KR100960611B1 - Modified flagellin improved Toll-like receptor 5 stimulating activity - Google Patents

Modified flagellin improved Toll-like receptor 5 stimulating activity Download PDF

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KR100960611B1
KR100960611B1 KR1020080011330A KR20080011330A KR100960611B1 KR 100960611 B1 KR100960611 B1 KR 100960611B1 KR 1020080011330 A KR1020080011330 A KR 1020080011330A KR 20080011330 A KR20080011330 A KR 20080011330A KR 100960611 B1 KR100960611 B1 KR 100960611B1
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이준행
이시은
김수영
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Abstract

본 발명은 톨-유사 수용체-5(이하 TLR5) 자극 활성이 강화된 플라젤린 변형체에 관한 것으로서, 보다 상세하게는 TLR5 작동제인 플라젤린의 일부 아미노산을 점돌연변이시킴으로써 TLR5 자극 활성을 향상시켜 플라젤린 고유의 활성을 강화시킨 플라젤린 변형체에 관한 것이다.The present invention relates to a flagellin variant with enhanced toll-like receptor-5 (hereinafter, TLR5) stimulating activity, and more particularly, to TLR5 stimulating activity by point mutation of some amino acids of flagellin, which is a TLR5 agonist, to enhance TLR5 stimulatory activity. It relates to a flagellin variant with enhanced activity.

플라젤린, 위치지정돌연변이, 톨-유사 수용체-5, NF-κB Flagellin, site mutation, toll-like receptor-5, NF-κB

Description

톨-유사 수용체-5 자극 활성이 증가된 플라젤린 변형체{Modified flagellin improved Toll-like receptor 5 stimulating activity}Modified flagellin improved Toll-like receptor 5 stimulating activity

본 발명은 톨-유사 수용체-5(이하 TLR5) 자극 활성이 강화된 플라젤린 변형체에 관한 것으로서, 보다 상세하게는 TLR5 작동제인 플라젤린의 일부 아미노산을 점돌연변이시킴으로써 TLR5 자극 활성을 향상시켜 플라젤린 고유의 활성을 강화시킨 플라젤린 변형체에 관한 것이다.The present invention relates to a flagellin variant with enhanced toll-like receptor-5 (hereinafter, TLR5) stimulating activity, and more particularly, to TLR5 stimulating activity by point mutation of some amino acids of flagellin, which is a TLR5 agonist, to enhance TLR5 stimulatory activity. It relates to a flagellin variant with enhanced activity.

편모(flagella)는 세균의 운동성을 결정하는 중요한 구성요소이며 크게 후크(hook), 기저체(basal body) 및 필라멘트(filament)로 구성되어 있다. 편모는 세균의 유영(swimming) 혹은 유주운동(swarming motility), 세균의 주성(taxis)을 결정하고, 바이오필름(biofilm)을 형성하여, 병원성 미생물의 부착능을 결정하는 기능이 있다고도 알려져 있다. 편모의 필라멘트를 구성하는 구성 단위 단백질을 플라젤린(flagellin)이라 하며, 플라젤린이 규칙적으로 조합되어(assembling) 필라멘트를 형성한다. Hayashi 등은 포유류가 발현하는 TLR5가 그람 음성 및 그람 양성 세 균의 플라젤린을 인지하여 NF-κB를 활성화시킨다고 보고하였다(Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A: Nature 410:1099-1103, 2001). Flagella (flagella) is an important component that determines the motility of the bacteria and consists largely of hooks (basal body) and filaments (filament). Flagella is known to have a function of determining the swimming or swarming motility of bacteria, the taxis of bacteria, and forming a biofilm to determine the adhesion of pathogenic microorganisms. The structural unit protein constituting the flagella filaments is called flagellin, and flagellin is regularly assembled to form filaments. Hayashi et al. Reported that mammalian expression of TLR5 activates NF-κB by recognizing Gram-negative and Gram-positive bacterial flagelins (Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng). JK, Akira S, Underhill DM, Aderem A: Nature 410: 1099-1103, 2001).

톨-유사수용체(TLR)는 대표적인 '패턴인식수용체(Pattern Recognition Receptor; PRR)'로서 이는 병원체에 존재하는 ‘병원체와 관련된 분자구조(Pathogen Associated Molecular Pattern; PAMP)'를 인식하는 수용체이며 포유동물뿐만 아니라 곤충, 식물세포의 표면에도 존재한다. 지금까지 모두 13 종류의 TLR이 밝혀져 있으며 각 TLR의 작동제에 대한 연구가 활발히 진행되고 있다(Akira S, Uematsu S, Takeuchi O: Cell 124(4): 783-801, 2006).Toll-like receptors (TLR) are typical "pattern recognition receptors (P attern R ecognition R eceptor; PRR) '; a as it' (PAMP P athogen A ssociated M olecular P attern) Molecular structures associated with pathogens, present on pathogens It is a recognized receptor and is present on the surface of insects and plant cells as well as mammals. Thirteen types of TLRs have been identified so far, and studies on agonists of each TLR have been actively conducted (Akira S, Uematsu S, Takeuchi O: Cell 124 (4): 783-801, 2006).

TLR과 같은 PRR은 숙주세포의 세포 표면 혹은 세포질 내에 분포하며, 다양한 PAMP의 자극에 의해 '선천성 면역 반응(innate immune response)'을 유도하고 나아가 '획득 면역 반응(adaptive immune response)'을 조절한다. 따라서, TLR 작동제(agonist)들은 다양한 ‘면역 조절제’, 특히 ‘백신 보조제’ 개발에 적합한 표적이 될 수 있다. PRRs, such as TLRs, are distributed on the cell surface or cytoplasm of host cells, and induce 'innate immune response' by stimulating various PAMPs and further regulate 'adaptive immune response'. Thus, TLR agonists can be suitable targets for the development of various 'immunomodulators', particularly 'vaccine adjuvants'.

백신 보조제란 백신과 함께 투여할 경우(coadministration) 백신 항원에 의해 유도되는 항원 특이적 면역 반응(Ag-specific immune response)을 향상(enhance), 지속(prolong) 또는 촉진(accelerate)시킬 수 있는 물질을 칭한다. 현재까지 사람에게 사용할 수 있도록 승인된 백신 보조제에는 'alum(알루미늄 포스페이트(aluminium phosphate) 및 알루미늄 히드록시드(aluminium hydroxide))' 및 '스쿠알렌 에멀젼(squalene emulsion)' 등이 있다. 백신 보조제는 다음 5가지의 조 건 중 1가지 이상을 충족해야만 한다: 1) 항원제공세포(antigen presenting cell) 표면의 공동자극 분자(co-stimulatory moleciule) 발현 조절, 항원 특이적 T 림프구 반응 유도 혹은 사이토카인(cytokine) 분비 조절과 같은 면역조절(immunomodulation), 2) 항원 제공(presentation), 3) CD8+ T 림프구 반응, 4) 표적(targeting), 5) 항원 축적능(depot generation). A vaccine adjuvant is a substance that can enhance, prolong or accelerate the antigen-specific immune response induced by the vaccine antigen when coadministration with the vaccine. It is called. Vaccine aids that have been approved for human use to date include 'alum' (aluminum phosphate and aluminum hydroxide) and 'squalene emulsion'. Vaccine adjuvant must meet at least one of the following five conditions: 1) regulate co-stimulatory moleciule expression on antigen presenting cell surface, induce antigen-specific T lymphocyte response, or Immunomodulation, such as cytokine secretion regulation, 2) antigen presentation, 3) CD8 + T lymphocyte response, 4) targeting, 5) antigen generation.

이상적인 백신 보조제는 1) 안전해야 하고, 2) 체내에서 생분해되어야 하며, 3) 항원 단독으로 투여했을 경우보다 높은 방어면역 혹은 치료 면역능을 보여야 하고, 4) 화학적 혹은 생물학적으로 확인된 물질이어야 하며, 5) 항원보다 낮은 농도에서 작용하는 것이 좋고, 6) 상업적 혹은 임상적으로 쉽게 적용할 수 있도록 반감기가 길어야 한다. The ideal vaccine adjuvant should be 1) safe, 2) biodegradable in the body, 3) show higher protective or therapeutic immunity than when administered alone, 4) chemically or biologically identified, and 5 It is recommended to work at concentrations lower than the antigen and 6) to have a long half-life for commercial or clinical application.

현재 백신 보조제로 사용되고 있거나 사용이 고려되고 있는 것으로는 1) 수산화 알루미늄 젤 등과 같은 미네랄 염(mineral salt), 2) 계면활성 물질, 3) 세균 유래 물질, 4) 사이토카인 혹은 호르몬, 6) 다가음이온(polyanion), 7) 폴리아크릴(polyacryl), 8) 담체(carrier), 9) 바이러스를 이용한 생 벡터(living vector), 및 10) 미네랄 오일(mineral oil)이나 리포좀(liposome)과 같은 운반제(vehicle) 등이 있다. 이 중에서 현재 활발한 연구가 진행되고 있으며 크게 주목받고 있는 단백질 유래 백신 보조제로는 Vibrio cholerae에서 유래한 콜레라 독소(cholera toxin, CT)와 대장균 유래의 이열성 독소(heat-labile toxin, LT)가 있다. 이들 백신 보조제는 점막 부위(mucosal area) 및 혈청(serum) 중에서 항원 특이적 항체(antigen-specific antibody) 생산을 유도하며, 항원제공세포(antigen presenting cell; APC) 표면의 B7-2 발현을 유도하여 CD4+ 세포의 공동자극 신호(co-stimulatory signaling)를 촉진하는 기전에 의한다는 보고가 있다. 그러나 이들은 장독성(enterotoxicity)이 높은 외독소이므로 독성(toxicity)은 줄이고 보조제적 특성(adjuvaticity)은 높이는 방향으로 연구가 진행 중이다. Currently used or contemplated for use as a vaccine adjuvant include: 1) mineral salts such as aluminum hydroxide gels, 2) surfactants, 3) bacteria-derived materials, 4) cytokines or hormones, 6) polyanions (polyanion), 7) polyacryl, 8) carriers, 9) living vectors using viruses, and 10) carriers such as mineral oils or liposomes. vehicle). Among these, active research is underway, and Vibrio , a protein-derived vaccine adjuvant, has attracted much attention. There are cholera toxin (CT) derived from cholerae and heat-labile toxin (LT) derived from E. coli. These vaccine adjuvants induce the production of antigen-specific antibodies in mucosal areas and serum and induce B7-2 expression on the surface of antigen presenting cells (APCs). It has been reported that it is a mechanism that promotes co-stimulatory signaling of CD4 + cells. However, since they are exotoxins with high enterotoxicity, research is being conducted to reduce toxicity and increase adjuvaticity.

