CN115991749A - Porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine - Google Patents
Porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine Download PDFInfo
- Publication number
- CN115991749A CN115991749A CN202211075065.3A CN202211075065A CN115991749A CN 115991749 A CN115991749 A CN 115991749A CN 202211075065 A CN202211075065 A CN 202211075065A CN 115991749 A CN115991749 A CN 115991749A
- Authority
- CN
- China
- Prior art keywords
- porcine
- pasteurellosis
- subunit vaccine
- proteins
- protein
- 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.)
- Pending
Links
- 229940031626 subunit vaccine Drugs 0.000 title claims abstract description 47
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 36
- 230000002458 infectious effect Effects 0.000 title claims abstract description 28
- 206010035664 Pneumonia Diseases 0.000 title claims abstract description 27
- 201000006509 pleuropneumonia Diseases 0.000 title claims abstract description 27
- 206010034107 Pasteurella infections Diseases 0.000 title claims abstract description 23
- 201000005115 pasteurellosis Diseases 0.000 title claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 74
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 55
- 241000606856 Pasteurella multocida Species 0.000 claims abstract description 31
- 108091007433 antigens Proteins 0.000 claims abstract description 31
- 102000036639 antigens Human genes 0.000 claims abstract description 31
- 229940051027 pasteurella multocida Drugs 0.000 claims abstract description 31
- 241000606748 Actinobacillus pleuropneumoniae Species 0.000 claims abstract description 28
- 241000204031 Mycoplasma Species 0.000 claims abstract description 11
- 239000013598 vector Substances 0.000 claims description 15
- 239000002671 adjuvant Substances 0.000 claims description 12
- 241000282898 Sus scrofa Species 0.000 claims description 9
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical group [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 claims description 9
- 230000000890 antigenic effect Effects 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 230000009465 prokaryotic expression Effects 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 abstract description 21
- 101000657454 Actinobacillus pleuropneumoniae RTX-I toxin determinant A from serotypes 1/9 Proteins 0.000 abstract description 12
- 230000003053 immunization Effects 0.000 description 34
- 238000002649 immunization Methods 0.000 description 34
- 241000699670 Mus sp. Species 0.000 description 23
- 239000013612 plasmid Substances 0.000 description 17
- 210000002966 serum Anatomy 0.000 description 14
- 239000000427 antigen Substances 0.000 description 13
- 239000002773 nucleotide Substances 0.000 description 12
- 125000003729 nucleotide group Chemical group 0.000 description 12
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 11
- 101150049752 ompD gene Proteins 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 241000282887 Suidae Species 0.000 description 10
- 238000010367 cloning Methods 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 10
- 101150067790 mlaA gene Proteins 0.000 description 10
- 101150008329 LPPB gene Proteins 0.000 description 8
- 101100399642 Salmonella paratyphi A (strain ATCC 9150 / SARB42) lpp3 gene Proteins 0.000 description 8
- 101150005075 lpp2 gene Proteins 0.000 description 8
- 101100350268 Actinobacillus pleuropneumoniae omlA gene Proteins 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 7
- 101150117480 bamE gene Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 101150060808 ompH gene Proteins 0.000 description 7
- 101150057202 ompW gene Proteins 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 101150038679 skp gene Proteins 0.000 description 7
- 230000004083 survival effect Effects 0.000 description 7
- 101100295756 Acinetobacter baumannii (strain ATCC 19606 / DSM 30007 / JCM 6841 / CCUG 19606 / CIP 70.34 / NBRC 109757 / NCIMB 12457 / NCTC 12156 / 81) omp38 gene Proteins 0.000 description 6
- 101710116435 Outer membrane protein Proteins 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 101150042295 arfA gene Proteins 0.000 description 6
- 244000309466 calf Species 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 101150087557 omcB gene Proteins 0.000 description 6
- 101150115693 ompA gene Proteins 0.000 description 6
- 231100000419 toxicity Toxicity 0.000 description 6
- 230000001988 toxicity Effects 0.000 description 6
- 101100245206 Dictyostelium discoideum psmC4 gene Proteins 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 229940031551 inactivated vaccine Drugs 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 101150095556 tbpB gene Proteins 0.000 description 5
- 239000003053 toxin Substances 0.000 description 5
- 231100000765 toxin Toxicity 0.000 description 5
- 108700012359 toxins Proteins 0.000 description 5
- 238000011725 BALB/c mouse Methods 0.000 description 4
- 108090001030 Lipoproteins Proteins 0.000 description 4
- 102000004895 Lipoproteins Human genes 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 238000010353 genetic engineering Methods 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000001018 virulence Effects 0.000 description 4
- 241000606750 Actinobacillus Species 0.000 description 3
- 201000008283 Atrophic Rhinitis Diseases 0.000 description 3
- 108010079246 OMPA outer membrane proteins Proteins 0.000 description 3
- 241000606860 Pasteurella Species 0.000 description 3
- 206010039088 Rhinitis atrophic Diseases 0.000 description 3
- 108010031127 Transferrin-Binding Protein B Proteins 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 101150003244 AN gene Proteins 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 208000004852 Lung Injury Diseases 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 206010069363 Traumatic lung injury Diseases 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 229940031567 attenuated vaccine Drugs 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 230000002008 hemorrhagic effect Effects 0.000 description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 2
- 231100000515 lung injury Toxicity 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- -1 omp Proteins 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 101150053844 APP1 gene Proteins 0.000 description 1
- 206010002515 Animal bite Diseases 0.000 description 1
- 206010002519 Animal scratch Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 208000012353 Contagious Pleuropneumonia Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000606807 Glaesserella parasuis Species 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 208000032969 Hemorrhagic Septicemia Diseases 0.000 description 1
- 101100189105 Homo sapiens PABPC4 gene Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 101710105045 Lipoprotein E Proteins 0.000 description 1
- 208000014645 Pasteurella hemorrhagic septicemia Diseases 0.000 description 1
- 241000606752 Pasteurellaceae Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 102100039424 Polyadenylate-binding protein 4 Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 206010042434 Sudden death Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 102100038359 Xaa-Pro aminopeptidase 3 Human genes 0.000 description 1
- 101710081949 Xaa-Pro aminopeptidase 3 Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 229910021502 aluminium hydroxide Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
- 231100000776 exotoxin Toxicity 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 231100000001 growth retardation Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 231100000668 minimum lethal dose Toxicity 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 101150003170 omlA gene Proteins 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229940124856 vaccine component Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine, and belongs to the field of veterinary vaccines. The bivalent subunit vaccine contains truncated actinobacillus pleuropneumoniae antigen proteins ApxI AN, apxII AN, apxIII AN and OmpD and porcine pasteurella multocida antigen proteins PlpE and VacJ, and can provide cross protection for porcine infectious actinobacillus pleuropneumoniae and porcine pasteurella multocida of different serotypes. The truncated proteins selected from the antigen protein combination can be efficiently and soluble expressed, and the antigen protein combination is easy to produce on a large scale and low in cost. The invention provides an effective technical means for preventing and treating porcine infectious pleuropneumonia and porcine pasteurellosis.
Description
Technical Field
The invention relates to the field of veterinary vaccines, in particular to a swine infectious pleuropneumonia-swine pasteurellosis bigeminal subunit vaccine.
Background
Porcine infectious pleuropneumonia (Porcine contagious pleuropneumonia, PCP) is caused by infection with actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae, APP), an acute contagious disease characterized by acute hemorrhagic cellulosic pleuropneumonia and chronic cellulosic necrotic pleuropneumonia (Chenyan. Veterinary infectious diseases [ M ]. Fifth edition. Beijing: china agricultural Press: 2011: 262-265.). Pigs at all ages can be infected with the pathogenic bacteria, and particularly pigs and nursery pigs are most susceptible. APP has extremely high acute infection mortality rate, usually causes sudden death of pigs without predictability, chronic infection causes growth retardation of pigs, reduces feed conversion rate, is easy to cause death due to other pathogenic infection, and causes extremely large economic loss to pig industry (Nahar N, turni C, tram G, blackall PJ, atack JM. Actinobacillus pleuropenoniae: the molecular determinants of virulence and pathogensis. Adv Microb Physiol.2021; 78:179-216.).
Actinobacillus pleuropneumoniae belongs to the genus actinobacillus of the family Pasteurellaceae, and is a gram-negative type of small bacillus. APP is currently divided into 19 serotypes, depending on the bacterial capsule and lipopolysaccharide antigen, with virulence varying between serotypes, and lack of cross-protection. The epidemic serotypes of APP are different in different regions in China, types 1, 3, 5 and 7 are dominant serotypes, types 5 and 7 are mainly popular in most northern regions in China, and types 1, 3 and 7 are mainly used in southern regions such as Fujian, guangdong and the like (Zhu Xiugao, li Yanqing. Actinobacillus pleuropneumoniae infection and serotype distribution [ J)]Animal medicine progress 2017,38 (10): 111-113.). Apx toxins are major virulence factors for APP, including ApxI, apxII, apxIII, apxIV, which vary in virulence but play a critical role in the pathogenesis of APP, wherein ApxIV is present in all APP serotypes, and serotypes 1, 5, 9, 11 secrete ApxI, apxII, serotypes 2, 3, 6, 8 secrete ApxII, apxIII, types 10 secrete ApxI, 7, 12 secrete ApxII (ITO H, SUEYOSHI M.the genetic organization of the capsular polysaccharide biosynthesis region of Actinobacillus pleuropneumoniae serotype [ J ]]Journal of Veterinary Medical Science,2015,77 (4): 483-486.). Subunit vaccines marketed have a large number of antigenic components, conferring cross-protection to some extent to all serotypes (Sassu EL,
Tobias TJ,Gottschalk M,Langford PR,Hennig-Pauka I.Update on Actinobacillus pleuropneumoniae-knowledge,gaps and challenges.Transboundary&emerging Diseases, 2017). Subunit vaccine (eplerk) produced by moesadong, the antigen of which comprises 3 exotoxins (ApxI, apxII, apxIII) and an Outer Membrane Protein (OMPs), and the OMPs with high purity can generate synergistic protection effect with the toxins and have the function of improving the cross protection. Coglaapix, manufactured by Ceva, provides protection for all serotypes of APP, the antigenic component of which comprises ApxI, apxII, apxIII. A commercial subunit vaccine (Porcilis APP) produced by nova corporation comprises rApxII, rOmlA, rTbpB, rCysL.
Pasteurella multocida (Pasteurella multocida, pm) belongs to the family Pasteurella, and is the most important pathogenic bacteria of livestock and poultry in more than 20 bacteria of the genus Pasteurella. The bacterium is a kind of short bacillus or club bacterium with both ends rounded, is a gram negative bacterium without flagellum, movement and spore formation, and is facultative anaerobic. Pasteurella multocida can be divided into five serogroups, A, B, D, E, F and 16, depending on the capsular polysaccharide (K antigen) and lipopolysaccharide components (Liu Chengping. Veterinary microbiology [ M ]. 4. Beijing: china agricultural Press, 2007:136-137.). Wherein type A mainly causes fowl cholera, type B, E mainly causes hemorrhagic septicemia, and type D mainly causes atrophic rhinitis of pigs. Animals infected with Pm often present sepsis, hemorrhagic inflammation, or suppurative lesions of subcutaneous connective tissue, joints, and organs. The strain can infect various hosts, and the symptoms of different animals after infection are different. Diseases caused after infection of pigs with Pm are swine plague and swine infectious atrophic rhinitis (Progressive atrophic rhinitis, PAR); humans can also be infected by animal scratch or bite (Al-Allaf AK, harvey TC, cunnington AR. Solid tamponade caused by Pasteurella multocida infection after a cat bit Postgrad Med J,2001, 77:199-200.). In addition, pm plays an important role in increasing the severity of primary lung injury caused by other pathogens.
