KR20010077206A - Monoclonal antibody specific for vp6 of porcine rotavirus and hybridoma cell line producing same - Google Patents
Monoclonal antibody specific for vp6 of porcine rotavirus and hybridoma cell line producing same Download PDFInfo
- Publication number
- KR20010077206A KR20010077206A KR1020000004851A KR20000004851A KR20010077206A KR 20010077206 A KR20010077206 A KR 20010077206A KR 1020000004851 A KR1020000004851 A KR 1020000004851A KR 20000004851 A KR20000004851 A KR 20000004851A KR 20010077206 A KR20010077206 A KR 20010077206A
- Authority
- KR
- South Korea
- Prior art keywords
- rotavirus
- monoclonal antibody
- cell line
- protein
- swine
- Prior art date
Links
- 241000702665 Porcine rotavirus Species 0.000 title claims abstract description 27
- 210000004408 hybridoma Anatomy 0.000 title 1
- 230000004927 fusion Effects 0.000 claims abstract description 34
- 241000702670 Rotavirus Species 0.000 claims description 50
- 241000282898 Sus scrofa Species 0.000 claims description 40
- 238000000034 method Methods 0.000 claims description 15
- 206010003445 Ascites Diseases 0.000 claims description 6
- 108700031314 Rotavirus VP6 Proteins 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 3
- 238000007912 intraperitoneal administration Methods 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- 210000000683 abdominal cavity Anatomy 0.000 abstract description 3
- 206010030113 Oedema Diseases 0.000 abstract 1
- 230000003187 abdominal effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 58
- 241000700605 Viruses Species 0.000 description 24
- 239000000243 solution Substances 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 12
- 239000000427 antigen Substances 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 239000002245 particle Substances 0.000 description 10
- 206010012735 Diarrhoea Diseases 0.000 description 9
- 239000008363 phosphate buffer Substances 0.000 description 9
- 241000282887 Suidae Species 0.000 description 8
- 238000003018 immunoassay Methods 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 206010035226 Plasma cell myeloma Diseases 0.000 description 6
- 230000007910 cell fusion Effects 0.000 description 6
- 201000000050 myeloid neoplasm Diseases 0.000 description 6
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 6
- 210000004988 splenocyte Anatomy 0.000 description 6
- 101710132601 Capsid protein Proteins 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 230000002458 infectious effect Effects 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 241000283707 Capra Species 0.000 description 4
- 208000005577 Gastroenteritis Diseases 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 229920002477 rna polymer Polymers 0.000 description 4
- 208000026775 severe diarrhea Diseases 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 102000013415 peroxidase activity proteins Human genes 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000004989 spleen cell Anatomy 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 102100034349 Integrase Human genes 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- 101710120037 Toxin CcdB Proteins 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 229940027941 immunoglobulin g Drugs 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000026267 regulation of growth Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- LVSPDZAGCBEQAV-UHFFFAOYSA-N 4-chloronaphthalen-1-ol Chemical compound C1=CC=C2C(O)=CC=C(Cl)C2=C1 LVSPDZAGCBEQAV-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000702658 Fijivirus Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241000617996 Human rotavirus Species 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 241000702259 Orbivirus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000702656 Phytoreovirus Species 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 241000702247 Reoviridae Species 0.000 description 1
- 241000702263 Reovirus sp. Species 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 206010067470 Rotavirus infection Diseases 0.000 description 1
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 1
- 101000980463 Treponema pallidum (strain Nichols) Chaperonin GroEL Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241001227561 Valgus Species 0.000 description 1
- 210000003969 blast cell Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/14—Reoviridae, e.g. rotavirus, bluetongue virus, Colorado tick fever virus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Public Health (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Analytical Chemistry (AREA)
- Veterinary Medicine (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
본 발명은 돼지 로타바이러스(porcine rotavirus)의 구조단백질인 VP6을 특이적으로 인식할 수 있는 단일클론 항체, 이를 분비하는 융합 세포주, 상기 융합 세포주를 이용하여 단일클론 항체를 생산하는 방법 및 상기 단일클론 항체를 이용한 돼지 로타바이러스의 검출방법에 관한 것이다.The present invention provides a monoclonal antibody that can specifically recognize VP6, a structural protein of porcine rotavirus, a fusion cell line secreting the same, a method for producing a monoclonal antibody using the fusion cell line, and the monoclonal The present invention relates to a method for detecting swine rotavirus using an antibody.
로타바이러스는 레오바이러스(Reovirus), 오르비바이러스(Orbivirus), 피토레오바이러스(Phytoreovirus), 피지바이러스(Fijivirus) 등과 함께 레오비리다에(Reoviridae)에 속하며, 혈청학적 분류에 의해 그룹 A에서 F까지 나뉘는데 그 중 그룹 A에 속하는 로타바이러스가 가장 광범위한 감염대상을 갖는 바이러스로서 사람, 원숭이, 말, 돼지, 개, 고양이, 토끼, 쥐, 소, 양 등에 감염되어 설사를 일으키며, 그룹 B에 속하는 로타바이러스는 기니아피그, 쥐 등을 숙주동물로 하며 이외의 정보는 아직 밝혀져 있지 않다. 로타바이러스는 동물에 감염시 심한 설사를 유발하는데, 사람 로타바이러스의 경우는 신생아와 유아에게서 심한 설사를 일으키고, 돼지 로타바이러스는 모든 연령의 돼지에게서 설사를 일으키며 특히 태어난지 5주 이내의 새끼 돼지에게 발병될 경우 치사율이 100%에 이르며 5주된 돼지 역시 90% 이상의 치사율을 나타낸다. 이 질병은 1963년 마엘베(Mahlerbe) 등에 의해 원숭이의 신장세포에서 배양증식이 가능하게 되었으나, 아직 그룹 B와 그룹 C의 로타바이러스는 실험실 배양방법이 정립되지 않은 상태이다. 이러한 로타바이러스에 의해 질병이 발생될 경우, 첫째, 새끼 돼지의 치사율이 매우 높고, 둘째, 적정한 치료대책이 없으며, 셋째, 바이러스에 대한 방역 대책이 어렵고, 넷째, 현재 개발된 백신들에 의한 방제효과가 낮다는 문제점이 있다.Rotavirus is Leo virus (Reovirus), ohreubi virus (Orbivirus), Pitot Leo virus (Phytoreovirus), Fiji belongs to the virus (Reoviridae) Leo birida together with (Fijivirus), divided in groups A by serological classification to F that Rotavirus, which belongs to group A, is the most widespread virus and infects humans, monkeys, horses, pigs, dogs, cats, rabbits, mice, cows, sheep, etc., and causes diarrhea. Pigs, mice, etc. as host animals, other information is not yet known. Rotavirus causes severe diarrhea when infected with animals, human rotavirus causes severe diarrhea in newborns and infants, swine rotavirus causes diarrhea in pigs of all ages, especially in piglets within 5 weeks of birth. The mortality rate is 100%, and the 5 week old pig also has a mortality rate of over 90%. The disease was allowed to grow in monkey kidney cells in 1963 by Mahlerbe et al., But the rotaviruses of group B and group C have yet to be established. When the disease is caused by rotavirus, firstly, the mortality rate of piglets is very high, secondly, there is no appropriate treatment measures, and thirdly, prevention measures against viruses are difficult, and fourth, the control effect by the vaccines currently developed. There is a problem that is low.
