KR20090037072A - The egg containing antibody igy charicterized by using salmonella and helicobacter pylori antigen & its production method - Google Patents
The egg containing antibody igy charicterized by using salmonella and helicobacter pylori antigen & its production method Download PDFInfo
- Publication number
- KR20090037072A KR20090037072A KR1020070102483A KR20070102483A KR20090037072A KR 20090037072 A KR20090037072 A KR 20090037072A KR 1020070102483 A KR1020070102483 A KR 1020070102483A KR 20070102483 A KR20070102483 A KR 20070102483A KR 20090037072 A KR20090037072 A KR 20090037072A
- Authority
- KR
- South Korea
- Prior art keywords
- salmonella
- antigen
- pylori
- antibody
- igy
- Prior art date
Links
- 241000607142 Salmonella Species 0.000 title claims abstract description 92
- 108091007433 antigens Proteins 0.000 title claims abstract description 73
- 102000036639 antigens Human genes 0.000 title claims abstract description 73
- 239000000427 antigen Substances 0.000 title claims abstract description 70
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 16
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 title abstract description 8
- 235000013601 eggs Nutrition 0.000 claims abstract description 53
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 239000002671 adjuvant Substances 0.000 claims abstract description 9
- 239000000872 buffer Substances 0.000 claims abstract description 8
- 239000000839 emulsion Substances 0.000 claims abstract description 5
- 241000287828 Gallus gallus Species 0.000 claims description 13
- 230000003053 immunization Effects 0.000 claims description 13
- 238000002649 immunization Methods 0.000 claims description 13
- 230000036039 immunity Effects 0.000 abstract description 4
- 102000002322 Egg Proteins Human genes 0.000 description 22
- 108010000912 Egg Proteins Proteins 0.000 description 22
- 235000013345 egg yolk Nutrition 0.000 description 22
- 210000002969 egg yolk Anatomy 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 20
- 235000013330 chicken meat Nutrition 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 101710116435 Outer membrane protein Proteins 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 206010016952 Food poisoning Diseases 0.000 description 8
- 208000019331 Foodborne disease Diseases 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 238000005259 measurement Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000004220 aggregation Methods 0.000 description 6
- 230000002776 aggregation Effects 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 108010046334 Urease Proteins 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 239000000020 Nitrocellulose Substances 0.000 description 4
- 230000004520 agglutination Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 210000000991 chicken egg Anatomy 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 230000037029 cross reaction Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229920001220 nitrocellulos Polymers 0.000 description 4
- 244000144977 poultry Species 0.000 description 4
- 235000013594 poultry meat Nutrition 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 229960005486 vaccine Drugs 0.000 description 4
- 238000012800 visualization Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 3
- 238000012790 confirmation Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000008029 eradication Effects 0.000 description 3
- 230000016784 immunoglobulin production Effects 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 229920000136 polysorbate Polymers 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 241000906901 Aracana Species 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 101000884675 Coxiella burnetii (strain RSA 493 / Nine Mile phase I) Co-chaperonin GroES Proteins 0.000 description 2
- 241000703769 Culter Species 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 102000004895 Lipoproteins Human genes 0.000 description 2
- 108090001030 Lipoproteins Proteins 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 2
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101000980463 Treponema pallidum (strain Nichols) Chaperonin GroEL Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000000679 carrageenan Substances 0.000 description 2
- 229920001525 carrageenan Polymers 0.000 description 2
- 229940113118 carrageenan Drugs 0.000 description 2
- 239000010779 crude oil Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002688 persistence Effects 0.000 description 2
- 230000002085 persistent effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-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
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 108010048581 Lysine decarboxylase Proteins 0.000 description 1
- 239000006154 MacConkey agar Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 108700028353 OmpC Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010013381 Porins Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 206010039438 Salmonella Infections Diseases 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241001467018 Typhis Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 241000607598 Vibrio Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 208000012873 acute gastroenteritis Diseases 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940069428 antacid Drugs 0.000 description 1
- 239000003159 antacid agent Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007374 clinical diagnostic method Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229940001442 combination vaccine Drugs 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 208000000718 duodenal ulcer Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003278 egg shell Anatomy 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012526 feed medium Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 150000002433 hydrophilic molecules Chemical class 0.000 description 1
- 238000013095 identification testing Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 239000006916 nutrient agar Substances 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 102000007739 porin activity proteins Human genes 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 206010039447 salmonellosis Diseases 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 229940082569 selenite Drugs 0.000 description 1
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
- 229940056729 sodium sulfate anhydrous Drugs 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000001325 yolk sac Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L15/00—Egg products; Preparation or treatment thereof
- A23L15/25—Addition or treatment with microorganisms or enzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/03—Antigenicity
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S424/00—Drug, bio-affecting and body treating compositions
- Y10S424/804—Drug, bio-affecting and body treating compositions involving IgG3, IgG4, IgA, or IgY
Abstract
Description
본 발명은 살모넬라와 헬리코박터 파일로리의 공동 특이 항원성을 이용한 항체 IgY를 포함하는 계란 및 그 생산방법에 관한 것이다.The present invention relates to an egg comprising the antibody IgY using the cospecific antigenicity of Salmonella and Helicobacter pylori and a method of producing the same.
살모넬라(Salmonella spp.)는 단간균으로 대부분 운동성이 있으며, 비포자형성 그람 음성균이다. 2400여 종이 보고되어 있으며, 이들 중 2,300여 종 이상이 사람에게 식중독을 유발한다. 감염량은 감염자의 건강 상태에 따라 다르며, 균종에 따라 다소 차이는 있지만 대개 15-20 cells 정도이다. 감염 후 8-72시간이 지나면 급성 위장염, 복통, 설사, 메스꺼움, 구토 오한 등의 증상을 나타내게 되고 특히 소아에게는 심한 탈수 증세를 일으켜 열과 패혈증으로 진행될 수 있다. 살모넬라속 균은 포유류나 조류의 소화관 내에서 검출되고 있고, 가금류인 닭의 경우에는 살모넬라가 주로 장내에 번식함으로서 도살시 닭의 고기는 물론 생산하는 계란의 난각 뿐만 아니라 난황까지 감염이 되며, 또한 살모넬라균은 계분과 함께 배출되어 오염된 계란에서 더 높은 비율로 발생하고 있다. 살모넬라를 보균한 닭은 난계대 전염율이 33.3%이며, S. typhimurium의 경우 닭에 이환되어 난소 등의 장기에 보균함으로써 생산된 계란에 균이 들어 있을 때가 많으며, 또한 불결한 양계장에서는 살모넬라에 오염된 흙이 계란에 부착되거나 보관 중에 균이 난각 표면에서 난각을 뚫고 계란 내부로 침투하면 계란의 부화율 감소와 내부 품질이 떨어지고 나아가 부패하게 된다. Salmonella spp. Is a simple bacillus that is mostly mobile and non-spore-forming gram-negative. More than 2400 species have been reported, and more than 2,300 of them cause food poisoning in humans. The amount of infection depends on the health of the infected person, and usually varies between 15-20 cells, depending on the species. After 8-72 hours after infection, acute gastroenteritis, abdominal pain, diarrhea, nausea, vomiting and chills may occur, and especially in children, severe dehydration may lead to fever and sepsis. Salmonella bacteria are detected in the digestive tracts of mammals and birds, and in the case of poultry chickens, salmonella breeds mainly in the intestine, which causes not only egg meat as well as egg yolks but also egg yolk during slaughter. The fungus is released with chicken meal and is occurring at higher rates in contaminated eggs. Salmonella-bearing chickens have an egg transmission rate of 33.3%, and S. typhimurium often contains germs in eggs produced by infecting the chickens with organs such as ovaries. If soil adheres to eggs or germs penetrate eggshells from egg surface during storage, they will decrease the hatching rate of the eggs and deteriorate their internal quality and further decay.
