KR101130884B1 - Recombinant fusion protein produced by fusing Vibrio vulnificus flagellin and pathogenic antigens and the mucosal vaccine containing the same as an active ingredient - Google Patents

Recombinant fusion protein produced by fusing Vibrio vulnificus flagellin and pathogenic antigens and the mucosal vaccine containing the same as an active ingredient Download PDF

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KR101130884B1
KR101130884B1 KR1020090033083A KR20090033083A KR101130884B1 KR 101130884 B1 KR101130884 B1 KR 101130884B1 KR 1020090033083 A KR1020090033083 A KR 1020090033083A KR 20090033083 A KR20090033083 A KR 20090033083A KR 101130884 B1 KR101130884 B1 KR 101130884B1
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이준행
이시은
김수영
누엔충
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Abstract

본 발명은 백신 보조제와 병원체의 항원을 융합시켜 제조한 재조합 융합 단백질 및 이를 포함하는 점막 투여용 백신에 관한 것으로, 보다 상세하게는 백신 보조제이면서 톨-유사수용체-5(TLR-5) 작동제인 패혈증 비브리오균(Vibrio vulnificus)의 플라젤린과 병원체의 항원 단백질을 융합시켜 제조한 재조합 융합 단백질 및 이를 포함하는 점막 투여용 백신에 관한 것이다.The present invention relates to a recombinant fusion protein prepared by fusing a vaccine adjuvant and an antigen of a pathogen and a vaccine for mucosal administration comprising the same, and more particularly, sepsis that is a vaccine adjuvant and a toll-like receptor-5 (TLR-5) agonist. The present invention relates to a recombinant fusion protein prepared by fusing the flagellin of Vibrio vulnificus and an antigenic protein of a pathogen, and a vaccine for mucosal administration comprising the same.

Description

패혈증 비브리오균의 플라젤린과 병원체의 항원 단백질을 융합시켜 제조한 재조합 융합 단백질 및 이를 유효성분으로 포함하는 점막 투여용 백신{Recombinant fusion protein produced by fusing Vibrio vulnificus flagellin and pathogenic antigens and the mucosal vaccine containing the same as an active ingredient}Recombinant fusion protein produced by fusing Vibrio vulnificus flagellin and pathogenic antigens and the mucosal vaccine containing the same as an active ingredient}

본 발명은 백신 보조제와 병원체의 항원을 융합시켜 제조한 재조합 융합 단백질 및 이를 포함하는 점막 투여용 백신에 관한 것으로, 보다 상세하게는 백신 보조제이면서 톨-유사수용체-5(TLR-5) 작동제인 패혈증 비브리오균(Vibrio vulnificus)의 플라젤린과 병원체의 항원 단백질을 융합시켜 제조한 재조합 융합 단백질 및 이를 포함하는 점막 투여용 백신에 관한 것이다.The present invention relates to a recombinant fusion protein prepared by fusing a vaccine adjuvant and an antigen of a pathogen and a vaccine for mucosal administration comprising the same, and more particularly, sepsis that is a vaccine adjuvant and a toll-like receptor-5 (TLR-5) agonist. The present invention relates to a recombinant fusion protein prepared by fusing the flagellin of Vibrio vulnificus and an antigenic protein of a pathogen, and a vaccine for mucosal administration comprising the same.

폐렴구균(Streptococcus pneumoniae)은 그람양성 쌍구균으로 소아 및 성인에서 폐렴, 중이염 등 점막 감염과 수막염, 균혈증 등의 침습성 감염의 가장 중요한 원인균이다. 더욱이 최근 항생제 내성 폐렴구균의 출현 빈도가 잦아짐에 따라 효과적인 백신 개발의 필요성이 증가하고 있다. 폐렴구균은 협막 다당질의 혈청학적 성 질에 따라 약 90여개의 혈청형으로 구분되며 이 중 약 10여 가지 혈청형에 의해 대부분의 감염증이 발병하며 각 지역, 시대 및 연령에 따라 혈청형에 차이가 있다. 폐렴구균 질환을 예방하기 위한 백신으로 각각의 혈청형의 폐렴구균에서 추출하여 정제한 다당질(polysaccharide)을 혼합하여 만든 다당질 백신이 사용되었으나 침습 폐렴구균 감염의 위험이 가장 높은 2세 미만 소아에서는 면역원성 및 예방 효과가 낮은 제한점이 있다. 이를 보완하기 위하여 폐렴구균의 다당질과 운반체 단백을 결합시킨 폐렴구균 다당질 단백 결합 백신이 개발되어 사용되고 있으나 이 또한 각각의 혈청형을 모두 커버하기엔 제한적인 단점이 있다. Streptococcus pneumoniae is a Gram-positive diplococci, the most important causative agent of mucosal infections such as pneumonia and otitis media and invasive infections such as meningitis and bacteremia in children and adults. In addition, the recent increase in the frequency of antibiotic resistant pneumococci has increased the need for effective vaccine development. Pneumococci are classified into about 90 serotypes according to the serological properties of the capsular polysaccharides. Among them, about 10 serotypes cause most infectious diseases, and the serotypes vary according to each region, age, and age. have. As a vaccine to prevent pneumococcal disease, a polysaccharide vaccine prepared by mixing polysaccharides extracted from each pneumococcal of each serotype was used, but immunogenicity was observed in children under 2 years of age with the highest risk of invasive pneumococcal infection. And low preventive effects. To compensate for this, a pneumococcal polysaccharide protein-binding vaccine has been developed that combines the polysaccharide and carrier protein of pneumococcal pneumoniae, but this also has a limited disadvantage to cover each serotype.

다당질 백신과 다당질 단백 결합 백신의 이러한 단점을 보완하기 위해 대다수의 폐렴구균에서 발현되는 뉴모라이신(pneumolysin), 폐렴구균 표면 부착인자 A(PsaA: pneumococcal surface protein A) 및 폐렴구균 표면 단백 A(PspA: pneumococcal surface protein A)와 같은 단백 항원들이 이용될 수 있다. 폐렴구균 단백질 백신은 현재 임상적으로 중요한 모든 폐렴구균에 대해 넓은 방어 면역능을 갖는 백신 제작에 이용될 수 있으며 다당질 단백 결합 백신보다 제조 및 생산적인 면에서 훨씬 용이한 장점이 있다. To overcome these shortcomings of polysaccharide vaccines and polysaccharide protein-binding vaccines, pneumolysin, pneumococcal surface protein A (PsaA) and pneumococcal surface protein A (PspA), which are expressed in the majority of pneumococci Protein antigens such as pneumococcal surface protein A) can be used. Pneumococcal protein vaccines can now be used in the production of vaccines with broad protective immunity against all clinically important pneumococci, and are much easier to manufacture and produce than polysaccharide protein-binding vaccines.

폐렴구균의 PspA는 폐렴구균의 표면에서 발견되는 잘 알려진 독력인자로서 동물 실험에서 PspA는 폐렴구균 보균과 호흡기 감염뿐만 아니라 패혈증에 대해서도 방어 면역능을 나타내는 것으로 알려져 있어 현재 폐렴구균의 백신 개발에 중요한 타겟이 되고 있다. PspA of pneumococci is a well-known virulence factor found on the surface of pneumococci. In animal experiments, PspA is known to show protective immunity against sepsis as well as pneumococcal carriers and respiratory infections. It is becoming.

편모(flagella)는 세균의 운동성을 결정하는 중요한 구성요소이며 크게 후 크(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 TLR-5가 그람 음성 및 그람 양성 세균의 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 is an important component in determining the motility of bacteria and is composed of hook, basal body and 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 TLR-5 recognizes Gram-negative and Gram-positive bacteria flagellin and activates NF-κB (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(PRR: Pattern Recognition Receptor)'로서 이는 병원체에 존재하는 '병원체와 관련된 분자구조(PAMP: Pathogen Associated Molecular Pattern)'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 receptor (PRR: P attern R ecognition R eceptor) ': for as it' (P athogen A ssociated M olecular P attern PAMP) molecules associated with pathogens structure, present in the pathogen 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', in particular 'vaccine adjuvants'.

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(APC: antigen presenting cell) 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 중이고, 최근 한 임상시험에서 비강으로 인플루엔자 항원과 함께 투여되었을 경우 안면신경마비(Bells's palsy) 발생이 증가한 보고도 있다(Musch M, Zhou W, Rhodes P, Bopp M, Chen RT, LInder T, Spyr C, Steffen R: N. Engl. J. Med. 350:896-903, 2004).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 currently underway, and protein-derived vaccine supplements are attracting much attention, such as cholera toxin (CT) derived from Vibrio 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, studies are being conducted to reduce toxicity and increase adjuvaticity, and in recent clinical trials, facial nerve palsy (when administered with influenza antigens in the nasal cavity) Bells's palsy) increased incidence (Musch M, Zhou W, Rhodes P, Bopp M, Chen RT, LInder T, Spyr C, Steffen R: N. Engl. J. Med. 350: 896-903, 2004) .

