CN107513506A - Mycoplasma hyopneumoniae, vaccine combination and its application - Google Patents

Mycoplasma hyopneumoniae, vaccine combination and its application Download PDF

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CN107513506A
CN107513506A CN201610435285.0A CN201610435285A CN107513506A CN 107513506 A CN107513506 A CN 107513506A CN 201610435285 A CN201610435285 A CN 201610435285A CN 107513506 A CN107513506 A CN 107513506A
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antigen
strains
pig
inactivation
haemophilus parasuis
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CN107513506B (en
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田克恭
金云云
孙进忠
张许科
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Pulaike Biological Engineering Co Ltd
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Abstract

The invention provides one plant of mycoplasma hyopneumoniae GZ strain, and it has more preferable immunogenicity, and the vaccine combination of preparation can produce protection completely to pig, and GZ strains titre in prepared by amplification is high, is easy to cultivate, can produce a large amount of antigens.Its antigen prepared can use simultaneously with other antigens, and each caused immune efficacy is mutually unaffected.

Description

Mycoplasma hyopneumoniae, vaccine combination and its application
Technical field
The present invention relates to a kind of mycoplasma hyopneumoniae, and the vaccine combination of its preparation and application, belong to biotechnology Field.
Background technology
Porcine mycoplasmal pneumonia (Mycoplasma Pneumoniae of swine, MPS) is also known as swine enzootic pneumonia, is by pig lung A kind of chronic respiratory infectious disease caused by scorching mycoplasma (Mycoplasma hyopneumoniae, Mhp), with hyperinfection Property, with the characteristics of high incidence and low actual, be mainly shown as cough, expiratory dyspnea, dissect visible lung tissue meat and become or Dali Stone sample lesion, based on pulmonary lesion, especially characterized by the change of pancreas sample and carnification occur in two lung lobus cardiacuses, middle leaf and sharp leaf.Its Main harm is to cause the growth retardation of pig and feed conversion rate to decline.Mycoplasma hyopneumoniae is exhaled by respiratory infectious, infection Respiratory tract cilium is caused to come off, damage after inhaling road epithelium, epithelial cell is downright bad, reduces the immunologic function of respiratory mucosa, easily Cause the scabies secondary infection of other respiratory pathogenses, cause its disease symptom to aggravate, death rate increase.Swine enzootic pneumonia is popular in the world Various regions, it is that one of most important disease of economic loss of raising pigs is caused in world wide.
According to documents and materials, the mycoplasma hyopneumoniae separated all over the world belongs to same serotype, but separation strains Between antigenicity very big difference be present, Frey confirmed antigen between mycoplasma hyopneumoniae difference separation strains in 1992 first The difference of property.Requirement of the mycoplasma hyopneumoniae to culture medium is high, slow-growing, and which greatly increases being produced into for vaccine This.Therefore, the new pig that immunogenicity is preferable, the speed of growth is very fast and viable bacteria titre is higher is isolated from domestic clinical case Preventing and treating of the M. hyopneunzoniae strain for China's porcine mycoplasmal pneumonia is particularly important.
The content of the invention
It is an object of the invention to provide one kind the more preferable mycoplasma hyopneumoniae strain of immunogenicity, institute are accredited as through separation It is mycoplasma hyopneumoniae GZ (Mycoplasma hyopneumoniae strain GZ) to state strain, and preserving number is：CCTCC NO： M2016212, depositary institution are China typical culture collection center, and preservation address is Wuhan, China Wuhan University, during preservation Between be on April 20th, 2016.
The vaccine strain of the strain not only more existing business of the present invention has more preferable immunogenicity, also has good Viral titer is cultivated, culture is easy, the advantages of being easily obtained substantial amounts of antigen.
The present invention relates to a kind of vaccine combination, wherein the vaccine combination includes the mycoplasma hyopneumoniae poison of immune amount The antigen and pharmaceutically acceptable carrier of strain or its culture.
The vaccine combination of the present invention has good immunogenicity, and infection that can be to mycoplasma hyopneumoniae is carried out completely Protection.
The invention further relates to the vaccine combination to prepare the relevant disease of prevention and treatment mycoplasma hyopneumoniae infection Medicine in application.
Vaccine strain culture titre provided by the invention is high, and immunogenicity is good, effectively reduces usage amount, the vaccine combination of preparation Thing clinically can effectively control the infection of mycoplasma pneumoniae in swinery.
Embodiment
Hereinafter, embodiments of the present invention are illustrated.
The present invention relates to mycoplasma hyopneumoniae GZ strains, also referred to as mycoplasma hyopneumoniae GZ, preserving number is CCTCC NO: M2016212。
Vaccine strain of the mycoplasma hyopneumoniae GZ strains with more existing business of the present invention with more preferable immunogenicity, And it is easily cultivated, and is suitably industrially applied.The present invention relates to a kind of vaccine combination, wherein, the vaccine combination includes The described mycoplasma hyopneumoniae GZ strains or inactivation antigen of its culture of immune amount, the full mycoplasma antigen of work or subunit Antigen, and pharmaceutically acceptable carrier.
" culture " is the different generation subcultures of virus, and those skilled in the art know its base between different generations Small variation can occur because sequence is only possible.
The composition of composition or the amount of component of the present invention is preferably therapeutically effective amount.The therapeutically effective amount refers to Their immunological role is played without causing excessive side effect institute necessary amounts in the host that composition is applied.Composition used and The accurate amount of composition to be administered is by according to factor such as the type of the disease for the treatment of, the type of animal to be treated and year Age, the mode of administration, and other compositions in composition and change.
As one embodiment of the present invention, mycoplasma hyopneumoniae GZ strains culture of the present invention is the training of 1~42 generation Support thing.
Term used herein " vaccine combination " refers to the pharmaceutical composition containing mycoplasma hyopneumoniae immunogenicity, the medicine Compositions can induce, stimulate or strengthen the immune response that pig is directed to mycoplasma pneumoniae.The vaccine combination includes immune Attenuated live vaccine, inactivated vaccine, subunit vaccine or the synthetic peptide vaccine of the mycoplasma hyopneumoniae strain of amount.
Term used herein " inactivated vaccine ", also referred to as inactivated vaccines, refer to being used as antigen to produce immunity Inactivation of viruses suspension.The example of inactivated vaccine includes whole virus vaccine and cracking type vaccine.Can be with using known method Easily produce inactivated vaccine.For example, inactivated virus vaccine can be obtained by using formalin processing virus.Cracking type epidemic disease Seedling can be prepared after being handled with ether by peplos.Such as inactivation can be passed through with the mycoplasma hyopneumoniae GZ strains of the present invention Method be prepared into inactivated vaccine.
As one embodiment of the present invention, in vaccine combination of the present invention, described mycoplasma hyopneumoniae GZ Strain or its culture inactivation antigen for inactivation full mycoplasma antigen or its schizolysis antigen, described mycoplasma hyopneumoniae GZ strains Or the full mycoplasma antigen of the work of its culture for attenuation work full mycoplasma antigen, described mycoplasma hyopneumoniae GZ strains or The subunit antigen of its culture is antigen protein P97, P110, P46 and P36.
As one embodiment of the present invention, the full branch of mycoplasma hyopneumoniae GZ strains of the present invention or the inactivation of its culture Mycoplasma antigen can be prepared using ablation method well known in the art.
As one embodiment of the present invention, the schizolysis antigen of mycoplasma hyopneumoniae GZ strains of the present invention or its culture can Handled using nonionic surfactant, with the antigenic component on acquisition, enrichment of cell film.
As one embodiment of the present invention, the antigen protein of mycoplasma hyopneumoniae GZ strains of the present invention or its culture P97, P110, P46 and P36 can use gene engineering method to prepare, can also directly synthesize.
As one embodiment of the present invention, in vaccine combination of the present invention, the mycoplasma hyopneumoniae GZ Strain or its culture inactivation antigen content for inactivation before >=107.0CCU/ml。
As a kind of preferred embodiment of the present invention, in vaccine combination of the present invention, the pig pneumonia branch is former The content of the inactivation antigen of body GZ strains or its culture is before inactivation 107.0~1010.0CCU/ml.Term " adjuvant ", which refers to, to be added to To increase the material of the immunogenicity of composition in the composition of the present invention.
It is described pharmaceutically acceptable in vaccine combination of the present invention as one embodiment of the present invention Carrier includes adjuvant；The adjuvant includes：(1) aluminium glue adjuvant, saponin(e, Avridine, DDA；(2) water-in-oil emulsion, oil-in-water Emulsion, W/O/W emulsion；Or polymer, maleic anhydride and the alkenyl of (3) acrylic or methacrylic acid derive The copolymer of thing；And RIBI adjuvant systems, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipids- Amine adjuvant, E.coli LT, cholera toxin, IMS 1314, muramyl dipeptide, one kind in Gel adjuvants or several Kind.
Preferably, saponin(e is Quil A, QS-21, GPI-0100.
