CN104894009A - Mycoplasma hyopneumoniae strain and application thereof - Google Patents
Mycoplasma hyopneumoniae strain and application thereof Download PDFInfo
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Abstract
The invention relates to a mycoplasma hyopneumoniae strain S. The strain is a mycoplasma hyopneumoniae new isolate strain and can grow stably on an acellular medium, the growth titer can reach 10<9>-10<10>CCU/mL, and rabbit and piglet immunity test prove that the strain has good immunogenicity. The R1 region amino acid of the strain's P97 gene contains 11 continuous amino acid sequences of AAPKE/V, and can serve as a unique marker of molecular biology. The mycoplasma hyopneumoniae strain can be used for preparation of mycoplasma hyopneumoniae inactivated vaccines.
Description
Technical field the present invention relates to strain mycoplasma hyopneumoniae bacterial classification and an application thereof, belongs to veterinary microbiology field.
Background technology
Porcine mycoplasmal pneumonia, also known as mycoplasma pneumonia of swine, is the contact chronic respiratory tract disease of the boar caused by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp), generally popular in the world.According to document announcement, the mycoplasma hyopneumoniae be separated all over the world all belongs to same serotype, but has larger difference between genome between different isolates.(the Frey such as Frey in 1992, J., Haldimann, A. & Nicolet, J. (1992) .Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains.Int J Syst Bacteriol 42, 275-280) confirm genomic difference between mycoplasma hyopneumoniae different isolates first, 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 that the viewpoint of Frey, 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, result of study 3300-3307.) has also drawn same conclusion.
Mycoplasma is can the minimum prokaryotic organism of self-replacation, and poor growth is higher to the requirement of substratum.Compared with other common mycoplasmas, Mhp growth is more slow, seems harsher, be called as " fastidious microorganism " the requirement of vitro culture.Domestic and international experts and scholars, by studying for a long period of time, have developed polytype nutritional medium for the separation of Mhp, cultivation, as A26 mycoplasma bodily form substratum, KM2 substratum, Friis substratum and OX-heart soup substratum etc.But, these cultivate the deficiency still existed more or less, and culture effect still can not be satisfactory.Therefore isolate antigenicity, vaccine strain tool that immunogenicity is good is of great significance.
Mycoplasma hyopneumoniae genome contains rare codon, and certified antigen protein only has a few so far.Think that the major antigen albumen of mycoplasma hyopneumoniae has P97, P65, P46, P36 etc. at present, wherein P46 is the membranin that Mhp has very strong species specificity, and it can cause body early immune to react.Nineteen ninety-five, (the Futo such as 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.J Bacteriol 177, 1915-1917.) find P46 gene (1257bp), 1 CGG (in Mhp encode arginine is comprised in its ORF, be nonsense codon in other mycoplasmas) and 3 TGA codons (in mycoplasma codes for amino acid tryptophan, be terminator codon in universal codons).P97 is the main adhesion factor of Mhp, its R1 district of different strains comprises the AAKPV/E primitive (Hsu of different number, T., Artiushin, S. & Minion, F.C. (1997) .Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae.J Bacteriol 179,1317-1323).Therefore the sequencing of P46 albumen and P97 proteinic genome, can be used as the mark of molecular biology of the different strain qualification of mycoplasma hyopneumoniae.
Summary of the invention
The object of this invention is to provide a strain mycoplasma hyopneumoniae bacterial classification, this bacterial strain well-grown on cell-free medium, and immunogenicity is strong.Mycoplasma hyopneumoniae bacterial strain is accredited as through mycoplasma hyopneumoniae 16s RNA Auele Specific Primer, by its called after mycoplasma hyopneumoniae S strain, also again mycoplasma hyopneumoniae is accredited as after mycoplasma hyopneumoniae P46 primer and the amplification of P97 R1 primer specificity, its base is as different in some position with JN F65 strain in 232 strains from other mycoplasma hyopneumoniae bacterial strains with aminoacid sequence, and the molecular biology identification that can be this bacterial strain does molecule marker; This bacterial strain can be used for preparing porcine mycoplasmal inactivated vaccine.
