CN109022368A - A kind of porcine circovirus type 2 strain, vaccine composition and its preparation method and application - Google Patents

A kind of porcine circovirus type 2 strain, vaccine composition and its preparation method and application Download PDF

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CN109022368A
CN109022368A CN201710433037.7A CN201710433037A CN109022368A CN 109022368 A CN109022368 A CN 109022368A CN 201710433037 A CN201710433037 A CN 201710433037A CN 109022368 A CN109022368 A CN 109022368A
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antigen
inactivation
type
plants
virus
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CN109022368B (en
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田克恭
姚亚丽
孙进忠
张许科
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Luoyang Huizhong Biotechnology Co Ltd
Pulaike Biological Engineering Co Ltd
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Abstract

The present invention relates to a kind of strain of gene hypotype that porcine circovirus 2 type is new, which has good, wide spectrum immunogenicity.Related disease caused by porcine circovirus type 2 infection can be prevented and/or be treated to its vaccine composition prepared, and can all have good immune protection effectiveness to the strain in different geographical source.

Description

A kind of porcine circovirus type 2 strain, vaccine composition and its preparation method and application
Technical field
The present invention relates to a kind of strains, the vaccine composition of its preparation, preparation method and application, belong to animal virology neck Domain.
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 polyphyly Unite exhaustion syndrome, pneumonia, pigskin inflammation and nephrotic syndrome and breeding difficulty, be mainly shown as breathing, uropoiesis, enteron aisle, lymph, Cardiovascular, nerve, propagating system and skin dysfunction causes great economic damage to global pig-breeding It loses.
There are mainly two types of gene hypotype PCV2a and PCV2b by PCV2, and the two gene group leader is respectively 1767 and 1768 nucleosides Acid only has been reported in Denmark there are also a kind of PCV2c.PCV2a is Major Epidemic strain before 2003, and PCV2b starts later Now and range is expanded rapidly, finds new gene hypotype PCV2d again within 2009.It is clinically, commonly used with PCV2 vaccine, Under immune pressure, the speed of mutation of PCV2 is accelerated.
In recent years, the strain of a kind of new gene hypotype between PCV2b and PCV2d comes into vogue, this viroid It is characterized in having mutation in ORF2 gene or there are the recombinations between different genes subtype gene.Since the new PCV2 gene is sub- The prevalence of type strain, and had differences between its antigen and existing gene hypotype, and existing commercialized vaccine is with PCV2a Or PCV2b is vaccine strain come what is prepared, cannot completely be protected to newfangled strain, therefore be directed to the cri dernier cri Strain prepare new vaccine, it is 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 new porcine circovirus type 2 infection Vaccine composition, the vaccine composition can for porcine circovirus 2 type virus offer be effectively protected, show and significantly exempt from Epidemic disease characteristic.
For this purpose, it is an object of the present invention to provide a kind of porcine circovirus type 2 strain, belong to currently popular new Gene hypotype, with good, broad spectrum activity immunogenicity.
It is another object of the present invention to provide a kind of epidemic diseases prevented and/or treat new porcine circovirus type 2 infection Seedling composition, the vaccine composition include the antigen and medicine of the porcine circovirus type 2 strain of the present invention of immune amount Acceptable carrier on.
It is another object of the present invention to provide a kind of prevention and/or treatment each gene hypotype senses of porcine circovirus 2 type Dye and the vaccine composition of their mixed infections.
It is another object of the present invention to provide a kind of prevention and/or the 2 porcine circovirus in treatment different geographical source The vaccine composition of each gene hypotype infection of type and their mixed infections.
It is a further object to provide a kind of methods for preparing the vaccine composition, comprising: step (1) proliferation The porcine circovirus type 2 strain；Step (2) inactivates the porcine circovirus type 2 strain of step (2) proliferation, and assistant is added Agent, emulsification.
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 2 type related diseases.
Invention advantage:
(1) strain of the present invention has good immunogenicity, is immunized once, body can be stimulated rapidly to generate immune Power, the attack of effective protection strain have good protective effect, can reach good immunoprotection with lower antigenic content Effect, further decreases production cost；
(2) present invention is for the first time using the vaccine composition of the strain preparation of the new gene hypotype of porcine circovirus 2 type, no It is only capable of providing protection for the strain of the new gene hypotype of porcine circovirus 2 type, moreover it is possible to sub- to porcine circovirus 2 type different genes The strain of type individually infects or mixed infection provides prevention and/or treatment；
(3) vaccine of the invention can provide protection completely, tool for the porcine circovirus type 2 strain in different geographical source There is the protective capability of wide spectrum.
Specific embodiment
Hereinafter, embodiments of the present invention will be described.
" porcine circovirus 2 type new gene hypotype " refers to a kind of new PCV2 gene hypotype, exists in ORF2 gene It is mutated or there are the recombination between different genes hypotype, there is the sequence label of PCV2b, but form one in phylogenetic analysis A individual branches, pig, which infects the Clinical signs occurred after the strain of the gene hypotype, to be had: duration high temperature, appetite stimulator, essence The depressed, coat of mind 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.
The present invention relates to HH3 plants of porcine circovirus 2 type (Porcine Circovirus type 2, strain HH3), protect It is hidden in China typical culture collection center, deposit number is CCTCC NO.V201726, and the deposit date is on June 4th, 2017, is protected Hide address: Wuhan, China Wuhan University.
Of the invention HH3 plants of porcine circovirus 2 type belong to porcine circovirus 2 type new gene hypotype, immunogenicity and existing There is the PCV2 gene hypotype in technology to have differences.The strain has good immunogenicity, and the vaccine of preparation can be effective Excite immanoprotection action.
A length of 1768 nucleotide of new porcine circovirus 2 type genome provided by the invention, the label sequence with PCV2b Column, but an individual branches are formed in phylogenetic analysis, there is mutation in ORF2 gene or there are different genes hypotypes Between recombination, be a kind of new PCV2 gene hypotype.
The invention further relates to a kind of vaccine compositions, wherein the vaccine composition contains the pig of the immune amount HH3 plants of antigens of circovurus type 2 and pharmaceutically acceptable carrier.
