CN104248753A - Vaccine composition and application thereof - Google Patents
Vaccine composition and application thereof Download PDFInfo
- Publication number
- CN104248753A CN104248753A CN201410204020.0A CN201410204020A CN104248753A CN 104248753 A CN104248753 A CN 104248753A CN 201410204020 A CN201410204020 A CN 201410204020A CN 104248753 A CN104248753 A CN 104248753A
- Authority
- CN
- China
- Prior art keywords
- albumen
- vaccine combination
- antigen
- nrdf
- swine
- 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 swine mycoplasmal pneumonia vaccine composition comprising a P46 protein, a NrdF protein and / or an adjuvant acceptable in veterinary medicine; and the vaccine composition can also include mycoplasmal whole bacteria antigen of swine pneumonia. The invention also discloses application of the vaccine composition to preparation of drugs for preventing and / or treating swine mycoplasmal pneumonia. The multiple immunogenic protein in the vaccine composition can be obtained through large amounts of recombinant expression on the vaccine composition by means of genetic engineering, and the method not only has short time consumption but also can be applied to mass production; and the combination of multiple immunogenic protein fragments can induce the synergic immune effect; when the vaccine composition containing multiple immunogenic protein and mycoplasmal whole bacteria antigen of swine pneumonia, the content of mycoplasmal whole bacteria antigen of swine pneumonia is decreased significantly than single immunization by full bacteria vaccine.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to mycoplasmal pneumonia of swine vaccine combination and application thereof.
Background technology
Mycoplasmal pneumonia of swine, also known as swine enzootic pneumonia or pig endemic conditions pneumonia, is a kind of contact chronic respiratory infectious disease caused by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp), is prevalent in all over the world.Ill pig main manifestations is cough and asthma, and growth retardation, feed conversion rate is low, and the development of pig industry in serious harm.
Research in recent years finds: mycoplasma hyopneumoniae infection case is increasing, and many Large-scale pig farm are subject to the serious threat of swine enzootic pneumonia.Although antibiotic is applied in treatment asthma certain effect, pay attention to day by day is abused for antibiosis by country, management increasingly stringent, therefore, and a kind of effective ways that vaccine immunity will be following this disease of effectively prevention.And the vaccine of domestic application is mainly based on attenuated vaccine and inactivated vaccine at present, domestic attenuated vaccine is not easily promoted due to needs intrathecal; Although inactivated vaccine is by intramuscular injection, operate easy, mainly monopolized by the offshore company headed by Pfizer, Hai Bolai, Bo Linge, and vaccine price costliness causes epidemic prevention cost to uprise.
In recent years, people start to attempt utilizing modern molecular biology means to start with from the pathogenesis and major antigen albumen studying Mhp, heterogenous expression carrier is utilized to obtain a large amount of Mhp associated antigen protein, simultaneously recombinant expressed with other related stimulus factors and be prepared into Novel pig mycoplasma pneumonia subunit vaccine.Containing the nucleic acid of whole-bacterial-vaccine, not only not there is not the potential safety hazard that deactivation does not thoroughly cause in this novel mycoplasmal pneumonia of swine subunit vaccine, and because it is not containing all antigens of full bacterium, is conducive to the purification of disease.But mycoplasmal pneumonia of swine subunit vaccine immune effect is not good at present; can not provide to pig and protect (Fagan PK completely; Djordievic SP.Chin J; et al.Oral immunization of mice with attenuated Salmonella typhimurium aroA expressing a recombinant Mycoplasma hyopeumoniae antigen (NrdF) .Infection and immunity; 1997,65:2502-2507).
Summary of the invention
In order to solve the deficiencies in the prior art, the invention provides mycoplasmal pneumonia of swine vaccine combination and application thereof.
Main purpose of the present invention is to provide a kind of mycoplasmal pneumonia of swine vaccine combination, and described vaccine combination comprises P46 albumen, NrdF albumen and veterinarily acceptable adjuvant.
Term used herein " mycoplasmal pneumonia of swine " symptom clinically includes, but is not limited to: respiratory symptom (the clinical pneumonia of serious acute, cough, dyspnea, breathing speed are compeled), heating, hyperinfection, high incidence, low actual, Necrotic cell bronchitis and secondary bacterial respiratory tract infection increase.
Term used herein " mycoplasmal pneumonia of swine vaccine combination " refers to the vaccine that can be used in preventing and/or treating disease or disease known to mycoplasma hyopneumoniae infection, this vaccine combination can comprise anyly effectively can prevent and/or treat pig by the vaccine of mycoplasma hyopneumoniae infection, can be used in mycoplasmal pneumonia of swine vaccine combination and includes, but is not limited to mycoplasmal pneumonia of swine cell preparation, deactivation or improvement live vaccine, subunit vaccine wholly or in part.
Mycoplasmal pneumonia of swine vaccine combination of the present invention is preferably subunit vaccine, described " subunit vaccine " comprises the derivative polypeptide of one or more mycoplasma hyopneumoniae or protein, or the immunogenic fragments of described polypeptide or protein, or described one or more mycoplasma hyopneumoniae of encoding derives polypeptide or protein, or one or more mycoplasma hyopneumoniae gene of described polypeptide or protein immunogenic fragment or nucleic acid, and described gene or nucleic acid can be expressed in pig body.The immunogenic fragments of mycoplasma hyopneumoniae polypeptide, protein, described peptide and protein or mycoplasma hyopneumoniae gene or nucleic acid can be used for techniques well known synthesis or restructuring preparation.
