CN102727883B - Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof - Google Patents
Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof Download PDFInfo
- Publication number
- CN102727883B CN102727883B CN201110140951.5A CN201110140951A CN102727883B CN 102727883 B CN102727883 B CN 102727883B CN 201110140951 A CN201110140951 A CN 201110140951A CN 102727883 B CN102727883 B CN 102727883B
- Authority
- CN
- China
- Prior art keywords
- vaccine
- porcine reproductive
- swine fever
- cell
- respiratory syndrome
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 150
- 241000282898 Sus scrofa Species 0.000 title claims abstract description 129
- 206010037660 Pyrexia Diseases 0.000 title claims abstract description 109
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 title claims abstract description 13
- 238000011081 inoculation Methods 0.000 claims abstract description 25
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 241000700605 Viruses Species 0.000 claims description 110
- 210000004027 cell Anatomy 0.000 claims description 105
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 claims description 54
- 239000003440 toxic substance Substances 0.000 claims description 46
- 238000011218 seed culture Methods 0.000 claims description 37
- 231100000614 poison Toxicity 0.000 claims description 36
- 239000007788 liquid Substances 0.000 claims description 34
- 238000000034 method Methods 0.000 claims description 31
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 22
- 230000001524 infective effect Effects 0.000 claims description 20
- 231100000167 toxic agent Toxicity 0.000 claims description 20
- 239000000427 antigen Substances 0.000 claims description 18
- 108091007433 antigens Proteins 0.000 claims description 18
- 102000036639 antigens Human genes 0.000 claims description 18
- 238000004108 freeze drying Methods 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 17
- 125000003729 nucleotide group Chemical group 0.000 claims description 17
- 239000002574 poison Substances 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 8
- 239000002671 adjuvant Substances 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 230000008859 change Effects 0.000 claims description 6
- 229960003444 immunosuppressant agent Drugs 0.000 claims description 6
- 230000001861 immunosuppressant effect Effects 0.000 claims description 6
- 239000003018 immunosuppressive agent Substances 0.000 claims description 6
- 208000015181 infectious disease Diseases 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 5
- 238000012423 maintenance Methods 0.000 claims description 5
- 230000002265 prevention Effects 0.000 claims description 4
- 210000003501 vero cell Anatomy 0.000 claims description 4
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 101800001728 Nsp1 Proteins 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 3
- 239000000413 hydrolysate Substances 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 238000005096 rolling process Methods 0.000 claims description 2
- 238000004114 suspension culture Methods 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 23
- 230000036039 immunity Effects 0.000 abstract description 15
- 238000002347 injection Methods 0.000 abstract description 14
- 239000007924 injection Substances 0.000 abstract description 14
- 238000003860 storage Methods 0.000 abstract description 12
- 229940031567 attenuated vaccine Drugs 0.000 abstract description 10
- 201000010099 disease Diseases 0.000 abstract description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 9
- 230000003053 immunization Effects 0.000 abstract description 8
- 238000002649 immunization Methods 0.000 abstract description 6
- 230000001717 pathogenic effect Effects 0.000 abstract description 6
- 230000001900 immune effect Effects 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000005847 immunogenicity Effects 0.000 abstract description 3
- 206010033799 Paralysis Diseases 0.000 abstract description 2
- 230000002480 immunoprotective effect Effects 0.000 abstract 2
- 206010062016 Immunosuppression Diseases 0.000 abstract 1
- 230000003247 decreasing effect Effects 0.000 abstract 1
- 230000001506 immunosuppresive effect Effects 0.000 abstract 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 74
- 230000001850 reproductive effect Effects 0.000 description 73
- 208000011580 syndromic disease Diseases 0.000 description 73
- 238000012360 testing method Methods 0.000 description 59
- 206010008631 Cholera Diseases 0.000 description 23
- 230000001681 protective effect Effects 0.000 description 22
- 238000010171 animal model Methods 0.000 description 20
- 206010015856 Extrasystoles Diseases 0.000 description 19
- 230000003612 virological effect Effects 0.000 description 18
- 238000004321 preservation Methods 0.000 description 14
- 241000710777 Classical swine fever virus Species 0.000 description 10
- 238000009781 safety test method Methods 0.000 description 8
- 231100000041 toxicology testing Toxicity 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 238000010255 intramuscular injection Methods 0.000 description 7
- 239000007927 intramuscular injection Substances 0.000 description 7
- 206010025482 malaise Diseases 0.000 description 7
- 239000002435 venom Substances 0.000 description 7
- 231100000611 venom Toxicity 0.000 description 7
- 210000001048 venom Anatomy 0.000 description 7
- 108700026244 Open Reading Frames Proteins 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 238000010166 immunofluorescence Methods 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 244000005700 microbiome Species 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 231100000957 no side effect Toxicity 0.000 description 5
- 239000003223 protective agent Substances 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 108010076039 Polyproteins Proteins 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 4
- 229910052785 arsenic Inorganic materials 0.000 description 4
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 4
- 229940031551 inactivated vaccine Drugs 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000002356 single layer Substances 0.000 description 4
- 208000035473 Communicable disease Diseases 0.000 description 3
- 206010021703 Indifference Diseases 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 101710172711 Structural protein Proteins 0.000 description 3
- 230000003712 anti-aging effect Effects 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 230000002238 attenuated effect Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000001079 digestive effect Effects 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 241001493065 dsRNA viruses Species 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000003340 mental effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 230000035911 sexual health Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000002255 vaccination Methods 0.000 description 3
- 239000000273 veterinary drug Substances 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 2
- 101150001779 ORF1a gene Proteins 0.000 description 2
- 241000710778 Pestivirus Species 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000010100 anticoagulation Effects 0.000 description 2
- 230000036528 appetite Effects 0.000 description 2
- 235000019789 appetite Nutrition 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 229940030156 cell vaccine Drugs 0.000 description 2
- 239000000701 coagulant Substances 0.000 description 2
- LXWYCLOUQZZDBD-LIYNQYRNSA-N csfv Chemical compound C([C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=C(O)C=C1 LXWYCLOUQZZDBD-LIYNQYRNSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229930182490 saponin Natural products 0.000 description 2
- 150000007949 saponins Chemical class 0.000 description 2
- 235000017709 saponins Nutrition 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000005723 virus inoculator Substances 0.000 description 2
