CN107537031A - A kind of vaccine combination for preventing porcine reproductive and respiratory syndrome and its preparation method and application - Google Patents
A kind of vaccine combination for preventing porcine reproductive and respiratory syndrome and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of vaccine combination for being used to prevent porcine reproductive and respiratory syndrome, wherein, the vaccine combination includes classical porcine reproductive and respiratory syndrome virus antigen and/or highly pathogenic PRRSV antigen and/or porcine reproductive and respiratory syndrome virus the class NADC30 strain antigens of immune amount, and pharmaceutically acceptable carrier.Vaccine combination provided by the invention is highly resistant to the attack of new epidemic strain, highly pathogenic PRRSV and classical porcine reproductive and respiratory syndrome virus, solves the problems, such as the porcine reproductive and respiratory syndrome virus infection of separate sources, the purification for porcine reproductive and respiratory syndrome virus has great significance.
Description
Technical field
The invention belongs to vaccine veterinary art, and in particular to prevent porcine reproductive and respiratory syndrome vaccine combination and its
Preparation method and application.
Background technology
Porcine reproductive and respiratory syndrome is by porcine reproductive and respiratory syndrome virus (porcine reproductive and
Respiratory syndrome virus, PRRSV) it is caused using farrowing sow breeding difficulty and piglet respiratory symptom as spy
A kind of infectious disease of sign.The disease is found in the North Carolina state (1987) in the U.S. earliest, hereafter in succession in Canada (1998
Year), German (nineteen ninety), Britain (1991) find this disease.1992 World Organization for Animal Health (OIE) by this disease be named as pig breeding
With respiration syndrome (PRRS).
Porcine reproductive and respiratory syndrome virus is single strand plus RNA virus, due to the point mutation of rna gene group, missing,
Insert and substitute and there is the very high frequency of mutation.China also reported this sick prevalence in 1996, and 2006 are even more to break out
Porcine reproductive and respiratory syndrome caused by the highly pathogenic PRRSV of variation, causes huge economic loss to pig industry.
However, with the application of porcine reproductive and respiratory syndrome live vaccine, pig breeding is controlled to a certain extent with exhaling
The outburst of syndrome is inhaled, but the problem of some are new also occurs, especially in this year, how regional epidemiology survey is found,
The pig farm of immune porcine reproductive and respiratory syndrome live vaccine, there is new PRRSV popular, compared with conventional epidemic strain, its gene
Larger change occurs for sequence.And currently available vaccines, existing vaccines is clinically not satisfactory for the preventive effect again of such case, moreover, newly
PRRSV epidemic strains and highly pathogenic PRRSV, classical PRRSV mixed infections, cause the complexity of the clinically state of an illness, therefore, need
The vaccine combination for the newest clinical setting in China is developed, effectively to prevent the new porcine reproductive and respiratory syndrome in China
Prevalence.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of vaccine group for being used to prevent porcine reproductive and respiratory syndrome
Compound, the vaccine combination are made by porcine reproductive and respiratory syndrome virus strain, the porcine reproductive and respiratory syndrome virus strain
It is obtained to provide effective immunoprotection with pop street strain, show significant immunological characteristic.
One aspect of the present invention is to provide a kind of vaccine combination for being used to prevent porcine reproductive and respiratory syndrome, its
In, classical the porcine reproductive and respiratory syndrome virus antigen and/or high-pathogenicity porcine that the vaccine combination includes immune amount are numerous
Grow and breath syndrome virus antigen and/or porcine reproductive and respiratory syndrome virus class NADC30 strain antigens, adjuvant and medicine
Acceptable carrier on.
One aspect of the present invention is to provide a kind of method for preparing the vaccine combination, wherein, methods described bag
Include：(1) porcine reproductive and respiratory syndrome virus described in Multiplying culture；(2) harvest, and centrifuge, abandoning supernatant；(3) utilize and contain
The isolate that the extraction step solution (2) of detergent obtains；(4) extract that step (3) obtains is centrifuged, harvests supernatant
Liquid；(5) adjuvant is added, is stirred.
One aspect of the present invention is that providing described vaccine combination is preparing prevention porcine reproductive and respiratory syndrome
Medicine in application.
One aspect of the present invention is to provide described vaccine combination in the breeding of preparation epidemic prevention pig and breathed comprehensive
Application in the medicine of the porcine reproductive and respiratory syndrome virus variant infection occurred after simulator sickness live vaccine.
Invention advantage
The present invention uses three kinds of different types of porcine reproductive and respiratory syndrome virus antigens, prepares polyvalent vaccine, has
Good immunogenicity, after being inoculated with pig, body can be stimulated rapidly to produce immunity, produce higher neutralizing antibody water
It is flat.Polyvalent vaccine prepared by the present invention can effectively protect caused by popular strain the singly phenomenon of infection or mixed infection, can
With relevant disease caused by effectively protecting swinery resistance porcine reproductive and respiratory syndrome virus, the productivity of swinery is improved.
Embodiment
Hereinafter, embodiments of the present invention are illustrated.
One aspect of the present invention is to provide a kind of vaccine combination for being used to prevent porcine reproductive and respiratory syndrome, its
In, classical the porcine reproductive and respiratory syndrome virus antigen and/or high-pathogenicity porcine that the vaccine combination includes immune amount are numerous
Grow and breath syndrome virus antigen and/or porcine reproductive and respiratory syndrome virus class NADC30 strain antigens, adjuvant and medicine
Acceptable carrier on.
Clinical symptoms caused by classical porcine reproductive and respiratory syndrome virus are Adult Pig apocleisis, expiratory dyspnea, fever, end
Tip cyanosis, dead, pneumonia before weaned piglet or after wean, sow generation miscarriage in antenatal 1 week, premature labor, stillbirth, again heat, after
Acroparalysis sustainable several months, still birth rate and the rise of the suckling pig death rate.Strain is represented as ATCC VR-2332 strains and CH-1a
Strain.
Clinical symptoms caused by highly pathogenic PRRSV are sow heating, miscarriage, and wean is forward and backward
The rise of the piglet death rate, all ages and classes pig infects, compared with classical porcine reproductive and respiratory syndrome virus, pig caused a disease
Power is significantly increased, and the Adult Pig death rate rises.Full genome sequencing result shows that high-pathogenicity porcine reproductive is sick with respiration syndrome
Compared with classical porcine reproductive and respiratory syndrome virus VR2332 strains, the 1594th~1680 nucleotides of its NSP2 gene lacks strain
Lose.Strain is represented as JXA1 strains, TJ strains and HuN4 strains.