Term used herein " prevents and/or treats " and to refer to when relating to mycoplasma hyopneumoniae infection and suppress the propagation copying, suppress mycoplasma hyopneumoniae of mycoplasma hyopneumoniae antibacterial or prevent mycoplasma hyopneumoniae from settling down in its host, and alleviate mycoplasma hyopneumoniae infection disease or the symptom of disease.If bacterial load amount reduce, pulmonary infection alleviate and/or food ration and/or growth increase, so just can think that described treatment reaches therapeutic effect.
Term used herein " pig " refers to any animal belonging to Suidae (Suidae) member, as pig.
Term used herein " P46 albumen " belongs to the Cell membrane antigens albumen of Mhp, causes early immune to react; This molecular weight of albumen reaches 46kD, is the ORFs (Open reading frames, i.e. open reading frame) containing 1257bp (coding 419AA); This albumen kind inner height is guarded, and in its gene order, have codon TGA (the Futo S of 3 codes for amino acid tryptophan (Trp), Seto Y, Mitsuse S, et al.Molecular cloning of a46-kilodalton surface antigen (p46) gene from mycoplasma hyopneumoniae:direct evidence of cgg codon usage for arginine, J Bacteriol, 1995,177 (7): 1915-1917).
Preferably, described P46 albumen is one or both protein mixtures that SEQ ID No.2 or SEQ ID No.4 encode.
The nucleotides sequence of described P46 albumen is classified as SEQ ID No.1, SEQ ID No.3.
Term used herein " NrdF albumen " is for having immunogenic protein fragments in NrdF whole protein, namely the R2 subunit C of NrdF whole protein holds (Fagan PK, Djordjevic SP, Eamens GJ, et al.Molecular characterization of a ribonucleotide reductase (NrdF) gene fragment of Mycoplasma hyopneumoniae and assessment of the recombinant product as an experimental vaccine for enzootic pneumonia.Infection and immunity, 1996, 64:1060-1064).
Preferably, described NrdF albumen is the albumen that SEQ ID No.6 encodes.
The nucleotides sequence of described NrdF albumen is classified as SEQ ID No.5.
Term used herein " adjuvant " can comprise aluminium glue adjuvant; Saponin (saponin), as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); Water in oil emulsion; Oil in water emulsion; W/O/W Emulsion; The polymer of acrylic or methacrylic acid; The compound that the copolymer of maleic anhydride and alkenyl (alkenyl) derivant is selected.
Term used herein " Emulsion " can especially based on light liquid paraffin oil (European Pharmacopea type); Because of the isoprenoid oil (isoprenoid oil) that olefin oligomerisation produces, as squalane (squalane) or Squalene oil (squalene oil), especially isobutene. or certain herbaceous plants with big flowers alkene; The ester containing linear alkyl of acid or alcohol, more specifically vegetable oil, ethyl oleate, propylene glycol two-(caprylate/certain herbaceous plants with big flowers acid esters), glycerol three-(caprylate/certain herbaceous plants with big flowers acid esters) or Rikemal PO 200; The ester of branched chain fatty acid or alcohol, especially isostearate.Oil and emulsifier combination use to form Emulsion.Emulsifying agent preferred nonionic surfactants, especially the ester of the ester of the ester of the ester (as anhydrous mannitol oleate) of the ester of sorbitan, mannide (mannide), aliphatic dihydroxy alcohol (glycol), the ester of polyglycereol (polyglycerol), the ester of propylene glycol and oleic acid, the ester of isostearic acid, the ester of castor oil acid or hydroxy stearic acid, their optional ethoxylations, also has Pluronic L121, especially Pluronic product, particularly L121.See " The theory and practical application of adjuvants " (Ed.by DES Stewart-Tull that Hunter etc. writes, John Wiley and Sons, New York, " Vaccine " (1997,15:564-570) of 1995:51-94) writing with Todd etc.Such as, " the Vaccine design that Powell M and Newman M writes can be used, the Subunit and adiuvant approach " (Plenum Press, 1995) the 147th page describe SPT Emulsion and the 183rd page describe MF59 Emulsion.
Term used herein " polymer of acrylic or methacrylic acid " is preferably crosslinked acrylic or methacrylic acid polymer, especially be cross-linked with the sugar poly alkenyl ether of (sugar) or polyalcohols, these compounds are known is called as carbomer (Carbomer, trade name Carbopol) (Phameuropa, 1996,8 (2)).Those skilled in the art also can see US Patent No. 2909462, which depict this kind of acrylate copolymer, itself and poly-hydroxylated compound crosslink, described compound has at least 3 hydroxyls, preferably more than 8, wherein the hydrogen atom of at least 3 hydroxyls is had the unsaturated lipid alkyl of at least 2 carbon atoms (aliphatic radical) replacement.Preferred group is that those contain the group of 2-4 carbon atom, such as vinyl, pi-allyl and other ethylenically unsaturated group (ethylenically unsaturated group).Described unsaturated group self can comprise other substituent group, as methyl.These products are sold with the name of carbopol, and (BF Goodrich, Ohio, USA) is suitable especially.They and allyl sucrose or be cross-linked with Allyl pentaerythritol (allyl pentaerythritol).This wherein can mention carbopol 974P, 934P and 971P, most preferably uses carbopol 971P.