Vaccine composition of the invention has good immunogenicity, is immunized once, body can be stimulated rapidly to generate Protection completely is realized in immune efficacy, the attack of effective protection strain.
The porcine circovirus 2 type HH3 plants of antigen can be HH3 plants of porcine circovirus 2 type or the inactivation of its culture is anti- Former, attenuation totivirus antigen.
" 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 porcine circovirus 2 type immunogenicity.The pharmaceutical composition can lure Hair, stimulation or enhancing pig are directed to the immune response of porcine circovirus 2 type.
" 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 having weakened with virulence but still the disease that can replicate in host or on cell Poison.Term used herein " attenuation " is for referring in a manner of making cause of disease lose pathogenic but holding immunogenicity to gene It is mutated and manually to reduce pathogen toxicity.In general, real by UV radiation, chemical treatment or external continuous high-order squamous subculture Now it is attenuated.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 porcine circovirus 2 type HH3 plants of antigen is porcine circovirus 2 type HH3 The totivirus inactivation antigen of strain or its culture.
As one embodiment of the present invention, the porcine circovirus 2 type HH3 plants of antigen is porcine circovirus 2 type HH3 The cracking type antigen of strain or its culture.
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, the porcine circovirus 2 type HH3 plants of cultures are the culture of >=1 generation Object.
As a kind of preferred embodiment of the invention, the porcine circovirus 2 type HH3 plants of cultures are >=5 generations Culture.
As a kind of more preferable embodiment of the invention, the porcine circovirus 2 type HH3 plants of cultures were 5~48 generations Secondary culture.
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 described porcine circovirus 2 type HH3 plants or its culture Antigenic content be inactivation before >=105.0TCID50/ml。
Porcine circovirus 2 type HH3 plants of inactivated whole virus antigen has good immunogene in vaccine composition of the invention Property, body can be stimulated rapidly to generate immunity, inactivate preceding 10 when using5.0TCID50When the content of/ml, can also it reach good Immanoprotection action, realize 100% protective rate.
As a kind of preferred embodiment of the invention, the inactivation of described porcine circovirus 2 type HH3 plants 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 described porcine circovirus 2 type HH3 plants 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 described porcine circovirus 2 type HH3 plants or its culture contains Amount range is further selected from 105.0~106.0TCID50/ ml or 106.0~107.0TCID50/ml。
As one embodiment of the present invention, the pharmaceutically acceptable carrier is adjuvant, and the 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, water Packet water-in-oil emulsion.
As a kind of preferred embodiment of the invention, the adjuvant is MontanideTMGel.
As one embodiment of the present invention, the adjuvant content is 5~20V/V%.
As a kind of preferred embodiment of the invention, the adjuvant content is 10V/V%.
Vaccine composition of the invention, which can be used, to be deployed with technology, and preferably pharmaceutically acceptable carrier is adjusted together Match.For example, oil can help to stablize composite, and additionally serve as vaccine adjuvant.Oil adjuvant, can also be with either natural source It is by artificial synthesized acquisition.
Term " adjuvant " refers to the substance for being added to and increasing the immunogenicity of composition in composition of the invention.It is known Adjuvant includes, but are not limited to: (1) aluminium hydroxide, saponin(e (Saponine) (such as QuilA), Avridine, DDA, (2) propylene The polymer of the polymer of acid or methacrylic acid, maleic anhydride and alkenyl derivative, (3) vaccine can be with water packet Oil, Water-In-Oil or W/O/W emulsion form are made, or (4) MontanideTMGel.
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 MontanideTMGel.
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.
The invention further relates to a kind of methods for preparing the vaccine composition, wherein the described method includes: step (1) increases Grow culture described porcine circovirus 2 type HH3 plants or its culture；And step (2) inactivates the pig of step (1) Multiplying culture HH3 plants of circovurus type 2 or its culture, HH3 plants of porcine circovirus 2 type of the step (3) after the inactivation or its culture Middle addition adjuvant, emulsification.
Vaccine composition of the invention further includes other pathogens or antigen composition using to prepare resistance The combined vaccine or combination vaccine of the disease of porcine circovirus type 2 infection.
Term " combined vaccine " refers to that porcine circovirus 2 type 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 porcine circovirus 2 type and bacterial antigens preparation of the invention.For example, Porcine circovirus 2 type 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.
As one embodiment of the present invention, the vaccine composition, which also contains, to be made of the following antigen of immune amount One of group is a variety of: CSFV antigen, porcine pseudorabies virus antigen, swine flue antigen, pig breeding and breathing Syndrome virus antigen, PPV Antigen Using, Latex agglutination test antigen, haemophilus parasuis antigen, Streptococcus suis are anti- Original, pig bordetella bacilli antigen, porcine contagious pleuropneumonia antigen, pig pasteurella multocida antigen, Salmonella choleraesuls are anti- Former, mycoplasma hyopneumoniae antigen, mycoplasma hyorhinis antigens.
Porcine circovirus 2 type antigen 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 contains 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 contains 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, in vaccine composition of the present invention, the swine fever virus subtracts Malicious antigen is swine fever virus Lapinized strain, porcine pseudorabies virus attenuation antigen is HN1201-R plants of low virulent strains, described Porcine pseudorabies virus inactivation antigen is HN1201 plants of inactivated whole virus antigens, porcine reproductive and respiratory syndrome virus attenuation Antigen is JXA1-R plants of attenuated strains, the pig parvoviral inactivation antigen is HN-2011 plants of inactivated whole virus antigens, the secondary pig Haemophilus inactivation antigen is that JS plants of 4 type of haemophilus parasuis serum or the full bacterium antigen of ZJ plants of 5 types inactivations, the pig pneumonia branch are former Body inactivation antigen is the HN0613 plants of full bacterium antigens of inactivation.
