CN CN201510620815.4A patent/CN105420141B/en active Active
- 2015-09-25 CN CN201510620812.0A patent/CN105148262B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130129682A1 (en) * | 2009-11-04 | 2013-05-23 | Regents Of The University Of Minnesota | Haemophilus parasuis polypeptides and methods of use |
| CN102908615A (en) * | 2011-08-01 | 2013-02-06 | 普莱柯生物工程股份有限公司 | Novel haemophilus parasuis disease trivalent inactivated vaccine and preparation method thereof |
| CN104248755A (en) * | 2013-10-30 | 2014-12-31 | 普莱柯生物工程股份有限公司 | Haemophilus parasuis disease vaccine composition, preparation method and application thereof |
| CN104452556A (en) * | 2014-12-02 | 2015-03-25 | 吉林大学 | Verification system for vehicle-mounted pavement primary crack acquisition system based on line structured light reference |
Non-Patent Citations (3)
| Title |
|---|
| MIAO LI 等: "Identification of secreted proteins as novel antigenic vaccine candidates of Haemophilus parasuis serovar 5", 《VACCINE》 * |
| 徐惠娟: "副猪嗜血杆菌病灭活疫苗菌株的筛选", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 樊祜卿: "副猪嗜血杆菌流行病学研究及疫苗候选株的筛选", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105148262A (en) | 2015-12-16 |
| CN105420141B (en) | 2019-05-14 |
| CN105543120A (en) | 2016-05-04 |
| CN105112342B (en) | 2019-03-15 |
| CN105524857A (en) | 2016-04-27 |
| CN105148262B (en) | 2018-07-10 |
| CN105112342A (en) | 2015-12-02 |
| CN105543120B (en) | 2020-06-19 |
| CN105505811A (en) | 2016-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102499982B (en) | Method for producing trivalent inactivated vaccine against Haemophilus parasuis infection | |
| CN103263666B (en) | Porcine circovirus 2 type, porcine mycoplasmal pneumonia bivalent inactivated vaccine and preparation method thereof | |
| CN101144062B (en) | Lactobacillus casei strain and application for products thereof in bird immunity | |
| CN106591244B (en) | A kind of Porcine epidemic diarrhea virus, inactivated vaccine and preparation method thereof | |
| CN102058880A (en) | Method for producing trivalent inactivated vaccines for porcine infectious pleuropneumonia | |
| CN106929451A (en) | One plant of Streptococcus suis and its application | |
| CN105420141A (en) | Haemophilus parasuis strain | |
| CN103160555A (en) | Culture medium, culture method and application of high-yield exotoxin of clostridium perfringens | |
| CN104163858B (en) | Pasteurella multocida acellular antigen, preparation method and applications thereof | |
| CN104611274A (en) | Haemophilus parasuis and application thereof | |
| CN109554420B (en) | Clostridium perfringens type B exotoxin and preparation method, toxin production medium and application thereof | |
| CN104069489B (en) | Newcastle disease and infectious bursa of Fabricius bivalent inactivated vaccine and preparation method thereof | |
| CN105754905A (en) | Pseudomonas aeruginosa of minks and application of pseudomonas aeruginosa | |
| AU2020103220A4 (en) | Exotoxin produced by veterinary Clostridium Novyi and preparation method thereof, culture medium for toxin production and use | |
| CN105169380A (en) | Bivalent propolis inactivated vaccine for rabbit hemorrhagic disease and multocida pasteurellosis and preparation method of bivalent propolis inactivated vaccine | |
| CN110075289A (en) | A kind of haemophilus parasuis, Streptococcus suis and Actinobacillus pleuropneumoniae triple inactivated vaccine and its application | |
| CN103409374A (en) | Trigeminy inactivated vaccine for porcine circovirus disease, porcine streptococcus suis disease and porcine haemophilus parasuis disease, preparation method of the vaccine and applications of the vaccine | |
| CN105749266A (en) | Mink hemorrhagic pneumonia and botulism combined inactivate vaccine and preparing method thereof | |
| CN108273051B (en) | The rapidly and efficiently preparation method of pig A type C.perfringens inactivated vaccine | |
| CN109943507B (en) | Preparation method and application of veterinary A-type clostridium perfringens toxin | |
| CN103182077B (en) | Application of serum 4 type haemophilus parasuis vaccine strain | |
| CN102600469A (en) | Classical swine fever live vaccine | |
| CN105169382A (en) | Inactivated quadrivalent propolis vaccine for haemophilus parasuis disease and preparation method of inactivated quadrivalent propolis vaccine | |
| CN103800899A (en) | Milk cattle mastitis vaccine | |
| CN100366291C (en) | O, A type bivalent inactivated vaccine for foot-and-mouth disease of ruminant as cattle and sheep |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |