CN108659116A - A kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody - Google Patents
A kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody Download PDFInfo
- Publication number
- CN108659116A CN108659116A CN201810491780.2A CN201810491780A CN108659116A CN 108659116 A CN108659116 A CN 108659116A CN 201810491780 A CN201810491780 A CN 201810491780A CN 108659116 A CN108659116 A CN 108659116A
- Authority
- CN
- China
- Prior art keywords
- aeromonas
- bsa
- pepf1
- smcc
- reaction
- 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.)
- Granted
Links
- 241000607534 Aeromonas Species 0.000 title claims abstract description 47
- 244000052616 bacterial pathogen Species 0.000 title claims abstract description 12
- 230000002163 immunogen Effects 0.000 title claims abstract description 10
- 238000001308 synthesis method Methods 0.000 title claims abstract description 6
- 229920001184 polypeptide Polymers 0.000 claims abstract description 28
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 28
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 28
- 108700006385 OmpF Proteins 0.000 claims abstract description 11
- 101710116435 Outer membrane protein Proteins 0.000 claims abstract description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000007822 coupling agent Substances 0.000 claims description 7
- 230000001588 bifunctional effect Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000013019 agitation Methods 0.000 claims description 5
- 235000018417 cysteine Nutrition 0.000 claims description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 238000011191 terminal modification Methods 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 238000001631 haemodialysis Methods 0.000 claims description 3
- 230000000322 hemodialysis Effects 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- PXXGVUVQWQGGIG-YUMQZZPRSA-N Glu-Gly-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N PXXGVUVQWQGGIG-YUMQZZPRSA-N 0.000 claims description 2
- UVTSZKIATYSKIR-RYUDHWBXSA-N Gly-Tyr-Glu Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O UVTSZKIATYSKIR-RYUDHWBXSA-N 0.000 claims description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 claims description 2
- 108010002311 N-glycylglutamic acid Proteins 0.000 claims description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 2
- VTCKHZJKWQENKX-KBPBESRZSA-N Tyr-Lys-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O VTCKHZJKWQENKX-KBPBESRZSA-N 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 2
- 150000002466 imines Chemical class 0.000 claims description 2
- 238000012986 modification Methods 0.000 claims description 2
- 230000004048 modification Effects 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 229910052698 phosphorus Inorganic materials 0.000 claims description 2
- 239000011574 phosphorus Substances 0.000 claims description 2
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 claims description 2
- 159000000000 sodium salts Chemical class 0.000 claims description 2
- 125000002730 succinyl group Chemical group C(CCC(=O)*)(=O)* 0.000 claims description 2
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical group CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 claims 1
- 238000003756 stirring Methods 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 13
- 102000036639 antigens Human genes 0.000 abstract description 13
- 108091007433 antigens Proteins 0.000 abstract description 13
- 210000002969 egg yolk Anatomy 0.000 abstract description 13
- 210000002966 serum Anatomy 0.000 abstract description 8
- 241000588724 Escherichia coli Species 0.000 abstract description 6
- 241000699670 Mus sp. Species 0.000 abstract description 4
- 238000005859 coupling reaction Methods 0.000 abstract description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 abstract description 2
- 241000519590 Pseudoalteromonas Species 0.000 abstract description 2
- 241000544286 Vibrio anguillarum Species 0.000 abstract description 2
- 241000607618 Vibrio harveyi Species 0.000 abstract description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 abstract description 2
- 241000607265 Vibrio vulnificus Species 0.000 abstract description 2
- 230000008878 coupling Effects 0.000 abstract description 2
- 238000010168 coupling process Methods 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 abstract description 2
- 241000193830 Bacillus <bacterium> Species 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 201000010099 disease Diseases 0.000 description 18
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 18
- 241000607528 Aeromonas hydrophila Species 0.000 description 11
- 230000003115 biocidal effect Effects 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 241000251468 Actinopterygii Species 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000001717 pathogenic effect Effects 0.000 description 6
- 241000287828 Gallus gallus Species 0.000 description 5
- 235000013330 chicken meat Nutrition 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 229960005486 vaccine Drugs 0.000 description 5
- 102000002322 Egg Proteins Human genes 0.000 description 4
- 108010000912 Egg Proteins Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 241000607516 Aeromonas caviae Species 0.000 description 3
- 102100033620 Calponin-1 Human genes 0.000 description 3
- 208000032843 Hemorrhage Diseases 0.000 description 3
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 3
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 206010040047 Sepsis Diseases 0.000 description 3
- 150000001413 amino acids Chemical group 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 241000607598 Vibrio Species 0.000 description 2
- 206010047400 Vibrio infections Diseases 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000001143 conditioned effect Effects 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 238000011194 good manufacturing practice Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000004681 ovum Anatomy 0.000 description 2
- 230000000505 pernicious effect Effects 0.000 description 2
- 208000013223 septicemia Diseases 0.000 description 2
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 241000252082 Anguilla anguilla Species 0.000 description 1
- 235000010894 Artemisia argyi Nutrition 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000463291 Elga Species 0.000 description 1
- 101710146739 Enterotoxin Proteins 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 241000252234 Hypophthalmichthys nobilis Species 0.000 description 1
- 108010042653 IgA receptor Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241000646357 Monopterus cuchia Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241000269799 Perca fluviatilis Species 0.000 description 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- -1 Tetramethyl benzidine Chemical compound 0.000 description 1
- 241001482311 Trionychidae Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 244000030166 artemisia Species 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
- 231100000776 exotoxin Toxicity 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 235000021474 generally recognized As safe (food) Nutrition 0.000 description 1
- 235000021473 generally recognized as safe (food ingredients) Nutrition 0.000 description 1
- 239000003228 hemolysin Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 229940126583 recombinant protein vaccine Drugs 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000010148 water-pollination Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
Abstract
The present invention relates to a kind of immunogen synthesis methods being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody, belong to technical field of immunoassay.It uses Aeromonas outer membrane protein（Outer membrane protein,Omp）Conservative polypeptide PepF1 on OmpF and bovine serum albumin(BSA)（BSA）Chemical covalent coupling is carried out, Aeromonas conservative polypeptide epitope antigen has been synthesized.The mice serum of the immunogen immune or 3 Hygrophilous monad of laying hen Yolk antibody pair and 4 plants of Aeromonas bacteriums have potency under the conditions of particular polypeptide sequence PepF1 and coupling reaction, simultaneously for common E.coli, E.coli O157：H7, Vibrio anguillarum, vibrio parahemolyticus, Vibrio harveyi, Vibrio vulnificus, comma bacillus, pseudoalteromonas, Wdwardsiella tarda do not have cross reaction.And the mice serum or Yolk antibody that thalline is immunized have cross reaction to common E.coli CICC21530 and DH5 α.The Novel free epidemic focus can be used for preparing aquatic pathogenic bacterium Aeromonas chiasma type monoclonal antibody or livitin.
Description
Technical field
The present invention relates to a kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody,
Belong to technical field of immunoassay.
Background technology
Aeromonas hydrophila is one of the main pathogenic bacteria of fish culture septicemia, is brought to fresh-water fishes, culture fishery
Huge economic losses.Aeromonas hydrophila is distributed widely in the various water bodys of nature, is that the primary of a variety of aquatic animals causes
Germ is conditioned pathogen, is that typical people-beast-fish is total to illness pathogen.It is very strong that Aeromonas hydrophila can generate toxicity
Exotoxin, such as：Hemolysin, histotoxin, necrotoxin, enterotoxin and protease etc. make aquatic livestock cause a disease.In actual production
It is more by the fulminant hemorrhage of infected by Aeromonas hydrophila, because there are many serotype of Aeromonas hydrophila, furthermore pair infected
As difference, so symptom is also different.Such as red fin disease of silver carp fulminant hemorrhage, soft-shelled turtle septicaemia, swamp eel hemorrhage, common eel.
The bacterium is conditioned pathogen, when environment cataclysm, water quality deterioration, the other bacterium of Chang Huiyu（Such as mild Aerononas punctata, vibrios）
Mixed infection aggravates disease.It is more violent by the general patient's condition of the disease of infected by Aeromonas hydrophila, mostly pernicious infectious disease, the death rate
It is very high.