Term used herein " copolymer of maleic anhydride and alkenyl derivative " also can consider the copolymer EMA (Monsanto) of maleic anhydride and ethylene, these polymer dissolve and produce acid solution in water, through neutralization, preferably be neutralized to physiological pH, to produce assist agent solution, immunogenicity, immunogenicity or vaccinal compositions itself can be mixed wherein.
Term used herein " adjuvant " also comprises, but be not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), Avridine lipid-amine adjuvant, E.coli LT (restructuring or other), cholera toxin, IMS1314, muramyldipeptide, Gel adjuvant etc.
Preferably, described adjuvant is one or more in the copolymer of aluminium glue adjuvant, saponin, water in oil emulsion, oil in water emulsion, W/O/W Emulsion, acrylic acid, the polymer of methacrylic acid, maleic anhydride and alkenyl (alkenyl) derivant, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E.coli LT, cholera toxin, IMS1314, muramyldipeptide or Gel adjuvant; Be preferably Gel adjuvant.
More preferably, the volume ratio of described adjuvant in described vaccine combination is 5%-30%.
As one embodiment of the present invention, the P46 albumen in described vaccine combination, NrdF albumen expressing in series.
As one embodiment of the present invention, described vaccine combination is made up of P46 albumen, NrdF albumen and veterinarily acceptable adjuvant.
Preferably, described vaccine combination is also containing the full bacterium antigen of mycoplasma hyopneumoniae.
Term used herein " mycoplasma hyopneumoniae full bacterium antigen " refers to and comprises at least one to the rear any compositions inducing, stimulate or strengthen the full bacterium antigen of the immunne response of opposing mycoplasma hyopneumoniae infection of pig inoculation.Described mycoplasma hyopneumoniae full bacterium antigen is complete mycoplasma hyopneumoniae, not only comprise the street strain of clinical separation well known to those skilled in the art, also comprise deactivation, the work of improvement or the mycoplasma hyopneumoniae of attenuation, be preferably deactivated form, be more preferably mycoplasma hyopneumoniae HN0603 strain or J strain.Mycoplasma hyopneumoniae full bacterium antigen can also comprise any one antigen of following compositions, as: mycoplasma hyopneumoniae is as Boehringer Ingelheim company
auspicious times of suitable Respisure and RespisureOne of M.hyo, company of Harbin Pharmaceutical Group, the MycoGard of Portec Inc. of the U.S., the RespiFend MH of Pfizer Inc., the pig gram of Cimmeria animal health company breathe heavily, 168 strain live vaccine of domestic production.
As one embodiment of the present invention, described vaccine combination is made up of P46 albumen, NrdF albumen, mycoplasma hyopneumoniae full bacterium antigen and veterinarily acceptable adjuvant.
Term used herein " mycoplasma hyopneumoniae " and " Mhp " are used interchangeably in the present invention.
Term used herein " open reading frame " or " ORF " refer to coding not containing the minimum nucleotide sequence needed for specific Mhp protein of the termination codon inserted.
The present invention's abbreviation used is: AA, aminoacid; Bp, base pair; KD, kilodalton.
Preferably, described vaccine combination also comprises other antigens, other antigens described comprise in pig circular ring virus antigen, porcine reproductive and respiratory syndrome virus antigen, haemophilus parasuis antigen, PPV Antigen Using, Actinobacillus pleuropneumoniae antigen, bacillus coli antigen, atrophic rhinitis antigen, porcine pseudorabies virus antigen, hog cholera pathogen antigen, swine flue antigen one or more.Preferably, other antigens described are pig circular ring virus antigen.
Another object of the present invention is to provide described vaccine combination preparing the application prevented and/or treated in the medicine of mycoplasmal pneumonia of swine.
The present invention has following outstanding advantage:
(1) described vaccine combination carries out recombinant expressed in a large number by genetic engineering means to the component of vaccine combination, not only consuming time short, also can be convenient to large-scale production;
(2) in described vaccine combination, the combination of many immunogenic protein fragment can be induced and be produced collaborative immune effect;
(3), when described vaccine combination is containing many immunogenic proteins and mycoplasma hyopneumoniae full bacterium antigen, than when being used alone whole-bacterial-vaccine immunity, the content of the full bacterium antigen of mycoplasma hyopneumoniae significantly reduces.
in sequence table:
Sequence 1 is the nucleotide sequence of P46 albumen;
Sequence 2 is the aminoacid sequence of P46 albumen;
Sequence 3 is the nucleotide sequence of P46 albumen;
Sequence 4 is the aminoacid sequence of P46 albumen;
Sequence 5 is the nucleotide sequence of NrdF albumen;
Sequence 6 is the aminoacid sequence of NrdF albumen.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Mycoplasma hyopneumoniae P46 albumen used in the embodiment of the present invention, the sequence of NrdF albumen, derive from the international standard strain J strain reported, the accession number in NCBI is AE017243.1.
In the embodiment of the present invention, express for coli expression carrier, obtain mycoplasma hyopneumoniae P46 albumen, NrdF albumen by genetic engineering means, but no matter this embodiment does not under any circumstance all form limitation of the invention.
