CN109010817A - A kind of vaccine composition and its preparation method and application prevented and/or treatment 3 type of pig circular ring virus infects - Google Patents

A kind of vaccine composition and its preparation method and application prevented and/or treatment 3 type of pig circular ring virus infects Download PDF

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Publication number
CN109010817A
CN109010817A CN201710433747.XA CN201710433747A CN109010817A CN 109010817 A CN109010817 A CN 109010817A CN 201710433747 A CN201710433747 A CN 201710433747A CN 109010817 A CN109010817 A CN 109010817A
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antigen
virus
circular ring
inactivation
pig
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CN109010817B (en
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田克恭
李向东
肖燕
孙进忠
张许科
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Pulaike Biological Engineering Co Ltd
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Pulaike Biological Engineering Co Ltd
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/552—Veterinary vaccine
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    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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Abstract

The present invention relates to the vaccine compositions of a kind of prevention and/or treatment pig circular ring virus mixed infection, wherein the vaccine composition includes the 3 type antigen of pig circular ring virus and carrier of immune amount.Vaccine composition of the invention has good, wide spectrum immunogenicity, can 3 type strain mixed infection of pig circular ring virus to different geographical source there is protective effect.

Description

A kind of vaccine composition and its system prevented and/or treatment 3 type of pig circular ring virus infects Preparation Method and application
Technical field
The present invention relates to the vaccine composition of a kind of prevention and/or treatment 3 type of pig circular ring virus infection, preparation method and answer With belonging to animal virology field.
Background technique
Pig circular ring virus (Porcine circoviruses, PCV) is the cricoid DNA virus of sub-thread, and genome length is about It is one of the smallest animal DNA virus for 1.7kb.Have determined the PCV there are two types of type, i.e. 1 type of pig circular ring virus (PCV1) With porcine circovirus 2 type (PCV2).PCV1 was sent out in PK cell culture as a kind of pollutants identification for the first time in 1974 It is existing, it is not pathogenic to pig.PCV2 was reported for the first time in 1998, can cause the pig circular ring virus 2 of pig in the clinical setting Malicious related disease (Porcine circovirus associated diseases, PCVAD), mainly causes weanling pig more System failure syndrome, pneumonia, pigskin inflammation and nephrotic syndrome and breeding difficulty, are mainly shown as breathing, uropoiesis, enteron aisle, leaching Bar, cardiovascular, nerve, propagating system and skin dysfunction, great economy is caused to global pig-breeding Loss.
However, it is separated to the circovirus that a strain virus genome is 2.0kb together in the breeding difficulty case of pig, Confirm no matter be respectively less than in the homology of nucleotide or amino acid sequence through follow-up test with known circovirus 50%, according to the standard of the international virology classification committee, same virus should have >'s 75% in Circovirus The homology of genome nucleotide sequence, the homology of amino acid sequence of the Cap protein with > 70%, can affirm this accordingly It is a kind of new pig circular ring virus.It can cause the dermatitis nephrotic syndrome of pig, Hypertrophic necrosis with a variety of cause of disease mixed infections The inflammatory reaction of property pneumonia, breeding difficulty and heart and multisystem.
Originally early in the systemic disease as caused by PCV2 in 1985 just with the outburst of fragmentary state, due to failing to pay attention to, Lead to the late nineteen nineties large-scale outbreak disease, and new pig circular ring virus PCV3 have in terms of PNDS and breeding difficulty with The similar pathogenic characteristic of PCV2, and can be prevented or be treated for PCV3 infection without vaccine product at present, therefore at this stage Exploitation can effectively prevent PCV3 infection or be in urgent need with the vaccine of the extensive mixed infection of other cause of diseases, for the new clinic Epidemic situation prepares new vaccine composition, particularly significant to pig farm disease control.
Summary of the invention
To solve the deficiencies in the prior art, the present invention provides a kind of prevention and/or treats the mixing sense of novel pig circular ring virus The vaccine composition of dye, the vaccine composition can provide novel pig circular ring virus mixed infection and be effectively protected, show aobvious The immunological characteristic of work.
For this purpose, it is an object of the present invention to provide a kind of prevention and/or treating novel pig circular ring virus mixed infection Vaccine composition can 3 type epidemic strain of effective protection pig circular ring virus and one kind containing 3 type strain antigen of pig circular ring virus Or the mixed infection of various other cause of diseases, and complete protection is provided for the infection of 3 type of pig circular ring virus of separate sources.
It is another object of the present invention to provide above-mentioned vaccine compositions in preparation prevention and/or treatment pig circular ring virus Application in the drug of 3 type mixed infection diseases.
Invention advantage:
(1) strain of the present invention has good immunogenicity, is immunized once, and body can be stimulated quickly to generate immune effect Power, the attack of effective protection epidemic strain have good protective effect, and can reach good immune with lower antigenic content Protective effect further decreases production cost；
(2) present invention is for the first time using the strain preparation prevention of pig circular ring virus 3 type prevalence new at present and/or treatment pig circle The vaccine composition of 3 type mixed infection of circovirus virus is not interfered in vaccine composition between each antigenic component, can be applied jointly With；
(3) vaccine of the invention can be provided for the 3 type virus strain infection of pig circular ring virus in different geographical source and be protected completely Shield, the protective capability with wide spectrum.
Specific embodiment
Hereinafter, embodiments of the present invention will be described.
3 type of pig circular ring virus be genome 2.0kb circovirus, with known circovirus no matter nucleotide also It is that the homology of amino acid sequence is respectively less than 50%, is a kind of new pig circular ring virus, can draw with a variety of cause of disease mixed infections Play the inflammatory reaction of the dermatitis nephrotic syndrome of pig, Hypertrophic necrotizing pneumonia, breeding difficulty and heart and multisystem.
SG plants of 3 type of pig circular ring virus (Porcine Circovirus type 3, Strain SG) of the invention, is preserved in China typical culture collection center, deposit number are CCTCC NO.V201712, and the deposit date is on March 23rd, 2017, preservations Address: Wuhan, China Wuhan University.
The present invention relates to the vaccine compositions of a kind of prevention and/or treatment 3 type mixed infection of pig circular ring virus, wherein institute Stating vaccine composition includes that the 3 type antigen of pig circular ring virus measured and pharmaceutically acceptable carrier is immunized；Wherein, the epidemic disease Seedling composition also includes one of group being made of the following antigen of immune amount or a variety of: CSFV antigen, pseudorabies Sick viral antigen, swine flue antigen, porcine reproductive and respiratory syndrome virus antigen, PPV Antigen Using, the B-mode brain of pig Scorching viral antigen, haemophilus parasuis antigen, Streptococcus suis antigen, pig bordetella bacilli antigen, porcine contagious pleuropneumonia antigen, Pig pasteurella multocida antigen, Salmonella choleraesuls antigen, mycoplasma hyopneumoniae antigen, mycoplasma hyorhinis antigens.
Vaccine composition of the invention has good immunogenicity, is only immunized once, and protection completely can be generated to pig, And antigenic content is lower can also reach good immanoprotection action.
Vaccine composition of the invention, may include 3 type of pig circular ring virus of immune amount or the inactivation antigen of its culture, It is attenuated totivirus antigen and pharmaceutically acceptable carrier.
" culture " is the different generation subcultures of virus, as known to those skilled in the art its base between different generations Small variation can occur because sequence is only possible.
" vaccine composition " refers to the pharmaceutical composition containing 3 type immunogenicity of pig circular ring virus.The pharmaceutical composition can lure Hair, stimulation or enhancing pig are directed to the immune response of 3 type of pig circular ring virus.
" inactivation antigen ", also referred to as inactivated vaccines refer to being used as suspension of the antigen to generate the inactivation of viruses of immunity Liquid.The example of inactivated vaccine includes whole virus vaccine and cracking type vaccine.Inactivation can be easily generated using known method Vaccine.For example, by handling the available inactivated virus vaccine of virus with formalin.Cracking type vaccine can be after being handled with ether It is prepared by peplos.
" attenuation totivirus antigen " refers to the virus that virulence has weakened but still can replicate in host or on cell. Term used herein " attenuation " carries out gene in a manner of making cause of disease lose pathogenic but holding immunogenicity for referring to Mutation manually to reduce pathogen toxicity.In general, being subtracted by UV radiation, chemical treatment or external continuous high-order squamous subculture realization Poison.Either artificial gene alteration, such as the specific nucleotide in known array is lacked so that virulence attenuation of.
As one embodiment of the present invention, the 3 type antigen of pig circular ring virus be SG plants of 3 type of pig circular ring virus or its Culture inactivated whole virus antigen, SG plants of deposit numbers are CCTCC NO. V201712.
As one embodiment of the present invention, in vaccine composition of the invention, the 3 type antigen of pig circular ring virus is SG plants of 3 type virus of pig circular ring virus or its culture cracking type antigen.
The different generation subculture gene orders of virus are only possible to occur small variation, can guarantee and similarly exempt from The inactivated vaccine of epidemic focus, preparation can have similar immune efficacy.
As one embodiment of the present invention, in vaccine composition of the invention, SG plants of 3 type of the pig circular ring virus trainings Support the culture that object is >=1 generation.
As a kind of preferred embodiment of the invention, in vaccine composition of the invention, the 3 type SG of pig circular ring virus The culture of strain is the culture of >=5 generations.
As a kind of more preferable embodiment of the invention, in vaccine composition of the invention, 3 type of pig circular ring virus SG plants of culture is the culture of 5~55 generations.
The ingredient of composition of the invention or the amount of component are preferably therapeutically effective amount.The therapeutically effective amount refers to Their immunological role is played without leading to excessive side effect institute necessary amounts in the host of composition application.Ingredient used and The accurate amount of composition to be administered by the type of the disease according to many factors such as treatment, the type of animal to be treated and Age, the mode of application and other ingredients in composition and change.
As one embodiment of the present invention, the inactivated whole virus of SG plants of 3 type of pig circular ring virus or its culture Antigenic content be inactivation before >=105.0TCID50/ml。
The inactivated whole virus antigen of SG plants of 3 type of pig circular ring virus or its culture has good in vaccine composition of the invention Good immunogenicity, can stimulate body rapidly to generate immunity, inactivate preceding 10 when using5.0TCID50When the content of/ml, Good immanoprotection action can be reached, realize 100% protective rate.
As a kind of preferred embodiment of the invention, the inactivation of SG plants of 3 type of pig circular ring virus or its culture is complete Viral antigen content is inactivation preceding 105.0~107.0TCID50/ml。
As a kind of more preferable embodiment of the invention, the inactivation of SG plants of 3 type of pig circular ring virus or its culture Totivirus antigenic content is inactivation preceding 106.0TCID50/ml。
In vaccine composition of the present invention, the inactivated whole virus antigen of SG plants of 3 type of pig circular ring virus or its culture contains Amount range is further selected from 105.0~106.0TCID50/ ml or 106.0~107.0TCID50/ml。
Vaccine composition of the invention further includes other pathogens or antigen composition using to prepare resistance The combined vaccine or combination vaccine of the various diseases of 3 type of pig circular ring virus infection.
Term " combined vaccine " refers to that 3 type of pig circular ring virus of the invention is mixed with the viral antigen of at least one different virus The vaccine of object preparation.Term " combination vaccine " refers to the vaccine of 3 type of pig circular ring virus and bacterial antigens preparation of the invention.For example, 3 type of pig circular ring virus of the invention can be with the antigen of following cause of disease: swine fever virus, porcine pseudorabies virus, swine influenza virus, pig Reproductive and respiratory syndrome virus, pig parvoviral, Latex agglutination test and/or haemophilus parasuis, Streptococcus suis, pig wave Family name bacillus, porcine contagious pleuropneumonia, pig pasteurella multocida, Salmonella choleraesuls, mycoplasma hyopneumoniae, hog snout branch are former Body mixing or combination.
3 type antigen of pig circular ring virus of the invention can form vaccine composition with a variety of antigenic components and be co-administered, each anti- Original is not interfered between each other, and antigenic component therein can generate protection completely for respective poison strain of attacking.
As a kind of preferred embodiment of the invention, the vaccine composition also includes by the following antigen group of immune amount At one of group or a variety of: swine fever virus is attenuated antigen, porcine pseudorabies virus attenuation antigen, pig breeding and integrates with breathing Levy virus attenuation antigen, pig parvoviral inactivation antigen, haemophilus parasuis inactivation antigen, mycoplasma hyopneumoniae inactivation antigen.
As a kind of preferred embodiment of the invention, the vaccine composition also includes by the following antigen group of immune amount At one of group or a variety of: swine fever virus is attenuated antigen, porcine pseudorabies virus inactivation antigen, pig breeding and integrates with breathing Levy virus attenuation antigen, pig parvoviral inactivation antigen, haemophilus parasuis inactivation antigen, mycoplasma hyopneumoniae inactivation antigen.
As a kind of more preferable embodiment of the invention, the swine fever virus attenuation antigen is the weak poison of swine fever virus rabbitization Strain, porcine pseudorabies virus attenuation antigen are HN1201-R plants of attenuated strains, the porcine pseudorabies virus inactivation antigen is HN1201 plants of inactivated whole virus antigens, porcine reproductive and respiratory syndrome virus attenuation antigen are JXA1-R plants of attenuated strains, institute State that pig parvoviral inactivation antigen is HN-2011 plants of inactivated whole virus antigens, the haemophilus parasuis inactivation antigen is secondary pig JS plants of 4 type of haemophilus serum or the full bacterium antigen of ZJ plants of 5 types inactivations, the mycoplasma hyopneumoniae inactivation antigen are HN0613 plants and go out Full bacterium antigen living.
As one embodiment of the present invention, in vaccine composition of the invention, the pig circular ring virus inactivation antigen Content is inactivation preceding 105.0~107.0TCID50/ ml, the swine fever virus attenuation antigenic content is 104.0~106.0TCID50/ ml, The porcine pseudorabies virus attenuation antigenic content is 106.0~107.0TCID50/ ml, the porcine pseudorabies virus inactivation are anti- Former content is inactivation preceding 106.0~107.0TCID50/ ml, the porcine reproductive and respiratory syndrome virus attenuation antigenic content are 105.0~107.0TCID50/ ml, the pig parvoviral inactivation antigen content are inactivation preceding 106.0~108.0TCID50/ ml, institute Haemophilus parasuis inactivation antigen content is stated as inactivation preceding 108.0~1010.0CFU/ml, the mycoplasma hyopneumoniae inactivation antigen Content is inactivation preceding 108.0~1010.0CCU/ml。
As a kind of preferred embodiment of the invention, in vaccine composition of the invention, the pig circular ring virus inactivation Antigenic content is inactivation preceding 105.0~107.0TCID50/ ml, the swine fever virus attenuation antigenic content is 105.0TCID50/ ml, institute Stating porcine pseudorabies virus attenuation antigenic content is 106.0TCID50/ ml, the porcine pseudorabies virus inactivation antigen content are Inactivation preceding 106.0TCID50/ ml, the porcine reproductive and respiratory syndrome virus attenuation antigenic content is 106.0TCID50/ ml, institute Pig parvoviral inactivation antigen content is stated as inactivation preceding 107.0TCID50/ ml, the haemophilus parasuis inactivation antigen content are to go out Living preceding 109.0CFU/ml, the mycoplasma hyopneumoniae inactivation antigen content are inactivation preceding 109.0CCU/ml。
As one embodiment of the present invention, in vaccine composition of the invention, the pharmaceutically acceptable is carried Body includes adjuvant, and the adjuvant includes white oil, Drake oil and animal oil, vegetable oil or mineral oil；Or aluminium hydroxide, phosphorus Sour aluminium and metal salt；Or MontanideTMGel, carbomer, saualane or squalene, ISA206 adjuvant, saponin(e, water in oil emulsion Agent, oil in water emulsion, W/O/W emulsion.
As a kind of preferred embodiment of the invention, in vaccine composition of the invention, the adjuvant is MontanideTM Gel。
As one embodiment of the present invention, in vaccine composition of the invention, the adjuvant content is 5~20V/ V%.
As a kind of preferred embodiment of the invention, in vaccine composition of the invention, the adjuvant content is 10V/ V%.
Vaccine composition of the invention, which can be used, to be deployed with technology, acceptable carrier one preferably on veterinary pharmaceutical Play allotment.For example, oil can help to stablize composite, and additionally serve as vaccine adjuvant.Oil adjuvant either natural source, It can be by artificial synthesized acquisition.Term " adjuvant ", which refers to, to be added in composition of the invention to increase the immune of composition The substance of originality.Known adjuvant includes, but are not limited to: (1) aluminium hydroxide, saponin(e (Saponine) (such as QuilA), Ah Husband halts, DDA, the polymer of the polymer of (2) acrylic or methacrylic acid, maleic anhydride and alkenyl derivative, (3) vaccine can be made in the form of oil-in-water, Water-In-Oil or W/O/W emulsion, or (4) MontanideTM Gel。
Especially, emulsion can be based on light liquid paraffin oil, isoprenoid oil, such as saualane or squalene；Alkene, Especially isobutene or the oil of decene oligomerizationization generation, the ester that the acid with straight chained alkyl or alcohol are formed, more particularly vegetable oil, oil Sour ethyl ester, propylene glycol two (caprylate/decylate), glycerol three (caprylate/decylate), Rikemal PO 200；Branch's rouge The ester of fat acid esters or alcohol, especially isostearate.Oil is used together to form emulsion with emulsifier.The preferred nonionic table of emulsifier Face activating agent, especially polyoxyethylated fatty acid (such as oleic acid), sorbitan, mannitol (such as anhydromannitol Oleate), glycerol, polyglycereol, propylene glycol and optionally oleic acid, isostearic acid, ricinoleic acid, the hydroxy stearic acid of ethoxylation The ether of the ester of formation, fatty alcohol and polyalcohol (such as oleyl alcohol), polyoxypropylene polyoxyethylene block copolymer, especially PluronicR, especially L121 are (referring to Hunter etc., 1995, " The Theory and Practical Application OfAdjuvants " (Steward-Tull, D.E.S chief editor) John Wiley andSons, NY, 51-94；Todd etc., Vaccine, 1997,15,564-570).
