KR102106168B1 - Combination Vaccines Against Streptococcal Disease in Fish - Google Patents
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Abstract
Description
본 발명은 스트렙토코커스 균주를 이용한 연쇄구균병 예방용 백신에 관한 것으로, 더욱 상세하게는 불활성화된 스트렙토코커스 파라우베리스 균주를 포함하는 어류의 연쇄구균병 예방용 다가백신, 그 제조방법 및 투여방법에 관한 것이다.The present invention relates to a vaccine for preventing streptococcal disease using a Streptococcus strain, and more specifically, a multivalent vaccine for preventing streptococcal disease in fish containing the inactivated Streptococcus parauberis strain, a method of manufacturing and administration thereof It is about.
연쇄구균병(Streptococcosis)은 자연산 및 양식산 담수어 및 해산어의 패혈증을 유발하는 질병이다. 해산어류인 방어(Yellowtail; Seriolae spp.), 뱀장어(Eel; Anguilla japonica) 등 전세계적으로 다양한 어종에 감염되어 질병을 유발한다. Streptococcus parauberis, Streptococcus iniae, Streptococcus agalactiae, Streptococcus dysgalactiae 종은 전세계 어느 지역에 상관없이 어류에 발생하여 심각한 피해를 주고 있다.Streptococcosis is a disease that causes sepsis in fresh and marine freshwater and marine fish. It is infected with various fish species worldwide, such as yellowtail ( Seriolae spp.) And eel (Eel; Anguilla japonica ), causing disease. Streptococcus parauberis , Streptococcus iniae , Streptococcus agalactia e, and Streptococcus dysgalactiae species occur severely in fish anywhere in the world.
2014년 세계 수산물 생산량은 1억9,572만톤이며 그 중 어획생산량은 9,463만톤으로 최근 몇 년간 비슷한 수준을 보이고 있는 반면, 양식생산량은 1억109만톤으로 해마다 증가하여, 2025년 양식 생산량은 1억200만톤으로 2013~2015년 수준에서 39% 증가할 것으로 예상하고 있다(FAO). 2017년 국내 어류양식 생산량은 86,387톤으로, 어종별 생산량은 2017년도에 넙치가 41,207톤(전체 생산량의 47.7%)이었으며, 같은 해 생산금액 역시 넙치가 5,845억원(57.9%)을 기록하여 넙치가 가장 대표적인 우리나라 어류 양식 품종임을 알 수 있다(어업생산통계, 통계청, 2018). 넙치는 해양수산부가 추진하는 수산분야의 10대 수출전략 품목으로 지정되었으며, 현재 양식 수산물 전략품목육성 연구개발 사업으로 추진되는 부가가치가 높은 품종이다.In 2014, the world production of marine products was 193.57 million tons, of which the production of fish was 94.3 million tons, similar to the level in recent years, while the production of aquaculture increased to 109.11 million tons annually. Therefore, it is expected to increase by 39% from 2013 to 2015 level (FAO). In 2017, domestic fish farming production amounted to 86,387 tons, and the production by fish type was 41,207 tons (47.7% of total production) in 2017, and in the same year, the production amount of halibut was 584 billion won (57.9%). It can be seen that it is a representative Korean fish farming variety (Fish Production Statistics, Statistics Korea, 2018). Olive flounder has been designated as one of the top 10 export strategic items in the fisheries sector promoted by the Ministry of Oceans and Fisheries, and is currently a high value-added variety promoted as a research and development project for aquaculture products.
양식 넙치의 질병으로 인한 피해는 연중 생산량의 30%에 달하는 연간 1.2만여톤 정도로 추정되며, 2018년 양식 넙치에 폐사를 가장 많이 유발한 질병으로 기생충성 질병인 스쿠티카병(44.5%), 여윔증(12%), 바이러스성출혈성패혈증(16.6%), 세균성 질병인 연쇄구균병(9.7%)으로 확인되고 있다(양식생물 폐사동향 조사 및 활어수송용 소독장치 효과 검증 연구, 국립수산과학원 연구용역, 2018). 최근 우리나라 넙치에 가장 많이 발생하는 질병으로 기생충성 질병인 스쿠티카병(61%), 바이러스성출혈성 패혈증(10.6%), S. parauberis의 의해 발생하는 연쇄구균병(9.8%)이 보고되어 있다(양식장 폐사현황 및 폐사체 처리방안 연구, 2014-2016, 국립수산과학원). 넙치 양식에서 문제가 되는 연쇄구균은 S. iniae, S. parauberis로 알려져 있으며, 2003년에서 2005년 제주지역 양식 넙치에서 분리된 연쇄구균을 동정한 결과, S. parauberis 54%, S. iniae 46%의 비율로 분리되었다. 그러나 2000년대 후반부터 현재까지 병든 넙치에서 S. iniae의 비율이 낮아져 최근에 거의 분리되지 않고 있으며, 대부분 S. parauberis가 분리되고 있는 실정이다.The damage caused by diseases of farmed halibut is estimated to be about 120,000 tons per year, which accounts for 30% of the annual production, and the most common cause of mortality in farmed halibut in 2018 is the parasite-related disease, Scutica disease (44.5%), nymphosis ( 12%), viral hemorrhagic septicemia (16.6%), and bacterial disease, streptococcal disease (9.7%) (investigation of mortality of aquatic organisms and research on the effectiveness of disinfection devices for live fish transportation, National Fisheries Research Institute, 2018) ). Recently, the most common diseases in Korean flounder have been reported, parasitic diseases such as Scutica disease (61%), viral hemorrhagic sepsis (10.6%), and streptococcal disease caused by S. parauberis (9.8%). Research on the status of mortality and treatment of dead bodies, 2014-2016, National Institute of Fisheries Science). Streptococcus that is problematic in flounder farming is known as S. iniae and S. parauberis, and as a result of identifying streptococcus isolated from cultured flounder in Jeju from 2003 to 2005, S. parauberis 54%, S. iniae 46% Was separated at the rate of. But in the diseased flounder from the late 2000s to the present low proportion of S. iniae are not separated in the last few, most of the situation with S. parauberis separated.
양식 어류의 병원체 검출률이 매년 지속적으로 증가하고 있으며, 현재 넙치에 상용화되어 있는 연쇄구균(S. parauberis) 백신 보급에도 불구하고 지속적으로 해당 질병이 발생하고 있어, 현 백신 항원의 개량이 필요한 실정이다. 항생·항균제의 오남용에 의한 약제 내성균 및 다제 내성균 출현으로 치료효능이 감소되고 있어, 질병이 발생한 후 화학요법제로 치료하는 것은 점차 어려운 실정이므로 질병을 사전 예방할 수 있는 백신개발이 필요하다. 실제로 우리나라에서는 넙치의 S. parauberis의 백신은 2009년부터 상업화되어 보급되었으나, 백신 보급 이후에도 S. parauberis는 지속적으로 발생하고 있다(수산생물질병특성연구, 2012-2017). 일반적으로 넙치에 가장 높은 폐사를 유발하는 스쿠티카병은 치어기에 발생하지만, 연쇄구균병은 중간육성어, 성어 등에 발생하여 보다 큰 경제적인 피해를 유발하고 있다. 최근 양식 넙치에 발생한 질병에 의한 폐사 원인 분석 결과, 세균성 질병에 의한 폐사가 13%로 이중 연쇄구균병에 의한 폐사가 67.1%로 가장 높게 나타났다.The detection rate of pathogens in aquaculture fish is constantly increasing every year, and despite the spread of the S. parauberis vaccine, which is currently commercialized for halibut, the disease continues to occur, and thus it is necessary to improve the current vaccine antigen. With the emergence of drug-resistant bacteria and multi-drug-resistant bacteria caused by misuse of antibiotics and antibacterial agents, treatment efficacy with chemotherapeutic agents after the disease is gradually becoming difficult, so it is necessary to develop a vaccine that can prevent disease in advance. Indeed, in Korea, the vaccine of S. parauberis from halibut has been commercialized and distributed since 2009, but S. parauberis has been continuously occurring even after the vaccine was supplied (Aquatic Products Disease Research, 2012-2017). In general, Scutica disease, which causes the highest mortality in halibut, occurs in sting, but streptococcal disease occurs in medium-growth fish, adult fish, and the like, causing greater economic damage. As a result of the analysis of the causes of mortality caused by diseases in aquaculture halibut, mortality caused by bacterial disease was 13%, and the death caused by streptococcal disease was the highest (67.1%).
국내에서는 S. parauberis 불활화 백신, 재조합 단백질백신, 약독화 백신 등의 다양한 연구가 꾸준히 진행되고 있으나, 현재 다른 세균 병원체와 혼합백신 형태로 제작된 불활화 백신만이 상용화되어 시판되고 있다(6개사 9개 제품). 스페인에서 터봇 대상으로 S. parauberis를 항원으로 하는 Hipra사의 ICTHIOVACⓡ-STR 불활화 주사 백신이 개발되어 판매되고 있으나 국내에서는 S. parauberis에 대한 단독백신은 판매되고 있지 않다. 어류 양식 산업의 특성상 저비용의 어류 백신 개발이 필수적이므로 여러 가지 세균 병원체를 한꺼번에 예방 가능한 혼합 백신이 상용화되어 있으나, 실제로 혼합 백신 사용 이후에 백신 효과에 대한 조사는 미미한 실정이다. 넙치에서 S. parauberis에 의한 연쇄구균병은 백신 보급 이후에도 지속적으로 발생하고 있어, 현재 상용화된 백신의 효능에 대한 검증이 필요하다.In Korea, various studies such as S. parauberis inactivated vaccine, recombinant protein vaccine, and attenuated vaccine have been conducted steadily, but currently only inactivated vaccines produced in the form of mixed vaccines with other bacterial pathogens are commercially available and commercially available (6 companies) 9 products). In Spain, Hipra's ICTHIOVAC (R) -STR inactivating vaccine, which targets S. parauberis as an antigen for turbots, has been developed and marketed, but in Korea, a single vaccine against S. parauberis has not been sold. Due to the nature of the fish farming industry, the development of low-cost fish vaccines is essential, so mixed vaccines that can prevent various bacterial pathogens are commercially available. However, the investigation of vaccine effects after using the mixed vaccines is insignificant. Streptococcus disease caused by S. parauberis in halibut continues to occur even after the vaccine is supplied, and it is necessary to verify the efficacy of the currently commercialized vaccine.
사람의 인플루엔자백신이나 폴리오백신과 같이 동종(同種)의 많은 형, 즉 인플루엔자의 경우 A1, A2, B형, 폴리오의 경우 I, I, III형을 섞어서 만드는 백신을 다가백신(polyvalent vaccine)이라고 하며, 이러한 병원체는 항원형이 몇 개로 나뉘어 있어, 따로 포함되어 있는 백신(단가백신)으로 예방접종한 경우, 질병 유형이 백신과 전혀 다른 유형으로 발생한다면 도움이 되지 않기 때문에 다가백신을 활용하고 있다. 사람에서 폐렴을 유발하는 연쇄구균인 S. pneumoniae의 경우 다양한 혈청형이 있어 이를 모두 예방할 수 있는 23가 다가 피막다당류 백신이 사용되고 있다(송준영, 정희진 2014 폐렴구균백신, J. Korean Med Assoc 57(9), 780-788).A vaccine made by mixing many types of the same type of human influenza vaccine or polio vaccine, that is, A1, A2, B type for influenza, and I, I, III type for polio, is called a polyvalent vaccine. , These pathogens are divided into several types of antigens, so if they are vaccinated with a vaccine (unit price vaccine) that is included separately, the multivalent vaccine is used because it does not help if the disease type occurs in a completely different type from the vaccine. In the case of S. pneumoniae , a streptococcus that causes pneumonia in humans, there are various serotypes, and 23 multivalent polysaccharide vaccines are used to prevent them (Jun-Young Song, Hee-Jin Jeong 2014, pneumococcal vaccine, J. Korean Med Assoc 57 (9 ), 780-788).
이에, 본 발명자들은 넙치에서 질병을 유발하는 S. parauberis에 대하여 Multilocus sqquecne typing(MLST) 방법을 통해 분자역학 및 혈청형 조사를 실시하여, 국내 발생하는 넙치 연쇄구균 S. parauberis 백신에 적합한 균주를 선정하기 위한 기초자료를 확보하였으며, 각각의 혈청형별 단가백신을 제작하여 교차 백신 효능을 평가하였다. 교차 백신 효능 평가 결과를 바탕으로 S. parauberis 다가백신 개발을 위해 혈청형이 다른 S. parauberis 균주를 혼합하여 백신 효능을 평가하고, 본 발명을 완성하였다.Accordingly, the present inventors conducted molecular epidemiology and serotype investigation through the Multilocus sqquecne typing (MLST) method for S. parauberis causing disease in halibut, selecting a suitable strain for the halibut streptococcus S. parauberis vaccine in Korea. Basic data for the following were secured, and cross-vaccination efficacy was evaluated by producing a unit price vaccine for each serotype. Based on the results of the cross vaccine efficacy evaluation, the vaccine efficacy was evaluated by mixing S. parauberis strains with different serotypes for the development of the S. parauberis multivalent vaccine, and the present invention was completed.
본 배경기술 부분에 기재된 상기 정보는 오직 본 발명의 배경에 대한 이해를 향상시키기 위한 것이며, 이에 본 발명이 속하는 기술분야에서 통상의 지식을 가지는 자에게 있어 이미 알려진 선행기술을 형성하는 정보를 포함하지 않을 수 있다.The above information described in this background section is only for improving the understanding of the background of the present invention, and thus does not include information that forms prior art already known to those of ordinary skill in the art. It may not.
본 발명의 목적은 불활성화된 스트렙토코커스 파라우베리스(Streptococcus parauberis) 균주를 항원으로 포함하는 연쇄구균병 예방용 불활성화 백신을 제공하는 데 있다.An object of the present invention is to provide an inactivated vaccine for preventing streptococcal disease comprising an inactivated Streptococcus parauberis strain as an antigen.
본 발명의 다른 목적은 연쇄구균병에 감염된 넙치로부터 분리된 스트렙토코커스 파라우베리스(Streptococcus parauberis) 균주를 포르말린으로 불활성화시켜 백신을 수득하는 단계를 포함하는 것을 특징으로 하는 연쇄구균병 예방용 불활성화 백신의 제조방법을 제공하는 데 있다.Another object of the present invention comprises the step of obtaining a vaccine by inactivating Streptococcus parauberis strain isolated from halibut infected with Streptococcus disease with formalin to obtain a vaccine. It is to provide a method for producing a vaccine.
본 발명의 또 다른 목적은 상기 백신을 어류에 투여하는 것을 포함하는 어류를 면역시키는 방법을 제공하는 데 있다.Another object of the present invention is to provide a method for immunizing fish comprising administering the vaccine to the fish.
상기 목적을 달성하기 위하여, 본 발명은 불활성화된 스트렙토코커스 파라우베리스(Streptococcus parauberis) KCTC 13800BP 균주, KCTC 13801BP 균주, KCTC 13802BP 균주 또는 KCTC 13803BP 균주를 항원으로 포함하는 연쇄구균병 예방용 불활성화 백신을 제공한다.In order to achieve the above object, the present invention is an inactivated Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain or KCTC 13803BP strain as an antigen, an inactivated vaccine for preventing streptococcal disease Gives
본 발명은 또한, 연쇄구균병에 감염된 넙치로부터 분리된 스트렙토코커스 파라우베리스(Streptococcus parauberis) KCTC 13800BP 균주, KCTC 13801BP 균주, KCTC 13802BP 균주 또는 KCTC 13803BP 균주를 포르말린으로 불활성화시켜 백신을 수득하는 단계를 포함하는 것을 특징으로 하는 연쇄구균병 예방용 불활성화 백신의 제조방법을 제공한다.The present invention also provides a step of obtaining a vaccine by inactivating Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain, or KCTC 13803BP strain isolated from halibut infected with streptococcal disease. It provides a method for producing an inactivated vaccine for preventing streptococcal disease characterized in that it comprises.
본 발명은 또한, 상기 백신을 어류에 투여하는 것을 포함하는 어류를 면역시키는 방법을 제공한다.The present invention also provides a method of immunizing fish comprising administering the vaccine to the fish.
