CN103585622A - Application of swine mycoplasma pneumonia vaccine strain - Google Patents
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Abstract
The invention relates to application of a swine mycoplasma pneumonia vaccine strain. The swine mycoplasma pneumonia vaccine strain No.NJ is classified and named as Mycoplasma hyopneumoniae, is preserved in China's typical culture collection center with a preservation number of CCTCC and the No. of M2012286 at July 16th, 2012. The drug is a vaccine. The inactivated vaccine provided by the invention is simple and convenient to produce, safe, reliable and excellent in protection effect, is equivalent to or superior to the existing commercial vaccines in the market as for the vaccine effect, and can effectively prevent prevalence of swine mycoplasma pneumonia.
Description
Technical field
The present invention relates to a kind of application of mycoplasmal pneumonia of swine vaccine strain, belong to veterinary biologics field.
Technical background
Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) be to cause mycoplasmal pneumonia of swine (Mycoplasmal pneumoniae of swine, Mps claims again swine enzootic pneumonia) main pathogen, be also a kind of important constitutional cause of disease of porcine respiratory disease syndrome.Mhp main infection porcine respiratory, pathogenicity own is not strong, clinical symptoms mainly take cough and asthma as main, characteristics of lesion is mainly sharp leaf, lobus cardiacus, the middle leaf of lung and is " meat sample " or " Macrobrachium nipponensis sample " consolidation every leaf leading edge.After Mhp infects body, cilium main and respiratory tract inwall sticks, cause ciliated cell pathological changes and apoptosis, cause cilium fracture and come off, cause body morbidity, and can have a strong impact on growth promoter and the feed conversion rate of pig, or destroy mucosa-cilium barrier, the easily infection (particularly to young pig) of other pathogenic bacterium of secondary, improve fatality rate, thereby cause huge economic loss to pig industry.At present the main prophylactico-therapeutic measures of mycoplasmal pneumonia of swine is vaccine and medicine, but effectively medicine lacks, and therefore, carrying out this sick vaccine development will have great importance for the prevention of primary disease and control.
Summary of the invention
Technical problem to be solved by this invention is to provide the application of mycoplasmal pneumonia of swine vaccine strain, and this vaccine strain has certain pathogenic and have a good immunogenicity to pig.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The application of a kind of mycoplasmal pneumonia of swine vaccine strain in preparation treatment mycoplasmal pneumonia of swine medicine;
Wherein, described mycoplasmal pneumonia of swine vaccine strain, its Classification And Nomenclature is mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), bacterial strain number is NJ, be preserved in Chinese Typical Representative culture collection center, address: China. Wuhan. Wuhan University, postcode 430072, deposit number: CCTCC NO:M2012286, preservation date: on July 16th, 2012.This bacterial strain is that inventor gathers voluntarily Screening and Identification and obtains a strain mycoplasmal pneumonia of swine vaccine strain in August, 2004 in pig farm, Nanjing.
Wherein, described medicine is vaccine.
Wherein, every part 1～2ml of described vaccine comprises following component:
Wherein, described immunological adjuvant is injection white oil, ISA206VG(SEPPIC), ISA201VG(SEPPIC), Gel01ST(SEPPIC) or ISA11R VG(SEPPIC) etc.
Wherein, described vaccine prepares as follows:
1) produce the preparation of strain: get lyophilizing mycoplasmal pneumonia of swine vaccine strain CCTCC NO:M2012286, with cultivating 3～10 after fluid medium dilution, purely after the assay was approved, as first order seed.At 37 ℃, cultivate pH value after 3～7 days and reduce to 6.6～7.0 results, through purely after the assay was approved, as secondary seed.
2) preparation of bacterium liquid for seedling: mycoplasmal pneumonia of swine vaccine strain secondary seed solution is inoculated in fluid medium and cultivates 3～10, then amplification culture in the same way, until the required amount of seedling, and guarantee that subculture was no more than for 6 generations;
3) seedling bacterium liquid deactivation and join Seedling: by step 2) obtaining, adds the thimerosal of formula ratio, 37 ℃ of deactivations; The aseptic PBS buffer and the immunological adjuvant that add formula ratio, mixing and emulsifying obtains mycoplasmal pneumonia of swine inactivated vaccine.