As one embodiment of the present invention, in vaccine composition of the present invention, the porcine circovirus 2 type HH3 The inactivated whole virus antigenic content of strain or its culture is inactivation preceding 105.0~107.0TCID50/ ml, the swine fever virus attenuation are anti- Former 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 antigen content are inactivation preceding 106.0~107.0TCID50/ ml, the pig breeding and breathing It is 10 that syndrome virus, which is attenuated antigenic content,5.0~107.0TCID50/ ml, the pig parvoviral inactivation antigen content are before inactivating 106.0~108.0TCID50/ ml, the haemophilus parasuis inactivation antigen content are inactivation preceding 108.0~1010.0CFU/ml, it is described Mycoplasma hyopneumoniae inactivation antigen content is inactivation preceding 108.0~1010.0CCU/ml。
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 a kind of preferred embodiment of the invention, in vaccine composition of the present invention, the porcine circovirus 2 type The inactivated whole virus antigenic content of HH3 plants or its culture is inactivation preceding 105.0~107.0TCID50/ ml, the swine fever virus subtract Malicious antigenic content is 105.0TCID50/ ml, the porcine pseudorabies virus attenuation antigenic content is 106.0TCID50/ ml, the pig Pseudorabies virus inactivation antigen content is inactivation preceding 106.0TCID50/ ml, the porcine reproductive and respiratory syndrome virus attenuation are anti- Former content is 106.0TCID50/ ml, the pig parvoviral inactivation antigen content are inactivation preceding 107.0TCID50/ ml, the pair pig Haemophilus inactivation antigen content is inactivation preceding 109.0CFU/ml, the mycoplasma hyopneumoniae inactivation antigen content are before inactivating 109.0CCU/ml。
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 caused by preparation prevention and/or treatment porcine circovirus type 2 infection Application in the drug of related disease, wherein related disease caused by the porcine circovirus type 2 infection is by PCV2 difference base Because hypotype individually infect or their mixed infections caused by related disease.
The porcine circovirus 2 type new gene hypotype antigen of vaccine composition of the invention can not only be directed to 2 porcine circovirus Type new gene hypotype strain carries out immunoprotection, moreover it is possible to carry out immunoprotection for PCV2 others gene hypotype strain, also can Mixed infection between effective protection difference PCV2 gene hypotype.
The porcine circovirus 2 type antigen of vaccine composition of the invention has antigen broad spectrum activity, can be directed to different geographical The porcine circovirus type 2 strain in source provides protection completely.
As one embodiment of the present invention, related disease caused by the porcine circovirus type 2 infection is served as reasons PCV2a, PCV2b, PCV2d, PCV2new gene hypotype individually infect or their mixed infections caused by related disease.
As one embodiment of the present invention, the porcine circovirus 2 type related disease includes postweaning multisystemic Exhaustion syndrome, dermatitis nephrotic syndrome, breeding difficulty and the heart of pig and the inflammatory reaction of multisystem.
Term used herein " porcine circovirus 2 type related disease " is for referring to as caused by porcine circovirus type 2 infection Disease.Nonexhaustive includes, pmws, pig dermatitis nephrotic syndrome, breeding difficulty and heart With the inflammatory reaction of multisystem, but not limited to this.
Term " prevention and/or treatment " refers to when being related to porcine circovirus type 2 infection inhibits answering for porcine circovirus 2 type System, the propagation for inhibiting porcine circovirus 2 type prevent porcine circovirus 2 type from settling down in its host, and mitigate pig annulus The disease of viral 2 types infection or the symptom of illness.If viral loads reduction, illness mitigation and/or food ration and/or growth increase Add, 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 porcine circovirus 2 type of embodiment is identified
1, pathological material of disease source
, sporadicly there is the phenomenon that Sow abortion, the mummification of fetus increase in the pig farm of an immune commercialization PCV2 vaccine at home. Impacted sow shows anorexia, related to PCV2 containing the mummified fetus of different gestational age in the nest of miscarriage The symptom of miscarriage is consistent.By immunohistochemistry and quantitative PCR detection, PCV2 is positive.After immune PCV2 commercialized vaccine still It detects that PCV2, cause value must go into seriously from morbidity porcine tissue, further to investigate thoroughly reason, chooses each tissue pathological material of disease and carry out cause of disease 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, in 37 DEG C of culture 1h, the DMEM culture solution for containing 2% calf serum is added in replacement, is cultivated 5 in 37 DEG C. The culture solution containing virus is harvested, culture solution is after 2 freeze thawing, harvest virus.
3, PCR and sequencing analysis identification virus
The viral cultures for taking above step to harvest extract the nucleic acid of viral sample with nucleic acid extraction kit, use PCV2 specific primer carries out PCR amplification identification, the results show that PCR amplification goes out 1.7kb purpose band.
PCR product send sequencing company to carry out nucleotide sequencing, and sequencing results carry out phylogenetic analysis.As a result The Strain whole genome sequence is shown with it has been reported that the homology of other PCV2 crossed is less than 96%, amino acid sequence is few In 94%, the strain is further analyzed between PCV2b and PCV2d, there is mutation in ORF2 gene or there are different genes The recombination of hypotype belongs to a new PCV2 gene hypotype in phylogenetic analysis.
The screening of 2 porcine circovirus type 2 vaccines strain of embodiment
Specific primer is designed according to the above-mentioned porcine circovirus 2 type that is separated to, by quantitative PCR analysis from all parts of the country 42 parts of doubtful PCV2 positive samples of the pig farm acquisition sporadicly fallen ill after immune commercialization PCV2 vaccine, screening isolate 16 plants PCV2 virus, between this 16 plants, genome nucleotide sequence homology is up to 99.6~100%, Cap protein amino acid sequence Column homology is up to 99.5~100%, and further analysis belongs to the same gene hypotype.It tests and is immunized through this pathogenicity Originality test, finishing screen select 1 plant of pathogenic PCV2 Strain strong, immunogenicity is good, broad spectrum activity is strong.The pig circular ring virus 2 type viral nomenclatures are porcine circovirus 2 type HH3 plants, submit preservation.
Embodiment HH3 plants of pathogenicities of 3 porcine circovirus 2 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 PCV2HH3 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.
HH3 plants of 1 porcine circovirus 2 type of table 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 porcine circovirus 2 type virus again, and blank control group is no different Often, show that porcine circovirus 2 type HH3 plants of inoculations piglet of the present invention can lead to piglet morbidity, clinical table disease is typical pig annulus Viral infection symptoms.