- DRHZYJAUECRAJM-DWSYSWFDSA-N (2s,3s,4s,5r,6r)-6-[[(3s,4s,4ar,6ar,6bs,8r,8ar,12as,14ar,14br)-8a-[(2s,3r,4s,5r,6r)-3-[(2s,3r,4s,5r,6s)-5-[(2s,3r,4s,5r)-4-[(2s,3r,4r)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxy-3,5-dihydroxyoxan-2-yl]oxy-3,4-dihydroxy-6-methyloxan-2-yl]oxy-5-[(3s,5s, Chemical compound O([C@H]1[C@H](O)[C@H](O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O1)O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@H]5CC(C)(C)CC[C@@]5([C@@H](C[C@@]4(C)[C@]3(C)CC[C@H]2[C@@]1(C=O)C)O)C(=O)O[C@@H]1O[C@H](C)[C@@H]([C@@H]([C@H]1O[C@H]1[C@@H]([C@H](O)[C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@](O)(CO)CO3)O)[C@H](O)CO2)O)[C@H](C)O1)O)O)OC(=O)C[C@@H](O)C[C@H](OC(=O)C[C@@H](O)C[C@@H]([C@@H](C)CC)O[C@H]1[C@@H]([C@@H](O)[C@H](CO)O1)O)[C@@H](C)CC)C(O)=O)[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O DRHZYJAUECRAJM-DWSYSWFDSA-N 0.000 description 1
- KRQUFUKTQHISJB-YYADALCUSA-N 2-[(E)-N-[2-(4-chlorophenoxy)propoxy]-C-propylcarbonimidoyl]-3-hydroxy-5-(thian-3-yl)cyclohex-2-en-1-one Chemical compound CCC\C(=N/OCC(C)OC1=CC=C(Cl)C=C1)C1=C(O)CC(CC1=O)C1CCCSC1 KRQUFUKTQHISJB-YYADALCUSA-N 0.000 description 1
- 241001516864 Allende Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000001726 Classical Swine Fever Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 206010011732 Cyst Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000710781 Flaviviridae Species 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 206010041349 Somnolence Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 229940061720 alpha hydroxy acid Drugs 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 206010003549 asthenia Diseases 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
- 239000000920 calcium hydroxide Substances 0.000 description 1
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 229940028617 conventional vaccine Drugs 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 208000031513 cyst Diseases 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 208000032625 disorder of ear Diseases 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000021472 generally recognized as safe Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- GZQKNULLWNGMCW-PWQABINMSA-N lipid A (E. coli) Chemical class O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O1 GZQKNULLWNGMCW-PWQABINMSA-N 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000004531 microgranule Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 239000007762 w/o emulsion Substances 0.000 description 1
Images
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 veterinary biologics technical field, more specifically relate to a kind of method and goods thereof of preparing 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, (claim again high-pathogenicity blue ear disease), cause huge economic loss to the pig industry of China, classified as one of epidemic disease of compulsory immunization by the Ministry of Agriculture.Simultaneously swine fever Ye Shi China classifies one of epidemic disease of compulsory immunization as, and therefore, in epidemic prevention process, spininess, multiple dose, panimmunity program are difficult to reasonable arrangement, both loaded down with trivial details, time-consuming, effort, increase cost, and easily cause with Louing and plant, directly affect immune effect.Adopt multiple vaccines immunity inoculation, can reach the object of " pin is anti-how sick ".Meanwhile, the immune programme for children that these two kinds of epidemic diseases are taked is more approaching, and this development for high-pathogenicity porcine reproductive and respiration syndrome and swine fever bigeminal live vaccine provides may.
Summary of the invention
One of object of the present invention be to provide a kind of immunity high, without the bigeminy vaccine compositions of porcine reproductive and respiratory syndrome virus vaccine and the swine Fever Vaccine of immune interference, reach the object of anti-two diseases of a pin.
Two of object of the present invention is to provide a kind of method of the bigeminy vaccine compositions of 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, the lethal pig infectious disease that cause, OIE disease register is listed in by OIE (OIE), is the infectious disease of legal essential report.In China, swine fever is one of great epidemic disease, in " the sick register of planting of one, two, three class animal epidemics ", lists swine fever one of in class animal epidemic, and the eruption and prevalence of swine fever causes serious economic loss to 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 has cyst membrane.The about 12.5kb of genome total length, only contain a large Open reading frame (ORF), this ORF approximately 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, being 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 controlling 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 because inactivated vaccine exists the shortcomings such as immunizing dose is large, duration of immunity is short, the time of generation immunity is slow, cost is higher, progressively replaced by swine fever attenuated vaccine later.Swine fever attenuated vaccine strain forms a little less than being caused by swine fever street strain, and abroad someone reports in succession, different swine fever virus is adapted to rabbit with distinct methods, become the variation strain of virulence attenuation of, but these variation strains still can bring out severe reaction and death.Through application 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. France's " Thiveosal " cold variation low virulent strain.System of China (C system) hog cholera lapinised virus vaccine that wherein Chinese scholar is succeeded in developing, from nineteen fifty-seven, except China's extensive use, and has been generalized to Eurasian a lot of country, makes these state controls or has eliminated swine fever.This vaccine is acknowledged as at present more satisfactory in the world swine Fever Vaccine.
Hog cholera lapinised virus (C strain) is divided into according to the difference of production technology: the first technique is to produce with rabbit body, gets lymph node or the spleen of inoculation rabbit or organizes seedling, and swine fever spleen drenches Seedling and swine fever breast rabbit Seedling.This technique can effectively avoid exogenous virus to pollute, and has guaranteed viral hereditary stability simultaneously, but need to use a large amount of rabbit, and quality control difficulty is larger, and production cost is higher.The second technique is application cattle, sheep primary cell or pig passage cell production seedling, i.e. swine fever cell vaccine.This technique 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 at present two kinds of genotype: Europe class and american type.In PRRSV genome, there are multiple open reading frame, the sequence that wherein first open reading frame (ORF1a and ORF1b) contains PRRSV genomic 80%, the necessary rna replicon enzyme of its coding PRRSV virus replication (Straw et al, Diseases of Swine, 9TH edition, chapter24 (2006)).ORF1a and ORF1b are translated into a polyprotein (poly-protein); the proteinase activity region that this polyprotein is wherein contained cuts into multiple non-structural proteins; comprise that Nsp1-Nsp12(is shown in; 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 essentially no immunosuppressant between described porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine.
" essentially no immunosuppressant " refers to, described porcine reproductive and respiratory syndrome virus vaccine and described swine Fever Vaccine can significantly not weaken the other side's immune effect after immune swine.
In some embodiments, in described vaccine combination, described swine Fever Vaccine can mix in the proper ratio with described porcine reproductive and respiratory syndrome virus vaccine.Described swine Fever Vaccine and described porcine reproductive and respiratory syndrome virus vaccine can be made respectively to the antigen liquid with certain virus titer, then calculate the ratio of mixing according to the virus titer recording.In some embodiments, the mixed proportion of described swine Fever Vaccine and described porcine reproductive and respiratory syndrome virus vaccine is that 1:2 is to 1:4.
In some embodiments, the described swine Fever Vaccine in vaccine combination provided by the invention is hog cholera lapinised virus cell vaccine.In some embodiments, preferably C strain of described swine fever attenuated virus.In some embodiments, the coded sequence of described swine Fever Vaccine and SEQ ID NO:1 have at least 80% homology.In some embodiments, the coded sequence of described swine Fever Vaccine is SEQ ID NO:1.