Wherein, described liquid culture based formulas is: EaglesShi liquid 1000mL, lactoalbumin hydrolysate 10.0g, porcine blood serum 200～400mL, fresh yeast diffusion juice 10～20mL, phosphate buffer 600mL, penicillin 200～1,000 ten thousand units, 0.4% phenol red 3.5mL is 7.4～7.6 with 10g/L NaOH adjust pH.
Beneficial effect: the present invention has advantages of following outstanding:
1) bacterial strain immunogenicity is good: mycoplasmal pneumonia of swine vaccine strain of the present invention is that local isolated strains obtains to have and cultivates the bacterial strain that titre is high and immunogenicity is good after the Screening and Identification that goes down to posterity, and has guaranteed specificity and the immune efficacy of vaccine.
2) production of vaccine is easy: mycoplasmal pneumonia of swine vaccine strain of the present invention is the local isolated strains through cultivating, and has the cultural character of high titre, has guaranteed the high titre of antigen for vaccine, has reduced concentration step; In the present invention, apply thimerosal deactivation, thimerosal itself is a kind of antiseptic, with it, comes deactivation to take into account deactivation and anticorrosion, has reduced consumption, has reduced the untoward reaction to animal; The immunological adjuvant composition using in inactivated vaccine of the present invention is simple, and seedling method is easy, has simplified production technology, has reduced and has produced the risk of polluting.
3) vaccine safety is efficient: mycoplasmal pneumonia of swine vaccine strain of the present invention is the Local Isolates through cultivating, there is the feature that titre is high, immunogenicity is good of cultivating, the adjuvant using is the ripe commercialization adjuvant through market test, has guaranteed the safety and efficiently of vaccine.Effect relatively finds that the vaccine immunity protection effect of preparing with this bacterial strain is significantly higher than import vaccine.
In a word, that inactivated vaccine of the present invention is produced is easy, vaccine safety is efficient, can meet preferably current pig farm in the urgent need to, be easy to apply on a large scale, there are wide market prospect and larger economical, societal benefits.
Accompanying drawing explanation
Fig. 1 mycoplasmal pneumonia of swine inactivated vaccine (NJ strain) preparation flow figure.
The NJ strain of Fig. 2 mycoplasma hyopneumoniae and the comparison of other bacterial strains 16s rRNA gene similarity.
The NJ strain of Fig. 3 mycoplasma hyopneumoniae and other bacterial strains 16s rRNA gene evolution tree.
The NJ strain of Fig. 4 mycoplasma hyopneumoniae and the comparison of other bacterial strains P46 gene similarity.
The NJ strain of Fig. 5 mycoplasma hyopneumoniae and other bacterial strains P46 gene evolution tree.
The specific embodiment
The following examples will further illustrate the present invention, and not limit the scope of the invention.
Embodiment 1: the preparation method of mycoplasmal pneumonia of swine inactivated vaccine (NJ strain).
1 strain culturing
Bacterial strain is selected mycoplasma hyopneumoniae NJ strain, and this bacterial strain, by our isolation identification, goes down to posterity and cultivates rear acquisition, and this bacterial strain send Chinese Typical Representative culture collection center (CCTCC) on July 16th, 2012, and preserving number is: CCTCCM2012286;
2 strain characteristics
2.1 forms and this bacterium of biochemical characteristic are the polymorphic microorganism of Gram-negative, have ring-type, spherical, point-like, shaft-like and the two poles of the earth shape, easy coloring not, acellular wall.Biochemical characteristic should meet the characteristic of this bacterium in systematic bacteriology.
In 2.2 cultural character liquid medium withins (pH value 7.4～7.6), put 37 ℃ and cultivate pH value after 3～7 days and reduce to 6.6～7.0, slight homogeneous is muddy, and viable bacteria titre can reach 10
9more than CCU/mL.Inoculation solid medium, puts 37 ℃ and cultivates after 3～10 days, visible fried egg sample bacterium colony.
Liquid culture based formulas is: EaglesShi liquid 1000mL, lactoalbumin hydrolysate 10.0g, porcine blood serum 200～400mL, fresh yeast diffusion juice 10～20mL, phosphate buffer 600mL, penicillin 200～1,000 ten thousand units, 0.4% phenol red 3.5mL is 7.4～7.6 with 10g/L NaOH adjust pH.
Solid culture formula: add Noble Agar in liquid medium within.