The preparation of 4 HH3 plants of antigen of porcine circovirus 2 type of embodiment
The culture for HH3 plants of the porcine circovirus 2 type 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, sets It is cultivated in 37 DEG C.Daily observation 1~2 time, cell well-grown harvest cell culture after cultivating 4~7 in 36~37 DEG C, After cell culture freeze thawing 2-3 times of harvest, virus liquid is harvested, measures viral titer in virus liquid.By the hollow fibre of virus liquid The filtering of (0.5 μm~2 μm) filter column is tieed up, to remove cell fragment, 0.1%~0.2% formalin is added and is inactivated at 37 DEG C For 24 hours, the viral antigen after inactivation completely is to prepare vaccine.
The preparation of 5 HH3 plants of inactivated vaccines of porcine circovirus 2 type of embodiment
Water-soluble adjuvant Gel adjuvant is added slowly to HH3 plants of inactivation antigens of porcine circovirus 2 type prepared by embodiment 4 In (France match BIC Corp), with revolving speed it is constantly 800rpm mulser stirring 12min during adding, mixes.Vaccine is specific Formula is shown in Table 2.
HH3 plants of inactivated vaccine formulas of 2 porcine circovirus 2 type of table and content
Embodiment HH3 plants of inactivated vaccine Study On Immunogenicities of 6 porcine circovirus 2 type
28~30 ages in days are randomly divided into 5 through ELISA detection PCV2, PCV3 antigen, sodium selenite 25 of negative antibody 5/group, HH3 plants of inactivated vaccines of porcine circovirus 2 type prepared by embodiment 5 are immunized in group.3rd~6 group of difference immune vaccine 1~ 4, the 7th group is not immunized as a control group.Each 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 HH3 plants of pig circular ring virus 105.0TCID50/ head is observed continuously respectively after attacking poison Piglet is determined that concrete outcome is shown in Table 3 according to each piglet clinical symptoms, pathological change and viral diagnosis result.
HH3 plants of inactivated vaccine Study On Immunogenicity results of 3 porcine circovirus 2 type of table
The results show that 100% (5/5) can be provided after piglet is immunized in HH3 plants of inactivated vaccines of porcine circovirus 2 type for piglet Protection, and attack after poison control piglet attacks poison and all fall ill.Show porcine circovirus 2 type HH3 plants of inactivated vaccines provided by the invention With good Vaccine effectiveness.
The HH3 plants of inactivated vaccine immunogenicity comparative tests of 7 porcine circovirus 2 type of embodiment
28~30 ages in days are randomly divided into through ELISA detection PCV2, PCV3 antigen, sodium selenite 130 of negative antibody 13 groups, 10/group, HH3 plants of inactivated vaccines 1 of porcine circovirus 2 type prepared by the 8th~10 group of immune embodiment 5, the 11st~13 group (PCV2a plant, content is >=10 before inactivating by immune commercialized vaccine D15.0TCID50/ ml), the 14th~16 group of immune commercialized vaccine D2 (PCV2b plants, content be inactivation before >=105.0TCID50/ ml), the 17th~19 group is not immune as attacking malicious control group, and the 20th group It is not immune to be used as blank control group.8th~10 group of immune group vaccinates 2ml/ head, and 1 time immune；11st~16 group of immune group note Vaccine 2ml/ head is penetrated, 2 times immune；17th group of control group is inoculated with DMEM culture medium 2ml/ head, 1 inoculation；18th~20 group of control Group inoculation DMEM culture medium 2ml/ head, 2 inoculations.It is immunized and carries out within 28th attacking poison afterwards, the 8th group, the 11st group, the 14th group, the 17th group Poison is attacked with HH3 plants of porcine circovirus 2 type；9th group, the 12nd group, the 15th group, the 18th group with the pig circular ring virus 2 separated from Jiangxi, China JX02 plants of velogen strains of malicious 2a type attack poison；10th group, the 13rd group, the 16th group, the 19th group with the pig circular ring virus 2 separated from Chinese Shanghai SH03 plants of velogen strains of malicious 2b type attack poison；Attacking toxic dose is 105.0TCID50Each piglet is observed continuously after attacking poison, according to each piglet in/head Clinical symptoms, pathological change and viral diagnosis are determined that concrete outcome is shown in Table 4.
4 porcine circovirus 2 type HH3 inactivated vaccine immunogenicity comparative test result of table
The results show that the 17th~19 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 porcine circovirus 2 type；
8th~10 group, 12~13 groups, 15~16 groups of each immune groups attack clinical symptoms without exception after poison, each organizer of dissect Official is also without exception, carries out PCR detection by each organs and tissues, shows PCV2 feminine gender；11st group 4, the 14th group 3 different journeys There is body temperature and increases 40.5 DEG C or more in degree, continues 3~5 days, and appetite stimulator, spirit are depressed, coat is thick disorderly, the thin and speed of growth The clinical symptoms such as slow down, the pathological change that different degrees of pulmonary consolidation occurs in dissect, lymph enlargement, kidney have necrosis to put, by each dirty Device tissue carries out PCR detection, can be separated to porcine circovirus 2 type virus again；
20th group of blank control group is without exception.Show that porcine circovirus 2 type HH3 plants of inactivated vaccines provided by the invention are immune Once, effective, complete immunoprotection can be also provided for PCV2a, PCV2b to pig, and from each organs and tissues The PCV2 strain for attacking poison cannot be detected；And the either PCV2a vaccine or PCV2b vaccine being commercialized can only be provided for existing There is the complete protection of gene hypotype PCV2a, PCV2b Strain, cannot provide popular for the new porcine circovirus 2 type of the present invention The complete protection of strain.
Vaccine composition of the invention has good immunogenicity, and the PCV2 of different genes hypotype can be provided Full guard.