Preferably, emulsion is that SPT emulsions, MF59 emulsions, or emulsion are formed by oil with emulsifier combination, and emulsion can be based on light Saxol, because caused by olefin oligomerisation isoprenoid oil (such as saualane or squalene oil, particularly alkene, isobutene Or it is oily caused by decene oligomerization), acid or alcohol containing linear alkyl ester (more specifically vegetable oil, ethyl oleate, propane diols two- (caprylate/certain herbaceous plants with big flowers acid esters), glycerine three-(caprylate/certain herbaceous plants with big flowers acid esters) or Rikemal PO 200), the ester of branched chain fatty acid or alcohol it is (outstanding Its isostearate)；Emulsifying agent is nonionic surfactant (the especially ester of polyoxyethylated fatty acid (such as oleic acid), mountain The ester of pears glycan, the ester (such as anhydrous mannitol oleate) of mannide, the ester of aliphatic dihydroxy alcohol, the ester, poly- sweet of glycerine Ester, the ester and the ester of oleic acid, the ester of isostearic acid, the ester of castor oil acid or the ester of hydroxy stearic acid of propane diols of oil, it is above-mentioned Ester can be through ethoxylation, the ether of fatty alcohol and polyalcohol (such as oleyl alcohol), Pluronic L121 (especially, particularly L121)).
Preferably, the polymer of acrylic or methacrylic acid is the acrylic or methacrylic acid polymer of crosslinking, especially It is the compound carbomer being crosslinked with the poly alkenyl ether of sugar or polyalcohols, is preferably carbopol 974P, 934P and 971P.
Preferably, the copolymer of maleic anhydride and alkenyl derivative is the copolymer of maleic anhydride and ethene EMA。
Preferably, the adjuvant includes Gel 01, ISA206, ISA760VG, carbomer, aluminium hydroxide.
The concentration range of the adjuvant is the preferably 10%V/V from 10% to 70%V/V.
Known adjuvant includes, but are not limited to：(1) aluminium hydroxide, saponin(e (Saponine) (such as QuilA), A Fuli Fixed, DDA, the polymer of the sour polymer of (2) acrylic or methacrylic, maleic anhydride and alkenyl derivative, or (3) Vaccine can be made in the form of oil-in-water, Water-In-Oil or W/O/W emulsion.
Especially, emulsion can be based on light liquid paraffin oil, isoprenoid oil, such as saualane or squalene；Alkene, Ester oily particularly caused by isobutene or decene oligomerization, that acid or alcohol with straight chained alkyl are formed, more particularly vegetable oil, oil Sour ethyl ester, propane diols two (caprylate/decylate), glycerine three (caprylate/decylate), Rikemal PO 200；Branch's fat The ester of fat acid esters or alcohol, particularly isostearate.Oil is used together to form emulsion with emulsifying agent.The preferred nonionic table of emulsifying agent Face activating agent, particularly polyoxyethylated fatty acid (such as oleic acid), sorbitan, mannitol (such as anhydromannitol Oleate), glycerine, polyglycereol, propane diols and the alternatively oleic acid of ethoxylation, isostearic acid, castor oil acid, hydroxy stearic acid The ether of the ester of formation, fatty alcohol and polyalcohol (such as oleyl alcohol), polyoxypropylene polyoxyethylene block copolymer, particularly PluronicR, especially L121 are (with reference to Hunter etc., 1995, " The Theory and Practical Application OfAdjuvants " (Steward-Tull, D.E.S are edited) John Wiley andSons, NY, 51-94；Todd etc., Vaccine, 1997,15,564-570).
Especially, acrylic or methacrylic acid polymer is crosslinked by the poly alkenyl ether of sugar or polyalcohol.These are changed Compound is referred to as carbomer.
Adjuvant used in the present invention also includes Gel 01 (French SEPPIC), ISA206 (French SEPPIC), ISA760VG One or more of combinations of (French SEPPIC).
Preferably, vaccine adjuvant of the present invention is carbomer (Carbomer) (trade name Carbopol), Gel 01 (French SEPPIC), aluminium hydroxide.
Most preferably, it is Gel 01 (French SEPPIC) to invent described vaccine adjuvant.
In final vaccine combination, the concentration range of adjuvant is the preferably 10%V/V from 10% to 70%V/V.
As one embodiment of the present invention, vaccine combination of the present invention also includes medicine, immunostimulant, Antioxidant, surfactant, colouring agent, ethereal oil, buffer, dispersant, propellant and preservative.
Preferably, immunostimulant includes alpha-interferon, beta-interferon, gamma interferon, granular leukocyte macrophage colony thorn Swash the factor, macrophage colony stimulatory factor and interleukin-22.Vaccine combination of the present invention can also be further by other reagents It is added to the composition of the present invention.In order to prepare such composition, method well known in the art can be used.As the present invention A kind of embodiment, vaccine combination of the present invention further includes other pathogen antigens, other described pathogen antigens Resist including CSFV antigen, PRV antigen, pig circular ring virus antigen, haemophilus parasuis antigen, Streptococcus suis Original, swine flu antigen, porcine contagious pleuropneumonia antigen, pig pasteurella multocida antigen, pig bordetella bacilli antigen, pig breeding Resist with breath syndrome virus antigen, Salmonella choleraesuls antigen, PPV Antigen Using, and/or Latex agglutination test It is former.As one embodiment of the present invention, the vaccine combination also includes any in following further antigens of immune amount A kind of antigen：Pig circular ring virus antigen, haemophilus parasuis antigen.
As one embodiment of the present invention, the pig circular ring virus antigen resists including porcine circovirus 2 type ZJ/H strains Original, pig gyrate virus II type DBN-SX07 strains antigen, porcine circovirus 2 type SD strains antigen, porcine circovirus 2 type ZJ/C strains resist Original, porcine circovirus type 2 strain PCV2SD strains antigen, porcine circovirus 2 type SH strains antigen or pig gyrate virus II type PCV2/ HZ09 strain antigens；The haemophilus parasuis antigen includes the type haemophilus parasuis GX0905 strains antigen of serum 13, Serotype 5 pair Haemophilus suis JX1002 strains antigen, the type haemophilus parasuis HN1009 strains antigen of serum 4, the type haemophilus parasuis of serum 4 YBH04 strains antigen, Serotype 5 haemophilus parasuis YBH05 strains antigen, the type haemophilus parasuis YBH13 strains antigen of serum 13, blood Clear 1 type haemophilus parasuis LC strain antigen, the type haemophilus parasuis SHCM10 strains antigen of serum 12, the bloodthirsty bar of Serotype 5 pair pig Bacterium JSYZ10 strains antigen, the type haemophilus parasuis FJMH10 strains antigen of serum 13, the type haemophilus parasuis FS0307 strains of serum 4 resist Original, Serotype 5 haemophilus parasuis XX0306 strains antigen, Serotype 5 haemophilus parasuis LZ-20100109 strains antigen, serum 5 Type haemophilus parasuis LX-5 strains antigen, the type JS strains antigen of haemophilus parasuis serum 4, haemophilus parasuis Serotype 5 ZJ strains Antigen, the type HeB strains antigen of haemophilus parasuis serum 12 or its combination.
Porcine circovirus 2 type ZJ/H strains are deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, Preserving number is CGMCC NO.6391, is disclosed in Chinese patent application CN102787100A；Pig gyrate virus II type DBN-SX07 strains China Committee for Culture Collection of Microorganisms's common micro-organisms center is deposited in, preserving number is CGMCC NO.3064, is disclosed in Chinese patent application CN101549155A；Porcine circovirus 2 type SD strains are deposited in China Committee for Culture Collection of Microorganisms Common micro-organisms center, preserving number are CGMCC NO.5774, are disclosed in Chinese patent application CN102732486A；Pig circular ring virus 2 Malicious 2 type ZJ/C strains are preserved in China typical culture collection center, and preserving number is CCTCC NO.V201251, are disclosed in Chinese special Profit application CN103285385A；Porcine circovirus type 2 strain PCV2SD strains are deposited in Chinese microorganism strain preservation conservator Meeting common micro-organisms center, preserving number is CGMCC NO.7707, is disclosed in Chinese patent application CN103421748A；Pig annulus Viral 2 type SH strains are deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC NO.2389, it is disclosed in Chinese patent application CN101240264A；Pig gyrate virus II type PCV2/HZ09 strains are preserved in Chinese allusion quotation Type culture collection, preserving number are CCTCC NO.V201312, are disclosed in Chinese patent application CN103436498A.