In some embodiments, the PRRSV that described PRRSV vaccine is attenuation.In this application, " attenuation PRRSV " refers to a kind of PRRSV, and it can infection host, but can not cause Porcine reproductive and respiratory syndrome, or its symptom causing is less and/or lighter.Attenuation PRRSV comprises attenuation PRRSV alive and the deactivation product being obtained by its deactivation." Porcine reproductive and respiratory syndrome " refers to a series of physiology of causing after natural PRRSV infected pigs and the symptom of pathology.It is slow or dead etc. that these symptoms include, but not limited to heating, drowsiness, inappetence, asthenia, dyspnea, cough, sow breeding difficulty, piglet growth.
In some embodiments, the PRRSV live vaccine that described PRRSV vaccine is attenuation.In some embodiments, described PRRSV vaccine contains Nsp1 nucleotide sequence, and the coded sequence of described sequence and SEQ ID NO:2 have at least 90% homology.In some embodiments, described PRRSV vaccine further contains Nsp2 nucleotide sequence, and the coded sequence of described Nsp2 nucleotide sequence and SEQ ID NO:3 have at least 90% homology.
" coded sequence " refers to a kind of DNA sequence in this application, and it can transcribedly obtain corresponding RNA sequence.PRRSV and swine fever virus are all 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 " refers to the similarity degree of two aminoacid sequences or two nucleotide sequences in this application.The homology of aminoacid sequence or nucleotide sequence can calculate by any suitable method well known in the art, for example, target amino acid (or nucleotide) sequence and reference aminoacid (or nucleotide) sequence can be carried out to sequence alignment, can introduce if desired vacancy, make aminoacid identical between the sequence of two comparisons (or nucleotide) number reach optimization, and calculate on this basis the percentage ratio of same amino acid (or nucleotide) between two aminoacid (or nucleotide) sequence.The comparison of aminoacid (or nucleotide) sequence and the calculating of homology can realize by software well known in the art, for example, but be not limited to, BLAST software (in the network address of state-run biotechnology information centre of the U.S. (NCBI), can obtain:
http:// blast.ncbi.nlm.nih.gov/Blast.cgi, or 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 (in European bio information institute network address, can obtain:
http:// www.ebi.ac.uk/lools/msa/clustalw2/, separately see, for example, Higgins D.G. et al, Methods in Enzymology, 266:383-402 (1996); Larkin M.A.et al, 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., separately see, 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)).Use software while carrying out sequence alignment, the default parameters that can use software to provide, or the parameter that also can provide software according to practical situation adjusts, and these are all in the knowledge of those skilled in the range.
In some embodiments, described 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, the nucleotide sequence that described porcine reproductive and respiratory syndrome virus vaccine contains porcine reproductive and respiratory syndrome virus, the coded sequence of this sequence and SEQ ID NO:4 have at least 90% homology.In some embodiments, the nucleotide sequence of described porcine reproductive and respiratory syndrome virus is encoded by SEQ IDNO:4.
In the vaccine combination providing in the application, can also further contain adjuvant.Adjuvant can protect vaccine not destroy in receptor, and/or stimulating immune system non-specifically, thereby contributes to strengthen the immunoreation to described vaccine.The example of adjuvant comprises, but be 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's adjuvant etc.), saponins (saponin) adjuvant (as: Stimulon
tMdeng), antibacterial or microorganism derivatives class (as, lipopolysaccharide, fat A derivant (lipid A derivatives) etc.) and microgranule (as poly--alpha-hydroxy acid etc.).
In the vaccine combination providing in the application, 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, Pidolidone sodium or lactoalbumin hydrolysate.
In some embodiments, in vaccine combination of the present invention, the ratio of two kinds of antigen liquids and freeze drying protectant is 1: 4.
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, described high-pathogenicity porcine reproductive and the preferably TJM strain of respiration syndrome attenuated virus, its microbial preservation number is: CGMCC NO.3121; The preferably C strain of described swine fever attenuated virus, through passage, should have hog cholera lapinised virus (spleen poison) limiting dilution assay to carry out two-wheeled clone purification and obtain hog cholera lapinised virus cell toxicant, and its microbial preservation number is: CGMCC NO.3891.
The concrete preservation information of PRRSV TJM strain is as follows: microbial preservation number: CGMCC No.3121; Classification And Nomenclature; 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's common micro-organisms center; The preservation time: on June 15th, 2009.
The concrete preservation information of C strain is as follows: microbial preservation number: CGMCC No.3891; Classification And Nomenclature: 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's common micro-organisms center; The preservation time: on May 27th, 2010.
Between PRRSV TJM strain and swine Fever Vaccine C strain, without immunosuppressant, and all there is good safety, immunogenicity and specificity, can be good at preventing high-pathogenicity porcine reproductive and respiration syndrome and the swine fever of Major Epidemic in current swinery.
The application also provides a kind of method of preparing above-mentioned porcine reproductive and respiratory syndrome virus vaccine and swine fever bigeminal live vaccine, comprising: (1) is gone down to posterity cell line and cultivated; (2) high-pathogenicity porcine reproductive and respiration syndrome TJM strain and fever virus lapinized Chinese Strain (spleen poison) are inoculated respectively to permissive cell system, cultivate by maintenance medium, seed culture of viruses is produced in preparation; (3) prepared production seed culture of viruses is inoculated respectively on the medium that covers with 90-100% cell, cultivated propagation by maintenance medium and obtain high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus antigen liquid and fever virus lapinized Chinese Strain cell toxicant antigen liquid; (4) high-pathogenicity porcine reproductive and respiration syndrome TJM strain antigen liquid and fever virus lapinized Chinese Strain cell toxicant antigen liquid are mixed in proportion, add freeze drying protectant, mix homogeneously, through lyophilisation, to obtain final product.
Wherein, the cell for the highly pathogenic PRRSV of growing described in step (1), (2), (3) includes but not limited to the primary cells such as continuous cell line or PAM cell such as Marc-145 cell line, MA-104 cell line, Vero cell line, CL-2621 cell line; Cell for the swine fever virus that grows includes but not limited to the primary cells such as continuous cell line or BT cell 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.Wherein PT cell line and ST cell line are pig testis continuous cell line.
Cell line described in step (1) go down to posterity and cultivation comprises: by cell line through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, with cell growth medium continue cultivate, when cell covers with 90-100%, go down to posterity or virus inoculation for continuing; 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 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 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
50; Hog cholera lapinised virus cell toxicant seed culture of viruses standard is that every 1ml should be not less than 10 containing 100,000 rabbit infective doses of viral > or application determination of immunofluorescence method viral level
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, cultivate 3-5 days after inoculation, gather in the crops virus liquid in the time that cytopathy reaches 70%; The infective dose (MOI) of fever virus lapinized Chinese Strain is that 0.1-0.5 or inoculum concentration are 3%-5% cell toxicant, inoculates latter 5 days and does to gather in the crops and change liquid for the first time, changes liquid every results on the 4th later, and results are no more than 5 times.