Currently, the main means of control aeromonas hydrophila disease include antibiotic and vaccine.China birds, aquatic products are supported at present
Prevalence and the abuse of antibiotics for growing industry disease are commonplace, cause the antibiotic residue problem in meat, fish, environment prominent
Go out, huge threat is brought to China's food security, the export trade, environmental protection, public health.It is reported that China in 2013
16.2 ten thousand tons of the usage amount total amount of antibiotic, wherein 48% is people's antibiotic, remaining is veterinary antibiotic.People is exhausted with antibiotic
It is mostly used in the treatment of disease and on a declining curve, and veterinary antibiotic greater portion is used in feed.Due to for animals
Antibiotic is cheap, in order to which many raisers of diseases prevention are not added with the use of restraining.And ox, sheep, chicken, duck, fish etc. have eaten for animals resist
It is last to react on the mankind again further through being discharged into water, soil, in crops after raw element, lead to drug tolerant bacteria and " super
Grade bacterium " problem, forms a pernicious ecological chain circulatory system.
Simultaneously as the pathogen serotype is various, the effect of common Bacteria vaccine is widely different, often to bacterial strain is immunized
Effect it is preferable, and there is no effect or effect general other serotypes.Recombinant protein vaccine based on conservative outer membrane protein
Effect is preferable, but it is mostly fish injecting immune one by one to be faced with route of inoculation, time-consuming and laborious, of high cost, easily cause fish death etc.
Problem；Moreover, the approval process of live vaccine is extremely complex, generally requires the time of 5-10, and wants mating medicine
Grade GMP (Good Manufacturing Practice) workshop, cannot meet the market demand.
Livitin（IgY）As food composition and Immune discrimination active material with green in terms of aquatic products disease control
Color, wide spectrum, sustainable feature.Fowl source antibody refers to after egg-laying bird is immunized with specific antigen, from the yolk of immune eggs
The middle immunoglobulin with antigentic specificity, rake tropism obtained with physics mode, while it is edible as raw-food material
Safety it is preferable；Fowl source antibody has been considered as the first choice " drug " of substitute antibiotics by U.S. FDA, and thinks the antibody conduct of fowl source
Food ratio is more advantageous as drug, and be included in " generally accepted safe material (Generally Accepted as Safe,
GRAS) " scope.Compared to Traditional control means using antibiotic, vaccine control relevant disease, fowl source antibody disease preventing and treating has
Following characteristics：（1）Efficiently, with strong points, using antigen and antibody specific immune response, only inhibit corresponding cause of disease.（2）Green
Safety, Yolk antibody are egg yolk constituent, in environment degradable, noresidue problem without side-effects to animal, human body.
（3）Compared to vaccine, research and development, application program simplify, and application method is simple in the prevention of specific pathogen, pass through oral, spraying more
Etc. modes carry out, use cost is low.（4）Have a wide range of application, Yolk antibody be high-volume development functionality food and medical diagnosis on disease,
Bacterial-infection resisting, viral infection resisting prophylactic treatment provide new selection.（5）It is easy to obtain and duration is good, obtaining
Live chickens can be immunized after pathogenic strain or conservative antigen, ovipository cycle is long, yield is high.The ovum gallinaceum xanthoglobulin that height is exempted from（IgY）
The uniform identity of aquatic pathogenic bacterium and specificity are increased as feed additive for the bio-identification of aquatic products disease, inhibition for it
It grows, green prevention and control is laid a good foundation.
Invention content
The object of the present invention is to provide a kind of immunogenes being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody
Synthetic method, to prepare, aquatic products disease bacterium Aeromonas hydrophila belongs to interior wide spectrum monoclonal antibody, Yolk antibody provides method
And thinking.
Technical scheme of the present invention, to achieve the above object, the present invention select the Aeromonas outer membrane of N-terminal modification Cys
The conservative polypeptide epitope PepF1 of albumen OmpF is used in combination SMCC methods to synthesize Aeromonas polypeptide antigen and carrier as antigen
The artificial antigen of protein B SA.
Aeromonas surface antigen type is various, concrete structure also changeable, conservative and table due to bacterial strain serotype is different
The polypeptides epitope that face can expose is the basis for preparing Aeromonas specific antibody.However, polypeptide antigen molecular weight is logical
Often between 1000-2000, belong to molecule haptens, it can not direct immunization.Pass through bifunctional coupling agent SMCC and carrier protein
It is just provided with immunogenicity after BSA couplings, antibody can be prepared.
By Aeromonas conservative polypeptide epitope PepF1-SMCC-BSA immunogen immune BALB/c small white mouses and laying hen
（Roche chicken）After can be obtained Aeromonas chiasma type mouse antibody or Yolk antibody.
A kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody, step are：
（1）The modification of polypeptide PepF1：The Aeromonas outer membrane protein OmpF conservative polypeptide PepF1 that screening obtains are repaiied in N-terminal
Adorn cysteine Cys；
（2）The derivative of BSA：By bifunctional coupling agent 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinyls
Imines ester sodium salt Sulfo-SMCC and bovine serum albumin(BSA) BSA are according to reacting molar ratio 40-80:1 in 0.05-0.15 M/L phosphorus
1-2h is reacted at room temperature in phthalate buffer, obtains SMCC-BSA, and ultrafiltration removes extra Sulfo-SMCC；
（3）The preparation of immunogene：By step（1）Gained polypeptide PepF1 and step（2）BSA after gained derives rubs according to reaction
You compare 30-60:1 reacts at room temperature 2-6h in 0.05-0.15 M/L phosphate buffers, and Aeromonas friendship is obtained after dialysis
The immunogene PepF1-SMCC-BSA of forked type antibody.
The Aeromonas outer membrane protein OmpF conservative polypeptides PepF1 is specially Tyr Lys Gly Glu Gly Arg
Gly Tyr Glu Leu Ala Ala。
The immunogen synthesis method is as follows：The PB buffer solutions of carrier protein BSA 10mg 0.1M are taken,
The bifunctional coupling agent Sulfo-SMCC that n,N-Dimethylformamide (DMF) dissolves will be used according to reaction molar ratio Sulfo-
SMCC:BSA is 40-80:1 is added in BSA solution, and 1-2h is reacted at room temperature under magnetic agitation；It uses after reaction
Millipore super filter tubes ultrafiltration 2-4 times, obtains SMCC-BSA, removes unreacted bifunctional coupling agent；By 0.05-0.15M/L's
The Aeromonas conservative polypeptide epitope PepF1 through N-terminal modification cysteine Cys of PB buffer solutions is according to reaction mole
Than polypeptide PepF1:BSA is 30-60:1 is added in reaction solution, and 2-6h is reacted at room temperature under magnetic agitation；Then use 0.01-
The PBS hemodialysis reaction solution of 0.05 M/L, it is primary that 4-6h replaces dialyzate, and dialysis 2-3 days is to get to Aeromonas chiasma type
The immunogene PepF1-SMCC-BSA of antibody.
Beneficial effects of the present invention：Wide spectrum in aquatic products disease bacterium Aeromonas, category are prepared the present invention provides a kind of outside
The mouse antibody of specificity or the immunogene preparation method of Yolk antibody；The present invention has screened the Aeromonas on outer membrane protein OMPF
The conservative amino acid sequence PepF1 of category.And polypeptide PepF1-SMCC-BSA artificial immunogens have been synthesized for the first time, by artificial polypeptide
Antigen PepF1-SMCC-BSA Immune Laying Hens and BALB/c small white mouses, indirect ELISA is experiments have shown that immune chicken yolk antibody IgY
And mice serum is to 3 Hygrophilous monads of test（CICC 10500, MCCC 1A00190, MCCC 1A0007）, 4 plants
Aeromonas bacterium（Podbielniak Aeromonas, aeromonas punctata, Zhaodong Aeromonas, bivalve Aeromonas）There is potency,
Simultaneously for common E.coli, E.coli O157：H7, Vibrio anguillarum, vibrio parahemolyticus, Vibrio harveyi, Vibrio vulnificus, suddenly
Random vibrios, pseudoalteromonas, Wdwardsiella tarda do not have cross reaction, specificity good.And the thermophilic aqueous vapor unit cell of direct immunization
It is anti-that the mice serum and Yolk antibody of bacterium thalline more serious intersection occur to common E.coli CICC 21530 and DH5 α
It answers, illustrates that the polypeptide immunogen that the present invention uses has the advantages that conservative, specificity.The ovum gallinaceum xanthoglobulin that height is exempted from（IgY）
It is aquatic products disease septicemia to safety of the uniform identity with specificity and as food composition in Aeromonas
Green prevention and control is laid a good foundation.
