CN102727883A - Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof - Google Patents
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Abstract
The invention provides a preparation method for a combined live vaccine used for preventing high pathogenic porcine reproductive and respiratory syndrome and swine fever and a product thereof. According to the invention, no immunosuppression occurs between an attenuated vaccine strain of high pathogenic porcine reproductive and respiratory syndrome and an attenuated vaccine strain of swine fever; the combined live vaccine prepared from the two attenuated vaccine strains is identical with each single vaccine in the aspects of security, immunogenicity, immunity duration and immuno-protective effects and has remarkable immuno-protective effects on preventing porcine reproductive and respiratory syndrome and swine fever. When the product provided in the invention is used to immunize and inoculate animals, two diseases can be prevented simultaneously with only one injection, which enables work load of immunization and inoculation to be mitigated, immunization frequency to be reduced, stress on swinery to be decreased, and immunological paralysis and immunological failure caused by frequent immunization to be avoided. The product of a vaccine preparation in the invention further has the advantages of a long storage life and storage stability.
Description
Technical field
The invention belongs to the veterinary biologics technical field, more specifically relate to a kind of method and goods thereof that prepare Porcine reproductive and respiratory syndrome and swine fever bigeminal live vaccine.
Background technology
Current; Porcine reproductive and respiratory syndrome (PRRS) and swine fever (CSF) are two kinds of important infectious disease of serious harm China pig industry; Since 2006 break out high-pathogenicity porcine reproductive and respiration syndrome, (claimed high-pathogenicity blue ear disease again); Caused enormous economic loss for the pig industry of China, classified as one of eqpidemic disease of compulsory immunization by the Ministry of Agriculture.Simultaneously swine fever also is that China classifies one of eqpidemic disease of compulsory immunization as, and therefore in the epidemic prevention process, spininess, multiple dose, panimmunity program are difficult to reasonable arrangement, both loaded down with trivial details, time-consuming, effort, increases cost, and causes easily with Louing and plant, and directly influences immune effect.Adopt the multiple vaccines immunity inoculation, can reach the purpose of " pin is anti-how sick ".Simultaneously, the immune programme for children that these two kinds of eqpidemic diseases are taked is more approaching, and this development for high-pathogenicity porcine reproductive and respiration syndrome and swine fever bigeminal live vaccine provides maybe.
Summary of the invention
One of the object of the invention provides a kind of immunity height, the porcine reproductive and respiratory syndrome virus vaccine of no immune interference and the bigeminy vaccine compositions of swine Fever Vaccine, reaches the purpose of anti-two diseases of a pin.
Two of the object of the invention provides a kind of bigeminy vaccine method for compositions for preparing porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine.
The application provides a kind of vaccine combination, and it contains porcine reproductive and respiratory syndrome virus (PRRSV) vaccine and swine Fever Vaccine.
Swine fever (Classical Swine Fever; CSF) be by swine fever virus (Classical Swine Fever Virus; CSFV) a kind of height contagiousness that causes, lethal pig infectious disease; In OIE disease register is listed it by OIE (OIE), is the infectious disease of legal essential report.In China, swine fever is one of great eqpidemic disease, in " one, two, three type of sick register of planting of animal epidemic ", lists swine fever one of in one type of animal epidemic, and the eruption and prevalence of swine fever causes serious economic loss for the pig industry of the world and China.
Swine fever virus belongs to flaviviridae, Pestivirus.This virus is a kind of positive chain RNA virus that cyst membrane is arranged.The about 12.5kb of genome total length; Only contain a big open reading frame (ORF); This ORF about 4000 amino acid residues of encoding; The polyprotein of the about 438kD of molecular weight, and further under the effect of virus and host cell proteins enzyme, be processed as 12 kinds of maturation proteins, all structural protein and the non-structural protein of CSFV are coded by this ORF.
Vaccine is the important means of control swine fever, comprises inactivated vaccine and attenuated vaccine.50～sixties of 20th century is the peak time of swine fever inactivated vaccine development, and during this period, formalin and crystal violet inactivated vaccine are widely used.But immunizing dose is big because inactivated vaccine exists, duration of immunity is short, slow, the more high shortcoming of cost of the time of generation immunity, is progressively replaced by the swine fever attenuated vaccine later on.The swine fever attenuated vaccine strain forms a little less than being caused by swine fever street strain, and abroad the someone reports in succession, with distinct methods different swine fever virus is adapted to rabbit, becomes the variation strain of virulence attenuation of, but these variation strains still can bring out severe reaction and death.Through using for many years, generally recognized as safe is effective, does not have the attenuated vaccine strain of remaining pathogenicity to have three kinds: 1. Chinese rabbitization attenuated vaccine; 2. Japanese GPE (-) cell weak-toxic vaccine; 3. French " Thiveosal " cold variation low virulent strain.China system (C system) hog cholera lapinised virus vaccine of succeeding in developing of Chinese scholar wherein from nineteen fifty-seven, except that China's extensive use, and has been generalized to Eurasian a lot of nationally, makes these state controls or has eliminated swine fever.This vaccine is acknowledged as more satisfactory in the world swine Fever Vaccine at present.
Hog cholera lapinised virus (C strain) is divided into according to the difference of production technology: first kind of technology is with the production of rabbit body, gets lymph node or the spleen of inoculation rabbit or organizes seedling, and promptly the swine fever spleen drenches Seedling and swine fever breast rabbit Seedling.This technology can effectively avoid exogenous virus to pollute, and has guaranteed viral hereditary stability simultaneously, but needs to use a large amount of rabbit, and the quality control difficulty is bigger, and production cost is higher.Second kind of technology is to use cattle, sheep primary cell or pig passage cell production seedling, i.e. swine fever cell vaccine.This technology avoids using in a large number laboratory animal, but the risk that exists exogenous virus to pollute.
PRRSV is a kind of positive chain RNA virus, has found two kinds of genotype at present: Europe class and american type.A plurality of ORFs are arranged in the PRRSV genome; Wherein first ORFs (ORF1a and ORF1b) contains the sequence of PRRSV genomic 80%; The necessary rna replicon enzyme of its coding PRRSV virus replication (Straw et al; Diseases of Swine, 9TH edition, chapter 24 (2006)).ORF1a and ORF1b are translated into a polyprotein (poly-protein), and the proteinase activity zone that this polyprotein is wherein contained cuts into a plurality of non-structural proteins, comprise that Nsp1-Nsp12 (sees; For example; Vries et al, Seminars in Virology, 8:33-47 (1997); Allende et al, Journal of General Virology, 80:307-315 (1999)).