Described in step (3), gathering in the crops antigen liquid 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
50; Hog cholera lapinised virus cell seed culture of viruses is identified and is met fever virus lapinized Chinese Strain seed culture of viruses standard completely, pig is had no side effect safely, the every 1ml of cell seed culture of viruses should be not less than 10 containing 100,000 rabbit infective doses of viral > or application determination of immunofluorescence method viral level
6.0tCID
50.
Antigen liquid volume parts described in step (4) is 75-80, and the volume parts of heat-resisting lyophilized protecting agent is 25-20.
The effect inspection standard of the making articles described in step (4) comprises: in every dosage vaccine, high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus antigenic content answer>=10
5.0tCID
50/ ml; Hog cholera lapinised virus (C strain) antigenic content is answered>=7500 rabbit infective doses or application determination of immunofluorescence method viral level>=10
4.0tCID
50/ ml.
The present invention also provides the vaccine combination obtaining by above-mentioned preparation method, and it contains porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine.
The purposes of vaccine combination described in the present invention also provides 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 bigeminy Seedling aspect prevention high-pathogenicity porcine reproductive and respiration syndrome and swine fever.The present invention uses between high-pathogenicity porcine reproductive and respiration syndrome attenuated vaccine strain and swine fever attenuated vaccine strain without immunosuppressant; apply its bigeminy Seedling of preparing and each single Seedling comparison, indifference in safety, immunogenicity, immune duration, immune protective effect and storage life test.Safety testing shows, vaccine single dose, single dose repeat, safety after overdose inoculation test animal, and body temperature, the mental status are normal, without any clinical symptoms; Potency test demonstration, vaccine of the present invention has good protective effect 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; Immune duration test demonstration, immune duration is 6 months, can guarantee provides effective protection to pig in duration of immunity; Of storage test demonstration, vaccine is 18 months 2 ℃～8 ℃ storage lives, has long shelf-life, the advantage of stable storage.Apply can a pin anti-two diseases of goods immunity inoculation animal of the present invention, alleviate immunity inoculation workload, reduce immune time, reduce to swinery stress, avoid because of frequent immune immunological paralysis and the immuning failure causing.
The present invention also provides the method for immune swine, comprises pig is used to vaccine combination of the present invention.Can be by the mode of for example injecting to pig immunity.The mode such as single dose administration, multiple dose repeat administration of can carrying out is carried out immunity.Concrete immunization ways and immunizing dose can be according to being adjusted according to practical situation by the personnel that have veterinary's experience.
Figure of description
Fig. 1 is high-pathogenicity porcine reproductive and respiration syndrome and 37 ℃ of anti-aging test swine fever virus titer determinations of swine fever bigeminal live vaccine.
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 Pestivirus titer determination of swine fever bigeminal live vaccine.
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 and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications and replacement all fall within the scope of protection of the present invention.
The method that embodiment 1 use cell line is produced high-pathogenicity porcine reproductive and respiration syndrome and swine fever bigeminal live vaccine
(1) seedling going down to posterity and cultivating with cell: will produce high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus Marc-145 cell line through pancreas enzyme-EDTA cell dispersion liquid had digestive transfer culture, to three goes down to posterity; To produce fever virus lapinized Chinese Strain virus BT cell line through pancreas enzyme-EDTA cell dispersion liquid had digestive transfer culture, one to five goes down to posterity; Add respectively cell growth medium and continue to cultivate in 37 ℃, while forming good monolayer, go down to posterity or virus inoculation for continuing;
(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, cultivated 3-5 days after inoculation, gather in the crops virus liquid in the time that cytopathy reaches 70%; Fresh hog cholera lapinised virus spleen poison is made to 0.3% viral suspension, inoculate well-grown BT cell line monolayer, put 37 ℃ and continue to cultivate.Cultivate venom as production seed culture of viruses every harvesting on the 5th.
Seed culture of viruses is identified: tests by " People's Republic of China's veterinary drug allusion quotation " appendix, and should be without antibacterial, mycete, mycoplasma growth.Cell venom has no side effect safely to pig, 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
50; Hog cholera lapinised virus cell seed culture of viruses is identified and is met fever virus lapinized Chinese Strain seed culture of viruses standard completely, pig is had no side effect safely, the every 1ml of cell seed culture of viruses should be not less than 10 containing 100,000 rabbit infective doses of viral > or the every 1ml viral level of application determination of immunofluorescence method
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, after inoculation, cultivate 3-5 days, in the time that cytopathy reaches 70%, gather in the crops virus liquid as seedling venom; Get and cover with 90-100% monolayer BT cell, discard cell culture fluid, wash 2 times with PBS, be that 3%-5% inoculates by fever virus lapinized Chinese Strain cell toxicant seed culture of viruses according to MOI 0.1-0.5 or inoculum concentration, inoculate latter 5 days and do to gather in the crops and change liquid for the first time, change liquid every results on the 4th, results are no more than 5 times later, using each receipts of results time virus liquid as seedling venom; The virus liquid of results is put-20 ℃ of following preservations.
The check of seedling venom: test by " People's Republic of China's veterinary drug allusion quotation " appendix, should be without antibacterial, mycete, mycoplasma growth.Seedling venom has no side effect safely to pig, 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
50; Hog cholera lapinised virus cell seed culture of viruses is identified and is met fever virus lapinized Chinese Strain seed culture of viruses standard completely, pig is had no side effect safely, the every 1ml of cell seed culture of viruses should be not less than 10 containing 100,000 rabbit infective doses of viral > or the every 1ml viral level of application determination of immunofluorescence method
6.0tCID
50.
Above-mentioned nutrient solution used formula is: 92%-95%MEM liquid or DMEM liquid, 5%-8% calf serum, add appropriate antibiotics, pH value is adjusted into 7.2.The formula of above-mentioned maintenance medium used is:
95%-98%MEM liquid or DMEM liquid, 2-5% calf serum, add appropriate antibiotics, pH value is adjusted into 7.2.
(4) heat-resisting lyophilized protecting agent preparation: for subsequent use through autoclaving after each heat-resisting lyophilized protecting agent component is prepared according to a certain percentage.
(5) join Seedling, lyophilizing and subpackage: the freeze drying protectant that the antigen liquid that is 75-80 by volume parts is 25-20 with volume parts mix homogeneously after quantitative separating to ampulla, add a cover and be placed in freeze dryer, through pre-cooling, dry run freeze dried vaccine.After vaccine freeze-drying, carry out steriling test, safety examination and efficacy test.
