CN109125719A - It is a kind of containing 3 type of pig circular ring virus, the immunogenic composition of porcine circovirus 2 type antigen and its application - Google Patents
It is a kind of containing 3 type of pig circular ring virus, the immunogenic composition of porcine circovirus 2 type antigen and its application Download PDFInfo
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- CN109125719A CN109125719A CN201710464584.1A CN201710464584A CN109125719A CN 109125719 A CN109125719 A CN 109125719A CN 201710464584 A CN201710464584 A CN 201710464584A CN 109125719 A CN109125719 A CN 109125719A
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- circular ring
- porcine circovirus
- ring virus
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N2750/00011—Details
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Abstract
The present invention relates to a kind of immunogenic composition containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen, wherein, which includes the porcine circovirus 2 type new gene hypotype antigen and pharmaceutically acceptable carrier of the 3 type antigen of pig circular ring virus of immune amount, immune amount.The immunogenic composition has good immunogenicity, and primary immunization can protect the mixed infection of pig circular ring virus 3 type, porcine circovirus 2 type completely.
Description
Technical field
The invention belongs to field of veterinary, and in particular to a kind of to exempt from containing pig circular ring virus 3 type, porcine circovirus 2 type antigen
Epidemic disease Immunogenic Compositions and its application.
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.
Clinically, commonly used with PCV2 vaccine, under immune pressure, the speed of mutation of PCV2 is accelerated, Yi Leijie
The strain of new gene hypotype between PCV2b and PCV2d comes into vogue, and this kind of virus characteristic is to exist to dash forward in ORF2 gene
It is recombinated between change or the ORF2 gene through different genes hypotype.Due to the prevalence of the new PCV2 gene hypotype strain,
It is had differences between antigen and existing gene hypotype, and existing commercialized vaccine is using PCV2a or PCV2b as vaccine strain
Come what is prepared, newfangled PCV2 strain cannot completely be protected.
Together in the breeding difficulty case of pig, it is separated to the circovirus that a strain virus genome is 2.0kb, through subsequent
Test confirms, no matter in the homology of nucleotide or amino acid sequence is respectively less than 50% with known circovirus, according to
The standard of the international virology classification committee, same virus should have the genome nucleotide of > 75% in Circovirus
The homology of acid sequence, the homology of amino acid sequence of the Cap protein with > 70% can affirm that this is a kind of new accordingly
Pig circular ring virus (PCV3).
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 has in terms of PNDS and breeding difficulty and PCV2
Similar pathogenic characteristic, albumen homology is very low between PCV2 and PCV3, using PCV2 vaccine cross protection cannot to PCV3 into
Row effectively prevention.
The complex situations of clinically PCV infection, therefore needle is further aggravated in new pig circular ring virus and PCV2 mixed infection
Prepared by new immunogenic composition to the new clinical epidemic situation, 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 treatment pig circular ring virus mixed infection
Immunogenic composition, the immunogenic composition can provide the mixed infection of porcine circovirus 2 type and 3 types effective protect
Shield, shows significant immunological characteristic.
It is an object of the present invention to provide a kind of prevention and/or treat the immune of different pig circular ring virus mixed infections
Immunogenic Compositions, the immunogenic composition include the pig circular ring virus of the 3 type antigen of pig circular ring virus of immune amount, immune amount
2 type antigens and pharmaceutically acceptable carrier.
It is an object of the present invention to provide a kind of prevention and/or the porcine circovirus 2 types for the treatment of different genes hypotype
With the immunogenic composition of 3 type mixed infection of pig circular ring virus.
It is an object of the present invention to provide a kind of prevention and/or the porcine circovirus 2 types in treatment different geographical source
With the immunogenic composition of 3 type mixed infection of pig circular ring virus.
It is an object of the present invention to provide above-mentioned immunogenic compositions in preparation prevention and/or treatment pig circular ring virus 2
Application in the drug of malicious related disease.
The present invention uses new at present 3 type immunogenic protein antigen of pig circular ring virus and porcine circovirus 2 type new for the first time
Gene hypotype strain immunogenic protein antigen, preparation prevention and/or treatment Infection of Porcine circovirus IMMUNOGENIC COMPOSITION
Object, can not only infection to 3 type of porcine circovirus 2 type and pig circular ring virus or mixed infection carry out immunoprotection, moreover it is possible to be directed to
Different genes hypotype porcine circovirus type 2 strain is protected.
Immunogenic composition of the invention can be directed to 3 type strain of pig circular ring virus, the pig circular ring virus 2 in different geographical source
Malicious 2 type strain mixed infections provide protection completely, the protective capability with wide spectrum.
Specific embodiment
Hereinafter, embodiments of the present invention will be described.
" 3 type of pig circular ring virus " is the circovirus of genome 2.0kb, with known circovirus either nucleotide
Or the homology of amino acid sequence is respectively less than 50%, is a kind of new pig circular ring virus, can be with a variety of cause of disease mixed infections
Cause the inflammatory reaction of the dermatitis nephrotic syndrome of pig, Hypertrophic necrotizing pneumonia, breeding difficulty and heart and multisystem.
" porcine circovirus 2 type new gene hypotype " refers to a kind of new PCV2 gene hypotype, exists in ORF2 gene
It is recombinated between mutation or the ORF2 gene through different genes hypotype, the sequence label with PCV2b, but in genetic evolution point
An individual branches are formed in analysis, pig, which infects the Clinical signs occurred after the strain of the gene hypotype, to be had: duration high temperature, food
Be intended to decline, spirit is depressed, coat is thick disorderly, the thin and speed of growth slows down, it is swollen that different degrees of pulmonary consolidation, lymph occurs in dissect
Greatly, kidney has downright bad point.
The present invention relates to a kind of immunogenic composition containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen, wherein
The immunogenic composition includes the porcine circovirus 2 type of the 3 type Cap protein antigen of pig circular ring virus of immune amount, immune amount
New gene hypotype Cap protein antigen and pharmaceutically acceptable carrier；Wherein, the 3 type Cap protein of pig circular ring virus is anti-
It originally was the live vector that 3 type Cap protein of pig circular ring virus or recombination have the 3 type Cap protein gene of pig circular ring virus, the pig circle
2 type new gene hypotype Cap protein antigen of circovirus virus is porcine circovirus 2 type new gene hypotype Cap protein or recombination have it is described
The live vector of carrying Cap gene of porcine circovirus type 2 gene.
