KR20140003826A - Inactivated vaccine of viral hemorrhagic septicemia - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
- A01K61/13—Prevention or treatment of fish diseases
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- 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
- A61K39/12—Viral antigens
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Abstract
Description
본 발명은 바이러스성 출혈성 패혈증(Viral hemorrhagic septicemia; VHS)에 감염된 넙치로부터 분리된 바이러스성 출혈성 패혈증 바이러스 (수탁번호 KCTC 11932BP) 주 및 이를 항원으로 포함하는 바이러스성 출혈성 패혈증 불활화 백신에 관한 것이다.
The present invention relates to a viral hemorrhagic sepsis inactivated vaccine comprising a viral hemorrhagic sepsis virus (Accession No. KCTC 11932BP) strain isolated from a flounder infected with a viral hemorrhagic septicemia (VHS) and an antigen thereof.
넙치에서 발생하는 주요 바이러스성 질병으로는 바이러스성 출혈성 패혈증(Viral hemorrhagic septicemia; VHS), 바이러스성 신경 괴사증 (Viral nervous necrosis; VNN), 해산 버나바이러스증(Marine birnavirus; MBV), 히라메 랍도바이러스증 (Hirame rhabdovirus; HRV), 이리도바이러스증 등이 있다. Viral hemorrhagic septicemia (VHS), Viral nervous necrosis (VNN), Marine birnavirus (MBV), Hiramabudo virus Hirame rhabdovirus (HRV), and Iridovirus.
국내 양식넙치 생산량의 절반가량을 차지하는 제주지역 넙치의 바이러스성 질병 발생조사를 위해 2007~2008년의 기간 동안 제주특별자치도 해양수산연구원에 질병검사를 의뢰한 결과, 가장 문제가 되는 질병은 바이러스성 출혈성 패혈증인 것으로 나타났다. 특히 바이러스성 출혈성 패혈증은 양식 넙치에서 병원성이 높아 대량폐사를 유발하며, 혼합 감염을 포함하면 전체바이러스성 질병 중 약 60%에 이르는 높은 발생률을 보이고 있다. In order to investigate the occurrence of viral diseases in flounder in Jeju region, which accounts for about half of the production of domestic cultured flounder, during the period from 2007 to 2008, a survey was conducted with the Jeju Special Self-Governing Province Marine Fisheries Research Institute. The most serious diseases were viral hemorrhagic Sepsis. In particular, viral hemorrhagic sepsis causes high mortality due to high virulence in cultured flounder, and it has a high incidence rate of about 60% of all viral diseases including mixed infection.
VHS는 유럽 대륙 인근의 대서양 대구 (Atlantic cod)에서 처음 분리된 이후, 유럽 전역뿐만 아니라 북미, 일본 및 우리나라 등 전 세계적으로 수십여 종의 해수 및 담수어류에 감염되어 많은 피해를 주고 있는 질병이다. 우리나라에서는 2001년 양식 넙치에서 처음 분리 보고된 이후, 양식 넙치뿐만 아니라 동해안과 남해안의 자연산 어류에게서도 검출되었다. Since its first isolation from the Atlantic cod in the European continent, VHS has been infecting dozens of species of seawater and freshwater fish worldwide, including Europe, North America, Japan and Korea. In Korea, it was first detected in cultured flounder in 2001, and then it was detected not only in cultured flounder but also in wild fish of the east coast and southern coast.
VHSV는 랍도바이러스과 (Rhabdoviridae ) 및 Novirhabdovirus에 속하는 바이러스로서, 음성 단일가닥 RNA를 가지고 있고 최적 온도가 9~12℃이며, 4~14℃의 수온 범위에서 일반적으로 발생하는 저수온성 질병이다(Smail, 1999; OIE). VHS발병은 연중 발생하며, 수온이 상승하고 변동하는 봄이 주요 발생시기이다. 저수온기 발병은 일반적으로 폐사율이 낮게 진행되나 누적 폐사율이 높으며, 고수온기에는 바이러스 성장이 억제되어 일정 수준의 누적 폐사만이 관찰된다(Wolf, 1988; Smail, 1999). VHS는 어린 치어기뿐 아니라 출하 시기의 큰 성어에서도 폐사를 일으키며 감염어는 체색흑화, 복막과 복강 내의 투명 복수액 저류, 간 울혈, 비장 비대, 신장종대(Issiki et al., 2001), 간, 신장, 비장 및 골격근에 괴사와 아가미 새엽의 비후, 간농축, 골격근 혈구 축적 등의 내부증상이 나타나며(Manual of diagnostic tests for aquatic animals, 2006), 피부 상피층 또는 아가미 내피 세포를 통해 바이러스가 어체로 침입하여 혈류를 타고 신장, 비장의 조혈 형성조직 등 내부장기로 빠르게 이동하는 것으로 보고되어있다. 또한 VHS는 'OIE notifiable disease'로 규정되어 수산물 유통 시 검역이 되는 질병(OIE, 2001)으로 넙치의 수출입에 있어서 중요한 질병이기도 하다. VHSV is a virus belonging to the bayireoseugwa (Rhabdoviridae) and Novirhabdovirus wax, have a negative single-stranded RNA, and is the optimum temperature for 9 ~ 12 ℃, a low temperature disease which generally occurs in the water temperature range of 4 ~ 14 ℃ (Smail, 1999; OIE). The onset of VHS occurs year-round, and spring is the main time of year when water temperature rises and fluctuates. In general, low-temperature epidemics are associated with low mortality rates but high cumulative mortality rates, while high-temperature epidemics inhibit viral growth and only a certain number of cumulative deaths are observed (Wolf, 1988; Smail, 1999). VHS is not only a young child, but it also causes mortality in large-sized sexually transmitted fishes. Infectious fish are black color, transparent multiple fluid reserves in peritoneal and abdominal cavity, liver congestion, spleen hypertrophy, kidney enlargement (Issiki et al. In this study, we investigated the clinical manifestations of the vagus nerves in the spleen and skeletal muscles, and the intrinsic symptoms such as thickening of the gill lobules, hepatic enrichment and skeletal muscle cell accumulation (Manual of diagnostic tests for aquatic animals, 2006) Have been reported to migrate rapidly to internal organs such as kidneys, hematopoietic tissues of the spleen, and the like. In addition, VHS is defined as 'OIE notifiable disease' and it is a disease which is quarantine in the circulation of marine products (OIE, 2001).