The outer membrane proteins of Pasteurella multocida play a key role in the process of disease progression caused by bacterial infection of the body. The PlpE (Pasteurellalipoprotein E) protein belongs to lipoprotein in the outer membrane protein of Pasteurella multocida, and it has been demonstrated that the PlpE protein can induce higher levels of specific antibodies in mice, with a certain immunoprotection (Wu JR, shaien JH, shieh HK, chen CF, chang PC.protective immunity conferred by recombinant Pasteurella multocida lipoprotein E (PlpE). Vaccine,2007, 25:4140-4148.). The Chinese scholars respectively study the PlpE proteins of the swine pasteurella multocida and the bovine pasteurella multocida, and the results show that the PlpE proteins have good antigenicity and immune protection. VacJ is a highly conserved and widely distributed outer membrane lipoprotein in the Pm strain, and many related studies have shown that lipoproteins can also induce higher levels of antibodies in the body (Sathish Bhadravati S, abhinenndra K, revaniah Y, viswas KN.immunology of highly conserved recombinant VacJ outer membrane lipoprotein of Pasteurella multocida. Vaccine,2014, 32:290-296.). In addition, outer membrane proteins OmpA, ompH, omp, ompW, etc. also have some immunoprotection (Dabo SM, taylor JD, refer AW.Pasteurella multocida and bovine respiratory disease. Animal Health Res Rev,2008, 8:129-150.).
In order to effectively prevent and control APP and Pm infection and improve breeding benefits, vaccine immunization is one of the most effective means, and the current researches on related vaccines mainly comprise three categories of inactivated vaccines, attenuated vaccines and genetic engineering vaccines, and the clinically common porcine infectious pleuropneumonia vaccines only comprise inactivated vaccines and foreign subunit vaccines. However, inactivated vaccines cannot provide cross protection against different APP serotypes of infection, moesate subunit vaccines are expensive, raising the cost of cultivation, and only the Outer Membrane Protein (OMP) of the vaccine component belongs to subunit components, the relevant toxin antigen components (Apx i, apx ii, apx iii) actually belong to toxoids. The vaccine for preventing the pasteurellosis is mainly an inactivated vaccine and a low-virulent vaccine clinically, however, the inactivated vaccine is limited by the limited protective force among the serotypes of Pm, and cannot be used universally. The immune effect of the attenuated vaccine is easily affected by factors such as environment and the like, and the risk of virulence return exists. In addition, whether inactivated or attenuated live vaccines, the protection period is short, and multiple immunizations are required. Along with the promotion of a series of measures such as resistance limiting and resistance prohibiting, the difficulty of prevention and control of the disease is increased along with the promotion, the research and development of effective vaccines are also urgent, and subunit vaccines have the advantages of high cross protection level, low safety, capability of effectively reducing lung injury and mortality and the like, so that the industry is in need.
As the main pathogen of porcine bacterial respiratory infectious diseases, porcine actinobacillus pleuropneumoniae and porcine pasteurella multocida belong to the family of Pasteurella, and infected pigs can cause similar respiratory symptoms, the use of the bivalent subunit vaccine can realize the effect of one needle with multiple prevention, so that the immunization times can be reduced, the production cost can be reduced, and a stronger cross protection level can be provided. Actinobacillus pleuropneumoniae and pasteurella multocida have numerous serotypes and have weak cross protection force among different serotypes. Thus, current vaccine studies are more biased towards bivalent subunit vaccines. In recent years, with the development of a plurality of strong immunogenic antigen proteins of actinobacillus pleuropneumoniae and pasteurella multocida, the creation of a porcine infectious pleuropneumonia-swine pasteurellosis bigeminal subunit vaccine is possible.
Given the large molecular weight of many antigenic proteins of bacteria, mostly above 100KDa, purification of these proteins using recombinant expression in vitro using genetic engineering methods is difficult and heavy, such as: the insoluble expression of the protein increases the purification difficulty and the production cost; low protein yield, inability to mass production, etc. Therefore, the antigen structure of the protein is predicted and analyzed by bioinformatics technology, and then the protein is truncated and expressed on the basis of retaining most of antigen epitopes, so that the efficient and soluble expression of the protein is realized, and the cost of downstream purification and mass production is reduced.
The swine streptococcosis and haemophilus parasuis bigeminal genetic engineering subunit vaccine of a new veterinary drug certificate in the country of acquisition and batching in 2019 is the first genetic engineering subunit vaccine aiming at the two bacterial diseases in the world, and the research and development of the current bigeminal subunit vaccine is proved to be a hot spot for vaccine research. At present, no bivalent subunit vaccine aiming at porcine infectious pleuropneumonia and porcine pasteurellosis exists at home and abroad.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides an antigen protein combination of actinobacillus pleuropneumoniae and pasteurella multocida and application thereof in preparing a porcine infectious pleuropneumonia-swine pasteurellosis bigeminal subunit vaccine.