Description of the drawings
Fig. 1 is polyacrylamide gel electrophoresis（SDS-PAGE）Artificial synthesized PepF1-SMCC-BSA is characterized manually to resist
It is former.1, BSA；2、BSA-SMCC；3、PepF1-SMCC-BSA；
Fig. 2 is the PepF1-SMCC-BSA artificial antigens immune serum and 3 Hygrophilous monads, 4 plants of gas unit cells of synthesis
The cross reaction of bacterium campylobacter bacteria.
Wherein 3 Hygrophilous monads（1, ASP CICC 10500,4, ASP MCCC 1A00190,2, ASP MCCC
1A0007）, 4 plants of Aeromonas bacteriums（5, Podbielniak Aeromonas, 3, aeromonas punctata, 6, Zhaodong Aeromonas, 7, bivalve
Aeromonas）.
Specific implementation mode
Aeromonas bacterial strain described in embodiment is purchased from Chinese industrial Organism Depositary（CICC）, Chinese common micro-
Biological deposits center（CGMCC）, Chinese Sea Organism Depositary（MCCC）.Polypeptide sequence is by giving birth to work bioengineering (Shanghai)
Limited liability company synthesizes.
It further illustrates the present invention by the following examples.
Instrument：Table-type high-speed refrigerated centrifuge, Hunan He Xi instrument and equipment Co., Ltd；ELGA ultrapure water machines, Britain Chinese mugwort
Erg；WD-9405B type horizontal shakers, Liuyi Instruments Plant, Beijing；96 hole 8 × 12 is high to adsorb removable ELISA Plate, and biology is contained by Wuxi state
Engineering Co., Ltd；Multiskan FC microplate reader, Thermo Fisher Scientific；200 μ L, 1mL liquid-transfering guns, 300 μ
8 channel liquid-transfering guns of L, Thermo Labsystems companies；XW-80A turbine mixers, Qingpu Shanghai Hu Xi instrument plants;
Millipore super filter tubes（Cut off:10000）, it is purchased from Millipore-Merck companies.
Reagent：Tetramethyl benzidine (TMB), Sigma reagents Co., Ltd；Other reagents are analytical reagents.
Steps are as follows：
1, the screening and design of polypeptide sequence：Outer membrane protein is the important feature of Gram-negative bacterial cell film, wherein outer membrane egg
White OmpF is the important adhesion factor of Aeromonas hydrophila, is that the bacterium survives vital two-component regulatory in adverse conditions
The key component of system.Meanwhile Outer Membrane Protein of Aeromonas Hydrophila OmpF Aeromonas hydrophila belong in homology 83% with
On.OmpF is more promising Immune target.By being compared to Aeromonas OmpF protein amino acid sequences, in conjunction with biology
Bioinformatic tool is to the antigenicity of OmpF amino acid sequences, alpha-helix, beta sheet region, hydrophily, hydrophobicity, surface exposed property
It is analyzed, has chosen conservative, the preferable polypeptide sequence of surface exposed property as candidate antigens, polypeptide sequence length is generally
10-20 AA.Selection N-terminal or C-terminal modification cysteine Cys are subsequently coupled.
2, the preparation of immunogene：The PB buffer solutions of carrier protein BSA 10mg 0.1M are taken, N, N- dimethyl will be used
The Sulfo-SMCC of formamide (DMF) dissolving is according to reaction molar ratio 70:1 is added in BSA solution, room temperature under magnetic agitation
React 1h.Millipore super filter tubes are used after reaction（Cut off: 10000 MW）Ultrafiltration 3 times, removes unreacted idol
Join agent.By the Aeromonas conservative polypeptide epitope PepF1 of the PB buffer solutions of 0.1M according to reaction molar ratio 60:1 is added
Into reaction solution, 1h is reacted at room temperature under magnetic agitation.The PBS hemodialysis reaction solution of 0.01M, 6h is then used to replace dialyzate one
Secondary, dialysis 2 days is to get to the immunogene of Aeromonas chiasma type antibody.
3, the preparation of immune serum or Yolk antibody
（1）Experimental animal：Select 6, the Roche laying hen of the BALB/c mouse of 8 week old and 6 20 week old is immunized.