The type haemophilus parasuis GX0905 strains of serum 13 are preserved in China typical culture collection center, and preserving number is CCTCC NO.M2014125, it is disclosed in Chinese patent application CN104498384A；Serotype 5 haemophilus parasuis JX1002 strains China typical culture collection center is preserved in, preserving number is CCTCC NO.M2014127, is disclosed in Chinese patent application CN104388340A；The type haemophilus parasuis HN1009 strains of serum 4 are preserved in China typical culture collection center, and preserving number is CCTCC NO.M2014126, it is disclosed in Chinese patent application CN104312964A；The type haemophilus parasuis YBH04 strains of serum 4, Serotype 5 haemophilus parasuis YBH05 strains, the type haemophilus parasuis YBH13 strains of serum 13 are deposited in Chinese microorganism strain guarantor Administration committee's common micro-organisms center is hidden, preserving number YBH04 strains are CGMCC NO.5479, YBH05 strains are CGMCC NO.5480, YBH13 strain are CGMCC NO.5501, are disclosed in Chinese patent application CN102499982A；Serum 1 type pair pig is bloodthirsty Bacillus LC strains are deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC NO.5257, it is disclosed in Chinese patent application CN102399724A；The type haemophilus parasuis SHCM10 strains of serum 12 are preserved in China Type Tissue Collection, preserving number are CCTCC NO.M2014261, are disclosed in Chinese patent application CN104450556A； Serotype 5 haemophilus parasuis JSYZ10 strains are preserved in China typical culture collection center, preserving number CCTCC NO.M2014260, it is disclosed in Chinese patent application CN104450557A；The type haemophilus parasuis FJMH10 strains of serum 13 are preserved in China typical culture collection center, preserving number are CCTCC NO.M2014262, are disclosed in Chinese patent application CN104450555A；The type haemophilus parasuis FS0307 strains of serum 4 are preserved in China typical culture collection center, and preserving number is CCTCC NO.M2013094, it is disclosed in Chinese patent application CN103194413A；Serotype 5 haemophilus parasuis XX0306 strains China typical culture collection center is preserved in, preserving number is CCTCC NO.M2013095, is disclosed in Chinese patent application CN103194412A；Serotype 5 haemophilus parasuis LZ-20100109 strains are deposited in Chinese microorganism strain preservation conservator Meeting common micro-organisms center, preserving number is CGMCC NO.5802, is disclosed in Chinese patent application CN102851249A；Serotype 5 Haemophilus parasuis LX-5 strains are deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC NO.10230, it is disclosed in Chinese patent application CN104611274A；The type JS strains of haemophilus parasuis serum 4, secondary pig are thermophilic Blood bacillus Serotype 5 ZJ strains, the type HeB strains of haemophilus parasuis serum 12 are preserved in China typical culture collection center, preservation Number 4 type JS strains are CCTCC NO.M2011172,5 type ZJ strains are CCTCC NO.M2011173,12 type HeB strains are CCTCC NO.M2011174, it is disclosed in Chinese patent application CN102908615A.
As one embodiment of the present invention, in vaccine combination of the present invention, the pig circular ring virus antigen For the pig circular ring virus antigen or subunit antigen of inactivation, the pig circular ring virus content of the inactivation for before inactivation 5 × 105.0TCID50/ ml, the pig circular ring virus subunit antigen content are 20 μ g/ml；The haemophilus parasuis antigen is inactivation Haemophilus parasuis antigen, the haemophilus parasuis antigenic content of the inactivation is before inactivation 2 × 109CFU/ml。
As one embodiment of the present invention, in vaccine combination of the present invention, the pig circular ring virus antigen For the pig circular ring virus SH strain antigens of inactivation, the haemophilus parasuis antigen of the inactivation is the haemophilus parasuis serum of inactivation 4 type JS strains antigens and the haemophilus parasuis Serotype 5 ZJ strain antigens of inactivation.As one embodiment of the present invention, this hair In bright described vaccine combination, full mycoplasma antigen, the pig circular ring virus 2 of the mycoplasma hyopneumoniae GZ strains inactivation comprising immune amount The totivirus antigen of malicious SH strains inactivation, the full mycoplasma antigen content of mycoplasma hyopneumoniae GZ strains of the inactivation for before inactivation 5 × 108CCU/ml, the pig circular ring virus content of the inactivation is before inactivation 5 × 105.0TCID50/ml。
As one embodiment of the present invention, in vaccine combination of the present invention, the pig pneumonia of immune amount is included The full mycoplasma antigen of mycoplasma GZ strains inactivation, pig circular ring virus subunit antigen, the mycoplasma hyopneumoniae GZ strains of the inactivation Full mycoplasma antigen content is before inactivation 5 × 108CCU/ml, the pig circular ring virus subunit antigen content are 20 μ g/ml.
As one embodiment of the present invention, in vaccine combination of the present invention, the pig pneumonia of immune amount is included Full mycoplasma antigen, totivirus antigen, the haemophilus parasuis serum 4 of pig circular ring virus SH strains inactivation of mycoplasma GZ strains inactivation Full bacterium antigen, the full bacterium antigen of haemophilus parasuis Serotype 5 ZJ strains inactivation of type JS strains inactivation, the pig pneumonia branch of the inactivation The full mycoplasma antigen content of substance GZ strains is before inactivation 5 × 108CCU/ml, the pig circular ring virus content of the inactivation are before inactivating 5×105.0TCID50/ ml, the type JS strains antigenic content of haemophilus parasuis serum 4 of the inactivation is before inactivation 2 × 109CFU/ Ml, the haemophilus parasuis Serotype 5 ZJ strains antigenic content of the inactivation is before inactivation 2 × 109CFU/ml。
As one embodiment of the present invention, in vaccine combination of the present invention, the pig pneumonia of immune amount is included The full mycoplasma antigen of mycoplasma GZ strains inactivation, pig circular ring virus subunit antigen, the type JS strains of haemophilus parasuis serum 4 inactivation Full bacterium antigen, the full bacterium antigen of haemophilus parasuis Serotype 5 ZJ strains inactivation, the mycoplasma hyopneumoniae GZ strains of the inactivation are complete Mycoplasma antigen content is before inactivation 5 × 108CCU/ml, the pig circular ring virus subunit antigen content is 20 μ g/ml, described The type JS strains antigenic content of haemophilus parasuis serum 4 of inactivation is before inactivation 2 × 109CFU/ml, the secondary pig of the inactivation are bloodthirsty Bacillus Serotype 5 ZJ strains antigenic content is before inactivation 2 × 109CFU/ml。
The invention further relates to described vaccine combination to prepare prevention and treatment porcine mycoplasmal pneumonia and pig pneumonia branch original Application in the medicine of body-sensing dye relevant disease.Term " prevention " refers to by its infection related to mycoplasma hyopneumoniae or disease Symptom be blocked or postpone；Term " treatment " refers to be alleviated by the symptom of the infection related to mycoplasma hyopneumoniae or disease Or the process being completely eliminated.
The invention will now be further described with reference to specific embodiments, and advantages of the present invention and feature will be with description more To be clear.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.Those skilled in the art It should be understood that the details and form of technical solution of the present invention can be carried out without departing from the spirit and scope of the invention Modifications or substitutions, but these modifications and replacement are each fallen within protection scope of the present invention.
The separation identification of the mycoplasma hyopneumoniae GZ strains of embodiment 1
1. pathological material of disease source
Pathological material of disease from 2016 from Henan Province pig farm collection doubtful porcine mycoplasmal pneumonia lesion lungs, totally 20 parts.
2. pathological material of disease identification with multi-plex PCR
2.1 design of primers and synthesis
Multiplex PCR (mycoplasma hyopneumoniae, pig mycoplasma flocculare and mycoplasma hyorhinis) primer bibliography (the .A multiplex PCR to Identify Porcine Mycoplasma Present such as T.stakenbory in Broth Cultures) synthesized：
mhp-f：5’-TTCAAAGGAGCCTTCAAGCTTC-3’；
mhr-f：5’-GGGAAGAAAAAAATTAGGTAGGG-3’；
mfl-f：5’-CGGGATGTAGCAATACATTCAG-3’；
m-r：5’-AGAGGCATGATGATTTGACGTC-3’；
It is expected that mycoplasma hyopneumoniae, mycoplasma hyorhinis, pig mycoplasma flocculare expanding fragment length be respectively 1000bp, 1129bp、754bp。
2.2DNA template extractions and PCR identifications
It is sterile to take disease to be good for intersection lung tissue, DNA profiling is extracted with animal tissue's genome extracts kit, according to following Reaction system enters performing PCR amplification：The 5.0 μ l of μ l, d NTP 10nmol/L, 10 × buffer of template DNA 2, sense primer are The μ l of 8pmol, anti-sense primer 12pmol, Ex Tag polymerase 0.5, distilled water is added to 50 μ l；PCR reaction conditions are：95℃ 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, expand 30 circulations；72 DEG C extend 10 minutes；2-8 DEG C of preservation.PCR primer is entered The agarose gel electrophoresis of row 1.0% detects, and it is positive and cotton-shaped without mycoplasma hyorhinis, pig as a result to filter out 5 parts of mycoplasma hyopneumoniaes The sick lung tissue of mycoplasma contamination.