Immunogenic composition of the invention can be generated through primary immunization, antigenic component therein for respective cause of disease
Protection completely, the mixed infection of the prevention of protection pig and resistance for pig circular ring virus 3 type and porcine circovirus 2 type.
Immunogenic composition of the invention can be directed to the 2 porcine circovirus of pig circular ring virus 3 type and different genes hypotype
The mixed infection of type generates protection completely.
Immunogenic composition of the invention can protect pig to 3 type of the pig circular ring virus virus in different geographical source and pig
The mixed infection of circovurus type 2 virus is protected completely.
Term " immunogenic composition " refers to containing 3 type of pig circular ring virus, the medicine group of porcine circovirus 2 type immunogenicity
Object is closed, which can induce, stimulates or enhance pig and exempt from for 3 type of pig circular ring virus, porcine circovirus 2 type are viral
Epidemic disease reaction.
Term " immune amount " should be understood as " immune effective dose " that also known as immunoprotection amount or generation immune response is effective
Amount, is the amount of antigen that immune response can be effectively induced in recipient's body, which is enough to prevent or improve sign or the disease of disease
Shape, including unfavorable health effect or its complication.The immune response may be sufficient to diagnostic purpose or other tests, or
It is likely to be suitable for the sign or symptom of prevention disease, including infecting caused unfavorable healthy result as caused by pathogen
Or its complication.Humoral immunity can be induced by cell-mediated immunity or this two.Animal is to immunogenicity group
The immune response for closing object can be for example, by measurement antibody titer, lymphocyte proliferation assays and indirect assessment, or with wild type
The protective immunity directly assessed after strain attack, and should provided by immunogenic composition by monitoring sign or symptom
It can be overall by measuring the clinical symptom such as reduction of health farrowing, the increase of stillborn foetus quantity, the animal subject of such as animal subject
Physiological status and general health and performance are assessed.The immune response may include but be not limited to inducing cell and/or body fluid
Immunity.
3 type Cap protein antigen of pig circular ring virus of the present invention, porcine circovirus 2 type Cap proteantigen can be
The Cap protein subunit antigen of recombinant expression, expression system can be prokaryotic expression system, eukaryotic expression system, can also be with
It is by artificial synthesized antigenic synthetic peptide；Or it can be the live vector that recombination has the pig circular ring virus Cap protein gene.
" subunit antigen " refers to that the protective antigen gene of pathogen is cloned into protokaryon using gene engineering method
Or in eukaryotic expression system, make its high efficient expression and manufactured antigen.A possibility that it causes side reaction than totivirus antigen is small.
" antigenic synthetic peptide " refers to a kind of small peptide for containing only immunologic determinants component, i.e., presses native protein by artificial means
The amino acid sequence of matter synthesizes protectiveness small peptide, and antigen made by adjuvant is added after connecting with carrier.
" live vector " refer to non-pathogenic microorganism by the method for genetic engineering be allowed to carry and express certain antigen or
The gene of antigenic determinant generates immunogenicity, and non-pathogenic microorganism can be bacterium and virus, and virus live vector is frequently as load
The virus of body has vaccinia virus, fowlpox virus, herpes turkey virus, adenovirus, Pseudorabies virus, retrovirus, slow virus；
Bacterial live vector can be Attenuated Salmonella, BCG vaccine, attenuation monocyte Li bacillus, attenuation comma bacillus, attenuation will and congratulate
Salmonella, Lactococcus, bud endosperm acidfast bacilli, Gao Shi streptococcus.
As one embodiment of the present invention, contain 3 type of pig circular ring virus, porcine circovirus 2 type antigen in of the invention
Immunogenic composition in, the 3 type Cap protein of pig circular ring virus be sequence SEQ.ID NO.1 coding albumen, it is described
Porcine circovirus 2 type new gene hypotype Cap protein is the albumen of SEQ.ID NO.2 coding.
Immunogenic protein immunogenicity of the invention is good, and primary immunization just can provide complete protection.
As one embodiment of the present invention, contain 3 type of pig circular ring virus, porcine circovirus 2 type antigen in of the invention
Immunogenic composition in, described 3 its encoding gene of type Cap protein of pig circular ring virus have SEQ.ID NO.1 shown in
Nucleotide sequence or its degenerate sequence, its encoding gene of the carrying Cap gene of porcine circovirus type 2 have SEQ.ID NO.2 institute
The nucleotide sequence shown or its degenerate sequence.
As one embodiment of the present invention, contain 3 type of pig circular ring virus, porcine circovirus 2 type antigen in of the invention
Immunogenic composition in, the 3 type Cap protein content of pig circular ring virus be >=20 μ g/ml；The porcine circovirus 2 type
New gene hypotype Cap protein content is >=20 μ g/ml.
In immunogenic composition of the invention, when antigenic content can also stimulate immune system generation to exempt from down to 20 μ g/ml
Epidemic disease reaction generates protection completely to pig.
As a kind of preferred embodiment of the invention, contain 3 type of pig circular ring virus, porcine circovirus 2 type in of the invention
In the immunogenic composition of antigen, the 3 type Cap protein content of pig circular ring virus is 20 μ of μ g/ml~100 g/ml；It is described
Porcine circovirus 2 type new gene hypotype Cap protein content is 20 μ of μ g/ml~100 g/ml.
As a kind of more preferable embodiment of the invention, contain 3 type of pig circular ring virus, 2 porcine circovirus in of the invention
In the immunogenic composition of type antigen, the 3 type Cap protein antigenic content of pig circular ring virus is 20 μ of μ g/ml~50 g/ml；
The porcine circovirus 2 type new gene hypotype Cap protein content is 20 μ of μ g/ml~50 g/ml.
As a kind of more preferable embodiment of the invention, contain 3 type of pig circular ring virus, 2 porcine circovirus in of the invention
In the immunogenic composition of type antigen, the 3 type Cap protein antigenic content of pig circular ring virus is 30 μ of μ g/ml~50 g/ml；
The porcine circovirus 2 type new gene hypotype Cap protein content is 30 μ of μ g/ml~50 g/ml.
In immunogenic composition of the invention, the 3 type Cap protein antigenic content of pig circular ring virus is also selected from
20 μ of μ g/ml~30 g/ml, 30 μ of μ g/ml~100 g/ml or the 50 μ g/ml content ranges of μ g/ml~100.