（2）Antigen configures：By immunogene normal saline dilution, it is configured to the solution of required concentration；
（3）Emulsification：Above-mentioned solution and equivalent is complete or incomplete freund adjuvant is emulsified with mixing method, emulsification is complete
Subcutaneous multi-point injection mouse or Roche laying hen afterwards；
（4）Immunization method：Mouse is immunized according to following immune flow：Head exempts from 80 μ g, and two exempt from 80 μ g, and three exempt from 40 μ g.Exempt from by following
Epidemic disease flow Immune Laying Hens：Head exempts from 500 μ g, and two exempt from 500 μ g, and three exempt from 300 μ g.3 exempt from after analyzed with indirect enzyme-linked immunosorbent（ELISA）
Method measures to bacterium in Aeromonas Pseudomonas and belongs to the potency of other outer bacteriums；
（5）Small white mouse is taken a blood sample：Second and third time carries out docking blood sampling in 1 week after being immunized；Laying hen IgY：It receives within second and third time immune latter 1 week
Collection egg simultaneously detaches yolk preparation IgY crude extracts.Antiserum titre and cross reaction are measured using indirect ELISA.
4, ELISA reaction process：
（1）Antibody titer determination step：Test bacterium solution is boiled into inactivation and is serially diluted 96 hole enzymes of coating with coating buffer solution
Target, 100 holes μ L/, in 4 DEG C of refrigerator overnights.Next day takes out ELISA Plate and is back to room temperature, and 200 μ L PBST solution are injected per hole, are shaken
3min is vibrated on bed, cleaning solution is firmly got rid of, is patted dry on blotting paper, continues washing 2 times.Following washing methods is identical；
（2）Fully after washing, ELISA Plate is closed with Block buffer, 200 holes μ L/ take after incubating 2 h in 37 DEG C of incubate box
It is for use to go out drying；
（3）Positive serum is serially diluted corresponding 7 ranks before being added to ELISA Plate, negative serum, 100 μ L/ are added in eighth row
Hole, 37 DEG C be incubated 1h after wash, pat dry；
（4）It is added 100 μ L per hole, 1:The sheep anti mouse secondary antibody or 1 of 3000 diluted HRP labels:The sheep of 3000 diluted HRP labels
Anti- chicken secondary antibody, 37 DEG C be incubated 1 h after wash, pat dry；
（5）100 μ L developing solutions are added per hole, and (TMB is 1 with substrate liquid proportional:5), 37 DEG C of dark place, 15 min of reaction, after taking-up
50 μ L terminate liquids (sulfuric acid of 2 mol/L) are added per hole, light absorption value A is measured with microplate reader450.Concrete outcome is as shown in Figure 2.
Including 3 Hygrophilous monads（CICC 10500, MCCC 1A00190, MCCC 1A0007）, 4 plants of gas lists
Born of the same parents' campylobacter bacteria（Podbielniak Aeromonas, aeromonas punctata, Zhaodong Aeromonas, bivalve Aeromonas）.
Claims (3)
1. a kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody, it is characterised in that step
Suddenly it is：
（1）The modification of polypeptide PepF1：The Aeromonas outer membrane protein OmpF conservative polypeptide PepF1 that screening obtains are repaiied in N-terminal
Adorn cysteine Cys；
（2）The derivative of BSA：By bifunctional coupling agent 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinyls
Imines ester sodium salt Sulfo-SMCC and bovine serum albumin(BSA) BSA are according to reacting molar ratio 40-80:1 in 0.05-0.15 M/L phosphorus
1-2h is reacted at room temperature in phthalate buffer, obtains SMCC-BSA, and ultrafiltration removes extra Sulfo-SMCC；
（3）The preparation of immunogene：By step（1）Gained polypeptide PepF1 and step（2）BSA after gained derives rubs according to reaction
You compare 30-60:1 reacts at room temperature 2-6h in 0.05-0.15 M/L phosphate buffers, and Aeromonas friendship is obtained after dialysis
The immunogene PepF1-SMCC-BSA of forked type antibody.