3. the separation and culture of bacterial strain
PCR is taken to be accredited as the lungs that mycoplasma hyopneumoniae is positive and is polluted without mycoplasma hyorhinis, pig mycoplasma flocculare, super Aseptic operation scissors clip site of pathological change edge tissues are used in net platform, are put into sterilized petri dishes, it is broken that sick lung tissue is cut into 1-2mm Block, it is inoculated with Friis meat soups, 37 DEG C of cultures, the daily pH value change for observing nutrient solution.When nutrient solution changes colour, the first generation is taken to pass through The culture 0.5ml of 0.45 μm of filter filtering is inoculated into 1.5ml 2000IU/ml containing penicillin Friis broth bouillons, 37 DEG C are continued to cultivate, and culture medium directly takes culture to carry out transplanting passage after turning yellow, and such as culture discoloration, carry out smear, respectively Carry out Gram's staining and Wright's staining, microscopy.As Gram's staining does not find bacterium, while Wright's staining discovery mycoplasma sample During thalline, take 0.2m1 cultures to be coated on Friis Solid media for plates surface, put 37 DEG C, 5%CO2Under the conditions of cultivate, often Day has seen whether mycoplasma sample bacterium colony.Take mycoplasma sample single bacterium colony to be inoculated in Friis meat soups, taken after culture medium its colour changed into yellow 0.2m1 cultures are coated on Friis Solid media for plates surface and put 37 DEG C, 5%CO2Under the conditions of carry out purifying culture, so Carry out purifying culture 2 times.Last time is purified to the mycoplasma sample colony inoculation Friis fluid nutrient mediums of culture growth, waits to train Strain is preserved after supporting base its colour changed into yellow.
4. mycoplasma hyopneumoniae GZ strain biological characteristicses
4.1 cultural character
GZ strains well-grown in Friis fluid nutrient mediums (pH value 7.4~7.6), 37 DEG C are cultivated 3~5 days, culture p H drops to 6.8~7.0, and slight homogeneous muddiness is presented.Viable bacteria titre can reach 10 in culture10~1011(CCU is CCU/ml Color changing units).Solid medium is inoculated with, after 37 DEG C are cultivated 3~5, colony growth enters inside culture medium, in typical fry Egg-shaped, it is consistent with mycoplasma colonial morphology.
4.2 forms and biochemical characteristic
This bacterium is Gram-negative, and after the dyeing of bacterium solution smear Ji's nurse Sa, oil mirror observes annular in shape, spherical, thread, point Shape, shaft-like or the two poles of the earth shape etc., acellular wall.
Biochemical test bibliography carries out (Cao Shu pools veterinary microbiologies and immunological technique [M] Beijing：Beijing Agriculture University press .1992.49~50,126~129,371~373.).It is substantially carried out digitonin sensitivity tests, urea Enzyme test, breakdown of glucose experiment, arginine hydrolysis experiment, benzyltriphenylphosphonium chloride tetrazole (TTC) reduction test, aesculin hydrolysis Experiment, mannitol decomposition run, film and spot formation experiment, the results are shown in Table 1.
The Mhp GZ strain biochemical results of table 1
Note:+ positive ,-negative；It is mycoplasma that digitonin sensitization test, which more than 1mm occurs to suppress band person,；TTC is reduced It is negative to test mycoplasma hyopneumoniae, mycoplasma hyorhinis is positive；(Cao Shu pools veterinary microbiologies and immunological technique [M] Beijing： Publishing house of Beijing Agricultural University .1992.49~50,126~129,371~373.).
4.3 serological characteristic
The mycoplasma hyopneumoniae culture of Friis plating 0.1ml exponential phases, by undiluted rabbit-anti pig pneumonia The μ l of mycoplasma serum 25 are adsorbed in the 6mm filter papers of sterilizing, and the filter paper is attached at into planar surface, cultivate visible to bacterium colony. The scraps of paper of normal rabbit serum processing are as negative control.There is the generation that bacterium colony suppresses ring around the culture observation circle scraps of paper.Suppress wide Degree reaches 2.42mm, can be judged to the mycoplasma hyopneumoniae positive.
5. isolated strains is pathogenic
Isolated strains culture transtracheal is injected into the susceptible pig 3 of 1~2 week old health, every 5ml (108CCU/ml).Separately If 3 top part identical control pigs, each tracheae injection of culture medium 5ml, as blank control.Test pig, control pig isolated rearing. Routinely raised after attacking poison, antibiotic-free in feed.Observation 28 days, surveys body temperature daily.Cut open inspection after poison is injected 28 is attacked, according to pig Eaton agent pneumonia tuberculosis varying index scoring criteria becomes to the tuberculosis of test pig scores.Test group carries out tuberculosis change with control group Index differential is analyzed.The separation of mycoplasma hyopneumoniae is carried out to test pig and control pig, and separation strains are entered with performing PCR identification.
Clinicing symptom observation result for attack poison 10 days after test pig in succession occur cough, asthma etc. symptom, control pig not There is the symptom；There is different degrees of lesion in the visible test pig lung of cut open inspection on the 28th after attacking poison, compares pig no abnormality seen.It is clinical Symptom Observation and pulmonary lesion the results are shown in Table 2.
Mycoplasma hyopneumoniae separation is carried out to 3 pigs of test group, mycoplasma hyopneumoniae is expanded to its change-colour fluid culture thing Specific P46 genes enter performing PCR identification, as a result amplify 1200bp purpose band, it was demonstrated that separation cause of disease is that pig pneumonia branch is former Body.
Virulence detection result of the Mhp GZ strains of table 2 to piglet
Note:In otherness statistical analysis, compare between group, the identical person of letter represents that difference is not notable, capitalization difference person Represent difference extremely significantly (P ＜ 0.01).
From be detected as with porcine mycoplasmal pneumonia typical cytopathic, through porcine mycoplasmal multiplex PCR mycoplasma hyopneumoniae it is positive and The microorganism of doubtful mycoplasma hyopneumoniae is isolated in the sick lung polluted without mycoplasma hyorhinis and pig mycoplasma flocculare, through items Identification meets the characteristic of mycoplasma hyopneumoniae, and growth inhibition test, PCR identifications and sequencing result show that the microorganism belongs to pig lung Scorching mycoplasma, it is named as mycoplasma hyopneumoniae GZ strains.It is reachable that content is cultivated in mycoplasma hyopneumoniae GZ strains in Friis culture mediums 1010~1011CCU, there is stronger virulence to the susceptible pig of health, the Eaton agent pneumonias such as its cough, asthma, expiratory dyspnea can be caused Typical clinical symptoms and pulmonary lesion.
The preparation of the porcine mycoplasmal pneumonia GZ strain inactivated vaccines of embodiment 2
1. the preparation of production strain
Mycoplasma hyopneumoniae GZ strains freeze-drying lactobacillus unpacking after, by 10% inoculum concentration be inoculated with fluid nutrient medium, 37 DEG C culture 3~ 5, harvested when pH value is down to 6.8~7.0, through purely examining, seed is produced as one-level.First order seed is taken by 5% inoculation Amount inoculation liquid supports base, and 37 DEG C are cultivated 3~5, are harvested when pH value is down to 6.8~7.0, qualified through purely examining, as two level Produce seed.
Liquid Culture based formulas:OX-heart leachate (BD companies) 300ml, distilled water 360m1, correction pH value to 7.4,121 DEG C sterilizing 15 minutes, add following filtration sterilization composition：Hank's balanced salt solutions (10 ×) 40m1,0.25 (W/V) is phenol red 10m1, horse serum 200m1,5% (W/V) lactoalbumin hydrolysate 100m1,25%W/V yeast leachate 20m1,10000IU/ml mould Plain 10ml.
2. prepared by the culture of bacterium solution
Qualified mycoplasma hyopneumoniae GZ strains secondary seed is inoculated in fluid nutrient medium by 5% (v/v) respectively, 37 DEG C culture 3~5 days, harvested when pH value is down to 6.8~7.0.
3. mycoplasma hyopneumoniae count plate determines
Fluid nutrient medium containing phenolic red indicator is dispensed into 10ml cillin bottles, if two rows, often arranges 13,1.8ml/ Bottle, takes 0.2ml cultures to be checked to be seeded to the 1st cillin bottle, 10 times of dilutions successively after mixing, until being diluted to the 12nd XiLin Bottle, the 13rd bottle is blank control, puts 37 DEG C of cultures, record produces the highest bottle number of color change untill 21 days, to judge CCU drops Degree, the average value of two row's results is taken, as a result viable bacteria titre is 1011CCU/ml。
4. the inactivation of bacterium solution
The bacterium solution of harvest adds final concentration of 0.01% thimerosal after purely inspection, count plate, after being well mixed It is placed in 2~8 DEG C to inactivate 12 hours, during which shakes 3 times.
5. inactivation is examined and steriling test
Inactivation is examined：1ml inactivated bacterial liquids are taken to be inoculated with 50ml fluid nutrient mediums, 37 DEG C of cultures, each transplanting one on the 5th, 10 Secondary, last time continues culture observation 11 days after transplanting, Medium's PH Value does not reduce, and culture medium color does not change.
Steriling test：By existing《Chinese veterinary pharmacopoeia》Annex is tested, no varied bacteria growing.
6. vaccine formulation
Take and examine the ratio of qualified mycoplasma hyopneumoniae GZ strain inactivation antigen liquid and Gel01 adjuvants according to 90: 10 (V/V) Mixing, 30min is stirred with 300rpm/min with after the sterile PBS supplement volumes that pH is 7.2, is configured to antigenic content difference respectively 3 kinds of inactivated vaccines, the specific formula and content of vaccine are shown in Table 3.
The porcine mycoplasmal pneumonia inactivated vaccine formula of table 3 and content
The inspection of the porcine mycoplasmal pneumonia GZ strain inactivated vaccines of embodiment 3
1. character is examined
Vaccine is the faint yellow aqueous solution, and a small amount of precipitation occurs after being long placed in, and is in homogenous suspension after shaking.
2. steriling test
By existing《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
3. safety verification
3.1BALB/c mouse safety examinations
16~18g BALB/c mouses 30, are divided into 3 groups, 10/group, the 1st group, the 2nd group, the 3rd group respectively every it is subcutaneous 1, vaccine 2,3 each 0.5ml of vaccine are vaccinated, observed after immunization 14 days, whether observation mouse is strong to be lived, including is searched for food, drunk water, essence God, behavioral aspect etc..
As a result 3 groups of BALB/c mouses that 3 kinds of vaccines are immunized respectively are shown, the state of mind is good, and feeding and drinking-water are normal, nothing Adverse reaction, no death condition.It is safe to mouse to prove porcine mycoplasmal pneumonia inactivated vaccine of the present invention.
The safety examination of 3.2 piglets
The negative sodium selenite 15 of 14~21 age in days mycoplasma hyopneumoniaes is chosen, is divided into 3 groups, 5/group, the 1st group, the 2nd Group, the 3rd group respectively every piglet musculi colli vaccinate 1, vaccine 2, the 4ml of vaccine 3, be immunized after all piglet Continuous Observations 14 days, including spirit, feeding, drinking-water, behavioral aspect etc. has without exception, while observes vaccine injection site to whether there is redness etc. different Often, the sampling of cut open inspection injection site makes histotomy after 14 days, and observation has no inflammation, vaccine residual, granuloma etc..As a result show Vaccine immunity piglet is observed 14 and is showed no adverse reaction, and the state of mind is good, and feeding, drinking-water are normal, no death condition.Cut open and kill All internal organs have no abnormal changes afterwards, and vaccine injection site cut open inspection has no that vaccine remains, and histotomy observation has no inflammation, granulation The abnormal conditions such as swollen, necrosis.Prove the porcine mycoplasmal pneumonia inactivated vaccine of the different antigenic contents of the present invention has preferably to piglet Security.
4. efficacy test
The efficacy test of 4.1 piglets
Choose 14~21 age in days mycoplasma hyopneumoniae ELISA negative antibodies, the healthy susceptible pig 20 of PRRSV antigen negatives Head, it is randomly divided into 4 groups.1st group of each musculi colli vaccinates 1 (1ml), and the 2nd group of each musculi colli vaccinates 2 (1ml), the 3 groups of each musculi collis vaccinate 2 (1ml), and malicious control is attacked in the 4th group of not immune conduct, the isolated rearing with the conditions of.35 after exempting from Day, all pig tracheaes are injected with the strong poison of CVCC354 strains tissue and (is purchased from China Veterinery Drug Inspection Office, the bacterial strain is in China The porcine mycoplasmal pneumonia vaccine potency inspection strain of veterinary medicament supervision institute of state preservation), 5ml/ heads (100MID), observation 28 Day, cut open to kill and take lung, the asthma pneumonia lesion of test pig is scored by 28 point-scores, calculating pneumonia lesion by following equation subtracts Few rate.
Pneumonia lesion slip=(attack poison control pig pneumonia lesion average mark-immune swine pneumonia disease become average mark)/and attack poison Compare pig pneumonia lesion average mark × 100%
Attack poison protection and the results are shown in Table 4.As a result show, vaccine 1, vaccine 2, the immune group of vaccine 3 are exempted from compared with attacking malicious control group Epidemic disease group becomes with the average tuberculosis of control group has pole significant difference, shows that mycoplasma hyopneumoniae GZ strains have good immunogenicity.
Poison protection result is attacked in the effect inspection of the piglet of table 4
Group Vaccine Size of animal Average tuberculosis varying index ± standard deviation Pneumonia lesion slip
1st group Vaccine 1 5 2.8±1.48Bb 79%
2nd group Vaccine 2 5 1.8±1.30Bb 84.2%
3rd group Vaccine 3 5 0.8±0.84Bc 92.9%
4th group Attack malicious control 5 11.4±5.12Aa /
Note：In otherness statistical analysis, compare between group, the identical person of letter represents that difference is not notable, capitalization difference person Represent difference extremely significantly (P ＜ 0.01), lowercase is different to represent significant difference (P ＜ 0.05)
The porcine mycoplasmal pneumonia inactivated vaccine of embodiment 4 (GZ strains) is compared with the effect of import seedling
14~21 age in days sodium selenite 5 is immunized in the vaccine 1 prepared in Example 2, and 1ml/ heads, porcine mycoplasmal pneumonia enters Mouth inactivated vaccine vaccine 4 (P strains, 7.5 × 108CCU/ heads part) 14~21 age in days sodium selenite 5 is immunized, 2ml/ head parts, set simultaneously Putting 5 pigs, immune be used as does not attack malicious control group, the isolated rearing with the conditions of.35 days after exempting from, all pig tracheaes are injected The strong poison of CVCC354 strains tissue (is purchased from China Veterinery Drug Inspection Office, the bacterial strain supervises institute's preservation for China veterinary medicament Porcine mycoplasmal pneumonia vaccine potency inspection strain), 5ml/ heads (100MID), observe 28, cut open to kill and take lung, by 28 point-scores to examination The asthma pneumonia lesion for testing pig is scored, and pneumonia lesion slip is calculated by following equation.
Pneumonia lesion slip=(attack poison control pig pneumonia lesion average mark-immune swine pneumonia disease become average mark)/and attack poison Compare pig pneumonia lesion average mark × 100%
Attack poison protection and the results are shown in Table 5.As a result show, vaccine 1, import seedling immune group are attacked malicious control group and compared, immune group with The average tuberculosis of control group, which becomes, has pole significant difference, and vaccine 1 is suitable with import seedling immune group protecting effect, slightly above import seedling. However, the antigenic content of the invention reached used in the effect above further illustrates epidemic disease of the invention well below control vaccine group Seedling strain has good immunogenicity.
The piglet immunological of table 5 protects result
The preparation of the porcine circovirus 2 type antigen of embodiment 5
1. the preparation that production is planted with bacterium (poison)
By seed culture of viruses PCV2SH strains with MEM fluid nutrient mediums (with the MEM dehydrated mediums purchased from Invitrogen companies of the U.S. By specification is prepared) make 1:9 dilutions, are then inoculated in PK15 (ATCC, preserving number CCL- by the 5% of cell culture fluid volume 33) cell monolayer, 37 DEG C adsorb 30 minutes, add cell maintenance medium (added in MEM fluid nutrient mediums 4% calf serum and 2mmol/L D- glucosamine hydrochloric acids), 37 DEG C are cultivated 4, freeze thawing 2~3 times, harvest virus, and virus titer is 106.5TCID50/ml。
2. the culture and preparation of virus liquid
With rolling bottle cell culture method.The PK15 cells of individual layer will be covered with, remove cell culture fluid (in MEM fluid nutrient mediums Add 6% calf serum and 2mmol/L D- glucosamine hydrochloric acids), seed culture of viruses liquid volume ratio is pressed into 0.1~0.2TCID50/ The inoculum concentration of cell is inoculated on PK15 cells, Spin cells bottle 2 weeks, and 37 DEG C adsorb 30 minutes, add cell maintenance medium, put 37 DEG C rotating and culturing (10~12 turn/hour).Daily observation 1~2 time, cell growth is good, 37 DEG C of cultures harvestings on the 4th and thin Cytosol, freeze thawing 3 times, put less than -20 DEG C preservations.
3. the processing and concentration of virus liquid
By doughnut filter column (10 μm and 0.45 μm of the Millipore companies aperture) filtering of above-mentioned virus liquid, remove thin Make 5 times (volume ratios) with Mi Libo (Millipore companies) film bag (molecule interception is 300Kda) after born of the same parents' fragment to concentrate.
4. the assay of pig circular ring virus SH strain virus liquid
Virus liquid is made into 10 times by those skilled in the art's universal method with MEM fluid nutrient mediums to be serially diluted, takes 10-5、 10-6、10-73 dilution factors, each dilution factor are inoculated with the hole of 96 well culture plate PK15 cell monolayers 4 respectively, per hole 0.1ml, simultaneously Negative control is set up, contains 5%CO at 37 DEG C2Incubator in continue culture 24 hours, change cell maintenance medium, it is 24 small to continue culture When；Cell is fixed with cold acetone, containing PCV2 positive cells with each dilution factor of indirect immunofluorescence assay (IFA) measure (is in Green) hole count, viral TCID is calculated according to KarberShi methods50, it is as a result 5 × 106.0TCID50/ml。
5. the inactivation of virus liquid and inactivating efficacy measure
Qualified virus liquid will be examined to add formalin, make final concentration of 0.2% (V/V) of formalin, 37 DEG C go out It is living 18 hours, stir 1 time, each 10min within every 4 hours, inactivate and inactivation of viruses liquid is put into 2~8 DEG C of preservations after terminating.
The inoculation of a small amount of inactivation of viruses liquid is taken to grow up to the PK15 cells of individual layer, 37 DEG C of absorption abandons virus liquid after 1 hour, adds New cell maintenance medium, 37 DEG C are cultivated 2, answer acellular lesion (CPE), continuous blind passage 3 times, grow up to after cell monolayer change into it is thin Born of the same parents' maintaining liquid, 37 DEG C are cultivated 2, are detected with IIF (IFA), and redgreen PCV2 positive cells produce, and explanation is gone out It is living thorough.
The preparation of the porcine circovirus 2 type subunit antigen of embodiment 6
PCV2 prion sample particulate antigens, content 1.3mg/ml are prepared according to patent CN103173470A.
The preparation of the haemophilus parasuis antigen of embodiment 7
1. the preparation that production is planted with bacterium (poison)
By the type JS strains of haemophilus parasuis 4 and the type ZJ strains streak inoculation of haemophilus parasuis 5 in containing 5% NBCS and Tryptose soy agar (the TSA, purchased from U.S. BD public affairs of 0.005% NADH (NAD, BBI companies of the U.S.) Department) flat board (abbreviation TSA/NAD flat boards), 37 DEG C are cultivated 24~48 hours, respectively select more than 5 colonies typicals, are purely examined and are closed After lattice, as first order seed.First order seed access contains 5% NBCS and 0.005% NADH The Tryptose soy meat soup (TSA, purchased from U.S. company BD) (abbreviation TSB/NAD fluid nutrient mediums) of (NAD, BBI companies of the U.S.), 37 DEG C of shaking table 180rpm shaken cultivations 12 hours,
Carry out Gram's staining is sampled simultaneously, observation ne ar is uniform under the microscope, meets haemophilus parasuis Morphological feature, without any varied bacteria growing, as secondary seed.
The preparation of haemophilus parasuis 2. (4 type JS strains and 5 type ZJ strains) bacterium solution
The secondary seed of the qualified type JS strains of haemophilus parasuis 4 and the type ZJ strains of haemophilus parasuis 5 is pressed respectively 1:100 (v/v) are inoculated in TSB/NAD fluid nutrient mediums, put 37 DEG C of shaking table 200rpm cultures.Culture was to 12~16 hours, harvest.
3. the processing of bacterium solution
After the type ZJ strains of haemophilus parasuis 4 and 5 type JS strains are centrifuged with continuous centrifuge (10000rpm/min) respectively again Volume to before centrifuging is redissolved using PBS (pH value is 7.2~7.4), then (molecule is cut with Mi Libo (Millipore companies) film bag Allowance is 300KDa) make 5 times (volume ratio) concentration.
4. the type ZJ strains of haemophilus parasuis 4 determine with 5 type JS strain bacterium solutions viable counts
Bacterium solution samples, and makees 10 times by method well known to those skilled in the art and is serially diluted, takes 10-6、10-72 dilutions Degree is inoculated in foregoing TSA/NAD solid mediums, each plating 0.1ml, after 37 DEG C are cultivated 24 hours, selects clump count to exist Flat board between 30 and 300 carries out bacterium colony counting, and 4 type ZJ strains and 5 type JS strain bacterium solution count results are 5 × 109CFU/ml。
5. the inactivation and inactivation of bacterium solution are examined
By the bacterium solution of two kinds of different serotypes of above-mentioned preparation, formalin is added, makes the final concentration of of formalin 0.3% (V/V), 37 DEG C inactivate 24 hours, are stirred 1 time, each 10min every 4 hours therebetween, inactivate inactivated bacterial liquid after terminating Put 2~8 DEG C of preservations.
The TSA/NAD solid mediums of 6 plates are prepared, 3 TSA/NAD consolidate wherein by the bacterium solution of inactivation for sterile working 1 drop is added dropwise on body culture medium plate, is rule with oese, puts 37 DEG C of common incubator cultures, while set up 3 TSA/ for not connecing bacterium NAD solid mediums compare.Observed after 24 hours, plate is without any bacterial growth, while control does not connect 2 culture mediums of bacterium Also without bacterial growth, continue to observe result into 48 hours 6 plates all without bacterial growth, illustrate that inactivation is thorough.
The pig circular ring virus antigen of embodiment 8, haemophilus parasuis antigen, the system of porcine mycoplasmal pneumonia antigenc vaccine compositions It is standby
Mycoplasma hyopneumoniae antigen, PCV2 antigens, PCV2 subunit antigens and secondary pig prepared by Example 2,4,5,6 Influenzae antigens, four kinds of antigen liquids are prepared into hybrid antigen liquid according to the final antigenic content of connection seedling or are directly prepared into anti- Stoste, antigen liquid and the adjuvants of Gel 01 (match Bick SEPPIC companies of France produce) are then pressed 90:10 (V/V) are mixed, and use pH 30min is stirred with 500rpm/min for 7.2 PBS liquid supplement volume, is configured to 5 kinds of different inactivated vaccines respectively, vaccine Specific formula and content are shown in Table 6.
The vaccine combination composition formula of table 6 and content
It is qualified that 5 kinds of vaccines of preparation are carried out with character, sterile and safety verification respectively, available for follow-up test.
The different vaccine of the porcine circovirus 2 type antigen of embodiment 9, haemophilus parasuis antigen, mycoplasma hyopneumoniae antigen Composition is tested the immune effect of pig
1. vaccine immunity piglet is used in experiment
14~21 age piglet 100 is selected, totally 20 groups, 5/group.15 groups of immune group, wherein, the 8th group, the 9th group of immune vaccine 5, the 10th group, the 11st group of immune vaccine 6, the 12nd group, the 13rd group and the 14th group immune vaccine 7, the 15th group, the 16th group, the 17th group With the 18th group of immune vaccine 8, the 19th group, the 20th group, the 21st group and the 22nd group immune vaccine 9；5 groups of control group, wherein, the 23rd To attack malicious control group, the 27th group is Normal group for group, the 24th group, the 25th group and the 26th group.Specifically immune packet is shown in Table 7.
The immune packet of table 7
2. poison is attacked after piglet immunological
2.1PCV2 attacks poison
The 35d after immune, takes immune group the 8th group, the 10th group, the 15th group and the 19th group 4 groups totally 20, the 23rd group of control group Piglet 1 group 5, immune group and control group piglet respectively (contain 10 with PCV2SH strains6.0TCID50/ ml) collunarium 1ml/ heads, intramuscular injection 2ml/ heads, attack after poison the 4th, 7, in the both sides oxter of every first tap poison pig and both sides buttocks, totally 4 points are inoculated with to all pigs respectively The keyhole hemocyanin (KLH/ICFA, 0.5mg/ml) emulsified with incomplete Freund's adjuvant, each point inoculation 1ml (4ml/ Head), at the same intraperitoneal inoculation thioglycollate medium, 10ml/ heads；Attack after poison the 11st, 19 day again intraperitoneal inoculation TGA train Support base, 10ml/ heads.Continuous Observation 25 days after poison are attacked, are slaughtered after being weighed in the 25th day after attacking poison, cut open inspection.According to body temperature, relative day Weightening and virus antigen detection result are judged.
2.2 haemophilus parasuises 4,5 types attack poison
The 35d after immune, takes immune group the 12nd group, the 16th group and the 20th group 3 groups totally 15, the 24th group of control group piglet 1 Group 5, carried out attacking poison with 4 type bacterial strains, 3ml bacterium solutions are injected intraperitoneally, it is 4 types 9.0 × 10 to attack toxic agent amount9CFU/ heads, 5 types 6.0 × 109CFU/ heads, its clinical manifestation is observed after attacking poison, observation is cutd open after 14 days and kills test pig, carries out pathological observation；Take immune group the 13rd Group, the 17th group and the 21st group 3 groups totally 15, the 25th group of control group piglet 1 group 5, carry out attacking poison with 5 type bacterial strains, intraperitoneal injection 3ml bacterium solutions, it is 5 types 6.0 × 10 to attack toxic agent amount9CFU/ heads, its clinical manifestation is observed after attacking poison, observation is cutd open after 14 days and kills experiment Pig, carry out pathological observation.
2.3 mycoplasma hyopneumoniaes attack poison
The 35d after immune, takes immune group the 9th group, the 11st group, the 14th group, the 18th group and the 22nd group 5 groups totally 25, the 26th Group control group piglet 5, progress mycoplasma hyopneumoniae challenge test, immune group and control group piglet are with CVCC354 strains (in being purchased from Veterinary medicament supervision institute of state, the porcine mycoplasmal pneumonia vaccine potency that the bacterial strain supervises institute's preservation for China veterinary medicament are examined With strain) tracheae injection 5ml/ heads (100MID), observed 28 days after attacking poison, cut open to kill and take lung, by asthma of 28 point-scores to test pig Sick pneumonia lesion is scored, and pneumonia lesion slip is calculated by following equation.