Technical scheme of the present invention
1. a mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) S strain, this bacterial strain delivers China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservation of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 1st, 2014, preserving number CGMCC No.10095.
2. i (mycoplasma hyopneumoniae) vaccine strain S of the present invention strain, the number of base sequence of its P46 gene is:
Corresponding aminoacid sequence is:
3. i (mycoplasma hyopneumoniae) vaccine strain S of the present invention strain, the R1 district base sequence of its P97 gene is:
Corresponding aminoacid sequence is:
4. the application of i (mycoplasma hyopneumoniae) vaccine strain S of the present invention strain, its feature can be used as production of vaccine bacterial strain for the preparation of mycoplasma hyopneumoniae inactivated vaccine at this bacterial strain.
Detailed description of the present invention
One, the Isolation and Identification of mycoplasma hyopneumoniae
1. pathological material of disease source:
Strains separation is from the lung tissue of pig farm, Jilin songyuan morbidity pig, this pathological material of disease is detected without pathogen infections such as blue otopathy, swine fever, pseudo-rabies, actinobacillus pleuropneumoniae, haemophilus parasuises through PCR, pathological material of disease is after homogenized, the homemade LPS substratum of our company (LPS medium component: PPLO 5g, yeast powder 10g is inoculated into by (V/V) 1: 10, glucose 5g, Hanks liquid 1000ml, porcine blood serum 10% ~ 20%; Optimum pH: 7.4 ~ 7.8, culture temperature is 37 DEG C), cultivate 7 ~ 10 for 37 DEG C, continuous passage 3 ~ 5 generation, isolate can on LPS substratum 37 DEG C cultivate 72 ~ 96 hours, Medium's PH Value is down to 6.8 ~ 6.9 by 7.6.
2. culture condition
Get above liquid culture, inoculate LPS substratum by 10% (V/V), cultivate 72 ~ 96 hours for 37 DEG C, Medium's PH Value is down to 6.6 ~ 6.8 by 7.6, and culture is homogeneous slightly muddy red-brown, without precipitation.Culture, through steriling test, answers asepsis growth.
3. serological identification
Get above culture, culture is diluted to 10
-4~ 10
-5cCU/mL, carries out metabolic inhibition test with mycoplasma hyopneumoniae positive serum respectively, and result display mycoplasma hyopneumoniae positive serum can suppress the growth of culture, shows that this culture be separated to is mycoplasma hyopneumoniae.
4. molecular biology identification
This above culture carries out 1000 times of dilutions, with pyrolysis method (Shen Qingchun, Qin Qingsong, Wang Qin etc. (2006) .PCR method measures mycoplasma hyopneumoniae culture bacterium number. Chinese Preventive Veterinary Medicine report, 55-57.)) extract DNA as template, pcr amplification is carried out respectively respectively with mycoplasma hyopneumoniae and mycoplasma hyorhinis 16s RNA Auele Specific Primer, there is the mycoplasma hyopneumoniae specific band (see Fig. 1) of 649bp in result display measuring samples swimming lane, and uses mycoplasma hyorhinis P37 protein-specific primer to fail to amplify any band (see Fig. 2).As can be seen here, isolate is mycoplasma hyopneumoniae, and not containing mycoplasma hyorhinis.
Show that this bacterial strain is mycoplasma hyopneumoniae by above serological identification and 16s RNA PCR primer qualification result, named mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) S strain, this bacterial strain delivers China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservation of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 1st, 2014, preserving number CGMCC No.10095.
5. sequential analysis
Get mycoplasma hyopneumoniae S strain bacterium liquid pyrolysis method and extract DNA as template, with using mycoplasma hyopneumoniae P46 PCR primer and P97R1 district PCR primer to carry out specific amplification respectively, obtaining specific band, after being cloned into carrier T, carrying out sequential analysis.
6. immunogenicity inspection
10 are reached by viable bacteria titre
9after the S strain bacterium liquid deactivation of CCU/mL, prepare inactivated vaccine with suitable adjuvant, inoculate rabbit and 7 age in days sodium selenites respectively, by the malicious protection of attacking measuring rabbit antibodies level and piglet, Evaluation of Immunogenicity is carried out to it.
Two, the preparation of mycoplasma hyopneumoniae inactivated vaccine
(1) preparation of vaccine manufacture antigen: secondary seed is pressed 1:20 and inoculate Lps-5 substratum, cultivate 3 ~ 6 at 37 DEG C, when pH value reaches about 6.9, results culture, again in 1:50 ratio Secondary Culture in the same way, until results bacterium liquid manufactures vaccine;
(2) deactivation: after cultivating, bacterium liquid puts inactivation treatment through beta-propiolactone, adds Thiomersalate as sanitas;
(3) seedling is joined: mixed in the ratio of (V/V) 1:1 with adjuvant (France matches oily adjuvant or the water adjuvant of BIC Corp) by bacterium liquid after deactivation, be prepared into mycoplasma hyopneumoniae inactivated vaccine.
The Microbial resources information that the present invention relates to
Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), S strain, this bacterial strain delivers China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservation of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 1st, 2014, preserving number CGMCC No.10095.
Accompanying drawing explanation
Fig. 1: mycoplasma hyopneumoniae 16s RNA primer amplified electrophorogram.
Fig. 2: mycoplasma hyorhinis P37 protein-specific primer amplification electrophorogram.
Fig. 3: mycoplasma hyopneumoniae S strain P46 gene amplification fragment electrophorogram.
Fig. 4: mycoplasma hyopneumoniae S strain P97 gene R1 district amplified fragments electrophorogram.
Positively effect of the present invention
The present invention relates to a strain mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) S strain, have typical mycoplasma hyopneumoniae infection simultaneously without the pig lungs of other pathogen infections obvious, by appropriate media continuous passage gained.By molecular biology and serological identification, confirm that this bacterial strain is the new strain isolated of mycoplasma hyopneumoniae, can on cell-free medium stable growth, growth titre can reach 10
9~ 10
10through rabbit and piglet immunological test, CCU/mL, confirms that its immunogenicity is good, can as the production bacterial classification of mycoplasma hyopneumoniae inactivated vaccine.Warp and other mycoplasma hyopneumoniae bacterial strains, as 232 strains and JNF65 strain compare, its P46 gene base sequence has part different with aminoacid sequence, but homology is higher, and P97 gene base sequence and aminoacid sequence are all distinguished larger, and the continuous amino acid sequence of P97 aminopeptidase gene acid containing 11 AAPKE/V, can be used as the mark of molecular biology of its uniqueness; This strain mycoplasma hyopneumoniae bacterial strain can be used for preparing mycoplasma hyopneumoniae inactivated vaccine.
Embodiment
Below for further illustrating the present invention, restriction is not made to the present invention.
Embodiment 1
---the cultivation of mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) S strain
By S strain bacterial classification 10% Pigs Inoculated mycoplasma pneumoniae LPS substratum, cultivate 72 ~ 96 hours for 37 DEG C, when pH value of solution to 6.6 ~ 6.8, yellowly or orange-yellow, evenly muddy without precipitation, results bacterium liquid, go down to posterity after 2 times, dilution bacterium liquid carries out CCU counting (colour change unit, CCU), and viable count can reach 10
9~ 10
10cCU/mL.
Embodiment 2
---the mycoplasma hyopneumoniae 16s RNA Auele Specific Primer PCR of mycoplasma hyopneumoniae S strain bacterium liquid measures
1. design of primers
According to the gene order of the 16s RNA of the mycoplasma hyopneumoniae that genebank announces, with reference to (2006) (Shen Qingchun such as Shen Qingchun, Qin Qingsong, Wang Qin etc. (2006) .PCR method measures mycoplasma hyopneumoniae culture bacterium number. Chinese Preventive Veterinary Medicine report, 55-57.) and synthetic primer.
Upstream primer P1 sequence is: 5'-GAGCCTTCAAGCTTCACCAAGA-3 ' (sequence 1)
Downstream primer P2 sequence is: 5'-TGTGTTAGTGACTTTTGCCACC-3 ' (sequence 2)
Estimate that amplified fragments total length is 649bp.
2. template extraction
Get cultured bacterium liquid 1mL and carry out the centrifugal 15min of 12000r/min, abandon supernatant.Precipitate with 100 μ l 1 × TEN solution suspension, boiling water bath 10min, room temperature leaves standstill 3min.The centrifugal 2min of 12000r/min, supernatant is template DNA.
3.PCR increases
PCR reagent purchased from Beijing Quanshijin Biotechnology Co., Ltd, PCR reaction system: 2 × EasyTaq PCR SuperMix 10 μ L, each 1 μ L of upstream and downstream primer, template 2 μ L, moisturizing to 20 μ L.Reaction conditions is 94 DEG C of denaturation 5min, 95 DEG C of sex change 50s, and 60 DEG C of annealing 30s, 72 DEG C extend 50s, and 35 circulations, 72 DEG C of ends extend 5min.
4. running gel imaging
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-specific primer PCR measures
1. design of primers
Gene order according to the P37 of the mycoplasma hyorhinis that genebank announces designs and synthesizes primer.
Upstream primer P1 sequence is: 5 '-GTAGTCAAGCAAGAGGATGT-3 ' (sequence 3)
Downstream primer P2 sequence is: 5 '-GCTGGAGTTATTATACCAGGA-3 ' (sequence 4), amplified fragments total length is 346bp.
2. template extraction
Get cultured bacterium liquid 1mL and carry out the centrifugal 15min of 12000r/min, abandon supernatant.Precipitate with 100 μ l 1 × TEN solution suspension, boiling water bath 10min, room temperature leaves standstill 3min.The centrifugal 2min of 12000r/min, supernatant is template DNA.
3.PCR increases
PCR reagent purchased from Beijing Quanshijin Biotechnology Co., Ltd, PCR reaction system: 2 × EasyTaq PCRSuperMix 10 μ L, each 1 μ L of upstream and downstream primer, template 2 μ L, moisturizing to 20 μ L.PCR reaction conditions: 94 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 50 DEG C of annealing 30s, 72 DEG C extend 50s, and 35 circulations, 72 DEG C of ends extend 5min.
4. running gel imaging
PCR primer carries out 1.0% agarose gel electrophoresis, and mycoplasma hyorhinis BTS-7 strain (purchased from China Veterinery Drug Inspection Office) amplified fragments 346bp, S strain, without specific amplification fragment, is shown in Fig. 2.
5. conclusion isolate is accredited as not containing mycoplasma hyorhinis (note: owing to being easy to be subject to the pollution of mycoplasma hyorhinis when mycoplasma hyopneumoniae is separated causes isolate impure and impact research work subsequently) through PCR.
Embodiment 4
---the P46 gene sequencing of S bacterium liquid
1. design of primers:
According to the primer sequence synthetic primer that gene order and the Zhang Fenggang etc. " being separated and PCR qualification of mycoplasma hyopneumoniae " of the P46 of the mycoplasma hyopneumoniae that genebank announces deliver, amplified fragments total length is 1162bp.Its primer:
Upstream primer P1 sequence is: 5'-CGGGATCCACTTCAGATTCTAAACCACAA-3 ' (sequence 5),
Downstream primer P2 sequence is: 5'-CCAAGCTTTTAGGCATCAGGATTATCAAC-3 ' (sequence 6).
2. template extraction
Get S bacterium liquid, 232 strain bacterium liquid and each 1mL of JNF65 strain bacterium liquid, carry out the centrifugal 15min of 12000r/min, abandon supernatant.Precipitate with 100 μ l 1 × TEN solution suspension, boiling water bath 10min, room temperature leaves standstill 3min.The centrifugal 2min of 12000r/min, supernatant is template DNA.
3.PCR increases
PCR reagent purchased from Beijing Quanshijin Biotechnology Co., Ltd, PCR reaction system: 2 × EasyTaq PCR SuperMix10 μ L, each 1 μ L of upstream and downstream primer, template 2 μ L, moisturizing to 20 μ L.PCR reaction conditions: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 50s, and 35 circulations, 72 DEG C of ends extend 10min.
4. running gel imaging
PCR primer carries out 1.0% agarose gel electrophoresis, amplified fragments 1162bp, in the same size with Mhp232 amplified fragments, sees Fig. 4.
The order-checking of 5.S strain P46 Gene Partial and sequence results
The PCR primer of S strain P46 checks order, and two-way survey logical order-checking base sequence is (sequence 7):
Translation becomes aminoacid sequence, and result is (sequence 8):
* in sequence, the amino acid of boldface is: the tryptophane of being encoded by TGA, and in universal codons, TGA is terminator codon.
By the BLAST software on-line analysis on NCBI website, result shows that the homology of the respective regions of above nucleotide sequence and mycoplasma hyopneumoniae international standard bacterial strain 232 strain (12 bases have difference), J strain (10 bases have difference), 7448 strains (14 bases have difference) etc. all reaches 99%; On aminoacid sequence, homology is higher, only differs an amino acid (221 Q-A) with 7448 strains, differs two amino acid (182 G-Q and 221 Q-A) with 232 strains.
6. conclusion
Seen by mycoplasma hyopneumoniae S strain P46 portion gene sequencing result, higher with the corresponding sequence homology of other bacterial strains, nucleotide sequence and aminoacid sequence all reach the homology of 99%, and this conforms to the well-conserved feature of P46 gene.
Embodiment 5
---the P97 gene R1 district order-checking of S strain
1. design of primers:
According to the nucleotide sequence in the P97 gene R1 district of mycoplasma hyopneumoniae 232 strain that genebank announces, design and synthesis 1 pair of primer, amplified fragments contains the total length in R1 district, and 232 strain amplified productions estimate that size is about 320bp, and different strains expansion fragment length is variant.
Upstream primer P1 sequence is: 5'-CAAAAGAAGG TAAAAGAGAA-3'(sequence 9)
Downstream primer P2 sequence is: 5'-AAGCCAGTAT TAGTAGCAAC-3'(sequence 10).
2. template extraction
Get S strain bacterium liquid 1mL, carry out the centrifugal 15min of 12000rpm, abandon supernatant.Precipitate with 100 μ l 1 × TEN solution suspension, boiling water bath 10min, room temperature leaves standstill 3min.The centrifugal 2min of 12000r/min, supernatant is template DNA.
3.PCR increases
PCR reagent purchased from Beijing Quanshijin Biotechnology Co., Ltd, PCR reaction system: 2 × EasyTaq PCR SuperMix10 μ L, each 1 μ L of upstream and downstream primer, template 2 μ L, moisturizing to 20 μ L.PCR reaction conditions: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 50s, and 35 circulations, 72 DEG C of ends extend 10min.
4. running gel imaging
Amplified production is through 1.0% agarose gel electrophoresis, and result shows that the amplified fragments size of S strain is about 180bp, sees Fig. 4.
The part order-checking of 5.P97 gene and sequence results (sequence 11)
Application DNAstar software translation is aminoacid sequence, and its result is (sequence 12):
* have the amino acid of underscore to be tumor-necrosis factor glycoproteins in sequence, amount to 11 Ala Ala (
val/Thr)lys Pro Glu (Val) repeats, and is abbreviated as AA (V/T) PKE/V.
The R1 district of the P97 gene of mycoplasma hyopneumoniae S strain comprises the aminoacid sequence repetition of 11 AA (V/T) PKE/V altogether, is different from other bacterial strains, identical with 168 strain repeat numbers, but wherein has nearly 7 amino acid whose difference, as following table 1.
The difference of the R1 region amino acid sequence of table 1 four strain mycoplasma hyopneumoniae P97 gene
Note: "-" represents disappearance; Underscore represents different at this place from S strain.