The HH3 plants of inactivated vaccine broad spectrum activity protection tests of 8 porcine circovirus 2 type of embodiment
28~30 ages in days are randomly divided into 10 through ELISA detection PCV2, PCV3 antigen, sodium selenite 50 of negative antibody Group, 5/group, HH3 plants of inactivated vaccines 1 of porcine circovirus 2 type prepared by the 21st~25 group of immune embodiment 5, the 26th~30 group is not Immune be used as attacks malicious control group.Each immune group vaccinates 2ml/ head, attacks malicious control group inoculation DMEM culture medium 2ml/ head.It is immune It carries out within 28th attacking poison afterwards, the 21st group, the 26th group is used the 2 porcine circovirus a gene hypotype HN06 separated from Henan, China recently Strain velogen strain attacks poison；22nd group, the 27th group is used JS04 plants of the 2 porcine circovirus b gene hypotype separated recently from Jiangsu Province, China Velogen strain attacks poison；23rd group, the 28th group is used JL13 plants of the 2 porcine circovirus d gene hypotype separated recently from Chinese Jilin Province by force Strain attacks poison；24th group, the 29th group is used CQ14 plants of the 2 porcine circovirus new gene hypotype separated recently from Chinese Chongqing City by force Strain attacks poison；25th group, the 30th group is used GD15 plants of the 2 porcine circovirus new gene hypotype separated recently from Guangdong province, China by force Strain attacks poison；Attacking toxic dose is 105.0TCID50Each piglet is observed continuously after attacking poison in/head, according to each piglet clinical symptoms, pathology Variation and viral diagnosis are determined that concrete outcome is shown in Table 5.
HH3 plants of inactivated vaccine broad spectrum activity protection test results of 5 porcine circovirus 2 type of table
The results show that the 26th~30 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 porcine circovirus 2 type virus；And the 21st~25 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 PCV2 feminine gender.
Show that porcine circovirus 2 type HH3 plants of inactivated vaccines provided by the invention can be directed to different geographical source not to pig The porcine circovirus 2 type of homogenic hypotype attacks poison and provides effective, complete immunoprotection, and cannot detect from each organs and tissues Attack the PCV2 of poison.Vaccine composition of the invention has the immunogenicity of wide spectrum, for different geographical source different genes hypotype Porcine circovirus 2 type complete protect can be provided.
The preparation of 9 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 cell count is carried out after dispersion, and 3% calf is added in inoculating cell culture bottle The DMEM cell culture fluid of serum, at the same according to M.O.I.=0.1 connect toxic dose be added hog cholera seed culture of viruses poison (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 culture three days, after receiving poison The cell maintenance medium containing 1.5% calf serum is added, it is later primary every 2 days receipts poison, it can continuously receive poison 5 times.After finally receiving poison The antigen that each batch is received poison is mixed and is placed in -20 DEG C of storages.
The preparation of 10 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 rotating and culturings is placed in, when lesion reaches When 80%, toxic cell culture fluid is harvested, cell culture fluid receives poison after 2 freeze thawing, measures malicious valence, sets cryo-conservation.
The preparation of 11 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%, the cell culture fluid of harvest after 2 freeze thawing, Poison is received, malicious valence is measured.10% (v/v) formalin is added into virus liquid makes final concentration of 0.2% (v/v) of formaldehyde, in 37 It DEG C inactivation 18 hours, stirs 1 time within every 4 hours, stirs 10min every time, it is spare after inactivating completely.
The preparation of 12 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 13 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, is placed in 37 DEG C of rotating and culturings, when lesion reaches 80%, receives Toxic cell culture fluid is obtained, for the cell culture fluid of harvest 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 14 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 company 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 first order seed trainings of JS plants of 4 type of haemophilus parasuis serum, 5 type of above-mentioned preparation are taken Object is supported, is added in TSB/NAD fluid nutrient medium by 1% amount, is cultivated 12~16 hours in 37 DEG C, is used as two after being examined purely Grade 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, by ZJ plants of JS plants of 4 type of haemophilus parasuis serum of above-mentioned preparation, 5 type second level kinds Daughter bacteria liquid is added in culture medium by 1% amount and is cultivated respectively, and mixing is placed on 37 DEG C and cultivates 16~18 hours, when the concentration of bacterium solution OD600, which reaches, stops culture when 2.5 or more, DO value is begun to ramp up, pH value is reduced to 6.5 or less.
After culture carries out count plate, (Yantai City is in pairs for the formalin of 0.2% (V/V) addition 37% in an amount Chemical 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 15 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, in 37 DEG C of 3~7d of shaken cultivation, training is harvested when pH value is down to 6.8 by 7.5 Object is supported, is purely examined, as first order seed；
The breeding of secondary seed: it takes the first order seed of above-mentioned preparation to be inoculated in fluid nutrient medium by 5% inoculum concentration, is placed in 37 DEG C 3~7d of shaken cultivation, 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 in shaken cultivation 3~7 days at 37 DEG C, to PH value harvests bacterium solution when being down to 6.8 by 7.5.
It takes and examines qualified mycoplasma hyopneumoniae bacterium solution, final concentration of 0.2% first is slowly added to by bacterium solution volume total amount Aldehyde solution (v/v) is placed in 37 DEG C of inactivations, primary every 3~4h stirring therebetween, takes out afterwards for 24 hours, spare after inactivating completely.
The preparation of the 16 strain vaccine composition of HH3 containing porcine circovirus 2 type of embodiment
The porcine pseudorabies virus that swine fever virus prepared by embodiment 9 is attenuated antigen, prepared by embodiment 10 is attenuated respectively Heat resisting protective is added in porcine reproductive and respiratory syndrome virus attenuation antigen prepared by antigen, embodiment 12, and (2wt% gelatin is water-soluble Liquid and 15wt% lactose aqueous solution are with the preparation of 1:1 (v/v) ratio) to be mixed well after the mixing of 1:1 (v/v) ratio, quantitative separating, Rapidly carry out vacuum freezedrying, as swine fever virus live vaccine part, porcine pseudorabies virus live vaccine part, pig breeding with Breath syndrome virus live vaccine part.
Water-soluble adjuvant Gel adjuvant is added slowly to HH3 plants of inactivation antigens of porcine circovirus 2 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 2 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 6 It is shown.
The 6 strain vaccine composition components of HH3 containing porcine circovirus 2 type of table match (live seedling part)
The pseudorabies of HH3 plants of inactivation antigens of porcine circovirus 2 type, the preparation of embodiment 11 that respectively prepared by Example 4 The haemophilus parasuis of pig parvoviral inactivation antigen, the preparation of embodiment 14 prepared by sick inactivation of virus antigen, embodiment 13 goes out Mycoplasma hyopneumoniae inactivation antigen prepared by active antigen, embodiment 15, by the antigen that five kinds of antigens are final according to composition in table 7 Content is prepared, and is added in water-soluble adjuvant Gel adjuvant (match BIC Corp of France), is constantly with revolving speed during adding 800rpm mulser stirs 12min, mixes.The specific formula and content of vaccine composition are shown in Table 7.