Clinical symptoms caused by porcine reproductive and respiratory syndrome virus class NADC30 strains are bred and breathed between classical pig
Between clinical symptoms caused by syndrome virus and highly pathogenic PRRSV, the more classical pig breeding of virulence
It is strong with breath syndrome virus, but it is weaker than highly pathogenic PRRSV, it is comprehensive with breathing for the breeding of classical pig
Simulator sickness virus and the Strain of highly pathogenic PRRSV producer restructuring.Represent strain as
HNjz15 strains, HNyc15 strains, JL580 strains, FJZ03 strains, HENXX-1 strains, HENXC-4 strains, HENAN-HEB strains, HENAN-XINX
Strain, FJY04 strains.
As one embodiment of the present invention, the vaccine of the present invention for being used to prevent porcine reproductive and respiratory syndrome
Composition, wherein, the classical porcine reproductive and respiratory syndrome virus antigen is porcine reproductive and respiratory syndrome virus VR2332
Strain antigen, the highly pathogenic PRRSV antigen are that porcine reproductive and respiratory syndrome virus JXA1 strains resist
Original, the porcine reproductive and respiratory syndrome virus class NADC30 strains antigen are porcine reproductive and respiratory syndrome virus HNjz15 strains
Antigen.
As one embodiment of the present invention, the vaccine of the present invention for being used to prevent porcine reproductive and respiratory syndrome
Composition, wherein, the highly pathogenic PRRSV antigen is porcine reproductive and respiratory syndrome virus
JXA1 strain antigens, the porcine reproductive and respiratory syndrome virus class NADC30 strains antigen is porcine reproductive and respiratory syndrome virus
HNjz15 strains or HNyc15 strains or JL580 strains or FJZ03 strains or HENXX-1 strains or HENXC-4 strains or HENAN-HEB strains or
HENAN-XINX strains or FJY04 strain antigens.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus VR2332 strains antigen includes pig
The inactivation antigen of Reproductive and respiratory syndrome virus VR2332 strains or its culture, attenuation antigen, subunit antigen or synthetic peptide
Antigen.
As one embodiment of the present invention, it is numerous that the porcine reproductive and respiratory syndrome virus JXA1 strains antigen includes pig
Grow and the inactivation antigen of breath syndrome virus JXA1 strains or its culture, attenuation antigen, subunit antigen or antigenic synthetic peptide.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus HNjz15 strains antigen includes pig
The inactivation antigen of Reproductive and respiratory syndrome virus HNjz15 strains or its culture, attenuation antigen, subunit antigen or synthetic peptide
Antigen.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus HNyc15 strains antigen includes pig
The inactivation antigen of Reproductive and respiratory syndrome virus HNyc15 strains or its culture, attenuation antigen, subunit antigen or synthetic peptide
Antigen.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus JL580 strains antigen includes pig
The inactivation antigen of Reproductive and respiratory syndrome virus JL580 strains or its culture, attenuation antigen, subunit antigen or synthetic peptide resist
It is former.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus FJZ03 strains antigen includes pig
The inactivation antigen of Reproductive and respiratory syndrome virus FJZ03 strains or its culture, attenuation antigen, subunit antigen or synthetic peptide resist
It is former.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus HENXX-1 strain antigens include
The inactivation antigen of porcine reproductive and respiratory syndrome virus HENXX-1 strains or its culture, attenuation antigen, subunit antigen or synthesis
Peptide antigen.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus HENXC-4 strain antigens include
The inactivation antigen of porcine reproductive and respiratory syndrome virus HENXC-4 strains or its culture, attenuation antigen, subunit antigen or synthesis
Peptide antigen.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus HENAN-HEB strains antigen bag
Include porcine reproductive and respiratory syndrome virus HENAN-HEB strains or its culture inactivation antigen, attenuation antigen, subunit antigen or
Antigenic synthetic peptide.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus HENAN-XINX strains antigen bag
Include the inactivation antigen, attenuation antigen, subunit antigen of porcine reproductive and respiratory syndrome virus HENAN-XINX strains or its culture
Or antigenic synthetic peptide.
As one embodiment of the present invention, the porcine reproductive and respiratory syndrome virus FJY04 strains antigen includes pig
The inactivation antigen of Reproductive and respiratory syndrome virus FJY04 strains or its culture, attenuation antigen, subunit antigen or synthetic peptide resist
It is former.
" culture " is the different generation subcultures of virus, and those skilled in the art know gene between different generations
Sequence is only possible to occur small variation.
" vaccine combination " means the pharmaceutical composition containing porcine reproductive and respiratory syndrome virus immunogenicity.The medicine
Composition can induce, stimulate or strengthen the immune response that pig is directed to porcine reproductive and respiratory syndrome virus.Vaccine combination bag
Include inactivation antigen, attenuation antigen, subunit antigen, the antigenic synthetic peptide of the porcine reproductive and respiratory syndrome virus strain of immune amount.
" inactivation antigen ", also referred to as inactivated antigens, refer to being used as antigen to produce the suspension of the inactivation of viruses of immunity
Liquid.The example of inactivation antigen includes totivirus inactivation antigen and cracking type antigen.Can easily it be produced using known method
Inactivation antigen.For example, totivirus inactivation antigen can be obtained by using formalin processing virus.Cracking type antigen can be at ether
It is prepared after reason by peplos.
The virus that " attenuation antigen " is referred to having been weakened with virulence but still can replicated in host or on cell.This hair
Bright term " attenuation " used is used to refer to be mutated in a manner of cause of disease is lost pathogenic but holding immunogenicity to gene
Manually reduce pathogen toxicity.Generally, radiated, be chemically treated or external continuous high-order squamous subculture realizes attenuation by UV.
Artificial gene alteration, such as the specific nucleotide in known array is lacked so that virulence attenuation of.
" 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.It causes the possibility of side reaction small than totivirus antigen.
" antigenic synthetic peptide " refers to a kind of small peptide of the only component containing immunologic determinants, i.e., presses native protein by artificial means
The amino acid sequence synthesis protectiveness small peptide of matter, the antigen after being connected with carrier plus made by adjuvant.
The composition of composition or the amount of component of the present invention is preferably therapeutically effective amount.The therapeutically effective amount refers to
Their immunological role is played without causing excessive side effect institute necessary amounts in the host that composition is applied.Composition used and
The accurate amount of composition to be administered is by according to factor such as the type of the disease for the treatment of, the type of animal to be treated and year
Age, the mode of administration, and other compositions in composition and change.
As one embodiment of the present invention, the antigenic content of the porcine reproductive and respiratory syndrome virus VR2332 strains
For before inactivation >=105.0TCID50/ ml porcine reproductive and respiratory syndrome virus VR2332 strains or its culture inactivation antigen.
As one embodiment of the present invention, the antigenic content of the porcine reproductive and respiratory syndrome virus VR2332 strains
For before inactivation 106.0~108.0TCID50/ ml porcine reproductive and respiratory syndrome virus VR2332 strains or its culture inactivation antigen.
As one embodiment of the present invention, the antigenic content of the porcine reproductive and respiratory syndrome virus VR2332 strains
For before inactivation 107.0TCID50/ ml porcine reproductive and respiratory syndrome virus VR2332 strains or its culture inactivation antigen.
As one embodiment of the present invention, the highly pathogenic PRRSV JXA1 strain antigens
For before inactivation >=105.0TCID50/ ml highly pathogenic PRRSV JXA1 strains or the inactivation of its culture
Antigen.
As one embodiment of the present invention, the highly pathogenic PRRSV JXA1 strain antigens
For before inactivation 106.0~108.0TCID50/ ml highly pathogenic PRRSV JXA1 strains or its culture
Inactivation antigen.
As one embodiment of the present invention, the highly pathogenic PRRSV JXA1 strain antigens
For before inactivation 107.0TCID50/ ml highly pathogenic PRRSV JXA1 strains or the inactivation of its culture resist
It is former.
As one embodiment of the present invention, the antigenic content of the porcine reproductive and respiratory syndrome virus HNjz15 strains
For before inactivation >=105.0TCID50/ ml porcine reproductive and respiratory syndrome virus HNjz15 strains or its culture inactivation antigen.
As one embodiment of the present invention, the antigenic content of the porcine reproductive and respiratory syndrome virus HNjz15 strains
For before inactivation 106.0~108.0TCID50/ ml porcine reproductive and respiratory syndrome virus HNjz15 strains or its culture inactivation antigen.
As one embodiment of the present invention, the antigenic content of the porcine reproductive and respiratory syndrome virus HNjz15 strains
For before inactivation 107.0TCID50/ ml porcine reproductive and respiratory syndrome virus HNjz15 strains or its culture inactivation antigen.
As one embodiment of the present invention, the vaccine of the present invention for being used to prevent porcine reproductive and respiratory syndrome
Composition, wherein, the vaccine combination includes before inactivating >=106.0TCID50/ ml porcine reproductive and respiratory syndrome virus
VR2332 strains or its culture inactivation antigen, before inactivation >=106.0TCID50/ ml high-pathogenicity porcine reproductive and respiration syndrome disease
The inactivation antigen of malicious JXA1 strains or its culture, before inactivation >=106.0TCID50/ ml porcine reproductive and respiratory syndrome virus
HNjz15 strains or its culture inactivation antigen.
As one embodiment of the present invention, the vaccine of the present invention for being used to prevent porcine reproductive and respiratory syndrome
Composition, wherein, the vaccine combination is included 10 before inactivation6.0~108.0TCID50/ ml porcine reproductive and respiratory syndrome disease
Malicious VR2332 strains or its culture inactivation antigen, 10 before inactivation6.0~108.0TCID50/ ml high-pathogenicity porcine reproductive is comprehensive with breathing
The inactivation antigen of simulator sickness virus JXA1 strains or its culture, 10 before inactivation6.0~108.0TCID50/ ml pig breeding and breathing integrates
Levy virus HN jz15 strains or its culture inactivation antigen.
As one embodiment of the present invention, the vaccine of the present invention for being used to prevent porcine reproductive and respiratory syndrome
Composition, wherein, the vaccine combination is included 10 before inactivation7.0TCID50/ ml porcine reproductive and respiratory syndrome virus
VR2332 strains or its culture inactivation antigen, 10 before inactivation7.0TCID50/ ml highly pathogenic PRRSV
The inactivation antigen of JXA1 strains or its culture, 10 before inactivation7.0TCID50/ ml porcine reproductive and respiratory syndrome virus HNjz15 strains
Or its culture inactivation antigen.
Porcine reproductive and respiratory syndrome virus VR2332 strains, are disclosed in Chinese patent application CN1190349A.
Porcine reproductive and respiratory syndrome virus JXA1 strains, also known as porcine reproductive and respiratory syndrome virus ultrastrong variation strain
NVDC-JXA1 strains, it is disclosed in Chinese patent application CN101045917A.
Porcine reproductive and respiratory syndrome virus HNjz15 strains (Porcine reproductive and respiratory
Syndrome virus, strain HNjz15) preserving number is：CCTCC NO.V201540, depositary institution are Chinese Typical Representative culture
Thing collection, preservation address are Wuhan, China Wuhan University, and the preservation time is September in 2015 21.
As one embodiment of the present invention, the adjuvant includes white oil, Drake oil (Drakeoil), and other
Animal oil, vegetable oil or mineral oil；Or aluminium hydroxide, aluminum phosphate and other metal salts；MontanideTMGel, carbomer, spiny dogfish
Alkane or squalene, ISA206 adjuvants, saponin(e, water-in-oil emulsion, oil in water emulsion, W/O/W emulsion, DDA.
Techniques well known can be used to allocate in the vaccine combination of the present invention, in the present invention, the pharmaceutically acceptable vaccine
Adjuvant includes oily adjuvant, and it is selected from white oil, saualane or squalene, Drake oil (Drakeoil), and other animal oil, plant
Thing oil or mineral oil.Above-mentioned oily adjuvant both can be natural origin or pass through artificial synthesized acquisition.In the present invention,
The vaccine combination is oil-in-water emulsion, water-in-oil emulsion or double emulsion, and the double emulsion is usually expressed as oil-in-water
Bag aqueous emulsion.
In an embodiment of the invention, the pharmaceutically acceptable carrier include suspending agent, surfactant,
Antigens inactive agent or preservative.The suspending agent may include, for example, aluminum stearate, and art it is available other
Suspending agent.The surfactant may include, for example, dehydration mountain plough alcohol monoleate (TWEEN series), department this (SPAN), with
And the available other surfaces activating agent of art.The antigens inactive agent includes but is not limited to, such as formalin,
Beta-propiolactone etc..The preservative includes, such as thimerosal.The application method and dosage of above-mentioned substance are this area skill
Known to art personnel.
Certain side reaction can be brought to animal body based on oily adjuvant, it is also an option that other adjuvants of this area, including
Aluminium hydroxide, aluminum phosphate and other metal salts, to prepare suspension, reduce immunostimulation
As one embodiment of the present invention, the adjuvant includes oil in water emulsion and DDA.
" oil in water emulsion " refers to be made up of oil, emulsifying agent, and described emulsifying agent includes hydrophilic surfactant active, oleophylic
Property surfactant, water composition.Wherein described " oil "：Being suitable for the oil of the present invention includes alkane, alkene, alkynes, acid corresponding to it and
Alcohol, and its ether and ester, and its mixture.The individual compound of oil is light hydrocarbon compounds, i.e. this kind of component has 6 to 30 carbon
Atom.The oil can be synthetically prepared or purified from petroleum product.This part can have straight or branched construction.It can be fully saturated
Or with one or more double bonds or ginseng key.Some nonmetabolizable oil for the present invention include, such as：Mineral oil, paraffin
Oil and cycloalkane.Including " light mineral oil ", i.e.,：Obtained in a similar fashion by distilling oil, but its proportion is compared with white mineral oil slightly
Low oil.Metabolizable oil includes metabolizable, nontoxic oil.Such as saualane, squalene.Typically, of the invention oily group
The amount divided was 1% to 50% (by volume)；Or it is 10% to 45%；Or it is 20% to 40%.Described " lipophile table
Face activating agent " is selected from including but not limited to Si Ben -80, mannitol oleate or mannide oleate AEO-3 one kind
Or several combinations；Preferably, described lipophilic surfactant preferably sucrose derivative has at least one but is not more than
The sucrose ester of N-1 fatty acid ester group；Preferably, sucrose derivative is sulfuric ester) 1- (lauroyl) 7- sucrose, (sulfuric ester)
1- (lauroyl) 7- sucrose, sucrose ester L195 (Mitsubishi-Kagaku food companies, Tokyo, Japan), (sulfuric ester) 2-
L195 one or several kinds of combinations.Described " hydrophilic surfactant active " is from including but not limited to Tween-80, sucrose ester
L1695Mitsubishi-Kagaku food companies, Tokyo, Japan), polyethylene mannitol oleate, lecithin, AE0-9,
AE0-12, poloxamer188 one or several kinds of mixing.
" DDA " is the abbreviation of DDA, its usage amount be usually per dosage about 1 microgram extremely
About 5000 micrograms.Its usage amount also can be per the microgram of dosage about 1 to about 4000 micrograms, per the microgram of dosage about 1 to about 3000 micrograms,
Per the microgram of dosage about 1 to about 2000 micrograms and per the microgram of dosage about 1 to about 1000 micrograms.Its usage amount also can be per dosage about 5
Microgram is micro- to about 750 micrograms, every microgram of dosage about 5 to about 500 micrograms, every microgram of dosage about 5 to about 200 micrograms, every dosage about 5
Gram to about 100 micrograms, per the microgram of dosage about 15 to about 100 micrograms and per the microgram of dosage about 30 to about 75 micrograms.
One aspect of the present invention is to provide a kind of method for preparing the vaccine combination, wherein, methods described bag
Include：(1) porcine reproductive and respiratory syndrome virus described in Multiplying culture；(2) harvest, and centrifuge, abandoning supernatant；(3) utilize and contain
The isolate that the extraction step solution (2) of detergent obtains；(4) extract that step (3) obtains is centrifuged, harvests supernatant
Liquid；(5) adjuvant is added, is stirred.
As one embodiment of the present invention, the method provided by the invention for preparing the vaccine combination, wherein, step
Suddenly it is by porcine reproductive and respiratory syndrome virus the step of porcine reproductive and respiratory syndrome virus described in Multiplying culture described in (1)
Vaccine strain is inoculated in respective permissive cell, and cultivates the permissive cell after the inoculation, harvesting culture；It is described susceptible
Cell can be continuous cell line or primary cell.It is suitable for the permissive cell of porcine reproductive and respiratory syndrome virus
Including but not limited to, (ATCC is numbered ST cell lines：CRL-1746), (ATCC is numbered PK-15 cell lines：CCL-33), Africa is green
(ATCC is numbered MK cells Marc-145 cell lines：CRL-12219), (ATCC is numbered bovine kidney cells MDBK cell lines：CCL-
22), (ATCC is numbered nose of an ox first osteocyte BT cell lines：CRL-1390), (ATCC is numbered Vero cell line：CCL-81)、BHK-
(ATCC is numbered 21 cell lines：CCL-10), porcine kidney cell system is (such as：IBRS-2, see for example, DECASTRO,
M.P.1964.Behavior offoot and mouth disease virus in cell culture：
susceptibility of the IB-RS-2swine cell line.ArquivosInstitutoBiologica 31：
63-78), rabbit kidney continuous cell line (RK, such as：ATCC is numbered：The continuous cell line such as CCL-106), or chicken embryo fibroblasts,
The primary cell such as PAM cells and porcine kidney cell.Primary cell can by means commonly known in the art, with the tissue in animal body
Separated and prepared.
As one embodiment of the present invention, the method provided by the invention for preparing the vaccine combination, wherein, step
Suddenly the solution that (3) include detergent can be adapted for any solution for extracting PRRSV antigens.One nonrestrictive example is to make
With non-ionic detergent, such as poly- (Ethylene Glycol) p- isooctyl-phenyl ether, (promise is P- to the stupid epoxide polyethoxy ethanol of octyl group
40 (Nonidet P-40)) or triton x-100 (Triton X-100).
In some embodiments of the present invention, the solution comprising detergent is 0.05M tri- (carboxymethyl) aminomethane
0.025M edta buffer liquid, the volume of buffer solution are 5~10 times of step (2) isolate.
In some embodiments of the present invention, the concentration of detergent is about 0.5% in solution, for example, about 0.5% triton
X-100 solution.
In some embodiments of the present invention, the extraction process of step (3) is stirred 2~15 hours under the conditions of 4 DEG C.
In some embodiments of the present invention, step (4) centrifugal rotational speed is 10000g.
One aspect of the present invention is that providing described vaccine combination is preparing prevention porcine reproductive and respiratory syndrome
Medicine in application.
One aspect of the present invention is to provide described vaccine combination in the breeding of preparation epidemic prevention pig and breathed comprehensive
Application in the medicine of the porcine reproductive and respiratory syndrome virus variant infection occurred after simulator sickness live vaccine.
" prevention " refers to suppresses porcine reproductive and respiratory syndrome virus infection by the vaccine combination given according to the present invention
Or postpone all behaviors of seizure of disease.
The invention will now be further described with reference to specific embodiments, and advantages of the present invention and feature will be with description more
To be clear.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.Those skilled in the art
It should be understood that the details and form of technical solution of the present invention can be carried out without departing from the spirit and scope of the invention
Modifications or substitutions, but these modifications and replacement are each fallen within protection scope of the present invention.
Heretofore described " TCID50" (50%tissue culture infective dose) refer to half cell
Culture infective dose, it is a kind of representation for representing virus infectivity.
DMEM fluid nutrient mediums (liquid) with purchased from Life Technologies companies of the U.S. DMEM dehydrated mediums according to
Its specification is prepared.
The DMEM culture mediums of the present invention are prepared with reference to GB/T18641-2002 appendix As compound method.
Adjuvant of the present invention includes oil in water emulsion and DDA.
Oil in water emulsion preparation method：By sucrose ester L195 10g (Mitsubishi-Kagaku food companies, Tokyo, days
This) and Tween-80 10g, injection white oil 40g for animals, 190g 0.01w/v% thimerosals (Sigma) phosphate buffered saline solution
(PBS- thimerosals；PH7.0) it is mixed to prepare.Under environment temperature, with least 400 bars of internal pressure, carried out three times by homogenizer
Mixture is emulsified.Each emulsion is detected under microscope.If in the case where amplifying 1000 times of microscope, every 10
There is oil droplet of more than 10 diameters more than 1 μm by Yezhong is inspected, that just repeats the emulsion process.The emulsion of gained is preserved
In 4 DEG C until using.
DDA solution manufacturing methods：By GERBU Adjuvant 100 (DDA；Fluka Analytical) it is dissolved in
In ethanol, the stock solution of 15 mg/mls is prepared, with 0.2 zut filter DDA stock solutions.
Adjuvant preparation method：It is well mixed that DDA solution is added to oil in water emulsion.
Embodiment 1
The preparation of porcine reproductive and respiratory syndrome virus JXA1 strain antigens
Porcine reproductive and respiratory syndrome virus JXA1 strain cultures are inoculated in Marc-145 cells, according to virus-culturing fluid
1% (V/V) accesses of amount are formed in the Marc-145 cell cultures of individual layer, put 37 DEG C of cultures, harvest contains within -48 hours 24 hours
Poison cell nutrient solution, make cell agglomerating through centrifugation, supernatant discarding.Cell mass is resuspended to 0.05M tri- (carboxymethyl) aminomethane
In 0.025MEDTA buffer solutions, wherein comprising 0.5% triton x-100, the volume of buffer solution is 5-10 times of concentrating cells.Mixing
Thing is stirred 2-15 hours at 4 DEG C, and 10000g is centrifuged 1 hour.It is standby to harvest supernatant.
Embodiment 2
The conventional preparation of porcine reproductive and respiratory syndrome virus JXA1 strain antigens
Porcine reproductive and respiratory syndrome virus JXA1 strain cultures are inoculated in Marc-145 cells, according to virus-culturing fluid
1% (V/V) accesses of amount are formed in the Marc-145 cell cultures of individual layer, put 37 DEG C of cultures, when lesion reaches 80%, are received
Toxic cell culture fluid is obtained, after 2 freeze thawing, poison is received, determines malicious valency.Adding 10% (v/v) formalin makes the end of formaldehyde dense
Spend for 0.2% (V/V), 37 DEG C inactivate 18 hours, stir 1 time within every 4 hours, stir 10min every time, standby after inactivating completely.
Embodiment 3
The preparation of porcine reproductive and respiratory syndrome virus JXA1 strain vaccine compositions
Porcine reproductive and respiratory syndrome virus JXA1 strains antigen prepared by embodiment 1 and embodiment 2 respectively with containing DDA's
Oil in water emulsion adjuvant is according to volume ratio 50:50 mixing, 120 revs/min are stirred 15 minutes under the conditions of 30 DEG C.Specific proportioning is shown in
Table 1.
The PRRSV JXA1 strain vaccines composition of table 1 matches
Embodiment 4
Porcine reproductive and respiratory syndrome virus JXA1 strain vaccine composition Study On Immunogenicities
43 age in days PRRSV antigen-antibody feminine genders piglets 35 are randomly divided into 7 groups, 5/group, vaccinated according to table 2,
Control group is inoculated with DMEM culture medium 2ml/ heads.
The PRRSV JXA1 strain vaccine composition Study On Immunogenicity animal packets of table 2
| Group | Vaccinate | Immunizing dose |
| 1 | Vaccine 1 | 2ml/ heads |
| 2 | Vaccine 2 | 2ml/ heads |
| 3 | Vaccine 3 | 2ml/ heads |
| 4 | Vaccine 4 | 2ml/ heads |
| 5 | Vaccine 5 | 2ml/ heads |
| 6 | Vaccine 6 | 2ml/ heads |
| 7 | DMEM culture mediums | 2ml/ heads |
Poison is attacked after immune within 28 days, attacks poison with porcine reproductive and respiratory syndrome virus JXA1 strains, attacking toxic agent amount is
105.5TCID50/ head, observation clinical symptoms are shown in Table 3.
Malicious situation is attacked after the immune piglet of the PRRSV JXA1 strain vaccines composition of table 3
As a result show, piglet is immunized in porcine reproductive and respiratory syndrome virus vaccine combination prepared by the inventive method, uses
Poison is attacked in JXA1 strains, energy blocking virus infection (clinical symptoms occur), 100% (5/5) protection is provided for piglet；Trained with regular growth
Piglet is immunized in porcine reproductive and respiratory syndrome virus vaccine combination prepared by the method for supporting, and poison is attacked with JXA1 strains, it is impossible to resistance completely
Disconnected virus infection (clinical symptoms occur), it is only capable of the limited protection of offer 40% (2/5)~80% (4/5)；It is especially of the invention
Provide lower antigenic content and can be achieved with the complete protection to porcine reproductive and respiratory syndrome virus JXA1 strains；It is right to attack poison
Attacked according to piglet after poison and clinical symptoms all occur, part pig is dead.
Above-mentioned experiment demonstrate the inventive method offer porcine reproductive and respiratory syndrome virus vaccine combination have it is good
Good immune effect, vaccine combination prepared by more existing regular growth cultural method have more preferable immune protective effect.
Embodiment 5
The preparation of porcine reproductive and respiratory syndrome virus VR2332 strain antigens
Porcine reproductive and respiratory syndrome virus VR2332 strain cultures are inoculated in Marc-145 cells, according to Virus culture
1% (V/V) accesses of liquid measure are formed in the Marc-145 cell cultures of individual layer, put 37 DEG C of cultures, are harvested within -48 hours 24 hours
Toxic cell culture fluid, make cell agglomerating through centrifugation, supernatant discarding.Cell mass is resuspended to 0.05M tri- (carboxymethyl) amino first
In alkane 0.025M edta buffer liquid, wherein comprising 0.5% triton x-100, the volume of buffer solution is 5-10 times of concentrating cells.
Mixture is stirred 2-15 hours at 4 DEG C, and 10000g is centrifuged 1 hour.It is standby to harvest supernatant.
Embodiment 6
The conventional preparation of porcine reproductive and respiratory syndrome virus VR2332 strain antigens
Porcine reproductive and respiratory syndrome virus VR2332 strain cultures are inoculated in Marc-145 cells, according to Virus culture
1% (V/V) accesses of liquid measure are formed in the Marc-145 cell cultures of individual layer, put 37 DEG C of cultures, when lesion reaches 80%,
Toxic cell culture fluid is harvested, after 2 freeze thawing, poison is received, determines malicious valency.Adding 10% (v/v) formalin makes the end of formaldehyde
Concentration is 0.2% (V/V), and 37 DEG C inactivate 18 hours, stir 1 time within every 4 hours, stir 10min every time, standby after inactivating completely.
Embodiment 7
The preparation of porcine reproductive and respiratory syndrome virus HNjz15 strain antigens
Porcine reproductive and respiratory syndrome virus HNjz15 strain cultures are inoculated in PAM cells, according to Virus culture liquid measure
1% (V/V) access formed in the PAM cell cultures of individual layer, put 37 DEG C of cultures, harvest contains poison cell within -48 hours 24 hours
Nutrient solution, make cell agglomerating through centrifugation, supernatant discarding.Cell mass is resuspended to (carboxymethyl) aminomethanes of 0.05M tri- 0.025M
In edta buffer liquid, wherein comprising 0.5% triton x-100, the volume of buffer solution is 5-10 times of concentrating cells.Mixture is 4
DEG C stirring 2-15 hours, 10000g centrifuge 1 hour.It is standby to harvest supernatant.Embodiment 8
The conventional preparation of porcine reproductive and respiratory syndrome virus HNjz15 strain antigens
Porcine reproductive and respiratory syndrome virus HNjz15 strain cultures are inoculated in PAM cells, according to Virus culture liquid measure
1% (V/V) access formed in the PAM cell cultures of individual layer, put 37 DEG C of cultures, when lesion reaches 80%, harvest it is toxic
Cell culture fluid, after 2 freeze thawing, poison is received, determines malicious valency.Adding 10% (v/v) formalin makes the final concentration of of formaldehyde
0.2% (V/V), 37 DEG C inactivate 18 hours, stir 1 time within every 4 hours, stir 10min every time, standby after inactivating completely.
Embodiment 9
Porcine reproductive and respiratory syndrome virus VR2332 strains, the preparation of HNjz15 strain vaccine compositions
Porcine reproductive and respiratory syndrome virus VR2332 prepared by embodiment 5, embodiment 6, embodiment 7 and embodiment 8
Strain, HNjz15 strains antigen are respectively with the oil in water emulsion adjuvant containing DDA according to volume ratio 50:50 mixing, 120 under the conditions of 30 DEG C
Rev/min stirring 15 minutes.Specific proportioning is shown in Table 4.
The PRRSV VR2332 strains of table 4, HNjz15 strain vaccines composition proportioning
Embodiment 10
Porcine reproductive and respiratory syndrome virus VR2332 strains, HNjz15 strain vaccine composition Study On Immunogenicities
43 age in days PRRSV antigen-antibody feminine genders piglets 50 are randomly divided into 10 groups, 5/group, vaccinated according to table 5,
Control group is inoculated with DMEM culture medium 2ml/ heads.
The PRRSV VR2332 strains of table 5, HNjz15 strain vaccine composition Study On Immunogenicity animal packets
Poison is attacked within 28 days after immune, the 8th group, the 9th group, the 10th group, the 11st group, the 16th group is used porcine reproductive and respiratory syndrome
Poison is attacked in VR2332 strains, and it is 10 to attack toxic agent amount5.5TCID50/ head, observation clinical symptoms are shown in Table 6.
Malicious situation is attacked after the immune piglet of the PRRSV VR2332 strain vaccines composition of table 6
| Group | Clinical symptoms | Protective rate |
| 8 | It is without exception | 100% (5/5) |
| 9 | It is without exception | 100% (5/5) |
| 10 | 3 body temperature rises, appetite reduce, and spirit is depressed | 40% (2/5) |
| 11 | 1 body temperature rise, appetite reduce, and spirit is depressed | 80% (4/5) |
| 16 | Whole body temperature rises, appetite reduce, and spirit is depressed | 0% (0/5) |
As a result show, piglet is immunized in porcine reproductive and respiratory syndrome virus vaccine combination prepared by the inventive method, uses
Poison is attacked in VR2332 strains, energy blocking virus infection (clinical symptoms occur), 100% (5/5) protection is provided for piglet；Use regular growth
Piglet is immunized in porcine reproductive and respiratory syndrome virus vaccine combination prepared by cultural method, attacks poison with VR2332 strains, it is impossible to complete
Full blocking virus infection (clinical symptoms occur), it is only capable of the limited protection of offer 40% (2/5)~80% (4/5)；Especially originally
Invention provides lower antigenic content and can be achieved with the complete protection to porcine reproductive and respiratory syndrome virus VR2332 strains；
Attack after poison control piglet attacks poison and clinical symptoms all occur.
Above-mentioned experiment demonstrate the inventive method offer porcine reproductive and respiratory syndrome virus vaccine combination have it is good
Good immune effect, vaccine combination prepared by more existing regular growth cultural method have more preferable immune protective effect.
Poison is attacked after immune within 28 days, attacks poison with porcine reproductive and respiratory syndrome HNjz15 strains, attacking toxic agent amount is
105.5TCID50/ head, observation clinical symptoms are shown in Table 7.
Malicious situation is attacked after the immune piglet of the PRRSV HNjz15 strain vaccines composition of table 7
As a result show, piglet is immunized in porcine reproductive and respiratory syndrome virus vaccine combination prepared by the inventive method, uses
Poison is attacked in HNjz15 strains, energy blocking virus infection (clinical symptoms occur), 100% (5/5) protection is provided for piglet；Use regular growth
Piglet is immunized in porcine reproductive and respiratory syndrome virus vaccine combination prepared by cultural method, attacks poison with HNjz15 strains, it is impossible to complete
Full blocking virus infection (clinical symptoms occur), it is only capable of the limited protection of offer 40% (2/5)~80% (4/5)；Especially originally
Invention provides lower antigenic content and can be achieved with the complete protection to porcine reproductive and respiratory syndrome virus HNjz15 strains；
Attack after poison control piglet attacks poison and clinical symptoms all occur.
Above-mentioned experiment further demonstrates the porcine reproductive and respiratory syndrome virus vaccine combination of the inventive method offer
With good immune effect, there is vaccine combination prepared by more existing regular growth cultural method more preferable immunoprotection to imitate
Fruit.
Embodiment 11
The preparation of porcine reproductive and respiratory syndrome virus vaccine combination
Porcine reproductive and respiratory syndrome virus JXA1 strains prepared by embodiment 1, embodiment 5, embodiment 7, VR2332 strains,
HNjz15 strains antigen is mixed again with the oil in water emulsion adjuvant containing DDA according to volume ratio 50:50 mixing, 120 under the conditions of 30 DEG C
Rev/min stirring 15 minutes.Specific proportioning is shown in Table 8.
The PRRSV vaccine combinations of table 8 match
Embodiment 12
Porcine reproductive and respiratory syndrome virus mixed infection is tested
43 age in days PRRSV antigen-antibody feminine genders piglets 25 are randomly divided into 5 groups, 5/group, according to the injecting virus of table 9,
Control group is inoculated with DMEM culture medium 2ml/ heads.
The PRRSV mixed infections experimental animal of table 9 is grouped
After each group attacks poison, observation clinical symptoms are shown in Table 10.
The PRRSV mixed infection result of the tests of table 10
As a result show, either attack poison with porcine reproductive and respiratory syndrome virus JXA1 strains, VR2332 strains or HNjz15 strains,
It can not make pig all dead, and poison is attacked with three kinds of porcine reproductive and respiratory syndrome virus hybrid viruses, even if general attack poison amount
Only 105.0TCID50(attack poison amount 10 less than single strain5.5TCID50), it will also result in and attack all death of malicious pig；Control group is no different
Often.Demonstrate, the present invention can cause porcine reproductive and respiratory syndrome illness to aggravate using three kinds of virus mixed infection pigs, three kinds
Viral coinfection effect.
Embodiment 13
Porcine reproductive and respiratory syndrome virus vaccine combination Study On Immunogenicity
43 age in days PRRSV antigen-antibody feminine genders piglets 25 are randomly divided into 5 groups, 5/group, vaccinated according to table 11,
Control group is inoculated with DMEM culture medium 2ml/ heads.
The PRRSV vaccine combination Study On Immunogenicity animal packets of table 11
| Group | Vaccinate | Immunizing dose |
| 23 | Vaccine 15 | 2ml/ heads |
| 24 | Vaccine 16 | 2ml/ heads |
| 25 | Vaccine 17 | 2ml/ heads |
| 26 | Vaccine 18 | 2ml/ heads |
| 27 | DMEM culture mediums | 2ml/ heads |
Poison is attacked after immune within 28 days, and with porcine reproductive and respiratory syndrome, (JXA1 strains, VR2332 strains and HNjz15 strains mix disease
Poison) poison is attacked, toxic agent amount is attacked as 105.0TCID50/ head, observation clinical symptoms are shown in Table 12.
The PRRSV vaccine combination Study On Immunogenicity results of table 12
As a result show, piglet is immunized in porcine reproductive and respiratory syndrome virus vaccine combination prepared by the inventive method, uses
JXA1 strains, VR2332 strains and the attack of HNjz15 strains hybrid virus, energy blocking virus infection (clinical symptoms occur), are provided for piglet
Protect 100% (5/5).
Embodiment 14
Porcine reproductive and respiratory syndrome virus vaccine combination immunogenicity contrast test
43 age in days PRRSV antigen-antibody feminine genders piglets 25 are randomly divided into 5 groups, 5/group, vaccinated according to table 13,
Control group is inoculated with DMEM culture medium 2ml/ heads.
The PRRSV vaccine combination immunogenicity contrast test animal packets of table 13
| Group | Vaccinate | Immunizing dose |
| 28 | Vaccine 15 | 2ml/ heads |
| 29 | Vaccine 4 | 2ml/ heads |
| 30 | Vaccine 8 | 2ml/ heads |
| 31 | Vaccine 12 | 2ml/ heads |
| 32 | DMEM culture mediums | 2ml/ heads |
Poison is attacked after immune within 28 days, and with porcine reproductive and respiratory syndrome, (JXA1 strains, VR2332 strains and HNjz15 strains mix disease
Poison) poison is attacked, toxic agent amount is attacked as 105.0TCID50/ head, observation clinical symptoms are shown in Table 14.
The PRRSV vaccine combination immunogenicity comparative test results of table 14
As a result show, porcine reproductive and respiratory syndrome virus vaccine combination prepared by the inventive method is with relatively low antigen
Piglet is immunized in content, with JXA1 strains, VR2332 strains and HNjz15 strains hybrid virus attack, also can blocking virus infection (face
Bed symptom), provide 100% (5/5) protection for piglet；And porcine reproductive and respiratory syndrome virus JXA1 prepared by the inventive method
Even if strain vaccine composition, VR2332 strain vaccines composition, HNjz15 strain vaccines composition are with the immune son of higher antigenic content
Pig, it can not also block and infect (clinical symptoms occur) caused by being attacked with JXA1 strains, VR2332 strains and HNjz15 strains hybrid virus,
The protection of offer 20%~40% is provided.Existing market can be resisted by illustrating the vaccine combination of three kinds of antigen provided by the invention
The infection of the different porcine reproductive and respiratory syndrome virus of upper three popular classes, particularly with complexity mixed infection situation,
There is preferable immanoprotection action, the purification for porcine reproductive and respiratory syndrome virus has great significance.
Embodiment 15
The vaccine combination of the present invention prophylactic tria that pig farm is fallen ill after immune porcine reproductive and respiratory syndrome live vaccine
This research includes 768 market pigs (porcine reproductive and respiratory syndrome live vaccine was immunized), by these pigs according to body
Again and whether a brood of give birth to distinguishes, random point of 2 groups, i.e. vaccine immunity group (402), control group (366).
A groups：On the day of research (the 0th day), the vaccine 16 of the inoculation present invention, 2ml/ heads are carried out to the pig of about 43 ages in days.
B groups：The non-immunized pig of control group.
Terminate research within 4th week after immune.
Pig farm incidence
According to raising and the morbidity history on farm, animal undergoes porcine reproductive and respiratory syndrome relevant clinical disease since the mid-term
Shape.Now, it is considered as being related to after Swinery immunity porcine reproductive and respiratory syndrome live vaccine with being led after poison, with wild poison again subinfection
Cause morbidity.
As a result
During whole experiment, immune group occurs without the phenomena of mortality, beginning pig body temperature rise in 2 weeks, part pig
Appetite slightly drops, and gradually recovers after 3 weeks；And control group, from beginning pig body temperature rise in 2 weeks, gradual expanded scope, skin hair
Red, spirit is depressed, and appetite declines, and respiratory symptom occurs, gradually has the phenomena of mortality after 3 weeks；Cutoff test terminates, and control group has
159 pig morbidities, wherein 50 dead.
By carrying out extraction of diseased material to dead pig, highly pathogenic PRRSV and pig are identified as
Reproductive and respiratory syndrome virus class NADC30 strain mixed infections.
As a result show, vaccine combination of the present invention can solve immune swine breeding to pig progress vaccine inoculation and breathe comprehensive
After simulator sickness live vaccine, the problem of pig farm is still fallen ill, for preventing the restructuring variation of porcine reproductive and respiratory syndrome virus and net
Change has great significance.
Embodiment 16
The preparation of porcine reproductive and respiratory syndrome virus class NADC30 strain antigens
It is comprehensive with breathing using pig breeding that the present embodiment prepares porcine reproductive and respiratory syndrome virus class NADC30 strains antigen
Simulator sickness virus HN yc15 strains (complete genome sequence accession number KT945018.1), JL580 strains (complete genome sequence accession number
KR706343.1), FJZ03 strains (complete genome sequence accession number KP860909.1), HENXX-1 strains (complete genome sequence accession number
KU950372.1), (complete genome sequence logs in for HENXC-4 strains (complete genome sequence accession number KU950371.1), HENAN-HEB strains
Number KJ143621.1), HENAN-XINX strains (complete genome sequence accession number KF611905.1), (complete genome sequence logs in for FJY04 strains
Number KP860910.1) prepared.
Above-mentioned porcine reproductive and respiratory syndrome virus class NADC30 strain cultures are inoculated in PAM cells respectively, according to
1% (V/V) accesses of Virus culture liquid measure are formed in the PAM cell cultures of individual layer, put 37 DEG C of cultures, -48 hours 24 hours
Toxic cell culture fluid is harvested, makes cell agglomerating through centrifugation, supernatant discarding.Cell mass is resuspended to 0.05M tri- (carboxymethyl) ammonia
In methylmethane 0.025M edta buffer liquid, wherein comprising 0.5% triton x-100, the volume of buffer solution is the 5-10 of concentrating cells
Times.Mixture is stirred 2-15 hours at 4 DEG C, and 10000g is centrifuged 1 hour.It is standby to harvest supernatant.
Embodiment 17
The preparation of porcine reproductive and respiratory syndrome virus (JXA1 strains, class NADC30 strains) vaccine combination
Porcine reproductive and respiratory syndrome virus JXA1 strains prepared by embodiment 1, embodiment 7, embodiment 16, class NADC30
Strain mixing is again with the oil in water emulsion adjuvant containing DDA according to volume ratio 50:50 mixing, 120 revs/min of stirrings under the conditions of 30 DEG C
15 minutes.Specific proportioning is shown in Table 15.
The PRRSV of table 15 (JXA1 strains, class NADC30 strains) vaccine combination matches
Embodiment 18
Porcine reproductive and respiratory syndrome virus (JXA1 strains, class NADC30 strains) vaccine combination Study On Immunogenicity
43 age in days PRRSV antigen-antibody feminine genders piglets 90 are randomly divided into 18 groups, 5/
Group, vaccinated according to table 16, control group inoculation DMEM culture medium 2ml/ heads.
The vaccine combination of table 16 (JXA1 strains, class NADC30 strains) Study On Immunogenicity animal packet
Poison is attacked after immune within 28 days, is attacked poison with porcine reproductive and respiratory syndrome (JXA1 strains, class NADC30 strains hybrid virus), is attacked
Toxic agent amount is 105.0TCID50/ head, observation clinical symptoms are shown in Table 17.
The vaccine combination of table 17 (JXA1 strains, class NADC30 strains) Study On Immunogenicity
As a result show, porcine reproductive and respiratory syndrome virus (JXA1 strains, class NADC30 strains) epidemic disease prepared by the inventive method
Piglet is immunized in seedling composition, is attacked with JXA1 strains and class NADC30 strains hybrid virus, and energy blocking virus infection (clinical condition occurs
Shape), provide 100% (5/5) protection for piglet.
Popular at least two classes of in the market can be resisted not by illustrating the vaccine combination of two kinds of antigen provided by the invention
The infection of same porcine reproductive and respiratory syndrome virus, particularly with the situation of the mixed infection of complexity, have preferably immune
Protective effect, the purification for porcine reproductive and respiratory syndrome virus have great significance.
Described above is only the preferred embodiments of the present invention, not does any formal limitation to the present invention, though
So the present invention is disclosed above with preferred embodiment, but is not limited to the present invention, any to be familiar with this professional technology people
Member, in the range of technical solution of the present invention is not departed from, when the technology contents using the disclosure above make a little change or repair
The equivalent embodiment for equivalent variations is adornd, as long as being the content without departing from technical solution of the present invention, the technology according to the present invention is real
Any simple modification, equivalent change and modification that confrontation above example is made, still fall within the scope of technical solution of the present invention
It is interior.
Claims (10)
1. a kind of vaccine combination for being used to prevent porcine reproductive and respiratory syndrome, wherein, the vaccine combination includes immune
Classical the porcine reproductive and respiratory syndrome virus antigen and/or highly pathogenic PRRSV antigen of amount
And/or porcine reproductive and respiratory syndrome virus class NADC30 strain antigens, adjuvant and pharmaceutically acceptable carrier.
2. vaccine combination according to claim 1, wherein, the highly pathogenic PRRSV resists
Originally it was porcine reproductive and respiratory syndrome virus JXA1 strain antigens.
3. vaccine combination according to claim 2, wherein, the porcine reproductive and respiratory syndrome virus JXA1 strain antigens
Inactivation antigen including porcine reproductive and respiratory syndrome virus JXA1 strains or its culture, attenuation antigen, subunit antigen or conjunction
Into peptide antigen.
4. vaccine combination according to claim 2, wherein, the highly pathogenic PRRSV
JXA1 strains antigen for inactivation before >=106.0TCID50/ ml highly pathogenic PRRSV JXA1 strains or its training
Support the inactivation antigen of thing.
5. vaccine combination according to claim 2, wherein, the highly pathogenic PRRSV
JXA1 strains antigen is before inactivation 106.0~108.0TCID50/ ml highly pathogenic PRRSV JXA1 strains or
The inactivation antigen of its culture.
6. vaccine combination according to claim 2, wherein, the highly pathogenic PRRSV
JXA1 strains antigen is before inactivation 107.0TCID50/ ml highly pathogenic PRRSV JXA1 strains or its culture
The inactivation antigen of thing.
7. vaccine combination according to claim 1, wherein, the adjuvant includes oil in water emulsion and DDA.
8. a kind of method for preparing vaccine combination described in claim 1, wherein, methods described includes：(1) described in Multiplying culture
Porcine reproductive and respiratory syndrome virus；(2) harvest, and centrifuge, abandoning supernatant；(3) the solution extraction step containing detergent is utilized
Suddenly the isolate that (2) obtain；(4) extract that step (3) obtains is centrifuged, harvests supernatant；(5) adjuvant is added, is stirred
Mix uniformly.
9. according to the method for claim 8, wherein, it is p- that the solution that step (3) includes detergent includes poly- (Ethylene Glycol)
The stupid epoxide polyethoxy ethanol of isooctyl-phenyl ether, octyl group (promise is P-40 (Nonidet P-40)) or triton x-100
(Triton X-100)。
10. the vaccine combination described in any one of claim 1~7 is in the medicine for preparing prevention porcine reproductive and respiratory syndrome
Application, and occur after epidemic prevention porcine reproductive and respiratory syndrome live vaccine is prepared porcine reproductive and respiratory syndrome disease
Application in the medicine of malicious variant infection.
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| CN113521271A (en) * | 2021-05-27 | 2021-10-22 | 杭州佑本动物疫苗有限公司 | Porcine reproductive and respiratory syndrome bivalent inactivated vaccine and preparation method thereof |
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| CN112226419B (en) * | 2020-10-26 | 2022-07-19 | 兆丰华生物科技(福州)有限公司 | Preparation method of porcine reproductive and respiratory syndrome live vaccine |
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| CN116036253B (en) * | 2022-11-24 | 2023-12-26 | 畜科生物工程有限公司 | Live vaccine for porcine reproductive and respiratory syndrome and preparation method thereof |
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