The aim of the invention is achieved by the following technical scheme:
an antigen protein combination of actinobacillus pleuropneumoniae and pasteurella multocida comprises proteins 1-6 with amino acid sequences shown in SEQ ID NO.1-6 respectively. Wherein, the protein 1 with the amino acid shown as SEQ ID NO.1 is the truncated protein of actinobacillus pleuropneumoniae Apx I AN, the protein 2 with the amino acid shown as SEQ ID NO.2 is the truncated protein of actinobacillus pleuropneumoniae Apx II AN, the protein 3 with the amino acid shown as SEQ ID NO.3 is the truncated protein of actinobacillus pleuropneumoniae Apx III AN, the protein 4 with the amino acid shown as SEQ ID NO.4 is the truncated protein of actinobacillus pleuropneumoniae OmpD, the protein 5 with the amino acid shown as SEQ ID NO.5 is the truncated protein of Pasteurella multocida PlpE, and the protein 6 with the amino acid shown as SEQ ID NO.6 is the truncated protein of Pasteurella multocida VacJ. Further, the nucleotide sequences of the encoded proteins 1-6 are shown in SEQ ID NO.7-12, respectively.
The protein 1-6 can be obtained through prokaryotic expression, the expression vector is preferably a pET-sumo vector, and the expression host is preferably E.coli BL21 (DE 3) or E.coli Transseta (DE 3).
The application of the antigen protein combination of the actinobacillus pleuropneumoniae and the pasteurella multocida in preparing the combined subunit vaccine of the porcine infectious pleuropneumonia and the swine pasteurellosis is provided.
A porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine comprising an antigenic protein combination of said porcine actinobacillus pleuropneumoniae and porcine pasteurellosis, further comprising an adjuvant, preferably an aluminium hydroxide adjuvant.
The application of the porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine in preparing medicines for preventing and treating porcine infectious pleuropneumonia and porcine pasteurellosis.
Compared with the prior art, the invention has the following advantages and effects:
(1) The truncated protein selected in the antigen protein combination of the invention is highly homologous in all serotypes of APP and Pm, the antigen proteins with strong immunity protection are combined by evaluating the immunity protection of single antigen proteins, an antigen combination scheme with the best protection effect is screened, and subunit vaccines prepared by the antigen combination scheme can provide cross protection for porcine infectious actinobacillus pleuropneumoniae and porcine pasteurella multocida with different serotypes, the toxicity attack protection rates of APP5 type and APP7 type strains are 100%, and the toxicity attack protection rates of Pm A type and Pm D type strains are 90%.
(2) The truncated proteins selected in the antigen protein combination are optimized to realize soluble expression successfully, so that the antigen protein combination is easy to express and purify in a large amount, and the production cost is reduced.
(3) The invention lays a foundation for the development of high-efficiency, low-cost and broad-spectrum vaccine for porcine infectious pleuropneumonia-porcine pasteurellosis and provides an effective technical means for the prevention and treatment of porcine infectious pleuropneumonia and porcine pasteurellosis.
Drawings
FIG. 1 is a graph showing the detection of specific antibody levels in serum from mice of different test groups.
FIG. 2 is a graph of survival after challenge for mice from different test groups.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
EXAMPLE 1 expression purification of proteins
1. Extraction of bacterial genome
The laboratory freeze-dried and preserved actinobacillus pleuropneumoniae APP1 and APP3 strains are streaked on a TSA plate containing 10% of new born calf serum and 0.01% of NAD, placed in a constant temperature incubator at 37 ℃ for overnight culture, single colony is selected and placed in 5mL of TSB culture medium containing 10% of new born calf serum and 0.01% of NAD, placed in a shaking table at 37 ℃ for 180rpm/min for culture, and then expanded and cultured to 10mL of TSB culture medium containing 10% of new born calf serum and 0.01% of NAD, and bacterial genome is extracted by using a bacterial genome extraction kit according to the specification. The extracted genome is frozen in a refrigerator at the temperature of minus 20 ℃ for standby.
The freeze-dried and preserved porcine Pasteurella multocida A strain in a laboratory is streaked on a TSA plate containing 10% of new born calf serum, placed in a constant temperature incubator at 37 ℃ for overnight culture, single colony is selected and placed in 5mL of TSB culture medium containing 10% of new born calf serum, placed in a shaking table at 37 ℃ and 180rpm/min for culture, and then expanded and cultured to 10mL of TSB culture medium containing 10% of new born calf serum, and bacterial genome is extracted by a bacterial genome extraction kit according to the specification. The extracted genome is frozen in a refrigerator at the temperature of minus 20 ℃ for standby.
2. Recombinant plasmid construction and verification
The invention utilizes bioinformatics software to analyze and predict the ApxI AN, apxII AN and ApxIII AN, ompD, lppB, omlA, tbpB proteins of actinobacillus pleuropneumoniae, the transmembrane region, signal peptide and epitope of PlpE, vacJ, ompA, ompH, ompW protein of Pasteurella multocida, respectively constructs truncated expressed proteins with different epitopes and different lengths, and finally realizes the efficient and soluble expression of target proteins by optimizing different expression vectors and host strains and culture and purification conditions, and is beneficial to mass preparation and purification.
