KR101491793B1 - Novel Actinobacillus pleuropneumoniae serotype 12 strain, diagnostic composition and vaccine composition comprising the same - Google Patents
Novel Actinobacillus pleuropneumoniae serotype 12 strain, diagnostic composition and vaccine composition comprising the same Download PDFInfo
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- KR101491793B1 KR101491793B1 KR20130108362A KR20130108362A KR101491793B1 KR 101491793 B1 KR101491793 B1 KR 101491793B1 KR 20130108362 A KR20130108362 A KR 20130108362A KR 20130108362 A KR20130108362 A KR 20130108362A KR 101491793 B1 KR101491793 B1 KR 101491793B1
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Abstract
본 발명은 돼지 흉막폐렴에 대한 예방 효과를 갖는 신규한 액티노바실러스 플로르뉴모니아 혈청형 12번 균주 KACC 91770P, 상기 균주를 포함하는 돼지 흉막폐렴 진단용 조성물 및 상기 균주를 포함하는 돼지 흉막폐렴 예방용 백신 조성물에 관한 것이다.The present invention relates to a novel actinobacillus flourneumoniae serotype 12 strain KACC 91770P having a preventive effect against swine flu pneumonia, a composition for diagnosing porcine pleural effusion comprising the strain, and a method for preventing porcine pleurisy including the strain Vaccine compositions.
Description
본 발명은 신규한 액티노바실러스 플로르뉴모니아 혈청형 12번 균주 및 이를 포함하는 진단용 조성물 및 백신 조성물에 관한 것으로, 보다 상세하게는 돼지 흉막폐렴에 대한 예방 효과를 갖는 신규한 액티노바실러스 플로르뉴모니아 혈청형 12번, 상기 균주를 포함하는 돼지 흉막폐렴 진단용 조성물 및 상기 균주를 포함하는 돼지 흉막폐렴 예방용 백신 조성물에 관한 것이다.The present invention relates to a novel
돼지 흉막폐렴은 액티노바실러스 플로르뉴모니아 (Actinobacillus pleuropneumoniae)에 의해 감염되는데 일반적으로 급성으로 진행되어 육성돈 또는 비육돈의 급사 등을 야기하는 질병으로서, 주요 증상은 식욕결핍, 호흡곤란, 구토, 청색증, 고열, 코와 입의 출혈, 급사 등이다. Pig pleural pneumonia is caused by Actinobacillus pleuropneumoniae , which generally progresses acutely and causes a sudden death of growers or finishing pigs. Major symptoms include appetite deficiency, dyspnea, vomiting, cyanosis, High fever, bleeding in the nose and mouth, sudden death.
감염 초기에 급성형으로 진행되는 경우가 많아서 치료가 어려우며, 만성으로 진행되는 경우일지라도 증체율 감소, 출하 지연 등으로 양돈 산업에 막대한 경제적 피해를 일으키는 질병이다(BossJT, Janson H, Sheehan BJ, Beddek AJ, Rycroft AN, Kroll JS, Langford PR. Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes Infect. 2002. 4: 225-235; Sebunya TN, Saunders JR, Osborne AD. Dose response relationship of Haemophilus pleuropneumoniae aerosols in pigs. Can J Comp Med. 1983. 47: 54-56). (BossJT, Janson H, Sheehan BJ, Beddek AJ, et al., 2000). This is a disease that causes severe economic damage to the swine industry due to the slow rate of delivery and delays in shipment, Rycroft AN, Kroll JS, Langford PR. Actinobacillus pleuropneumoniae: pathobiology and pathogenesis of infection. Microbes Infect. 2002. 4: 225-235; Sebunya TN, Saunders JR, Osborne AD. Dose response relationship of Haemophilus pleuropneumoniae aerosols in pigs. Comp Med 1983: 47: 54-56).
돼지 흉막폐렴의 원인균인 액티노바실러스 플로르뉴모니아는 Apx 독소를 포함하여 다양한 병원성 인자를 산생하며, 지금까지 전 세계적으로 15 종류의 혈청형이 밝혀져 있는데, 각 혈청형간 교차 방어능이 완벽하지 않기 때문에 유행 혈청형을 파악하는 것이 해당 국가의 흉막폐렴 관리를 위하여 매우 유용하다(Barbara E. Straw et al. Disease of Swine 9th Edition. 2006. p. 563~576. Blackwell publishing). Actinobacillus flourneumonia, a causative organism of swine pneumonia, produces various pathogenic factors including Apx toxin. Up to now, 15 types of serotypes have been identified worldwide. Since the cross-reactivity between the serotypes is not perfect, Identifying the serotype is very useful for the management of pleural pneumonia in that country (Barbara E. Straw et al., Disease of Swine 9th Edition, 2006. p.
액티노바실러스 플로르뉴모니아는 국가마다 유행하는 혈청형 분포가 다양한데 유럽의 경우 혈청형 2번이 우세하며, 캐나다 및 미국의 경우에는 혈청형 1번과 5번에 의한 흉막폐렴이 가장 많이 발생하고 있다. 따라서 해당 국가에서는 효과적인 진단 및 예방을 위하여 유행하는 병원체 특성을 파악한 후 이에 적합한 대표균주를 선발하여 다방면에 활용하고 있다. Actinobacillus fluorescensis has a wide distribution of serotypes prevalent among countries. Serotype 2 is the most prevalent in Europe, while in Canada and the United States, pleural pneumonia is most commonly caused by serotypes 1 and 5 . Therefore, in order to effectively diagnose and prevent diseases, the pathogen characteristics of the pathogens are identified, and representative strains suitable for them are selected and utilized in various fields.
국내 양돈장의 경우 15종의 혈청형 중 2번 및 5번에 의한 흉막폐렴이 가장 많이 발생하고 있는 실정이다. 한편 종돈 수입과 관련하여, 최근 국내에서는 혈청형 1, 3, 4, 6, 7, 10, 12번에 의한 흉막폐렴의 발생 보고가 있어 이에 대한 대책도 시급히 필요하다 할 것이다.In domestic pig farms, the highest number of pleural pneumonia due to 2nd and 5th out of 15 serotypes is present. In addition, regarding the importation of breeder pigs, there is a report on the occurrence of pleural pneumonia due to
따라서, 국내 흉막폐렴의 피해를 최소화하기 위하여, 국내에서 유행하고 있는 혈청형 12번을 이용한 진단액 생산 및 예방약 개발이 절실한 실정이다. Therefore, in order to minimize the damage of domestic pleural pneumonia, it is urgent to develop a diagnostic fluid and to develop a preventive
본 발명자들은 상기와 같은 종래기술의 과제를 해결하기 위한 연구를 지속적으로 진행하여, 국내 돼지 흉막폐렴 병변에서 신규한 액티노바실러스 플로르뉴모니아 혈청형 12번 균주를 분리하였고, 이들 분리주의 병원성, 발육양상, 유전학적인 특성을 비교 분석하여 돼지 흉막폐렴에 대한 예방 효과를 갖는 신규한 액티노바실러스 플로르뉴모니아 혈청형 12번 균주를 수득하게 되었다.DISCLOSURE OF THE INVENTION The inventors of the present invention have continued to carry out studies to solve the problems of the prior art as described above and have succeeded in isolating a novel strain of
따라서, 본 발명의 목적은 국내 돼지유래 흉막폐렴의 진단 및 백신에 유용한 새로운 액티노바실러스 플로르뉴모니아 혈청형 12번 균주를 제공하는 것이다.Accordingly, it is an object of the present invention to provide a novel strain of actinobacillus fluorescenoma
상기와 같은 목적을 달성하기 위한 본 발명은 돼지 흉막폐렴에 대한 예방 효과를 갖는 액티노바실러스 플로르뉴모니아 혈청형 12번 균주 KACC 91770P를 제공한다.In order to achieve the above object, the present invention provides KACC 91770P
또한, 본 발명은 상기 액티노바실러스 플로르뉴모니아 혈청형 12번 균주를 포함하는 돼지 흉막폐렴 진단용 조성물을 제공한다.In addition, the present invention provides a composition for diagnosing porcine pleuropneumonia, comprising the above-mentioned strain of Actinobacillus flourneumoniae
또한, 본 발명은 상기 액티노바실러스 플로르뉴모니아 혈청형 12번 균주를 포함하는 돼지 흉막폐렴 예방용 백신 조성물을 제공한다.In addition, the present invention provides a vaccine composition for preventing swine pneumonia in pigs comprising the Actinobacillus flourneumonia
본 발명에 의한 액티노바실러스 플로르뉴모니아 혈청형 12번 균주는 야외 감염 돼지를 색출하기 위한 진단용 항원으로 활용되거나, 이를 포함하는 백신형태로 제조되어 돼지 흉막폐렴에 대한 예방약 개발에 활용될 수 있다.The Actinobacillus flourneumoniae
또한, 국내 실정에 적합하고 효과적인 백신 개발이 가능하며, 흉막폐렴에 대한 면역효과 증진과 흉막폐렴 예방을 통해 양돈 농가에서 가축질병으로 인한 경제적 피해를 크게 감소시킬 것으로 판단된다.In addition, it is possible to develop an effective vaccine suitable for domestic situation, and it is considered that the economic damage caused by livestock diseases in swine farms will be greatly reduced through improvement of immunity effect on pleural pneumonia and prevention of pleural pneumonia.
더욱이 국내 백신 제조업체의 신제품 개발 및 경쟁력 강화로 수입 백신 대체에 따른 외화 절감 효과 및 국내 백신의 해외 수출 효과 등 국가 경제에 미치는 긍정적 효과가 클 것으로 기대된다.Furthermore, the development of new products and strengthening competitiveness of domestic vaccine manufacturers are expected to have a positive effect on the national economy, such as foreign currency savings resulting from the replacement of imported vaccines and the effect of exporting domestic vaccines.
도 1은 돼지 흉막폐렴을 예방할 수 있는 액티노바실러스 플로르뉴모니아 KACC 91770P의 16s rDNA 염기서열 결과를 나타낸 것이다.
도 2는 액티노바실러스 플로르뉴모니아 국내 분리주들에 대해 PFGE법을 사용하여 유전적인 상관관계를 비교분석한 결과를 나타낸 것이다.
도 3은 돼지 흉막폐렴을 예방할 수 있는 액티노바실러스 플로르뉴모니아 KACC 91770P의 발육 곡선을 나타낸 것이다.1 shows the results of 16s rDNA sequencing of Actinobacillus fluorescensia KACC 91770P which can prevent pig pleural pneumonia.
Figure 2 shows the results of comparative analysis of genetic correlations using the PFGE method for domestic isolates of Actinobacillus fluorescens.
Figure 3 shows the growth curve of Actinobacillus flourneumonia KACC 91770P, which can prevent pig pleural pneumonia.
본 발명은 돼지 흉막폐렴에 대한 예방 효과를 갖는 액티노바실러스 플로르뉴모니아 혈청형 12번 균주 KACC 91770P에 관한 것이다. The present invention relates to an actinobacillus flourneumoniae
본 발명의 상기 액티노바실러스 플로르뉴모니아 혈청형 12번 균주는 서열번호 1로 이루어지는 16s rDNA 염기서열을 갖는 것을 특징으로 한다.The Actinobacillus floxunion
본 발명의 상기 액티노바실러스 플로르뉴모니아 균주는 농업생명공학연구원의 농업유전자원센터(Korean Agricultural Culture Collection; KACC)에 2013년 02월 26일자로 수탁하고, 수탁번호 KACC 91770P를 부여받았다.The Actinobacillus flourneumoniae strain of the present invention was deposited with the Korean Agricultural Culture Collection (KACC) of the Institute of Agricultural Biotechnology on Feb. 26, 2013, and received the accession number KACC 91770P.
본 발명의 상기 액티노바실러스 플로르뉴모니아 혈청형 12번 균주는 종래의 액티노바실러스 플로르뉴모니아 백신 및 진단액에 사용되지 않은 혈청형이며, 신규한 액티노바실러스 플로르뉴모니아 균주로서, 유전학적으로 국내 분리주의 대표적인 특성을 나타낸다.The Actinobacillus flourneumoniae
본 발명의 상기 균주는 국내 야외 분리주에 비하여 발육속도가 빠른 것을 특징으로 하는데, 상기 균주는 배양 12시간부터 최대 흡광도를 나타낼 수 있다.The strain of the present invention is characterized in that the growth rate is faster than that of a domestic field isolate, and the strain exhibits the maximum absorbance from 12 hours of culture.
또한, 본 발명의 상기 균주는 10배 농축액에서 고병원성을 갖는 것을 특징으로 하는데, 상기 균주는 10배 농축액에서 60%의 폐사율을 나타낼 수 있다.In addition, the strain of the present invention is characterized by high disease activity in a 10-fold concentrated solution, which can exhibit a mortality of 60% in a 10-fold concentrated solution.
본 발명에서, 상기 균주는 다양한 항생제 내성을 갖는 것을 특징으로 하는데, 상기 항생제는 네오마이신(neomycin), 옥시테트라싸이클린(oxytetracycline), 스펙티노마이신(spectinomycin) 및 타일로신(tylosin)으로 이루어진 군으로부터 선택되는 1종 이상일 수 있으나, 이에 한정되는 것은 아니다. In the present invention, the strain is characterized by having various antibiotic resistance, wherein the antibiotic is selected from the group consisting of neomycin, oxytetracycline, spectinomycin and tylosin But is not limited thereto.
본 발명에서, 상기 동물은 특별한 제한은 없으나, 흉막폐렴균이 감염될 수 있는 돼지, 바람직하게는 돼지일 수 있다.In the present invention, the animal is not particularly limited, but may be a pig, preferably a pig, from which the pneumococcus can be infected.
또한, 본 발명은 상기 액티노바실러스 플로르뉴모니아 혈청형 12번 균주를 포함하는 돼지 흉막폐렴 진단용 조성물에 관한 것이다. In addition, the present invention relates to a composition for diagnosing porcine pleural effusion comprising the above-mentioned strain of Actinobacillus flourneumonia
본 발명의 상기 흉막폐렴 진단용 조성물에서, 상기 균주는 불활성화되어 병원성을 상실한 것일 수 있다.In the composition for diagnosing pleural effusion of the present invention, the strain may be inactivated and lost pathogenicity.
본 발명의 상기 흉막폐렴 진단용 조성물에서, 상기 균주는 약독화된 것일 수 있다. In the composition for diagnosing pleural effusion of the present invention, the strain may be attenuated.
본 발명은 상기 액티노바실러스 플로르뉴모니아 혈청형 12번 균주를 포함하는 돼지 흉막폐렴 예방용 백신 조성물에 관한 것이다.The present invention relates to a vaccine composition for preventing swine flu pneumonia, which comprises the above-mentioned Actinobacillus flourneumoniae
본 발명의 상기 백신 조성물에서, 상기 균주는 불활성화되어 병원성을 상실한 것일 수 있다. In the vaccine composition of the present invention, the strain may be inactivated and lost pathogenicity.
본 발명의 상기 백신 조성물에서, 상기 균주는 약독화된 것일 수 있다. In the vaccine composition of the present invention, the strain may be attenuated.
본 발명에 따른 상기 액티노바실러스 플로르뉴모니아 혈청형 12번 균주는 국내 흉막폐렴 감염증에 대한 특이적 진단 및 예방용 백신주로서 매우 유용하게 사용될 수 있다.The Actinobacillus flourneumoniae
상기 액티노바실러스 플로르뉴모니아 혈청형 12번 균주를 이용한 백신은 주로 불활성화된 상태로 포함될 수 있으며, 상기 균주를 이용하여 제작한 약독화 균주도 포함될 수 있다. The vaccine using Actinobacillus fluorescenumi
상기 불활성화 방법은 당업계의 통상적인 방법에 의할 수 있으며, 예를 들어 KACC 91770P의 배양액에 불활성화제 (예: 1% formalin)를 첨가하여 불활성화시키고 여기에 보조제 (예: aluminum hydroxide gel)를 첨가하여 불활성화 사균백신을 제조할 수 있다. The inactivating method may be carried out according to a conventional method in the art. For example, an inactivating agent (for example, 1% formalin) is added to the culture solution of KACC 91770P to inactivate the active agent (for example, aluminum hydroxide gel) Can be added to produce an inactivated killed bacterial vaccine.
또한, 상기 약독화 균주는 당업계에 통상적인 약독화 방법에 의할 수 있으며, 예를 들어 약독화를 일으키는 화학물질, 박테리오파지, 백혈구 등을 이용하거나 유전공학적 기법으로 단일 또는 복수의 유전자를 결손 또는 손상시켜 병원성이 소실 또는 약화된 약독화 균주를 생산할 수 있다. In addition, the attenuated strain may be attenuated by a conventional attenuation method, for example, by using attenuating chemicals, bacteriophage, leukocyte, etc., or by using genetic engineering techniques, Can be used to produce attenuated strains that have lost or attenuated virulence.
또한, 본 발명에 따른 상기 KACC 91770P 균주를 돼지 등과 같은 동물의 흉막폐렴 백신에 사용하는 경우, 상기 백신은 당업계의 통상적인 방법으로 제조할 수 있으며, 상기 균주를 액티노바실러스 플로르뉴모니아 다른 혈청형의 균주 및 기타 질병 원인체와 혼합 및 복합으로 사용할 수 있다. In addition, when the KACC 91770P strain according to the present invention is used in an animal pleural fluid pneumonia vaccine such as a pig, etc., the vaccine can be prepared by a conventional method in the art, and the strain is treated with Actinobacillus flourneumoniae It can be used in combination with and mixed with serotype strains and other disease agents.
본 발명의 상기 액티노바실러스 플로르뉴모니아 혈청형 12번 균주의 유전자를 사용하여 생산한 서브유니트(subunit) 백신, 벡터(vector) 백신, 키메라(chimera) 백신, 또는 DNA 백신 등과 같은 다양한 형태로 제조될 수 있으므로, 그 활용도가 매우 높다. A subunit vaccine, a vector vaccine, a chimera vaccine, or a DNA vaccine produced by using the gene of the Actinobacillus floxunion
상기 백신은 당업계에서 통상적으로 사용하는 수의학적으로 수용 가능한 매개체 또는 부형제를 포함할 수 있으며, 어쥬번트를 추가로 첨가할 수 있다. 또한 상기 백신은 면역증강용 보조제를 더 포함할 수 있다. The vaccine may include a veterinarily acceptable mediator or excipient conventionally used in the art, and an adjuvant may be further added. The vaccine may further comprise an adjuvant for enhancing immunity.
본 발명에 따른 KACC 91770P 균주를 흉막폐렴 진단에 사용하는 경우, 당업계의 통상적인 방법으로 제조할 수 있다. When the KACC 91770P strain according to the present invention is used for the diagnosis of pleural pneumonia, it can be produced by a conventional method in the art.
진단액의 제조는 당업계의 통상적인 방법에 의할 수 있으며, 예를 들어 KACC 91770P 균주의 배양액에 불활성화제(예: 1% formalin)를 첨가하여 불활성화시켜 응집반응용 진단액으로 사용할 수 있다. The preparation of the diagnostic solution can be carried out according to a conventional method in the art, for example, by adding an inactivating agent (for example, 1% formalin) to the culture solution of KACC 91770P strain and inactivating it and using it as a diagnostic fluid for flocculation reaction .
또한, 상기 약독화 균주를 증식시켜 외막단백질을 추출하여 ELISA 항원으로 사용하여 진단액을 제조할 수 있다.In addition, the attenuated strain may be propagated to extract the outer membrane protein and used as an ELISA antigen to produce a diagnostic solution.
이하, 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 다만, 하기 실시예들은 본 발명을 예시하는 것으로서, 본 발명의 내용이 이들의 실시예에 의해 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by these examples.
<실시예 1> 액티노바실러스 플로르뉴모니아 분리 및 동정Example 1 Isolation and Identification of Actinobacillus fluorescens
액티노바실러스 플로르뉴모니아 KACC 91770P 균주는 전형적인 흉막폐렴 병변을 나타낸 돼지의 폐를 수거하여 초코렛 아가에서 혐기 상태로 37℃, 24~48시간 정치배양한 후, 유사한 순수 집락을 획득하여 생화학적인 성상검사로 균을 동정하였다. 상기 혐기 상태는 Advanced사의 Anoxomat™을 이용하여 형성하였다.Actinobacillus flourneumonia KACC 91770P was collected from pig lungs showing typical pleural pneumonia lesions and cultured in a choclet agar anaerobically at 37 ° C for 24 to 48 hours and then obtained a similar pure colonization to obtain biochemical properties The bacteria were identified by inspection. The anaerobic state was formed using Advanced Anoxomat (TM).
액티노바실러스 플로르뉴모니아의 Apx toxin 유전자를 이용한 PCR 방법을 통하여 혈청형을 확인하였다. 하지만 혈청형 2번, 8번, 15번과 혈청형 9번, 11번, 12번, 13번이 정확히 구별되지 않아 특이유전자인 omlA 및 cps 유전자를 이용한 PCR 방법으로 균종 및 혈청형 동정을 실시하였다(Rayamajhi et al., J Vet Diagn Invest. 17:359362, 2005). Serotype was confirmed by PCR using the Apx toxin gene of Actinobacillus fluorescens. However, because the
특이유전자인 omlA 및 cps 유전자를 이용한 PCR 방법으로 혈청형 2번, 5번, 6번과 혈청형 1번, 7번, 12번을 정확하게 동정하였다(Øystein Angen et al., Veterinary Microbiology. 132:312318, 2008). 혈청형 동정을 한 결과, 상기 KACC 71770P 균주가 액티노바실러스 플로르뉴모니아 혈청형 12번임을 확인하였다.
KACC 91770P 균주의 DNA를 추출(Genomic cell/tissue spin mini kit, Genotech)하여 16S rDNA에 대한 DNA 단편을 PCR하였다. DNA fragments of 16S rDNA were PCR-amplified by extracting DNA of KACC 91770P (Genomic cell / tissue spin kit, Genotech).
PCR에 사용된 프라이머는 2F: 5’-AGAGTTTGATCCTGGCTCAG-3’(서열번호 2), 2R: 5’-ACGGCTACCTTGTTACGACTT-3’(서열번호 3)이며, PCR의 반응 조건은 95℃: 전변성(predenaturation) 5분, 95℃: 변성(denaturation) 45초, 55℃: 어닐링(annealing) 45초, 72℃: 익스텐션(extension) 60초, 30 싸이클, 72℃: 최종 익스텐션(last extension) 10분이었다. The primers used in PCR were 2F: 5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 2), 2R: 5'- ACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 5 minutes 95 ° C Denaturation 45 sec 55 ° C Annealing 45 sec 72 ° C Extension 60
상기 염기서열 분석에 사용된 프라이머는 3F: 5’-AGTTTGATCCTGGCTCAG-3’(서열번호 4), 3F': 5’-CCAGCAGCCGCGGTAATACG-3’(서열번호 5), 3R: 5’-GGTTACCTTGTTACGACTT-3’(서열번호 6)이며, ABI BigDye terminatorcycle sequencing ready reaction kit v3.1(ABI)를 이용한 싸이클 시퀀싱(cycle sequencing) 방법으로 염기서열을 해독하였다.The primers used in the above sequence analysis were 3F: 5'-AGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 4), 3F': 5'-CCAGCAGCCGCGGTAATACG-3 '(SEQ ID NO: 5), 3R: 5'- GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 6), and the nucleotide sequence was decoded by a cycle sequencing method using ABI BigDye terminator cycle sequencing ready reaction kit v3.1 (ABI).
상기 염기서열 분석시 PCR 반응 조건은 95℃: 전변성(predenaturation) 2분, 96℃: 변성(denaturation) 10초, 50℃: 어닐링(annealing) 10초, 60℃: 익스텐션(extension) 3분, 30 싸이클이다. PCR was carried out at 95 ° C for 2 minutes, 96 ° C for denaturation for 10 seconds, annealing at 50 ° C for 10 seconds, extension at 60 ° C for 3 minutes, It is 30 cycles.
분석된 염기서열을 NCBI의 데이터베이스와 비교 분석한 결과, 액티노바실러스 플로르뉴모니아 특이 염기서열을 확인하였다.The analyzed nucleotide sequence was compared with the database of NCBI. As a result, the specific nucleotide sequence of Actinobacillus fluorescens was confirmed.
도 1은 돼지 흉막폐렴을 예방할 수 있는 액티노바실러스 플로르뉴모니아 KACC 91770P의 16s rDNA 염기서열 결과를 나타낸 것으로서, NCBI의 데이터베이스와 비교한 결과, 액티노바실러스 플로르뉴모니아와 99% 상동성을 보였다.
1 shows the result of 16s rDNA sequencing of Actinobacillus fluorescensia KACC 91770P, which can prevent pig pleural pneumonia. As compared with NCBI database, it has 99% homology with Actinobacillus flourneumonia It looked.
<실시예 2> 국내 분리주간의 유전형 비교≪ Example 2 >
액티노바실러스 플로르뉴모니아 국내 분리균주를 대상으로 미국 CDC에서 유전형 비교 분석법으로 널리 사용되고 있는 pulsed field gel electrophoresis (PFGE)법으로 국내 분리주간의 유전형을 비교 분석하였다.The genotypes of domestic isolates were compared and analyzed by pulsed field gel electrophoresis (PFGE) method, which is widely used as a comparative genotyping method in US CDC for Actinobacillus fluorescensis isolates.
PFGE는 분리균주의 전체 염색체 DNA를 apaI 효소로 처리하여 CHEF mapper apparatus (Bio-rad, hercules, USA)에 6.0V/m로 20시간 동안 4~40초까지 램프 펄스(ramp pulse)로 전기영동하여 밴드를 확인하였다. PFGE is the complete chromosomal DNA of the isolate apaI enzyme, and the band was confirmed by electrophoresis on a CHEF mapper apparatus (Bio-rad, Hercules, USA) at 6.0 V / m for 20 to 4 seconds using a ramp pulse.
각 밴드는 BioNumerics (V5.10, Applied Maths, Belgium) software를 이용하여 unweighted pair group method with arithmetic means (UPGMA) 방법으로 분석하였다.Each band was analyzed by the unweighted pair group method with arithmetic means (UPGMA) using BioNumerics (V5.10, Applied Maths, Belgium) software.
도 2는 액티노바실러스 플로르뉴모니아 국내 분리주들에 대해 PFGE법을 사용하여 유전적인 상관관계를 비교분석한 결과를 나타낸 것이다. Figure 2 shows the results of comparative analysis of genetic correlations using the PFGE method for domestic isolates of Actinobacillus fluorescens.
도 2의 결과와 같이, 국내에서 분리된 액티노바실러스 플로르뉴모니아 혈청형 12번 균주는 90% 근연관계를 기준으로 분류하였을 때 크게 2가지 유전형이 있으며, 본 발명의 상기 KACC 91770P 균주가 포함된 그룹이 국내 분리주의 대표적인 유전형임을 확인하였다.
As shown in FIG. 2, when the isolate of actinobacillus flourneumonia serotype 12 isolated from Korea was classified on the basis of 90% relational relationship, there were two genotypes, and the KACC 91770P strain of the present invention was included Were identified as a representative genotype of domestic isolates.
<실시예 3> 분리균주의 발육 양상 관찰 ≪ Example 3 > Observation of growth pattern of isolated strains
다양한 국내 분리주(P-APP-9-12-3 및 P-APP-11-6-2)와 액티노바실러스 플로르뉴모니아 KACC 91770P 균주를 BHI(Brain Heart Infusion broth) 배지에 37℃, 33시간 배양하면서 일정 시간별로 흡광도를 측정하였다. A variety of domestic isolates (P-APP-9-12-3 and P-APP-11-6-2) and Actinobacillus flourneumonia KACC 91770P strain were inoculated into BHI (Brain Heart Infusion broth) The absorbance was measured at a constant time while culturing.
다양한 국내 분리주에 비해, 액티노바실러스 플로르뉴모니아 KACC 91770P 균주들의 발육 속도가 상대적으로 빨랐다. 즉, 돼지 흉막폐렴을 예방할 수 있는 액티노바실러스 플로르뉴모니아 KACC 91770P 균주의 발육 곡선을 나타낸 도 3에서 보는 바와 같이, KACC 91770P 균주들은 배양 12시간부터 최대 흡광도를 나타내었다.
Actinobacillus flourneumonia KACC 91770P strains were relatively fast compared to various domestic isolates. That is, as shown in FIG. 3 showing the growth curve of Actinobacillus flourneumonia KACC 91770P strain, which can prevent pig pleural pneumonia, KACC 91770P strains showed maximum absorbance from 12 hours of culture.
<실시예 4> 시험 균주간의 병원성 비교Example 4: Pathogenicity comparison between test strains
KACC 91770P균주의 병원성을 확인하기 위하여, 5주령 마우스에 0.2ml씩, 복강주사하여 병원성을 비교하였다. In order to confirm the pathogenicity of KACC 91770P strain, the pathogenicity was compared by intraperitoneal injection in 0.2 ml of 5-week-old mice.
그 결과를 하기의 표 1에 나타내었는데, 본 발명의 KACC 91770P 균주는 10배 농축액에서 고병원성을 갖는 것을 특징으로 하는데, 상기 균주는 10배 농축액에서 60%의 폐사한 반면, 국내 분리주 P-APP-9-12-3은 10마리 모두 생존하여 높은 병원성의 차이를 관찰할 수 있었다.The results are shown in Table 1 below. The KACC 91770P strain of the present invention is characterized by high disease activity in a 10-fold concentrated solution. The strain was killed at 60% in a 10-fold concentrated solution, while the strain P-APP- 9-12-3 were able to observe high pathogenicity differences in all 10 animals.
(405nm)Inoculation OD
(405 nm)
a생존 마리수/접종 마리수
a Survival rate / Inoculation rate
<실시예 5> 항생제 감수성 양상 조사Example 5: Investigation of antibiotic susceptibility pattern
KACC 91770P 균주의 항생제 감수성 평가는 17종의 항생제를 사용하여 최소발육 억제농도(minimal inhibitory concentration; MIC)를 측정하였다. The antimicrobial susceptibility of KACC 91770P strain was evaluated by using 17 antibiotics to determine the minimal inhibitory concentration (MIC).
측정 결과를 하기 표 2에 나타내었는데, 상기 17종의 항생제 중, 총 4개의 항생제(네오마이신, 옥시테트라싸이클린, 스펙티노마이신, 타일로신)에 내성을 보임을 알 수 있다.The results of the measurement are shown in Table 2 below. Of the 17 antibiotics, the antibiotics are resistant to four antibiotics (neomycin, oxytetracycline, spectinomycin, tylosin).
본 발명에 의하면, 병원성이 강한 혈청형 12번을 활용하여 국내 실정에 적합한 효과적인 백신 개발이 가능하며, 흉막폐렴에 대한 면역효과 증진과 흉막폐렴 예방을 통해 양돈 농가에서 가축질병으로 인한 경제적 피해를 크게 감소시킬 것으로 판단된다.According to the present invention, it is possible to develop an effective vaccine suitable for the domestic situation by utilizing the
또한, 국내 백신 제조업체의 신제품 개발 및 경쟁력 강화로 수입 백신 대체에 따른 외화 절감 효과 및 국내 백신의 해외 수출 효과 등 국가 경제에 미치는 긍정적 효과가 클 것으로 기대된다.In addition, the development of new products and strengthening competitiveness of domestic vaccine manufacturers are expected to have a positive effect on the national economy, such as foreign currency savings resulting from the replacement of import vaccines and the effect of exporting domestic vaccines.
<110> Animal, Plant and Fisheries Quarantine and Inspection Agency <120> Novel Actinobacillus pleuropneumoniae serotype 12 strain, diagnostic composition and vaccine composition comprising the same <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 1360 <212> DNA <213> Actinobacillus pleuropneumoniae <220> <221> gene <222> (1) <223> 16s rDNA of Actinobacillus pleuropneumoniae serotype 12 strain <400> 1 gcccttccct cgaacgaaag aaacggctgc tcaccgcctg cccactcatt acgaaccctt 60 agaccgaata cctcccccta ttgatgccct ttgacatcga ttatggcgca ttatagatac 120 ctaatttccc accctgaaag cccggtggac ggtattctac tcgggttcac cctaatccat 180 caaccaatcc atttccgact ggttcggctg ctagagatcg accagactct cctactggtc 240 ggtgtgacct tgactctgtg ccaggtctga ggatgccctc cgtcgtcacc ccttataacg 300 tgttaccccc cttgggacta cgtcggtacg gcgcacttac ttcttccgga agcccaacat 360 ttcaagaaag ccatcgctcc ttccatagtt taaattatct aaaccattaa ctgcaattga 420 tgtcttcttc gtggccgatt gaggcacggt cgtcggcgcc attatgcctc ccacgctcgc 480 aattagcctt attgacccgc atttcccgtg cgtccgccaa ctaattcact ctacactttc 540 ggggcccgaa ttggaccctt aacgtaaagt atgaccagtt gatctcatga aatccctccc 600 catcttaagg tgcacatcgc cactttacgc atctctacac ctccttatgg cttccgcttc 660 cgtcggggaa cccttacatg actgcgagta cacgctttcg cacccctcgt ttgtcctaat 720 ctatgggacc atcaggtgcg acatttgcga cagctaaacc cctaacctga cactcagacc 780 acgggcttcg attgcactat ttagctggcg gacccctcat gccggcgttc caattttgag 840 tttacttaac tgcccccggg cgtgttcgcc acctcgtaca ccaaattaag ctacgttgcg 900 cttcttggaa tggatgagaa ctgtaggtac cttagaacat ctctatgctc tcacggaagc 960 ccttggtact ctgtccacga cgtaccgaca gcagtcgagc acaacacttt acaacccaat 1020 tcagggcgtt gctcgcgttg ggaataggaa acaacggtcg ctaatccagc ccttgagttt 1080 cctctgacgg ccactatttg gcctccttcc acccctactg cagttcagta gtaccgggaa 1140 tgctcatccc gatgtgtgca cgatgttacc gcatatgtct cccttcgttc taccgctgta 1200 cctcgtttag agtgtttcat gcagattcag gcctaacctc agacgttgag ctgaggtact 1260 tcagccttag cgatcattag cgtttagtct tacaacgcca cttatgcaag ggcccggaac 1320 atgtgtggcg ggcagtgtgg taccctcacc caacatggtc 1360 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 acggctacct tgttacgact t 21 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 agtttgatcc tggctcag 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 ccagcagccg cggtaatacg 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 ggttaccttg ttacgactt 19 <110> Animal, Plant and Fisheries Quarantine and Inspection Agency <120> Novel Actinobacillus pleuropneumoniae serotype 12 strain, diagnostic composition and vaccine composition comprising the same <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 1360 <212> DNA <213> Actinobacillus pleuropneumoniae <220> <221> gene <222> (1) <223> 16s rDNA of Actinobacillus pleuropneumoniae serotype 12 strain <400> 1 gcccttccct cgaacgaaag aaacggctgc tcaccgcctg cccactcatt acgaaccctt 60 agaccgaata cctcccccta ttgatgccct ttgacatcga ttatggcgca ttatagatac 120 ctaatttccc accctgaaag cccggtggac ggtattctac tcgggttcac cctaatccat 180 caaccaatcc atttccgact ggttcggctg ctagagatcg accagactct cctactggtc 240 ggtgtgacct tgactctgtg ccaggtctga ggatgccctc cgtcgtcacc ccttataacg 300 tgttaccccc cttgggacta cgtcggtacg gcgcacttac ttcttccgga agcccaacat 360 ttcaagaaag ccatcgctcc ttccatagtt taaattatct aaaccattaa ctgcaattga 420 tgtcttcttc gtggccgatt gaggcacggt cgtcggcgcc attatgcctc ccacgctcgc 480 aattagcctt attgacccgc atttcccgtg cgtccgccaa ctaattcact ctacactttc 540 ggggcccgaa ttggaccctt aacgtaaagt atgaccagtt gatctcatga aatccctccc 600 catcttaagg tgcacatcgc cactttacgc atctctacac ctccttatgg cttccgcttc 660 cgtcggggaa cccttacatg actgcgagta cacgctttcg cacccctcgt ttgtcctaat 720 ctatgggacc atcaggtgcg acatttgcga cagctaaacc cctaacctga cactcagacc 780 acgggcttcg attgcactat ttagctggcg gacccctcat gccggcgttc caattttgag 840 tttacttaac tgcccccggg cgtgttcgcc acctcgtaca ccaaattaag ctacgttgcg 900 cttcttggaa tggatgagaa ctgtaggtac cttagaacat ctctatgctc tcacggaagc 960 ccttggtact ctgtccacga cgtaccgaca gcagtcgagc acaacacttt acaacccaat 1020 tcagggcgtt gctcgcgttg ggaataggaa acaacggtcg ctaatccagc ccttgagttt 1080 cctctgacgg ccactatttg gcctccttcc acccctactg cagttcagta gtaccgggaa 1140 tgctcatccc gatgtgtgca cgatgttacc gcatatgtct cccttcgttc taccgctgta 1200 cctcgtttag agtgtttcat gcagattcag gcctaacctc agacgttgag ctgaggtact 1260 tcagccttag cgatcattag cgtttagtct tacaacgcca cttatgcaag ggcccggaac 1320 atgtgtggcg ggcagtgtgg taccctcacc caacatggtc 1360 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 acggctacct tgttacgact t 21 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 agtttgatcc tggctcag 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 5 ccagcagccg cggtaatacg 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 6 ggttaccttg ttacgactt 19
Claims (15)
상기 균주는 10배 농축액에서 100%의 마우스 폐사율을 나타내고,
상기 균주는 국내 분리주인 액티노바실러스 플로르뉴모니아(Actinobacillus pleuropneumoniae) P-APP-9-12-3에 비하여 발육속도가 빠르며,
상기 균주는 스펙티노마이신 및 타일로신으로 이루어진 군으로부터 선택되는 1종 이상의 항생제에 대하여 내성을 갖는 것을 특징으로 하는 액티노바실러스 플로르뉴모니아(Actinobacillus pleuropneumoniae) KVCC-BA1200404 혈청형 12번 균주 (KACC 91770P). Actinobacillus pleuropneumoniae KVCC-BA1200404 serotype 12 strain (KACC 91770P), isolated from pigs and causing pleural pneumonia in pigs,
The strain exhibited mouse mortality of 100% in a 10-fold concentrated liquid,
The strain has a higher rate of growth than Actinobacillus pleuropneumoniae P-APP-9-12-3, which is a domestic isolate,
The strain is resistant to one or more antibiotics selected from the group consisting of spectinomycin and tylosin. Actinobacillus pleuropneumoniae KVCC-BA1200404 Serotype 12 strain (KACC 91770P).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2831240C1 (en) * | 2023-12-22 | 2024-12-03 | Общество с ограниченной ответственностью "Центр биотехнологической обработки продуктов питания при институте микроэкологии" (ООО "ЦБО Микроэкологии") | Bacteria strains actinobacillus pleuropneumoniae serotype 11, intended for producing mono- and polyvalent immunogenic compositions aimed at specific prevention of porcine actinobacillus pleuropneumoniae |
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| KR100602460B1 (en) | 1999-06-09 | 2006-07-19 | 유니버시티 오브 로드 아일랜드 | Live attenuated bacteria for use in a vaccine |
| KR101164045B1 (en) | 2002-11-20 | 2012-07-18 | 라보라토리오스 이프라 에스.에이. | Live attenuated vaccine against porcine pleuropneumonia |
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| KR100510906B1 (en) | 1997-04-10 | 2006-01-12 | 악조 노벨 엔.브이. | Attenuated live bacteria of Actino Bacillus pluronomymoniae |
| KR100602460B1 (en) | 1999-06-09 | 2006-07-19 | 유니버시티 오브 로드 아일랜드 | Live attenuated bacteria for use in a vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2831240C1 (en) * | 2023-12-22 | 2024-12-03 | Общество с ограниченной ответственностью "Центр биотехнологической обработки продуктов питания при институте микроэкологии" (ООО "ЦБО Микроэкологии") | Bacteria strains actinobacillus pleuropneumoniae serotype 11, intended for producing mono- and polyvalent immunogenic compositions aimed at specific prevention of porcine actinobacillus pleuropneumoniae |
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