2. being used to prepare the immunogene synthesis side of aquatic pathogenic bacterium Aeromonas chiasma type antibody according to claim 1
Method, it is characterised in that：The Aeromonas outer membrane protein OmpF conservative polypeptides PepF1 is specially Tyr Lys Gly Glu
Gly Arg Gly Tyr Glu Leu Ala Ala。
3. being used to prepare the immunogene synthesis side of aquatic pathogenic bacterium Aeromonas chiasma type antibody according to claim 1
Method, it is characterised in that be as follows：
The PB buffer solutions for taking carrier protein BSA 10mg 0.1M, it is double by being dissolved with n,N-Dimethylformamide DMF
Function coupling agent Sulfo-SMCC is according to reaction molar ratio Sulfo-SMCC:BSA is 40-80:1 is added in BSA solution, magnetic
Power stirring is lower to react at room temperature 1-2h；Millipore super filter tubes ultrafiltration 2-4 times is used after reaction, obtains SMCC-BSA, is removed not
The bifunctional coupling agent of reaction；By the gas list through N-terminal modification cysteine Cys of the PB buffer solutions of 0.05-0.15M/L
Born of the same parents bacterium conservative polypeptide epitope PepF1 is according to reaction molar ratio polypeptide PepF1:BSA is 30-60:1 is added in reaction solution,
2-6h is reacted at room temperature under magnetic agitation；The PBS hemodialysis reaction solution of 0.01-0.05 M/L, 4-6h is then used to replace dialyzate one
Secondary, dialysis 2-3 days is to get to the immunogene PepF1-SMCC-BSA of Aeromonas chiasma type antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810491780.2A CN108659116B (en) | 2018-05-22 | 2018-05-22 | Immunogen synthesis method for preparing aquatic pathogenic bacterium aeromonas cross-type antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810491780.2A CN108659116B (en) | 2018-05-22 | 2018-05-22 | Immunogen synthesis method for preparing aquatic pathogenic bacterium aeromonas cross-type antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108659116A true CN108659116A (en) | 2018-10-16 |
| CN108659116B CN108659116B (en) | 2021-04-02 |
Family
ID=63776444
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810491780.2A Active CN108659116B (en) | 2018-05-22 | 2018-05-22 | Immunogen synthesis method for preparing aquatic pathogenic bacterium aeromonas cross-type antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108659116B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0587636A1 (en) * | 1991-06-07 | 1994-03-23 | Secretary Of State For Scotland In Her Britannic Majesty's Gov. Of The United Kingdom Of Great Britain And Northern Ireland | Fish vaccine for aeromonas salmonicida infection |
| CN101074260A (en) * | 2007-03-20 | 2007-11-21 | 福建省农业科学院生物技术研究所 | Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal |
| CN104059142A (en) * | 2014-07-03 | 2014-09-24 | 江南大学 | Synthetic method of immunogen for preparing salmonella cross-type antiboby |
| CN106996971A (en) * | 2017-04-28 | 2017-08-01 | 江南大学 | Detect the kit and method of human peripheral EVs TRPC5 contents |
| WO2017161181A1 (en) * | 2016-03-16 | 2017-09-21 | Spogen Biotech Inc. | Fusion proteins, recombinant bacteria, and exosporium fragments for animal health and aquaculture |
-
2018
- 2018-05-22 CN CN201810491780.2A patent/CN108659116B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0587636A1 (en) * | 1991-06-07 | 1994-03-23 | Secretary Of State For Scotland In Her Britannic Majesty's Gov. Of The United Kingdom Of Great Britain And Northern Ireland | Fish vaccine for aeromonas salmonicida infection |
| CN101074260A (en) * | 2007-03-20 | 2007-11-21 | 福建省农业科学院生物技术研究所 | Production of hygrophilous monospermous bacterium main-protective antigen univalent and multivalent vitelline antibody and use in aquatic animal |
| CN104059142A (en) * | 2014-07-03 | 2014-09-24 | 江南大学 | Synthetic method of immunogen for preparing salmonella cross-type antiboby |
| WO2017161181A1 (en) * | 2016-03-16 | 2017-09-21 | Spogen Biotech Inc. | Fusion proteins, recombinant bacteria, and exosporium fragments for animal health and aquaculture |
| CN106996971A (en) * | 2017-04-28 | 2017-08-01 | 江南大学 | Detect the kit and method of human peripheral EVs TRPC5 contents |
Non-Patent Citations (3)
| Title |
|---|
| JON R.BACKSTROM ET AL.: "Generation of anti-peptide antibodies against serotonin 5-HT2A and 5-HT2C receptors", 《JOURNAL OF NEUROSCIENCE METHODS》 * |