In immunogenic composition of the invention, the porcine circovirus 2 type new gene hypotype Cap protein antigen contains
Amount is also selected from 20 μ of μ g/ml~30 g/ml, 30 μ of μ g/ml~100 g/ml or the 50 μ g/ml content ranges of μ g/ml~100.
In immunogenic composition of the invention, the 3 type Cap protein antigenic content of pig circular ring virus and pig circle
2 type new gene hypotype Cap protein antigenic content of circovirus virus can be independently selected from respective any of the above-described content range.
As one embodiment of the present invention, contain 3 type of pig circular ring virus, porcine circovirus 2 type antigen in of the invention
Immunogenic composition in, it is described recombinate have 3 type Cap protein gene of pig circular ring virus live vector be recombinant attenuated sramana
Salmonella, recombinant Newcastle disease virus, recombinant poxvirus or recombined adhenovirus；The recombination has the porcine circovirus 2 type new base
Because the live vector of subtype C ap protein gene is recombination attenuation salmonella, recombinant Newcastle disease virus, recombinant poxvirus or recombination
Adenovirus.
As one embodiment of the present invention, contain 3 type of pig circular ring virus, porcine circovirus 2 type antigen in of the invention
Immunogenic composition in, the 3 type Cap protein gene of pig circular ring virus and the porcine circovirus 2 type new gene hypotype
In the same live vector, the live vector is recombination attenuation salmonella, recombinant Newcastle disease virus, again for Cap protein genetic recombination
Group poxvirus or recombined adhenovirus.
The advantages of live vector immunogenic composition of the invention is because of with inactivated vaccine and live vaccine, in immune effect
It can guarantee to protect pig in power, and its immune efficacy is stronger, adjuvant can not be added.
As one embodiment of the present invention, the present invention exempts from containing pig circular ring virus 3 type, porcine circovirus 2 type antigen
In epidemic disease Immunogenic Compositions, the pharmaceutically acceptable carrier includes adjuvant, the adjuvant include white oil, Drake oil, and
Animal oil, vegetable oil or mineral oil；Or aluminium hydroxide, aluminum phosphate and metal salt；Or MontanideTMGel, carbomer, spiny dogfish
Alkane or squalene, ISA206 adjuvant, saponin(e, water-in-oil emulsion, oil in water emulsion, W/O/W emulsion.
As a kind of preferred embodiment of the invention, of the invention is anti-containing 3 type of pig circular ring virus, porcine circovirus 2 type
In former immunogenic composition, the adjuvant 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
Amount needed for side effect.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.
It is of the invention containing pig circular ring virus 3 type, porcine circovirus 2 type antigen as one embodiment of the present invention
In immunogenic composition, the adjuvant content is 5~20V/V%.
As a kind of preferred embodiment of the invention, of the invention is anti-containing 3 type of pig circular ring virus, porcine circovirus 2 type
In former immunogenic composition, the adjuvant content is 10V/V%.
Immunogenic composition of the invention, which can be used, to be deployed with technology, preferably pharmaceutically acceptable carrier one
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 " 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。
Other reagents can also be further added in composition of the invention by immunogenic composition of the present invention.Example
Such as, composition of the invention can also include following reagent, such as: drug, immunostimulant (such as: alpha-interferon, beta-interferon,
Gamma interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and white
Interleukin 2 (IL2)), antioxidant, surfactant, colorant, ethereal oil, buffer, dispersing agent, propellant and anti-corrosion
Agent.In order to prepare such composition, method well known in the art can be used.
Peroral dosage form or non-oral dosage forms can be prepared into immunogenic 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 immunogenic compositions in preparation prevention and/or to treat porcine circovirus associated diseases
Application in drug, wherein the porcine circovirus associated diseases are by PCV2 and 3 type of pig circular ring virus individually infects or it
Related disease caused by mixed infection.
As one embodiment of the present invention, the medicine of the preparation prevention and/or treatment porcine circovirus associated diseases
Application in object is preparation prevention and/or treatment pmws, the dermatitis nephrotic syndrome of pig, increasing
Application in the drug of the inflammatory reaction of natural disposition necrotizing pneumonia, breeding difficulty and heart and multisystem.
Term used herein " porcine circovirus associated diseases " is for referring to by 3 type of pig circular ring virus and 2 type mixed infections
Caused disease.Nonexhaustive includes, pmws, pig dermatitis nephrotic syndrome, breeding difficulty
And the inflammatory reaction of heart and multisystem, but not limited to this.
Term " prevention and/or treatment " refers to inhibition pig circular ring virus 2 when being related to 3 type of pig circular ring virus and 2 type mixed infection
The duplication of malicious 3 types and 2 types inhibits the propagation of 3 type of pig circular ring virus and 2 types or prevents 3 type of pig circular ring virus and 2 types in its host
It settles down in vivo, and mitigates the disease of 3 type of pig circular ring virus and the infection of 2 types or the symptom of illness.If viral loads reduction,
Illness mitigation and/or food ration and/or growth increase, then can think that the treatment has reached therapeutic effect.
The invention further relates to above-mentioned immunogenic compositions in preparation prevention and/or to treat pig circular ring virus mixed infection
Application in drug.
As one embodiment of the present invention, the present invention prepares prevention and/or treats pig circular ring virus mixed infection
In application in drug, the PCV2 gene hypotype is PCV2a, PCV2b, PCV2d, PCV2new gene hypotype.
Immunogenic composition of the invention can provide effectively for different PCV2 gene hypotypes and PCV3, expand epidemic disease
The application range of seedling can be prevented and/or be treated to the independent infection and mixed infection of PCV2 different genes hypotype and PCV3.
Immunogenic composition of the invention has good immunogenicity, can not only prevent pig circular ring virus correlation disease
Disease, moreover it is possible to mitigate the Clinical signs for having infected the pig of pig circular ring virus, namely there is 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；Again through 0.22 μm of filter membrane filter, filtrate exists supernatant
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 identify viral species
The viral cultures for taking above step extract the nucleic acid of viral sample with nucleic acid extraction kit, use annulus disease
Seed culture of viruses Genus-specific primers carry out PCR amplification identification, the results show that PCR amplifies 2000bp purpose band.PCR product send survey
Sequence company carries out nucleotide sequencing, and sequencing results carry out phylogenetic analysis.The Strain full genome as the result is shown
Group 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 international virus
The standard of the credit class committee, same virus should have the genome nucleotide sequence of > 75% in Circovirus
Homology, the homology of amino acid sequence of the Cap protein with > 70% can affirm that it is a kind of new pig circle accordingly
Circovirus virus, and the third circovirus found on pig body at present.
The building of embodiment 2pET28a-PCV3-Cap expression vector
1, the extraction of PCV3 viral DNA
Plasmid extraction kit is purchased from Tiangeng biology；T4DNA Ligase is purchased from BioLab；PET28a plasmid is purchased from
Novagen；For Ago-Gel plastic recovery kit purchased from day pool biology, other reagents are that analysis is pure.
According to viral DNA extracts kit specification, 3 type virus liquid of 0.2ml pig circular ring virus is taken to be centrifuged in sterile 1.5ml
Guan Zhong adds 0.4ml VB in virus liquid, and vortex mixes, and is stored at room temperature 10 minutes.Add 0.45ml AD buffer in virus liquid
In, firmly mix.VB column is put into 2ml collecting pipe, the above-mentioned virus liquid for being mixed with reagent of 0.6ml is taken to be added in VB column,
14000g is centrifuged 1 minute, then the remaining above-mentioned virus liquid for being mixed with reagent is added in VB column, and 14000g is centrifuged 1 minute,
2ml collecting pipe is discarded, VB column is put into new 2ml collecting pipe, 0.4ml W1buffer is added, 14000g is centrifuged 30 seconds, then
Add 0.6ml Wash buffer in VB column, 14000g is centrifuged 30 seconds, vacant centrifugation 3 minutes again buffer is then not added, by this
VB column is put into new 1.5ml EP pipe, and 50 μ l RNase free water are added and are placed in film center, stand 3 minutes, 14000g
It is centrifuged 1 minute, being centrifuged the liquid to get off in EP pipe is 3 type viral DNA genome solution of pig circular ring virus.
2, Cap gene magnification
Oligonucleotide primer is synthesized according to the conserved region sequence of Cap gene 5 ' and 3 ' ends, carries out PCR.Primer sequence
It is shown in Table 1.
1 pig circular ring virus of table, 3 type Cap gene magnification primer
| PCV3-Cap-F | CCA CAG AAG GCG CTA TGT C |
| PCV3-Cap-R | CCG CAT AAG GGT CGT CTT G |
PCR product is sent to Invitrogen for sequencing, codon optimization is carried out to Cap gene according to sequencing result, it is excellent
Cap gene order after change is as shown in sequence table SEQ ID NO.1.
3, expression vector establishment
Cap gene send the Suzhou biotech inc Hong Xun to carry out complete sequence synthesis after optimization, and is connected to
On pET28a plasmid.Plasmid and molecular chaperones plasmid pG-Tf2 cotransformation e. coli bl21 (DE3) after connection, picking list
It is cloned in overnight incubation in the LB culture medium containing 100 μ g/ml kanamycins and 20 μ g/ml chloramphenicol, is sequenced after extracting plasmid
Analysis, determines that composition sequence is correct, and positive colony is pET28a-PCV3-Cap/pG-Tf2 expression bacterial strain pET28a-PCV3-
Cap/pG-Tf2/E.Coli BL21(DE3)。
The expression of 3 pig circular ring virus of embodiment, 3 type Cap protein
By pET28a-PCV3-Cap/pG-Tf2/E.Coli BL21 (DE3) strain inoculated prepared in embodiment 2 to containing
In the LB culture medium of 50-100 μ g/ml kanamycins and 20 μ g/ml chloramphenicol, while containing 5-10ng/ml tetra- in LB culture medium
Ring element is used for the inducing expression of molecular chaperone protein, and inoculum concentration is 1% (V/V), in 37 DEG C of shaken cultivations.Work as OD600=0.4~
When 0.6, placed 30 minutes in 28 DEG C.Be added isopropyl-beta D-thio galactopyranoside (IPTG), make its final concentration of 0.1
~1.0mM, in 28 DEG C shaken cultivation 24 hours.After culture, thallus is collected, and with PBS (PBS component: sodium chloride 8g, chlorine
Change potassium 0.2g, disodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.24g, adjust pH to 7.4, constant volume 1L) thallus, ultrasonication is resuspended
Thallus, the lysate centrifuging and taking supernatant after ultrasonication.Soluble destination protein content is higher in expression product, and expression quantity is reachable
The 25% of mycoprotein total amount, endotoxin content are 0.28 × 105EU/ml。
4 Bacillus coli expression pig circular ring virus of embodiment, 3 type Cap protein Endotoxin removal
The 0.5ml supernatant solution and final concentration of 1% to be processed containing soluble Cap protein is added in 1.5ml centrifuge tube
(v/v) Qula leads to X-114 (Triton X-114) (5 μ l of additional amount), vortex oscillation.The sample that vortex oscillation mixes is on ice
It places 5 minutes.After sample is cooling, centrifuge tube is immediately placed in warm bath 5min in 37 DEG C of water-baths, so that generating new two-phase.Then,
Sample is centrifuged 60s under the conditions of 37 DEG C of temperature.After centrifugation, destination protein will be left in upper layer, and containing it is endotoxic it is non-from
Sub- surfactant will remain in centrifugation bottom of the tube with the shape of oil droplet, separate two-phase.It is above-mentioned entirely to remove endotoxic behaviour
Make circulation 3 times.After measured, purity of protein does not reduce final purified product, and endotoxin content falls to 0.008 × 105EU/
ml；Meanwhile by 200KV transmission electron microscope, 60000 times of amplification factor, observation through 5% phosphotungstic acid negative staining, be fixed on the copper of spray carbon
Online PCV3 virus-like particle, a large amount of virus-like particle as the result is shown, and it is uniform in size, it is rendered as hollow granule state.
The experimental results showed that Triton X-114 can remove remaining endotoxin in recombinant protein, and to the purity of albumen
Do not influence；Meanwhile also without influence PCV3 virus-like particle formation and stable form.
The separation of 5 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；Again through 0.22 μm of filter membrane filter, filtrate exists supernatant
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, 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 public
Department carries out nucleotide sequencing, and sequencing results carry out phylogenetic analysis.The Strain full-length genome sequence as the result is shown
Column are with it has been reported that the homology of other PCV2 crossed is less than 96%, and amino acid sequence is less than 94%, further full genome
Group sequence between PCV2b and PCV2d, in ORF2 gene analysis shows there is mutation or through different genes hypotype in the strain
It is recombinated between ORF2 gene, is determined through phylogenetic analysis, belong to a new PCV2 gene hypotype.
The building of 6 pET28a-PCV2-Cap expression vector of embodiment
1, the extraction of PCV2 viral DNA
Plasmid extraction kit is purchased from Tiangeng biology；T4 DNA Ligase is purchased from BioLab；PET28a plasmid is purchased from
Novagen；For Ago-Gel plastic recovery kit purchased from day pool biology, other reagents are that analysis is pure.
According to viral DNA extracts kit specification, the porcine circovirus 2 type disease for taking 0.2ml new referring to 2 method of embodiment
Venom extracts DNA genome.
2, porcine circovirus 2 type Cap gene magnification
Oligonucleotide primer is synthesized according to the conserved region sequence of Cap gene 5 ' and 3 ' ends, carries out PCR.Primer sequence
It is shown in Table 2.
2 porcine circovirus 2 type Cap gene magnification primer of table
| PCV2-Cap-F | CCCATGCCCTGAATTTCCA |
| PCV2-Cap-R | CAGCGCACTTCTTTCGTTTTCAG |
PCR product is sent to Invitrogen for sequencing, as the result is shown with the Cap protein ammonia of PCV2 in the prior art
Base acid sequence is compared, and the amino acid sequence of Cap gene coding is in 52~64,106~108,131~137 generation genes
Mutation or recombination carry out codon optimization to Cap gene according to sequencing result, the Cap gene order such as sequence table after optimization
Shown in SEQ ID NO.2.
3, expression vector establishment
Cap gene send the Suzhou biotech inc Hong Xun to carry out complete sequence synthesis after optimization, and is connected to
On pET28a plasmid.Plasmid and molecular chaperones plasmid pG-Tf2 cotransformation e. coli bl21 (DE3) after connection, picking list
It is cloned in overnight incubation in the LB culture medium containing 100 μ g/ml kanamycins and 20 μ g/ml chloramphenicol, is sequenced after extracting plasmid
Analysis, determines that composition sequence is correct, and positive colony is pET28a-PCV2-Cap/pG-Tf2 expression bacterial strain pET28a-PCV2-
Cap/pG-Tf2/E.Coli BL21(DE3)。
The expression of 7 carrying Cap gene of porcine circovirus type 2 of embodiment
By pET28a-PCV2-Cap/pG-Tf2/E.Coli BL21 (DE3) bacterial strain prepared in embodiment 6 referring to implementation
Example 3 carries out protein induced expression.Soluble destination protein content is higher in expression product, and expression quantity is up to mycoprotein total amount
50%, endotoxin content is 0.26 × 105EU/ml。
8 Bacillus coli expression Cap protein Endotoxin removal of embodiment
The supernatant solution containing soluble Cap protein that embodiment 7 is expressed carries out Endotoxin removal referring to embodiment 4.Through
Measurement, purity of protein do not reduce, and endotoxin content falls to 0.008 × 105EU/ml；Meanwhile by 200KV transmission electron microscope,
60000 times of amplification factor, as a result observation is shown through 5% phosphotungstic acid negative staining, the PCV2 virus-like particle being fixed on the copper mesh for spraying carbon
Show a large amount of virus-like particle, and uniform in size, is rendered as hollow granule state.
The result shows that Triton X-114 can remove remaining endotoxin in recombinant protein, and do not have to the purity of albumen
It influences；Meanwhile also without influence PCV2 virus-like particle formation and stable form.
The preparation of 9 pig circular ring virus of embodiment 3 type, porcine circovirus 2 type immunogenic composition
The porcine circovirus 2 type Cap egg that the 3 type Cap protein of pig circular ring virus, the embodiment 8 that purify by embodiment 4 are purified
It is added slowly to after white mixing in proportion in water-soluble adjuvant Gel adjuvant (match BIC Corp of France), is constantly used during adding
Revolving speed is that 800rpm mulser stirs 12min, is mixed.The specific formula of immunogenic composition is shown in Table 3.
Table 3 is matched containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen immunogenicity composition
Embodiment 10 contains 3 type of pig circular ring virus, porcine circovirus 2 type antigen immunogenicity composition Study On Immunogenicity
28~30 ages in days are randomly divided into 12 through ELISA detection PCV2, PCV3 antigen, sodium selenite 60 of negative antibody
5/group, 3 type of pig circular ring virus, 2 type immunogenic compositions prepared by embodiment 9 are immunized in group.1st~2 group of immune combination
The 1, the 3rd~4 group of immune composition 2 of object, the 5th~6 group of immune composition 3, the 7th~8 group of difference immune composition 4, the 9th~10
Immune be used as does not attack malicious control group to group for immunogenic composition 5, the 6, the 11st~12 group of composition respectively.Each immune group injecting immune
Immunogenic Compositions 2ml/ head, control group are inoculated with DMEM culture medium 2ml/ head.Carry out within 28th attacking poison after immune, the 1st group, the 3rd group, the
5 groups, the 7th group, the 9th group, the 11st group are attacked malicious SG plants of (Porcine Circovirus type 3, Strain of 3 type of pig circular ring virus
SG is preserved in China typical culture collection center, and deposit number is CCTCC NO:V201712, and the deposit date is in March, 2017
23 days, preservation address: Wuhan, China Wuhan University), 105.0TCID50/ head, the 2nd group, the 4th group, the 6th group, the 8th group, the 10th
Group, the 12nd group attack malicious porcine circovirus 2 type HH3 plants (Porcine Circovirus type 2, Strain HH3 be preserved in
State's Type Tissue Collection, deposit number is CCTCC NO:V201726, the deposit date is on June 4th, 2017, preservation
Location: Wuhan, China Wuhan University), 105.0TCID50Each piglet is observed continuously after attacking poison in/head, according to each piglet clinical symptoms,
Pathological change and viral diagnosis result are determined that concrete outcome is shown in Table 4.