The embodiment of the present invention is with Montanide
tMgel01 (French SEPPIC company) as adjuvant, and is the adjuvant of 10% with volume ratio, and preparing mycoplasmal pneumonia of swine vaccine combination is that example is set forth, but no matter this embodiment does not under any circumstance all form limitation of the invention.
In the embodiment of the present invention adopt mycoplasma hyopneumoniae HN0613 strain, preserving number is CCTCC No.M2012230, preservation date is on June 13rd, 2012, and depositary institution is China typical culture collection center, and this strain is open in patent application CN103083655A.
The counteracting toxic substances strain adopted in the embodiment of the present invention is mycoplasma hyopneumoniae CVCC354 strain, and the preserving number of this strain is CVCC354, and depositary institution is National Veterinary Microbiological Culture Collection administrative center.
In the embodiment of the present invention, pneumonopathy varying index adopts 28 point-scores to pass judgment on.Described 28 point-scores observe pathological changes according to the typical pulmonary of mycoplasmal pneumonia of swine, carries out quantification judge occurring degree, comprises the essence sample pathological changes of apex pulmonis leaf, lobus cardiacus, lobus diaphragmaticus, the appearance of middle leaf, as " carnification ", " change of pancreas sample ".Pneumonia pathological changes standards of grading are: 2 sharp leaves, 2 lobus cardiacuses, 2 lobus diaphragmaticus, 1 middle leaf amount to 7 lobes of the lung, and each lobe of the lung full marks are 4 points, amount to 28 points.Shared by the lobe of the lung area of generation essence pathological changes, the ratio of this leaf, gives a mark to each lobe of the lung respectively.0 point is designated as without pneumonia pathological changes; Lesion area ratio is 1%-25%, is designated as 1 point; Lesion area ratio 26%-50%, is designated as 2 points; Lesion area ratio is 51%-75%, is designated as 3 points; Lesion area ratio is 76%-100%, is designated as 4 points.As lobe of the lung tow sides all have pathological changes, score with the one side that lesion area is large.The pneumonia disease that each lobe of the lung marking summation is this morbidity strain becomes point.After respectively immune group pig and matched group pig being scored, the lobe of the lung pathological changes slip according to following formulae discovery immune group pig:
Embodiment of the present invention statistical analysis technique is: the pneumonopathy varying index of statistics 7 lobes of the lung, determines lesion degree.Carry out ANOVA analysis with SPSS computer software, more each group difference, determine the effectiveness of pathological changes difference.
PH7.2PBS formula of liquid used in the present invention is: add NaCl9g, Na in 1000mL distilled water
2hPO
412H
2o6g, NaH
2pO
42H
2o0.4g, chemical reagent used in the present invention is analytical pure, purchased from traditional Chinese medicines group.
For making the present invention easier to understand, below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for the present invention to limit the scope of the invention.Experimental technique of the present invention, if without specified otherwise, is conventional method; Described biomaterial, if without specified otherwise, all can obtain from commercial channels.
The expression of embodiment 1 mycoplasma hyopneumoniae albumen, qualification and purification
The structure of 1.1P46 recombinant vector and the expression of albumen, qualification and purification
According to NCBI (
http:// www.ncbi.nlm.nih.gov) in report mycoplasma hyopneumoniae J strain (accession number: AE017243.1) in P46 protein nucleotide sequence (see SEQ ID No.1, SEQ ID No.3), with primer and the rite-directed mutagenesis primer sequence of Primer Premier5 software design P46 albumen, details are in table 1.
Table 1P46 protein primer sequence table
By Ma Fengying (Ma Fengying, Yao Ruiyu, Zou Hao is brave. the immunne response of mycoplasmal pneumonia of swine subunit vaccine. and Chinese veterinary's journal, 2012,32 (4): 529-533) method etc. set up, build P46 prokaryotic expression carrier, and be transformed into escherichia coli, express mycoplasma hyopneumoniae P46 albumen.
Result: pcr amplification product, after electrophoresis, occurs object band at about 1260bp, about 1152bp respectively, conforms to expection size; After PCR primer being reclaimed, be connected to prokaryotic expression carrier and check order, sequencing result shows, recombiant plasmid full mutation is correct.Recombiant plasmid is imported escherichia coli express, use affinity column to carry out purification in the P46 albumen after expressing, purified product is through SDS-PAGE electrophoresis, and destination protein fragment conforms to expection size.Concentrate after being dialysed by purifying protein, protein concentrate content is 1mg/mL, and frozen in-20 DEG C.
The structure of 1.2NrdF recombinant expression carrier and the expression of albumen, qualification and purification
According to NCBI (
http:// www.ncbi.nlm.nih.gov) in report mycoplasma hyopneumoniae J strain (accession number: AE017243.1) in NrdF protein nucleotide sequence (see SEQ ID No.9), with the primer sequence of Primer Premier5 software design NrdF albumen, and add BamHI, HindIII restriction enzyme site respectively at upstream and downstream primer, Details as Follows:
Forward primer: 5'-CGGGATCCTTAAAACTCCCAATCTT-3'
Downstream primer: 5'-CCCAAGCTTGATCTATTATATAAACTAAT-3'
By Chen (Chen AY, Fry SR, Forbes-Faulkner J, et al.Comparative immunogenicity of M.hyopneumoniae NrdF encoded in different expression systems delivered orally via attenuated S.tyhimurium aroA in mice.Veterinary microbiology, 2006, method 114:252-259) etc. set up, build NrdF prokaryotic expression carrier, and be transformed into escherichia coli, express mycoplasma hyopneumoniae NrdF albumen.