High-pathogenicity porcine reproductive prepared by the present embodiment and respiration syndrome and swine fever bigeminal live vaccine, in every dosage vaccine, high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus antigenic content answer>=10
5.0tCID
50; Hog cholera lapinised virus (C strain) antigenic content is answered>=7500 rabbit infective doses or application determination of immunofluorescence method viral level>=10
4.0tCID
50; Character 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, be that (or fluorescent quantitation is 10 to 7500 rabbit infective doses containing hog cholera lapinised virus cell toxicant basis seed culture of viruses dosage
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 swine fever feminine gender is not inoculated matched group, uses 3 experimental animals, musculi colli injection Cell sap; The 7th group is blank group, uses 2 experimental animals, does not inoculate any material to off-test.
Latter 7 days of first 3 days of vaccination and inoculation, measure all piglet rectal temperatures every day and carry out clinical observation; Before inoculation, after 3d and inoculation, 0d, 3d, 7d, 10d, 14d, 21d, 28d, 31d, 35d, 38d, 42d gather all piglet anticoagulations and coagulant blood, and anticoagulation is for CD3
+, CD4
+, CD8
+t cell detection, coagulant blood separation of serum is for antibody titer monitoring.
Immunity is carried out challenge test in latter 28 days, get first group, the 3rd group, the 5th group experimental animal respectively collunarium add intramuscular injection high-pathogenicity porcine reproductive and respiration syndrome and check with a strong malicious 3ml/ pig, collunarium 2ml (1ml/ nostril), intramuscular injection 1ml; Getting second group, the 4th group, the 6th group experimental animal intramuscular injection swine fever checks with strong arsenic bloom door blood poison, 1ml/ pig; The 7th group of blank experimental animal do not attacked any strong poison.Attack after every day viewing test animal clinical symptoms, comprise appetite, breathing, the mental status etc., every day thermometric.High-pathogenicity porcine reproductive and respiration syndrome strong virus attack are tested and are finished for 21 days after counteracting toxic substances, and swine fever strong virus attack is tested and finished for 16 days after counteracting toxic substances, after challenge trial finishes, calculates every treated animal clinical protection rate, M & M.
Result: leukocyte testing result shows: in latter 28 days of immunity, 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 after two kinds of basic seeds culture of viruses of immunity, non-interference antibody generates, and the 3rd group consistent with the anti-highly pathogenic PRRSV antibody Fluctuation of the 4th group of experimental animal anti-highly pathogenic PRRSV antibody Fluctuation and first group of experimental animal, the 3rd group consistent with the swine fever virus resistant antibody Fluctuation of the 4th group of experimental animal and the swine fever virus resistant antibody Fluctuation of second group of experimental animal; High-pathogenicity porcine reproductive and respiration syndrome check show by strong malicious counteracting toxic substances result: first group of counteracting toxic substances protective rate is 5/5, and 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 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 counteracting toxic substances sickness rate is 5/5, counteracting toxic substances mortality rate 3/5; Swine fever check shows by strong arsenic bloom door blood poison counteracting toxic substances result: second group of counteracting toxic substances protective rate is 5/5, and 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 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 counteracting toxic substances sickness rate is 3/3, counteracting toxic substances mortality rate 3/3; In sum, result of the test shows, between the basic seed culture of viruses of high-pathogenicity porcine reproductive and respiration syndrome TJM strain basis seed culture of viruses and hog cholera lapinised virus cell toxicant without immunosuppressant.
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 to safety testing.Content of the test comprises that fattening piglet 1 single dose inoculation safety testing, single dose repeated inoculation safety testing, overdose inoculates safety testing, non-use age in days piglet overdose inoculation safety testing and different cultivars pig overdose are inoculated to safety testing.
Result shows, after single dose inoculation, single dose repeated inoculation and overdose inoculation, each group experimental animal body temperature is normal, the mental status and appetite are good, injection site and whole body have no untoward reaction, on fertility performance, without impact, bigeminy Seedling overdose is inoculated non-use age in days piglet, different cultivars pig overdose is inoculated to 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) prepared by laboratory are carried out respectively potency test, when carrying out every batch of laboratory products potency test, design two kinds of single Seedling controlled trial groups of high-pathogenicity porcine reproductive and respiration syndrome TJM strain and fever virus lapinized Chinese Strain (C strain) cell toxicant, join the effect comparative study of Seedling and single Seedling.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, test points six groups.5 of first group, second group each service test animals, musculi colli injection inoculation bigeminal live vaccine, dosage is 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
50.5 of the 3rd group of service test animals, musculi colli injection inoculation fever virus lapinized Chinese Strain (C strain) cell toxicant vaccine, dosage is 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 1ml/ head, vaccine contg is 10
5.0tCID
50.The 5th group is that 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 experimental animal vaccination 28 days, get first group, the 3rd group, the 5th group experimental animal strong arsenic bloom door blood poison of intramuscular injection swine fever check respectively, 1ml/ pig, swine fever challenge test finishes for 16 days after counteracting toxic substances, get second group, the 4th group, the 6th group experimental animal respectively collunarium add the check of intramuscular injection high-pathogenicity porcine reproductive and respiration syndrome with a strong malicious 3ml/ pig, collunarium 2ml (1ml/ nostril), intramuscular injection 1ml, high-pathogenicity porcine reproductive and respiration syndrome challenge test finish for 21 days after counteracting toxic substances.Result shows, after the 3 batches of high-pathogenicity porcine reproductives and respiration syndrome and swine fever bigeminal live vaccine inoculation test animal, bigeminy Seedling immune group all can produce good protective effect to high-pathogenicity porcine reproductive and respiration syndrome strong virus attack and swine fever strong virus attack, with single Seedling matched group indifference, wherein, three batches of bigeminy Seedlings are respectively 5/5 to the protective rate of swine fever, 5/5 and 5/5, three batches of swine fever list Seedlings are respectively 5/5 to the protective rate of swine fever, 5/5 and 5/5, three batches of bigeminy Seedlings are respectively 4/5 to the protective rate of high-pathogenicity porcine reproductive and respiration syndrome, 4/5 and 5/5, the three batches of high-pathogenicity porcine reproductives and respiration syndrome list Seedling are respectively 5/5 to the protective rate of high-pathogenicity porcine reproductive and respiration syndrome, 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 obvious clinical symptoms, the sickness rate standard that conforms with the regulations.
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, high-pathogenicity porcine reproductive prepared by laboratory and respiration syndrome and swine fever bigeminal live vaccine carry out immune duration test, simultaneously compare research with high-pathogenicity porcine reproductive and respiration syndrome TJM strain and two kinds of single Seedling immune durations of fever virus lapinized Chinese Strain (C strain) cell toxicant.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 is 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
50.10 of the 3rd group of service test animals, musculi colli injection inoculation fever virus lapinized Chinese Strain (C strain) cell toxicant vaccine, dosage is 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 1ml/ head, vaccine contg is 10
5.0tCID
50.The 5th group is that 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.After vaccination, blood sampling in 1 month, 2 months, 3 months, 4 months, 5 months, 6 months detects antibody titer; The each group of experimental animal that extracts respectively half quantity for after vaccine immunity 3 months and 6 months carries out challenge test, wherein first group, the 3rd group, the 5th group experimental animal be intramuscular injection swine fever strong arsenic bloom door blood poison for check respectively, 1ml/ pig, second group, the 4th group, the 6th group experimental animal respectively collunarium injection high-pathogenicity porcine reproductive checked with a strong malicious 2ml/ pig, 1ml/ nostril with respiration syndrome.March, 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, 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 to 6 months, can guarantee provides effective protection to pig in duration of immunity.This result of the test shows to join immune duration effect and the two kinds of single Seedlings generation immune duration effect indifferences that Seedling produces simultaneously.