즉, 로타바이러스는 사람을 포함하여 여러 동물에서 장염을 유발하는 주요 병인으로서 공통적인 특징을 가지고, 입자의 크기는 직경이 70nm이며, 유전체는 이중나선의 리보핵산으로 구성되어 있으며, 특히 돼지 로타바이러스는 VP1, VP2,VP3, VP4, VP6 및 VP7을 주요 구조단백질로 하여 3중 구조로 구성되어 있다. 구체적으로, VP1과 VP3는 리보핵산을 보호하고 있고, VP2는 핵산을 둘러싸고 있는 코어(core)단백질의 주성분이며, VP6와 VP7은 각각 안쪽 및 바깥쪽의 외단백질을 구성하고 있다. VP4는 바이러스의 감염을 촉진하는 역할을 하며 VP7 단백질의 외부에 위치한다. 돼지 로타바이러스에 의한 돼지의 설사 질병을 감소시키는 가장 효과적인 예방방법은 돼지 로타바이러스 감염에 대한 조기 진단을 통해 방역 대책을 확립하는 것이다. 바이러스에 대한 진단방법은 바이러스를 인식하는 항체를 생산하고 이 항체를 이용하여 바이러스 감염을 측정하는 방법이 일반적으로 사용되고 있는데, 이 때 복합클론항체보다는 단일클론 항체를 이용하는 것이 특이성이 뛰어나다.In other words, rotavirus is a common etiology that causes enteritis in many animals including humans. The size of the particles is 70 nm in diameter, and the genome consists of double-stranded ribonucleic acid. Is composed of triple structure with VP1, VP2, VP3, VP4, VP6 and VP7 as main structural proteins. Specifically, VP1 and VP3 protect ribonucleic acid, VP2 is the main component of the core protein surrounding the nucleic acid, and VP6 and VP7 constitute the inner and outer outer proteins, respectively. VP4 promotes viral infection and is located outside of the VP7 protein. The most effective prophylactic method of reducing diarrheal disease in pigs by swine rotavirus is to establish anti-defective measures through early diagnosis of swine rotavirus infection. As a diagnostic method for a virus, a virus-aware antibody is produced and a method of measuring viral infection using the antibody is generally used. In this case, it is more specific to use a monoclonal antibody than a polyclonal antibody.
그런데 코어단백질은 바이러스 입자 내부에 위치하고 있어서 이에 대한 항체는 살아있는 바이러스 입자를 인식할 수 없으며, 외피 단백질에 대한 항체는 바이러스 입자의 인식이 가능하다. 외피 단백질 VP6, VP7 및 VP4 중 VP7 단백질은 로타바이러스의 아형(strain)에 따라 염기서열이 달라 서로 다른 에피토프(epitope)를 구성하고 있고, VP4 단백질은 로타바이러스가 완전한 형태의 구조일 경우에만 존재하는 단백질로서 이들에 대한 항체를 이용하는 것은 극히 제한되어 있다. 반면 VP6 단백질은 로타바이러스의 생성 또는 증식과정에서 가장 많이 나타나는 주외피 단백질로서 그룹 A에 속하는 로타바이러스에서 공통적인 항원구조를 갖는 바이러스 입자로 이에 대한 항체를 이용하는 것이 가장 유력한 방법이다. 구체적으로, VP6 단백질은 약 45 kDa 분자량을 갖는 단백질로 로타바이러스 아형에서 자주 변하지 않는 염기서열을 가지고 있으며 돼지 이외의 동물에 감염되는 로타바이러스의 VP6 단백질과도 동질성이 매우 높다. 따라서 VP6 단백질에 대한 단일클론 항체는 로타바이러스의 탐지에 매우 유용하게 이용될 수 있다.However, since the core protein is located inside the virus particle, the antibody against it cannot recognize the living virus particle, and the antibody against the envelope protein can recognize the virus particle. Among the coat proteins VP6, VP7, and VP4, the VP7 protein is composed of different epitopes due to different sequences of the rotavirus subtypes, and the VP4 protein is present only when the rotavirus has a complete structure. The use of antibodies against them as proteins is extremely limited. On the other hand, the VP6 protein is the most frequently expressed envelope protein during the production or propagation of rotavirus, and the most effective method is to use an antibody against the virus particles having a common antigen structure in rotavirus belonging to group A. Specifically, the VP6 protein is a protein having a molecular weight of about 45 kDa, which has a nucleotide sequence that does not change frequently in the rotavirus subtype, and is highly homologous to the VP6 protein of rotavirus that infects animals other than pigs. Therefore, monoclonal antibodies against VP6 protein can be very useful for the detection of rotavirus.
전염성 위장염 바이러스(1992년, Saif와 wesley) 및 돼지 유행성 설사증 바이러스(1981년, Pansaert 등)는 동일한 리보핵산으로 구성된 바이러스들로서 돼지 로타바이러스와 같이 돼지에 감염되어 심한 설사를 유발하는 바이러스들이다. 따라서, 이들 설사를 유발하는 바이러스들의 정확한 발병원인을 진단하여 감염원에 따른 백신을 개발하여야 하는데 아직 이들 바이러스와 로타바이러스를 구별하는 항체는 개발된 바 없다.Infectious gastroenteritis viruses (Saif and wesley in 1992) and swine pandemic diarrhea virus (Pansaert et al., 1981) are viruses composed of the same ribonucleic acid, which are swine-infected viruses such as swine rotavirus that cause severe diarrhea. Therefore, it is necessary to develop a vaccine according to an infectious agent by diagnosing the exact cause of viruses causing these diarrhea, but no antibody has yet been developed to distinguish between these viruses and rotaviruses.