Particularly, acrylic or methacrylic acid polymer is crosslinked by the poly alkenyl ether of sugar or polyalcohol.These are changed It closes object and is referred to as carbomer.
Preferably, the adjuvant that the present invention selects is MontanideTM Gel。
The amount for being suitable for the invention the adjuvant of composition is preferably effective quantity." effective quantity " refers to adjuvant same Antigen of the present invention played in host when being administered in combination for their immunological role must or it is enough excessive without causing Side effect institute necessary amounts.The accurate amount of adjuvant to be administered will be according to the disease of many factors ingredient for example used and treatment Type, the type of animal to be treated and age, the mode of application and other ingredients in composition and change.
Other reagents can also be further added in composition of the invention by vaccine composition of the present invention.For example, Composition of the invention can also include following reagent, and such as: drug, immunostimulant is (such as: alpha-interferon, beta-interferon, γ- Interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and interleukin 2 (IL2)), antioxidant, surfactant, colorant, ethereal oil, buffer, dispersing agent, propellant and preservative.In order to Such composition is prepared, method well known in the art can be used.
Peroral dosage form or non-oral dosage forms can be prepared into vaccine composition according to the present invention.
Preferably can by intradermal, muscle, peritonaeum, intravenous, subcutaneous, intranasal or epidural pathways give it is non- Peroral dosage form.
The invention further relates to above-mentioned vaccine compositions in preparation prevention and/or to treat 3 type related disease of pig circular ring virus Application in drug.
In vaccine composition of the present invention antigen in above-mentioned content range can effective challenge, resistance be respectively directed to Cause of disease attack poison, do not generate interference between each antigenic component in vaccine composition, can protect completely, protective rate 100%.
As one embodiment of the present invention, in the application of vaccine composition of the present invention.The pig circular ring virus 2 Malicious 3 type related diseases include the pmws of pig, dermatitis nephrotic syndrome, Hypertrophic gangrenosum acne lung The inflammatory reaction of scorching, breeding difficulty and heart and multisystem.
The invention further relates to above-mentioned vaccine compositions to lead in preparation prevention and/or treatment 3 type mixed infection of pig circular ring virus Application in the drug of the disease of cause.
Term used herein " 3 type mixed infection disease of pig circular ring virus " is for referring to by 3 type of pig circular ring virus and one kind Or disease caused by a variety of cause of disease co-infections.Nonexhaustive includes, pmws, pig dermatitis kidney The inflammatory reaction of sick syndrome, Hypertrophic necrotizing pneumonia, breeding difficulty and heart and multisystem, but not limited to this.
Term " prevention and/or treatment " refers to that inhibition includes pig circular ring virus 2 when being related to 3 type mixed infection of pig circular ring virus Duplication, the inhibition of malicious 3 types include the propagation of 3 type of pig circular ring virus or prevent from including that 3 type of pig circular ring virus is default in its host It occupies, and mitigating includes the disease of 3 type of pig circular ring virus infection or the symptom of illness.If viral loads are reduced, illness mitigates And/or food ration and/or growth increase, then can think that the treatment has reached therapeutic effect.
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be with description more It is clear.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art It should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form carry out Modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Used chemical reagent is that analysis is pure in the embodiment of the present invention, is purchased from Chinese medicines group.
To keep the present invention easier to understand with reference to specific embodiments the present invention is further explained.It is of the present invention Experimental method, if being conventional method without specified otherwise；The biomaterial, if without specified otherwise, it can be from business way Diameter obtains.
The separation of 1 pig circular ring virus of embodiment, 3 type is identified
1, pathological material of disease source
There is the sow death rate and increases by 9.4% compared with history average in a commercialization pig farm at home, pregnancy rate drop Low 1.2%, the phenomenon that the mummification of fetus increases by 8.2%.In clinical manifestation, impacted sow shows anorexia, in multifocal Papule, the symptom of spot and surface dermatitis.Containing the mummified fetus of different gestational age in the nest of miscarriage, with PCV2 related streams The symptom of production is consistent.Although the overall clinical performance and miscarriage symptom observed in sow cause with 2 porcine circovirus type Breeding difficulty disease it is consistent, but different tissues of all sows, including kidney, lymph node, lung, skin and stillborn foetus, by exempting from Epidemic disease group and quantitative PCR are feminine gender to PCV2, PRRSV, PPV, CSFV, mycoplasma hyopneumoniae detection.Further to investigate thoroughly original Cause chooses each tissue pathological material of disease and carries out pathogen separation.
2, the separation and culture of Strain
DMEM culture solution is added with 1:10 (volume ratio) in pathological material of disease, grinding prepares tissue suspension；Tissue suspension is through repeatedly 3 After secondary freeze thawing, it is centrifuged 15min in 12000r/min, collects supernatant；Supernatant is again through 0.22 μm of filter membrane filter；Filtrate exists It is passed on PK15 cell, 37 DEG C of culture 1h, the DMEM culture solution for containing 2% calf serum is added in replacement, is placed in 37 DEG C and cultivates 5. The culture solution containing virus is harvested, culture solution is after 2 freeze thawing, harvest virus.
3, PCR and sequencing analysis identify viral species
The viral cultures for taking above step to harvest extract the nucleic acid of viral sample with nucleic acid extraction kit, use circle Circovirus virus species specificity primer carries out PCR amplification identification, the results show that PCR amplification goes out 2000bp purpose band.PCR product Sequencing company is sent to carry out nucleotide sequencing, sequencing results carry out phylogenetic analysis.The Strain is complete as the result is shown Genome sequence and amino acid sequence are with it has been reported that the homology for other circovirus crossed is less than 50%, and according to the world Virology is classified the standard of the committee, and same virus should have the genome nucleotide sequence of > 75% in Circovirus The homology of column, the homology of amino acid sequence of the Cap protein with > 70%, therefore can affirm, it is a kind of new pig Circovirus, and the third circovirus found on pig body at present.
The screening of 2 pig circular ring virus of embodiment, 3 type vaccine strain
Specific primer is designed according to above-mentioned 3 type of pig circular ring virus being separated to, it is each from the whole nation by quantitative PCR analysis 235 parts of doubtful PCV3 positive samples of ground acquisition, 121 plants of PCV3 viruses are isolated in screening, in this 121 plants, its base between each strain Because a group nucleotide sequence homology is up to 98.9~99.6%, Cap protein amino acid sequence homology is up to 97.7~ 99.5%.It is tested through this pathogenicity and immunogenicity test, finishing screen selects 1 plant of pathogenic strong, immunogenicity well, extensively The strong PCV3 Strain of spectrality.The 3 type viral nomenclature of pig circular ring virus is SG plants of 3 type of pig circular ring virus, submits preservation.
3 pig circular ring virus of embodiment SG plants of pathogenicities of 3 type
Two are randomly divided into through ELISA detection PCV2, PCV3 antigen, sodium selenite 10 of negative antibody with 28~30 ages in days Group, 5/group, the 1st group (contains 10 with PCV3SG plants5.0TCID50/ head) poison, intramuscular injection are attacked, blank control group is inoculated with DMEM training Support base, each group piglet isolated rearing.Each group piglet is observed continuously after attacking poison, is examined according to its clinical symptoms, pathological change and virus Survey is determined that concrete outcome is shown in Table 1.
SG plants of 1 pig circular ring virus of table, 3 type is to piglet pathogenicity test results
There is 40.5 DEG C of duration or more high temperature on the 3rd~5, appetite stimulator, spirit the results show that attacking poison and organizing all pigs Depressed, coat is thick disorderly, the thin and speed of growth slows down, and different degrees of pulmonary consolidation occurs in dissect, lymph enlargement, kidney have necrosis Point carries out PCR detection by each organs and tissues, can be separated to pig circular ring virus 3 type virus again, and blank control group is no different Often, show that SG plants of inoculation piglets of 3 type of pig circular ring virus of the present invention can lead to piglet morbidity, clinical table disease is typical pig annulus Viral infection symptoms.
The preparation of 4 pig circular ring virus of embodiment, 3 SG plants of antigen of type
The culture for SG plants of 3 type of the pig circular ring virus different generations that embodiment 2 is screened is according to Virus culture liquid measure 1% (V/V) access is formed in the PK15 passage cell of single layer, is placed in 37 DEG C and is adsorbed 30 minutes, cell maintenance medium is then added, It is placed in 37 DEG C of cultures.Daily observation 1~2 time, cell well-grown harvest cell culture after cultivating 4~7 in 36~37 DEG C Object after cell culture freeze thawing 2-3 times of harvest, harvests virus liquid, and measure viral titer.By virus liquid doughnut The filtering of (0.5 μm~2 μm) filter column adds 0.1%~0.2% formalin and inactivates at 37 DEG C to remove cell fragment For 24 hours, the viral antigen after inactivation completely is to prepare vaccine.
The preparation of 5 CSFV antigen of embodiment
By grow up to good single layer the high sensitive ST cell of swine fever virus Lapinized strain is used containing 0.125% pancreatin and The digestive juice of 0.03%EDTA carries out digestion dispersion, and Tissue Culture Flask is inoculated with after cell count, and 3% calf serum is added DMEM cell culture fluid, while connecing toxic dose according to M.O.I.=0.1 and hog cholera seed culture of viruses poison is added (purchased from China Institute of Veterinary Drug Control, deposit number AVCC NO.AV1412), it is placed in 37 DEG C of incubators and is cultivated.It carries out receiving poison for the first time after cultivating three days, adds and contain after receipts poison The cell maintenance medium of 1.5% calf serum, it is later primary every 2 days receipts poison, it can continuously receive poison 5 times.Finally by each batch after receipts poison The antigen mixing for receiving poison is placed in -20 DEG C of storages.
The preparation of 6 porcine pseudorabies virus of embodiment attenuation antigen
By HN1201-R plants of (being disclosed in Chinese patent application CN105087506A) cultures of porcine pseudorabies virus by disease 1% (V/V) access of malicious Culture liquid measure is formed in the ST cell culture of single layer, 37 DEG C of cultures is placed in, when lesion reaches 80% When, toxic cell culture fluid is harvested, cell culture fluid receives poison after 2 freeze thawing, measures malicious valence, is placed in cryo-conservation.
The preparation of 7 porcine pseudorabies virus inactivation antigen of embodiment
HN1201 plants of porcine pseudorabies virus of gE gene-deleted strain (is disclosed in Chinese patent application CN103923884A) culture is formed in the ST cell culture of single layer by 1% (V/V) access of Virus culture liquid measure, is placed in 37 DEG C of rotating and culturings harvest toxic cell culture fluid when lesion reaches 80%, after 2 freeze thawing, harvest virus liquid, measurement Malicious valence.10% (v/v) formalin is added into virus liquid makes final concentration of 0.2% (V/V) of formaldehyde, inactivates 18 in 37 DEG C Hour, it stirs 1 time within every 4 hours, stirs 10min every time, it is spare after inactivating completely.
The preparation of 8 porcine reproductive and respiratory syndrome virus of embodiment attenuation antigen
Required amount of Marc-145 cell (cell spinner bottle for having grown up to single layer) is chosen, growth-promoting media is discarded, by 1% (V/ V it) is inoculated with JXA1-R plants of production seed culture of viruses (being disclosed in Chinese patent application CN101307305A), is added containing 2% fetal calf serum DMEM cell culture fluid is cultivated in 37 DEG C of rotations (9~12 turn/hour), observes cytopathy caused by virus under the microscope Become (CPE), harvests cell culture when CPE reaches 70% or more, cell culture freeze thawing 1 time, and be centrifuged or crossed and filtered out Cell fragment is removed, is frozen in -40 DEG C or less.
The preparation of 9 pig parvoviral inactivation antigen of embodiment
By HN-2011 plants of pig parvoviral (being disclosed in Chinese patent application CN102886043A) cultures by M.O.I. =0.01, which connects toxic dose, accesses in the ST cell culture to form single layer, 37 DEG C of rotating and culturings is placed in, when lesion reaches 80% When, toxic cell culture fluid is harvested, for cell culture fluid after 2 freeze thawing, harvest virus measures malicious valence.It is added into virus liquid 10% (v/v) formalin makes final concentration of 0.2% (V/V) of formaldehyde, inactivates 18 hours, stirs 1 time in 37 DEG C within every 4 hours, Stirring 10min every time, it is spare after inactivating completely.
The preparation of 10 haemophilus parasuis inactivation antigen of embodiment
The breeding of first order seed: JS plants of 4 type of haemophilus parasuis serum, ZJ plants of 5 type (are disclosed in Chinese patent application CN102908615A) freeze-drying lactobacillus, in TSA/NAD, (TSA is the production of BD company, and NAD is raw for Roche company for streak inoculation respectively Produce) on plate, it is placed in 37 DEG C and cultivates 18~24 hours, choose satisfactory bacterium colony, being inoculated in TSB/NAD, (TSB is BD public Department's production, NAD are that Roche company produces) in fluid nutrient medium, in 37 DEG C culture 12~16 hours, as first order seed；
The breeding of secondary seed: ZJ plants of JS plants of 4 type of haemophilus parasuis serum of above-mentioned preparation, 5 type first order seeds are taken Culture is added in TSB/NAD fluid nutrient medium by 1% amount, is cultivated 12~16 hours in 37 DEG C, the conduct after examining purely Secondary seed；
In TSB culture medium, calf serum (the Zhejiang day Hangzhoupro biotechnology of 0.01~0.05%NAD, 5~10% is added Co., Ltd), 0.1~5% glucose, ZJ plants of JS plants of 4 type of haemophilus parasuis serum, 5 type secondary seed bacterium solutions are pressed 1% amount is added in culture medium cultivates respectively, and mixing is placed on 37 DEG C and cultivates 16~18 hours, when the concentration OD600 of bacterium solution reaches It begun to ramp up to 2.5 or more, DO value, stop culture when pH value is reduced to 6.5 or less.
After culture count plate, 37% formalin (Yantai City's chemical industry in pairs is added in 0.2% (V/V) in an amount Co., Ltd), it is placed in 37 DEG C of inactivations for 24 hours, during which stirs 3~5 times, it is spare after inactivating completely.
The preparation of 11 mycoplasma hyopneumoniae inactivation antigen of embodiment
The breeding of first order seed: HN0613 plants of freeze-drying lactobacillus (being disclosed in Chinese patent application CN103031258A) unpackings Afterwards, it is inoculated with fluid nutrient medium by 10% inoculum concentration, is placed in 37 DEG C of 3~7d of shaken cultivation, harvest when pH value is down to 6.8 by 7.5 Culture is purely examined, as first order seed；
The breeding of secondary seed: the first order seed of above-mentioned preparation is taken to be inoculated in fluid nutrient medium, 37 DEG C of vibrations by 5% inoculum concentration 3~7d of culture is swung, harvests culture when pH value is down to 6.8 by 7.5, is purely examined, as secondary seed；
The formula (based on 1065ml) of fluid nutrient medium: cattle heart leachate 300ml, ddH2O (secondary distilled water) 360ml, PH value is corrected to sterilize 15 minutes to 7.4,121 DEG C.Add the composition of following filtration sterilization: Hank ' s balanced salt solution (10 ×) (10 times concentration) 40ml, 0.25% phenol red 10ml, horse serum 200ml, 5% lactoalbumin hydrolysate 100ml, 25% yeast leachate 20ml, 10000IU/ml penicillin 10ml, 1% thaliium acetate solution 25m1；
Secondary seed solution is inoculated in fluid nutrient medium with 5% (v/v), is placed at 37 DEG C and cultivates 3~7, to pH value Bacterium solution is harvested when being down to 6.8 by 7.5.
The mycoplasma hyopneumoniae bacterium solution for examining qualified above-mentioned preparation is taken, is slowly added to by bacterium solution volume total amount final concentration of 0.2% formalin (v/v) is placed in 37 DEG C of inactivations, primary every 3~4h stirring therebetween, takes out afterwards for 24 hours, inactivation is complete It is spare afterwards.
The preparation of the 3 type SG strain vaccine composition containing pig circular ring virus of embodiment 12
The porcine pseudorabies virus that swine fever virus prepared by embodiment 5 is attenuated antigen, prepared by embodiment 6 is attenuated respectively anti- Heat resisting protective (2wt% aqueous gelatin solution is added in porcine reproductive and respiratory syndrome virus attenuation antigen prepared by former, embodiment 8 With 15wt% lactose aqueous solution with 1:1 (v/v) ratio preparation) mixed with 1:1 (v/v) ratio after mix well, quantitative separating is fast Speed carries out vacuum freezedrying, and as swine fever virus live vaccine part, porcine pseudorabies virus live vaccine part, pig breeds and exhale Inhale syndrome virus virus live vaccine part.
Water-soluble adjuvant Gel adjuvant is added slowly to pig circular ring virus SG plants of inactivation antigens of 3 type prepared by embodiment 4 In (France match BIC Corp), with revolving speed it is constantly 800rpm mulser stirring 12min during adding, mixes, as pig circle 3 type inactivated vaccine part of circovirus virus.With inactivated vaccine part of dilution live vaccine part when use, in two kinds of antigen ratios such as table 2 It is shown.
The proportion containing 3 type SG strain vaccine composition components of pig circular ring virus of table 2 (live seedling part)
The porcine pseudorabies of SG plants of inactivation antigens of 3 type of pig circular ring virus, the preparation of embodiment 7 that respectively prepared by Example 4 The haemophilus parasuis inactivation of pig parvoviral inactivation antigen, the preparation of embodiment 10 prepared by inactivation of virus antigen, embodiment 9 is anti- Mycoplasma hyopneumoniae inactivation antigen prepared by former, embodiment 11 prepares five kinds of antigens according to the final antigenic content of composition, It is added in water-soluble adjuvant Gel adjuvant (France match BIC Corp), with revolving speed is constantly 800rpm mulser during adding 12min is stirred, is mixed.The specific formula and content of vaccine composition are shown in Table 3.
The proportion containing 3 type SG strain vaccine composition components of pig circular ring virus of table 3 (inactivated vaccine part)
The 3 type SG strain vaccine composition Study On Immunogenicity containing pig circular ring virus of embodiment 13
1,3 type partial immunity originality of pig circular ring virus is tested
28~30 ages in days are randomly divided into 9 through ELISA detection PCV2, PCV3 antigen, sodium selenite 45 of negative antibody 5/group, vaccine composition prepared by embodiment 12 is immunized in group.3rd~10 group of difference immune vaccine 1~8, the 11st group is not immunized As attacking malicious control group.Each immune group vaccinates 2ml/ head, attacks malicious control group inoculation DMEM culture medium 2ml/ head.After immune It carries out within 28th attacking poison, attacking toxic dose is SG plants of pig circular ring virus 105.0TCID50Each piglet is observed continuously after attacking poison, according to each in/head Piglet clinical symptoms, pathological change and viral diagnosis result are determined that concrete outcome is shown in Table 4.
The vaccine composition PCV3 partial immunity originality test result of the SG plants of antigens of 3 type containing pig circular ring virus of table 4
The results show that after the vaccine composition primary immunization piglet of SG plants of antigens of 3 type containing pig circular ring virus, it can be for pig circle The infection of 3 type of circovirus virus provides 100% (5/5) protection for piglet, and attacks after malicious control group piglet attacks poison and all fall ill.Show this hair 3 type antigen part of pig circular ring virus has good in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus of bright offer Vaccine effectiveness can provide pig 100% protection, and viral diagnosis is feminine gender.
It is not interfered between SG plants of antigens of 3 type of pig circular ring virus of the invention and a variety of viral antigens, bacterial antigens, it can be common It is applied after composition vaccine composition.
2, swine fever partial immunity originality is tested
20 ages in days are randomly divided into 2 through ELISA detection PCV2, PCV3, CSFV antigen, sodium selenite 8 of negative antibody Group, 4/group.Malicious control group is attacked in vaccine 1 prepared by the 12nd group of immune embodiment 12, the 13rd group of not immune conduct.By every part 2ml vaccine 1 dilutes 3000 times, and intramuscular injection, every 1ml attacks malicious control group inoculation DMEM culture medium 1ml/ head.10~14 days Afterwards, together with the identical control group of condition 4, swine fever crossdrift system is injected blood poison 1.0ml/ (105MLD), observe 16.As a result It is shown in Table 5.
The vaccine composition CSFV partial immunity originality test result of the SG plants of antigens of 3 type containing pig circular ring virus of table 5
Group Immune head part Protective rate Clinical symptoms
12 1/3000 100% (4/4) No body temperature reaction
13 DMEM culture medium 1ml 0% (0/4) 4 dead
According to Chinese veterinary pharmacopoeia standard, classical swine fever virus vaccine immune efficacy is detected, vaccine 300 times or more need to be diluted and exempted from After epidemic disease, then detected, and the present embodiment the results show that the vaccine composition 1/ of the 3 SG plants of antigen of type containing pig circular ring virus After 3000 immune piglets of part (3000 times of dilution), 100% (4/4) protection can be provided for piglet for swine fever virus infection, and There is no body temperature reaction, and attacks malicious control group piglet and attack all morbidity death after poison.Show provided by the invention containing pig circular ring virus 3 CSFV antigen part in the vaccine composition of SG plants of antigen of type has good Vaccine effectiveness and safety.
3, porcine pseudorabies virus partial immunity originality is tested
9 ages in days are randomly divided into 3 through ELISA detection PCV2, PCV3, PRV antigen, sodium selenite 15 of negative antibody Group, 5/group, vaccine 2 prepared by the 14th group of immune embodiment 12, vaccine 4 prepared by the 15th group of immune embodiment 12, the 16th group Immune be used as does not attack malicious control group.Immune group vaccinates 2ml/ head, attacks malicious control group inoculation DMEM culture medium 2ml/ head.It is immune It carries out within 21st attacking poison afterwards, attacking toxic dose is porcine pseudorabies virus HN1201 strain 107.0TCID50/ head attacks and measures son after poison daily Temperature of pig body, observes clinical symptoms and death condition, concrete outcome are shown in Table 6.
The vaccine composition PRV partial immunity originality test result of the SG plants of antigens of 3 type containing pig circular ring virus of table 6
The results show that after piglet is immunized in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus, it can blocking virus infection (clinical symptoms occur) can infect for porcine pseudorabies virus and provide 100% (5/5) protection for piglet, and attack malicious control group Piglet attacks all death on the 4th after poison.In the vaccine composition for showing the 3 SG plants of antigen of type provided by the invention containing pig circular ring virus Porcine pseudorabies virus inactivation antigen or live virus antigen part all have good Vaccine effectiveness.
4, porcine reproductive and respiratory syndrome virus partial immunity originality is tested
4~6 week old are divided at random through the sodium selenite 10 of ELISA detection PCV2, PCV3, PRRSV antigen, negative antibody At 2 groups, 5/group, malicious control group is attacked in vaccine 3 prepared by the 17th group of immune embodiment 12, the 18th group of not immune conduct.Immune group 2ml/ head is vaccinated, malicious control group inoculation DMEM culture medium 2ml/ head is attacked.It carries out within 28th attacking poison after immune, attacking toxic dose is pig Reproductive and respiratory syndrome virus NVDC-JXA1 strain 105.0TCID50/ head, daily thermometric simultaneously observe clinical symptoms and death condition. Concrete outcome is shown in Table 7.
The vaccine composition PRRSV partial immunity originality test result of the SG plants of antigens of 3 type containing pig circular ring virus of table 7
The results show that the 3 SG plants of antigen of type containing pig circular ring virus vaccine composition be immunized piglet after, can for pig breeding and Breath syndrome virus infection is that piglet provides 100% (5/5) protection, and attacks malicious control group piglet and attack after poison all morbidities and extremely Die 3.Show that the pig breeding in the vaccine composition of the 3 SG plants of antigen of type provided by the invention containing pig circular ring virus is integrated with breathing Levying viral antigen part has good Vaccine effectiveness.
5, pig parvoviral partial immunity originality is tested
10~20kg is randomly divided into through ELISA detection PCV2, PCV3, PPV antigen, sodium selenite 10 of negative antibody 2 groups, 5/group, vaccine 5 prepared by the 19th group of immune embodiment 12, the 20th group not immune as blank control group.Immune group note Vaccine 2ml/ head is penetrated, blank control group is inoculated with DMEM culture medium 2ml/ head.It 28 days after immune, takes a blood sample together with control group, measurement is anti- Body, control group should be negative (HI antibody titer≤1:8), and immune group at least 4 antibody response occur, antibody titer >=1 HI: 64.Concrete outcome is shown in Table 8.
The vaccine composition PPV partial immunity originality test result of the SG plants of antigens of 3 type containing pig circular ring virus of table 8
The results show that the HI antibody of PPV is equal after piglet is immunized in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus Greater than 1:64,100% (5/5) protection can be provided for piglet for porcine parvovirus infection.Show provided by the invention containing pig circle PPV Antigen Using part in the vaccine composition of 3 SG plants of antigen of type of circovirus virus has good Vaccine effectiveness.
6, haemophilus parasuis partial immunity originality is tested
By 14~21 age in days piglets through ELISA detection PCV2, PCV3, HPs antigen, negative antibody sodium selenite 30 with Machine is divided into 6 groups, and 5/group, the vaccine 6 of the 21st group, the 22nd group immune preparation of embodiment 12, the 23rd group, the 24th group is immunized embodiment The vaccines 8 of 12 preparations, the 25th group, the 26th group is not immunized conduct and attacks malicious control group.Immune group vaccinates 2ml/ head, and it is right to attack poison According to a group inoculation DMEM culture medium 2ml/ head.35 days after immune, the 21st group of immune group, the 23rd group are taken, attacks the 25th group of malicious control group, It is carried out attacking poison with JS plants of 4 type, 3ml bacterium solution is injected intraperitoneally, attacking toxic dose is 9.0 × 109CFU/ head；Take the 22nd group of immune group, 24 groups, attack the 26th group of malicious control group, attack poison with ZJ plants of 5 type, 3ml bacterium solution is injected intraperitoneally, attack toxic dose be 6.0 × 109CFU/ head；Its clinical manifestation is observed after attacking poison, observation is cutd open after 14 days and kills test pig, and pathological observation is carried out.Concrete outcome is shown in Table 9。
The vaccine composition HPs partial immunity originality test result of the SG plants of antigens of 3 type containing pig circular ring virus of table 9
Group Piglet number 4 JS plants of types attack malicious protective rate 5 ZJ plants of types attack malicious protective rate
21 5 5/5 —
22 5 — 5/5
23 5 5/5 —
24 5 — 5/5
25 5 0/5 —
26 5 — 0/5
Note: Haemophilus parasuis morbidity standard: morbid pig appearance fever (40.5 DEG C of body temperature or more, continue 1~5), Apathetic, cough, expiratory dyspnea, it is thin, walk lamely and the thick clinical symptoms such as disorderly of coat.To dying pig dissect, it is seen that multiple The lesions such as scrositis (pleurisy, pericarditis, peritonitis), arthritis and meningitis, each serosal surface (joint capsule, pericardium, pleura And peritonaeum) there is serosity or fibrinous exudate.
The results show that after piglet is immunized in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus, it can be bloodthirsty for secondary pig 4 type of bacillus, the infection of 5 type bacterium provide 100% (5/5) protection for piglet, and attack after malicious control group piglet attacks poison and all fall ill.Show 4 types, 5 type haemophilus parasuis antigen portions in the vaccine composition of the 3 SG plants of antigen of type provided by the invention containing pig circular ring virus Divide and all has good Vaccine effectiveness.
7, mycoplasma hyopneumoniae partial immunity originality is tested
By 14~21 age in days piglets through ELISA detection PCV2, PCV3, Mhp antigen, negative antibody sodium selenite 15 with Machine is divided into 3 groups, and 5/group, vaccine 7 prepared by the 27th group of immune embodiment 12, the 28th group is immunized vaccine prepared by embodiment 12 Malicious control group is attacked in 8, the 29th group of not immune conduct.Immune group vaccinates 2ml/ head, attacks malicious control group inoculation DMEM culture medium 2ml/ head.35 days after immune, the 27th group of immune group, the 28th group are taken, attacks the 29th group of malicious control group, with CVCC354 plants (purchased from China Veterinary medicament supervises institute, which examines for the porcine mycoplasmal pneumonia vaccine potency that China veterinary medicament supervises institute's preservation and use Strain) tracheae injects 5ml/ (100MID), and it is observed 28 days after attacking poison, cuts open to kill and take lung, by 28 point-scores to the asthma of test pig Pneumonia lesion scores, and pneumonia lesion slip is calculated according to the following formula.Concrete outcome is shown in Table 10.
Pneumonia lesion slip=(attack poison control pig pneumonia lesion average mark-immune swine pneumonia disease flattens respectively)/and attack poison Compare pig pneumonia lesion average mark × 100%
The vaccine composition Mhp partial immunity originality test result of the SG plants of antigens of 3 type containing pig circular ring virus of table 10
Note: in otherness statistical analysis, comparison among groups, the identical person of letter indicates that difference is not significant, and letter is different to indicate poor Different significant (P < 0.05)
The results show that after piglet is immunized in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus, it is former for pig pneumonia branch Body-sensing dye, each immune group compared with attacking malicious control group, immune group and attack malicious control group be averaged tuberculosis change there are significant differences.Show Mycoplasma hyopneumoniae antigen part in the vaccine composition of the 3 SG plants of antigen of type provided by the invention containing pig circular ring virus has Good Vaccine effectiveness.
In conclusion from the above different vaccine compositions for 4 type bacterium of PCV3, CSFV, PRV, PRRSV, PPV, HPs, 5 type bacterium of HPs, Mhp Study On Immunogenicity result from the point of view of, each vaccine composition can reach preferable protecting effect, show this Inventing antigenic component different in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus provided has protection well Effect.Illustrate that SG plants of inactivation antigens of 3 type of pig circular ring virus of the invention can form immune composition with Different Kinds of Pathogens antigenic component It is co-administered.
The 3 type SG strain vaccine composition broad spectrum activity protection test containing pig circular ring virus of embodiment 14
28~30 ages in days are randomly divided into through ELISA detection PCV2, PCV3 antigen, sodium selenite 225 of negative antibody 45 groups, 5/group, vaccine 1 prepared by the 30th~34 group of immune embodiment 12, epidemic disease prepared by the 35th~39 group of immune embodiment 12 Vaccine 3 prepared by the 2, the 40th~44 group of seedling immune embodiment 12, vaccine 4 prepared by the 45th~49 group of immune embodiment 12, the 50th Vaccine 6 prepared by the 5, the 55th~59 group of vaccine immune embodiment 12 prepared by~54 groups of immune embodiments 12, the 60th~64 group is exempted from Vaccine 8 prepared by the 7, the 65th~69 group of vaccine immune embodiment 12 prepared by epidemic disease embodiment 12, the 70th~74 group is not immunized conduct Attack malicious control group.Each immune group vaccinates 2ml/ head, attacks malicious control group inoculation DMEM culture medium 2ml/ head.After immune 28 days into Row attacks poison, the 30th group, the 35th group, the 40th group, the 45th group, the 50th group, the 55th group, the 60th group, the 65th group, the 70th group with recently from Pig circular ring virus HN12 plants of velogen strains of 3 type of Henan, China separation attack poison；31st group, the 36th group, the 41st group, the 46th group, 51 groups, the 56th group, the 61st group, the 66th group, the 71st group are used JS08 plants of 3 type of pig circular ring virus separated recently from Jiangsu Province, China by force Strain attacks poison；32nd group, the 37th group, the 42nd group, the 47th group, the 52nd group, the 57th group, the 62nd group, the 67th group, the 72nd group is used recently The pig circular ring virus JL11 plants of velogen strains of 3 type separated from Chinese Jilin Province attack poison；33rd group, the 38th group, the 43rd group, the 48th group, 53rd group, the 58th group, the 63rd group, the 68th group, the 73rd group is used the 3 type CQ04 of pig circular ring virus separated recently from Chinese Chongqing City Strain velogen strain attacks poison；34th group, the 39th group, the 44th group, the 49th group, the 54th group, the 59th group, the 64th group, the 69th group, the 74th group of use Recently poison is attacked from pig circular ring virus GD05 plants of velogen strains of 3 type that Guangdong province, China separates；Attacking toxic dose is 105.0TCID50/ Each piglet is observed continuously after attacking poison in head, is determined according to each piglet clinical symptoms, pathological change and viral diagnosis, specific to tie Fruit is shown in Table 11~19.
11 vaccine of table, 1 broad spectrum activity protection test result
Group Clinical symptoms Pathological change Viral diagnosis (positive rate) Protective rate
30 It is without exception It is without exception 0% (0/5) 100% (5/5)
31 It is without exception It is without exception 0% (0/5) 100% (5/5)
32 It is without exception It is without exception 0% (0/5) 100% (5/5)
33 It is without exception It is without exception 0% (0/5) 100% (5/5)
34 It is without exception It is without exception 0% (0/5) 100% (5/5)
12 vaccine of table, 2 broad spectrum activity protection test result
Group Clinical symptoms Pathological change Viral diagnosis (positive rate) Protective rate
35 It is without exception It is without exception 0% (0/5) 100% (5/5)
36 It is without exception It is without exception 0% (0/5) 100% (5/5)
37 It is without exception It is without exception 0% (0/5) 100% (5/5)
38 It is without exception It is without exception 0% (0/5) 100% (5/5)
39 It is without exception It is without exception 0% (0/5) 100% (5/5)
13 vaccine of table, 3 broad spectrum activity protection test result
14 vaccine of table, 4 broad spectrum activity protection test result
Group Clinical symptoms Pathological change Viral diagnosis (positive rate) Protective rate
45 It is without exception It is without exception 0% (0/5) 100% (5/5)
46 It is without exception It is without exception 0% (0/5) 100% (5/5)
47 It is without exception It is without exception 0% (0/5) 100% (5/5)
48 It is without exception It is without exception 0% (0/5) 100% (5/5)
49 It is without exception It is without exception 0% (0/5) 100% (5/5)
15 vaccine of table, 5 broad spectrum activity protection test result
Group Clinical symptoms Pathological change Viral diagnosis (positive rate) Protective rate
50 It is without exception It is without exception 0% (0/5) 100% (5/5)
51 It is without exception It is without exception 0% (0/5) 100% (5/5)
52 It is without exception It is without exception 0% (0/5) 100% (5/5)
53 It is without exception It is without exception 0% (0/5) 100% (5/5)
54 It is without exception It is without exception 0% (0/5) 100% (5/5)
16 vaccine of table, 6 broad spectrum activity protection test result
Group Clinical symptoms Pathological change Viral diagnosis (positive rate) Protective rate
55 It is without exception It is without exception 0% (0/5) 100% (5/5)
56 It is without exception It is without exception 0% (0/5) 100% (5/5)
57 It is without exception It is without exception 0% (0/5) 100% (5/5)
58 It is without exception It is without exception 0% (0/5) 100% (5/5)
59 It is without exception It is without exception 0% (0/5) 100% (5/5)
17 vaccine of table, 7 broad spectrum activity protection test result
Group Clinical symptoms Pathological change Viral diagnosis (positive rate) Protective rate
60 It is without exception It is without exception 0% (0/5) 100% (5/5)
61 It is without exception It is without exception 0% (0/5) 100% (5/5)
62 It is without exception It is without exception 0% (0/5) 100% (5/5)
63 It is without exception It is without exception 0% (0/5) 100% (5/5)
64 It is without exception It is without exception 0% (0/5) 100% (5/5)
18 vaccine of table, 8 broad spectrum activity protection test result
Group Clinical symptoms Pathological change Viral diagnosis (positive rate) Protective rate
65 It is without exception It is without exception 0% (0/5) 100% (5/5)
66 It is without exception It is without exception 0% (0/5) 100% (5/5)
67 It is without exception It is without exception 0% (0/5) 100% (5/5)
68 It is without exception It is without exception 0% (0/5) 100% (5/5)
69 It is without exception It is without exception 0% (0/5) 100% (5/5)
Malicious control group test result is attacked in 19 broad spectrum activity protection test of table
The results show that the 70th~74 group is attacked malicious control group and attacked after poison and occur 40.5 DEG C of body temperature raising or more in various degree, Continue 3~5 days, appetite stimulator, the depressed, coat of spirit are thick disorderly, the thin and speed of growth slows down etc., and clinical symptoms, dissect occur The pathological change that different degrees of pulmonary consolidation, lymph enlargement, kidney have necrosis to put, carries out PCR detection by each organs and tissues, can be again It is separated to pig circular ring virus 3 type virus；And the 30th~69 group of immune group attacks clinical symptoms without exception after poison, each histoorgan of dissect Also without exception, PCR detection is carried out by each organs and tissues, shows PCV3 feminine gender.Show provided by the invention containing pig circular ring virus 2 The vaccine composition of malicious 3 SG plants of antigens of type can attack poison for 3 type of pig circular ring virus in different geographical source to pig and provide effectively , complete immunoprotection, and the PCV3 strain for attacking poison cannot be detected from each organs and tissues.Vaccine composition of the invention has wide The immunogenicity of spectrum can provide protection completely for 3 type of pig circular ring virus in different geographical source.