PCT 국제특허공개공보 제WO 2005/070455호에서 본 발명자들은 최근 패혈증 비브리오균의 숙주 부착 및 침습인자(adhesion/invasion factor)를 스크리닝하기 위하여 ‘전위자 돌연변이 라이브러리(transposon mutant library)’를 제작하여 숙주 세포에 대한 부착능(adhesion)과 운동성(motility)이 상실된 3 클론의 Tn-플랭킹 영역(Tn-flanking region)을 분석하는 과정 중에 56개의 유전자로 구성된 2개의 편모관련 오페론(flagella operon)을 동정하였다. 이 과정에서 패혈증 비브리오균은 총 여섯 개의 플라젤린 유전자를 가지고 있으며(flaA, flaB , flaF , flaC , flaD , flaE) 이 중 flaB 유전자가 플라젤린을 구성하는 주요 인자임을 확인하였다. 본 발명자는 패혈증 비브리오균 극성 플라젤린(polar flagellin)을 구성하는 성분인 플라젤린 재조합 단백질(FlaB)을 패혈증 비브리오균에 대한 ‘성분 백신(component vaccine)’으로서 응용 가능성을 추진하던 중에, 플라젤린 백신 보조제 효능이 있음을 알게 되었고 이를 규명하고자 연구를 진행하였다. 그 결과, 백신 항원인 테타누스 유독소를 플라젤린과 함께 실험동물의 비강에 투여하면 플라젤린이 숙주의 TLR5에 신호를 보내어 면역체계를 활성화시킴으로써 백신의 효능을 증폭시키며, 테타누스 유독소와 플라젤린을 함께 투여하여 면역한 마우스에 치사량의 테타누스 독소를 주사할 경우에 100% 방어 면역능을 유도함으로써 탁월한 ‘점막 백신 보조제’ 효능이 있음을 증명하였다(Lee SE, Kim SY, Jeong BC, Kim YR, Bae SJ, Ahn OS, Lee JJ, Song HC, Kim JM, Choy HE, Chung SS, Kweon MN, Rhee JH.: Infect. Immun. 74: 694-702, 2006). In PCT International Patent Publication No. WO 2005/070455, the present inventors recently prepared a 'transposon mutant library' to screen for host adhesion and invasion factor of sepsis vibrio. Identifying two flagella-related operons consisting of 56 genes during the analysis of Tn-flanking regions of three clones that have lost adhesion and motility to cells It was. In this process, sepsis vibrioviruses have a total of six flagellin genes ( flaA , flaB , flaF , flaC , flaD , flaE ), and the flaB gene is the major constituent of flagellin. The present inventors are pursuing the application of flagellin recombinant protein (FlaB), which is a component of the sepsis vibrio polar flagellin, as a 'component vaccine' against sepsis vibrio, We found that there was an adjuvant effect and we studied to find out. As a result, when the vaccine antigen tetanus toxicant is administered to the nasal cavity of experimental animals together with flagellin, flagellin signals the host's TLR5 to activate the immune system and amplifies the efficacy of the vaccine. Injecting lethal doses of tetanus toxin into mice immunized with JELIN proved to have an excellent 'mucosal vaccine adjuvant' efficacy by inducing 100% protective immunity (Lee SE, Kim SY, Jeong BC, Kim YR). , Bae SJ, Ahn OS, Lee JJ, Song HC, Kim JM, Choy HE, Chung SS, Kweon MN, Rhee JH .: Infect. Immun. 74: 694-702, 2006).

다양한 TLR 작동제 중에서 TLR5를 자극하는 플라젤린은 다른 TLR 작동제[CpG-DNA, MALP (mycoplasmal lipopeptide)]와는 달리 단백질 성분이기 때문에 재조합 단백질을 합성하여 지속적인 품질 관리(quality control)가 가능할 뿐만 아니라 TLR5 자극 활성이 강화된 다양한 재조합 융합 단백질을 제작할 수 있다. Flagellin, which stimulates TLR5 among various TLR agonists, is a protein component unlike other TLR agonists [CpG-DNA, MALP (mycoplasmal lipopeptide)], so it is not only possible to synthesize recombinant proteins for continuous quality control but also TLR5. Various recombinant fusion proteins with enhanced stimulatory activity can be produced.

플라젤린의 3차원 구조에 관한 연구 보고에 의하면 플라젤린 단량체(monomer) 사이에서는 극성을 띄는 아미노산 잔기와 전하를 띄는 아미노산 잔기가 서로 반응(polar-charge reaction)하여 각 단량체가 축 상호작용(axial interaction)하여 다량체를 이루어 플라젤라 고유의 필라멘트 구조를 형성한다(Yonekura K, Maki-Yonekura S, Namba K: Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature. 2003 424(6949):643-50; Samatey FA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K: Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature. 2001 410(6826):331-7). Smith 등의 연구에 의하면 TLR5는 다량체화된 필라멘트 타입의 플라젤린을 인식하는 것이 아니라 플라젤린 단량체(monomer)를 인식하는 것으로 보고되었다(Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, Barrett SL, Cookson BT, Aderem A.Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol. 2003 4(12):1247-53).According to a study on the three-dimensional structure of flagellin, a polar-charge reaction between a polar amino acid residue and a charged amino acid residue reacts with each other in a polar-charge reaction. (Yonekura K, Maki-Yonekura S, Namba K: Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy.Nature. 2003 424 (6949): 643-50) Samatey FA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K: Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling.Nature. 2001 410 (6826): 331-7). According to Smith et al., TLR5 was reported to recognize flagelin monomers rather than multimerized filament type flagelins (Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA). , Barrett SL, Cookson BT, Aderem A. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility.Nat Immunol. 2003 4 (12): 1247-53).

현재 임상에서 사용되고 있는 ‘예방 및 치료 백신제제들’(감염질환, 자가면역 질환, 알러지 질환, 암 백신)에서 관찰되는 공통적인 문제점은 해당 반응을 증폭시켜 주는 목적으로 사용되는 백신 보조제로부터 유래한다는 것이다. 즉, 좀 더 안전하고, 효율이 좋은 백신 보조제의 개발이 강력하게 요망되고 있다.A common problem observed in 'preventive and therapeutic vaccines' (infectious diseases, autoimmune diseases, allergic diseases, cancer vaccines) currently being used in the clinic is that they are derived from vaccine aids used to amplify the response. . That is, the development of safer and more efficient vaccine adjuvant is strongly desired.

이에 본 발명자들은 패혈증 비브리오균 플라젤린 유전자 flaB의 플라젤린 단량체(monomer)간의 축상호반응(axial interaction)에 관여할 것으로 예견되는 아미노산 서열을 돌연변이시켜 축상호반응에 관여하는 극성-전하 반응(polar-charge reaction)을 억제시킬 수 있는 재조합 플라젤린 변형체를 제조함으로써 기존의 플라젤린에 비해 TLR5 자극 활성이 월등히 향상된 플라젤린 변형체를 제작할 수 있음을 발견하고 본 발명을 완성하였다. Therefore, the present inventors mutated an amino acid sequence predicted to be involved in the axial interaction between the flagellin monomers of the sepsis Vibrio flagellin gene flaB and involved in the axial interaction. By preparing a recombinant flagellin variant capable of inhibiting charge reaction, the present inventors have found that a flagellin variant with significantly improved TLR5 stimulating activity compared to conventional flagellin can be prepared.

따라서, 본 발명의 목적은 TLR5 자극 활성이 향상된 플라젤린 변형체를 제공하는 것이다.Accordingly, it is an object of the present invention to provide flagellin variants with enhanced TLR5 stimulatory activity.

또한 본 발명의 목적은 상기 플라젤린 변형체를 유효성분으로 함유하는 백신 보조제를 제공하는 것이다.It is also an object of the present invention to provide a vaccine adjuvant containing the flagellin variant as an active ingredient.

상기 목적을 달성하기 위하여, 본 발명에서는 TLR5 작동제인 플라젤린의 플라젤린 단량체(monomer)간의 상호반응을 억제하는 플라젤린 변형체를 제공한다.In order to achieve the above object, the present invention provides a flagellin variant that suppresses the interaction between flagellin monomer (flameline) monomer of flagellin TLR5 agonist.

또한 본 발명은 상기 기능이 향상된 플라젤린 변형체를 유효성분으로 함유하는 백신 보조제를 제공한다.In another aspect, the present invention provides a vaccine adjuvant containing the improved flagellin variant as an active ingredient.