미국에서는 살모넬라균에 의한 식중독 사고가 연간 2백만 ～ 4백만 건 발생되고 있다(FDA, 1998A). 스코틀랜드에서는 1980년-1985년 224건의 살모넬라균에 의한 식중독에 의하여 2,245명의 환자가 발생하였고, 그 중 12명이 사망했는데 그 원인을 분석한 결과 가금류에 오염된 살모넬라균의 확산을 막지 못하였기 때문이었다. 한편 우리나라에서는 1993년도부터 1997년까지의 식중독 발생 원인은 살모넬라에 의한 것이 46.5%, 장염 비브리오균과 황색 포도상구균에 의한 것이 각각 1%와 19.2%로 나타났으며, 1999년과 2000년 역시 살모넬라균에 의한 원인이 28.8%～35.6%로서 가장 큰 비율을 차지하였다. Salmonella는 자연에 널리 분포하고 있으며 식품에 오염되어 식중독을 유발하는 중요한 세균이다. Salmonella에 의한 식중독을 방지하기 위하여 생산자와 소비자는 식품의 위생적인 관리에 유의하여야 한다. 우유를 비롯한 축산 식품의 오염을 방지하고 공중보건을 유지하기 위한 수단으로서 오염을 조기에 예방하는 방법이 필요하다. 특히 시유가 제조되는 공정 중에는 원유 또는 충전기와 같은 유가공 기계에 의한 살균 후에 2차 오염이 일어날 수 있다. 2 차 오염에 의한 시유의 저장성이 감소될 뿐만 아니라 Salmonella와 같은 식중독균에 의해 안전성이 위협될 수 있다. 실제로 오염된 시유에 의해 미국 일리노이(Illinois)시에서 16,000명의 salmonellosis 식중독 환자가 발생하였다. 또한 Salmonella에 감염된 젖소는 패혈증, 설사 및 유산을 유발하여 우유 생산성을 떨어뜨릴 뿐만 아니라 위생적인 우유의 생산에 장애 요인이 될 수 있다.In the United States, two to four million cases of food poisoning by Salmonella occur each year (FDA, 1998A). In Scotland, 2,245 patients died from food poisoning caused by 224 Salmonella in 1980-1985, and 12 of them died because the analysis did not prevent the spread of Salmonella contaminated with poultry. In Korea, the cause of food poisoning from 1993 to 1997 was 46.5% due to Salmonella, 1% and 19.2% due to enteritis Vibrio and Staphylococcus aureus, respectively. The most common cause was by 28.8% ~ 35.6%. Salmonella is widely distributed in nature and is an important bacterium that causes food poisoning by contaminating food. In order to prevent food poisoning by Salmonella , producers and consumers should take care of hygiene management of food. There is a need for early prevention of pollution as a means to prevent contamination of livestock foods, including milk, and to maintain public health. In particular, secondary contamination may occur after sterilization by a dairy machine such as crude oil or a filling machine during the process in which the crude oil is produced. In addition to diminishing the shelf-life of secondary milk due to secondary contamination, safety can be threatened by food poisoning bacteria such as Salmonella . In fact, 16,000 salmonellosis food poisoning cases occurred in Illinois, USA, due to contaminated oil. In addition, Salmonella -infected cows can cause sepsis, diarrhea and miscarriage, which can reduce milk productivity and can be a barrier to hygienic milk production.
한편 OMP(Outer membrane protein)들은 그람음성세균의 외막에 존재하는 단백질들로서 친수성 분자들을 통과시키는 생리학적으로 중요한 역할을 한다. 이 단백질들의 발현은 세균의 생장 환경에 따라 다르다. Salmonella의 OMP들을 이용하여 백신을 개발할 수 있으며 임상진단법에 응용할 수 있다고 보고된 바 있다. Arockilamy와 Krishnaswamy은 Salmonella typhi의 OMP 중에서 다수를 차지하며 세균의 외벽에 노출되어 있는 porin 단백질인 OmpC를 순수분리하였다.On the other hand, OMP (Outer membrane protein) are proteins in the outer membrane of Gram-negative bacteria and play an important physiological role in passing hydrophilic molecules. The expression of these proteins depends on the growth environment of the bacteria. It has been reported that vaccines can be developed using Salmonella 's OMPs and applied to clinical diagnostic methods. Arockilamy and Krishnaswamy Salmonella OmpC, a porin protein that is occupied by typhi and exposed to the outer wall of bacteria, was isolated.
Helicobacter pylori는 나선형(curved or spiral)의 그람음성 미호기성 세균으로, 1983년 위장 질환 환자의 위 조직 검체에서 최초로 분리됨이 보고되었다. 이후 다양한 연구들이 수행되어 현재에는 위염, 위 십이지장궤양은 물론 위암의 발생과도 밀접한 관련이 있는 것으로 알려져 있다. 이에 따라 이러한 질병들의 치료방법들도 주로 제산제를 사용하던 증상 치료로부터 H. pylori 박멸로 바뀌게 되었다. H. pylori의 박멸을 위해 현재 여러 가지 항생물질들이 사용되고 있고, 그 중 일부는 높은 박멸율을 보이고 있지만, 값이 비싸고, 종종 항생물질에 대해 내성이 유발되며, 일단 감염된 후에는 단지 일시적인 치료에 그칠 뿐 재발률이 높아 새로운 치료법이 요구되고 있다. 또한 1996년 현재 세계 인구의 1/3～1/2이 감염된 사실에 비해 실제로 질병으로 발전된 경우는 매우 낮은 비율인 경우에서 알 수 있듯이, H. pylori 감염은 장기간 동안 증상을 보이지 않다가 증세가 악화된 후에야 자각 증상이 나타나는 잠복성 질환이므로 치료법의 개선과 더불어 백신 등의 개발이 절실히 필요하다고 하겠다. Helicobacter Pylori is a gram-negative microaerobic bacterium in the form of a spiral or spiral, and was first isolated from gastric tissue samples in patients with gastrointestinal disease in 1983. Since then, various studies have been carried out and are now known to be closely related to gastritis, gastric duodenal ulcer and gastric cancer. As a result, treatments for these diseases have shifted from the treatment of antacids to H. pylori eradication. Several antibiotics are currently used for the eradication of H. pylori , some of which have high eradication rates, but are expensive, often resistant to antibiotics, and once infected, are only temporary treatment. High recurrence rates also require new treatments. In addition, H. pylori infection does not show symptoms for a long time but worsens, as can be seen in a very low rate of disease development compared with the fact that as of 1996, one third to one half of the world's population is infected. Since it is a latent disease that manifests symptoms, it is urgently needed to improve the treatment and develop vaccines.
백신제조 시 균체 자체를 사용할 경우 면역계 조절이 불가능하므로, 현재는 대신 균체의 성분 일부를 재조합 방법으로 생산하여 사용하고 있으며, 실제로 urease를 이용한 실험이 가장 활발히 진행되어 현재 임상 실험 중에 있으며, 재조합 urease B subunit(UreB)와 재조합 heat shock protein A(HspA)로 구성된 복합 백신 사용 시 동물 실험에서 100% 예방됨이 관찰된다는 보고가 있다. 감염성 세균의 경우, 외막 단백질(outer membrane proteins, OMPs)을 포함한 표면성분들이 주로 숙주에의 부착(colonization), 지속적 감염유지(persistence) 및 염증 반응(infammatory response)등을 유발시키는 것으로 알려져 있어서 이들을 이용한 백신 개발이 수행되고 있다.When the vaccine itself is used, it is impossible to control the immune system. Currently, some of the components of the bacteria are produced by recombinant methods.In fact, experiments using urease are most actively conducted and are currently in clinical trials.Recombinant urease B It has been reported that a combination vaccine consisting of a subunit (UreB) and recombinant heat shock protein A (HspA) is 100% prevented in animal experiments. In infectious bacteria, surface components, including outer membrane proteins (OMPs), are known to induce host colonization, persistent persistence, and infammatory response. Vaccine development is underway.