2.3 serological characteristics are identified with metabolic inhibition test, are inoculated in the fluid medium that is added with anti-mycoplasma hyopneumoniae serum, cultivate 10 at 37 ℃, contrast and compare invariant color with culture medium.
2.4 virulence through the healthy susceptible 5mL of intratracheal injection 7～15 ages in days, are cutd open inspection after 28 days by strain culture, and typical mycoplasmal pneumonia of swine pathological changes appears in Pigs Inoculated pulmonary as seen.
2.5 immunogenicities are made vaccine by the present invention, the healthy susceptible pigs of intramuscular injection immunity 7～15 ages in days, booster immunization again after 14 days; Head exempted from after 35 days, together with the identical contrast pig of condition, with mycoplasma hyopneumoniae Js, organized strong malicious intratracheal injection counteracting toxic substances, and counteracting toxic substances cutd open inspection after 28 days, visible immune swine mycoplasmal pneumonia of swine pneumonopathy varying index slip >=60%.
2.6 gene analysis
According to mycoplasma hyopneumoniae 16s rRNA and the P46 gene order of Genbank report, design respectively primer, after pcr amplification, order-checking, the sequence of mensuration is carried out ClustalW with the whole genome sequence of having announced and is compared.
For 16s rRNA gene, NJ strain and GenBank report that the similarity of other bacterial strains is 99.7%～100.0%, and similarity is relatively shown in Fig. 2, and cladogram as shown in Figure 3, be shown in shown in sequence table SEQ ID No:1 by NJ strain 16s rRNA sequence.For P46 gene, NJ strain and GenBank report that the similarity of other bacterial strains is 98.7%～99.9%, and similarity is relatively shown in Fig. 4, and cladogram as shown in Figure 5, be shown in shown in sequence table SEQ ID No:2 by NJ strain P46 sequence.This explanation mycoplasma hyopneumoniae NJ strain is mycoplasma hyopneumoniae, and different from other bacterial strains.
3 produce the preparation of strain
Get mycoplasma hyopneumoniae NJ strain freeze-drying lactobacillus, with after fluid medium dilution, inoculation solid medium is cultivated 10 at 37 ℃, selects well-grown bacterium colony, purely after the assay was approved, and as first order seed.Get again first order seed inoculation fluid medium, at 37 ℃, cultivate pH value after 3～7 days and reduce to 6.6～7.0 results, through purely after the assay was approved, as secondary seed.
The preparation of bacterium liquid for 4 seedlings
Get secondary seed inoculation fluid medium, cultivate 4～7 for 37 ℃, carry out amplification culture after culture variable color, subculture was no more than for 6 generations, until pH value, reduced to 6.6～7.0 o'clock results, according to the regulation of Chinese veterinary pharmacopoeia, purely checked and assay.
The deactivation of 5 antigens
It is 0.005% thimerosal that the seedling being up to the standards is added to final concentration with bacterium liquid, and 37 ℃ are carried out deactivation.Get deactivation sample 1mL and join in the fluid medium of 50mL, put 37 ℃ of cultivations, observe 5, then get 0.5mL and be inoculated in 4.5mL fluid medium, put 37 ℃ of cultivations, observe 10, invariant color as equal in twice cultivation, is judged to deactivation and is up to the standards.
6 join Seedling and mix
Every part is no less than 2.86 * 10 containing mycoplasma hyopneumoniae before deactivation
8cCU, joins Seedling ratio and determines (weight percentage of immunological adjuvant in vaccine is 5%～75%) by adjuvant used, and antigen insufficient section is supplied with PBS.
6.1 injection white oils join Seedling and 94 parts of injection white oils (weight ratio) are got in mixing, Si Ben-80 6 part, add in oil phase tank, stir, and through 121 ℃ of sterilizings 30 minutes, are cooled to room temperature standby.Get 96 parts of the bacterium liquid that deactivation is up to the standards, 4 parts of tween 80s, add in water tank, and stirring and evenly mixing is standby.2～3 parts of oil phases are added in emulsion tank, first stirring at low speed 30 minutes, more slowly add 1 part of water, continue to stir, with high-speed shearing machine, carry out emulsifying, before emulsifying finishes, adding final concentration is 0.005%(g/mL) thimerosal solution, make water-in-oil emulsion.