Based on the above study, primers (Table 1) were designed for truncated expressed proteins and pET-sumo plasmids, and PCR was performed according to the procedure of Table 2 using bacterial genomes as templates to amplify the target gene fragments ApxI AN, apxII AN, apxIII AN, ompD, lppB, omlA, tbpB and PlpE, vacJ, ompA, ompH, ompW, pET-sumo, respectively. Because PrimerSTAR Max DNA Polymerase has the characteristics of high specificity, high reaction sensitivity and high amplification efficiency, the high annealing efficiency greatly shortens the annealing and extension time, and according to the specification, the PCR of the target gene can be pre-denatured for 3min at 95 ℃; denaturation at 95℃for 10s; annealing at 58 ℃ for 10s; extending at 72 ℃ for 30s; finally, the reaction sequence was extended for 2min at 72℃for 30 cycles. After PCR, nucleic acid electrophoresis was performed, and the results showed that the amplified ApxI AN, apxII AN, apxIII AN, ompD, lppB, omlA, tbpB and PlpE, vacJ, ompA, ompH, ompW and pET-sumo band sizes were located near the expected positions, respectively. Wherein, a truncated gene ApxI AN has a nucleotide sequence shown in SEQ ID NO. 7; a truncated gene ApxIIAN has a nucleotide sequence shown in SEQ ID NO. 8; a truncated gene ApxIII AN, the nucleotide sequence of which is shown as SEQ ID NO. 9; a truncated gene OmpD, the nucleotide sequence of which is shown in SEQ ID NO. 10; a truncated gene LppB, the nucleotide sequence of which is shown in SEQ ID No. 13; a truncated gene OmlA, the nucleotide sequence of which is shown in SEQ ID NO. 14; a truncated gene TbpB, the nucleotide sequence of which is shown as SEQ ID NO. 15; a truncated gene PlpE, the nucleotide sequence of which is shown in SEQ ID No. 11; a truncated gene VacJ, the nucleotide sequence of which is shown in SEQ ID No. 12; a truncated gene OmpA has a nucleotide sequence shown in SEQ ID NO. 16; a truncated gene OmpH, the nucleotide sequence of which is shown as SEQ ID NO. 17; a truncated gene OmpW has a nucleotide sequence shown as SEQ ID NO. 18.
TABLE 1 primer sequences
Note that: underlined indicates homology arms.
TABLE 2 PCR reaction System
| Reaction components | Volume of |
| 2×Phanta Max Buffer | 25μL |
| dNTP Mix | 1μL |
| Upstream primer | 2μL |
| Downstream primer | 2μL |
| Phanta Max super-Fidelity DNA polymerase | 1μL |
| Template | 1.5μL |
| ddH 2 O | 17.5μL |
| Totals to | 50μL |
The linearized pET-sumo plasmid was subjected to efficient recombinant ligation with recovered ApxI AN, apxII AN, apxIII AN, ompD, lppB, omlA, rTbpB and PlpE, vacJ, ompA, ompH, ompW using a clonExpress II recombinant ligation kit according to the system shown in Table 3. The product was transferred into E.coli DH 5. Alpha. Competent cells, and the bacterial solution was plated on LA plates containing 75. Mu.g/. Mu.LAmp for resistance screening. The obtained monoclonal is inoculated into LB culture medium containing 75 mug/mug of Amp, shake culture is carried out for 12 hours at 37 ℃, and then the plasmid is subjected to PCR or double enzyme digestion identification and sent to the Optimago company for sequencing identification. Specifically, the truncated ApxI AN gene is cloned to a pET-sumo vector to construct a recombinant plasmid pET-sumo-ApxI AN; cloning the truncated ApxIIAN gene to a pET-sumo vector to construct a recombinant plasmid pET-sumo-ApxIIAN; cloning the truncated ApxIII AN gene to a pET-sumo vector to construct a recombinant plasmid pET-sumo-ApxIII AN; cloning the truncated OmpD gene to a pET-sumo vector, and constructing a recombinant plasmid pET-sumo-OmpD; cloning the truncated LppB gene to a pET-sumo vector to construct a recombinant plasmid pET-sumo-LppB; cloning the truncated omlA gene to a pET-sumo vector, and constructing a recombinant plasmid pET-sumo-omlA; cloning the truncated TbpB gene to a pET-sumo vector to construct a recombinant plasmid pET-sumo-TbpB; cloning the truncated PlpE gene to a pET-sumo vector to construct a recombinant plasmid pET-sumo-PlpE; cloning the truncated VacJ gene to a pET-sumo vector to construct a recombinant plasmid pET-sumo-VacJ; cloning the truncated OmpA gene to a pET-sumo vector, and constructing a recombinant plasmid pET-sumo-OmpA; cloning the truncated OmpH gene to a pET-sumo vector, and constructing a recombinant plasmid pET-sumo-OmpH; the truncated OmpW gene is cloned to a pET-sumo vector, and a recombinant plasmid pET-sumo-OmpW is constructed.
The recombinant expression plasmids are identified by PCR and sequencing, and the sequences are consistent with expectations, and the sequence is free from mutation, so that the successful insertion of the genes into the corresponding vectors is proved.