On the one hand, the invention provides vaccine combination, it contains porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine, and has basically no immunosuppressant between described porcine reproductive and respiratory syndrome virus vaccine and the swine Fever Vaccine.
" have basically no immunosuppressant " and be meant, said porcine reproductive and respiratory syndrome virus vaccine and said swine Fever Vaccine can significantly not weaken the other side's immune effect later at immune swine.
In some embodiments, in the said vaccine combination, said swine Fever Vaccine can mix with said porcine reproductive and respiratory syndrome virus vaccine in the proper ratio.Can said swine Fever Vaccine and said porcine reproductive and respiratory syndrome virus vaccine be processed the antigen liquid with certain virus titer respectively, calculate blended ratio according to the virus titer that records again.In some embodiments, the mixed proportion of said porcine reproductive and respiratory syndrome virus vaccine and said swine Fever Vaccine is 1: 2 to 1: 4.
In some embodiments, the said swine Fever Vaccine in the vaccine combination provided by the invention is the hog cholera lapinised virus cell vaccine.In some embodiments, preferably C strain of said swine fever attenuated virus.In some embodiments, the coded sequence of said swine Fever Vaccine and SEQ ID NO:1 have at least 80% homology.In some embodiments, the coded sequence of said swine Fever Vaccine is SEQ ID NO:1.
In some embodiments, the said PRRSV vaccine PRRSV that is attenuation.In this application, " attenuation PRRSV " is meant a kind of PRRSV, and it can infection host, but can not cause Porcine reproductive and respiratory syndrome, and perhaps its symptom that causes is less and/or lighter.Attenuation PRRSV comprises attenuation PRRSV alive and the deactivation product that is obtained by its deactivation." Porcine reproductive and respiratory syndrome " is meant a series of physiology that cause after the natural PRRSV infected pigs and the symptom of pathology.These symptoms include, but not limited to heating, drowsiness, inappetence, asthenia, dyspnea, cough, sow breeding difficulty, piglet growth slowly or death etc.
In some embodiments, the said PRRSV vaccine PRRSV live vaccine that is attenuation.In some embodiments, said PRRSV vaccine contains the Nsp1 nucleotide sequence, and the coded sequence of said sequence and SEQ ID NO:2 have at least 90% homology.In some embodiments, said PRRSV vaccine further contains the Nsp2 nucleotide sequence, and the coded sequence of said Nsp2 nucleotide sequence and SEQ ID NO:3 have at least 90% homology.
" coded sequence " is meant a kind of DNA sequence in this application, and it can be transcribed obtains corresponding RNA sequence.PRRSV and swine fever virus all are positive chain RNA virus, and the coded sequence of its gene is DNA, and it can be transcribed into positive chain RNA, and the sequence in the genome of this positive chain RNA and virus itself is identical.
" homology " is meant the similarity degree of two aminoacid sequences or two nucleotide sequences in this application.Aminoacid sequence or homology of nucleotide sequence can calculate through any appropriate method well known in the art; For example; Can target amino acid (or nucleotide) sequence and reference aminoacid (or nucleotide) sequence be carried out sequence alignment; Can introduce vacancy in case of necessity, make that identical aminoacid (or nucleotide) number reaches optimization between the sequence of two comparisons, and calculate the percentage ratio of same amino acid (or nucleotide) between two aminoacid (or nucleotide) sequence on this basis.The comparison of aminoacid (or nucleotide) sequence and the calculating of homology can realize through software well known in the art, for example, but is not limited to, BLAST software (on the network address of the U.S. state-run biotechnology information centre (NCBI), can obtain:
Http:// blast.ncbi.nlm.nih.gov/Blast.cgi, perhaps see, for example, Altschul S.F. et al, J.Mol.Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 software (on European bio information institute network address, can obtain:
Http:// www.ebi.ac.uk/lools/msa/clustalw2/, other sees, for example, and Higgins D.G. et al, Methods in Enzymology, 266:383-402 (1996); Larkin M.A.et al, and Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007)); (on the website of Sweden's bioinformatics institute, can obtain: http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cg i with TCoffee software etc.; Other sees; For example; Poirot O.et al, Nucleic Acids Res., 31 (13): 3503-6 (2003); Notredame C.et al, J.Mol.Boil., 302 (1): 205-17 (2000)).When using software to carry out sequence alignment, the default parameters that can use software to provide perhaps also can be adjusted the parameter that software provides according to practical situation, and these are all in those skilled in the art's the ken.
In some embodiments, said porcine reproductive and respiratory syndrome virus vaccine contains the Nsp1 nucleotide sequence by SEQ ID NO:2 coding, and contains the Nsp2 nucleotide sequence by SEQ ID NO:3 coding.
In some embodiments, said porcine reproductive and respiratory syndrome virus vaccine contains the nucleotide sequence of porcine reproductive and respiratory syndrome virus, and the coded sequence of this sequence and SEQ ID NO:4 have at least 90% homology.In some embodiments, the nucleotide sequence of said porcine reproductive and respiratory syndrome virus is encoded by SEQ IDNO:4.
In the vaccine combination that the application provides, can also further contain adjuvant.Adjuvant can protect vaccine not destroy in the receptor, and/or non-specific ground stimulating immune system, thereby helps to strengthen the immunoreation to said vaccine.The example of adjuvant includes, but not limited to mineral salts (for example, aluminium hydroxide, aluminum phosphate, calcium hydroxide), water in oil emulsion (for example, Freund's complete adjuvant, incomplete Freund etc.), saponins (saponin) adjuvant (as: Stimulon
TMDeng), antibacterial or microorganism derivatives class (as, lipopolysaccharide, fat A derivant (lipid A derivatives) etc.) and microgranule (as gather-alpha-hydroxy acid etc.).
In the vaccine combination that the application provides, can also further contain freeze drying protectant.Freeze drying protectant can be protected the stability of biological product in freeze-drying process, reduces the bioactive destruction of freeze-drying process to vaccine.The example of freeze drying protectant comprises sucrose, L-sodium glutamate or lactoalbumin hydrolysate.
