KR101990850B1 - Monoclonal antibody against infectious pancreatic necrosis virus and nervous necrosis virus and use thereof - Google Patents
Monoclonal antibody against infectious pancreatic necrosis virus and nervous necrosis virus and use thereof Download PDFInfo
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- KR101990850B1 KR101990850B1 KR1020170175234A KR20170175234A KR101990850B1 KR 101990850 B1 KR101990850 B1 KR 101990850B1 KR 1020170175234 A KR1020170175234 A KR 1020170175234A KR 20170175234 A KR20170175234 A KR 20170175234A KR 101990850 B1 KR101990850 B1 KR 101990850B1
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Abstract
Description
본 발명은 전염성 췌장괴사증 바이러스(Infectious Pancreatic Necrosis Virus, IPNV)에 특이적인 단클론항체에 관한 것으로, 보다 구체적으로는 전염성 췌장괴사증 바이러스 및 신경괴사증 바이러스(Nervous Necrosis Virus, NNV) 감염의 진단, 예방 및 치료 등에 유용하게 이용될 수 있는 전염성 췌장괴사증 바이러스 및 신경괴사증 바이러스에 특이적인 단클론항체 및 이의 용도에 관한 것이다.The present invention relates to a monoclonal antibody specific to the infectious pancreatic necrosis virus (IPNV), more specifically to the diagnosis, prevention and treatment of infectious pancreatic necrosis virus and Nervous Necrosis virus (NNV) infection The present invention relates to a monoclonal antibody specific for infectious pancreatic necrosis virus and neurodegenerative virus,
전염성 췌장괴사증 (Infectious pancreatic necrosis)은 1940년대 초 일본에서 양식중인 넙치(Paralichthys olivaceus)가 대량으로 폐사하면서 원인 바이러스가 처음으로 분리되었다. 이후 일본의 조피볼락 (Sebastes schlegelii) 및 은어 (Plecoglossus altivelis), 중국의 돌가자미 (Kareius bicoloratus) 등 아시아 지역에서만 분리되는 바이러스가 2012년도 유럽의 회색숭어(Thymallus thymallus)에서도 검출되었다. 국내의 경우 1998년도에 넙치로부터 바이러스가 분리되었으며, 참돔과 감성돔에 대하여 병원성을 지니는 것을 확인하였다.Infectious pancreatic necrosis was first isolated in Japan in the early 1940s when the flounder ( Paralichthys olivaceus ) cultured in Japan was killed in large quantities. Subsequently, viruses isolated only in Asia, such as Japanese rockfish ( Sebastes schlegelii ) and swine ( Plecoglossus altivelis ) and Chinese stone flounder ( Kareius bicoloratus ), were also detected in the European gray mullet ( Thymallus thymallus ) In Korea, viruses were isolated from flounder in 1998 and confirmed pathogenicity to red sea bream and rhubarb.
전염성 췌장괴사증의 원인바이러스는 전염성 췌장괴사증 바이러스 (Infectious pancreatic necrosis virus, IPNV)로 이중가닥의 RNA 바이러스이며 Rhabdoviridae과, Aquabirnavirus속의 외막이 없는 바이러스이다. 직경 70 nm의 정 20면체의 바이러스 입자의 모형을 보인다.Infectious pancreatic necrosis virus (IPNV) is a double-stranded RNA virus and is a virus free from the outer membrane of Rhabdoviridae and Aquabirnavirus. A model of the virus particle of a regular icosahedron with a diameter of 70 nm is shown.
IPNV는 다른 어류바이러스인 Marine birnavirus (MABV)와 함께 family Birnaviridae, genus Aquabirnavirus에 속하는 RNA virus로 VP1, VP2 (outer capsid protein), VP3 (inner surface of the viral capsid), VP4 (non-structural protein), VP5 (non-structural protein)의 단백질로 구성되어 있는 것이 특징이다.IPNV is an RNA virus belonging to the family Birnaviridae and genus Aquabirnavirus together with Marine birnavirus (MABV) which is another fish virus. VP1, VP2 (outer capsid protein), VP3 (inner surface of the viral capsid), VP4 VP5 (non-structural protein) protein.
바이러스성 신경괴사증(viral nervous necrosis, VNN)은 전 세계적으로 양식산 해산어에 심각한 피해를 유발 하는 질병으로 알려져 있다(Munday et al., 2002). 본 질병은 1980년대 말 일본에서 양식되는 줄전갱이(Pseudocaranx den\-tex )에서 발생된 이래로 돌돔(Oplegnathus fasciatus), 붉바리 (E. akaara), 자바리(E. moara), 넙치(Paralichthys olivaceus) 등 에서 발생되었고, 또한 유럽과 타 아시아 국가에서도 유사한 시기에 barramundi (Lates calcarifer), 터봇(Scophthalmus maxi\-mus), 능성어류 등에서 발생되었다(Egusa et al., 2006). 최근까 지의 연구에 따르면 약 30종 이상의 해산어에서 VNN이 발생 된다고 보고되어 있다(Egusa et al., 2006; Munday et al., 2002; OIE, 2009). VNN은 종묘생산단계의 자어부터 성어까지 광범위하게 발생하는 질병으로서 주로 고수온기에 발병하며, 병어는 신경세포의 이상으로 인한 이상 유영(힘없이 유영하거나 선회 등의 비정상적인 유영 행동)이 나타나며, 병리조직학적으로 척수, 척수신경절, 뇌 및 망막의 신경조직에 공포변성이 나타난다(Egusa et al., 2006; OIE, 2009). Viral nervous necrosis (VNN) is known worldwide as a serious disease causing serious damage to fish cultured (Munday et al., 2002). This disease has been reported in the late 1980s since it originated in Japan ( Pseudocaranx den \ -tex ), and has been reported to be caused by Olegnathus fasciatus , E. akaara, E. moara , and Paralichthys olivaceus (Egusa et al., 2006) and also in Europe and other Asian countries at similar times in barramundi ( Lates calcarifer ), turbot ( Scophthalmus maxi-musc ), and fish. Recent studies have reported that VNN occurs in more than 30 marine fishes (Egusa et al., 2006; Munday et al., 2002; OIE, 2009). VNN is a disease that occurs extensively in the seedling stage of the seedling production stage and mainly affects the warmth of the fish. The disease is caused by abnormally swimming (abnormal swimming behavior such as swimming or turning without power) due to nerve cell abnormality, (Egusa et al., 2006; OIE, 2009). In this study,
VNN의 원인 병원체인 신경괴사증바이러스(nervous necrosis virus, NNV)는 크기가 약 30 nm 정도로 외막이 없는 정이십면체의 RNA 바이러스로서 노다바이러스과 베타노다바이 러스속에 속한다(Schneemann et al., 2005). NNV는 5개의 유 전자형인 striped jack NNV (SJNNV) type, tiger puffer NNV (TPNNV) type, barfin flounder NNV (BFNNV) type, redspot\-ted grouper NNV (RGNNV) type 및 turbot NNV (TNV) type 으로 구분되며(Johansen et al., 2004; Nishizawa et al., 1997), 국내 양식산 능성어 및 연안에서 서식하는 자연산 어류에서 검 출되는 NNV는 모두 RGNNV type에 속한다고 보고되어 있다 (Gomez et al., 2008; Kim et al., 2012). The nervous necrosis virus (NNV), a causative agent of VNN, is an outer membrane-free, dichromic RNA virus of approximately 30 nm in size, belonging to the genus Nodavirus and Beta Nodavirus (Schneemann et al., 2005). NNV is divided into five types: striped jack NNV (SJNNV) type, tiger puffer NNV (TPNNV) type, barfin flounder NNV (BFNNV) type, redspot \ -ted grouper NNV (RGNNV) type and turbot NNV Nanni et al., 2004), and NNVs detected in wild-caught fish and wild fish in coastal areas are all reported to belong to the RGNNV type (Gomez et al., 2008; Kim et al., 2012).