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 laboratory to prepare the 3 batches of high-pathogenicity porcine reproductives and respiration syndrome and the swine fever bigeminal live vaccine (200904,200905,200906) of different size, complete 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).3 batches of bigeminy Seedling goods are placed in 2～8 ℃ of preservations and sample and carry out character, vacuum, residual moisture content, potency test and 37 ℃ of anti-aging tests respectively for 3,6,9,12,18 months.Result shows that 3 batches of goods preserve after 18 months at 2～8 ℃, and character be still white loose agglomerate, adds dissolving rapidly after diluent; Vacuum detects as white or purple aura; Average residual moisture all meets " People's Republic of China's veterinary drug allusion quotation " required standard; In 3 batches of bigeminy Seedlings (200904,200905,200906), high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus are tired and are respectively 10
5.3tCID
50/ ml, 10
5.3tCID
50/ ml, 10
5.2tCID
50/ ml, the 3 batches of high-pathogenicity porcine reproductives and respiration syndrome TJM strain list Seedling (200907,200908,200909) virus titer are respectively 10
5.2tCID
50/ ml, 10
5.3tCID
50/ ml, 10
5.1tCID
50/ ml, in 3 batches of bigeminy Seedlings (200904,200905,200906), hog cholera lapinised virus cell toxicant is tired and is respectively 10
4.2tCID
50/ ml, 10
4.1tCID
50/ ml, 10
4.3tCID
50/ ml (all>=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>=7500 rabbit infective doses), 2～8 ℃ of storage life result of the tests show bigeminy Seedling and relatively difference with insignificance of two kinds of single Seedlings.Place virus titer on the 14th for 37 ℃ and still maintain higher level, in 3 batches of bigeminy Seedlings (200904,200905,200906), high-pathogenicity porcine reproductive and respiration syndrome TJM strain virus are tired and are respectively 10
5.3tCID
50/ ml, 10
5.2tCID
50/ ml, 10
5.2tCID
50/ ml, the 3 batches of high-pathogenicity porcine reproductives and respiration syndrome TJM strain list Seedling (200907,200908,200909) virus titer are respectively 10
5.2tCID
50/ ml, 10
5.4tCID
50/ ml, 10
5.3tCID
50/ ml, in 3 batches of bigeminy Seedlings (200904,200905,200906), hog cholera lapinised virus cell toxicant is tired and is respectively 10
4.2tCID
50/ ml, 10
4.2tCID
50/ ml, 10
4.3tCID
50/ ml (all>=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>=7500 rabbit infective doses), can reach vaccine quality standard.Illustrate that this heat-resisting lyophilized protecting agent all has good protective effect to high-pathogenicity porcine reproductive and respiration syndrome TJM strain and fever virus lapinized Chinese 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 practical application, make stable the reaching or approaching similar vaccine level in the world of preservation of this live vaccine.
Claims (22)
1. a vaccine combination, contain porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine, the PRRSV live vaccine that wherein said porcine reproductive and respiratory syndrome virus vaccine is attenuation, and wherein said porcine reproductive and respiratory syndrome virus vaccine contains the Nsp1 nucleotide sequence by SEQ ID NO:2 coding, and contain the Nsp2 nucleotide sequence by SEQ ID NO:3 coding.
2. vaccine combination according to claim 1, is characterized in that, essentially no immunosuppressant between described porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine.
3. vaccine combination according to claim 1, the mixed proportion of wherein said swine Fever Vaccine and described porcine reproductive and respiratory syndrome virus vaccine is that 1:2 is to 1:4.
4. vaccine combination according to claim 1, the TCID of wherein said swine Fever Vaccine and described porcine reproductive and respiratory syndrome virus vaccine
50mixed proportion be 1:10.
5. vaccine combination according to claim 1, the content of wherein said swine Fever Vaccine is that 7500 rabbit infective dose/ml or fluorescent quantitation are 10
4.0tCID
50/ ml, 10
4.1tCID
50/ ml, 10
4.2tCID
50/ ml or 10
4.3tCID
50/ ml, and/or the content of described porcine reproductive and respiratory syndrome virus vaccine is 10
5.0tCID
50/ ml, 10
5.3tCID
50/ ml or 10
5.2tCID
50/ ml.
6. vaccine combination according to claim 1, the coded sequence of wherein said swine Fever Vaccine is as shown in SEQ ID NO:1.
7. vaccine combination according to claim 1, the nucleotide sequence that wherein said porcine reproductive and respiratory syndrome virus vaccine contains porcine reproductive and respiratory syndrome virus, this sequence is encoded by SEQ ID NO:4.
8. vaccine combination according to claim 1, it further contains adjuvant.
9. vaccine combination according to claim 1, it further contains freeze drying protectant.
10. vaccine combination according to claim 9, the volume parts of described freeze drying protectant is 25-20.
11. vaccine combinations according to claim 9, wherein said freeze drying protectant comprises sucrose, Pidolidone sodium or lactoalbumin hydrolysate.
Prepare the method for vaccine combination claimed in claim 1, comprising for 12. 1 kinds:
(1) cell line gone down to posterity and cultivate;
(2) porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine are inoculated respectively to permissive cell, cultivate by maintenance medium, kind of a poison is produced in preparation;
(3) prepared production kind poison is inoculated respectively on the medium that covers with 90-100% cell, cultivated propagation by maintenance medium and obtain porcine reproductive and respiratory syndrome virus vaccine antigen liquid and swine Fever Vaccine antigen liquid;
(4) porcine reproductive and respiratory syndrome virus vaccine antigen liquid and swine Fever Vaccine antigen liquid are mixed.
13. in accordance with the method for claim 12, it is characterized in that: the cell for the porcine reproductive and respiratory syndrome virus vaccine of growing described in step (1), (2), (3) is the continuous cell line of Marc-145 cell line, MA-104 cell line, Vero cell line, CL-2621 cell line or the primary cell of PAM cell.
14. in accordance with the method for claim 12, it is characterized in that: the cell for the swine Fever Vaccine of growing described in step (1), (2), (3) is 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 the continuous cell line of PT cell line or the primary cell of BT cell.
15. in accordance with the method for claim 12, it is characterized in that: the cell culture processes described in step (1) for following any one: in rolling bottle, cultivate, its cell density 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.