이에 본 발명자들은 바이러스성 출혈성 패혈증에 의한 양식 어가의 경제적 손실을 줄이고 넙치의 원활한 수출 및 유통을 위해 효과적인 VHS 예방용 백신을 개발하고자 노력한 결과, 바이러스성 출혈성 패혈증에 감염된 넙치로부터 신규 바이러스성 출혈성 패혈증 바이러스 주를 분리하고, 이를 불활화하여 백신으로 이용할 수 있음을 확인함으로써, 본 발명을 완성하였다.
Accordingly, the present inventors have made efforts to develop an effective VHS prevention vaccine for reducing the economic loss of cultured fishes due to viral hemorrhagic sepsis and for smooth export and distribution of flounder. As a result, it has been found that a new viral hemorrhagic sepsis virus And isolating the strain and inactivating it and confirming that it can be used as a vaccine, thus completing the present invention.
본 발명의 목적은 신규 바이러스성 출혈성 패혈증 바이러스 주를 제공하는 것이다. It is an object of the present invention to provide a novel viral hematopoietic sepsis virus strain.
본 발명의 또 다른 목적은 불활화된 상기 바이러스성 출혈성 패혈증 바이러스 주를 항원으로 포함하는, 바이러스성 출혈성 패혈증 예방 또는 치료용 백신 조성물을 제공하는 것이다. It is still another object of the present invention to provide a vaccine composition for preventing or treating viral hemorrhagic sepsis, which comprises the inactivated viral hemorrhagic sepsis virus strain as an antigen.
본 발명의 또 다른 목적은 불활화된 상기 바이러스성 출혈성 패혈증 바이러스 주를 넙치에 투여하여 바이러스성 출혈성 패혈증을 예방하는 방법을 제공하는 것이다.
Yet another object of the present invention is to provide a method for preventing viral hemorrhagic sepsis by administering the inactivated viral hemorrhagic sepsis virus strain to flounder.
상기 목적을 달성하기 위하여, 본 발명은 신규 바이러스성 출혈성 패혈증 바이러스 주를 제공한다. In order to achieve the above object, the present invention provides a novel viral hematopoietic sepsis virus strain.
또한 본 발명은 불활화된 상기 바이러스성 출혈성 패혈증 바이러스 주를 항원으로 포함하는, 바이러스성 출혈성 패혈증 예방 또는 치료용 백신 조성물을 제공한다. The present invention also provides a vaccine composition for the prevention or treatment of viral hemorrhagic sepsis comprising the inactivated viral hemorrhagic sepsis virus strain as an antigen.
또한 본 발명은 불활화된 상기 바이러스성 출혈성 패혈증 바이러스 주를 넙치에 투여하여 바이러스성 출혈성 패혈증을 예방하는 방법을 제공한다.
The present invention also provides a method for preventing viral hemorrhagic sepsis by administering the inactivated viral hemorrhagic sepsis virus strain to a flounder.
본 발명에 따른 백신은 바이러스성 출혈성 패혈증을 효과적으로 예방하여 바이러스성 출혈성 패혈증으로 인한 어류의 폐사를 현저하게 감소시킬 수 있으며, 이를 통해 어류의 생산성 증대 및 항생제 사용으로 인한 비용을 감소시키는 우수한 효과를 가지고 있다.
The vaccine according to the present invention can effectively prevent viral hemorrhagic sepsis and significantly reduce fish mortality due to viral hemorrhagic sepsis and thus has an excellent effect of increasing fish productivity and reducing costs due to the use of antibiotics have.
도1은 바이러스성 출혈성 패혈증에 대한 양성 여부를 확인하기 위한 PCR 실험 결과를 나타낸 도이다.
도 2는 VHSV 접종 전 EPC 세포의 모습을 나타낸 도이다.
도 3은 VHSV 접종 후 (약 48시간 후) EPC 세포의 모습을 나타낸 도이다.
도 4는 VHSV 국내 분리주의 계통수 분석 결과를 나타낸 도이다.FIG. 1 is a graph showing the results of PCR tests to confirm the positive for viral hemorrhagic sepsis.
Fig. 2 is a view showing the appearance of EPC cells before VHSV inoculation.
FIG. 3 shows the appearance of EPC cells after VHSV inoculation (about 48 hours).
FIG. 4 is a diagram showing the results of analysis of the VHSV domestic separation system.
본 발명은 수탁번호가 KCTC 11932BP인 바이러스성 출혈성 패혈증(Viral hemorrhagic septicemia; VHS) 바이러스 주를 제공한다. The present invention provides Viral hemorrhagic septicemia (VHS) virus strains with accession number KCTC 11932BP.
또한 본 발명은 불활화된 상기 바이러스성 출혈성 패혈증 바이러스 주를 항원으로 포함하는, 바이러스성 출혈성 패혈증 예방 또는 치료용 불활화 백신 조성물을 제공한다. The present invention also provides an inactivated vaccine composition for the prevention or treatment of viral hemorrhagic sepsis comprising the inactivated viral hemorrhagic sepsis virus strain as an antigen.
또한 본 발명은 불활화된 상기 바이러스성 출혈성 패혈증 바이러스 주를 어류에 투여하여 바이러스성 출혈성 패혈증을 예방하는 방법을 제공한다.
The present invention also provides a method for preventing viral hemorrhagic sepsis by administering the inactivated viral hemorrhagic sepsis virus strain to fish.
이하, 본 발명에 대하여 보다 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명에 따른 신규한 바이러스성 출혈성 패혈증(Viral hemorrhagic septicemia; VHS) 바이러스 주는 국내의 바이러스성 출혈성 패혈증에 감염된 넙치로부터 분리된 것으로서, 2011년 5월 12일 수탁번호 KCTC 11932BP로 한국생명공학연구원에 기탁되었다. The novel viral hemorrhagic septicemia virus (VHS) virus according to the present invention was isolated from the flounder infected with viral hemorrhagic septicemia in Korea and deposited on May 12, 2011 with the deposit number KCTC 11932BP .
상기 수탁번호가 KCTC 11932BP 인 바이러스성 출혈성 패혈증(Viral hemorrhagic septicemia; VHS) 바이러스 주를 불활화하여 제조한 백신은 바이러스성 출혈성 패혈증을 효과적으로 예방하여 바이러스성 출혈성 패혈증으로 인한 어류의 폐사를 현저하게 감소시킬 수 있으며, 이를 통해 어류의 생산성 증대 및 항생제 사용으로 인한 비용을 감소시키는 우수한 효과를 가지고 있다.
The vaccine prepared by inactivating the viral hemorrhagic septicemia virus strain (KCTC 11932BP) is effective in preventing viral hemorrhagic sepsis and significantly reducing fish mortality due to viral hemorrhagic sepsis Which has an excellent effect of increasing fish productivity and reducing the cost of using antibiotics.
본 발명에서 용어, "예방"이란 조성물의 투여로 바이러스성 출혈성 패혈증의 발병을 억제 또는 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" means any act that inhibits or delays the onset of viral hemorrhagic sepsis upon administration of the composition.
본 발명에서 용어, "치료"란 조성물의 투여로 바이러스성 출혈성 패혈증에 의해 이미 유발된 질환의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다.The term "treatment" in the present invention means any action that improves or alleviates symptoms of a disease already caused by viral hemorrhagic sepsis upon administration of the composition.
본 발명에서 용어, "백신"이란 감염 질환을 예방하기 위한 면역을 위하여 쓰이는 항원을 의미한다. 백신에 의해 자극을 받으면 개체 내의 면역 시스템이 작동하여 항체를 생성하게 되고, 그 감수성을 유지하다가 재감염이 일어나는 경우, 빠른 시간 내에 항체를 효과적으로 생성함으로써 질환을 극복할 수 있게 된다.The term "vaccine" in the present invention means an antigen used for immunization to prevent an infectious disease. When stimulated by a vaccine, the immune system in the individual operates to generate antibodies. If the sensitization is maintained and reinfection occurs, the antibody can be effectively generated within a short time, thereby overcoming the disease.
본 발명의 백신은 추가로 약리학적으로 허용가능한 담체 또는 희석제를 포함한다. 백신에 적합한 담체는 기술분야의 당업자에게 공지되어 있으며, 단백질, 설탕 등을 포함하지만, 이로 한정되는 것은 아니다. 상기의 담체는 수용액 또는 비-수용액, 현탁액, 및 에멀전일 수 있다. 비-수용액 담체는 프로필렌 글리콜, 폴리에틸렌 글리콜, 식용유 예컨대 올리브 오일, 및 주사 가능한 유기 에스테르 예컨대 에틸 올레이트를 포함한다. 수용액 담체는 식염수 및 완충배지를 포함하는, 물, 알콜/수용액, 에멀전 또는 현탁액을 포함한다. The vaccine of the present invention further comprises a pharmacologically acceptable carrier or diluent. Suitable carriers for the vaccine are known to those skilled in the art and include, but are not limited to, proteins, sugars and the like. Such carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Non-aqueous carriers include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media.
비경구 담체는 염화나트륨 용액, 링거 덱스트로오스, 덱스트로오스 및 염화나트륨, 유산처리 링거 또는 고정 오일을 포함한다. 정맥 주사용 담체는 예컨대 링거 덱스트로오스를 기본으로 하는 것과 같은 전해질 보충제, 액체 및 영양 보충제 등을 포함한다. 방부제 및 기타 첨가제 예컨대 예를 들면 항미생물제제, 항산화제, 킬레이트제, 불활성 가스 등과 같은 것이 추가로 존재할 수 있다. 바람직한 방부제는 포르말린, 티메로살, 네오마이신, 폴리믹신 B 및 암포테리신 B를 포함한다.Parenteral carriers include sodium chloride solution, Ringer ' s dextrose, dextrose and sodium chloride, lactic acid treatment linger or fixed oils. The intravenous carrier includes electrolyte supplements, liquids and nutritional supplements, such as those based on Ringer ' s dextrose. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like may be additionally present. Preferred preservatives include formalin, thimerosal, neomycin, polyamicin B and amphotericin B.
어주번트는 면역반응의 향상 및/또는 접종 후 흡수 속도를 촉진하는 화합물 또는 혼합물을 칭하는 것으로 임의의 흡수-촉진제를 포함한다. 허용 가능한 어주번트는 프로인트 완전어주번트, 프로인트 불완전어주번트, 사포닌, 미네랄 젤 예컨대 수산화 알루미늄, 계면활성제 예컨대 리소레시틴, 플루론 폴리올, 다중음이온, 펩타이드, 오일 또는 탄화수소 에멀전, 키홀림펫 헤모시아닌, 디니트로페놀 등을 포함하나, 이에 제한되지 않는다. Adjuvant includes any absorption-enhancing agent to refer to a compound or mixture that enhances the immune response and / or promotes the rate of absorption after inoculation. Acceptable ejuvants include, but are not limited to, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oils or hydrocarbon emulsions, But are not limited to, dinitrophenol and the like.
본 발명의 백신 조성물의 투여량은 바람직하게는 0.01mL/미 내지 1mL/미의 범위이나, 개체의 종류에 따라 달라질 수 있으며, 이에 제한되지 않는다. The dose of the vaccine composition of the present invention is preferably in a range of 0.01 mL / mL to 1 mL / well, but it is not limited to this.
본 발명의 백신 조성물은 필요에 따라 기술분야의 당업자에게 공지된 방법, 예를 들면 근육, 피하, 복강, 정맥, 경구, 진피, 또는 안구 접종될 수 있다.
The vaccine composition of the present invention may be administered by a method known to those skilled in the art, for example, muscle, subcutaneous, intraperitoneal, intravenous, oral, dermal, or ocularly as needed.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.
실시예Example 1. 신규 바이러스성 출혈성 패혈증 바이러스의 분리 및 동정 1. Isolation and identification of new viral hematopoietic sepsis virus
2007년~2011년까지 녹십자수의약품 어병진단센터 및 제주자치도 해양수산연구원에서 병성감정한 VHSV양성 가검물을 이용하여 녹십자수의약품 수의연구소에서 EPC (Epithelioma Papulosum Cyprini)세포를 이용하여 바이러스를 분리하였다. VHSV 양성을 확인한 PCR 결과를 도 1에, VHSV 접종 전, 후의 EPC 세포를 관찰한 결과를 도 2 및 도3에 나타내었다. From 2007 to 2011, viruses were isolated from EPC (Epithelioma Papulosum Cyprini) cells at the Green Cross Drug Drug Research Center using the VHSV-positive specimens diagnosed at the Green Cross Drug Fishery Diagnosis Center and the Jeju Autonomous Prefecture Marine Fisheries Research Institute. The results of PCR confirmed that VHSV was positive are shown in Fig. 1, and the results of observation of EPC cells before and after VHSV inoculation are shown in Fig. 2 and Fig.
도 1에 나타낸 바와 같이, VHSV에 양성을 나타내는 가검물을 분리한 후, 도 2 및 도3에 나타낸 바와 같이, VHSV를 EPC 세포에 접종하여 CPE (Cytopathic effect, 세포 병변효과)를 확인하고, 이로부터 총 30개의 분리주를 확보하였다. 이를 표 1에 나타내었다. As shown in Fig. 1, after isolating positive samples showing positive VHSV, VHSV was inoculated into EPC cells as shown in Fig. 2 and Fig. 3 to confirm CPE (cytopathic effect, cytopathic effect) A total of 30 isolates were obtained. This is shown in Table 1.
분리된 VHSV의 유전형을 확인하기 위해 당단백질 유전자 (Glycoprotein gene)와 뉴클레오캡시드 유전자(Nucleocapsid gene)를 부분적으로 시퀀싱하여, Genebank에 등제된 VHSV의 염기서열과 비교하였다. 그 결과를 도 4에 나타내었다. Glycoprotein gene and Nucleocapsid gene were partially sequenced and compared to the nucleotide sequence of VHSV that was sequenced in Genebank to confirm the genotype of isolated VHSV. The results are shown in Fig.
도 4에 나타낸 바와 같이, 현재 VHSV는 크게 4가지의 Genotype으로 구분되는데, 표 1에 나타낸 국내 분리주는 모두 Genotype Ⅳ (Genotype Ⅳa)임을 확인하였으며, 일본 및 북미의 해수어에서 분리되는 분리주와 높은 상동성을 나타내었다. 특히 국내 분리주들은 98.6 %이상의 높은 상동성을 나타내었으며, 분리년도에 따른 염기서열의 변이는 거의 없었다. 30여 개의 분리주 중 GCVP-02주는 세포배양 시 가장 높은 바이러스 역가로 배양되었고, 넙치에서 가장 높은 병원성(폐사율)을 나타내어 이를 백신주로 선발하여, 2011년 5월 12일 한국생명공학연구원에 기탁하였다 (수탁번호 KCTC 11932BP). GCVP-02의 당단백질 유전자 서열을 서열변호 1에, 뉴클레오캡시드 유전자 서열을 서열번호 2에 나타내었다.
As shown in FIG. 4, the present VHSV is largely classified into four genotypes. It is confirmed that all of the domestic isolates shown in Table 1 are Genotype IV (Genotype IVa), and the isolates isolated from Japanese and North American sea- Respectively. Especially, Korean isolates showed high homology of 98.6% or more, and there was little variation in the sequence according to the year of isolation. Among the 30 isolates, GCVP-02 was the most virulent strain cultivated in cell culture, and the highest virulence rate (fluticidal rate) was found in flounder, and it was selected as a vaccine and donated to the Korea Biotechnology Research Institute on May 12, 2011 Accession No. KCTC 11932BP). The glycoprotein gene sequence of GCVP-02 is shown in SEQ ID NO: 1, and the nucleocapsid gene sequence is shown in SEQ ID NO: 2.
실시예Example 2. 바이러스성 출혈성 패혈증 불활화 백신 제조 2. Production of viral hemorrhagic sepsis inactivated vaccine
상기 실시예 1에서 분리한 바이러스성 출혈성 패혈증바이러스 GCVP-02 (기탁번호: KCTC 11932BP)를 마스터 시드 바이러스 (Master Seed Virus, MSV)로 이용하였으며, 이를 불활화하여 백신을 제조하였다. GCVP-02에 대한 정보를 하기 표 2에 나타내었다. The viral hemorrhagic sepsis virus GCVP-02 (accession number: KCTC 11932BP) isolated in Example 1 was used as Master Seed Virus (MSV) and the vaccine was inactivated. Information on GCVP-02 is shown in Table 2 below.
백신의 어주번트는 이뮤노졸과 겔을 혼합하여 사용하였으며, VHSV 배양을 위해 EPC 세포 (Epithelioma Papulosum Cyprini cell, ATCC No. CRL-2872)를 사용하였다. 세포 및 바이러스 증식용 배지 및 배양용 배지의 조성을 하기 표 3 및 표4에 나타내었다.
The vaccine was prepared by mixing an immunosol and a gel, and EPC cells (Epithelioma Papulosum Cyprini cell, ATCC No. CRL-2872) were used for VHSV culture. The composition of the cell and virus growth medium and the culture medium are shown in Tables 3 and 4 below.
바이러스 벌크를 준비하기 위하여, MSV(Master Seed Virus)를 EPC 세포에서 최대 4대까지 계대한 VHSV를 WSV (Working Seed Virus)로 하였다. WSV는 -80℃ 이하에서 보존하거나, 동결건조하여 -20℃에 보관하였다. 종독을 희석하여 단층이 형성된 플라스크에 접종하고, 20℃에서 48～72시간 배양하여 CPE가 80% 이상 형성된 시점에서 바이러스를 채독한 후 불활화 전까지 -40℃에 보관하였다. To prepare the virus bulk, MSV (Master Seed Virus) was used as a Working Seed Virus (VHSV) for up to 4 units in EPC cells. WSV was stored at -80 캜 or lower, lyophilized and stored at -20 캜. The virus was inoculated into the monolayer flask diluted with the genus, incubated at 20 ° C for 48 to 72 hours, and then stored at -40 ° C until the inactivation.
채독한 바이러스 벌크 중 일부를 채취하여 표준운영지침서 SOP Bio 7-013에 의거하여 무균시험을 실시하고, 표준운영지침서 SOP Bio 1-009에 의거하여 바이러스 함량시험을 실시한 결과, 107.5~108.1 TCID50/mL의 함량을 보였다. Some of the viruses that were taken were taken and sterilized according to the standard operating instructions SOP Bio 7-013 and subjected to the virus content test according to the standard operating instructions SOP Bio 1-009, resulting in 10 7.5 to 10 8.1 TCID 50 / mL.
또한, 채독된 벌크를 표준운영지침서 SOP Bio 1-011, “바이러스 채독액의 BEI 불활화”에 의거하여 불활화시켰다. 불활화 후 얻은 물질은 멸균 가제나 동망을 사용하여 세포 잔해물 (cell debris)을 제거한 후 어주번트 혼합 전까지 2～8℃ 냉실에 보관하였다. 보존제로 티메로살 용액을 사용하였으며, 최종 농도가 0.01%가 되도록 한 후, 벌크로부터 무균시험 및 불활화 확인 실험을 수행하였다. 불활화 확인 실험을 위해, 불활화된 VHSV 벌크 10배 희석액을 EPC 세포 또는 이와 감수성이 동등한 세포에 접종하여 20℃에서 1시간 감작한 후 7일간 배양하였다. 이를 연속 2회 실시한 후 CPE가 확인되지 않아야 불활화가 이루어졌다고 판단하였다. In addition, the collected bulk was inactivated according to the standard operating instructions SOP Bio 1-011, " BEI inactivation of viral clearance solution ". After inactivation, the cell debris was removed using a sterile gauze or a copper mesh, and then stored in a cold room at 2-8 ° C until the mixture was mixed. The thimerosal solution was used as a preservative, and the final concentration was adjusted to 0.01%. Then, aseptic test and inactivation confirmation experiment were performed from the bulk. For inactivation confirmation experiments, an inactivated VHSV bulk 10-fold dilution was inoculated into EPC cells or cells whose sensitivity was equivalent thereto, sensitized for 1 hour at 20 ° C, and then cultured for 7 days. After two consecutive sessions, it was judged that the CPE had not been confirmed before it was inactivated.
불활화된 바이러스성 출혈성 패혈증 바이러스 (항원농도: 108.0TCID50/㎖) 벌크, 겔 또는 이뮤노졸(Seppic)을 9:1, 8:2의 부피 비율로 혼합하여, 최종 백신을 제조하였다. 상기 백신은 폴리프로필렌 또는 이와 유사 재질의 용기를 사용하였으며, 고무마개 및 타공 알루미늄 캡, 그리고 마지막으로 PP캡을 사용하여 밀봉한 후 2~5℃의 냉암소에 보관하였다.
A final vaccine was prepared by mixing bulk, gel or immunosol (Seppic) at volume ratios of 9: 1, 8: 2 inactivated viral hemorrhagic sepsis virus (antigen concentration: 10 8.0 TCID 50 / ml). The vaccine used was a polypropylene or similar container, sealed with a rubber stopper and a perforated aluminum cap, and finally with a PP cap and stored in a cold dark place at 2-5 ° C.
실험예Experimental Example 1. 바이러스성 출혈성 패혈증 불활화 백신의 자가실험 1. Self-testing of viral hemorrhagic sepsis inactivated vaccine
상기 실시예 2에서 제조한 바이러스성 출혈성 패혈증 불활화 백신을 이용하여 하기 표 5와 같이 시험 백신을 제조하였다.
Using the viral hemorrhagic sepsis inactivated vaccine prepared in Example 2, a test vaccine was prepared as shown in Table 5 below.
1-1. 무균시험1-1. Aseptic test
상기 표 5의 시험 백신을 이용하여 무균시험을 수행하였다. 그 결과를 표 6에 나타내었다.
A sterile test was carried out using the test vaccines of Table 5 above. The results are shown in Table 6.
～ 10. 12Oct 05, 2011
~ 10. 12
～ 12. 21
~ 12. 21
～ 2. 20Feb 13, 2012
~ 2.20
표 6에 나타낸 바와 같이, 무균실험 결과 시험 백신에서 아무런 이상이 발생하지 않음을 확인하였다.
As shown in Table 6, the aseptic experiment confirmed that no abnormality occurred in the test vaccine.
1-2. 1-2. 수소이온농도Hydrogen ion concentration 시험 exam
상기 표 5의 시험 백신을 이용하여 동물용생물학적제제 일반검정 기준 수소이온농도 시험법에 따라 수소이온농도를 측정하였다. 그 결과를 표 7에 나타내었다.
Using the test vaccine of Table 5, the hydrogen ion concentration was measured according to the test method for hydrogen ion concentration in general test for animal biological preparations. The results are shown in Table 7.
표 7에 나타난 바와 같이, 시험 백신의 수소이온농도는 동물용의약품 생물학적제제 일반검정 기준에 따라 pH6.0~8.0범위 안에 있음을 확인하였다.
As shown in Table 7, the hydrogen ion concentration of the test vaccine was confirmed to be in the range of pH 6.0 to 8.0 according to the general test standard for veterinary pharmaceuticals.
1-3. 방부제 정량 시험1-3. Preservative Quantitative Test
상기 표 5의 시험 백신을 이용하여 국가검정동물용의약품 검정기준의 동물용 생물학적제제 일반시험법에 따라 포르말린 정량시험을 수행하였다. 그 결과를 표 8에 나타내었다.
Formalin quantitative test was carried out using the test vaccine of Table 5 according to the General Test Methods for Animal Biological Agents of the National Test for Veterinary Drugs. The results are shown in Table 8.
Porpholine Content
표 8 나타낸 바와 같이, 방부제 정량 시험 결과, 시험 백신에서 규정량 이하를 포함하고 있음을 확인하였다.
As shown in Table 8, it was confirmed that the preservative dosing test contained less than the specified amount in the test vaccine.
1-4. 불활화 확인 시험1-4. Inactivation confirmation test
상기 표 5의 시험 백신을 이용하여 국가 검정법에 따라 불활화 확인 시험을 수행하였다. 그 결과를 표 9에 나타내었다.
Inactivation confirmation tests were performed according to the National Assay Method using the test vaccines of Table 5 above. The results are shown in Table 9.
～ 10. 19Oct 05, 2011
~ 10. 19
～ 12. 28
~ 12. 28
～ 2. 27Feb 13, 2012
~ 2. 27
표 9에 나타낸 바와 같이, 무균실험 결과 시험 백신에서 CPE가 나타나지 않아, 불활화가 정상적으로 이루어졌음을 확인하였다.
As shown in Table 9, aseptic test results showed that the CPE did not appear in the test vaccine, indicating that the inactivation was normal.
1-5. 안전성 시험1-5. Safety test
넙치에 상기 표 5의 시험 백신을 처리한 후 21일 동안 임상증상을 관찰하였다. 그 결과를 표 10에 나타내었다.
The flounder was treated with the test vaccine of Table 5 above and the clinical symptoms were observed for 21 days. The results are shown in Table 10.
~ 11.21October 24, 2011
~ 11.21
~ 1.25April 14, 2012
~ 1.25
~ 3.21February 29, 2012
~ 3.21
표 10에 나타낸 바와 같이, 안전 시험 결과, 모든 넙치가 아무 이상 없이 생존하며, 백신으로 이용될 수 있는 안전성을 가짐을 확인하였다.
As shown in Table 10, as a result of the safety test, it was confirmed that all flounder survives without any abnormality and has safety that can be used as a vaccine.
1-6. 효력 시험1-6. Effectiveness test
백신군에 상기 표 5의 시험 백신을 2회 접종하고 3주 후에, 백신군 및 대조군 각 20미에 미리 확인한 공격접종량 결정시험 결과에 따라 바이러스성 출혈성 패혈증 바이러스를 105.0 TCID50/fish의 농도로 복강 접종하였다. 이 후, 대조군의 최소 누적폐사율이 60% 되는 시점에서 백신군의 상대생존율을 조사하였다. 그 결과를 표 11에 나타내었다.
The vaccine group was inoculated twice with the test vaccine of Table 5, and after 3 weeks, the viral hemorrhagic septic virus was infused at a concentration of 10 5.0 TCID 50 / Respectively. Subsequently, the relative survival rate of the vaccine group was examined when the minimum cumulative mortality rate of the control group was 60%. The results are shown in Table 11.
표 11에 나타낸 바와 같이, 백신군에서 대조군에 비하여 70% 이상의 높은 상대생존율을 보임을 확인하였으며, 이를 통해 본 발명의 바이러스성 출혈성 패혈증바이러스 GCVP-02 (기탁번호: KCTC 11932BP)가 불활화 백신으로 이용될 수 있음을 확인하였다.
As shown in Table 11, the vaccine group showed a high relative survival rate of 70% or more as compared with the control group. As a result, the viral hemorrhagic sepsis virus GCVP-02 (accession number: KCTC 11932BP) It can be used.
<110> Jeju special Self-Governing Province, Ocean and Fisheries Reseach Institute GREEN CORSS VETERINARY PORDUCTS CO. <120> Inactivated vaccine of Viral hemorrhagic septicemia <130> 110 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 563 <212> DNA <213> Paralichthys olivaceus <400> 1 aggtgtcccc atgagttcga ggacacaaac aagggcttgg tctctgtccc agcccagatc 60 atccatcttc cgttatcagt caccagcgtc tcagcagtcg caaatggcca ctacctacac 120 agagtgacct accgggtcac ctgctcaacc aatttcttcg gaggacaaac cattgaaaaa 180 accatcctgg aggcaaagct gtcccgtcaa gaggccatca atgaggctgg caaggatcac 240 gagtaccctt tcttccccga accttcctgc atctggatga aggacaatgt ccacaaggac 300 ataacccact attacaagac cccaaagaca gtgtccattg acctctacag tagaaagttt 360 ctaaaccctg acttcataga gggggtttgt acaacatcac cctgcccaac ccactggcaa 420 ggagtctact ggatcggcgc tacacctcag gcccattgcc ctacctcaga aacgcttaag 480 gggcatctgt tcaccaggac acatgatcac agggtggtca aggcaatcgt tgcgggtcac 540 cacccctggg ggctcacaat ggc 563 <210> 2 <211> 421 <212> DNA <213> Paralichthys olivaceus <400> 2 atggaagggg gaatccgcgc agctttttca ggcctgaacg atgtcaggat cgaccccact 60 ggaggagagg gacgggtgct tgtccctggt gaagtggaac tcatcgtgta tgctggtccc 120 tttggtgcag atgatgggaa ggtgatcgtg gatgcacttg ccgcacttgg aggaccccag 180 actgtgcaag cactgtccgt acttctctcc tttgtattcc aaggaagtgc acaaggagat 240 ctggaggcaa agtgcaagat cctcactgac atgggcttca aggtgacaca gtcaccccgg 300 gcaactggta tcgaagccgg aatccttatg ccgatgaggg aactggccca gactgtcaac 360 aacgacaacc tcatggacat cgtcaagggg gccctgatga cgtgttccct tttgaccaag 420 t 421 <110> Jeju special Self-Governing Province, Ocean and Fisheries Reseach Institute GREEN CORSS VETERINARY PORDUCTS CO. <120> Inactivated vaccine of Viral hemorrhagic septicemia <130> 110 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 563 <212> DNA <213> Paralichthys olivaceus <400> 1 aggtgtcccc atgagttcga ggacacaaac aagggcttgg tctctgtccc agcccagatc 60 atccatcttc cgttatcagt caccagcgtc tcagcagtcg caaatggcca ctacctacac 120 agagtgacct accgggtcac ctgctcaacc aatttcttcg gaggacaaac cattgaaaaa 180 accatcctgg aggcaaagct gtcccgtcaa gaggccatca atgaggctgg caaggatcac 240 gagtaccctt tcttccccga accttcctgc atctggatga aggacaatgt ccacaaggac 300 ataacccact attacaagac cccaaagaca gtgtccattg acctctacag tagaaagttt 360 ctaaaccctg acttcataga gggggtttgt acaacatcac cctgcccaac ccactggcaa 420 ggagtctact ggatcggcgc tacacctcag gcccattgcc ctacctcaga aacgcttaag 480 gggcatctgt tcaccaggac acatgatcac agggtggtca aggcaatcgt tgcgggtcac 540 cacccctggg ggctcacaat ggc 563 <210> 2 <211> 421 <212> DNA <213> Paralichthys olivaceus <400> 2 atggaagggg gaatccgcgc agctttttca ggcctgaacg atgtcaggat cgaccccact 60 ggaggagagg gacgggtgct tgtccctggt gaagtggaac tcatcgtgta tgctggtccc 120 tttggtgcag atgatgggaa ggtgatcgtg gatgcacttg ccgcacttgg aggaccccag 180 actgtgcaag cactgtccgt acttctctcc tttgtattcc aaggaagtgc acaaggagat 240 ctggaggcaa agtgcaagat cctcactgac atgggcttca aggtgacaca gtcaccccgg 300 gcaactggta tcgaagccgg aatccttatg ccgatgaggg aactggccca gactgtcaac 360 aacgacaacc tcatggacat cgtcaagggg gccctgatga cgtgttccct tttgaccaag 420 t 421
Claims (5)
Viral hemorrhagic septicemia (VHS) virus strain with accession number KCTC 11932BP.
The virus strain of claim 1, wherein the virus strain is isolated from flounder infected with viral hemorrhagic sepsis.
A vaccine composition for preventing or treating viral hemorrhagic sepsis, comprising the inactivated viral hemorrhagic sepsis virus strain of claim 1 as an antigen.
The vaccine composition of claim 3, further comprising a carrier or adjuvant.
A method for preventing viral hemorrhagic sepsis by administering the inactivated viral hemorrhagic sepsis virus strain to the flounder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020120070671A KR101464310B1 (en) | 2012-06-29 | 2012-06-29 | Inactivated vaccine of Viral hemorrhagic septicemia |
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| KR1020120070671A KR101464310B1 (en) | 2012-06-29 | 2012-06-29 | Inactivated vaccine of Viral hemorrhagic septicemia |
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| KR101464310B1 KR101464310B1 (en) | 2014-11-21 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20180092588A (en) * | 2017-02-10 | 2018-08-20 | 부경대학교 산학협력단 | Process for the production of fish pathogenic viruses using cell on microcarrier bead |
| WO2020008219A1 (en) | 2018-07-03 | 2020-01-09 | Genima Innovations Marketing Gmbh | Heater for beverages in open cups |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR102301833B1 (en) * | 2019-10-30 | 2021-09-14 | 주식회사 바이오쓰리에스 | Composition for treating or preventing cancer containing viral haemorrhagic septicaemia virus |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010514412A (en) * | 2006-12-22 | 2010-05-06 | ソリューシオネス バイオテクノロジカス イノヴァシオン リミターダ | DNA fish vaccine |
| JP2010065027A (en) | 2008-08-12 | 2010-03-25 | Mie Univ | Inactivated vaccine to fish viral hemorrhagic septicemia and formula for the same |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20180092588A (en) * | 2017-02-10 | 2018-08-20 | 부경대학교 산학협력단 | Process for the production of fish pathogenic viruses using cell on microcarrier bead |
| WO2020008219A1 (en) | 2018-07-03 | 2020-01-09 | Genima Innovations Marketing Gmbh | Heater for beverages in open cups |
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| KR101464310B1 (en) | 2014-11-21 |
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