The 7 strain vaccine composition components of HH3 containing porcine circovirus 2 type of table match (inactivated vaccine part)
The vaccine composition Study On Immunogenicity of 17 HH3 containing porcine circovirus 2 type plants of antigen of embodiment
1, porcine circovirus 2 type partial immunity originality 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 16 is immunized in group.31st~38 group of difference immune vaccine 5~12, the 39th group unavoidably Epidemic disease is used as and attacks malicious control group.Each immune group vaccinates 2ml/ head, and control group is inoculated with DMEM culture medium 2ml/ head.28 days after immune It carries out attacking poison, attacking toxic dose is HH3 plants of pig circular ring virus 105.0TCID50Each piglet is observed continuously after attacking poison, according to each son in/head Pig clinical symptoms, pathological change and viral diagnosis result are determined that concrete outcome is shown in Table 8.
The vaccine composition PCV2 partial immunity originality test result of 8 HH3 containing porcine circovirus 2 type plants of antigens of table
The results show that pig can be directed to after a piglet is immunized in the vaccine composition of HH3 containing porcine circovirus 2 type plants of antigen Circovurus type 2 infection provides 100% (5/5) protection for piglet, and attacks after malicious control group piglet attacks poison and all fall ill.Show this Inventing porcine circovirus 2 type antigen part in the strain vaccine composition of HH3 containing porcine circovirus 2 type provided has protection well Effect can provide pig 100% protection, and viral diagnosis is feminine gender.HH3 plants of porcine circovirus 2 type of the invention with it is more It does not interfere between kind viral antigen, bacterial antigens, is applied after vaccine composition can be collectively constituted.
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 5 prepared by the 40th group of immune embodiment 16, the 41st group of not immune conduct.By every part 2ml vaccine 5 dilutes 3000 times, and intramuscular injection, every 1ml attacks malicious control group inoculation DMEM culture medium 1ml/ head.10~14 days Afterwards, malicious control group 4, blood poison 1.0ml/ (10, injection swine fever crossdrift system are attacked together with condition is identical5MLD), observe 16.Knot Fruit is shown in Table 9.
The vaccine composition CSFV partial immunity originality test result of 9 HH3 containing porcine circovirus 2 type plants of antigens of table
Group Immune head part Protective rate Clinical symptoms
40 1/3000 100% (4/4) No body temperature reaction
41 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 is immunized Afterwards, then detected, and the present embodiment the results show that the vaccine composition 1/3000 of HH3 containing porcine circovirus 2 type plants of antigen After head part (3000 times of dilution) immune piglet, 100% (4/4) protection can be provided for piglet for swine fever virus infection, and do not have Body temperature reaction, and attack poison control piglet and attack all morbidity death after poison.Show HH3 containing porcine circovirus 2 type provided by the invention CSFV antigen part in the vaccine composition of strain antigen 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 6 prepared by the 42nd group of immune embodiment 16, vaccine 8 prepared by the 43rd group of immune embodiment 16, the 44th 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 10.
The vaccine composition PRV partial immunity originality test result of 10 HH3 containing porcine circovirus 2 type plants of antigens of table
The results show that after piglet is immunized in the vaccine composition of HH3 containing porcine circovirus 2 type plants of antigen, it can blocking virus sense It contaminates (clinical symptoms occur), can be infected 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.Show the vaccine composition of HH3 containing porcine circovirus 2 type plants of antigen provided by the invention In 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 7 prepared by the 45th group of immune embodiment 16, the 46th 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 11.
The vaccine composition PRRSV partial immunity originality test result of 11 HH3 containing porcine circovirus 2 type plants of antigens of table
The results show that can be bred for pig after piglet is immunized in the vaccine composition of HH3 containing porcine circovirus 2 type plants of antigen With breath syndrome virus infection be that piglet provides 100% (5/5) protection, and attack malicious control group piglet attack after poison all fall ill and Dead 3.Show pig breeding and the breathing in the vaccine composition of HH3 containing porcine circovirus 2 type plants of antigen provided by the invention Syndrome virus 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 9 prepared by the 47th group of immune embodiment 16, the 48th 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, surveys together with blank control group Determine antibody, blank control group should be negative (HI antibody titer≤1:8), and immune group at least 4 antibody response, HI antibody effect occur Valence >=1:64.Concrete outcome is shown in Table 12.
The vaccine composition PPV partial immunity originality test result of 12 HH3 containing porcine circovirus 2 type plants of antigens of table
The results show that the HI antibody of PPV is equal after piglet is immunized in the vaccine composition of HH3 containing porcine circovirus 2 type plants of antigen 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 2 HH3 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 10 of the 49th group, the 50th group immune preparation of embodiment 16, the 51st group, the 52nd group is immunized implementation Vaccine 12 prepared by example 16, the 53rd group, the 54th group is not immunized conduct and attacks malicious control group.Immune group vaccinates 2ml/ head, attacks poison Control group is inoculated with DMEM culture medium 2ml/ head.35 days after immune, the 49th group of immune group, the 51st group are taken, attacks the 53rd 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 50th group of immune group, 52 groups, attack the 54th 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 13。
The vaccine composition HPs partial immunity originality test result of 13 HH3 containing porcine circovirus 2 type plants of antigens of table
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 HH3 containing porcine circovirus 2 type plants of antigen, it can be thermophilic for secondary pig 4 type of blood 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.Table Haemophilus parasuis antigen part tool in the vaccine composition of bright HH3 containing porcine circovirus 2 type provided by the invention plants of antigen There is 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 11 prepared by the 55th group of immune embodiment 16, the 56th group is immunized vaccine prepared by embodiment 16 Malicious control group is attacked in 12, the 57th 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 55th group of immune group, the 56th group are taken, attacks the 57th 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 14.
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 14 HH3 containing porcine circovirus 2 type plants of antigens of table
Group Piglet number Average tuberculosis varying index ± standard deviation Pneumonia lesion slip
55 5 1.8±1.14b 84.6%
56 5 2.0±0.76b 85.4%
57 5 11.7±3.94a /
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 strain vaccine composition of HH3 containing porcine circovirus 2 type, for mycoplasma hyopneumoniae sense 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 this hair Mycoplasma hyopneumoniae antigen part in the vaccine composition of the strain antigen of HH3 containing porcine circovirus 2 type of bright offer has fine Vaccine effectiveness.
To sum up, 4 type bacterium of PCV2, CSFV, PRV, PRRSV, PPV, HPs, HPs 5 are directed to from the above different vaccine compositions Type bacterium, Mhp Study On Immunogenicity result from the point of view of, each vaccine composition can reach good protecting effect, show the present invention Different antigenic components has good Vaccine effectiveness in the vaccine composition of the strain antigen of HH3 containing porcine circovirus 2 type provided. The inactivation antigen of HH3 plants of antigen of porcine circovirus 2 type of the invention can collectively constitute immune combination with Different Kinds of Pathogens antigenic component Object is co-administered.
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. HH3 plants of porcine circovirus 2 type (Porcine Circovirus type 2, strain HH3), is preserved in Chinese Typical Representative Culture collection, deposit number are CCTCC NO.V201726, and the deposit date is on June 4th, 2017, preservation address: China Wuhan Wuhan University.
2. a kind of vaccine composition, wherein the vaccine composition contains the pig circular ring virus described in claim 1 of immune amount 2 HH3 plants of type antigens and pharmaceutically acceptable carrier.
3. vaccine composition according to claim 2, wherein the porcine circovirus 2 type HH3 plants of antigen is pig circular ring virus 2 The totivirus inactivation antigen of HH3 plants of malicious 2 type or its culture.
4. vaccine composition according to claim 3, wherein the porcine circovirus 2 type HH3 plants of cultures were >=1 generation Secondary culture；Preferably, the porcine circovirus 2 type HH3 plants of cultures are the culture of >=5 generations；It is highly preferred that described HH3 plants of cultures of porcine circovirus 2 type are the culture of 5~48 generations.
5. vaccine composition according to claim 3, wherein described porcine circovirus 2 type HH3 plants or its culture go out Totivirus antigenic content living be inactivation before >=105.0TCID50/ml；Preferably, described porcine circovirus 2 type HH3 plants or its culture The inactivated whole virus antigenic content of object is inactivation preceding 105.0~107.0TCID50/ml；It is highly preferred that the porcine circovirus 2 type The inactivated whole virus antigenic content of HH3 plants or its culture is inactivation preceding 106.0TCID50/ml。
6. vaccine composition according to claim 2, wherein the pharmaceutically acceptable carrier is adjuvant, the assistant Agent 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, water Packet water-in-oil emulsion；Preferably, the adjuvant is MontanideTMGel；
The adjuvant content is 5~20V/V%；It is highly preferred that the adjuvant content is 10V/V%.
7. vaccine composition according to claim 2, wherein the vaccine composition also contains by the following anti-of immune amount One of group of original composition is a variety of: CSFV antigen, porcine pseudorabies virus antigen, swine flue antigen, pig are numerous It grows and breath syndrome virus antigen, PPV Antigen Using, Latex agglutination test antigen, haemophilus parasuis antigen, pig Streptococcal antigens, pig bordetella bacilli antigen, porcine contagious pleuropneumonia antigen, pig pasteurella multocida antigen, hog cholera are husky Door Salmonella antigen, mycoplasma hyopneumoniae antigen, mycoplasma hyorhinis antigens；
Preferably, the vaccine composition also contains one of group being made of the following antigen of immune amount or a variety of: swine fever Virus attenuation antigen, porcine pseudorabies virus attenuation antigen, porcine reproductive and respiratory syndrome virus are attenuated antigen, pig parvoviral Inactivation antigen, haemophilus parasuis inactivation antigen, mycoplasma hyopneumoniae inactivation antigen；
Preferably, the vaccine composition also contains 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；
It is highly preferred that the swine fever virus attenuation antigen is swine fever virus Lapinized strain, porcine pseudorabies virus attenuation Antigen is HN1201-R plants of attenuated strains, the porcine pseudorabies virus inactivation antigen is HN1201 plants of inactivated whole virus antigens, institute State porcine reproductive and respiratory syndrome virus attenuation 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 are JS plants of 4 type of haemophilus parasuis serum or 5 type ZJ Strain inactivates full bacterium antigen, the mycoplasma hyopneumoniae inactivation antigen is the HN0613 plants of full bacterium antigens of inactivation.
8. vaccine composition according to claim 6, wherein described porcine circovirus 2 type HH3 plants or its culture go out Totivirus antigenic content living 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 pseudorabies Sick inactivation of virus antigenic content is inactivation preceding 106.0~107.0TCID50/ ml, the porcine reproductive and respiratory syndrome virus attenuation are anti- Former 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 antigen content are inactivation preceding 108.0~1010.0CFU/ml, the pig pneumonia Mycoplasma inactivation antigen content is inactivation preceding 108.0~1010.0CCU/ml；
Preferably, the inactivated whole virus antigenic content of described porcine circovirus 2 type HH3 plants or its culture is inactivation preceding 105.0~ 107.0TCID50/ ml, the swine fever virus attenuation antigenic content is 105.0TCID50/ ml, the porcine pseudorabies virus attenuation are anti- Former content is 106.0TCID50/ ml, the porcine pseudorabies virus inactivation antigen content are inactivation preceding 106.0TCID50/ ml, it is described It is 10 that porcine reproductive and respiratory syndrome virus, which is attenuated antigenic content,6.0TCID50/ ml, the pig parvoviral inactivation antigen content are Inactivation preceding 107.0TCID50/ ml, the haemophilus parasuis inactivation antigen content are inactivation preceding 109.0CFU/ml, the pig pneumonia Mycoplasma inactivation antigen content is inactivation preceding 109.0CCU/ml。
9. preventing and/or treating porcine circovirus 2 type in preparation according to the described in any item vaccine compositions of claim 2~8 Application in the drug of related disease caused by infecting, wherein related disease caused by the porcine circovirus type 2 infection is served as reasons PCV2 different genes hypotype individually infect or their mixed infections caused by related disease.