이에 본 발명에서는 Salmonella와 H.pylori의 그람음성세균 외막에 존재하는 단백질인 OMP와 우레아제(urease) 생산이라는 공통된 특성에 착안하여 두 균의 항원 특성과 암탉을 이용한 IgY 생산으로 얻어진 항체를 이용, 서로 다른 균에 대해서 각 균이 특이적인 면역반응을 나타냄을 실험하여 두 균에 대한 면역원을 동시에 가진 anti-Salmonella + H. pylori IgY 계란을 생산하고자 하였다.Therefore, the present invention focuses on the common characteristics of OMP and urease production, proteins present in the outer membrane of Gram-negative bacteria of Salmonella and H. pylori , using the antigen characteristics of the two bacteria and antibodies obtained by IgY production using hens. We tried to produce anti- Salmonella + H. pylori IgY eggs with immunogen against both bacteria by experimenting with the specific immune response against each other.
암탉의 혈청(blood serum) 내의 IgG는 난황을 통하여 획득된 면역기능을 후대에 물려준다는 특성이 있는바, 전에는 주로 토끼(rabbit)에서 얻어지던 anti-HRV antibody를 난황을 이용할 경우에는 약 30배 이상의 수율이 증가되며 토끼(rabbit)는 혈청(blood serum)을 얻어내기 위해서 죽여야 하지만 계란은 그렇지 않기 때문에 환경 및 동물 보호의 면에서도 유리하다고 할 수 있어 본 발명에서는 암탉을 이용하였다.IgG in hen's blood serum is characterized by inheriting the immune function acquired through egg yolk, which is about 30 times higher than that of egg yolk, which was previously obtained mainly from rabbits. The yield is increased and rabbits must be killed to obtain blood serum, but since eggs are not, it can be said to be advantageous in terms of environment and animal protection.
상기한 목적을 달성하기 위한 본 발명은 살모넬라균과 헬리코박터 파일로리균을 동시 항원으로 하거나 살모넬라균을 단독항원으로 하여 생산한 항 헬리코박터파일로리 항체 IgY를 포함한 계란인 것을 특징으로 한다.The present invention for achieving the above object is characterized in that the egg containing the anti-Helicobacter pylori antibody IgY produced by using Salmonella and Helicobacter pylori as a simultaneous antigen or Salmonella as the sole antigen.
본 발명은 또한,The present invention also provides
동결 건조한 살모넬라균 단독 항원으로 하거나 혹은 살모넬라균과 파일로리 균 항원을 혼합하여 멸균된 PBS 버퍼에 부유시키고,Lyophilized Salmonella alone or mixed with Salmonella and Pylori bacteria and suspended in sterile PBS buffer,
1차 면역에는 incomplete freund's adjuvant(프로인트 불완전 애쥬번트)를 사용하고, 2, 3차 면역에는 complete freund's adjuvant(프로인트 완전 애쥬번트)를 1차 면역 후 각각 3주, 5주차에 항원과 동량으로 한 유상액을 닭에 주사한 후,Incomplete freund's adjuvant is used for primary immunization and complete freund's adjuvant for second and third immunization is equal to antigen 3 weeks and 5 weeks after primary immunization, respectively. After injecting an emulsion into the chicken,
그 닭으로부터 항체 IgY를 포함한 계란을 얻는 것을 특징으로 한다.It is characterized by obtaining the egg containing the antibody IgY from the chicken.
본 발명자는 본 발명을 통해 그람음성균인 Salmonella와 H.pylori의 공통 항원 특이성을 확인하였으며, 각 항원에 대해 다른 항원으로 생성된 항체가 특이적으로 반응하여 각각의 항원, 항체가 교차 반응한다는 것을 확인하였는바, 이에 따르면 우수하고 효율적인 면역효과를 갖는 계란을 획득할 수 있다.The present inventors confirmed the common antigen specificity of the Gram-negative bacteria Salmonella and H.pylori , and confirmed that each antigen and antibody cross-react by specifically reacting antibodies generated by different antigens for each antigen. According to this, it is possible to obtain an egg having an excellent and efficient immune effect.
이하, 본 발명을 실시한 예를 구체적으로 설명한다.Hereinafter, the example which implemented this invention is demonstrated concretely.
Ⅰ. 재료 및 방법I. Materials and methods
1. 항원의 준비1. Preparation of the antigen
본 실험에서 항원으로 사용한 균주는 Salmonella typhimurium(ATCC 13311), Helicobacter pylori(KCTC 12083) 등으로 한국유전자은행에서 구입하였다.The strain used as antigen in this experiment was Salmonella as typhimurium (ATCC 13311), Helicobacter pylori (KCTC 12083) was purchased, such as gene banks in South Korea.
Salmonella typhimurium는 Nutrient agar에 35℃ 24시간, Helicobacter pylori는 fetal bovine serum(FBS)이 5% 첨가된 BA배지에서 10% CO2하에서 3～4일 동안 각각 배양시켰다. 균체는 3시간 동안 0.5% formalin으로 처리하여, 멸균한 phosphate buffer saline(PBS, pH 7.2)으로 3회 세척 후 동결 건조하여 -70℃에 저장하여 사용하였다. Salmonella typhimurium was cultured in Nutrient agar at 35 ° C for 24 hours, and Helicobacter pylori was incubated in BA medium containing 5% fetal bovine serum (FBS) for 3-4 days under 10% CO 2 . The cells were treated with 0.5% formalin for 3 hours, washed three times with sterile phosphate buffer saline (PBS, pH 7.2), lyophilized and stored at -70 ° C.
2. 실험동물 및 재료2. Laboratory Animals and Materials
본 실험에 사용된 실험동물은 경북 영천에서 박승용씨 농장에서 사육중인 15~20주령의 아라카나 교잡종 32마리로서 8마리씩 4군으로 나누어, 일반 산란계와 동일한 농장의 사육조건 사료(농협사료), 온도(18~25℃)에서 실시하였다.The experimental animals used in this experiment were 32 15--20-week-old Aracana hybrids in Yeongcheon, Gyeongbuk, divided into four groups of eight animals. It carried out at (18-25 degreeC).
분석용 시약인 incomplete freund's adjuvant, λ-caraageenan, rabbit anti chicken IgG-AP conjugated, chicken IgG, disodium hydrogenposphate 등은 Sigam(USA)사 제품을 사용하였으며, 그 외 일반시약은 특급 또는 일급품으로 사용하였다.As analytical reagents, incomplete freund's adjuvant, λ-caraageenan, rabbit anti chicken IgG-AP conjugated, chicken IgG, and disodium hydrogenposphate were used by Sigam (USA).
3. 면역3. Immunity
동물의 면역은 동결 건조한 항원(Salmonella , H. pylori , Salmonella +H. pylori)을 멸균된 PBS(approximately 2×109 organism per mL)에 부유시키고, 1차면역에는 incomplete freund's adjuvant(Sigma Co., USA)를 2, 3차 면 역은 complete freund's adjuvant롤 각각 3주, 5주령차에 항원과 동량으로 하여, 이 유상액을 1mL씩 양다리의 근육 내에 각 0.5mL씩 주사하였다. 계란은 매일 수집한 후, 4℃에 보관하여 실험하였다.Animal immunity was suspended in lyophilized antigens ( Salmonella , H. pylori , Salmonella + H. pylori ) in sterile PBS (approximately 2 × 10 9 organism per mL) and incomplete freund's adjuvant (Sigma Co., USA), the second and third immunizations were complete freund's adjuvant rolls at 3 and 5 weeks of age, respectively, with the same amount of the antigen, and 1 mL of this emulsion was injected into the muscles of both legs. Eggs were collected daily and then stored at 4 ° C for testing.