TABLE 3 homologous recombination reaction System
| Reaction components | Volume of |
| Linearization carrier | 2μL |
| Target fragment | 2μL |
| 5×CE MultiS Buffer | 4μL |
| Exnase MultiS | 2μL |
| ddH 2 O | 10μL |
| Totals to | 20μL |
3. Expression purification of recombinant proteins of interest
Transferring the recombinant plasmid with correct sequencing verification into E.coli BL21 (DE 3) or E.coli transduction (DE 3) competent cells, coating thalli on LA (containing 75 mug/mL Amp) plates for resistance screening, the recombinant strains pET-sumo-ApxI AN-Transseta (DE 3), pET-sumo-ApxII AN-Transseta (DE 3), pET-sumo-ApxIII AN-Transseta (DE 3), pET-sumo-OmpD-BL21 (DE 3), pET-sumo-LppB-BL21 (DE 3), pET-sumo-OmlA-BL21 (DE 3), pET-sumo-TbpB-BL21 (DE 3), pET-sumo-PlpE-BL21 (DE 3), pET-sumo-VacJ-BL21 (DE 3), pET-sumo-OmpA-BL21 (DE 3), pET-sumo-OmpH-BL21 (DE 3), pET-sumo-OmpW-BL21 (DE 3) were picked up and inoculated into LB (75. Mu.g/mL liquid medium, respectively, and transferred to liquid medium (Amp) of 75. Mu.g/mL, respectively, and the liquid medium was prepared 600 When the temperature reaches 0.6-0.8, a proper amount of bacterial liquid is taken as a blank control (without adding IPTG), the IPTG with the final concentration of 1mM is added into the rest bacterial liquid, and each recombinant bacterium is subjected to protein induction under the conditions of 4h at 37 ℃, 8h at 28 ℃ and 16h at 16 ℃. After the induction, the cells were collected by centrifugation, resuspended, and the supernatant and pellet were collected and subjected to SDS-PAGE. The cells were collected and examined for expression of the target protein by SDS-PAGE.
After the conditions for inducible expression were established, the newly streaked recombinant strain was inoculated and transferred to an expansion culture in 1L LB (75. Mu.g/mLAMP) liquid medium, and shaken at 37℃at 200rpm to OD 600 At 0.6-0.8, IPTG with a final concentration of 1mM is added, and each recombinant bacterium is induced under the optimal induction condition. After the induction is completed, the thalli are collected, crushed under high pressure, centrifuged at low temperature, and the supernatant and the sediment are separated. And then carrying out affinity chromatography on the supernatant by using Ni resin to purify the protein.
All recombinant proteins can be expressed in a soluble form through optimization of expression vectors and expression conditions. The recombinant protein was purified by Ni resin affinity chromatography and subjected to SDS-PAGE detection. The results showed that the purified ApxIAN, apxIIAN, apxIIIAN, ompD, lppB, omlA, tbpB and PlpE, vacJ, ompA, ompH, ompW sizes were 52kDa, 26kDa, 51kDa, 55kDa, 54kDa, 50kDa, 70kDa and 52kDa, 38kDa, 48kDa, 35kDa, 32kDa, respectively, as expected. Further, western Blot analysis shows that the recombinant protein is successfully expressed and has good antigenicity.
Example 2 screening of Actinobacillus pleuropneumoniae and Pasteurella multocida antigen proteins in pigs and evaluation of optimal antigen combinations
1. Screening of actinobacillus pleuropneumoniae antigen proteins
The purified actinobacillus pleuropneumoniae protein OmpD, lppB, omlA, tbpB of example 1 is taken, diluted appropriately according to the concentration, mixed with the aluminum hydroxide adjuvant 1:1 in equal volume, and fully combined to prepare the subunit vaccine of single protein, and finally the content of each protein in each dose of vaccine is 50 mug. SPF-class 5-week-old female BALB/c mice were randomly divided into 6 groups of 8 mice. Immunization was performed according to the mouse test protocol of table 4. The second immunization is carried out after 14d of the first immunization, the immunization mode is multipoint injection of the cervical and dorsal skin, the immunization dosage and the route are the same as those of the first immunization, finally, the APP5 strain is used for toxicity attack, and the survival condition of each group of mice is counted after continuous observation for one week. The results show that: the control mice all died, the survival rate of the moesadong commercial vaccine group mice was 67.5%, while the survival rate of the OmpD, omlA, lppB groups mice was 37.5% higher than that of the TbpB group with 12.5% (see table 5 for results).
Table 4 mouse test protocol
Table 5 immune protection ratio after each group had been subjected to toxicity attack by APP5 type strain
2. Evaluation of optimal antigen combination protocol for Actinobacillus pleuropneumoniae
Taking the purified actinobacillus pleuropneumoniae protein ApxIAN, apxIIAN, apxIIIAN, ompD, lppB of the example 1, properly diluting according to the concentration, uniformly mixing the uniformly mixed protein solution with an aluminum hydroxide adjuvant 1:1 in equal volume, fully combining to prepare subunit vaccines containing a plurality of proteins, and finally enabling the content of each protein in each dose of vaccine to be 20 mug. SPF-class 5-week-old female BALB/c mice were randomly divided into 6 groups of 20 mice. Immunization was performed according to the mouse test protocol of table 6. And (3) carrying out secondary immunization after 14d of the primary immunization, wherein the immunization mode is subcutaneous multipoint injection of the cervical and the back, the immunization dosage and the immunization route are the same as those of the primary immunization, and finally, carrying out toxicity attack by using APP5 and APP7 strains, continuously observing for one week, and counting the survival condition of each group of mice. The results show that: the highest protection against challenge by vaccine two groups (three toxins+ompd) was 90% for both APP5 and APP7 strains (results shown in tables 7 and 8). Thus, the combination regimen of ApxIAN, apxIIAN, apxIIIAN, ompD was chosen as an antigen combination for porcine infectious pleuropneumonia subunit vaccine.