본 발명에서는 PCT 국제특허공개공보 제WO 2005/070455호에 의해 플라젤린이 TLR5를 자극하여 강력한 점막백신 보조제 효능이 있는 것으로 밝혀진 플라젤린의 TLR5 자극 활성을 더욱 향상시킨 플라젤린 변형체를 제공함으로써 보다 업그레이드된 플라젤린 백신 보조제를 개발하였다. 이는 새로운 패러다임의 ‘감염질환’, ‘자가 면역 질환’ 및 ‘알러지 질환’ 등의 치료와 나아가 ‘항암면역 치료’와 같은 응용 연구(Applied Science research)는 차세대의 주목 받는 연구 테마이며, 기초 연구와 베드-사이드(bed-side)의 임상연구를 연결해 주는 중요한 고리를 제공할 것이다. 따라서, 본 발명에 의한 결과를 각종 백신제제에 적용한다면 엄청난 부가가치를 창출할 수 있을 것이다.In the present invention, it is further upgraded by providing a flagellin variant which further improves the TLR5 stimulating activity of flagellin, which was found to be a potent mucosal vaccine adjuvant by stimulating TLR5 by PCT WO 2005/070455. Flagellin vaccine adjuvant was developed. This is the new paradigm's treatment of 'infectious diseases', 'autoimmune diseases' and 'allergic diseases', and 'applied science research' such as 'anti-cancer immunity treatment' is the next generation's attention research theme. It will provide an important link between bed-side clinical research. Therefore, if the result of the present invention is applied to various vaccine preparations, it will be able to create enormous added value.

이하 본 발명을 더욱 상세하게 설명하면 다음과 같다.Hereinafter, the present invention will be described in more detail.

본 발명은 TLR5 작동제인 플라젤린의 일부 아미노산을 점돌연변이시킴으로써 플라젤린 단량체(monomer)의 다량체화(multimerization)를 억제하여 TLR5 자극 활성이 강화된 플라젤린 변형체에 관한 것이다.The present invention relates to flagellin variants having enhanced TLR5 stimulatory activity by inhibiting multimerization of flagellin monomers by point mutation of some amino acids of flagellin, a TLR5 agonist.

본 발명은 TLR5 작동제인 플라젤린의 플라젤린 단량체 간의 축 상호반응에 관여할 것으로 분석된 야생형 패혈증 비브리오의 플라젤린 FlaB의 93 번째 아미노산인 아르기닌(Arginine; R93)을 알라닌(alanine; A)으로 위치 지정 돌연변이시켜 제조한 서열번호 2의 플라젤린 변형체(R93A), 97 번째 아미노산인 루신(leucine; L97)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 4의 플라젤린 변형체(L97W), 101 번째 아미노산인 아스파라긴(asparagine; N)을 알라닌(alanine; A)으로 위치 지정 돌연변이시켜 제조한 서열번호 6의 플라젤린 변형체(N101A), 103 번째 아미노산인 세린(serine; S103)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 8의 플라젤린 변형체(S103W), 108 번째 아미노산인 글루타민산(glutamic acid; E)을 알라닌(alanine; A)으로 위치 지정 돌연변이시켜 제조한 서열번호 10의 플라젤린 변형체(E108A), 151 번째 아미노산인 알라닌(alanine; A151)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 12의 플라젤린 변형체(A151W), 153 번째 아미노산인 아스파라긴(aspargine; N153)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 14의 플라젤린 변형체(N153W) 및 291 번째 아미노산인 발린(valine; V291)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 16의 플라젤린 변형체(V291W)에 관한 것이다. In the present invention, arginine (R93), the 93rd amino acid of flagellin FlaB of wild-type sepsis Vibrio, which is analyzed to be involved in the axial interaction between flagellin monomers of flagellin, a TLR5 agonist, is assigned to alanine (A). Flageline variant (R93A) of SEQ ID NO: 2 prepared by mutation, flageline variant (L97W) of SEQ ID NO: 4 prepared by site-directed mutation of leucine (L97), the 97th amino acid, with tryptophan (W), Plageline variant (N101A) of SEQ ID NO: 6 prepared by positional mutation of asparagine (N), the 101st amino acid, with alanine (A), serine (S103), the 103rd amino acid, tryptophan (tryptophan; W) flageline variant (S103W) prepared by positional mutation in position 8) and the amino acid glutamic acid (glutamic acid; E) to alanine (A) Flageline variant (A108W) of SEQ ID NO: 10 (A108W) prepared by mutating the positional mutation of Alanine (A151), the 151th amino acid, tryptophan (W) Plageline variant (N153W) of SEQ ID NO: 14 prepared by positional mutation of asparagine (N153), the 153th amino acid, with tryptophan (W), and valine (V291), the 291th amino acid, were tryptophan; It relates to a flagellin variant (V291W) of SEQ ID NO: 16 prepared by positional mutation in W).

본 명세서에서 언급하는 플라젤린은 세균의 운동성을 결정하는 섬모인 플라젤라를 구성하는 단위 분자이다. Flagellin as referred to herein is a unit molecule constituting flagella, the cilia that determine the motility of bacteria.

본 발명에서 제공하는 상기 플라젤린 변형체들은 TLR5 자극 활성이 월등히 향상되었으며, 이러한 현상은 수용액 상에서 플라젤린 변형체가 기존의 플라젤린에 비해서 다량체화(multimerization)가 현저히 감소되어 플라젤린 중합이 억제되어 나타남을 알 수 있었다.The flagellin variants provided in the present invention have significantly improved TLR5 stimulating activity, and this phenomenon shows that the flagellin variant is significantly reduced in multimerization compared to conventional flagellin in aqueous solution, indicating that flagellin polymerization is suppressed. Could know.

따라서 본 발명에 의해 제조된 TLR5 자극활성이 월등히 향상된 플라젤린 변형체는 기존의 플라젤린을 대체하여 보다 강력한 TLR5 작동제를 제공함으로써 기존 의 플라젤린이 갖는 효율보다 강화된 백신 보조제를 제공할 수 있다.Therefore, the flagellin variant with the improved TLR5 stimulatory activity produced by the present invention can provide a more powerful TLR5 agonist in place of the existing flagellin, thereby providing an enhanced vaccine adjuvant than the existing flagellin.

본 발명에 의한 플라젤린 변형체를 이용한 TLR5 자극 활성 강화를 증명하는 과정은 다음과 같다:The process of demonstrating enhanced TLR5 stimulation activity using the flagellin variant according to the present invention is as follows:

1) 서열번호 2, 4, 6, 8, 10, 12, 14 및 16의 플라젤린 변형체를 제조하는 단계;1) preparing flagellin variants of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16;

2) 상기 플라젤린 변형체가 수용액 상에서 플라젤린 중합을 억제시킴을 확인하는 단계; 및2) confirming that the flagellin variant inhibits flagellin polymerization in an aqueous solution; And

3) 대장균에서 유도된 각 재조합 플라젤린 변형체의 TLR5 활성화를 확인하는 단계;3) confirming TLR5 activation of each recombinant flagellin variant derived from E. coli;

를 포함한다.It includes.

이하, 실시예 및 시험예를 들어 본 발명을 보다 자세하게 설명한다. 그러나, 이들 실시예 또는 시험예들은 본 발명을 구체적으로 설명하려는 것이지, 이러한 실시예 또는 시험예에 의하여 본 발명의 권리범위가 제한되는 것은 아니다.Hereinafter, an Example and a test example are given and this invention is demonstrated in detail. However, these examples or test examples are intended to describe the present invention in detail, and the scope of the present invention is not limited by these examples or test examples.

본 발명에서 사용하는 균주 및 플라스미드의 특성은 표 1에 정리하였다. 각각의 제조방법과 자세한 특성은 해당 실시예 및 시험예에서 적시하였다.The characteristics of the strains and plasmids used in the present invention are summarized in Table 1. Each manufacturing method and detailed characteristics are indicated in the corresponding examples and test examples.

균주 혹은 Strain or
플라스미드Plasmid 특 징Characteristic 출 처source EscherichiaEsherichia colicoli DH5αDH5α F recA1; restriction negativeF recA 1; restriction negative 실험실 기존 보유Existing lab ER2566
ER2566
F-λ- fhuA2 [ lon ] ompT lacZ :: T7 gene1 gal sulA11 ( mcrC - mrr )114:: IS10
R( mcr -73:: miniTn10 - TetS )2 R(zgb-210::Tn10)(TetS) endA1 [ dcm ] F- λ- fhuA2 [ lon ] ompT lacZ :: T7 gene1 gal sulA1 1 (mcrC - mrr ) 114 :: IS10
R ( mcr -73 :: miniTn10 - TetS ) 2 R (zgb-210 :: Tn10) (TetS) endA1 [ dcm ] New
England
BiolabNew
England
Biolab BL21(DE3)BL21 (DE3) F-ompT hsdS B ( r B - m B -) gal dcm (DE3)F- ompT hsdS B ( r B - m B- ) gal dcm (DE3) NovagenNovagen PlasmidsPlasmids pCR2.1 TOPO pCR2.1 TOPO PCR TA cloning vector, AmpR,KmR PCR TA cloning vector, Amp R , Km R InvitrogenInvitrogen pTYB12pTYB12 IMPACT(Intein Mediated Purification with an Affinity Chitin-binding Tag) 발현 벡터, AmpR Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) expression vector, Amp R New
England
iolabNew
England
iolab

<각 균주의 배양 및 보관><Cultivation and storage of each strain>

하기 실시예 및 시험예에서 사용한 대장균 균주들은 Luria Bertani 배지(Difco Co.)에서 배양하였다. 사용한 균주들은 배양 후 글리세롤을 30% 되게 첨가하여 -80℃ 초저온 냉동고에서 보관하였다.E. coli strains used in the following examples and test examples were cultured in Luria Bertani medium (Difco Co.). The used strains were added to glycerol 30% after incubation and stored in -80 ℃ cryogenic freezer.