Result: pcr amplification product, after electrophoresis, occurs object band at about 291bp respectively, conforms to expection size; After PCR primer being reclaimed, be connected to prokaryotic expression carrier and check order, sequencing result shows, construction of recombinant plasmid is correct.Recombiant plasmid is imported escherichia coli express, use affinity column to carry out purification in the NrdF albumen after expressing, purified product is through SDS-PAGE electrophoresis, and destination protein fragment conforms to expection size.Concentrate after being dialysed by purifying protein, protein concentrate content is 1mg/mL, and frozen in-20 DEG C.
The structure of 1.3P46-NrdF recombinant expression carrier and the expression of fusion rotein, qualification and purification
According to NCBI (
http:// www.ncbi.nlm.nih.gov) in report mycoplasma hyopneumoniae J strain (accession number: AE017243.1) in the nucleotide sequence (seeing SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 successively) of P46I, P46II, NrdF albumen, the primer sequence of P46I-NrdF, P46II-NrdF is designed respectively with PrimerPremier5 software, and add BamHI, Sal I restriction enzyme site respectively at upstream and downstream primer, Details as Follows:
(1)P46I-NrdF
P46I-1：5'-CGGGATCCGCAGAAGAAGGCAAGAGT-3'，
P46I-2：5'-TAGGCATCAGGATTAGATTGGGAGTTTTAA-3'，
NrdF-1：5'-TAATCCTGATGCCTTAAAACTCCCAATC-3'，
NrdF-2：5'-CCCAAGCTTGATCTATTATATAAACTAAT-3'，
P46I-NrdF forward primer: 5'-CGGGATCCGCAGAAGAAGGCAAGAGT-3',
P46I-NrdF downstream primer: 5'-GCGTCGACTAGCTCTAAACCTTGATCAGTT-3'.
(2)P46II-NrdF
P46II-1：5'-CCCGGATCCGGTTCGACTTCAGATTC-3'，
P46II-2：5'-TAGGCATCAGGATTAGATTGGGAGTTTTAA-3'，
NrdF-1：5'-TAATCCTGATGCCTTAAAACTCCCAATC-3'，
NrdF-2：5'-CCCAAGCTTGATCTATTATATAAACTAAT-3'，
P46II-NrdF forward primer: 5'-CCCGGATCCGGTTCGACTTCAGATTC-3',
P46II-NrdF downstream primer: 5'-GCGTCGACTAGCTCTAAACCTTGATCAGTT-3'.
With mycoplasma hyopneumoniae J strain DNA for template, with P46I-1, P46I-2 primer amplification P46I gene, with NrdF-1 and NrdF-2 primer amplification NrdF gene, then with above-mentioned gene outcome for template, the genes of interest of fragment is gone out to merge with P46I-NrdF primer amplification, build P46I-NrdF prokaryotic expression carrier, proceed to escherichia coli, express P46I-NrdF albumen.
According to the method described above, P46II-NrdF fusion rotein is prepared.
Result is identified: pcr amplification product, after electrophoresis, occurs object band at about 1500bp and 1400bp respectively, conforms to expection size; After PCR primer being reclaimed, be connected to prokaryotic expression carrier and check order, sequencing result shows, construction of recombinant plasmid is correct.Recombiant plasmid is imported escherichia coli express, use affinity column to carry out purification respectively two kinds of fusion rotein after expressing, purified product is through SDS-PAGE electrophoresis, and destination protein fragment conforms to expection size.
Concentrate after being dialysed by purifying protein, protein concentrate content is 1mg/mL, frozen in-20 DEG C.
The preparation of embodiment 2 mycoplasma hyopneumoniae antigen
Diluted by mycoplasma hyopneumoniae HN0613 strain freeze-drying lactobacillus fluid medium, streak inoculation is on solid medium plate, and put 37 DEG C and cultivate 7 days, the bacterium colony that growth selection is good, is inoculated in culture medium slant, cultivates 7 days, as first order seed for 37 DEG C.
The slant culture of first order seed washed by the fluid medium that takes a morsel, and is inoculated in fluid medium bassoon, puts 37 DEG C and cultivates 7 days, through checking purely as secondary seed.
The secondary seed solution of being cultivated by fluid medium by volume 10% (V/V) is inoculated in fluid medium.3-6 days is cultivated, band its colour changed into yellow, results bacterium liquid when pH value drops to 6.8-7.0 at 37 DEG C, purely after the assay was approved, then amplification culture (subculture was no more than for 6 generations) in the same way.Culture saves backup through the rearmounted 2-8 DEG C that is purely up to the standards.
To the bacterium liquid sampling of results, carry out 10 times of serial dilutions by Friis culture medium and carry out count plate, cultivate 20 days for 37 DEG C, observe the change of culture medium color, the CCU titre sample being sample with most highly diluted multiple.Arrange 2 to repeat to average, be final CCU value (5.5 × 10
9cCU/mL).
Above-mentioned culture is added the thimerosal of 1%, make its final concentration be 0.01%, 4 DEG C of deactivations 12 hours, make it deactivation complete.
The preparation of embodiment 3 mycoplasmal pneumonia of swine vaccine combination
Mycoplasma hyopneumoniae P46 albumen, NrdF albumen, P46I-NrdF and P46II-NrdF fusion rotein prepared by embodiment 1, and mycoplasma hyopneumoniae antigen prepared by embodiment 2, use pH7.2PBS solution dilution, by the albumen after dilution or antigenic solution and Montanide
tMgel01 adjuvant mixes in component and ratio contained by mycoplasmal pneumonia of swine vaccine combination in table 2,10-15min is stirred with the rotating speed of 500-800r/min, 1% (volume ratio) thimerosal solution is added before termination is stirred, make its final concentration be no more than ten thousand/, abundant vibration mixing, after subpackage, 2-8 DEG C saves backup.
Component and ratio contained by table 2 mycoplasmal pneumonia of swine vaccine combination
Note: the P36 albumen reference Hu Maozhi in vaccine J etc. are at document (Hu Maozhi, Jiao Xinan, Zhang little Rong etc. the cloning and expression of P 36 gene of Mycoplasma hyopneumoniae. Chinese Preventive Veterinary Medicine report, 2004,26 (3): 188-191) method described in, and with NCBI (
http:// www.ncbi.nlm.nih.gov) in the 169238-170185 position nucleotides sequence of mycoplasma hyopneumoniae J strain (accession number: AE017243.1) of report be classified as template, preparation P36 albumen; P65 albumen with reference to Ma Fengying etc. document (Ma Fengying, Yao Ruiyu, Zou Haoyong etc. the immunne response of mycoplasmal pneumonia of swine subunit vaccine. Chinese veterinary's journal, 2012,32 (4): 529-533) in the method that describes, and with NCBI (
http:// www.ncbi.nlm.nih.gov) in the 81104-82906 position nucleotides sequence of mycoplasma hyopneumoniae J strain (accession number: AE017243.1) of report be classified as template, preparation P65 albumen.
The mycoplasmal pneumonia of swine vaccine combination potency test of embodiment 4 different component
By the vaccine combination prepared by embodiment 3, immunity selects 14-21 age in days piglet 70 (getting rid of Porcine reproductive and respiratory syndrome, pig annulus 2 type and swine fever), totally 12 groups, 5/group, 1mL/ head respectively, arranges J strain with reference to vaccine contrast simultaneously.Latter 14 days of first immunity, carry out two according to same dose and approach and exempt from, two exempt from latter 28 days, injecting mycoplasma hyopneumoniae Jinan to all pig tracheas is strong malicious CVCC354 strain, and 100MID/ head, observes and dissect after 28 days, and observe pulmonary lesion, according to 28 point-scores, pulmonary lesion is marked.
Result of the test is as follows:
(1) immunity and counteracting toxic substances after clinical symptoms
That afternoon after immunity, all there is the transient rising of temperature (39.7-40.1 DEG C) in 14 groups of all pigs, and normally, red and swollen grade for other clinical symptoms do not appear in injection site for 1-12 group and counteracting toxic substances matched group temperature.After counteracting toxic substances about 10 days, there is the symptoms such as cough and asthma in counteracting toxic substances matched group pig (wherein 4) successively, and last till to cut open and kill the date, and fur is rough, the minimizing of more blank group of daily inleting appetite.
(2) pneumonia lesion score
After counteracting toxic substances 28 days, cut open and kill all pigs, according to 28 points of scoring methods, the pneumonopathy of every pig is become and marks, the results are shown in Table 3.
Table 3 mycoplasmal pneumonia of swine vaccine combination efficacy test results
Note 1: the 10 group is inactivated vaccine matched group, and selected vaccine is the mycoplasmal pneumonia of swine inactivated vaccine (J strain) in existing product, namely pacifies hectogram (M+PAC); 11st group is prior art matched group, institute's immune component is P36, P46, P65, P97R1-NrdF compositions, reference Ma Fengying etc. are at document (Ma Fengying, Yao Ruiyu, Zou Hao is brave. the immunne response of mycoplasmal pneumonia of swine subunit vaccine. and Chinese veterinary's journal, 2012,32 (4): 529-533) method described in is prepared from.
Note 2: in diversity statistical analysis, compare between group, alphabetical identical person represents that difference is not remarkable, and alphabetical different person represents significant difference (p < 0.05).
As shown in Table 3:
1-4 group (i.e. vaccine A-D group) is respectively the vaccine combination containing P46, NrdF two kinds of albumen or the vaccine combination containing two kinds of albumen and mycoplasma hyopneumoniae antigen, carry out cuing open after Immunization killing, observe and cut open the display of the result after killing: pneumonopathy becomes the equal > 60% of slip, especially, the protected effect of 3-4 group is better than 1-2 group, and any two groups are compared difference all significantly (p > 0.05); Compared with 9-11 group, immune effect is better, and difference is all significantly (p < 0.05) compared with 9-11 group; Compared with the 12nd group (i.e. inactivated vaccine matched group), 1-2 group immune effect is poor, and 3-4 group immune effect is slightly poor, but difference is not all significantly (p > 0.05); Compared with counteracting toxic substances matched group, difference all significantly (p < 0.05).
6-8 group (i.e. vaccine E-H group) is respectively the vaccine combination containing P46-NrdF fusion rotein or the vaccine combination containing P46-NrdF fusion rotein and mycoplasma hyopneumoniae antigen, counteracting toxic substances result display after immunity: pneumonopathy becomes slip and all reaches > 60%, especially, the immune effect of 7-8 group is better than 5-6 group, and any two groups are compared difference all significantly (p > 0.05); Compared with 1-4 group, immune effect is better, but is only slightly better than 3-4 group, and compared with 1-4 group difference all remarkable (p > 0.05); Compared with 9-11 group, immune effect is better, and difference is all significantly (p < 0.05) compared with 9-11 group; Compared with the 12nd group (i.e. inactivated vaccine matched group), 5-6 group immune effect is poor, and 7-8 group immune effect is suitable, but difference is not all significantly (p > 0.05); Compared with counteracting toxic substances matched group, difference all significantly (p < 0.05).
In sum:
(1) containing the counteracting toxic substances result display after the vaccine combination immunity pig of P46, NrdF two kinds of albumen: all pig pneumonia pathological changes slips, all higher than 60%, all can reach the effect of immunoprotection; Compared with inactivated vaccine in existing product, though difference is not remarkable, the effect produced after immunity has a certain distance.
(2) show containing the counteracting toxic substances result after the vaccine combination immunity pig of P46, NrdF two kinds of albumen and mycoplasma hyopneumoniae antigen: all pig pneumonia pathological changes slips, all higher than 68%, all can obtain good immune protective effect; Compared with only containing the vaccine combination of P46, NrdF two kinds of albumen, immune effect is more excellent, but difference is not remarkable; Compared with inactivated vaccine in existing product, difference is not remarkable, and immune effect is slightly poor or suitable.
The above is only the preferred embodiments of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, not departing from the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be the content not departing from technical solution of the present invention, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (10)
1. a mycoplasmal pneumonia of swine vaccine combination, is characterized in that, described vaccine combination contains P46 albumen, NrdF albumen and veterinarily acceptable adjuvant.
2. vaccine combination according to claim 1, is characterized in that, described P46 albumen is one or both protein mixtures that SEQ ID No.2 or SEQ ID No.4 encode.
3. vaccine combination according to claim 1, is characterized in that, described NrdF albumen is the albumen that SEQ ID No.6 encodes.
4. the vaccine combination according to any one of claim 1-3, is characterized in that, the P46 albumen in described vaccine combination, NrdF albumen expressing in series.
5. a mycoplasmal pneumonia of swine vaccine combination, is characterized in that, described vaccine combination is made up of P46 albumen, NrdF albumen and veterinarily acceptable adjuvant.
6. the vaccine combination according to any one of claim 1-5, is characterized in that, described vaccine combination is also containing the full bacterium antigen of mycoplasma hyopneumoniae.
7. the vaccine combination according to any one of claim 1-6, it is characterized in that, described adjuvant is one or more in the copolymer of aluminium glue adjuvant, saponin, water in oil emulsion, oil in water emulsion, W/O/W Emulsion, acrylic acid, the polymer of methacrylic acid, maleic anhydride and alkenyl (alkenyl) derivant, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E.coli LT, cholera toxin, IMS1314, muramyldipeptide or Gel adjuvant.
8. vaccine combination according to claim 7, is characterized in that, described adjuvant is Gel adjuvant.
9. vaccine combination according to any one of claim 1-8, it is characterized in that, described vaccine combination also comprises other antigens, other antigens described comprise in pig circular ring virus antigen, porcine reproductive and respiratory syndrome virus antigen, haemophilus parasuis antigen, PPV Antigen Using, Actinobacillus pleuropneumoniae antigen, bacillus coli antigen, atrophic rhinitis antigen, porcine pseudorabies virus antigen, hog cholera pathogen antigen, swine flue antigen one or more; Preferably, other antigens described are pig circular ring virus antigen.