In some embodiments, the ratio of two kinds of antigen liquids and freeze drying protectant is 1: 4 in the vaccine combination of the present invention.
The seed culture of viruses of porcine reproductive and respiratory syndrome virus vaccine of the present invention and swine Fever Vaccine is respectively high-pathogenicity porcine reproductive and respiration syndrome low virulent strain and swine fever low virulent strain; Wherein, Said high-pathogenicity porcine reproductive and the preferably TJM strain of respiration syndrome attenuated virus, its microbial preservation number are: CGMCC NO.3121; The preferably C strain of said swine fever attenuated virus through passage, should have hog cholera lapinised virus (spleen poison) limiting dilution assay to carry out the two-wheeled clone purification and obtain the hog cholera lapinised virus cell toxicant, and its microbial preservation number is: CGMCC NO.3891.
The concrete preservation information of PRRSV TJM strain is following: microbial preservation number: CGMCC No.3121; The classification name; Porcine reproductive and respiratory syndrome virus; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation time: on June 15th, 2009.
The concrete preservation information of C strain is following: microbial preservation number: CGMCC No.3891; Classification name: swine fever virus; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; The preservation time: on May 27th, 2010.
Do not have immunosuppressant between PRRSV TJM strain and the swine Fever Vaccine C strain, and all have good safety, immunogenicity and specificity, can be good at preventing main popular high-pathogenicity porcine reproductive and respiration syndrome and swine fever in the current swinery.
The application also provides a kind of method for preparing above-mentioned porcine reproductive and respiratory syndrome virus vaccine and swine fever bigeminal live vaccine, and comprising: (1) is gone down to posterity cell line and cultivated; (2) permissive cell system is inoculated in high-pathogenicity porcine reproductive and respiration syndrome TJM strain and hog cholera lapinised virus strain (spleen poison) respectively, cultivate preparation production seed culture of viruses with keeping liquid; (3) prepared production seed culture of viruses is inoculated respectively on the medium that covers with the 90-100% cell, obtained high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus antigen liquid and hog cholera lapinised virus strain cell toxicant antigen liquid with keeping the liquid multiplication by culture; (4) with high-pathogenicity porcine reproductive and respiration syndrome TJM strain antigen liquid and hog cholera lapinised virus strain cell toxicant antigen liquid proportional mixing, add freeze drying protectant, mix homogeneously through lyophilisation, promptly gets.
Wherein, the grow cell of high-pathogenicity porcine reproductive and breath syndrome virus includes but not limited to primary cells such as continuous cell lines such as Marc-145 cell line, MA-104 cell line, Vero cell line, CL-2621 cell line or PAM cell being used to described in step (1), (2), (3); The cell that is used for the growing swine Pestivirus includes but not limited to primary cells such as continuous cell lines such as BT cell line, Vero cell line, MPK cell line, SK6 cell line, PK2a cell line, CPK cell line, RKC cell line, MDBK cell line, mdck cell system, CRFK cell line, PT cell line and ST cell line or BT cell.Wherein PT cell line and ST cell line are the pig testis continuous cell line.
Cell line described in the step (1) go down to posterity and cultivation comprises: cell line through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, continue is cultivated with cell growth medium, when cell covers with 90-100%, is used to continue to go down to posterity or virus inoculation; Wherein, described cell culture processes be preferably following any one: in rolling bottle, cultivate, make its cell density reach 5 * 10
7/ ml-1 * 10
8/ ml; In bioreactor, add and adhere to carrier and carry out suspension culture, make its cell density reach 5 * 10
8/ ml-1 * 10
9/ ml, the described carrier that adheres to is preferably the microcarrier or the scraps of paper.
Preferably 33-37 ℃ of cultivation temperature described in step (1), (2), (3), described cell culture environment is 5%CO
2
Production seed culture of viruses viral level standard described in the step (2) comprises: high-pathogenicity porcine reproductive and respiration syndrome TJM strain seed culture of viruses standard are that every 1ml viral level should be not less than 10
7.0TCID
50Hog cholera lapinised virus cell toxicant seed culture of viruses standard is that every 1ml contains virus＞100,000 a rabbit infective dose or application determination of immunofluorescence method viral level should be not less than 10
6.0TCID
50
High-pathogenicity porcine reproductive described in step (2) or (3) and respiration syndrome TJM strain infective dose (MOI) are 0.01-0.5, and cultivated 3-5 days the inoculation back, when cytopathy reaches 70%, gathers in the crops viral liquid; The infective dose of hog cholera lapinised virus strain (MOI) is that 0.1-0.5 or inoculum concentration are the 3%-5% cell toxicant, inoculates the back 5 days work results first time and changes liquid, and every later on the results at a distance from 4 days changed liquid, gathers in the crops to be no more than 5 times.
The antigen liquid of results described in the step (3) viral level standard comprises: high-pathogenicity porcine reproductive and respiration syndrome TJM strain seed culture of viruses standard are that every 1ml viral level should be not less than 10
7.0TCID
50Hog cholera lapinised virus cell seed culture of viruses is identified and is met hog cholera lapinised virus strain seed culture of viruses standard fully, and pig safety is had no side effect, and the every 1ml of cell seed culture of viruses contains virus＞100,000 a rabbit infective dose or application determination of immunofluorescence method viral level should be not less than 10
6.0TCID
50
Antigen liquid volume parts described in the step (4) is 75-80, and the volume parts of heat-resisting lyophilized protecting agent is 25-20.
The effect inspection standard of processing goods described in the step (4) comprises: high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus antigenic content answer>=10 in every dosage vaccine
5.0TCID
50/ ml; Hog cholera lapinised virus (C strain) antigenic content is answered>=7500 rabbit infective doses or is used determination of immunofluorescence method viral level>=10
4.0TCID
50/ ml.
The present invention also provides the vaccine combination that obtains through above-mentioned method for preparing, and it contains porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine.
The present invention also provides the purposes of described vaccine combination in the biological product of preparation prevention or treatment Porcine reproductive and respiratory syndrome and swine fever.