[실시예 1] 위치 지정 돌연변이 재조합 플라젤린 변형체의 제조Example 1 Preparation of Positional Mutant Recombinant Flagellin Variant

기존에 보고된 살모넬라균의 플라젤린 3차원 구조 분석 데이터(Yonekura K, Maki-Yonekura S, Namba K: Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature. 2003 424(6949):643-50; Samatey FA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K: Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature. 2001 410(6826):331-7)와 본 발명자의 생물정보학적 분석(bioinformatics analysis)에 의하면 패혈증 비브리오균 플라젤린 FlaB 단량체 간의 축 상호반응(axial interaction)에 필요한 아미노산 잔기는 다음과 같다. 즉, 축 반응에 관여하는 각 단량체 중 상위에 존재하는 단량체(upper subunit)의 70번째 아미노산인 아스파르트산(aspartic acid; D70), 135번째 아미노산인 아스파라긴(asparagine; D135), 151번째 아미노산인 알라닌(alanine; A151)과 153번째 아미노산인 아스파라긴(asparagine; N153)이 하위에 존재하는 단량체(lower subunit)의 각각 100번째 아미노산인 아스파라긴(asparagine; N100), 108번째 아미노산인 글루타민산(glutamic acid; E108), 93번째 아미노산인 아르기닌(arginine; R93)과 292번째 아미노산인 알라닌 (alanine; A292)이 극성-전하 반응(polar-charge reaction)을 통해 다량체(multimer)를 형성할 것으로 분석되었다(즉, 상위 플라젤린 D70:하위 플라젤린 N100; 상위 플라젤린 N135:하위 플라젤린 E107; 상위 플라젤린 A151:하위 플라젤린 R93; 상위 플라젤린 N153:하위 플라젤린 A292).Previously reported flagellin three-dimensional structural analysis data of Salmonella (Yonekura K, Maki-Yonekura S, Namba K: Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature. 2003 424 (6949): 643-50; Samatey FA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K: Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling.Nature. 2001 410 (6826): 331-7) and inventors According to the bioinformatics analysis, amino acid residues required for axial interaction between sepsis Vibrio flagellin FlaB monomers are as follows. That is, aspartic acid (D70), the 70th amino acid of the upper subunit of each monomer involved in the axial reaction, asparagine (D135), the 135th amino acid, and alanine (the 151th amino acid) alanine (A151) and asparagine (N153), the 153th amino acid, asparagine (N100), the 100th amino acid, and glutamic acid (E108), the 108th amino acid, respectively, of the lower subunit. Arginine (R93), the 93rd amino acid, and alanine (A292), the 292th amino acid, were analyzed to form multimers through a polar-charge reaction (i.e., a higher flan). Zelin D70: Lower Flagellin N100; Upper Flagellin N135: Lower Flagellin E107; Upper Flagellin A151: Lower Flagellin R93; Upper Flagellin N153: Lower Flagellin A292).

본 발명에서는 플라젤린 단량체 간의 축 상호반응에 관여할 것으로 분석된 93 번째 아미노산인 아르기닌(Arginine; R93)을 알라닌(alanine; A)으로 위치 지정 돌연변이시켜 서열번호 2의 플라젤린 변형체(R93A), 101 번째 아미노산인 아스파라긴(asparagine; N)을 알라닌(alanine; A)으로 위치 지정 돌연변이시켜 서열번호 6의 플라젤린 변형체(N101A), 108 번째 아미노산인 글루타민산(glutamic acid; E)을 알라닌(alanine; A)으로 위치 지정 돌연변이시켜 서열번호 10의 플라젤린 변형체(E108A), 151 번째 아미노산인 알라닌(alanine; A151)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 서열번호 12의 플라젤린 변형체(A151W), 153 번째 아미노산인 아스파라긴(aspargine; N153)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 서열번호 14의 플라젤린 변형체(N153W)를 제조하였다. 또한 추가적으로 플라젤린 단량체 간의 축 상호반응에 관여할 것으로 추정된 97 번째 아미노산인 루신(leucine; L97)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 서열번호 4의 플라젤린 변형체(L97W), 103 번째 아미노산인 세린(serine; S103)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 서열번호 8의 플라젤린 변형체(S103W)와 291 번째 아미노산인 발린(valine; V291)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 서열번호 16의 플라젤린 변형체(V291W)를 제조하였다.In the present invention, the arginine (R93), the 93th amino acid analyzed to be involved in the axial interaction between the flagellin monomers, is positionally mutated to alanine (A) to flageline variant (R93A), 101. Positional mutation of asparagine (N), the first amino acid, to alanine (A), to transform the flageline variant (N101A) of SEQ ID NO: 6, glutamic acid (E), the 108th amino acid, into alanine (A) Positional mutagenesis of the flageline variant (E108A) of SEQ ID NO: 10 and alanine (A151), the 151th amino acid, to positional mutation of tryptophan (W) to the flageline variant of SEQ ID NO: 12 (A151W), 153 The flageline variant of SEQ ID NO: 14 (N153W) was prepared by site-directed mutation of asparagine (N153) as the first amino acid with tryptophan (W). In addition, the 97th amino acid leucine (L97), which is believed to be involved in the axial interactions between flagellin monomers, was mutated to tryptophan (W) by positional mutation, thereby making the flageline variant (L97W) of SEQ ID NO: 4, 103rd The amino acid serine (S103) is positioned and mutated to tryptophan (W) to place the flageline variant of SEQ ID NO: 8 (S103W) and the 291th amino acid valine (V291) to tryptophan (W) Assignment mutations produced the flagellin variant (V291W) of SEQ ID NO: 16.

먼저 위치 지정 돌연변이(site-directed mutagenesis)를 위한 주형 (template) DNA를 얻기 위해 서열번호 17의 PCR 프라이머 FlaB-V5 (5'-gcggccgcatggcagtgaatgtaaatgtaaatacaaac-3'; 클로닝을 위해 삽입한 제한효소 NotI 제한효소 인식부위를 밑줄로 그어 표시하였음)와 서열번호 18의 PCR 프라이머 FlaB-V4 (5'-cccggggcctagtagacttagcgctga-3': 클로닝을 위해 삽입한 제한효소 SmaI 제한효소 인식부위를 밑줄로 그어 표시하였음)를 사용하여 PCR을 통해 flaB 유전자의 ORF을 함유하고 있는 1,142 bp의 DNA 조각을 증폭하였다. 즉, 프라이머 FlaB-V5 및 FlaB-V4을 사용한 PCR 반응은 5℃에서 1분간 초기변성한 후, 95℃에서 30초간 변성, 70℃에서 30초간 어닐링, 및 72℃에서 1분간 확장하는 사이클을 30회 반복한 다음 마지막 72℃에서 10분간 반응시키는 조건으로 실시하였다. 이렇게 증폭된 flaB DNA 조각은 PCR 2.1-TOPO 클로닝 벡터(Invitrogen Co.)에 클로닝하여 pCMM270으로 명명하였으며, pCMM270 플라스미드는 다음의 위치 지정 돌연변이를 위한 주형 DNA(template DNA)로 사용되었다. PCR primer FlaB-V5 of SEQ ID NO: 17 (5'- gcggccgc atggcagtgaatgtaaatgtaaatacaaac-3 '; restriction enzyme Not I restriction enzyme inserted for cloning first to obtain template DNA for site-directed mutagenesis) Recognition sites are underlined) and PCR primer FlaB-V4 of SEQ ID NO: 18 (5'- cccggg gcctagtagacttagcgctga-3 ': underlined restriction enzyme Sma I restriction enzyme insertion site inserted for cloning ) FlaB via PCR using DNA fragments of 1,142 bp containing the ORF of the gene were amplified. In other words, PCR reactions using primers FlaB-V5 and FlaB-V4 were initially denatured at 5 ° C for 1 minute, then denatured at 95 ° C for 30 seconds, annealed at 70 ° C for 30 seconds, and extended at 72 ° C for 1 minute. After repeating the reaction was carried out under the conditions of reacting for 10 minutes at the last 72 ℃. The flaB DNA fragment thus amplified was cloned into PCR 2.1-TOPO cloning vector (Invitrogen Co.) and named pCMM270. The pCMM270 plasmid was used as template DNA for the following positional mutations.

패혈증 비브리오균 플라젤린 유전자 flaB 의 위치지정돌연변이 제작을 위한 PCR 조건 및 클로닝된 플라스미드를 하기 표 2에 정리하였다.Sepsis Vibrio flagellin gene PCR conditions and cloned plasmids for the directed mutation of flaB are summarized in Table 2 below.