Joining Seedling and mixing and to join to specifications Seedling and mixing 6.2ISA206VG(SEPPIC), ISA201VG(SEPPIC), Gel01ST(SEPPIC), ISA11R VG(SEPPIC).As ISA201VG, by antigen and adjuvant weight ratio 50:50, join Seedling, adjuvant is preheated to 31 ℃, with low-shearing force, stir, maintain 30 ℃, add antigen (water) in adjuvant, stir homogeneous.As ISA11R VG, in the ratio proportioning of adjuvant and antigen weight ratio 15:85, with low shear force agitator, stir homogeneous in antigen water being added to adjuvant under room temperature or lower temperature.As Gel01ST, adjuvant is joined Seedling by 5%～20%, stirs homogeneous in antigen water being added to adjuvant under room temperature or lower temperature with low shear force agitator.
7 product inspections
7.1 character should meet affiliated dosage form.As should be water-in-oil type with injection white oil, as should be oil-in-water water-in type again with ISA201VG, as should be oil-in-water type with ISA11R VG, as should be water aqua type with Gel01ST.
7.2 stability are drawn vaccine 10mL and are added centrifuge tube, and with 3000r/min centrifugal 15 minutes, the water that separate out at the pipe end should be no more than 0.5mL.
7.3 viscositys are tested by existing < < Chinese veterinary pharmacopoeia > > appendix, are no more than 200cP.
7.4 steriling tests are tested by existing < < Chinese veterinary pharmacopoeia > > appendix, asepsis growth.
7.5 safety verifications are with 5 of the healthy susceptible pigs of 7～15 ages in days, and each intramuscular injection vaccine 2mL, observed after 14 days, except occurring that one crosses gonosome temperature rise, other parts and systemic adverse reactions that the vaccinate of having no way of causes.
7.6 efficacy tests, with 15 of the healthy susceptible pigs of 7～15 ages in days, are divided into 3 groups, are respectively counteracting toxic substances matched group, normal healthy controls group and vaccine immunity group, 5 every group.1 part of each intramuscular injection vaccine of vaccine immunity group, after 14 days, then 1 part of each intramuscular injection vaccine.Head exempts from latter 35 days, together with 5 of counteracting toxic substances contrast pigs, and with the strong poison of mycoplasma hyopneumoniae Js strain tissue of normal saline dilution, through intratracheal injection counteracting toxic substances, every 5mL.After counteracting toxic substances 28 days, cut open all test pig of inspection, the left sharp leaf of observed and recorded lung tissue, left lobus cardiacus, left lobus diaphragmaticus, right sharp leaf, right lobus cardiacus, right lobus diaphragmaticus, accessory lobes pathological changes percentage, be designated as 1 by 0～25%; 26～50% are designated as 2; 51～75% are designated as 3; 76～100% are designated as 4, add up each test swine diseases pneumonopathy varying index (being up to 28).Pneumonopathy varying index slip=(counteracting toxic substances matched group pneumonopathy varying index meansigma methods-immune group pneumonopathy varying index meansigma methods)/(counteracting toxic substances matched group pneumonopathy varying index meansigma methods-normal healthy controls group pneumonopathy varying index meansigma methods) * 100%, calculates mycoplasmal pneumonia of swine vaccine immunity group thumps varying index slip by above-mentioned formula.
The 7.7 thimerosal determination of residual amount are measured by existing < < Chinese veterinary pharmacopoeia > > appendix, should meet the regulation of veterinary biologics general rule.
1 part of every intramuscular injection of 8 intramuscular injection piglets.
Embodiment 2: vaccine.
1 vaccine
The oily adjuvant of mycoplasmal pneumonia of swine inactivated vaccine (NJ strain), mycoplasmal pneumonia of swine inactivated vaccine (NJ strain) ISA11RVG, mycoplasmal pneumonia of swine inactivated vaccine (NJ strain) Gel01ST, the mycoplasmal pneumonia of swine inactivated vaccine that abroad certain company produces.
Strong poison for 2 checks
The strong poison of mycoplasma hyopneumoniae Js strain tissue, is prepared by Jiangsu Province Agriculture Science Institute veterinary institute.
3 test pig
30 of the healthy susceptible pigs of 7～15 ages in days, derive from Jiangsu Province agriculture and animal husbandry tomorrow Science and Technology Ltd..