Pig provided by the invention is showing significant technique effect with the bigeminy Seedling aspect prevention high-pathogenicity porcine reproductive and respiration syndrome and the swine fever.The present invention is with not having immunosuppressant between high-pathogenicity porcine reproductive and respiration syndrome attenuated vaccine strain and the swine fever attenuated vaccine strain; Bigeminy Seedling and each the single Seedling of using its preparation compare, indifference in safety, immunogenicity, immune duration, immune protective effect and storage life test.Safety testing shows, vaccine single dose, single dose repeat, safety behind the overdose inoculation test animal, and body temperature, the mental status are normal, do not have any clinical symptoms; Potency test shows that vaccine of the present invention has good protective action to the strong virus attack of high-pathogenicity porcine reproductive and respiration syndrome and swine fever, can effectively prevent high-pathogenicity porcine reproductive and respiration syndrome and swine fever; The immune duration test shows that immune duration is 6 months, and can guarantee provides effective protection to pig in the duration of immunity; The test of vaccine storage life shows that vaccine is 18 months 2 ℃～8 ℃ storage lives, has long shelf-life, the advantage of stable storage.Use can a pin anti-two diseases of goods immunity inoculation animal of the present invention, alleviate the immunity inoculation workload, reduce immune time, reduce to swinery stress, avoid because of frequent immune immunological paralysis and the immuning failure that is caused.
The present invention also provides the method for immune swine, comprises pig is used vaccine combination of the present invention.Can be immune to pig through the mode of for example injection.The modes such as single dose administration, multiple dose repeat administration of can carrying out are carried out immunity.Concrete immunization ways and immunizing dose can be according to by there being the empirical personnel of veterinary to adjust according to practical situation.
Figure of description
Fig. 1 is high-pathogenicity porcine reproductive and respiration syndrome and 37 ℃ of anti-aging test swine fever virus of swine fever bigeminal live vaccine titer determination.
Fig. 2 is high-pathogenicity porcine reproductive and respiration syndrome and 37 ℃ of anti-aging test high-pathogenicity porcine reproductives of swine fever bigeminal live vaccine and respiration syndrome TJM strain virus titer determination.
Fig. 3 is high-pathogenicity porcine reproductive and respiration syndrome and 2-8 ℃ of storage life test pig of swine fever bigeminal live vaccine Pestivirus titer determination.
Fig. 4 is high-pathogenicity porcine reproductive and respiration syndrome and 2-8 ℃ of storage life test high-pathogenicity porcine reproductive of swine fever bigeminal live vaccine and respiration syndrome TJM strain virus titer determination.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
(1) seedling going down to posterity and cultivating with cell: will produce high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus with Marc-145 cell line through pancreas enzyme-EDTA cell dispersion liquid had digestive transfer culture, one to three goes down to posterity; To produce the hog cholera lapinised virus strain virus with BT cell line through pancreas enzyme-EDTA cell dispersion liquid had digestive transfer culture, one to five goes down to posterity; Respectively refinement intracellular growth liquid continues to cultivate in 37 ℃, when forming good monolayer, is used to continue to go down to posterity or virus inoculation;
(2) breeding of cell seed culture of viruses: high-pathogenicity porcine reproductive and respiration syndrome TJM strain seed culture of viruses are inoculated according to the inoculum concentration of MOI0.01-0.5, and cultivated 3-5 days the inoculation back, when cytopathy reaches 70%, gathers in the crops viral liquid; The fresh spleen poison of hog cholera lapinised virus is processed 0.3% viral suspension, inoculate well-grown BT cell line monolayer, put 37 ℃ and continue to cultivate.Cultivate venom at a distance from harvesting on the 5th and use seed culture of viruses as production.
Seed culture of viruses is identified: test by " People's Republic of China's veterinary drug allusion quotation " appendix, should not have antibacterial, mycete, mycoplasma growth.The cell venom has no side effect to pig safety, and wherein high-pathogenicity porcine reproductive and the every 1ml viral level of respiration syndrome TJM strain seed culture of viruses should be not less than 10
7.0TCID
50Hog cholera lapinised virus cell seed culture of viruses is identified and is met hog cholera lapinised virus strain seed culture of viruses standard fully, and pig safety is had no side effect, and the every 1ml of cell seed culture of viruses contains virus＞100,000 a rabbit infective dose or the every 1ml viral level of application determination of immunofluorescence method should be not less than 10
6.0TCID
50
(3) breeding of seedling venom: get and cover with 90-100% monolayer Marc-145 cell; Discard cell culture fluid; Wash 2 times with PBS; High-pathogenicity porcine reproductive and respiration syndrome TJM strain seed culture of viruses are inoculated according to the inoculum concentration of MOI0.01-0.5, and cultivated 3-5 days the inoculation back, when cytopathy reaches 70%, gathers in the crops viral liquid and use venom as seedling; Get and cover with 90-100% monolayer BT cell; Discarding cell culture fluid, wash 2 times with PBS, is that 3%-5% inoculates with hog cholera lapinised virus strain cell toxicant seed culture of viruses according to MOI 0.1-0.5 or inoculum concentration; Inoculate back 5 days and do to gather in the crops for the first time to change liquid; Whenever change liquid at a distance from results on the 4th later on, results are no more than 5 times, each that gather in the crops received time viral liquid use venom as seedling; The viral liquid of results is put below-20 ℃ and is preserved.
The check of seedling venom: test by " People's Republic of China's veterinary drug allusion quotation " appendix, should not have antibacterial, mycete, mycoplasma growth.The seedling venom has no side effect to pig safety, and wherein high-pathogenicity porcine reproductive and the every 1ml viral level of respiration syndrome TJM strain seed culture of viruses should be not less than 10
7.0TCID
50Hog cholera lapinised virus cell seed culture of viruses is identified and is met hog cholera lapinised virus strain seed culture of viruses standard fully, and pig safety is had no side effect, and the every 1ml of cell seed culture of viruses contains virus＞100,000 a rabbit infective dose or the every 1ml viral level of application determination of immunofluorescence method should be not less than 10
6.0TCID
50
Above-mentioned nutrient solution used prescription is: 92%-95%MEM liquid or DMEM liquid, 5%-8% calf serum, add an amount of antibiotics, pH value is adjusted into 7.2.The above-mentioned used prescription of keeping liquid is:
95%-98%MEM liquid or DMEM liquid, 2-5% calf serum, add an amount of antibiotics, pH value is adjusted into 7.2.
(4) heat-resisting lyophilized protecting agent preparation: each component of heat-resisting lyophilized protecting agent is prepared after autoclaving is subsequent use according to a certain percentage.
(5) join Seedling, lyophilizing and packing: with volume parts is quantitatively to divide behind the antigen liquid of 75-80 and the freeze drying protectant mix homogeneously that volume parts is 25-20 to be filled in the peace bottle, adds a cover to be placed in the freeze dryer, through pre-cooling, dry run freeze dried vaccine.Carry out steriling test, safety examination and efficacy test behind the vaccine freeze-drying.
High-pathogenicity porcine reproductive and the respiration syndrome and the swine fever bigeminal live vaccine of present embodiment preparation, high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus antigenic content answer>=10 in every dosage vaccine
5.0TCID
50Hog cholera lapinised virus (C strain) antigenic content is answered>=7500 rabbit infective doses or is used determination of immunofluorescence method viral level>=10
4.0TCID
50Character check, safety verification, efficacy test etc. are all qualified.
Immune inhibition test between Test Example 1 high-pathogenicity porcine reproductive and respiration syndrome TJM strain basis seed culture of viruses and hog cholera lapinised virus cell toxicant basis seed culture of viruses
30 of test use 21-28 age in days high-pathogenicity porcine reproductive and respiration syndrome and swine fever antigen, antibody jack to jack adapter Sexual health piglets, test is divided into 7 groups.5 of first group of service test animals, musculi colli injection high-pathogenicity porcine reproductive and respiration syndrome TJM strain basis seed culture of viruses, inoculating seed culture of viruses dosage is 10
5.0TCID
50/ ml, 1ml/ pig; 5 of second group of service test animals, musculi colli injection hog cholera lapinised virus cell toxicant basis seed culture of viruses, inoculation seed culture of viruses dosage is that (or fluorescent quantitation is 10 to 7500 rabbit infective dose/ml
4.0TCID
50/ ml), 1ml/ pig; 5 of the 3rd group and the 4th group of each service test animals; Musculi colli injection high-pathogenicity porcine reproductive and respiration syndrome TJM strain basis seed culture of viruses and hog cholera lapinised virus cell toxicant basis seed culture of viruses mixed liquor; 1ml/ pig contains high-pathogenicity porcine reproductive and respiration syndrome TJM strain basis seed culture of viruses 10 in every 1ml mixed liquor
5.0TCID
50, containing hog cholera lapinised virus cell toxicant basis seed culture of viruses dosage is that (or fluorescent quantitation is 10 to 7500 rabbit infective doses
4.0TCID
50); The 5th group is that high-pathogenicity porcine reproductive and respiration syndrome feminine gender are not inoculated matched group, uses 5 experimental animals, musculi colli injection Cell sap; The 6th group is that the swine fever feminine gender is not inoculated matched group, uses 3 experimental animals, musculi colli injection Cell sap; The 7th group is the blank group, uses 2 experimental animals, does not inoculate any material to off-test.
Back 7 days of preceding 3 days of vaccination and inoculation are measured all piglet rectal temperatures every day and are carried out clinical observation; 3d and inoculation back 0d, 3d, 7d, 10d, 14d, 21d, 28d, 31d, 35d, 38d, 42d gather all piglet anticoagulations and coagulant blood before inoculation, and anticoagulation is used for CD3
+, CD4
+, CD8
+The T cell detection, the coagulant blood system is used for the antibody titer monitoring from serum.
Immunity was carried out challenge test in back 28 days, get first group, the 3rd group, the 5th group experimental animal respectively collunarium add the intramuscular injection high-pathogenicity porcine reproductive and check with a strong malicious 3ml/ pig collunarium 2ml (1ml/ nostril), intramuscular injection 1ml with respiration syndrome; Get second group, the 4th group, the 6th group experimental animal intramuscular injection swine fever check with strong arsenic bloom door blood poison, 1ml/ pig; The 7th group of blank experimental animal do not attacked any strong poison.Attack back viewing test animal clinical symptoms every day, comprise appetite, breathing, the mental status etc., every day thermometric.High-pathogenicity porcine reproductive and respiration syndrome strong virus attack are tested end in 21 days behind counteracting toxic substances, and the swine fever strong virus attack is tested end in 16 days behind counteracting toxic substances, and challenge trial is calculated every treated animal clinical protection rate, M & M after finishing.
The result: the leukocyte testing result shows: in back 28 days of immunity, and each immune group and matched group experimental animal CD3
+, CD4
+, CD8
+T cellular change rule is similar, and difference is not remarkable; The 3rd group with the 4th group of experimental animal simultaneously behind two kinds of basic seeds culture of viruses of immunity; Non-interference antibody generates; And the 3rd group of anti-high-pathogenicity porcine reproductive and the breath syndrome virus antibody growth and decline rule with the 4th group of anti-high-pathogenicity porcine reproductive of experimental animal and breath syndrome virus antibody growth and decline rule and first group of experimental animal is consistent, and the 3rd group of swine fever virus resistant antibody growth and decline rule with the swine fever virus resistant antibody growth and decline rule of the 4th group of experimental animal and second group of experimental animal is consistent; High-pathogenicity porcine reproductive and respiration syndrome check show with strong malicious counteracting toxic substances result: first group of counteracting toxic substances protective rate is 5/5, and the counteracting toxic substances sickness rate is 0/5, counteracting toxic substances mortality rate 0/5; The 3rd group of counteracting toxic substances protective rate is 5/5, and the counteracting toxic substances sickness rate is 0/5, counteracting toxic substances mortality rate 0/5; The 5th group of counteracting toxic substances protective rate is 0/5, and the counteracting toxic substances sickness rate is 5/5, counteracting toxic substances mortality rate 3/5; The swine fever check shows with strong arsenic bloom door blood poison counteracting toxic substances result: second group of counteracting toxic substances protective rate is 5/5, and the counteracting toxic substances sickness rate is 0/5, counteracting toxic substances mortality rate 0/5; The 4th group of counteracting toxic substances protective rate is 5/5, and the counteracting toxic substances sickness rate is 0/5, counteracting toxic substances mortality rate 0/5; The 6th group of counteracting toxic substances protective rate is 0/3, and the counteracting toxic substances sickness rate is 3/3, counteracting toxic substances mortality rate 3/3; In sum, result of the test shows not have immunosuppressant between high-pathogenicity porcine reproductive and respiration syndrome TJM strain basis seed culture of viruses and hog cholera lapinised virus cell toxicant basis seed culture of viruses.
Test Example 2 high-pathogenicity porcine reproductives and respiration syndrome and swine fever bigeminal live vaccine safety testing
3 batches of laboratory products (lot number is respectively 200904,200905,200906) have been carried out safety testing.Content of the test comprises that fattening 1 single dose of piglet inoculates safety testing, single dose repeated inoculation safety testing, overdose inoculation safety testing, non-use age in days piglet overdose inoculation safety testing and different cultivars pig overdose are inoculated safety testing.
The result shows; It is normal respectively to organize experimental animal body temperature after single dose inoculation, single dose repeated inoculation and the overdose inoculation; The mental status and appetite are good; Injection site and whole body are not seen untoward reaction, and fertility performance is not had influence, and bigeminy Seedling overdose is inoculated non-use age in days piglet, different cultivars pig overdose is inoculated all safety.
Test Example 3 high-pathogenicity porcine reproductives and respiration syndrome and swine fever bigeminal live vaccine potency test
3 batches of laboratory products (lot number is respectively 200904,200905,200906) to prepared in laboratory are carried out potency test respectively; Design high-pathogenicity porcine reproductive and respiration syndrome TJM strain and two kinds of single Seedling controlled trial groups of hog cholera lapinised virus strain (C strain) cell toxicant join the effectiveness comparative study of Seedling and single Seedling when carrying out every batch of laboratory products potency test.The every batch of laboratory products is all used 28 of high-pathogenicity porcine reproductive and respiration syndrome and swine fever antigen, antibody jack to jack adapter Sexual health piglets, and test divides six groups.5 of first group, second group each service test animals, musculi colli injection inoculation bigeminal live vaccine, dosage are the 1ml/ head, hog cholera lapinised virus vaccine content is that (or fluorescent quantitation is 10 to 7500 rabbit infective doses
4.0TCID
50), high-pathogenicity porcine reproductive and respiration syndrome TJM strain vaccine content are 10
5.0TCID
505 of the 3rd group of service test animals, musculi colli injection inoculation hog cholera lapinised virus strain (C strain) cell toxicant vaccine, dosage is the 1ml/ head, vaccine contg is that (or fluorescent quantitation is 10 to 7500 rabbit infective doses
4.0TCID
50).5 of the 4th group of service test animals, musculi colli injection high-pathogenicity porcine reproductive and respiration syndrome TJM strain vaccine, dosage is the 1ml/ head, vaccine contg is 10
5.0TCID
50The 5th group is that the swine fever feminine gender is not inoculated matched group, 3 of service test animals.The 6th group is that high-pathogenicity porcine reproductive and respiration syndrome feminine gender are not inoculated matched group, 5 of service test animals.After the experimental animal vaccination 28 days; The check of intramuscular injection swine fever is malicious with strong arsenic bloom door blood respectively to get first group, the 3rd group, the 5th group experimental animal; 1ml/ pig; The swine fever challenge test finished behind counteracting toxic substances in 16 days, get second group, the 4th group, the 6th group experimental animal respectively collunarium add the intramuscular injection high-pathogenicity porcine reproductive and check with a strong malicious 3ml/ pig collunarium 2ml (1ml/ nostril) with respiration syndrome; Intramuscular injection 1ml, high-pathogenicity porcine reproductive and respiration syndrome challenge test be end in 21 days behind counteracting toxic substances.The result shows; Behind the 3 batches of high-pathogenicity porcine reproductives and respiration syndrome and the swine fever bigeminal live vaccine inoculation test animal; Bigeminy Seedling immune group all can produce good protective action to high-pathogenicity porcine reproductive and respiration syndrome strong virus attack and swine fever strong virus attack; With single Seedling matched group indifference; Wherein, the three batches of bigeminy Seedlings are respectively 5/5,5/5 and 5/5, three batch of swine fever list Seedling to the protective rate of swine fever the protective rate of swine fever are respectively 5/5,5/5 and 5/5; The three batches of bigeminy Seedlings are respectively 4/5,4/5 and 5/5, three batch of high-pathogenicity porcine reproductive and respiration syndrome list Seedling to the protective rate of high-pathogenicity porcine reproductive and respiration syndrome the protective rate of high-pathogenicity porcine reproductive and respiration syndrome are respectively 5/5,4/5 and 4/5.Swine fever negative control group and high-pathogenicity porcine reproductive and respiration syndrome negative control treated animal are all fallen ill, and show tangible clinical symptoms, sickness rate standard up to specification.
Test Example 4 high-pathogenicity porcine reproductives and respiration syndrome and the test of swine fever bigeminal live vaccine immune duration
Use 56 of high-pathogenicity porcine reproductive and respiration syndrome and swine fever antigen, antibody jack to jack adapter Sexual health piglets; The high-pathogenicity porcine reproductive of prepared in laboratory and respiration syndrome and swine fever bigeminal live vaccine are carried out the immune duration test, compare research with high-pathogenicity porcine reproductive and respiration syndrome TJM strain and two kinds of single Seedling immune durations of hog cholera lapinised virus strain (C strain) cell toxicant simultaneously.Test is divided into six groups, 10 of first group, second group each service test animals, and musculi colli injection inoculation bigeminal live vaccine, dosage are the 1ml/ head, hog cholera lapinised virus vaccine content is that (or fluorescent quantitation is 10 to 7500 rabbit infective doses
4.0TCID
50), high-pathogenicity porcine reproductive and respiration syndrome TJM strain vaccine content are 10
5.0TCID
5010 of the 3rd group of service test animals, musculi colli injection inoculation hog cholera lapinised virus strain (C strain) cell toxicant vaccine, dosage is the 1ml/ head, vaccine contg is that (or fluorescent quantitation is 10 to 7500 rabbit infective doses
4.0TCID
50).10 of the 4th group of service test animals, musculi colli injection high-pathogenicity porcine reproductive and respiration syndrome TJM strain vaccine, dosage is the 1ml/ head, vaccine contg is 10
5.0TCID
50The 5th group is that the swine fever feminine gender is not inoculated matched group, 6 of service test animals.The 6th group is that high-pathogenicity porcine reproductive and respiration syndrome feminine gender are not inoculated matched group, 10 of service test animals.Blood sampling in 1 month, 2 months, 3 months, 4 months, 5 months, 6 months detects antibody titer after the vaccination; The experimental animal of respectively organizing that extracted half quantity in 3 months and 6 months respectively behind the vaccine immunity carries out challenge test; The check of intramuscular injection swine fever is malicious with strong arsenic bloom door blood respectively for wherein first group, the 3rd group, the 5th group experimental animal; 1ml/ pig; Second group, the 4th group, the 6th group experimental animal collunarium injection high-pathogenicity porcine reproductive respectively checked with a strong malicious 2ml/ pig, 1ml/ nostril with respiration syndrome.March, the counteracting toxic substances result showed, bigeminy Seedling group swine fever protective rate, swine fever list Seedling protective rate are respectively 5/5,5/5, and bigeminy Seedling group high-pathogenicity porcine reproductive and respiration syndrome protective rate, high-pathogenicity porcine reproductive and respiration syndrome list Seedling protective rate are respectively 5/5,4/5.June, the counteracting toxic substances result showed, bigeminy Seedling swine fever protective rate, swine fever list Seedling protective rate are respectively 5/5,5/5, and the pathogenic Porcine reproductive and respiratory syndrome protective rate of bigeminy height of seedling, high-pathogenicity porcine reproductive and respiration syndrome list Seedling protective rate are respectively 5/5 and 4/5.Therefore immune duration is decided to be 6 months, can guarantee provides effective protection to pig in the duration of immunity.This result of the test shows that simultaneously immune duration effect and two kinds of single Seedlings of joining the Seedling generation produce immune duration effect indifferences.
Test Example 5 high-pathogenicity porcine reproductives and respiration syndrome and the test of swine fever bigeminal live vaccine storage life
These goods add novel heat-resisting lyophilized protecting agent, adopt improved freeze-dry process to be prepared from.In stability (storage life) test of vaccine; Use the 3 batches of high-pathogenicity porcine reproductives and the respiration syndrome and the swine fever bigeminal live vaccine (200904,200905,200906) of prepared in laboratory different size; Accomplish vaccine stability and storage life research, and carried out the contrast test same period with two kinds of single Seedlings (200901,200902,200903,200907,200908,200909).The 3 batches of bigeminy Seedling goods place 2～8 ℃ preserve 3,6,9,12,18 months respectively sampling carry out character, vacuum, residual moisture content, potency test and 37 ℃ of anti-aging tests.The result show 3 batches of goods 2～8 ℃ preserve 18 months after, character still be the white loose agglomerate, dissolves rapidly after adding diluent; Vacuum detects and is white or purple aura; Average residual moisture all meets " People's Republic of China's veterinary drug allusion quotation " required standard; High-pathogenicity porcine reproductive and respiration syndrome TJM strain virus are tired and are respectively 10 in 3 batches of bigeminy Seedlings (200904,200905,200906)
5.3TCID
50/ ml, 10
5.3TCID
50/ ml, 10
5.2TCID
50/ ml, single Seedling (200907,200908, the 200909) virus titer of the 3 batches of high-pathogenicity porcine reproductives and respiration syndrome TJM strain is respectively 10
5.2TCID
50/ ml, 10
5.3TCID
50/ ml, 10
5.1TCID
50The hog cholera lapinised virus cell toxicant is tired and is respectively 10 among the/ml, 3 batches of bigeminy Seedlings (200904,200905,200906)
4.2TCID
50/ ml, 10
4.1TCID
50/ ml, 10
4.3TCID
50/ ml (all>=and 7500 rabbit infective doses), 3 batches of hog cholera lapinised virus cell toxicant list Seedlings (200901,200902,200903) virus titer is respectively 10
4.1TCID
50/ ml, 10
4.2TCID
50/ ml, 10
4.2TCID
50/ ml (all>=and 7500 rabbit infective doses), 2～8 ℃ of storage life result of the tests show that bigeminy Seedling and two kinds of single Seedlings compare difference with insignificance.Place virus titer on the 14th for 37 ℃ and still keep higher level, high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus are tired and are respectively 10 in 3 batches of bigeminy Seedlings (200904,200905,200906)
5.3TCID
50/ ml, 10
5.2TCID
50/ ml, 10
5.2TCID
50/ ml, single Seedling (200907,200908, the 200909) virus titer of the 3 batches of high-pathogenicity porcine reproductives and respiration syndrome TJM strain is respectively 10
5.2TCID
50/ ml, 10
5.4TCID
50/ ml, 10
5.3TCID
50The hog cholera lapinised virus cell toxicant is tired and is respectively 10 among the/ml, 3 batches of bigeminy Seedlings (200904,200905,200906)
4.2TCID
50/ ml, 10
4.2TCID
50/ ml, 10
4.3TCID
50/ ml (all>=and 7500 rabbit infective doses), 3 batches of hog cholera lapinised virus cell toxicant list Seedlings (200901,200902,200903) virus titer is respectively 10
4.2TCID
50/ ml, 10
4.1TCID
50/ ml, 10
4.2TCID
50/ ml (all>=and 7500 rabbit infective doses), can reach the vaccine quality standard.Explain that this heat-resisting lyophilized protecting agent all has the better protect effect to high-pathogenicity porcine reproductive and respiration syndrome TJM strain and hog cholera lapinised virus strain in lyophilizing and preservation process.Overcome freeze drying protectant that conventional vaccine adds and freeze-dry process and can only preserve difficulty in subzero (20 ℃); Fundamentally solve vaccine and preserve key technology in transportation and the practical application, make stable the reaching or approaching similar vaccine level in the world of preservation of this live vaccine.
Claims (24)
1. a vaccine combination contains porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine.
2. vaccine combination according to claim 1 is characterized in that, has basically no immunosuppressant between described porcine reproductive and respiratory syndrome virus vaccine and the swine Fever Vaccine.
3. vaccine combination according to claim 1, the mixed proportion of wherein said swine Fever Vaccine and said porcine reproductive and respiratory syndrome virus vaccine is 1: 2 to 1: 4.
4. vaccine combination according to claim 1, the coded sequence of wherein said swine Fever Vaccine and SEQ IDNO:1 have at least 80% homology.
5. vaccine combination according to claim 4, the coded sequence of wherein said swine Fever Vaccine is shown in SEQ IDNO:1.