4. 4. 난황으로부터From egg yolk 계란항체( Egg Antibody ( IgYIgY )의 분리정제Separation and purification of
계란으로부터 IgY의 분리정제는 Hatta 등의 방법에 따라 다음과 같이 실시하였다. 계란에서 난황을 난백과 분리한 후 난황의 부피를 측정하여 2배 희석하였다. 난황의 lipoprotein을 제거하기 위하여 희석액 2배의 0.15% λ-carrageenan(Sigma Co., USA)용액을 혼합하고 실온에 30분간 방치하였다. 혼합물을 10,000 × g로 15분간 원심분리한 후, filter paper No. 2(Advantec Toyo)로 여과하였다. 이 여과액에 20mM disodium hydrogenphopshate를 용해하고 나서 3N HCl을 사용하여 pH 8.0으로 맞추었다. 이 용액을 DEAE-Sephacel column(Φ 5.0×10.0㎝)에 분당 5㎖로 통과시켰다. Column 부피의 20배되는 20mM phosphat buffer(pH 8.0)로 column을 세척한 후, column의 5배 부피의 0.2M phosphate buffer(pH 8.0)로 용출시켜 단백질을 분리하였다. 수집된 peak fraction은 spectrophotometer Model U-2000(Hitachi Co., Japan)으로 280nm에서 흡광도를 측정하였다. 20℃에서 용출액을 15%(w/v)가 되도록 sodium sulfate anhydrous powder를 가하여, 30분 동안 천천히 용해한 후 10,000 × g로 15분간 원심분리 하였다. 침전물을 다시 phosphate buffer(10mM, pH 8.0) 100㎖에 용해한 후, 위의 염석과정을 2회 반복하였다. 최종 침전물을 phosphate buffer(10mM, pH 8.0)에 용해한 후, 같은 buffer로 투석을 실시하였다. 투석된 용 액을 0.45㎛ membrane filter로 여과 후 동결 건조하여 보관하였다.Separation and purification of IgY from eggs was carried out according to the method of Hatta et al. After egg yolk was separated from egg white, egg yolk was measured and diluted twice. To remove egg yolk lipoprotein, 0.15% λ-carrageenan (Sigma Co., USA) solution of 2 times dilution was mixed and left at room temperature for 30 minutes. After centrifuging the mixture at 10,000 x g for 15 minutes, filter paper No. Filtration with 2 (Advantec Toyo). 20 mM disodium hydrogenphopshate was dissolved in the filtrate and adjusted to pH 8.0 using 3N HCl. This solution was passed through a DEAE-Sephacel column (Φ 5.0 × 10.0 cm) at 5 ml per minute. After washing the column with 20mM phosphat buffer (pH 8.0) of 20 times the volume of the column, eluted with 0.2M phosphate buffer (pH 8.0) of 5 times the volume of the column was separated. The collected peak fraction was measured at 280 nm by spectrophotometer Model U-2000 (Hitachi Co., Japan). Sodium sulfate anhydrous powder was added to the eluate at 20 ° C. at 15% (w / v), slowly dissolved for 30 minutes, and centrifuged at 10,000 × g for 15 minutes. The precipitate was dissolved again in 100 ml of phosphate buffer (10 mM, pH 8.0), and the above salting process was repeated twice. The final precipitate was dissolved in phosphate buffer (10 mM, pH 8.0), and then dialyzed with the same buffer. The dialysed solution was filtered through a 0.45㎛ membrane filter and then lyophilized and stored.
water-soluble protein fraction(WSF) powder는 면역된 계란 내의 난황을 분리하여 2배 희석한 다음, 난황의 lipoprotein을 제거하기 위하여 희석액 2배의 0.15% λ-carrageenan(Sigma Co., USA) 용액을 혼합하고 실온에 30분간 방치하였다. 혼합물을 10,000 × g로 15분간 원심분리한 후, 상청액을 filter paper No. 2(Advantec Toyo)로 여과하였다. 이 여과액에 100mM disodium hydrogenphopshate(Sigma Co., USA)를 용해한 후, 여과하고 3M NaOH를 사용하여 pH 8.0으로 조정하여 동결 건조하였다. 동결 건조된 WSF powder는 응집반응실험에 사용하였다.The water-soluble protein fraction (WSF) powder is diluted twice by separating egg yolk from immunized eggs, and then mixed with 0.15% lambda-carrageenan (Sigma Co., USA) solution in 2-fold dilution to remove egg yolk lipoprotein. And left at room temperature for 30 minutes. The mixture was centrifuged at 10,000 x g for 15 minutes, and then the supernatant was filtered out of filter paper No. Filtration with 2 (Advantec Toyo). After dissolving 100 mM disodium hydrogenphopshate (Sigma Co., USA) in this filtrate, it was filtered, adjusted to pH 8.0 with 3M NaOH, and lyophilized. Freeze-dried WSF powder was used in the flocculation reaction experiment.
5. 계란항체(5. Egg antibody IgYIgY ) 함량 측정 및 항체 생성 확인 실험) Content measurement and antibody production confirmation experiment
(1) Enzyme-linked immunosorbent assay(ELISA)(1) Enzyme-linked immunosorbent assay (ELISA)
난황과 혈청의 항체활성을 측정하기 위하여 효소면역측정법(enzyme-linked immunosorbent assay: ELISA)을 사용하였다. 각 항원을 405nm에서 coating buffer(50mM carbonate buffer pH 9.6)로 조절한 다음 immunoglobulin 96well plate에 100㎕씩 넣고, 4℃에서 overnight 반응시켜 coating하였다. PBS-T(phosphate buffer saline, 0.05% Tween-20, pH 7.4)로 세 번 세척한 후, blocking액(PBS pH 7.4, 5% skim milk)을 150㎕씩 넣고 실온에서 30분 이상 blocking시킨 후, PBS-T로 세 번 세척하였다. 희석된 난황과 혈청을 100㎕씩 넣고 25℃에서 2시간 동안 반응시킨 후 PBS-T로 세 번 세척하였다. Sencondary antibody로서 혈청은 rabbit anti-chicken IgG-alkaline phosphatase(Sigma Co., USA)용액을, 난황은 anti-chicken IgY-alkaline phosphatase(Progmega Co., USA)용액을 0.01M PBS로 5,000배 희석한 용액을 well당 100㎕씩 넣고, 실온에서 1시간 반응시켰다. PBS-T로 세 번 세척한 후, 기질요액(10% diethanolamine buffer, 1% phosphatase substrate(Sigma Co., USA))을 100㎕씩 넣고 37℃에서 30분 반응 후, 반응 정지액(3N NaOH) 50㎕으로 반응을 정지시킨 후, 405nm의 파장에서 ELISA reader(Model 550 BIO-RAD, USA)로 각 well의 흡광도를 측정하였다.Enzyme-linked immunosorbent assay (ELISA) was used to measure the antibody activity of egg yolk and serum. Each antigen was adjusted to a coating buffer (50mM carbonate buffer pH 9.6) at 405 nm, and then 100 µl each of the immunoglobulin 96well plate was coated by overnight reaction at 4 ° C. After washing three times with PBS-T (phosphate buffer saline, 0.05% Tween-20, pH 7.4), 150 μl of blocking solution (PBS pH 7.4, 5% skim milk) was added and blocked for 30 minutes at room temperature. Wash three times with PBS-T. 100 μl of diluted egg yolk and serum were added and reacted at 25 ° C. for 2 hours, and then washed three times with PBS-T. As a sencondary antibody, serum was diluted 5,000 times with rabbit anti-chicken IgG-alkaline phosphatase (Sigma Co., USA) solution and egg yolk with anti-chicken IgY-alkaline phosphatase (Progmega Co., USA) solution in 0.01M PBS. 100 μl per well was added and allowed to react at room temperature for 1 hour. After washing three times with PBS-T, 100 μl each of the substrate urea (10% diethanolamine buffer, 1% phosphatase substrate (Sigma Co., USA)) was added and the reaction stopper (3N NaOH) after 30 minutes at 37 ° C. After stopping the reaction in 50ul, the absorbance of each well was measured by ELISA reader (Model 550 BIO-RAD, USA) at a wavelength of 405nm.