4 EXPERIMENTAL DESIGN
With the oily adjuvant of mycoplasmal pneumonia of swine inactivated vaccine (NJ strain), mycoplasmal pneumonia of swine inactivated vaccine (NJ strain) ISA11RVG, each 1 batch of import mycoplasmal pneumonia of swine inactivated vaccine, the every batch of vaccine is with 5 of 7～15 age in days piglets, the vaccine of every intramuscular injection 1mL, after 14 days with Isodose booster immunization once.Set up pig that condition is identical to make each 5 of counteracting toxic substances matched group and normal healthy controls groups simultaneously.Vaccine immunity group head exempts to observe clinical response in latter 28 days.After head exempts from 35 days, vaccine immunity group and counteracting toxic substances matched group test pig were carried out challenge test, and counteracting toxic substances cutd open inspection after 28 days, and statistics pulmonary lesion is scored, and calculated pneumonopathy varying index slip.
5 clinical responses are observed
Test pig is observed 28 after vaccinate, and the test pig of 3 immune group all occurs after immunity that one crosses heat pyrexia as a result, but all recovers normal in immunity in latter three days.Have no other abnormal clinical response.
6 effect comparative tests
Test pig counteracting toxic substances after 28 days in, observe finding has 4 appearance cough in various degree in 5 first taps poison matched group pigs, respectively has 1～2 to occur slight cough in 3 immune group, normal healthy controls has no the mycoplasmal pneumonia of swine such as cough characteristic symptom.Counteracting toxic substances cutd open inspection after 28 days, and each organizes pneumonopathy varying index, calculated pneumonopathy varying index slip, and effect comparative test the results are shown in Table 1.Have result visible, the immune group pneumonopathy varying index of 3 different adjuvants of mycoplasmal pneumonia of swine inactivated vaccine (NJ) is all significantly lower than counteracting toxic substances matched group and import vaccine group.
The similar vaccine potency comparative test of table 1 result
Note: a～d represents to compare between two between every group, and alphabetical identical person represents that difference is not remarkable, and alphabetical different persons represent significant difference (p<0.05).
Claims (6)
1. a mycoplasmal pneumonia of swine vaccine strain is treated the application in mycoplasmal pneumonia of swine medicine in preparation;
Wherein, described mycoplasmal pneumonia of swine vaccine strain, its Classification And Nomenclature is mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), bacterial strain number is NJ, be preserved in Chinese Typical Representative culture collection center, deposit number: CCTCC NO:M2012286, preservation date: on July 16th, 2012.
2. application according to claim 1, is characterized in that, described medicine is vaccine.
3. application according to claim 2, is characterized in that, every part 1～2ml of described vaccine comprises following component:
4. application according to claim 3, is characterized in that, described immunological adjuvant is injection white oil, MONTANIDE ISA206VG, ISA201VG, Gel01ST, ISA11R or ISA760VG etc.
5. application according to claim 3, is characterized in that, described vaccine prepares as follows:
1) produce the preparation of strain: get lyophilizing mycoplasmal pneumonia of swine vaccine strain CCTCC NO:M2012286, after diluting with fluid medium, cultivate after 3～10 days as first order seed; At 37 ℃, cultivate pH value after 3～7 days and reduce to 6.6～7.0 results as secondary seed;
2) preparation of bacterium liquid for seedling: mycoplasmal pneumonia of swine vaccine strain secondary seed solution is inoculated in fluid medium and cultivates 3～7, then amplification culture in the same way, until the required amount of seedling, and guarantee that subculture was no more than for 6 generations;
3) seedling bacterium liquid deactivation and join Seedling: respectively by step 2) obtaining, adds the thimerosal of formula ratio, 37 ℃ of deactivations; The aseptic PBS buffer and the immunological adjuvant that add formula ratio, mixing and emulsifying obtains mycoplasmal pneumonia of swine inactivated vaccine.
6. application according to claim 5, it is characterized in that described liquid culture based formulas is: EaglesShi liquid 1000mL, lactoalbumin hydrolysate 10.0g, porcine blood serum 200～400mL, fresh yeast diffusion juice 10～20mL, phosphate buffer 600mL, penicillin 200～1,000 ten thousand units, 0.4% phenol red 3.5mL is 7.4～7.6 with 10g/L NaOH adjust pH.
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| CN104399070A (en) * | 2014-10-31 | 2015-03-11 | 哈药集团生物疫苗有限公司 | Swine mycoplasma pneumonia inactivated vaccine and preparation method thereof |
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