최근, 소비자들의 안전한 먹거리에 대한 수요 증가로 인해 전 세계적으로 수산생물의 방역 (Aquatic animal Disease Control), 예찰 (Surveillance) 및 모니터링 (Monitoring)의 중요성이 더욱 커지고 있다. 또한, 국가적인 예찰 및 방역활동 뿐만 아니라 지역적 (regional) 이고 구역화 (zoining)된 수산생물의 질병 예찰 및 모니터링 연구결과의 분석은 향후 수산생물질병의 역학조사 및 질병 관리에 필요한 유용한 자료를 제공 할 뿐만 아니라 자국 내 특정 질병에 대한 무병(청정)지역을 증명할 수 있는 매우 중요한 역할을 한다. 따라서 IPNV 및 NNV의 국내 분리주의 서열변이에 대한 지속적인 연구 및 다른 국가 분리주와의 서열비교와 지속적인 모니터링도 중요하게 되었다.In recent years, the increasing demand for safe food for consumers has increased the importance of Aquatic animal disease control, surveillance and monitoring worldwide. In addition, national analysis and analysis of regional and zoined marine biological disease prediction and monitoring studies as well as national surveillance and disinfection activities will provide useful data for future epidemiological studies and disease management of marine biological diseases But it plays a very important role in proving a disease-free (clean) area for a specific disease in your country. Therefore, it is important to continuously study IPNV and NNV sequencing of domestic isolates, and to compare and monitor the sequence with other isolates.
국립수산과학원에 의해 실시된 우리나라 양식장의 병원체 감염률 조사에서 2006년 이후 바이러스성 신경괴사 바이러스의 검출률이 가장 높게 나타났으며(60% 이상), 이어 바이러스성 출혈성 패혈증바이러스에 의한 감염이 뒤를 따랐다(28%). 이와 같이 바이러스성 출혈성 패혈증은 높은 감염률을 나타내고 있으며, 밀집양식에서 전염성이 높아지고, 한번 감염으로 대량폐사를 일으켜 수산어가에 큰 피해를 입히고 있으나, 감염 시 초기 진단이 어려운 점이 있어 이의 용이한 진단방법 및 치료와 예방에 대한 연구가 시급하다.In 2006, the detection rate of viral nerve necrosis virus was the highest (over 60%), followed by infection with viral hemorrhagic septicemia virus in the survey of pathogen infection rate in Korean farms conducted by the National Fisheries Research Institute %). As described above, viral hemorrhagic sepsis shows a high infection rate, the infectiousness is increased in dense mode, and it causes massive death due to infection once, which causes great damage to fish food. However, since early diagnosis is difficult in infection, Research on treatment and prevention is urgent.
따라서, 본 발명의 목적은 종묘 생산시기에 대량 발생하여 높은 폐사율을 보이는 어병의 원인바이러스인 전염성 췌장괴사증 바이러스는 물론 신경괴사증바이러스에도 감수성 및 특이성이 있는 단클론항체를 제공하는 것이다.Accordingly, an object of the present invention is to provide a monoclonal antibody having susceptibility and specificity to infectious pancreatic necrosis virus as well as neurodegenerative virus, which is a virus causing a high mortality rate in a seedling production period and causing a fish disease.
본 발명의 다른 목적은 전염성 췌장괴사증 바이러스에 감수성 및 특이성이 높고, 신경괴사증바이러스에도 특이성이 있는 단클론항체를 생산하는 융합세포주를 제공하는 것이다.It is another object of the present invention to provide a fusion cell line producing a monoclonal antibody having a high sensitivity and specificity to infectious pancreatic necrosis virus and also having specificity for neurodegenerative virus.
본 발명의 또 다른 목적은 어류의 전염성 췌장괴사증(IPN) 및 바이러스성신경괴사증(VNN)의 진단, 치료 및 예방에 용이하게 이용할 수 있는 IPN 및 VNN 진단용 조성물 및 진단키트를 제공하는 것이다.It is another object of the present invention to provide a composition and diagnostic kit for IPN and VNN which can be easily used for diagnosis, treatment and prevention of infectious pancreatic necrosis (IPN) and viral neurotrophic disease (VNN) of fish.
본 발명의 목적들은 이상에서 언급한 목적들로 제한되지 않으며, 언급되지 않은 또 다른 목적들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.The objects of the present invention are not limited to the above-mentioned objects, and other objects not mentioned can be clearly understood by those skilled in the art from the following description.
상술된 본 발명의 목적을 달성하기 위해, 본 발명은 전염성 췌장 괴사증 바이러스(Infectious Pancreatic Necrosis Virus, IPNV)의 항원단백질 VP2와 특이적으로 결합하는 단일클론 항체 19F2를 제공한다.In order to achieve the object of the present invention described above, the present invention provides a monoclonal antibody 19F2 that specifically binds to the antigen protein VP2 of Infectious Pancreatic Necrosis Virus (IPNV).
바람직한 실시예에 있어서, 상기 단일클론항체 19F2는 하이브리도마 세포(KCLRF-BP-00414)에 의해 생산된다. In a preferred embodiment, the monoclonal antibody 19F2 is produced by hybridoma cells (KCLRF-BP-00414).
바람직한 실시예에 있어서, 상기 단일클론항체 19F2는 신경괴사증바이러스 (nervous necrosis virus, NNV)에 특이적으로 결합한다. In a preferred embodiment, the monoclonal antibody 19F2 specifically binds to nervous necrosis virus (NNV).
또한, 본 발명은 상술된 어느 하나의 단일클론 항체 19F2를 생산하는 하이브리도마 세포주( KCLRF-BP-00414)를 제공한다. The present invention also provides a hybridoma cell line (KCLRF-BP-00414) which produces any of the monoclonal antibodies 19F2 described above.
또한, 본 발명은 상술된 어느 하나의 단일클론항체 19F2를 포함하고 전염성 췌장괴사증 및 신경괴사증 중 1개 이상의 질병을 진단하는 진단 조성물을 제공한다.The invention also provides a diagnostic composition comprising any one of the monoclonal antibodies 19F2 described above and diagnosing at least one of infectious pancreatic necrosis and neurodegenerative disease.
또한, 본 발명은 상술된 진단 조성물을 포함하는 어류의 전염성 췌장괴사증 및 신경괴사증 진단 키트를 제공한다.In addition, the present invention provides a kit for infectious pancreatic necrosis and neuro-necrosis of fish comprising the diagnostic composition described above.
또한, 본 발명은 상술된 진단 조성물을 포함하는 어류의 전염성 췌장괴사증 및 신경괴사증 진단용 마이크로어레이를 제공한다.In addition, the present invention provides a microarray for the diagnosis of infectious pancreatic necrosis and neurotoxicity of fish comprising the above-described diagnostic composition.
먼저, 본 발명에 의하면 전염성 췌장괴사증 바이러스에 감수성 및 특이성이 높고, 신경괴사증바이러스에도 특이성이 있는 단클론항체 및 이를 생산하는 융합세포주를 제공할 수 있다.First, according to the present invention, a monoclonal antibody having high susceptibility and specificity to infectious pancreatic necrosis virus and having specificity for neurodegenerative virus, and a fused cell line producing the same can be provided.
또한 본 발명에 의하면 어류의 전염성 췌장괴사증 및/또는 바이러스성신경괴사증(VNN)의 진단, 치료 및 예방에 용이하게 이용할 수 있는 전염성췌장괴사증 및 VNN 진단용 조성물 및 진단키트를 제공함으로써 양식장에서 대량폐사를 일으켜 막대한 손해를 입히고 있는 전염성췌장괴사증 및/또는 바이러스성신경괴사증의 진단과 치료 및 예방에 이용할 수 있다. According to the present invention, there is provided a kit for diagnosing, treating and preventing infectious pancreatic necrosis (VNN) and / or infectious pancreatic necrosis (VNN) in fish, and a diagnostic kit for diagnosing infectious pancreatic necrosis and VNN, And can be used for the diagnosis, treatment and prevention of infectious pancreatic necrosis and / or viral neurotrophic disease which are causing great damage.
본 발명의 효과는 이상에서 언급한 효과로 제한되지 않으며, 언급되지 않은 또 다른 효과들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.
도 1은 SDS-PAGE에서 관찰되는 정제된 IPNV의 구조 단백질을 나타낸 사진이다.
도 2는 본 발명에 따른 단클론항체 19F2의 IPNV 부위 확인을 나타낸 웨스턴 블로팅 결과를 나타낸 것이다(M: marker (kDa), 1: 1B3).
도 3은 본 발명에 따른 단클론항체 19F2의 어류바이러스들에 대한 특이성을 나타내는 웨스턴 블로팅 사진이다(P: 양성대조구 (Purified IPNV), M: marker (kDa), 1: IPNV, 2: MABV, 3: VHSV, 4: IHNV, 5: HIRRV, 6: SVCV, 7: NNV, 8: CHSE-214, 9: FHM, 10: EPC, 11: SSN-1).
도 4는 본 발명에 따른 단클론 항체 19F2의 어류바이러스들에 대한 특이성을 확인한 ELISA 결과를 나타낸 그래프이다. Figure 1 is a photograph showing the structural protein of purified IPNV observed on SDS-PAGE.
Figure 2 shows Western blotting results showing the identification of the IPNV site of monoclonal antibody 19F2 according to the invention (M: marker (kDa), 1: 1B3).
FIG. 3 is a Western blotting photograph showing the specificity of the monoclonal antibody 19F2 according to the present invention for fish viruses (P: Positive control (Purified IPNV), M: marker (kDa), 1: IPNV, 2: MABV, 3 : VHSV, 4: IHNV, 5: HIRRV, 6: SVCV, 7: NNV, 8: CHSE-214, 9: FHM, 10: EPC, 11: SSN-1).
FIG. 4 is a graph showing ELISA results confirming the specificity of the monoclonal antibody 19F2 according to the present invention for fish viruses.
본 발명에서 사용되는 용어는 가능한 현재 널리 사용되는 일반적인 용어를 선택하였으나, 특정한 경우는 출원인이 임의로 선정한 용어도 있는데 이 경우에는 단순한 용어의 명칭이 아닌 발명의 상세한 설명 부분에 기재되거나 사용된 의미를 고려하여 그 의미가 파악되어야 할 것이다.Although the terms used in the present invention have been selected as general terms that are widely used at present, there are some terms selected arbitrarily by the applicant in a specific case. In this case, the meaning described or used in the detailed description part of the invention The meaning must be grasped.
이하, 첨부한 도면 및 바람직한 실시예들을 참조하여 본 발명의 기술적 구성을 상세하게 설명한다.Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.
그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화 될 수도 있다. 명세서 전체에 걸쳐 본 발명을 설명하기 위해 사용되는 동일한 참조번호는 동일한 구성요소를 나타낸다.However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.
본 발명의 기술적 특징은 전염성 췌장 괴사증 바이러스(Infectious Pancreatic Necrosis Virus, IPNV)는 물론 신경괴사증바이러스 (nervous necrosis virus, NNV)에 특이적으로 결합하는 단일클론항체 및 상기 단일클론항체를 생산하는 융합세포주를 개발한 것에 있다.Technical features of the present invention include a monoclonal antibody that specifically binds to nervous necrosis virus (NNV) as well as Infectious Pancreatic Necrosis Virus (IPNV) and a fusion cell line that produces the monoclonal antibody I have developed it.
따라서, 본 발명은 전염성 췌장 괴사증 바이러스(Infectious Pancreatic Necrosis Virus, IPNV)의 항원단백질 VP2와 특이적으로 결합할 뿐만 아니라, 신경괴사증바이러스 (nervous necrosis virus, NNV)에 특이적으로 결합하는 단일클론 항체 19F2를 제공한다.Accordingly, the present invention provides a monoclonal antibody 19F2 that specifically binds to the antigen protein VP2 of Infectious Pancreatic Necrosis Virus (IPNV), as well as specifically binds to nervous necrosis virus (NNV) Lt; / RTI >
본 발명에서 "19F2 항체"또는 "단클론항체 19F2"는 하이브리도마 세포주( KCLRF-BP-00414)에 의해 생산되는 단클론 항체를 의미한다.In the present invention, "19F2 antibody" or "monoclonal antibody 19F2" means a monoclonal antibody produced by a hybridoma cell line (KCLRF-BP-00414).
단일클론항체는 당업자에 알려진 융합에 의한 불멸화된 세포주 생성기술 (Koeher and Milstein, Nature, 256:495 (1975))에 의하여 제조될 수 있다. 그 제조방법을 간단히 설명하면 다음과 같다. 먼저 순수한 단백질을 약 20 ㎍을 얻어서 쥐에 면역화를 시키거나 펩타이드를 합성하여 소혈청 알부민과 결합시켜 쥐에 면역화시킨다. 그 후 쥐에서 분리된 항원-생산 B 임파구를 인간 또는 마우스의 골수종세포와 융합하여 불멸화된 하이브리도마를 생성한다. 이어, ELISA (Enzymelinked immunosorbent assay)로 하이브리도마 세포의 단일클론항체의 생성여부를 알아보고 양성 클론을 택하여 배양한 후 항체를 분리 정제하거나 쥐의 복강에 주입한 후 복수를 채취한다.Monoclonal antibodies can be prepared by immortalized cell line generation techniques (Koeher and Milstein, Nature, 256: 495 (1975)) by fusion as known to those of skill in the art. The manufacturing method will be briefly described as follows. First, about 20 μg of pure protein is obtained and the mice are immunized or the peptide is synthesized and bound to bovine serum albumin to immunize mice. The antigen-producing B lymphocytes isolated from the mice are then fused with human or mouse myeloma cells to produce immortalized hybridomas. Next, an ELISA (Enzymelinked Immunosorbent Assay) is used to determine whether a monoclonal antibody is produced. Hybridoma cells are cultured in a selective clone. The antibody is separated or purified or injected into the abdominal cavity of a mouse.
먼저 항체를 생산하는 하이브리도마 세포의 제조에 필요한 마우스를 면역화시켰다. 구체적으로는, IPNV를 배양하여 농축 및 정제한 후 정제된 바이러스 구조단백질을 인산염 완충액(phosphate buffered saline; PBS)에 희석하고 동일한 부피의 프로인트 완전 아주반트(complete Freund's adjuvant, Sigma)와 혼합하여 에멀젼을 제조한 후, 상기 에멀젼을 마우스의 발바닥에 1차 주사하였다. 1차 면역화 후 2주 후에 마우스로부터 채혈한 후 ELISA를 실시하여 항체가를 측정하였으며, 마우스의 면역성을 높이기 위하여 정제된 바이러스 구조단백질만을 사용하여 2차 면역하였다. 2차 면역화 후 1주 후에 3차 면역화하고, 마지막 면역화한 후 3일 후에 면역화된 마우스로부터 비장조직을 적출하였다. 적출된 비장조직과 골수종 세포인 Sp2/0Ag14 세포를 세포수를 기준으로 10:1의 비율로 혼합하고 폴리에틸렌 글리콜(PEG)을 넣어 세포 융합시키고 배양하였다.First, mice required for the production of antibody-producing hybridoma cells were immunized. Specifically, IPNV was cultured and purified, and the purified virus structural protein was diluted in phosphate buffered saline (PBS) and mixed with the same volume of complete Freund's adjuvant (Sigma) to obtain an emulsion And then the emulsion was firstly injected into the soles of the mice. Two weeks after the primary immunization, blood samples were collected from the mice and ELISA was performed to measure antibody titers. Second immunization was performed using only the purified virus structural proteins to increase the immunity of the mice. Three-fold immunization was done one week after the second immunization and spleen tissue was excised from the immunized mice three days after the last immunization. The extracted spleen tissue and myeloma cells, Sp2 / 0Ag14 cells, were mixed at a ratio of 10: 1 based on the number of cells, and the cells were fused with polyethylene glycol (PEG).
상기 융합세포군 중에서 IPNV에만 특이적으로 반응하는 융합세포들을 선별하고, 선별된 융합세포가 단일클론이 되도록 제한 희석법(limiting dilution)을 사용하여 융합세포를 희석한 후, 하나의 세포로부터 증식한 융합세포의 흡광도(O.D 값)를 ELISA로 확인하여 흡광도가 가장 우수한 융합세포를 선택하였다. 최종적으로 선택한 융합세포 즉 하이브리도마 세포를 "IPNV 19F2"로 명명하였으며, 한국세포주연구재단에 2017년 10월 18일자로 기탁하였다(수탁번호: KCLRF-BP-00414 ).Among the fusion cell groups, the fusion cells that specifically react only with IPNV are selected, and the fusion cells are diluted using limiting dilution so that the selected fusion cell becomes a single clone, and the fusion cell (OD value) was selected by ELISA to select the best-absorbed fusion cell. The final selected fusion cell or hybridoma cell was designated as "IPNV 19F2 " and deposited on Oct. 18, 2017 (Accession No .: KCLRF-BP-00414) in Korean Cell Line Research Foundation.
상기 얻어진 하이브리도마 세포(KCLRF-BP-00414)를 마우스에 주사하고, 마우스의 혈청으로부터 IPNV에 특이적으로 결합하는 단일클론항체 19F2를 분리하였다.The obtained hybridoma cells (KCLRF-BP-00414) were injected into mice and monoclonal antibody 19F2, which specifically binds to IPNV, was isolated from the mouse serum.
또한, 본 발명의 하이브리도마 세포 및 이로부터 생산된 단일클론항체는 IPNV 특히 IPNV의 항원단백질 VP2에 대한 결합 특이성이 높았고, 더 나아가 신경괴사증바이러스 (nervous necrosis virus, NNV)에 대한 결합 특이성도 있음을 확인하였다.In addition, the hybridoma cells of the present invention and the monoclonal antibodies produced therefrom have high binding specificity to IPNV, especially IPNV, antigen protein VP2, and further have binding specificity to nervous necrosis virus (NNV) Respectively.
또한, 본 발명의 상기 단클론항체를 포함하는 진단 조성물 및 진단키트를 제공한다.In addition, a diagnostic composition and a diagnostic kit comprising the monoclonal antibody of the present invention are provided.
본 발명에서 사용된 용어, "진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단은 전염성 췌장괴사증 바이러스의 감염 여부를 확인하는 것이다.As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the infection of the infectious pancreatic necrosis virus.
본 발명의 항체를 이용하여 시료, 예를 들면 어류조직에 포함된 IPNV 또는 NNV를 분석하는 방법으로는 통상의 항원-항체반응의 분석법을 사용할 수 있다. 예컨대, 면역 검정(측정)법으로는 효소 면역 측정법(ELISA), 형광면역 측정법(FIA), 방사선 면역 측정법(RIA), 발광 면역 측정법, 면역블롯(immunoblot)법, 웨스턴 블롯(western blot)법, 면역 염색법 등이 있다.As a method of analyzing IPNV or NNV contained in a sample, for example, a fish tissue using the antibody of the present invention, a conventional assay method of antigen-antibody reaction can be used. Examples of the immunoassay method include enzyme immunoassay (ELISA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescent immunoassay, immunoblot, Western blot, And immunostaining.
본 발명의 진단용 조성물은 특이적인 단일클론항체 이외에 면역학적 분석에 사용되는 당업계에 공지된 시약 등을 추가로 포함할 수 있다.The diagnostic composition of the present invention may further include reagents and the like known in the art used for immunological analysis in addition to the specific monoclonal antibody.
면역학적 분석은 항원과 항체의 결합을 측정할 수 있는 방법이라면 모두 포함될 수 있다. 이러한 방법들은 당 분야에 공지되어 있으며 비제한적인 예로서, 면역세포화학 및 면역조직화학, 방사선 면역 분석법, ELISA, 면역블롯, 파아르 분석법, 면역침강, 라텍스 응집, 적혈구 응집, 비탁계법, 면역확산법, 카운터-전류 전기영동법, 단일 라디칼 면역확산법, 면역크로마토그래피법, 단백질 칩 및 면역형광법 등이 있다.Immunological assays can involve any method that can measure the binding of an antigen to an antibody. Such methods are well known in the art and include, but are not limited to, immunocytochemistry and immunohistochemistry, radioimmunoassay, ELISA, immunoblot, Farar assay, immunoprecipitation, latex coagulation, erythrocyte coagulation, , Counter-current electrophoresis, single-radical immunodiffusion, immunochromatography, protein chip and immunofluorescence.
면역학적 분석에 사용되는 시약으로는 검출 가능한 신호를 생성할 수 있는 표지, 용해제, 세정제가 포함되며, 표지물질이 효소인 경우에는 효소활성을 측정할 수 있는 기질 및 반응 정지제를 포함할 수 있다.Reagents used in immunological assays include labels, solubilizers, and detergents capable of producing a detectable signal, and, if the label is an enzyme, a substrate capable of measuring enzyme activity and a quencher .
상기에서 검출 가능한 신호를 생성할 수 있는 표지는 항원-항체 복합체의 형성을 정성 또는 정량적으로 측정가능하게 하는데, 이의 예로는 효소, 형광물질, 리간드, 발광물, 미소입자(microparticle), 레독스 분자 및 방사성 동위원소 등이 있다. 효소로는 β-글루쿠로니다제, β-D-글루코시다제, 우레아제, 퍼옥시다아제, 알칼라인포스파타아제, 아세틸콜린에스테라아제, 글루코즈 옥시다아제, 헥소키나제, 말레이트 디하이드로게나아제, 글루코스-6-인산디하이드로게나아제, 인버타아제 등을 사용할 수 있다. 형광물로는 플루오레신, 이소티오시아네이트, 로다민, 피코에리테린, 피코시아닌, 알로피코시아닌, 플루오르신이소티옥시아네이트 등을 사용할 수 있다. 리간드로는 바이오틴 유도체 등이 있으며, 발광물로는 아크리디늄 에스테르, 루시페린, 루시퍼라아제 등이 있다. 미소입자로는 콜로이드 금, 착색된 라텍스 등이 있고 레독스 분자로는 페로센, 루테늄 착화합물, 바이올로젠, 퀴논, Ti 이온, Cs 이온, 다이이미드, 1,4-벤조퀴논, 하이드로퀴논 등이 있다. 방사성 동위원소로는 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re 등이 있다. 그러나 상기 예시된 것들 외에 면역학적 분석법에 사용할 수 있는 것이라면 어느 것이라도 사용할 수 있다.The marker capable of generating a detectable signal as described above enables the formation of an antigen-antibody complex to be qualitatively or quantitatively measurable. Examples thereof include an enzyme, a fluorescent substance, a ligand, a luminescent material, a microparticle, a redox molecule And radioactive isotopes. Examples of the enzymes include? -Glucuronidase,? -D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase, malate dehydrogenase, glucose- Phosphoric acid dehydrogenase, invertase and the like can be used. Examples of the minerals include fluorescein, isothiocyanate, rhodamine, picoeriterine, phycocyanin, allophycocyanin, fluorine synthase, and the like. Examples of the ligand include biotin derivatives, and examples of the luminescent material include acridinium ester, luciferin, and luciferase. Examples of the fine particles include colloidal gold and colored latex. Examples of redox molecules include ferrocene, ruthenium complex, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone and the like. Radioactive isotopes include 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re. However, in addition to those exemplified above, any of them can be used as long as they can be used for immunological assays.
본 발명의 진단용 조성물은 진단의 신속도 및 편리성을 높이기 위해, 공지된 다양한 방법을 이용하여 적합한 담체 또는 지지체상에 고정된 상태로 제공될 수 있다. 적합한 담체 또는 지지체의 예로는 아가로스, 셀룰로즈, 니트로셀룰로즈, 덱스트란, 세파덱스, 세파로즈, 리포솜, 카르복시메틸셀룰로즈, 폴리아크릴아미드, 폴리스테린, 반려암, 여과지, 이온교환수지, 플라스틱 필름, 플라스틱 튜브, 유리, 폴리아민-메틸 비닐-에테르-말레산 공중합체, 아미노산 공중합체, 에틸렌-말레산 공중합체, 나일론, 컵, 플랫 팩(flat packs) 등이 포함된다. 그 외의 다른 고체 기질로는 세포 배양 플레이트, ELISA 플레이트, 튜브 및 폴리머성 막이 있다. 상기 지지체는 임의의 가능한 형태, 예를 들어 구형(비드), 원통형(시험관 또는 웰 내면), 평면형(시트, 시험 스트립)을 가질 수 있다.The diagnostic composition of the present invention may be provided in a fixed state on a suitable carrier or support using various known methods in order to increase the speed and convenience of diagnosis. Examples of suitable carriers or supports are agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposomes, carboxymethylcellulose, polyacrylamide, polysterine, gabapentin, filter paper, , Glass, polyamine-methyl vinyl ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, cup, flat packs and the like. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (test tube or well inner surface), planar (sheet, test strip).
본 발명의 진단용 조성물은 진단용 키트 또는 마이크로어레이 형태로 제공될 수 있다. 진단용 키트로는 예를 들면, 시료 중의 특정 단백질을 검출하기 위해 면역크로마토그래피법을 기초로 하는 측방 유동 검정 키트(lateral flow assay kit)의 형태로 제공될 수 있다. 측방 유동 검정 키트는 통상 시료가 적용되는 샘플패드(sample pad), 탐지용 항체가 코팅되어 있는 방출패드(releasing pad), 시료가 이동하여 분리되고 항원-항체 반응이 일어나는 전개용 막(예를 들어 니트로셀룰로스) 또는 스트립, 그리고 흡수패드(absorptionpad)로 이루어진다.The diagnostic composition of the present invention may be provided in the form of a diagnostic kit or microarray. The diagnostic kit may be provided in the form of, for example, a lateral flow assay kit based on immunochromatography to detect a specific protein in a sample. Lateral flow assay kits typically include a sample pad to which a sample is applied, a releasing pad coated with a detection antibody, a developing membrane that separates the sample and separates the antigen-antibody reaction (for example, Nitrocellulose) or strip, and an absorption pad.
마이크로어레이는 일반적으로 특정 시약으로 처리된 슬라이드글라스 표면 위에 항체를 부착하여 항원-항체 반응에 의해 상기 항체에 특이적으로 부착하는 단백질을 검출할 수 있도록 하는 것이다.The microarray generally attaches an antibody on a surface of a slide glass treated with a specific reagent so that a protein specifically binding to the antibody can be detected by an antigen-antibody reaction.
본 발명에서 IPNV 및/또는 NNV를 인식하는 항체를 이용하는 면역 검정의 바람직한 예로서, ELISA 법을 들 수 있다. ELISA 법은 일반적인 경합법, 샌드위치법 등의 수법에 따라 실시할 수 있고, 액상계 또는 고체상태계에서도 실시할 수 있다.In the present invention, ELISA is a preferable example of immunoassay using an antibody recognizing IPNV and / or NNV. The ELISA method can be carried out according to a conventional method such as a compaction method, a sandwich method, or the like, and can also be carried out in a liquid-phase system or a solid-state system.
본 발명의 전염성 췌장괴사증 및 바이러스성 신경괴사증 진단용 키트(kit)는 본 발명의 단일클론항체 19F2를 포함하는 것으로, 상기 진단용 키트는 필요에 따라 면역 반응을 이용하고, 웰, 염색제, 검출용의 효소 표지 항체, 세척액, 항체 희석액, 검체 희석액, 효소 기질, 효소 기질액 희석액, 그밖의 시약을 포함할 수 있다.The kit for the diagnosis of infectious pancreatic necrosis and viral neuroticism of the present invention comprises the monoclonal antibody 19F2 of the present invention, wherein the diagnostic kit uses an immune response if necessary, and comprises an enzyme for a well, a stain, Labeling antibodies, washing solutions, antibody dilutions, sample dilutions, enzyme substrates, enzyme substrate solutions, and other reagents.
본 발명에서 사용할 수 있는 진단키트는 IPNV 및/또는 NNV에 특이적인 단일클론항체 19F2; 기질과의 반응에 의해서 발색하는 표지체가 접합된 2차항체 접합체 (Conjugate); 상기 표지체와 발색 반응할 발색 기질 용액; 세척액; 및 효소반응 정지용액을 포함할 수 있다.Diagnostic kits that can be used in the present invention include monoclonal antibody 19F2 specific for IPNV and / or NNV; A secondary antibody conjugate (conjugate) conjugated with a labeling substance that reacts with a substrate; A color developing substrate solution to be colored with the label; Washing liquid; And an enzyme reaction stop solution.
또한, 본 발명의 진단키트는 IPNV 및/또는 NNV 표준 항원을 포함하는 양성 대조군과 상기 항원이 주입되지 않은 동물의 항혈청을 포함하는 음성 대조군을 추가적으로 포함할 수 있다.In addition, the diagnostic kit of the present invention may further comprise a positive control comprising IPNV and / or NNV standard antigen and a negative control comprising antiserum of the animal not infected with said antigen.
본 발명의 진단키트는 항원-항체 결합반응을 통하여 항체 단백질에 대한 항원을 정량분석 또는 정성분석함으로써 전염성 췌장괴사증 및/또는 VNN을 진단할 수 있으며, 상기 항원-항체 결합반응은 통상의 ELISA, RIA (Radioimmnoassay), 샌드위치 측정법 (Sandwich assay), 폴리아크릴아미드 겔 상의 웨스턴 블롯, 면역 블롯 분석 또는 면역조직화학염색 방법(Immnohistochemical staining)으로 측정할 수 있다. 예를 들어, 검체 및 대조군을 표면에 코팅시킨 96 웰 마이크로타이터 플레이트 등을 이용하여 재조합 모노클로날 항체 단백질과 반응하는 ELISA를 수행하도록 상기 진단키트를 제공할 수 있다.The diagnostic kit of the present invention can diagnose infectious pancreatic necrosis and / or VNN by quantitative analysis or qualitative analysis of an antigen against an antibody protein through an antigen-antibody binding reaction, and the antigen-antibody binding reaction can be detected by conventional ELISA, RIA (Radioimmnoassay), sandwich assay, Western blotting on polyacrylamide gel, immunoblot analysis or immunohistochemical staining. For example, the diagnostic kit can be provided to perform an ELISA that reacts with a recombinant monoclonal antibody protein using, for example, a 96-well microtiter plate coated with a sample and a control.
항원-항체 결합 반응을 위한 고정체로는 니트로셀룰로오즈 막, PVDF 막, 폴리비닐 (Polyvinyl) 수지 또는 폴리스티렌(Polystyrene) 수지로 합성된 웰 플레이트 (Well plate) 및 유리로 된 슬라이드 글래스 등이 사용될 수 있다. 2차 항체의 표지체는 발색반응을 하는 통상의 발색제가 바람직하며, HRP (Horseradish peroxidase), 염기성 탈인산화효소 (Alkaline phosphatase), 콜로이드 골드 (Coloid gold), FITC (Poly L-lysine-fluorescein isothiocyanate), RITC(Rhodamine-B-isothiocyanate) 등의 형광물질 (Fluorescein) 및 색소 (Dye) 등의 표지체가 사용될 수 있다. 발색을 유도하는 기질은 발색반응을 하는 표지체에 따라 사용하는 것이 바람직하며, TMB(3,3',5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine) 등을 사용할 수 있다. 이 때, 발색제 기질은 완충용액 (0.1 M NaAc, pH 5.5)에 용해된 상태로 제공되는 것이 더욱 바람직하다.As a fixture for the antigen-antibody binding reaction, a nitrocellulose membrane, a PVDF membrane, a well plate synthesized from a polyvinyl resin or a polystyrene resin, and a glass slide glass can be used. The marker of the secondary antibody is preferably a conventional coloring agent that performs a color reaction. Examples of the marker include HRP (Horseradish peroxidase), basic dephosphorylase (Alkaline phosphatase), colloidal gold, FITC (Poly-Lysine-fluorescein isothiocyanate) , Fluorescein such as Rhodamine-B-isothiocyanate (RITC), and coloring matter (Dye) may be used. The substrate that induces color development is preferably used according to the labeling substance that undergoes a color reaction. TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline -6-sulfonic acid], and OPD (o-phenylenediamine). At this time, it is more preferable that the color developer substrate is provided in a dissolved state in a buffer solution (0.1 M NaAc, pH 5.5).
TMB와 같은 발색기질은 2차 항체 접합체의 표지체로 사용된 HRP에 의해 분해되어 발색 침적체를 생성하고 그 발색 침적체의 침적 정도를 육안으로 확인함으로써폐암 특이적 마커 단백질 항원의 존재 유무를 검출한다. 세척액은 인산염 완충용액, NaCl 및 트윈 20을 포함하는 것이 바람직하며, 0.02 M 인산염 완충용액, 0.13 MNaCl, 및 0.05% 트윈 20 (Tween 20)으로 구성된 완충용액 (PBST)이 더욱 바람직하다. 세척액은 항원-항체 결합반응 후 항원-항체 결합체에 2차 항체를 반응시킨 다음 적당량을 고정체에 첨가하여 3 내지 6회 세척한다. 반응정지용액은 황산용액(H2SO4)이 바람직하게 사용될 수 있다.The chromogenic substrate such as TMB is decomposed by HRP used as a marker of the secondary antibody conjugate to generate a chromogenic precipitate and the presence or absence of a specific marker protein of lung cancer is detected by visually observing the degree of deposition of the chromogenic precipitate . The wash liquor preferably comprises a phosphate buffer solution, NaCl and
한편, IPNV 및/또는 NNV에 특이적인 항체인 19F2를 생물학적 마이크로칩 (Biological microchip) 상에 고정시킨 후 대상어류로부터 분리된 검체 시료와 반응시켜 상기 항체 단백질에 대한 항원을 검출할 수 있는 생물학적 마이크로칩(Biological microchip) 및 자동화된 미세배열 시스템 (Microarray system)을 이용하면, 대량으로 시료를 분석할 수 있다.On the other hand, a 19F2 antibody specific to IPNV and / or NNV was immobilized on a biological microchip and then reacted with a specimen sample separated from the target fish to obtain a biological microchip capable of detecting an antigen for the antibody protein (Biological microchip) and automated microarray system can be used to analyze a large amount of sample.
실시예Example
1. 바이러스의 배양1. Culture of virus
실험에 사용된 전염성췌장괴사증바이러스 (nfectious pancreatic necrosis virus, IPNV)는 VR-299의 serotype이며, 바이러스 (VR-299)를 대량으로 배양하기 위하여 75㎠ tissue culture flask (Nunc, Denmark)에 epithelioma papilosum cyprin (EPC)을 단층으로 배양한 후 바이러스를 접종하여 20℃에서 10일간 배양하면서 cytopathic effect (CPE)를 관찰하였다. 세포에 바이러스가 감염되어 90% 이상 용해된 세포 배양액을 4℃에서 12,000 rpm으로 30분간 원심 분리하여 세포 잔여물을 제거한 후 상층액을 분리하였다. 분리된 바이러스 상층액은 실험에 사용되기 전까지 ??80℃에서 보존하였다.In order to cultivate a large amount of virus (VR-299), 75cm2 tissue culture flask (Nunc, Denmark) was infected with epithelioma papilosum cyprin (Nunc, Denmark) (EPC) was cultured in a single layer, and the cytopathic effect (CPE) was observed when the virus was inoculated and cultured at 20 ° C for 10 days. The cells were incubated at 4 ° C for 30 minutes at 12,000 rpm for 30 minutes to remove cell debris, and the supernatant was separated. The isolated virus supernatant was stored at -80 ° C until used in the experiment.
2. 바이러스의 농축 및 정제2. Concentration and purification of virus
바이러스 배양액에 polyethylene glycol (PEG)-6000 (Sigma, USA)과 NaCl을 각각 7.5% (w/v), 2.3% (w/v)로 첨가한 후 4℃에서 overnight 하였다. PEG가 처리가 된 바이러스 배양액을 4℃에서 12,000 rpm으로 30분간 원심 분리한 후 pellet을 phosphate buffer saline (PBS: 0.13M NaCl, 2.7mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4)완충용액으로 현탁하였다. 현탁액은 30,000 rpm에서 2시간 동안 초원심을 실시한 후 pellet을 PBS로 재부유 시켜 바이러스를 농축하였다. 바이러스를 정제하기 위해, 농축된 바이러스를 step sucrose gradient (10%, 55% sucrose 용액 (w/w)) 위에 넣은 후 21,000 rpm으로 2시간 동안 초원심분리를 실시하였다. 20%와 35% 경계 지점에 형성된 바이러스로 추정되는 band를 주사기를 이용하여 취한 후, PBS로 현탁하여 30,000 rpm으로 2시간 동안 초원심 분리하였다. 원심 분리 후 얻어진 침전물은 PBS 완충용액으로 다시 재현탁하여 실험에 사용되기 전까지 ??80℃에 보존하였다.To the viral culture, polyethylene glycol (PEG) -6000 (Sigma, USA) and NaCl were added at 7.5% (w / v) and 2.3% (w / v), respectively. PEG-treated viral cultures were centrifuged at 12,000 rpm for 30 min at 4 ° C. The pellet was resuspended in phosphate buffer saline (PBS: 0.13M NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 ) And suspended in buffer solution. The suspension was subjected to ultracentrifugation at 30,000 rpm for 2 hours and the pellet was resuspended in PBS to concentrate the virus. To purify the virus, concentrated virus was placed on a step sucrose gradient (10%, 55% sucrose solution (w / w)) and ultracentrifuged for 2 hours at 21,000 rpm. The band, which was presumed to be the virus at the border between 20% and 35%, was taken with a syringe, suspended in PBS and ultracentrifuged at 30,000 rpm for 2 hours. The precipitate obtained after centrifugation was resuspended in PBS buffer and stored at -80 ° C until used in the experiment.
3. 바이러스 면역 및 hybridoma 제작3. Virus immunization and hybridoma production
면역은 정제 재조합 단백질 (약 100 ㎍)와 complete freunds adjuvant를 1:1 비율로 혼합하여 BALB/c 마우스의 발바닥에 1차 면역하였다. 2주 후 마우스로부터 채혈한 후 ELISA를 실시하여 항체가를 측정하였으며, 재조합 단백질만을 사용하여 2차 면역하였다. 1주후 3차 면역시켰고, 면역 3일 후 마우스의 비장 조직을 분리한 다음 PEG 1500 (Roche, USA)를 이용하여 myeloma cell (Sp2/0Ag14)과 융합시킨 후 hypoxanthine, thymidine (HT) (gibco, USA) 배지를 사용하여 96 well plate (TPP, Switzerland)에 분주하여 CO2배양기에서 37℃로 배양하여 hybridoma를 제작하였다. Immunization was performed by mixing the purified recombinant protein (about 100 μg) and complete freunds adjuvant at a ratio of 1: 1, and then immunizing the BALB / c mice with a primary antibody. After 2 weeks, blood was drawn from the mouse, ELISA was performed to measure antibody titer, and secondary immunization was performed using only the recombinant protein. After 3 days of immunization, the mouse spleen was isolated and then fused with myeloma cell (Sp2 / 0Ag14) using PEG 1500 (Roche, USA) and then hypoxanthine, thymidine (HT) ) Medium and seeded in 96 well plates (TPP, Switzerland) and cultured in a CO 2 incubator at 37 ° C to prepare hybridomas.
4. Hybridoma의 screening4. Screening of Hybridoma
바이러스에 대하여 특이적으로 반응하는 항체를 생산하는 hybridoma를 screening 하기 위하여 enzyme-linked immunosorbent assay (ELISA)를 실시하였다. An enzyme-linked immunosorbent assay (ELISA) was performed to screen hybridomas producing antibodies that specifically react with viruses.
항원은 정제된 바이러스액을 96 well plate (corning, USA)에 well 당 50 ㎕ (250 ng/well)씩 분주하여 4℃에서 overnight 또는 37℃에서 2시간 coating하였다. 2% skim milk/TBST (Tris-buffered saline-Tween 20) 200 ㎕를 분주하여 blocking 한 후, TBST로 1회 세정하고 1차 항체로서 hybridoma 배양 상등액을 well 당 50 ㎕씩 분주하여 37℃에서 2시간 반응하였다. TBST로 3회 세정한 후 2차 항체로는 horseradish peroxidase (HRP)가 표식되어 있는 goat anti-mouse IgG (Pierce, USA)를 2% skim milk/TBST로 5,000배 희석하여 well 당 50 ㎕씩 분주하여 37℃에서 1시간 반응하였다. TBST로 5회 세정한 후 tetramethylbenzidine base (TMB, color reagents) (surmodics, TMBC)를 well 당 50 ㎕ 분주하여 발색하였다. 각 well에 1N H2SO4를 50 ㎕씩 넣어 발색 반응을 중지시킨 후, microplate photometer (Multiskan, USA)로 450 ㎚에서 흡광도를 측정하였다. Antigen was coated with either 50 μl (250 ng / well) per well in 96 well plates (corning, USA) at 4 ° C overnight or at 37 ° C for 2 hours. After blocking with 200 μl of 2% skim milk / TBST (Tris-buffered saline-Tween 20), the cells were washed once with TBST, and 50 μl of the hybridoma culture supernatant as the primary antibody was dispensed at 37 ° C for 2 hours Respectively. After washing three times with TBST, goat anti-mouse IgG (Pierce, USA) with horseradish peroxidase (HRP) labeled as a secondary antibody was diluted 5,000 times with 2% skim milk / TBST and 50 ㎕ per well And reacted at 37 ° C for 1 hour. After washing 5 times with TBST, 50 μl of tetramethylbenzidine base (TMB, color reagents) (Surmodics, TMBC) was added to each well. 50 μl of 1N H 2 SO 4 was added to each well to stop the color reaction and the absorbance was measured at 450 nm using a microplate photometer (Multiskan, USA).
상술된 바와 같이 제작된 Hybridoma로부터 생성되는 항체를 ELISA와 western blot으로 스크린 한 결과, 4개 시료에서 양성 반응을 나타내었다. 4개의 시료를 대상으로 ELISA로 3회 클로닝을 실시한 결과, 흡광도가 가장 우수한 융합세포를 선택하여 최종적으로 1개의 clone (19F12)을 선별하였다. 최종적으로 선별한 융합세포 즉 하이브리도마 세포를 "IPNV 19F2"로 명명하였으며, 한국세포주연구재단에 2017년 10월 18일자로 기탁하였다(수탁번호: KCLRF-BP-00414 ). Antibodies generated from the hybridoma prepared as described above were screened by ELISA and western blot, and the results were positive in four samples. Four clones were cloned by ELISA three times, and one clone (19F12) was finally selected by selecting the best-absorbing fusion cell. Finally, the selected hybridized cell or hybridoma cell was designated as "IPNV 19F2 " and deposited on Oct. 18, 2017 (Accession No .: KCLRF-BP-00414) in Korean Cell Line Research Foundation.
기탁된 clone으로부터 생산된 항체를 사용하여 (항체의 이름 : clone의 이름과 동일하게 사용) 특이성 조사를 실시하였다. Specificity studies were conducted using antibodies produced from the deposited clones (using the same name as the antibody name: clone).
실험예 1Experimental Example 1
정제된 바이러스의 구조단백질을 분석하기 위해 sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE)를 실시하였다. 정제된 바이러스에 동량의 SDS-sample buffer (1.59％ SDS, 0.5M Tris-HCl (pH 6.8), 14% glycerol (w/v), 0.01% bromophenol blue (w/v), 4% 2-mecaptoethanol (v/v))를 첨가하여 100℃에서 3분간 열처리한 후 SDS-PAGE (12％ acrylamide separating gel, 4％ acrylamide stacking gel, 30 mA, 2 hours)를 실시하였다. 전기영동 후, 겔은 0.2％ coomassie brilliant blue R-250 (Wako, Japan)로 염색하여 확인하고 그 결과를 도 1에 나타내었다.To analyze the structural proteins of the purified virus, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed. To the purified virus, an equal volume of SDS-sample buffer (1.59% SDS, 0.5M Tris-HCl, pH 6.8, 14% glycerol (w / v), 0.01% bromophenol blue (w / v), 4% 2-mecaptoethanol v / v) was added and heat-treated at 100 ° C for 3 minutes. SDS-PAGE (12% acrylamide separating gel, 4% acrylamide stacking gel, 30 mA, 2 hours) was performed. After electrophoresis, the gel was stained with 0.2% coomassie brilliant blue R-250 (Wako, Japan) and the results are shown in Fig.
도 1은 10%와 55% 사이의 sucrose 농도에서 관찰되는 바이러스 밴드를 취한 후 SDS-PAGE를 실시하여 관찰되는 정제된 IPNV의 구조 단백질을 나타낸 사진이다. CHSE-214 세포에 대량 배양한 IPNV를 사용하여 PEG로 농축한 후, step sucrose gradient에서 초원심을 실시한 결과, 10%와 55% 사이의 sucrose 농도에서 바이러스로 추정되는 밴드가 관찰되었다. 바이러스로 추정되는 밴드를 초원심으로 수집한 후, SDS-PAGE를 실시한 결과, 도 1에 도시된 바와 같이 93 kDa (VP1 protein), 59-55 kDa (VP2 protein), 33 kDa (VP3 protein)의 단백질이 확인되었다.FIG. 1 is a photograph showing the structural protein of purified IPNV observed by performing SDS-PAGE after taking a virus band observed at a sucrose concentration of between 10% and 55%. After concentrating with PEG using IPNV, which was mass-cultured in CHSE-214 cells, supernatant was performed in a step sucrose gradient and a band estimated as a virus was observed at sucrose concentrations of between 10% and 55%. As a result of SDS-PAGE, a band estimated to be a virus was collected by ultracentrifuge, and as a result, 93 kDa (VP1 protein), 59-55 kDa (VP2 protein) and 33 kDa (VP3 protein) The protein was identified.
실험예 2Experimental Example 2
항원에 대한 항체의 인식 능력을 확인하기 위하여 western immunoblot 분석을 실시하고 그 결과를 도 2에 나타내었다. SDS-PAGE는 위와 동일한 방법으로 실시하였다. 정제된 바이러스를 사용하여 전기영동 한 후, gel에 있는 단백질을 transblot 장치 (ATTO, Japan)를 이용하여 144 ㎃에서 1시간 동안 nitrocellurose membrane (advantec, Japan)에 blotting하였다. Blotting 후 2% skim milk로 blocking하여 1시간 반응시킨 후, buffer 1 (0.1M maleic acid, 0.15M NaCl, pH 7.5)으로 15분간 세정하였다. 1차 항체로는 본 연구에서 제작한 hybridoma 배양 상등액을 2% skim milk로 2배 희석하여 1시간 반응시킨 후 위와 동일한 방법으로 세정하였다. 2차 항체로는 alkaline phosphatase (AP)가 표식되어 있는 goat polyclonal anti-mouse IgG antibody (novus, USA)를 2% skim milk로 1,000배 희석하여 1시간 반응시킨 후 5회 세정하고 발색제 (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2용액 (pH 9.5) 20㎖, NBT (75 ㎎/㎖ 4-nitrotetrazolium blue chloride) 90 ㎕, BCIP (50 ㎎/㎖ 5-Bromo-4-chloro-3-indolyl phosphate p-toluidine salt/dimethyl-formamide) 70㎕)로 발색하여 육안으로 확인하였다. 발색이 완료된 후 발색 정지액 (1mM EDTA, 10mM Tris-HCl)을 첨가하였다.Western immunoblot analysis was performed to confirm the recognition ability of the antibody against the antigen, and the results are shown in FIG. SDS-PAGE was performed in the same manner as above. After electrophoresis using the purified virus, the proteins in the gel were blotted on a nitrocellulose membrane (Advantec, Japan) for 1 hour at 144 mA using a transblot device (ATTO, Japan). After blotting, the cells were blocked with 2% skim milk, reacted for 1 hour, and then washed with buffer 1 (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) for 15 minutes. As a primary antibody, the hybridoma supernatant prepared in this study was diluted 2-fold with 2% skim milk, reacted for 1 hour, and then washed in the same manner as above. As a secondary antibody, a goat polyclonal anti-mouse IgG antibody (novus, USA) labeled with alkaline phosphatase (AP) was diluted 1,000 times with 2% skim milk, reacted for 1 hour, washed 5 times, -HCl, 100 mM NaCl, 50
도 2은 단클론 항체의 IPNV 인식 부위를 나타낸 웨스턴 블로팅 결과이다(M: marker (kDa), 1: 19F2).단클론 항체의 IPNV 인식 부위를 조사하기 위해, 정제된 IPNV를 사용하여 western blot을 실시한 결과, IPNV의 VP2 protein (59-55 kDa)을 인식하는 것이 확인되었다.2 is a Western blotting result showing the IPNV recognition site of the monoclonal antibody (M: marker (kDa), 1: 19F2). To examine the IPNV recognition site of the monoclonal antibody, Western blot using purified IPNV As a result, it was confirmed that VP2 protein (59-55 kDa) of IPNV was recognized.
실험예 3Experimental Example 3
VR-299의 serotype에 대한 단클론 항체가 다양한 어류바이러스에 대해 반응하는지를 조사하기 위해, IPNV (VR-299), 해양버나바이러스 (marine birnavirus, MABV), 바이러스성출혈성패혈증바이러스 (Viral hemorrhagic septicemia virus, VHSV), 전염성조혈기괴사증바이러스 (Infectious hematopoietic necrosis virus, IHNV), 넙치랩도바이러스 (hirame rhabdovirus, HIRRV), 잉어봄바이러스혈증바이러스 (spring viraemia of carp virus, SVCV) 및 신경괴사증바이러스 (nervous necrosis virus, NNV)를 사용하여 다음과 같이 western blot을 실시하고, 그 결과를 도 3에 나타내었다.To investigate whether monoclonal antibodies against VR-299 serotypes reacted with various fish viruses, we used IPNV (VR-299), marine birnavirus (MABV), viral hemorrhagic septicemia virus ), Infectious hematopoietic necrosis virus (IHNV), hirame rhabdovirus (HIRRV), spring viraemia of carp virus (SVCV) and nervous necrosis virus (NNV ), The following western blotting was carried out, and the results are shown in Fig.
Western blot에 사용된 바이러스 항원은 7종의 어류바이러스를 chinook salmon embryo (CHSE-214) (IHNV, IPNV, MABV), fathead minnow (FHM) (VHSV), EPC (HIRRV, SVCV) 또는 striped snakehead (SSN-1) (NNV) 세포에 접종한 후 3일째 SDS-sample buffer를 첨가하여 세포를 lysis 시킨 후 100℃에서 3분간 열처리하여 제작하였다. Western blot은 위와 동일한 방법으로 실시하였다.The viral antigens used in the Western blot included seven species of fish viruses: chinook salmon embryo (CHSE-214) (IHNV, IPNV, MABV), fathead minnow (VHV), EPC (HIRRV, SVCV) or striped snakehead -1) (NNV) cells. After 3 days, cells were lysed by adding SDS-sample buffer and heat-treated at 100 ° C for 3 minutes. Western blot was performed in the same manner as above.
도 3은 단클론 항체의 어류바이러스들에 대한 특이성을 나타내는 웨스턴 블로팅 사진이다(P: 양성대조구 (Purified IPNV), M: marker (kDa), 1: IPNV, 2: MABV, 3: VHSV, 4: IHNV, 5: HIRRV, 6: SVCV, 7: NNV, 8: CHSE-214, 9: FHM, 10: EPC, 11: SSN-1). FIG. 3 is a Western blotting photograph showing the specificity of the monoclonal antibody against fish viruses (P: positive control (Purified IPNV), M: marker (kDa), 1: IPNV, 2: MABV, 3: VHSV, IHNV, 5: HIRRV, 6: SVCV, 7: NNV, 8: CHSE-214, 9: FHM, 10: EPC, 11: SSN-1).
도 3에 도시된 바와 같이 본 발명의 단클론 항체19F2를 사용하여 어류바이러스들에 대한 특이성 조사하기 위해, IPNV, MABV, VHSV, IHNV, HIRRV, SVCV 및 NNV에 감염된 세포와 정상 세포 (CHSE-214, FHM, EPC, SSN-1)를 사용하여 western blot을 실시한 결과, 단클론항체 19F2는 Purified IPNV와 IPNV 감염세포에만 반응하였고, MABV, VHSV, IHNV, HIRRV, SVCV, NNV, CHSE-214, FHM, EPC, SSN-1에는 반응하지 않았다.In order to investigate the specificity for fish viruses using the monoclonal antibody 19F2 of the present invention as shown in Fig. 3, cells infected with IPNV, MABV, VHSV, IHNV, HIRRV, SVCV and NNV and normal cells (CHSE- FHM, EPC, and SSN-1), monoclonal antibody 19F2 reacted only with purified IPNV and IPNV-infected cells and MABV, VHSV, IHNV, HIRRV, SVCV, NNV, CHSE-214, , But not SSN-1.
실험예 4Experimental Example 4
VR-299의 serotype에 대한 단클론 항체가 다양한 어류바이러스에 대해 반응하는지를 조사하기 위해, IPNV (VR-299), 해양버나바이러스 (marine birnavirus, MABV), 바이러스성출혈성패혈증바이러스 (Viral hemorrhagic septicemia virus, VHSV), 전염성조혈기괴사증바이러스 (Infectious hematopoietic necrosis virus, IHNV), 넙치랩도바이러스 (hirame rhabdovirus, HIRRV), 잉어봄바이러스혈증바이러스 (spring viraemia of carp virus, SVCV) 및 신경괴사증바이러스 (nervous necrosis virus, NNV)를 사용하여 다음과 같이 ELISA를 실시하고, 그 결과를 도 4에 나타내었다.To investigate whether monoclonal antibodies against VR-299 serotypes reacted with various fish viruses, we used IPNV (VR-299), marine birnavirus (MABV), viral hemorrhagic septicemia virus ), Infectious hematopoietic necrosis virus (IHNV), hirame rhabdovirus (HIRRV), spring viraemia of carp virus (SVCV) and nervous necrosis virus (NNV ), The ELISA was carried out as follows, and the results are shown in Fig.
ELISA는 Purified IPNV와 IPNV (109.55 TCID50/㎖), MABV (109.3 TCID50/㎖), VHSV (109.3 TCID50/㎖), IHNV (107.3 TCID50/㎖), HIRRV CA9703 (108.3 TCID50/㎖), SVCV (107.55 TCID50/㎖) 그리고 NNV (108.05 TCID50/㎖) 상등액을 사용하여 실시하였다. 바이러스 상등액을 증류수로 320배 희석하여 96 well ELISA microplates (Greiner-bio-one, Germany)에 각각 50 ㎕씩 분주한 후 37℃에서 overnight하여 항원을 코팅하였다. T-PBS (0.05% Tween-20/PBS (v/v))로 3회 세정하였고 5% skim milk를 380 ㎕씩 분주하여 25℃에서 1시간 동안 blocking하였다. T-PBS로 3회 세정한 후 1차 항체로는 본 연구에서 제작한 단클론 항체를 시료당 2개의 well에 50 ㎕씩 분주하여 25℃에서 1시간 반응하였다. T-PBS로 3회 세정한 후 horseradish peroxidase (HRP)가 표식되어 있는 goat anti-mouse IgG (Youngin, Korea)를 5% skim milk로 1,000배 희석하여 well 당 50 ㎕씩 분주하였으며, 25℃에서 1시간 반응하였다. T-PBS로 5회 세정한 후 ELISA 발색액 (100mM Na2HPO4,50mMcitricacid,1㎎/㎖ o-phenylen diamine, 0.1% H2O2)을 well 당 50 ㎕ 분주하여 발색하였다. 각 well에 1N H2SO4를 50 ㎕씩 넣어 발색 반응을 중지시킨 후, 490 ㎚에서 흡광도를 측정하였다.ELISA was Purified IPNV with IPNV (10 9.55 TCID 50 / ㎖ ), MABV (10 9.3
도 4는 단클론 항체의 어류바이러스들에 대한 특이성을 확인한 ELISA 결과를 나타낸 그래프이다 (1: Purified IPNV, 2: IPNV, 3: MABV, 4: VHSV, 5: IHNV, 6: HIRRV, 7: SVCV, 8: NNV, 9: CHSE-214, 10: FHM, 11: EPC, 12: MEM10, 13: 2차 증류수). FIG. 4 is a graph showing ELISA results confirming the specificity of a monoclonal antibody against fish viruses (1: Purified IPNV, 2: IPNV, 3: MABV, 4: VHSV, 5: IHNV, 6: HIRRV, 8: NNV, 9: CHSE-214, 10: FHM, 11: EPC, 12: MEM 10 , 13: secondary distilled water).
바이러스 배양액 (IPNV, MABV, VHSV, IHNV, HIRRV, SVCV 및 NNV)과 CHSE-214 배양액, MEM10, 2차 증류수 사용하여 ELISA를 실시한 결과가 도시된 도 4는 단클론 항체19F2가 Purified IPNV와 IPNV에 반응하였고, VHSV, IHNV, HIRRV, SVCV에는 반응하지 않았음을 보여주지만, 특이하게도 NNV에 대해 높은 OD값이 관찰되었음을 보여준다. 이러한 결과는 본 발명의 단클론 항체 19F2가 ELISA법으로는 IPNV 뿐만 아니라 NNV 검출에도 유용함을 보여주었다. Figure 4, which shows the results of ELISA using the culture media of virus (IPNV, MABV, VHSV, IHNV, HIRRV, SVCV and NNV) and CHSE-214 medium, MEM 10 and secondary distilled water, shows that monoclonal antibody 19F2 is expressed in purified IPNV and IPNV And did not respond to VHSV, IHNV, HIRRV, or SVCV, but unusually high OD values were observed for NNV. These results show that the monoclonal antibody 19F2 of the present invention is useful for detection of NNV as well as IPNV by the ELISA method.
본 발명은 이상에서 살펴본 바와 같이 바람직한 실시 예를 들어 도시하고 설명하였으나, 상기한 실시 예에 한정되지 아니하며 본 발명의 정신을 벗어나지 않는 범위 내에서 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 다양한 변경과 수정이 가능할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, Various changes and modifications will be possible.
Claims (7)
A monoclonal antibody that specifically binds to the antigen protein VP2 of the infectious pancreatic necrosis virus (IPNV) produced by a hybridoma cell line deposited with accession number KCLRF-BP-00414.
상기 단일클론항체는 신경괴사증바이러스 (nervous necrosis virus, NNV)에 특이적으로 결합하는 것을 특징으로 하는 단일클론 항체.
The method according to claim 1,
Wherein said monoclonal antibody specifically binds to nervous necrosis virus (NNV).
Accession No. KCLRF-BP-00414 which produces a monoclonal antibody that specifically binds to any one or more of antigen protein VP2 and nervous necrosis virus (NNV) of Infectious Pancreatic Necrosis Virus (IPNV) Lt; / RTI > cell line.
A diagnostic kit for communicable pancreatic necrosis and neurogenic fish in a fish comprising the diagnostic composition of claim 5.
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| KR102091731B1 (en) | 2018-06-07 | 2020-03-20 | 전남대학교산학협력단 | Monoclonal antibody against marine birnavirus and nervous necrosis virus, and use thereof |
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