(2) SDS-polyacrylamide gel electrophoresis(SDS-PAGE)에 의한 분자량 측정(2) Molecular weight measurement by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Salmonella, H. pylori, Salmonella+H. pylori, 항원과 정제된 anti-Salmonella , anti-H. pylori, anti- Salmonella+H.pylori, 항체의 특성을 조사하기 위하여 분자량을 측정 SDS-polyacrylamide slab gel 전기영동을 Laemmli의 방법으로 실시하였다. 0.5M Tris-HCl과 10% SDS, 30% acrylamide를 함유한 4.0%(v/v) 농축겔을 사용하고, 분리겔은 1.5M Tris-HCl과 10% SDS, 30% acrylamide을 함유한 10%(v/v)겔을 사용하였다. 분리정제항체 단백질은 30㎍/㎕(D.W)을 sample buffer로 1:4로 희석하여 각 well에 20㎕씩 loading하였다. 각 균은 667nm O.D 1에 맞추어 sonicate(pulse 20, duty cycle 50)후 40㎍/㎖(D.W)를 sample buffer로 1:4로 희석 후 20㎕씩 loading하였다. marker는 wide range(Sigma Co., USA)와 low range(Sigma Co., USA) 두 가지를 5㎕씩 loading하였다. 단백질 band는 Coomassie blue로 염색하였다. Salmonella , H. pylori , Salmonella + H. pylori , In order to investigate the characteristics of the antigen, purified anti- Salmonella , anti- H. Pylori , anti- Salmonella + H.pylori and antibody, molecular weight was measured by SDS-polyacrylamide slab gel electrophoresis. 4.0% (v / v) concentrated gel containing 0.5M Tris-HCl, 10% SDS and 30% acrylamide was used, and the separation gel was 10% containing 1.5M Tris-HCl, 10% SDS and 30% acrylamide. (v / v) gel was used. The isolated and purified antibody protein was diluted 30 μg / μl (DW) 1: 4 with sample buffer and loaded 20 μl into each well. Each bacterium was sonicate (pulse 20, duty cycle 50) in accordance with 667nm OD 1 and then diluted 40㎍ / mL (DW) 1: 4 with sample buffer and loaded 20μl. The markers were loaded with 5 μl of wide range (Sigma Co., USA) and low range (Sigma Co., USA). Protein bands were stained with Coomassie blue.
(3) 웨스턴 블로팅(Western blotting)(3) Western blotting
면역된 닭으로부터 얻은 특이 IgY 항체의 항원특이성을 알아보기 위해 western blotting을 실시하였다. 먼저 separating gel 농도 10%의 SDS-PAGE에 Samonella, H. pylori , Salmonella + H. pylori , 항원 파쇄액을 전개시키고, gel을 Towbin등의 방법에 의해 nitrocellulose(NC) paper(Gelman Sciences Co.)에 transfer한 다음 5% Skim milk로 blocking하여 0.08% Tween을 첨가한 PBS에 3회 세척하였다. Primary antibody은 1/2000으로 희석하여 4℃에서 overnight한 후 다음날 Tween을 첨가한 PBS에 3회 세척하였다. NC membrane을 1/5000로 희석한 secondary antibody(Anti-Chiken IgY AP Conjugate Promega USA)에 넣어준 후 2시간동안 Incubation한다. Tween을 첨가한 PBS에 3회 세척한 후 Lumi-PhosTMWB(PIERCE USA.)을 NC membrane에 뿌린 후 1분 후에 wrap으로 싼 다음 그 위에 X-ray film을 올리고 cassette에 넣어 감광시켰다.Western blotting was performed to determine the antigenic specificity of specific IgY antibodies from immunized chickens. First, Samonella, H. pylori , Salmonella + H. pylori , antigen crushing solution was developed, gel was transferred to nitrocellulose (NC) paper (Gelman Sciences Co.) by Towbin method, and then blocked with 5% Skim milk to PBS added with 0.08% Tween. Wash three times. The primary antibody was diluted to 1/2000, overnight at 4 ° C, and washed three times in PBS with Tween the next day. Add NC membrane to secondary antibody (Anti-Chiken IgY AP Conjugate Promega USA) diluted to 1/5000 and incubate for 2 hours. After washing three times in PBS added with Tween, Lumi-Phos TM WB (PIERCE USA.) Was sprinkled on the NC membrane, wrapped in a wrap after 1 minute, and then placed on an X-ray film and placed in a cassette.
기본적인 각 항체의 항원특이성을 알아보기 위해 1차 실험을 실시하였고, 서로 다른 항원, 항체에 대한 특이성을 알아보고자 Primary antibody를 바꿔서 실험을 실시하였다.In order to examine the antigen specificity of each basic antibody, a first experiment was conducted, and the experiment was performed by changing the primary antibody to determine specificity of different antigens and antibodies.
6. 항균 활성(계란 항체에 의한 항원의 응집반응)6. Antibacterial activity (aggregation reaction of antigen by egg antibody)
특이항체(IgY)가 항원으로 사용된 균체를 응집시키는 정도를 측정하였다. 조난황 specific IgY 분말을 20%, 10%, 5%가 되게 멸균 증류수에 녹인 후 membrane filter로 여과 제균 한 후 PBS에 현탁한 각 균체(O.D.=1.0)를 동량혼합하고 실온에서 방치하여 반응시키고 광학현미경으로 응집이 나타나는지를 관찰하였다.The extent to which the specific antibody (IgY) aggregated the cells used as antigen was measured. Dissolve the yolk-specific IgY powder in sterile distilled water to 20%, 10%, and 5%, filter and sterilize it with a membrane filter, and then mix and mix the same cells (OD = 1.0) suspended in PBS, and react at room temperature. It was observed under the microscope whether aggregation appeared.
7. 계란 안전성 실험(면역된 계란 중 7. Egg Safety Experiments (of Immunized Eggs) SalmonellaSalmonella 안전성 확인시험) Safety confirmation test)
각 검체 10g(계란자체)을 취하여 90mL의 Selenite F 배지(배지 28)에 가한 후 35℃에서 24시간 증균배양하였다. 증균배양액을 MacConkey 한천배지(배지30)에 접종하여 35℃에서 24시간 배양한 후, 의심되는 집락을 확인시험하였다. 생화학적 확인시험은 분리된 평판배지상의 집락을 보통한천배지에 옮겨 35℃에서 18시간 배양한 후, TSI 사면배지의 사면과 고층부에 접종하고 35℃에서 18시간 배양하여 생물학적 성상을 검사하였다. 살모넬라는 유당, 서당 비분해(사면부 적색), 가스생성(균열확인) 양성인 균에 대하여 그람음성 간균임을 확인하고 우레아제(urease) 음성, 라이신 디카복실레이스(lysine decarboxylase) 양성 등의 특성을 확인하였다. 10 g of each sample (egg) was taken, added to 90 mL of Selenite F medium (medium 28), and enriched for 24 hours at 35 ° C. The enrichment medium was inoculated in MacConkey agar medium (medium 30) and incubated at 35 ° C. for 24 hours, and then the suspected colonies were identified. In the biochemical identification test, the colonies on the separated plate medium were transferred to ordinary agar medium and incubated at 35 ° C for 18 hours, and then inoculated on the slope and the upper part of TSI slope medium and cultured at 35 ° C for 18 hours to examine their biological properties. Salmonella was found to be Gram-negative bacillus against lactose, sucrose undigested (red slope), and gas-producing (cracked) positive bacteria, and confirmed characteristics such as urease negative and lysine decarboxylase positive.