| MAHIMA SHARMA APARNA DIXIT: "Identification and immunogenic potential of B cell epitopes of outer membrane protein OmpF of Aeromonas hydrophila in translational fusion with a carrier protein", 《APPL MICROBIOL BIOTECHNOL》 * |
| ROB PATRO: "A computational method for designing diverse linear epitopes including citrullinated peptides with desired binding affinities to intravenous immunoglobulin", 《BMC BIOINFORMATICS》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108659116B (en) | 2021-04-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kovacs-Nolan et al. | Avian egg antibodies: basic and potential applications | |
| CN107033250B (en) | Bovine coronavirus recombinant multi-epitope antigen and application thereof | |
| CN1062605C (en) | Escherichia coli vaccine | |
| CN101955545A (en) | Multi-target recombination gene and application of protein thereof in preventing and treating infection of helicobacter pylori | |
| CN109970852A (en) | Secrete hybridoma cell strain and the application of rabies poison M protein monoclonal antibody | |
| CN101016541A (en) | Method of producing brucella vaccine antigen protein | |
| CN105198989A (en) | Shewanella-smarisflavi-resistant egg yolk antibody and preparation method thereof | |
| CN110845605A (en) | Anti-canine parvovirus genetic engineering antibody and application thereof | |
| CN102031245B (en) | Monoclonal antibody of avian reticuloendothelial hyperplasia protein gp90 and application thereof | |
| CN109293753A (en) | A kind of enterotoxigenicEscherichia coli tetravalence dynein immunogene and preparation method thereof | |
| CN105541977A (en) | Riemerella anatipestifer OmpH intercepted recombinant protein, and preparation method and application thereof | |
| CN103837684A (en) | Antibody reagent for rapidly detecting salmonellas and detection method thereof | |
| CN115141810B (en) | Hybridoma cell strain secreting monoclonal antibody against mycoplasma synoviae and application | |
| CN108752470B (en) | Egg yolk antibody for resisting carp herpesvirus II type ORF72 and preparation method thereof | |
| CN108659116A (en) | A kind of immunogen synthesis method being used to prepare aquatic pathogenic bacterium Aeromonas chiasma type antibody | |
| CN106397602B (en) | A kind of reinforced chicken Marek's disease protein engineering vaccine of molecule adjuvant | |
| CN113754782A (en) | Helicobacter pylori egg yolk antibody and preparation method and application thereof | |
| CN104031152A (en) | Recombined pig/cow source escherichia coli heat stable enterotoxin fusion protein STp5-His, monoclonal antibody for resisting protein and application of protein | |
| CN109608541B (en) | Yolk antibody for resisting swine enterotoxigenic escherichia coli and preparation method thereof | |
| WO2021087441A2 (en) | Composition and methods for preventing and treating african swine fever in wild and domestic swine | |
| Fan et al. | Preparation and physicochemical property of chicken yolk immunoglobulin (IgY) against porcine transmissible gastroenteritis virus (TGEV) | |
| CN110376385B (en) | Preparation method and application of protein antigen for expressing QX type infectious bronchitis virus S1 by genetic engineering | |
| CN104479012B (en) | The preparation method of PPR virus Yolk antibody | |
| CN113144183B (en) | Broad-spectrum vibrio vaccine containing epitope K7 and preparation and application thereof | |
| CN110759988B (en) | Application of porcine NLRP3 truncated fragment as antigen structural protein |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 222005 No. 59 Cangwu Road, Haizhou District, Lianyungang City, Jiangsu Province Patentee after: Jiangsu Ocean University Address before: 222005 No. 59 Cangwu Road, Haizhou District, Lianyungang City, Jiangsu Province Patentee before: HUAIHAI INSTITUTE OF TECHNOLOGY |
|
| CP01 | Change in the name or title of a patent holder | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221125 Address after: 276800 Room 201, Building 2, 50 meters north of the intersection of Zhaoyang Road and Liaocheng Road, High tech Zone, Rizhao City, Shandong Province Patentee after: Shandong Xinpin Biomedical Technology Co.,Ltd. Address before: 222005 No. 59 Cangwu Road, Haizhou District, Lianyungang City, Jiangsu Province Patentee before: Jiangsu Ocean University |
|
| TR01 | Transfer of patent right |