6. vaccine combination according to claim 1, wherein said porcine reproductive and respiratory syndrome virus vaccine contains the Nsp1 nucleotide sequence, and the coded sequence of said sequence and SEQ ID NO:2 have at least 90% homology.
7. vaccine combination according to claim 6, wherein said porcine reproductive and respiratory syndrome virus vaccine further contains the Nsp2 nucleotide sequence, and the coded sequence of said Nsp2 nucleotide and SEQ ID NO:3 have at least 90% homology.
8. vaccine combination according to claim 7, wherein said porcine reproductive and respiratory syndrome virus vaccine contain the Nsp1 nucleotide sequence by SEQ ID NO:2 coding, and contain the Nsp2 nucleotide sequence by SEQ ID NO:3 coding.
9. vaccine combination according to claim 1, wherein said porcine reproductive and respiratory syndrome virus vaccine contains the nucleotide sequence of porcine reproductive and respiratory syndrome virus, and the coded sequence of this sequence and SEQ ID NO:4 have at least 90% homology.
10. vaccine combination according to claim 9, wherein said porcine reproductive and respiratory syndrome virus vaccine contains the nucleotide sequence of porcine reproductive and respiratory syndrome virus, and this sequence is encoded by SEQ IDNO:4.
11. vaccine combination according to claim 1, it further contains adjuvant.
12. vaccine combination according to claim 1, it further contains freeze drying protectant.
13. vaccine combination according to claim 1, wherein said freeze drying protectant comprises sucrose, L-sodium glutamate or lactoalbumin hydrolysate.
14. a method for preparing the described vaccine combination of claim 1 comprises:
(1) cell line is gone down to posterity and cultivate;
(2) porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine are inoculated permissive cell respectively, cultivate with keeping liquid, kind of a poison is produced in preparation;
(3) prepared production kind poison is inoculated respectively on the medium that covers with the 90-100% cell, obtained porcine reproductive and respiratory syndrome virus vaccine antigen liquid and swine Fever Vaccine antigen liquid with keeping the liquid multiplication by culture;
(4) porcine reproductive and respiratory syndrome virus vaccine antigen liquid and swine Fever Vaccine antigen liquid are mixed.
15. according to the described method of claim 14, it is characterized in that: the cell that is used for growing swine reproductive and respiratory syndrome viral vaccine described in step (1), (2), (3) is primary cells such as continuous cell lines such as Marc-145 cell line, MA-104 cell line, Vero cell line, CL-2621 cell line or PAM cell.
16., it is characterized in that according to the described method of claim 14: being used to described in step (1), (2), (3) the grow cell of swine Fever Vaccine be primary cells such as continuous cell lines such as BT cell line, Vero cell line, MPK cell line, SK6 cell line, PK2a cell line, CPK cell line, RKC cell line, MDBK cell line, mdck cell system, CRFK cell line, ST cell line and PT cell line or BT cell.
17., it is characterized in that according to the described method of claim 14: the cell culture processes described in the step (1) be following any one; In rolling bottle, cultivate, its cell density is reached: 5 * 10
7/ ml-1 * 10
8/ ml; In bioreactor, add and adhere to carrier and carry out suspension culture, make its cell density reach 5 * 10
8/ ml-1 * 10
9/ ml.
18. according to the described method of claim 14; It is characterized in that: the porcine reproductive and respiratory syndrome virus vaccine infection dosage (MOI) described in step (2) or (3) is 0.01-0.5; Cultivated 3-5 days the inoculation back; When cytopathy reaches 70%, gather in the crops viral liquid, produce seed culture of viruses and antigen liquid in order to preparation.
19. according to the described method of claim 14; It is characterized in that: the infective dose (MOI) of the swine Fever Vaccine described in step (2) or (3) is that 0.1-0.5 or inoculum concentration are the 3%-5% cell toxicant; Inoculate back 5 days and do to gather in the crops for the first time to change liquid, whenever change liquid at a distance from results on the 4th later on, results are no more than 5 times.
20., it is characterized in that according to the described method of claim 14: the freeze drying protectant described in the step (4) by mixed dissolutions such as sucrose, L-sodium glutamate, lactoalbumin hydrolysates after obtain freeze drying protectant behind the autoclaving.
21., it is characterized in that: be that the antigen liquid of 75-80 and freeze drying protectant mix homogeneously that volume parts is 25-20 prepare said preparation with volume parts in the step (4) according to the described method of claim 14.
22. the vaccine combination that any one method for preparing of claim 14-21 obtains, it contains porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine.
23. the purposes of the described vaccine combination of claim 1 in the biological product of preparation prevention or treatment Porcine reproductive and respiratory syndrome and swine fever.
24. the method for an immune swine comprises pig is used the described vaccine combination of claim 1.
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| CN201110140951.5A CN102727883B (en) | 2011-05-27 | 2011-05-27 | Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof |
| MX2013013906A MX347210B (en) | 2011-05-27 | 2012-05-25 | Combined vaccines for prevention of porcine virus infections. |
| RU2013158322A RU2628313C2 (en) | 2011-05-27 | 2012-05-25 | Combined vaccines for prevention of pigs viral infections |
| US14/122,627 US9592286B2 (en) | 2011-05-27 | 2012-05-25 | Combined vaccines for prevention of porcine virus infections |
| BR112013030321A BR112013030321A2 (en) | 2011-05-27 | 2012-05-25 | vaccine composition, method for preparing the vaccine composition, use of the vaccine composition, method for immunizing a pig, csfv vaccine strain, and use of a cell in cultivating a csfv vaccine strain. |
| TW101118902A TWI579297B (en) | 2011-05-27 | 2012-05-25 | Combined vaccines for prevention of porcine virus infections |
| JP2014511724A JP6096176B2 (en) | 2011-05-27 | 2012-05-25 | Combination vaccine for the prevention of swine virus infection |
| KR1020137034298A KR20140036262A (en) | 2011-05-27 | 2012-05-25 | Combined vaccines for prevention of porcine virus infections |
| CA2837125A CA2837125A1 (en) | 2011-05-27 | 2012-05-25 | Combined vaccines for prevention of porcine virus infections |
| PCT/CN2012/076125 WO2012163258A1 (en) | 2011-05-27 | 2012-05-25 | Combined vaccines for prevention of porcine virus infections |
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| CN201110140951.5A CN102727883B (en) | 2011-05-27 | 2011-05-27 | Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof |
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