Embodiment 6
---the preparation of porcine mycoplasmal pneumonia inactivated vaccine (S strain) and inspection
1. bacterium liquid is cultivated
Mycoplasma hyopneumoniae S strain freeze-drying lactobacillus mycoplasma hyopneumoniae LPS substratum is redissolved, 1:10 Pigs Inoculated mycoplasma pneumoniae LPS substratum, cultivates 72 ~ 96 hours, when solution colour is red-brown (pH to 6.6 ~ 6.8) for 37 DEG C, even muddy without precipitation, results bacterium liquid.S strain bacterium liquid was gone down to posterity for 2 generations continuously on LPS substratum, makes viable count reach 10
9cCU/mL, the qualified rear bacterium liquid of steriling test is for the preparation of vaccine.
2. prepare vaccine
By after the S strain bacterium liquid deactivation that is up to the standards, mix carry out emulsification seedling with adjuvant (France matches oily adjuvant or the water adjuvant of BIC Corp), vaccine bacteria containing amount can reach 5 × 10
8cCU/mL, is prepared into mycoplasma hyopneumoniae inactivated vaccine vaccine.
3. vaccine test
3.1 proterties inspection vaccine outward appearances should be creamy white emulsion.
Formulation water-in-oil-in water.Get a clean suction pipe, draw a little vaccine and drip in cold water surface, should the diffusion in cloud.
Stability is drawn vaccine 10mL and is added in centrifuge tube, and with the centrifugal 15min of 3000r/min, the aqueous phase of sucking-off at the bottom of pipe should no more than 0.5mL.
Viscosity according to existing " Chinese veterinary pharmacopoeia ", (compile by the Chinese veterinary pharmacopoeia council.Veterinary drug allusion quotation 2010 Nian Bansanbu Chinese agriculture press of the People's Republic of China (PRC), 2011, the present invention was hereinafter referred to as " Chinese veterinary pharmacopoeia ") carry out, should conform with the regulations.
3.2 steriling tests carry out according to existing " Chinese veterinary pharmacopoeia ", answer asepsis growth.
3.3 safety verification
Mouse often criticizes vaccine abdominal injection 15 ~ 22g Balb/C mouse 8, and 0.5mL/ only, observes 10, observes mouse survival situation.
Piglet often criticizes vaccine intramuscular injection 7 age in days piglet 5,2mL/ head, and head exempts from latter 14 days, intramuscular injection 2mL again, observes 30, observes piglet survival condition and with or without side reaction, and within 0 day, 7 days and two, exempt to survey body temperature continuously in latter 7 days respectively at immunity, observation body temperature situation.
3.4 efficacy test
Piglet extracts 1 batch of porcine mycoplasmal pneumonia inactivated vaccine (S strain) and 1 batch of external import vaccine intramuscular injection 7 age in days healthy susceptible SPF piglet 5 respectively, 1mL/ head, intramuscular injection vaccine 1mL again after 14 days.After first immunisation 21 days, together with contrast pig 5, respectively strong poison gas pipe is organized to inject with mycoplasma hyopneumoniae ZS strain, every 5mL (containing 40 ~ 200 minimum morbidity dosage).Attack poison after 28 days, cut open inspection, observe pig pulmonary lesion situation, use 55 point-scores to judge
Rabbit is by 3 batches of porcine mycoplasmal pneumonia inactivated vaccines (S strain) intramuscular injection 1.5 ~ 2kg rabbit 5, and only, intramuscular injection vaccine 0.1mL again after 14 days, separately establishes 5 contrasts to 0.1mL/.After head exempts from, blood sampling on the 21st, measures serum I HA antibody titer, and contrast rabbit IHA tires not higher than 1:10, and the IHA of immunize rabbit tires at least 4 and is not less than 1:40.
4. result
4.1 proterties inspections
Appearance milky white emulsion.
Formulation water-in-oil-in water.Get a clean suction pipe, draw a little vaccine and drip in cold water surface, spread in cloud.
Stability is drawn vaccine 10mL and is added in centrifuge tube, with the centrifugal 15min of 3000r/min, and the no more than 0.5mL of the aqueous phase of sucking-off at the bottom of pipe.