10. application according to claim 9, wherein the porcine circovirus 2 type related disease includes weanling pig polyphyly System exhaustion syndrome, dermatitis nephrotic syndrome, breeding difficulty and the heart of pig and the inflammatory reaction of multisystem.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112386685A (en) * 2020-12-31 2021-02-23 北京科牧丰生物制药有限公司 PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccine

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN102988978A (en) * 2011-08-01 2013-03-27 普莱柯生物工程股份有限公司 Vaccine composition containing porcine circovirus type 2 antigen and haemophilus parasuis antigen, as well as preparation method and application thereof
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
CN103127497A (en) * 2011-11-25 2013-06-05 普莱柯生物工程股份有限公司 Bigeminy inactivated vaccine of porcine circovirus type 2 and swine mycoplasma hyopneumoniae and preparation method of bigeminy inactivated vaccine
CN103263666A (en) * 2013-05-24 2013-08-28 北京大北农科技集团股份有限公司动物医学研究中心 Duplex inactivated vaccine of porcine circovirus type 2 and porcine mycoplasma hyopneumoniae and preparation method of duplex inactivated vaccine
CN103301455A (en) * 2013-07-01 2013-09-18 青岛农业大学 Porcine circovirus-haemophilus parasuis bivalent inactivated vaccine and preparation process thereof
CN103409374A (en) * 2013-07-23 2013-11-27 武汉科前动物生物制品有限责任公司 Trigeminy inactivated vaccine for porcine circovirus disease, porcine streptococcus suis disease and porcine haemophilus parasuis disease, preparation method of the vaccine and applications of the vaccine
CN103623403A (en) * 2012-08-27 2014-03-12 普莱柯生物工程股份有限公司 Vaccine composition for resisting swine fever virus and porcine circovirus 2 infection, and preparation and application thereof
CN103667196A (en) * 2012-09-25 2014-03-26 普莱柯生物工程股份有限公司 Vaccine composition containing porcine circovirus-2 antigen and swine flu antigen
CN103908665A (en) * 2013-01-05 2014-07-09 普莱柯生物工程股份有限公司 Vaccine composition, preparation method and application thereof
CN104288762A (en) * 2013-07-19 2015-01-21 普莱柯生物工程股份有限公司 Vaccine composition, and preparation method and application thereof
CN104288760A (en) * 2013-07-18 2015-01-21 普莱柯生物工程股份有限公司 Vaccine composition, and preparation method and application thereof
CN104450559A (en) * 2012-06-29 2015-03-25 普莱柯生物工程股份有限公司 New mycoplasma hyopneumoniae strain and vaccine composite of new mycoplasma hyopneumoniae
CN104862423A (en) * 2015-06-09 2015-08-26 四川农业大学 Visible gene chip and kit for pig pseudorabies virus, porcine parvovirus and porcine circovirus type-II
CN105709220A (en) * 2014-12-03 2016-06-29 普莱柯生物工程股份有限公司 Vaccine composition for porcine circovirus and swine influenza and its preparation method and use
CN105749273A (en) * 2012-01-13 2016-07-13 普莱柯生物工程股份有限公司 Bivalent inactivated vaccine for porcine circovirus type 2 and porcine parvovirus and preparation method thereof

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102988978A (en) * 2011-08-01 2013-03-27 普莱柯生物工程股份有限公司 Vaccine composition containing porcine circovirus type 2 antigen and haemophilus parasuis antigen, as well as preparation method and application thereof
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
CN103127497A (en) * 2011-11-25 2013-06-05 普莱柯生物工程股份有限公司 Bigeminy inactivated vaccine of porcine circovirus type 2 and swine mycoplasma hyopneumoniae and preparation method of bigeminy inactivated vaccine
CN105749273A (en) * 2012-01-13 2016-07-13 普莱柯生物工程股份有限公司 Bivalent inactivated vaccine for porcine circovirus type 2 and porcine parvovirus and preparation method thereof
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
CN104450559A (en) * 2012-06-29 2015-03-25 普莱柯生物工程股份有限公司 New mycoplasma hyopneumoniae strain and vaccine composite of new mycoplasma hyopneumoniae
CN103623403A (en) * 2012-08-27 2014-03-12 普莱柯生物工程股份有限公司 Vaccine composition for resisting swine fever virus and porcine circovirus 2 infection, and preparation and application thereof
CN103667196A (en) * 2012-09-25 2014-03-26 普莱柯生物工程股份有限公司 Vaccine composition containing porcine circovirus-2 antigen and swine flu antigen
CN103908665A (en) * 2013-01-05 2014-07-09 普莱柯生物工程股份有限公司 Vaccine composition, preparation method and application thereof
CN103263666A (en) * 2013-05-24 2013-08-28 北京大北农科技集团股份有限公司动物医学研究中心 Duplex inactivated vaccine of porcine circovirus type 2 and porcine mycoplasma hyopneumoniae and preparation method of duplex inactivated vaccine
CN103301455A (en) * 2013-07-01 2013-09-18 青岛农业大学 Porcine circovirus-haemophilus parasuis bivalent inactivated vaccine and preparation process thereof
CN104288760A (en) * 2013-07-18 2015-01-21 普莱柯生物工程股份有限公司 Vaccine composition, and preparation method and application thereof
CN104288762A (en) * 2013-07-19 2015-01-21 普莱柯生物工程股份有限公司 Vaccine composition, and preparation method and application thereof
CN103409374A (en) * 2013-07-23 2013-11-27 武汉科前动物生物制品有限责任公司 Trigeminy inactivated vaccine for porcine circovirus disease, porcine streptococcus suis disease and porcine haemophilus parasuis disease, preparation method of the vaccine and applications of the vaccine
CN105709220A (en) * 2014-12-03 2016-06-29 普莱柯生物工程股份有限公司 Vaccine composition for porcine circovirus and swine influenza and its preparation method and use
CN104862423A (en) * 2015-06-09 2015-08-26 四川农业大学 Visible gene chip and kit for pig pseudorabies virus, porcine parvovirus and porcine circovirus type-II

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112386685A (en) * 2020-12-31 2021-02-23 北京科牧丰生物制药有限公司 PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis quadruple inactivated vaccine

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