Table 6 mouse test protocol
Table 7 immune protection rates of groups after challenge to APP5 strain
Table 8 immune protection ratio after each group had been detoxified against APP7 strain
3. Screening of antigen proteins of Pasteurella multocida in pigs
The purified porcine Pasteurella multocida protein OmpD, plpE, vacJ, ompA, ompH, ompW of example 1 is taken, diluted appropriately according to the concentration, evenly mixed with the aluminum hydroxide adjuvant in an equal volume of 1:1, and fully combined to prepare a subunit vaccine of single protein, and finally the content of each protein in each vaccine dose is 50 mug. SPF-class 5-week-old female BALB/c mice were randomly divided into 8 groups of 16 mice. Immunization was performed according to the mouse test protocol of table 9. The second immunization is carried out after 14d of the first immunization, the immunization mode is multipoint injection of the cervical and dorsal skin, the immunization dosage and the route are the same as those of the first immunization, and finally PmA type and PmD type are used for attacking toxin, and the survival condition of each group of mice is counted by continuously observing for one week. The results show that: the VacJ group showed better cross protection of PmA and PmD, 50% and 62.5%, respectively, whereas the PlpE group showed extremely high protection of PmA, and only 25% for PmD, while the other groups showed lower protection (results shown in tables 10 and 11). Thus, vacJ and PlpE were selected as antigen combinations for swine pasteurella multocida subunit vaccines.
Table 9 mouse test protocol
Immune protection rate of each group of PmA strain after challenge in Table 10
Table 11 immune protection rates after challenge of PmD strains
Example 3 preparation of porcine infectious pleuropneumonia-porcine pasteurella multocida bivalent subunit vaccine and evaluation of immune Effect
1. Preparation of porcine infectious pleuropneumonia-porcine pasteurella multocida bigeminal subunit vaccine
The protein ApxIAN, apxIIAN, apxIIIAN, ompD and the PlpE and VacJ purified in example 1 are taken, diluted properly according to the concentration, and then mixed uniformly, the mixed protein solution is mixed uniformly with the aluminum hydroxide adjuvant 1:1 in equal volume, and the content of each protein in each dose of vaccine is 25 mug after full combination.
2. Evaluation of the immune Effect of a bivalent subunit vaccine
SPF-class 5-week-old female BALB/c mice were randomly assigned to control and bivalent subunit vaccine groups of 40 animals each. Immunization was performed according to the mouse test protocol of table 12. The second immunization was performed 14d after the first immunization by multipoint injection of the cervical back skin, at the same dose and route as the first immunization. Before the first immunization and the second immunization and the toxicity attack, the orbital vein blood sampling is carried out on each group of mice, and the serum is split and frozen for standby. The control group and the bivalent subunit vaccine group were each divided into 4 groups of 10 mice before challenge, and APP5, APP7, pmA, pmD strains were used for challenge, respectively.
Table 12 mouse test protocol
3. Mouse serum specific antibody level detection
Recombinant proteins ApxIAN, apxIIAN, apxIIIAN, ompD and PlpE, vacJ were each diluted with coating buffer (25 mmol/L carbonate buffer, ph=9.6) to 4 μg/mL coating ELISA plate, 100 μl/well, overnight coated at 4 ℃. Taking the serum of a bivalent subunit vaccine mouse before primary immunization, before booster immunization and before virus attack and the serum (1:500) of a control group (PBS+aluminum hydroxide adjuvant group) mouse as primary antibodies, taking goat anti-mouse IgG-HRP (1:5000) as secondary antibodies, fully incubating, using TMB chromogenic solution for developing for 30min, using chromogenic stop solution for stopping developing, and then using an enzyme-labeled instrument for reading OD 450 Is measured and compared for differences in specific antibody levels of ApxIAN, apxIIAN, apxIIIAN, ompD and PlpE, vacJ in the serum of each group of mice.
The results show that the specific antibody levels of ApxIAN, apxIIAN, apxIIIAN, ompD and PlpE, vacJ in the serum of each group of mice are significantly increased (p < 0.01) after booster immunization with the bivalent subunit vaccine compared to before the primary immunization; the antibody level in the serum of the mice of the PBS+aluminum hydroxide adjuvant control group is not obviously different before and after immunization; there was a significant difference in the levels of each antibody in the bivalent subunit vaccine group and the control group after boost (p <0.05, p < 0.01) (see figure 1). It was shown that the bivalent subunit vaccine can stimulate mice to produce high-level specific antibodies to the 6 antigens.
4. Toxicity test
After 14d booster immunization, groups of mice were challenged intraperitoneally with freshly cultured strain APP5, strain APP7, strain PmA and strain PmD, respectively, at the minimum lethal dose MLD of the respective strain (APP 5: 2X 10) 8 CFU/APP 7: 3X 10 8 CFU/per, pmA:30 CFU/per, pmD: 2X 10 6 CFU/only). And observing clinical symptoms and death conditions of the mice within one week, and statistically analyzing the immune protection rate of the bivalent subunit vaccine.
The test results showed that the PBS control mice all died during the observation period, and the protection rates of the bivalent subunit vaccine against the APP5, APP7, pmA and PmD strains were 100% (10/10), 90% (9/10) and 90% (9/10), respectively (see Table 13 for the results). According to the statistical Log-Rank algorithm, the difference in survival rates between PBS + aluminum hydroxide adjuvant control and bivalent subunit vaccine mice was very significant (p < 0.01) (see fig. 2). The results show that the bivalent subunit vaccine has good immunoprotection effect on mice respectively infected by APP5 type, APP7 type, pmA type and PmD type strains.
Table 13 immune protection ratio of bivalent subunit vaccine after challenge to different strains
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (9)
1. An antigenic protein combination of actinobacillus pleuropneumoniae and pasteurella multocida swine, characterized in that: comprises proteins 1-6 with amino acid sequences shown as SEQ ID NO.1-6 respectively.
2. The antigen protein combination of claim 1, wherein: the nucleotide sequences of the coded proteins 1-6 are shown as SEQ ID NO.7-12 respectively.
3. The antigen protein combination of claim 1, wherein: proteins 1-6 are obtained by prokaryotic expression.
4. A combination of antigenic proteins of a. Pleuropneumoniae and pasteurella multocida according to claim 3 wherein: the prokaryotic expression vector is pET-sumo vector, and the host is colibacillus.