Viscosity is according to existing " Chinese veterinary pharmacopoeia ", and use viscosity apparatus to measure, viscosity number (CP) is respectively 26, all conforms with the regulations.
4.2 steriling tests extract 2 bottles and carry out steriling test, observe 7, equal asepsis growth.
4.3 safety examination results
3 batches of vaccine abdominal injection 15 ~ 22g Balb/C mouse 8 respectively, observe 10, and mouse is all strong to live; Overdose intramuscular injection piglet, observes 30, and piglet is all strong to live, except injection site occurs slight red and swollen, without other side reactions;
Within after vaccinate 4 ~ 5 days first, often organize each temperature of pig body and slightly raise, continue all to recover normal after 1 ~ 3 day, two exempt from the reaction of rear body temperature concentrates the 1st day after injection, with and recover normal.
4.4 efficacy test results
4.4.1 rabbit efficacy test result
The comparison of antibody I HA titration result after table 2 rabbit immunization
As can be seen from the above table, after porcine mycoplasmal pneumonia inactivated vaccine (S strain) immunizing rabbit, IHA antibody titer can reach more than 1:40, and with control group significant difference, composite antibody level is a little more than similar import vaccine antibody level.
4.4.2 piglet immunological attacks toxic effect force inspecting result
Table 3 piglet attacks the comparison of the rear pulmonary lesion situation of poison
Immunization test-results shows that porcine mycoplasmal pneumonia inactivated vaccine (S strain) has stronger immune protective efficiency, and reach the level of more than 80%, vaccine difference similar with import is not obvious.
Claims (4)
1. a strain mycoplasma hyopneumoniae bacterial classification, it is characterized in that this strain mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) called after S strain, deliver China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservation of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on December 1st, 2014, preserving number CGMCC No.10095.
2. a strain mycoplasma hyopneumoniae bacterial classification described in claim 1, is characterized in that the number of base sequence of the P46 gene of this bacterial strain is:
Corresponding aminoacid sequence is:
3. pig one strain mycoplasma hyopneumoniae bacterial classification described in claim 1, is characterized in that the R1 district base sequence of this bacterial strain P97 gene is:
Corresponding aminoacid sequence is:
4. the application of a strain mycoplasma hyopneumoniae bacterial classification described in claim 1, its feature can be used as production of vaccine bacterial strain for the preparation of i (mycoplasma hyopneumoniae) vaccine at this bacterial strain.
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| 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 |
| CN107513506A (en) * | 2016-06-17 | 2017-12-26 | 普莱柯生物工程股份有限公司 | Mycoplasma hyopneumoniae, vaccine combination and its application |
| 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 |
| CN108623663A (en) * | 2018-05-14 | 2018-10-09 | 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） | The immunogenicity functional polypeptide and its encoding gene of mycoplasma hyopneumoniae P97 albumen and application |
| CN109207502A (en) * | 2018-10-10 | 2019-01-15 | 杭州洪桥中科基因技术有限公司 | Porcine mycoplasmal pneumonia and porcine circovirus 2 type recombinant protein and prepare bigeminy vaccine |
| CN117843737A (en) * | 2024-03-04 | 2024-04-09 | 江苏省农业科学院 | Mycoplasma hyopneumoniae antigen content detection kit and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107513506A (en) * | 2016-06-17 | 2017-12-26 | 普莱柯生物工程股份有限公司 | Mycoplasma hyopneumoniae, vaccine combination and its application |
| 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 |
| CN108623663A (en) * | 2018-05-14 | 2018-10-09 | 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） | The immunogenicity functional polypeptide and its encoding gene of mycoplasma hyopneumoniae P97 albumen and application |
| 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 |
| CN109207502A (en) * | 2018-10-10 | 2019-01-15 | 杭州洪桥中科基因技术有限公司 | Porcine mycoplasmal pneumonia and porcine circovirus 2 type recombinant protein and prepare bigeminy vaccine |
| CN117843737A (en) * | 2024-03-04 | 2024-04-09 | 江苏省农业科学院 | Mycoplasma hyopneumoniae antigen content detection kit and application thereof |
| 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 |
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