본 발명은 어류, 특히 양식 넙치의 연쇄구균병을 효과적으로 예방하여 연쇄구균병으로 인한 어류의 폐사를 현저하게 줄일 수 있는 효과를 가진다. 따라서, 어류의 생산성 증대는 물론, 항생제 사용으로 인한 비용을 절감시킬 수 있다. 또한 본 발명은 어류의 세균성 질병을 예방할 수 있는 백신을 사용함으로써, 항생제를 사용하지 않은 건강한 어류 생산을 가능하게 하여, 양식업의 발전에 이바지하고 어류 소비를 크게 향상시킬 수 있다.The present invention has the effect of significantly preventing the death of fish caused by streptococcal disease by effectively preventing streptococcal disease of fish, especially cultured halibut. Therefore, it is possible to reduce the cost of using antibiotics as well as increasing the productivity of fish. In addition, the present invention, by using a vaccine that can prevent the bacterial disease of fish, by enabling the production of healthy fish without antibiotics, can contribute to the development of aquaculture and significantly improve fish consumption.
도 1은 S. parauberis 64개 균주의 유전자 5종[DNA gyrase subunit B(gyrB), Surface M-protein(simA), autolysin, capsular polysaccharide biosynthesis protein, tyrosine-protein kinase(wze)]에 대한 염기서열(약 4,120bp)의 계통 주 분석 결과를 나타낸 도면이다.
도 2는 S. parauberis 혈청형 분석을 나타낸 것으로, 도 2(A)는 슬라이드 응집반응(I: 양성반응, II: 음성반응), 도 2(B)는 Microtiter 응집반응을 나타낸 도면이다.
도 3은 국내 2002년-2017년 S. parauberis 균의 혈청형 분포를 나타낸 그래프이다.
도 4는 국내 2002년-2017년 S. parauberis 균의 지역별, 분리연도별 분리 비율을 나타낸 도면이다.
도 5는 국내 시판되고 있는 S. parauberis 항원을 포함하는 불활화 백신의 serotype-PCR 결과를 나타낸 도면이다.
도 6은 S. parauberis 7개 균주의 인위감염 후 넙치의 누적폐사량을 나타낸 그래프이다.
도 7은 S. parauberis 4개 균주의 농도별 넙치의 누적폐사량을 나타낸 그래프이다.
도 8은 넙치의 S. parauberis 백신 접종 및 인위감염 실험 일정을 도식화한 도면이다.
도 9는 S. parauberis 다가백신 접종 2주 후 항체가 측정 결과를 나타낸 그래프이다.
도 10은 S. parauberis 다가백신 접종 1주 후 내부 장기의 병리조직 검사 결과이다.
도 11은 S. parauberis 다가백신 접종 2주 후 내부 장기의 병리조직 검사 결과이다.
도 12는 S. parauberis 다가백신 접종 3주 후 내부 장기의 병리조직 검사 결과이다.Figure 1 shows the nucleotide sequence of 5 genes of S. parauberis 64 strains [DNA gyrase subunit B ( gyrB ), Surface M-protein (simA), autolysin, capsular polysaccharide biosynthesis protein, tyrosine-protein kinase (wze)] This is a diagram showing the results of analysis of the main strain of about 4,120 bp).
Figure 2 shows the S. parauberis serotype analysis, Figure 2 (A) is a slide aggregation reaction (I: positive reaction, II: negative reaction), Figure 2 (B) is a diagram showing the microtiter aggregation reaction.
Figure 3 is a graph showing the serotype distribution of S. parauberis bacteria in Korea 2002-2017.
Figure 4 is a diagram showing the separation ratio by region and year of separation of S. parauberis in Korea between 2002 and 2017.
5 is a diagram showing the serotype-PCR results of an inactivated vaccine containing the S. parauberis antigen commercially available in Korea.
6 is a graph showing the cumulative mortality of flounder after artificial infection of 7 strains of S. parauberis .
7 is a graph showing the cumulative mortality of flounder by concentration of four strains of S. parauberis .
8 is a diagram schematically showing the schedule of S. parauberis vaccination and artificial infection experiments of halibut.
9 is a graph showing the results of
10 is a result of the pathological histologic examination of the
Figure 11 shows the pathological histologic results of
Figure 12 is the pathological histologic results of the
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known in the art and commonly used.
본 발명의 일 실시예에서, 연쇄구균병에 감염된 넙치에서 분리된 Streptococcus parauberis 19FBSPa0003(PR5) 균주, Streptococcus parauberis FP2331 균주, Streptococcus parauberis FPa4365 균주 및 Streptococcus parauberis FPa4870 균주를 불활성화시킨 후, 이를 항원으로 포함하는 백신을 어류에 접종한 결과, 모든 백신접종구에서 낮은 폐사율을 나타냈으며, 혈청형별 교차 백신 효능도 있음을 확인하였다. 또한, 상기 상이한 혈청형별 항원들을 포함하는 다가백신의 경우, 단일 혈청형 백신에 비해 백신 효능이 더욱 증가됨을 확인하였다.In one embodiment of the present invention, after inactivating Streptococcus parauberis 19FBSPa0003 (PR5) strain, Streptococcus parauberis FP2331 strain, Streptococcus parauberis FPa4365 strain and Streptococcus parauberis FPa4870 strain isolated from halibut infected with streptococcal disease, comprising this as an antigen As a result of inoculating the vaccine with fish, all vaccinations showed low mortality, and it was confirmed that there was also a cross vaccine efficacy by serotype. In addition, it was confirmed that in the case of a polyvalent vaccine containing antigens according to the different serotypes, the vaccine efficacy was further increased compared to a single serotype vaccine.
따라서, 본 발명은 일 관점에서, 불활성화된 스트렙토코커스 파라우베리스(Streptococcus parauberis) KCTC 13800BP 균주, KCTC 13801BP 균주, KCTC 13802BP 균주 및 KCTC 13803BP 균주로 구성된 군에서 선택되는 어느 둘 이상의 균주를 항원으로 포함하는 연쇄구균병 예방용 불활성화 백신에 관한 것이다.Therefore, in one aspect, the present invention includes, as an antigen, any one or more strains selected from the group consisting of inactivated Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain, and KCTC 13803BP strain. It relates to an inactivated vaccine for preventing streptococcal disease.
본 발명에 있어서, 상기 KCTC 13800BP 균주는 한국생물자원센터(Korean Collection for Type Culture)에 2019년 1월 30일자로 기탁된 Streptococcus parauberis 19FBSPa0003 균주이며, 본 명세서에서 19FBSPa0003는 PR5와 동일한 균주를 의미한다.In the present invention, the KCTC 13800BP strain is a Streptococcus parauberis 19FBSPa0003 strain deposited as of January 30, 2019 at the Korean Collection for Type Culture, where 19FBSPa0003 means the same strain as PR5.
본 발명에 있어서, 상기 KCTC 13801BP 균주는 한국생물자원센터에 2019년 1월 30일자로 기탁된 Streptococcus parauberis FP2331 균주이며, 상기 KCTC 13802BP 균주는 한국생물자원센터에 2019년 1월 30일자로 기탁된 Streptococcus parauberis FPa4365 균주이고, 상기 KCTC 13803BP 균주는 한국생물자원센터에 2019년 1월 30일자로 기탁된 Streptococcus parauberis FPa4870 균주이다.In the present invention, the KCTC 13801BP strain is a Streptococcus parauberis FP2331 strain deposited in January 30, dated 2019 in Korea, Biological Resource Center, the KCTC 13802BP strains of Streptococcus deposited in January 30, dated 2019 in Korea, Biological Resource Center The parauberis FPa4365 strain, and the KCTC 13803BP strain is a Streptococcus parauberis FPa4870 strain deposited with the Korea Biological Resource Center on January 30, 2019.
본 발명에 있어서, 상기 백신은 KCTC 13802BP 균주 및 KCTC 13803BP 균주가 1:1의 비율로 혼합되는 것을 특징으로 할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the vaccine may be characterized in that the KCTC 13802BP strain and the KCTC 13803BP strain are mixed in a ratio of 1: 1, but is not limited thereto.
본 발명에 있어서, 상기 백신은 KCTC 13800BP 균주, KCTC 13802BP 균주 및 KCTC 13803BP 균주가 1:1:1의 비율로 혼합되는 것을 특징으로 할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the vaccine may be characterized in that the KCTC 13800BP strain, KCTC 13802BP strain and KCTC 13803BP strain are mixed in a ratio of 1: 1: 1, but is not limited thereto.
본 발명에 있어서, 상기 백신의 항원 농도는 1 내지 30mg/fish인 것을 특징으로 할 수 있다.In the present invention, the antigen concentration of the vaccine may be characterized in that 1 to 30mg / fish.
본 발명의 일 실시예에서, Streptococcus parauberis 19FBSPa0003(PR5) 균주, FP2331 균주, FPa4365 균주 또는 FPa4870 균주를 포함하는 백신접종 후 항원 종류별(혈청형 Ia~II)로 혈청의 항체가를 측정한 결과, 대조구(PBS)와 비교하여 모두 유의적으로 높은 항체가를 형성하였으며, 각각의 백신구는 동일한 혈청형의 항원으로 항체가를 측정할 경우 가장 높은 항체가를 나타냈다.In one embodiment of the present invention, as a result of measuring the antibody titer of serum by the type of antigen (serum type Ia ~ II) after vaccination including Streptococcus parauberis 19FBSPa0003 (PR5) strain, FP2331 strain, FPa4365 strain or FPa4870 strain, control Compared to (PBS), all antibody titers formed significantly higher, and each vaccine cell showed the highest antibody titer when measuring the antibody titer with the same serotype antigen.
또한, S. parauberis 혈청형 Ia+Ic 백신구와 Ia+Ib+Ic 백신구로 인위감염하면 대조구와 비교할 때 높은 생존율을 나타내었으며, S. parauberis 단일 혈청형 백신구와 비교하여도 높은 상대생존율을 나타내었다.In addition, artificial infection with S. parauberis serotype Ia + Ic vaccine and Ia + Ib + Ic vaccine showed high survival rate compared to control, and high relative survival rate compared to S. parauberis single serotype vaccine.
본 발명에서 용어, “백신”이란 감염 질환을 예방하기 위한 면역을 위하여 쓰이는 항원으로서, 약독화시킨 미생물 또는 바이러스로 제조되며, 특정 감염증에 대해 인공적으로 면역원성을 획득하기 위하여 약화시키거나 사멸시킨 병원 미생물을 개체 내에 투여하는 것을 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.In the present invention, the term "vaccine" is an antigen used for immunity to prevent an infectious disease, and is made of attenuated microorganisms or viruses, and is a hospital that is attenuated or killed to artificially obtain immunogenicity against a specific infectious disease It means that the microorganism is administered in the individual. When stimulated by a vaccine, the immune system in the individual acts to produce antibodies, and if it maintains sensitivity and re-infection occurs, it is possible to overcome the disease by effectively generating antibodies in a short time.
상기 불활성화 백신은 당해 기술분야에 널리 공지된 방법을 이용하여 제조될 수 있으며, 예를 들어, 당해 기술분야에 알려진 불활성화제를 사용하여 제조될 수 있다. 바람직하게는 포르말린 처리에 의해 불활성화시키는 것을 특징으로 할 수 있으나, 이에 제한되는 것은 아니다.The inactivated vaccine may be prepared using methods well known in the art, for example, using an inactivating agent known in the art. Preferably, it can be characterized in that it is inactivated by formalin treatment, but is not limited thereto.
본 발명에 따른 혼합 불활성화 백신은 보조제(adjuvant)를 추가로 포함할 수 있다. 상기 보조제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있으나, 바람직하게는 몬타나이드(Montanide) ISA35 또는 Gel01이 사용될 수 있다.The mixed inactivated vaccine according to the present invention may further include an adjuvant. The adjuvant can be used without limitation as long as it is known in the art, but preferably, Montanide ISA35 or Gel01 can be used.
또한, 안정화제 또는 첨가제로서, 당류나 아미노산류, 광물유, 식물유, 백반, 인산알루미늄, 벤토나이트, 실리카, 무라밀디펩티드(muramyl dipeptide) 유도체, 사이모신, 인터류킨 등이 사용될 수도 있다.In addition, as stabilizers or additives, sugars or amino acids, mineral oil, vegetable oil, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, cymosin, interleukin, and the like may be used.
본 발명에 있어서, 상기 백신은 적당한 용적의, 예를 들어 약 10~500㎖ 부피의 바이알에 백신액을 분주한 다음, 밀봉하여 사용될 수 있다. 이러한 백신은 액상뿐만 아니라, 나누어 주입한 다음에 동결건조함으로써 건조 제제로 사용될 수도 있다. 상기 건조 제제는 사용 직전에 첨가한 감균액으로 건조물질을 완전하게 재용해하여 사용될 수 있다.In the present invention, the vaccine may be used by dispensing the vaccine solution into a vial having an appropriate volume, for example, about 10 to 500 ml, and sealing the vaccine solution. These vaccines can be used as dry preparations, as well as in liquid form, by injecting and then lyophilizing. The dry formulation may be used by completely re-dissolving the dry substance with the antiseptic solution added immediately before use.
본 발명에 있어서, 상기 혼합 불활성화 백신은 감염의 위험성이 있는 임의의 연령의 어류에 사용할 수 있다. 이에 제한되는 것은 아니나, 치어를 포함한 중간육성어, 성어에 사용 가능하다.In the present invention, the mixed inactivated vaccine can be used for fish of any age at risk of infection. It is not limited to this, but can be used for medium-grown fish, including pompoms, and adult words.
본 발명은 다른 관점에서, 연쇄구균병에 감염된 넙치로부터 분리된 스트렙토코커스 파라우베리스(Streptococcus parauberis) KCTC 13800BP 균주, KCTC 13801BP 균주, KCTC 13802BP 균주 및 KCTC 13803BP 균주로 구성된 군에서 선택되는 어느 둘 이상의 균주를 포르말린으로 불활성화시켜 백신을 수득하는 단계를 포함하는 것을 특징으로 하는 연쇄구균병 예방용 불활성화 백신의 제조방법에 관한 것이다.According to another aspect of the present invention, Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain, and KCTC 13803BP strain isolated from flounder infected with streptococcal disease It relates to a method for producing an inactivated vaccine for preventing streptococcal disease, characterized in that it comprises the step of obtaining a vaccine by inactivating with formalin.
본 발명에 있어서, 상기 백신은 Streptococcus parauberis KCTC 13800BP 균주, KCTC 13801BP 균주, KCTC 13802BP 균주 또는 KCTC 13803BP 균주를 배양한 후 불활성화시킨 다음, 농도를 조절하여 제조한다. 본 발명의 일 실시예에서, 동결 보존된 S. parauberis 4개 균주를 배양하고, 배양된 균액에 37%(v/v) 포르말린(Merck, Germany)을 각 배양액의 0.5%(v/v)가 되도록 첨가하여 교반 배양기에서 150rpm, 20℃로 24시간 동안 불활화하였다. 불활화가 확인된 균액을 원심분리한 다음, 멸균 생리 식염수로 세척한 후, 균체를 수거하고 최종적으로 습균체 무게를 측정하여 실험구에 따라 농도를 달리하여 백신을 제조하였다.In the present invention, the vaccine is prepared by incubating Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain or KCTC 13803BP strain, and then adjusting the concentration. In one embodiment of the present invention, 4 strains of cryopreserved S. parauberis were cultured, and 37% (v / v) formalin (Merck, Germany) was added to the cultured bacterial solution, and 0.5% (v / v) of each culture solution was obtained. The mixture was inactivated at 150 rpm and 20 ° C. in a stirred incubator for 24 hours. After centrifugation of the incubation solution, the inactivation was confirmed, washed with sterile physiological saline, the cells were collected, and finally, the weight of the cells was measured, and the vaccine was prepared by varying the concentration according to the experimental group.
본 발명은 또 다른 관점에서, 상기 연쇄구균병 예방용 불활성화 백신을 어류에 투여하는 것을 포함하는 어류를 면역시키는 방법에 관한 것이다.In another aspect, the present invention relates to a method for immunizing fish comprising administering to the fish an inactivated vaccine for preventing streptococcal disease.
본 발명에 있어서, 상기 백신을 어류의 복강에 주사하는 것을 특징으로 할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the vaccine may be characterized in that it is injected into the abdominal cavity of fish, but is not limited thereto.
본 발명에 있어서, 상기 어류는 감염의 위험성이 있는 넙치 등의 어류를 포함하나, 이에 제한되는 것은 아니다.In the present invention, the fish include, but are not limited to, fish, such as flounder, which are at risk of infection.
본 발명의 일 실시예에서, 4가지 S. parauberis 백신 처리 후, 각각의 혈청형에 대한 인위감염 시험을 수행한 결과, 백신을 접종하지 않은 대조구에 비하여 모든 백신접종구에서 낮은 폐사율을 나타냈으며, 혈청형별 교차 백신 효능도 있음을 확인하였다. 또한, S. parauberis 혈청형 Ia 내지 Ic를 포함하는 다가백신접종구의 경우, 단일 혈청형 백신접종구와 비교하여 높은 백신 효능을 나타내는 것을 확인하였다.In one embodiment of the present invention, after treatment with four S. parauberis vaccines, as a result of performing an artificial infection test for each serotype, it showed a low mortality rate in all vaccinated cells compared to the control group not vaccinated, It was confirmed that there was also a cross vaccine efficacy by serotype. In addition, it was confirmed that the multivalent vaccine comprising S. parauberis serotypes Ia to Ic showed higher vaccine efficacy compared to the single serotype vaccine.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
실시예 1: 연쇄구균 Example 1: Streptococcus S. parauberisS. parauberis 의 분리 및 동정Separation and identification
국립수산과학원 병리연구과에서 보존하고 있던 2002년~2017년 국내의 병든 양식 넙치에서 분리된 S. parauberis 199개 균주 중에서 분리연도 및 지역별로 대표균주 64개 균주를 선정하여 분석에 사용하였다.Among the 199 S. parauberis strains isolated from domestic diseased flounder from 2002 to 2017, which were preserved by the National Institute of Fisheries Science and Pathology, 64 representative strains were selected and used for analysis.
균주를 1% NaCl을 첨가한 tryptic soy agar(TSA, Becton Dickinson, USA) 또는 brain heart infusion agar(BHIA, Becton Dickinson, USA) 배지에 도말한 후, 25℃에서 24시간 배양하여 균의 성상을 확인하고 이를 계대배양하여 표현형, 혈청형, 유전학적 분석 등을 실시하였다.After spreading the strain on a tryptic soy agar (TSA, Becton Dickinson, USA) or brain heart infusion agar (BHIA, Becton Dickinson, USA) medium with 1% NaCl, the cultures were incubated at 25 ° C for 24 hours to confirm the properties of the bacteria. Then, it was subcultured to perform phenotype, serotype, and genetic analysis.
분석한 균주의 분리지역 및 연도를 표 1에 나타내었다.Table 1 shows the separation regions and years of the analyzed strains.
실시예 2:Example 2: S. parauberis S. parauberis 의 표현형 분석Phenotypic analysis of
생화학적 특성을 분석하기 위해 S. parauberis 균주를 1% NaCl이 첨가된 BHIA배지에서 25℃, 24시간 배양하였다. API 20strep kit(BIOMERIEUX, France)와 API ZYM kit(BIOMERIEUX, France) 실험방법은 순수배양된 균 집락을 제조사의 방법에 따라 스트립에 접종 배양하여 시험하였다.In order to analyze the biochemical properties, S. parauberis strains were incubated for 24 hours at 25 ° C. in BHIA medium with 1% NaCl added. The API 20strep kit (BIOMERIEUX, France) and API ZYM kit (BIOMERIEUX, France) test methods were tested by inoculating and culturing the pure cultured colonies on a strip according to the manufacturer's method.
S. parauberis 64개 균주의 API 20strep, API zym kit 분석 결과, 조사한 모든 균주는 비운동성의 gram-positive cocci로 catalase, oxidase 음성으로 확인되었다(표 2). As a result of analysis of API 20strep and API zym kit of 64 strains of S. parauberis , all the investigated strains were identified as catalase and oxidase negative as motility gram-positive cocci (Table 2).
PA: alkaline phospatase, AD: arginine dihydrorase, RI: ribose acidification, MA: mannitol acidification, SO: sorbitol acidification, LA: lactose acidification, TR: trehalose acidification, IN: inulin acidification, AM: starch acidification, 2: alkaline phospatase, 3: esterase(C4), 4: esterase lipase(C8), 5: lipase(C14), 7: valine arylamidase, 8: crystine arylamidase, 10: α-chymotrypsin, 11: acid phosphatase, 14: β-galactosidase, 16: α-glucosidase.PA: alkaline phospatase, AD: arginine dihydrorase, RI: ribose acidification, MA: mannitol acidification, SO: sorbitol acidification, LA: lactose acidification, TR: trehalose acidification, IN: inulin acidification, AM: starch acidification, 2: alkaline phospatase, 3: esterase (C4), 4: esterase lipase (C8), 5: lipase (C14), 7: valine arylamidase, 8: crystine arylamidase, 10: α-chymotrypsin, 11: acid phosphatase, 14: β-galactosidase, 16 : α-glucosidase.
API 20strep 생화학적 특성 분석한 결과 모든 균주는 pyruvate acetoin production(VP), hippurate hydrolysis(HIP), β-glucosidase(ESC), pyrrolidonyl arylamidase(PYRA), leucine arylamidase(LAP) 양성, α-galactosidase(αGAL), β-galactosidase(βGUR), L-arabinose acidification(ARA), Raffinose acidification(RAF), Glycogen acidification(GLYG) 음성 반응을 보였다(표 3).As a result of API 20strep biochemical characterization, all strains are pyruvate acetoin production (VP), hippurate hydrolysis (HIP), β-glucosidase (ESC), pyrrolidonyl arylamidase (PYRA), leucine arylamidase (LAP) positive, α-galactosidase (αGAL) , β-galactosidase (βGUR), L-arabinose acidification (ARA), Raffinose acidification (RAF), and Glycogen acidification (GLYG) showed negative reactions (Table 3).
Ia는 serotype Ia이며, II는 serotype II이다. Ia is serotype Ia, and II is serotype II.
API zym kit 생화학적 특성 분석한 결과, 모든 균주는 leucine acrylamidase, Naphtol-AS-BI-phosphohydrolase 양성, typsin, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase 음성 반응을 보였다(표 4).As a result of API zym kit biochemical analysis, all strains are leucine acrylamidase, Naphtol-AS-BI-phosphohydrolase positive, typsin, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase , α-fucosidase showed a negative reaction (Table 4).
Ia는 serotype Ia이며, II는 serotype II이다. Ia is serotype Ia, and II is serotype II.
실시예 3:Example 3: S. parauberis S. parauberis 의 유전형 분석Genotyping
실시예 3-1: primer 제작 및 유전자 염기서열 분석Example 3-1: Primer preparation and gene sequence analysis
유전적 특성 분석을 위하여 각 분리 균주를 1% NaCl이 첨가된 BHIB배지에서 24시간 배양한 후 13,000rpm에서 2분간 원심분리, 상층액을 제거하여 세균 pellet을 제작하였다. 세균 pellet은 Promega Genomic DNA purification kit(Promega, USA)를 사용하여 DNA를 추출하였다. Housekeeping gene groEL, gyrB는 기 보고된 Primer를 사용하였으며, S. parauberis KCTC 11537(Nho et al., 2011) 및 KCTC 11980(Park et al., 2013) genomes에서 항원성·병원성 관련이 있을 것으로 추정되는 유전자 6종(capsule polysaccharide 4종, autolysin 1종, M-like protein 1종)을 선정하여 특이적으로 증폭할 수 있는 primer를 제작하여 분석하였다(표 5).To analyze the genetic properties, each isolate was cultured in BHIB medium with 1% NaCl added for 24 hours, followed by centrifugation at 13,000 rpm for 2 minutes and removal of the supernatant to prepare a bacterial pellet. For bacterial pellets, DNA was extracted using a Promega Genomic DNA purification kit (Promega, USA). Housekeeping gene groEL, gyrB was used as a Primer reporting period, S. parauberis KCTC 11537 (Nho et al., 2011) and KCTC 11980 (Park et al., 2013) are estimated to be antigenic, pathogenic genomes from related Six genes (4 capsule polysaccharides, 1 autolysin, and 1 M-like protein) were selected and primers capable of specifically amplifying were prepared and analyzed (Table 5).
(5´to 3´)Sequence
(5´to 3´)
(bp)PCR product size
(bp)
(Genbank accession no./protein ID)References
(Genbank accession no./protein ID)
(서열번호 1)5'-CAGGCCTAACACATGCAAGTC-3 '
(SEQ ID NO: 1)
(서열번호 2)5'-ACGGGCGGTGTGTRC-3 '
(SEQ ID NO: 2)
(서열번호 3)5'-GAHGTNGTiGAAGGiATGCA-3 '
(SEQ ID NO: 3)
(서열번호 4)5'-ATTTGRCGiAYWGGYTCTTC-3 '
(SEQ ID NO: 4)
(서열번호 5)5'-GGTTCiTCNGTiGTTAATGC-3 '
(SEQ ID NO: 5)
(서열번호 6)5'-GTTCRTGNGTTCCiCCTTCAT-3 '
(SEQ ID NO: 6)
(서열번호 7)5'-GCTAAGGTCATGACCCATTC-3 '
(SEQ ID NO: 7)
(CP002471.1/AEF25898.1)This study
(CP002471.1 / AEF25898.1)
(서열번호 8)5'-AGGCGTGCAATTTCAGCTTG-3 '
(SEQ ID NO: 8)
(type I)Autolysin
(type I)
(서열번호 9)5'-CTCAAACTGAGGCTAGTAAT-3 '
(SEQ ID NO: 9)
(CP002471.1/AEF25898.1)This study
(CP002471.1 / AEF25898.1)
(서열번호 10)5'-GTCCCGGCTAATCCAACTGT-3 '
(SEQ ID NO: 10)
(서열번호 11)5'-CTTGGTGGATCCCAAGCAC-3 '
(SEQ ID NO: 11)
(CP002471.1/AEF25344.1)This study
(CP002471.1 / AEF25344.1)
(서열번호 12)5'-CCAAACAACCACTCTCCAGC-3 '
(SEQ ID NO: 12)
(서열번호 13)5'-GCAAGCGGTCACGGAAAAGT-3 '
(SEQ ID NO: 13)
(CP002471.1/AEF25347.1)This study
(CP002471.1 / AEF25347.1)
(서열번호 14)5'-CACCTACATAGGTTCCACTA-3 '
(SEQ ID NO: 14)
(서열번호 15)5'-GCAAATCTGTCGGCTTTTCG-3 '
(SEQ ID NO: 15)
(CP002471.1/AEF25350.1)This study
(CP002471.1 / AEF25350.1)
(서열번호 16)5'-ACGACTACCAAGGACGTTAC-3 '
(SEQ ID NO: 16)
(서열번호 17)5'-CCGAAGAGTACTATAACTCC-3 '
(SEQ ID NO: 17)
(CP002471.1/AEF25351.1)This study
(CP002471.1 / AEF25351.1)
(서열번호 18)5'-TGGCTTACCATAATTCCCGT-3 '
(SEQ ID NO: 18)
(서열번호 19)ATTGTTAGTCATTCAGTTGT
(SEQ ID NO: 19)
(서열번호 20)AATTATAGTCAACAGTCCAG
(SEQ ID NO: 20)
(서열번호 21)ATTTCTACCAGGTTACTTTG
(SEQ ID NO: 21)
(서열번호 21)ATTTCTACCAGGTTACTTTG
(SEQ ID NO: 21)
(서열번호 22)GAACTACTTAGGTTTAGCAT
(SEQ ID NO: 22)
(서열번호 23)AACTTGTAAATAGGATTGCT
(SEQ ID NO: 23)
상기 표 5에서 염기서열은 IUPAC nucleotide code(Nucleotide ambiguity code)로 표시하였으며, R은 A 또는 G, i(서열목록에는 S로 표기)는 Inosine, H는 A, C 또는 T를 의미한다. N은 any base, Y는 C 또는 T, W는 A 또는 T를 의미한다.In Table 5, the base sequence is represented by an IUPAC nucleotide code (Nucleotide ambiguity code), R is A or G, i (indicated by S in the sequence list), Inosine, H is A, C or T. N means any base, Y means C or T, and W means A or T.
제작한 primer로 PCR 증폭하였으며 PCR 산물은 EtBr이 포함된 1.5% agarose gel(Bioneer, Korea)을 사용하여 전기영동한 후 UV transilluminator(Alpha Innotech, USA)를 이용해 band를 확인하였고, 염기서열을 분석하였다(표 6 내지 표 14).PCR was amplified with the prepared primer, and the PCR product was electrophoresed using a 1.5% agarose gel (Bioneer, Korea) containing EtBr, and then the band was confirmed using a UV transilluminator (Alpha Innotech, USA), and the sequence was analyzed. (Tables 6 to 14).
유전자 염기서열분석은 MEGA6 program과 GENETYX Ver.8.0(SDC Software Development, Japan)을 사용하였으며, 결정된 각 염기서열은 National Center for Biotechnology Institute(NCBI)에서 제공하는 Basic Local Alignment Search Tool(BLAST)을 이용하여 기존에 보고된 S. parauberis 균주의 유전자 및 본 실시예에서 분석한 균주의 유전자를 비교 분석하였다. Blast search의 염기서열 정보를 바탕으로 Bioedit Ver.7.2.1를 사용하여 multiple alignments를 실시하였고, MEGA6 program(http://www.megasoftware.net; Tamura et al., 2013)을 사용한 근린결합분석(NJ; neighbor-joining analysis, 1,000 rounds of boostrap)을 통하여 각 염기서열간 유전적 거리와 계통도(phylogenetic tree)를 작성하였다(도 1).For gene sequencing, MEGA6 program and GENETYX Ver.8.0 (SDC Software Development, Japan) were used, and each determined sequencing was performed using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Institute (NCBI). Genes of the previously reported S. parauberis strain and genes of the strain analyzed in this example were compared and analyzed. Based on the base sequence information of the Blast search, multiple alignments were performed using Bioedit Ver.7.2.1, and neighborhood binding analysis using the MEGA6 program (http://www.megasoftware.net; Tamura et al., 2013) ( The genetic distance and phylogenetic tree between each sequence were prepared through NJ; neighbor-joining analysis, 1,000 rounds of boostrap (FIG. 1).
실시예 3-2: 유전형 분석 결과Example 3-2: Results of genotyping
넙치에서 분리된 64개 균주의 16S rRNA 분석결과 S. parauberis로 확인되었으며, 동일한 염기서열로 확인되었다. S. parauberis 64개 균주에 대한 multilocus sequence typing(MLST) 분석을 실시하였다. Sugar O-acryltransferase sialic acid(Soa), Polysaccharide biosynthesis protein(PB) 유전자는 일부 균주에서는 증폭이 되지 않았다. 분석한 64개 균주에서 16S rRNA, groEL 유전자는 모두 염기서열이 동일하였으며, 균주마다 염기서열이 차이가나는 gyrB 및 항원성·병원성 관련 유전자 4종에 대해서만 MLST 분석을 수행하였다. 넙치에서 분리되는 S. parauberis 64개 균주는 크게 2가지 MLST 유전형으로 나누어지며, MLST-1 group 36개 균주, MLST-2 group 28개 균주로 확인되었다(도 1). 균주별 유전형 분석결과는 하기 표 15에 나타내었다.As a result of 16S rRNA analysis of 64 strains isolated from halibut, it was identified as S. parauberis , and was identified with the same nucleotide sequence. Multilocus sequence typing (MLST) analysis was performed for 64 S. parauberis strains. Sugar O-acryltransferase sialic acid (Soa) and polysaccharide biosynthesis protein (PB) genes were not amplified in some strains. In the 64 strains analyzed, 16S rRNA and groEL genes all had the same base sequence, and MLST analysis was performed only on gyrB and antigen-pathogenicity-related genes with different base sequences for each strain. The 64 strains of S. parauberis isolated from halibut were largely divided into two MLST genotypes, and were identified as 36 strains of MLST-1 group and 28 strains of MLST-2 group (FIG. 1). The results of genotyping for each strain are shown in Table 15 below.
CPB 및 SOA 항목에서 -로 표시된 것은 PCR 증폭이 되지 않은 것이다.In the CPB and SOA categories, the ones marked with-are not PCR amplified.
실시예 4:Example 4: S. parauberis S. parauberis 의 혈청형 분석Serotype analysis
실시예 4-1: 혈청형 분석 방법Example 4-1: Serotype analysis method
슬라이드 응집반응으로 혈청형을 분석하였으며, 참조균주(S. parauberis serotype I FPa4164 및 S. parauberis serotype O2 S53)에 대한 토끼항혈청을 제작하여 분석에 사용하였다. 슬라이드글라스에 S. parauberis에 대한 2종류의 토끼 항혈청을 각각 70μL씩 떨어뜨리고 넙치 유래 S. parauberis 64개 균주를 McFarland no. 6 standard로 멸균 생리식염수에 현탁한 균액을 각 10μL 떨어뜨려 잘 섞어 30초 이내에 응집 형성 유무를 확인하였다(도 2(A)). 응집은 어두운 배경에서 슬라이드를 측면에서 관찰하였으며, 대조군은 균을 현탁하지 않은 멸균 생리식염수를 사용하였다.The serotype was analyzed by the slide agglutination reaction, and rabbit antiserum against the reference strains ( S. parauberis serotype I FPa4164 and S. parauberis serotype O2 S53) was prepared and used for analysis. Two kinds of rabbit antisera against S. parauberis were added to the slide glasses by 70 μL each, and 64 strains of S. parauberis from halibut were added to McFarland no. With 6 standard, each 10 μL of the suspension suspended in sterile physiological saline was mixed well to confirm the presence or absence of aggregation within 30 seconds (FIG. 2 (A)). Aggregation was observed from the side of the slide on a dark background, and a sterile physiological saline solution without suspension was used as a control.
S. parauberis의 serotype I 안에 subserotypes을 구분하기 위해서 microtiter 응집반응을 통해 S. parauberis 64개 균주의 혈청형 분석을 시도하였다(도 2(B)). S. parauberis 유전형과 표현형의 특성이 다른 4가지 균주 DK14, DW1, FPa4870, FP4743에 대한 토끼 항혈청을 제작하여 Kanai et al.(2015)의 방법으로 microtiter 응집반응을 통한 sub-serotyping을 수행하였다. 96 well plates에 토끼항혈청을 PBS로 2배씩 순차적으로 희석한 후 4가지 S. parauberis FKC 현탁액(2mg/mL PBS)을 동일한 용량 25㎕씩 첨가 후 혼합 4℃에서 O/N incubation한 후 응집항체가를 측정하였다. 항혈청의 cross-absorbed test를 위하여 absorbed-항혈청을 제작하였으며(Kanai at al., (2015) Fish pathology 50(2), 75-80), absorbed-항혈청은 응집항체가 4 미만으로 제작하였다. Tu et al.(2015)의 방법에 따라 S. parauberis에 대한 serotyping PCR을 실시하였다. PCR primer의 염기서열은 상기 표 2에 나타낸 바와 같다.To distinguish subserotypes in serotype I of S. parauberis , a serotype analysis of 64 strains of S. parauberis was attempted through microtiter aggregation (FIG. 2 (B)). Rabbit antisera was prepared for four strains DK14, DW1, FPa4870, and FP4743 with different S. parauberis genotypes and phenotypic characteristics, and sub-serotyping through microtiter aggregation reaction was performed by the method of Kanai et al. (2015). After serially diluting rabbit antisera twice with PBS in 96 well plates, add 4 S. parauberis FKC suspensions (2mg / mL PBS) at the same volume of 25 µl and mix and then incubate at 4 ° C. Was measured. For the cross-absorbed test of antisera, absorbed-antisera was prepared (Kanai at al., (2015) Fish pathology 50 (2), 75-80), and absorbed-antisera produced less than 4 aggregated antibodies. Serotyping PCR for S. parauberis was carried out according to the method of Tu et al. (2015). The base sequence of the PCR primer is shown in Table 2 above.
실시예 4-2: 혈청형 분석 결과Example 4-2: Serotype analysis results
슬라이드 응집반응 결과, S. parauberis 균주는 serotype I 58개 균주, serotype II 4개 균주, untypable serotype 2개 균주로 확인되었다. 64개 균주의 Serotyping-PCR 결과는 상기 표 15에 나타낸 바와 같다.As a result of the slide aggregation reaction, S. parauberis strains were identified as 58 strains of serotype I, 4 strains of serotype II, and 2 strains of untypable serotype. Serotyping-PCR results of 64 strains are shown in Table 15 above.
S parauberis 4가지 균주 DK14, DW1, FPa4870, FP4743에 대한 토끼 항혈청을 제작하여 microtiter 응집반응을 통한 sub-serotyping을 수행한 결과, Serotype I 58개 균주는 anti-DK14, anti-DW1, anti-FPa4870 항혈청과 응집반응을 나타내지만, 응집가는 항혈청 종류에 따라 차이가 있어 serotype I 안에 subserotype의 존재하는 것이 시사되었다. 응집가에 따라 S. parauberis serotype I의 subserotyping한 결과, Ia 25개 균주, Ib 25개 균주, Ic 8개 균주, Non-typeable 2개 균주로 확인되었다(표 16).S parauberis 4 strains DK14, DW1, FPa4870, and FP4743 were prepared with rabbit antiserum, and sub-serotyping through microtiter aggregation reaction resulted in 58 strains of Serotype I anti-DK14, anti-DW1, and anti-FPa4870 antisera It shows hyperagglutination reaction, but the agglutination value differs depending on the type of antiserum, suggesting the presence of subserotype in serotype I. As a result of subserotyping of S. parauberis serotype I according to the cohesive value, it was identified as 25 strains of Ia, 25 strains of Ib, 8 strains of Ic, and 2 strains of non-typeable (Table 16).
(Antiserum)Antiserum
(Antiserum)
Ia (n=25)Subserotype
Ia (n = 25)
Ib (n=25)Subserotype
Ib (n = 25)
Ic (n=8)Subserotype
Ic (n = 8)
(n=2)Non-typeable
(n = 2)
S. parauberis serotype I 안에서 subserotype의 분류를 위하여 anit-DK14, anti-DW1, anti-FPa4870 항혈청을 교차응집소흡수방법으로 균주별 흡수 항혈청(absorbed-antiserum)을 제작하여 다시 응집가를 측정하였다. 교차응집소흡수방법을 통해서 분석한 결과, serotype I에서 3가지 subtype 존재하며, subserotype Ic type의 경우 Ia type 보다 Ib type에서 높은 응집가를 나타내었다(표 17). In order to classify subserotypes in S. parauberis serotype I, anit-DK14, anti-DW1, and anti-FPa4870 antiserum were prepared by cross-agglomerate absorption method, and absorbed-antiserum for each strain was prepared to measure the aggregation value again. As a result of analyzing through the cross-aggregation absorption method, there are three subtypes in serotype I, and in the case of subserotype Ic type, it shows a higher aggregation value in Ib type than Ia type (Table 17).
(Antiserum)Antiserum
(Antiserum)
(Ia)Anti-DK14
(Ia)
(Ib)Anti-DW1
(Ib)
(Ic)Anti-FPa4870
(Ic)
Serotype Ib의 균주는 모두 MLST-2 group에 포함되었으며, serotype II 및 Ia은 MLST-1 group에 포함되었고, serotype Ic는 유전형과 상관성이 없었다(도 1).All strains of Serotype Ib were included in the MLST-2 group, serotype II and Ia were included in the MLST-1 group, and serotype Ic was not correlated with genotype (FIG. 1).
S. parauberis 분리지역에 따른 혈청형(유전형) 분포를 분석한 결과, 분리지역에 따른 특정 혈청형을 확인할 수 없었으며, 분리 지역에 관계없이 다양한 혈청형이 확인되었다(표 18, 도 3 및 도 4). As a result of analyzing the distribution of serotypes (genotypes) according to the S. parauberis isolates, specific serotypes could not be identified according to the isolates, and various serotypes were identified regardless of the isolates (Table 18, FIGS. 3 and 3). 4).
S. parauberis 분리 연도에 따른 혈청형 분포를 분석한 결과, 2010년 이전에는 serotype Ib가 우점적이며, 2010년 이후에는 serotype Ia(MLST-1 group)가 지속적으로 분리되었고, 최근에 serotype Ic 분리율이 증가함을 확인하였다(표 19). As a result of analyzing the serotype distribution according to the S. parauberis separation year, serotype Ib was dominant before 2010, serotype Ia (MLST-1 group) was continuously isolated after 2010, and recently the serotype Ic separation rate was It was confirmed that the increase (Table 19).
Ib=2Ia = 10,
Ib = 2
Ib=3Ia = 1,
Ib = 3
특히 serotype Ia와 Ic의 경우, 2009년 S. parauberis의 넙치 백신이 상용화된 이후부터 분리비율이 증가하는 것으로 나타났다. 이는 상용화된 백신의 효과가 없다기 보다는 백신의 사용으로 인한 S. parauberis의 혈청형의 변화로 추정된다(도 3 및 도 4).In particular, in the case of serotype Ia and Ic, the separation ratio has been increased since the flounder vaccine of S. parauberis was commercialized in 2009. This is presumed to be a change in the serotype of S. parauberis due to the use of the vaccine rather than the effect of the commercialized vaccine (FIGS. 3 and 4).
실시예 5: 시판 상업용 백신의 혈청형 조사Example 5: Serotype investigation of commercial commercial vaccines
2018년 우리나라에서 판매되고 있는 넙치 S. parauberis 항원을 포함하는 불활화 백신 7종을 구입하였다. 원심분리하고 Promega Genomic DNA purification kit(Promega, USA)를 사용하여 DNA를 추출한 후, serotype-PCR(Tu et al., 2015)로 혈청형을 분석하였다. PCR 증폭하였으며 PCR 산물은 EtBr이 포함된 1.5% agarose gel(Bioneer, Korea)을 사용하여 전기영동한 후 UV transilluminator(Alpha Innotech, USA)를 이용해 band를 확인하였다.In 2018, 7 types of inactivated vaccines containing the halibut S. parauberis antigen sold in Korea were purchased. After centrifugation and DNA extraction using Promega Genomic DNA purification kit (Promega, USA), serotype was analyzed by serotype-PCR (Tu et al., 2015). The PCR was amplified and the PCR product was electrophoresed using a 1.5% agarose gel (Bioneer, Korea) containing EtBr, and then the band was confirmed using a UV transilluminator (Alpha Innotech, USA).
그 결과, 모두 serotype Ib과 serotype Ib+II으로 확인되었다(도 5 및 표 20).As a result, both were identified as serotype Ib and serotype Ib + II (FIG. 5 and Table 20).
실시예 6: Example 6: S. parauberisS. parauberis 의 병원성 분석Pathogenicity analysis
혈청형이 다른 대표 균주 7개 균주(FPa4870, FP4743, FP2331, FPa4365, PR5(19FBSPa0003), DK14, DW)를 선정하여 넙치(16.5±0.7cm)에 106cfu/fish 농도로 인위감염실험(수온 23±1℃, 피하주사법, 시험구당 10마리)을 수행하였다. 피하주사를 통한 인위감염 실험은 Mori et al. (2010) Fish Pathology, 45(1), 37-42의 방법으로 수행하였다.7 strains (FPa4870, FP4743, FP2331, FPa4365, PR5 (19FBSPa0003), DK14, DW) of 7 different strains of serotypes were selected, and artificial infection experiments at 10 6 cfu / fish concentration in flounder (16.5 ± 0.7cm) (water temperature) 23 ± 1 ° C., subcutaneous injection, 10 animals per test sphere). Experiments on artificial infections by subcutaneous injection were conducted by Mori et al. (2010) Fish Pathology, 45 (1), 37-42.
1차 감염실험에서 혈청형별로 폐사율이 비교적 높게 나타난 PR5, FPa4365, FPa4870, FP2331 균주를 가지고 2차 감염실험을 수행하였다(도 6). 4개 균주를 넙치(11.6±0.5cm)에 105~1010cfu/fish 인위감염(수온 25±0.5℃, 피하주사법)시킨 후 2주간의 누적 폐사율을 측정하였다. 그 결과 S. parauberis의 serotype I에 포함되는 FPa4365(Ia), PR5(Ib), FPa4870(Ic)는 모두 넙치에 높은 병원성을 나타냈다(도 7). Serotype II의 FP2331(II) 균주를 1010cfu/fish로 넙치에 피하주사하였으나, 고농도에서도 넙치에서는 폐사가 발생하지 않았으므로, FP2331 균주는 넙치에 병원성이 없거나 낮을 것으로 판단된다. 최종적으로 혈청형 Ia, Ib, Ic 균주는 108cfu/fish, 혈청형 II는 1010cfu/fish의 농도를 백신 효능 검증을 위한 인위감염 농도로 설정하였다.In the first infection experiment, a second infection experiment was performed with PR5, FPa4365, FPa4870, and FP2331 strains with relatively high mortality rates for each serotype (FIG. 6). After 4 strains of halibut (11.6 ± 0.5cm) were artificially infected with 10 5 ~ 10 10 cfu / fish (water temperature 25 ± 0.5 ℃, subcutaneous injection method), the cumulative mortality for 2 weeks was measured. As a result, all of FPa4365 (Ia), PR5 (Ib), and FPa4870 (Ic) included in serotype I of S. parauberis showed high pathogenicity to halibut (FIG. 7). The FP2331 (II) strain of Serotype II was injected subcutaneously to the halibut with 10 10 cfu / fish, but mortality did not occur in the halibut even at high concentrations, so the FP2331 strain is judged to have no or low pathogenicity to the halibut. Finally, serotypes Ia, Ib, Ic strain is 10 8 cfu / fish, serotype II was set to the artificial infection levels for vaccine efficacy validated the concentration of 10 10 cfu / fish.
실시예 7: 혈청형간 Example 7: Serotype liver S. parauberisS. parauberis 백신의 효능 분석 Vaccine efficacy analysis
실시예 7-1: 백신 제작 및 효능 분석 방법Example 7-1: vaccine production and efficacy analysis method
S. parauberis 혈청형 Ia, Ib, Ic, II 균주(FPa4870, FP2331, FPa4365, PR5(19FBSPa0003))에 formalin을 처리한 불활화 백신(FKC) 제작하여 넙치에 복강 접종(1mg/fish)하고, 대조구는 PBS를 복강 주사하였다. S. parauberis serotypes Ia, Ib, Ic, and II strains (FPa4870, FP2331, FPa4365, PR5 (19FBSPa0003)) were prepared by inactivation vaccine (FKC) treated with formalin, and intraperitoneally inoculated (1 mg / fish) to the flounder, control PBS was injected intraperitoneally.
동결 보존된 S. parauberis 4개 균주를 1.5%(v/v) NaCl이 첨가된 BHIA 배지에서 30℃ 온도로 24시간 동안 배양하였다. 배양된 평판배지의 집락을 무균적으로 1.5% NaCl이 첨가된 BHIB에 접종 후 교반 배양기(JS Research Inc.)에서 150rpm, 30℃ 온도로 48시간 동안 배양하였다. 배양된 균액에 37%(v/v) 포르말린(Merck, Germany)을 각 배양액의 0.5%(v/v)가 되도록 첨가하여 교반 배양기에서 150rpm, 20℃로 24시간 동안 불활화하였다. 배양균의 불활화를 BHIA(Difco, USA)에서 최종 확인하였다. 불활화가 확인된 균액을 12,000RCF로 원심분리한 다음, 멸균 생리 식염수로 3회 세척한 후 균체를 수거하고 최종적으로 습균체 무게를 측정하여 실험구에 따라 농도를 달리하여 현탁하였다. 백신접종 2주 후에 미부정맥에서 혈액을 채취하여 ELISA법으로 항체가를 측정하였다. ELISA법을 이용한 항체가 조사 방법으로 96-well plate에 S. parauberis FKC를 10μg/well의 농도가 되도록 조절하여 분주하고 4℃ over night하여 plate 바닥에 항원을 부착하였다. S. parauberis 혈청형별 ELISA값을 측정하기 위하여 4가지 다른 혈청형의 S. parauberis의 FKC를 제작하여 각각의 혈청에 대한 항체가를 조사하였다. 3% skim milk를 이용하여 실온에서 1시간 동안 blocking을 수행하고, 1차 항체로 넙치 혈청과 장 점액을 10배 희석하여 분주한 후 1시간 동안 반응시킨다. 그 후, PBST를 이용하여 3회 세척한 뒤 2차 항체는 Anti-Japanese flounder IgM Mab를 1:1000로 희석하여 분주한 후 1시간 동안 37℃에서 반응시킨다. 세척 후, AP-conjugated anti-mouse IgG(whole molecule)를 1:1000으로 희석하여 분주한 후 37℃에서 1시간 반응시키고, 세척한 다음, 기질(PNPP substrate solution)을 첨가하여 20분 후 405nm에서 흡광도를 확인하였다. 백신 접종 4주 후 각각의 백신접종구에 혈청형 Ia, Ib, Ic, II 균주로 각각 인위감염 실험하여 3주간 누적폐사율을 측정하였다(도 8). 상대생존율은 아래와 같이 계산하였다.Four strains of cryopreserved S. parauberis were cultured in BHIA medium to which 1.5% (v / v) NaCl was added at a temperature of 30 ° C. for 24 hours. Colonies of the cultured plate medium were inoculated into BHIB to which 1.5% NaCl was added aseptically, and then incubated for 48 hours at 150 rpm and 30 ° C. in a stirred incubator (JS Research Inc.). 37% (v / v) formalin (Merck, Germany) was added to the cultured bacterial solution to be 0.5% (v / v) of each culture, and inactivated at 150 rpm and 20 ° C. in a stirred incubator for 24 hours. The inactivation of the culture was finally confirmed in BHIA (Difco, USA). The inactivated bacteria were centrifuged at 12,000 RCF, washed three times with sterile physiological saline, the cells were collected, and finally, the weight of the cells was measured and suspended at different concentrations according to the experimental group. After 2 weeks of vaccination, blood was collected from the arrhythmia and the antibody titer was measured by ELISA. Antibodies using the ELISA method were used to control S. parauberis FKC to a concentration of 10 μg / well in 96-well plates and dispensed, and antigen was attached to the bottom of the plate at 4 ° C over night. In order to measure the ELISA value of each S. parauberis serotype, FKCs of four different serotypes of S. parauberis were prepared and antibody titers for each serum were examined. Blocking is performed at room temperature for 1 hour using 3% skim milk, and after diluting and dissolving halibut serum and
상대생존율(%) = {1-(시험구의 누적폐사율/대조구의 누적폐사율)} × 100Relative survival rate (%) = {1- (cumulative mortality rate of the test zone / control mortality rate)} × 100
실시예 7-2: 혈청형별 백신의 효능 및 교차 백신 효능 분석Example 7-2: Efficacy of cross-type vaccine and analysis of cross vaccine efficacy
혈청형별 백신접종 2주 후 채혈하여 항원 종류별(혈청형 Ia~II)로 혈청의 항체가를 측정한 결과, 대조구와 비교하여 모두 유의적으로 높은 항체가를 형성하였으며, 각각의 백신구는 동일한 혈청형의 항원으로 항체가를 측정할 경우 가장 높은 항체가를 나타내었다(표 21).After 2 weeks of vaccination by serotype, blood was collected and the antibody titer of the serum was measured by antigen type (serum type Ia ~ II). When the antibody titer was measured with the antigen of, it showed the highest antibody titer (Table 21).
백신구FPa4365 (Ia)
Vaccine
백신구PR5 (Ib)
Vaccine
백신구FPa4870 (Ic)
Vaccine
백신구FP2331 (II)
Vaccine
(PBS)Control
(PBS)
넙치에 대한 4가지 혈청형이 다른 S. parauberis 백신 처리 후, 각각의 혈청형에 대한 인위감염 시험을 수행한 결과, 백신을 접종하지 않은 대조구에 비하여 모든 백신접종구에서 낮은 폐사율을 나타내었다(표 22).After treatment with S. parauberis vaccine with 4 different serotypes for halibut, an artificial infection test for each serotype resulted in a lower mortality rate in all vaccinated cells compared to the unvaccinated control group (Table). 22).
혈청형간 교차 백신 효능을 대조구와 비교한 상대생존율을 비교한 결과 모든 시험구에서 동일한 혈청형의 백신 접종 후 동일한 혈청형의 균주를 인위감염하였을 때 가장 높은 상대생존율을 나타내었다(표 23). 혈청형 Ib와 Ic는 상호 간의 교차 백신 효능 반응이 높은 것(상대생존율: 73.9~82.5%)으로 확인되었으나, Ia와 Ib, Ic와는 비교적 낮은 교차 백신 효능(상대생존율: 0~30.8%)을 나타내었다(표 23).As a result of comparing the relative survival rate of the cross vaccine efficacy between the serotypes and the control group, the highest relative survival rate was shown when all strains were artificially infected with the same serotype strain after vaccination of the same serotype (Table 23). Serotypes Ib and Ic were found to have a high cross vaccine efficacy response (relative survival rate: 73.9 to 82.5%), but relatively low cross vaccine efficacy (relative survival rate: 0 to 30.8%) to Ia, Ib, and Ic. (Table 23).
실시예 8: Example 8: S. parauberisS. parauberis 다가백신의 효능 분석 Analysis of the efficacy of polyvalent vaccines
실시예 8-1: 다가백신 제작 및 효능 분석 방법Example 8-1: Multivalent vaccine production and efficacy analysis method
S. parauberis 혈청형 II는 넙치에 대한 병원성 없고 최근 넙치 양식장에서는 분리되고 있지 않기 때문에, 다가백신 개발을 위한 백신 대상균주에서 제외하였다. S. parauberis 3가지 혈청형(혈청형 Ia(FPa4365), 혈청형 Ib(PR5), 혈청형 Ic(FPa4870)) 균주를 formalin 처리한 불활화 백신(FKC)을 제작하여 실험을 진행하였다. Since S. parauberis serotype II is not pathogenic to halibut and is not isolated from halibut farms recently, it was excluded from the vaccine strain for multivalent vaccine development. Three types of S. parauberis (serum type Ia (FPa4365), serotype Ib (PR5), and serotype Ic (FPa4870)) strains were prepared by formalin-treated inactivation vaccine (FKC).
동결 보존된 S. parauberis 균주를 1.5%(v/v) NaCl이 첨가된 BHIA 배지에서 30℃, 24시간 동안 배양하였다. 배양된 평판배지의 집락을 무균적으로 1.5% NaCl이 첨가된 BHIB에 접종 후 교반 배양기(JS Research Inc.)에서 150rpm, 30℃, 48시간 동안 배양하였다. 배양된 균액에 37%(v/v) 포르말린(Merck, Germany)을 각 배양액의 0.5%(v/v)가 되도록 첨가하여 교반 배양기에서 150rpm, 20℃로 24시간 동안 불활화하였다. 배양균의 불활화를 BHIA(Difco, USA)에서 최종 확인하였다. 불활화가 확인된 균액을 12,000RCF로 원심분리한 다음, 멸균 생리 식염수로 3회 세척한 후 균체를 수거하고 최종적으로 습균체 무게를 측정하여 실험구에 따라 농도를 달리하여 현탁하였다. 각 S. parauberis 혈청형별 불활화 단독 백신 연구 결과를 기초로, 혈청형 Ib와 Ic는 상호 교차 백신 효능이 있는 것으로 확인되었으며, 표 24의 항원 조합과 농도로 넙치(평균전장: 15.05±0.9cm, 평균체중: 26.7±4.74g)에 복강주사 방법으로 백신을 접종하였다. 실험구당 총 80마리의 넙치를 실험에 사용하였다.The cryopreserved S. parauberis strain was cultured in BHIA medium to which 1.5% (v / v) NaCl was added at 30 ° C. for 24 hours. Colonies of the cultured plate medium were aseptically inoculated into BHIB to which 1.5% NaCl was added, and then incubated at 150 rpm, 30 ° C., 48 hours in a stirred incubator (JS Research Inc.). 37% (v / v) formalin (Merck, Germany) was added to the cultured bacterial solution to be 0.5% (v / v) of each culture solution, and inactivated at 150 rpm and 20 ° C. for 24 hours in a stirred incubator. The inactivation of the culture was finally confirmed in BHIA (Difco, USA). The inactivated bacteria were centrifuged at 12,000 RCF, washed three times with sterile physiological saline, the cells were collected, and finally, the weight of the cells was measured and suspended at different concentrations according to the experimental group. Based on the results of the inactivated single vaccine study for each S. parauberis serotype, serotypes Ib and Ic were found to have mutual cross-vaccination efficacy and flounder (average length: 15.05 ± 0.9cm, with the antigen combination and concentration in Table 24). The average body weight: 26.7 ± 4.74g) was vaccinated by an intraperitoneal injection method. A total of 80 halibut per experiment were used for the experiment.
백신 접종한 2주 후 미부정맥에서 혈액을 채혈(n=10)하여, ELISA법으로 S. parauberis에 대한 항체가를 측정하였다. ELISA법을 이용한 항체가 조사 방법으로 96-well plate에 S. parauberis FKC를 10μg/well의 농도가 되도록 조절하여 분주하고 4℃ over night하여 plate 바닥에 항원을 부착하였다. S. parauberis 혈청형별 ELISA값을 측정하기 위하여 3가지 다른 혈청형의 S. parauberis의 FKC를 제작하여 각각의 혈청에 대한 항체가를 조사하였다. 3% skim milk를 이용하여 실온에서 1시간 동안 blocking을 수행하고, 1차 항체로 넙치 혈청과 장 점액을 10배 희석하여 분주한 후 1시간 동안 반응시킨다. 그 후, PBST를 이용하여 3회 세척한 뒤 2차 항체는 Anti-Japanese flounder IgM Mab를 1:1000로 희석하여 분주한 후 1시간 동안 37℃에서 반응시킨다. 세척 후, AP-conjugated anti-mouse IgG(whole molecule)를 1:1000으로 희석하여 분주한 후 37℃에서 1시간 반응시키고, 세척한 다음, 기질(PNPP substrate solution)을 첨가하여 20분 후 405nm에서 흡광도를 확인하였다. 백신 접종 2주 후에 항체가를 측정한 결과, 대조구에서 항체가가 확인되지 않았으며, Ia+Ic 구 및 Ia+Ib+Ic 구에서 대조구와 비교하여 유의성 있게 높은 항체가를 형성하였다(표 25, 도 9).After 2 weeks of vaccination, blood was collected from the arrhythmia ( n = 10), and the antibody titer to S. parauberis was measured by ELISA. Antibodies using the ELISA method were used to control S. parauberis FKC to a concentration of 10 μg / well in 96-well plates and dispensed, and antigen was attached to the bottom of the plate at 4 ° C over night. In order to measure ELISA values by S. parauberis serotype, FKCs of S. parauberis of three different serotypes were prepared and antibody titers for each serum were examined. Blocking is performed at room temperature for 1 hour using 3% skim milk, and after diluting and dissolving halibut serum and
실시예 8-2: 다가백신의 효능 분석Example 8-2: Analysis of the efficacy of the polyvalent vaccine
백신 접종 3주 후에 S. parauberis Ia FPa4365(5.2×108cfu/fish), Ib PR5(7×108cfu/fish), Ic FPa4870(1.4×108cfu/fish) 3가지 균주로 수온 26~27℃를 유지하면서 인위감염 실험을 수행하였으며, 3주간 폐사율을 확인하였다(표 26).Three weeks after vaccination, the water temperature is 26 ~ with three strains of S. parauberis Ia FPa4365 (5.2 × 10 8 cfu / fish), Ib PR5 (7 × 10 8 cfu / fish), and Ic FPa4870 (1.4 × 10 8 cfu / fish). An artificial infection experiment was performed while maintaining 27 ° C. and mortality was confirmed for 3 weeks (Table 26).
대조구와 비교한 상대생존율은 아래와 같이 계산하였다.The relative survival rate compared to the control was calculated as follows.
상대생존율(%) = {1-(시험구의 누적폐사율/대조구의 누적폐사율)} × 100Relative survival rate (%) = {1- (cumulative mortality rate of the test zone / control mortality rate)} × 100
S. parauberis 혈청형 Ia+Ic 백신구와 Ia+Ib+Ic 백신구에 3가지 다른 혈청형의 S. parauberis로 인위감염하면 대조구와 비교하여 높은 생존율을 나타내었으며, 표 23의 S. parauberis 단일 혈청형 Ia 및 Ib 백신구와 비교하여 높은 상대생존율을 나타냈다(표 27). When S. parauberis serotype Ia + Ic and Ia + Ib + Ic vaccines were artificially infected with three different serotypes of S. parauberis , the survival rate was higher than that of the control group, and S. parauberis single serotype of Table 23. It showed a high relative survival rate compared to the Ia and Ib vaccine groups (Table 27).
예를 들어, Ia 단일 혈청형 백신 접종 후 S. parauberis Ia 혈청형의 FPa4365 균주로 인위감염하면 대조구와 비교하여 55.6%의 상대생존율을 나타내었으나, Ia+Ic 백신구와 Ia+Ib+Ic 구에 FPa4365 균주로 인위감염하면 대조구와 비교하여 93.4% 및 66.7%의 상대생존율을 나타내었다. 또한, Ic 단일 혈청형 백신 접종 후 S. parauberis Ic 혈청형의 FPa4870 균주로 인위감염하면 대조구와 비교하여 75.1%의 상대생존율을 나타내었으나, Ia+Ic 백신구와 Ia+Ib+Ic 구에 FPa4365 균주로 인위감염하면 대조구와 비교하여 50% 및 30%의 상대생존율을 나타내어 Ic 단일 혈청형 백신에 비해 낮은 효능을 나타내었다. 혈청형을 혼합한 다가백신 사이의 백신효능 비교에서는 Ia+Ic 백신구는 Ia+Ib+Ic 백신구에 비하여 3가지 모든 혈청형의 균주에 비해 높은 상대생존율을 나타내었으며, S. parauberis에 대한 특이항체가는 Ia+Ib+Ic 백신구가 Ia+Ic 백신구에 비해 높은 항체가를 나타내었다.For example, when artificially infected with the FPa4365 strain of the S. parauberis Ia serotype after vaccination of a single Ia serotype, the relative survival rate of 55.6% was compared to that of the control group, but FPa4365 in the Ia + Ic vaccine and Ia + Ib + Ic spheres. When artificially infected with the strain, the relative survival rates of 93.4% and 66.7% were shown compared to the control. In addition, when artificially infected with FPa4870 strain of S. parauberis Ic serotype after inoculation of Ic single serotype vaccination, the relative survival rate of 75.1% was compared with that of the control group, but the Ia + Ic vaccine and Ia + Ib + Ic spheres were used as FPa4365 strains. In the case of artificial infection, the relative survival rates of 50% and 30% were compared to those of the control group, and the efficacy was lower than that of the Ic single serotype vaccine. In comparison of vaccine efficacy between multivalent vaccines mixed with serotypes, the Ia + Ic vaccines showed higher relative survival rates compared to all three serotype strains compared to the Ia + Ib + Ic vaccines, and specific antibodies to S. parauberis Thin Ia + Ib + Ic vaccines showed higher antibody titers than Ia + Ic vaccines.
실시예 9: Example 9: S. parauberisS. parauberis 다가백신의 안정성 분석 Stability analysis of polyvalent vaccine
실시예 9-1: 다가백신 안정성 분석 방법Example 9-1: Multivalent vaccine stability analysis method
다가백신 안전성 조사를 위해서 넙치(평균전장: 15.83±1.04cm, 평균체중: 27.2±4.75g)에 백신효능 조사 시 사용한 항원 농도의 2, 5, 10배 농도로 복강으로 주사접종하여 안전성을 평가하였다(표 28). 복강접종 후 3주까지의 폐사율을 확인하였으며, 매주 넙치의 미부정맥에서 채혈하여(n=5), 혈액 생화학적 성상 및 병리조직학적 검사를 통하여 안전성을 확인하였다. 혈액 성상은 자동혈액분석기를 이용하여 glucose, ALT, AST 및 total protein의 농도를 측정하였으며, 병리조직학적 조사는 신장, 비장, 간 등을 중성포르말린에 고정 후 상법에 따라 조사하였다.To investigate the safety of the multivalent vaccine, safety was assessed by intraperitoneal injection of flounder (average length: 15.83 ± 1.04 cm, average weight: 27.2 ± 4.75 g) at a concentration of 2, 5, and 10 times the antigen concentration used for vaccine efficacy investigation. Table 28. Mortality was confirmed up to 3 weeks after intraperitoneal vaccination, and blood was collected from the arrhythmia of the flounder weekly ( n = 5) to confirm safety through blood biochemical properties and histopathological examination. Blood properties were measured using an automatic blood analyzer, and the concentrations of glucose, ALT, AST, and total protein were measured. Histopathological investigation was performed by fixing kidney, spleen, liver, etc. in neutral formalin and following the conventional method.
실시예 9-2: 다가백신의 안정성 분석 결과Example 9-2: Results of stability analysis of polyvalent vaccine
S. parauberis Ia+Ic 백신구, Ia+Ib+Ic 백신구의 항원량을 2배, 5배, 10배의 고농도로 백신을 주사한 후 3주간 모든 실험구에서 폐사가 발생하지 않았다. 혈액생화학적 성상(AST, ALT, GLU, TP)은 모든 백신 접종구에서 백신 접종 후 1, 2, 3주에 대조구와 유의적인 차이가 보이지 않아, 투여 농도는 넙치에 안전한 것으로 확인되었다(P>0.05, 표 29). No death occurred in all experimental groups for 3 weeks after the vaccine was injected at high concentrations of 2, 5, and 10 times the antigen levels of the S. parauberis Ia + Ic vaccine and Ia + Ib + Ic vaccine. Blood biochemical properties (AST, ALT, GLU, TP) did not show a significant difference from the control group at 1, 2, or 3 weeks after vaccination in all vaccination groups, and the concentration was confirmed to be safe for flounder (P> 0.05, Table 29).
항목Item
(Ia+Ic 2배)(Ia + Ic 2x)
(Ia+Ic 5배)(Ia +
(Ia+Ic 10배)(Ia +
(Ia+Ib+Ic 2배)(2 times Ia + Ib + Ic)
(Ia+Ib+Ic 5배)(Ia + Ib +
(Ia+Ib+Ic 10배)(Ia + Ib +
(대조구)(Control)
(U/L)(U / L)
(U/L)(U / L)
(mg/dl)(mg / dl)
(g/dl)(g / dl)
다가백신 접종 후 병리조직학적 검사 결과, 백신구와 대조구의 간, 신장 및 비장 병리조직학적 검사에서 차이가 보이지 않아 다가백신은 넙치에 안전한 것으로 확인되었다(도 10 내지 도 12).As a result of the histopathological examination after inoculation of the polyvalent vaccine, no differences were observed in the liver, kidney and spleen pathological histologic examinations of the vaccine and control groups, and the polyvalent vaccine was confirmed to be safe for flounder (FIGS. 10 to 12).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Since specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> Republic of Korea(National Institute of Fisheries Science) <120> Combination Vaccines Against Streptococcal Disease in Fish <130> P19-B252 <160> 45 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 caggcctaac acatgcaagt c 21 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acgggcggtg tgtrc 15 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gahgtngtsg aaggsatgca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atttgrcgsa ywggytcttc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggttcstcng tsgttaatgc 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gttcrtgngt tccsccttca t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gctaaggtca tgacccattc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aggcgtgcaa tttcagcttg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ctcaaactga ggctagtaat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gtcccggcta atccaactgt 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cttggtggat cccaagcac 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ccaaacaacc actctccagc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gcaagcggtc acggaaaagt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cacctacata ggttccacta 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gcaaatctgt cggcttttcg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 acgactacca aggacgttac 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ccgaagagta ctataactcc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tggcttacca taattcccgt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 attgttagtc attcagttgt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 aattatagtc aacagtccag 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 atttctacca ggttactttg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gaactactta ggtttagcat 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 aacttgtaaa taggattgct 20 <210> 24 <211> 1287 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA <400> 24 gcctagtcag atgagttgcg aacgggtgag taacgcgtag gtaacctacc tcatagcggg 60 ggataactat tggaaacgat agctaatacc gcatgacaat taagtactca tgtactaaat 120 ttaaaaggag caattgcttc actatgagat ggacctgcgt tgtattagct agttggtgag 180 gtaacggctc accaaggcca cgatacatag ccgacctgag agggtgatcg gccacactgg 240 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc ggcaatgggg 300 gcaaccctga ccgagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt aaagctctgt 360 tgttagagaa gaacggtaat gggagtggaa aatccattac gtgacggtaa ctaaccagaa 420 agggacggct aactacgtgc cagcagccgc ggtaatacgt aggtctcgag cgttgtccgg 480 atttattggg cgtaaagcga gcgcaggcgg ttatttaagt ctgaagttaa aggccgtggc 540 tcaaccatgg ttcgctttgg aaactggata acttgagtgc agaaggggag agtggaattc 600 catgtgtagc ggtgaaatgc gtagatatat ggaggaacac cggtggcgaa agcggctctc 660 tggtctgtaa ctgacgctga ggctcgaaag cgtggggagc aaacaggatt agataccctg 720 gtagtccacg ccgtaaacga tgagtgctag gtgttaggcc ctttccgggg cttagtgccg 780 cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga 840 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 900 ccttaccagg tcttgacatc cctctgaccg tcctagagat aggactttcc ttcgggacag 960 aggtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020 gcaacgagcg caacccctat tgttagttgc catcattaag ttgggcactc tagcgagact 1080 gccggtaata aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1140 gggctacaca cgtgctacaa tggttggtac aacgagtcgc aagccggtga cggcaagcta 1200 atctcttaaa gccaatctca gttcggattg taggctgcaa ctcgcctaca tgaagtcgga 1260 atcgctagta atcgcggatc agcacgc 1287 <210> 25 <211> 465 <212> DNA <213> Artificial Sequence <220> <223> gyrB-A <400> 25 tcatctcagt tagatgttcg tgtcttcaaa aatggtagca ttcattatca agagtttaaa 60 cgtggaatcg tttatgatga tttgaaaatt attggagaaa cggatttaac aggaacaaca 120 gttcacttta ctccggaccc agaaattttt acagaaacag ttgaatttga ttttgaaaag 180 cttgctaagc gtgtccaaga attagcattt ttaaaccgtg gattacgaat ctcaattgcc 240 gataaacgta atggaattga acaagtcaaa gattaccatt atgaaggtgg tatcgctagc 300 tatgttgaat ttttaaatga gaaaaaagat gttattatcg aacatcctat ctttactgat 360 ggagagatgg atggcattgc tgtcgaagtt gcgatgcaat atacaacagg ctatcatgaa 420 aatgtaatga gttttgccaa caatattcat acacatgaag gcgga 465 <210> 26 <211> 465 <212> DNA <213> Artificial Sequence <220> <223> gyrB-B <400> 26 tcatctcagt tagatgttcg tgtcttcaaa aatggtagca ttcattatca agagtttaaa 60 cgtggaatcg tttatgatga tttgaaaatt attggagaaa cggatttaac aggcacaaca 120 gttcacttta ctccggaccc agaaattttt acagaaacag ttgaatttga ttttgaaaag 180 cttgctaagc gtgtccaaga attagcattt ttaaaccgtg gattacgaat ctcaattgcc 240 gataaacgta atggaattga acaagtcaaa gattaccatt atgaaggtgg tatcgctagc 300 tatgttgaat ttttaaatga gaaaaaagat gttattatcg aacatcctat ctttactgat 360 ggagagatgg atggcattgc tgtcgaagtt gcgatgcaat atacaacagg ctatcatgaa 420 aatgtaatga gttttgccaa caatattcat acacatgaag gcgga 465 <210> 27 <211> 769 <212> DNA <213> Artificial Sequence <220> <223> groE-A <400> 27 ggatgcagtt tgaccgtggt tacctttctc aatacatggt aacagataac gaaaaaatgg 60 ttgctgactt agaaaatcca tttattttga tcactgataa aaaagtatca aacattcaag 120 aaattcttcc actcttagaa gaagtcttaa aaacaaaccg cccattattg attattgctg 180 atgatgttga tggtgaagcc ttaccaacat tagttttaaa taaaattcgc ggaacattta 240 atgttgttgc cgttaaagca ccaggatttg gcgaccgtcg taaggctatg ttagaggata 300 ttgccattct aacaggtgga actgtgatta ctgaagatct tggtttagag cttaaggatg 360 cgacaatagc agcgctaggc caagcagcta aaatctctgt ggataaagat tcaacagtta 420 ttgttgaagg ttctggcggt gcggatgcta ttgccaatcg tgttggccta attaaatccc 480 aattagaaac aacaacgtct gagttcgacc gtgagaaatt gcaagaacgt ctagctaagt 540 tagcgggtgg tgtggcagtt atcaaggttg gtgctgcgac agaaactgag ttgaaagaaa 600 tgaagctccg cattgaagat gctttgaatg ctactcgtgc tgctgttgaa gaagggatcg 660 tagctggtgg aggaactgcc tttatttcag tgattgagaa agtgcaagct ctagaacttg 720 aaggtgataa tgcgactggt cgtaacattg tccttcgtgc ccttgaaga 769 <210> 28 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> simA-A <400> 28 gctaaagagg ctgaagttgc taacaatcgt gttattgttt taagtaatgc tttaaatcaa 60 gctaaagaag aatttgattt gaaagctgct gaagcacaaa aactgaataa taacgtcact 120 gtattatcaa atgccctaca acaagctaaa gaagaatttg acaaaaaagc ccaggactat 180 caagcagctc ttgcagaaaa agatgctatt gacggacaat tagaaactgc tttgactaaa 240 gtttcagacc tagaaagtca attggcaagt ttaaaagaag ctaatgaagc tgctaatcaa 300 aagattactc aattagaaca agaagttgct gatggtgggg ttgcctttaa agaccttaaa 360 gggcaaaaag aaaaatcaga cgcacaagtt aaagctcaag cagataagat taaagaactg 420 gaatctgact tagaagcgac tcaatcagaa ttaaaccagg tgaaagagca acgtgctaat 480 ttagaacaag aagttgctga gcttggaact aaggtaaaag atttagaaaa agaaaaagct 540 gatttagaga aaaaagcgat agacttggaa gctgacaaag cggaatttga agcagaaatg 600 gatcgtttaa aagccttact agctgataaa gacttagcaa atgataaatt acttaaagaa 660 atggaagatc tacaaaaaga ctttgatact aagaaaaatc atagtgacag agaa 714 <210> 29 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> simA-B <400> 29 gctaaagagg ctgaagttgc taacaatcgt gttattgttt taagtaatgc tttaaatcaa 60 gctaaagaag aatttgattt gaaagctgct gaagcacaaa aactgaataa taacgtcact 120 gtattatcaa atgccctaca acaagctaaa gaagaatttg acaaaaaagc ccaggactat 180 caagcagctc ttgcagaaaa agatgctatt gacggacaat tagaaactgc tttgactaaa 240 gtttcagacc tagaaagtca attggcaagt ttaaaagaag ctaatgaagc tgctaatcaa 300 aagattactc aattagaaca agaagttgct gatggtgggg ttgcctttaa agaccttaaa 360 gggcaaaaag aaaaatcaga cgcacaagtt aaagctcaag cagataagat taaagaactg 420 gaatctgact tagaagcgac tcaatcagaa ttaaaccagg tgaaagagca acgtgctaat 480 ttagaacaag aagttgctga gcttggaacc aaggtaaaag atttagaaaa agaaaaagct 540 gatttagaga aaaaagcgat agacttggaa gctgacaaag cggaacttga agcagaaatg 600 gatcgtttaa aagccttact agctgataaa gacttagcaa atgataaatt acttaaagaa 660 atggaagatc tacaaaaaga ctttgatact aagaaaaatc atagtgacag agaa 714 <210> 30 <211> 1237 <212> DNA <213> Artificial Sequence <220> <223> autolysin-A <400> 30 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tcagttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc gaaacagtct 180 cagctgatgt gagtggtcat gttttaaaaa ttgtctataa tgggcaacta gaaccaacta 240 agaagataaa gtatgcagtt tggacagata atgctggcca agatgattta gtttggtata 300 ctgctgatca agttggtgcg gcatatattg atttatctaa aaaacataga gcatatgggc 360 tatataatat tcatacctat tcgcaagatg taacaggtaa gatgtctggc ttaaatgcga 420 gacaatttac tattcttaaa ccaacagtaa gtacatcgtt taacgtacaa actaatggtc 480 ttgttgatat tgtcgtttca aatgtgagag gtgatattag ctcaattaaa gttcctgttt 540 ggtcagattt aggtggccaa aacgatatta aatggtatca agctactcaa tcaactgatg 600 gaacatacaa agtttcagta aagatttcag atcattcaaa tgatactggc cattttgctg 660 ttcatgttta tgggaatagt acaatcacga atagtcaaat cggattaggg acaacagaag 720 gttttacaat tgctacccca gacccccaaa atgtagtgtc agtcgtagta gctaatgatg 780 gtttccacct agcattaaat tcgaatgttg ttaaagaatt taccaaagtg aaatttgctg 840 tttggtcgga tcaagctggt caagatgacc ttcattggta cacagccaat gctcagggtc 900 aagtcattgt tccttatgtt aatcatagta attatggtct atacaacatc catacctata 960 gttttgagtc gggaagtgct aaaggtctaa atacaagaac aattacagta ccaaatccaa 1020 ctgctagcgc agccattaca cagaaatcag atcttgaatt tttagtatca gttacgaatg 1080 tcccagctta tatcacaaaa gttatgttac caagttggag tgaaattaat ggccaagatg 1140 atatcaagtg ggtgacggca agtaagctag ctgataatag ttaccaagcc attattaata 1200 ttggagatca taagtataat ttaggtcatt atttagt 1237 <210> 31 <211> 1237 <212> DNA <213> Artificial Sequence <220> <223> autolysin-A1 <400> 31 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tcccttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc gaaacagtct 180 cagctgatgt gagtggtcat gttttaaaaa ttgtctataa tgggcaacta gaaccaacta 240 agaagataaa gtatgcagtt tggacagata atgctggcca agatgattta gtttggtata 300 ctgctgatca agttggtgcg gcatatattg atttatctaa aaaacataga gcatatgggc 360 tatataatat tcatacctat tcgcaagatg taacaggtaa gatgtctggc ttaaatgcga 420 gacaatttac tattcttaaa ccaacagtaa gtacatcgtt taacgtacaa actaatggtc 480 ttgttgatat tgtcgtttca aatgtgagag gtgatattag ctcaattaaa gttcctgttt 540 ggtcagattt aggtggccaa aacgatatta aatggtatca agctactcaa tcaactgatg 600 gaacatacaa agtttcagta aagatttcag atcattcaaa tgatactggc cattttgctg 660 ttcatgttta tgggaatagt acaatcacga atagtcaaat cggattaggg acaacagaag 720 gttttacaat tgctacccca gacccccaaa atgtagtgtc agtcgtagta gctaatgatg 780 gtttccacct agcattaaat tcgaatgttg ttaaagaatt taccaaagtg aaatttgctg 840 tttggtcgga tcaagctggt caagatgacc ttcattggta cacagccaat gctcagggtc 900 aagtcattgt tccttatgtt aatcatagta attatggtct atacaacatc catacctata 960 gttttgagtc gggaagtgct aaaggtctaa atacaagaac aattacagta ccaaatccaa 1020 ctgctagcgc agccattaca cagaaatcag atcttgaatt tttagtatca gttacgaatg 1080 tcccagctta tatcacaaaa gttatgttac caagttggag tgaaattaat ggccaagatg 1140 atatcaagtg ggtgacggca agtaagctag ctgataatag ttaccaagcc attattaata 1200 ttggagatca taagtataat ttaggtcatt atttagt 1237 <210> 32 <211> 1228 <212> DNA <213> Artificial Sequence <220> <223> autolysin-B <400> 32 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tcagttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc tcagctgatg 180 tgagtggtca tgttttaaaa attgtctata atgggcaact agaaccaact aagaagataa 240 agtatgcagt ttggacagat aatggtggcc aagatgattt agtttggtat actgctgatc 300 aagttggtgc ggcatatatt gatttatcta aaaaacatag agcatatggg ctatataata 360 ttcataccta ttcgcaagat gtaacaggta agatgtctgg cttaaatgcg agacaattta 420 ctattcttaa accaacagta agtacatcgt ttaacgtaca aactaatggt attgttgata 480 ttgtcgtttc aaatgtgaga ggtgatatta gctcaattaa agttcctgtt tggtcagatt 540 taggtggcca aaacgatatt aaatggtatc aagctactca atcaactgat ggaacataca 600 aagtttcagt aaagatttca gatcattcaa atgatactgg ccattttgct gttcatgttt 660 atgggaatag tacaatcacg aatagtcaaa tcggattagg gacaacagaa ggttttacaa 720 ttgctacccc agaccccaaa aatgtagtgt cagccgtagt agctaatgat ggtttccacc 780 tagcattaaa ttcgaatgtt gttaaagaat ttaccaaagt gaaatttgct gtttggtcgg 840 atcaagctgg tcaagatgac cttcattggt acacagccaa tgctcagggt caagtcattg 900 ttccttatgt taatcatagt aattatggtc tatacaacat ccatacctat agttttgagt 960 cgggaagtgc taaaggtcta aatacaagaa caattacagt accaaatcca actgctagcg 1020 cagccattac acagaaatca gatcttgaat ttttagtatc agttacgaat gtcccagctt 1080 atatcacaaa agttatgtta ccaagttgga gtgaaattaa tggccaagat gatatcaagt 1140 gggtgacggc aagtaagcta gctgataata gttaccaagc cattattaat attggagatc 1200 ataagtataa tttaggtcat tatttagt 1228 <210> 33 <211> 1228 <212> DNA <213> Artificial Sequence <220> <223> autolysin-B1 <400> 33 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tccgttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc tcagctgatg 180 tgagtggtca tgttttaaaa attgtctata atgggcaact agaaccaact aagaagataa 240 agtatgcagt ttggacagat aatggtggcc aagatgattt agtttggtat actgctgatc 300 aagttggtgc ggcatatatt gatttatcta aaaaacatag agcatatggg ctatataata 360 ttcataccta ttcgcaagat gtaacaggta agatgtctgg cttaaatgcg agacaattta 420 ctattcttaa accaacagta agtacatcgt ttaacgtaca aactaatggt attgttgata 480 ttgtcgtttc aaatgtgaga ggtgatatta gctcaattaa agttcctgtt tggtcagatt 540 taggtggcca aaacgatatt aaatggtatc aagctactca atcaactgat ggaacataca 600 aagtttcagt aaagatttca gatcattcaa atgatactgg ccattttgct gttcatgttt 660 atgggaatag tacaatcacg aatagtcaaa tcggattagg gacaacagaa ggttttacaa 720 ttgctacccc agaccccaaa aatgtagtgt cagccgtagt agctaatgat ggtttccacc 780 tagcattaaa ttcgaatgtt gttaaagaat ttaccaaagt gaaatttgct gtttggtcgg 840 atcaagctgg tcaagatgac cttcattggt acacagccaa tgctcagggt caagtcattg 900 ttccttatgt taatcatagt aattatggtc tatacaacat ccatacctat agttttgagt 960 cgggaagtgc taaaggtcta aatacaagaa caattacagt accaaatcca actgctagcg 1020 cagccattac acagaaatca gatcttgaat ttttagtatc agttacgaat gtcccagctt 1080 atatcacaaa agttatgtta ccaagttgga gtgaaattaa tggccaagat gatatcaagt 1140 gggtgacggc aagtaagcta gctgataata gttaccaagc cattattaat attggagatc 1200 ataagtataa tttaggtcat tatttagt 1228 <210> 34 <211> 405 <212> DNA <213> Artificial Sequence <220> <223> CPB-A <400> 34 ccaagcacta ataatgaata tttacagaaa tattgatcgt acacaaattc aatttgattt 60 tataatcgat catccaagta tgaactacta tcaggatgaa atagaaagac taggtggacg 120 agtatattct tttccaacat ttacaggaag aaatgttcgt aatgtgcgta atgaatggga 180 taagtttttt aaagaacatg tagagtactc gattattcat tttcatgtta gaagttatat 240 ttctttatta attcctattg caaaaagata cggtttaata acaatatcac atagtcatag 300 tatttctaat ggactaggac taaaatcaaa gattaagaat cttttacaaa ttccgattag 360 atatcaagct gattatcttt ttgcttgctc taaagaggct ggaga 405 <210> 35 <211> 533 <212> DNA <213> Artificial Sequence <220> <223> SOA-A <400> 35 gaaaagttgt tgcagaaatt gctaaattat ctggttataa cgacattatt tttcttgatg 60 attattctaa cgaaaaactt tgttctggtt atccagttgt cggaaaagtt tctgaaattg 120 ttaatttcaa aaatgaagat gtttttatag ctattggatc aagtgctgtt agagagaaaa 180 ttgctaaaca tttgaaggac cacaaaatag tatccttaat acatccggcc gcagttgtaa 240 gtgaaaaagc taaaattggc aaagggagtg ttattatggc tggtgctgta gtaaaccctg 300 atactgaaat tggtgaattt tgtatagtca atacttgttc ttctgtagat catgattgta 360 ttataggtga tttttcacac gtctcagttg gcagtcacgt tgcaggaaca gtaacagttg 420 gttctcatgt atggataggt gctggggcga caataattaa taacatcgaa acacacaata 480 atatttgtat tggagcagga gcaacggtta taaataattt agtagatagt gga 533 <210> 36 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-A <400> 36 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatccct attaagttat ttggtggacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgctggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacacgggat taaagtactg ggaaataaaa attccattcc 420 aagattacta gcagaatatg aagttaaaca ggttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatctg ccgtaacgca aacgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctcaac tgacaaagct gttaacccac ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 37 <211> 1112 <212> DNA <213> Artificial Sequence <220> <223> PB-A1 <400> 37 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatccct attaagttat ttggtggacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggtgct ggtgaaggtg gtagtgcctt tattcaaaca 300 attctgaata agagtaaaga tattgacatt gtcggtattg ttgatgctga tatcaataaa 360 ttaggcacct acttacacgg gattaaagta ctgggaaata aaaattccat tccaagatta 420 ctagcagaat atgaagttaa acaggttacg attgccatcc caagtttatc tggggaagaa 480 cgagaatcaa tcttagatat ctgccgtaac gcaaacgttc acgtaaacaa tatgcctagt 540 attgagaata tcgttctagg taatgtgtca cttaataaat ttaaagaaat tgagattgct 600 gacttacttg gacgaaagga agtagtatta gatcaaacgt ctttaaattc attctttaac 660 gggaaaacag ttcttgtaac tggtgcagga ggatcaattg gttcagaaat ctgtcgtcaa 720 gtttctaaat ttaatccagc acgcatttta cttttagggc atggtgaaaa ttccatttat 780 ctgattcatc gtgaattatc agcactgtta aagggacgaa ttgacattgt cccaattatc 840 gcggacattc aagatcgaga cttgattttt gaaattatgg cgaattatcg acctgatata 900 gtctatcatg ctgcagcaca taaacatgtg ccattgatgg aatacaatcc aaaagaagct 960 gttaaaaata atatctttgg gacaaaaaat gtggcggaag cagccaaggc tgctgggatt 1020 cctaaattta tcatggtctc aactgacaaa gctgttaacc cacctaatgt gatgggtgca 1080 actaagcgct tcgctgaaat gatcgtcact gg 1112 <210> 38 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B <400> 38 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 39 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B1 <400> 39 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agacgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 40 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B2 <400> 40 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtgttgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 41 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B3 <400> 41 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 42 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-A <400> 42 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacaac atccgcaaat attgctatct catttgctaa 120 agcaggtcta aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctgtcaggta atgcagaatt 240 atcagcagtt atctctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatcgactc 420 agcgattatt tcacaaaaag ctgacgcaaa catcttagta acagaagctg gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtgctt tgttcttggg 540 tgttatttta aataaagtag aagaaacact tgattcatat ggtggttatg gtag 594 <210> 43 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-A1 <400> 43 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacaac atccgcaaat attgctatct catttgctaa 120 agcaggtcta aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctgtcaggta atgcagaatt 240 atcagcagtt atctctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatcgactc 420 agcgattatt tcacaaaaag ctgacgcaaa catcttagta acagaagctg gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtgctt tgttcttggg 540 tgttatttta aataaagtag aagaatcact tgattcatat ggtggttatg gtag 594 <210> 44 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-B <400> 44 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacatt atccgcaaat attgctatct catttgctaa 120 agcaggtctt aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctatcaggta atgcagaatt 240 atcagcagtt atttctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatagactc 420 agcgattatt tcacaaaaag ctgacgcaaa cattttagta acagaagcag gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtgcct tgttcttggg 540 tgttatttta aataaagtag aagaaacact tgattcatat ggtggttatg gtag 594 <210> 45 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-B1 <400> 45 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacatt atccgcaaat attgctatct catttgctaa 120 agcaggtctt aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctatcaggta atgcagaatt 240 atcagcagtt atttctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatagactc 420 agcgattatt tcacaaaaag ctgacgcaaa cattttagta acagaagcag gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtacct tgttcttggg 540 tgttatttta aataaagtag aagaaacact tgattcatat ggtggttatg gtag 594 <110> Republic of Korea (National Institute of Fisheries Science) <120> Combination Vaccines Against Streptococcal Disease in Fish <130> P19-B252 <160> 45 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 caggcctaac acatgcaagt c 21 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acgggcggtg tgtrc 15 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gahgtngtsg aaggsatgca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atttgrcgsa ywggytcttc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggttcstcng tsgttaatgc 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gttcrtgngt tccsccttca t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gctaaggtca tgacccattc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aggcgtgcaa tttcagcttg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ctcaaactga ggctagtaat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gtcccggcta atccaactgt 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cttggtggat cccaagcac 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ccaaacaacc actctccagc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gcaagcggtc acggaaaagt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cacctacata ggttccacta 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gcaaatctgt cggcttttcg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 acgactacca aggacgttac 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ccgaagagta ctataactcc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tggcttacca taattcccgt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 attgttagtc attcagttgt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 aattatagtc aacagtccag 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 atttctacca ggttactttg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gaactactta ggtttagcat 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 aacttgtaaa taggattgct 20 <210> 24 <211> 1287 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA <400> 24 gcctagtcag atgagttgcg aacgggtgag taacgcgtag gtaacctacc tcatagcggg 60 ggataactat tggaaacgat agctaatacc gcatgacaat taagtactca tgtactaaat 120 ttaaaaggag caattgcttc actatgagat ggacctgcgt tgtattagct agttggtgag 180 gtaacggctc accaaggcca cgatacatag ccgacctgag agggtgatcg gccacactgg 240 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc ggcaatgggg 300 gcaaccctga ccgagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt aaagctctgt 360 tgttagagaa gaacggtaat gggagtggaa aatccattac gtgacggtaa ctaaccagaa 420 agggacggct aactacgtgc cagcagccgc ggtaatacgt aggtctcgag cgttgtccgg 480 atttattggg cgtaaagcga gcgcaggcgg ttatttaagt ctgaagttaa aggccgtggc 540 tcaaccatgg ttcgctttgg aaactggata acttgagtgc agaaggggag agtggaattc 600 catgtgtagc ggtgaaatgc gtagatatat ggaggaacac cggtggcgaa agcggctctc 660 tggtctgtaa ctgacgctga ggctcgaaag cgtggggagc aaacaggatt agataccctg 720 gtagtccacg ccgtaaacga tgagtgctag gtgttaggcc ctttccgggg cttagtgccg 780 cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga 840 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 900 ccttaccagg tcttgacatc cctctgaccg tcctagagat aggactttcc ttcgggacag 960 aggtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020 gcaacgagcg caacccctat tgttagttgc catcattaag ttgggcactc tagcgagact 1080 gccggtaata aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1140 gggctacaca cgtgctacaa tggttggtac aacgagtcgc aagccggtga cggcaagcta 1200 atctcttaaa gccaatctca gttcggattg taggctgcaa ctcgcctaca tgaagtcgga 1260 atcgctagta atcgcggatc agcacgc 1287 <210> 25 <211> 465 <212> DNA <213> Artificial Sequence <220> <223> gyrB-A <400> 25 tcatctcagt tagatgttcg tgtcttcaaa aatggtagca ttcattatca agagtttaaa 60 cgtggaatcg tttatgatga tttgaaaatt attggagaaa cggatttaac aggaacaaca 120 gttcacttta ctccggaccc agaaattttt acagaaacag ttgaatttga ttttgaaaag 180 cttgctaagc gtgtccaaga attagcattt ttaaaccgtg gattacgaat ctcaattgcc 240 gataaacgta atggaattga acaagtcaaa gattaccatt atgaaggtgg tatcgctagc 300 tatgttgaat ttttaaatga gaaaaaagat gttattatcg aacatcctat ctttactgat 360 ggagagatgg atggcattgc tgtcgaagtt gcgatgcaat atacaacagg ctatcatgaa 420 aatgtaatga gttttgccaa caatattcat acacatgaag gcgga 465 <210> 26 <211> 465 <212> DNA <213> Artificial Sequence <220> <223> gyrB-B <400> 26 tcatctcagt tagatgttcg tgtcttcaaa aatggtagca ttcattatca agagtttaaa 60 cgtggaatcg tttatgatga tttgaaaatt attggagaaa cggatttaac aggcacaaca 120 gttcacttta ctccggaccc agaaattttt acagaaacag ttgaatttga ttttgaaaag 180 cttgctaagc gtgtccaaga attagcattt ttaaaccgtg gattacgaat ctcaattgcc 240 gataaacgta atggaattga acaagtcaaa gattaccatt atgaaggtgg tatcgctagc 300 tatgttgaat ttttaaatga gaaaaaagat gttattatcg aacatcctat ctttactgat 360 ggagagatgg atggcattgc tgtcgaagtt gcgatgcaat atacaacagg ctatcatgaa 420 aatgtaatga gttttgccaa caatattcat acacatgaag gcgga 465 <210> 27 <211> 769 <212> DNA <213> Artificial Sequence <220> <223> groE-A <400> 27 ggatgcagtt tgaccgtggt tacctttctc aatacatggt aacagataac gaaaaaatgg 60 ttgctgactt agaaaatcca tttattttga tcactgataa aaaagtatca aacattcaag 120 aaattcttcc actcttagaa gaagtcttaa aaacaaaccg cccattattg attattgctg 180 atgatgttga tggtgaagcc ttaccaacat tagttttaaa taaaattcgc ggaacattta 240 atgttgttgc cgttaaagca ccaggatttg gcgaccgtcg taaggctatg ttagaggata 300 ttgccattct aacaggtgga actgtgatta ctgaagatct tggtttagag cttaaggatg 360 cgacaatagc agcgctaggc caagcagcta aaatctctgt ggataaagat tcaacagtta 420 ttgttgaagg ttctggcggt gcggatgcta ttgccaatcg tgttggccta attaaatccc 480 aattagaaac aacaacgtct gagttcgacc gtgagaaatt gcaagaacgt ctagctaagt 540 tagcgggtgg tgtggcagtt atcaaggttg gtgctgcgac agaaactgag ttgaaagaaa 600 tgaagctccg cattgaagat gctttgaatg ctactcgtgc tgctgttgaa gaagggatcg 660 tagctggtgg aggaactgcc tttatttcag tgattgagaa agtgcaagct ctagaacttg 720 aaggtgataa tgcgactggt cgtaacattg tccttcgtgc ccttgaaga 769 <210> 28 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> simA-A <400> 28 gctaaagagg ctgaagttgc taacaatcgt gttattgttt taagtaatgc tttaaatcaa 60 gctaaagaag aatttgattt gaaagctgct gaagcacaaa aactgaataa taacgtcact 120 gtattatcaa atgccctaca acaagctaaa gaagaatttg acaaaaaagc ccaggactat 180 caagcagctc ttgcagaaaa agatgctatt gacggacaat tagaaactgc tttgactaaa 240 gtttcagacc tagaaagtca attggcaagt ttaaaagaag ctaatgaagc tgctaatcaa 300 aagattactc aattagaaca agaagttgct gatggtgggg ttgcctttaa agaccttaaa 360 gggcaaaaag aaaaatcaga cgcacaagtt aaagctcaag cagataagat taaagaactg 420 gaatctgact tagaagcgac tcaatcagaa ttaaaccagg tgaaagagca acgtgctaat 480 ttagaacaag aagttgctga gcttggaact aaggtaaaag atttagaaaa agaaaaagct 540 gatttagaga aaaaagcgat agacttggaa gctgacaaag cggaatttga agcagaaatg 600 gatcgtttaa aagccttact agctgataaa gacttagcaa atgataaatt acttaaagaa 660 atggaagatc tacaaaaaga ctttgatact aagaaaaatc atagtgacag agaa 714 <210> 29 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> simA-B <400> 29 gctaaagagg ctgaagttgc taacaatcgt gttattgttt taagtaatgc tttaaatcaa 60 gctaaagaag aatttgattt gaaagctgct gaagcacaaa aactgaataa taacgtcact 120 gtattatcaa atgccctaca acaagctaaa gaagaatttg acaaaaaagc ccaggactat 180 caagcagctc ttgcagaaaa agatgctatt gacggacaat tagaaactgc tttgactaaa 240 gtttcagacc tagaaagtca attggcaagt ttaaaagaag ctaatgaagc tgctaatcaa 300 aagattactc aattagaaca agaagttgct gatggtgggg ttgcctttaa agaccttaaa 360 gggcaaaaag aaaaatcaga cgcacaagtt aaagctcaag cagataagat taaagaactg 420 gaatctgact tagaagcgac tcaatcagaa ttaaaccagg tgaaagagca acgtgctaat 480 ttagaacaag aagttgctga gcttggaacc aaggtaaaag atttagaaaa agaaaaagct 540 gatttagaga aaaaagcgat agacttggaa gctgacaaag cggaacttga agcagaaatg 600 gatcgtttaa aagccttact agctgataaa gacttagcaa atgataaatt acttaaagaa 660 atggaagatc tacaaaaaga ctttgatact aagaaaaatc atagtgacag agaa 714 <210> 30 <211> 1237 <212> DNA <213> Artificial Sequence <220> <223> autolysin-A <400> 30 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tcagttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc gaaacagtct 180 cagctgatgt gagtggtcat gttttaaaaa ttgtctataa tgggcaacta gaaccaacta 240 agaagataaa gtatgcagtt tggacagata atgctggcca agatgattta gtttggtata 300 ctgctgatca agttggtgcg gcatatattg atttatctaa aaaacataga gcatatgggc 360 tatataatat tcatacctat tcgcaagatg taacaggtaa gatgtctggc ttaaatgcga 420 gacaatttac tattcttaaa ccaacagtaa gtacatcgtt taacgtacaa actaatggtc 480 ttgttgatat tgtcgtttca aatgtgagag gtgatattag ctcaattaaa gttcctgttt 540 ggtcagattt aggtggccaa aacgatatta aatggtatca agctactcaa tcaactgatg 600 gaacatacaa agtttcagta aagatttcag atcattcaaa tgatactggc cattttgctg 660 ttcatgttta tgggaatagt acaatcacga atagtcaaat cggattaggg acaacagaag 720 gttttacaat tgctacccca gacccccaaa atgtagtgtc agtcgtagta gctaatgatg 780 gtttccacct agcattaaat tcgaatgttg ttaaagaatt taccaaagtg aaatttgctg 840 tttggtcgga tcaagctggt caagatgacc ttcattggta cacagccaat gctcagggtc 900 aagtcattgt tccttatgtt aatcatagta attatggtct atacaacatc catacctata 960 gttttgagtc gggaagtgct aaaggtctaa atacaagaac aattacagta ccaaatccaa 1020 ctgctagcgc agccattaca cagaaatcag atcttgaatt tttagtatca gttacgaatg 1080 tcccagctta tatcacaaaa gttatgttac caagttggag tgaaattaat ggccaagatg 1140 atatcaagtg ggtgacggca agtaagctag ctgataatag ttaccaagcc attattaata 1200 ttggagatca taagtataat ttaggtcatt atttagt 1237 <210> 31 <211> 1237 <212> DNA <213> Artificial Sequence <220> <223> autolysin-A1 <400> 31 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tcccttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc gaaacagtct 180 cagctgatgt gagtggtcat gttttaaaaa ttgtctataa tgggcaacta gaaccaacta 240 agaagataaa gtatgcagtt tggacagata atgctggcca agatgattta gtttggtata 300 ctgctgatca agttggtgcg gcatatattg atttatctaa aaaacataga gcatatgggc 360 tatataatat tcatacctat tcgcaagatg taacaggtaa gatgtctggc ttaaatgcga 420 gacaatttac tattcttaaa ccaacagtaa gtacatcgtt taacgtacaa actaatggtc 480 ttgttgatat tgtcgtttca aatgtgagag gtgatattag ctcaattaaa gttcctgttt 540 ggtcagattt aggtggccaa aacgatatta aatggtatca agctactcaa tcaactgatg 600 gaacatacaa agtttcagta aagatttcag atcattcaaa tgatactggc cattttgctg 660 ttcatgttta tgggaatagt acaatcacga atagtcaaat cggattaggg acaacagaag 720 gttttacaat tgctacccca gacccccaaa atgtagtgtc agtcgtagta gctaatgatg 780 gtttccacct agcattaaat tcgaatgttg ttaaagaatt taccaaagtg aaatttgctg 840 tttggtcgga tcaagctggt caagatgacc ttcattggta cacagccaat gctcagggtc 900 aagtcattgt tccttatgtt aatcatagta attatggtct atacaacatc catacctata 960 gttttgagtc gggaagtgct aaaggtctaa atacaagaac aattacagta ccaaatccaa 1020 ctgctagcgc agccattaca cagaaatcag atcttgaatt tttagtatca gttacgaatg 1080 tcccagctta tatcacaaaa gttatgttac caagttggag tgaaattaat ggccaagatg 1140 atatcaagtg ggtgacggca agtaagctag ctgataatag ttaccaagcc attattaata 1200 ttggagatca taagtataat ttaggtcatt atttagt 1237 <210> 32 <211> 1228 <212> DNA <213> Artificial Sequence <220> <223> autolysin-B <400> 32 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tcagttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc tcagctgatg 180 tgagtggtca tgttttaaaa attgtctata atgggcaact agaaccaact aagaagataa 240 agtatgcagt ttggacagat aatggtggcc aagatgattt agtttggtat actgctgatc 300 aagttggtgc ggcatatatt gatttatcta aaaaacatag agcatatggg ctatataata 360 ttcataccta ttcgcaagat gtaacaggta agatgtctgg cttaaatgcg agacaattta 420 ctattcttaa accaacagta agtacatcgt ttaacgtaca aactaatggt attgttgata 480 ttgtcgtttc aaatgtgaga ggtgatatta gctcaattaa agttcctgtt tggtcagatt 540 taggtggcca aaacgatatt aaatggtatc aagctactca atcaactgat ggaacataca 600 aagtttcagt aaagatttca gatcattcaa atgatactgg ccattttgct gttcatgttt 660 atgggaatag tacaatcacg aatagtcaaa tcggattagg gacaacagaa ggttttacaa 720 ttgctacccc agaccccaaa aatgtagtgt cagccgtagt agctaatgat ggtttccacc 780 tagcattaaa ttcgaatgtt gttaaagaat ttaccaaagt gaaatttgct gtttggtcgg 840 atcaagctgg tcaagatgac cttcattggt acacagccaa tgctcagggt caagtcattg 900 ttccttatgt taatcatagt aattatggtc tatacaacat ccatacctat agttttgagt 960 cgggaagtgc taaaggtcta aatacaagaa caattacagt accaaatcca actgctagcg 1020 cagccattac acagaaatca gatcttgaat ttttagtatc agttacgaat gtcccagctt 1080 atatcacaaa agttatgtta ccaagttgga gtgaaattaa tggccaagat gatatcaagt 1140 gggtgacggc aagtaagcta gctgataata gttaccaagc cattattaat attggagatc 1200 ataagtataa tttaggtcat tatttagt 1228 <210> 33 <211> 1228 <212> DNA <213> Artificial Sequence <220> <223> autolysin-B1 <400> 33 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tccgttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc tcagctgatg 180 tgagtggtca tgttttaaaa attgtctata atgggcaact agaaccaact aagaagataa 240 agtatgcagt ttggacagat aatggtggcc aagatgattt agtttggtat actgctgatc 300 aagttggtgc ggcatatatt gatttatcta aaaaacatag agcatatggg ctatataata 360 ttcataccta ttcgcaagat gtaacaggta agatgtctgg cttaaatgcg agacaattta 420 ctattcttaa accaacagta agtacatcgt ttaacgtaca aactaatggt attgttgata 480 ttgtcgtttc aaatgtgaga ggtgatatta gctcaattaa agttcctgtt tggtcagatt 540 taggtggcca aaacgatatt aaatggtatc aagctactca atcaactgat ggaacataca 600 aagtttcagt aaagatttca gatcattcaa atgatactgg ccattttgct gttcatgttt 660 atgggaatag tacaatcacg aatagtcaaa tcggattagg gacaacagaa ggttttacaa 720 ttgctacccc agaccccaaa aatgtagtgt cagccgtagt agctaatgat ggtttccacc 780 tagcattaaa ttcgaatgtt gttaaagaat ttaccaaagt gaaatttgct gtttggtcgg 840 atcaagctgg tcaagatgac cttcattggt acacagccaa tgctcagggt caagtcattg 900 ttccttatgt taatcatagt aattatggtc tatacaacat ccatacctat agttttgagt 960 cgggaagtgc taaaggtcta aatacaagaa caattacagt accaaatcca actgctagcg 1020 cagccattac acagaaatca gatcttgaat ttttagtatc agttacgaat gtcccagctt 1080 atatcacaaa agttatgtta ccaagttgga gtgaaattaa tggccaagat gatatcaagt 1140 gggtgacggc aagtaagcta gctgataata gttaccaagc cattattaat attggagatc 1200 ataagtataa tttaggtcat tatttagt 1228 <210> 34 <211> 405 <212> DNA <213> Artificial Sequence <220> <223> CPB-A <400> 34 ccaagcacta ataatgaata tttacagaaa tattgatcgt acacaaattc aatttgattt 60 tataatcgat catccaagta tgaactacta tcaggatgaa atagaaagac taggtggacg 120 agtatattct tttccaacat ttacaggaag aaatgttcgt aatgtgcgta atgaatggga 180 taagtttttt aaagaacatg tagagtactc gattattcat tttcatgtta gaagttatat 240 ttctttatta attcctattg caaaaagata cggtttaata acaatatcac atagtcatag 300 tatttctaat ggactaggac taaaatcaaa gattaagaat cttttacaaa ttccgattag 360 atatcaagct gattatcttt ttgcttgctc taaagaggct ggaga 405 <210> 35 <211> 533 <212> DNA <213> Artificial Sequence <220> <223> SOA-A <400> 35 gaaaagttgt tgcagaaatt gctaaattat ctggttataa cgacattatt tttcttgatg 60 attattctaa cgaaaaactt tgttctggtt atccagttgt cggaaaagtt tctgaaattg 120 ttaatttcaa aaatgaagat gtttttatag ctattggatc aagtgctgtt agagagaaaa 180 ttgctaaaca tttgaaggac cacaaaatag tatccttaat acatccggcc gcagttgtaa 240 gtgaaaaagc taaaattggc aaagggagtg ttattatggc tggtgctgta gtaaaccctg 300 atactgaaat tggtgaattt tgtatagtca atacttgttc ttctgtagat catgattgta 360 ttataggtga tttttcacac gtctcagttg gcagtcacgt tgcaggaaca gtaacagttg 420 gttctcatgt atggataggt gctggggcga caataattaa taacatcgaa acacacaata 480 atatttgtat tggagcagga gcaacggtta taaataattt agtagatagt gga 533 <210> 36 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-A <400> 36 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatccct attaagttat ttggtggacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgctggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacacgggat taaagtactg ggaaataaaa attccattcc 420 aagattacta gcagaatatg aagttaaaca ggttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatctg ccgtaacgca aacgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctcaac tgacaaagct gttaacccac ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 37 <211> 1112 <212> DNA <213> Artificial Sequence <220> <223> PB-A1 <400> 37 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatccct attaagttat ttggtggacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggtgct ggtgaaggtg gtagtgcctt tattcaaaca 300 attctgaata agagtaaaga tattgacatt gtcggtattg ttgatgctga tatcaataaa 360 ttaggcacct acttacacgg gattaaagta ctgggaaata aaaattccat tccaagatta 420 ctagcagaat atgaagttaa acaggttacg attgccatcc caagtttatc tggggaagaa 480 cgagaatcaa tcttagatat ctgccgtaac gcaaacgttc acgtaaacaa tatgcctagt 540 attgagaata tcgttctagg taatgtgtca cttaataaat ttaaagaaat tgagattgct 600 gacttacttg gacgaaagga agtagtatta gatcaaacgt ctttaaattc attctttaac 660 gggaaaacag ttcttgtaac tggtgcagga ggatcaattg gttcagaaat ctgtcgtcaa 720 gtttctaaat ttaatccagc acgcatttta cttttagggc atggtgaaaa ttccatttat 780 ctgattcatc gtgaattatc agcactgtta aagggacgaa ttgacattgt cccaattatc 840 gcggacattc aagatcgaga cttgattttt gaaattatgg cgaattatcg acctgatata 900 gtctatcatg ctgcagcaca taaacatgtg ccattgatgg aatacaatcc aaaagaagct 960 gttaaaaata atatctttgg gacaaaaaat gtggcggaag cagccaaggc tgctgggatt 1020 cctaaattta tcatggtctc aactgacaaa gctgttaacc cacctaatgt gatgggtgca 1080 actaagcgct tcgctgaaat gatcgtcact gg 1112 <210> 38 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B <400> 38 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 39 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B1 <400> 39 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agacgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 40 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B2 <400> 40 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtgttgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 41 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B3 <400> 41 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 42 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-A <400> 42 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacaac atccgcaaat attgctatct catttgctaa 120 agcaggtcta aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctgtcaggta atgcagaatt 240 atcagcagtt atctctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatcgactc 420 agcgattatt tcacaaaaag ctgacgcaaa catcttagta acagaagctg gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtgctt tgttcttggg 540 tgttatttta aataaagtag aagaaacact tgattcatat ggtggttatg gtag 594 <210> 43 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-A1 <400> 43 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacaac atccgcaaat attgctatct catttgctaa 120 agcaggtcta aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctgtcaggta atgcagaatt 240 atcagcagtt atctctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatcgactc 420 agcgattatt tcacaaaaag ctgacgcaaa catcttagta acagaagctg gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtgctt tgttcttggg 540 tgttatttta aataaagtag aagaatcact tgattcatat ggtggttatg gtag 594 <210> 44 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-B <400> 44 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacatt atccgcaaat attgctatct catttgctaa 120 agcaggtctt aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctatcaggta atgcagaatt 240 atcagcagtt atttctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatagactc 420 agcgattatt tcacaaaaag ctgacgcaaa cattttagta acagaagcag gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtgcct tgttcttggg 540 tgttatttta aataaagtag aagaaacact tgattcatat ggtggttatg gtag 594 <210> 45 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-B1 <400> 45 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacatt atccgcaaat attgctatct catttgctaa 120 agcaggtctt aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctatcaggta atgcagaatt 240 atcagcagtt atttctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatagactc 420 agcgattatt tcacaaaaag ctgacgcaaa cattttagta acagaagcag gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtacct tgttcttggg 540 tgttatttta aataaagtag aagaaacact tgattcatat ggtggttatg gtag 594
Claims (7)
Inactivated Streptococcus parauberis KCTC 13800BP, KCTC 13801BP, KCTC 13802BP and KCTC 13803BP As described above, the KCTC 13800BP strain, the KCTC 13801BP strain, the KCTC 13802BP strain, and the KCTC 13803BP strain are inactivated for the prevention of streptococcal disease, which are strains of serotype Ib, serotype II, serotype Ia and serotype Ic, respectively. vaccine.
According to claim 1, wherein the vaccine is an inactivated vaccine for preventing streptococcal disease, characterized in that the KCTC 13802BP strain and KCTC 13803BP strain are mixed in a ratio of 1: 1.
According to claim 1, wherein the vaccine is an inactivated vaccine for preventing streptococcal disease, characterized in that the KCTC 13800BP strain, KCTC 13802BP strain and KCTC 13803BP strain are mixed in a ratio of 1: 1: 1.
The method of claim 1, wherein the antigen concentration of the vaccine is 1 to 30mg / fish inactivated vaccine for preventing streptococcal disease.
Vaccines by inactivating any two or more strains selected from the group consisting of Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain and KCTC 13803BP strain isolated from halibut infected with streptococcal disease As a method for preparing an inactivated vaccine for preventing streptococcal disease, comprising the steps of obtaining, the KCTC 13800BP strain, the KCTC 13801BP strain, the KCTC 13802BP strain and the KCTC 13803BP strain are serotype Ib and serotype II, respectively. , Serotype Ia and serotype Ic strains, characterized in that the method of producing an inactivated vaccine for preventing streptococcal disease.
A method of immunizing fish comprising administering the vaccine of claim 1 to fish.
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KR102471093B1 (en) | 2020-07-14 | 2022-11-25 | 제주대학교 산학협력단 | Octavalent vaccine composition for preventing diseases in fish and manufacturing method thereof |
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