Table 4 contains 3 type of pig circular ring virus, 2 type antigen immunogenicity composition Study On Immunogenicity results
The results show that can be piglet after containing 3 type of pig circular ring virus, 2 type antigen immunogenicity composition primary immunization piglets
100% (5/5) protection is provided, and attacks after malicious control group piglet attacks poison and all falls ill.Show provided by the invention containing pig circular ring virus 2
Malicious 3 types, 2 type antigen immunogenicity compositions have good Vaccine effectiveness.And different content of the present invention contain pig circular ring virus 3
Virus cannot be detected in pig body after type, 2 type antigen immunogenicity composition Immunizations, this explanation is as the immune present invention
Immunogenic composition after, even if the pig that wild virus infection is immune, will not development growth to pig, feed getting fat generate
It influences.
Embodiment 11 contains 3 type of pig circular ring virus, porcine circovirus 2 type antigen immunogenicity composition broad spectrum activity protection test
28~30 ages in days are randomly divided into through ELISA detection PCV2, PCV3 antigen, sodium selenite 100 of negative antibody
20 groups, 5/group, immunogenic composition 1 prepared by the 13rd~22 group of immune embodiment 9, the 23rd~32 group of not immune conduct is attacked
Malicious control group.Each immune group injecting immune Immunogenic Compositions 2ml/ head attacks malicious control group inoculation DMEM culture medium 2ml/ head.It is immune
It carries out within 28th attacking poison afterwards, the 13rd group, the 23rd group is used the 2 porcine circovirus a gene hypotype HN06 separated from Henan, China recently
Strain velogen strain attacks poison；14th group, the 24th group is used JS04 plants of the 2 porcine circovirus b gene hypotype separated recently from Jiangsu Province, China
Velogen strain attacks poison；15th group, the 25th group is used JL13 plants of the 2 porcine circovirus d gene hypotype separated recently from Chinese Jilin Province
Velogen strain attacks poison；16th group, the 26th group is used CQ14 plants of the 2 porcine circovirus new gene hypotype separated recently from Chinese Chongqing City
Velogen strain attacks poison；17th group, the 27th group is used GD15 plants of the 2 porcine circovirus new gene hypotype separated recently from Guangdong province, China
Velogen strain attacks poison；18th group, the 28th group with recently from Henan, China separate pig circular ring virus HN12 plants of velogen strains of 3 type attack
Poison；19th group, the 29th group with recently from Jiangsu Province, China separate pig circular ring virus JS08 plants of velogen strains of 3 type attack poison；20th
Group, the 30th group with recently from Chinese Jilin Province separate pig circular ring virus JL11 plants of velogen strains of 3 type attack poison；21st group, the 31st group
With recently from Chinese Chongqing City separate pig circular ring virus CQ04 plants of velogen strains of 3 type attack poison；22nd group, the 32nd group is used recently therefrom
Pig circular ring virus GD05 plants of velogen strains of 3 type of Guangdong Province of state separation attack poison；Attacking toxic dose is 105.0TCID50/ head is attacked after poison continuously
Each piglet is observed, is determined that concrete outcome is shown in Table 5~6 according to each piglet clinical symptoms, pathological change and viral diagnosis.
Table 5 infects broad spectrum activity protection test knot for PCV2 containing 3 type of pig circular ring virus, 2 type antigen immunogenicity compositions
Fruit
The results show that the 23rd~27 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 13rd~17 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.The experimental results showed that provided by the invention contain
3 type of pig circular ring virus, 2 type antigen immunogenicity composition primary immunizations, can be to pig for different geographical source, different bases
Poison is attacked because of the porcine circovirus 2 type of hypotype, effective, complete immunoprotection is provided, and cannot detect from each organs and tissues and attack poison
PCV2.
Table 6 infects broad spectrum activity protection test knot for PCV3 containing 3 type of pig circular ring virus, 2 type antigen immunogenicity compositions
Fruit
The results show that the 28th~32 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 18th~22 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.The experimental results showed that provided by the invention contain
3 type of pig circular ring virus, 2 type antigen immunogenicity compositions can attack pig for 3 type of pig circular ring virus in different geographical source
Poison provides effective, complete immunoprotection, and the PCV3 strain for attacking poison cannot be detected from each organs and tissues.Show that the present invention mentions
The immunogenic composition of confession has the immunogenicity of wide spectrum, and popular 3 type of pig circular ring virus, the wild poison of 2 types can be provided
Protection completely.It, will not be to pig even if the pig that wild virus infection is immune and after immunogenic composition of the invention is immunized
Development growth, feed getting fat have an impact.
Embodiment 12 contains 3 type of pig circular ring virus, 2 type antigen immunogenicity composition mixed infection protection tests
28~30 ages in days are randomly divided into 4 through ELISA detection PCV2, PCV3 antigen, sodium selenite 20 of negative antibody
Group, 5/group.Immunogenic composition 1 prepared by the 33rd group of immune embodiment 9, the 34th group of immune embodiment 9 prepare immune
Malicious control is attacked in Immunogenic Compositions 5, immunogenic composition 6 prepared by the 35th group of immune embodiment 9, the 36th group of not immune conduct
Group.Each immune group injecting immune Immunogenic Compositions 2ml/ head, control group are inoculated with DMEM culture medium 2ml/ head.It carries out within 28 days after immune
Poison is attacked, all groups are attacked SG plants of malicious 3 type of pig circular ring virus and porcine circovirus 2 type HH3 plants of hybrid virus liquid, and attacking toxic dose is
105.0TCID50Each piglet is observed continuously after attacking poison in/head, is determined according to each piglet clinical symptoms, pathological change result, tool
Body the results are shown in Table 7.
Table 7 contains 3 type of pig circular ring virus, 2 type antigen immunogenicity composition mixed infection protection test results
The results show that the 36th 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
5 days, there are different journeys in clinical symptoms, the dissects such as appetite stimulator, spirit are depressed, coat is thick disorderly, the thin and speed of growth slows down
The pathological change that degree pulmonary consolidation, lymph enlargement, kidney have necrosis to put；33rd group of immune group attacks clinical symptoms without exception after poison, dissect
Each histoorgan is also without exception；And the 34th group, the 35th group of immune group not can effectively prevent the mixed infection of PCV2, PCV3, still
In morbidity state.The experimental results showed that provided by the invention primary containing 3 type of pig circular ring virus, 2 type antigen immunogenicity compositions
It is immunized that pig can be combined for PCV2, PCV3 and effective, the complete immunoprotection of poison offer is provided.
Embodiment 13 contains 3 type of pig circular ring virus, 2 type antigen immunogenicity composition application tests
There is the sow death rate and increases by 9.8% 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 8.6%.In clinical manifestation, impacted sow shows anorexia, is in multifocal mound
Rash, the symptom of spot and surface dermatitis.Containing the mummified fetus of different gestational age in the nest of miscarriage, there will be clinical manifestation
32 farrowing sows are chosen in the sow of symptom and are randomly divided into two groups of A groups, B group, and 16/group, A group connects for immunogenic composition
Immunogenic composition 1 prepared by embodiment 9 is immunized in kind group, A group, and B group is blank control group.Immune group injecting immune originality group
Object 2ml/ head is closed, blank control group is inoculated with DMEM culture medium 2ml/ head.Two groups of sow produce surviving of son situations are counted, the results are shown in Table 8.
8 immune group of table and blank control group sow produce surviving of son statistical result
The results show that immune group sow produce surviving of son is without exception, sodium selenite, average 11.7/nest are produced, healthy rate is up to
99.5%, and apparent the mummification of fetus and weak pigle situation occurs in blank control group, production sodium selenite be averaged 6.9/nest, strong
Health rate 59.0%, wherein there are also three sows abortion situation occurs, full nest the mummification of fetus, immune group and blank control group difference are aobvious
It writes.
The result of table 8 demonstrates the present invention containing 3 type of pig circular ring virus, 2 type antigen immunogenicity compositions to infection pig circle
The sow of circovirus virus has good immanoprotection action, and can be protected for the sow for having infected PCV, that is, has
Therapeutic effect.
Meanwhile the farrowed pig of B group control group is individually insulated and is raised by nest, 13 nests are divided into two groups of B1 groups (including B-1 nest
To B-11 nest, in addition to B-5 nest is disregarded because of full nest the mummification of fetus, totally 10 nest piglet), (including B-12 removes B- to B-16 nest to B2 group
14 nests and B-16 nest disregard outer, totally 3 nest piglet because of full nest the mummification of fetus), B1 group exempting from of preparing of immune embodiment 9 before eating breast milk
Epidemic disease Immunogenic Compositions 1, B2 group are blank control group.Immune group injecting immune Immunogenic Compositions 2ml/ head, blank control group inoculation
DMEM culture medium 2ml/ head.Each piglet is observed continuously, is sentenced according to each piglet clinical symptoms, pathological change and viral diagnosis
Fixed, concrete outcome is shown in Table 9.
Table 9 is containing 3 type of pig circular ring virus, 2 type antigen immunogenicity compositions to the Immunoprotection test of piglet
The results show that immune group piglet clinical symptoms without exception, random each histoorgan of dissect is also without exception, passes through dissect
Each organs and tissues of pig carry out PCR detection, show PCV3, PCV2 feminine gender, and blank control group piglet occurs in various degree
Body temperature increases 40.5 DEG C or more, continues 5 days, and appetite stimulator, spirit are depressed, coat is thick disorderly, the thin and speed of growth slows down etc. faces
Bed symptom, part pig is dead, and the pathological change that different degrees of pulmonary consolidation occurs in dissect, lymph enlargement, kidney have necrosis to put is led to
It crosses each organs and tissues and carries out PCR detection, 3 type of pig circular ring virus and porcine circovirus 2 type virus can be separated to again.
Since PCV can demonstrate annulus containing pig of the invention in swinery by vertical transmission, the result of table 9
Viral 3 types, 2 type antigen immunogenicity compositions have the piglet of 3 type of mixed infection pig circular ring virus and 2 types good immune
Protective effect, and can be protected for the piglet of mixed infection PCV3 and PCV2 virus, that is, there is therapeutic effect, protect
Shield rate is 100%.
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.
SEQUENCE LISTING
<110>this Australia's biotechnology (Suzhou) Co., Ltd of Pulaike Biological Engineering Co., Ltd.
<120>a kind of containing 3 type of pig circular ring virus, the immunogenic composition of porcine circovirus 2 type antigen and its application
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 645
<212> DNA
<213>3 type of pig circular ring virus
<400> 1
atgcgtcacc gtgctatctt ccgtcgtcgt ccgcgtccgc gtcgtcgtcg tcgtcaccgt 60
cgtcgttacg ctcgtcgtaa actgttcatc cgtcgtccga ccgctggtac ctactacacc 120
aaaaaatact ctaccatgaa cgttatctct gttggtaccc cgcagaacaa caaaccgtgg 180
cacgctaacc acttcatcac ccgtctgaac gaatgggaaa ccgctatctc tttcgaatac 240
tacaaaatcc tgaaaatgaa agttaccctg tctccggtta tctctccggc tcagcagacc 300
aaaaccatgt tcggtcacac cgctatcgac ctggacggtg cttggaccac caacacctgg 360
ctgcaggacg acccgtacgc tgaatcttct acccgtaaag ttatgacctc taaaaaaaaa 420
cactctcgtt acttcacccc gaaaccgctg ctggctggta ccacctctgc tcacccgggt 480
cagtctctgt ctttcttctc tcgtccgacc ccgtggctga acacctacga cccgaccgtt 540
cagtggggtg ctctgctgtg gtctatctac gttccggaaa aaaccggtat gaccgacttc 600
tacggtacca aagaagtttg gatccgttac aaatctgttc tgtaa 645
<210> 2
<211> 702
<212> DNA
<213>porcine circovirus 2 type new gene hypotype
<400> 2
atgacctacc cgcgtcgtcg ttaccgtcgt cgtcgtcacc gtccgcgttc tcacctgggt 60
cagatcctgc gtcgtcgtcc gtggctggtt cacccgcgtc accgttaccg ttggcgtcgt 120
aaaaacggta tcttcaacac ccgtctgtct cgttctttcg gttacaccgt tgttacctct 180
accgttaccc cgccgtcttg ggctgttgac atgatgcgtt tcaacatcaa cgacttcctg 240
ccgccgggtg gtggttctaa cccgcgttct gttccgttcg aatactaccg tatccgtaaa 300
gttaaagttg aattcttcgc tcgttctccg atcacccagg gtgaccgtgg tgttggttct 360
tctgctgtta tcctggacga caacttcgtt aacaaaacca acgctctgtc ttacgacccg 420
tacgttaact actcttctcg tcacaccatc acccagccgt tctcttacca ctctcgttac 480
ttcaccccga aaccggttct ggactctacc atcgactact tccagccgaa caacaaacgt 540
aaccagctgt ggctgcgtct gcagaccacc ggtaacgttg accacgttgg tctgggtacc 600
gctttcgaac actctatcta cgaccaggct tacaacatcc gtgttaccat gtacgttcag 660
ttccgtgaat tcaacctgaa agacccgccg ctgaacccgt aa 702
Claims (10)
1. a kind of immunogenic composition containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen, wherein the immunogenicity
Composition includes the porcine circovirus 2 type new gene hypotype of the 3 type Cap protein antigen of pig circular ring virus of immune amount, immune amount
Cap protein antigen and pharmaceutically acceptable carrier；Wherein, the 3 type Cap protein antigen of pig circular ring virus is pig circular ring virus 2
Malicious 3 type Cap proteins or recombination have the live vector of the 3 type Cap protein gene of pig circular ring virus, the porcine circovirus 2 type new
Gene hypotype Cap protein antigen is that porcine circovirus 2 type new gene hypotype Cap protein or recombination have the porcine circovirus 2 type
The live vector of Cap protein gene.
2. the immunogenic composition according to claim 1 containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen,
In, the 3 type Cap protein of pig circular ring virus is the albumen of SEQ.ID NO.1 coding, and the porcine circovirus 2 type new gene is sub-
Type Cap protein is the albumen of SEQ.ID NO.2 coding.
3. the immunogenic composition according to claim 1 containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen,
In, the 3 type Cap protein content of pig circular ring virus is >=20 μ g/ml；The porcine circovirus 2 type new gene hypotype Cap egg
Bai Hanliang is >=20 μ g/ml.
4. the immunogenic composition according to claim 1 containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen,
In, the 3 type Cap protein content of pig circular ring virus is 20 μ of μ g/ml~100 g/ml；The porcine circovirus 2 type new gene is sub-
Type Cap protein content is 20 μ of μ g/ml~100 g/ml.
5. the immunogenic composition according to claim 1 containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen,
In, the 3 type Cap protein content of pig circular ring virus is 20 μ of μ g/ml~50 g/ml；The porcine circovirus 2 type new gene is sub-
Type Cap protein content is 20 μ of μ g/ml~50 g/ml.
6. the immunogenic composition according to claim 1 containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen,
In, the 3 type Cap protein content of pig circular ring virus is 30 μ of μ g/ml~50 g/ml；The porcine circovirus 2 type new gene is sub-
Type Cap protein content is 30 μ of μ g/ml~50 g/ml.
7. the immunogenic composition according to claim 1 containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen,
In, the 3 type Cap protein gene of pig circular ring virus and the porcine circovirus 2 type new gene hypotype Cap protein genetic recombination
In the same live vector, the live vector is recombination attenuation salmonella, recombinant Newcastle disease virus, recombinant poxvirus or recombination
Adenovirus.
8. the immunogenic composition according to claim 1 containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen,
In, the pharmaceutically acceptable carrier includes 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, W/O/W emulsion；
The adjuvant content is 5~20V/V%；It is highly preferred that the adjuvant content is 10V/V%.
9. the immunogenic composition according to claim 1 containing 3 type of pig circular ring virus, porcine circovirus 2 type antigen,
In, the pharmaceutically acceptable carrier includes adjuvant, and the adjuvant is MontanideTMGel.The adjuvant content be 5~
20V/V%；It is highly preferred that the adjuvant content is 10V/V%.
10. described in any item containing 3 type of pig circular ring virus, the immunogene of porcine circovirus 2 type antigen according to claim 1~9
Property composition preparation prevention and/or treatment porcine circovirus associated diseases drug in application；Preferably, the pig annulus
Virus associated-diseases include pmws, the dermatitis nephrotic syndrome of pig, Hypertrophic necrotizing pneumonia,
The inflammatory reaction of breeding difficulty and heart and multisystem.
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| CN201710464584.1A CN109125719B (en) | 2017-06-19 | 2017-06-19 | Immunogenic composition containing porcine circovirus type 3 and porcine circovirus type 2 antigens and application thereof |
| JP2020519165A JP6882602B2 (en) | 2017-06-19 | 2017-12-26 | Immunogenic compositions containing porcine circovirus type 3 and porcine circovirus type 2 antigens and their uses. |
| PCT/CN2017/118696 WO2018233264A1 (en) | 2017-06-19 | 2017-12-26 | Immunogenic composition comprising porcine circovirus type 3 and porcine circovirus type 2 antigens and use thereof |
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Cited By (2)
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|---|---|---|---|---|
| CN109762052A (en) * | 2019-01-18 | 2019-05-17 | 南京农业大学 | 3 type Cap recombinant protein of pig circular ring virus and its encoding gene and application |
| CN111558037A (en) * | 2020-06-08 | 2020-08-21 | 武汉科前生物股份有限公司 | Bivalent subunit vaccine and preparation method and application thereof |
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| CN109010818B (en) * | 2017-06-09 | 2021-11-09 | 普莱柯生物工程股份有限公司 | Bivalent vaccine composition for preventing and/or treating porcine circovirus infection and preparation method and application thereof |
| MX2021012051A (en) | 2019-04-04 | 2022-01-18 | Boehringer Ingelheim Animal Health Usa Inc | Porcine circovirus type 3 (pcv3) vaccines, and production and uses thereof. |
| CN110174510A (en) * | 2019-04-26 | 2019-08-27 | 华南农业大学 | A kind of preparation method of the fluorescence immune chromatography detection card of PCV3 |
| CN111187781B (en) * | 2019-09-12 | 2023-09-15 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Optimized porcine circovirus type 3 capsid protein gene and application thereof in preparation of virus-like particles |
| CN110981945B (en) * | 2019-12-08 | 2021-10-19 | 中牧实业股份有限公司 | Expression preparation and application of porcine circovirus type 2 recombinant Cap protein |
| PE20211141A1 (en) * | 2019-12-19 | 2021-06-25 | Farm Veterinarios S A C | SALMONELLA ENTERITIDIS RECOMBINANTE AND ITS USE AS A SWINE VACCINE |
| CN115819567B (en) * | 2022-11-19 | 2023-09-01 | 深圳盛源生物技术有限公司 | Monoclonal antibody and application thereof in detection kit |
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| Publication number | Publication date |
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| CN109125719B (en) | 2022-06-14 |
| WO2018233264A1 (en) | 2018-12-27 |
| JP2020524182A (en) | 2020-08-13 |
| JP6882602B2 (en) | 2021-06-02 |
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