Ⅱ. 결과 및 고찰II. Results and Discussion
1. 항체의 생성변화 추이1. Changes in Production of Antibodies
Salmonella와 H. pylori, Salmonella+H. pylori 등으로 면역된 산란계에서 얻은 특이항체(IgY)의 항체 생성을 확인하기 위하여 ELISA로 확인한 결과 Slmonella 로 면역된 산란계는 10주차까지 높은 역가를 나타내었으며, Salmonella+H. pylori 복합투여 군에서도 단일 항원 투여군보다 더 고른 항체 증가율을 나타내었다. 또한 Salmonella와 H. pylori의 그람음성세균 외막에 존재하는 단백질인 OMP와 urease 생산이라는 공통된 항원특성에 착안하여 각각의 균을 교차면역반응 한 결과 H. pylori 항원에 1차 항체를 Salmonella로 사용한 경우 높은 역가를 보였으며, Salmonella 항원에 H. pylori를 1차 항체로 사용한 결과도 비슷한 경향을 나타내었다. 이는 감염성 세균의 경우, 외막단백질(outer membrane proteins, OMPs)을 포함한 표면성분들이 주로 숙주에의 부착(colonization), 지속적 감염유지(persistence) 및 염증반응(inflammatory response) 등을 유발시키는 것으로 특이적인 항원, 항체 결합을 유도하는 것으로 볼 수 있겠다.It exhibited a high titer to Salmonella and H. pylori, Salmonella + H. pylori as a result of immunization with Slmonella confirmed by ELISA to determine the antibody production of antibodies (IgY) derived from the immunized laying hens laying hens or the like 10 weeks, Salmonella + H. pylori combination group showed more even antibody growth rate than single antigen group. In addition, when using a high primary antibody to H. pylori and Salmonella of Gram-negative bacteria outer membrane protein OMP and urease production that results H. pylori antigen crossed immune response to each of the bacteria in view of the common antigens present in the nature in Salmonella Titers showed similar trends in the results of using H. pylori as a primary antibody for Salmonella antigen. In infectious bacteria, surface components, including outer membrane proteins (OMPs), mainly cause colonization, persistent persistence, and inflammatory responses to the host. It can be seen as inducing antibody binding.
Fig. 1. 살모넬라로 면역된 경우 항체의 생성변화 추이(Changes of antibody contents in chicken egg yolk immunized by salmonella during the immunization period.)Fig. 1.Changes of antibody contents in chicken egg yolk immunized by salmonella during the immunization period.
Fig. 2 살모넬라와 헬리코박터 파일로리로 면역된 경우 항체의 생성 변화(Changes of antibody contents in chicken egg yolk immunized by salmonella +H. pylori during the immunization period.)Fig. 2 Changes of antibody contents in chicken egg yolk immunized by salmonella + H. pylori during the immunization period.
Fig. 3. 헬리코박터 파일로리 항원에 1차 항체를 살모넬라로 한 경우 항체의 생성변화 추이[Changes of antibody contents in chicken egg yolk immunized by salmonella during the immunization period(antigen : H. pylori, antibody : Salmonella)]Fig. 3. Changes of antibody contents in chicken egg yolk immunized by salmonella during the immunization period (antigen: H. pylori , antibody: Salmonella )]
Fig. 4. 살모넬라 항원에 1차 항체를 헬리코박터 파일로리로 한 경우 항체의 생성변화 추이[Changes of antibody contents in chicken egg yolk immunized by H. pylori during the immunization period(antigen : Salmonella, antibody : H. pylori)]Fig. 4. Changes of antibody contents in chicken egg yolk immunized by H. pylori during the immunization period (antigen: Salmonella , antibody: H. pylori )]
2. 항균 활성2. antibacterial activity
난황 내의 특이 항체에 대한 응집가는 Fig. 5, 6, 7, 8에서와 같다. 항체에 의해 면역되지 않은 계란 항체는 응집을 일으키지 않지만, 농도별에 따른 Salmonella와 H. pylori 특이항체(IgY)는 매우 높은 응집가를 나타내었다.Agglomerates for specific antibodies in egg yolk are shown in Fig. Same as in 5, 6, 7, 8. Egg antibodies not immunized by the antibodies did not cause aggregation, but Salmonella and H. pylori- specific antibodies (IgY) by concentration showed very high aggregation values.
또한 항원 공통성에 기인한 교차 반응에서 Salmonella 항원에 H. pylori 항체, H.pylori 항원에 Salmonella 항체를 투여한 결과 항원, 항체가 동일한 실험보 다는 다소 떨어지는 경향을 보였으나 면역화 되지 않은 계란 항체 실험보다는 높은 응집가를 나타내었다. 이는 그람음성세균이라는 공통된 특성을 반영한 결과라고 보여진다. Salmonella also in cross-reactions caused by antigenic commonality Result of administration of the Salmonella antibody to H. pylori antibodies, H.pylori antigen to antigen antigen, exhibited a slightly higher than eungjipga egg antibody experiments showed non-immunized or a falling trend antibodies rather than the same experiment. This may be a result of reflecting the common characteristics of Gram-negative bacteria.
Fig. 5. 난황 내의 특이 항체(살모넬라 특이 항체)에 대한 응집가[Visualization of agglutination by IgY using light microscope. Salmonella antigen (Salmonella specific IgY)]Fig. 5. Visualization of agglutination by IgY using light microscope. Salmonella antigen ( Salmonella specific IgY)]
Fig. 6. 난황 내의 특이 항체(헬리코박터 파일로리 특이 항체)에 대한 응집가[Visualization of agglutination by IgY using light microscope. H. pylori antigen(H. pylori specific IgY)]Fig. 6. Visualization of agglutination by IgY using light microscope. H. pylori antigen ( H. pylori specific IgY)]
Fig. 7. 난황 내의 특이 항체(살모넬라 항원에 헬리코박터 파일로리 항체)에 대한 응집가[Visualization of agglutination by IgY using light microscope. Salmonella antigen(H. pylori specific IgY)]Fig. 7. Visualization of agglutination by IgY using light microscope for specific antibodies in the yolk sac (Helicobacter pylori antibody to Salmonella antigens). Salmonella antigen ( H. pylori specific IgY)]
Fig. 8. 난황 내의 특이 항체(헬리코박터 파일로리 항원에 살모넬라 항체)에 대한 응집가[Visualization of agglutination by IgY using light microscope. H. pylori antigen(Salmonella specific IgY)]Fig. 8. Visualization of agglutination by IgY using light microscope for specific antibodies in eggs (Salmonella antibodies to Helicobacter pylori antigen). H. pylori antigen ( Salmonella specific IgY)]
3. 항원·항체 특이성 및 분자량 측정결과3. Results of antigen and antibody specificity and molecular weight
(1) SDS-PAGE(SDS-polyacrylamide gel electrophoresis)에 의한 분자량 측정(1) Molecular weight measurement by SDS-PAGE (SDS-polyacrylamide gel electrophoresis)
Fig. 9의 lane 2-4, 6-8은 sonicating(pulse 20, duty cycle 50) 처리한 Salmonella , H.pylori, Salmonella +H. pylori 항원과 분리 정제된 각각의 특이항체(IgY)를 SDS-PAGE 전기영동법으로 측정한 분자량 결과이다. 항원의 분자량 측정에서는 50kDa 전후의 주요 항원을 포함한 다양한 크기의 단백질 항원들을 확인할 수 있었다. Salmonella +H. pylori 항원에서는 각각의 단일 항원band에서 나타났던 band를 모두 확인할 수 있었다.Fig. Lanes 2-4 and 6-8 of 9 were treated with sonicating (pulse 20, duty cycle 50) Salmonella , H. pylori, Salmonella + H. The specific molecular weight (IgY) isolated from the pylori antigen was purified by SDS-PAGE electrophoresis. In the molecular weight measurement of antigens, protein antigens of various sizes were identified, including major antigens before and after 50 kDa. Salmonella + H. In the pylori antigen, all bands in each single antigen band were identified.
분리 정제된 난황항체의 분자량 측정을 위한 SDS-PAGE 측정에서는 Salmonella , H. pylori , Salmonella+H.pylori에서 세 특이항체가 비슷하게 band를 나타내었다(Fig.9의 lane 6-8).In the SDS-PAGE measurement of the molecular weight of purified yolk antibody, three specific antibodies were similarly banded in Salmonella , H. pylori , and Salmonella + H.pylori (lane 6-8 in Fig. 9).
Shimizu 등은 포유동물의 IgG의 heavy chain 분자량이 50KD인데 비해서 IgY는 68KD인 것으로 IgG와 IgY의 분자량의 차이점을 보고한 바 있다. 또한 토끼의 IgG의 heavy chain의 아미노산 잔기는 440, light chain의 아미노산 잔기는 214개인 것에 반해, IgY는 각각 559개와 206개임을 확인하였으며, 이들 chain의 S-S결합의 위치나 수량 및 당분자와의 결합위치 등에서 차이점이 있음을 확인한 바 있다.Shimizu et al. Reported the difference between the molecular weight of IgG and IgY as that of IgY is 68KD compared to the heavy chain molecular weight of 50KD in mammalian IgG. The amino acid residues of rabbit IgG heavy chain were 440 and light chain amino acid residues were 214, while IgY was 559 and 206, respectively. The difference was found in the back.
(2) 항체의 항원 특이성 측정결과 Western blotting(2) Western blotting of antigen specificity of antibody
각 항원에 의해 생성된 항체가 각 항원에 특이적으로 반응하는지를 알아보기 위해 western blotting을 실시하였다(Fig. 9의 lane 9-14). 각각의 항원에 대해서는 40kDa 부위에서 공통으로 확인되었으며, Salmonella 항원에 의해 생성된 항체에 대해 H. pylori 항원에 교차 반응하였을 때 100～40kDa 부위에서 다양한 항원성을 확인할 수 있었으며, H. pylori 항원에 의해 생성된 항체에 대해 Salmonella 항원에 교차 반응하였을 때도 100～40kDa 부위에서 다양한 항체의 항원 특이적인 반응을 나타내었다. Western blotting was performed to determine whether the antibodies produced by each antigen specifically reacted to each antigen (lanes 9-14 in Fig. 9). For each antigen was identified in common at the 40 kDa site and H. pylori for antibodies produced by Salmonella antigen When antigens were cross-reacted, various antigenicities were confirmed at 100-40kDa sites. Antigen-specific responses of various antibodies at 100-40kDa sites were also observed when cross-reacted with Salmonella antigen against antibodies produced by H. pylori antigen. Indicated.
4. 계란 안전성 실험(면역된 계란 중 4. Egg Safety Experiments (of Immunized Eggs) SalmonellaSalmonella 안전성 확인시험) Safety confirmation test)
면역된 닭에서 생산된 계란에 대한 안전성을 실험하기 위해서 면역된 계란 자체에 대한 안전성 실험을 한 결과 음성인 것으로 확인되었다. 가금류인 닭의 경우에는 살모넬라가 주로 장내에서 번식함으로서 도살시 닭의 고기는 물론이고 생산되는 계란의 난각뿐만 아니라 난황까지 감염이 되며, 또한 살모넬라균은 계분과 함께 배출되어 오염된 계란에서 더 높은 비율로 발생하는데 특이적으로 면역된 계란 난황에 포함된 특이항체로 인해 Salmonella 오염율이 낮은 안전한 계란을 생산할 수 있을 것으로 생각된다.In order to test the safety of the eggs produced in the immunized chicken, the safety test on the immunized egg itself was confirmed as negative. In the case of poultry chickens, salmonella breeds mainly in the intestine, which causes not only egg meat but also egg yolk as well as egg meat when slaughtered, and Salmonella bacteria are discharged together with poultry and have a higher rate in contaminated eggs. The specific antibodies contained in egg yolk, which are specifically immunized, may produce safe eggs with low Salmonella contamination.
Fig. 10. 안전성 확인시험 처리과정(biochemical confirmatory test. treatment of Salmonella specific IgY Egg .)Fig. 10. Biochemical confirmatory test.treatment of Salmonella specific IgY Egg . )
Fig. 11. 안전성 확인시험(biochemical confirmatory test of Salmonella specific IgY Egg .)Fig. 11.Biochemical confirmatory test of Salmonella specific IgY Egg . )
Enrichment culter test - A : Salmonella specific IgY Egg. B : H. pylori specific IgY Egg. C : Salmonella + H. pylori specific IgY Egg. D : Salmonella, H. pylori , Salmonella + H. pylori specific IgY. Enrichment culter test -A: Salmonella specific IgY Egg. B: H. pylori specific IgY Egg. C: Salmonella + H. pylori specific IgY Egg. D: Salmonella, H. pylori , Salmonella + H. pylori specific IgY.
Isolation culter test - E : Salmonella specific IgY Egg. F : H. pylori specific IgY Egg. Isolation culter test -E: Salmonella specific IgY Egg. F: H. pylori specific IgY Egg.
G : Salmonella + H. pylori specific IgY Egg.G: Salmonella + H. pylori specific IgY Egg.
Ⅲ. 요약III. summary
앞에서 본 실험은 그람음성균인 Salmonella와 H, pylori의 공통 특이 항원성을 이용한 항원항체 교차 반응 실험에 관한 것으로 Salmonella , H. pylori 항원을 아라카나 교잡종인 청색 산란계에 면역시킨 후 생산된 특이항체(IgY)를 이용 항체의 교차 반응 실험, 항원성 및 분자량 확인, 항균력, 계란 안전성을 측정한 결과 다음과 같은 결과를 얻었다. This experiment was related to the antigen-antibody cross-reaction experiment using the common specific antigens of Gram-negative bacteria Salmonella and H, pylori . Salmonella , H. pylori After the immunization of the antigen to the blue laying hens, the Aracana hybrid, the specific antibodies (IgY) were used to measure cross-reactivity, antigenicity and molecular weight, antimicrobial activity, and egg safety.
(1) Salmonella , H. pylori , Salmonella +H. pylori , 각 군에 대한 항원 투여 항체 생산을 ELISA로 확인한 결과 10주령까지 고른 항체 증가율을 나타내었다. Salmonella , H. pylori 각각의 항원에 대해 교차 항체를 사용하여 ELISA test 한 결과도 높은 역가를 나타내었다.(1) Salmonella , H. pylori , Salmonella + H. pylori, it exhibited a growth rate picked antibody antigen administered antibody production by ELISA as a result of 10-week-old check for each group. The ELISA test using cross-antibodies against Salmonella and H. pylori antigens also showed high titers.
(2) 농도별에 따른 Salmonella와 H. pylori 특이항체(IgY)는 매우 높은 응집가를 나타내었으며, 항원 특이성에 기인한 교차반응에서도 항원, 항체가 동일한 실 험보다는 다소 떨어지는 경향을 나타내었으나, 면역화 되지 않은 계란 항체 실험보다는 높은 응집가를 나타내었다. (2) Salmonella and H. pylori specific antibodies (IgY) by concentrations showed very high aggregation values, and even in cross-reactions due to antigen specificity, antigens and antibodies tended to be somewhat lower than the same experiments, but they were not immunized. It showed a higher aggregation value than the egg antibody experiment.
(3) 항원.항체 특이성을 알아보기 위해 SDS-PAGE 실험 결과 각 항원의 분자량 측정에서 50kDa 전후의 주요 항원을 포함한 다양한 크기의 단백질 항원을 확인할 수 있었다. 또한 분리정제 된 난황항체의 분자량 측정 결과 Salmonella , H. pylori에서 비슷한 특이항체 band를 확인할 수 있었다.(3) Antigen and Antibody Specificity As a result of SDS-PAGE experiments, it was possible to identify protein antigens of various sizes including major antigens before and after 50 kDa in molecular weight measurement of each antigen. Molecular weight measurements of the isolated and purified yolk antibodies showed similar specific antibody bands in Salmonella and H. pylori .
(4) 항체의 항원 특이성을 알아보기 위해 western blotting을 실험 한 결과 각각의 항원에 대해서는 40kDa부위에서 공통으로 확인되었으며, Salmonella , H. pylori 교차 항체 투여에 대한 실험결과에서는 100～40kDa 부위에서 다양한 항원, 항체 특이성을 확인할 수 있었다.(4) As a result of Western blotting experiments to determine the antigen specificity of the antibody, each antigen was commonly identified at 40kDa site, and Salmonella and H. pylori cross-antibody administration resulted in various antigens at 100 ~ 40kDa site. , Antibody specificity could be confirmed.
(5) Salmonella 항원으로 면역된 계란에 대한 안전성 실험을 한 결과 생물학적 성상 등의 검사에서 음성으로 확인되어 안전한 것으로 나타났다.(5) Safety experiments on eggs immunized with Salmonella antigen showed negative safety in tests such as biological properties.
이상의 여러 실험 결과에서 그람음성균인 Salmonella와 H. pylori의 공통 항원 특이성을 확인하였으며, 각 항원에 대해 다른 항원으로 생성된 항체가 특이적으로 반응하여 각각의 항원, 항체가 교차 반응한다는 것을 확인하였다.The results of the above experiments confirmed the common antigen specificity of Gram-negative bacteria Salmonella and H. pylori , and it was confirmed that the antibodies generated by different antigens reacted specifically to each antigen, resulting in the cross-reaction of each antigen and antibody.
암탉의 혈청(blood serum) 내의 IgG는 난황을 통하여 획득된 면역기능을 후대에 물려준다는 특성이 있고 난황을 이용할 경우에는 수율이 우수하며 계란은 일 상적으로 접하는 음식이라는 점에서 본 발명의 산업화상의 이점을 찾을 수 있다. 특히 본 발명에 따르면 항원의 공통 특이성을 이용함으로써 우수하고 효율적인 면역효과를 갖는 계란을 획득할 수 있다.The IgG in the blood serum of the hen is characterized by inheriting the immune function obtained through egg yolk to later generations, and when egg yolk is used, the yield is excellent and the egg is a food commonly encountered in the industrial aspect of the present invention. This can be found. In particular, according to the present invention, by using the common specificity of the antigen it is possible to obtain an egg having an excellent and efficient immune effect.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020070102483A KR100906257B1 (en) | 2007-10-11 | 2007-10-11 | The egg containing antibody IgY charicterized by using Salmonella and Helicobacter pylori antigen ? its production method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020070102483A KR100906257B1 (en) | 2007-10-11 | 2007-10-11 | The egg containing antibody IgY charicterized by using Salmonella and Helicobacter pylori antigen ? its production method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20090037072A true KR20090037072A (en) | 2009-04-15 |
| KR100906257B1 KR100906257B1 (en) | 2009-07-07 |
Family
ID=40761884
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020070102483A KR100906257B1 (en) | 2007-10-11 | 2007-10-11 | The egg containing antibody IgY charicterized by using Salmonella and Helicobacter pylori antigen ? its production method |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR100906257B1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111205367A (en) * | 2020-02-23 | 2020-05-29 | 珠海华敏医药科技有限公司 | Helicobacter pylori yolk antibody freeze-dried powder, preparation method and application preparation |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100364198B1 (en) * | 2000-06-09 | 2002-12-12 | 영농조합법인다한 | An Improved Method for Inducing IgY to Helicobacter pylori and an Egg Prepared by Using the Same |
| KR20020032772A (en) * | 2000-10-27 | 2002-05-04 | 김정우 | Egg yolk antibody against salmonella |
| KR100422074B1 (en) * | 2001-02-23 | 2004-03-10 | 주식회사 에그 바이오택 | The method for production of egg containing anti-E.coli IgY and anti-Helicobacter pylori IgY and anti-Salmonella enteritidis and Salmonella typhimurium simultaneously, and egg, yolk, yogurt and ice-cream containing specific IgY for anti-E.coli and anti-Helicobacter pylori and anti-Salmonella enteritidis and anti-Salmonella typhimurium |
| KR100548898B1 (en) * | 2003-07-22 | 2006-02-02 | 주식회사 단바이오텍 | Soft Boiled Egg Containing Egg Yolk Antibody |
-
2007
- 2007-10-11 KR KR1020070102483A patent/KR100906257B1/en not_active IP Right Cessation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111205367A (en) * | 2020-02-23 | 2020-05-29 | 珠海华敏医药科技有限公司 | Helicobacter pylori yolk antibody freeze-dried powder, preparation method and application preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| KR100906257B1 (en) | 2009-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200299360A1 (en) | Compositions and methods for treatment in broad-spectrum, undifferentiated or mixed clinical applications | |
| Porter et al. | Intestinal antibody secretion in the young pig in response to oral immunization with Escherichia coli | |
| Esmailnejad et al. | Production, purification, and evaluation of quail immunoglobulin Y against Salmonella typhimurium and Salmonella enteritidis | |
| KR100906257B1 (en) | The egg containing antibody IgY charicterized by using Salmonella and Helicobacter pylori antigen ? its production method | |
| KR20040083748A (en) | The method for production of spectific yolk antibody containing anti-helicobacter pylori IgY, anti-salmonella enteritidis IgY and anti-staphylococcus aureus IgY and have the anti-enzyme with anti-enzyme. | |
| RU2683027C2 (en) | Polyvalent combined immunizing and/or therapeutic preparation for use in bacterial infections or food poisoning, particularly salmonellosis, method for production of this preparation, its use and vaccine comprising this preparation | |
| Barman et al. | Protective efficacy of maternal antibodies induced by Salmonella toxoid (vaccine) | |
| Gordon et al. | Generation and characterization of IgY antibodies from Lohmann Brown hens immunized with Salmonella spp. for their subsequent application in nanotherapy | |
| KR100548898B1 (en) | Soft Boiled Egg Containing Egg Yolk Antibody | |
| Chae et al. | Effects of egg yolk antibodies produced in response to different antigenic fractions of E. coli O157: H7 on E. coli suppression | |
| KR100280331B1 (en) | Production of egg yolk antibody(anti-K88) against fimbrial antigen of E. coli strain K88 | |
| KR100280332B1 (en) | Production of egg yolk antibody(anti-K99) against fimbrial antigen of E. coli strain K99 | |
| KR100573424B1 (en) | Immune milk containing Helicobacter pylori antibody ? its manufacturing method | |
| KR100489615B1 (en) | Antibody For Prevention And Treatment Of Listeria Monocytogenes Infection, Eggs Containing Thereof And Method For Producing Thereof | |
| KR100280333B1 (en) | Production of egg yolk antibody(anti-K987P) against fimbrial antigen of E. coli strain K987P | |
| KR20020032772A (en) | Egg yolk antibody against salmonella | |
| KR101496937B1 (en) | Method for Separating Yolk Antibody against Pili Antigen of E. coli or Flagella Protein Antigen of Salmonella sp. | |
| Yousif et al. | Detection of specific antibodies of Escherichia coli O157: H7 isolated from children by using Eliza test | |
| OA17508A (en) | Compositions and methods for treatment in broad-spectrum, undifferentiated or mixed clinical applications. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant | ||
| LAPS | Lapse due to unpaid annual fee |