16. in accordance with the method for claim 12, 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, after inoculation, cultivate 3-5 days, in the time that cytopathy reaches 70%, gather in the crops virus liquid, produce seed culture of viruses and antigen liquid in order to preparation.
17. in accordance with the method for claim 12, 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 3%-5% cell toxicant, inoculate latter 5 days and do to gather in the crops and change liquid for the first time, change liquid every results on the 4th, results are no more than 5 times later.
18. in accordance with the method for claim 12, it is characterized in that: further in step (4), add freeze drying protectant, described freeze drying protectant obtains after by sucrose, Pidolidone sodium or lactoalbumin hydrolysate mixed dissolution after autoclaving.
19. in accordance with the method for claim 18, it is characterized in that: the freeze drying protectant that the antigen liquid that is 75-80 by volume parts in step (4) is 25-20 with volume parts is mixed homogeneously and prepared described vaccine combination.
The vaccine combination that 20. any one preparation method of claim 12-19 obtain, it contains porcine reproductive and respiratory syndrome virus vaccine and swine Fever Vaccine.
The purposes of 21. vaccine combinations claimed in claim 1 in the biological product of preparation prevention or treatment Porcine reproductive and respiratory syndrome and swine fever.
22. vaccine combinations claimed in claim 1 are being prepared the purposes of pig vaccine, and described pig vaccine is for immune swine.
Priority Applications (10)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 |
| PCT/CN2012/076125 WO2012163258A1 (en) | 2011-05-27 | 2012-05-25 | Combined vaccines for prevention of porcine virus infections |
| TW101118902A TWI579297B (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 |
| KR1020137034298A KR20140036262A (en) | 2011-05-27 | 2012-05-25 | Combined vaccines for prevention of porcine virus 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. |
| 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 |
| JP2014511724A JP6096176B2 (en) | 2011-05-27 | 2012-05-25 | Combination vaccine for the prevention of swine virus infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102727883A CN102727883A (en) | 2012-10-17 |
| CN102727883B true CN102727883B (en) | 2014-05-14 |
Family
ID=46984644
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110140951.5A Active CN102727883B (en) | 2011-05-27 | 2011-05-27 | Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102727883B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102973933B (en) * | 2011-12-29 | 2014-08-27 | 普莱柯生物工程股份有限公司 | Bivalent vaccine for porcine reproductive and respiratory syndrome and classical swine fever prevention or treatment, and preparation method thereof |
| CN103041384B (en) * | 2012-12-29 | 2016-03-16 | 普莱柯生物工程股份有限公司 | A kind of vaccine combination and its preparation method and application |
| CN104561092B (en) * | 2014-12-19 | 2018-12-11 | 中国农业科学院上海兽医研究所 | Express construction method and the application of CSFV E 2 protein recombination PRRS viral genetic engineering vaccine |
| CN116763914B (en) * | 2023-02-06 | 2024-02-23 | 广东永顺生物制药股份有限公司 | Swine fever and highly pathogenic porcine reproductive and respiratory syndrome bivalent heat-resistant protective agent live vaccine and preparation method thereof |
| CN116286677B (en) * | 2023-04-07 | 2023-12-12 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | African swine fever virus strain and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006099561A1 (en) * | 2005-03-14 | 2006-09-21 | Boehringer Ingelheim Vetmedica, Inc. | Immunogenic compositions comprising lawsonia intracellularis |
| EP1792996A1 (en) * | 2005-12-01 | 2007-06-06 | Consejo Superior de Investigaciones Cientificas | Nucleic acid sequences encoding vaccines against Porcine reproductive and respiratory syndrome virus (PRRSV) |
| WO2007076520A2 (en) * | 2005-12-29 | 2007-07-05 | Boehringer Ingelheim Vetmedica, Inc. | Multivalent pcv2 immunogenic compositions and methods of producing such compositions |
| CN101332298A (en) * | 2007-06-29 | 2008-12-31 | 薛景山 | Bivalent vaccine for pig and preparation method thereof |
| WO2009056541A1 (en) * | 2007-10-31 | 2009-05-07 | Novartis Ag | Protection of an animal against pestivirus infection |
| CN101633909A (en) * | 2009-08-13 | 2010-01-27 | 武华 | Attenuated live vaccine strain for preventing pig-pig infection breeding and respiratory syndrome |
| CN101690808A (en) * | 2009-10-14 | 2010-04-07 | 广东永顺生物制药有限公司 | Method for preparing swine fever-pseudorabies bigeminal live vaccine and product thereof |
| CN101879311A (en) * | 2010-06-28 | 2010-11-10 | 武华 | Method for preparing live vaccines of hog cholera and product thereof |
| CN102100910A (en) * | 2009-12-21 | 2011-06-22 | 北京清大天一科技有限公司 | Method for producing viral vaccines |
-
2011
- 2011-05-27 CN CN201110140951.5A patent/CN102727883B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006099561A1 (en) * | 2005-03-14 | 2006-09-21 | Boehringer Ingelheim Vetmedica, Inc. | Immunogenic compositions comprising lawsonia intracellularis |
| EP1792996A1 (en) * | 2005-12-01 | 2007-06-06 | Consejo Superior de Investigaciones Cientificas | Nucleic acid sequences encoding vaccines against Porcine reproductive and respiratory syndrome virus (PRRSV) |
| WO2007076520A2 (en) * | 2005-12-29 | 2007-07-05 | Boehringer Ingelheim Vetmedica, Inc. | Multivalent pcv2 immunogenic compositions and methods of producing such compositions |
| CN101332298A (en) * | 2007-06-29 | 2008-12-31 | 薛景山 | Bivalent vaccine for pig and preparation method thereof |
| WO2009056541A1 (en) * | 2007-10-31 | 2009-05-07 | Novartis Ag | Protection of an animal against pestivirus infection |
| CN101633909A (en) * | 2009-08-13 | 2010-01-27 | 武华 | Attenuated live vaccine strain for preventing pig-pig infection breeding and respiratory syndrome |
| CN101690808A (en) * | 2009-10-14 | 2010-04-07 | 广东永顺生物制药有限公司 | Method for preparing swine fever-pseudorabies bigeminal live vaccine and product thereof |
| CN102100910A (en) * | 2009-12-21 | 2011-06-22 | 北京清大天一科技有限公司 | Method for producing viral vaccines |
| CN101879311A (en) * | 2010-06-28 | 2010-11-10 | 武华 | Method for preparing live vaccines of hog cholera and product thereof |
Non-Patent Citations (7)
| Title |
|---|
| Factors critical for successful vaccination against classical swine fever in endemic areas;S.Suradhat et.al.;《Veterinary Microbiology》;20061012;第119卷(第1期);第1-9页 * |
| Negative impact of porcine reproductive and respiratory syndrome virus infection on the efficacy of classical swine fever vaccine;S.Suradhat et.al.;《Vaccine》;20051219;第24卷(第14期);第2634-2642页 * |