Pneumonia lesion slip=(attack poison control pig pneumonia lesion average mark-immune swine pneumonia disease become average mark)/and attack poison Compare pig pneumonia lesion average mark × 100%
3. attack malicious result
3.1PCV2 attacks poison
After PCV2 attacks poison, Temperature changing situation is shown in Table 8, and vaccine 5, vaccine 6, vaccine 8 and the immune group piglet of vaccine 9 are only individual There is transient body temperature rise phenomenon in other pig, and body temperature recovers normal quickly after raising one day, without other clinical symptoms；Control group All pig body temperature raise more than 40.5 DEG C after pig attacks poison, continue 3~5 days, and anorexia, spirit are depressed, hair is thick disorderly, disappear The thin and speed of growth slows down.
Attack malicious sequela and protection situation is shown in Table 9,10.Vaccine 5, vaccine 6, vaccine 8 and vaccine 9 are immunized piglet and attack nothing after poison Obvious clinical symptoms, specific pathologies change is not observed, antigen PCR detections are feminine gender, and protecting effect is up to 5/5；And compare Group piglet all falls ill, and clinical symptoms, pathological change is obvious, and cause of disease PCR detections are the positive.Terminate to poison observation is attacked, vaccine 5th, piglet average daily gain is immunized without significant difference, compared with control group, vaccine 5, vaccine 6, epidemic disease in vaccine 6, vaccine 8 and vaccine 9 Seedling 8 and vaccine 9 are immunized piglet average daily gain pole and are significantly higher than control group.Show vaccine 5, vaccine 6, vaccine 8 and epidemic disease After seedling 9 is immune to PCV2 to attack malicious protecting effect good.
The PCV2 of table 8 attacks the number of days that each group experiment temperature of pig body surpasses 40.5 DEG C after poison and compared
The PCV2 of table 9 attacks each group experimental animal morbidity result of determination after poison
PCV2 attacks after poison any 2 met in following 3, you can is judged to fall ill.
A：Clinical symptoms：Piglet body temperature raises (>=40 DEG C), should at least continue 3, and it is heavy obvious anorexia, spirit occur Strongly fragrant, hair is slightly random, becomes thin and slows down with the speed of growth；
B：Pathological change：Groin and lymphoglandulae tracheales oedema, lungs Mild edema, kidney turn to be yellow or have a spotty necrosis.
Histologic lesion is that lymph has obvious lymphocyte intrusion, or has multinucleate giant cell；
C：Viral diagnosis：Lymph node tissue is detected with PCR, detects PCV2.
Table 10 is immunized after piglet PCV2 attacks poison and protects situation
Group Piglet quantity Attack malicious protective rate Average daily gain (kg)
8th group 5 5/5 0.0251Bb
10th group 5 5/5 0.0255Bb
15th group 5 5/5 0.0249Bb
19th group 5 5/5 0.0253Bb
23rd group 5 0/5 0.0149Aa
Note：In otherness statistical analysis, compare between group, the identical person of letter represents that difference is not notable, capitalization difference person Represent difference extremely significantly (P ＜ 0.01), lowercase is different to represent significant difference (P ＜ 0.05)
3.2 haemophilus parasuises attack poison
After haemophilus parasuis 4,5 type bacterial strains attack poison, result of the test such as table 11.As a result it is that vaccine 3, vaccine 4 and vaccine 5 are right The poison protection of attacking of 4 type JS strains and 5 type ZJ strains is respectively 4/5 (80%)~5/5 (100%).
Piglet protection situation is immunized after attacking poison in the haemophilus parasuis of table 11
Group Size of animal (head) Malicious protective rate is attacked in 4 type JS strains Malicious protective rate is attacked in 5 type ZJ strains
12nd group 5 5/5 /
13rd group 5 / 4/5
16th group 5 5/5 /
17th group 5 / 5/5
20th group 5 5/5 /
21st group 5 / 5/5
24th group 5 0/5 /
25th group 5 / 0/5
Note：Haemophilus parasuis morbidity standard：Morbid pig appearance heating (more than 40.5 DEG C of body temperature, continuing 1~5), It is One's spirits are drooping, cough, have difficulty in breathing, becoming thin, walking lamely and the thick clinical symptoms such as disorderly of hair.To dying pig cut open inspection, it is seen that multiple The lesions such as scrositis (pleurisy, pericarditis, peritonitis), arthritis and meningitis, each serosal surface (capsular ligament, pericardium, pleura And peritonaeum) there is serosity or fibrinous exudate.
3.3 mycoplasma hyopneumoniaes attack poison
Mycoplasma hyopneumoniae attacks poison protection and the results are shown in Table 12.As a result show, each vaccine combination immune group is with attacking malicious compare Group is compared, and immune group becomes with the average tuberculosis of control group has pole significant difference.
Poison protection result is attacked in the effect inspection of the piglet of table 12
Group Size of animal Average tuberculosis varying index ± standard deviation Pneumonia lesion slip
9th group 5 2.4±1.14Bb 85.6%
11st group 5 2.0±0.71Bb 85.0%
14th group 5 2.2±0.84Bb 84.3%
18th group 5 1.8±1.30Bb 84.7%
22nd group 5 1.6±1.14Bb 86.4%
26th group 5 11.8±3.96Aa /
Note：In otherness statistical analysis, compare between group, the identical person of letter represents that difference is not notable, capitalization difference person Represent difference extremely significantly (P ＜ 0.01), lowercase is different to represent significant difference (P ＜ 0.05)
To sum up, more than different vaccine combinations to PCV2, the type JS strains of haemophilus parasuis 4 and 5 type ZJ strains, pig pneumonia branch Substance is attacked from the point of view of poison protection result, and each vaccine combination can reach preferable protecting effect, illustrate newly to separate in the present invention Mycoplasma hyopneumoniae GZ strains and PCV2 antigens, PCV2 subunit antigens, the type JS strains of haemophilus parasuis 4 and 5 type ZJ strain antigens Remain to meet or exceed the immune protective effect of single seedling when being prepared into different vaccine combinations, do not influenceed by other antigens.
Described above is only the preferred embodiments of the present invention, not does any formal limitation to the present invention, though So the present invention is disclosed above with preferred embodiment, but is not limited to the present invention, any to be familiar with this professional technology people Member, in the range of technical solution of the present invention is not departed from, when the technology contents using the disclosure above make a little change or repair The equivalent embodiment for equivalent variations is adornd, as long as being the content without departing from technical solution of the present invention, the technology according to the present invention is real Any simple modification, equivalent change and modification that confrontation above example is made, still fall within the scope of technical solution of the present invention It is interior.

Claims (10)

1. mycoplasma hyopneumoniae GZ strains, preserving number is CCTCC NO:M2016212.
2. a kind of vaccine combination, wherein, the pig pneumonia branch that the vaccine combination is included described in the claim 1 of immune amount is former The inactivation antigen of body GZ strains or its culture, the full mycoplasma antigen of work or subunit antigen, and pharmaceutically acceptable load Body.
3. vaccine combination according to claim 2, wherein, described mycoplasma hyopneumoniae GZ strains culture is 1~42 The culture of generation；Described mycoplasma hyopneumoniae GZ strains or the inactivation antigen of its culture for inactivation full mycoplasma antigen or The full mycoplasma antigen of its schizolysis antigen, described mycoplasma hyopneumoniae GZ strains or the work of its culture is the full branch for the work being attenuated The subunit antigen of mycoplasma antigen, described mycoplasma hyopneumoniae GZ strains or its culture be antigen protein P97, P110, P46 and P36。
4. vaccine combination according to claim 2, wherein, the mycoplasma hyopneumoniae GZ strains or the inactivation of its culture The content of antigen for inactivation before >=107.0CCU/ml；Preferably, the mycoplasma hyopneumoniae GZ strains or the inactivation of its culture resist Former content is before inactivation 107.0~1010.0CCU/ml。
5. vaccine combination according to claim 2, wherein, described pharmaceutically acceptable carrier includes adjuvant, institute Stating adjuvant includes：(1) aluminium glue adjuvant, saponin(e, Avridine, DDA；(2) water-in-oil emulsion, oil in water emulsion, W/O/W Emulsion；Or polymer, the copolymer of maleic anhydride and alkenyl derivative of (3) acrylic or methacrylic acid；And RIBI adjuvant systems, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipids-amine adjuvant, Escherichia coli One or more in heat-labile toxin, cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvants；
Preferably, saponin(e is Quil A, QS-21, GPI-0100；
Preferably, emulsion is that SPT emulsions, MF59 emulsions, or emulsion are formed by oil with emulsifier combination, and emulsion can be based on light liquid Paraffin oil, because caused by olefin oligomerisation isoprenoid oil (such as saualane or squalene oil, particularly alkene, isobutene or the last of the ten Heavenly stems It is oily caused by alkene oligomerization), ester (more specifically vegetable oil, ethyl oleate, propane diols two-(octanoic acid containing linear alkyl of acid or alcohol Ester/certain herbaceous plants with big flowers acid esters), glycerine three-(caprylate/certain herbaceous plants with big flowers acid esters) or Rikemal PO 200), the ester of branched chain fatty acid or alcohol it is (especially different Stearate)；Emulsifying agent is that (ester, the sorb of especially polyoxyethylated fatty acid (such as oleic acid) gather nonionic surfactant The ester of sugar, the ester (such as anhydrous mannitol oleate) of mannide, the ester of aliphatic dihydroxy alcohol, the ester of glycerine, polyglycereol Ester, the ester of the ester of propane diols and oleic acid, the ester of isostearic acid, the ester of the ester of castor oil acid or hydroxy stearic acid, above-mentioned ester can Through ethoxylation, the ether of fatty alcohol and polyalcohol (such as oleyl alcohol), Pluronic L121 (especiallyParticularly L121))；
Preferably, the polymer of acrylic or methacrylic acid is the acrylic or methacrylic acid polymer of crosslinking, especially With sugar poly alkenyl ether or polyalcohols crosslinking compound carbomer, be preferably carbopol 974P, 934P and 971P；
Preferably, the copolymer of maleic anhydride and alkenyl derivative is the copolymer EMA of maleic anhydride and ethene；
Preferably, the adjuvant includes Gel 01, ISA206, ISA760VG, carbomer, aluminium hydroxide；
The concentration range of the adjuvant is the preferably 10%V/V from 10% to 70%V/V.
6. vaccine combination according to claim 2, wherein, described vaccine combination also includes medicine, immunostimulation Agent, antioxidant, surfactant, colouring agent, ethereal oil, buffer, dispersant, propellant and preservative；Preferably, exempt from Epidemic disease stimulant includes alpha-interferon, beta-interferon, gamma interferon, granulocyte macrophage colony stimulating factor, macrophage collection G-CSF and interleukin-22.
7. vaccine combination according to claim 2, wherein, the vaccine combination further resists comprising other cause of diseases Original, other described pathogen antigens include CSFV antigen, PRV antigen, pig circular ring virus and resist former and deputy pig bloodthirsty Bacteroides antigen, Streptococcus suis antigen, swine flu antigen, porcine contagious pleuropneumonia antigen, pig pasteurella multocida antigen, pig Bordetella bacilli antigen, porcine reproductive and respiratory syndrome virus antigen, Salmonella choleraesuls antigen, PPV Antigen Using and/ Or Latex agglutination test antigen.
8. vaccine combination according to claim 7, wherein, the pig circular ring virus antigen includes porcine circovirus 2 type ZJ/H strains antigen, pig gyrate virus II type DBN-SX07 strains antigen, porcine circovirus 2 type SD strains antigen, porcine circovirus 2 type ZJ/C strains antigen, porcine circovirus type 2 strain PCV2SD strains antigen, porcine circovirus 2 type SH strains antigen or pig gyrate virus II Type PCV2/HZ09 strain antigens；
It is bloodthirsty that the haemophilus parasuis antigen includes the type haemophilus parasuis GX0905 strains antigen of serum 13, Serotype 5 pair pig Bacillus JX1002 strains antigen, the type haemophilus parasuis HN1009 strains antigen of serum 4, the type haemophilus parasuis YBH04 strains of serum 4 resist Original, Serotype 5 haemophilus parasuis YBH05 strains antigen, the type haemophilus parasuis YBH13 strains antigen of serum 13, serum 1 type pair pig Haemophilus LC strains antigen, the type haemophilus parasuis SHCM10 strains antigen of serum 12, Serotype 5 haemophilus parasuis JSYZ10 strains Antigen, the type haemophilus parasuis FJMH10 strains antigen of serum 13, the type haemophilus parasuis FS0307 strains antigen of serum 4, Serotype 5 Haemophilus parasuis XX0306 strains antigen, Serotype 5 haemophilus parasuis LZ-20100109 strains antigen, Serotype 5 pair pig are bloodthirsty Bacillus LX-5 strains antigen, the type JS strains antigen of haemophilus parasuis serum 4, haemophilus parasuis Serotype 5 ZJ strains resist former and deputy pig thermophilic The type HeB strains antigen of blood bacillus serum 12 or its combination.
9. vaccine combination according to claim 7, wherein, the pig circular ring virus antigen is the pig circular ring virus of inactivation Antigen or subunit antigen, the pig circular ring virus content of the inactivation is before inactivation 5 × 105.0TCID50/ ml, the pig circular ring virus 2 Malicious subunit antigen content is 20 μ g/ml；The haemophilus parasuis antigen is the haemophilus parasuis antigen of inactivation, described to go out Haemophilus parasuis antigenic content living is before inactivation 2 × 109CFU/ml；Preferably, the pig circular ring virus antigen is inactivation Pig circular ring virus SH strain antigens, the haemophilus parasuis antigen of the inactivation resists for the type of haemophilus parasuis serum 4 of inactivation Former JS strains antigen and the haemophilus parasuis Serotype 5 ZJ strain antigens of inactivation.
10. the vaccine combination according to any one of claim 2~9 is preparing prevention and treatment porcine mycoplasmal pneumonia and pig Application in the medicine of Mycoplasma Pneumoniae Associated Diseases.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109055283A (en) * 2018-09-30 2018-12-21 华中农业大学 A kind of mycoplasma hyopneumoniae and its preparing the application in inactivated vaccine
CN109745555A (en) * 2019-02-22 2019-05-14 河南省农业科学院畜牧兽医研究所 A kind of mycoplasma hyopneumoniae and haemophilus parasuis bivalent inactivated vaccine and its application
CN109837226A (en) * 2019-02-21 2019-06-04 华中农业大学 The M. bovis genes mutant strain and adhesion protein that Adhering capacity reduces
CN111856006A (en) * 2020-06-22 2020-10-30 华中农业大学 Application of mycoplasma bovis secretory protein MbovP274
CN112513638A (en) * 2018-08-06 2021-03-16 积水医疗株式会社 Method for immunologically detecting mycoplasma pneumoniae
CN113832213A (en) * 2021-11-10 2021-12-24 北京兴德通医药科技股份有限公司 Mycoplasma detection culture medium and preparation method thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103083655A (en) * 2011-11-02 2013-05-08 普莱柯生物工程股份有限公司 Vaccine composition for preventing and treating porcine circovirus type 2, haemophilus parasuis and mycoplasma hyopneumoniae infection and preparation method thereof
CN103184171A (en) * 2011-12-29 2013-07-03 北京大北农科技集团股份有限公司 Mycoplasma hyopneumoniae DJ-166 strain and application thereof
CN104248753A (en) * 2014-05-15 2014-12-31 普莱柯生物工程股份有限公司 Vaccine composition and application thereof
CN104685057A (en) * 2012-07-10 2015-06-03 海博莱科学有限公司 Mutant strains of mycoplasma hyopneumoniae
CN104894009A (en) * 2015-05-15 2015-09-09 北京中海生物科技有限公司 Mycoplasma hyopneumoniae strain and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103083655A (en) * 2011-11-02 2013-05-08 普莱柯生物工程股份有限公司 Vaccine composition for preventing and treating porcine circovirus type 2, haemophilus parasuis and mycoplasma hyopneumoniae infection and preparation method thereof
CN103184171A (en) * 2011-12-29 2013-07-03 北京大北农科技集团股份有限公司 Mycoplasma hyopneumoniae DJ-166 strain and application thereof
CN104685057A (en) * 2012-07-10 2015-06-03 海博莱科学有限公司 Mutant strains of mycoplasma hyopneumoniae
CN104248753A (en) * 2014-05-15 2014-12-31 普莱柯生物工程股份有限公司 Vaccine composition and application thereof
CN104894009A (en) * 2015-05-15 2015-09-09 北京中海生物科技有限公司 Mycoplasma hyopneumoniae strain and application thereof

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112513638A (en) * 2018-08-06 2021-03-16 积水医疗株式会社 Method for immunologically detecting mycoplasma pneumoniae
CN109055283A (en) * 2018-09-30 2018-12-21 华中农业大学 A kind of mycoplasma hyopneumoniae and its preparing the application in inactivated vaccine
CN109837226A (en) * 2019-02-21 2019-06-04 华中农业大学 The M. bovis genes mutant strain and adhesion protein that Adhering capacity reduces
CN109745555A (en) * 2019-02-22 2019-05-14 河南省农业科学院畜牧兽医研究所 A kind of mycoplasma hyopneumoniae and haemophilus parasuis bivalent inactivated vaccine and its application
CN109745555B (en) * 2019-02-22 2022-01-28 河南省农业科学院畜牧兽医研究所 Mycoplasma hyopneumoniae and haemophilus parasuis combined inactivated vaccine and application thereof
CN111856006A (en) * 2020-06-22 2020-10-30 华中农业大学 Application of mycoplasma bovis secretory protein MbovP274
CN113832213A (en) * 2021-11-10 2021-12-24 北京兴德通医药科技股份有限公司 Mycoplasma detection culture medium and preparation method thereof
CN113832213B (en) * 2021-11-10 2024-03-12 北京兴德通医药科技股份有限公司 Mycoplasma detection culture medium and preparation method thereof

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