In conclusion from the different vaccine compositions of SG plants of antigens of 3 type containing pig circular ring virus above for different geographical come From the point of view of the 3 type Study On Immunogenicity result of pig circular ring virus in source, each vaccine composition can reach preferable protecting effect, show The vaccine composition of the 3 SG plants of antigen of type provided by the invention containing pig circular ring virus has good broad spectrum activity Vaccine effectiveness.
The 3 type SG strain vaccine composition mixed infection protection test containing pig circular ring virus of embodiment 15
1,3 type of pig circular ring virus, swine fever virus mixed infection protection test
28~30 ages in days are random through ELISA detection PCV2, PCV3, CSFV antigen, the sodium selenite 15 of negative antibody It is divided into 3 groups, 5/group.Vaccine 1 prepared by the 75th group of immune embodiment 12, the 76th group, the 77th group is not immunized conduct and attacks malicious control Group.Immune group vaccinates 2ml/ head, attacks malicious control group inoculation DMEM culture medium 2ml/ head.It carries out within 28th attacking poison after immune, attack Toxic dose is SG plants of pig circular ring virus 105.0TCID50/ head, blood poison 1.0ml/ (10, swine fever crossdrift system5MLD), the 75th group simultaneously Attack malicious pig circular ring virus and swine fever virus, the 76th group is attacked malicious control group for pig circular ring virus, and the 77th group for swine fever virus to attack poison right According to group, each piglet is observed continuously after attacking poison, is determined that concrete outcome is shown in Table 20 according to each piglet clinical symptoms, pathological change.
20 pig circular ring virus of table, 3 type, swine fever virus mixed infection protection test result
The results show that pig circular ring virus 2 can be directed to after piglet is immunized in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus Malicious 3 types, swine fever virus mixed infection provide 100% (5/5) protection for piglet, and react without body temperature, and two groups are attacked malicious control Group piglet attacks all morbidities or dead after poison.Show the vaccine combination of the 3 SG plants of antigen of type provided by the invention containing pig circular ring virus Object has good Vaccine effectiveness and safety, can provide effective protection to 3 type of pig circular ring virus, swine fever virus mixed infection.
2,3 type of pig circular ring virus, porcine pseudorabies virus mixed infection protection test
9 ages in days are randomly divided into 4 through ELISA detection PCV2, PCV3, PRV antigen, sodium selenite 20 of negative antibody Group, 5/group.Vaccine 2 prepared by the 78th group of immune embodiment 12, vaccine 4 prepared by the 79th group of immune embodiment 12, the 80th group, Malicious control group is attacked in 81st group of not immune conduct.Immune group vaccinates 2ml/ head, attacks malicious control group inoculation DMEM culture medium 2ml/ Head.It carries out within 28th attacking poison after immune, attacking toxic dose is SG plants of pig circular ring virus 105.0TCID50/ head, porcine pseudorabies virus HN1201 strain 107.0TCID50/ head, the 78th group, the 79th group is attacked malicious pig circular ring virus and porcine pseudorabies virus simultaneously, and the 80th group is Pig circular ring virus attacks malicious control group, and the 81st group is attacked malicious control group for porcine pseudorabies virus, and each piglet, root is observed continuously after attacking poison Determined that concrete outcome is shown in Table 21 according to each piglet clinical symptoms, pathological change and viral diagnosis result.
21 pig circular ring virus of table, 3 type, porcine pseudorabies virus mixed infection protection test result
The results show that pig circular ring virus 2 can be directed to after piglet is immunized in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus Malicious 3 types, porcine pseudorabies virus mixed infection provide 100% (5/5) protection for piglet, and two groups are attacked malicious control group piglet and attacked All morbidities or dead after poison.Show that the vaccine composition of the 3 SG plants of antigen of type provided by the invention containing pig circular ring virus has very Good Vaccine effectiveness and safety can provide effective protection to 3 type of pig circular ring virus, porcine pseudorabies virus mixed infection.
3,3 type of pig circular ring virus, porcine reproductive and respiratory syndrome virus mixed infection protection test
28~30 ages in days are random through ELISA detection PCV2, PCV3, PRRSV antigen, the sodium selenite 15 of negative antibody It is divided into 3 groups, 5/group.Vaccine 3 prepared by the 82nd group of immune embodiment 12, the 83rd group, the 84th group is not immunized conduct and attacks malicious control Group.Immune group vaccinates 2ml/ head, attacks malicious control group inoculation DMEM culture medium 2ml/ head.It carries out within 28th attacking poison after immune, attack Toxic dose is SG plants of pig circular ring virus 105.0TCID50/ head, NVDC-JXA1 plants of porcine reproductive and respiratory syndrome virus 105.0TCID50/ head, the 82nd group is attacked malicious pig circular ring virus and porcine reproductive and respiratory syndrome virus simultaneously, and the 83rd group is pig annulus Virus attacks malicious control group, and the 84th group is attacked malicious control group for porcine reproductive and respiratory syndrome virus, and each son is observed continuously after attacking poison Pig is determined that concrete outcome is shown in Table 22 according to each piglet clinical symptoms, pathological change.
22 pig circular ring virus of table, 3 type, porcine reproductive and respiratory syndrome virus mixed infection protection test result
The results show that pig circular ring virus 2 can be directed to after piglet is immunized in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus Malicious 3 types, porcine reproductive and respiratory syndrome virus mixed infection provide 100% (5/5) protection for piglet, and two groups are attacked malicious control Group piglet attacks all morbidities or dead after poison.Show the vaccine combination of the 3 SG plants of antigen of type provided by the invention containing pig circular ring virus Object has good Vaccine effectiveness and safety, can be to 3 type of pig circular ring virus, porcine reproductive and respiratory syndrome virus mixed infection Effective protection is provided.
4,3 type of pig circular ring virus, haemophilus parasuis, mycoplasma hyopneumoniae mixed infection protection test
21 ages in days are divided at random through the sodium selenite 25 of ELISA detection PCV2, PCV3, HPs, Mhp antigen, negative antibody At 5 groups, 5/group.Vaccine 8 prepared by the 85th group of immune embodiment 12, the 86th group, the 87th group, the 88th group, the 89th group is not immunized As attacking malicious control group.Immune group vaccinates 2ml/ head, and control group is inoculated with DMEM culture medium 2ml/ head.It carries out within 35 days after immune Poison is attacked, attacking toxic dose is SG plants of pig circular ring virus 105.0TCID50/ head, 4 type JS strain 9.0 × 10 of haemophilus parasuis9CFU/, 5 Type ZJ strain 6.0 × 109CFU/ head, CVCC354 plants of 100MID/ heads of mycoplasma hyopneumoniae, the 85th group attack simultaneously malicious pig circular ring virus, Haemophilus parasuis and mycoplasma hyopneumoniae, the 86th group is attacked malicious control group for pig circular ring virus, and the 87th group is haemophilus parasuis 4 Type attacks malicious control group, and the 88th group is attacked malicious control group for 5 type of haemophilus parasuis, and the 89th group is attacked malicious control for mycoplasma hyopneumoniae Each piglet is observed continuously after attacking poison in group, is determined that concrete outcome is shown in Table 23 according to each piglet clinical symptoms, pathological change.
23 pig circular ring virus of table, 3 type, haemophilus parasuis, mycoplasma hyopneumoniae mixed infection protection test result
Note: in otherness statistical analysis, comparison among groups, the identical person of letter indicates that difference is not significant, and letter is different to indicate poor Different significant (P < 0.05)
The results show that pig circular ring virus 2 can be directed to after piglet is immunized in the vaccine composition of the 3 SG plants of antigen of type containing pig circular ring virus Malicious 3 types, haemophilus parasuis, mycoplasma hyopneumoniae mixed infection are that piglet provides 100% (5/5) protection, and four groups attack it is malicious right It attacks after poison according to a group piglet and all falls ill.Show the vaccine composition tool of 3 type SG plants of antigens provided by the invention containing pig circular ring virus There are good Vaccine effectiveness and safety, it can be to 3 type of pig circular ring virus, haemophilus parasuis, mycoplasma hyopneumoniae mixed infection Effective protection is provided.
In conclusion being directed to pig circular ring virus 3 from the different vaccine compositions of SG plants of antigens of 3 type containing pig circular ring virus above For type with from the point of view of the mixed infection Study On Immunogenicity result of other cause of diseases, each vaccine composition can reach preferable protection effect Fruit shows that different antigenic components all have very in the vaccine composition of the 3 SG plants of antigen of type provided by the invention containing pig circular ring virus Good Vaccine effectiveness.
The above is only the preferred embodiment of the present invention, not does limitation in any form to the present invention, though So the present invention is disclosed above with preferred embodiment, and however, it is not intended to limit the invention, any technology people for being familiar with this profession Member, in the range of not departing from technical solution of the present invention, when the technology contents using the disclosure above make a little change or repair Decorations are the equivalent embodiment of equivalent variations, but anything that does not depart from the technical scheme of the invention content, technology according to the present invention are real Matter any simple modification, equivalent change and modification to the above embodiments, still fall within the range of technical solution of the present invention It is interior.

Claims (10)

1. the vaccine composition of a kind of prevention and/or treatment 3 type mixed infection of pig circular ring virus, wherein the vaccine composition 3 type antigen of pig circular ring virus comprising immune amount and pharmaceutically acceptable carrier；
Wherein, the vaccine composition also includes one of group being made of the following antigen of immune amount or a variety of: hog cholera Malicious antigen, porcine pseudorabies virus antigen, swine flue antigen, porcine reproductive and respiratory syndrome virus antigen, the tiny disease of pig Malicious antigen, Latex agglutination test antigen, haemophilus parasuis antigen, Streptococcus suis antigen, pig bordetella bacilli antigen, pig are infected Property pleuropneumonia antigen, pig pasteurella multocida antigen, Salmonella choleraesuls antigen, mycoplasma hyopneumoniae antigen, hog snout Mycoplasma antigen.
2. vaccine composition according to claim 1, wherein the 3 type antigen of pig circular ring virus is 3 type of pig circular ring virus SG plants or its culture inactivated whole virus antigen, SG plants of deposit numbers are CCTCC NO.V201712.
3. vaccine composition according to claim 2, wherein SG plants of cultures of 3 type of pig circular ring virus are >=1 generation Culture；Preferably, SG plants of 3 type of pig circular ring virus of the culture is the culture of >=5 generations；It is highly preferred that described The culture of SG plants of 3 type of pig circular ring virus is the culture of 5~55 generations.
4. vaccine composition according to claim 2, wherein SG plants of 3 type of pig circular ring virus or its culture go out Totivirus antigenic content living be inactivation before >=105.0TCID50/ml；Preferably, SG plants of 3 type of pig circular ring virus or its culture Inactivated whole virus antigenic content be inactivation preceding 105.0~107.0TCID50/ml；It is highly preferred that described SG plants of 3 type of pig circular ring virus Or the inactivated whole virus antigenic content of its culture is inactivation preceding 106.0TCID50/ml。
5. vaccine composition according to claim 1, wherein the vaccine composition also includes by the following anti-of immune amount One of group of original composition is a variety of: swine fever virus is attenuated antigen, porcine pseudorabies virus attenuation antigen, pig breeding and breathing Syndrome virus, which is attenuated antigen, pig parvoviral inactivation antigen, haemophilus parasuis inactivation antigen, mycoplasma hyopneumoniae and inactivates, to be resisted It is former；Or
Preferably, the vaccine composition also includes one of group being made of the following antigen of immune amount or a variety of: swine fever Virus attenuation antigen, porcine pseudorabies virus inactivation antigen, porcine reproductive and respiratory syndrome virus are attenuated antigen, pig parvoviral Inactivation antigen, haemophilus parasuis inactivation antigen, mycoplasma hyopneumoniae inactivation antigen.
6. vaccine composition according to claim 5, wherein the swine fever virus attenuation antigen is that swine fever virus rabbitization is weak Strain, porcine pseudorabies virus attenuation antigen are HN1201-R plants of attenuated strains, the porcine pseudorabies virus inactivation antigen For HN1201 plants of inactivated whole virus antigens, the porcine reproductive and respiratory syndrome virus be attenuated antigen be JXA1-R plants of attenuated strains, The pig parvoviral inactivation antigen is HN-2011 plants of inactivated whole virus antigens, the haemophilus parasuis inactivation antigen is secondary JS plants of 4 type of haemophilus suis serum or the full bacterium antigen of ZJ plants of 5 types inactivations, the mycoplasma hyopneumoniae inactivation antigen are HN0613 plants Inactivate full bacterium antigen.
7. vaccine composition according to claim 5, wherein the pig circular ring virus inactivation antigen content is before inactivating 105.0~107.0TCID50/ ml, the swine fever virus attenuation antigenic content is 104.0~106.0TCID50/ ml, the pseudorabies Sick virus attenuation antigenic content is 106.0~107.0TCID50/ ml, the porcine pseudorabies virus inactivation antigen content are before inactivating 106.0~107.0TCID50/ ml, the porcine reproductive and respiratory syndrome virus attenuation antigenic content is 105.0~107.0TCID50/ Ml, the pig parvoviral inactivation antigen content are inactivation preceding 106.0~108.0TCID50/ ml, the haemophilus parasuis inactivation Antigenic content is inactivation preceding 108.0~1010.0CFU/ml, the mycoplasma hyopneumoniae inactivation antigen content are inactivation preceding 108.0~ 1010.0CCU/ml；
Preferably, the pig circular ring virus inactivation antigen content is inactivation preceding 105.0~107.0TCID50/ ml, the swine fever virus Being attenuated antigenic content is 105.0TCID50/ ml, the porcine pseudorabies virus attenuation antigenic content is 106.0TCID50/ ml, it is described Porcine pseudorabies virus inactivation antigen content is inactivation preceding 106.0TCID50/ ml, the porcine reproductive and respiratory syndrome virus attenuation Antigenic content is 106.0TCID50/ ml, the pig parvoviral inactivation antigen content are inactivation preceding 107.0TCID50/ ml, the pair Haemophilus suis inactivation antigen content is inactivation preceding 109.0CFU/ml, the mycoplasma hyopneumoniae inactivation antigen content are before inactivating 109.0CCU/ml。
8. vaccine composition according to claim 1, wherein the pharmaceutically acceptable carrier includes adjuvant, institute Stating adjuvant includes white oil, Drake oil and animal oil, vegetable oil or mineral oil；Or aluminium hydroxide, aluminum phosphate and metal salt； Or MontanideTMGel, carbomer, saualane or squalene, ISA206 adjuvant, saponin(e, water-in-oil emulsion, oil in water emulsion, W/O/W emulsion；Preferably, the adjuvant is MontanideTMGel；
The adjuvant content is 5~20V/V%；It is highly preferred that the adjuvant content is 10V/V%.
9. described in any item vaccine compositions are in preparation prevention and/or treatment 3 type of pig circular ring virus according to claim 1~8 Application in the drug of related disease；Preferably, the 3 type related disease of pig circular ring virus includes postweaning multisystemic failure The inflammatory reaction of syndrome, the dermatitis nephrotic syndrome of pig, Hypertrophic necrotizing pneumonia, breeding difficulty and heart and multisystem.
10. described in any item vaccine compositions are in preparation prevention and/or treatment 3 type of pig circular ring virus according to claim 1~8 Application in the drug of disease caused by mixed infection.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102488895A (en) * 2011-12-30 2012-06-13 重庆大学 Porcine circovirus, porcine parvovirus and porcine reproductive and respiratory syndrome virus triple virus-like particle vaccine and its preparation method
CN102961742A (en) * 2012-01-13 2013-03-13 普莱柯生物工程股份有限公司 Bivalent inactivated vaccine of porcine circovirus type 2 and porcine parvovirus and preparation method thereof
WO2017066772A1 (en) * 2015-10-16 2017-04-20 Kansas State University Research Foundation Porcine circovirus type 3 immunogenic compositions and methods of making and using the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102488895A (en) * 2011-12-30 2012-06-13 重庆大学 Porcine circovirus, porcine parvovirus and porcine reproductive and respiratory syndrome virus triple virus-like particle vaccine and its preparation method
CN102961742A (en) * 2012-01-13 2013-03-13 普莱柯生物工程股份有限公司 Bivalent inactivated vaccine of porcine circovirus type 2 and porcine parvovirus and preparation method thereof
WO2017066772A1 (en) * 2015-10-16 2017-04-20 Kansas State University Research Foundation Porcine circovirus type 3 immunogenic compositions and methods of making and using the same

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