돌연변이 Mutation
위치location 프라이머primer
(서열번호)(SEQ ID NO) PCR 어닐링 온도PCR annealing temperature pCR2.1- TOPOpCR2.1- TOPO
클론 이름Clone name pTYB12pTYB12
클론 이름Clone name R93AR93A FlaB-SD13-2 (서열번호 19)
5'-catcctacaacgtatggctgacctatctctacaatc-3'
FlaB-SD14-2 (서열번호 20)
5'-gattgtagagataggtcagccatacgttgtaggatg-3'FlaB-SD13-2 (SEQ ID NO: 19)
5'-catcctacaacgtatggctgacctatctctacaatc-3 '
FlaB-SD14-2 (SEQ ID NO: 20)
5'-gattgtagagataggtcagccatacgttgtaggatg-3 ' 63℃63 ℃ pCMM281pCMM281 pCMM291pCMM291 L97WL97W FlaB-SD17 (서열번호 21)
5'-gcgtgacctatcttggcaatccgcgaacgg-3'
FlaB-SD18 (서열번호 22)
5'-ccgttcgcggattgccaagataggtcacgc-3'FlaB-SD17 (SEQ ID NO: 21)
5'-gcgtgacctatcttggcaatccgcgaacgg-3 '
FlaB-SD18 (SEQ ID NO: 22)
5'-ccgttcgcggattgccaagataggtcacgc-3 ' 66℃66 ℃ pCMM282pCMM282 pCMM292pCMM292 N101AN101A FlaB-SD9-3 (서열번호 23)
5'-ctacaatccgcggccggctcaaactcaaaatc-3'
FlaB-SD10-3 (서열번호 24)
5'-gattttgagtttgagccggccgcggattgtag-3'FlaB-SD9-3 (SEQ ID NO: 23)
5'-ctacaatccgcggccggctcaaactcaaaatc-3 '
FlaB-SD10-3 (SEQ ID NO: 24)
5'-gattttgagtttgagccggccgcggattgtag-3 ' 64℃64 ℃ pCMM283pCMM283 pCMM293pCMM293 S103WS103W FlaB-SD15 (서열번호 25)
5'-caatccgcgaacggctggaactcaaaatcag-3'
FlaB-SD16 (서열번호 26)
5'-ctgattttgagttccagccgttcgcggattg-3'FlaB-SD15 (SEQ ID NO: 25)
5'-caatccgcgaacggctggaactcaaaatcag-3 '
FlaB-SD16 (SEQ ID NO: 26)
5'-ctgattttgagttccagccgttcgcggattg-3 ' 63℃63 ℃ pCMM284pCMM284 pCMM294pCMM294 E108AE108A FlaB-SD11 (서열번호 27)
5'-caaactcaaaatcagcgcgcgtggcgattc-3'
FlaB-SD12 (서열번호 28)
5'-gaatcgccacgcgcgctgattttgagtttg-3'FlaB-SD11 (SEQ ID NO: 27)
5'-caaactcaaaatcagcgcgcgtggcgattc-3 '
FlaB-SD12 (SEQ ID NO: 28)
5'-gaatcgccacgcgcgctgattttgagtttg-3 ' 63℃63 ℃ pCMM285pCMM285 pCMM295pCMM295 A151WA151W FlaB-SD5 (서열번호 29) 5'-gcaatgcaaattggttgggataacggtgaagcg-3'
FlaB-SD6 (서열번호 30)
5'-cgcttcaccgttatcccaaccaatttgcattgc-3'FlaB-SD5 (SEQ ID NO: 29) 5'-gcaatgcaaattggttgggataacggtgaagcg-3 '
FlaB-SD6 (SEQ ID NO: 30)
5'-cgcttcaccgttatcccaaccaatttgcattgc-3 ' 63℃63 ℃ pCMM286pCMM286 pCMM296pCMM296 N153WN153W FlaB-SD7 (서열번호 31)
5'-caaattggtgcggattggggtgaagcggtcatg-3'
FlaB-SD8 (서열번호 32) 5'-catgaccgcttcaccccaatccgcaccaatttg-3'FlaB-SD7 (SEQ ID NO: 31)
5'-caaattggtgcggattggggtgaagcggtcatg-3 '
FlaB-SD8 (SEQ ID NO: 32) 5'-catgaccgcttcaccccaatccgcaccaatttg-3 ' 67℃67 ℃ pCMM287pCMM287 pCMM297pCMM297 V291WV291W FlaB-SD19 (서열번호 33) 5'-gcgcaagagtcgtgggcgattgtggatgcg-3'
FlaB-SD20 (서열번호 34) 5'-cgcatccacaatcgcccacgactcttgcgc-3'FlaB-SD19 (SEQ ID NO: 33) 5'-gcgcaagagtcgtgggcgattgtggatgcg-3 '
FlaB-SD20 (SEQ ID NO: 34) 5'-cgcatccacaatcgcccacgactcttgcgc-3 ' 67℃67 ℃ pCMM288pCMM288 pCMM298pCMM298

각각의 위치지정 돌연변이 flaB(R93A, L97W, N101A, S103W, E108A, A151W, N153W 및 V291W) 제작을 위하여 상기 표 2에 기재된 각각의 프라이머쌍, PCR 주형인 pCMM270 및 교정(proof reading) 활성이 있는 고유의 Pfu 폴리머라아제(Stratagene Co.)를 사용하여 제조사의 지침에 따라 돌연변이 가닥을 합성하였다. 즉, 각각의 PCR 반응은 95℃에서 45초간 초기변성한 후, 95℃에서 45초간 변성, 상기 표 2에 기재된 어닐링 온도에서 1분간 어닐링, 및 68℃에서 10분간 확장하는 사이클을 16회 반복한 다음 마지막 68℃에서 10분간 반응시키는 조건으로 실시하였다. 이렇게 증폭된 서열번호 1, 3, 5, 7, 9, 11, 13 및 15의 위치지정 돌연변이 flaB DNA는 PCR 2.1-TOPO 클로닝 벡터에 클로닝하여 각각 pCMM281, pCMM282, pCMM283, pCMM284, pCMM285, pCMM286, pCMM287 및 pCMM288로 명명하였다(표 2). Each primer pair described in Table 2 above, pCMM270, a PCR template, and a unique with proof reading activity for the construction of each of the positional mutant flaBs (R93A, L97W, N101A, S103W, E108A, A151W, N153W and V291W) Pfu Mutagen strands were synthesized using polymerase (Stratagene Co.) according to the manufacturer's instructions. That is, each PCR reaction was initially denatured at 95 ° C. for 45 seconds, followed by 16 cycles of denaturation at 95 ° C. for 45 seconds, annealing for 1 minute at annealing temperatures shown in Table 2, and expansion at 68 ° C. for 10 minutes. Then, the reaction was carried out under the condition of reacting at the last 68 ° C for 10 minutes. Positioning mutant flaB DNA of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 and 15 amplified in this way was cloned into the PCR 2.1-TOPO cloning vector and respectively pCMM281, pCMM282, pCMM283, pCMM284, pCMM285, pCMM286, pCMM287 And pCMM288 (Table 2).

위치지정 돌연변이 flaB(R93A, L97W, N101A, S103W, E108A, A151W, N153W 및 V291W) 유전자가 포함된 서열번호 1, 3, 5, 7, 9, 11, 13 및 15의 DNA 조각을 제한효소 NotI과 SmaI으로 잘라낸 후 동일한 제한효소로 자른 pTYB12 플라스미드(New England Biolabs)에 클로닝한 후 각각 pCMM291, pCMM292, pCMM293, pCMM293, pCMM294, pCMM295, pCMM296, pCMM297 및 pCMM298로 명명하였다. 이들 플라스미드를 ER2566 대장균에 일렉트로포레이션 방법으로 넣어 주고 5-브로모-인돌-3-클로로-이소프로필-β-D-갈락토피라노시드(IPTG) 0.5mM을 가하여 발현을 유도하였다. 제조사(New England Biolabs Inc.)의 지침에 따라 키틴 비드 칼럼과 1,4-디티오트레이톨(1,4-DTT)을 이용하여 인테인 융합 단백질로부터 점돌연변이 플라젤린(R93A, L97W, N101A, S103W, E108A, A151W, N153W 및 V291W) 단백질들만을 얻었다. 분리된 점돌연변이 플라젤린 단백질 내에 함유되어 있는 내독소(endotoxin)는 AffinityPakTM Detoxi GelTM Endotoxin Removing gel(Pirece 사)을 이용하여 제거하여 사용하였다. 이렇게 분리한 재조합 단백질은 Superdex120(Amersham 사) 컬럼을 이용하여 젤-여과(Amersham사의 AKTA-Prime을 이용)을 실시하여 서열번호 2, 4, 6, 8, 10, 12, 14 및 16의 재조합 플라젤린 변형체를 고순도로 분리 정제하였다.Positioning mutation flaB (R93A, L97W, N101A, S103W, E108A, A151W, N153W and V291W) a gene comprising SEQ ID NO 1, 3, 5, limit the DNA fragment of 7, 9, 11, 13, and 15 enzyme Not I And cloned into pTYB12 plasmid (New England Biolabs) cut with Sma I and cut with the same restriction enzyme and named pCMM291, pCMM292, pCMM293, pCMM293, pCMM294, pCMM295, pCMM296, pCMM297 and pCMM298, respectively. These plasmids were placed in ER2566 Escherichia coli by electroporation and 0.5 mM of 5-bromo-indole-3-chloro-isopropyl-β-D-galactopyranoside (IPTG) was added to induce expression. Point mutations from flaget fusion proteins using chitin bead columns and 1,4-dithiothreitol (1,4-DTT) according to the manufacturer (New England Biolabs Inc.) (R93A, L97W, N101A, S103W, E108A, A151W, N153W and V291W) proteins only. Endotoxins contained in isolated point-mutated flagellin proteins were identified as AffinityPak ™ Detoxi Gel ™. Endotoxin removing gel (Pirece) was used to remove. The recombinant protein thus isolated was subjected to gel-filtration (using Amersham's AKTA-Prime) using a Superdex120 (Amersham) column to obtain a recombinant plaque of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. The jellyline variants were isolated and purified in high purity.

[시험예 1] 플라젤린 변형체 재조합 플라젤린 변형체 생산 및 수용액 내에서의 생화학적 특성 [Test Example 1] Flagelin Modification Production of recombinant flagellin variant and biochemical properties in aqueous solution

플라젤린 변형체 재조합 플라젤린 변형체를 제조하기 위하여, 플라젤린 변형체를 인코딩하는 서열번호 1, 3, 5, 7, 9, 11, 13 및 15의 유전자를 각각 pTYB12 플라스미드에 클로닝한 후 ER2566 대장균에 형질전환하고 배지에 0.3mM IPTG(Isopropyl-β-D-1-thiogalactoside)를 첨가하여 재조합 플라젤린 변형체를 유도한 후 SDS-폴리아크릴아미드 젤 전기영동 및 네이티브(Native)-젤 전기영동을 실시하여 확인하였으며, 그 결과를 도 3에 나타내었다. Flageline variants To prepare recombinant flageline variants, the genes of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15 encoding flageline variants were cloned into the pTYB12 plasmid, respectively, and then transformed into E. coli ER2566. In addition, 0.3mM IPTG (Isopropyl-β-D-1-thiogalactoside) was added to the medium to induce recombinant flagellin variants, and SDS-polyacrylamide gel electrophoresis and native-gel electrophoresis were confirmed. The results are shown in FIG. 3.

도 3을 살펴보면, 플라젤린(FlaB)과 플라젤린 변형체(mutated FlaB)를 SDS-PAGE한 결과, 서열번호 2, 4, 6, 8, 10, 12, 14 및 16의 점돌연변이 플라젤린(R93A, L97W, N101A, S103W, E108A, A151W, N153W 및 V291W)과 기존의 야생형 플라젤린(FlaB)은 모두 41.5 kDa에서 주요 밴드를 나타내었다. 네이티브(Native)-젤 전기영동 결과, FlaB는 매우 큰 다량체를 형성하여 대부분의 단백질은 퇴적용 젤(stacking gel)에 그대로 남아 있으며 분해 젤(resolving gel) 내로 진입한 일부의 FlaB 단백질은 66 kDa과 140 kDa 사이에서 흐릿하게 분포하고 있음을 관찰하였다. 반면 본 발명에 의한 점돌연변이 플라젤린 변형체(R93A, L97W, N101A, S103W, E108A, A151W, N153W 및 V291W) 단백질은 140 kDa 근처에서 다량체를 형성하였다. 이상의 결과는 FlaB 단백질은 수용액 상태에서 매우 큰 다량체(huge multimer)를 이루지만 본 발명에 의한 점돌연변이 플라젤린 변형체들(R93A, L97W, N101A, S103W, E108A, A151W, N153W 및 V291W)은 이보다 규모가 작은 다량체(multimer)로 존재함을 알 수 있었다. 즉, 본 발명에 의한 서열번호 2, 4, 6, 8, 10, 12, 14 및 16의 플라젤린 변형체(R93A, L97W, N101A, S103W, E108A, A151W, N153W 및 V291W)가 플라젤린의 중합을 억제하는 것을 확인할 수 있었다.Referring to Figure 3, SDS-PAGE of the flagellin (FlaB) and flagellin variant (mutated FlaB), the point mutations of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 and 16 flagellin (R93A, L97W, N101A, S103W, E108A, A151W, N153W and V291W) and the existing wild-type flagellin (FlaB) all showed major bands at 41.5 kDa. As a result of native-gel electrophoresis, FlaB forms very large multimers, so most of the protein remains in the stacking gel and some of the FlaB proteins entering the resolving gel are 66 kDa. Was observed to be blurred between and 140 kDa. On the other hand, the point mutant flagellin variants according to the present invention (R93A, L97W, N101A, S103W, E108A, A151W, N153W and V291W) protein formed a multimer near 140 kDa. The above results indicate that FlaB protein forms a very large multimer in aqueous solution, but point mutation flageline variants (R93A, L97W, N101A, S103W, E108A, A151W, N153W and V291W) according to the present invention are larger than this. Was found to exist as a small multimer. That is, the flagellin variants (R93A, L97W, N101A, S103W, E108A, A151W, N153W, and V291W) of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 according to the present invention undergo polymerization of flagellin. It was confirmed that it was suppressed.

[시험예 2] 플라젤린 변형체의 TLR5 자극 활성Test Example 2 TLR5 Stimulating Activity of the Flagellin Variant

야생형의 FlaB 및 본 발명에 의한 서열번호 2, 4, 6, 8, 10, 12, 14 및 16의 플라젤린 변형체의 TLR5 자극 활성을 측정하기 위하여 TLR5 매개 신호전달에 관여하는 주요 인자인 NF-κB의 전사 활성을 측정하기 위해 24-웰 평판 배양기에 293T 세포를 1x105 세포씩 분주하여 하룻저녁 배양한 후 FuGENE6(Roche 사)을 이용하여 NK-κB 전사활성을 관찰할 수 있는 리포터 플라스미드인 NF-κB-Luc(한양대학교 의과대학 미생물학 교실의 김정목 교수로부터 획득)와 TLR5 유전자가 클로닝된 p3xFlag-hTLR5 플라스미드(미국 Wake Forest University School of Medicine, Departments of Microbiology and Immunology의 Steven B. Mizel로부터 획득) 및 베타-갈락토시다아제(beta-galactosidase) 발현 대조군 플라스미드(Clontech 사)를 동시에 세포내로 도입시켰다. 24시간 추가 배양 후 새로운 배지를 교체하고, 실시예 1에서 분리한 서열번호 2, 4, 6, 8, 10, 12, 14 및 16의 플라젤린 변형체(R93A, L97W, N101A, S103W, E108A, A151W, N153W 및 V291W)를 18-24 시간 처리한 후 루시퍼라아제(luciferase) 활성을 측정하여 NF-κB 전사 정도를 발광분석기(luminometer, Berthold사)를 이용해 측정하였으며 그 결과를 도 4에 나타내었다. 도 4를 살펴보면, 야생형 FlaB 재조합 단백질(WT)을 20 ng/ml 처리한 군은 인산완충액으로만 처리한 대조군에 비해 TLR5 매개 NF-κB 전사 활성이 각각 약 6.6배 증가하였다. 반면 FlaB 재조합 플라젤린 변형체인 R93A, L97W, N101A, S103W, E108A, A151W, N153W 및 V291W를 20 ng/ml 처리한 군은 인상완충액으로만 처리한 대조군에 비해 TLR5 매개 NF-κB 전사 활성이 각각 약 14배, 25배, 22.6배, 23.7배, 21배, 22.5배, 24.5배 및 16.6배 증가하였다.NF-κB, a major factor involved in TLR5-mediated signaling in order to measure TLR5 stimulatory activity of wild-type FlaB and flagellin variants SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16 according to the present invention In order to measure the transcriptional activity of 293T cells in a 24-well plate incubator by 1x10 5 cells were incubated overnight, and then reporter plasmid NF-, which can observe NK-κB transcriptional activity using FuGENE6 (Roche). κB-Luc (obtained from Professor Kim Jung-mok of the Department of Microbiology, Hanyang University Medical School) and the p3xFlag-hTLR5 plasmid cloned from the TLR5 gene (obtained from Steven B. Mizel of Wake Forest University School of Medicine, Departments of Microbiology and Immunology, USA) -Galactosidase expression control plasmid (Clontech) was introduced into the cell simultaneously. The fresh medium was replaced after 24 hours of additional culture, and the flagellin variants (R93A, L97W, N101A, S103W, E108A, A151W of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16 isolated in Example 1) , N153W and V291W) was treated for 18-24 hours and luciferase activity was measured to measure the degree of NF-κB transcription using a luminometer (luminometer, Berthold) and the results are shown in FIG. 4. Referring to Figure 4, 20 ng / ml of wild-type FlaB recombinant protein (WT) treated group was increased by about 6.6 times the TLR5-mediated NF-κB transcription activity, respectively, compared to the control group treated only with phosphate buffer. On the other hand, 20 ng / ml of the FlaB recombinant flagellin variants R93A, L97W, N101A, S103W, E108A, A151W, N153W, and V291W had a weak TLR5-mediated NF-κB transcriptional activity, respectively, compared to the control treated only with impression buffer. 14, 25, 22.6, 23.7, 21, 22.5, 24.5, and 16.6 times increased.

도 1은 살모넬라균 플라젤린 St-FliC, 대장균 플라젤린 Ec-FliC 및 패혈증 비브리오균 플라젤린 Vv-FlaB의 아미노산 상동성을 비교한 것이며, 각 균주의 플라젤린 단백질의 도메인을 박스로 표시하였다(Samatey FA, et al., Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature. 2001 410(6826):331-7).Figure 1 compares the amino acid homology of Salmonella flagellin St-FliC, Escherichia coli flagellin Ec-FliC, and sepsis Vibrio flagellin Vv-FlaB, and the domains of flagellin proteins of each strain are boxed (Samatey). FA, et al., Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling.Nature. 2001 410 (6826): 331-7).

도 2는 본 발명에 따른 플라젤린 변형체 제작에 있어서 패혈증 비브리오균 플라젤린 유전자인 flaB의 다양한 위치지정 돌연변이들을 유도하는 과정을 보여주는 모식도이다(Samatey FA, et al ., Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature. 2001 410(6826):331-7). Figure 2 is a schematic diagram showing the process of inducing various positional mutations of the sepsis Vibrio flagellin gene flaB in the production of flagellin according to the present invention (Samatey FA, et al ., Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature. 2001 410 (6826): 331-7).

도 3은 본 발명에 따른 재조합 플라젤린 변형체가 수용액 상태에서 플라젤린 중합(polymerization)에 미치는 영향을 보여주는 결과이다.3 is a result showing the effect of the recombinant flagellin variant according to the invention on the flagellin polymerization (polymerization) in the aqueous solution.