5. Use of the antigenic protein combination of any one of claims 1-4 in the preparation of a porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine.
6. A porcine infectious pleuropneumonia-porcine pasteurellosis bivalent subunit vaccine, characterized in that: a combination comprising the antigenic proteins of any one of claims 1-4.
7. The porcine infectious pleuropneumonia-porcine pasteurellosis bivalent subunit vaccine as claimed in claim 6, wherein: an adjuvant is also included.
8. The porcine infectious pleuropneumonia-porcine pasteurellosis bivalent subunit vaccine as claimed in claim 7, wherein: the adjuvant is aluminum hydroxide adjuvant.
9. Use of a porcine infectious pleuropneumonia-porcine pasteurellosis bivalent subunit vaccine as claimed in any one of claims 6-8 in the manufacture of a medicament for the prevention and treatment of porcine infectious pleuropneumonia and porcine pasteurellosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211075065.3A CN115991749A (en) | 2022-09-02 | 2022-09-02 | Porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211075065.3A CN115991749A (en) | 2022-09-02 | 2022-09-02 | Porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115991749A true CN115991749A (en) | 2023-04-21 |
Family
ID=85993106
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211075065.3A Pending CN115991749A (en) | 2022-09-02 | 2022-09-02 | Porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115991749A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117304338A (en) * | 2023-09-15 | 2023-12-29 | 兆丰华生物科技(南京)有限公司 | Fusion protein antigen composition and preparation method and application thereof |
| CN118388664A (en) * | 2024-05-21 | 2024-07-26 | 广东省农业科学院动物卫生研究所 | Haemophilus parasuis fusion antigen and application thereof |
-
2022
- 2022-09-02 CN CN202211075065.3A patent/CN115991749A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117304338A (en) * | 2023-09-15 | 2023-12-29 | 兆丰华生物科技(南京)有限公司 | Fusion protein antigen composition and preparation method and application thereof |
| CN117304338B (en) * | 2023-09-15 | 2024-10-01 | 兆丰华生物科技(南京)有限公司 | Fusion protein antigen composition and preparation method and application thereof |
| CN118388664A (en) * | 2024-05-21 | 2024-07-26 | 广东省农业科学院动物卫生研究所 | Haemophilus parasuis fusion antigen and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115991749A (en) | Porcine infectious pleuropneumonia-porcine pasteurellosis bigeminal subunit vaccine | |
| CN113350495B (en) | Streptococcus suis-haemophilus parasuis disease-porcine infectious pleuropneumonia triple subunit vaccine and preparation method thereof | |
| CN108218965B (en) | Preparation method and application of salmonella abortus flagellin FliC | |
| CN115725002B (en) | Coli specific antigen fusion protein and recombinant lactococcus lactis thereof | |
| CN102276730A (en) | Preparation method for staphylococcus aureus Iron-regulated surface determinant B immunodominant fragment (IsdBid)-target of RNAIII activating protein (TRAP) fusion protein and application thereof | |
| CN107338208B (en) | Porcine atrophic rhinitis D type virus-producing pasteurella multocida vaccine strain and application thereof | |
| CN103421832B (en) | Comprise yolk antibody and the preparation and application of florfenicol Drug Resistance Gene Associated Proteins | |
| KR101151004B1 (en) | Vaccine composition for protection and treatment against pathogenic Escherichia coli and Salmonella in cattle by administration of attenuated Salmonella expressing adhesin gene of pathogenic Escherichia coli and vaccination method thereof | |
| CN103275228A (en) | K99-987P-F41 recombinant protein and application thereof | |
| WO2012009774A2 (en) | Recombinant microorganisms, methods for preparing vaccine strains, antigens, and vector vaccine compositions of same, uses thereof, and related antibodies, diagnostic kit, and treatment and/or prophylactic methods | |
| CN103421731B (en) | A kind of haemophilus parasuis attenuation salmonella seedling | |
| CN117427153A (en) | Vaccine prepared from truncated erysipelothrix rhusiopathiae SpaA protein and application thereof | |
| CN101294144A (en) | Type II streptococcus suis sa1KR gene knockout mutant strain, preparation method and application thereof | |
| CN103421733B (en) | A kind of haemophilus parasuis-Salmonella choleraesuls bigeminy gene engineering vaccine | |
| CN116350763A (en) | Attenuated strain vaccine composition of bordetella bronchiseptica and preparation method thereof | |
| CN116785418A (en) | Fish-source streptococcus agalactiae subunit vaccine and preparation method and application thereof | |
| Nefedchenko et al. | Prevalence of different OmpH-types among Pasteurella multocida isolated from lungs of calves with respiratory problems | |
| CN108904791A (en) | A kind of clostridium perfringens alpha toxin recombinant subunit vaccine and its production method | |
| EP1342784A1 (en) | ExPEC-specific proteins, genes encoding them and uses thereof | |
| Jiao et al. | Immunization effect of recombinant Lactobacillus casei displaying Aeromonas veronii Aha1 with an LTB adjuvant in carp | |
| CN115819625B (en) | Escherichia coli tetravalent antigen fusion polypeptide | |
| CN105497885A (en) | Subunit vaccine, and preparation method and application thereof | |
| CN110240657A (en) | A kind of haemophilus parasuis fusion protein AfuA-OppA2 with immune protective | |
| CN114262719B (en) | Preparation method and application of haemophilus parasuis trivalent genetic engineering subunit vaccine | |
| CN102206258A (en) | NmpC subunit vaccine of salmonella paratyphi A and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |