KR101037566B1 - 감쇠된 그람 음성 박테리아 - Google Patents
감쇠된 그람 음성 박테리아 Download PDFInfo
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- KR101037566B1 KR101037566B1 KR1020047015890A KR20047015890A KR101037566B1 KR 101037566 B1 KR101037566 B1 KR 101037566B1 KR 1020047015890 A KR1020047015890 A KR 1020047015890A KR 20047015890 A KR20047015890 A KR 20047015890A KR 101037566 B1 KR101037566 B1 KR 101037566B1
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Abstract
개시 및 청구된 것은 그람 음성 박테리아의 돌연변이체로; 상기 박테리아는 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 또는 93 로 식별되는 뉴클레오티드 서열로 이루어진 군에서 선택되는 뉴클레오티드 서열로 코딩되는 아미노산 서열과 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 가진 폴리펩티드를 코딩하는 뉴클레오티드 서열 중 하나 이상의 돌연변이를 포함하며; 상기 돌연변이는 박테리아의 발병력을 감쇠시킨다. 상기 돌연변이체를 함유하는 면역원성 조성물 및 백신이 또한 개시 및 청구된다.
Description
관련 출원/참고문헌으로 포함되는 것
본 출원은, 참고문헌으로 본원에 포함되는, 2002 년 4 월 5 일에 출원된 US 가출원 시리즈 제 60/370,282 호를 우선권으로 청구한다. 상기 출원, 및 그에 인용되거나 또는 그의 기소 중인 모든 문헌들 ("출원 인용 문헌") 및 출원 인용 문헌에서 인용 및 참고된 모든 문헌, 및 본원에 인용된 모든 문헌들 ("본원 인용 문헌"), 및 본원 인용 문헌에 인용 또는 참조된 모든 문헌들은 본원에 업급되는 임의의 제품에 대한 임의의 제조자의 지시사항, 설명, 제품 사양 및 제품 시트지 또는 본원에 인용되어 포함되는 임의의 문헌에 기재된 것과 함께 본원에 참고문헌으로 포함되며, 본 발명의 실시에 있어 이용될 수 있다.
본 발명은 살아있는 감쇠된 그람 음성 박테리아에 관한 것이다. 감쇠된 그람 음성 박테리아는 면역원성 조성물 또는, 예컨대 박테리아 감염을 방지할 뿐 아니라 연구에서의 백신 조성물에서 연구자 및 접촉할 수 있는 대상체 (예를 들어, 동물, 인간) 에 대한 더 높은 안전성이 있는 감쇠된 균주로서 이용될 수 있다.
따라서, 본 발명은 본 발명의 그람 음성 박테리아; 예를 들어 살아있는 감쇠된 그람 음성 박테리아를 함유하는 면역원 또는 백신 조성물에 관한 것이다. 상기 박테리아는 또한 조성물에서 불활성화될 수 있으나; 이는 박테리아가 살아있는 감쇠된 그람 음성 박테리아인 것이 유리할 수 있다. 따라서, 본 발명은 추가로 상기 조성물을 제조 및/또는 제형화하는 방법에 관한 것이다; 예를 들어 적합한 배지 상에서 또는 내에서 박테리아를 배양 또는 성장 또는 증식시키고, 상기 박테리아를 수합하고, 임의로는 상기 박테리아를 불활성화시키며, 수의학적으로 또는 약제학적으로 허용되는 적합한 담체, 부형제, 희석제 또는 비히클 및/또는 아쥬반트 및/또는 안정화제와 혼합하거나; 또는 상기 박테리아를 수의학적으로 또는 약제학적으로 허용되는 적합한 담체, 부형제, 희석제 또는 비히클 및/또는 아쥬반트 및/또는 안정화제와 혼합한다. 그러므로, 본 발명은 또한 이러한 조성물의 제형화에서의 박테리아의 용도에 관한 것이다.
감쇠된 박테리아는 또한, 핵산 분자 이종체, 예를 들어, 감쇠된 박테리아 이외의 병원과 같은 병원성 제제 유래의 면역원, 항원 또는 에피토프를 코딩하는 핵산 분자를 감쇠된 박테리아로 복제 및/또는 발현시키기 위한 발현 또는 복제 벡터로서 작용할 수 있다. 벡터로서 감쇠된 박테리아를 사용하는 것은, 감쇠된 박테리아로 작업하는 연구자 또는 기술자 및 이들과 접촉할 수 있는 대상체 (예를 들어, 동물, 인간) 에게 더 큰 안전도를 제공한다.
따라서, 본 발명은 또한, 상기 벡터를 제조하는 방법, 예를 들어 박테리아가 이종성 핵산 분자를 포함 및 임의로는 발현하도록 박테리아를 형질전환하는 방법에 관한 것이다.
본 발명은 또한 발현에 적합한 조건 하에 유전자 생성물을 코딩하는 이종성 핵산 분자를 포함 및 발현하도록 형질전환된 박테리아를 배양, 성장 또는 증식시키 고, 임의로는 유전자 생성물을 수합 또는 단리 또는 분리하거나; 또는 이를 발현하도록 형질전환된 박테리아로부터 유전자 생성물을 수합 또는 단리 또는 분리하는 것을 포함하는, 유전자 생성물, 예를 들어 박테리아에 대한 면역원, 에피토프 또는 항원, 이종체와 같은 폴리펩티드를 생산하는 방법; 또는, 예를 들어, 병원 및/또는 박테리아에 의해 감염되기 쉬운 동물, 예를 들어, 소 또는 칠면조와 같은 동물에게 유전자 생성물을 발현하도록 형질전환한 박테리아를 투여하는 것을 포함하는, 유전자 생성물이 유래되거나 또는 수득되는 병원 및/또는 박테리아에 대한 보호적인 면역 응답성 또는 유전자 생성물 및/또는 박테리아에 대한 면역학적 응답성을 도출하는 방법; 또는 벡터 또는 형질전환된 박테리아를 약제학적으로 또는 수의학적으로 허용되는 담체, 희석제, 비히클 또는 부형제 및/또는 아쥬반트 및/또는 안정화제와 혼합하는 것을 포함하는 면역원성, 면역학적 또는 백신 조성물의 제조 방법에 관한 것이다.
본 발명은 또한, 박테리아의 감쇠를 위한 표적, 예를 들어 박테리아의 감쇠를 위한 표적을 코딩하는 돌연변이화된 뉴클레오티드 서열 또는 유전자, 및 박테리아의 감쇠를 위해 폴리펩티드를 표적화하는 방법 및 감쇠된 박테리아를 생성하는 방법에 관한 것이다. 감쇠에 대한 표적은 예를 들어 면역원성 조성물에서 또는 백신 조성물에서 면역원성 화합물로서, 또는 면역원성 또는 백신 조성물에서의 사용을 위한 에피토프의 생성을 위해 사용될 수 있다. 따라서, 본 발명은 조성물 중, 예를 들어, 약제학적으로 또는 수의학적으로 허용되는 담체, 희석제, 부형제 또는 비히클 및/또는 아쥬반트 및/또는 안정화제와 혼합한 조성물의 제조에서 감쇠를 위한 표적의 용도에 관한 것이다.
본 발명은 더욱이 대상체 (예를 들어, 동물) 에 파스튜렐라 (Pasteurella) 와 같은 그람 음성 박테리아에 의해 감염되기 쉬운 동물, 예컨대 칠면조 또는 소에서 면역학적 또는 면역원성 또는 보호적 면역 응답성을 유도하는 방법에 관한 것으로, 상기 동물에서 본 발명의 백신 또는 면역원성 조성물을 투여하는 것을 포함한다.
더욱이, 본 발명은 상기 감쇠된 박테리아, 예를 들어, 파스튜렐라(Pasteurella) 와 같은 그람 음성 박테리아의 제조에 관한 것으로; 예를 들어, 하나 이상의 트랜스포존성 구성원 (transposable element) 을 박테리아에 도입하고 표적 종에서 치사 (mortality) 를 초래하지 않는 (따라서 감쇠되는) 트랜스포존성 구성원을 포함하는 박테리아를 단리하는 것을 포함하는 방법에 관한 것이다. 추가로, 임의로는 박테리아에서 돌연변이체를 동정함으로써 감쇠된 박테리아 제조를 위한 대안적인 수단을 수득할 수도 있다.
본 발명은 또한 감쇠된 박테리아 제조를 위한 상기 대안적인 수단에 관한 것이다. 돌연변이체는 동정 또는 특징화되므로, 돌연변이체는 하나 이상의 트랜스포존성 구성원을 박테리아로 도입하는 기술, 예컨대 동종 재조합법, 예를 들어 박테리아 게놈의 일부가 최소한 그곳에 추가되거나 (삽입) 또는 그로부터 결실되거나 (2 회 이상의 부가 및/또는 결실이 또한 포함된다) 또는 치환되도록 하는 (예컨대 또다른 것에 의한 하나 이상의 뉴클레오티드의 치환) 동종 재조합으로 도입할 수 있다. 따라서, 본 발명은 공지되거나 또는 선행기술에서 동정된 개질 또는 돌연변이, 예를 들어 본원에서 동정된 개질 또는 돌연변이를 포함하는 감쇠된 박테리아의 제조 방법으로서, 유리하게는 재조합을 통해 결실 또는 삽입 또는 치환을 박테리아 게놈에 도입하고, 임의로는 개질 또는 돌연변이를 포함하는 박테리아를 동정 및/또는 단리하는 것을 포함하는 방법에 관한 것이다.
따라서, 본 발명은 추가로 그람 음성 박테리아의 돌연변이체에 관한 것으로서, 상기 박테리아가 서열 번호: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 로 동정된 뉴클레오티드 서열로 이루어진 군에서 선택된 뉴클레오티드의 서열에 의해 코딩되는 아미노산 서열과 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 가진 폴리펩티드를 코딩하는 뉴클레오티드 서열에서 하나 이상의 돌연변이를 가지며; 상기 돌연변이가 박테리아의 발병력을 감쇠시키게 되는 돌연변이체에 관한 것이다. 또한, 본 발명은 본원에 기재된 상기 박테리아와 관련된 용도, 조성물 및 방법에 관한 것이다.
살아있는 감쇠된 미생물이 매우 유효한 백신이 될 수 있다는 것은 잘 확립되어 있다; 상기 백신에 의해 유도되는 면역 응답성은 종종 비복제성 면역원에 의해 생산되는 것보다 더 큰 규모이며 더 긴 지속 기간이 있다. 상기에 관해서는, 살아있는 감쇠된 균주가 숙주 내에서 제한된 감염을 확립하고 자연적인 감염의 조기 단계를 모방한다는 한 가지 설명이 가능하다. 또한, 멸균되거나 또는 불활성화된 제제와는 달리, 생백신은 마크로파지와 같은 항원-제시 세포를 복제하는 이들의 능 력과 연계될 수 있는 강력한 세포-매개 응답성을 유도할 수 있다.
특히 화학 돌연변이유발 기술을 이용한, 동물 및 인간에서의 살아있는 감쇠된 백신의 이용의 긴 역사가 있다. 그러나, 경험에 의하면 감쇠된 백신은 발병력으로 변환될 수 있다.
박테리아 병리학의 지식 증가와 더불어 현대 분자생물학 기술은 생체내 미생물의 성장 및 생존에 수반되는 각종 유전자의 동정을 유도해왔다. 이는 감쇠를 위한 신규한 유전자 표적 및 장래 백신 균주가 발병력에 수반되는 것으로 공지된 선택된 유전자에 정해진 비-변환 돌연변이를 도입함으로써 '분별있게' 감쇠될 수 있다는 개념을 제공했으며, 예로써 참고 문헌은 다음과 같다: WO-A-00/61724, WO-A-00/68261 및 EP-A-0889120.
다수의 감쇠된 균주가 실험실에서 생산되었으나, 단지 일부만이 동물용의 잠재적인 백신 후보물질로 자격이 있다. 이는, 부분적으로는 백신의 면역원성과 반응성 및 병원성으로 되는 미생물의 변환 능력을 균형짓기 위한 필요성에 기인할 것이다.
적합한 백신 후보물질을 제공할, 감쇠에 적합한 유전자의 선별은 간단하지 않으며 쉽게 예측할 수 없다. 다수의 인자들이 백신으로서의 감쇠된 돌연변이체의 접근성에 영향을 줄 수 있으며, 결과적으로는 적합한 감쇠 유전자를 동정 및 선별하기 위해서는 연구 노력이 필요하다. 다수의 감쇠 실험이 시험관 내에서만 수행되었고, 이들의 결과는 생체 내에서 특히 백신접종된 동물에 대한 수득되는 돌연변이의 잔류하는 병원성과 연관해서는 추정될 수 없다.
참고 문헌은 하기와 같다:
Kachlany 의 문헌은 Tad 유전자에 관한 것이다. Kachlany 의 문헌에서 돌연변이화된 Tad 유전자와 감쇠 사이에는 관련성이 없다. Kachlany 의 문헌에서는 동물에 대한 실험이 없고, Tad 유전자는 본 발명에서 선별되지 않는다. Fuller 의 논문은 본 발명에서 선별되지 않는 서열에 관한 것이다. Kehrenberg 는 감쇠된 돌연변이체, 또는 Signature Tagged Mutagenesis 또는 STM 기술에 대한 것이 아니고; 오히려, Kehrenberg 의 문헌은 트랜스포존 (transposon) 의 지시 삽입 (동일한 삽입 구성원의 이용) 에 관한 것이다. DeAngelis 1998a 에서는 STM 기술에 대한 일반적인 서술만을 제공할 뿐, 돌연변이체에 대한 것은 없다. DeAngelis 1998b 은 트랜스포존을 HA 생합성 부위 (HA biosynthesis locus; Genbank AF036004) 로 삽입하는 STM 기술의 이용에 관한 것이다. 상기 서열은 PM70 의 서열 Pm0775 에 대한 동종체이다. Pm0775 을 코딩하는 서열은 본 발명에서는 선별되지 않는다. Lee 의 문헌은 Tn10 트랜스포존을 이용한 STM 기술의 용도에 관한 것이다; Lee 는 동물에 대한 임의의 시험 또는 감쇠된 돌연변이체에 대한 임의의 연구를 개시 또는 제안하지 못했다; 오히려, Lee 의 문헌은 자가영양성 돌연변이체에 관한 것이다. Choi 의 문헌에서는 파스튜렐라 멀토시다 (Pasteurella multocida) 트랜스포존 삽입 돌연변이체를 언급하는데, 상기 돌연변이체에 의해 유도되는 치사는 없는 것으로 추측되며, Choi 의 문헌은 트랜스포존 삽입의 위치에 대한 설명이 포함되어 있지 않아서, 재생산가능한 것으로 언급될 수 없다. Nnalue 의 문헌은 유사하게 즉제의 발명을 교시 또는 제안하지 못했다. Stocker 의 특허는 Tn10 트랜스포존을 aroA 유전자에 삽입하는 것에 관한 것이다. AroA 유전자는 본 발명에서 선별되지 않는다. Highlander 의 문헌은 Tn1545 트랜스포존을 lktC 유전자로 삽입하여 류코톡신을 불활성화시키는 것에 관한 것이다. LktC 유전자는 본 발명에서 선별되지 않는다. 따라서, 즉제의 발명이 당업계에 교시 또는 제안된 것으로 여겨지지 않는다.
더욱이, 감쇠에 수반되는 유전자 또는 핵산 서열을 특징화하여, 이를 근거로 감쇠된 박테리아 뿐만 아니라 감쇠된 백신 또는 면역원성 조성물, 예컨대 높은 수준의 면역원성을 가지면서 부작용이 제한되거나 또는 없는 우수한 안전성을 나타내는 것을 개발하는 것이 요망된다.
발명의 개요
본 발명은 제 1 폴리펩티드를 코딩하는 제 1 뉴클레오티드 서열에 돌연변이가 있어 감쇠된 발병력을 가진 박테리아를 제공하는 그람 음성 박테리아의 돌연변이체로서:
제 1 폴리펩티드가 아미노산 서열을 가지며;
제 2 폴리펩티드가 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 또는 93 으로 식별되는 뉴클레오티드 서열에 의해 코딩되는 아미노산 서열을 가지며;
제 1 폴리펩티드의 아미노산 서열이 제 2 폴리펩티드의 것과 동일하거나, 또는 제 1 폴리펩티드의 아미노산 서열이 제 2 폴리펩티드의 아미노산 서열과 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 같는 돌연변이 를 제공한다.
돌연변이체 박테리아는 파스튜렐라세애 (Pasteurellaceae) 일 수 있으며, 예를 들어 상기 박테리아는 파스튜렐라세애 멀토시다, 파스튜렐라세애 해몰리티카, 파스튜렐라세애 아나티페스티페르 또는 악티노바실러스 플레우로뉴모니애일 수 있으며; 유리하게는 파스튜렐라 멀토시다이다.
돌연변이는 제 1 뉴클레오티드 서열에서의 결실, 또는 그곳으로의 삽입이거나 또는 핵산의 치환, 예컨대 전체 제 1 뉴클레오티드 서열의 결실이거나; 또는 서열 번호 2 에서 뉴클레오티드 180-181 또는 뉴클레오티드 182-183 또는 뉴클레오티드 190-191, 서열 번호 6 에서 뉴클레오티드 77-78 또는 뉴클레오티드 1026-1027 또는 뉴클레오티드 1027-1028, 서열 번호 9 에서 뉴클레오티드 416-417, 서열 번호 12 에서 뉴클레오티드 389-390, 서열 번호 16 에서 뉴클레오티드 381-382, 서열 번호 19 에서 뉴클레오티드 219-220, 서열 번호 22 에서 뉴클레오티드 1353-1354, 서열 번호 25 에서 뉴클레오티드 136-137, 서열 번호 28 에서 뉴클레오티드 384-385, 서열 번호 31 에서 뉴클레오티드 222-223 또는 뉴클레오티드 225-226, 서열 번호 34 에서 뉴클레오티드 217-218, 서열 번호 37 에서 뉴클레오티드 1411-1412, 서열 번호 40 에서 뉴클레오티드 943-944, 서열 번호 43 에서 뉴클레오티드 855-856, 서열 번호 46 에서 뉴클레오티드 369-370, 서열 번호 49 에서 뉴클레오티드 111-112, 서열 번호 52 에서 뉴클레오티드 443-444, 서열 번호 55 에서 뉴클레오티드 4-5, 서열 번호 61 에서 뉴클레오티드 573-574, 서열 번호 64 에서 뉴클레오티드 875-876, 서열 번호 70 에서 뉴클레오티드 218-219, 서열 번호 75 에서 뉴클레오티드 1072-1087, 서열 번호 78 에서 뉴클레오티드 64-65, 서열 번호 81 에서 뉴클레오티드 282-283, 서열 번호 84 에서 뉴클레오티드 1431-1432, 서열 번호 87 에서 뉴클레오티드 974-975, 서열 번호 90 에서 뉴클레오티드 802-803, 서열 번호 92 에서 뉴클레오티드 850-851; 또는 서열 번호 58 에서 바로 상류 뉴클레오티드 1; 또는 서열 번호: 67 에서 바로 상류 뉴클레오티드 1 사이의 삽입일 수 있다.
돌연변이체는 이종성 핵산 서열, 예컨대 발병력 바이러스, 기생체 또는 박테리아 제제 유래의 면역원, 치료용 단백질, 알레르겐 (allergen), 성장 인자 또는 싸이토카인을 코딩하는 이종성 핵산 서열을 포함할 수 있다.
본 발명은 또한 본 발명에 따른 돌연변이체 및 약제학적으로 또는 수의학적으로 허용되는 희석제, 담체, 비히클 또는 부형제를 함유하며, 임의로는 아쥬반트를 추가로 함유하는 면역원성 조성물 또는 백신을 제공한다.
본 발명은 또한 특정 아미노산 서열을 가진 단리된 제 1 폴리펩티드를 제공하는데, 여기서:
제 2 폴리펩티드는 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 또는 93 로 식별되는 뉴클레오티드 서열로 코딩되는 아미노산 서열을 가지며;
제 1 폴리펩티드의 아미노산 서열은 제 2 폴리펩티드의 것과 동일하거나 또는 제 1 폴리펩티드의 아미노산 서열은 제 2 폴리펩티드의 아미노산 서열과 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 갖는다.
본 발명은 단리된 제 1 폴리펩티드, 및 약제학적으로 또는 수의학적으로 허 용되는 희석제, 담체, 비히클 또는 부형제, 및 임의로는 아쥬반트를 함유하는 면역원성 또는 백신 조성물에 관한 것이다.
추가로, 본 발명은 단리된 제 1 폴리펩티드에 특이적인 항체를 포함하는 항체 제제에 관한 것이다.
본 발명은 또한 샘플 중에서 단리된 제 1 폴리펩티드 또는 단리된 상기 제 1 폴리펩티드에 특이적인 항체를 검출하는 것을 포함하는, 그람 음성 박테리아에 의한 감염을 검출하는 진단법을 포함한다.
본 발명은 추가로 상기 항체 제제를 투여하는 것을 포함하는 수동 면역화 방법에 관한 것이다.
본 발명은 또한 서열 번호: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 또는 93 로 식별되거나, 또는 서열 번호 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96 또는 97 로 식별되는 서열을 가진 단리된 핵산 분자 뿐만 아니라, 서열 번호: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 또는 93 로 식별되거나, 또는 서열 번호: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96 또는 97 로 식별되는 뉴클레오티드 서열의 10 개 이상 연속된 핵산인 서열을 가진 단리된 핵산 분자를 포함하는 그람 음성 박테리아를 검출하기 위한 PCR 프라이머를 제공한다. 프로브 또는 프라이머는, 증폭하기 원하는 서열에 대해 특이적이거나 또는 증폭하기 원하는 서열 내에 있고, 적어도 보존성인, 예를 들어 그람 음성 박테리아 사이에서 또는 그람 음성 박테리아의 특별한 패밀리 사이에서 또는 종 사이에서, 예컨대 파스튜렐라 (Pasteurella) 중에서, 또는 파스튜렐라 멀토시다, 파스튜렐라 해몰리티카, 파스튜렐라 아나티페스티페르 또는 악티노바실러스 플레우로뉴모니애 중에서; 유리하게는 파스튜렐라 멀토시다 중에서 보존성인, 8 개 이상, 바람직하게는 10 개 이상, 더욱 바람직하게는, 12, 13, 14 또는 15 개 이상, 예컨대 20 개 이상, 예를 들어 23 또는 25 개 이상, 예를 들어 27 또는 30 개 이상의 뉴클레오티드의 임의의 스트렛치일 수 있다. PCR 또는 혼성화 프라이머 또는 프로브에 있어서 최적에 길이에 관하서는 문헌 [Kajimura 등, GATA 7(4):71-79 (1990)] 을 참조한다.
용어 "면역원성 조성물" 및 "면역학적 조성물" 및 "면역원성 또는 면역학적 조성물" 은 표적 병원에 대한 면역 응답성, 예를 들어, 동물 (예컨대 조류, 예를 들어, 칠면조 또는 소, 예를 들어 젖소) 에 투여 또는 주사한 후, 표적 병원 (예를 들어, 파스튜렐라 멀토시다) 에 대한 면역 응답성을 유도하는 임의의 조성물을 총괄한다. 용어 "백신성 조성물" 및 "백신" 및 "백신 조성물" 은 표적 병원에 대한 보호적인 면역 응답성을 유도하거나 또는 병원에 대항하여 효능적으로 보호하는, 예를 들어 동물 (예컨대 조류, 예를 들어, 칠면조 또는 소, 예를 들어 젖소) 에 투여 또는 주사한 후, 표적 병원에 대한 보호적인 면역 응답성을 유도하거나 또는 병원 (예를 들어, 파스튜렐라 멀토시다) 에 대한 효능적인 보호를 제공하는 임의의 조성물을 총괄한다. 병원의 서브유닛은 병원, 예를 들어 파스튜렐라 멀토시다와 같은 박테리아로부터 단리된 항원 또는 면역원 또는 에피토프의 서브유닛; 및 서브유닛 조성물은 명원, 예를 들어 그람 음성 박테리아, 예를 들어 파스튜렐라 멀토시다와 같은 박테리아로부터 단리된 하나 이상의 항원, 면역원 또는 에피토프를 함유하거나 본질적으로 이들로 이루어진다.
본 발명에서 사용되는 "포함하다 (comprise)", "포함된", "포함하는" 등의 용어는 US 특허법에서 이에 부여하는 의미를 가질 수 있으며; 예를 들어, 이들은 "포함하다 (include)", "포함된", "포함하는" 등의 의미를 가질 수 있고; "본질적으로 ~ 로 이루어지다" 및 "본질적으로 ~ 로 이루어지는" 과 같은 용어는 US 특허법에 제시된 의미를 가질 수 있으며; 예를 들어 이들은 요소들이 명백하게 인용되도록 하고 있진 않지만, 발명의 기본적으로 신규한 특성에 영향을 미치는 선행기술에서 발견되는 요소들은 배제한다는 점에 주의해야 한다.
상기 및 기타 구현예들은 하기의 상세한 설명으로 개시되거나 또는 이로부터 명백해지며 이로부터 설명된다.
발명의 상세한 설명
본 발명은 예를 들어 그람 음성 박테리아 (예를 들어, 파스튜렐라 멀토시다)와 같은 박테리아인 미생물의 감쇠, 뉴클레오티드 서열에 의해 코딩되는 생성물 (예를 들어 단백질, 항원, 면역원, 에피토프), 그러한 뉴클레오티드 서열, 생성물, 미생물의 제조 방법, 및 예를 들어 발현 벡터와 같은 벡터 (예를 들어 시험관 내 또는 생체 내 발현 벡터) 또는 면역원성 또는 면역 반응 유도를 위한 백신 또는 면 역원성 조성물 제조와 같은 그들의 용도를 제공한다.
미생물의 뉴클레오티드 서열 및 유전자에 도입된 돌연변이는, 신규하고 진보한 감쇠 돌연변이체를 생성한다. 이들 돌연변이체는, 높은 면역원성 정도를 가지는 살아 있는 감쇠 백신 또는 살아 있는 감쇠된 면역원성 조성물의 제조에 유용하다.
이들 돌연변이체는, 발현 생성물의 시험관 내 발현 및, 뉴클레오티드 서열의 복제 (예를 들어, DNA 의 복제) 또는 재생성, 그리고 생체 내 발현 생성물에 유용할 수 있는, 벡터로서 또한 유용하다.
돌연변이의 동정은, 신규하며 진보성있는 뉴클레오티드 서열 및 유전자와, 뉴클레오티드 서열 및 유전자에 의해 코딩된 신규하며 진보성 있는 유전자 생성물을 제공한다.
그러한 유전자 생성물은, 항원, 면역원 및 에피토프를 제공하며, 분리된 유전자 생성물로 유용하다.
그러한 분리된 유전자 생성물 및 그들의 에피토프는, 항체 생성에 유용하며 진단 적용에 유용하다.
에피토프, 항원 또는 면역원을 제공 또는 생성 가능한 그러한 유전자 생성물은, 백신으로 뿐만 아니라, 면역원 또는 면역 조성물로 또한 유용하다.
한 국면에서, 본 발명은, 뉴클레오티드 서열 또는 유전자 중에 감쇠 돌연변이를 함유하는 박테리아를 제공하며, 여기에서 돌연변이는, 유전자에 의해 코딩되는 단백질 또는 폴리펩티드의 생물학적 활성 및/또는 발현을 감소 또는 제거함으로 써, 박테리아의 독성을 감소시킨다.
돌연변이는 그 기능을 방해하여 감쇠에 이르게하는 코딩 서열 또는 유전자 내에 반드시 위치할 필요는 없다. 돌연변이는, 예를 들어, 전사 개시, 번역 및 전사 종결을 조절하는 영역 내에서 유전자 발현의 조절에 관계된 뉴클레오티드 서열에서 또한 만들어질 수 있다. 따라서, 또한 포함되는 것으로는, 프로모터 및 리보솜 결합 영역 (보통 이들 조절 구성원은, 코딩 서열 또는 유전자의 개시 코돈의 약 60-250 뉴클레오티드 상류에 존재한다; Doree S M 등, J.Bacteriol. 2001, 183(6): 1983-9 ; Pandher K 등, Infect. Imm. 1998, 66(12): 5613-9 ; Chung J Y 등, FEMS Microbiol letters 1998, 166: 289-296), 전사 종결자 (보통 종결자는, 코딩 서열 또는 유전자의 종결 코돈의 약 50 뉴클레오티드 하류 내에 존재한다; Ward C K 등, Infect. Imm. 1998, 66(7): 3326-36)가 있다. 오페론의 경우, 그러한 조절 영역은 코딩 서열 또는 유전자의 상류의 더 먼 곳에 존재 가능하다. 유전자간 영역에서의 돌연변이 또한 감쇠에 이르게 할 수 있다.
코딩 서열 또는 유전자와 관련된 그러한 조절 서열내의 돌연변이는, 이러한 뉴클레오티드 서열의 돌연변이가 유전자에 의해 코딩되는 단백질 또는 폴리펩티드의 생활성 및/또는 발현을 변형, 억제 또는 제거하여, 감쇠되는 박테리아의 독성을 초래하며, 본 발명에서 동정된 코딩 서열 또는 유전자 내의 돌연변이와 균등하게 된다.
감쇠는, 박테리아의 병원성을 감소 또는 제거하고, 임상 증후 또는 상처의 정도를 감소 또는 제거하며, 박테리아의 성장 속도를 감소시키고, 박테리아에 의한 사망을 방지한다.
본 발명은 예를 들어 파스튜렐라 멀토시다, 파스튜렐라 해몰리티카, 파스튜렐라세애 아나티페스티페르 및 악티노바실러스 플레우로뉴모니애와 같은 파스튜렐라 (Pasteurella) 패밀리 박테리아와 같은 그람 음성 박테리아인 박테리아와 같은 미생물에 관한 것이다. 유리하게 박테리아는 파스튜렐라 멀토시다이다.
파스튜렐라 멀토시다는 그람 음성 박테리아로, 이는 생산성 동물의 각종 질병 원인제 및 기회성 인간 병원체이다. 이는 가금 및 야생 조류의 조류 콜레라, 소 출혈성 패혈증 및 돼지 위축성 비염과 같은 심한 파스퇴렐라증의 병인제이다 (Hunt ML 등, Vet Microbiol 2000, 72(1-2): 3-25). 분리물은, 캡슐 항원에 따라 혈청학적으로 혈청군 (A, B, D, E 및 F)으로 분류 또는 신체 LPS 항원에 따라 16 개의 혈청타입으로 분류 가능하다.
박테리아의 감쇠 관련 잠재적 뉴클레오티드 서열은, Signature Tagged Mutagenesis (STM)에 의해 동정되었다. 이 방법은, 여기에 언급된 문헌에 논의된 것으로, 또한 WO-A-96/17951을 언급할 수 있다.
STM은, 미생물의 게놈으로의, 고유하며, 사인 태깅된 트랜스포존의 삽입과 관련된다.
삽입 부위에서, 게놈 뉴클레오티드 서열은 파괴된다. 본 발명에서, 수득하는 돌연변이 (따라서 돌연변이를 가지는 돌연변이체)를 감쇠에 대해 분석한다.
감쇠된 각 돌연변이체에 대한 파괴된 영역의 서열 (즉, 유전자 또는 코딩 서열 또는 오픈 리딩 프레임 (ORF))은 PCR 증폭 (폴리머라아제 연쇄 반응), 트랜스포존 가장 자리 DNA 영역의 클로닝 및 서열 분석에 의해 결정한다.
본 발명의 구현에서, WO-A-96/17951에 언급된 STM 방법은 파스튜렐라 멀토시다에서 기능성이 되도록 적응화된다. 이들 적응은, Tn5 대신 Tn10 트랜스포존의 사용 및, 스트렙토마이신 내성 선별 대신 류신 결핍 CDM 매질의 선별을 사용하는 것을 특징적으로 포함한다. 더 자세한 것은 실시예에 언급한다.
STM 방법에 의해 동정된 잠재적 유전자에서 감쇠 관련 유전자 또는 뉴클레오티드 서열을 추가로 선별하는 것은, 동물에게 돌연변이 박테리아를 주사한 후 치사가 발생하지 않는 것을 근거로 하는 것이다.
수의학적 적용에서, 본 발명의 한 유리한 면은, 동물 모델에서가 아니라 목표 동물 내에서 직접 실험 선별하는 것을 시행함을 포함한다. 본 방법은, 돌연변이 박테리아의 적절한 돌연변이에 대한 보다 정확한 선별을 가능하게 한다. 파스튜렐라 멀토시다에서, 실험은 파스튜렐라 멀토시다의 자연 목표 숙주의 하나인 칠면조에 대해 직접 실행한다.
칠면조에게, 사인-태깅된 파스튜렐라 멀토시다 (P. multocida) 돌연변이체의 충분 공급량을 근육내 주사한다 (즉, 0.5 ml, 동물 한 마리당 107 CFU). 주사 이후 특정 시점에서 재분리되지 않은 돌연변이체는 잠재적으로 감쇠된 것으로 간주한다. 재분리되지 않은 돌연변이체는, PCR 증폭 및 사인 태그 분석에 의해 재분리된 집합 (pool) 의 것과 구분된다.
각각의 잠재 감쇠된 돌연변이체는 이후 근육내 경로를 통해 칠면조에 주사한 다 (즉, 0.5 ml, 동물 한 마리당 104 CFU). 칠면조의 치사율을 주사 이후 7일 동한 매일 기록한다. 살아남은 칠면조는 감쇠된 것으로 간주한다.
특정 방법을 파스튜렐라 멀토시다 균주 P-1059 상에서 수행하여 다수의 감쇠된 돌연변이체를 수득했다. 이들 중 다섯 가지는 Pasteur Institute (파리, 프랑스)의 CNCM (Collection Nationale de Cultures de Microorganismes)에 2003 년 4 월 1 일자로 기탁된 것이다. 4G11 돌연변이체는 기탁번호 CNCM I-2999로 입수가능하다. 5D5 돌연변이체는 기탁번호 CNCM I-3000으로 입수가능하다. 9C8 돌연변이체는 기탁번호 CNCM I-3001로 입수가능하다. 9H4 돌연변이체는 기탁번호 CNCM I-3002로 입수가능하다. 13E1 돌연변이체는 기탁번호 CNCM I-3003으로 입수가능하다.
트랜스포존 삽입 부위 가장 자리의 뉴클레오티드 서열은, 서열 번호: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96, 97로 지정된다.
트랜스포존은, 파스튜렐라 멀토시다 균주 P-1059에서 서열 1, 8, 11, 14, 15, 27, 33, 42, 54, 57, 66, 72, 73, 77, 80, 95 및 97의 5' 말단에 바로 삽입되며, 서열 4, 5, 18, 21, 24, 30, 36, 39, 45, 48, 51, 60, 63, 69, 83, 86, 89 및 96의 3' 말단에 바로 삽입된다. 돌연변이체 9H4에서, 트랜스포존은, 서열 번호: 92 서열의 850-851 위치의 뉴클레오티드 사이에 삽입된다.
본 발명의 특징적인 면은, 서열 서열 번호: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96 및 97 에서 선택되는 서열을 포함하는 이들의 조절 영역 및/또는 유전자 또는 ORF 내의 감쇠된 돌연변이 함유 파스튜렐라 멀토시다 균주 P-1059의 감쇠된 돌연변이체이다.
추가의 본 발명의 특별한 구현예는, 부다페스트 조약 조항에 따라 CNCM에 기탁된 여기에 언급한 감쇠된 돌연변이체와 같은 본 발명에 따른 감쇠된 돌연변이체를 포함한다.
감쇠된 P-1059 돌연변이체는, 예를 들어, 트랜스포존 삽입 또는 직접 돌연변이 (결실, 삽입, 치환)에 의해 수득 가능하다. 감쇠 돌연변이는, 이들 뉴클레오티드 서열 또는 유전자 내에서 또는, 이들의 상보 서열 내에서 만들어질 수 있다. 감쇠 돌연변이는 또한, 상기 유전자 또는 뉴클레오티드 서열의 조절 영역 관련 뉴클레오티드 서열 내에서 만들어질 수 있다.
상기 서열 또는 그 일부 (적어도 그의 10, 15 또는 20 뉴클레오티드로서, 예를 들어, 그의 적어도 연속 10개 뉴클레오티드 또는, 그의 적어도 연속 15개 뉴클레오티드 및 보다 유리하게는 그의 적어도 연속 20개 뉴클레오티드 내지 서열의 전장까지)는, 트랜스포존 삽입된 돌연변이체의 검출 및 선별을 위한 PCR 프라이머로 사용 가능하다. PCR 은, 한 쌍의 프라이머, 예를 들어 트랜스포존 특이성인 것과 돌연변이시킬 유전자 또는 뉴클레오티드 서열에 특이적인 것을 수반한다. PCR 증폭 생성물의 기대 크기에 따라, 상기 방법은 PCR 단편의 증폭 및/또는 검출을 가능케한다. 해당 유전자 또는 ORF 및/또는 생물체 (예를 들어, 파스튜렐라 (Pasteurella) 와 같은 그람 음성 박테리아, 예컨대 파스튜렐라 멀토시다 균주 PM70 또는 P-1059 (참고로 하기를 참조)와 같은 파스튜렐라 멀토시다 내 이들의 조절 영역에 대한 지식; 예를 들어, 해당 유전자 또는 ORF 및/또는 그들 조절 영역의 크기는, PCR 프라이머 디자인, 증폭된 PCR 단편의 스크리닝 및, 돌연변이체의 선별을 가능하게 하는 올바른 크기의 단편 검출에 이용 가능하다.
파스튜렐라 멀토시다 균주 PM70의 전체 게놈은, EMBL 데이터베이스 및 문헌에서 얻을 수 있다 (May BJ 등, Proc. Natl. Acad. Sci. USA, 2001, 98(6) 3460-5). 서열 번호: 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96, 97 서열에 대한 블라스트는, PM70 게놈 상의 동종 서열을 찾아내어, PM70 내의 해당 유전자 또는 ORF를 측정 가능하게 한다.
파스튜렐라 멀토시다 균주 PM70 내의 뉴클레오티드 서열은, 서열 번호: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61,64, 67, 7O, 75, 78, 81, 84, 87, 90 으로 정한다.
P-1059 균주의 9H4 돌연변이체에 대해서는, PM70에서 동종 서열이 발견되지 않았다. P-1059 ORF 는 서열분석되어 서열 번호 93 으로 표시된다.
본 발명의 다른 면은, 서열 번호: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87 및 90에서 선택되는 뉴클레오티드 서열 및/또는 그들의 조절 영역 함유 유전자 또는 ORF 내의 하나 이상의 감쇠 돌연변이를 가지는 PM70 주의 감쇠된 돌연변이체이다.
감쇠 돌연변이는, 이들 뉴클레오티드 서열 또는 유전자 및 이들의 상보 서열 내에서 만들 수 있다. 감쇠 돌연변이는 또한, 상기 유전자의 조절 영역에 수반되는 뉴클레오티드 서열 내에서 만들어질 수 있다. 감쇠 돌연변이체는 예를 들어, 트랜스포존 삽입 또는 직접 돌연변이 (결실, 삽입, 치환)에 의해 수득 가능하다.
"상보성" 이라는 용어는 이중 가닥 게놈의 나머지 가닥의 뉴클레오티드 서열을 말하며 따라서, 센스 가닥에 상응하는 안티 센스 가닥을 포함하며, 역에 대해서도 성립하다. "뉴클레오티드" 라는 용어는 또한 디옥시리보뉴클레오티드 (디옥시리보핵산 또는 DNA로 된 것), 리보뉴클레오티드 (리보핵산 또는 RNA로 된 것) 및 메신저 리보뉴클레오티드 (mRNA)를 포함한다.
보다 일반적으로 감쇠 돌연변이는, 그람 음성 박테리아, 예를 들어 파스튜렐라 멀토시다 (P. multocida), 파스튜렐라 해몰리티카 (P. haemolytica), 파스튜렐라세애 아나티페스티페르 (P. anatipestifer) 및 악티노바실러스 플레우로뉴모니애 (A. pleuropneumoniae) 와 같은 파스튜렐라 (Pasteurella) 패밀리 박테리아와 같은 박테리아의 게놈 내로 도입되며, 유리하게는, 파스튜렐라 멀토시다 (P. multocida) 의 다양한 주 중 임의의 게놈 내의 박테리아 (즉, P-1059 균주 및 PM70 균주)이고, 돌연변이는, 서열 번호: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 로 식별되는 뉴클레오티드 서열에 의해 코딩되는 아미노산 서열 하나에 대해 약 70% 이상 동일성, 약 75% 이상 동일성, 약 80% 이상 동일성, 약 85% 이상 동일성, 약 90% 이상 동일성, 유리하게는 약 95, 96, 97, 98 또는 99% 이상 동일성 또는 그 이상의 동일성을 가지는 아미노산 서열을 코딩하는 하나 이상의 뉴클레오티드 서열에 존재하는 것이다. 감쇠 돌연변이는, 이들 뉴클레오티드 서열 또는 유전자 및 이들의 상보 서열 내에서 만들 수 있다. 감쇠 돌연변이는 또한, 상기 유전자의 조절 영역 관련 뉴클레오티드 서열 내에서 만들어질 수 있다. 감쇠 돌연변이체는 예를 들어, 트랜스포존 삽입 또는 직접 돌연변이 (결실, 삽입, 치환)에 의해 수득 가능하다. 수득한 감쇠 돌연변이체는 본 발명의 구현예이다. 특별한 구현예는 P-1059 감쇠 돌연변이체이다.
두 아미노산 서열간의 동일성 백분율은 표준 파라미터 (즉, “National Center for Biotechnology Information"(NCBI, Bethesda, MD., USA) 서버 및 Altschul 등, J. Mol. Biol. 1990. 215. 403-410에서 입수할 수 있는 BLAST 또는 BLASTX 알고리즘을 주목; 이러한 문헌은 따라서 "블라스트 (blast)" 라는 용어로 알고리즘 또는 BLAST 또는 BLASTX 및 BLOSUM62 매트릭스의 사용을 언급한다) 를 사용한 NCBI(National Cancer for Biotechnology Information) Pairwise blast 및 blosum62 matrix로 확립할 수 있다.
본원에 사용된 동사 "코드”는 핵산 서열이 실제의 코딩 서열에만 국한되는 것을 의미하는 것은 아니며, 비-코딩 서열인 이의 조절서열을 포함하는 전체 유전자를 또한 포괄한다.
핵산 서열 상동성과 같은 서열 상동성 또는 동일성은 또한 NCBI에서도 이용할 수 있는 Myers 및 Miller의 "Align" 프로그램 ("Optimal Alignments in Linear Space", CABIOS 4, 11-17, 1988, 본원에 참조로 도입됨), 및 NCBI 사이트와 같은 사이트 상에서 인터넷을 통해 사용할 수 있는 상기와 동일하거나 또는 기타 다른 프로그램을 사용하여 결정할 수 있다.
대안 또는 부가적으로, 핵산 또는 아미노산 서열과 관련되어, 용어 "상동성" 또는 "동일성"은, 예를 들면 두 서열간의 상동성의 양적 측정을 나타낼 수 있다. 서열 상동성 백분율은 다음과 같이 계산될 수 있다:
(N ref -N dif )*100/N ref , 여기에서 N dif 는 정렬된 경우 두 서열에서의 비동일한 잔기의 총수이며 N ref 는 서열 중 하나의 잔기의 갯수이다. 그러므로, DNA 서열 AGTCAGTC는 서열 AATCAATC와 75%의 서열 동일성을 가질 것이다(N ref = 8; N dif
= 2).
대안 또는 부가적으로, 서열과 관련되어 용어 "상동성" 또는 "동일성"은 두개의 서열 중에 더 짧은 것의 핵산 또는 아미노산의 수로 나눈 동일한 핵산 또는 아미노산을 갖는 위치의 수를 언급할 수 있으며 여기에서 상기 두 서열의 정렬은, 예를 들면 윈도우크기 20개 핵산, 단어길이 4개 핵산, 및 갭(gap)페널티 4를 사용한 Wilbur 및 Lipman 알고리즘(Wilbur 및 Lipman, 1983 PNAS USA 80:726, 본원에 참조로 도입됨)에 따라 결정될 수 있으며, 정렬을 포함한 서열 데이터의 컴퓨터-보조 분석 및 해석은 시중에서 구입할 수 있는 프로그램(예를 들면, IntelligeneticsTM Suite, Intelligenetics Inc. CA)을 사용하여 편리하게 수행될 수 있다. RNA서열이 DNA서열과 유사한 것으로, 또는 어느 정도의 서열 동일성 또는 상동성을 갖는 것으로 언급되는 경우에, DNA 서열의 티미딘(T)은 RNA 서열의 우라실(U)과 같은 것으로 간주된다. 따라서, RNA 서열도 본 발명의 범위내이고 DNA 서열의 티미딘(T)이 RNA 서열의 우라실(U)과 같은 것으로 간주함으로써 DNA 서열로부터 유추될 수 있다.
유리하게는, 아미노산 서열 동일성 또는 상동성과 같은 서열 동일성 또는 상동성은 NCBI에서도 이용할 수 있는 BlastP 프로그램 (Altschul 등, Nucl. Acids Res. 25, 3389-3402, 본원에 참조로 도입됨), 및 NCBI 사이트와 같은 사이트 상에서 인터넷을 통해 사용할 수 있는 상기와 동일하거나 또는 기타 다른 프로그램을 사용하여 결정할 수 있다.
하기의 문헌(각각이 본원에 참조로 도입됨)은 두 단백질의 아미노산 잔기와 같은 서열의 상대적 동일성 또는 상동성 비교용 알고리즘을 제공하며, 앞서 기술한 것과 관련하여 대안 또는 부가적으로, 이들 참조문헌의 교시는 상동성 또는 동일성 백분율 결정에 사용될 수 있다 : Needleman SB 및 Wunsch CD, "A general method applicable to the search for similarities in the amino acid sequences of two proteins", J. Mol. Biol. 48:444-453(1970); Smith TF 및 Waterman MS, "Comparison of Biosequences", Advances in Applied Mathematics 2:482-489(1981); Smith TF, Waterman MS 및 Sadler JR, "Statistical characterization of nucleic acid sequence functional domains", Nucleic Acid Res., 11:2205-2220(1983); Feng DF 및 Dolittle RF, "Progressive sequence alignment as a prerequisite to correct phylogenetic tree", J. of Molec. Evol., 25:351-360(1987); Higgins DG 및 Sharp PM, "Fast and sensitive multiple sequence alignment on a microcomputer", CABIOS, 5:151-153(1989); Thompson JD, Higgins DG 및 Gibson TJ, "Cluster W: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, positions-specific gap penalties and weight matrix choice", Nucleic Acid Res., 22:4673-480(1994); 및 Devereux J, Haeberlie P 및 Smithies O, "A comprehensive set of sequence analysis program for the VAX", Nucleic Acid Res., 12:387-395(1984). 그리고 불필요한 실험 없이도 당업자라면 상동성 백분율을 결정하기 위해 많은 다른 프로그램 또는 참조문헌을 참고할 수 있다.
본 발명은 동일한 생물학적 기능을 가지는 폴리펩티드 또는 단백질을 코딩하는 핵산서열 또는 유전자의 돌연변이에 관한것이다. 기능의 유사성은 활성 부위의 보존에 의해 분석 또는 규명 또는 결정 또는 검토될 수 있다. 이는 NCBI DART 연구(Domain Architecture Retrieval Tool)로 할 수 있다.
본 발명은 그러므로 본원에서 정의된 감쇠 돌연변이를 포함하는 본원에서 기술된 박테리아의 감쇠된 돌연변이체를 제공한다.
감쇠된 그람음성 박테리아 돌연변이체는 하나의 돌연변이를 포함하며, 여기에서 하나 이상의 특정 유전자 또는 핵산 서열의 전부 또는 일부는 본원에서 논의된 대로 돌연변이 된다. 상기 특정 유전자 또는 핵산 서열은 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 또는 93 의 서열, 또는 이들의 조절영역을 포함하는 것 또는 이들에 상동성이 있는 (예를 들면, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 또는 99%의 상동성) 것을 포함한다. 유리하게는, 상기 특정 유전자 또 는 핵산 서열은 서열 번호 2, 6, 9, 12, 25, 31, 37, 40, 43, 46, 70, 75, 78, 81, 84, 87, 90 또는 93 의 서열, 또는 이들의 조절영역을 포함하는 것 또는 이들에 상동성이 있는 것을 포함한다. 더욱 유리하게는, 상기 특정 유전자 또는 핵산 서열은 서열 번호 6, 12, 25, 31, 37, 40, 46, 70, 75, 84, 87, 90 또는 93 의 서열, 또는 이들의 조절영역을 포함하는 것 또는 이들에 상동성이 있는 것을 포함한다. 그리고 더욱 특히 유리하게는, 상기 특정 유전자 또는 핵산 서열은 서열 번호 37, 40, 75, 90 또는 93 의 서열, 또는 이들의 상동 서열을 포함하는 것 또는 이들에 상동성이 있는 것을 포함한다. 바람직하게 상기 돌연변이체는 파스튜렐라(Pasteurella), 예컨대 파스튜렐라 멀토시다(P. multocida), 예를 들면 P-1059 또는 PM70이다.
돌연변이는 임의의 공지의 기술, 예컨대, 예를 들면, 재조합 DNA 기술을 사용하여 선택된 유전자 또는 핵산 서열에 잘 정의된 돌연변이를 도입(유도 돌연변이화) 하기위해 미생물로 도입될 수 있다. 이러한 돌연변이는 동종 또는 이종 핵산서열의 도입, 결실, 치환, 예를 들면, 하나 이상의 핵산을 다른 것으로 또는 이들의 조합에 의한 치환일 수 있다. 구현예에서, 상기 돌연변이는 결실 돌연변이로, 여기에서 상기 유전자 또는 핵산 서열의 손상 (disruption) 은 일부의 결실, 및 유리하게는 전체 핵산 서열 또는 유전자의 결실로 야기된다. 핵산의 결실은 병원성으로의 복귀는 피한다. 다른 구현예에서 상기 돌연변이는 본원, 예를 들면 실시예에서 기술된 트랜스포존 삽입 위치에 해당하는 위치로의 삽입이다. 이러한 위치들은, P-1059 균주를 참조로하여, 유리하게는 서열 번호 1, 8, 11, 14, 15, 27, 33, 42, 54, 57, 66, 72, 73, 77, 80, 95 및 97 의 서열의 바로 5' 말단 및 서열 번호 4, 5, 18, 21, 24, 30, 36, 39, 45, 48, 51, 60, 63, 69, 83, 86, 89 및 96 의 서열의 바로 3' 말단에 위치한다. 이러한 위치들은 또한 PM70 주에서 하기의 서열 사이에 위치하는 것들이다: 서열 번호 2 의 뉴클레오티드 180-181 또는 182-183 또는 190-191, 서열 번호 6 의 뉴클레오티드 77-78 또는 1026-1027 또는 1027-1028, 서열 번호 9 의 뉴클레오티드 416-417, 서열 번호 12의 뉴클레오티드 389-390, 서열 번호 16 의 뉴클레오티드 381-382, 서열 번호 19의 뉴클레오티드 219-220, 서열 번호 22의 뉴클레오티드 1353-1354, 서열 번호 25의 뉴클레오티드 136-137, 서열 번호 28의 뉴클레오티드 384-385, 서열 번호 31의 뉴클레오티드 222-223 또는 225-226, 서열 번호 34의 뉴클레오티드 217-218, 서열 번호 37의 뉴클레오티드 1411-1412, 서열 번호 40의 뉴클레오티드 943-944, 서열 번호 43의 뉴클레오티드 855-856, 서열 번호 46의 뉴클레오티드 369-370, 서열 번호 49의 뉴클레오티드 111-112, 서열 번호 52의 뉴클레오티드 443-444, 서열 번호 55의 뉴클레오티드 4-5, 서열 번호 61의 뉴클레오티드 573-574, 서열 번호 64의 뉴클레오티드 875-876, 서열 번호 70의 뉴클레오티드 218-219, 서열 번호 75의 뉴클레오티드 1072-1087, 서열 번호 78의 뉴클레오티드 64-65, 서열 번호 81의 뉴클레오티드 282-283, 서열 번호 84의 뉴클레오티드 1431-1432, 서열 번호 87의 뉴클레오티드 974-975, 서열 번호 90의 뉴클레오티드 802-803, 서열 번호 92의 뉴클레오티드 850-851; 또는, 서열 번호 58의 뉴클레오티드 1 의 바로 상류; 또는 서열 번호 67의 뉴클레오티드 1 의 바로 상류. 이러한 위치들은 또한, 이의 상동성 백분율로 본원에서 정의된 상동 아 미노산 서열을 코딩하는 예컨대 파스튜렐라세아 (Pasteurellacaea) 패밀리의 일원, 예를 들면 파스튜렐라 멀토시다, 파스튜렐라 헤모리티카, P. 아나티페스티페르 (P. anatipestifer), A. 플루로뉴모니애(A. pleuropneumoniae)와 같은 다른 그람 음성 박테리아의 핵산 서열에서 유사한 서열 쌍(PM70에 대해 언급된 것 보다)간에 위치하는 것들이다. 따라서, 돌연변이체는 그람음성 박테리아이며 유리하게는 파스튜렐라, 예컨대 P. 멀토시다, P. 헤모리티카, P. 아나티페스티페르, A. 플루로뉴모니애, 예를 들면 P-1059 또는 PM70과 같은 P. 멀토시다일 수 있다.
정의에 의하면, 결실 돌연변이체는 본 발명에 따른 핵산서열에 또는 핵산서열의 하나 이상의 결실을 포함한다. 이러한 결실 돌연변이체는 특정 유전자 서열 또는 특정 핵산 서열의 전부 또는 일부가 결실된 것들을 포함한다. 하나의 양태에서, 상기 돌연변이는 유전자 또는 특정 핵산 서열의 적어도 하나의 핵산, 약 10% 이상, 약 20% 이상, 약 30% 이상, 약 40% 이상, 약 50% 이상, 약 60% 이상, 약 70% 이상, 약 80% 이상, 약 90% 이상, 약 95% 이상, 약 98% 이상, 또는 약 99% 이상의 결실을 초래한다. 바람직하게 전체 유전자 또는 특정 핵산서열이 결실된다.
돌연변이체는 하나 이상의 돌연변이를 포함할 수 있으며, 이는 부가적 또는 상승적 정도의 감쇠를 초래할 수 있으며, 감쇠의 복귀 예방에 더 좋을 수 있다.
이러한 다수 돌연변이는 예컨대 aro 유전자, 예를 들면 aroA (Homchampa P. 등, Veterinary Microbiology, 1994, 42:35-44)와 같은 이들의 감쇠 성질이 알려진 유전자 또는 핵산 서열로의 돌연변이 및 본 발명에 따른 핵산 서열 또는 유전자로의 돌연변이를 연합할 수 있다.
하나의 구현예에서 돌연변이체는 두개 이상의 돌연변이를 포함하며, 여기에서 각각의 돌연변이의 경우 특정 유전자 또는 핵산 서열의 전부 또는 일부가 본원에서 언급된 대로 돌연변이 된다. 이러한 특정 유전자 또는 핵산 서열은 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 또는 93 의 서열, 또는 이들에 상동성이 있는 것 또는 이들의 조절영역을 포함하는 것을 포함한다. 따라서, 두개 이상의 앞서 언급한 돌연변이된 서열, 예를 들면 본원에서 논의된 대로 결실된 돌연변이를 갖는 돌연변이체가 본 발명에 포괄된다. 유리하게는, 돌연변이체는 두개 이상의 하기의 서열 또는 예를 들면, 본원에서 논의된 대로 결실된, 하기의 돌연변이된 서열을 포함하거나 또는 이에 상동인 서열을 가진다: 서열 번호 2, 6, 9, 12, 25, 31, 37, 40, 43, 46, 70, 75, 78, 81, 84, 87, 90 또는 93 의 서열, 또는 이들의 조절 영역. 더욱 유리하게는 돌연변이된 상기 특정 유전자 또는 핵산 서열(예를 들면, 돌연변이된 두개 이상)은 서열 번호 6, 12, 25, 31, 37, 40, 46, 70, 75, 84, 87, 90 또는 93 의 서열, 또는 이들의 조절영역을 포함하는 것 또는 이들에 상동인 것을 포함한다. 상기 돌연변이체는 그람음성 박테리아일 수 있으며, 유리하게는 상기 돌연변이체는 파스튜렐라, 예컨대 P. 멀토시다, 예를 들면 P-1059 또는 PM70이다.
유리하게는, 본원에서 논의된 대로 예를 들면 결실로 돌연변이된 두개 이상의 하기 서열, 또는 이들의 조절영역을 갖는 돌연변이체가 본 발명에 포함된다: 서열 번호 37, 40, 75, 90 및 93, 또는 이들의 상동 핵산서열.
다양한 구현예는 서열 서열 번호 37 및 40; 서열 번호 37 및 75; 서열 번호 37 및 90; 서열 번호 37 및 93; 서열 번호 40 및 75; 서열 번호 40 및 90; 서열 번호 40 및 93; 서열 번호 75 및 90; 서열 번호 75 및 93; 서열 번호 90 및 93, 또는 이들의 조절 영역을 포함하는 또는 이들에 상동인 유전자 또는 핵산서열의 또는 핵산서열에 결실이 있는 돌연변이체를 포함한다. 상기 돌연변이체는 그람음성 박테리아일 수 있으며 유리하게는, 파스튜렐라, 예컨대 P. 멀토시다, 예를 들면 P-1059 또는 PM70이다.
특정한 게놈 부위에 돌연변이를 도입하는 방법은 공지되어 있으며, 당업계의 상기 문헌 및 지식으로부터 당업자에게 자명할 것이다. 예를 들면, 돌연변이되는 모든 유전자 또는 서열 또는 단편은 벡터로 클로닝되고, 변형되어 이의 발현 및/또는 이의 생물학적 활성을 파괴한다. 벡터는, 예를 들어 전기천공에 의해서 (예, Jablonski L. et al., Microbial Pathogenesis, 1992, 12, 63-68), 또는 접합에 의해서 (Lee M.D. et al., Vet. Microbiol., 1996, 50, 143-148) 박테리아에 도입된다. 변형된 DNA 단편은 유전자 재조합에 의해서, 유리하게는 박테리아 염색체와 벡터간의 동종 재조합에 의해서 박테리아 게놈에 재도입된다. 예로서, 벡터는 Cardenas (Cardenas M et al., Vet Microbiol 2001 May 3; 80(1):53-61) 에 기재된 바와 같은 자살 플라스미드 (suicide plasmid) 일 수 있다. 유리하게는, 상기 벡터는 (동종 재조합에 사용되는) 2 개의 측면 팔 또는 영역 사이에, 임의의 가능한 번역을 차단하기 위해 폴리스톱 서열 (예를 들어, 6 개의 정지 코돈, 각 리딩 프레임 중 하나) 을 추가로 포함한다.
본 발명의 감쇠 미생물, 예를 들면 파스튜렐라 멀토시다 (P. multocida) 와 같은 그램 음성 박테리아는 이의 게놈에 삽입된 하나 이상의 동종 또는 이종 핵산 서열을 추가로 포함할 수 있다. 이것은 생체내 또는 생체외에서, 이종 핵산 분자의 재생 또는 복제 및/또는 이종 핵산 분자의 발현에 유용하다. 이종 핵산 서열은 유리하게는, 감쇠 미생물에 의해 자연적으로 발현되는 것과는 상이한 병원성 바이러스, 기생체 또는 박테리아제로부터 면역원, 항원 또는 에피토프를 코딩한다. 상기 이종 서열은 미생물 또는 박테리아의 또다른 균주, 예를 들면 또다른 파스튜렐라 멀토시다 (P. multocida) 균주로부터 면역원, 항원 또는 에피토프를 코딩할 수 있다. 면역원 또는 항원은 병원성제 또는 병원성 제제의 분비된 항원에 대해 면역 응답성을 유도할 수 있는 단백질 또는 폴리펩티드이며, 하나 이상의 에피토프를 함유하고; 에피토프는 병원성제 또는 병원성제의 분비된 항원에 대해 면역 응답성을 유도할 수 있는 펩티드 또는 폴리펩티드이다.
이러한 벡터에서의 상기 사용에 적합한 이종 핵산 서열은 당업자에게는 자명할 것이며 (Fedorova ND and Highlander SK, Infect Immun 1997, 65(7):2593-8), 예를 들면 파스튜렐라세애 패밀리 멤버 (특히 파스튜렐라 멀토시다, 파스튜렐라 해몰리티카, 파스튜렐라 안티페스티페르, 악티노바실러스 플레우로뉴모니애) 에서 유래하거나, E. coli, Salmonella, Campylobacter 와 같은 박테리아에서 유래하는 것을 포함한다.
이종성 서열은 유리하게는 감쇠 미생물 및 상기 발현된 면역원 모두에 대해 면역 응답성을 나타내기 위해 투여될 때, 숙주내 미생물에 의해 발현되도록 삽입된 다. 이종성 서열은 유리하게는 프로모터와 같은, 발현을 허용하는 조절 요소와 함께 삽입되거나, 상기 구성원에 작동적으로 연결되거나, 상기 구성원의 하류에 있다. 단백질의 배치 (address) 및 분비에 유용한 뉴클레오티드 서열이 또한 첨가될 수 있다. 따라서, 리더 또는 시그날 서열은 세포 벽을 통한 전달 및/또는 분비를 용이하게 하기 위해 발현 생성물내에 포함될 수 있다.
한가지 양태에 있어서, 동종성 또는 이종성 서열은 감쇠에 사용되는 선택된 뉴클레오티드 서열 또는 선택된 유전자내에 삽입되고; 유리하게는 동종 또는 이종 서열은 본원에서 확인된 트랜스포존 삽입 위치에 해당하는 위치중 하나에 삽입된다.
발현을 향상시키기 위해서, 코돈 사용이 사용되는 박테리아 벡터에 적응될 수 있다.
본 발명의 감쇠 돌연변이체는 또한 치료 단백질, 알레르겐, 성장 인자 또는 시토킨 또는 면역조절제 또는 면역자극제, 예를 들면 GM-CSF, 예컨대 표적 종에 조화되는 GM-CSF 를 코딩하는 핵산 서열을 포함한다 (예를 들어, 감쇠 벡터가 소에 투여하기 위한 파스튜렐라 멀토시다인 경우, 소 GM-CSF 는, 예를 들면 또다른 이종 단백질, 펩티드, 폴리펩티드, 항원, 면역원 또는 에피토프의 벡터에 의한 발현과 함께, 벡터에 의해서 발현될 수 있다).
본 발명의 또다른 양태에 따르면, 감쇠 미생물은 살아있는 감쇠 면역원 조성물 또는 살아있는 감쇠 백신 조성물을 제조하는데 사용된다.
본 발명의 유리한 양태에 따르면, 감쇠 미생물은 본 발명에 따라서 돌연변이 된, 그램 음성 박테리아, 예를 들면 파스튜렐라 (Pasteurella), 예컨대 파스튜렐라 멀토시다 (P. multocida), 예를 들어 P-1059 또는 PM70 이다.
유리하게는 본원에 기재된 바와 같이, 미생물은 Pasteurella 이외의 다른 작용제에 의해 야기되는 감염에 대해서 동물 또는 사람을 면역화 및/또는 백신화하기 위한 재조합 벡터로서 작용할 수 있다.
면역원 조성물 또는 백신 조성물은 감쇠 돌연변이체 및 약학적 또는 수의학적으로 허용 가능한 담체, 부형제, 희석제 또는 전색제, 및 임의로 안정화제 및/또는 보조제를 포함한다. 감쇠 돌연변이체는 이종성 에피토프, 항원, 면역원, 및/또는 성장 인자, 시토킨, 면역조절제 또는 면역자극제와 같은, 벡터에 이종성인 핵산 분자를 추가로 발현하는 벡터일 수 있다.
용어 "면역원 조성물" 은 본원에서 동물 또는 사람에 주입되면, 대상 병원균에 대해 면역 응답을 도출할 수 있는 임의의 조성물을 포함한다. 용어 "백신 조성물" 또는 "백신" 은 본원에서 동물 또는 사람에 주입되면, 대상 병원균에 대해 보호 면역 응답을 유도할 수 있는 임의의 조성물을 포함한다.
약제학적으로 또는 수의학적으로 허용 가능한 부형제는 물 또는 염수일 수 있으나, 예를 들면 또한 박테리아 배지를 포함할 수 있다.
본 발명에 따른 살아있는 감쇠 박테리아는 유리하게는 안정화제로 냉동 건조될 수 있다. 냉동 건조는 충분히 공지된 표준 냉동 건조 과정에 따라서 실시될 수 있다. 약학적 또는 수의학적으로 허용 가능한 안정화제는 탄수화물 (예를 들어, 소르비톨, 만니톨, 락토오스, 수크로오스, 글루코오스, 덱스트란, 트레할로오 스), 나트륨 글루타메이트 (Tsvetkov T et al., Cryobiology 1983, 20(3):318-23; Israeli E et al., Cryobiology 1993, 30(5):519-23), 단백질, 예를 들어 펩톤, 알부민, 락트알부민 또는 카세인, 단백질 함유 시약, 예를 들어 탈지 우유 (Mills CK et al., Cryobiology 1988, 25(2):148-52; Wolff E et al., Cryobiology 1990, 27(5):569-75, 및 완충제 (예를 들어, 포스페이트 완충제, 알칼리 금속 포스페이트 완충제) 일 수 있다.
아쥬반트는 냉동 건조된 제제를 용해시키는데 사용될 수 있다.
아쥬반트의 예는 수중유, 수중유중수 에멀젼이며, 이는 광유 및/또는 식물성유 및 비이온성 계면활성제, 예컨대 블록 공중합체, Tween
, Span
을 기재로 한다. 다른 적합한 아쥬반트는, 예를 들어 비타민 E, 사포닌, 및 Carbopol
, 수산화알루미늄 또는 인산 알루미늄 ("Vaccine Design, The subunit and adjuvant approach", Pharmaceutical Biotechnology, vol. 6, Edited by Michael F. Powell and Mark J. Newman, 1995, Plenum Press New York) 이다.
살아있는 감쇠 박테리아는 글리세롤 함유 매질내에서 -70 ℃ 에서 보관될 수 있다.
임의로는, 면역원 조성물 또는 백신은 비활성화 또는 생 형태의 다른 병원성 미생물 또는 바이러스에서 선택되는 하나 이상의 면역원, 항원 또는 에피토프와 조합될 수 있다.
본 발명의 다른 양태는 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 및 93 으로 지정된 본 발명에 따른 뉴클레오티드 서열 또는 유전자, 유리하게는 서열 번호 1, 4, 5, 8, 11, 14, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 73, 77, 80, 83, 86, 89, 92, 95, 96, 97 로 지정된 것과 같은, 본 발명에 따른 뉴클레오티드 서열 또는 유전자이다.
본 발명의 다른 양태는 펩티드, 폴리펩티드 또는 단백질, 또는 더욱 통상적으로는 발현 생성물, 예를 들어 면역원, 항원 또는 에피토프의 발현 및 제조를 위한 본 발명의 뉴클레오티드 서열 또는 유전자의 사용이다. 한가지 양태에 있어서, 이들 뉴클레오티드 서열 또는 유전자에 의해 인코드된 폴리펩티드 또는 펩티드 또는 단백질은 면역원 조성물 또는 백신내에서 아단위 면역원 또는 항원 또는 에피토프로서 사용될 수 있다. 에피토프 결정 과정, 예를 들어 중복 펩티드 라이브러리의 생성 (Hemmer B. et al., Immunology Today, 1998,19(4),163-168), 펩스칸 (Pepscan) (Geysen H. M. et al., Proc. Nat. Acad. Sci. USA, 1984, 81(13), 3998-4002; Geysen H. M. et al., Proc. Nat. Acad. Sci. USA, 1985, 82(1), 178-182; Van der Zee R. et al., Eur. J. Immunol., 1989, 19(1), 43-47; Geysen H. M., Southeast Asian J. Trop. Med. Public Health, 1990, 21(4), 523-533; Multipin
Peptide Synthesis Kits de Chiron) 및 알고리즘 (De Groot A. et al., Nature Biotechnology, 1999, 17, 533-561) 이 과도한 실험없이 본 발명의 실시에 사용될 수 있다.
유리한 폴리펩티드는 서열 번호 3, 7, 10, 13, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 76, 79, 82, 85, 88, 91, 94 로 식별되는 아미노산 서열을 갖는 것, 또는 뉴클레오티드 서열 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 에 의해서 코딩된 것이다. 이들 폴리펩티드로부터의 에피토프가 또한 유리하게 사용될 수 있다.
본 발명은 또다른 박테리아, 예를 들면 그램 음성 박테리아, 유리하게는 파스튜렐라세애 (Pasteurellacaea) 패밀리 멤버, 예를 들어 파스튜렐라 멀토시다, 파스튜렐라 해몰리티카, 파스튜렐라 아나티페스티페르, 악티노바실러스 플레우로뉴모니애로부터의 동등한 폴리펩티드를 포함하며, 더욱 유리하게는 파스튜렐라 멀토시다의 다양한 균주중 임의의 하나의 게놈에서, 아미노산 서열이 서열 번호 3, 7, 10, 13, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 76, 79, 82, 85, 88, 91, 94 로 식별되는 아미노산 서열중 하나에 대해 약 70 % 이상, 약 75 % 이상, 약 80 % 이상, 약 85 % 이상, 약 90 % 이상, 약 95 % 이상, 및 약 96, 97, 98 또는 99 % 이상의 동일성을 갖는 동등한 폴리펩티드 및/또는 SEQ 를 갖는 상기 확인된 폴리펩티드와 동일한 생물학적 기능을 갖는 폴리펩티드가 포함된다. 동일성 확립 기준 또는 동일한 생물학적 기능은 상기에서 기재하였다.
본 발명은 또한 폴리펩티드의 10 개 이상, 20 개 이상, 예를 들면 30 개 이상, 유리하게는 50 개 이상, 더욱 유리하게는 70 개 이상의 아미노산의 사슬을 갖는 이들 폴리펩티드의 면역원 단편, 예를 들면 폴리펩티드의 10 개 이상의 인접 아미노산, 유리하게는 폴리펩티드의 20 개 이상의 인접 아미노산, 예를 들면 폴리펩티드의 30 개 이상, 더욱 유리하게는 50 개 이상의 인접 아미노산, 더욱 더 유리하 게는 폴리펩티드의 70 개 이상의 인접 아미노산을 함유하는 폴리펩티드의 단편을 포함한다. 물론, 단편은 전체 폴리펩티드보다 작다. 단편은 다른 폴리펩티드, 예를 들면 융합 폴리펩티드와 조합될 수 있다; 예를 들면, 본 발명의 폴리펩티드 또는 이의 단편은 또다른 부분 (또다른 폴리펩티드), 예를 들어 면역원성을 향상시키는 리포단백질 부분 또는 시그널 또는 리더 서열 부분과 같은 면역원성 촉진 부분 및/또는 분비 촉진 부분을 포함하는 융합 폴리펩티드의 일부일 수 있다. 따라서, 본 발명은 융합물로서, 예를 들면 융합 폴리펩티드의 일부로서, 예컨대 유리하게는 리포단백질 부분과 같은 면역원성 촉진 부분 및/또는 시그널 또는 리더 서열 부분과 같은 분비 촉진 부분을 포함하는 융합 폴리펩티드의 일부로서, 폴리펩티드, 단백질, 항원, 면역원 또는 에피토프 - 본원에서 동정된 서열 또는 이의 단편 또는 본 발명의 벡터에 이종성인 것 - 의 발현을 계획한다.
폴리펩티드 또는 단편은 유리하게는 시험관내 발현에 의해서 생성된다. 본 발명에 따른 뉴클레오티드 서열 (예를 들면, 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93) 또는 이의 단편은 프로모터, 리보솜 결합 부위 및 종결자와 같은 조절 구성원에 작동적으로 연결된 벡터에 삽입되어, 코돈을 개시하고 코돈을 종결한다. 유리한 벡터는 박테리아, 즉 대장균 (Escherichia coli) (Mahona F et al., Biochimie 1994, 46(1): 9-14 ; Watt M A et al., Cell Stress Chaperones 1997, 2(3): 180-90; Frey J Res. Microbiol. 1992, 143(3): 263-9) 에서 시험관내 발현에 유용한 플라스미드이다.
이들 폴리펩티드는 또한 화학적으로 합성될 수 있다 (Luo Y et al., Vaccine 1999, 17(7-8): 821-31).
따라서, 본 발명의 양태는 본 발명에 따른 하나 이상의 폴리펩티드 또는 단편 (서브유닛 면역원성 조성물 또는 백신) 또는 본원에 기재된 하나 이상의 생체내 발현 벡터 (생 재조합 면역원성 조성물 또는 백신), 및 약학적 또는 수의학적으로 허용 가능한 담체, 부형제, 희석제 또는 전색제, 및 임의의 보조제를 포함하는 면역원 조성물 또는 백신이다. 이러한 성분의 예는 생 백신과 관련해서 본원에 기재하였다.
또다른 양태에 있어서, 이들 뉴클레오티드 서열 또는 이들의 단편은 상기 뉴클레오티드 서열 또는 단편에 의해서 코딩되는 폴리펩티드를 숙주내에서 생체내 발현할 수 있는 생 재조합 면역원성 조성물 또는 백신을 생성하도록 재조합 백터에 삽입될 수 있다.
생체내 발현 벡터는 폴리뉴클레오티드 벡터 또는 플라스미드 (EP-A2-1001025; Chaudhuri P Res. Vet. Sci. 2001, 70(3), 255-6), 바이러스 (예컨대, 아데노바이러스, 폭스바이러스, 예를 들어 파울폭스 (US-A-5,174,993, US-A-5,505,941 및 US-A-5,766,599) 또는 카나리폭스 (US-A-5,756,103)) 또는 박테리아, 즉 Escherichia coli 또는 Salmonella sp. 일 수 있다.
본 발명의 폴리펩티드 및 단편은 또한 치료에 사용될 수 있다.
폴리펩티드 및 단편은 또한 항체-항원 반응에서 시약으로서 사용될 수 있다. 따라서, 본 발명의 또다른 양태는 그램 음성 박테리아에 의한 감염 검출용 진단 방 법 및/또는 키트이다. 키트, 예를 들어 ELISA 는 본 발명에 따른 하나 이상의 폴리펩티드 또는 단편 (예를 들면, 본원에서 서열에 의해 확인된 하나 이상의 폴리펩티드 또는 본원에서 논의된 이의 단편) 을 포함할 수 있다.
본원에서 폴리펩티드 또는 단편 (본원에서 서열에 의해 확인된 폴리펩티드 또는 본원에서 논의된 이의 단편) 에 대한 항체는 진단 시약으로서, 또는 수동 면역화 또는 백신화에, 또는 치료에 사용될 수 있다. 수동 면역화에 투여되는 항체의 양은 당업계에서 사용되는 양과 동일 또는 비슷할 수 있으며, 따라서 당업계의 지식으로부터, 당업자는 과도한 실험없이 수동 면역화를 실시할 수 있다.
본 발명의 또다른 양태는 본 발명에 따른 폴리펩티드 또는 단편에 특이적인 항체를 포함하는 항체 제조 및 이것을 이용한 진단법이다. 폴리펩티드에 특이적인 항체에 관해서, 이것은 항체가 바람직하게는 폴리펩티드에 결합하는 것, 예를 들면 항체가 폴리펩티드에 결합하고 다른 폴리펩티드에는 결합하지 않거나, 또는 폴리펩티드에 허용 가능하게 특별한 폴리펩티드에 대한 특이성을 가짐으로써, Sambrook 의 상기 문헌을 포함한, 당업계에 공지된 또는 본원에서 인용된 문헌에 논의된 기술을 사용하여, 항체가 샘플로부터 폴리펩티드를 분리하거나, 샘플내 그의 존재를 5 % 이하의 거짓 포지티브로 검출할 수 있도록 하는 것을 의미한다.
항체는 폴리클로날 또는 모노클로날일 수 있다.
항체의 제조 방법은 당업자에게 널리 알려져 있다.
폴리클로날 항체가 요구되는 경우, 선택된 동물 (예, 쥐, 토끼, 염소, 말 등) 은 폴리펩티드 또는 단편에 의해서 면역시킨다. 면역된 동물로부터 혈청을 수 집하여, 공지의 과정에 따라 처리하고, 가능하게는 정제한다. 예를 들어, Jurgens et al. J. Chrom., 1985, 348: 363-370 참조.
하이브리도마 기술을 이용한 모노클로날 항체의 일반적인 제조 방법론은 충분히 알려져 있다. 불멸 항체 생성 세포 라인은 세포 융합에 의해서, 그리고 또한 종양 유전자 DNA 로의 B 림프구의 직접 형질전환, 또는 Epstein-Barr 바이러스로의 트랜스펙션과 같은 다른 기술에 의해서 생성될 수 있다. 예를 들어, J. E. Liddell "A practical guide to monoclonal antibodies" ed. John Wiley and sons, 1991, p. 188; S. J. de StGroth et al. J. Immunol. Methods, 1980, 35(1-2), 1-21 참조.
본 발명에 따른 뉴클레오티드 서열 및 이들의 단편은, 예를 들어 진단 방법에서 혼성화용 프로브로서 사용될 수 있다.
엄격한 혼성화 조건이 유리하게 사용된다. Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104 에 의해 기재된 것을 언급할 수 있다. 엄격한 조건하에서의 혼성화는 포지티브 혼성화 시그널이, 1 ×SSC 완충액 및 0.1 % SDS 로 55 ℃, 유리하게는 62 ℃, 더욱 유리하게는 68 ℃ 에서 1 시간 세정, 예를 들면 0.2 ×SSC 완충액 및 0.1 % SDS 중에서 55 ℃, 예컨대 62 ℃, 유리하게는 68 ℃ 에서 1 시간 세정후에도 여전히 관찰됨을 의미한다.
또한, 엄격한 혼성화 조건 하에서 결합하는 능력에 의해 뉴클레오티드 서열의 특성을 나타낼 수 있다. 따라서, 본 발명은 엄격한 혼성화 조건 하에서 이에 결합하는, 본 명세서에서 확인된 핵산 서열 및 핵산 분자를 기대할 수 있다.
본 발명에 따른 뉴클레오티드 서열 및 이의 단편은, 예를 들어 모든 매질, 예를 들어 조직 샘플, 생물학적 유체, 물, 식품 중의 그람 음성 박테리아의 검출을 위하여, PCR을 위한 또는 증폭 및/또는 혼성화를 포함하는 유사한 방법에서의 프라이머로서 사용될 수 있다.
유리하게는, 본 발명에 따른 뉴클레오티드 서열 또는 유전자, 예를 들어, SEQ ID NO: 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 의 20 개 이상의 인접한 핵산, 예컨대 30개 이상의 인접한 핵산, 예를 들어 50 개 이상의 인접한 핵산, 예를 들어 70 개 이상의 인접한 핵산 또는 더 유리하게는 100 개 이상의 인접한 핵산이 사용될 수 있다.
또한, 본 발명은 동물, 유리하게는 박테리아에 감염되기 쉬운 동물, 예를 들어, 조류, 토끼, 소 및 돼지 종, 및 더 유리하게는 조류 종, 예를 들어 닭, 칠면조 및 오리(브리더(breeders), 브로일러(broilers) 및 레이어(layers)를 포함) 또는 인간에 있어서 박테리아 감염에 대해 면역성을 부여하거나 이를 예방하기 위해서, 또는 박테리아 감염을 막기 위한 방법에 관한 것이다.
상기 방법에 따라서, (1) 본 발명의 살아있는 감쇠된 면역원성 조성물 또는 백신, 또는 (2) 본 발명의 서브 유닛 면역원성 조성물 또는 백신, 또는 (3) 본 발명의 살아있는 재조합 면역원성 조성물 또는 백신, 또는 이의 배합물이 투여된다. 물론, 본 발명의 구현은 본 발명이 아닌 다른 백신 또는 면역원성 조성물과 함께 사용될 수 있고, 예를 들어 프라임-부스트 방법 (prime-boost process)에서, 본 발명의 백신 또는 면역원성 조성물이 먼저 투여되고 상이한 백신 또는 면역원성 조성물이 그 이후에 투여되거나, 또는 반대의 순서로 투여된다.
특히 근육내(IM), 진피내(ID) 또는 피하(SC) 주사에 의해, 또는 비강내, 기관내 또는 경구 투여를 통해 투여될 수 있다. 본 발명에 따른 면역원성 조성물 또는 백신은 유리하게는 주사기, 무침 장치 (예를 들어, Pigjet, Avijet, Dermojet 또는 Biojector (Bioject, Oregon, USA)), 스프레이, 음용수, 점안제(eye-drop)에 의해 투여된다.
살아있는 감쇠된 면역원성 조성물 또는 백신의 유리한 투여는 구강(예를 들어 음용수, 전신 스프레이), 눈(예를 들어 점안제, 전신 스프레이), 기관내(예를 들어 스프레이), 진피내, 피하(SC) 또는 기관내(IM) 경로를 통한 in ovo 투여이다.
살아있는 감쇠된 미생물의 양은 숙련자에 의해, 과도한 실험 없이, 본 개시 및 종래 기술의 지식으로부터 결정 및 최적화될 수 있다. 일반적으로 동물(사람 포함)은 단일 제형 단위에서, 대략 104 내지 109 CFU, 유리하게는 대략 105 내지 108 CFU 및 더 유리하게는 대략 106 내지 107 CFU가 투여될 수 있다.
근육내 경로에 의해서, 조류 동물은 단일 제형 단위에서, 대략 104 내지 107 CFU, 유리하게는 대략 105 내지 106 CFU가 투여될 수 있다. 단일 제형 단위의 부 피는 약 0.2 ml 내지 약 0.5 ml일 수 있고 유리하게는 약 0.3 ml이다. 구강, 기관내 또는 눈 경로에 의해, 조류 동물은 단일 제형 단위에서, 대략 105 내지 108 CFU, 유리하게는 대략 106 내지 107 CFU가 투여될 수 있다. 스프레이 투여에 대하여, 부피는 장치 및 물방울의 크기에 맞추어지고, 약 1000 마리 동물에 대하여 약 30 내지 약 600 ml이고 유리하게는 동물 당 약 0.2 ml이다.
소 및 돼지 동물에 대하여, 유리한 경로는 IM 및 SC이다. 상기 동물은 단일 제형 단위에서, 대략 104 내지 109 CFU, 유리하게는 대략 105 내지 10
8 CFU가 투여될 수 있다. 단일 제형 단위의 부피는 약 0.2 ml 내지 약 5.0 ml이고 유리하게는 약 0.5 ml 내지 약 2.0 ml이며 더 유리하게는 1.0 ml이다.
토끼는 IM 또는 SC 경로를 통해, 단일 제형 단위에서, 대략 104 내지 108 CFU, 유리하게는 대략 105 내지 107 CFU가 투여될 수 있다. 단일 제형 단위의 부피는 약 0.2 ml 내지 약 0.5 ml이고 유리하게는 약 0.5 ml이다. 이들은 또한 ID 경로를 통해 단일 제형 단위에서, 대략 104 내지 108 CFU, 유리하게는 105 내지 107 CFU가 투여된다. 단일 제형 단위의 부피는 약 0.1 ml 내지 약 0.2 ml일 수 있다.
본 발명은 이제 하기의 비제한적인 실시예에 의해 추가로 기술될 것이다.
실시예 1: 시그너쳐(signature) 태그된
P. multocida
트랜스포존 돌연변이체 의 라이브러리의 구축(STM 스크리닝)
태그된 SM10λpir pLOF/km 형질전환주의 구축
태그를 [Hensel et. al., (Science, 1995, 269:400-403)]에 기술된 바와 같이 제조하였다. 먼저, 혼성화를 잘 하지만 서로 크로스-혼성화 하지 않는 태그를 포함하는, 태그된-pUTminiTn5Km2 플라스미드를 선별하였다. 태그된 pUTminiTn5Km2 벡터 내의 mini-Tn5 트랜스포존은 몇몇의 파스튜렐라 멀토시다 균주에서 교차하지 않는 것으로 발견되었다. 반대로 트랜스포존 mini-Tn10은 파스튜렐라 멀토시다 (P. multocida) 에서 기능하는 것으로 보여졌다(Lee et. al., Vet Microbiol, 1996, 50:143-8). 따라서, 미리 선별된 태그를 pUTminiTn5 벡터로부터 플라스미드 pLOF/Km를 포함하는 mini-Tn10로 이동시켰다 (Herrero et al., J. Bacteriology, 1990, 172: 6557-6567). 태그를, 이 태그가 클로닝되는, pUTn-miniTn5Km2 벡터, KpnI 부위의 어느 한 쪽에 결합하는 프라이머를 이용하여 PCR에 의해 증폭하였다. 상기 프라이머는 제한 효소 SalI에 대한 서열을 포함하였다. 상기 PCR 산물을 SalI로 절단하고, SalI 부위가 유일한 SfiI 클로닝 부위로 대체되는, pLOF/Km 벡터의 개질된 버전의 SalI 부위로 클로닝하였다. 태그된 pLOF/Km 플라스미드를 그 후 E. coli 균주 SM10λpir (Kmr, thi, thr, leu, tonA, lacY, supE, recA::RP4-2-Tc::Muλpir)으로 형질전환하였다(Miller V.L. et al., J. Bacteriol., 1988, 170: 2575-83). 상기 균주는 접합에 의해서 수용자 박테리아 내로 pLOF/Km 와 같은 플라스미드를 동원할 수 있다.
선별 및 반-선별 (counter selection) 의 방법
접합 방법은 플라스미드 pLOF/KM 및 E. coli SM10λpir 공여자 박테리아에 대한 반-선별 방법을 수득한, 수용자 박테리아 P. multocida에 대한 선별 방법을 요구한다.
상기 선별 방법에서, 수용자 박테리아 파스튜렐라 멀토시다의 선별 방법은 mini-Tn10 트랜스포존에 의해 코딩되는, 카나마이신 사용에 대해 선된다.
먼저 접합시 E. coli 공여자 박테리아에 대한 반-선별을 위하여, 자발적 스트렙토마이신 내성 돌연변이체인 파스튜렐라 균주 P-1059을 사용하였다. 그러나, 상기 균주는 칠면조에 대하여 발병력이 감쇠하는 것으로 보였고, 따라서 여기서 사용될 수 없었다. 파스튜렐라 멀토시다 균주는 화학 합성배지(CDM)에서 증식할 수 있다 (Hu et. al., Infection and Immunity 1986, 804-810). 한천을 포함하지만 류신을 포함하지 않는 합성배지의 개질된 버전이 E. coli 공여자 박테리아에 대한 상대-선별 가능하게 하기 위하여 사용되었다. 파스튜렐라 균주는 상기 배지에서 증식할 수 있었고, 이의 조성은 표 1에서 제시되며, 반면 류신 영양요구주인 E. coli SM10λpir은 증식할 수 없었다.
| 성분 | 농도 g/리터 |
| 노블 한천(noble agar) | 20 |
| Na2HP04.12H20 | 32.31 |
| KH2PO4 | 1.368 |
| NaCl | 1.196 |
| 글루코즈 | 6.0 |
| L-아르기닌 히드로클로라이드 | 0.24 |
| L-시스테인 히드로클로라이드 | 0.12 |
| L-세린 | 0.2 |
| L-글루타민산 | 0.15 |
| L-이소루신 | 0.064 |
| L-페닐알라닌 | 0.095 |
| L-아스파르트산 | 1.6 |
| L-티로신 | 0.08 |
| 티아민 히드로클로라이드 | 0.0002 |
| MgSO4.7H20 | 0.246 |
| 판토텐산칼슘 | 0.004 |
| 니코틴아미드 | 0.01 |
| 오르토산 | 0.003 |
P. multocida
균주의 CDM 배지 상의 계대
P. multocida 균주의 동결건조된 앰플 (USDA P-1059, American Type Culture Collection 사 제, 접근 번호 ATCC 15742)을 200 ㎕의 BHI(뇌-심장 융합물)의 첨가에 의해 소생시켰고, 현탁액의 분취물을 BHI 한천 플레이트 상에 도말(streak)하였으며, 플레이트를 37℃에서 밤새 인큐베이션하였다. 상기 플레이트로부터의 콜로니 물질을 사용하여 BHI 브로쓰 배양물로 접종하였으며, 이를 37℃에서 밤새 진탕하여 인큐베이션하였다. 글리세롤을 첨가하여 15% v/v의 최종 농도가 되게 하였으며, 분취물을 -80℃에서 동결 저장하였다. 상기 동결된 분취물 중의 하나로부터의 샘플을 BHI 한천 플레이트 상에 도말하었으며, 밤새 인큐베이션하였다. 그 후, 상기 BHI 플레이트로부터의 콜로니 물질을 표 1에 제시된 조성을 가진 CDM 한천 플레이트 상에 도말하였으며, 37℃에서 3일동안 인큐베이션하였다. 상기 CDM 플레이트로부터의 콜로니 물질을 BHI 브로쓰 배양물 내로 인큐베이션하였으며, 37 ℃에서 밤새 진탕하여 인큐베이션하였다. 글리세롤을 상기 배양물 내로 첨가하여 15% v/v의 최종 농도가 되게 하였고, 분취물을 -80℃에서 동결하였다. 상기 균주를 16084 (CDM)로 명명하였다.
돌연변이체 은행의 구축
태그된 SM10λpir pLOF/km 형질전환주를 16084 (CDM) P. multocida 균주와 접합하였다. 동기 돌연변이체(접합 과정시 돌연변이체의 복제로 인하여 발생하는, 동일한 위치에 있는 트랜스포존을 가지는 돌연변이체)의 분리를 최소화하기 위하여, 각 태그된 SM10λpir 형질전환주를 3개 이상의 분리된 접합 부위에서 파스튜렐라 멀토시다 균주와 접합하였다. 파스튜렐라 (Pasteurella) 트랜스포존 균주를 50 ㎍/ml 카나마이신으로 보충된 CDM 한천 플레이트 상에서 선별하였다.
각각의 태그된 트랜스포존에 대한 카나마이신 내성 돌연변이체를, BHI 카나마이신 50 ㎍/ml 한천 플레이트 상에서 두 번 도말하여 단일 콜로니를 형성하였다. 그 후, 단일 콜로니를 BHI 브로쓰 배양물 내로 인큐베이션하였고, 37℃에서 밤새 진탕하여 인큐베이션하였다. 그 후, 글리세롤을 첨가하여 15 % v/v의 최종 농도가 되게 하였고 돌연변이체를 각 바이알에서 -80℃로 저장하였다.
실시예 2: 칠면조에 대한 발병력이 감쇠된 돌연변이체에 대한, 시그너쳐-태그된 파스튜렐라 (Pasteurella) 돌연변이체 은행의 스크리닝
실시예 1에서 수득된, 20 ㎕의 각각의 글리세롤 보존 돌연변이체를 50 ㎍/ml의 카나마이신이 보충된, 200 ㎕의 BHI 배양 배지와 혼합함에 의해, 칠면조의 접종을 위한 P. multocida 돌연변이체의 배양물을 증식하였고, 96 웰 마이크로타이터 디쉬에 위치시켰다. 상기 마이크로타이터 디쉬를 37℃에서 약 18 시간동안 정적 조건에서 인큐베이션하였다. 그 후 상기 돌연변이체의 18 시간 배양된 10 ㎕ 분취물을, 신선한 마이크로타이터 플레이트 내에서 50 ㎍/ml의 카나마이신으로 보충된 200 ㎕의 BHI 배양 배지와 혼합하였고, 상기 플레이트를 대략 4시간동안 37℃에서 인큐베이션하였다. 상기 배양을 대수증식기에서 중단하였고, 각 돌연변이체의 100 ㎕ 배양물을 신선한 마이크로타이터 플레이트로 이동시켰으며, 650 nm에서의 흡광도(OD)의 측정을 위해 사용하였다.
접종물 또는 도입 풀(input pool)을, 남아있는 100 ㎕의 4시간 배양물을 혼합함에 의해 형성하였다. 각각의 도입 풀은 48개의 상이한 돌연변이체로 이루어져 있다. 상기 풀링된 현탁액의 타이터를 100 ㎕ 분취물의 FACS (형광표지세포분리기) 분석에 의해 측정하였다. 풀링된 현탁액의 분취물(1 ml)을 그 후 생리학적 완충수에서 희석하여, 2.107cfu/ml의 타이터를 가지는 현탁액을 수득하였다. 그 후, 5 마리의 3주령 칠면조의 군에 상기 현탁액의 0.5ml 분취물(한 마리 당 107cfu)을 근육내 접종하였다. 접종 전 칠면조의 혈청 상태를, 접종 하루 전 채취된 혈액 샘플 내의 Pasteurella 에 대한 항체 존재에 대한 검사에 의해 측정하였다. 나머지의 도입 풀로부터의 세포를 원심분리에 의해 수득하였고 세포 펠렛으로부터 염색체 DNA를 추출하였다.
접종 후 대략 14시간에, 1 ml의 혈액 샘플을 5 마리 칠면조 중 3 마리로부터 채취하였다. 혈액 샘플의 일련의 희석물(10-1 내지 10-7)을, 5% 양(sheep) 혈액으 로 보충된 Columbia 한천 플레이트 상에 위치시켰다. 상기 플레이트를 24시간 동안 37℃에서 인큐베이션하였고, 그 이후 대략 10000 Pasteurella 콜로니를 BHI 배지에 재현탁하였다. 도출 풀(output pool)이라고 명명되는 상기 현탁액을, 그 후 원심분리하고 염색체 DNA를 세포 펠렛으로부터 추출하였다.
도입 풀에는 존재하지만 칠면조로부터 재분리되지 않은 Pasteurella 돌연변이체를, 문헌 [Hensel et al., Science 1995, 269:400-403]에 기술된 바와 같이, 도입 및 도출 풀로부터의 DNA 샘플에 존재하는 시그너쳐 태그의 PCR 증폭, 및 시그너쳐 태그를 암호화하는 DNA로 로드된 도트 블롯(dot blot)에 대한, 증폭된 PCR 산물의 혼성화에 의해 확인하였다. 상기 돌연변이체는 발병력이 잠재적으로 감쇠된 것으로 생각되었다. 이러한 감쇠는 칠면조의 잠재적 돌연변이체의 단일 감염 이후에 치사율의 감소에 대한 스크리닝에 의해 확인된다.
실시예 3 : 파스튜렐라 멀토시다 돌연변이체의 칠면조에 대한 발병력 감쇠의 확인
글리세롤 원액 20 ㎕ 와 카나미아신 50 ㎍/mL로 보충된 BHI 배양 배지 200 ㎕ 를 마이크로타이터 접시 내에서 혼합함으로써, 실시예 2에서 잠재적으로 감쇠된 것으로 확인된 트랜스포존 돌연변이체 또는 제한된 배양 성장 능력을 갖는 돌연변이를 소생시켰다. 상기 마이크로타이터 접시를 37℃에서 18 시간 동안 정적 조건에서 배양시켰다. 그 후, 상기 배양물의 각 돌연변이체 10 ㎕ 분취량을 취하여 새로운 마이크로타이터플레이트 내에서카나미아신 50㎍/mL 이 보충된 BHI 배지 200 ㎕ 와 혼합시켜, 상기 플레이트를 약 4 시간 동안 정적 조건에서 배양하였다. 상기 배양물을 성장의 대수증식기(exponential phase)에서 정지시키고 각 돌연변이체의 배양물 100 ㎕를 새로운 마이크로타이터 플레이트로 이동시켜 650 nm에서 광학밀도(OD)의 측정을 위해 사용하였다. 이어서, 상기 돌연변이체 각각의 배양물을 생리적 완충수 내에서 10000 분의 1로 희석시켜 약 2.104 cfu/mL의 농도를 수득하였다. 그 후, 상기 희석액의 분취량 (0.5 mL)을 2 마리의 5주령 칠면조에게 근육 내로 접종하였다 (동물 당 104 cfu). 칠면조의 각 군의 몇몇의 동물의 혈청 상태를 접종 전에 취한 혈액 시료로부터 측정하였다. 상기 칠면조에 대하여 그 다음 7일 동안 치사율을 관측하였다. 상기 시험된 돌연변이체들 중 72 개가 접종된 두 조류의 어느 것에서도 사망률을 나타내지 않았다. 상기 72 개 돌연변이체는 발병력이 감쇠된 것으로 간주되었다.
실시예 4: 칠면조에서 스크리닝 후에 확인된 트랜스포존 삽입 돌연변이체의 특성분석
감쇠된 P.멀토시다 돌연변이체의 게놈에서 트랜스포존 삽입 부위는, 역방향 PCR 또는 임의적 프라이밍 PCR (arbitrarily primed PCR)에 의해 트랜스포존 삽입의 한 측면의 DNA를 클로닝함으로써 확인되었다.
상기 돌연변이체는, BHI 카나미아신 50 ㎍/mL 한천 플레이트 상으로 분취량을 도말(streaking)함으로써 -80℃ 글리세롤 원액으로부터 소생되었다. 그 후, 단일 콜로니들을 사용하여 염색체 DNA가 제조되는 BHI 브로쓰 배양물을 접종하였다.
역방향 PCR를 위하여, 4 염기쌍 인식 부위, 예컨대 Tsp509I, αTaqI 또는 RsaI를갖는 제한효소로 염색체 DNA를 절단했다. 이어서 상기 DNA를 큰 부피로 라이게이션시켜 분자내 라이게이션을 촉진시켰다. 그 후, 상기 트랜스포존 주변의 DNA 를, 트랜스포존의 공지의 서열에 결합하는 바깥쪽으로 향하는 프라이머, 예컨대 StipJ (서열 번호: 98,20 머) (5'ATC TGA TCC TTC AAC TCA GC 3'), StipA (서열 번호: 99,19 머) (5'CGC AGG GCT TTA TTG ATT C 3'), KTGRI (서열 번호: 100,27 머) (5'GCG GAA TTC GAT GAA TGT TCC GTT GCG 3'), Tn10IR1 (서열 번호: 101,20 머) (5'TTT ACC AAA ATC ATT AGG GG 3') 및 Tn10IR4 (서열 번호: 102,19 머) (5'GAT CAT ATG ACA AGA TGT G 3')를 사용하여 상기 결찰된 DNA 주형으로부터 확장시켰다. 이어서 상기 역방향 PCR 생성물을 클로닝하고 서열을 분석하였다.
임의적 프라이밍 PCR을 위하여, 트랜스포존에 결합하는 하나의 바깥쪽으로 향하는 프라이머, 예컨대 StipA, 및 임의 프라이머, 예컨대 arb1 (서열 번호: 103, 35 머) (5'GGC CAC GCG TCG ACT AGT ACN NNN NNN NNN GAT AT 3') 또는 arb6 (서열 번호: 104, 35 머) (5'GGC CAC GCG TCG ACT AGT ACN NNN NNN NNN CAG CC 3')와의 제 1 회 PCR 반응에서 염색체 DNA를 주형으로서 사용하였다. 상기 제 1 회 PCR반응의 결합 온도는 초기에 매우 낮게 설정되고, 연속되는 사이클에서 결합 온도를 승온시킨다. 이어서, 상기 제 1 회 PCR의 생성물 부분을 제 2 회 PCR에서 주형으로서 사용하였다. 상기 제 2 회 PCR은 또 다른 바깥쪽을 향하는 프라이머, 예컨대 KTGRI (상기 제 1 회 PCR에서 사용되는 프라이머보다 트랜스포존의 말단에 더 가까운 위치에서 트랜스포존에 어닐링함)를 사용한다. 상기 제 2 회 PCR에서 사 용되는 다른 프라이머는 상기 제 1 회 PCR에서 사용되는 임의 프라이머, 예컨대 arb2 (서열 번호: 105,20 머)(5'GGC CAC GCG TCG ACT AGT AC 3')의 5' 말단에서 공지 서열의 20 개 염기들과 동일한 서열을 갖는다. 이어서 상기 제 2 회 PCR의 PCR 생성물을 클로닝하고 서열을 분석하였다.
그 후 상기 수득된 서열은 트랜스포존에 의해 파괴될 수 있는 오픈 리딩 프레임 (open reading frames: ORF) 을 확인하기 위해 분석되었고, 또한 EMBL 데이터베이스 및 University of Minnesota [May BJ 등, Proc. Natl. Acad. Sci. USA, 2001,98 (6): 3460-5]에 의해 측정된 파스튜렐라 멀토시다 균주 PM70의 게놈 서열에서 현재 이용가능한 유사 서열을 검색하기 위해 사용되었다.
참고로 하기 뉴클레오티드 서열에서, N은 임의 핵산 (A 또는 C 또는 G 또는 T)에 대응한다.
돌연변이체 1G4, 3F4, 3G12, 12D6 및 14C10
돌연변이체 1G4, 3F4 및 12D6는 정확하게 동일한 트랜스포존 삽입 부위를 갖는다. 상기 트랜스포존 삽입 부위 측면의 DNA 서열은 서열 번호: 1 (775 머)로 나타낸다. 상기 트랜스포존은 상기 서열의 5' 말단에서 즉시 삽입된다.
개시 코돈은 서열 서열 번호: 1 서열의 위치 179-181에 위치한다.
돌연변이체 3G12에 대하여, 상기 트랜스포존 삽입 부위를 플랭킹하는 DNA서열은 서열 번호: 95 (101 머)로 주어진다. 상기 트랜스포존은 상기 서열의 5' 말단에서 즉시 삽입된다.
돌연변이체 14C10에 대하여, 상기 트랜스포존 삽입 부위를 플랭킹하는 DNA서 열은 서열 번호: 96 (220 머)로 주어진다. 상기 트랜스포존은 상기 서열의 3' 말단에서 즉시 삽입된다.
4 개의 다른 파스튜렐라 단백질 및 유전자는 서열 번호: 1로 코딩되는 60 개 아미노산 서열로 블라스트 (blast) 를 수행하여 확인되었다.
돌연변이체 1G4, 3F4 및 12D6에서 트랜스포존의 위치는 Genbank 접근 번호 AE006115 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 8507-8508 에 해당된다. 돌연변이체 3G12에서 트랜스포존의 위치는 상기 AE006115 서열의 위치 8609-8610 에 해당된다. 돌연변이체 14C10 에서 트랜스포존의 위치는 상기 AE006115 서열의 위치 8517-8518 에 해당된다. 트랜스포존은 PM70 유전자 PM0773, PhyA 의 상동성을 손상시킨다. 상기 PhyA 유전자는 캡슐 합성에 수반되는 것으로 예측된다. PM0773 의 뉴클레오티드 서열은 본원에서 서열 번호 2 로 식별되며, 그의 아미노산 서열은 서열 번호 3 로 식별된다.
돌연변이체 1G8 9D1 및 9D8
돌연변이체 1G8에서, 트랜스포존은 서열 서열 번호: 4 (226 머)의 3' 말단에서 즉시 삽입된다. 상기 서열은 잠재적인 더 긴 단백질을 코딩하는 2 개의 오픈 리딩 프레임 (+2 및 -2) 을 갖는다. 본 발명에 따른 상기 ORF는 프레임 -2 내에 있다.
돌연변이체 9D1에서 삽입되는 트랜스포존은 서열 서열 번호: 5 (87 머)의 3' 말단에서 즉시 삽입된다.
돌연변이체 9D8에서 삽입되는 트랜스포존은 서열 서열 번호 : 4의 위치 225 다음에서 삽입된다.
돌연변이체 1G8, 9D1 및 9D8에서 트랜스포존은 PM70 유전자, PM0871 의 상동성을 파괴한다. 상기 돌연변이체에서 트랜스포존의 위치는 Genbank 접근 번호 AE006125 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 9849-9850 (돌연변이체 1G8), 8899-8900 (돌연변이체 9D1) 또는 9848-9849 (돌연변이체 9D8) 에 해당된다. PM0871 의 뉴클레오티드 서열은 본원에서 서열 번호 6 로 식별되며, 그의 아미노산 서열은 서열 번호 7 로 식별된다.
또다른 파스튜렐라세애 유전자/단백질은 서열 번호 7 로 블라스트를 수행하여동정했다. 상기는 해모필루스 인플루엔자 (Haemophilus influenzae) HI1586 (Genbank 접근 번호 U32832 및 AAC23234)이다. 본 출원인은 PM0871과 HI1586 간의 507 개 아미노산에 대하여 72% 의 동일성을 발견하였다.
돌연변이체 2F2
상기 트랜스포존 삽입 부위 측면의 DNA 서열은 서열 번호 8 (78 머)로 나타낸다. 상기 트랜스포존은 상기 서열의 5' 말단 바로 곁에 존재한다. 상기 서열은 잠재적인 더 긴 단백질을 코딩하는 3 개의 오픈 리딩 프레임 (+1, +2 및 -1) 을 갖는다. 본 발명에 따른 상기 ORF는 프레임 +2 내에 있다.
돌연변이체 2F2에서 트랜스포존은 PM70 유전자, PM1727의 상동성을 손상시킨다. 상기 트랜스포존은 Genbank 접근 번호 AE006210 (PM1727) 인 파스튜렐라 멀토시다 PM70 서열의 위치 644-645 에 해당하는 위치에 위치한다. PM1727의 뉴클레오티드 서열은 본원에서 서열 번호 9 로 식별되며, 그의 아미노산 서열은 서열 번호 10 로 식별된다.
또다른 파스튜렐라세애 유전자/단백질이 서열 번호 10 으로 블라스트를 수행하여 동정되었다. 상기는 해모필루스 인플루엔자 HI0621 (Genbank 접근 번호 U32744 및 AAC22281) 이다. 본 출원인은 PM1727과 HI0621 간의 183 개 아미노산에 대하여 77%의 동일성을 발견하였다.
PM1727은 가수분해효소의 슈퍼패밀리(superfamily)의 일원이며, 특히 히스티디놀 인산 포스파타제 (histidinol phosphate phosphatases)와 연관된다.
돌연변이체 3A2
상기 트랜스포존 삽입 부위 측면의 DNA 서열은 서열 번호 11 (467 머)로 나타낸다. 상기 트랜스포존은 상기 서열의 5' 말단 바로 곁에 위치한다.
정지 코돈은 위치 428-430에 위치한다.
돌연변이체 3A2 에서 트랜스포존은 Genbank 접근 번호 AE006094 (PM0586) 인 파스튜렐라 멀토시다 PM70 서열의 위치 5103-5104 에 해당하는 위치에 위치한다. 본원에서 PM0586의 뉴클레오티드 서열은 서열 번호 12 로서 식별되며, 그의 아미노산 서열은 서열 번호 13 로 식별된다.
두 개의 다른 파스튜렐라세 유전자 및 단백질이 서열 번호 13 로 블라스트를 수행하여 동정되었다. 상기 유전자 및 단백질은 파스튜렐라 해몰리티카 A1 PlpD (Genbank 접근 번호 AF058703 및 AAC32565) 및 해모필루스 솜너스 (Haemophilus somnus) 31 kDa (Genbank 접근 번호 L07795 및 AAA24941) 이다. 본 출원인은 PM0586과 P. 해몰리티카 PlpD 간의 276 개 아미노산에 대하여 73%, 및 PM0586과 H. 솜너스 31 kDa 간의 273 개 아미노산에 대하여 71% 동일성을 발견하였다.
PlpD 및 PM0586은 ompA 단백질 패밀리의 일원이다.
돌연변이체 3D3
상기 트랜스포존 삽입 부위 주변의 DNA 서열은 서열 번호 14 (204 머, 5' 말단에 트랜스포존) 및 서열 번호 15 (35 머, 5' 말단에 트랜스포존) 로 나타낸다.
정지 코돈은 서열 번호 14 의 위치 7-9 및 서열 번호 15의 위치 33-35 에 위치한다.
또다른 파스튜렐라세애 유전자/단백질이 서열 번호 14 및 그의 코딩된 아마노산 서열 (65 개 아미노산) 로 블라스트를 수행하여 동정되었다. 본 출원인은 PM0064 단백질과 65 개 아미노산에 대하여 100% 동일성을 발견하였다. 돌연변이체 3D3에서 트랜스포존의 위치는 Genbank 접근 번호 AE006042 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 4778-4779 또는 4787-4788, 각각 서열 번호: 14 및 15 으로부터 유래되는 위치에 해당한다. 이러한 차이는 트랜스포존 삽입 부위에서 수 개의 뉴클레오티드의 복제를 야기하는 트랜스포존의 삽입에 기인하는 것으로 보인다. 위치 4788은 PM0064 유전자 내에 위치한다. 위치 4778은 PM0064의 정지 코돈의 6 bp 하류(downstream)이다. 본원에서 PM0064의 뉴클레오티드 서열은 서열 번호 16 로 식별되며, 그의 아미노산 서열은 서열 번호 17 로 식별된다.
다른 그램 음성 박테리아 유전자 및 단백질은 서열 번호 17 로 수행된 블라스트로 동정되었다.
| 균주 | 유전자 (Genbank ref.) | 단백질 (Genbank ref.) | 동일성% |
| 헤모필러스 인플루엔자 (Haemophilus influenzae) | HI0017 (U32687) | HI0017 (AAC21695) | 127 개 아미노산에 걸쳐 81% |
| 대장균 (Escherichia coli) K12 | YfiD (AE000344) | yfiD (AAC75632) | 127 개 아미노산에 걸쳐 81% |
| 살모넬라 타이피 (Salmonella typhi) | STY2839 (AL627275) | yfiD (CAD02795) | 127 개 아미노산에 걸쳐 81% |
| 살모넬라 타이피뮤리움 (Salmonella typhimurium) | STM2646 (AE008820) | yfiD (AAL21540) | 127 개 아미노산에 걸쳐 81% |
| 세라티아 리쿠어파시엔스 (Serratia liquefaciens) | 0rfX (X66505) | 0rfX (CAA47136) | 127 개 아미노산에 걸쳐 79% |
| 예르시니아 페스티스 (Yersinia pestis) | YPO2705 (AJ414153) | YPO2705 (CAC92944) | 127 개 아미노산에 걸쳐 79% |
| 비브리오 콜레라 (Vibrio cholerae) | VC2361 (AE004306) | VC2361 (AAF95504) | 127 개 아미노산에 걸쳐 73% |
돌연변이체 3D8
트랜스포존 (transposon) 삽입 부위 측면의 DNA 서열을 서열 번호 18 (75 머) 에 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다.
돌연변이체 3D8 중 트랜스포존의 위치는 Genbank 접근 번호 AE006080 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 7769-7770 사이에 해당한다. 트랜스포존은 PM70 유전자 PM0445 의 상동성을 손상시킨다. PM0445 의 뉴클레오티드 서열은 본원에서 서열 번호 19 로 식별되며, 그 아미노산 서열은 서열 번호 20 로 식별된다.
돌연변이체 3E1
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 21 (229 머) 에 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다.
돌연변이체 3E1 중 트랜스포존의 위치는 Genbank 접근 번호 AE006133 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 9195-9196 사이에 해당한다. 트랜스포존은 PM70 유전자 PM0940 의 상동성을 손상시킨다. PM0940 의 뉴클레오티드 서열은 본원에서 서열 번호 22 로 식별되며, 그 아미노산 서열은 서열 번호 23 으로 식별된다.
다른 그램 음성 박테리아 유전자 및 단백질은 서열 번호 17 로 수행된 블라스트로 동정되었다.
| 균주 | 유전자 (Genbank ref.) | 단백질 (Genbank ref.) | 동일성% |
| 헤모필러스 인플루엔자 | HI0075 (U32693) | nrdD (AAC21751) | 713 개 아미노산에 걸쳐 87% |
| 대장균 K12 | nrdD (AE000495) | nrdD (AAC77195) | 709 개 아미노산에 걸쳐 76% |
| 살모넬라 타이피 | STY4791 (AE627283) | nrdD (CAD06912) | 709 개 아미노산에 걸쳐 76% |
| 살모넬라 타이피뮤리움 | STM4452 (AE008908) | nrdD (AAL23272) | 709 개 아미노산에 걸쳐 76% |
| 예르시니아 페스티스 | YPO3464 (AJ414157) | nrdD (CAC92683) | 709 개 아미노산에 걸쳐 75% |
| 비브리오 콜레라 | VCA0511 (AE004381) | VCA0511 (AAF96414) | 709 개 아미노산에 걸쳐 74% |
돌연변이체 3H2
트랜스포존 (transposon) 삽입 부위 측면의 DNA 서열을 서열 번호 24 (58 머) 에 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다. 상기 서열은 잠재적으로 더 긴 단백질을 코딩하는 2 개의 오픈 리딩 프레임 (+1 및 -3) 을 갖는다. 본 발명에 따른 ORF 는 프레임 +1 이다.
하나의 다른 파스튜렐라세애 유전자는 서열 번호 24 및 그 코딩되는 아미노산 서열 (19 개 아미노산) 으로의 블라스트로 동정되었다. PM1951 단백질과 19 개 아미노산에 걸쳐 100% 동일성을 발견하였다. 돌연변이체 3H2 중 트랜스포존의 위치는 Genbank 접근 번호 AE006231 (PM1951, uvrA) 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 9418-9419 사이에 해당한다. PM1951 의 뉴클레오티드 서열은 본원에서 서열 번호 25 로 식별되며, 그 아미노산 서열은 서열 번호 26 로 식별된다.
다른 그램 음성 박테리아 유전자 및 단백질은 서열 번호 26 으로 수행된 블라스트로 동정되었다.
| 균주 | 유전자 (Genbank ref.) | 단백질 (Genbank ref.) | 동일성% |
| 헤모필러스 인플루엔자 | UvrA (U32711) | UvrA (AAC21915) | 943 개 아미노산에 걸쳐 89% |
| 대장균 K12 | UvrA (AE000479) | UvrA (AAC77028) | 940 개 아미노산에 걸쳐 80% |
| 예르시니아 페스티스 | UvrA (AJ414142) | UvrA (CAC89185) | 943 개 아미노산에 걸쳐 80% |
| 비브리오 콜레라 | UvrA (AE004127) | UvrA (AAF93567) | 940 개 아미노산에 걸쳐 80% |
| 살모넬라 타이피 |
UvrA (AL627282) | UvrA (CAD09238) | 941 개 아미노산에 걸쳐 80% |
| 살모넬라 타이피뮤리움 | UvrA (AE008898) | UvrA (AAA27250) | 941 개 아미노산에 걸쳐 80% |
| 슈도모나스 아에루기노사 (Pseudomonas aeruginosa) | UvrA (AE004840) | UvrA (AAGO7622) | 943 개 아미노산에 걸쳐 75% |
UvrA 는 DNA 보수 ABC 절단 뉴클레아제이다.
돌연변이체 4D6
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 27 (54 머) 에 나타낸 다. 트랜스포존은 상기 서열의 5' 말단 바로 곁에 존재한다. 상기 서열은 잠재적으로 더 긴 단백질을 코딩하는 2 개의 오픈 리딩 프레임 (+1 및 -2) 을 갖는다. 본 발명에 따른 ORF 는 틀 +1 이다.
돌연변이체 4D6 중 트랜스포존의 위치는 Genbank 접근 번호 AE006036 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 6492-6493 사이에 해당한다. 트랜스포존은 PM70 유전자 PM0032 또는 hktE 의 상동성을 손상시킨다. PM0032 의 뉴클레오티드 서열은 본원에서 서열 번호 28 로 식별되며, 그 아미노산 서열은 서열 번호 29 로 식별된다.
하나의 다른 파스튜렐라세애 유전자/단백질은 서열 번호 29 로의 블라스트로 동정되었다. 이는 액티노바실러스 액티노마이세템코미탄스 (Actinobacillus actinomycetemcomitans) 카탈라아제 (Genbank 접근 번호 AF162654 및 AAF17882) 이다. PM0032 와 A. 액티노마이세템코미탄스 카탈라아제 사이의 482 개 아미노산에 걸쳐 85% 동일성을 발견하였다.
HktE 는 카탈라아제이다.
돌연변이체 4F4 및 12A5
돌연변이체 4F4 에 대해서, 트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 30 (172 머) 에 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다.
돌연변이체 12A5 에 대해서, 트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 97 (546 머) 에 나타낸다. 트랜스포존은 상기 서열의 5' 말단 바로 곁에 존 재한다.
4 개의 다른 파스튜렐라세애 유전자 및 단백질은 서열 번호 30 으로 코딩되는 57 개의 아미노산 서열로 수행된 블라스트로 동정되었다.
| 균주 | 유전자 (Genbank ref.) | 단백질 (Genbank ref.) | 동일성% |
| P. 멀토시다 (multocida) 택손 747 | HyaC (AF067175) | HyaC (AAC67251) | 57 개 아미노산에 걸쳐 96% |
| P. 멀토시다 PM70 | PM0776 (AE006116) | AAK02860 | 57 개 아미노산에 걸쳐 96% |
| P. 멀토시다 P4218 | FcbC (AF302467) | FcbC (AAK17922) | 57 개 아미노산에 걸쳐 91% |
| P. 멀토시다 P934 | DcbC (AF302465) | DcbC (AAK17904) | 57 개 아미노산에 걸쳐 88% |
돌연변이체 4F4 중 트랜스포존의 위치는 Genbank 접근 번호 AE006116 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 5272-5273 사이에 해당한다. 돌연변이체 12A5 중 트랜스포존의 위치는 AE006116 서열의 위치 5275-5276 사이에 해당한다. 트랜스포존은 PM70 유전자 PM0776 의 상동성을 손상시킨다. PM0776 의 뉴클레오티드 서열은 본원에서 서열 번호 31 로 식별되며, 그 아미노산 서열은 서열 번호 32 로 식별된다. 상기 단백질은 UDP 글루코오스 탈수소효소이다.
돌연변이체 4F12
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 33 (226 머) 에 나타낸다. 트랜스포존은 상기 서열의 5' 말단 바로 곁에 존재한다.
돌연변이체 4F12 중 트랜스포존의 위치는 Genbank 접근 번호 AE006038 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 9263-9264 사이에 해당한다. 트랜스포존은 PM70 유전자 PM0048 또는 fadR 의 상동성을 손상시킨다. PM0048 의 뉴클레오티드 서열은 본원에서 서열 번호 34 로 식별되며, 그 아미노산 서열은 서열 번 호 35 로 식별된다. FadR 은 지방산 대사의 전사 조절자이며, 여러 지방산 생합성 (fab) 및 지방산 분해 (fad) 유전자에 영향을 미치는 대장균 단백질의 상동체이다.
돌연변이체 4G11
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 36 (214 머) 에 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다.
하나의 다른 파스튜렐라세애 유전자는 서열 번호 36 및 그 코딩되는 아미노산 서열 (70 개 아미노산) 로 수행된 블라스트로 동정되었다. PM1024 단백질의 70 개 아미노산에 걸쳐 100% 동일성을 발견하였다. 돌연변이체 4G11 중 트랜스포존의 위치는 Genbank 접근 번호 AE006143 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 3532-3533 사이에 해당한다. 트랜스포존은 PM70 유전자 PM1024 또는 HtpG 의 상동성을 손상시킨다. PM1024 의 뉴클레오티드 서열은 본원에서 서열 번호 37 로 식별되며, 그 아미노산 서열은 서열 번호 38 로 식별된다.
다른 그램 음성 박테리아 유전자 및 단백질은 서열 번호 38 로 수행된 블라스트로 동정되었다.
| 균주 | 유전자 (Genbank ref.) | 단백질 (Genbank ref.) | 동일성% |
| 액티노바실러스 액티노마이세템코미탄스 | HtpG (U26968) | HtpG (AAC44732) | 625 개 아미노산에 걸쳐 88% |
| 헤모필러스 인플루엔자 | HtpG (U32695) | HtpG (AAC21778) | 625 개 아미노산에 걸쳐 86% |
| 대장균 K12 | HtpG (AEOOO153) | HtpG (AAC73575) | 621 개 아미노산에 걸쳐 76% |
| 예르시니아 페스티스 | HtpG (AJ414155) | HtpG (CAC92355) | 622 개 아미노산에 걸쳐 76% |
| 살모넬라 타이피 |
STY0531 (AL627267) | HtpG (CAD04972) | 621 개 아미노산에 걸쳐 76% |
| 살모넬라 타이피뮤리움 | HtpG (AE008718) | HtpG (AAL19441) | 621 개 아미노산에 걸쳐 76% |
HtpG 는 열 쇼크 (heat shock) 단백질이다.
4G11 돌연변이체는 파스퇴르 연구소 은행에 부다페스트 조약 하에 기탁되었으며, 접근 번호 CNCM I-2999 로 이용가능하다.
돌연변이체 5D5
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 39 (252 머) 에 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다.
돌연변이체 5D5 중 트랜스포존의 위치는 Genbank 접근 번호 AE006188 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 5695-5696 사이에 해당한다. 트랜스포존은 PM70 유전자 PM1517 또는 PlpE 의 상동성을 손상시킨다. PM1517 의 뉴클레오티드 서열은 본원에서 서열 번호 40 으로 식별되며, 그 아미노산 서열은 서열 번호 41 로 식별된다.
PlpE 는 막 지질단백질로 예측된다.
5D5 돌연변이체는 파스퇴르 연구소 은행에 부다페스트 조약 하에 기탁되었으며, 접근 번호 CNCM I-3000 으로 이용가능하다.
돌연변이체 5F11
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 42 (546 머) 에 나타낸다. 트랜스포존은 상기 서열의 5' 말단 바로 곁에 존재한다.
종결 코돈은 위치 148-150 에 위치한다.
돌연변이체 5F11 중 트랜스포존의 위치는 Genbank 접근 번호 AE006150 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 572-573 사이에 해당한다. 트랜스포존은 PM70 유전자 PM1087 또는 NifR3 의 상동성을 손상시킨다. PM1087 의 뉴클레오티드 서열은 본원에서 서열 번호 43 으로 식별되며, 그 아미노산 서열은 서열 번호 44 로 식별된다.
하나의 다른 파스튜렐라세애 유전자/단백질은 서열 번호 44 로 수행된 블라스트로 동정되었다. 이는 헤모필러스 인플루엔자 HI0979 (Genbank 접근 번호 U32778 및 AAC22639) 였다. PM1087 및 HI0979 사이의 332 개 아미노산에 걸쳐 78% 동일성을 발견하였다.
NifR3 은 니트로게나아제 조절 유전자이다.
돌연변이체 5G9
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 45 (43 머) 에 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다. 상기 서열은 잠재적으로 더 긴 단백질을 코딩하는 3 개의 오픈 리딩 프레임 (+2, +3 및 -1) 을 갖는다. 본 발명에 따른 ORF 는 틀 +2 이다.
4 개의 다른 파스튜렐라세애 유전자 및 단백질은 서열 번호 45 로 코딩되는 14 개의 아미노산 서열로 수행된 블라스트로 동정되었다.
| 균주 | 유전자 (Genbank ref.) | 단백질 (Genbank ref.) | 동일성% |
| P. 멀토시다 P4218 | FcbE (AF302467) | FcbE (AAK17920) | 14 개 아미노산에 걸쳐 100% |
| P. 멀토시다 PM70 | PM0774 (AE006116) | HyaE (AAK02858) | 14 개 아미노산에 걸쳐 100% |
| P. 멀토시다 택손 747 | HyaE (AF067175) | HyaE (AAC67249) | 14 개 아미노산에 걸쳐 100% |
| P. 멀토시다 P934 | DcbE (AF302465) |
DcbE (AAK17906) | 14 개 아미노산에 걸쳐 71% |
돌연변이체 5G9 중 트랜스포존의 위치는 Genbank 접근 번호 AE006116 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 573-574 사이에 해당한다. 트랜스포존은 PM70 유전자 PM0774 또는 HyaE 의 상동성을 손상시킨다. PM0774 의 뉴클레오티드 서열은 본원에서 서열 번호 46 으로 식별되며, 그 아미노산 서열은 서열 번호 47 로 식별된다. 상기 유전자는 캡슐 합성에 관여한다.
돌연변이체 6E5
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 48 (279 머) 에 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다.
개시 코돈은 위치 169-171 에 위치한다.
돌연변이체 6E5 중 트랜스포존의 위치는 Genbank 접근 번호 AE006182 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 6673-6674 사이에 해당한다. 트랜스포존은 PM70 유전자 PM1459 또는 pgtB 의 상동성을 손상시킨다. PM1459 의 뉴클레오티드 서열은 본원에서 서열 번호 49 로 식별되며, 그 아미노산 서열은 서열 번호 50 으로 식별된다.
PgtB 는 포스포글리세레이트 수송 조절 단백질이다.
돌연변이체 6E6
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 51 (93 머) 에 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다.
종결 코돈은 위치 12-14 에 위치한다.
돌연변이체 6E6 중 트랜스포존의 위치는 Genbank 접근 번호 AE006096 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 9051-9052 사이에 해당한다. 트랜스포존은 PM70 유전자 PM0605 의 상동성을 손상시킨다. PM0605 의 뉴클레오티드 서열은 본원에서 서열 번호 52 로 식별되며, 그 아미노산 서열은 서열 번호 53 으로 식별된다.
돌연변이체 6F12
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 54 (772 머) 에 나타낸다. 트랜스포존은 상기 서열의 5' 말단 바로 곁에 존재한다.
개시 코돈은 위치 2-4 에 위치한다.
돌연변이체 6F12 중 트랜스포존의 위치는 Genbank 접근 번호 AE006192 인 파스테우렐라 멀토시다 PM70 게놈 서열의 위치 5362-5363 사이에 해당한다. 트랜스포존은 PM70 유전자 PM1556 또는 comF 유전자의 상동성을 손상시킨다. PM1556 의 뉴클레오티드 서열은 본원에서 서열 번호 55 로 식별되며, 그 아미노산 서열은 서열 번호 56 으로 식별된다.
ComF 는 적격성 (competence) 단백질 F 이다.
돌연변이체 6G4
트랜스포존 삽입 부위 측면의 DNA 서열은 서열 번호 57 (700 머) 로 나타낸다. 상기 트랜스포존은 상기 서열의 5' 말단의 바로 곁에 존재한다.
돌연변이체 6G4 에서의 트랜스포존의 위치는 파스튜렐라 멀토시다 PM70 게놈 서열인, Genbank 접근 번호 AE006206 의 위치 3758-3759 사이에 상응한다. 삽입은 PM1696 및 PM1697 유전자 사이에 있다. 트랜스포존은 프로모터 영역 및 PM1696 의 개시 코돈 사이에 삽입된다.
PM1696 의 개시 코돈은 서열 번호 57 서열에서의 위치 26-28 에 위치한다.
PM1696 의 뉴클레오티드 서열은 본원에서 서열 번호 58 로 식별되며, 그의 아미노산 서열은 서열 번호 59 로 식별된다.
기타 그람 음성 박테리아 유전자 및 단백질은 서열 번호 59 를 이용한 블라스트 (blast) 로 식별했다.
| 균주 | 유전자 (Genbank 번호) | 단백질 (Genbank 번호) | 상동성% |
| 해모필러스 인플루엔자 (Haemophilus influenzae) | HI0266 (U32713) | HI0266 (AAC21932) | 184 개 아미노산에 걸쳐 87% |
| 살모넬라 타이피 (Salmonella typhi) | STY3386 (AL627278) | STY3386 (CAD07732) | 185 개 아미노산에 걸쳐 71% |
| 살모넬라 타이피뮤리움 (Salmonella typhimurium) | STM3207 (AE008847) |
ygiH (AAL22081) | 185 개 아미노산에 걸쳐 71% |
돌연변이체 6H1
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 60 (188 머) 로 나타낸다. 트랜스포존은 상기 서열의 3' 바로 곁에 존재한다.
돌연변이체 6H1 중 트랜스포존의 위치는 Genbank 접근 번호 AE006119 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 4139-4140 사이에 해당한다. 트랜스포존은 PM70 유전자 PM0806 또는 speF 유전자의 상동성을 손상시킨다. PM0806 의 뉴클레오티드 서열은 본원에서 서열 번호 61 으로 식별되며, 그의 아미노산 서열은 서열 번호 62 로 식별된다.
두 개의 다른 파스튜렐라세애 및 비브리오나세애(Vibrionaceae) 유전자는 서열 번호 62 로 수행된 블라스트로 동정되었다. 상기 유전자들은 해모필러스 인플루엔자 (Haemophilus influenzae) speF (Genbank 접근 번호 U32740 및 AAC22248) 및 비브리오 콜레라 오르니틴 (Vibrio cholerae ornithine) 탈카르복실화효소 (decarboxylase) (AE004431 및 AAF96957) 이다. PM0806 과 H. 인플루엔자 (H. influenzae) speF 사이에서는 719 개의 아미노산에 걸쳐 83% 의 상동성을 발견했다.
SpeF 는 오르니틴 탈카르복실화효소이다.
돌연변이체 6H6
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 63 (101 머) 로 나타낸다. 트랜스포존은 상기 서열의 3' 말단의 바로 곁에 존재한다. 상기 서열은 잠재적인 더 긴 단백질을 코딩하는 2 개의 오픈 리딩 프레임 (+1 및 -1) 을 갖는다. 본 발명에 따른 ORF 는 프레임 +1 내에 있다.
돌연변이체 6H6 중 트랜스포존의 위치는 Genbank 접근 번호 AE006155 인 파스튜렐라세애 멀토시다 PM70 게놈 서열의 위치 983-984 사이에 해당한다. 트랜스포존은 PM70 유전자 PM 1138 의 상동성을 손상시킨다. 본원에서 PM 1138 의 뉴클레오티드 서열은 서열 번호 64 로 식별되며, 그의 아미노산 서열은 서열 번호 65 로 식별된다.
돌연변이체 7A7
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 66 (222 머) 로 나타낸다. 트랜스포존은 상기 서열의 5' 말단 바로 곁에 존재한다. 돌연변이체 7A7 의 트랜스포존의 위치는 Genbank 접근 번호 AE006170 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 7853-7854 에 해당한다 (PMI1321 및 PM1322 사이의 유전자간 영역 내). 트랜스포존은 PM1322 의 종결 영역 및 정지 코돈 사이에 삽입된다.
정지 코돈은 서열 번호 66 서열 내의 위치 25-27 에 위치한다. PM 1322 의 뉴클레오티드 서열은 본원에서 서열 번호 67 로 식별되며, 그의 아미노산 서열은 서열 번호 68 로 식별된다.
돌연변이체 7F8
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 69 (55 머) 로 나타냈다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다. 상기 서열은 잠재적인 더 긴 단백질을 코딩하는 3 개의 오픈 리딩 프레임 (+1, +3 및 -3) 을 갖는다. 본 발명에 따른 ORF 는 프레임 +3 이다.
돌연변이체 7F8 내의 트랜스포존의 위치는 Genbank 접근 번호 AE006224 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 8292-8293 에 해당한다. 트랜스포존은 PM70 유전자 PM 1866 의 상동성을 손상시킨다. PM1866 의 뉴클레오티드 서열은 서열 번호 70 으로 식별되며, 그의 아미노산 서열은 서열 번호 71 로 식별된다.
돌연변이체 9C8
트랜스포존 삽입 부위의 양면의 DNA 서열을 서열 번호 72 (598 머, 5' 말단 에 트랜스포존) 및 서열 번호 73 (561 머, 5' 말단에 트랜스포존) 으로 나타낸다.
정지 코돈은 서열 번호 72 의 위치 26-28 에 위치한다. 서열 번호 72 및 73 의 서열은 함께 조합되어 ORF 를 한정한다. 생성되는 서열은 서열 번호 74 (575 머) 로 정한다.
돌연변이체 9C8 의 트랜스포존의 위치는 Genbank 접근 번호 AE006132 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 2224-2225 또는 2210-2211 에 해당하며, 상기 위치들은 각각 서열 번호 72 및 73 으로부터 추론된다. 두 위치가 모두 PM0926 (fimA) 유전자의 내측에 있다. PM0926 의 뉴클레오티드 서열은 본원에서 서열 번호 75 로 식별되며, 그의 아미노산 서열은 서열 번호 76 으로 식별된다.
또다른 파스튜렐라에 유전자/단백질은 서열 번호 76 으로 수행된 블라스트로 동정되었다. 이는 해모필러스 인플루엔자 (Haemophilus influenzae) FimA (Genbank 접근 번호 AF053125 및 AAC08991) 이다. PM0926 와 H. 인플루엔자 (H. influenzae) FimA 사이에는 171 개의 아미노산 사이에 걸쳐 77% 의 상동성을 발견했다.
FimA 은 섬모 (fimbrial) 단백질인 어드헤신 (adhesin) 이다.
9C8 돌연변이체는 부다페스트 조약 하에 파스퇴르 연구소 (Pasteur Institute Collection) 에 기탁되었으며, 접근번호 CNCM I-3001 로 입수가능하다.
돌연변이체 9H4
트랜스포존 삽입 부위 양면의 DNA 서열을 서열 번호 92 (1391 머) 로 나타낸다. 트랜스포존은 상기 서열의 위치 850-851 에서 삽입되었다. 상기 서열은 단지 1 개의 리딩 프레임을 갖는다. 본 발명에 따른 ORF 는 프레임 -2 중에 있다.
개시 코돈은 서열 번호 92 의 위치 1318-1316 에 위치하며, 종결 코돈은 상기의 위치 29-31 에 위치한다. ORF 제공 서열을 서열 번호 93 (1290 머) 로 정하고, 그의 아미노산 서열을 서열 번호 94 로 정한다.
서열 번호 92 및 서열 번호 94 로 수행된 블라스트는 임의의 동종 유전자 또는 단백질을 동정하지 못했다.
9H4 돌연변이체는 부다페스트 조약 하에 파스퇴르 연구소에 기탁되었으며, 접근 번호 CNCM I-3002 로 입수가능하다.
돌연변이체 10G11
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 77 (70 머) 로 나타낸다. 트랜스포존은 상기 서열의 5' 말단 바로 곁에 존재한다. 개시 코돈은 위치 62-64 에 위치한다.
돌연변이체 10G11 내 트랜스포존의 위치는 Genbank 접근 번호 AE006056 의 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 2938-2939 에 해당한다. 트랜스포존은 PM70 유전자 PM0220 (rpL3 1_1) 의 상동성을 손상시킨다. PM0220 의 뉴클레오티드 서열은 본원에서 서열 78 로 식별되며, 그의 아미노산 서열은 서열 번호 79 로 식별된다.
돌연변이체 10G11 내 트랜스포존의 위치는 Genbank 접근 번호 AE006056 의 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 2938-2939 에 해당한다. 트랜스포존은 PM70 유전자 PM0220 (rpL3 1_1) 의 상동성을 손상시킨다. PM0220 의 뉴클레오티드 서열은 본원에서 서열 78 로 식별되며, 그의 아미노산 서열은 서열 번호 79 로 식별된다.
RpL3 1-1 은 50S 리보솜 단백질이다.
돌연변이체 11E8
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 80 (506 머) 로 나타낸다. 트랜스포존의 상기 서열의 5' 말단 바로 곁에 존재한다.
개시 코돈은 서열 번호 80 의 위치 195-197 에 위치한다.
돌연변이체 11E8 중 트랜스포존의 위치는 Genbank 접근 번호 AE006085 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 282-283 에 해당한다. 트랜스포존은 PM70 유전자 PM0488 의 상동성을 손상시킨다. PM0488 의 뉴클레오티드 서열은 본원에서 서열 번호 81 로 식별되며, 그의 아미노산 서열은 서열 번호 82 로 식별된다.
돌연변이체 11E8 중 트랜스포존의 위치는 Genbank 접근 번호 AE006085 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 282-283 에 해당한다. 트랜스포존은 PM70 유전자 PM0488 의 상동성을 손상시킨다. PM0488 의 뉴클레오티드 서열은 본원에서 서열 번호 81 로 식별되며, 그의 아미노산 서열은 서열 번호 82 로 식별된다.
돌연변이체 12A1
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 83 (243 머) 로 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다.
또다른 파스튜렐라세애 유전자는 서열 번호 83 및 그의 코딩되는 아미노산 서열 (81 개의 아미노산)으로 수행된 블라스트로 동정되었다. PM0063 단백질과 81 개의 아미노산에 걸쳐 100% 의 상동성을 발견했다. 돌연변이체 12A1 중의 트랜스포존의 위치는 Genbank 접근 번호 AE006042 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 2880-2881 에 해당한다. 트랜스포존은 PM70 유전자 PM0063 또는 lepA 유전자의 상동성을 손상시킨다. PM0063 의 뉴클레오티드 서열은 본원에서 서열 번호 84 로 식별되며, 그의 아미노산 서열은 서열 번호 85 로 식별된다.
다른 그람 음성 박테리아 유전자 및 단백질은 서열 번호 85 로 수행된 블라스트로 동정되었다.
| 균주 | 유전자 (Genbank ref.) |
단백질 (Genbank ref.) |
동일성% |
| 헤모필러스 인플루엔자 (Haemophilus influenzae) | H10016 (U32687) | LepA (AAC21694) | 598 개의 아미노산에 걸쳐 95% |
| 예르시니아 페스티스(Yersinia pestis) | YPO2716 (AJ414153) | LepA (CAC92955) | 597 개의 아미노산에 걸쳐 88% |
| 대장균 K 12 (Escherichia coli K12) | LepA (AE000343) | LepA (AAC75622) | 597 개의 아미노산에 걸쳐 89% |
| 살모넬라 타이피 (Salmonella typhi) | STY2829 (AL627275) | LepA (CAD02785) | 597 개의 아미노산에 걸쳐 89% |
| 살로넬라 타이피뮤리움 (Salmonella typhimurium) | LepA (AE008817) | LepA (AAL21477) | 597 개의 아미노산에 걸쳐 89% |
| 비브리오 콜레라 (Vibrio cholerae) | VC2463 (AE004316) | LepA (AAF95605) | 597 개의 아미노산에 걸쳐 84% |
| 슈도모나스 애루기노사 (Pseudomonas aeruginosa) | PA0767 (AE004511) | LepA (AAGO4156) | 594 개의 아미노산에 걸쳐 75% |
LepA 은 GTP-결합 멤브레인 단백질이다.
돌연변이체 12B3
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 서열 번호 86 (147 머) 로 나타낸다. 트랜스포존은 상기 서열의 3' 말단 바로 곁에 존재한다.
돌연변이체 12B3 중 트랜스포존의 위치는 Genbank 접근 번호 AE006152 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 4028-4029 에 해당한다. 트랜스포존은 PM70 유전자 PM1112 또는 deaD 유전자의 상동성을 손상시킨다. PM 1112 의 뉴클레오티드 서열은 본원에서 서열 번호 87 로 식별되며, 그의 아미노산 서열은 서열 번호 88 로 식별된다.
또다른 파스튜렐라세애 유전자/단백질은 서열 번호 88 로 수행된 블라스트로 동정되었다. 이는 해모필러스 인플루엔자 (Haemophilus influenzae) HI0231 (Genbank 접근 번호 U32709 및 AAC21900) 이다. PM1112 및 HI0231 사이의 605 개 아미노산에 걸쳐 80% 의 상동성을 발견했다.
DeaD 는 RNA 헬리케이즈이다.
돌연변이체 13E1
트랜스포존 삽입 부위 측면의 DNA 서열을 서열 번호 89 (187 머) 로 나타낸다. 트랜스포존의 상기 서열의 3' 말단 바로 곁에 존재한다. 상기 서열은 잠재적으로 더 긴 단백질을 코딩하는 2 개의 오픈 리딩 프레임 (+1 및 -3) 을 갖는다. 본 발명에 따른 ORF 는 프레임 -3 에 있다.
삭제
돌연변이체 13E1 내의 트랜스포존의 위치는 Genbank 접근 번호 AE006138 (PM0989) 인 파스튜렐라 멀토시다 PM70 게놈 서열의 위치 2173-2174 사이에 해당한다. PM0989 의 뉴클레오티드 서열은 본원에서 서열 번호 90 로 식별되며, 그의 아미노산 서열은 서열 번호 91 로 식별된다.
또다른 파스튜렐라세애 유전자/단백질은 서열 번호 91 로 수행된 블라스트로 동정되었다. 이는 해모필러스 인플루엔자 (Haemophilus influenzae) HI0325 (Genbank 접근 번호 U32717 및 AAC21988) 이다. PM0989 및 HI0325 사이의 414 개 아미노산에 결쳐 79% 의 상동성을 발견했다.
13E1 돌연변이체는 부다페스트 조약 하에 파스퇴르 연구소에 기탁되었으며, 접근 번호 CNCM I-3003 으로 입수가능하다.
실시예 5: 트랜스포존 삽입 돌연변이체의 PCR 선별
트랜스포존은 박테리아의 게놈 내의 임의의 위치에 삽입될 수 있다. 그러나, 올바른 돌연변이체의 선별은 PCR 을 이용하여 수행될 수 있다.
한 쌍의 프라이머가 사용되는데, 하나는 트랜스포존에 특이적인 것으로, 예컨대 Tn10IR1 (서열 번호 101), Th10IR4 (서열 번호 102), KTGRI (서열 번호 100), StipA (서열 번호 99) 및 StipJ (서열 번호 98) 이며, 다른 하나는 돌연변이화된 유전자 또는 서열에 대해 특이적이다. 상기 유전자 또는 이의 일부의 뉴클레오티드 서열 (예를 들어, 상기 서열분석된 트랜스포존의 삽입 위치 부근 영역의 서열) 은 상기 프라이머의 고안에 도움이 된다. 이는 파스튜렐라 멀토시다의 기타 균주, 또는 기타 그람 음성 박테리아, 예컨대 파스튜렐라세애 패밀리의 박테리아, 특히 파스튜렐라 해몰리티카, 파스튜렐라 아나티페스티페르 및 악티노바실러스 플레우로뉴모니애의 유전자 또는 뉴클레오티드 서열에 적용될 수 있다.
파스튜렐라 멀토시다 균주 PM70 또는 P-1059 (실시예 4) 내에서의 해당하는 유전자 또는 ORF 및/또는 이들의 조절 영역에 대한 지식, 예컨대 그의 크기는, 증폭된 PCR 단편을 스크리닝하고, 돌연변이화된 유전자 또는 서열 내에 삽입된 트랜스포존에 해당하는 크기를 가진 것을 검출하기 위해 이용된다. 트랜스포존이 유전자의 외부에 삽입된 경우, 증폭된 PCR 단편이 없을 수 있거나, 또는 크기가 너무 크게 단편을 증폭할 수 있다. 따라서, PCR 은 돌연변이의 선별을 가능케한다.
파스튜렐라 멀토시다 P-1059 균주에 대해, 상기 유전자 특이적 프라이머는 하기일 수 있다:
선행기술에서 수득한 돌연변이체의 경우, PCR 은 하기의 프라이머쌍으로 수행되었으며, 증폭된 PCR 단편은 하기의 크기를 가진다:
실시예 6: 상동성 공격 (homologous challenge) 에 대한 트랜스포존 삽입 돌연변이의 효능 및 보호
파스튜렐라 멀토시다 16084 균주 유래의 트랜스포존 삽입 돌연변이체 (실시 예 1) 를 3 주령의 일반적인 칠면조에게 점안에 의해 투여했다. 효능은 파스튜렐라 멀토시다 16084 균주로 인한 안내 상동성 공격 (homologous challenge) 에 대해 연구되었다.
24 개 군의 3 주령인 일반적인 칠면조를 정했다. 제 0 일째에, 군들에게 하기 표에 나타낸 돌연변이체를 약 108 CFU 으로 점안하여 접종했다.
한 군의 3 주령인 일반적인 칠면조를 백신접종하지 않고 대조군으로 제공했다 (군 25).
모든 칠면조를, 새 한 개체마다 108 CFU 의 점안으로 파스튜렐라 멀토시다 16084 균주를 사용하여 제 23 일째에 공격했다.
치사율은 공격 후 2 주 동안 매일 기록했다. 연구 종결시 (제 37 일), 임상적인 시험은 생존한 새의 건강 상태를 측정하여 수행했다.
공격당한 새의 개체수 및 제 37 일째에 건강한 새의 개체수를 고려하여 보호 율을 계산했다.
일부의 군에 대해서, 상기의 실험을 재현하고, 보호율을 누적했다. 본 발명의 일부 돌연변이체는 상기 실험에서 시험되지 않았다.
실시예 7: 이종성 공격에 대한 트랜스포존 삽입 돌연변이체의 효능 및 보호
파스튜렐라 멀토시다 16084 균주로부터 유도된 트랜스포존 삽입 돌연변이체 (실시예 1) 를 3 주령의 일반적인 칠면조에 점안으로 투여했다. 효능은 파스튜렐라 멀토시다 X73 균주 (USDA) 로 안내 이종성 공격 (ocular heterogous challenge) 에 대해 연구했다.
3 주령인 10 마리의 일반적인 칠면조 5 개군을 정했다. 제 0 일 째에, 군들은 하기 표에 표시한 바와 같이 약 108 CFU 의 돌연변이체를 점안하여 접종했다.
3 주령인 10 마리의 일반적인 칠면조 한 군을 백신접종하지 않고 남겨두고 대조군으로서 제공했다 (군 6).
모든 칠면조를 새 1 마리 당 106 CFU 로 점안하여 투여되는 파스튜렐라 멀토시다 X73 균주를 이용하여 제 21 일째에 공격했다.
치사율은 공격 후 2 주 동안 매일 기록했다. 연구 종결시 (제 36 일째), 임상 시험을 수행하여 생존한 새의 건강 상태를 측정했다.
보호율은 공격한 새의 개체수 및 제 36 일째에 건강한 새의 개체수를 고려하여 계산했다.
실시예 8: 콘쥬게이션에 의한 예정 결실 돌연변이체의 구축
초기에, 표적 유전자 + 부근 DNA 서열을 고성능 폴리머라제를 이용한 PCR 로 증폭하고 적합한 클로닝 벡터로 클로닝했다. 초기 구축물을 생성하기 위한 역방향 PCR 에 사용되는 경우 유전자를 결실시키는 PCR 프라이머를 고안했다. 상기 PCR 프라이머는 XbaI 부위를 포함하여 신규한 제한효소 부위를 도입하고, 따라서 유전자 결실에 대한 마커를 제공한다. 이어서, 결실 구축물은 파스튜렐라세애 염색체로의 이동을 위해 자살 벡터 (suicide vector) pCVD442 로 이동된다 (Donnenberg 등, Infection and Immunity, 1991, 59: 4310-4317). 이어서, pCVD442 플라스미드는 E. coli 균주 SM10λpir 로 형질전환된다. 상기 구축물은 E. coli SM10λpir/pCVD442 로 콘쥬게이션되어 16084 (CDM) P. multocida 균주로 도입된다. 상동 부위에 염색체로 통합된 플라스미드를 포함하는 형질전환체 및 재조합체 (자오선 이성질체 (merodiploids)) 는 pCVD442 플라스미드 상에 존재하는 항생제 내성 마커 (앰피실린 내성 유전자) 를 이용하여 선별된다. Pasteurella 돌연변이체는 1 ㎍/ml 의 앰피실린이 보충된 BHI 아가 플레이트 상에서 선별된다.
pCVD442 플라스미드는 복제를 위해서는 Pir 단백질을 필요로 한다. 상기 단백질은 pir 유전자에 의해 코딩되는데, 이는 공여자 균주 SM10λpir 내의 람다 파지 라이소겐으로서 존재하나, 수용자 파스튜렐라세애 물토시타 (P. multocida) 에는 존재하지 않는다. 따라서, pCVD442 플라스미드는 수용자 파스튜렐라세애 물토시타 (P. multocida) 균주에서는 복제하지 않는다: 따라서, 항생제 내성 콜로니는 플라스미드가 염색체에 통합된 경우에만 수득된다. 상기 자살 벡터는 또한 효소 레반 수크라아제 (enzyme levan sucrase) 를 코딩하는 sacB 유전자를 포함하는데, 이는 수크로오스 존재 하의 대부분 그람 음성 박테리아에 대해 독성이다. 따라서, 수크로오스 선별은 제 2 재조합 발생이 일어나 염색체로부터 플라스미드가 소실된 콜로니를 단리하기 위한 상대 선별법으로서 이용될 수 있다. 상기 제 2 재조합 발생은 2 가지의 결과, 즉 야생형 대립유전자의 재생성 또는 결실 돌연변이체의 생성을 초래할 수 있다. 결실 돌연변이체를 포함하는 콜로니는 콜로니 PCR 로 식별된다.
실시예 9: 전기천공에 의한 예정 결실 돌연변이의 구축
초기에, 표적 유전자 + 부근 DNA 서열을 고성능 폴리머라제를 이용한 PCR 로 증폭시키고, 적합한 클로닝 벡터에 클로닝했다. 초기 구축물 생성을 위한 역방향 PCR 에 사용되는 경우 유전자를 결실시키는 PCR 프라이머를 고안했다. 상기 PCR 프라이머는 XbaI 부위를 포함하여, 신규한 제한효소 부위를 도입함으로써, 유전자 결실에 대한 마커를 제공한다. 이어서, 결실 구축물은 파스튜렐라 (Pasteurella) 염색체로의 이동을 위해 자살 벡터 pCVD442 로 이동시킨다. 상기 구축물은 전기천공으로 16084 (CDM) P. multocida 균주에 도입된다. 16084 의 실질적인 세포외 캡슐을 제거하기 위해, 정상 (stationary phase) 세포를 양의 고환 히알루로니다아제 (타입 V, 사용 전 멸균여과, 최종 농도 25 ㎍/ml) 로 1 시간 동안 처리한 후, 수합하여 세포를 세척한다. pCVD442 (1.5 ㎍) 을 10% 글리세롤 (0.05 ml, 1010 세포/ml) 중 세포 현탁액과 혼합한 직후, 혼합물을 빙냉 1 mm 전기천용 큐벳 (Biorad, Hercules, CA, USA) 으로 피펫팅한다. GenePulser (Biorad) 를 이용하여 세포를 펄스한다 (12.5 kV/cm, 250 ohms, 40 ㎌). 펄스 직후, 세포를 1 ml BHI 로 희석하고, 배양물 일부 (5 - 50 ㎕) 를 재빨리 (1 - 5 분 이내) 1 ㎍/ml 의 앰피실린을 함유하는 BHI 아가 플레이트 상에 도말한다. 상동성 부위에서 염색체 에 통합된 플라스미드를 포함하는 재조합체 (자오선 이성체) 를 플라스미드 상에 존재하는 항생제 내성 마커 (앰피실린 내성 유전자) 를 이용하여 선별한다.
pCVD442 플라스미드는 수용자 파스튜렐라 멀토시다 (P. multocida) 균주 내에서 복제하지 않으며, 따라서 항생제 내성 콜로니는 플라스미드가 염색체에 통합되는 경우에만 수득된다. 상기 자살 벡터는 또한 효소 레반 수크라아제를 코딩하는 sacB 유전자를 포함하는데, 이는 수크로오스 존재 하의 대부분 그람 음성 박테리아에 대해 독성이다. 따라서, 수크로오스 선별은 제 2 재조합 발생이 일어나 염색체로부터 플라스미드가 소실된 콜로니를 단리하기 위한 상대 선별법으로서 이용될 수 있다. 상기 제 2 재조합 발생은 2 가지의 결과, 즉 야생형 대립유전자의 재생성 또는 결실 돌연변이체의 생성을 초래할 수 있다.
콜로니는 37℃에서 2-4 일 동안 인큐베이션한 후 나타난다. 콜로니를 유사한 플레이트에 도말하면 개별적인 형질전환체가 단리된다. 결실 돌연변이를 포함하는 콜로니는 콜로니 PCR 로 식별된다.
실시예 10: 백신 및 효능의 시험
실시예 8 또는 9 에서 수득된 감쇠된 결실 돌연변이를 CDM 배양 배지 (Hu 등, Infection and Immunity 1986, 804-81 0) 에서 24 시간 내지 48 시간 동안의 진탕 조건 하에 배양한다.
배양물은 흡광도 또는 pH 측정을 하여 성장이 정지할 때 수합한다.
박테리아 농도는 흡광도로 측정되며, 필요한 경우 농도는 신선한 배양 배지 ml 당 109 CFU 의 최종 농도로 조정한다.
백신의 효능은 백신접종 및 공격에 의해 3 주령 칠면조에서 시험한다.
칠면조는 백신접종 전에 혈액 샘플의 ELISA 로 Pasteurella 항체의 부재성 여부에 대해 검사한다.
제 1 군의 칠면조는 안구 경로를 통해 0.1 ml 로 108 CFU 를 주사하여 백신접종했다.
제 2 군은 백신접종하지 않고 남겨두었다 (대조군).
모든 동물들은 제 21 일 또는 제 23 일째에 안구 경로를 통해 P. multocida P-1059 균주 (1 마리 당 108 CFU, 0.1 ml) 로 공격했다.
치사율은 제 35 일째까지 매일 관찰했다.
대조군에 비해 백신접종된 동물에서 더 낮은 치사율이 관찰된다.
본 발명은 하기의 항목들로 더 기술된다.
1. 그람 음성 박테리아의 돌연변이체로서, 상기 박테리아가 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90 또는 93 로 식별되는 뉴클레오티드 서열로 이루어진 군으로부터 선택되는 뉴클레오티드 서열에 의해 코딩되는 아미노산 서열과 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 갖는 폴리펩티드를 코딩하는 뉴클레오티드 서열 중 돌연변이가 있고, 상기 돌연변이가 박테리아의 발병력 감쇠를 초래하는 돌연변이체.
2. 항목 1 에 있어서, 박테리아가 파스튜렐라세애인 돌연변이체.
3. 항목 2 에 있어서, 박테리아가 파스튜렐라 멀토시다, 파스튜렐라 해몰리티카, 파스튜렐라 아나티페스티페르 및 악티노바실러스 플레우로뉴모니애로 이루어진 군으로부터 선택되는 돌연변이체.
4. 항목 3 에 있어서, 박테리아가 파스튜렐라 멀토시다인 돌연변이체.
5. 항목 1 내지 4 중 어느 하나에 있어서, 돌연변이가 뉴클레오티드 서열 중의 결실, 또는 그곳으로의 삽입 또는 핵산의 치환인 돌연변이체.
6. 항목 5 에 있어서, 돌연변이가 전체 뉴클레오티드 서열의 결실인 돌연변이체.
7. 항목 5 에 있어서, 서열 번호 2 에서 뉴클레오티드 180-181 또는 182-183 또는 190-191, 서열 번호 6 에서 뉴클레오티드 77-78 또는 1026-1027 또는 1027-1028, 서열 번호 9 에서 뉴클레오티드 416-417, 서열 번호 12 에서 뉴클레오티드 389-390, 서열 번호 16 에서 뉴클레오티드 381-382, 서열 번호 19 에서 뉴클레오티드 219-220, 서열 번호 22 에서 뉴클레오티드 1353-1354, 서열 번호 25 에서 뉴클레오티드 136-137, 서열 번호 28 에서 뉴클레오티드 384-385, 서열 번호 31 에서 뉴클레오티드 222-223 또는 225-226, 서열 번호 34 에서 뉴클레오티드 217-218, 서열 번호 37 에서 뉴클레오티드 1411-1412, 서열 번호 40 에서 뉴클레오티드 943-944, 서열 번호 43 에서 뉴클레오티드 855-856, 서열 번호 46 에서 뉴클레오티드 369-370, 서열 번호 49 에서 뉴클레오티드 111-112, 서열 번호 52 에서 뉴클레오티드 443-444, 서열 번호 55 에서 뉴클레오티드 4-5, 서열 번호 58 에서 뉴클레오티 드 1 의 바로 상류, 서열 번호 61 에서 뉴클레오티드 573-574, 서열 번호 64 에서 뉴클레오티드 875-876, 서열 번호 67 에서 뉴클레오티드 1 의 바로 상류, 서열 번호 70 에서 뉴클레오티드 218-219, 서열 번호 75 에서 뉴클레오티드 1072-1087, 서열 번호 78 에서 뉴클레오티드 64-65, 서열 번호 81 에서 뉴클레오티드 282-283, 서열 번호 84 에서 뉴클레오티드 1431-1432, 서열 번호 87 에서 뉴클레오티드 974-975, 서열 번호 90 에서 뉴클레오티드 802-803, 서열 번호 92 에서 뉴클레오티드 850-851 사이에서 삽입이 있는 돌연변이체.
8. 항목 1 내지 7 중 어느 한 항목에 있어서, 병원성 바이러스, 기생체 또는 박테리아 제제 유래의 면역원, 치료용 단백질, 알레르겐 (allergen), 성장 인자 또는 싸이토카인을 코딩하는 이종성 핵산 서열을 포함하는 돌연변이체.
9. 항목 1 내지 8 중 어느 한 항목에 따른 감쇠된 돌연변이체 및 약제학적으로 허용되는 희석제, 담체, 비히클 또는 부형제를 함유하는 면역원성 조성물.
10. 항목 9 에 있어서, 아쥬반트를 추가로 함유하는 면역원성 조성물.
11. 항목 1 내지 8 중 어느 한 항목에 따른 감쇠된 돌연변이체 및 약제학적으로 허용되는 희석제, 담체, 비히클 또는 부형제를 함유하는 백신.
12. 항목 11 에 있어서, 아쥬반트를 추가로 함유하는 백신.
13. 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 으로 식별되는 뉴클레오티드 서열로 이루어진 군으로부터 선택되는 뉴클레오티드 서열로 코딩되는 아미노산 서열과 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 가진 폴리펩티드 및 약제학적으로 허용되는 희석제, 담체, 비히클 또는 부형제, 및 임의로는 아쥬반트를 함유하는 면역원성 조성물.
14. 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 로 식별되는 뉴클레오티드 서열로 이루어진 군으로부터 선택되는 뉴클레오티드 서열로 코딩되는 아미노산 서열과 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 갖는 폴리펩티드에 특이적인 항체를 함유하는 항체 제제.
15. 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 로 식별되는 뉴클레오티드 서열로 이루어진 군으로부터 선택되는 뉴클레오티드 서열에 의해 코딩되는 아미노산 서열과 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% 또는 99% 이상의 상동성을 갖는 폴리펩티드 또는 상기 폴리펩티드에 특이적인 항체를 이용하여 그람 음성 박테리아에 의한 감염을 검출하기 위한 진단 방법.
16. 그람 음성 박테리아에 대한 수동 면역 조성물 또는 치료용 조성물 제조를 위한 항목 14 에 따른 항체 제제의 용도.
17. 배지 중 그람 음성 박테리아 검출용 PCR 을 위한 프라이머로서의, 서열 번호 2, 6, 9, 12, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 75, 78, 81, 84, 87, 90, 93 으로 식별되는 뉴클리오티드 서열로 이루어진 군으로부터 선택되는 뉴클레오티드 서열, 또는 20 개 이상의 뉴클레오티드의 단편의 용도.
***
본 발명의 바람직한 구현예가 상기 기재되었지만, 이들의 다수의 자명한 변형예가 본 발명의 진의를 벗어나지 않고 이용가능하기 때문에 상기 항목에 의해 정해지는 본 발명이 상기 기재된 특정 사항으로만 한정되지 않는다.
<110> MERIAL LIMITED
<120> ATTENUATED GRAM NEGATIVE BACTERIA
<130> 454313-3171.1
<140> 10/406,686
<141> 2003-04-03
<150> 60/370,282
<151> 2002-04-05
<160> 118
<170> PatentIn Ver. 2.1
<210> 1
<211> 775
<212> DNA
<213> Pasteurella multocida
<220>
<221> modified_base
<222> (580)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (599)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (617)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (637)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (668)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (698)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (703)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (707)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (715)..(716)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (718)..(719)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (722)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (743)..(745)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (747)..(750)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (756)..(757)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (767)..(768)
<223> a, t, c, g, other or unknown
<400> 1
gtgctttata tccccattct aaaatacatg ttctctcctt tttccatgtg acaaatggag 60
agaacatttt caagcgttgg gtaaaaaagc cgcttaaata aggaattttt aacatccctt 120
tagaaaaaat aagaaactct tgatacatat ttaatctaat atagtcatat aaagttgaca 180
tatcatatat taaacatgac tagttaatca ttaaatatta aacaacctca acttaataaa 240
acaaataata aacaaacaag gtaaaaaaca aactaatact gagcaaataa aaaacggatt 300
aatataataa cgatatatca acctctaaaa cagaccaaaa ataaatcaca cgagacaaaa 360
gaacaattat aatccaaata ttaattaata aataaacacc tagcgcaacg aataatcaaa 420
caaaatcaca tttagattta tttaaattaa aaatatagat tatattttaa atataatgct 480
agaattcggc accaaaattt ttctccagct gtaaattaga gataaagata tgaaaaaggt 540
tattatcatg ggacataaac agtctaacta tcaagatgtn gaaaaggttt ttcaatgtna 600
tgggatgaat ccccgcntcc atcaaaacgt gaaaaangtc cccatcgaac ttttgctgag 660
tgaggatnag atagggcaaa tctgcaaatt catccacngc cgncgcngac tcatnnanng 720
cnaatcgcca tagtagttat acnnncnnnn gtttanngtt gaccgcnnag gcgag 775
<210> 2
<211> 2091
<212> DNA
<213> Pasteurella multocida
<400> 2
atgtcaattt tatatgacta tattagatta aatatgtatc aagagtttct tattttttct 60
aaagggatgt taaaaattcc ttatttaagc ggttttttta cccaacgctt gaaaatgttc 120
tctccatttg tcacatggaa aaaggagaga acatgtattt tagaatgggg atataaagca 180
tcatcaaaga aagcgaggca ttttgcacaa caacatgatt taccttatgc gacgatagaa 240
gatggttttt tacgttctat tggactgggt gtggatgggt atccaccttt ctcattagtg 300
tacgatgata ttggtattta ttatgatatc aatcagcctt ctcgtttaga aaacttaatt 360
ctttctcaag atgtattact tcaggaaaaa gtcgatcaag ttgaatatgc aattgaatta 420
atttgtacac ataacctttc caaatataac cacgctattg atacaccttt acagaatacg 480
aagaggccga ttgtcttagt cgttgatcaa acatatggcg atatggcagt tactttcggg 540
aatgccgagc agagtgattt tctacatatg ttagaacgtg cgattattga aaatccaaca 600
gcagaaatct ggttaaaaac ccatcctgat gtaatgtgtg gtaaaaaaca aggctattta 660
acggaatatc agcaatttcc aagagtaaaa gtattatcag aagattttaa ttctatctca 720
ctactaaagc atgttgataa agtttattgc gtgacatctc acactgggtt tgaggccctt 780
ttgttaggaa aaactgtcgt gacttttggg gctgcctggt tttctggttg gggattgacg 840
gatgatcggc atgcgtatat tcgtcagcta aaacagagta agagaagagc gaagcgttca 900
ttgttgcagt tattctatgc tgcttatttc caatattgtc gttatattaa tcctaatacg 960
ggtaaatcag gcacattgtt tgatgtcatt gattatctga ttcaagcaaa aaaagtaacc 1020
aatcagttag ctggtgatat ttattgtgtg ggtatgcgct tttggaaacg taaagtggtt 1080
caaccctttt ttcaatttcc acgctgtcgt ttacattttg tgctgaatgt gcatgagcta 1140
aagcgatgta ttcacgagaa atctcaggct aaaatagtgg tgtggggaca ttcacacatt 1200
gaagtggttg aatatgccaa gcaacagcaa cttcctcttt tgagaatgga agatggtttt 1260
ttacgttcag ttgggttagg gtctaattta acgccaccga tatcattagt tttagatgac 1320
gttggcattt attttgacgc ccaatctcgt tcccgattag aggatattct acagcatcaa 1380
tcctttactc taaaggattt acagcgcgca gaaacgttaa agaaaacact gattgagcaa 1440
catattggta agtataatgt gggacataca cacttatgcc taacacacat cagacaaaat 1500
aaacttttag ttgtgggaca agtggaaaat gatgcttcaa ttcaatatgg ttcaccgcat 1560
attcgtacga atgcagagtt attatgtacg gtcagaaaaa ataatcccca agcctatatt 1620
atttataaac ctcatcctga tgtggttgca ggcaatcgta aaaacacaga tcgtctagat 1680
gattatcgac agtatgctga tttcgtggtt gagaaagcca atatattgga ttgcattaac 1740
caagtggatg aagtgcatac gatgacctct ttagcggggt ttgaagcgtt actgcgcgag 1800
aaaaaagtac attgttatgg cttgcctttt tattctaact gggggctaac agtggatcat 1860
ctttctctaa accgaagaag tcggaagtta agtcttttag aattaattgc tggcgtgctg 1920
atttattacc cacaatatat tgacccaaaa acaaaaacaa tgatcgatgt gcagcgagcg 1980
gttgatattc tgatcgagca acgtcgaaaa ataaaaaata ataaattaca tacaaattat 2040
tttatgaaca tttttatgaa attaaaaaat gtttattctg ttttgaggta g 2091
<210> 3
<211> 696
<212> PRT
<213> Pasteurella multocida
<400> 3
Met Ser Ile Leu Tyr Asp Tyr Ile Arg Leu Asn Met Tyr Gln Glu Phe
1 5 10 15
Leu Ile Phe Ser Lys Gly Met Leu Lys Ile Pro Tyr Leu Ser Gly Phe
20 25 30
Phe Thr Gln Arg Leu Lys Met Phe Ser Pro Phe Val Thr Trp Lys Lys
35 40 45
Glu Arg Thr Cys Ile Leu Glu Trp Gly Tyr Lys Ala Ser Ser Lys Lys
50 55 60
Ala Arg His Phe Ala Gln Gln His Asp Leu Pro Tyr Ala Thr Ile Glu
65 70 75 80
Asp Gly Phe Leu Arg Ser Ile Gly Leu Gly Val Asp Gly Tyr Pro Pro
85 90 95
Phe Ser Leu Val Tyr Asp Asp Ile Gly Ile Tyr Tyr Asp Ile Asn Gln
100 105 110
Pro Ser Arg Leu Glu Asn Leu Ile Leu Ser Gln Asp Val Leu Leu Gln
115 120 125
Glu Lys Val Asp Gln Val Glu Tyr Ala Ile Glu Leu Ile Cys Thr His
130 135 140
Asn Leu Ser Lys Tyr Asn His Ala Ile Asp Thr Pro Leu Gln Asn Thr
145 150 155 160
Lys Arg Pro Ile Val Leu Val Val Asp Gln Thr Tyr Gly Asp Met Ala
165 170 175
Val Thr Phe Gly Asn Ala Glu Gln Ser Asp Phe Leu His Met Leu Glu
180 185 190
Arg Ala Ile Ile Glu Asn Pro Thr Ala Glu Ile Trp Leu Lys Thr His
195 200 205
Pro Asp Val Met Cys Gly Lys Lys Gln Gly Tyr Leu Thr Glu Tyr Gln
210 215 220
Gln Phe Pro Arg Val Lys Val Leu Ser Glu Asp Phe Asn Ser Ile Ser
225 230 235 240
Leu Leu Lys His Val Asp Lys Val Tyr Cys Val Thr Ser His Thr Gly
245 250 255
Phe Glu Ala Leu Leu Leu Gly Lys Thr Val Val Thr Phe Gly Ala Ala
260 265 270
Trp Phe Ser Gly Trp Gly Leu Thr Asp Asp Arg His Ala Tyr Ile Arg
275 280 285
Gln Leu Lys Gln Ser Lys Arg Arg Ala Lys Arg Ser Leu Leu Gln Leu
290 295 300
Phe Tyr Ala Ala Tyr Phe Gln Tyr Cys Arg Tyr Ile Asn Pro Asn Thr
305 310 315 320
Gly Lys Ser Gly Thr Leu Phe Asp Val Ile Asp Tyr Leu Ile Gln Ala
325 330 335
Lys Lys Val Thr Asn Gln Leu Ala Gly Asp Ile Tyr Cys Val Gly Met
340 345 350
Arg Phe Trp Lys Arg Lys Val Val Gln Pro Phe Phe Gln Phe Pro Arg
355 360 365
Cys Arg Leu His Phe Val Leu Asn Val His Glu Leu Lys Arg Cys Ile
370 375 380
His Glu Lys Ser Gln Ala Lys Ile Val Val Trp Gly His Ser His Ile
385 390 395 400
Glu Val Val Glu Tyr Ala Lys Gln Gln Gln Leu Pro Leu Leu Arg Met
405 410 415
Glu Asp Gly Phe Leu Arg Ser Val Gly Leu Gly Ser Asn Leu Thr Pro
420 425 430
Pro Ile Ser Leu Val Leu Asp Asp Val Gly Ile Tyr Phe Asp Ala Gln
435 440 445
Ser Arg Ser Arg Leu Glu Asp Ile Leu Gln His Gln Ser Phe Thr Leu
450 455 460
Lys Asp Leu Gln Arg Ala Glu Thr Leu Lys Lys Thr Leu Ile Glu Gln
465 470 475 480
His Ile Gly Lys Tyr Asn Val Gly His Thr His Leu Cys Leu Thr His
485 490 495
Ile Arg Gln Asn Lys Leu Leu Val Val Gly Gln Val Glu Asn Asp Ala
500 505 510
Ser Ile Gln Tyr Gly Ser Pro His Ile Arg Thr Asn Ala Glu Leu Leu
515 520 525
Cys Thr Val Arg Lys Asn Asn Pro Gln Ala Tyr Ile Ile Tyr Lys Pro
530 535 540
His Pro Asp Val Val Ala Gly Asn Arg Lys Asn Thr Asp Arg Leu Asp
545 550 555 560
Asp Tyr Arg Gln Tyr Ala Asp Phe Val Val Glu Lys Ala Asn Ile Leu
565 570 575
Asp Cys Ile Asn Gln Val Asp Glu Val His Thr Met Thr Ser Leu Ala
580 585 590
Gly Phe Glu Ala Leu Leu Arg Glu Lys Lys Val His Cys Tyr Gly Leu
595 600 605
Pro Phe Tyr Ser Asn Trp Gly Leu Thr Val Asp His Leu Ser Leu Asn
610 615 620
Arg Arg Ser Arg Lys Leu Ser Leu Leu Glu Leu Ile Ala Gly Val Leu
625 630 635 640
Ile Tyr Tyr Pro Gln Tyr Ile Asp Pro Lys Thr Lys Thr Met Ile Asp
645 650 655
Val Gln Arg Ala Val Asp Ile Leu Ile Glu Gln Arg Arg Lys Ile Lys
660 665 670
Asn Asn Lys Leu His Thr Asn Tyr Phe Met Asn Ile Phe Met Lys Leu
675 680 685
Lys Asn Val Tyr Ser Val Leu Arg
690 695
<210> 4
<211> 226
<212> DNA
<213> Pasteurella multocida
<400> 4
agcaaaagtt cgattactac cagagacagt aagaagtgcg gttaaaatac ctaacaagaa 60
gagaaataac acaatattaa tgttatttga attaagtgcg ttatcactgt atgccaatga 120
aataatatta tttttgagat acattagcgt atgcgaaata ttaaaatctg caagcattaa 180
agcacctaca ataataccaa cactcaatga taaaataaca cggcgc 226
<210> 5
<211> 87
<212> DNA
<213> Pasteurella multocida
<400> 5
cgacactatg cattcttatt gatcgtcaag tgagtttggc ggaatacggt aaatcctgga 60
ttttaggcgt gaagtcaatg ctcggtg 87
<210> 6
<211> 1524
<212> DNA
<213> Pasteurella multocida
<400> 6
atggaactta ttgattattc gacgtctatt tggtctgtcg taccccctat tttagcatta 60
ttattagcca ttgggacacg ccgtgttatt ttatcattga gtgttggtat tattgtaggc 120
gctttaatgc ttgcagattt taatatttcg catacgctaa tgtatctcaa aaataatatt 180
atttcattgg catacagtga taacacactt aattcaaata acattaatat tgtgttattt 240
ctcttcttgt taggtatttt aaccgcactt cttactgtct caggcagtaa tcgagccttt 300
gcagaatggg cacaaaaacg aattaaagat agaaaagggg ctaaattatt agccgcatcg 360
ctcgtgtttg tgactttcat tgacgattat tttcatagct tagcggtggg agcgattgcc 420
agcccagtta cagataaatt taaagtttca cgcccaaaac ttgcctatat tcttgattca 480
accgctgcgc caatgtgtgt gttgatgcct gtatcaagtt ggggcgccta tattattaca 540
cttattgcag gacttcttgc gacttattcg atcaccgagt attcccctat cggtgcattt 600
atgacaatga gtgcaatgaa cttttatgct attttttcta ttttaatggt gttctttgta 660
tcttattatt cgtttgatat tggttcaatg gcgcgtcacg aaagaatggc cctagcgcgt 720
gtaacagaag aagaaaaact ggaaagtagt aataaagggc atgttctcta tttaatttta 780
ccgattactg tcctgatttt agcaaccgtt ggtatgatga tgtacacggg ctatgaagca 840
ttagcggcgg atggaaaacc ttttgatgtg ttaggcgcgt ttgagaatac tacagtaggg 900
atttcattgg ttgtgggggg attaagtgcg gtcttgattt cgacactatg cattcttatt 960
gatcgtcaag tgagtttggc tgaatacggt aaatcctgga ttttaggcgt gaagtcaatg 1020
ctcggtgcgg tattgatttt attgtttgct tggactatta ataccatcgt tggagatgtc 1080
aaaacaggga tttatttatc ttcattagta tcggatagtt taccgattgc tttgttgcct 1140
gcgttattat ttattttaac tggaatcatg gcattctcga caggaacaag ctggggaact 1200
tttgggatta tgttaccgat cgcggcagcg attgcagcga atactgcacc agaattgatg 1260
ttaccttgtt tatccgcagt catggctggt gcagtttgtg gtgatcattg ctcaccgatt 1320
tcggatacca cgattttatc ttctaccggg gcaaaatgta atcatatcga ccatgtaaca 1380
acacagttac cttatgcgat gttaattgcg acagcgtcta ttgctggcta tttagtacta 1440
gggttcagcc agtcaggcat actgggtttt gtgacaacgg gtgtggtttt atcagtactt 1500
gtttttatat ttagaaaaaa ataa 1524
<210> 7
<211> 507
<212> PRT
<213> Pasteurella multocida
<400> 7
Met Glu Leu Ile Asp Tyr Ser Thr Ser Ile Trp Ser Val Val Pro Pro
1 5 10 15
Ile Leu Ala Leu Leu Leu Ala Ile Gly Thr Arg Arg Val Ile Leu Ser
20 25 30
Leu Ser Val Gly Ile Ile Val Gly Ala Leu Met Leu Ala Asp Phe Asn
35 40 45
Ile Ser His Thr Leu Met Tyr Leu Lys Asn Asn Ile Ile Ser Leu Ala
50 55 60
Tyr Ser Asp Asn Thr Leu Asn Ser Asn Asn Ile Asn Ile Val Leu Phe
65 70 75 80
Leu Phe Leu Leu Gly Ile Leu Thr Ala Leu Leu Thr Val Ser Gly Ser
85 90 95
Asn Arg Ala Phe Ala Glu Trp Ala Gln Lys Arg Ile Lys Asp Arg Lys
100 105 110
Gly Ala Lys Leu Leu Ala Ala Ser Leu Val Phe Val Thr Phe Ile Asp
115 120 125
Asp Tyr Phe His Ser Leu Ala Val Gly Ala Ile Ala Ser Pro Val Thr
130 135 140
Asp Lys Phe Lys Val Ser Arg Pro Lys Leu Ala Tyr Ile Leu Asp Ser
145 150 155 160
Thr Ala Ala Pro Met Cys Val Leu Met Pro Val Ser Ser Trp Gly Ala
165 170 175
Tyr Ile Ile Thr Leu Ile Ala Gly Leu Leu Ala Thr Tyr Ser Ile Thr
180 185 190
Glu Tyr Ser Pro Ile Gly Ala Phe Met Thr Met Ser Ala Met Asn Phe
195 200 205
Tyr Ala Ile Phe Ser Ile Leu Met Val Phe Phe Val Ser Tyr Tyr Ser
210 215 220
Phe Asp Ile Gly Ser Met Ala Arg His Glu Arg Met Ala Leu Ala Arg
225 230 235 240
Val Thr Glu Glu Glu Lys Leu Glu Ser Ser Asn Lys Gly His Val Leu
245 250 255
Tyr Leu Ile Leu Pro Ile Thr Val Leu Ile Leu Ala Thr Val Gly Met
260 265 270
Met Met Tyr Thr Gly Tyr Glu Ala Leu Ala Ala Asp Gly Lys Pro Phe
275 280 285
Asp Val Leu Gly Ala Phe Glu Asn Thr Thr Val Gly Ile Ser Leu Val
290 295 300
Val Gly Gly Leu Ser Ala Val Leu Ile Ser Thr Leu Cys Ile Leu Ile
305 310 315 320
Asp Arg Gln Val Ser Leu Ala Glu Tyr Gly Lys Ser Trp Ile Leu Gly
325 330 335
Val Lys Ser Met Leu Gly Ala Val Leu Ile Leu Leu Phe Ala Trp Thr
340 345 350
Ile Asn Thr Ile Val Gly Asp Val Lys Thr Gly Ile Tyr Leu Ser Ser
355 360 365
Leu Val Ser Asp Ser Leu Pro Ile Ala Leu Leu Pro Ala Leu Leu Phe
370 375 380
Ile Leu Thr Gly Ile Met Ala Phe Ser Thr Gly Thr Ser Trp Gly Thr
385 390 395 400
Phe Gly Ile Met Leu Pro Ile Ala Ala Ala Ile Ala Ala Asn Thr Ala
405 410 415
Pro Glu Leu Met Leu Pro Cys Leu Ser Ala Val Met Ala Gly Ala Val
420 425 430
Cys Gly Asp His Cys Ser Pro Ile Ser Asp Thr Thr Ile Leu Ser Ser
435 440 445
Thr Gly Ala Lys Cys Asn His Ile Asp His Val Thr Thr Gln Leu Pro
450 455 460
Tyr Ala Met Leu Ile Ala Thr Ala Ser Ile Ala Gly Tyr Leu Val Leu
465 470 475 480
Gly Phe Ser Gln Ser Gly Ile Leu Gly Phe Val Thr Thr Gly Val Val
485 490 495
Leu Ser Val Leu Val Phe Ile Phe Arg Lys Lys
500 505
<210> 8
<211> 78
<212> DNA
<213> Pasteurella multocida
<400> 8
gggaaaagca gcaaatatca aaaatactgt tttagtgaaa acaggaaaac cgattacagc 60
agaaggcgta cacccacc 78
<210> 9
<211> 555
<212> DNA
<213> Pasteurella multocida
<400> 9
atgaaaaaag caattttttt agatcgagat ggcacattaa atattgatca tggctatgtt 60
catgaaattg atcagtttca atttattgac ggtagcattg aagcgttaca acaactgaaa 120
gcgatgggct atttattggt acttgtaaca aatcagtcag gtattgcgcg tggatatttt 180
agcgaagatc aatttttaca gctgacagaa tggatggatt ggtctcttgc agatcgtgga 240
gtggatttag atggcatcta ttattgccca caccacacag aaggaaaagg tgagtattgc 300
caagactgcg attgccgtaa gccaaaacct ggtatgttac tgcaggcaat taaggaactt 360
aatatagatc ccaatacctc ttttatggtg ggtgataaag tggaagatat gttagcaggt 420
aaaggtgcca aaattaaaaa tactgtttta gtgaaaacag gcaagcctat tacggaggat 480
ggcaaaaaac aggcaaacta tgtattagag tccattgcgg atctaccaaa actgataaaa 540
ggattaaaaa gttaa 555
<210> 10
<211> 184
<212> PRT
<213> Pasteurella multocida
<400> 10
Met Lys Lys Ala Ile Phe Leu Asp Arg Asp Gly Thr Leu Asn Ile Asp
1 5 10 15
His Gly Tyr Val His Glu Ile Asp Gln Phe Gln Phe Ile Asp Gly Ser
20 25 30
Ile Glu Ala Leu Gln Gln Leu Lys Ala Met Gly Tyr Leu Leu Val Leu
35 40 45
Val Thr Asn Gln Ser Gly Ile Ala Arg Gly Tyr Phe Ser Glu Asp Gln
50 55 60
Phe Leu Gln Leu Thr Glu Trp Met Asp Trp Ser Leu Ala Asp Arg Gly
65 70 75 80
Val Asp Leu Asp Gly Ile Tyr Tyr Cys Pro His His Thr Glu Gly Lys
85 90 95
Gly Glu Tyr Cys Gln Asp Cys Asp Cys Arg Lys Pro Lys Pro Gly Met
100 105 110
Leu Leu Gln Ala Ile Lys Glu Leu Asn Ile Asp Pro Asn Thr Ser Phe
115 120 125
Met Val Gly Asp Lys Val Glu Asp Met Leu Ala Gly Lys Gly Ala Lys
130 135 140
Ile Lys Asn Thr Val Leu Val Lys Thr Gly Lys Pro Ile Thr Glu Asp
145 150 155 160
Gly Lys Lys Gln Ala Asn Tyr Val Leu Glu Ser Ile Ala Asp Leu Pro
165 170 175
Lys Leu Ile Lys Gly Leu Lys Ser
180
<210> 11
<211> 467
<212> DNA
<213> Pasteurella multocida
<400> 11
gagctctgca tttagtttaa gtggtgattt cttatttgac ttcaataaag attcattaac 60
agcaaaaggt aaagaagttg ttgacagcgt tgcaacacaa ttaaaagcct ctgatgcaaa 120
agaagtgaaa gtcgcaggct ttactgaccg tttaggttca gaagcgtata acttaaaact 180
ttctcaacgt cgtgcagatc gtgttaaagc gcgtttaatt gagcaaggtg ttgccgcaaa 240
tattcatgct gtaggctatg gtaaagcaca acaagtgaaa gcttgtgatg atgtacaagg 300
tgcagcatta agagactgtt tacgtcctaa ccgtcgtgtt gaaattaccg cttctggtac 360
tgtgttaaaa caaggttcac aaggtatgga agcagggaca acaggaccag caccacttta 420
tagaaaataa tttttctcaa tgaaatagaa gggcgcttta atagcgc 467
<210> 12
<211> 819
<212> DNA
<213> Pasteurella multocida
<400> 12
atgaaattat ctcgcgtttt attaacagtt gttgctgcga cgacattggc tgcctgcggt 60
aatttaagta aagttactcc agaaggtaca tctgacaatt tagtgtggcc aaaaattgat 120
gaatcagtct ttaatcatga tggtagccaa tttggttctt ggccaaactg ggataacgta 180
cgcatggttg agcgtggtat gaataaagac caactttata atttgttagg tcgtccacac 240
ttctctgaag gcttatacgg tgtgcgtgaa tgggactatg tgtttaacta tcgtgagaat 300
ggtgtacata aagtatgtca atataaagtc ttatttgaca aaaatatgaa tgcacaaagt 360
ttcttctggt atccaaatgg ctgtaacggt agctctgcat ttagtttaag tggtgatttc 420
ttatttgact tcaataaaga ttcattaaca gcaaaaggta aagaagttgt tgacagcgtt 480
gcaacacaat taaaagcctc tgatgcaaaa gaagtgaaag tcgcaggctt tactgaccgt 540
ttaggttcag aagcgtataa cttaaaactt tctcaacgtc gtgcagatcg tgttaaagcg 600
cgtttaattg agcaaggtgt tgccgcaaat atccatgctg taggctatgg taaagcacaa 660
caagtgaaag cttgtgatga tgtacaaggt gcagcattaa gagattgttt acgtcctaac 720
cgtcgtgttg aaattaccgc ttctggtact gtgttaaaac aaggttcaca aggtatggaa 780
gcagggacaa caggaccagc accactttat agaaaataa 819
<210> 13
<211> 272
<212> PRT
<213> Pasteurella multocida
<400> 13
Met Lys Leu Ser Arg Val Leu Leu Thr Val Val Ala Ala Thr Thr Leu
1 5 10 15
Ala Ala Cys Gly Asn Leu Ser Lys Val Thr Pro Glu Gly Thr Ser Asp
20 25 30
Asn Leu Val Trp Pro Lys Ile Asp Glu Ser Val Phe Asn His Asp Gly
35 40 45
Ser Gln Phe Gly Ser Trp Pro Asn Trp Asp Asn Val Arg Met Val Glu
50 55 60
Arg Gly Met Asn Lys Asp Gln Leu Tyr Asn Leu Leu Gly Arg Pro His
65 70 75 80
Phe Ser Glu Gly Leu Tyr Gly Val Arg Glu Trp Asp Tyr Val Phe Asn
85 90 95
Tyr Arg Glu Asn Gly Val His Lys Val Cys Gln Tyr Lys Val Leu Phe
100 105 110
Asp Lys Asn Met Asn Ala Gln Ser Phe Phe Trp Tyr Pro Asn Gly Cys
115 120 125
Asn Gly Ser Ser Ala Phe Ser Leu Ser Gly Asp Phe Leu Phe Asp Phe
130 135 140
Asn Lys Asp Ser Leu Thr Ala Lys Gly Lys Glu Val Val Asp Ser Val
145 150 155 160
Ala Thr Gln Leu Lys Ala Ser Asp Ala Lys Glu Val Lys Val Ala Gly
165 170 175
Phe Thr Asp Arg Leu Gly Ser Glu Ala Tyr Asn Leu Lys Leu Ser Gln
180 185 190
Arg Arg Ala Asp Arg Val Lys Ala Arg Leu Ile Glu Gln Gly Val Ala
195 200 205
Ala Asn Ile His Ala Val Gly Tyr Gly Lys Ala Gln Gln Val Lys Ala
210 215 220
Cys Asp Asp Val Gln Gly Ala Ala Leu Arg Asp Cys Leu Arg Pro Asn
225 230 235 240
Arg Arg Val Glu Ile Thr Ala Ser Gly Thr Val Leu Lys Gln Gly Ser
245 250 255
Gln Gly Met Glu Ala Gly Thr Thr Gly Pro Ala Pro Leu Tyr Arg Lys
260 265 270
<210> 14
<211> 204
<212> DNA
<213> Pasteurella multocida
<400> 14
tgccaattat agactttgtg tgaatgtacg agtaatcaca tcacgttgtt gttcaggtgt 60
taatgagttg aaacgtacag cgtaacctga aacacggatt gttaattgtg ggtatttttc 120
tgggttattg accgcatctt ctaaggtttc gcggcgtaat acgttaacgt ttaagtgttg 180
accaccttct actttgactg ttgg 204
<210> 15
<211> 35
<212> DNA
<213> Pasteurella multocida
<400> 15
aggtgttttt ttaagaggta aatggatgcc aatta 35
<210> 16
<211> 384
<212> DNA
<213> Pasteurella multocida
<400> 16
atgattaaag gtattcaaat tacccaagcg gctaatgaca atttattaaa ctcattttgg 60
ttattagata gcgaaaaagg tgaagcgcgt tgtttatgtg ctaaaggtga cttcgttgaa 120
gatcaaatcg ttgcagtaag tgaattaggt caaatcgaat atcgcgaatt accagttgat 180
atcgccccaa cagtcaaagt agaaggtggt caacacttaa acgttaacgt attacgccgc 240
gaaaccttag aagatgcggt caataaccca gaaaaatacc cacaattaac aatccgtgtt 300
tcaggttacg ctgtacgttt caactcatta acacctgaac aacaacgtga tgtgattact 360
cgtacattca cacaaagtct ataa 384
<210> 17
<211> 127
<212> PRT
<213> Pasteurella multocida
<400> 17
Met Ile Lys Gly Ile Gln Ile Thr Gln Ala Ala Asn Asp Asn Leu Leu
1 5 10 15
Asn Ser Phe Trp Leu Leu Asp Ser Glu Lys Gly Glu Ala Arg Cys Leu
20 25 30
Cys Ala Lys Gly Asp Phe Val Glu Asp Gln Ile Val Ala Val Ser Glu
35 40 45
Leu Gly Gln Ile Glu Tyr Arg Glu Leu Pro Val Asp Ile Ala Pro Thr
50 55 60
Val Lys Val Glu Gly Gly Gln His Leu Asn Val Asn Val Leu Arg Arg
65 70 75 80
Glu Thr Leu Glu Asp Ala Val Asn Asn Pro Glu Lys Tyr Pro Gln Leu
85 90 95
Thr Ile Arg Val Ser Gly Tyr Ala Val Arg Phe Asn Ser Leu Thr Pro
100 105 110
Glu Gln Gln Arg Asp Val Ile Thr Arg Thr Phe Thr Gln Ser Leu
115 120 125
<210> 18
<211> 75
<212> DNA
<213> Pasteurella multocida
<400> 18
acattcttga tgcaataagt cataacgttt tttgagaaac tggagcttat taaagaaaaa 60
gcgtacatgc cctgt 75
<210> 19
<211> 390
<212> DNA
<213> Pasteurella multocida
<400> 19
atgacacgga ttaatctcat cgcccccgct gaactttgtg atcaacatct gttagcagaa 60
cacagagaac tgacacgtat tcccaatgct gtggcaaaag ggaaatttag cctcctcggt 120
cagccagaag attataaatt aggtacaggg catgtacgct ttttctttaa taagctccag 180
tttctcaaaa aacgttatga cttattgcat caagaatgtc gagctcgagg ttttaatgtg 240
caatatattt ggcccgacaa gttgccggag gacgataacc tctggttaga ctacatccct 300
actgagcatg ccttagccgc caatagagcg cgtattttag aaagaatgcc tgtcaaagcc 360
cgctttacac caagtaaagc tacaacttaa 390
<210> 20
<211> 129
<212> PRT
<213> Pasteurella multocida
<400> 20
Met Thr Arg Ile Asn Leu Ile Ala Pro Ala Glu Leu Cys Asp Gln His
1 5 10 15
Leu Leu Ala Glu His Arg Glu Leu Thr Arg Ile Pro Asn Ala Val Ala
20 25 30
Lys Gly Lys Phe Ser Leu Leu Gly Gln Pro Glu Asp Tyr Lys Leu Gly
35 40 45
Thr Gly His Val Arg Phe Phe Phe Asn Lys Leu Gln Phe Leu Lys Lys
50 55 60
Arg Tyr Asp Leu Leu His Gln Glu Cys Arg Ala Arg Gly Phe Asn Val
65 70 75 80
Gln Tyr Ile Trp Pro Asp Lys Leu Pro Glu Asp Asp Asn Leu Trp Leu
85 90 95
Asp Tyr Ile Pro Thr Glu His Ala Leu Ala Ala Asn Arg Ala Arg Ile
100 105 110
Leu Glu Arg Met Pro Val Lys Ala Arg Phe Thr Pro Ser Lys Ala Thr
115 120 125
Thr
<210> 21
<211> 229
<212> DNA
<213> Pasteurella multocida
<400> 21
cgatttcaat ccctttttgg cggagttgtt catcatcata aatatgttta tcgccgtaga 60
gggcattgat tgtttcatgg ataccaatat agcctaatga aatagaggca cgcccatttt 120
taaagatttg tgccacattg tcatcagcct ttaagcgtac accacaggca ccctccatat 180
aaagaattgg ggcaacgcga gctttggtat gttctaaacg tgcaatgcg 229
<210> 22
<211> 2142
<212> DNA
<213> Pasteurella multocida
<400> 22
atggcaactt tctttgtaat taaacgagat gggtcacgaa cggggtttga aatccaacgt 60
atcattaatg caattaaaaa agcggcacaa gcggtgaata ttgaagatga acgttattgt 120
catacaatgg gacagcaggt atgtaatgac atctttactc gttaccaaca agaaattgat 180
atcagccaca ttcaaaaaat cgtagaaaat accttaatgg cgggaaaata ccctgaaata 240
gcgcgagctt atatcgaata ccgccatgat cgggatctcg cgcgagaaaa acgcagtcaa 300
ctcacaaaag aaatcgaagg attaattcag caaagtaatg ttgaactcct caatgaaaat 360
gccaataaag atgcgaaagt tatcccaact caacgcgatc tcttagcggg tattgtggca 420
aaacattacg ctaaacgtta tattctgcca cgcgatgtcg tagacgcaca tgaaaaaggg 480
gaaattcatt atcacgattt agactatgcc ccatttttcc caatgtttaa ctgcatgctt 540
gtcgatctca aagggatgct aagcaatggt ttcaaaatgg gtaatgccga aattgaacca 600
ccgaaatcga tcacaacagc aaccgcagtc agtgcacaaa ttatcgcaca agtcgcgagc 660
catatttacg gtggtaccac gattaaccgt atagatgaaa tccttgcccc ttatgtgcaa 720
ttaagttatg aaaaacattt aaaaaatgca gcggaatgga aagttcccga accagaagcc 780
tacgcgaaag cactcattga aaaagaatgt ttcgacgctt ttcaatcctt agaatatgaa 840
gtcaatacgc tgcatacttc aaatgggcaa accccttttg tcacttttgg ctttggctta 900
ggaacgacgt ggcaatcgag acttatccag cgctcaattc tgaaaaatcg tattcgtggt 960
ttaggcaaaa atcacaaaac ccctgtcttc ccaaaactgg tgttcactat taaaaaaggc 1020
attaaccata gcccgagtga tcctaactac gacattaaac aactggcttt agaatgtgcc 1080
tccaaacgga tgtatcctga tattctcaat tatgatcagg tggtgaaagt cacgggttct 1140
tttaaagcac caatgggatg ccgtagtttc ttaggtgctt atcaggagca aggacaggaa 1200
atccatgatg gacgtaataa cttaggcgta gtgagtttga atttaccgcg tatagcaatt 1260
gaagccaacg ccacgaattc agcccaaagt gcggtcgagt tttataaaat tttagatcaa 1320
cgtcttgcga ttgccaaaaa agccttaatg acacgcattg cacgtttaga acataccaaa 1380
gctcgcgttg ccccaattct ttatatggag ggtgcctgtg gtgtacgctt aaaggctgat 1440
gacaatgtgg cacaaatctt taaaaatggg cgtgcctcta tttcgttagg ctatattggt 1500
atccatgaaa caatcaatgc cctctacggc gataaacata tttatgatga tgaacaactc 1560
cgccaaaaag ggattgaaat cgtcgaatat ttacacgaga ccgtgcaacg ttggaaacaa 1620
gaaacaggtt atgctttcag cctatattcc acaccaagtg aaaacctttg tgaccgtttc 1680
tgtcgcttgg atactaagca atttgggctt atcgaaggtg tcacagataa aggctactat 1740
actaatagct accacttaga cgtagagaaa aaagtcaatc cttatgacaa gatagatttt 1800
gaattgcctt atccaccgtt cgcaagcggc gggtttattt gctatggtga atacccaaat 1860
gttcagcata accttaaagc attagaggac gtttgggatt atagctatga cagagtgcct 1920
tactatggga ccaatacacc gattgatgaa tgctatgaat gtggtttcag tggtgaattt 1980
gaatgtacca gtaaagggtt tacttgtccg aaatgtggta accatgacag tgagaaagtc 2040
tccgtgaccc gacgtgtctg tggctatctt ggcagtccag atgccagacc atttaatgcc 2100
ggtaaacaag aagaagtcaa gcgcagagta aaacatctct aa 2142
<210> 23
<211> 713
<212> PRT
<213> Pasteurella multocida
<400> 23
Met Ala Thr Phe Phe Val Ile Lys Arg Asp Gly Ser Arg Thr Gly Phe
1 5 10 15
Glu Ile Gln Arg Ile Ile Asn Ala Ile Lys Lys Ala Ala Gln Ala Val
20 25 30
Asn Ile Glu Asp Glu Arg Tyr Cys His Thr Met Gly Gln Gln Val Cys
35 40 45
Asn Asp Ile Phe Thr Arg Tyr Gln Gln Glu Ile Asp Ile Ser His Ile
50 55 60
Gln Lys Ile Val Glu Asn Thr Leu Met Ala Gly Lys Tyr Pro Glu Ile
65 70 75 80
Ala Arg Ala Tyr Ile Glu Tyr Arg His Asp Arg Asp Leu Ala Arg Glu
85 90 95
Lys Arg Ser Gln Leu Thr Lys Glu Ile Glu Gly Leu Ile Gln Gln Ser
100 105 110
Asn Val Glu Leu Leu Asn Glu Asn Ala Asn Lys Asp Ala Lys Val Ile
115 120 125
Pro Thr Gln Arg Asp Leu Leu Ala Gly Ile Val Ala Lys His Tyr Ala
130 135 140
Lys Arg Tyr Ile Leu Pro Arg Asp Val Val Asp Ala His Glu Lys Gly
145 150 155 160
Glu Ile His Tyr His Asp Leu Asp Tyr Ala Pro Phe Phe Pro Met Phe
165 170 175
Asn Cys Met Leu Val Asp Leu Lys Gly Met Leu Ser Asn Gly Phe Lys
180 185 190
Met Gly Asn Ala Glu Ile Glu Pro Pro Lys Ser Ile Thr Thr Ala Thr
195 200 205
Ala Val Ser Ala Gln Ile Ile Ala Gln Val Ala Ser His Ile Tyr Gly
210 215 220
Gly Thr Thr Ile Asn Arg Ile Asp Glu Ile Leu Ala Pro Tyr Val Gln
225 230 235 240
Leu Ser Tyr Glu Lys His Leu Lys Asn Ala Ala Glu Trp Lys Val Pro
245 250 255
Glu Pro Glu Ala Tyr Ala Lys Ala Leu Ile Glu Lys Glu Cys Phe Asp
260 265 270
Ala Phe Gln Ser Leu Glu Tyr Glu Val Asn Thr Leu His Thr Ser Asn
275 280 285
Gly Gln Thr Pro Phe Val Thr Phe Gly Phe Gly Leu Gly Thr Thr Trp
290 295 300
Gln Ser Arg Leu Ile Gln Arg Ser Ile Leu Lys Asn Arg Ile Arg Gly
305 310 315 320
Leu Gly Lys Asn His Lys Thr Pro Val Phe Pro Lys Leu Val Phe Thr
325 330 335
Ile Lys Lys Gly Ile Asn His Ser Pro Ser Asp Pro Asn Tyr Asp Ile
340 345 350
Lys Gln Leu Ala Leu Glu Cys Ala Ser Lys Arg Met Tyr Pro Asp Ile
355 360 365
Leu Asn Tyr Asp Gln Val Val Lys Val Thr Gly Ser Phe Lys Ala Pro
370 375 380
Met Gly Cys Arg Ser Phe Leu Gly Ala Tyr Gln Glu Gln Gly Gln Glu
385 390 395 400
Ile His Asp Gly Arg Asn Asn Leu Gly Val Val Ser Leu Asn Leu Pro
405 410 415
Arg Ile Ala Ile Glu Ala Asn Ala Thr Asn Ser Ala Gln Ser Ala Val
420 425 430
Glu Phe Tyr Lys Ile Leu Asp Gln Arg Leu Ala Ile Ala Lys Lys Ala
435 440 445
Leu Met Thr Arg Ile Ala Arg Leu Glu His Thr Lys Ala Arg Val Ala
450 455 460
Pro Ile Leu Tyr Met Glu Gly Ala Cys Gly Val Arg Leu Lys Ala Asp
465 470 475 480
Asp Asn Val Ala Gln Ile Phe Lys Asn Gly Arg Ala Ser Ile Ser Leu
485 490 495
Gly Tyr Ile Gly Ile His Glu Thr Ile Asn Ala Leu Tyr Gly Asp Lys
500 505 510
His Ile Tyr Asp Asp Glu Gln Leu Arg Gln Lys Gly Ile Glu Ile Val
515 520 525
Glu Tyr Leu His Glu Thr Val Gln Arg Trp Lys Gln Glu Thr Gly Tyr
530 535 540
Ala Phe Ser Leu Tyr Ser Thr Pro Ser Glu Asn Leu Cys Asp Arg Phe
545 550 555 560
Cys Arg Leu Asp Thr Lys Gln Phe Gly Leu Ile Glu Gly Val Thr Asp
565 570 575
Lys Gly Tyr Tyr Thr Asn Ser Tyr His Leu Asp Val Glu Lys Lys Val
580 585 590
Asn Pro Tyr Asp Lys Ile Asp Phe Glu Leu Pro Tyr Pro Pro Phe Ala
595 600 605
Ser Gly Gly Phe Ile Cys Tyr Gly Glu Tyr Pro Asn Val Gln His Asn
610 615 620
Leu Lys Ala Leu Glu Asp Val Trp Asp Tyr Ser Tyr Asp Arg Val Pro
625 630 635 640
Tyr Tyr Gly Thr Asn Thr Pro Ile Asp Glu Cys Tyr Glu Cys Gly Phe
645 650 655
Ser Gly Glu Phe Glu Cys Thr Ser Lys Gly Phe Thr Cys Pro Lys Cys
660 665 670
Gly Asn His Asp Ser Glu Lys Val Ser Val Thr Arg Arg Val Cys Gly
675 680 685
Tyr Leu Gly Ser Pro Asp Ala Arg Pro Phe Asn Ala Gly Lys Gln Glu
690 695 700
Glu Val Lys Arg Arg Val Lys His Leu
705 710
<210> 24
<211> 58
<212> DNA
<213> Pasteurella multocida
<400> 24
attgtgatta cgggattatc gggatcaggt aaatcttctt tagcctttga taccctgt 58
<210> 25
<211> 2832
<212> DNA
<213> Pasteurella multocida
<400> 25
atggataaga ttgaggtacg tggggcgaga acccataatt taaaaaatat taatttaact 60
attcctcgtg ataaattaat tgtgattacg ggattatcgg gatcaggtaa atcttcttta 120
gcctttgata ccctgtatgc tgagggacaa cgccgttatg tagaatcctt gtcggcatat 180
gcacgtcaat tcttatccct aatggaaaag ccggatgtgg atcatattga aggattatcg 240
ccggcgattt ctattgaaca aaaatctacc tcacataatc cgcgttcaac ggtgggtacg 300
gtcaccgaaa ttcatgatta cttacgtctt ctttttgctc gagtagggga accgcgttgt 360
ccgaatcatg atgtgccttt agcggcacaa accattagcc agatggtgga taaagtattg 420
agtttgcctg aagaaagcaa aatgatgttg ctggcgccag tggtgaaaga acgtaagggt 480
gaacatgtta aattattaga gcaaattgcc gcccaaggtt atattcgagc cagaattgac 540
ggtgaaattt gtgatttatc tgatgcacct aaattagaat tacataaaaa acatactatt 600
gaagtggttg tggaccgttt taaggtgcgg tctgatttag ccacaagatt agcagaatcc 660
tttgaaacag cattggaatt atctggtggc accgctgtag ttgcttccat ggatgagcct 720
gaaacggaag aattggtctt ttcggctaat tttgcttgtc cacattgtgg gtattctgtc 780
ccagaattag aacctcgttt attttctttt aataatcccg ccggggcgtg tccgacttgc 840
gatggcttag gtgtacaaca atattttgat gaaaaacgcg tggtgcaaaa cccgagtatt 900
tcgttagcca gtggcgcagt aaaaggctgg gatcgtcgta acttctatta ctaccaaatg 960
ctcacctcct tagcgaagca ttatgaattt gatattgaat caccttttga ggcactgccg 1020
aaaaaaatcc agcagattat tttaaatggt tcaggtaagg aagaaattga gtttcaatac 1080
atgaatgatc gcggcgatgt agtcgtgcgt catcatgcat ttgaaggcat tctaaataat 1140
atggcgcgcc gttataaaga aacggaatcg ctgtctgtgc gtgaagaatt agcgaaaaat 1200
atcagtacct gtccttgcca tgattgtggg ggttcacgtt tacgtcaaga ggcacgtcat 1260
gtgtatattg gcaccaccac tttacctgat gtggcagaaa agagtattgg cgaaaccttg 1320
catttcttta gtgaattgca tttaagcggg caaagagctc aaattgccga gaaaatctta 1380
aaagaaatta aagagcgctt acaattttta gtcaatgtag ggttggatta tctttccctt 1440
tctcgttcag cagaaacctt gtctggtggg gaggcacagc gaattcgttt agccagtcaa 1500
attggtgcgg gtttagtggg ggtgatgtat gtgctagatg agccgtctat tggtttgcat 1560
caacgtgata atgagcgatt actgaataca ttgcttcact tacgtaactt agggaacacc 1620
gtgattgtgg tagaacatga tgaagatgcc attatggcgg cagatcatat tattgatatt 1680
ggtcccgggg caggagttca tggtgggcaa attgtggcag aaggttcggc aaaggcgatt 1740
atggctaatc cacactcaat tacggggaaa tttttatctg gggtcgagaa aatcgaaatt 1800
cccgcaaaac ggaccgcact tgataagaaa aaaatgttga aattagaagg ggcaacgggg 1860
aataatctga aatcagtgaa tttagccatt ccagtaggat tgtttacctg tgtgacaggt 1920
gtttcggggt cagggaaatc gaccttgatt aatgatacgt tgttcccatt agcacaaaat 1980
gccttgaatc gtgcggaaaa tacgcaattt gcgccttatc aatccatttc gggtttggaa 2040
ttttttgata aagtaattga tattgaccaa agtccaattg gtcgtacacc gcgttcgaat 2100
cctgccactt atactggctt atttacgccg attcgagaat tatttgcggg cgtgcctgag 2160
tcgagagccc ggggttataa tcccggacgt tttagtttta atgtacgcgg tggacgctgt 2220
gaggcctgtc aaggcgatgg tgtgattaaa gtagagatgc actttttgcc cgatgtgtat 2280
gtgccttgtg agcaatgtaa gggaaaacgt tataatcgag agaccttaga gatccgttac 2340
aaaggtaaaa cgattcatca agtgttagaa atgacggtag aagaagcgcg cgagtttttt 2400
gatgcgattc cgcagatcgc ccgtaaatta caaactttaa tggatgttgg tttatcctat 2460
attcgtttag gacaatcttc gaccacgtta tcgggtgggg aagcgcaacg agtgaaatta 2520
gcaacggagc tttcaaaacg tgatacaggg aaaactttgt atgtattaga tgaaccgacg 2580
acaggtttac attttgctga tattaaacag ctattaacag tcttgcatcg tttacgtgat 2640
caaggcaata cgatagtggt gattgagcac aatttagatg tgatcaaaac agccgattgg 2700
attattgatt taggtcctga aggggggaat ggcggtggac aaattattgc cacaggcaca 2760
ccagaacagg tcgctgaagt gaaaggttca cataccgcac gcttcttaaa aacgctttta 2820
caaaagcgct aa 2832
<210> 26
<211> 943
<212> PRT
<213> Pasteurella multocida
<400> 26
Met Asp Lys Ile Glu Val Arg Gly Ala Arg Thr His Asn Leu Lys Asn
1 5 10 15
Ile Asn Leu Thr Ile Pro Arg Asp Lys Leu Ile Val Ile Thr Gly Leu
20 25 30
Ser Gly Ser Gly Lys Ser Ser Leu Ala Phe Asp Thr Leu Tyr Ala Glu
35 40 45
Gly Gln Arg Arg Tyr Val Glu Ser Leu Ser Ala Tyr Ala Arg Gln Phe
50 55 60
Leu Ser Leu Met Glu Lys Pro Asp Val Asp His Ile Glu Gly Leu Ser
65 70 75 80
Pro Ala Ile Ser Ile Glu Gln Lys Ser Thr Ser His Asn Pro Arg Ser
85 90 95
Thr Val Gly Thr Val Thr Glu Ile His Asp Tyr Leu Arg Leu Leu Phe
100 105 110
Ala Arg Val Gly Glu Pro Arg Cys Pro Asn His Asp Val Pro Leu Ala
115 120 125
Ala Gln Thr Ile Ser Gln Met Val Asp Lys Val Leu Ser Leu Pro Glu
130 135 140
Glu Ser Lys Met Met Leu Leu Ala Pro Val Val Lys Glu Arg Lys Gly
145 150 155 160
Glu His Val Lys Leu Leu Glu Gln Ile Ala Ala Gln Gly Tyr Ile Arg
165 170 175
Ala Arg Ile Asp Gly Glu Ile Cys Asp Leu Ser Asp Ala Pro Lys Leu
180 185 190
Glu Leu His Lys Lys His Thr Ile Glu Val Val Val Asp Arg Phe Lys
195 200 205
Val Arg Ser Asp Leu Ala Thr Arg Leu Ala Glu Ser Phe Glu Thr Ala
210 215 220
Leu Glu Leu Ser Gly Gly Thr Ala Val Val Ala Ser Met Asp Glu Pro
225 230 235 240
Glu Thr Glu Glu Leu Val Phe Ser Ala Asn Phe Ala Cys Pro His Cys
245 250 255
Gly Tyr Ser Val Pro Glu Leu Glu Pro Arg Leu Phe Ser Phe Asn Asn
260 265 270
Pro Ala Gly Ala Cys Pro Thr Cys Asp Gly Leu Gly Val Gln Gln Tyr
275 280 285
Phe Asp Glu Lys Arg Val Val Gln Asn Pro Ser Ile Ser Leu Ala Ser
290 295 300
Gly Ala Val Lys Gly Trp Asp Arg Arg Asn Phe Tyr Tyr Tyr Gln Met
305 310 315 320
Leu Thr Ser Leu Ala Lys His Tyr Glu Phe Asp Ile Glu Ser Pro Phe
325 330 335
Glu Ala Leu Pro Lys Lys Ile Gln Gln Ile Ile Leu Asn Gly Ser Gly
340 345 350
Lys Glu Glu Ile Glu Phe Gln Tyr Met Asn Asp Arg Gly Asp Val Val
355 360 365
Val Arg His His Ala Phe Glu Gly Ile Leu Asn Asn Met Ala Arg Arg
370 375 380
Tyr Lys Glu Thr Glu Ser Leu Ser Val Arg Glu Glu Leu Ala Lys Asn
385 390 395 400
Ile Ser Thr Cys Pro Cys His Asp Cys Gly Gly Ser Arg Leu Arg Gln
405 410 415
Glu Ala Arg His Val Tyr Ile Gly Thr Thr Thr Leu Pro Asp Val Ala
420 425 430
Glu Lys Ser Ile Gly Glu Thr Leu His Phe Phe Ser Glu Leu His Leu
435 440 445
Ser Gly Gln Arg Ala Gln Ile Ala Glu Lys Ile Leu Lys Glu Ile Lys
450 455 460
Glu Arg Leu Gln Phe Leu Val Asn Val Gly Leu Asp Tyr Leu Ser Leu
465 470 475 480
Ser Arg Ser Ala Glu Thr Leu Ser Gly Gly Glu Ala Gln Arg Ile Arg
485 490 495
Leu Ala Ser Gln Ile Gly Ala Gly Leu Val Gly Val Met Tyr Val Leu
500 505 510
Asp Glu Pro Ser Ile Gly Leu His Gln Arg Asp Asn Glu Arg Leu Leu
515 520 525
Asn Thr Leu Leu His Leu Arg Asn Leu Gly Asn Thr Val Ile Val Val
530 535 540
Glu His Asp Glu Asp Ala Ile Met Ala Ala Asp His Ile Ile Asp Ile
545 550 555 560
Gly Pro Gly Ala Gly Val His Gly Gly Gln Ile Val Ala Glu Gly Ser
565 570 575
Ala Lys Ala Ile Met Ala Asn Pro His Ser Ile Thr Gly Lys Phe Leu
580 585 590
Ser Gly Val Glu Lys Ile Glu Ile Pro Ala Lys Arg Thr Ala Leu Asp
595 600 605
Lys Lys Lys Met Leu Lys Leu Glu Gly Ala Thr Gly Asn Asn Leu Lys
610 615 620
Ser Val Asn Leu Ala Ile Pro Val Gly Leu Phe Thr Cys Val Thr Gly
625 630 635 640
Val Ser Gly Ser Gly Lys Ser Thr Leu Ile Asn Asp Thr Leu Phe Pro
645 650 655
Leu Ala Gln Asn Ala Leu Asn Arg Ala Glu Asn Thr Gln Phe Ala Pro
660 665 670
Tyr Gln Ser Ile Ser Gly Leu Glu Phe Phe Asp Lys Val Ile Asp Ile
675 680 685
Asp Gln Ser Pro Ile Gly Arg Thr Pro Arg Ser Asn Pro Ala Thr Tyr
690 695 700
Thr Gly Leu Phe Thr Pro Ile Arg Glu Leu Phe Ala Gly Val Pro Glu
705 710 715 720
Ser Arg Ala Arg Gly Tyr Asn Pro Gly Arg Phe Ser Phe Asn Val Arg
725 730 735
Gly Gly Arg Cys Glu Ala Cys Gln Gly Asp Gly Val Ile Lys Val Glu
740 745 750
Met His Phe Leu Pro Asp Val Tyr Val Pro Cys Glu Gln Cys Lys Gly
755 760 765
Lys Arg Tyr Asn Arg Glu Thr Leu Glu Ile Arg Tyr Lys Gly Lys Thr
770 775 780
Ile His Gln Val Leu Glu Met Thr Val Glu Glu Ala Arg Glu Phe Phe
785 790 795 800
Asp Ala Ile Pro Gln Ile Ala Arg Lys Leu Gln Thr Leu Met Asp Val
805 810 815
Gly Leu Ser Tyr Ile Arg Leu Gly Gln Ser Ser Thr Thr Leu Ser Gly
820 825 830
Gly Glu Ala Gln Arg Val Lys Leu Ala Thr Glu Leu Ser Lys Arg Asp
835 840 845
Thr Gly Lys Thr Leu Tyr Val Leu Asp Glu Pro Thr Thr Gly Leu His
850 855 860
Phe Ala Asp Ile Lys Gln Leu Leu Thr Val Leu His Arg Leu Arg Asp
865 870 875 880
Gln Gly Asn Thr Ile Val Val Ile Glu His Asn Leu Asp Val Ile Lys
885 890 895
Thr Ala Asp Trp Ile Ile Asp Leu Gly Pro Glu Gly Gly Asn Gly Gly
900 905 910
Gly Gln Ile Ile Ala Thr Gly Thr Pro Glu Gln Val Ala Glu Val Lys
915 920 925
Gly Ser His Thr Ala Arg Phe Leu Lys Thr Leu Leu Gln Lys Arg
930 935 940
<210> 27
<211> 54
<212> DNA
<213> Pasteurella multocida
<400> 27
caggacttag tgggtaacaa cacaccagtc ttctttatcc gtgatccatt gaaa 54
<210> 28
<211> 1455
<212> DNA
<213> Pasteurella multocida
<400> 28
atgtctaaat gcccatttga ccatggttca aaaaccttaa cgaatgcagc tggtgcacct 60
attgttgaaa acgacaacac catgtctgcg ggtcctaaag gtccattact tttacaagat 120
gtttggttcc aagagaaatt agcacacttt gcacgtgagc gtattcctga gcgtgttgtt 180
catgcaaaag gttcagcagc gtacggtaca ttcactgtga cacacgatat cagcaaatac 240
accaaagcgg atttattcaa tggtatcggt aaacaaacac aagtcttatt acgtttctca 300
acagtagcgg gtgagcgcgg tgcggcagac gctgagcgtg atgtgcgtgg tttcgcatta 360
aaattctaca ctgaacaagg taactgggac ttagtgggta acaacacacc cgtattcttt 420
atccgtgatc cattgaaatt cccagatttc attcatactc aaaaacgtaa tccacaaact 480
aacttgcgtg atgcaaacgc ggcatgggat ttctggtcac gtcatcctga atcattacac 540
caaatcatga ttcttttcag tgatcgtggt attccaactg acttacgtca tatgaacggt 600
tacggtagcc atacatatag ctttattaac gcacaaaatg agcgtttctg ggtgaaattc 660
cacttcaaaa cacaacaagg tcacaaattc tatactaatg aagaagcggc taaagtggtg 720
ggtgaaaacc gtgagtcaag ccaacaagat ttatacgaag cgattgagcg tggcgaattc 780
ccacgttgga atgttcaagt gcaaatcatg ccagaagcag atgcacacaa acataactat 840
gcgtttgact taactaaagt atggccacac aaagattatc cgatgatcga agtgggtgta 900
ttagagttaa accaaaaccc aattaactac ttcgcagaag tggaacaagc tgcgtttgca 960
ccttctaaca tcgtaccggg aattggtttc tcaccagacc gtatgttaca aggtcgtctt 1020
ttctcatacc aagacgcgca acgttatcgt ttaggggtta accatcacca aatcccagtg 1080
aacgcaccaa aatgcccata ccacaccact caccgtgatg gcgcaatgcg tgtagataac 1140
aatggtggta cacaccctaa ctatgcaccg aaccgttttg atacttatgt gccgactcac 1200
caacaagagc ctgcattaga gttagagcgt tcagcagcac actttaactt ccgtgagtat 1260
gatgaagact actacacaca acctgccgca ctttacaact tattcgatgt ggatcaaaaa 1320
gcacgtgtgg cagccaactt cgcagcgggc ttagcaggtg ttacagaacc tgcgattgtt 1380
gaaagacaat tagcccactt cgacaaagta agcaaagaat tagctgatgc aattcgtgcg 1440
aacttagcga aataa 1455
<210> 29
<211> 484
<212> PRT
<213> Pasteurella multocida
<400> 29
Met Ser Lys Cys Pro Phe Asp His Gly Ser Lys Thr Leu Thr Asn Ala
1 5 10 15
Ala Gly Ala Pro Ile Val Glu Asn Asp Asn Thr Met Ser Ala Gly Pro
20 25 30
Lys Gly Pro Leu Leu Leu Gln Asp Val Trp Phe Gln Glu Lys Leu Ala
35 40 45
His Phe Ala Arg Glu Arg Ile Pro Glu Arg Val Val His Ala Lys Gly
50 55 60
Ser Ala Ala Tyr Gly Thr Phe Thr Val Thr His Asp Ile Ser Lys Tyr
65 70 75 80
Thr Lys Ala Asp Leu Phe Asn Gly Ile Gly Lys Gln Thr Gln Val Leu
85 90 95
Leu Arg Phe Ser Thr Val Ala Gly Glu Arg Gly Ala Ala Asp Ala Glu
100 105 110
Arg Asp Val Arg Gly Phe Ala Leu Lys Phe Tyr Thr Glu Gln Gly Asn
115 120 125
Trp Asp Leu Val Gly Asn Asn Thr Pro Val Phe Phe Ile Arg Asp Pro
130 135 140
Leu Lys Phe Pro Asp Phe Ile His Thr Gln Lys Arg Asn Pro Gln Thr
145 150 155 160
Asn Leu Arg Asp Ala Asn Ala Ala Trp Asp Phe Trp Ser Arg His Pro
165 170 175
Glu Ser Leu His Gln Ile Met Ile Leu Phe Ser Asp Arg Gly Ile Pro
180 185 190
Thr Asp Leu Arg His Met Asn Gly Tyr Gly Ser His Thr Tyr Ser Phe
195 200 205
Ile Asn Ala Gln Asn Glu Arg Phe Trp Val Lys Phe His Phe Lys Thr
210 215 220
Gln Gln Gly His Lys Phe Tyr Thr Asn Glu Glu Ala Ala Lys Val Val
225 230 235 240
Gly Glu Asn Arg Glu Ser Ser Gln Gln Asp Leu Tyr Glu Ala Ile Glu
245 250 255
Arg Gly Glu Phe Pro Arg Trp Asn Val Gln Val Gln Ile Met Pro Glu
260 265 270
Ala Asp Ala His Lys His Asn Tyr Ala Phe Asp Leu Thr Lys Val Trp
275 280 285
Pro His Lys Asp Tyr Pro Met Ile Glu Val Gly Val Leu Glu Leu Asn
290 295 300
Gln Asn Pro Ile Asn Tyr Phe Ala Glu Val Glu Gln Ala Ala Phe Ala
305 310 315 320
Pro Ser Asn Ile Val Pro Gly Ile Gly Phe Ser Pro Asp Arg Met Leu
325 330 335
Gln Gly Arg Leu Phe Ser Tyr Gln Asp Ala Gln Arg Tyr Arg Leu Gly
340 345 350
Val Asn His His Gln Ile Pro Val Asn Ala Pro Lys Cys Pro Tyr His
355 360 365
Thr Thr His Arg Asp Gly Ala Met Arg Val Asp Asn Asn Gly Gly Thr
370 375 380
His Pro Asn Tyr Ala Pro Asn Arg Phe Asp Thr Tyr Val Pro Thr His
385 390 395 400
Gln Gln Glu Pro Ala Leu Glu Leu Glu Arg Ser Ala Ala His Phe Asn
405 410 415
Phe Arg Glu Tyr Asp Glu Asp Tyr Tyr Thr Gln Pro Ala Ala Leu Tyr
420 425 430
Asn Leu Phe Asp Val Asp Gln Lys Ala Arg Val Ala Ala Asn Phe Ala
435 440 445
Ala Gly Leu Ala Gly Val Thr Glu Pro Ala Ile Val Glu Arg Gln Leu
450 455 460
Ala His Phe Asp Lys Val Ser Lys Glu Leu Ala Asp Ala Ile Arg Ala
465 470 475 480
Asn Leu Ala Lys
<210> 30
<211> 172
<212> DNA
<213> Pasteurella multocida
<400> 30
atttctcacg cattttttcg gtaaaaccaa cgggaatcgt tgattttata ataatcgttg 60
cttgtggatt gattgagagg gtttgttcaa tgacagcttc aacagtggat gtattaaaat 120
aacctgtttc ggtattatag tctgttggcg ttgcgatgat gacaaagtcc tg 172
<210> 31
<211> 1173
<212> DNA
<213> Pasteurella multocida
<400> 31
atgaagaaaa ttacaattgc tggggctggc tatgttggtt tatccaatgc agtattatta 60
gctcaacacc acaatgtgat cttattagat attgatcaaa ataaagttga tttaattaat 120
aataaaaaat cgcccatcac agataaagaa atcgaagatt tcttacaaaa taaatcactg 180
acaatgatgg caacaacaga taaagaagtg gcattaaaaa acgcagactt tgtcatcatc 240
gcaacgccaa cagactataa taccgaaaca ggttatttta atacatccac tgttgaagct 300
gtcattgaac aaaccctttc aatcaatcca caagcaacga ttattataaa atcaacaatt 360
cccgttggtt ttaccgaaaa catgcgtgaa aaatttaata ccccaaatct tatcttttca 420
cctgaatttc taagagaggg aaaagccctt tacgataatt tgtatccaag cagaattatt 480
gttggcagta cttcttatca agcaaaagta tttgccgata tgttgacaca gtgtgccaga 540
aaaaaagatg taactgtttt atttacacac aatactgagg ccgaagctgt taaattattt 600
gcaaatacgt atctcgcaat gcgagttgcc ttttttaatg aattagatac ttatgcgagt 660
cttcaccatt taaatacaaa agacattatc aatggtattt ctactgatcc tcgcattggt 720
acacactaca ataacccaag tttcggctat ggcggttatt gtttacccaa agacactaaa 780
cagttactgg ctaactatgc tgacgtgcct caaaatctca ttgaagccat tgtcaaatct 840
aatgaaacca gaaaacgttt cattactcat gatgtattaa ataagaaacc taaaactgtt 900
ggtatttatc gtttaatcat gaagtcaggt tctgataact tcagagcttc tgctattctc 960
gatattatgc cgcatctcaa agaaaacggt gttgagattg tgatttatga gccaacctta 1020
aatcaacagg catttgagga ctaccccgtt attaatcaac tctctgaatt tattaatcgc 1080
tctgatgtca ttctcgctaa tcgttctgag ccagatttaa atcaatgttc ccataaaatc 1140
tatacaagag atatttttgg cggtgatgct taa 1173
<210> 32
<211> 390
<212> PRT
<213> Pasteurella multocida
<400> 32
Met Lys Lys Ile Thr Ile Ala Gly Ala Gly Tyr Val Gly Leu Ser Asn
1 5 10 15
Ala Val Leu Leu Ala Gln His His Asn Val Ile Leu Leu Asp Ile Asp
20 25 30
Gln Asn Lys Val Asp Leu Ile Asn Asn Lys Lys Ser Pro Ile Thr Asp
35 40 45
Lys Glu Ile Glu Asp Phe Leu Gln Asn Lys Ser Leu Thr Met Met Ala
50 55 60
Thr Thr Asp Lys Glu Val Ala Leu Lys Asn Ala Asp Phe Val Ile Ile
65 70 75 80
Ala Thr Pro Thr Asp Tyr Asn Thr Glu Thr Gly Tyr Phe Asn Thr Ser
85 90 95
Thr Val Glu Ala Val Ile Glu Gln Thr Leu Ser Ile Asn Pro Gln Ala
100 105 110
Thr Ile Ile Ile Lys Ser Thr Ile Pro Val Gly Phe Thr Glu Asn Met
115 120 125
Arg Glu Lys Phe Asn Thr Pro Asn Leu Ile Phe Ser Pro Glu Phe Leu
130 135 140
Arg Glu Gly Lys Ala Leu Tyr Asp Asn Leu Tyr Pro Ser Arg Ile Ile
145 150 155 160
Val Gly Ser Thr Ser Tyr Gln Ala Lys Val Phe Ala Asp Met Leu Thr
165 170 175
Gln Cys Ala Arg Lys Lys Asp Val Thr Val Leu Phe Thr His Asn Thr
180 185 190
Glu Ala Glu Ala Val Lys Leu Phe Ala Asn Thr Tyr Leu Ala Met Arg
195 200 205
Val Ala Phe Phe Asn Glu Leu Asp Thr Tyr Ala Ser Leu His His Leu
210 215 220
Asn Thr Lys Asp Ile Ile Asn Gly Ile Ser Thr Asp Pro Arg Ile Gly
225 230 235 240
Thr His Tyr Asn Asn Pro Ser Phe Gly Tyr Gly Gly Tyr Cys Leu Pro
245 250 255
Lys Asp Thr Lys Gln Leu Leu Ala Asn Tyr Ala Asp Val Pro Gln Asn
260 265 270
Leu Ile Glu Ala Ile Val Lys Ser Asn Glu Thr Arg Lys Arg Phe Ile
275 280 285
Thr His Asp Val Leu Asn Lys Lys Pro Lys Thr Val Gly Ile Tyr Arg
290 295 300
Leu Ile Met Lys Ser Gly Ser Asp Asn Phe Arg Ala Ser Ala Ile Leu
305 310 315 320
Asp Ile Met Pro His Leu Lys Glu Asn Gly Val Glu Ile Val Ile Tyr
325 330 335
Glu Pro Thr Leu Asn Gln Gln Ala Phe Glu Asp Tyr Pro Val Ile Asn
340 345 350
Gln Leu Ser Glu Phe Ile Asn Arg Ser Asp Val Ile Leu Ala Asn Arg
355 360 365
Ser Glu Pro Asp Leu Asn Gln Cys Ser His Lys Ile Tyr Thr Arg Asp
370 375 380
Ile Phe Gly Gly Asp Ala
385 390
<210> 33
<211> 226
<212> DNA
<213> Pasteurella multocida
<400> 33
gcaccaaagt gaataatatt tgggaaacct cgggcttgaa tattttagag gtattagtac 60
gtttagatag caccaagtta cctagtttca tttctaacat cctgtccgcg cgaaccaata 120
tttcggcaat ctatattcaa aaagccttca aagtagaacc acagaaatca ctcgaagcgt 180
ttaaggatct tgatactcta gcagatacag cagaagctta tactaa 226
<210> 34
<211> 726
<212> DNA
<213> Pasteurella multocida
<400> 34
atgaataatg atcaacctct tttaaaagca cagagtccgg ctggtttagc ggaagaatat 60
attgtcagaa gtatctggaa taatcatttc ccaccaggct ccgatttgcc ggctgaacgt 120
gagttggcag agaaaattgg ggtgacgcgt accacgttac gtgaagtgct acaacgcctg 180
gcgcgtgacg gttggttgaa tattcaacat gggaaaccaa ccaaagtgaa taatatttgg 240
gaaacttcgg gcttgaatat tttagaggta ttagtacgtt tagatagcac caagttacct 300
agtttcattt ctaacatcct gtccgcgcga accaatattt cggcaatcta tattcaaaaa 360
gccttcaaag tagaaccaca gaaatcactc gaagcgttta aggatcttga tactctagca 420
gatacagcag aagcttatac taattttgat tatgatcttt tccgtaaatt agcatttgca 480
tctgataatc ctgtgtatgg tttgatttta aatagtttga aagggttata tacacgtgta 540
gggttgtttt attttgccaa tccatcggcg cgtgagttag cgaaacgctt ttatctttcg 600
ctaaagacgt tgtgtcaaac acagcaagtg aacgatgtca aagagtgtat ccgtcaatat 660
ggtaaagaca gtggggtgat ttgggcaaat atgcaggcat atttaccggc taattttaat 720
gaatag 726
<210> 35
<211> 241
<212> PRT
<213> Pasteurella multocida
<400> 35
Met Asn Asn Asp Gln Pro Leu Leu Lys Ala Gln Ser Pro Ala Gly Leu
1 5 10 15
Ala Glu Glu Tyr Ile Val Arg Ser Ile Trp Asn Asn His Phe Pro Pro
20 25 30
Gly Ser Asp Leu Pro Ala Glu Arg Glu Leu Ala Glu Lys Ile Gly Val
35 40 45
Thr Arg Thr Thr Leu Arg Glu Val Leu Gln Arg Leu Ala Arg Asp Gly
50 55 60
Trp Leu Asn Ile Gln His Gly Lys Pro Thr Lys Val Asn Asn Ile Trp
65 70 75 80
Glu Thr Ser Gly Leu Asn Ile Leu Glu Val Leu Val Arg Leu Asp Ser
85 90 95
Thr Lys Leu Pro Ser Phe Ile Ser Asn Ile Leu Ser Ala Arg Thr Asn
100 105 110
Ile Ser Ala Ile Tyr Ile Gln Lys Ala Phe Lys Val Glu Pro Gln Lys
115 120 125
Ser Leu Glu Ala Phe Lys Asp Leu Asp Thr Leu Ala Asp Thr Ala Glu
130 135 140
Ala Tyr Thr Asn Phe Asp Tyr Asp Leu Phe Arg Lys Leu Ala Phe Ala
145 150 155 160
Ser Asp Asn Pro Val Tyr Gly Leu Ile Leu Asn Ser Leu Lys Gly Leu
165 170 175
Tyr Thr Arg Val Gly Leu Phe Tyr Phe Ala Asn Pro Ser Ala Arg Glu
180 185 190
Leu Ala Lys Arg Phe Tyr Leu Ser Leu Lys Thr Leu Cys Gln Thr Gln
195 200 205
Gln Val Asn Asp Val Lys Glu Cys Ile Arg Gln Tyr Gly Lys Asp Ser
210 215 220
Gly Val Ile Trp Ala Asn Met Gln Ala Tyr Leu Pro Ala Asn Phe Asn
225 230 235 240
Glu
<210> 36
<211> 214
<212> DNA
<213> Pasteurella multocida
<400> 36
acaaccgctg gcgtatccgt taaacggtgg gttaagcgca cttctttcac gcgctcacca 60
agcaaggttt tcacacgttc cacaaaagaa gcatattgct catcttgtgc tttttgactg 120
tcttcctctt tatccgctaa atcacctaga tctaaatccg ctttactgat ggtttgcagt 180
ggcttaccgt caaattccgt taagtaactt aaca 214
<210> 37
<211> 1896
<212> DNA
<213> Pasteurella multocida
<400> 37
atgtcgacga atcaagaaac gcgtggtttt caatcagaag tcaaacaact tcttcaacta 60
atgatccatt ctctctattc caataaagaa attttcttac gtgaattaat ttccaatgcc 120
tctgatgcgg cagataaatt gcgttttaaa gccttgtctg tgccagagct ttatgaaggt 180
gatggggatt taaaagtgcg tattcgtttt gatgaagaga aaggtacctt aaccattagt 240
gataatggca ttgggatgac gcgtgatgaa gtaatcgatc atttaggtac cattgccaaa 300
tcgggtacca aagaattttt aagtgcatta ggacaagatc aagccaaaga tagccaatta 360
attggtcagt ttggggtcgg tttttattcc gcctttattg tggcagataa agtcactgtg 420
aaaacgcgtg cagcaggcgt aagtgcagat aaagcggtgc tttgggaatc ggcaggcgaa 480
ggtgagtatt ctgtggcgga tattgacaaa aaagaacgcg gtaccgaaat tacccttcac 540
ttacgtgaag atgaaaaagc ctttttaaat gattggcgct tacgtgaaat tatcggcaaa 600
tattcggatc atattggttt gccagtagaa attctagcca aagaatatga cgatgaaggc 660
aaagaaaccg gcattaaatg ggaaaaaatc aataaagcgc aagccttgtg gacacgtgca 720
aaaaatgaga tttcggagga agaatatcaa gagttctata agcatttaag tcatgatttt 780
accgatccgt tactttgggc acacaataaa gtagaaggaa atcaagaata taccagttta 840
ctttatgtgc cagcaaaagc cccttgggat ttatttaatc gcgaacataa acacggctta 900
aagctgtatg tgcaacgtgt ctttattatg gatgatgcgc aagtctttat gccaaattat 960
ctgcgtttta tgcgtggttt attagattcc aatgatttgc cactgaatgt atcgcgcgaa 1020
attttacaag ataacaaagt cacgagtgct ttacgtaaag ccctaacgaa acgtgcattg 1080
caaatgctcg aaaaattagc caaagacgat gcagagaaat accaacgctt ttggcaagag 1140
tttggtttgg tgttaaaaga aggtccagca gaagattttg caaataaaga aacgattgca 1200
aaattattac gttttgcttc aacacacaat gacagcagcc aacaaagcgt gtcgttagaa 1260
gactatgtgg cacgtatgaa agaaggacaa aaggcgattt attatattac ggcagatact 1320
tatgtcgccg cgaaaaactc accgcactta gaattgttca ataagaaagg cattgaagta 1380
ttattgttgt ccgatcgtat tgatgaatgg atgttaagct acttaacgga atttgatggt 1440
aagccactgc aaaccatcag taaagcggat ttagatctag gtgatttagc ggataaagag 1500
gaagacagtc aaaaagcaca agatgagcaa tatgcttctt ttgtggaacg tgtgaaaacc 1560
ttgcttggcg agcgcgtgaa agaagtgcgc ttaactcacc gtttaacgga tacgccagcg 1620
gttgtttcga cgggtgatga ccagatgacc acccaaatgg cgaaattgtt cgctgcggcg 1680
ggtcaagcga tgccagaggt taaatacacc ttcgaattaa atccagaaca tggtttagta 1740
caaaaagtag cagaaattgc cgatgagcag caatttgccg attggattga attgctactt 1800
gaacaagcaa tgttggctga gcgtggtagc cttgaaaatc cagttgcctt tattaaacgc 1860
atgaacacct tgttaagtaa actcacaagt cattaa 1896
<210> 38
<211> 631
<212> PRT
<213> Pasteurella multocida
<400> 38
Met Ser Thr Asn Gln Glu Thr Arg Gly Phe Gln Ser Glu Val Lys Gln
1 5 10 15
Leu Leu Gln Leu Met Ile His Ser Leu Tyr Ser Asn Lys Glu Ile Phe
20 25 30
Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Ala Asp Lys Leu Arg
35 40 45
Phe Lys Ala Leu Ser Val Pro Glu Leu Tyr Glu Gly Asp Gly Asp Leu
50 55 60
Lys Val Arg Ile Arg Phe Asp Glu Glu Lys Gly Thr Leu Thr Ile Ser
65 70 75 80
Asp Asn Gly Ile Gly Met Thr Arg Asp Glu Val Ile Asp His Leu Gly
85 90 95
Thr Ile Ala Lys Ser Gly Thr Lys Glu Phe Leu Ser Ala Leu Gly Gln
100 105 110
Asp Gln Ala Lys Asp Ser Gln Leu Ile Gly Gln Phe Gly Val Gly Phe
115 120 125
Tyr Ser Ala Phe Ile Val Ala Asp Lys Val Thr Val Lys Thr Arg Ala
130 135 140
Ala Gly Val Ser Ala Asp Lys Ala Val Leu Trp Glu Ser Ala Gly Glu
145 150 155 160
Gly Glu Tyr Ser Val Ala Asp Ile Asp Lys Lys Glu Arg Gly Thr Glu
165 170 175
Ile Thr Leu His Leu Arg Glu Asp Glu Lys Ala Phe Leu Asn Asp Trp
180 185 190
Arg Leu Arg Glu Ile Ile Gly Lys Tyr Ser Asp His Ile Gly Leu Pro
195 200 205
Val Glu Ile Leu Ala Lys Glu Tyr Asp Asp Glu Gly Lys Glu Thr Gly
210 215 220
Ile Lys Trp Glu Lys Ile Asn Lys Ala Gln Ala Leu Trp Thr Arg Ala
225 230 235 240
Lys Asn Glu Ile Ser Glu Glu Glu Tyr Gln Glu Phe Tyr Lys His Leu
245 250 255
Ser His Asp Phe Thr Asp Pro Leu Leu Trp Ala His Asn Lys Val Glu
260 265 270
Gly Asn Gln Glu Tyr Thr Ser Leu Leu Tyr Val Pro Ala Lys Ala Pro
275 280 285
Trp Asp Leu Phe Asn Arg Glu His Lys His Gly Leu Lys Leu Tyr Val
290 295 300
Gln Arg Val Phe Ile Met Asp Asp Ala Gln Val Phe Met Pro Asn Tyr
305 310 315 320
Leu Arg Phe Met Arg Gly Leu Leu Asp Ser Asn Asp Leu Pro Leu Asn
325 330 335
Val Ser Arg Glu Ile Leu Gln Asp Asn Lys Val Thr Ser Ala Leu Arg
340 345 350
Lys Ala Leu Thr Lys Arg Ala Leu Gln Met Leu Glu Lys Leu Ala Lys
355 360 365
Asp Asp Ala Glu Lys Tyr Gln Arg Phe Trp Gln Glu Phe Gly Leu Val
370 375 380
Leu Lys Glu Gly Pro Ala Glu Asp Phe Ala Asn Lys Glu Thr Ile Ala
385 390 395 400
Lys Leu Leu Arg Phe Ala Ser Thr His Asn Asp Ser Ser Gln Gln Ser
405 410 415
Val Ser Leu Glu Asp Tyr Val Ala Arg Met Lys Glu Gly Gln Lys Ala
420 425 430
Ile Tyr Tyr Ile Thr Ala Asp Thr Tyr Val Ala Ala Lys Asn Ser Pro
435 440 445
His Leu Glu Leu Phe Asn Lys Lys Gly Ile Glu Val Leu Leu Leu Ser
450 455 460
Asp Arg Ile Asp Glu Trp Met Leu Ser Tyr Leu Thr Glu Phe Asp Gly
465 470 475 480
Lys Pro Leu Gln Thr Ile Ser Lys Ala Asp Leu Asp Leu Gly Asp Leu
485 490 495
Ala Asp Lys Glu Glu Asp Ser Gln Lys Ala Gln Asp Glu Gln Tyr Ala
500 505 510
Ser Phe Val Glu Arg Val Lys Thr Leu Leu Gly Glu Arg Val Lys Glu
515 520 525
Val Arg Leu Thr His Arg Leu Thr Asp Thr Pro Ala Val Val Ser Thr
530 535 540
Gly Asp Asp Gln Met Thr Thr Gln Met Ala Lys Leu Phe Ala Ala Ala
545 550 555 560
Gly Gln Ala Met Pro Glu Val Lys Tyr Thr Phe Glu Leu Asn Pro Glu
565 570 575
His Gly Leu Val Gln Lys Val Ala Glu Ile Ala Asp Glu Gln Gln Phe
580 585 590
Ala Asp Trp Ile Glu Leu Leu Leu Glu Gln Ala Met Leu Ala Glu Arg
595 600 605
Gly Ser Leu Glu Asn Pro Val Ala Phe Ile Lys Arg Met Asn Thr Leu
610 615 620
Leu Ser Lys Leu Thr Ser His
625 630
<210> 39
<211> 252
<212> DNA
<213> Pasteurella multocida
<400> 39
gagttggttc agaatatatt gctcagtatg gcaatgtgag tcttactata caaaatggta 60
aaattcatgg tgagatttat aggcataacc gagggtacga tgatctattt aagctctctg 120
gagaaggccg gaatttaata ttaacgccac ataaaaataa ccctcatgat ctttccccaa 180
caggacccga caacatgaca atggagctga attttatcaa cgcagaaaag actgataaaa 240
aatacgttgt tc 252
<210> 40
<211> 1008
<212> DNA
<213> Pasteurella multocida
<400> 40
atgaaacaaa tcgttttaaa aacaagctta ttgatgaccc tctcttcatt attagttgca 60
tgtagcggcg gtggcggtag cgctggaaat cgtgctgacc gtgtagagga aaaagcacaa 120
ccggttcaat caaatagtga gccttcttcc gctccaatca aaaatcctac taataccgct 180
acgaatgatt ctcttcatga caaactttca atgtcttctc atgacacatc caaagaaaat 240
agtcaacaat cctcctttaa agcccctcta gaacaagaaa aaaaccaacc tgcacaagaa 300
aatctcactt ggacaggtta tcatgtttca gaagtgggaa atgcgagtaa taatgtagat 360
aaagataacg ttacggtatt cactttcgta aaatataatt ctcaatacaa tgatgatcca 420
gtttttgata aaacaaaaac acaaagtaaa acaatatcat tagttgacgg aaaaaatgag 480
aataaagagg attattataa ctttacgtta aaagacgctt tattttatta tggaagttat 540
ggacaacctt cagcagatta caaaaaagta gaaaaaaatt atatttatgc aattaaacca 600
gatgcaataa ataatgagaa cctcaatgca ctaactgcaa cttattatca agaagatggt 660
tttatatatt ccgtattaag tgatgtaaat cgagttggtt cagaatatat tcctcagtat 720
ggcaatgtga ctcttacttt ccgaaatggc aagatttatg gtgaaatcta cagatataat 780
agaggacgtg atgatttgtt tcagctctca ggagaaggac aaaacttaac tataacacca 840
cacaaggaca atccccataa actatcccct acaggacccg acaacatggc aatggagctg 900
aattttatca acgcagaaaa aactgataaa aaatacgttg ttggtgtagg aaaagctgaa 960
aaatattatg ggttattatt tgctgaaaaa agtcaccaag cacaataa 1008
<210> 41
<211> 335
<212> PRT
<213> Pasteurella multocida
<400> 41
Met Lys Gln Ile Val Leu Lys Thr Ser Leu Leu Met Thr Leu Ser Ser
1 5 10 15
Leu Leu Val Ala Cys Ser Gly Gly Gly Gly Ser Ala Gly Asn Arg Ala
20 25 30
Asp Arg Val Glu Glu Lys Ala Gln Pro Val Gln Ser Asn Ser Glu Pro
35 40 45
Ser Ser Ala Pro Ile Lys Asn Pro Thr Asn Thr Ala Thr Asn Asp Ser
50 55 60
Leu His Asp Lys Leu Ser Met Ser Ser His Asp Thr Ser Lys Glu Asn
65 70 75 80
Ser Gln Gln Ser Ser Phe Lys Ala Pro Leu Glu Gln Glu Lys Asn Gln
85 90 95
Pro Ala Gln Glu Asn Leu Thr Trp Thr Gly Tyr His Val Ser Glu Val
100 105 110
Gly Asn Ala Ser Asn Asn Val Asp Lys Asp Asn Val Thr Val Phe Thr
115 120 125
Phe Val Lys Tyr Asn Ser Gln Tyr Asn Asp Asp Pro Val Phe Asp Lys
130 135 140
Thr Lys Thr Gln Ser Lys Thr Ile Ser Leu Val Asp Gly Lys Asn Glu
145 150 155 160
Asn Lys Glu Asp Tyr Tyr Asn Phe Thr Leu Lys Asp Ala Leu Phe Tyr
165 170 175
Tyr Gly Ser Tyr Gly Gln Pro Ser Ala Asp Tyr Lys Lys Val Glu Lys
180 185 190
Asn Tyr Ile Tyr Ala Ile Lys Pro Asp Ala Ile Asn Asn Glu Asn Leu
195 200 205
Asn Ala Leu Thr Ala Thr Tyr Tyr Gln Glu Asp Gly Phe Ile Tyr Ser
210 215 220
Val Leu Ser Asp Val Asn Arg Val Gly Ser Glu Tyr Ile Pro Gln Tyr
225 230 235 240
Gly Asn Val Thr Leu Thr Phe Arg Asn Gly Lys Ile Tyr Gly Glu Ile
245 250 255
Tyr Arg Tyr Asn Arg Gly Arg Asp Asp Leu Phe Gln Leu Ser Gly Glu
260 265 270
Gly Gln Asn Leu Thr Ile Thr Pro His Lys Asp Asn Pro His Lys Leu
275 280 285
Ser Pro Thr Gly Pro Asp Asn Met Ala Met Glu Leu Asn Phe Ile Asn
290 295 300
Ala Glu Lys Thr Asp Lys Lys Tyr Val Val Gly Val Gly Lys Ala Glu
305 310 315 320
Lys Tyr Tyr Gly Leu Leu Phe Ala Glu Lys Ser His Gln Ala Gln
325 330 335
<210> 42
<211> 546
<212> DNA
<213> Pasteurella multocida
<220>
<221> modified_base
<222> (428)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (470)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (497)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (499)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (504)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (507)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (512)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (519)
<223> a, t, c, g, other or unknown
<400> 42
gcttggtatt tacagggaat ccaacctaat cccgttttta gacaggcttt taatgcaatt 60
actgatccca aagaacaatt aattgcttta gaaggttttt ttaatttgat tctgatggat 120
aaagaaaaaa atgttagaac aacaacgtaa tcctgctgat gcactaactg tatcagtgtt 180
aaattcacaa tctcaagtca caaataaacc attgcgtgat tctgtgaaac aagcattgag 240
aaattatttg tcgcagttag atggccaaga tgtcaatgag ctttatgaat tagtattagc 300
agaagttgag catcctatgt tagatatggt tatgcaatat acacgtggaa atcaaactcg 360
tgcagcgaca atgttaggga ttaaccgtgg cactttacgt aagaaattaa aaaagtacgg 420
tatgggtnaa cggaccattg tagtatttaa actagttttg gttatagaan ggcggactta 480
ggtccgcctt tttaatntnc attnccnttt cntttttcna aacaatgatt tttacgccct 540
caaatg 546
<210> 43
<211> 1005
<212> DNA
<213> Pasteurella multocida
<400> 43
atgacagtgc ggataggttc ttatcagctt aaaaatcgca tttttcttgc tcctatggct 60
ggcatcactg accaaccatt tcggcgaatc tgcactcatt atggggcagg tttaactttt 120
tctgaaatga tgtcaacgaa tccgcaagtc tggcataccg aaaaatcgaa actgcgcttg 180
gctcatcatc aagaagcagg aattaatgct gtgcaaatag ctggttgtga tcccgatgag 240
atggcgaaag ctgctcaaat caatgtagaa tatggggcag aaattattga tatcaatatg 300
ggctgcccag ccaaaaaagt gaatcgtaaa atggcgggct ctgcgctgtt acaatatcct 360
gatttggtca aacaaattct taataaagtt gtgaaatctg ttactgtacc agtgacatta 420
aagataagaa caggctggga taaagacaac cgaaattgtt tagaaatcgc taaaattgca 480
gagcaatgtg gtattcaagc actgaccatc cacggacgaa caaggagttg tatgtttgag 540
ggggaggctg aatatgacaa tatcaaggcg gtcaaagagc aactttctat tccgattatt 600
gccaatggcg atattacttc cgctgaaaaa gcaaagtatg ttcttgatta taccaacgca 660
gatgcaataa tgatcggacg tggttcatta ggcaatccgt ggcttttccg agttatggaa 720
agcttaattg aaaaagactc gattgtttta gagccaagtt taaacgagaa atgtaatgtg 780
attttacagc atatccaaga actgcatcaa ttttatggtg tggagaaagg atgtcgtatt 840
gcacgtaaac acgttgcttg gtatttacag ggaatccaac ctaatcccgt ttttagacag 900
gcttttaatg caattactga tcccaaagaa caattaattg ctttagaagg tttttttaat 960
ttgattctga tggataaaga aaaaaatgtt agaacaacaa cgtaa 1005
<210> 44
<211> 334
<212> PRT
<213> Pasteurella multocida
<400> 44
Met Thr Val Arg Ile Gly Ser Tyr Gln Leu Lys Asn Arg Ile Phe Leu
1 5 10 15
Ala Pro Met Ala Gly Ile Thr Asp Gln Pro Phe Arg Arg Ile Cys Thr
20 25 30
His Tyr Gly Ala Gly Leu Thr Phe Ser Glu Met Met Ser Thr Asn Pro
35 40 45
Gln Val Trp His Thr Glu Lys Ser Lys Leu Arg Leu Ala His His Gln
50 55 60
Glu Ala Gly Ile Asn Ala Val Gln Ile Ala Gly Cys Asp Pro Asp Glu
65 70 75 80
Met Ala Lys Ala Ala Gln Ile Asn Val Glu Tyr Gly Ala Glu Ile Ile
85 90 95
Asp Ile Asn Met Gly Cys Pro Ala Lys Lys Val Asn Arg Lys Met Ala
100 105 110
Gly Ser Ala Leu Leu Gln Tyr Pro Asp Leu Val Lys Gln Ile Leu Asn
115 120 125
Lys Val Val Lys Ser Val Thr Val Pro Val Thr Leu Lys Ile Arg Thr
130 135 140
Gly Trp Asp Lys Asp Asn Arg Asn Cys Leu Glu Ile Ala Lys Ile Ala
145 150 155 160
Glu Gln Cys Gly Ile Gln Ala Leu Thr Ile His Gly Arg Thr Arg Ser
165 170 175
Cys Met Phe Glu Gly Glu Ala Glu Tyr Asp Asn Ile Lys Ala Val Lys
180 185 190
Glu Gln Leu Ser Ile Pro Ile Ile Ala Asn Gly Asp Ile Thr Ser Ala
195 200 205
Glu Lys Ala Lys Tyr Val Leu Asp Tyr Thr Asn Ala Asp Ala Ile Met
210 215 220
Ile Gly Arg Gly Ser Leu Gly Asn Pro Trp Leu Phe Arg Val Met Glu
225 230 235 240
Ser Leu Ile Glu Lys Asp Ser Ile Val Leu Glu Pro Ser Leu Asn Glu
245 250 255
Lys Cys Asn Val Ile Leu Gln His Ile Gln Glu Leu His Gln Phe Tyr
260 265 270
Gly Val Glu Lys Gly Cys Arg Ile Ala Arg Lys His Val Ala Trp Tyr
275 280 285
Leu Gln Gly Ile Gln Pro Asn Pro Val Phe Arg Gln Ala Phe Asn Ala
290 295 300
Ile Thr Asp Pro Lys Glu Gln Leu Ile Ala Leu Glu Gly Phe Phe Asn
305 310 315 320
Leu Ile Leu Met Asp Lys Glu Lys Asn Val Arg Thr Thr Thr
325 330
<210> 45
<211> 43
<212> DNA
<213> Pasteurella multocida
<400> 45
gcaaaatttt tggggatggt ctgatcctaa tgcaattcaa ata 43
<210> 46
<211> 1869
<212> DNA
<213> Pasteurella multocida
<400> 46
atgaaaaagg ttattatcat tggacataaa cagtctaact atcaagatgt tgaaaaggtt 60
tttcaatgtt atgggatgaa tcccccgctt ccatcaaaac gtgaaaaaat gtcccccatc 120
gaaattggcc atgtacttaa taaagtatta ccaagtcttg agcacacacc taaaaatgta 180
tctttacttt ctaataagaa aagcaaaata aaaaaaggga attcagccaa aaataaatct 240
cataagcacg ctaaaacgaa cacaatacaa acgacttcga gcatctggga taacttatct 300
ctcgatttga tgctcgcgaa tatcgagcaa aatttttggg gatggtctga tcctaatgca 360
attcaaatat tagattattg ggctaacctt gacccaaaca ttcatttcgt ctttgtttat 420
gataagccag aaaatttatt ccaatatcat agcttagaag aggctctcaa attagataaa 480
cacaccgtac aagaaaaatt tgaagagtgg caaacctaca atgaaaaaat cctaacttac 540
tttaataaat ataaagatcg tagtgtatta ctgaatacac aacaactcca aaatacgaaa 600
aaaacatcac tgtctgaaat ttataaacat atttctgcac ctgatgcatt agtcaaaaaa 660
ctgaatgaac cttctctaaa taaagagatg gaaattattg aagtaaacca agatttatct 720
caccaagaag aatgtccact gtctaacttt attgttagcc aaattataaa aaattctcct 780
actgttacgc aggtatatga agaattacag tcgcatgctg atctgcctta tatttcagaa 840
caaaaattag taaatgatgc cgattttgct ctccttgcat ggaaagatat gattcaaaaa 900
aaagtcgatg taaatcaata tcaacatgaa aaagaattag aacttagcac aataaaagaa 960
cgtcaattag aggtcacaga gagatatcaa ttgacggaac aaaaactgtc agaaacacaa 1020
aaagaaatcg aacaaattaa agatgaaaat agaaaagtaa aatctgaaaa agcaaaactc 1080
actgcatctg ttcaatcaac gagcaaaata ctttctgaga aagaaaaaga gatttcttgc 1140
ataaaaagtg aaaatacaaa gattaaagaa gaaaaaatta aaattgatga agcataccac 1200
ttaaccaaga aaaccttgtc ggataaagaa aaagccctca aaacgcatca agatgaaatt 1260
gaagcgctca agataatttt taatgaaaat atttccgtac aagaagatat gcaagaaaaa 1320
tttcaggaag ccaataaaag aaaacaagaa cttgaacaag agctaaaagc catatcggat 1380
aagaaagcat tattagaaac agaaaacagc caaaaaaccc aagtatctga gtctttagaa 1440
aatgaaaata aagtgttatt agctcaactc caactcattc aagaagaatt agaaaaactt 1500
tatattgaca atcaagtatt aaaagctaaa ccacgccttt acggtgcagc tgatcgcata 1560
aaaaaccaat taacttatcg actaggttac aaaatacaaa gacatggaag aagtctattt 1620
ggtctcattt ttcttccttt catcttattt ttcacctatc tgggctttaa aagagagatg 1680
aaaaagtacg agtggaatac gctcccacca attcatgaat atgaagatgc gcatgaagcc 1740
aatcgcatta aaagccattt atcttataaa ttgggcgtcc tctttttgca agaaatcaac 1800
aatccgttta agtggcttac tctcccttat aaactgatta aagaaggtaa acgattcaag 1860
caaggttaa 1869
<210> 47
<211> 622
<212> PRT
<213> Pasteurella multocida
<400> 47
Met Lys Lys Val Ile Ile Ile Gly His Lys Gln Ser Asn Tyr Gln Asp
1 5 10 15
Val Glu Lys Val Phe Gln Cys Tyr Gly Met Asn Pro Pro Leu Pro Ser
20 25 30
Lys Arg Glu Lys Met Ser Pro Ile Glu Ile Gly His Val Leu Asn Lys
35 40 45
Val Leu Pro Ser Leu Glu His Thr Pro Lys Asn Val Ser Leu Leu Ser
50 55 60
Asn Lys Lys Ser Lys Ile Lys Lys Gly Asn Ser Ala Lys Asn Lys Ser
65 70 75 80
His Lys His Ala Lys Thr Asn Thr Ile Gln Thr Thr Ser Ser Ile Trp
85 90 95
Asp Asn Leu Ser Leu Asp Leu Met Leu Ala Asn Ile Glu Gln Asn Phe
100 105 110
Trp Gly Trp Ser Asp Pro Asn Ala Ile Gln Ile Leu Asp Tyr Trp Ala
115 120 125
Asn Leu Asp Pro Asn Ile His Phe Val Phe Val Tyr Asp Lys Pro Glu
130 135 140
Asn Leu Phe Gln Tyr His Ser Leu Glu Glu Ala Leu Lys Leu Asp Lys
145 150 155 160
His Thr Val Gln Glu Lys Phe Glu Glu Trp Gln Thr Tyr Asn Glu Lys
165 170 175
Ile Leu Thr Tyr Phe Asn Lys Tyr Lys Asp Arg Ser Val Leu Leu Asn
180 185 190
Thr Gln Gln Leu Gln Asn Thr Lys Lys Thr Ser Leu Ser Glu Ile Tyr
195 200 205
Lys His Ile Ser Ala Pro Asp Ala Leu Val Lys Lys Leu Asn Glu Pro
210 215 220
Ser Leu Asn Lys Glu Met Glu Ile Ile Glu Val Asn Gln Asp Leu Ser
225 230 235 240
His Gln Glu Glu Cys Pro Leu Ser Asn Phe Ile Val Ser Gln Ile Ile
245 250 255
Lys Asn Ser Pro Thr Val Thr Gln Val Tyr Glu Glu Leu Gln Ser His
260 265 270
Ala Asp Leu Pro Tyr Ile Ser Glu Gln Lys Leu Val Asn Asp Ala Asp
275 280 285
Phe Ala Leu Leu Ala Trp Lys Asp Met Ile Gln Lys Lys Val Asp Val
290 295 300
Asn Gln Tyr Gln His Glu Lys Glu Leu Glu Leu Ser Thr Ile Lys Glu
305 310 315 320
Arg Gln Leu Glu Val Thr Glu Arg Tyr Gln Leu Thr Glu Gln Lys Leu
325 330 335
Ser Glu Thr Gln Lys Glu Ile Glu Gln Ile Lys Asp Glu Asn Arg Lys
340 345 350
Val Lys Ser Glu Lys Ala Lys Leu Thr Ala Ser Val Gln Ser Thr Ser
355 360 365
Lys Ile Leu Ser Glu Lys Glu Lys Glu Ile Ser Cys Ile Lys Ser Glu
370 375 380
Asn Thr Lys Ile Lys Glu Glu Lys Ile Lys Ile Asp Glu Ala Tyr His
385 390 395 400
Leu Thr Lys Lys Thr Leu Ser Asp Lys Glu Lys Ala Leu Lys Thr His
405 410 415
Gln Asp Glu Ile Glu Ala Leu Lys Ile Ile Phe Asn Glu Asn Ile Ser
420 425 430
Val Gln Glu Asp Met Gln Glu Lys Phe Gln Glu Ala Asn Lys Arg Lys
435 440 445
Gln Glu Leu Glu Gln Glu Leu Lys Ala Ile Ser Asp Lys Lys Ala Leu
450 455 460
Leu Glu Thr Glu Asn Ser Gln Lys Thr Gln Val Ser Glu Ser Leu Glu
465 470 475 480
Asn Glu Asn Lys Val Leu Leu Ala Gln Leu Gln Leu Ile Gln Glu Glu
485 490 495
Leu Glu Lys Leu Tyr Ile Asp Asn Gln Val Leu Lys Ala Lys Pro Arg
500 505 510
Leu Tyr Gly Ala Ala Asp Arg Ile Lys Asn Gln Leu Thr Tyr Arg Leu
515 520 525
Gly Tyr Lys Ile Gln Arg His Gly Arg Ser Leu Phe Gly Leu Ile Phe
530 535 540
Leu Pro Phe Ile Leu Phe Phe Thr Tyr Leu Gly Phe Lys Arg Glu Met
545 550 555 560
Lys Lys Tyr Glu Trp Asn Thr Leu Pro Pro Ile His Glu Tyr Glu Asp
565 570 575
Ala His Glu Ala Asn Arg Ile Lys Ser His Leu Ser Tyr Lys Leu Gly
580 585 590
Val Leu Phe Leu Gln Glu Ile Asn Asn Pro Phe Lys Trp Leu Thr Leu
595 600 605
Pro Tyr Lys Leu Ile Lys Glu Gly Lys Arg Phe Lys Gln Gly
610 615 620
<210> 48
<211> 279
<212> DNA
<213> Pasteurella multocida
<400> 48
accgcttcct gagctacgcc aaattctcac tgccttacca gtatccgcag aacaagcaga 60
aaatgatgat tacttaaccc attttaatcg cagccaagaa ttacttaatt ggcaacattt 120
ttttattgcc cagcaacttg ctttcgttaa cgcattggaa aatcaagaat gaaaaaatgg 180
ttgaaacatt tagatttgag cactggctta caactgtctt ttctgatcag tgggctactt 240
tgtctgtttg tcggtggcgt cgggctttat acttggcac 279
<210> 49
<211> 1983
<212> DNA
<213> Pasteurella multocida
<400> 49
atgaaaaaat ggttgaaaca tttagatttg agcactggct tacaactgtc ttttctgatc 60
agtgggctac tttgtctgtt tgtcggtggc gtcgggcttt atacttggca gcaacaacgc 120
acggaaatca atttcgcact cgataaagat tttcctaaag tgcaagctgc gtttcaaaca 180
gaagaacaaa ttaatatcct ccatcatgcc tttatccatt tggtcaatgt caaaaacacc 240
aatgagaaag tcgaacgtta caaccatgca aagcaacagc tttcgacgtt aaaagaactg 300
atcattgaat tagacgaaaa tttagatgag gatttgatgg cattattaca acaacaagcc 360
tcacttttag aacaaatatc acaaaatatc acaggtacgc ttacgttaaa cgatgaactg 420
aataaaacca tttctcaaat caactggtta cataatgatt ttcacaatga attcaccgca 480
cttttgcaag aaatgagctg gcaacaatct actctggcta acaatattgt tcaacagcca 540
cacaacaaac aaaaaatcga acaattaaaa aaactacaac aagaattatt gttagtttac 600
gatttcacta cttatgaaga gcaaattatc acggaattac gcacccagat aacagagcca 660
actgaaagca atgtcattcg actacacaat tatttgagct atttatcgtt attaattact 720
aaccgaattc agttgcttgg tcttcattcc tccacgtcaa ccattaaaca aattttagat 780
gaactgatta actttggctt aaacccacaa gcactccccg ccctatttgc aatccgtacc 840
gaactgaacc aacaacgaga acagctgatt caacaaagtg ataagatatt cgaggcattt 900
cgcgagcaaa tcagtactca aattggtaac agtaaacaac aattacattt actgcataat 960
attgtcgaaa aaagtactac attcaacggc gcattaattt tattggtgat gctatttgcg 1020
ggaatttttg tcatcggtat taacttcttt tatattcgtt tacgtctctt aaaacgtttt 1080
caacaactta accacgccgt agttcaatta accaatggcg agcccaacgt caaaatcgcc 1140
atttatggca atgatgaatt agggcggatt gctaaattat tgcgcttatt tctgttcgaa 1200
atgaatcaca aaacagaaga gttaaaatcg cgtaatcaag ttctcttaga ggaaatcgaa 1260
caccgtattg aagtacaaac cgcattagaa aatgcccaaa atgaactaac ccaagccgca 1320
aaactggctg ctgtcggtaa aaccttgact tcgattagcc atgaaattac acaaccactt 1380
aatgccatga acgcttattt gtttagtgcg aaaaaagccg tgagtaaaca aaacagtgag 1440
gcagcacttg aatacttaaa taaaatcaat catttagttg aacgcacggc gctgattgtc 1500
aaacgcttac ggcaattctc acgccaaggg agcggcaaaa tacaagctgt caatttaatg 1560
gattgtattc aaagcgcgtg ggaattattg gaatcacaac ataaaccgcg tcaaagtcag 1620
ctcatcacgc ccacagattt accactcgta ttaggtgaag atgtccttat cgaacaagtg 1680
tttgtcaatc tcttcctcaa tgctttagaa gccattgaac acacaccgcc ccaaattcat 1740
attgacgttg acagcgataa tgcggaagac ctctgtttat ggatcaccga caatggtcaa 1800
ggttggccct taactgacaa gttattgcaa cctttttcga gcagtaaatc gatcaattta 1860
ggtttaggac tgtccattag tcaatccatc atggagcaat gtcaaggatc attgaccatt 1920
gcctctactc tcacccataa tgcattagtg atattaaaat ttaaggtggc tcaacatgtt 1980
taa 1983
<210> 50
<211> 660
<212> PRT
<213> Pasteurella multocida
<400> 50
Met Lys Lys Trp Leu Lys His Leu Asp Leu Ser Thr Gly Leu Gln Leu
1 5 10 15
Ser Phe Leu Ile Ser Gly Leu Leu Cys Leu Phe Val Gly Gly Val Gly
20 25 30
Leu Tyr Thr Trp Gln Gln Gln Arg Thr Glu Ile Asn Phe Ala Leu Asp
35 40 45
Lys Asp Phe Pro Lys Val Gln Ala Ala Phe Gln Thr Glu Glu Gln Ile
50 55 60
Asn Ile Leu His His Ala Phe Ile His Leu Val Asn Val Lys Asn Thr
65 70 75 80
Asn Glu Lys Val Glu Arg Tyr Asn His Ala Lys Gln Gln Leu Ser Thr
85 90 95
Leu Lys Glu Leu Ile Ile Glu Leu Asp Glu Asn Leu Asp Glu Asp Leu
100 105 110
Met Ala Leu Leu Gln Gln Gln Ala Ser Leu Leu Glu Gln Ile Ser Gln
115 120 125
Asn Ile Thr Gly Thr Leu Thr Leu Asn Asp Glu Leu Asn Lys Thr Ile
130 135 140
Ser Gln Ile Asn Trp Leu His Asn Asp Phe His Asn Glu Phe Thr Ala
145 150 155 160
Leu Leu Gln Glu Met Ser Trp Gln Gln Ser Thr Leu Ala Asn Asn Ile
165 170 175
Val Gln Gln Pro His Asn Lys Gln Lys Ile Glu Gln Leu Lys Lys Leu
180 185 190
Gln Gln Glu Leu Leu Leu Val Tyr Asp Phe Thr Thr Tyr Glu Glu Gln
195 200 205
Ile Ile Thr Glu Leu Arg Thr Gln Ile Thr Glu Pro Thr Glu Ser Asn
210 215 220
Val Ile Arg Leu His Asn Tyr Leu Ser Tyr Leu Ser Leu Leu Ile Thr
225 230 235 240
Asn Arg Ile Gln Leu Leu Gly Leu His Ser Ser Thr Ser Thr Ile Lys
245 250 255
Gln Ile Leu Asp Glu Leu Ile Asn Phe Gly Leu Asn Pro Gln Ala Leu
260 265 270
Pro Ala Leu Phe Ala Ile Arg Thr Glu Leu Asn Gln Gln Arg Glu Gln
275 280 285
Leu Ile Gln Gln Ser Asp Lys Ile Phe Glu Ala Phe Arg Glu Gln Ile
290 295 300
Ser Thr Gln Ile Gly Asn Ser Lys Gln Gln Leu His Leu Leu His Asn
305 310 315 320
Ile Val Glu Lys Ser Thr Thr Phe Asn Gly Ala Leu Ile Leu Leu Val
325 330 335
Met Leu Phe Ala Gly Ile Phe Val Ile Gly Ile Asn Phe Phe Tyr Ile
340 345 350
Arg Leu Arg Leu Leu Lys Arg Phe Gln Gln Leu Asn His Ala Val Val
355 360 365
Gln Leu Thr Asn Gly Glu Pro Asn Val Lys Ile Ala Ile Tyr Gly Asn
370 375 380
Asp Glu Leu Gly Arg Ile Ala Lys Leu Leu Arg Leu Phe Leu Phe Glu
385 390 395 400
Met Asn His Lys Thr Glu Glu Leu Lys Ser Arg Asn Gln Val Leu Leu
405 410 415
Glu Glu Ile Glu His Arg Ile Glu Val Gln Thr Ala Leu Glu Asn Ala
420 425 430
Gln Asn Glu Leu Thr Gln Ala Ala Lys Leu Ala Ala Val Gly Lys Thr
435 440 445
Leu Thr Ser Ile Ser His Glu Ile Thr Gln Pro Leu Asn Ala Met Asn
450 455 460
Ala Tyr Leu Phe Ser Ala Lys Lys Ala Val Ser Lys Gln Asn Ser Glu
465 470 475 480
Ala Ala Leu Glu Tyr Leu Asn Lys Ile Asn His Leu Val Glu Arg Thr
485 490 495
Ala Leu Ile Val Lys Arg Leu Arg Gln Phe Ser Arg Gln Gly Ser Gly
500 505 510
Lys Ile Gln Ala Val Asn Leu Met Asp Cys Ile Gln Ser Ala Trp Glu
515 520 525
Leu Leu Glu Ser Gln His Lys Pro Arg Gln Ser Gln Leu Ile Thr Pro
530 535 540
Thr Asp Leu Pro Leu Val Leu Gly Glu Asp Val Leu Ile Glu Gln Val
545 550 555 560
Phe Val Asn Leu Phe Leu Asn Ala Leu Glu Ala Ile Glu His Thr Pro
565 570 575
Pro Gln Ile His Ile Asp Val Asp Ser Asp Asn Ala Glu Asp Leu Cys
580 585 590
Leu Trp Ile Thr Asp Asn Gly Gln Gly Trp Pro Leu Thr Asp Lys Leu
595 600 605
Leu Gln Pro Phe Ser Ser Ser Lys Ser Ile Asn Leu Gly Leu Gly Leu
610 615 620
Ser Ile Ser Gln Ser Ile Met Glu Gln Cys Gln Gly Ser Leu Thr Ile
625 630 635 640
Ala Ser Thr Leu Thr His Asn Ala Leu Val Ile Leu Lys Phe Lys Val
645 650 655
Ala Gln His Val
660
<210> 51
<211> 93
<212> DNA
<213> Pasteurella multocida
<400> 51
atagaaatgg tttatttcca aatttcctca aatttcacct tggcttttaa gaattttggc 60
gttgccacta aattacagta gctgttttgt gct 93
<210> 52
<211> 525
<212> DNA
<213> Pasteurella multocida
<400> 52
atgacacaac aagcgatctt tgccggcggc tgtttttggt gcgttgaggc agtatttaat 60
caaattaaag gcgttgaaaa agcgacttca ggttatatta acgggacgac tgaaaatcca 120
acttacaaag aagtatgtac cggtgaaacg ggtcatgcgg aagcggtaaa agtggaattc 180
gatgcgacag tgattagtta tgaaaaatta ttagacatct tcttttctat ccataatcca 240
acccaattaa atcaccaggg cgaagatgtg ggaacgcaat atcgcacagg gatttactat 300
ttaaatgatg aacaagaaca gctggcaaat aagaaaattg cagaattaca accgcacttt 360
gccgaaaaaa ttgtcactga agtgctgcca gcacaaactt tttatcccgc agaagattat 420
caccaaggct atttattgca gaacccacaa aacagctact gtaatttagt ggcaacgcca 480
aaattcttaa aagccaaggt gaaatttgag gaaatttgga agtaa 525
<210> 53
<211> 174
<212> PRT
<213> Pasteurella multocida
<400> 53
Met Thr Gln Gln Ala Ile Phe Ala Gly Gly Cys Phe Trp Cys Val Glu
1 5 10 15
Ala Val Phe Asn Gln Ile Lys Gly Val Glu Lys Ala Thr Ser Gly Tyr
20 25 30
Ile Asn Gly Thr Thr Glu Asn Pro Thr Tyr Lys Glu Val Cys Thr Gly
35 40 45
Glu Thr Gly His Ala Glu Ala Val Lys Val Glu Phe Asp Ala Thr Val
50 55 60
Ile Ser Tyr Glu Lys Leu Leu Asp Ile Phe Phe Ser Ile His Asn Pro
65 70 75 80
Thr Gln Leu Asn His Gln Gly Glu Asp Val Gly Thr Gln Tyr Arg Thr
85 90 95
Gly Ile Tyr Tyr Leu Asn Asp Glu Gln Glu Gln Leu Ala Asn Lys Lys
100 105 110
Ile Ala Glu Leu Gln Pro His Phe Ala Glu Lys Ile Val Thr Glu Val
115 120 125
Leu Pro Ala Gln Thr Phe Tyr Pro Ala Glu Asp Tyr His Gln Gly Tyr
130 135 140
Leu Leu Gln Asn Pro Gln Asn Ser Tyr Cys Asn Leu Val Ala Thr Pro
145 150 155 160
Lys Phe Leu Lys Ala Lys Val Lys Phe Glu Glu Ile Trp Lys
165 170
<210> 54
<211> 772
<212> DNA
<213> Pasteurella multocida
<220>
<221> modified_base
<222> (537)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (540)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (545)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (581)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (613)..(614)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (665)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (668)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (685)..(686)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (700)..(704)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (721)..(723)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (732)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (734)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (737)..(738)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (748)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (752)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (755)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (770)..(772)
<223> a, t, c, g, other or unknown
<400> 54
gcataggtat cctttgcttg acataaaatg actacaggct aaagcacagg atattaaaag 60
ggcaaaagaa taagttactt ttgcgacacg catcgcaaaa gtaaaataaa tttagtcaat 120
caatttagct tgttttaaga aatactcaat gccatgttct ttgatcggta aggtgacatg 180
atcggctact gtttttagct gatgatgtgc attccccatt gcaacaccca ctcctgccgt 240
gcttaacatt tcaatatcat tcaagccatc accaaatgcc atcacatttt ccattgcaaa 300
gccaaaatgt tgaattgcac aagcgatacc cgtagctttt gagatttttt catcaaataa 360
atcaaccgag tatttatgcc agcgtaccga ttgtaatcct ttcagtacac cagaatcttg 420
gacaaattga tcttgcgtag catcataaaa agccagtatc tgaaaaacat catgactgtt 480
taaaatagtc tttatctaca tgataatgcc cttttagcgg atccaatgca tcacganctn 540
gatcngttat cgctgaaact gcggtatctg tcggtgacac ntgcgcataa caatctgatg 600
ttgatcacaa aannacgaac tctggatttt gcttagataa ggatctccga tggctatcta 660
tactnatntg acatcatgta cacanncatg tcgtgccatn nnnnacttag cgaagtgcag 720
nnnccgtcat cngncannca gacatcantc cnacntgcta ttaggataan nn 772
<210> 55
<211> 687
<212> DNA
<213> Pasteurella multocida
<400> 55
atgcggggat ttggtttttg ttgtgttcac tgccagcaac cgttagccat tgcacatcat 60
ggattatgta gtcgctgtaa tcagcaaatc agacgttttg cttattgtgg ccattgtggt 120
aaggaattaa cacgagatgc actacgttgt gggcattgtt tacaacataa agccagttgg 180
gatcgcatgg tgatcgttgg tcactatgtc gatcccttat cttgtttaat tcaccgtttt 240
aaattccaac atgccttctt tttagaccgt actttagcac gcctgctatt attagcgctc 300
tatcatgcaa gacgtactca tggacttatt tggccagaag tacttttgcc ggtgccttta 360
catcgtttac gtcattggca acgtggttac aatcaatctg cgttgattgc aaactatctt 420
gcgcattggc taaagatacc ctgcgatcat gattttctac agcgtattaa acatactcat 480
acgcaacgtg gtttaagtgc aacggaacga agaaaaaatt tacgccacgc atttcgtctt 540
catccaaaaa gtcaaacgca tcgctatcaa tctgttgcat taattgatga tgtaattaca 600
acaggtgcaa cgttgaatga gttggcactc ttattaaaaa aagcaggtgt tgagcatatt 660
caagtttggg gattagcaaa aacgtaa 687
<210> 56
<211> 228
<212> PRT
<213> Pasteurella multocida
<400> 56
Met Arg Gly Phe Gly Phe Cys Cys Val His Cys Gln Gln Pro Leu Ala
1 5 10 15
Ile Ala His His Gly Leu Cys Ser Arg Cys Asn Gln Gln Ile Arg Arg
20 25 30
Phe Ala Tyr Cys Gly His Cys Gly Lys Glu Leu Thr Arg Asp Ala Leu
35 40 45
Arg Cys Gly His Cys Leu Gln His Lys Ala Ser Trp Asp Arg Met Val
50 55 60
Ile Val Gly His Tyr Val Asp Pro Leu Ser Cys Leu Ile His Arg Phe
65 70 75 80
Lys Phe Gln His Ala Phe Phe Leu Asp Arg Thr Leu Ala Arg Leu Leu
85 90 95
Leu Leu Ala Leu Tyr His Ala Arg Arg Thr His Gly Leu Ile Trp Pro
100 105 110
Glu Val Leu Leu Pro Val Pro Leu His Arg Leu Arg His Trp Gln Arg
115 120 125
Gly Tyr Asn Gln Ser Ala Leu Ile Ala Asn Tyr Leu Ala His Trp Leu
130 135 140
Lys Ile Pro Cys Asp His Asp Phe Leu Gln Arg Ile Lys His Thr His
145 150 155 160
Thr Gln Arg Gly Leu Ser Ala Thr Glu Arg Arg Lys Asn Leu Arg His
165 170 175
Ala Phe Arg Leu His Pro Lys Ser Gln Thr His Arg Tyr Gln Ser Val
180 185 190
Ala Leu Ile Asp Asp Val Ile Thr Thr Gly Ala Thr Leu Asn Glu Leu
195 200 205
Ala Leu Leu Leu Lys Lys Ala Gly Val Glu His Ile Gln Val Trp Gly
210 215 220
Leu Ala Lys Thr
225
<210> 57
<211> 700
<212> DNA
<213> Pasteurella multocida
<220>
<221> modified_base
<222> (498)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (540)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (562)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (565)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (572)..(573)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (577)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (584)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (587)..(588)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (591)..(592)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (594)..(595)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (599)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (604)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (619)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (623)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (625)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (627)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (633)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (638)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (644)..(645)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (653)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (657)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (661)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (664)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (666)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (671)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (673)..(674)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (678)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (682)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (686)..(687)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (694)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (696)..(697)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (699)..(700)
<223> a, t, c, g, other or unknown
<400> 57
tgccgaccac tccaaaggac aaaaaatgag cctatttgcg attttctatc tgttcctggc 60
gtatttatta ggatctgttt ctagtgcaat tttattgtgt cgtttagcgg ggttgcctga 120
tcctagagaa agtggttctc ataatcccgg tgcaaccaat gtattgcgta ttggtgggcg 180
ttgggtggca ttgagtgtac tcctgtttga tatgctcaaa ggtatgttac ctgtttggtt 240
aggctattat cttggtttga ctcattttga gttagggatg gtggcattag gtgcttgttt 300
agggcacatt ttcccaatct tctttaaatt taaaggcgga aaaggggtag caacggcatt 360
tggtgctatt gcgccgattt catggggtgt cgcaggcagt atgctgggca cttggttatt 420
gattttcttc gtgagtggtt attcttcgct cagtgcagtg atgaccgcgc ttctggtacc 480
tttctatgtg tggtggtnta agcccgagtt tactttccct gtcgcttagt gtgttgcttn 540
tcgattatcg ccatcatgac anatncagcg tnngtgngtg ggcnagnnga nnanngtgna 600
atanactgaa aacaaaaang atnantnagc tanttacnaa aaanngacag acngtcnttt 660
natncncgtt nanntatnga cntatnngat ggcntnncnn 700
<210> 58
<211> 606
<212> DNA
<213> Pasteurella multocida
<400> 58
atgagcctat ttgcgatttt ctatctgttc ctggcgtatt tattaggatc tgtttctagt 60
gcaattttat tgtgtcgttt agcggggttg cctgatccaa gagaaagtgg ttctcataat 120
cccggtgcaa ccaatgtctt gcgtattggt gggcgttggg tggcattgag tgtactcctg 180
tttgatatgc ttaaaggtat gttacctgtt tggttaggct attatcttgg tttgactcat 240
tttgaattag ggatggtggc attaggtgct tgtttagggc acatttttcc aatcttcttt 300
aaatttaaag gcggaaaagg ggtggcaacg gcatttggtg ctattgcgcc gatctcatgg 360
ggtgtcgctg gcagtatgct aggcacttgg ttattgattt tcttcgtgag tggttattct 420
tcgctcagtg cggtgatgac cgcgcttctg gtacctttct atgtgtggtg gtttaagccc 480
gagtttactt tccctgtcgc tttagtgtgt tgcttgttga tttatcgcca tcatgacaat 540
attcagcgtt tgtggcgtgg gcaagaagac aaagtgtgga ataaactgaa aaacaaaaaa 600
gattaa 606
<210> 59
<211> 201
<212> PRT
<213> Pasteurella multocida
<400> 59
Met Ser Leu Phe Ala Ile Phe Tyr Leu Phe Leu Ala Tyr Leu Leu Gly
1 5 10 15
Ser Val Ser Ser Ala Ile Leu Leu Cys Arg Leu Ala Gly Leu Pro Asp
20 25 30
Pro Arg Glu Ser Gly Ser His Asn Pro Gly Ala Thr Asn Val Leu Arg
35 40 45
Ile Gly Gly Arg Trp Val Ala Leu Ser Val Leu Leu Phe Asp Met Leu
50 55 60
Lys Gly Met Leu Pro Val Trp Leu Gly Tyr Tyr Leu Gly Leu Thr His
65 70 75 80
Phe Glu Leu Gly Met Val Ala Leu Gly Ala Cys Leu Gly His Ile Phe
85 90 95
Pro Ile Phe Phe Lys Phe Lys Gly Gly Lys Gly Val Ala Thr Ala Phe
100 105 110
Gly Ala Ile Ala Pro Ile Ser Trp Gly Val Ala Gly Ser Met Leu Gly
115 120 125
Thr Trp Leu Leu Ile Phe Phe Val Ser Gly Tyr Ser Ser Leu Ser Ala
130 135 140
Val Met Thr Ala Leu Leu Val Pro Phe Tyr Val Trp Trp Phe Lys Pro
145 150 155 160
Glu Phe Thr Phe Pro Val Ala Leu Val Cys Cys Leu Leu Ile Tyr Arg
165 170 175
His His Asp Asn Ile Gln Arg Leu Trp Arg Gly Gln Glu Asp Lys Val
180 185 190
Trp Asn Lys Leu Lys Asn Lys Lys Asp
195 200
<210> 60
<211> 188
<212> DNA
<213> Pasteurella multocida
<400> 60
atcccaccga taaaaccaaa tggattacgc gcagtttcca aataaacagg ggtagcacca 60
gcttgaatta atgcaccatg atggatagat ttgtgattgt tacgatcaaa gagcacaaga 120
tcacccggtg tgagtaacgc attggtgacg actttatttg cagaagatgt cccatttaag 180
acgaagta 188
<210> 61
<211> 2163
<212> DNA
<213> Pasteurella multocida
<400> 61
atgctaaatt taaaaattgc atatagccca ttaattcgac cttattttca tacaaataga 60
gaactcgttt ctgttcaaga aacagatttc accgacattg gtgcaattat cctgagttct 120
gaagatattg aagattatat tgatagtata caagcaactg aatttaatat tccggtcttt 180
gttgctgtca ttgaaggtca atttttagat cctcaattct ttgacaaagt ctatcacgtt 240
caagatctca acaactacga catcaaccta tacagtcgcc aaattgaaac tgcggcgcgg 300
ttttacgaag aaaaaatcct cccacctttc tttaaaatgc taagtgaata tgtggaaatg 360
gggaattctg cttttgattg tccgggacac caaggtggac aatatttccg taaacatcct 420
gcaggacgtt atctctatga tttctacggt gaaaatattt tccgctcaga tatctgtaat 480
gccgatgtaa aattaggcga tttgctaatc catgaaggag ccgcttgtga tgctcaaaaa 540
cacgctgctc aagtctttaa tgctgataaa acctacttcg tcttaaatgg gacatcttct 600
gcaaataaag tcgtcaccaa tgcgttactc acaccgggtg atcttgtgct ctttgatcgt 660
aacaatcaca aatctatcca tcacggtgca ttaattcaag ctggtgctac ccctgtttat 720
ttggaaactg cgcgtaatcc atttggtttt atcggtggga tcgatagcca ttgttttgat 780
gaagattatt tgaaatcttt aattaaagat gttgcgcctg aaaaactaac acaagcacgt 840
cctttccgtt tagccgttat tcagctcggc acttatgacg gaaccatcta taatgcgcgc 900
caagtcgtag ataaaattgg tcatttatgt gactacatct tgtttgattc tgcgtgggta 960
ggttatgaac aattcattcc aatgatgaaa gattgctcac cgctcttgct tgaattaaat 1020
gaaaatgatc ccggcatcat cgtgacacaa tcagtacaca aacaacaagc cggcttctca 1080
caagcctcac aaattcacaa aaaagacaag cacattaaag gtcaacagcg ctactgtaat 1140
cataaacgct ttaataatgc attcatgtta cacgcctcca ccagcccatt ctaccctctt 1200
tttgccacac ttgatgtcaa tgcaaaaatt caaggtaccc ctgcgggtat tcgtttatgg 1260
catgactgtg tcaaaatcgg gatagaagca cgtaaaatgg tgctgaatag ttgtgatctg 1320
atcaaaccgt ttattccgcc ttatgtcaat ggcaaaaaat ggcaagacta cgatacagaa 1380
gaaatggcaa atgatttaac attcttcaaa ttccatgctg atgataaatg gcatcaattt 1440
gaaggctatg tagataacca atattttgtt gatccatgta aattcatgct aacgacgccg 1500
ggtattgata ttgaaacagg tgaatacgaa gacttcggtg tccctgctac gattcttgct 1560
aattatttac gtgaaaacgg cattattccg gaaaaatgtg acttaaactc aattctcttc 1620
ttattaacgc cagcagaaac cctcaccaaa atgcaaagtt tggttgcaca aattgcggca 1680
tttgaacaac acatcaaaaa agattcctta ctaaaagaag tcttaccaag tgtttatcac 1740
aacaatgaaa aacgctatga aggttatacc atccgtcgtc tttgccaaga aatgcatgat 1800
ttgtatgtca gccgtaacgt gaaaacttta caacgcaact tattcagaaa agcgaccttg 1860
cctgaatatg tgatgaatcc acatcaagct aatcttgaat ttgttcgtaa tcgtgtagaa 1920
ctggttccac taaccgaaat cgttaatcgc attgcggcag aaggagcact tccttatcca 1980
ccgggtgtgc tttgtgtcgt accgggtgaa aaatggagtc agactgcaca ggaatatttc 2040
ttagcactcg aagaaggcat taatttatta ccaggtttcg caccagaaat tcaaggggta 2100
tatctacaac aagatgcaga tggacgtatt cgtgcttatg gctacgtatt aactgaaaac 2160
taa 2163
<210> 62
<211> 720
<212> PRT
<213> Pasteurella multocida
<400> 62
Met Leu Asn Leu Lys Ile Ala Tyr Ser Pro Leu Ile Arg Pro Tyr Phe
1 5 10 15
His Thr Asn Arg Glu Leu Val Ser Val Gln Glu Thr Asp Phe Thr Asp
20 25 30
Ile Gly Ala Ile Ile Leu Ser Ser Glu Asp Ile Glu Asp Tyr Ile Asp
35 40 45
Ser Ile Gln Ala Thr Glu Phe Asn Ile Pro Val Phe Val Ala Val Ile
50 55 60
Glu Gly Gln Phe Leu Asp Pro Gln Phe Phe Asp Lys Val Tyr His Val
65 70 75 80
Gln Asp Leu Asn Asn Tyr Asp Ile Asn Leu Tyr Ser Arg Gln Ile Glu
85 90 95
Thr Ala Ala Arg Phe Tyr Glu Glu Lys Ile Leu Pro Pro Phe Phe Lys
100 105 110
Met Leu Ser Glu Tyr Val Glu Met Gly Asn Ser Ala Phe Asp Cys Pro
115 120 125
Gly His Gln Gly Gly Gln Tyr Phe Arg Lys His Pro Ala Gly Arg Tyr
130 135 140
Leu Tyr Asp Phe Tyr Gly Glu Asn Ile Phe Arg Ser Asp Ile Cys Asn
145 150 155 160
Ala Asp Val Lys Leu Gly Asp Leu Leu Ile His Glu Gly Ala Ala Cys
165 170 175
Asp Ala Gln Lys His Ala Ala Gln Val Phe Asn Ala Asp Lys Thr Tyr
180 185 190
Phe Val Leu Asn Gly Thr Ser Ser Ala Asn Lys Val Val Thr Asn Ala
195 200 205
Leu Leu Thr Pro Gly Asp Leu Val Leu Phe Asp Arg Asn Asn His Lys
210 215 220
Ser Ile His His Gly Ala Leu Ile Gln Ala Gly Ala Thr Pro Val Tyr
225 230 235 240
Leu Glu Thr Ala Arg Asn Pro Phe Gly Phe Ile Gly Gly Ile Asp Ser
245 250 255
His Cys Phe Asp Glu Asp Tyr Leu Lys Ser Leu Ile Lys Asp Val Ala
260 265 270
Pro Glu Lys Leu Thr Gln Ala Arg Pro Phe Arg Leu Ala Val Ile Gln
275 280 285
Leu Gly Thr Tyr Asp Gly Thr Ile Tyr Asn Ala Arg Gln Val Val Asp
290 295 300
Lys Ile Gly His Leu Cys Asp Tyr Ile Leu Phe Asp Ser Ala Trp Val
305 310 315 320
Gly Tyr Glu Gln Phe Ile Pro Met Met Lys Asp Cys Ser Pro Leu Leu
325 330 335
Leu Glu Leu Asn Glu Asn Asp Pro Gly Ile Ile Val Thr Gln Ser Val
340 345 350
His Lys Gln Gln Ala Gly Phe Ser Gln Ala Ser Gln Ile His Lys Lys
355 360 365
Asp Lys His Ile Lys Gly Gln Gln Arg Tyr Cys Asn His Lys Arg Phe
370 375 380
Asn Asn Ala Phe Met Leu His Ala Ser Thr Ser Pro Phe Tyr Pro Leu
385 390 395 400
Phe Ala Thr Leu Asp Val Asn Ala Lys Ile Gln Gly Thr Pro Ala Gly
405 410 415
Ile Arg Leu Trp His Asp Cys Val Lys Ile Gly Ile Glu Ala Arg Lys
420 425 430
Met Val Leu Asn Ser Cys Asp Leu Ile Lys Pro Phe Ile Pro Pro Tyr
435 440 445
Val Asn Gly Lys Lys Trp Gln Asp Tyr Asp Thr Glu Glu Met Ala Asn
450 455 460
Asp Leu Thr Phe Phe Lys Phe His Ala Asp Asp Lys Trp His Gln Phe
465 470 475 480
Glu Gly Tyr Val Asp Asn Gln Tyr Phe Val Asp Pro Cys Lys Phe Met
485 490 495
Leu Thr Thr Pro Gly Ile Asp Ile Glu Thr Gly Glu Tyr Glu Asp Phe
500 505 510
Gly Val Pro Ala Thr Ile Leu Ala Asn Tyr Leu Arg Glu Asn Gly Ile
515 520 525
Ile Pro Glu Lys Cys Asp Leu Asn Ser Ile Leu Phe Leu Leu Thr Pro
530 535 540
Ala Glu Thr Leu Thr Lys Met Gln Ser Leu Val Ala Gln Ile Ala Ala
545 550 555 560
Phe Glu Gln His Ile Lys Lys Asp Ser Leu Leu Lys Glu Val Leu Pro
565 570 575
Ser Val Tyr His Asn Asn Glu Lys Arg Tyr Glu Gly Tyr Thr Ile Arg
580 585 590
Arg Leu Cys Gln Glu Met His Asp Leu Tyr Val Ser Arg Asn Val Lys
595 600 605
Thr Leu Gln Arg Asn Leu Phe Arg Lys Ala Thr Leu Pro Glu Tyr Val
610 615 620
Met Asn Pro His Gln Ala Asn Leu Glu Phe Val Arg Asn Arg Val Glu
625 630 635 640
Leu Val Pro Leu Thr Glu Ile Val Asn Arg Ile Ala Ala Glu Gly Ala
645 650 655
Leu Pro Tyr Pro Pro Gly Val Leu Cys Val Val Pro Gly Glu Lys Trp
660 665 670
Ser Gln Thr Ala Gln Glu Tyr Phe Leu Ala Leu Glu Glu Gly Ile Asn
675 680 685
Leu Leu Pro Gly Phe Ala Pro Glu Ile Gln Gly Val Tyr Leu Gln Gln
690 695 700
Asp Ala Asp Gly Arg Ile Arg Ala Tyr Gly Tyr Val Leu Thr Glu Asn
705 710 715 720
<210> 63
<211> 101
<212> DNA
<213> Pasteurella multocida
<400> 63
gaaaaattag agaaacaaat agaatcactc aatctacaag aagattgttt tcttttagga 60
aataaagata atccgtatcc attaataaaa aatgctaagc t 101
<210> 64
<211> 1179
<212> DNA
<213> Pasteurella multocida
<400> 64
atgaatattc tatttgtaca taaaagcctt gtcgtcggag gcgctgaaag aattctaatt 60
aactatttaa atattctatc tggatttaat gaattcaaag ttacattact tttactagaa 120
aataaaggtg aagataacaa aaacatcaat caaatcaata aaaatattaa tatagatttt 180
attctagaca atagtgagtc aagaaaatat actgaatttg aaaataaaat aaatcagcgc 240
agcatcttca gaaaaatata taaatataaa ctatcaaaaa ttaataagat agaagaaaat 300
agaataaaaa aatacattaa aaacaaggaa tttgatttaa ttgttaattt taactcacac 360
cttgatttct tcttatcaaa caatcaaatt aacatcccga taattcgttg gatacacggt 420
caagctcatt tagatgactg gtgcaacaga agagaatggt accaaaacat tcttcctaaa 480
cacacttatt tctttgcaat tacaaaagaa atgcaaaaaa atgctcaaaa aatcttacta 540
tcttacggga tccaagaaga aagaatacat atcttataca atcctattga tattaatttt 600
gtccaggaac aatcaatcaa aaatactcat gacattcatc ataaacaata cttaattaac 660
gtttctcgtt tagatataga taagaatcat gaacaaatga ttaatattta ttatcaatta 720
aaaaaacgag gtatccaaga aaaattatat attgttgggg atggtgagtg tcgagaaaaa 780
ttagagaaac aaatagaatc actcaatcta caagaagatt gctttctttt aggaaataaa 840
gataatccgt atccattaat aaaaaatgct aagctattct tacacacctc tttgaaagag 900
gggttaccga cagttatcct agaaagcatg gcctgcggta cacctgtaat atccatggac 960
tgccctaccg gtccgaaaga aattctccga ggaggagaat ttggaggatt agtaaattta 1020
ggtgacgaga atgcttttat acaaaaaaca ctctctttcc ttcaaaatca agatgaatac 1080
aaccattatt gtaataaatt agaacaagct atttctcctt ttcgctttga agaaatcagc 1140
actatactct tatctcattt acaaaaattc aatagttaa 1179
<210> 65
<211> 392
<212> PRT
<213> Pasteurella multocida
<400> 65
Met Asn Ile Leu Phe Val His Lys Ser Leu Val Val Gly Gly Ala Glu
1 5 10 15
Arg Ile Leu Ile Asn Tyr Leu Asn Ile Leu Ser Gly Phe Asn Glu Phe
20 25 30
Lys Val Thr Leu Leu Leu Leu Glu Asn Lys Gly Glu Asp Asn Lys Asn
35 40 45
Ile Asn Gln Ile Asn Lys Asn Ile Asn Ile Asp Phe Ile Leu Asp Asn
50 55 60
Ser Glu Ser Arg Lys Tyr Thr Glu Phe Glu Asn Lys Ile Asn Gln Arg
65 70 75 80
Ser Ile Phe Arg Lys Ile Tyr Lys Tyr Lys Leu Ser Lys Ile Asn Lys
85 90 95
Ile Glu Glu Asn Arg Ile Lys Lys Tyr Ile Lys Asn Lys Glu Phe Asp
100 105 110
Leu Ile Val Asn Phe Asn Ser His Leu Asp Phe Phe Leu Ser Asn Asn
115 120 125
Gln Ile Asn Ile Pro Ile Ile Arg Trp Ile His Gly Gln Ala His Leu
130 135 140
Asp Asp Trp Cys Asn Arg Arg Glu Trp Tyr Gln Asn Ile Leu Pro Lys
145 150 155 160
His Thr Tyr Phe Phe Ala Ile Thr Lys Glu Met Gln Lys Asn Ala Gln
165 170 175
Lys Ile Leu Leu Ser Tyr Gly Ile Gln Glu Glu Arg Ile His Ile Leu
180 185 190
Tyr Asn Pro Ile Asp Ile Asn Phe Val Gln Glu Gln Ser Ile Lys Asn
195 200 205
Thr His Asp Ile His His Lys Gln Tyr Leu Ile Asn Val Ser Arg Leu
210 215 220
Asp Ile Asp Lys Asn His Glu Gln Met Ile Asn Ile Tyr Tyr Gln Leu
225 230 235 240
Lys Lys Arg Gly Ile Gln Glu Lys Leu Tyr Ile Val Gly Asp Gly Glu
245 250 255
Cys Arg Glu Lys Leu Glu Lys Gln Ile Glu Ser Leu Asn Leu Gln Glu
260 265 270
Asp Cys Phe Leu Leu Gly Asn Lys Asp Asn Pro Tyr Pro Leu Ile Lys
275 280 285
Asn Ala Lys Leu Phe Leu His Thr Ser Leu Lys Glu Gly Leu Pro Thr
290 295 300
Val Ile Leu Glu Ser Met Ala Cys Gly Thr Pro Val Ile Ser Met Asp
305 310 315 320
Cys Pro Thr Gly Pro Lys Glu Ile Leu Arg Gly Gly Glu Phe Gly Gly
325 330 335
Leu Val Asn Leu Gly Asp Glu Asn Ala Phe Ile Gln Lys Thr Leu Ser
340 345 350
Phe Leu Gln Asn Gln Asp Glu Tyr Asn His Tyr Cys Asn Lys Leu Glu
355 360 365
Gln Ala Ile Ser Pro Phe Arg Phe Glu Glu Ile Ser Thr Ile Leu Leu
370 375 380
Ser His Leu Gln Lys Phe Asn Ser
385 390
<210> 66
<211> 222
<212> DNA
<213> Pasteurella multocida
<400> 66
agcagagtaa gttctttttg cttgttaagt aacaaagctt attgtgacga cacgcgggtc 60
taaattgtgt tttccccagc gagtagcgta aagtaatctt gtccagcaag gatagcgatc 120
ccgacagaca tcgcttatgt aatggactga gcgtaatcta attgccgcat gccatgtttc 180
aatttctttg aactcttgta tcgtccatga aaattcaggg cg 222
<210> 67
<211> 831
<212> DNA
<213> Pasteurella multocida
<400> 67
atgtctgaca aaatttcacc caataagata tctgcgcttt cttctacttt attaatcact 60
ctttgggcaa aagcagttga atatgataaa gccaatccat tactgaaaga tcgcgaagca 120
gcaagaatga aaaaacagat tgactatgac tttcaaaagt ttgaatctgc tcatttatca 180
caagtgggat gttgtggacg cgcaaaatta tttgatcaag aaagcttaaa atttctttca 240
cagcaccaag acgcggttgt tgtgcagctt ggtgcgggct tagatgcacg ctttgaacgc 300
ttaggcaaac cacaagtcag tgcgtggtat gatttagact tacctgaagt catcaatata 360
cgtcgccaac ttttaccaga aacgagtaat cattatttgg ctgactcact tttcaataca 420
gattggatga aaacagttag tcaacataac aaacccgttt tattaattct tgaaggcgta 480
ttgatgtttt ttcctaaaga acaagtcaaa cagtttattg cctctgtggc tgaaaactta 540
cctaacagca caatgatttt cgatattgtg cccccaatgg cagtcggtcg tagtaaatac 600
cacgatgcac tcaaaaaaat agacagtcaa gaacgccctg aattttcatg gacaatacaa 660
gagatcaaag aaattgaaac atggcatgcg gcaattaaat tacgctcagt ccattacata 720
agcgatgtct gtcgggatcg ctatccttgc tggacaagat tactttacgc tactcgctgg 780
ggaaaacaca atttagaccc gcgtgtcgtc acaataagct ttgttactta a 831
<210> 68
<211> 276
<212> PRT
<213> Pasteurella multocida
<400> 68
Met Ser Asp Lys Ile Ser Pro Asn Lys Ile Ser Ala Leu Ser Ser Thr
1 5 10 15
Leu Leu Ile Thr Leu Trp Ala Lys Ala Val Glu Tyr Asp Lys Ala Asn
20 25 30
Pro Leu Leu Lys Asp Arg Glu Ala Ala Arg Met Lys Lys Gln Ile Asp
35 40 45
Tyr Asp Phe Gln Lys Phe Glu Ser Ala His Leu Ser Gln Val Gly Cys
50 55 60
Cys Gly Arg Ala Lys Leu Phe Asp Gln Glu Ser Leu Lys Phe Leu Ser
65 70 75 80
Gln His Gln Asp Ala Val Val Val Gln Leu Gly Ala Gly Leu Asp Ala
85 90 95
Arg Phe Glu Arg Leu Gly Lys Pro Gln Val Ser Ala Trp Tyr Asp Leu
100 105 110
Asp Leu Pro Glu Val Ile Asn Ile Arg Arg Gln Leu Leu Pro Glu Thr
115 120 125
Ser Asn His Tyr Leu Ala Asp Ser Leu Phe Asn Thr Asp Trp Met Lys
130 135 140
Thr Val Ser Gln His Asn Lys Pro Val Leu Leu Ile Leu Glu Gly Val
145 150 155 160
Leu Met Phe Phe Pro Lys Glu Gln Val Lys Gln Phe Ile Ala Ser Val
165 170 175
Ala Glu Asn Leu Pro Asn Ser Thr Met Ile Phe Asp Ile Val Pro Pro
180 185 190
Met Ala Val Gly Arg Ser Lys Tyr His Asp Ala Leu Lys Lys Ile Asp
195 200 205
Ser Gln Glu Arg Pro Glu Phe Ser Trp Thr Ile Gln Glu Ile Lys Glu
210 215 220
Ile Glu Thr Trp His Ala Ala Ile Lys Leu Arg Ser Val His Tyr Ile
225 230 235 240
Ser Asp Val Cys Arg Asp Arg Tyr Pro Cys Trp Thr Arg Leu Leu Tyr
245 250 255
Ala Thr Arg Trp Gly Lys His Asn Leu Asp Pro Arg Val Val Thr Ile
260 265 270
Ser Phe Val Thr
275
<210> 69
<211> 55
<212> DNA
<213> Pasteurella multocida
<400> 69
tcgatgaaaa acgccattat ggtcatggaa tcagctgcaa aattctcact ccaca 55
<210> 70
<211> 1314
<212> DNA
<213> Pasteurella multocida
<400> 70
atggtattac actatacccc tcatcaatcc gccccacgca acacaacatt cgttgcggaa 60
attcttgatc ttgattatca aggacgtggt gtagccaaag tacaaggcaa aacgtggttc 120
attgaaaatg cactgccaca agaaaaagtg gaagtgcgca ttgtcgatga aaaacgccat 180
tatggtcatg ggatcagctg caaaattctc actccacatc cagatcgcca gtcagcaaaa 240
tgtgcttact atgcccagtg cggtggttgc caaagtcaac atattccaat tgacatgcaa 300
cgtcaggcta aacaacaagc cttattccaa cgcttacaac aattacaacc tcaagcgacc 360
ttcatgccca tgatcgtcgc agcgccttgg cattatcgcc gtcgtgtgcg tttaagcgtg 420
cggtttcatc ccaaaagcaa acaacttgcg atgggtttgc gtcagagaaa tactcaacaa 480
atcgtgaatc tgcagcattg tgatgtgctt gaaatcccct taagtcaact cttacctaaa 540
ctacatttgt tgttttcaac atggtccctg cctaaaaacc tagggcatgt ggagttagtg 600
catgcggata atggaattgc gatgttatta cgccatacag gaaatttagc gcaaactgac 660
cgcactttat taaccaattt tgcgcaacaa gaaaacttaa tgttgtttgt acaagatgat 720
caacagatca cccaactaca tggcgaggca ccttactaca tactacgcga tggcaccaaa 780
ttacagtttg atatccgtga ctttatccaa gtgaatgctg ttgtaaatca gaaaatgatt 840
gatactgctc ttgagtggtt ggaactcaca tcgaacgata acgtattaga tttgttttgt 900
ggtatgggaa acttcaccct cccaatcagt cgtcaggtca atcaggttgt gggcattgaa 960
ggcgtaggag aaatggtgga gaaagcaaaa cgaaatgcgg aacaaaatca atgtgataat 1020
gtccaattct atcaggcgaa tttagatcaa ccttttgtgc aacaacattg ggcgagccaa 1080
cattttaata aaattttact ggacccacca cgtacaggcg cggcatttgc cttacatgcc 1140
ttatgtgaat tgggcgcaga aaaaatctta tatgtttcct gcaatcctgc tacattagta 1200
cgtgatacag cgattttatt acaatttaac taccgactta agaaagtcgc aatgatcgat 1260
atgttcccca atacaggaca tttagaatcc atcagtttat ttgaaaaaga atag 1314
<210> 71
<211> 437
<212> PRT
<213> Pasteurella multocida
<400> 71
Met Val Leu His Tyr Thr Pro His Gln Ser Ala Pro Arg Asn Thr Thr
1 5 10 15
Phe Val Ala Glu Ile Leu Asp Leu Asp Tyr Gln Gly Arg Gly Val Ala
20 25 30
Lys Val Gln Gly Lys Thr Trp Phe Ile Glu Asn Ala Leu Pro Gln Glu
35 40 45
Lys Val Glu Val Arg Ile Val Asp Glu Lys Arg His Tyr Gly His Gly
50 55 60
Ile Ser Cys Lys Ile Leu Thr Pro His Pro Asp Arg Gln Ser Ala Lys
65 70 75 80
Cys Ala Tyr Tyr Ala Gln Cys Gly Gly Cys Gln Ser Gln His Ile Pro
85 90 95
Ile Asp Met Gln Arg Gln Ala Lys Gln Gln Ala Leu Phe Gln Arg Leu
100 105 110
Gln Gln Leu Gln Pro Gln Ala Thr Phe Met Pro Met Ile Val Ala Ala
115 120 125
Pro Trp His Tyr Arg Arg Arg Val Arg Leu Ser Val Arg Phe His Pro
130 135 140
Lys Ser Lys Gln Leu Ala Met Gly Leu Arg Gln Arg Asn Thr Gln Gln
145 150 155 160
Ile Val Asn Leu Gln His Cys Asp Val Leu Glu Ile Pro Leu Ser Gln
165 170 175
Leu Leu Pro Lys Leu His Leu Leu Phe Ser Thr Trp Ser Leu Pro Lys
180 185 190
Asn Leu Gly His Val Glu Leu Val His Ala Asp Asn Gly Ile Ala Met
195 200 205
Leu Leu Arg His Thr Gly Asn Leu Ala Gln Thr Asp Arg Thr Leu Leu
210 215 220
Thr Asn Phe Ala Gln Gln Glu Asn Leu Met Leu Phe Val Gln Asp Asp
225 230 235 240
Gln Gln Ile Thr Gln Leu His Gly Glu Ala Pro Tyr Tyr Ile Leu Arg
245 250 255
Asp Gly Thr Lys Leu Gln Phe Asp Ile Arg Asp Phe Ile Gln Val Asn
260 265 270
Ala Val Val Asn Gln Lys Met Ile Asp Thr Ala Leu Glu Trp Leu Glu
275 280 285
Leu Thr Ser Asn Asp Asn Val Leu Asp Leu Phe Cys Gly Met Gly Asn
290 295 300
Phe Thr Leu Pro Ile Ser Arg Gln Val Asn Gln Val Val Gly Ile Glu
305 310 315 320
Gly Val Gly Glu Met Val Glu Lys Ala Lys Arg Asn Ala Glu Gln Asn
325 330 335
Gln Cys Asp Asn Val Gln Phe Tyr Gln Ala Asn Leu Asp Gln Pro Phe
340 345 350
Val Gln Gln His Trp Ala Ser Gln His Phe Asn Lys Ile Leu Leu Asp
355 360 365
Pro Pro Arg Thr Gly Ala Ala Phe Ala Leu His Ala Leu Cys Glu Leu
370 375 380
Gly Ala Glu Lys Ile Leu Tyr Val Ser Cys Asn Pro Ala Thr Leu Val
385 390 395 400
Arg Asp Thr Ala Ile Leu Leu Gln Phe Asn Tyr Arg Leu Lys Lys Val
405 410 415
Ala Met Ile Asp Met Phe Pro Asn Thr Gly His Leu Glu Ser Ile Ser
420 425 430
Leu Phe Glu Lys Glu
435
<210> 72
<211> 598
<212> DNA
<213> Pasteurella multocida
<220>
<221> modified_base
<222> (412)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (451)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (471)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (503)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (546)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (562)..(564)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (576)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (588)..(590)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (595)
<223> a, t, c, g, other or unknown
<400> 72
tcagtacttt gcttgcttaa gcaagtaaaa agtgcggtca tttttagcaa aaataaaggg 60
cttctgtttg gaagcccttt gtgtattgca agttagtctt ttttatgagt gtattcttta 120
tacatcgctt caatttgtgc tttatagcgt tccaaaatca ctttacgacg gagtttcaat 180
gtcggagtaa tttcttccat ttttgtggta aatgcctgag gtaataaagt gaatttttta 240
atttgctcaa agctaggcaa ttctttttgt aaatcattaa tacgctgttc aaacatttga 300
agaatatcag aatgtttaat gagttctaaa cgatcgtgat attttatatt taattgtttg 360
gcgtattctt caagactatt aaagcaaggc acaataagcg ctgagacata tnttttggca 420
tccgcaatga ctgcaatttg ttcaataaat ntatctttac ccactttggt ntcaatatat 480
tgtggagcaa tatattttcc atnggaggtt ttcattaact ctttgatacg atctgtaata 540
tataantacc ttgtggatca annncccagc atcacnagtt tttaaaannn gtctnctg 598
<210> 73
<211> 561
<212> DNA
<213> Pasteurella multocida
<220>
<221> modified_base
<222> (560)..(561)
<223> a, t, c, g, other or unknown
<400> 73
gagcaaagta gctgtccgcc gtatattgta agaagttggc ataagaatta acgcctaact 60
tgaccgcatc gcccatcgga tctaaacgac ctacatgtac gccggtacct ttacttaaac 120
tttcaattac ttttggtgtg aattgtggct ccgcaaataa gcaattcact ttatgttctt 180
taatttcccg cttaattttc gctaacgtct tagctcccgg cgccaccaac ggattaattg 240
tgaaataacc ggtttgtttt aagccataag cattattgaa ataactatac gcatcatgga 300
aaacataaaa ccctttttct ttaactggtg cgagttgctg tttaattttc tcgctttgtt 360
cagctaaagt gcggttaaat tctgccaaat tttgcgcaat tttctctttt ctctctggat 420
aagcttccgt taaacgtgtt gctaagcgtg tcgcgacaat tttgctaatc tctggcgaat 480
accacacatg ccagttagta ctgtgatcat gctcgtgttc atgtgcgtgg tcatgtttat 540
gctcatggtc gtgtttgtgn n 561
<210> 74
<211> 575
<212> DNA
<213> Pasteurella multocida
<220>
<221> modified_base
<222> (574)..(575)
<223> a, t, c, g, other or unknown
<400> 74
ttacttgctt aagcaagcaa agtagctgtc cgccgtatat tgtaagaagt tggcataaga 60
attaacgcct aacttgaccg catcgcccat cggatctaaa cgacctacat gtacgccggt 120
acctttactt aaactttcaa ttacttttgg tgtgaattgt ggctccgcaa ataagcaatt 180
cactttatgt tctttaattt cccgcttaat tttcgctaac gtcttagctc ccggcgccac 240
caacggatta attgtgaaat aaccggtttg ttttaagcca taagcattat tgaaataact 300
atacgcatca tggaaaacat aaaacccttt ttctttaact ggtgcgagtt gctgtttaat 360
tttctcgctt tgttcagcta aagtgcggtt aaattctgcc aaattttgcg caattttctc 420
ttttctctct ggataagctt ccgttaaacg tgttgctaag cgtgtcgcga caattttgct 480
aatctctggc gaataccaca catgccagtt agtactgtga tcatgctcgt gttcatgtgc 540
gtggtcatgt ttatgctcat ggtcgtgttt gtgnn 575
<210> 75
<211> 1101
<212> DNA
<213> Pasteurella multocida
<400> 75
atggcacgtt tcattaagac attgaaaaaa accgcattag cggcaagtat tgcttcttta 60
gcaactgtgg caaatgcgac gattgtgact tcgattaaac cattaggttt tattgcttca 120
tcgattgctg atggggtaac agacactgaa gtattagttc ctgcgggtgc ttcaccacat 180
gattacagct taaaaccctc agatatacaa aaattacagg gggcggaatt aatcctgtgg 240
gtcggggaag acattgatgc tttccttgat aaaacattac gtccaatgcc ttttaaaaag 300
gtgttaagta ttgctgattt tgcggaaatt ggtggtttgc ttgaaggtga agcacatgat 360
cataaacatg agcatgatca tactcacaaa cacgaccacg atcacaaaca cgaccacgat 420
cacaaacacg accacgatca caaacatgag cacgatcata aacacgacca cgatcacaaa 480
catgaccacg atcacaaaca cgaccatgct cacaagcatg agcacgatca caaacacgac 540
catgagcata aacatgacca cgcacatgga cacgagcatg atcacagtac taactggcat 600
gtgtggtatt cgccagagat tagcaaaatt gtcgcgacac gcttagcaac acgtttaacg 660
gaagcttatc cagagaaaaa agagaaaatt gcgcaaaatt tggcagaatt taaccgtact 720
ttagctgaac aaagcgagaa aattaaacag caactcgcac cagttaaaga aaaagggttt 780
tatgttttcc atgatgcgta tagctatttc aataatgctt atggcttaaa acaaaccggt 840
tatttcacaa ttaatccgtt ggtggcgccg ggagctaaga cgttagcgaa aattaagcag 900
gaaattaaag aacataaagt gaattgctta tttgcggagc cacaattcac accaaaagta 960
attgaaagtt taagtaaagg taccggtgta catgtaggtc gtttagatcc gatgggcgat 1020
gcggtcaagt taggcgttaa ttcttatgcc aacttcttac aatatacggc ggacagctac 1080
tttgcttgct taagcaagta a 1101
<210> 76
<211> 366
<212> PRT
<213> Pasteurella multocida
<400> 76
Met Ala Arg Phe Ile Lys Thr Leu Lys Lys Thr Ala Leu Ala Ala Ser
1 5 10 15
Ile Ala Ser Leu Ala Thr Val Ala Asn Ala Thr Ile Val Thr Ser Ile
20 25 30
Lys Pro Leu Gly Phe Ile Ala Ser Ser Ile Ala Asp Gly Val Thr Asp
35 40 45
Thr Glu Val Leu Val Pro Ala Gly Ala Ser Pro His Asp Tyr Ser Leu
50 55 60
Lys Pro Ser Asp Ile Gln Lys Leu Gln Gly Ala Glu Leu Ile Leu Trp
65 70 75 80
Val Gly Glu Asp Ile Asp Ala Phe Leu Asp Lys Thr Leu Arg Pro Met
85 90 95
Pro Phe Lys Lys Val Leu Ser Ile Ala Asp Phe Ala Glu Ile Gly Gly
100 105 110
Leu Leu Glu Gly Glu Ala His Asp His Lys His Glu His Asp His Thr
115 120 125
His Lys His Asp His Asp His Lys His Asp His Asp His Lys His Asp
130 135 140
His Asp His Lys His Glu His Asp His Lys His Asp His Asp His Lys
145 150 155 160
His Asp His Asp His Lys His Asp His Ala His Lys His Glu His Asp
165 170 175
His Lys His Asp His Glu His Lys His Asp His Ala His Gly His Glu
180 185 190
His Asp His Ser Thr Asn Trp His Val Trp Tyr Ser Pro Glu Ile Ser
195 200 205
Lys Ile Val Ala Thr Arg Leu Ala Thr Arg Leu Thr Glu Ala Tyr Pro
210 215 220
Glu Lys Lys Glu Lys Ile Ala Gln Asn Leu Ala Glu Phe Asn Arg Thr
225 230 235 240
Leu Ala Glu Gln Ser Glu Lys Ile Lys Gln Gln Leu Ala Pro Val Lys
245 250 255
Glu Lys Gly Phe Tyr Val Phe His Asp Ala Tyr Ser Tyr Phe Asn Asn
260 265 270
Ala Tyr Gly Leu Lys Gln Thr Gly Tyr Phe Thr Ile Asn Pro Leu Val
275 280 285
Ala Pro Gly Ala Lys Thr Leu Ala Lys Ile Lys Gln Glu Ile Lys Glu
290 295 300
His Lys Val Asn Cys Leu Phe Ala Glu Pro Gln Phe Thr Pro Lys Val
305 310 315 320
Ile Glu Ser Leu Ser Lys Gly Thr Gly Val His Val Gly Arg Leu Asp
325 330 335
Pro Met Gly Asp Ala Val Lys Leu Gly Val Asn Ser Tyr Ala Asn Phe
340 345 350
Leu Gln Tyr Thr Ala Asp Ser Tyr Phe Ala Cys Leu Ser Lys
355 360 365
<210> 77
<211> 70
<212> DNA
<213> Pasteurella multocida
<400> 77
gctttgcatt tgagtcataa aatagtacag tacggtaatt ttctggatga ataccttttt 60
tcatattggc 70
<210> 78
<211> 267
<212> DNA
<213> Pasteurella multocida
<400> 78
atgaaaaaag gtattcatcc agaaaattac cgtactgtac tattttatga ctcaaatgca 60
aagcaaggtt ttttaatccg ctcttgcgcc agaaccacaa cgaccatgaa atgggaagat 120
ggtcatgaat atcctgtctt tatgtgtgat acctcctcag catcacaccc gtactataca 180
ggtaaaacac gtcaaattgc gaatgaaggt cgtgcaagcg actttgtcaa tcgctacggc 240
aaatttggca cattaaaatc aaaataa 267
<210> 79
<211> 88
<212> PRT
<213> Pasteurella multocida
<400> 79
Met Lys Lys Gly Ile His Pro Glu Asn Tyr Arg Thr Val Leu Phe Tyr
1 5 10 15
Asp Ser Asn Ala Lys Gln Gly Phe Leu Ile Arg Ser Cys Ala Arg Thr
20 25 30
Thr Thr Thr Met Lys Trp Glu Asp Gly His Glu Tyr Pro Val Phe Met
35 40 45
Cys Asp Thr Ser Ser Ala Ser His Pro Tyr Tyr Thr Gly Lys Thr Arg
50 55 60
Gln Ile Ala Asn Glu Gly Arg Ala Ser Asp Phe Val Asn Arg Tyr Gly
65 70 75 80
Lys Phe Gly Thr Leu Lys Ser Lys
85
<210> 80
<211> 506
<212> DNA
<213> Pasteurella multocida
<400> 80
tgctccaact ctactttcaa cctatcctct gtccatgttc ttggaaacat cgtggataca 60
cctttatttc cctttttctc caaaacttcg gggcagtagg agatcaacac cctcgcttca 120
tagaccccat ttgggtattc cttaatcacc ttatctacaa tcacattgcc taagatggtg 180
tgtcttaacg ctcccatgta aaaaaatggt caatttctca aaacaaaact ttttcaaaat 240
tgaccgcact ttttcttcta actgttcctt ttcagaaaat caacaccttc acttaagaaa 300
acccctacgc atatttctcc atcagggcaa tgatagcttg agagctagga cgatgggact 360
catatttttt tatcccctca agtaattcat gttgtccatt aaaataatgt acgtttccac 420
ctttatccag catcaattta agcagatcta gcgctttcag ggacataacc tgtcattgcc 480
aatggaatca cttggtctcg atttgg 506
<210> 81
<211> 348
<212> DNA
<213> Pasteurella multocida
<400> 81
atgagagcgt taagacacac caccttaggc aatgtgattg tggataaggt gattaaggaa 60
tacccaaatg gggtttatga agcgagggtg ttgatcccta acccgaaagc ccaaaccgat 120
cctaccgccc cgaagttttt ggagaaaagg ggaaataaag gtgtatccac gatgtttcca 180
agaacatgga cagaggatag gttgaaagtg gagttggagc atgcgtttaa aaatggtata 240
cacgataaag ggcaagtatg gactgggata actaaatcag gtgttaaagt acaatggtat 300
agaagtgaaa aaggtgagat aaccagtgtt catccaatct tagaataa 348
<210> 82
<211> 115
<212> PRT
<213> Pasteurella multocida
<400> 82
Met Arg Ala Leu Arg His Thr Thr Leu Gly Asn Val Ile Val Asp Lys
1 5 10 15
Val Ile Lys Glu Tyr Pro Asn Gly Val Tyr Glu Ala Arg Val Leu Ile
20 25 30
Pro Asn Pro Lys Ala Gln Thr Asp Pro Thr Ala Pro Lys Phe Leu Glu
35 40 45
Lys Arg Gly Asn Lys Gly Val Ser Thr Met Phe Pro Arg Thr Trp Thr
50 55 60
Glu Asp Arg Leu Lys Val Glu Leu Glu His Ala Phe Lys Asn Gly Ile
65 70 75 80
His Asp Lys Gly Gln Val Trp Thr Gly Ile Thr Lys Ser Gly Val Lys
85 90 95
Val Gln Trp Tyr Arg Ser Glu Lys Gly Glu Ile Thr Ser Val His Pro
100 105 110
Ile Leu Glu
115
<210> 83
<211> 243
<212> DNA
<213> Pasteurella multocida
<400> 83
gccgatatgg tacgtgtcga cattatgatc aatggtgagc gtgtcgatgc gttagcgtta 60
atcgtgcata aagataatgc accttatcgt ggtcgtgaat tagtggaaaa aatgcgtgag 120
ctcattccac gtcaacaatt tgatattgcg attcaagcgg cgattggtaa ccacattatt 180
gcccgttcta ccgtcaaaca attacgtaaa aacgtattag caaaatgtta tggtggtgac 240
gtg 243
<210> 84
<211> 1797
<212> DNA
<213> Pasteurella multocida
<400> 84
atgaagaata tacgtaactt ttctattatt gcacacattg accacggtaa atcgacactc 60
tctgaccgcc ttattcaaac ttgcggtggc ttatctgatc gtgaaatgga agcccaagtg 120
ttggattcca tggatcttga acgtgaacgt gggattacga tcaaagcaca aagtgtgacc 180
ttaaattaca aagcgaaaga tggcgaaacc tatcaattaa atttcatcga tacgccaggt 240
cacgttgact tctcttatga agtatcgcgt tctttagccg cttgtgaagg cgcattatta 300
gtggtggatg cgggacaagg tgtcgaggca caaactttgg ctaactgcta taccgcaatt 360
gaaatgaatt tagaagtggt gccgatttta aacaaaatcg acttgcccgc ggcagatcct 420
gaacgcgttg cagaagaaat tgaagacatt gtcggtattg acgcgatgga agcggtgcgc 480
tgttcagcaa aaaccggtgt gggtattgaa gatgtgttgg aagaaattgt gcataaaatc 540
cctgcacccg aaggggatcc gaatgcacca ttacaagcct tgattatcga ctcgtggttt 600
gataactact taggcgtagt atctttagtg cgcattaaaa acggcacatt acgcaaaggc 660
gataaaatca aagtgatgtc tacagggcaa tcttacaatg tggatcgtct tggtattttc 720
accccaaaac aagtcgatac caccatttta aattgtggtg aagtgggttg ggtggtgtgc 780
gccattaaag atattttagg ggcacccgtg ggtgatacgc ttacttcgca caacaatcca 840
gcttcttctg tcctgccggg ttttaagaaa gttaagccac aggtgtatgc cggtttattc 900
ccaattagct ctgatgatta tgaagcattc cgtgatgcgc tcggtaaact tagtctaaac 960
gatgcgtcat tattctatga accagaaaac tccaccgcac ttggtttcgg tttccgttgt 1020
ggtttcttag gacttctcca catggagatt attcaagagc gtttagagcg cgaatacgat 1080
cttgatctga ttaccacagc accgacagta gtgtatgaag tggaaaaaac cgacggtgaa 1140
gtgatttatg tggatagccc atcaaaatta ccgccactca acaacattac ggagattcgt 1200
gaaccgattg cagaatgtaa catgctgtta ccacaaacct acttaggtaa cgtcattacg 1260
ctctgtgtag aaaaacgcgg tgtacaaacc aatatggttt accatggtaa ccaagtggca 1320
ttgacctatg aaatcccaat gggcgaagtg gtactggatt tcttcgaccg cttaaaatca 1380
acttctcgtg gttatgcttc cttagattat ggtttcaaac gtttccaagc cgccgatatg 1440
gtacgtgtcg acattatgat caatggtgag cgtgtcgatg cgttagcgtt aatcgtgcat 1500
aaagataatg caccttatcg tggtcgtgaa ttagtggaaa aaatgcgtga gctcattcca 1560
cgtcaacaat ttgatattgc gattcaagcg gcgattggta accacattat tgcccgttct 1620
actgtcaaac aattacgtaa aaacgtatta gcaaaatgtt atggtggtga cgttagccgt 1680
aagaaaaaac tcttacagaa acaaaaagaa ggtaaaaaac gcatgaagtc tttgggtaac 1740
gtcgaagtac cacaagaagc cttcttagcg attttacatg tcggaaaaga caaataa 1797
<210> 85
<211> 598
<212> PRT
<213> Pasteurella multocida
<400> 85
Met Lys Asn Ile Arg Asn Phe Ser Ile Ile Ala His Ile Asp His Gly
1 5 10 15
Lys Ser Thr Leu Ser Asp Arg Leu Ile Gln Thr Cys Gly Gly Leu Ser
20 25 30
Asp Arg Glu Met Glu Ala Gln Val Leu Asp Ser Met Asp Leu Glu Arg
35 40 45
Glu Arg Gly Ile Thr Ile Lys Ala Gln Ser Val Thr Leu Asn Tyr Lys
50 55 60
Ala Lys Asp Gly Glu Thr Tyr Gln Leu Asn Phe Ile Asp Thr Pro Gly
65 70 75 80
His Val Asp Phe Ser Tyr Glu Val Ser Arg Ser Leu Ala Ala Cys Glu
85 90 95
Gly Ala Leu Leu Val Val Asp Ala Gly Gln Gly Val Glu Ala Gln Thr
100 105 110
Leu Ala Asn Cys Tyr Thr Ala Ile Glu Met Asn Leu Glu Val Val Pro
115 120 125
Ile Leu Asn Lys Ile Asp Leu Pro Ala Ala Asp Pro Glu Arg Val Ala
130 135 140
Glu Glu Ile Glu Asp Ile Val Gly Ile Asp Ala Met Glu Ala Val Arg
145 150 155 160
Cys Ser Ala Lys Thr Gly Val Gly Ile Glu Asp Val Leu Glu Glu Ile
165 170 175
Val His Lys Ile Pro Ala Pro Glu Gly Asp Pro Asn Ala Pro Leu Gln
180 185 190
Ala Leu Ile Ile Asp Ser Trp Phe Asp Asn Tyr Leu Gly Val Val Ser
195 200 205
Leu Val Arg Ile Lys Asn Gly Thr Leu Arg Lys Gly Asp Lys Ile Lys
210 215 220
Val Met Ser Thr Gly Gln Ser Tyr Asn Val Asp Arg Leu Gly Ile Phe
225 230 235 240
Thr Pro Lys Gln Val Asp Thr Thr Ile Leu Asn Cys Gly Glu Val Gly
245 250 255
Trp Val Val Cys Ala Ile Lys Asp Ile Leu Gly Ala Pro Val Gly Asp
260 265 270
Thr Leu Thr Ser His Asn Asn Pro Ala Ser Ser Val Leu Pro Gly Phe
275 280 285
Lys Lys Val Lys Pro Gln Val Tyr Ala Gly Leu Phe Pro Ile Ser Ser
290 295 300
Asp Asp Tyr Glu Ala Phe Arg Asp Ala Leu Gly Lys Leu Ser Leu Asn
305 310 315 320
Asp Ala Ser Leu Phe Tyr Glu Pro Glu Asn Ser Thr Ala Leu Gly Phe
325 330 335
Gly Phe Arg Cys Gly Phe Leu Gly Leu Leu His Met Glu Ile Ile Gln
340 345 350
Glu Arg Leu Glu Arg Glu Tyr Asp Leu Asp Leu Ile Thr Thr Ala Pro
355 360 365
Thr Val Val Tyr Glu Val Glu Lys Thr Asp Gly Glu Val Ile Tyr Val
370 375 380
Asp Ser Pro Ser Lys Leu Pro Pro Leu Asn Asn Ile Thr Glu Ile Arg
385 390 395 400
Glu Pro Ile Ala Glu Cys Asn Met Leu Leu Pro Gln Thr Tyr Leu Gly
405 410 415
Asn Val Ile Thr Leu Cys Val Glu Lys Arg Gly Val Gln Thr Asn Met
420 425 430
Val Tyr His Gly Asn Gln Val Ala Leu Thr Tyr Glu Ile Pro Met Gly
435 440 445
Glu Val Val Leu Asp Phe Phe Asp Arg Leu Lys Ser Thr Ser Arg Gly
450 455 460
Tyr Ala Ser Leu Asp Tyr Gly Phe Lys Arg Phe Gln Ala Ala Asp Met
465 470 475 480
Val Arg Val Asp Ile Met Ile Asn Gly Glu Arg Val Asp Ala Leu Ala
485 490 495
Leu Ile Val His Lys Asp Asn Ala Pro Tyr Arg Gly Arg Glu Leu Val
500 505 510
Glu Lys Met Arg Glu Leu Ile Pro Arg Gln Gln Phe Asp Ile Ala Ile
515 520 525
Gln Ala Ala Ile Gly Asn His Ile Ile Ala Arg Ser Thr Val Lys Gln
530 535 540
Leu Arg Lys Asn Val Leu Ala Lys Cys Tyr Gly Gly Asp Val Ser Arg
545 550 555 560
Lys Lys Lys Leu Leu Gln Lys Gln Lys Glu Gly Lys Lys Arg Met Lys
565 570 575
Ser Leu Gly Asn Val Glu Val Pro Gln Glu Ala Phe Leu Ala Ile Leu
580 585 590
His Val Gly Lys Asp Lys
595
<210> 86
<211> 147
<212> DNA
<213> Pasteurella multocida
<400> 86
aaaagttcga cttccgtaat cggtttttta gttaattgtt caatattgcg taataaacga 60
cgttctcttg gttcaacaaa taacaatgca cgccctgtac gtccggcacg ccctgtacga 120
ccaatacggt ggacataaga ctcagca 147
<210> 87
<211> 1833
<212> DNA
<213> Pasteurella multocida
<400> 87
atgactgaaa caacaatgac tttcaatgat ttaggcttgc ctgaatttct tcttaacgcc 60
gtctctgact taggctttga aaccccttct ccaattcaac aaagttgtat cccaaacctg 120
ttaaatgggc atgatgtgct aggtatggca caaactggaa gtggtaaaac cgccgccttt 180
tcactccctt tattagcaca aattgattta gataaaaaat atccacaaat gttagtgatg 240
gcaccgacac gtgagttagc catccaagta gcagatgcct gtgagcactt ttgcaaatat 300
gcgaaaaata ccaatattgt taccctttat ggtggtcaac gctatgacat tcaattgcgt 360
gctttacgcc aaggtgctca ggttgtagtg gggacacctg gtcgtatttt agatcacatt 420
cgtcgtggca ctttagattt gtctaattta cgttttatgg tgttagatga agcggacgaa 480
atgttacgta tgggctttat tgatgatgtt gaaacggtga tggcagaatt accagaacaa 540
catcagactg cacttttctc agccaccatg ccagatccaa ttcgtcgtat tactaagcgt 600
tttatgaaag atccgaaaga gattaaaatt aaatcgacgc aaacgacgaa tccagatatt 660
acacagagtt gttggtatgt gcatggtttc cgtaaaaatg atgccttatt acgtttctta 720
gaagtagaaa aatttgatgc cgcgattatc tttactcgta ctaaaacggg gacattagat 780
gtaacggaat tgttggaaaa acatggtttc cgtgccgcag cattaaatgg cgatatgaca 840
caacaattac gtgaacaaac gcttgatcgt ttaagaaatg gtagtttaga tatccttgtg 900
gcaaccgatg tggcggcgcg tggtttagat gtggagcgca ttagcctcgt agtgaactat 960
gatattccat tagatgctga gtcttatgtt caccgtattg gtcgtacagg gcgtgcagga 1020
cgtacagggc gtgcattgtt atttgttgaa ccaagagaac gtcgtttatt acgtaatatt 1080
gaacaattaa ctaaaaaacc gattacggaa gtcgaagtgc caaatcatga ggtactacaa 1140
gcttgtcgcc gtgagaaatt taaagccaaa attacagtcc aattagagca tcatgattta 1200
ggactttatc gtagcttact agaagatatg ttcaccgcgg atcaagatca ggaagatatt 1260
gcggcggcga tgttgatgtt gttgcaaggt aaacaaaagc ttattttacc agccgatcca 1320
attattgatc gtaaaacttc acgtggtgat cgtggcgagc gtcgtgaacg tggtggacgt 1380
gaaaatccac gttcagcaga gcgtcgtggt tacggtacac cgcaggcgat ggatttatat 1440
cgtattgaag taggacgttt agatggcgcg gaagtccgtc atattgttgg ggcgattgcc 1500
aatgaaggtg atatcaatag tcgttatatt ggtcatatta aattatatga tgattacacc 1560
acgattgaat taccacaagg tatgccgaaa gaattattag gtgtatttgc gaaaacacgc 1620
gtgatgaaca aacaaatgca gatgtcattt gtgggagcgt ctaatgcagg ttcaagccgt 1680
gatcgcgatg atttcgctga ccgccgtggt ggaaaacgta aaggacgcgg cgatgaacca 1740
cgttttgggc gtgaagatcg taaatttaaa gaaaaaagtc agcgcacttt taatgatcgc 1800
ccacgcagag aaagacgtga acgccaaaag taa 1833
<210> 88
<211> 610
<212> PRT
<213> Pasteurella multocida
<400> 88
Met Thr Glu Thr Thr Met Thr Phe Asn Asp Leu Gly Leu Pro Glu Phe
1 5 10 15
Leu Leu Asn Ala Val Ser Asp Leu Gly Phe Glu Thr Pro Ser Pro Ile
20 25 30
Gln Gln Ser Cys Ile Pro Asn Leu Leu Asn Gly His Asp Val Leu Gly
35 40 45
Met Ala Gln Thr Gly Ser Gly Lys Thr Ala Ala Phe Ser Leu Pro Leu
50 55 60
Leu Ala Gln Ile Asp Leu Asp Lys Lys Tyr Pro Gln Met Leu Val Met
65 70 75 80
Ala Pro Thr Arg Glu Leu Ala Ile Gln Val Ala Asp Ala Cys Glu His
85 90 95
Phe Cys Lys Tyr Ala Lys Asn Thr Asn Ile Val Thr Leu Tyr Gly Gly
100 105 110
Gln Arg Tyr Asp Ile Gln Leu Arg Ala Leu Arg Gln Gly Ala Gln Val
115 120 125
Val Val Gly Thr Pro Gly Arg Ile Leu Asp His Ile Arg Arg Gly Thr
130 135 140
Leu Asp Leu Ser Asn Leu Arg Phe Met Val Leu Asp Glu Ala Asp Glu
145 150 155 160
Met Leu Arg Met Gly Phe Ile Asp Asp Val Glu Thr Val Met Ala Glu
165 170 175
Leu Pro Glu Gln His Gln Thr Ala Leu Phe Ser Ala Thr Met Pro Asp
180 185 190
Pro Ile Arg Arg Ile Thr Lys Arg Phe Met Lys Asp Pro Lys Glu Ile
195 200 205
Lys Ile Lys Ser Thr Gln Thr Thr Asn Pro Asp Ile Thr Gln Ser Cys
210 215 220
Trp Tyr Val His Gly Phe Arg Lys Asn Asp Ala Leu Leu Arg Phe Leu
225 230 235 240
Glu Val Glu Lys Phe Asp Ala Ala Ile Ile Phe Thr Arg Thr Lys Thr
245 250 255
Gly Thr Leu Asp Val Thr Glu Leu Leu Glu Lys His Gly Phe Arg Ala
260 265 270
Ala Ala Leu Asn Gly Asp Met Thr Gln Gln Leu Arg Glu Gln Thr Leu
275 280 285
Asp Arg Leu Arg Asn Gly Ser Leu Asp Ile Leu Val Ala Thr Asp Val
290 295 300
Ala Ala Arg Gly Leu Asp Val Glu Arg Ile Ser Leu Val Val Asn Tyr
305 310 315 320
Asp Ile Pro Leu Asp Ala Glu Ser Tyr Val His Arg Ile Gly Arg Thr
325 330 335
Gly Arg Ala Gly Arg Thr Gly Arg Ala Leu Leu Phe Val Glu Pro Arg
340 345 350
Glu Arg Arg Leu Leu Arg Asn Ile Glu Gln Leu Thr Lys Lys Pro Ile
355 360 365
Thr Glu Val Glu Val Pro Asn His Glu Val Leu Gln Ala Cys Arg Arg
370 375 380
Glu Lys Phe Lys Ala Lys Ile Thr Val Gln Leu Glu His His Asp Leu
385 390 395 400
Gly Leu Tyr Arg Ser Leu Leu Glu Asp Met Phe Thr Ala Asp Gln Asp
405 410 415
Gln Glu Asp Ile Ala Ala Ala Met Leu Met Leu Leu Gln Gly Lys Gln
420 425 430
Lys Leu Ile Leu Pro Ala Asp Pro Ile Ile Asp Arg Lys Thr Ser Arg
435 440 445
Gly Asp Arg Gly Glu Arg Arg Glu Arg Gly Gly Arg Glu Asn Pro Arg
450 455 460
Ser Ala Glu Arg Arg Gly Tyr Gly Thr Pro Gln Ala Met Asp Leu Tyr
465 470 475 480
Arg Ile Glu Val Gly Arg Leu Asp Gly Ala Glu Val Arg His Ile Val
485 490 495
Gly Ala Ile Ala Asn Glu Gly Asp Ile Asn Ser Arg Tyr Ile Gly His
500 505 510
Ile Lys Leu Tyr Asp Asp Tyr Thr Thr Ile Glu Leu Pro Gln Gly Met
515 520 525
Pro Lys Glu Leu Leu Gly Val Phe Ala Lys Thr Arg Val Met Asn Lys
530 535 540
Gln Met Gln Met Ser Phe Val Gly Ala Ser Asn Ala Gly Ser Ser Arg
545 550 555 560
Asp Arg Asp Asp Phe Ala Asp Arg Arg Gly Gly Lys Arg Lys Gly Arg
565 570 575
Gly Asp Glu Pro Arg Phe Gly Arg Glu Asp Arg Lys Phe Lys Glu Lys
580 585 590
Ser Gln Arg Thr Phe Asn Asp Arg Pro Arg Arg Glu Arg Arg Glu Arg
595 600 605
Gln Lys
610
<210> 89
<211> 187
<212> DNA
<213> Pasteurella multocida
<400> 89
tctacgttaa cgccacccgt tgtattaata acattggcaa agccagaagc agcgatcatc 60
acaaaaccaa tcatcgccat taaacgtaag ccttgttgga aaatgtcatt actttctttt 120
aatttgaaaa taccacaaac agcaaaaata atcagaccgg ctaatccacc aataatagtt 180
gaactct 187
<210> 90
<211> 1359
<212> DNA
<213> Pasteurella multocida
<400> 90
atgttattaa ctaaccctgt cgtgatttcc attgtggttc tacttgcgct cagtttattg 60
cgtattaatg ttgtcatcgc actcgttatt tccgcattag tggcaggttt aactggcaat 120
ttgggcgtca gtgaaacaat aaaaacgttt acgaatggac taggcggagg tgcagaggtc 180
gccatgaatt atgcgatttt aggcgcgttt gcggttgcca tttcaaaatc aggcattact 240
gatttacttg cctataaagt cattaaacgt ttgggcaata caccaagcag tcgctcaatg 300
gcgggtttta aatattttat cttaacaatc ctcacgctgt ttgccgtttc atcgcaaaac 360
ttattacctg tccatatcgc gtttattcct attgtgattc ccccgcttct tgcgattttc 420
aataaactaa aattggatcg tcgtgccgtt gcttgtgttt taacttttgg tttaaccgcc 480
acttatatgt tattaccagt agggtttggg aaaattttta ttgaaagtat cctcgttaag 540
aatatcaatc aagccggcgc gactttaggc ttacagacat ctgtggctga agtgtcatta 600
gctatggcag tcccagtgat tggcatgatt cttggtttac tgacagcgat ctttattagc 660
tatcgtaaac cgagagaata tgccatgatg cgcagcgaaa tcagcacgca agatattgaa 720
tcacatgttg ctcaaatcaa gccgttccat gtcggcgcaa gtttagtggc aatcattgtt 780
acttttgccc ttcagctctt taccagttca accattattg gtggattagc cggtctgatt 840
atttttgctg tttgtggtat tttcaaatta aaagaaagta atgacatttt ccaacaaggc 900
ttacgtttaa tggcgatgat tggttttgtg atgatcgctg cttctggctt tgccaatgtt 960
attaatacaa cgggtggtgt aacggcgtta gttgaaacct tcagtcaagg ttttggcgca 1020
gaaaataaag ggattgcagc ctttttaatg ctgttagttg gcttatttat tactatgggg 1080
attggctcat cattctcaac ggtacctatt attgcctcta tttatgtacc actttgtctt 1140
tctcttggtt tctcaccttt agcaacggtt tcgcttattg gggtatccgc tgcgcttggt 1200
gatgcgggtt cgcctgcctc tgactcaaca ttaggaccaa cctcgggttt aaatgcagat 1260
ggtaaacatg atcatatttg ggattctgtc gtcccaacat ttatccatta taatatccca 1320
ctcattcttt tcggttggtt agccgccatg tatctgtaa 1359
<210> 91
<211> 452
<212> PRT
<213> Pasteurella multocida
<400> 91
Met Leu Leu Thr Asn Pro Val Val Ile Ser Ile Val Val Leu Leu Ala
1 5 10 15
Leu Ser Leu Leu Arg Ile Asn Val Val Ile Ala Leu Val Ile Ser Ala
20 25 30
Leu Val Ala Gly Leu Thr Gly Asn Leu Gly Val Ser Glu Thr Ile Lys
35 40 45
Thr Phe Thr Asn Gly Leu Gly Gly Gly Ala Glu Val Ala Met Asn Tyr
50 55 60
Ala Ile Leu Gly Ala Phe Ala Val Ala Ile Ser Lys Ser Gly Ile Thr
65 70 75 80
Asp Leu Leu Ala Tyr Lys Val Ile Lys Arg Leu Gly Asn Thr Pro Ser
85 90 95
Ser Arg Ser Met Ala Gly Phe Lys Tyr Phe Ile Leu Thr Ile Leu Thr
100 105 110
Leu Phe Ala Val Ser Ser Gln Asn Leu Leu Pro Val His Ile Ala Phe
115 120 125
Ile Pro Ile Val Ile Pro Pro Leu Leu Ala Ile Phe Asn Lys Leu Lys
130 135 140
Leu Asp Arg Arg Ala Val Ala Cys Val Leu Thr Phe Gly Leu Thr Ala
145 150 155 160
Thr Tyr Met Leu Leu Pro Val Gly Phe Gly Lys Ile Phe Ile Glu Ser
165 170 175
Ile Leu Val Lys Asn Ile Asn Gln Ala Gly Ala Thr Leu Gly Leu Gln
180 185 190
Thr Ser Val Ala Glu Val Ser Leu Ala Met Ala Val Pro Val Ile Gly
195 200 205
Met Ile Leu Gly Leu Leu Thr Ala Ile Phe Ile Ser Tyr Arg Lys Pro
210 215 220
Arg Glu Tyr Ala Met Met Arg Ser Glu Ile Ser Thr Gln Asp Ile Glu
225 230 235 240
Ser His Val Ala Gln Ile Lys Pro Phe His Val Gly Ala Ser Leu Val
245 250 255
Ala Ile Ile Val Thr Phe Ala Leu Gln Leu Phe Thr Ser Ser Thr Ile
260 265 270
Ile Gly Gly Leu Ala Gly Leu Ile Ile Phe Ala Val Cys Gly Ile Phe
275 280 285
Lys Leu Lys Glu Ser Asn Asp Ile Phe Gln Gln Gly Leu Arg Leu Met
290 295 300
Ala Met Ile Gly Phe Val Met Ile Ala Ala Ser Gly Phe Ala Asn Val
305 310 315 320
Ile Asn Thr Thr Gly Gly Val Thr Ala Leu Val Glu Thr Phe Ser Gln
325 330 335
Gly Phe Gly Ala Glu Asn Lys Gly Ile Ala Ala Phe Leu Met Leu Leu
340 345 350
Val Gly Leu Phe Ile Thr Met Gly Ile Gly Ser Ser Phe Ser Thr Val
355 360 365
Pro Ile Ile Ala Ser Ile Tyr Val Pro Leu Cys Leu Ser Leu Gly Phe
370 375 380
Ser Pro Leu Ala Thr Val Ser Leu Ile Gly Val Ser Ala Ala Leu Gly
385 390 395 400
Asp Ala Gly Ser Pro Ala Ser Asp Ser Thr Leu Gly Pro Thr Ser Gly
405 410 415
Leu Asn Ala Asp Gly Lys His Asp His Ile Trp Asp Ser Val Val Pro
420 425 430
Thr Phe Ile His Tyr Asn Ile Pro Leu Ile Leu Phe Gly Trp Leu Ala
435 440 445
Ala Met Tyr Leu
450
<210> 92
<211> 1391
<212> DNA
<213> Pasteurella multocida
<400> 92
ctctagatag aggagcttat atatttactt aagaataagt tgctggtaaa tattcgttgt 60
gtttctcttt taagtactca tcaacactat tatgatcaac gttataagac aattgttctt 120
tgtaaaaatc taatcttgct cttgcattat taataatttc agcccaagtc atcacaataa 180
cttctacatt gtactctaga tcgtctgata ccacaccttt acgctttcct cgttgattgg 240
attctcgttt tgcgaattgg tcaagctcat ttgaaactgc tataaatgtc cactttgttt 300
tactatgatc gaatcgttca tcagatgaca ctgcgtaagc atagttttta atttgagtaa 360
ttacttcaga attaattttc tgacttggac gctttaattc tacaactaaa tattctttat 420
aaccttggct aggctttctt gctttatgaa aaaataaatc aactcttcct tgttttccat 480
cagaaagaaa tactggttta tctgcatcaa aactatcttt atcataataa tctaaatgtg 540
ttgcatgaat ctttaaaaca tcatttagtg tattttcact tcctgaaaaa ttaaaatctt 600
ccataaaaac ccaagtttca ttttctaaga ttttatgtaa ctgatctctt tccaaaagag 660
cttttttatt ctctttatca aaaagaagat tttctaatcc tttcaaaaaa ttaagtctat 720
ctgcaactat ctttgaagaa cggattatag atgttagaga tgtattctct aataatttag 780
aaaacatttc cttctcgtta tcattcaatt ttaatacctc ttccagaatt ctttgcattg 840
atgctggatt ctcttttatc gcattagata gcaattggaa agttaatctc tttgattcaa 900
tagaactaga gctaaatcta ggtaggttat cctcaacctt aacagctacg atatcaaaaa 960
gatttttttc tattttttca acggatgtat attcgtttgc tacatacgga taaatatcca 1020
aatctatcca agattttatt ctttttgcat tttcttcttc tctttgttgt ctaagatatt 1080
catttaattt tgttattgct tctgtaataa gttttctcgc attttcatcc atatcaacta 1140
tgctcaaatt atcactttca tttaagctgt taatagtctc tccacataaa tagacagtat 1200
agttatatcc ttgctttcta attctatttt tagtgtcata atcacaaatg aaggaataat 1260
tctctttgca tagataaaaa tctgaaacat ctttcttatc ccaaagaata attttcattt 1320
tcccatgaat atcagactct tcacctaaaa taatttcagt ttcagtgtta attaattctc 1380
gagggtctag a 1391
<210> 93
<211> 1290
<212> DNA
<213> Pasteurella multocida
<400> 93
ttaagaataa gttgctggta aatattcgtt gtgtttctct tttaagtact catcaacact 60
attatgatca acgttataag acaattgttc tttgtaaaaa tctaatcttg ctcttgcatt 120
attaataatt tcagcccaag tcatcacaat aacttctaca ttgtactcta gatcgtctga 180
taccacacct ttacgctttc ctcgttgatt ggattctcgt tttgcgaatt ggtcaagctc 240
atttgaaact gctataaatg tccactttgt tttactatga tcgaatcgtt catcagatga 300
cactgcgtaa gcatagtttt taatttgagt aattacttca gaattaattt tctgacttgg 360
acgctttaat tctacaacta aatattcttt ataaccttgg ctaggctttc ttgctttatg 420
aaaaaataaa tcaactcttc cttgttttcc atcagaaaga aatactggtt tatctgcatc 480
aaaactatct ttatcataat aatctaaatg tgttgcatga atctttaaaa catcatttag 540
tgtattttca cttcctgaaa aattaaaatc ttccataaaa acccaagttt cattttctaa 600
gattttatgt aactgatctc tttccaaaag agctttttta ttctctttat caaaaagaag 660
attttctaat cctttcaaaa aattaagtct atctgcaact atctttgaag aacggattat 720
agatgttaga gatgtattct ctaataattt agaaaacatt tccttctcgt tatcattcaa 780
ttttaatacc tcttccagaa ttctttgcat tgatgctgga ttctctttta tcgcattaga 840
tagcaattgg aaagttaatc tctttgattc aatagaacta gagctaaatc taggtaggtt 900
atcctcaacc ttaacagcta cgatatcaaa aagatttttt tctatttttt caacggatgt 960
atattcgttt gctacatacg gataaatatc caaatctatc caagatttta ttctttttgc 1020
attttcttct tctctttgtt gtctaagata ttcatttaat tttgttattg cttctgtaat 1080
aagttttctc gcattttcat ccatatcaac tatgctcaaa ttatcacttt catttaagct 1140
gttaatagtc tctccacata aatagacagt atagttatat ccttgctttc taattctatt 1200
tttagtgtca taatcacaaa tgaaggaata attctctttg catagataaa aatctgaaac 1260
atctttctta tcccaaagaa taattttcat 1290
<210> 94
<211> 429
<212> PRT
<213> Pasteurella multocida
<400> 94
Met Lys Ile Ile Leu Trp Asp Lys Lys Asp Val Ser Asp Phe Tyr Leu
1 5 10 15
Cys Lys Glu Asn Tyr Ser Phe Ile Cys Asp Tyr Asp Thr Lys Asn Arg
20 25 30
Ile Arg Lys Gln Gly Tyr Asn Tyr Thr Val Tyr Leu Cys Gly Glu Thr
35 40 45
Ile Asn Ser Leu Asn Glu Ser Asp Asn Leu Ser Ile Val Asp Met Asp
50 55 60
Glu Asn Ala Arg Lys Leu Ile Thr Glu Ala Ile Thr Lys Leu Asn Glu
65 70 75 80
Tyr Leu Arg Gln Gln Arg Glu Glu Glu Asn Ala Lys Arg Ile Lys Ser
85 90 95
Trp Ile Asp Leu Asp Ile Tyr Pro Tyr Val Ala Asn Glu Tyr Thr Ser
100 105 110
Val Glu Lys Ile Glu Lys Asn Leu Phe Asp Ile Val Ala Val Lys Val
115 120 125
Glu Asp Asn Leu Pro Arg Phe Ser Ser Ser Ser Ile Glu Ser Lys Arg
130 135 140
Leu Thr Phe Gln Leu Leu Ser Asn Ala Ile Lys Glu Asn Pro Ala Ser
145 150 155 160
Met Gln Arg Ile Leu Glu Glu Val Leu Lys Leu Asn Asp Asn Glu Lys
165 170 175
Glu Met Phe Ser Lys Leu Leu Glu Asn Thr Ser Leu Thr Ser Ile Ile
180 185 190
Arg Ser Ser Lys Ile Val Ala Asp Arg Leu Asn Phe Leu Lys Gly Leu
195 200 205
Glu Asn Leu Leu Phe Asp Lys Glu Asn Lys Lys Ala Leu Leu Glu Arg
210 215 220
Asp Gln Leu His Lys Ile Leu Glu Asn Glu Thr Trp Val Phe Met Glu
225 230 235 240
Asp Phe Asn Phe Ser Gly Ser Glu Asn Thr Leu Asn Asp Val Leu Lys
245 250 255
Ile His Ala Thr His Leu Asp Tyr Tyr Asp Lys Asp Ser Phe Asp Ala
260 265 270
Asp Lys Pro Val Phe Leu Ser Asp Gly Lys Gln Gly Arg Val Asp Leu
275 280 285
Phe Phe His Lys Ala Arg Lys Pro Ser Gln Gly Tyr Lys Glu Tyr Leu
290 295 300
Val Val Glu Leu Lys Arg Pro Ser Gln Lys Ile Asn Ser Glu Val Ile
305 310 315 320
Thr Gln Ile Lys Asn Tyr Ala Tyr Ala Val Ser Ser Asp Glu Arg Phe
325 330 335
Asp His Ser Lys Thr Lys Trp Thr Phe Ile Ala Val Ser Asn Glu Leu
340 345 350
Asp Gln Phe Ala Lys Arg Glu Ser Asn Gln Arg Gly Lys Arg Lys Gly
355 360 365
Val Val Ser Asp Asp Leu Glu Tyr Asn Val Glu Val Ile Val Met Thr
370 375 380
Trp Ala Glu Ile Ile Asn Asn Ala Arg Ala Arg Leu Asp Phe Tyr Lys
385 390 395 400
Glu Gln Leu Ser Tyr Asn Val Asp His Asn Ser Val Asp Glu Tyr Leu
405 410 415
Lys Glu Lys His Asn Glu Tyr Leu Pro Ala Thr Tyr Ser
420 425
<210> 95
<211> 101
<212> DNA
<213> Pasteurella multocida
<400> 95
cttatttaag cggttttttt acccaacgct tgaaaatgtt ctctccattt gtcacatgga 60
aaaaggagag aacatgtatt ttagaatggg gatataaagc a 101
<210> 96
<211> 220
<212> DNA
<213> Pasteurella multocida
<400> 96
ctttttcctg aagtaataca tcttgagaaa gaattaagtt ttctaaacga gaaggctgat 60
tgatatcata ataaatacca atatcatcgt acactaatga gaaaggtgga tacccatcca 120
cacccagtcc aatagaacgt aaaaaaccat cttctatcgt cgcataaggt aaatcatgtt 180
gttgtgcaaa atgcctcgct ttctttgatg atgctttata 220
<210> 97
<211> 546
<212> DNA
<213> Pasteurella multocida
<220>
<221> modified_base
<222> (529)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (532)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (535)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (538)
<223> a, t, c, g, other or unknown
<220>
<221> modified_base
<222> (543)
<223> a, t, c, g, other or unknown
<400> 97
gactttgtca tcatcgcaac gccaacagac tataataccg aaacaggtta ttttaataca 60
tccactgttg aagctgtcat tgaacaaacc ctttcaatca atccacaagc aacgattatt 120
ataaaatcaa cgattcccgt tggttttacc gaaaaaatgc gtgagaaatt tcataccaag 180
aacattattt tttctcctga gtttttaaga gaaggaaaag cacttcatga caatttgttt 240
ccaagcagaa ttattgttgg cagtacttct tatcaagcaa aagtatttgc cgatatgttg 300
acacagtgtg ccagaaaaaa agatgtaact gttttattta cacacaatac tgaggctgaa 360
gctgttaaat tatttgcaaa tacgtatctc gcaatgcgag ttgccttttc taatgaatta 420
gatacttatg cgagtcttca ccatttaaat acaaaagaca ttatcaatgg tatttctact 480
gatcctcgca ttggtacaca ctacaataac ccaagtttcg gctatggcng tnatngtnta 540
ccnaag 546
<210> 98
<211> 20
<212> DNA
<213> Pasteurella multocida
<400> 98
atctgatcct tcaactcagc 20
<210> 99
<211> 19
<212> DNA
<213> Pasteurella multocida
<400> 99
cgcagggctt tattgattc 19
<210> 100
<211> 27
<212> DNA
<213> Pasteurella multocida
<400> 100
gcggaattcg atgaatgttc cgttgcg 27
<210> 101
<211> 20
<212> DNA
<213> Pasteurella multocida
<400> 101
tttaccaaaa tcattagggg 20
<210> 102
<211> 19
<212> DNA
<213> Pasteurella multocida
<400> 102
gatcatatga caagatgtg 19
<210> 103
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<220>
<221> modified_base
<222> (21)..(30)
<223> a, t, c, g, other or unknown
<400> 103
ggccacgcgt cgactagtac nnnnnnnnnn gatat 35
<210> 104
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<220>
<221> modified_base
<222> (21)..(30)
<223> a, t, c, g, other or unknown
<400> 104
ggccacgcgt cgactagtac nnnnnnnnnn cagcc 35
<210> 105
<211> 20
<212> DNA
<213> Pasteurella multocida
<400> 105
ggccacgcgt cgactagtac 20
<210> 106
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 106
tacgttaacg ccacccgttg 20
<210> 107
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 107
gcttccatac cttgtgaacc 20
<210> 108
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 108
gggtgtacgc cttctgctg 19
<210> 109
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 109
attgcagtca ttgcggatgc 20
<210> 110
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 110
cgatatggta cgtgtcgac 19
<210> 111
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 111
aaaaggcgga cctaagtccg 20
<210> 112
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 112
ccgacaacat gacaatggag 20
<210> 113
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 113
tttgcagtgg cttaccgtc 19
<210> 114
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 114
cctgacgacc aatacggtg 19
<210> 115
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 115
ggatggtctg atcctaatgc 20
<210> 116
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 116
cgttcatcag atgacactgc 20
<210> 117
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 117
gtgattacgg gattatcggg 20
<210> 118
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Primer
<400> 118
tgaagtggta acgaggcttg 20
Claims (36)
- 서열번호 75 로 식별되는 뉴클레오티드 서열로 코딩되는 아미노산 서열을 갖는 폴리펩티드를 코딩하는 뉴클레오티드 서열 내 돌연변이를 갖는 그람 음성 박테리아의 돌연변이체로서, 상기 돌연변이가 서열 번호 75 에서 뉴클레오티드 1072-1087 사이의 삽입이고, 상기 돌연변이로 인해 감쇠된 발병력을 가진 박테리아를 제공하는 그람 음성 박테리아의 돌연변이체.
- 제 1 항에 있어서, 박테리아가 파스튜렐라세애 (Pasteurellaceae) 인 돌연변이체.
- 제 2 항에 있어서, 박테리아가 파스튜렐라 멀토시다 (Pasteurella multocida), 파스튜렐라 해몰리티카 (Pasteurella haemolytica), 파스튜렐라 아나티페스티페르 (Pasteurella anatipestifer) 또는 악티노바실러스 플레우로뉴모니애 (Actinobacillus pleuropneumoniae) 인 돌연변이체.
- 제 3 항에 있어서, 박테리아가 파스튜렐라 멀토시다인 돌연변이체.
- 삭제
- 삭제
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- 삭제
- 제 1 항에 따른 돌연변이체 및 약제학적으로 또는 수의학적으로 허용되는 희석제, 담체, 비히클 또는 부형제를 함유하는 면역원성 조성물 또는 백신.
- 제 10 항에 있어서, 아쥬반트를 추가로 함유하는 면역원성 조성물 또는 백 신.
- 삭제
- 삭제
- 서열 번호 75 로 식별되는 뉴클레오티드 서열을 가진 폴리펩티드로서, 상기 서열 번호 75 가 뉴클레오티드 1072-1087 사이의 삽입인 돌연변이를 갖는 것인 폴리펩티드.
- 제 14 항의 단리된 폴리펩티드 및 약제학적으로 또는 수의학적으로 허용되는 희석제, 담체, 비히클 또는 부형제, 및 임의로는 아쥬반트를 함유하는 면역원성 또는 백신 조성물.
- 제 14 항의 단리된 폴리펩티드에 특이적인 항체를 함유하는 수동 면역화를 위한 항체 제제.
- 제 14 항의 단리된 폴리펩티드 또는 상기 단리된 폴리펩티드에 특이적인 항체를 샘플 중에서 검출하는 것을 포함하는, 그람 음성 박테리아에 의한 감염의 검출 방법.
- 삭제
- 서열 번호 75 로 식별되는 서열을 가진 단리된 핵산 분자로서, 상기 서열 번호 75 가 뉴클레오티드 1072-1087 사이의 삽입인 돌연변이를 갖는 것인 핵산분자.
- 서열 번호 75 로 식별되는 뉴클레오티드 서열의 10 개 이상의 연속적인 핵산인 서열을 가진 단리된 핵산 분자를 함유하고, 상기 서열번호 75 의 뉴클레오티드 1072-1087 사이의 삽입인 돌연변이를 갖는 그람 음성 박테리아의 검출용 PCR 프라이머로서, 서열번호 109 로 식별되는 뉴클레오티드 서열을 갖는 그람 음성 박테리아 검출용 PCR 프라이머.
- 삭제
- 삭제
- 제 1 항에 있어서, 접근 번호 CNCM I-3001 로 입수가능한 돌연변이체 9C8 또는 그의 모든 동정된 특성을 가진 박테리아인 돌연변이체.
- 삭제
- 삭제
- 삭제
- 삭제
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Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US37028202P | 2002-04-05 | 2002-04-05 | |
| US60/370,282 | 2002-04-05 | ||
| US10/406,686 US7449178B2 (en) | 2002-04-05 | 2003-04-03 | Attenuated gram negative bacteria |
| US10/406,686 | 2003-04-03 | ||
| PCT/US2003/010308 WO2003086277A2 (en) | 2002-04-05 | 2003-04-04 | Attenuated gram negative bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20050016334A KR20050016334A (ko) | 2005-02-21 |
| KR101037566B1 true KR101037566B1 (ko) | 2011-05-27 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
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| KR1020047015890A KR101037566B1 (ko) | 2002-04-05 | 2003-04-04 | 감쇠된 그람 음성 박테리아 |
Country Status (16)
| Country | Link |
|---|---|
| US (3) | US7449178B2 (ko) |
| EP (1) | EP1575483B1 (ko) |
| JP (1) | JP4338528B2 (ko) |
| KR (1) | KR101037566B1 (ko) |
| CN (2) | CN102864090A (ko) |
| AT (1) | ATE518955T1 (ko) |
| AU (2) | AU2003223447C1 (ko) |
| BR (1) | BRPI0309001B1 (ko) |
| CA (1) | CA2481186C (ko) |
| CO (1) | CO5640065A1 (ko) |
| ES (1) | ES2371498T3 (ko) |
| HK (1) | HK1078271A1 (ko) |
| MX (1) | MXPA04009730A (ko) |
| NZ (1) | NZ535993A (ko) |
| PT (1) | PT1575483E (ko) |
| WO (1) | WO2003086277A2 (ko) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2370541C2 (ru) * | 1994-12-09 | 2009-10-20 | Имерджент Продакт Дивелопмент Юк Лимитед | Vgc2 днк salmonella typhimurium, мутантная бактерия, обладающая пониженной способностью к адаптации к условиям окружающей среды, и способ ее получения |
| EP1108034B1 (en) * | 1998-09-04 | 2008-08-06 | Emergent Product Development UK Limited | Attenuated salmonella spi2 mutants as antigen carriers |
| GB9910812D0 (en) * | 1999-05-10 | 1999-07-07 | Microscience Ltd | Vaccine composition |
| EP1350796B1 (en) * | 2002-04-05 | 2009-01-07 | Merial | Attenuated gram negative bacteria |
| US7449178B2 (en) * | 2002-04-05 | 2008-11-11 | Merial Limited | Attenuated gram negative bacteria |
| ATE347373T1 (de) | 2003-07-02 | 2006-12-15 | Biotechnology Res & Dev | Akapsuläre i p. multocida hyae /i deletionsmutanten |
| JP4981684B2 (ja) * | 2004-12-22 | 2012-07-25 | インターベツト・インターナシヨナル・ベー・ベー | パストゥレラ・マルトシダワクチン |
| WO2009158240A1 (en) | 2008-06-16 | 2009-12-30 | Emergent Product Development Uk Limited | Salmonella vectored vaccines against chlamydia and methods of use |
| US8917497B2 (en) * | 2012-07-23 | 2014-12-23 | Knaack Llc | Jobsite storage cabinet for housing electronic equipment |
| ES2824402T3 (es) | 2013-11-01 | 2021-05-12 | Boehringer Ingelheim Animal Health Usa Inc | Vacunas atenuadas contra Pasteurella multocida y procedimientos de fabricación y uso de las mismas |
| WO2016049917A1 (en) * | 2014-09-30 | 2016-04-07 | Bgi Shenzhen Co., Limited | Biomarkers for obesity related diseases |
| WO2022072431A1 (en) * | 2020-09-30 | 2022-04-07 | Zoetis Services Llc | Novel pasteurella multocida strains and vaccines having hyac and nanp deletions |
| TW202219269A (zh) * | 2020-09-30 | 2022-05-16 | 美國農業部 | 新型多殺性巴斯德氏菌株及具有ｈｙａＣ與ｎａｎＰ缺失之疫苗 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000061724A2 (en) | 1999-04-09 | 2000-10-19 | Pharmacia & Upjohn Company | Anti-bacterial vaccine compositions |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994011024A1 (en) * | 1992-11-06 | 1994-05-26 | Regents Of The University Of Minnesota | Composition protective against p. multocida pasteurellosis infection |
| US6793927B1 (en) * | 1993-12-06 | 2004-09-21 | The United States Of America As Represented By The Secretary Of The Department Of Agriculture | Construction of Pasteurella haemolytica vaccines |
| RU2370541C2 (ru) | 1994-12-09 | 2009-10-20 | Имерджент Продакт Дивелопмент Юк Лимитед | Vgc2 днк salmonella typhimurium, мутантная бактерия, обладающая пониженной способностью к адаптации к условиям окружающей среды, и способ ее получения |
| US6770275B1 (en) * | 1996-05-31 | 2004-08-03 | Akzo Nobel N.V. | Live attenuated RTC-producing bacteria of the family |
| US6086894A (en) * | 1996-06-27 | 2000-07-11 | Virginia Tech Intellectual Properties, Inc. | Recombinant vaccine for diseases caused by encapsulated organisms |
| US6783764B1 (en) * | 1997-04-10 | 2004-08-31 | Akzo Nobel Nv | Actinobacillus pleuropneumoniae subunit vaccine |
| CH692179A5 (de) | 1997-06-25 | 2002-03-15 | Kueschall Design Ag | Rollstuhl und Sitzmodul für den Rollstuhl, |
| US20010018055A1 (en) * | 1997-09-25 | 2001-08-30 | Briggs Robert E. | Deletion mutants of virulence factors of pasteurellaceae |
| US6790950B2 (en) * | 1999-04-09 | 2004-09-14 | Pharmacia & Upjohn Company | Anti-bacterial vaccine compositions |
| GB9910812D0 (en) * | 1999-05-10 | 1999-07-07 | Microscience Ltd | Vaccine composition |
| US6780405B1 (en) * | 2000-04-28 | 2004-08-24 | Avant Immunotherapeutics, Inc. | Regulated antigen delivery system (RADS) |
| WO2002018601A2 (en) * | 2000-08-25 | 2002-03-07 | Abbott Laboratories | Essential bacteria genes and genome scanning in haemophilus influenzae for the identification of 'essential genes' |
| US20040029129A1 (en) * | 2001-10-25 | 2004-02-12 | Liangsu Wang | Identification of essential genes in microorganisms |
| EP1350796B1 (en) * | 2002-04-05 | 2009-01-07 | Merial | Attenuated gram negative bacteria |
| US7449178B2 (en) * | 2002-04-05 | 2008-11-11 | Merial Limited | Attenuated gram negative bacteria |
| WO2004087749A2 (en) * | 2003-03-27 | 2004-10-14 | Children's Hospital, Inc. | Nontypeable haemophilus influenzae virulence factors |
| UA100370C2 (uk) * | 2006-12-11 | 2012-12-25 | Мериал Лимитед | Спосіб вакцинації птахів проти salmonella |
| TWI328458B (en) * | 2007-03-09 | 2010-08-11 | Univ Nat Chunghsing | Subunit vaccine of pasteurella multocida in veterinary uses |
-
2003
- 2003-04-03 US US10/406,686 patent/US7449178B2/en not_active Expired - Lifetime
- 2003-04-04 PT PT03719577T patent/PT1575483E/pt unknown
- 2003-04-04 MX MXPA04009730A patent/MXPA04009730A/es active IP Right Grant
- 2003-04-04 ES ES03719577T patent/ES2371498T3/es not_active Expired - Lifetime
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- 2003-04-04 NZ NZ535993A patent/NZ535993A/en not_active IP Right Cessation
- 2003-04-04 CN CN2011104311167A patent/CN102864090A/zh active Pending
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- 2003-04-04 KR KR1020047015890A patent/KR101037566B1/ko active IP Right Grant
- 2003-04-04 AT AT03719577T patent/ATE518955T1/de active
- 2003-04-04 CN CNA038123800A patent/CN1934240A/zh active Pending
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- 2003-04-04 CA CA2481186A patent/CA2481186C/en not_active Expired - Lifetime
- 2003-04-04 JP JP2003583304A patent/JP4338528B2/ja not_active Expired - Lifetime
- 2003-04-04 BR BRPI0309001-9A patent/BRPI0309001B1/pt not_active IP Right Cessation
-
2004
- 2004-11-04 CO CO04111105A patent/CO5640065A1/es not_active Application Discontinuation
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2005
- 2005-12-02 HK HK05111055.4A patent/HK1078271A1/xx not_active IP Right Cessation
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2008
- 2008-10-07 US US12/246,809 patent/US7943125B2/en not_active Expired - Lifetime
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- 2010-08-04 AU AU2010206113A patent/AU2010206113B2/en not_active Expired
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- 2011-04-20 US US13/091,046 patent/US8329163B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000061724A2 (en) | 1999-04-09 | 2000-10-19 | Pharmacia & Upjohn Company | Anti-bacterial vaccine compositions |
Non-Patent Citations (1)
| Title |
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