도 4는 본 발명에 따른 플라젤린 변형체의 TLR5 자극 활성을 측정한 결과를 보여주는 그래프이다.4 is a graph showing the results of measuring the TLR5 stimulatory activity of the flagellin variant according to the present invention.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Modified flagellin improved Toll-like receptor 5 stimulating activity <160> 34 <170> KopatentIn 1.71 <210> 1 <211> 1134 <212> DNA <213> R93A FlaB gene of V. vulnificus <400> 1 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaac cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatggctg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 2 <211> 377 <212> PRT <213> R93A FlaB amino acid of V. vulnificus <400> 2 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg 1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu 20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Ala Asp Leu Ser 85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile 100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu 115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr 130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp 180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu 195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr 210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys 260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln 275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His 290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln 340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala 355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly 370 375 <210> 3 <211> 1134 <212> DNA <213> L97W FlaB gene of V. vulnificus <400> 3 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaac cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatcttg gcaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 4 <211> 377 <212> PRT <213> L97W FlaB amino acid of V.vulnificus <400> 4 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg 1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu 20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser 85 90 95 Trp Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile 100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu 115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr 130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp 180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu 195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr 210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys 260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln 275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His 290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln 340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala 355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly 370 375 <210> 5 <211> 1134 <212> DNA <213> N101A FlaB gene of V. vulnificus <400> 5 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaac cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 gccggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 6 <211> 377 <212> PRT <213> N101A FlaB amino acid of V. vulnificus <400> 6 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg 1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu 20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser 85 90 95 Leu Gln Ser Ala Ala Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile 100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu 115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr 130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp 180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu 195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr 210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys 260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln 275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His 290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln 340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala 355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly 370 375 <210> 7 <211> 1134 <212> DNA <213> S103W FlaB gene of V.vulnificus <400> 7 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaac cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctgga actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 8 <211> 377 <212> PRT <213> S103W FlaB amino acid of V.vulnificus <400> 8 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg 1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu 20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser 85 90 95 Leu Gln Ser Ala Asn Gly Trp Asn Ser Lys Ser Glu Arg Val Ala Ile 100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu 115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr 130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp 180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu 195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr 210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys 260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln 275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His 290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln 340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala 355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly 370 375 <210> 9 <211> 1134 <212> DNA <213> E108A FlaB gene of V.vulnificus <400> 9 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaac cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agcgcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 10 <211> 377 <212> PRT <213> E108A FlaB amino acid of V.vulnificus <400> 10 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg 1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu 20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser 85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Ala Arg Val Ala Ile 100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu 115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr 130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp 180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu 195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr 210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys 260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln 275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His 290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln 340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala 355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly 370 375 <210> 11 <211> 1134 <212> DNA <213> A151W FlaB gene of V.vulnificus <400> 11 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaac cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt tgggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 12 <211> 377 <212> PRT <213> A151W FlaB amino acid of V.vulnificus <400> 12 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg 1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu 20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser 85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile 100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu 115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr 130 135 140 Lys Ala Met Gln Ile Gly Trp Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp 180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu 195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr 210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys 260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln 275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His 290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln 340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala 355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly 370 375 <210> 13 <211> 1134 <212> DNA <213> N153W FlaB gene of V.vulnificus <400> 13 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaac cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggattggg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 14 <211> 377 <212> PRT <213> N153W FlaB amino acid of V.vulnificus <400> 14 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg 1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu 20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser 85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile 100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu 115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr 130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Trp Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp 180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu 195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr 210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys 260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln 275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His 290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln 340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala 355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly 370 375 <210> 15 <211> 1134 <212> DNA <213> V291W FlaB gene of V.vulnificus <400> 15 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaac cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg tgggcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 16 <211> 377 <212> PRT <213> V291W FlaB amino acid of V.vulnificus <400> 16 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg 1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu 20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu 35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala 50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly 65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser 85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile 100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu 115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr 130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp 180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu 195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr 210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys 260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln 275 280 285 Glu Ser Trp Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His 290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln 340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala 355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly 370 375 <210> 17 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> FlaB-V5 primer <400> 17 gcggccgcat ggcagtgaat gtaaatgtaa atacaaac 38 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> FlaB-V4 primer <400> 18 cccggggcct agtagactta gcgctga 27 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD13-2 primer <400> 19 catcctacaa cgtatggctg acctatctct acaatc 36 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD14-2 primer <400> 20 gattgtagag ataggtcagc catacgttgt aggatg 36 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD17 primer <400> 21 gcgtgaccta tcttggcaat ccgcgaacgg 30 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD18 primer <400> 22 ccgttcgcgg attgccaaga taggtcacgc 30 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD9-3 primer <400> 23 ctacaatccg cggccggctc aaactcaaaa tc 32 <210> 24 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD10-3 primer <400> 24 gattttgagt ttgagccggc cgcggattgt ag 32 <210> 25 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD15 primer <400> 25 caatccgcga acggctggaa ctcaaaatca g 31 <210> 26 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD16 primer <400> 26 ctgattttga gttccagccg ttcgcggatt g 31 <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD11 primer <400> 27 caaactcaaa atcagcgcgc gtggcgattc 30 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD12 primer <400> 28 gaatcgccac gcgcgctgat tttgagtttg 30 <210> 29 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD5 primer <400> 29 gcaatgcaaa ttggttggga taacggtgaa gcg 33 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD6 primer <400> 30 cgcttcaccg ttatcccaac caatttgcat tgc 33 <210> 31 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD7 primer <400> 31 caaattggtg cggattgggg tgaagcggtc atg 33 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD8 primer <400> 32 catgaccgct tcaccccaat ccgcaccaat ttg 33 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD19 primer <400> 33 gcgcaagagt cgtgggcgat tgtggatgcg 30 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD20 primer <400> 34 cgcatccaca atcgcccacg actcttgcgc 30 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Modified flagellin improved Toll-like receptor 5 stimulating          activity <160> 34 <170> KopatentIn 1.71 <210> 1 <211> 1134 <212> DNA <213> R93A FlaB gene of V. vulnificus <400> 1 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaa cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatggctg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 2 <211> 377 <212> PRT <213> R93A FlaB amino acid of V. vulnificus <400> 2 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Ala Asp Leu Ser                  85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly     370 375 <210> 3 <211> 1134 <212> DNA <213> L97W FlaB gene of V. vulnificus <400> 3 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaa cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatcttg gcaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 4 <211> 377 <212> PRT <213> L97W FlaB amino acid of V.vulnificus <400> 4 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser                  85 90 95 Trp Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly     370 375 <210> 5 <211> 1134 <212> DNA <213> N101A FlaB gene of V. vulnificus <400> 5 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaa cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 gccggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 6 <211> 377 <212> PRT <213> N101A FlaB amino acid of V. vulnificus <400> 6 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser                  85 90 95 Leu Gln Ser Ala Ala Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly     370 375 <210> 7 <211> 1134 <212> DNA <213> S103W FlaB gene of V.vulnificus <400> 7 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaa cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctgga actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 8 <211> 377 <212> PRT <213> S103W FlaB amino acid of V.vulnificus <400> 8 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser                  85 90 95 Leu Gln Ser Ala Asn Gly Trp Asn Ser Lys Ser Glu Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly     370 375 <210> 9 <211> 1134 <212> DNA <213> E108A FlaB gene of V.vulnificus <400> 9 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaa cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agcgcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 10 <211> 377 <212> PRT <213> E108A FlaB amino acid of V.vulnificus <400> 10 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser                  85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Ala Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly     370 375 <210> 11 <211> 1134 <212> DNA <213> A151W FlaB gene of V.vulnificus <400> 11 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaa cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt tgggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 12 <211> 377 <212> PRT <213> A151W FlaB amino acid of V.vulnificus <400> 12 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser                  85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Trp Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly     370 375 <210> 13 <211> 1134 <212> DNA <213> N153W FlaB gene of V.vulnificus <400> 13 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaa cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggattggg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg gtagcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 14 <211> 377 <212> PRT <213> N153W FlaB amino acid of V.vulnificus <400> 14 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser                  85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Trp Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Val Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly     370 375 <210> 15 <211> 1134 <212> DNA <213> V291W FlaB gene of V.vulnificus <400> 15 atggcagtga atgtaaatac aaacgtagca gcaatgacag cacagcgtta cctgaataac 60 gcaaacagcg cacaacaaac ttcgatggag cgtctgtctt caggtttcaa aatcaacagt 120 gcaaaagatg acgcagccgg tctgcaaatc tctaaccgct tgaacgtaca aagtcgcggt 180 ctagacgttg cggtacgtaa cgccaacgac ggtatctcaa tcgcacaaa cgcagaaggt 240 gcgatgaacg agaccaccaa catcctacaa cgtatgcgtg acctatctct acaatccgcg 300 aacggctcaa actcaaaatc agagcgcgtg gcgattcaag aagaagtgac agcattgaat 360 gacgagctaa accgtattgc agaaaccacg tcttttggtg gtaacaagct gctaaacggt 420 acttacggca cgaaagcaat gcaaattggt gcggataacg gtgaagcggt catgctttca 480 ctgaaagaca tgcgctctga caacgtgatg atgggcggcg tgagctacca agctgaagaa 540 ggcaaagaca agaactggaa tgtggccgca ggcgacaacg acttgacgat tgcactgaca 600 gacagctttg gtaacgagca agagatcgaa atcaacgcga aagcgggtga tgacatcgaa 660 gagctagcga cgtacatcaa cggtcaaact gaccttgtaa aagcgtcagt gggtgaaggc 720 ggcaagctac agatctttgc tggtaacaac aaagttcaag gtgaaattgc tttctcaggt 780 agcctagctg gtgaacttgg cctaggcgaa ggcaaaaacg tcacggtaga cacgattgac 840 gtgacaaccg tacaaggtgc gcaagagtcg tgggcgattg tggatgcggc actgaaatac 900 gtagacagcc accgtgcaga gctgggtgca ttccagaacc gtttcaacca tgcaatcagc 960 aacttggaca acatcaacga aaacgtgaac gcgtcgaaga gccgaatcaa agataccgac 1020 ttcgcgaaag aaacgactca gttgaccaag acacaaattc tatcgcaagc atcaagttcc 1080 attcttgcgc aagcgaaaca agcgccaaac tcagcgctaa gtctactagg ctaa 1134 <210> 16 <211> 377 <212> PRT <213> V291W FlaB amino acid of V.vulnificus <400> 16 Met Ala Val Asn Val Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg   1 5 10 15 Tyr Leu Asn Asn Ala Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu              20 25 30 Ser Ser Gly Phe Lys Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu          35 40 45 Gln Ile Ser Asn Arg Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala      50 55 60 Val Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly  65 70 75 80 Ala Met Asn Glu Thr Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser                  85 90 95 Leu Gln Ser Ala Asn Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile             100 105 110 Gln Glu Glu Val Thr Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu         115 120 125 Thr Thr Ser Phe Gly Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr     130 135 140 Lys Ala Met Gln Ile Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser 145 150 155 160 Leu Lys Asp Met Arg Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr                 165 170 175 Gln Ala Glu Glu Gly Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp             180 185 190 Asn Asp Leu Thr Ile Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu         195 200 205 Ile Glu Ile Asn Ala Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr     210 215 220 Tyr Ile Asn Gly Gln Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly 225 230 235 240 Gly Lys Leu Gln Ile Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile                 245 250 255 Ala Phe Ser Gly Ser Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys             260 265 270 Asn Val Thr Val Asp Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln         275 280 285 Glu Ser Trp Ala Ile Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His     290 295 300 Arg Ala Glu Leu Gly Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser 305 310 315 320 Asn Leu Asp Asn Ile Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile                 325 330 335 Lys Asp Thr Asp Phe Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln             340 345 350 Ile Leu Ser Gln Ala Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala         355 360 365 Pro Asn Ser Ala Leu Ser Leu Leu Gly     370 375 <210> 17 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> FlaB-V5 primer <400> 17 gcggccgcat ggcagtgaat gtaaatgtaa atacaaac 38 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> FlaB-V4 primer <400> 18 cccggggcct agtagactta gcgctga 27 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD13-2 primer <400> 19 catcctacaa cgtatggctg acctatctct acaatc 36 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD14-2 primer <400> 20 gattgtagag ataggtcagc catacgttgt aggatg 36 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD17 primer <400> 21 gcgtgaccta tcttggcaat ccgcgaacgg 30 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD18 primer <400> 22 ccgttcgcgg attgccaaga taggtcacgc 30 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD9-3 primer <400> 23 ctacaatccg cggccggctc aaactcaaaa tc 32 <210> 24 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD10-3 primer <400> 24 gattttgagt ttgagccggc cgcggattgt ag 32 <210> 25 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD15 primer <400> 25 caatccgcga acggctggaa ctcaaaatca g 31 <210> 26 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD16 primer <400> 26 ctgattttga gttccagccg ttcgcggatt g 31 <210> 27 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD11 primer <400> 27 caaactcaaa atcagcgcgc gtggcgattc 30 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD12 primer <400> 28 gaatcgccac gcgcgctgat tttgagtttg 30 <210> 29 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD5 primer <400> 29 gcaatgcaaa ttggttggga taacggtgaa gcg 33 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD6 primer <400> 30 cgcttcaccg ttatcccaac caatttgcat tgc 33 <210> 31 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD7 primer <400> 31 caaattggtg cggattgggg tgaagcggtc atg 33 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD8 primer <400> 32 catgaccgct tcaccccaat ccgcaccaat ttg 33 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD19 primer <400> 33 gcgcaagagt cgtgggcgat tgtggatgcg 30 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> FlaB-SD20 primer <400> 34 cgcatccaca atcgcccacg actcttgcgc 30  