이에 본 발명자들은 돼지에서 심한 설사를 유발하는 돼지 로타바이러스의 정확한 진단시스템을 확립하기 위하여, 돼지 로타바이러스 입자로 생쥐(Balb/c)를 면역시킨 후, 생쥐의 비장(spleen)세포를 분리하여 생쥐의 골수종 세포(myeloma)와 융합시킴으로써, 돼지 로타바이러스의 단백질에 대한 단일클론 항체를 생산하는 융합 세포주를 제조하고, 이 융합 세포주를 배양하여 그 배양액으로부터 돼지 로타바이러스에 높은 특이성을 갖는 생쥐 단일클론 항체를 대량으로 생산할 수 있었으며, 이 단일클론항체가 로타바이러스를 돼지 전염성 위장염 바이러스, 돼지 유행성 설사 바이러스와 구분할 수 있는 특성을 가지고 있음을 확인함으로써 본 발명을 완성하게 되었다.In order to establish an accurate diagnosis system for swine rotavirus that causes severe diarrhea in pigs, the present inventors immunized mice (Balb / c) with swine rotavirus particles, and then separated spleen cells of mice. A fusion cell line producing a monoclonal antibody against the protein of porcine rotavirus by fusion with myeloma of the mouse, and culturing the fusion cell line and murine monoclonal antibody having high specificity for porcine rotavirus from the culture The present invention was completed by confirming that the monoclonal antibody has the characteristics of distinguishing rotavirus from the swine infectious gastroenteritis virus and the swine epidemic diarrhea virus.
본 발명의 목적은 돼지 로타바이러스 VP6 단백질을 특이적으로 인식하는 단일클론 항체, 이를 생산할 수 있는 융합 세포주 및 상기 융합 세포주를 배양하여 상기 단일클론 항체를 대량 생산하는 방법을 제공하는 것이다.An object of the present invention is to provide a monoclonal antibody that specifically recognizes swine rotavirus VP6 protein, a fusion cell line capable of producing the same, and a method for mass production of the monoclonal antibody by culturing the fusion cell line.
본 발명의 다른 목적은 상기 단일클론 항체를 이용하여 돼지 로타바이러스를 검출하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for detecting porcine rotavirus using the monoclonal antibody.
도 1은 본 발명의 단일클론 항체의 돼지 로타바이러스(porcine rotavirus)에 대한 특이성을 SDS-폴리아크릴아마이드 젤 전기영동으로 확인한 결과이다.1 is a result of confirming the specificity for porcine rotavirus of the monoclonal antibody of the present invention by SDS-polyacrylamide gel electrophoresis.
도 2는 본 발명의 단일클론 항체의 돼지 로타바이러스에 대한 특이성을 면역 블랏팅으로 확인한 결과를 나타낸 것이다.Figure 2 shows the results confirmed by immunoblotting the specificity for the swine rotavirus of the monoclonal antibody of the present invention.
도 3은 본 발명의 단일클론 항체의 돼지 로타바이러스에 대한 특이도를 효소면역학 방법으로 검증한 결과를 나타낸다.Figure 3 shows the results of verifying the specificity for the swine rotavirus of the monoclonal antibody of the present invention by the enzyme immunoassay method.
상기 목적을 달성하기 위하여, 본 발명에서는 생쥐 유래의 융합 세포주 NS1-RIO(KCTC 0654BP)에 의해 생산되고, 돼지 로타바이러스의 VP6 단백질에 특이적으로 결합하는 단일클론 항체 및 이를 생산하는 융합 세포주 NS1-RIO(KCTC 0654BP)를 제공한다.In order to achieve the above object, in the present invention, a monoclonal antibody produced by the mouse-derived fusion cell line NS1-RIO (KCTC 0654BP) and specifically binding to the VP6 protein of porcine rotavirus and the fusion cell line NS1-producing the same RIO (KCTC 0654BP) is available.
또한 본 발명에서는 상기 융합 세포주를 생쥐의 복강내로 주사한 후 복수액을 채취하고, 그로부터 돼지 로타바이러스에 대한 단일클론 항체를 분리하는 것을 포함하는, 돼지 로타바이러스에 대한 단일클론 항체를 생산하는 방법 및 상기 단일클론 항체를 이용하는 것을 특징으로 하는, 돼지 로타바이러스의 검출방법을 제공한다.In addition, the present invention comprises the method of producing a monoclonal antibody against swine rotavirus, comprising injecting the fusion cell line intraperitoneally of the mouse and then collecting ascites fluid and isolating the monoclonal antibody against swine rotavirus therefrom; Provided is a method for detecting swine rotavirus, characterized in that using the monoclonal antibody.
본 발명의 항체는 다음과 같이 제조할 수 있다. 즉, MA104 세포(돼지 췌장세포주, 입수처: 충북대 수의학과)를 우태아 혈청(fetal bovine serum)이 함유된 최소배지에서 배양한 다음, 돼지 로타바이러스를 MA104 세포에 감염시켜서 배양 플라스크에서 세포가 90% 이상 분리되었을 때 배양을 중지하고 배양 상등액을 원심분리하여 돼지 로타바이러스를 분리한다. 분리된 돼지 로타바이러스 입자를 인산완충액에 현탁시키고 동량의 프로인드 완전 보조항원(complete Freund adjuvant) 용액과 혼합하여 생쥐에 면역시키고, 면역된 비장세포와 골수종 세포를 융합시켜 융합 세포주(hybridoma cell line)를 얻은 다음, 돼지 로타바이러스 항원에만 특이적으로 반응하는 융합 세포주를 효소 면역 분석방법으로 선별한다.The antibody of the present invention can be prepared as follows. That is, MA104 cells (porcine pancreatic cell line, obtained from Chungbuk National University) were cultured in a minimal medium containing fetal bovine serum, and then infected with porcine rotavirus to MA104 cells, which resulted in 90 When more than% is isolated, stop the culture and centrifuge the culture supernatant to isolate swine rotavirus. The isolated porcine rotavirus particles were suspended in phosphate buffer and mixed with the same amount of complete Freund adjuvant solution to immunize mice, and the fused cell lines were fused by fusing the immunized splenocytes and myeloma cells. Next, the fusion cell lines that specifically react only with the swine rotavirus antigen are selected by enzyme immunoassay.
생쥐의 면역은 통상적인 방법에 따라 수행한다. 즉, 항원으로서의 돼지 로타바이러스를 복강내, 정맥내, 피하내로 1차 투여하고, 14 내지 21일 후 2차 투여한 후, 또 한 번 항원을 투여하여, 총 투여량이 생쥐당 5 내지 20㎍이 되도록 한다. 통상적인 보조항원으로 프로인드 완전 보조항원(complete Freund adjuvant)과 불완전 보조항원을 필요에 따라 적절하게 사용할 수 있다. 면역 항원의 최종 투여 후 3 내지 4 일째에 비장세포를 수득하여 면역세포로 사용한다. 이와 세포 융합을 시키기 위하여 세포융합 모세포, 예를 들어 SP2/0·Ag14 골수종 세포(myeloma cell)를 사용한다.Immunization of mice is performed according to conventional methods. In other words, porcine rotavirus as an antigen was first administered intraperitoneally, intravenously, subcutaneously, secondly after 14 to 21 days, and then another antigen was administered. Be sure to As a common auxiliary antigen, complete Freund adjuvant and incomplete auxiliary antigen can be used as appropriate. Splenocytes are obtained 3 to 4 days after the final administration of the immune antigen and used as immune cells. Cell fusion blast cells, for example SP2 / 0 · Ag14 myeloma cells, are used for cell fusion.
면역된 비장 세포들과 골수종 세포 사이의 세포 융합은, 예를 들면 문헌(Kohler G & Milstein C.,Nature,256, 495)과 같은 공지된 방법들 또는 이들을 변형한 방법들에 의하여 수행된다. 면역된 비장 세포들과 SP2/0·Ag14 골수종 세포를 20 : 1 내지 5 : 1의 비율, 바람직하게는 10 : 1로 혼합하고, 원심분리하여 융합한다. 융합된 세포는 HAT 배지와 같은 통상적인 선별 배지에서 배양하여 융합되지 않은 세포들을 소멸시키고, 융합된 세포들을 선별적으로 자라게 하여 분리할수 있다.Cell fusion between immunized spleen cells and myeloma cells is carried out by known methods such as, for example, Kohler G & Milstein C., Nature , 256 , 495 or modified methods thereof. Immunized spleen cells and SP2 / 0 · Ag14 myeloma cells are mixed at a ratio of 20: 1 to 5: 1, preferably 10: 1, and centrifuged to fuse. The fused cells can be isolated by culturing in a conventional selection medium such as HAT medium to destroy unfused cells and selectively growing the fused cells.
이렇게 하여 얻어진 융합세포는 공지의 제한 희석법(Current Protocols in Immunology)으로 처리하여 원하는 항체를 생산하는 개개의 클론을 획득하여, 결국 단일클론 항체를 생산할 수 있게 된다. 상기에서 선별된 융합 세포주를 "NS1-R1O"라 명명하고 이를 생명공학연구소 유전자은행에 1999년 8월 19일자로 기탁번호 제 KCTC 0654BP 호로서 기탁하였다.The fusion cells thus obtained can be treated with known restriction protocols (Current Protocols in Immunology) to obtain individual clones that produce the desired antibodies, resulting in the production of monoclonal antibodies. The selected fusion cell line was named "NS1-R1O" and was deposited with the Biotechnology Research Institute Gene Bank on August 19, 1999 as Accession No. KCTC 0654BP.
원하는 항체를 생산하는 융합세포의 클론은 효소 면역 측정법(Enzyme immunoassay)에 의해 검색할 수 있다.Clones of fusion cells producing the desired antibody can be searched by enzyme immunoassay.
본 발명의 단일클론 항체의 대량생산은 다음과 같이 이루어질 수 있다. 미리 0.5㎖의 프리스탄(pristane; 2,6,10,14-tetramethyl pentadecane)을 주사한 생쥐의 복강에 융합 세포주를 5 x 106세포/마리가 되도록 주사하고 약 10일 후 복수를 채취한 다음 원심분리하여 복수 상층액을 얻어서 프로테인-지 비드(Protain-G bwad,Sigma co.)가 충전되어 있는 유리관(1×10cm)을 통과시키고 글리신-에치시엘 (0.1M Glycine-HCl. pH 2.7)용액으로 항체를 용리하여 수득할 수 있다.Mass production of the monoclonal antibody of the present invention can be carried out as follows. Inject the fusion cell line into 5 x 10 6 cells / colum in the abdominal cavity of mice injected with 0.5 ml of pristane (2,6,10,14-tetramethyl pentadecane) in advance and harvest ascites approximately 10 days later. Centrifugation yielded a plurality of supernatants, passed through a glass tube (1 × 10 cm) filled with Protein-G bwad (Sigma co.), And glycine-etchiel (0.1 M Glycine-HCl. PH 2.7) solution. It can be obtained by eluting the antibody.
본 발명에서 확립한 세포주가 분비하는 돼지 로타바이러스에 대한 단일클론 항체는 항체의 아형이 면역글로블린 G3타입(IgG3)이며, 웨스턴 블랏팅 방법으로 확인한 결과 로타바이러스의 VP6 단백질을 특이적으로 인지하는 항체로서 효소면역측정법으로 항체의 특이성을 확인한 결과, 돼지에 설사질환을 유발하는 로타바이러스와 같이 리보핵산으로 구성되어 있는 바이러스들인 돼지 전염성 위장염 바이러스,돼지 유행성 설사증 바이러스와는 반응하지 않고 돼지 로타바이러스에 대해 높은 특이성을 나타낸다.In the monoclonal antibody against swine rotavirus secreted by the cell line established in the present invention, the subtype of the antibody is immunoglobulin G 3 type (IgG 3 ), and it is confirmed by Western blotting to specifically recognize the VP6 protein of rotavirus. As a result of confirming the specificity of the antibody by enzyme-immunoassay, the pig rotavirus does not react with swine infectious gastroenteritis virus and swine epidemic diarrhea virus, which are composed of ribonucleic acid such as rotavirus that causes diarrheal disease in pig High specificity for.
본 발명의 단일클론 항체를 이용하면 돼지의 분비물, 분변, 혈액으로부터 돼지 로타바이러스를 특이적으로 검출함으로써 돼지 로타바이러스의 감염여부를 정확하고 신속하게 진단할 수 있으므로 이의 전파경로 확인과 조기발견에 의한 방역효과도 높일 수 있으며, 포유 동물 세포의 생리활성, 분화 및 성장조절을 연구하는 기초 생물학 연구의 좋은 수단으로 이용될 수 있다.By using the monoclonal antibody of the present invention, specific detection of swine rotavirus from pig secretions, feces, and blood can accurately and quickly diagnose whether swine rotavirus is infected. It can also be used as a good means of basic biology research to study the physiological activity, differentiation and growth regulation of mammalian cells.
이하 본 발명을 하기 실시예에 의하여 더욱 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.
실시예 1: 돼지 로타바이러스에 대한 단일클론 항체를 생산하는 융합 세포주의 제조Example 1 Preparation of Fusion Cell Lines Producing Monoclonal Antibodies to Porcine Rotavirus
(단계 1) 돼지 로타바이러스의 배양 및 증식(Step 1) cultivation and propagation of porcine rotavirus
MA104 세포(돼지 췌장세포주, 입수처: 충북대 수의학과)를 10% 소태아 혈청(fetal bovine serum)이 포함된 최소배지가 들어 있는 T75 플라스크에 접종하여 플라스크 바닥에 세포가 70 내지 90%로 채워질 때까지 배양하였다. 배양된 세포를 생리식염수로 2회 세척하고 1㎖의 돼지 로타바이러스 용액(100 c.f.u)을 넣고 1 시간 동안 감염시킨 다음 20㎖의 최소배지(50㎍/ℓ 트립신이 포함된 배지)를 첨가하고 16 시간 동안 배양하였다.When MA104 cells (porcine pancreatic cell line, obtained from Chungbuk National University) are inoculated into a T75 flask containing a minimum medium containing 10% fetal bovine serum, and the bottom of the flask is filled with 70 to 90% of cells. Incubated until. The cultured cells were washed twice with physiological saline, inoculated with 1 ml of pig rotavirus solution (100 cfu) and infected for 1 hour, followed by the addition of 20 ml of medium (50 µg / l trypsin). Incubated for hours.
(단계 2) 돼지 로타바이러스의 분리(Step 2) Isolation of Porcine Rotavirus
상기 단계 1의 로타바이러스가 포함된 배지용액을 110,000 x g로 1 시간 동안 원심분리한 후 바이러스 침전물을 200㎕의 생리식염수에 용해시켰다. 이 용액을 60% 당이 포함된 인산완충액 위에 천천히 부은 다음, 130,000 x g로 2 시간 30분 동안 원심분리하여 바이러스 밴드를 용리한 후, 인산완충액에 현탁시키고 다시 130,000 x g로 2 시간 30분 동안 원심분리하여 정제된 돼지 로타바이러스 침전물을 수득하였다.After centrifuging the rotavirus medium of step 1 containing 110,000 x g for 1 hour, the virus precipitate was dissolved in 200 µl of saline. Slowly pour this solution onto the phosphate buffer containing 60% sugar, then centrifuge at 130,000 x g for 2 hours 30 minutes to elute the virus bands, then suspend in phosphate buffer and again. Centrifugation at 130,000 x g for 2 hours 30 minutes yielded purified swine rotavirus precipitate.
(단계 3) 생쥐의 면역화(Step 3) Immunization of Mice
융합 세포주의 개발에 필요한 면역화된 생쥐를 얻기 위하여, 상기 단계 2에서 분리된 돼지 로타바이러스 입자(20ug)를 포함하는 인산완충액과 동량의 프로인드 완전 보조항원(complete Freund adjuvant)를 유상화가 될 때까지 잘 혼합한 다음, 생후 6∼8 주된 발브씨(Balb/c) 생쥐의 복강내에 주사하였다. 2 주 후 처음 주사의 경우와 같은 양의 로타바이러스 입자를 포함하는 인산완충액을 프로인드 불완전 보조항원과 혼합하여 동일부위에 주사하였다. 이어서, 4 내지 5일 후에 생쥐의 꼬리부위 혈관에서 채혈한 혈액에서 혈청을 분리하여 항체가를 확인한 후 세포융합을 하기 3 내지 4일 전에 10㎍의 돼지 로타바이러스 입자를 포함하는 0.85% 생리식염수 10ug를 정맥 및 복강내에 주사하였다.In order to obtain the immunized mice required for the development of the fusion cell line, the phosphate buffer containing porcine rotavirus particles (20 ug) isolated in step 2 and the same amount of complete Freund adjuvant until emulsified The mixture was mixed well and then injected intraperitoneally in 6-8 week old Balb / c mice. Two weeks later, the same phosphate buffer containing rotavirus particles in the same amount as the first injection was mixed with Freund's incomplete auxiliary antigen. Subsequently, serum was separated from blood collected from the blood vessels of the tail of the mouse after 4 to 5 days, and the antibody titer was confirmed. Then, 3 to 4 days before the cell fusion, 10 ug of 0.85% saline containing 10 μg of pig rotavirus particles Was injected intravenously and intraperitoneally.
(단계 4) 세포 융합 및 배양(Step 4) Cell Fusion and Culture
항원으로 마지막 면역한 지 3 내지 4일 후에 면역된 생쥐를 에테르로 마취한 후 몸통의 좌측에 위치한 비장(spleen)을 꺼내서 조직분쇄기(tissue homogenizer)로 곱게 갈아서 한스완충액(Hans' buffered saline solution, HBSS)를 이용하여 현탁액을 만들고, 이 때 얻어진 비장세포를 15㎖의 원심분리관에 넣어서 원심분리하였다. 이 과정을 2회 반복하여 비장세포를 HBSS로 충분히 세척하였다.Three to four days after the last immunization with the antigen, anesthetized mice with ether, and the spleen located on the left side of the body were taken out, and finely ground with a tissue homogenizer, a Hans' buffered saline solution (HBSS). ) To make a suspension, and the splenocytes obtained at this time were put into a 15 ml centrifuge tube and centrifuged. This procedure was repeated twice to wash the splenocytes sufficiently with HBSS.
한편, 세포융합의 모세포(parent cell)로서 SP2/0·Ag14 세포주를 최대 밀도를 5 x 105/㎖ 정도로 유지시키면서 10% 우태아 혈청(FBS)을 함유하는 RPMI 배지에서 배양하였다. 모세포인 SP2/0·Ag14도 HBSS 완충액을 이용하여 2회 원심분리하여 세척하였다.On the other hand, SP2 / 0.Ag14 cell line as a parent cell for cell fusion was cultured in RPMI medium containing 10% fetal calf serum (FBS) while maintaining a maximum density of about 5 x 10 5 / ml. The parental cells, SP2 / 0.Ag14, were also washed by centrifugation twice with HBSS buffer.
이어서, 비장세포와 모세포를 각각 10㎖가 되도록 HBSS 완충액에 다시 현탁시킨 후 각 현탁액 내의 세포 수를 세어서 생쥐의 비장세포 1 x 108개와 SP2/0·Ag14 골수종 세포 1 x 107개가 되도록 두 현탁액을 50㎖의 원심분리관에서 섞은 다음 다시 원심분리하여 침전시켰다. 원심분리관 내의 침전물을 손가락으로 가볍게 두드려서 분산시키고, 37℃에 1분간 정치시킨 후 HBSS 완충액에 들어있는 45%(w/v) 폴리에틸렌글리콜(poly(ethylene glycol), PEG)-5%(w/v) DMSO의 혼합액 1㎖를 1분 동안 천천히 가하고 다시 1분간 약간 흔들어 주었다. 그 후 배양용 배지(RPMI)를 첨가하고 이 현탁액을 다시 원심분리한 후 세포 펠렛(pellet)을 HAT배지에 1∼2 x 105/㎖ 정도로 재현탁시키고, 96웰(well) 마이크로 타이터 플레이트(microtiter plate)에 0.2㎖씩 분주한 후 온도가 37℃로 조정된 CO2습도 배양기 안에서 배양하였다.Then, the cells were resuspended in HBSS buffer so that the splenocytes and the parental cells were each 10 ml, and the cells in each suspension were counted to obtain 1 x 10 8 splenocytes and 1 x 10 7 SP2 / 0.Ag14 myeloma cells. The suspension was mixed in 50 ml centrifuge tubes and then centrifuged again to precipitate. The precipitate in the centrifuge tube was dispersed by tapping lightly with a finger and allowed to stand at 37 ° C. for 1 minute and then 45% (w / v) polyethylene glycol (PE) -5% (w / v) contained in HBSS buffer. v) 1 ml of the mixed solution of DMSO was slowly added for 1 minute and shaken slightly for 1 minute. Thereafter, culture medium (RPMI) was added and the suspension was centrifuged again, and the cell pellet was resuspended in HAT medium at 1-2 × 10 5 / ml, and the 96 well micro titer plate was used. 0.2ml aliquots were placed on a microtiter plate and incubated in a CO 2 humidity incubator whose temperature was adjusted to 37 ° C.
(단계 5) 단일클론 항체를 생산하는 융합 세포의 선별(Step 5) Selection of Fusion Cells Producing Monoclonal Antibodies
융합 세포의 선별은 돼지 로타바이러스를 코팅한 마이크로플레이트를 이용한 효소 면역 측정법(Enzyme immunoassay)으로 수행하였다. 마이크로플레이트에 돼지 전염성 로타바이러스 항원을 각 웰(well)당 50㎕(2㎍/㎖)씩 가하여 플레이트 표면에 부착시키고, 반응하지 않는 항원은 인산 완충용액-트윈 20(PBST) 용액으로 세척하여 제거하였다. 융합 세포 배양액을 각 웰에 50㎕씩 가하여 1 시간 동안 반응시킨 후 인산 완충용액-트윈 20 용액으로 충분히 세척하여 반응하지 않은 배양액을 제거하였다. 여기에 염소 항-생쥐 IgG-호스래디쉬 퍼옥시다제(goat anti-mouse IgG-HRP, Sigma co.)를 가하여 1 시간 동안 실온에서 반응시킨 다음, PBST 용액으로 충분히 세척하였다. 이어서 퍼옥시다제의 기질용액(OPD)을 넣어 반응시키고, 그 반응정도를 효소면역 해독기(ELISA reader)로 492nm 파장에서 흡광도를 측정하였다.Selection of the fusion cells was carried out by enzyme immunoassay (Enzyme immunoassay) using a microplate coated with porcine rotavirus. 50 μl (2 μg / ml) of porcine infectious rotavirus antigen was added to the microplate and attached to the plate surface. The unreacted antigen was removed by washing with phosphate buffer-Tween 20 (PBST) solution. It was. 50 µl of the fusion cell culture was added to each well and allowed to react for 1 hour, followed by washing sufficiently with phosphate buffer-Tween 20 solution to remove unreacted culture. Goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP, Sigma co.) Was added thereto and reacted at room temperature for 1 hour, followed by washing with PBST solution sufficiently. Subsequently, the substrate solution (OPD) of peroxidase was added and reacted, and the degree of reaction was measured at 492 nm using an ELISA reader.
이어서, 돼지 로타바이러스 항원과 특이적으로 높은 결합력을 갖는 항체를 생산하는 융합 세포주군을 먼저 선별한 다음, 단일클론을 제한 희석(limiting dilution)하여 단일클론 항체를 생성하는 단일융합 세포주를 선별하여 4개의 클론을 얻었다. 이들 클론중 가장 높은 역가를 나타내는 클론을 선택하여 동결보존하였고 이중 면역 확산법으로 클론이 분비하는 항체의 아형이 면역글로블린 G3타입(IgG3)임을 확인하였다. 이 융합 세포주를 "NS1-R10"로 명명하고 생명공학연구소 유전자은행에 기탁번호 제 KCTC 0654BP 호로서 1999년 8월 19일자로 기탁하였다.Subsequently, a group of fusion cell lines that produce antibodies having a specifically high binding capacity with the swine rotavirus antigen is selected first, followed by a selection of monofusion cell lines that produce monoclonal antibodies by limiting dilution of monoclonal antibodies. Clones were obtained. The clones showing the highest titers among these clones were selected and cryopreserved, and the double immunodiffusion method confirmed that the subtype of the antibody secreted by the clone was immunoglobulin G 3 type (IgG 3 ). This fusion cell line was named "NS1-R10" and was deposited on August 19, 1999 as Accession No. KCTC 0654BP to the Bank of Biotechnology.
실시예 2: 돼지 로타바이러스에 특이적인 단일클론 항체의 대량생산Example 2 Mass Production of Monoclonal Antibodies Specific to Porcine Rotavirus
상기 실시예 1에서 확립한 융합 세포주로부터 돼지 로타바이러스에 대한 단일클론 항체를 대량으로 생산하기 위하여, 우선 발브씨 생쥐 한 마리당 0.5㎖의 프리스탄(pristane)을 복강내로 주사하였다. 1 주일 후 상기 실시예 1에서 수득한 융합 세포주를 생쥐 1 마리당 5 x 106개씩 주사하고, 복강이 부풀어 오른 생쥐에서 복수액을 채취하였다. 이 복수액은 높은 농도로 자란 융합세포를 포함하고 있으므로, 채취된 복수액을 10,000 x g로 원심분리하여 융합 세포를 침전시킨 후 프로테인-지 유리관(1×10cm)을 통과시키고 글리신 에치시엘 용액으로 항체를 용리하고 50배의 인산완충액에 16시간 투석한 다음 원심분리하여 상등액을 -20℃에서 보관하였다.In order to mass produce monoclonal antibodies against swine rotavirus from the fusion cell line established in Example 1 above, 0.5 ml of pristane was injected intraperitoneally per valgus mice. One week later, 5 x 10 6 cells were injected per mouse into the fusion cell line obtained in Example 1, and ascites fluid was collected from the inflated abdominal cavity. Since the ascites solution contained fusion cells grown at high concentrations, the ascites solution was centrifuged at 10,000 xg to precipitate the fusion cells, which were then passed through a Protein-G glass tube (1 × 10 cm) and the antibody was used as a glycine etchiel solution. Was eluted and dialyzed in 50-fold phosphate buffer for 16 hours and then centrifuged to store the supernatant at -20 ° C.
실시예 3: 웨스턴 블랏팅에 의한 돼지 로타바이러스에 특이적인 단일클론 항체의 특이성 검정Example 3: Specificity Assay of Monoclonal Antibodies Specific to Porcine Rotavirus by Western Blotting
(단계 1) SDS-폴리아크릴아미드 겔 전기영동(Step 1) SDS-polyacrylamide gel electrophoresis
돼지 로타바이러스로 MA104 세포를 감염시키고 인산 완충용액-트윈 20(PBST) 용액(0.05% 트윈 20)으로 희석시킨 후, 단백질 농도를 측정하여 10% SDS-폴리아크릴아마이드 젤에 전기영동하여 그 결과를 도 1에 나타내었다. 비교군으로서 비감염 MA104 세포를 사용하였다. 도 1에서 제 M 열은 표준분자 단백질이고, 제 1 열은 비감염 MA104 세포이고, 제 2 열은 로타바이러스 감염 MA104 세포이다.After infecting MA104 cells with porcine rotavirus and diluting with phosphate buffer-Tween 20 (PBST) solution (0.05% Tween 20), the protein concentration was measured and electrophoresed on 10% SDS-polyacrylamide gel. 1 is shown. Uninfected MA104 cells were used as a comparative group. In Figure 1, row M is the standard molecular protein, row 1 is uninfected MA104 cells, and row 2 is rotavirus infected MA104 cells.
(단계 2) 웨스턴 블랏팅 검정(Step 2) Western Blotting Assay
상기 단계 1에서 젤 속에 분리된 단백질을 니트로셀룰로즈 막에 전기적인 방법으로 옮겼다. 막에 옮긴 단백질의 비특이적인 반응을 줄이기 위해 니트로셀룰로즈 막에 3% 소혈청 알부민 용액을 12 내지 14 시간 동안 처리하였다. 돼지 로타바이러스에 특이적으로 반응하는 단일클론 항체를 확인하기 위하여, 본 발명의 NS1-R10 단일클론 항체를 가하여 실온에서 2 시간 동안 반응시켰다. 이 니트로셀룰로즈 막을 0.5% 트윈이 포함된 인산 완충액으로 3회 세척한 다음 염소 항-생쥐 IgG-호스래디쉬 퍼옥시다제(goat anti-mouse IgG-HRP, 입수처:Sigma co.)를 사용하여 실온에서 반응시키고, 0.5% 트윈이 포함된 인산 완충액으로 세척한 후 0.018%(v/v) 4-클로로-1-나프톨과 0.045%(v/v) H2O2가 인산 완충용액과 메틸알콜의 혼합용액에 용해된 기질을 이용하여 발색반응시켰다.The protein separated in the gel in step 1 was transferred to the nitrocellulose membrane by electric method. Nitrocellulose membranes were treated with 3% bovine albumin solution for 12-14 hours to reduce the nonspecific response of the proteins transferred to the membrane. In order to identify monoclonal antibodies that specifically respond to porcine rotavirus, NS1-R10 monoclonal antibodies of the present invention were added and reacted for 2 hours at room temperature. This nitrocellulose membrane was washed three times with phosphate buffer containing 0.5% Tween followed by goat anti-mouse IgG-HRP (goat anti-mouse IgG-HRP, obtained from Sigma co.). And phosphate buffer containing 0.5% Tween and 0.018% (v / v) 4-chloro-1-naphthol and 0.045% (v / v) H 2 O 2 were dissolved in phosphate buffer and methyl alcohol. Color reaction was carried out using a substrate dissolved in a mixed solution.
그 결과를 도 2에 나타내었다. 여기에서, 제 M 열은 표준분자 단백질이고,제 1 열은 비감염 MA104 세포이고, 제 2 열은 로타바이러스 감염 MA104 세포로서, 본 발명의 단일클론 항체가 돼지 로타바이러스로 감염된 세포의 단백질중 45 kDa 분자량인 VP6 단백질에 특이적으로 반응하는 것을 확인하였다.The results are shown in FIG. Here, column M is a standard molecular protein, column 1 is an uninfected MA104 cell, and column 2 is a rotavirus infected MA104 cell, wherein the monoclonal antibody of the present invention is 45 kDa in the protein of a cell infected with porcine rotavirus. It was confirmed that the compound specifically reacts with the molecular weight of the VP6 protein.
실시예 4: 효소면역분석방법에 의한 단일클론 항체의 돼지 로타바이러스에 대한 특이성 검정Example 4: Specificity Assay for Swine Rotavirus of Monoclonal Antibodies by Enzyme Immunoassay
돼지에 감염하여 설사를 유발하는 돼지 로타바이러스, 돼지 전염성 위장염 바이러스(TGEV, transmissible gatroenteritis virus) 및 돼지 유행성 설사바이러스(PEDV, porcine epidemic diarrhea virus)를 마이크로플레이트에 웰(well)당 50㎕(2㎍/㎖)씩 가하여 플레이트 표면에 부착시키고, 반응하지 않은 항원은 세척하여 제거하였다. 실시예 1에서 수득한 융합 세포 배양액을 각 웰에 50㎕를 가하여 1 시간 동안 반응시킨 후 인산 완충용액-트윈 20(PBST) 용액으로 충분히 세척하였다. 여기에 항-생쥐 IgG 호스래디쉬 퍼옥시다제를 가하여 1 시간 동안 실온에서 반응시킨 다음, PBST 용액으로 충분히 세척하였다. 이어서 퍼옥시다제의 기질용액을 반응시키고, 그 반응정도는 효소면역 측정기로 492nm 파장에서 흡광도를 측정하였다.Porcine rotavirus, swine infectious gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) that infect pigs and cause diarrhea in microplates 50 μl (2 μg) per well / Ml) was added to the plate surface and unreacted antigens were washed away. 50 μl of the fusion cell culture obtained in Example 1 was added to each well, followed by reaction for 1 hour, followed by sufficient washing with phosphate buffer-Tween 20 (PBST) solution. Anti-mouse IgG horseradish peroxidase was added thereto and reacted at room temperature for 1 hour, followed by sufficient washing with PBST solution. Subsequently, the substrate solution of peroxidase was reacted, and the degree of reaction was measured at 492 nm with an enzyme immunoassay.
그 결과를 도 3에 나타내었으며, 여기에서 보듯이 본 발명의 단일클론 항체는 돼지 로타바이러스 VP6 단백질에만 특이적으로 반응하는 항체임을 확인할 수 있다.The results are shown in FIG. 3, and as shown here, the monoclonal antibody of the present invention can be confirmed to be an antibody that specifically reacts only with swine rotavirus VP6 protein.
이와 같이 본 발명의 로타바이러스 VP6 단백질을 특이적으로 인식하는 단일클론 항체와 이를 생산하는 융합 세포주를 이용하면 돼지의 분비물 샘플검사에서 돼지 로타바이러스의 감염여부를 정확하고 신속하게 진단할 수 있어 로타바이러스의 전파경로 확인과 조기발견에 의한 방역효과도 높일 수 있으며, 본 발명의 항체는 돼지 이외의 포유 동물의 로타바이러스에 대해 특이적인 반응성을 갖고 있어 포유 동물 세포의 생리활성, 분화 및 성장조절을 연구하는 기초 생물학 연구의 좋은 수단으로 이용될 수 있다.Thus, using the monoclonal antibody that specifically recognizes the rotavirus VP6 protein of the present invention and the fusion cell line producing the same, it is possible to accurately and quickly diagnose the infection of swine rotavirus in the test of the secretion of swine from rotavirus. It is possible to enhance the prevention effect by confirming the propagation path and early detection, and the antibody of the present invention has a specific reactivity against rotavirus in mammals other than pigs to study the physiological activity, differentiation and growth regulation of mammalian cells. It can be used as a good means of basic biology research.
Claims (5)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2000-0004851A KR100369533B1 (en) | 2000-02-01 | 2000-02-01 | Monoclonal antibody specific for vp6 of porcine rotavirus and hybridoma cell line producing same |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR10-2000-0004851A KR100369533B1 (en) | 2000-02-01 | 2000-02-01 | Monoclonal antibody specific for vp6 of porcine rotavirus and hybridoma cell line producing same |
Publications (2)
Publication Number | Publication Date |
---|---|
KR20010077206A true KR20010077206A (en) | 2001-08-17 |
KR100369533B1 KR100369533B1 (en) | 2003-01-30 |
Family
ID=19643338
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
KR10-2000-0004851A KR100369533B1 (en) | 2000-02-01 | 2000-02-01 | Monoclonal antibody specific for vp6 of porcine rotavirus and hybridoma cell line producing same |
Country Status (1)
Country | Link |
---|---|
KR (1) | KR100369533B1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100460709B1 (en) * | 2001-08-14 | 2004-12-09 | 네오바이오다임 주식회사 | A monoclonal antibody against a porcine rotavirus, a hybridoma cell line producing it, a method of immunoassay using it and a kit |
CN110470829A (en) * | 2019-09-07 | 2019-11-19 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | A kind of 6 gene of the rotavirus vp that amino acid sequence morphs and immuno-chromatographic test paper strip |
CN113817054A (en) * | 2021-10-09 | 2021-12-21 | 武汉科前生物股份有限公司 | Mouse monoclonal antibody 5B11 specifically binding porcine rotavirus VP6 protein and application thereof |
-
2000
- 2000-02-01 KR KR10-2000-0004851A patent/KR100369533B1/en not_active IP Right Cessation
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100460709B1 (en) * | 2001-08-14 | 2004-12-09 | 네오바이오다임 주식회사 | A monoclonal antibody against a porcine rotavirus, a hybridoma cell line producing it, a method of immunoassay using it and a kit |
CN110470829A (en) * | 2019-09-07 | 2019-11-19 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | A kind of 6 gene of the rotavirus vp that amino acid sequence morphs and immuno-chromatographic test paper strip |
CN113817054A (en) * | 2021-10-09 | 2021-12-21 | 武汉科前生物股份有限公司 | Mouse monoclonal antibody 5B11 specifically binding porcine rotavirus VP6 protein and application thereof |
CN113817054B (en) * | 2021-10-09 | 2023-09-01 | 武汉科前生物股份有限公司 | Murine monoclonal antibody 5B11 specifically binding porcine rotavirus VP6 protein and application thereof |
Also Published As
Publication number | Publication date |
---|---|
KR100369533B1 (en) | 2003-01-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Greenberg et al. | Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies | |
Coulson et al. | Derivation of neutralizing monoclonal antibodies to human rotaviruses and evidence that an immunodominant neutralization site is shared between serotypes 1 and 3 | |
Padilla-Noriega et al. | Serologic analysis of human rotavirus serotypes P1A and P2 by using monoclonal antibodies | |
CN109265542B (en) | Antibody specifically binding norovirus GII.4 genotype VP1 protein or VLP, and preparation method and application thereof | |
CN112876559B (en) | Monoclonal antibody specifically binding to porcine rotavirus and application thereof | |
CN107312088B (en) | Porcine epidemic diarrhea virus specificity SIgA ELISA detection kit and application thereof | |
CN111793130B (en) | Haemophilus parasuis CdtB hybridoma cell and application of monoclonal antibody | |
KR100369533B1 (en) | Monoclonal antibody specific for vp6 of porcine rotavirus and hybridoma cell line producing same | |
FR2966461A1 (en) | Use of VP2 capsid protein of Bluetongue virus of serotype 8, a composition or compound having the protein, for the preparation of a reagent or a kit for in vitro diagnosis of an infection by BTV virus in an animal e.g. bovine | |
CN105542000B (en) | Monoclonal antibody and application thereof | |
CN115806611B (en) | Antibodies against novel coronavirus N proteins and uses thereof | |
Zhou et al. | Generation of internal image monoclonal anti-idiotypic antibodies against idiotypic antibodies to GP5 antigen of porcine reproductive and respiratory syndrome virus | |
Brüssow et al. | Polypeptide specificity of antiviral serum antibodies in children naturally infected with human rotavirus | |
CN116655779A (en) | Novel coronavirus antibodies and uses thereof | |
Yolken et al. | Identification of a group-reactive epitope of group B rotaviruses recognized by monoclonal antibody and application to the development of a sensitive immunoassay for viral characterization | |
KR101876535B1 (en) | Antibody for detecting of bovine viral diarrhea virus(bvdv), bvdv antigen detecting method and test kit using thereof | |
CN113817054A (en) | Mouse monoclonal antibody 5B11 specifically binding porcine rotavirus VP6 protein and application thereof | |
Kuzuya et al. | Rapid detection of human group C rotaviruses by reverse passive hemagglutination and latex agglutination tests using monoclonal antibodies | |
FR2966460A1 (en) | Differentiating a vaccinated animal against Bluetongue virus of infected animal from biological sample comprises contacting biological sample with NS1 protein of BTV virus and revealing antigen-antibody complex and anti-NS1 antibody | |
Kang et al. | Production and characterization of monoclonal antibodies against an avian group A rotavirus | |
CN114231496B (en) | Salmonella equine abortus competition ELISA antibody detection kit and application thereof | |
Ojeh et al. | Development of a biotin-streptavidin-enhanced enzyme-linked immunosorbent assay which uses monoclonal antibodies for detection of group C rotaviruses | |
KR100330312B1 (en) | Monoclonal antibody to saliva protein of porcine infectious gastroenteritis virus, hybridoma cell line producing the same, and production method thereof | |
JP3483586B2 (en) | Monoclonal antibodies against group C rotavirus inner core common antigen | |
KR102241521B1 (en) | Method of detecting antibody produced by foot-and-mouth disease vaccination using an antibody having immune reactivity to FMDV type O |
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 | ||
FPAY | Annual fee payment |
Payment date: 20080115 Year of fee payment: 6 |
|
LAPS | Lapse due to unpaid annual fee |