| S.Suradhat et.al..Factors critical for successful vaccination against classical swine fever in endemic areas.《Veterinary Microbiology》.2006,第119卷(第1期),第1-9页. |
| S.Suradhat et.al..Negative impact of porcine reproductive and respiratory syndrome virus infection on the efficacy of classical swine fever vaccine.《Vaccine》.2005,第24卷(第14期),第2634-2642页. |
| 张长龙等.猪瘟疫苗（兔源）猪O型口蹄疫疫苗、猪蓝耳病疫苗有效免疫试验报告.《畜禽业》.2008,(第233期),第40-43页. * |
| 沈惠忠等.猪繁殖与呼吸障碍综合征、猪瘟二种疫苗同时分点免疫方法研究.《上海畜牧兽医通讯》.2005,(第5期), |
| 猪繁殖与呼吸障碍综合征、猪瘟二种疫苗同时分点免疫方法研究;沈惠忠等;《上海畜牧兽医通讯》;20051117(第5期);第41页左栏，第3.2节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102727883A (en) | 2012-10-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104862286B (en) | Porcine pseudorabies virus gene-deleted strain, vaccine composition and its preparation method and application | |
| US8197823B2 (en) | Vaccine for porcine reproductive and respiratory syndrome, a preparion method and use thereof | |
| JP6096176B2 (en) | Combination vaccine for the prevention of swine virus infection | |
| CN102250843B (en) | Genetic engineering marked attenuated vaccine strain of porcine reproductive and respiratory syndrome virus and application thereof | |
| CN104784686B (en) | TGEV, PEDV bigeminal live vaccine and preparation method thereof | |
| CN104498441B (en) | Duck hepatitis A virus (HAV) type III low virulent strain and live vaccine prepared therefrom and application | |
| CN104004774A (en) | Swine pseudorabies virus and vaccine composition as well as preparation method and application thereof | |
| CN104513827A (en) | Porcine epizootic diarrhea virus strain, attenuated vaccine strain thereof and application thereof | |
| CN104162154B (en) | Bivalent inactivated vaccine against bovine viral diarrhea and infectious bovine rhinotracheitis, and preparation method and application thereof | |
| CN102727883B (en) | Combined live vaccine against porcine reproductive and respiratory syndrome and swine fever, and application thereof | |
| CN108486067A (en) | The inactivated vaccine and application of Porcine epidemic diarrhea virus variant and its preparation | |
| CN106148287A (en) | Porcine epidemic diarrhea virus strain and vaccine combination, preparation method and application | |
| CN103725651A (en) | Porcine epidemic diarrhea virus stain and application thereof | |
| CN102727884B (en) | Combined live vaccine against porcine reproductive and respiratory syndrome and pseudorabies, and preparation method thereof | |
| CN113491767A (en) | Triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and preparation method thereof | |
| CN104774811A (en) | Swine pseudorabies virus PRV-YF strain and application thereof | |
| CN107823639B (en) | Inactivated vaccine for bovine viral diarrhea virus and preparation method thereof | |
| CN108452298A (en) | A kind of technique producing yellow fever attenuated live vaccine with SPF chick-embryo cells | |
| CN103923885B (en) | Infections chicken cloacal bursa virus Vero cell adapted strain and application thereof | |
| CN102727882B (en) | Combined live vaccine against porcine reproductive and respiratory syndrome, swine fever and pseudorabies, and preparation method thereof | |
| CN105802921B (en) | Recombinant pseudorabies virus variant strain for expressing classical swine fever virus E2protein and construction method and application thereof | |
| CN105385661A (en) | Porcine circovirus type 2 large-scale cultivation method and applications thereof | |
| CN104288760A (en) | Vaccine composition, and preparation method and application thereof | |
| CN104388396B (en) | Porcine pseudorabies poison strain and inactivated vaccine prepared therefrom and application | |
| CN102965345B (en) | Porcine parvovirus, vaccine composition and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: SINOVET (JIANGSU) BIOPHARMACEUTICAL CO., LTD. Effective date: 20130329 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20130329 Address after: 100085 B302 room three, B District, Zhongguancun biomedicine Park, 5 Haidian District Road, Beijing, China Applicant after: Sinovet (Beijing) Biotechnology Co., Ltd. Applicant after: SINOVET (JIANGSU) BIOTECHNOLOGY CO., LTD. Address before: 100085 B302 room three, B District, Zhongguancun biomedicine Park, 5 Haidian District Road, Beijing, China Applicant before: Sinovet (Beijing) Biotechnology Co., Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wu Hua Inventor after: Xia Mingqi Inventor after: Wang Wei Inventor after: He Yanliang Inventor after: Wang Xin Inventor before: Wu Hua Inventor before: Xia Mingqi Inventor before: He Yanliang |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: WU HUA XIA MINGQI HE YANLIANG TO: WU HUA XIA MINGQI WANG WEI HE YANLIANG WANG XIN |
|
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wu Hua Inventor after: Xia Mingqi Inventor after: Wang Wei Inventor after: He Yanliang Inventor after: Wang Xin Inventor after: Zhao Jianzeng Inventor before: Wu Hua Inventor before: Xia Mingqi Inventor before: Wang Wei Inventor before: He Yanliang Inventor before: Wang Xin |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: WU HUA XIA MINGQI WANG WEI HE YANLIANG WANG XIN TO: WU HUA XIA MINGQI WANG WEI HE YANLIANG WANG XIN ZHAO JIANZENG |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20170904 Address after: 388 room 1, building 225300, No.1 drug City Avenue, Taizhou, Jiangsu Patentee after: SINOVET (JIANGSU) BIOTECHNOLOGY CO., LTD. Address before: 100085 B302 room three, B District, Zhongguancun biomedicine Park, 5 Haidian District Road, Beijing, China Co-patentee before: SINOVET (JIANGSU) BIOTECHNOLOGY CO., LTD. Patentee before: Sinovet (Beijing) Biotechnology Co., Ltd. |
|
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 225300, 409 Gao Jiang Road, Jiangsu, Taizhou Patentee after: SINOVET (JIANGSU) BIOTECHNOLOGY CO., LTD. Address before: 388 room 1, building 225300, No.1 drug City Avenue, Taizhou, Jiangsu Patentee before: SINOVET (JIANGSU) BIOTECHNOLOGY CO., LTD. |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Combined vaccine of porcine reproductive and respiratory syndrome and classical swine fever and its application Effective date of registration: 20220107 Granted publication date: 20140514 Pledgee: China Merchants Bank Co.,Ltd. Taizhou Branch Pledgor: SINOVET (JIANGSU) BIOLOGICAL PHARMACEUTICAL Co.,Ltd. Registration number: Y2022980000222 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20220518 Granted publication date: 20140514 Pledgee: China Merchants Bank Co.,Ltd. Taizhou Branch Pledgor: SINOVET (JIANGSU) BIOLOGICAL PHARMACEUTICAL Co.,Ltd. Registration number: Y2022980000222 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |