CN104894009B - One plant of mycoplasma hyopneumoniae strain and its application - Google Patents
One plant of mycoplasma hyopneumoniae strain and its application Download PDFInfo
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Abstract
The present invention relates to one plant of mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) strain, S strains, the bacterial strain is the new separation strains of mycoplasma hyopneumoniae, stable on cell-free medium can be grown, growth titre can reach 109~1010CCU/mL, confirm that its immunogenicity is good through rabbit and piglet immunological experiment；The R1 areas amino acid of bacterial strain P97 genes contains 11 AA (V/T) PKE/V continuous amino acid sequence, mark of molecular biology that can be unique as its, and this plant of mycoplasma hyopneumoniae strain can be used for preparing mycoplasma hyopneumoniae inactivated vaccine.
Description
Technical field belongs to veterinary microbiology field the present invention relates to one plant of mycoplasma hyopneumoniae strain and its application.
Background technology
Porcine mycoplasmal pneumonia is also known as mycoplasma pneumonia of swine, be by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae,
Mhp the contact chronic respiratory disease of a boar caused by), it is generally popular in the world.According to document announcement, separate all over the world
Mycoplasma hyopneumoniae belong to same serotype, but have larger difference between genome between different separation strains.Frey in 1992
Deng (Frey, J., Haldimann, A.＆Nicolet, J. (1992) .Chromosomal heterogeneity of various
Mycoplasma hyopneumoniae field strains.Int J Syst Bacteriol42,275-280) demonstrate,prove first
The difference of genome between real mycoplasma hyopneumoniae difference separation strains, Artiushin and Minion (1996) (Artiushin,
S.&Minion,F.C.(1996).Arbitrarily primed PCR analysis of Mycoplasma
hyopneumoniae field isolates demonstrates genetic heterogeneity.Int J Syst
Bacteriol 46,324-328.) further demonstrate Frey viewpoint, Kokotovic etc. (1999) (Kokotovic,
B.,Friis,N.F.,Jensen,J.S.&Ahrens,P.(1999).Amplified-fragment length
polymorphism fingerprinting of Mycoplasma species.J Clin Microbiol 37,3300-
3307.) result of study has also drawn same conclusion.
Mycoplasma is can be with the minimum prokaryotes of self-replacation, and slow-growing, the requirement to culture medium is higher.With other
Common mycoplasma is compared, and Mhp growths are more slow, and the requirement in vitro culture seems harsher, is referred to as " fastidious micro- life
Thing ".Domestic and international experts and scholars have developed polytype nutrient medium and be used for Mhp separation, culture by studying for a long period of time,
Such as A26 mycoplasmas bodily form culture medium, KM2 culture mediums, Friis culture mediums and OX-heart soup culture medium.But, these are cultivated still
It is still unsatisfactory in the presence of more or less deficiency, culture effect.Therefore the good vaccine of antigenicity, immunogenicity is isolated
Strain tool is of great significance.
Mycoplasma hyopneumoniae genome contains rare codon, and certified antigen protein only has a few so far.Mesh
Before think that the major antigen albumen of mycoplasma hyopneumoniae has P97, P65, P46, P36 etc., wherein P46 is that Mhp has very strong kind
Specific memebrane protein, it can cause body early immune to react.Nineteen ninety-five, Futo etc. (Futo, S., Seto, Y.,
Mitsuse,S.,Mori,Y.,Suzuki,T.&Kawai,K.(1995).Molecular cloning of a 46-
kilodalton surface antigen(P46)gene from Mycoplasma hyopneumoniae:direct
Evidence of CGG codon usage for arginine.JBacteriol 177,1915-1917.) find P46 bases
Because of (1257bp), 1 CGG (arginine being encoded in Mhp, be nonsense codon in other mycoplasmas) is included in its ORF
With 3 TGA codons (codes for amino acid tryptophan, is terminator codon in universal codons in mycoplasma).P97 is Mhp master
Adhesion factor is wanted, its R1 area of different strains includes AAKPV/E primitives (Hsu, T., Artiushin, the S.＆ of different numbers
Minion,F.C.(1997).Cloning and functional analysis of the P97swine cilium
adhesin gene of Mycoplasma hyopneumoniae.JBacteriol 179,1317-1323).Therefore P46 eggs
White and P97 proteinic genomes sequencing, the mark of molecular biology that can be identified as mycoplasma hyopneumoniae difference strain.
The content of the invention
It is an object of the invention to provide one plant of mycoplasma hyopneumoniae strain, the bacterial strain grows good on cell-free medium
It is good, and immunogenicity is strong.Mycoplasma hyopneumoniae bacterial strain is accredited as through mycoplasma hyopneumoniae 16s RNA specific primers, by it
Mycoplasma hyopneumoniae S strains are named as, are also identified again after mycoplasma hyopneumoniae P46 primers and the amplification of P97R1 primer specificities
For mycoplasma hyopneumoniae, its base and amino acid sequence are with such as 232 plants of other mycoplasma hyopneumoniae bacterial strains and JN F65 strains at certain
A little positions are different, can be that the molecular biology identification of the bacterial strain does molecular labeling；The bacterial strain can be used for preparing porcine mycoplasmal inactivation
Vaccine.
Technical scheme
1. a kind of mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) S strains, the bacterial strain is in 2014
Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain was delivered on December 1 to protect
Hide administration committee's common micro-organisms center preservation, preserving number CGMCC No.10095.
2. i (mycoplasma hyopneumoniae) vaccine strain S strains of the present invention, the number of base sequence of its P46 gene are：
Corresponding amino acid sequence is：
3. i (mycoplasma hyopneumoniae) vaccine strain S strains of the present invention, the R1 areas base sequence of its P97 gene are：
Corresponding amino acid sequence is：
4. the application of i (mycoplasma hyopneumoniae) vaccine strain S strains of the present invention, its feature can be used as production of vaccine in the bacterial strain
Bacterial strain is used to prepare mycoplasma hyopneumoniae inactivated vaccine.
The detailed description of the present invention
First, the separation and identification of mycoplasma hyopneumoniae
1. pathological material of disease source：
Strain isolation from the lung tissue of Jilin songyuan pig farm morbid pig, through PCR detect the pathological material of disease without blue otopathy, swine fever,
The pathogen infections such as pseudo- mad dog, actinobacillus pleuropneumoniae, haemophilus parasuis, pathological material of disease is after homogenized, by (V/V)
Be inoculated into our company homemade LPS culture medium (LPS medium component at 1: 10：PPLO 5g, dusty yeast 10g, glucose 5g,
Hanks liquid 1000ml, Swine serum 10%~20%；Optimum pH：7.4~7.8, cultivation temperature is 37 DEG C), 37 DEG C of cultures 7~
10 days, the generation of continuous passage 3~5, isolate can on LPS culture mediums 37 DEG C cultivate 72~96 hours, Medium's PH Value by 7.6 drop
To 6.8~6.9.
2. condition of culture
Above liquid culture is taken, is inoculated with LPS culture mediums by 10% (V/V), 37 DEG C are cultivated 72~96 hours, medium pH
Value is down to 6.6~6.8 by 7.6, and culture is in homogeneous slightly muddy brownish red, no precipitation.Culture, should be sterile through steriling test
Growth.
3. serological Identification
Above culture is taken, culture is diluted to 10-4~10-5CCU/mL, respectively with mycoplasma hyopneumoniae positive serum
Metabolic inhibition test is carried out, as a result shows that mycoplasma hyopneumoniae positive serum can suppress the growth of culture, shows what is be separated to
The culture is mycoplasma hyopneumoniae.
4. molecular biology identification
The above culture carries out 1000 times of dilutions, with pyrolysis method (Shen Qingchun, Qin Qingsong, Wang Qin etc. (2006) .PCR
Method measure mycoplasma hyopneumoniae culture bacterium number China Preventive Veterinary Medicine report, 55-57.)) DNA is extracted as template, difference
Enter performing PCR respectively with mycoplasma hyopneumoniae and mycoplasma hyorhinis 16s RNA specific primers to expand, as a result show that measuring samples are swum
There is 649bp mycoplasma hyopneumoniae specific band (see Fig. 1) in road, and uses mycoplasma hyorhinis P37 protein-specific primers
Fail to amplify any band (see Fig. 2).As can be seen here, isolate is mycoplasma hyopneumoniae, and does not contain mycoplasma hyorhinis.
Show that the bacterial strain is mycoplasma hyopneumoniae by above serological Identification and 16s RNAPCR primer qualification results, will
It names mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae Mhp) S strains, and the bacterial strain is sent on December 1st, 2014
Hand over Chinese microorganism strain preservation management committee of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, preserving number CGMCC No.10095.
5. sequence analysis
Mycoplasma hyopneumoniae S strains bacterium solution is taken to extract DNA as template by the use of pyrolysis method, with using mycoplasma hyopneumoniae respectively
P46PCR primers and P97R1 areas PCR primer carry out specific amplification, obtain specific band, after being cloned into carrier T, enter
Row sequence analysis.
6. immunogenicity is examined
With viable bacteria titre up to 109After CCU/mL S strains bacterium solution inactivation, inactivated vaccine is prepared with suitable adjuvant, is inoculated with respectively
Rabbit and 7 age in days sodium selenites, by determining that rabbit antibodies are horizontal and the malicious protection of attacking of piglet carries out immunogenicity to it and commented
Valency.
2nd, the preparation of mycoplasma hyopneumoniae inactivated vaccine
(1) preparation of vaccine manufacture antigen：Secondary seed is pressed 1:20 inoculation Lps-5 culture mediums, 37 DEG C cultivate 3~
6, when pH value reaches 6.9 or so, culture is harvested, then by 1:50 ratios Secondary Culture in the same way, until harvesting bacterium
Liquid manufactures vaccine；
(2) inactivate：Bacterium solution puts inactivation treatment through beta-propiolactone after culture, adds thimerosal as preservative；
(3) seedling is matched somebody with somebody：Bacterium solution presses (V/V) 1 with adjuvant (oily adjuvant or the water adjuvant of match BIC Corp of France) after inactivating:1
Ratio mixing, be prepared into mycoplasma hyopneumoniae inactivated vaccine.
Microbial resources information of the present invention
Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae Mhp), S strains, the bacterial strain is December 1 in 2014
Day delivers Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica Chinese microorganism strain preservation pipe
The common micro-organisms center preservation of the reason committee, preserving number CGMCC No.10095.
Brief description of the drawings
Fig. 1：Mycoplasma hyopneumoniae 16s RNA primer amplified electrophoretograms.
Fig. 2：Mycoplasma hyorhinis P37 protein-specifics primer expands electrophoretogram.
Fig. 3：Mycoplasma hyopneumoniae S strain P46 gene amplification fragment electrophoretograms.
Fig. 4：Mycoplasma hyopneumoniae S strain P97 gene R1 areas amplified fragments electrophoretogram.
The positive effect of the present invention
The present invention relates to one plant of mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) S strains, there is typical pig pneumonia
Pig lungs of the mycoplasma infection simultaneously without other obvious pathogen infections, as obtained by appropriate media continuous passage.Pass through molecule
Biology and serological Identification, it was demonstrated that the bacterial strain is the new separation strains of mycoplasma hyopneumoniae, can the stable life on cell-free medium
Long, growth titre can reach 109~1010CCU/mL, confirm that its immunogenicity is good through rabbit and piglet immunological experiment, can be as
The production strain of mycoplasma hyopneumoniae inactivated vaccine.Through entering with other mycoplasma hyopneumoniae bacterial strains, such as 232 plants and JNF65 strains
Row compares, and its P46 genes base sequence and amino acid sequence have part difference, but homology is higher, and P97 gene base sequences
Larger, and continuous amino acid sequence of the P97 aminopeptidase genes acid containing 11 AAPKE/V is distinguished with amino acid sequence, can conduct
Its unique mark of molecular biology；This plant of mycoplasma hyopneumoniae bacterial strain can be used for preparing mycoplasma hyopneumoniae inactivated vaccine.
Embodiment
Below to further illustrate the present invention, the present invention is not defined.
Embodiment 1
--- the culture of mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) S strains
S strains strain 10% is inoculated with mycoplasma hyopneumoniae LPS culture mediums, 37 DEG C are cultivated 72~96 hours, when pH value of solution extremely
When 6.6~6.8, yellowly or orange-yellow is uniformly muddy without precipitation, harvests bacterium solution, after passing on 2 times, dilution bacterium solution carries out CCU meters
Number (colour change unit, CCU), viable count is up to 109~1010CCU/mL。
Embodiment 2
--- the mycoplasma hyopneumoniae 16s RNA specific primers PCR measure of mycoplasma hyopneumoniae S strain bacterium solutions
1. design of primers
According to the 16s RNA for the mycoplasma hyopneumoniae announced on genebank gene order, with reference to Shen Qingchun etc. (2006
Year) (Shen Qingchun, Qin Qingsong, Wang Qin etc. (2006) .PCR methods measure mycoplasma hyopneumoniae culture bacterium number China prevention animal doctors
Journal, 55-57.) synthetic primer.
Sense primer P1 sequences are：5'-GAGCCTTCAAGCTTCACCAAGA-3 ' (sequence 1)
Anti-sense primer P2 sequences are：5'-TGTGTTAGTGACTTTTGCCACC-3 ' (sequence 2)
It is expected that amplified fragments total length is 649bp.
2. template extraction
Take cultured bacterium solution 1mL to carry out 12000r/min centrifugation 15min, abandon supernatant.Precipitation is molten with 100 1 × TEN of μ l
Liquid suspends, and boiling water bath 10min, is stored at room temperature 3min.12000r/min centrifuges 2min, and supernatant is template DNA.
3.PCR is expanded
PCR reagent is purchased from Beijing Quanshijin Biotechnology Co., Ltd, PCR reaction systems：2×EasyTaq PCR
The μ L of SuperMix 10, each 1 μ L of upstream and downstream primer, the μ L of template 2, moisturizing to 20 μ L.Reaction condition is 94 DEG C of pre-degeneration 5min, 95
DEG C denaturation 50s, 60 DEG C annealing 30s, 72 DEG C extension 50s, 35 circulation, 72 DEG C eventually extension 5min.
4. running gel is imaged
PCR primer carries out 1.0% agarose gel electrophoresis, and amplified fragments 649bp is shown in Fig. 1.
5. conclusion isolate is accredited as containing mycoplasma hyopneumoniae through PCR.
Embodiment 3
--- mycoplasma hyorhinis P37 protein-specifics primer PCR determines
1. design of primers
Primer is designed and synthesized according to the P37 for the mycoplasma hyorhinis announced on genebank gene order.
Sense primer P1 sequences are：5 '-GTAGTCAAGCAAGAGGATGT-3 ' (sequence 3)
Anti-sense primer P2 sequences are：5 '-GCTGGAGTTATTATACCAGGA-3 ' (sequence 4), amplified fragments total length are
346bp。
2. template extraction
Take cultured bacterium solution 1mL to carry out 12000r/min centrifugation 15min, abandon supernatant.Precipitation is molten with 100 1 × TEN of μ l
Liquid suspends, and boiling water bath 10min, is stored at room temperature 3min.12000r/min centrifuges 2min, and supernatant is template DNA.
3.PCR is expanded
PCR reagent is purchased from Beijing Quanshijin Biotechnology Co., Ltd, PCR reaction systems：2×EasyTaq PCR
The μ L of SuperMix 10, each 1 μ L of upstream and downstream primer, the μ L of template 2, moisturizing to 20 μ L.PCR reaction conditions：94 DEG C of pre-degeneration 5min,
95 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 50s, 35 circulate, 72 DEG C of whole extension 5min.
4. running gel is imaged
PCR primer carries out 1.0% agarose gel electrophoresis, and mycoplasma hyorhinis BTS-7 strains (are supervised purchased from Chinese veterinary medicament
Institute) amplified fragments 346bp, S strain without specific amplification fragment, is shown in Fig. 2.
5. conclusion isolate is accredited as through PCR does not contain mycoplasma hyorhinis (note：Hold very much when being separated due to mycoplasma hyopneumoniae
It is vulnerable to the pollution of mycoplasma hyorhinis, causes isolate impure and influence subsequent research work).
Embodiment 4
--- the P46 gene sequencing of S bacterium solutions
1. design of primers：
According to the P46 for the mycoplasma hyopneumoniae announced on genebank gene order and Zhang Fenggang etc.《Pig pneumonia branch is former
The separation of body and PCR identifications》On the primer sequence synthetic primer delivered, amplified fragments total length is 1162bp.Its primer：
Sense primer P1 sequences are：5'-CGGGATCCACTTCAGATTCTAAACCACAA-3 ' (sequence 5),
Anti-sense primer P2 sequences are：5'-CCAAGCTTTTAGGCATCAGGATTATCAAC-3 ' (sequence 6).
2. template extraction
S bacterium solutions, 232 plants of bacterium solutions and each 1mL of JNF65 strain bacterium solutions are taken, 12000r/min centrifugation 15min is carried out, abandons supernatant.It is heavy
100 μ l 1 × TEN solution suspensions of shallow lake, boiling water bath 10min, are stored at room temperature 3min.12000r/min centrifuges 2min, and supernatant is mould
Plate DNA.
3.PCR is expanded
PCR reagent is purchased from Beijing Quanshijin Biotechnology Co., Ltd, PCR reaction systems：2×EasyTaq PCR
SuperMix10 μ L, each 1 μ L of upstream and downstream primer, the μ L of template 2, moisturizing to 20 μ L.PCR reaction conditions：94 DEG C of pre-degeneration 4min, 94
DEG C denaturation 30s, 57 DEG C annealing 30s, 72 DEG C extension 50s, 35 circulation, 72 DEG C eventually extension 10min.
4. running gel is imaged
PCR primer carries out 1.0% agarose gel electrophoresis, amplified fragments 1162bp, with Mhp232 amplified fragments size one
Cause, see Fig. 4.
5.S strain P46 Gene Partials are sequenced and sequence results
S strains P46 PCR primer is sequenced, and the logical sequencing base sequence of two-way survey is (sequence 7)：
Translation turns into amino acid sequence, is as a result (sequence 8)：
* the amino acid of boldface is in sequence：The tryptophan encoded by TGA, TGA is close to terminate in universal codons
Numeral.
Pass through the BLAST software on-line analyses on NCBI websites, the results showed that above nucleotide sequence and mycoplasma hyopneumoniae
232 plants of international standard bacterial strain (12 bases have difference), J strains (10 bases have difference), 7448 plants (14 bases have difference)
Deng respective regions homology up to 99%；Homology is higher on amino acid sequence, and an amino acid is differed only by with 7448 plants
(221 Q-A), two amino acid (182 G-Q and 221 Q-A) are differed with 232 plants.
6. conclusion
Seen by mycoplasma hyopneumoniae S strain P46 portion gene sequencing results, with the corresponding sequence homologys of other bacterial strains compared with
Height, nucleotide sequence and amino acid sequence reach 99% homology, and this is consistent with the well-conserved feature of P46 genes.
Embodiment 5
--- the P97 gene R1 areas sequencing of S strains
1. design of primers：
According to the nucleotide sequence in 232 plants of P97 gene R1 areas of the mycoplasma hyopneumoniae announced on genebank, design is closed
Into 1 pair of primer, amplified fragments contain the total length in R1 areas, and the estimated size of 232 plants of amplified productions is about 320bp, and different strains expand
It is variant to open up fragment length.
Sense primer P1 sequences are：5'-CAAAAGAAGG TAAAAGAGAA-3'(sequences 9)
Anti-sense primer P2 sequences are：5'-AAGCCAGTAT TAGTAGCAAC-3'(sequences 10).
2. template extraction
S strain bacterium solution 1mL are taken, 12000rpm centrifugation 15min is carried out, abandons supernatant.100 μ l 1 × TEN solution suspensions of precipitation,
Boiling water bath 10min, is stored at room temperature 3min.12000r/min centrifuges 2min, and supernatant is template DNA.
3.PCR is expanded
PCR reagent is purchased from Beijing Quanshijin Biotechnology Co., Ltd, PCR reaction systems：2×EasyTaq PCR
The μ L of SuperMix 10, each 1 μ L of upstream and downstream primer, the μ L of template 2, moisturizing to 20 μ L.PCR reaction conditions：94 DEG C of pre-degeneration 4min,
94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extension 50s, 35 circulate, 72 DEG C of whole extension 10min.
4. running gel is imaged
Amplified production is through 1.0% agarose gel electrophoresis, the results showed that the amplified fragments size of S strains is about 180bp, sees figure
4。
The part sequencing of 5.P97 genes and sequence results (sequence 11)
It is amino acid sequence using DNAstar software translations, its result is (sequence 12)：
* the amino acid for having underscore in sequence is repetitive sequence, altogether 11 Ala Ala(Val/Thr)Lys Pro Glu
(Val) repeat, be abbreviated as AA (V/T) PKE/V.
The R1 areas of the P97 genes of mycoplasma hyopneumoniae S strains amount to the amino acid sequence weight for including 11 AA (V/T) PKE/V
It is multiple, other bacterial strains are different from, it is identical with 168 plant weight plural numbers, but wherein have the difference of up to 7 amino acid, such as table 1 below.
The difference of the R1 region amino acid sequences of 1 four plants of mycoplasma hyopneumoniae P97 genes of table
Note："-" represents missing；Underscore represents different in this place from S strains.
Embodiment 6
--- the preparation and inspection of porcine mycoplasmal pneumonia inactivated vaccine (S strains)
1. bacterium solution culture
Mycoplasma hyopneumoniae S strains freeze-drying lactobacillus mycoplasma hyopneumoniae LPS culture mediums are redissolved, 1:10 inoculation pig pneumonia branch
Substance LPS culture mediums, 37 DEG C are cultivated 72~96 hours, when solution colour be in brownish red (pH to 6.6~6.8), uniform muddy nothing
Precipitation, harvest bacterium solution.S strains bacterium solution was continuously passed on into for 2 generations on LPS culture mediums, makes viable count up to 109CCU/mL, steriling test
Bacterium solution is used to prepare vaccine after qualified.
2. prepare vaccine
After examining qualified S strains bacterium solution to inactivate, mixed with adjuvant (oily adjuvant or the water adjuvant of match BIC Corp of France)
Emulsification seedling is carried out, vaccine bacteria containing amount is up to 5 × 108CCU/mL, it is prepared into mycoplasma hyopneumoniae inactivated vaccine vaccine.
3. vaccine test
3.1 characters examine vaccine outward appearance to be creamy white emulsion.
Formulation water-in-oil-in water.A cleaning suction pipe is taken, a little vaccine is drawn and drips in cold water surface, should be in that cloud expands
Dissipate.
Stability is drawn vaccine 10mL and added in centrifuge tube, centrifuges 15min with 3000r/min, the aqueous phase that ttom of pipe suctions out should
Not more than 0.5mL.
Viscosity is according to existing《Chinese veterinary pharmacopoeia》(the Chinese veterinary pharmacopoeia committee compiles.Republic of China Veterinary Pharmacopoeia 2010
Nian Bansanbu Chinese agriculture publishing houses, 2011, the present invention was hereinafter referred to as《Chinese veterinary pharmacopoeia》) carry out, regulation should be met.
3.2 steriling tests are according to existing《Chinese veterinary pharmacopoeia》Carry out, answer asepsis growth.
3.3 safety verification
Every batch of vaccine intraperitoneal injection 15~22g Balb/C mouse 8 of mouse, 0.5mL/ only, is observed 10, observes mouse
Survival condition.
7 age in days piglet of every batch of vaccine intramuscular injection of piglet 5,2mL/ heads, head exempt from after 14 days, again intramuscular injection 2mL,
Observation 30 days, observe piglet survival condition and whether there is side reaction, and exempt from continuously to survey body in latter 7 days respectively at being immunized 0 day, 7 days and two
Temperature, observe body temperature situation.
3.4 efficacy test
Piglet extracts 1 batch of porcine mycoplasmal pneumonia inactivated vaccine (S strains) and 1 batch of external import vaccine distinguishes intramuscular injection 7 days
The age susceptible SPF piglets 5 of health, 1mL/ heads, intramuscular injection vaccine 1mL again after 14 days.21 days after first immunisation, together with control pig
5, respectively injected with the strong poison gas pipe of mycoplasma hyopneumoniae ZS strain tissues, every 5mL (containing 40~200 minimum dosage of falling ill).Attack
Poison is after 28 days, cut open inspection, observes pig pulmonary lesion situation, is judged using 55 point-scores
Rabbit by 3 batches of porcine mycoplasmal pneumonia inactivated vaccines (S strains) 1.5~2kg of intramuscular injection rabbit 5,0.1mL/ only,
Intramuscular injection vaccine 0.1mL again after 14 days, separately sets 5 controls.Take a blood sample within 21 days after head exempts from, determine serum I HA antibody titers,
Compare rabbit IHA potency and be not higher than 1:10, the IHA potency at least 4 that rabbit is immunized is not less than 1:40.
4. result
4.1 characters are examined
Appearance milky white emulsion.
Formulation water-in-oil-in water.A cleaning suction pipe is taken, a little vaccine is drawn and drips in cold water surface, spread in cloud.
Stability is drawn vaccine 10mL and added in centrifuge tube, centrifuges 15min with 3000r/min, the aqueous phase that ttom of pipe suctions out is not
More than 0.5mL.
Viscosity is according to existing《Chinese veterinary pharmacopoeia》, it is measured using viscosity apparatus, viscosity number (CP) is respectively 26, is accorded with
Close regulation.
4.2 steriling tests extract 2 bottles of progress steriling tests, observe 7, equal asepsis growth.
4.3 safety examination results
15~22g Balb/C mouse 8 are injected intraperitoneally in 3 batches of vaccines respectively, observe 10, and mouse is strong to live；Overdose flesh
Meat injects piglet, observes 30, and piglet is strong to live, except injection site appearance is slight red and swollen, without other side reactions；
Every group of each temperature of pig body on the 4th~5 slightly raises after vaccinating first, recovers normal after continuing 1~3, two exempt from
Body temperature reaction afterwards is concentrated after injection the 1st day, with and recover normal.
4.4 efficacy test results
4.4.1 rabbit efficacy test result
The comparison of antibody I HA titration results after the rabbit immunization of table 2
As can be seen from the above table, after porcine mycoplasmal pneumonia inactivated vaccine (S strains) immunizing rabbit, IHA antibody titers can reach
1:More than 40, it is horizontal with control group significant difference, the horizontal slightly above similar import vaccine antibody of composite antibody.
4.4.2 piglet immunological attacks toxic effect force inspecting result
The piglet of table 3 attacks the comparison of pulmonary lesion situation after poison
Immunization result of the test shows that porcine mycoplasmal pneumonia inactivated vaccine (S strains) has stronger immune protective efficiency, reaches
More than 80% level, vaccine difference unobvious similar with import.
Claims (4)
1. one plant of mycoplasma hyopneumoniae, it is characterised in that this plant of mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae,
Mhp S strains) are named as, deliver the micro- life of the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences on December 1st, 2014
The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center of thing research institute, preserving number CGMCC No.10095.
2. one plant of mycoplasma hyopneumoniae described in claim 1, it is characterised in that the number of base sequence of the P46 genes of this plant of mycoplasma
It is classified as：
Corresponding amino acid sequence is：
3. one plant of mycoplasma hyopneumoniae described in claim 1, it is characterised in that the R1 areas base sequence of the P97 genes of this plant of mycoplasma
It is classified as：
Corresponding amino acid sequence is：
4. the application of one plant of mycoplasma hyopneumoniae strain described in claim 1, its feature is in this plant of mycoplasma as production of vaccine
Bacterial strain is used to prepare i (mycoplasma hyopneumoniae) vaccine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107513506B (en) * | 2016-06-17 | 2021-11-09 | 普莱柯生物工程股份有限公司 | Mycoplasma hyopneumoniae, vaccine composition and application thereof |
| CN106635901A (en) * | 2016-12-19 | 2017-05-10 | 四川省华派生物制药有限公司 | Mycoplasma hyopneumoniae strain, vaccine and application thereof |
| CN108623663B (en) * | 2018-05-14 | 2021-09-21 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Immunogenic functional polypeptide of mycoplasma hyopneumoniae P97 protein and encoding gene and application thereof |
| CN108392628A (en) * | 2018-06-01 | 2018-08-14 | 北京万牧源农业科技有限公司 | A kind of porcine mycoplasmal pneumonia inactivated vaccine and preparation method thereof |
| CN109207502B (en) * | 2018-10-10 | 2021-08-10 | 浙江洪晟生物科技股份有限公司 | Porcine mycoplasma pneumonia and porcine circovirus type 2 recombinant protein and preparation of bivalent vaccine |
| CN117843737B (en) * | 2024-03-04 | 2024-05-03 | 江苏省农业科学院 | Mycoplasma hyopneumoniae antigen content detection kit and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1928076A (en) * | 2006-09-28 | 2007-03-14 | 中国兽医药品监察所 | Live vaccine for mycoplasma pneumonia of swine and production method thereof |
| CN103182076A (en) * | 2011-12-29 | 2013-07-03 | 北京大北农科技集团股份有限公司 | Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof |
-
2015
- 2015-05-15 CN CN201510249939.6A patent/CN104894009B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1928076A (en) * | 2006-09-28 | 2007-03-14 | 中国兽医药品监察所 | Live vaccine for mycoplasma pneumonia of swine and production method thereof |
| CN103182076A (en) * | 2011-12-29 | 2013-07-03 | 北京大北农科技集团股份有限公司 | Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins;Simionatto S 等;《Comp Immunol Microbiol Infect Dis》;20121231;第36卷(第2期);209-216 * |
| 猪肺炎支原体的研究进展;沈青春 等;《中国兽药杂志》;20031231;第37卷(第6期);26-30 * |
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