Claims (9)

톨-유사 수용체-5(TLR-5) 자극 활성이 강화된 패혈증 비브리오균의 플라젤린 변형체로서, 야생형 패혈증 비브리오균의 플라젤린 중 FlaB의 93 번째 아미노산인 아르기닌(Arginine; R93)을 알라닌(alanine; A)으로 위치 지정 돌연변이시켜 제조한 서열번호 2의 아미노산 서열을 가지는 패혈증 비브리오균의 플라젤린 변형체.Flageline variant of sepsis vibrio with enhanced Toll-like receptor-5 (TLR-5) stimulating activity, and Arginine (R93), the 93rd amino acid of FlaB, in the flagellin of wild-type sepsis Vibrio is alanine (alanine; A flagellin variant of sepsis vibrio bacterium having the amino acid sequence of SEQ ID NO: 2 prepared by positional mutation in A). 톨-유사 수용체-5(TLR-5) 자극 활성이 강화된 패혈증 비브리오균의 플라젤린 변형체로서, 야생형 패혈증 비브리오균의 플라젤린 중 FlaB의 97 번째 아미노산인 루신(leucine; L97)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 4의 플라젤린 변형체(L97W)임을 특징으로 하는 패혈증 비브리오균의 플라젤린 변형체.A flagellin variant of sepsis vibrio with enhanced toll-like receptor-5 (TLR-5) stimulatory activity, the leucine (L97), the 97th amino acid of FlaB, in the flagellin of wild-type sepsis vibrio is tryptophan; W) flagellin variant of sepsis vibrio, characterized in that it is a flagellin variant (L97W) of SEQ ID NO: 4 prepared by positional mutation. 톨-유사 수용체-5(TLR-5) 자극 활성이 강화된 패혈증 비브리오균의 플라젤린 변형체로서, 야생형 패혈증 비브리오균의 플라젤린 중 FlaB의 101 번째 아미노산인 아스파라긴(asparagine; N)을 알라닌(alanine; A)으로 위치 지정 돌연변이시켜 제조한 서열번호 6의 아미노산 서열을 가지는 패혈증 비브리오균의 플라젤린 변형체.Flagellin variant of sepsis vibrio with enhanced toll-like receptor-5 (TLR-5) stimulatory activity, and asparagine (N), the 101st amino acid of FlaB, in the flagellin of wild-type sepsis vibrio, alanine (alanine; A flagellin variant of sepsis vibrio bacterium having the amino acid sequence of SEQ ID NO: 6 prepared by positional mutation in A). 톨-유사 수용체-5(TLR-5) 자극 활성이 강화된 패혈증 비브리오균의 플라젤린 변형체로서, 야생형 패혈증 비브리오균의 플라젤린 중 FlaB의 103 번째 아미노산인 세린(serine; S103)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 8의 플라젤린 변형체(S103W)임을 특징으로 하는 패혈증 비브리오균의 플라젤린 변형체.Flageline variant of sepsis vibrio with enhanced Toll-like receptor-5 (TLR-5) stimulatory activity, serine (S103), the 103rd amino acid of FlaB in the flagellin of wild-type sepsis vibrio, tryptophan; W) flagellin variant of sepsis vibrio, characterized in that it is a flagellin variant (S103W) of SEQ ID NO: 8 prepared by positional mutation. 톨-유사 수용체-5(TLR-5) 자극 활성이 강화된 패혈증 비브리오균의 플라젤린 변형체로서, 야생형 패혈증 비브리오균의 플라젤린 중 FlaB의 108 번째 아미노산인 글루타민산(glutamic acid; E)을 알라닌(alanine; A)으로 위치 지정 돌연변이시켜 제조한 서열번호 10의 플라젤린 변형체(E108A)임을 특징으로 하는 패혈증 비브리오균의 플라젤린 변형체.Flageline variant of sepsis vibrio with enhanced Toll-like receptor-5 (TLR-5) stimulating activity, and glutamic acid (E), the 108th amino acid of FlaB, in the flagellin of wild-type sepsis vibrio A flagellin variant of sepsis vibrio, characterized in that it is a flagellin variant (E108A) of SEQ ID NO: 10 prepared by positional mutation in A). 톨-유사 수용체-5(TLR-5) 자극 활성이 강화된 패혈증 비브리오균의 플라젤린 변형체로서, 야생형 패혈증 비브리오균의 플라젤린 중 FlaB의 151 번째 아미노산인 알라닌(alanine; A151)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 12의 플라젤린 변형체(A151W)임을 특징으로 하는 패혈증 비브리오균의 플라젤린 변형체.Flageline variant of sepsis vibrio with enhanced toll-like receptor-5 (TLR-5) stimulating activity, and alanine (A151), the 151th amino acid of FlaB in the flagellin of wild-type sepsis vibrio, tryptophan; W) flagellin variant of sepsis vibrio, characterized in that it is a flagellin variant (A151W) of SEQ ID NO: 12 prepared by positional mutation. 톨-유사 수용체-5(TLR-5) 자극 활성이 강화된 패혈증 비브리오균의 플라젤린 변형체로서, 야생형 패혈증 비브리오균의 플라젤린 중 FlaB의 153 번째 아미노산인 아스파라긴(aspargine; N153)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 14의 플라젤린 변형체(N153W)임을 특징으로 하는 패혈증 비 브리오균의 플라젤린 변형체.Flageline variant of sepsis vibrio with enhanced Toll-like receptor-5 (TLR-5) stimulatory activity, and aspargine (N153), the 153th amino acid of FlaB in wild-type sepsis vibrio, is tryptophan; W) flagellin variant of sepsis vibrio, characterized in that it is a flagellin variant of SEQ ID NO: 14 (N153W) prepared by positional mutation. 톨-유사 수용체-5(TLR-5) 자극 활성이 강화된 패혈증 비브리오균의 플라젤린 변형체로서, 야생형 패혈증 비브리오균의 플라젤린 중 FlaB의 291 번째 아미노산인 발린(valine; V291)을 트립토판(tryptophan; W)으로 위치 지정 돌연변이시켜 제조한 서열번호 16의 플라젤린 변형체(V291W)임을 특징으로 하는 패혈증 비브리오균의 플라젤린 변형체.A flagellin variant of sepsis vibrio with enhanced toll-like receptor-5 (TLR-5) stimulating activity, which is characterized by tryptophan (valine; V291), the 291th amino acid of FlaB in the flagellin of wild-type sepsis vibrio W) flagellin variant of sepsis vibrio, characterized in that it is a flagellin variant of SEQ ID NO: 16 (V291W) prepared by positional mutation. 제 1항 내지 제 8항 중 어느 한 항에 의한 플라젤린 변형체를 유효성분으로 포함하는 백신 보조제.A vaccine adjuvant comprising the flagellin variant according to any one of claims 1 to 8 as an active ingredient.
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