10. the vaccine combination described in any one of claim 1-9 is preparing the application prevented and/or treated in the medicine of mycoplasmal pneumonia of swine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410204020.0A CN104248753B (en) | 2014-05-15 | 2014-05-15 | A kind of vaccine combination and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410204020.0A CN104248753B (en) | 2014-05-15 | 2014-05-15 | A kind of vaccine combination and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104248753A true CN104248753A (en) | 2014-12-31 |
| CN104248753B CN104248753B (en) | 2017-07-28 |
Family
ID=52184227
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410204020.0A Active CN104248753B (en) | 2014-05-15 | 2014-05-15 | A kind of vaccine combination and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104248753B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087616A (en) * | 2015-09-10 | 2015-11-25 | 河北师范大学 | Fusion gene of mycoplasma hyopneumoniae and preparation method of fusion gene |
| CN106139137A (en) * | 2015-03-30 | 2016-11-23 | 普莱柯生物工程股份有限公司 | A kind of vaccine combination 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 |
| CN112891524A (en) * | 2020-12-09 | 2021-06-04 | 扬州优邦生物药品有限公司 | Porcine mycoplasma pneumoniae subunit vaccine composition and preparation method and application thereof |
| CN113730565A (en) * | 2021-08-12 | 2021-12-03 | 通用生物(安徽)股份有限公司 | Fusion protein vaccine composition and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1612749A (en) * | 2001-07-02 | 2005-05-04 | 辉瑞产品公司 | Mycoplasma hyopneumoniae vaccine and methods for reducing mycoplasma bovis pneumonia in cattle |
| EP2027873A1 (en) * | 2001-07-02 | 2009-02-25 | Pfizer Products Inc. | One dose vaccination with mycoplasma hyopneumoniae |
| CN102732473A (en) * | 2011-04-08 | 2012-10-17 | 华中农业大学 | Recombinant salmonella choleraesuis expressing mycoplasma hyopneumoniae p46 protein, preparation method and application thereof |
| CN103031258A (en) * | 2012-06-29 | 2013-04-10 | 普莱柯生物工程股份有限公司 | Novel mycoplasma hyopneumoniae bacterial strain and vaccine composition thereof |
-
2014
- 2014-05-15 CN CN201410204020.0A patent/CN104248753B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1612749A (en) * | 2001-07-02 | 2005-05-04 | 辉瑞产品公司 | Mycoplasma hyopneumoniae vaccine and methods for reducing mycoplasma bovis pneumonia in cattle |
| EP2027873A1 (en) * | 2001-07-02 | 2009-02-25 | Pfizer Products Inc. | One dose vaccination with mycoplasma hyopneumoniae |
| CN102732473A (en) * | 2011-04-08 | 2012-10-17 | 华中农业大学 | Recombinant salmonella choleraesuis expressing mycoplasma hyopneumoniae p46 protein, preparation method and application thereof |
| CN103031258A (en) * | 2012-06-29 | 2013-04-10 | 普莱柯生物工程股份有限公司 | Novel mycoplasma hyopneumoniae bacterial strain and vaccine composition thereof |
Non-Patent Citations (2)
| Title |
|---|
| AUSTEN Y. CHEN等: "Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice", 《VACCINE》 * |
| 马丰英等: "猪支原体肺炎亚单位疫苗的免疫应答", 《中国兽医学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106139137A (en) * | 2015-03-30 | 2016-11-23 | 普莱柯生物工程股份有限公司 | A kind of vaccine combination and application thereof |
| CN105087616A (en) * | 2015-09-10 | 2015-11-25 | 河北师范大学 | Fusion gene of mycoplasma hyopneumoniae and preparation method of fusion gene |
| 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 |
| CN112891524A (en) * | 2020-12-09 | 2021-06-04 | 扬州优邦生物药品有限公司 | Porcine mycoplasma pneumoniae subunit vaccine composition and preparation method and application thereof |
| CN113730565A (en) * | 2021-08-12 | 2021-12-03 | 通用生物(安徽)股份有限公司 | Fusion protein vaccine composition and preparation method thereof |
| CN113730565B (en) * | 2021-08-12 | 2023-07-25 | 通用生物(安徽)股份有限公司 | Fusion protein vaccine composition and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104248753B (en) | 2017-07-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104862286B (en) | Porcine pseudorabies virus gene-deleted strain, vaccine composition and its preparation method and application | |
| CN105693827B (en) | Porcine pseudorabies virus subunit vaccine and preparation method and application thereof | |
| CN103031258B (en) | Novel mycoplasma hyopneumoniae bacterial strain and vaccine composition thereof | |
| CN104248753B (en) | A kind of vaccine combination and its application | |
| CN104248762A (en) | Porcine epidemic diarrhea vaccine composition, and preparation method and application thereof | |
| CN105018433B (en) | Porcine pseudorabies virus gene-deleted strain, vaccine composition and its preparation method and application | |
| CN107320720A (en) | A kind of vaccine combination, kit and application | |
| CN101880647B (en) | Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application | |
| CN104277115A (en) | Preparation method and application of fusion protein and vaccine composition containing same | |
| Li et al. | Surface Display of porcine circovirus type 2 antigen protein cap on the spores of Bacillus subtilis 168: An effective mucosal vaccine candidate | |
| CN111233984B (en) | O-type foot-and-mouth disease virus-like particle antigen, vaccine composition thereof, preparation method and application | |
| CN103667196A (en) | Vaccine composition containing porcine circovirus-2 antigen and swine flu antigen | |
| CN102727884A (en) | Combined live vaccine against porcine reproductive and respiratory syndrome and pseudorabies, and preparation method thereof | |
| AU2013342269B2 (en) | Attenuated Mannheimia haemolytica vaccines and methods of making and use | |
| CN106139137A (en) | A kind of vaccine combination and application thereof | |
| CN105012948B (en) | A kind of vaccine composition and its application | |
| CN105797152A (en) | Vaccine composition and its preparation method and use | |
| CN107868130B (en) | Fusion protein containing mycoplasma hyopneumoniae antigen, vaccine composition and application | |
| CN105754905A (en) | Pseudomonas aeruginosa of minks and application of pseudomonas aeruginosa | |
| CN106929480A (en) | Porcine reproductive and respiratory syndrome virus strain and its application | |
| CN103261214B (en) | There is the vaccine for systemic administration of bacteria distribution thing alive | |
| CN107537033B (en) | Vaccine composition, kit and application thereof | |
| WO2010097977A1 (en) | Oral polyvalent vaccine against swine erysipelas/mycoplasmal pneumonia | |
| WO2018223668A1 (en) | Bivalent vaccine composition for preventing and/or treating porcine circovirus infections and preparation method and use thereof | |
| CN105497885B (en) | A kind of subunit vaccine and its preparation method and application |
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 |