이에 본 발명자들은 패혈증 비브리오균 플라젤린 FlaB와 폐렴구균의 표면 단백질 PspA로 구성된 백신보조제-항원 융합 재조합 단백질(PspA-FlaB 또는 FlaB-PspA)을 제작 및 분리하여 마우스의 비강내로 면역한 후 폐렴구균의 감염에 대한 방어면역능을 타진한 결과, PspA 단독으로 면역한 군에 비해 PspA-FlaB 또는 FlaB-PspA로 면역한 군에서 혈청 및 점막분비물에서 PspA 특이적 항체(IgG 및 IgA)의 생성이 유의하게 증가하였고 치사량의 폐렴구균을 주사한 경우 월등한 방어면역능을 나타내는 것을 확인하고 본 발명을 완성하게 되었다. Therefore, the present inventors prepared and isolated vaccine adjuvant-antigen fusion recombinant protein (PspA-FlaB or FlaB-PspA) consisting of sepsis Vibrio flagellin FlaB and surface protein PspA of pneumococcal and immunized into the nasal cavity of mouse, As a result of screening the defense against infection, the production of PspA-specific antibodies (IgG and IgA) in serum and mucosal secretions was significantly increased in the group immunized with PspA-FlaB or FlaB-PspA compared to the group immunized with PspA alone. When injected with a lethal dose of pneumococcal, it was confirmed that it shows superior defense immunity and completed the present invention.

따라서, 본 발명의 목적은 병원체에 대한 방어면역능이 개선된 재조합 융합 단백질 및 이를 포함하는 점막 투여용 백신을 제공하는 것이다. Accordingly, it is an object of the present invention to provide a recombinant fusion protein having improved protective immunity against a pathogen and a vaccine for mucosal administration comprising the same.

상기 목적을 달성하기 위하여, 본 발명에서는 패혈증 비브리오균의 플라젤린 단백질과 병원체의 단백질 항원을 융합하여 제조한 재조합 융합 단백질을 제공한다.In order to achieve the above object, the present invention provides a recombinant fusion protein prepared by fusing the flagellin protein of sepsis Vibrio and the protein antigen of the pathogen.

또한 본 발명에서는 재조합 융합 단백질을 유효성분으로 포함하는 점막 투여용 백신을 제공한다.In another aspect, the present invention provides a vaccine for mucosal administration comprising a recombinant fusion protein as an active ingredient.

본 발명에서 백신 보조제와 항원을 융합시켜 제조한 재조합 융합 단백질은 혈청 및 점막분비물에서 PspA 특이적 항체(IgG 및 IgA)의 생성을 유의적으로 증가시켰고, 치사량의 폐렴구균에 대해서도 월등한 방어면역능을 보였다.In the present invention, the recombinant fusion protein prepared by fusing the vaccine adjuvant and the antigen significantly increased the production of PspA-specific antibodies (IgG and IgA) in serum and mucosal secretions, and showed superior protective immunity against lethal pneumococci. Seemed.

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

본 발명자들은 PCT 국제특허공개공보 제WO 2005/070455호에서 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 전위자 삽입 주위 영역(Transposon insertion 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 발명자들은 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 활성화시킴 으로써 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). The inventors of the present inventors have prepared a 'transposon mutant library' in order to screen the host adhesion and invasion factor of sepsis Vibrio in recent PCT WO 2005/070455. Identifying two flagella related operons consisting of 56 genes during the analysis of transposon insertion 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 flaB gene is the major constituent of flagellin. The inventors of the present invention are pursuing the possibility of applying the flagellin recombinant protein (FlaB), 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 toxin is administered to the nasal cavity of experimental animals together with flagellin, flagellin signals the host's TLR5 to activate the immune system, which amplifies the efficacy of the vaccine. Administration of flagellin with immunized mice injected with a lethal dose of tetanus toxin induces 100% protective immunity, demonstrating an excellent mucosal vaccine adjuvant effect (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) 등]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. Among the various TLR agonists, flagellin that stimulates TLR5 is a protein component unlike other TLR agonists (CpG-DNA, mycoplasmal lipopeptide (MALP), etc.), so it is not only possible to synthesize recombinant proteins for continuous quality control. Various recombinant fusion proteins with enhanced TLR5 stimulatory activity can be constructed.

따라서, 플라젤린과 다양한 질환(감염, 암 또는 알러지 등)의 면역 에피토프를 융합한 백신보조제-항원으로 구성된 재조합 융합 단백질의 제작은 향상된 면역능과 단백질 백신으로서의 장점을 갖는 효율적인 단백질 백신의 생산을 가능하게 한다. Thus, the production of recombinant fusion proteins consisting of vaccine adjuvant-antigens fused with flagellin and immune epitopes of various diseases (such as infections, cancers or allergies) enables the production of efficient protein vaccines with improved immunity and advantages as protein vaccines. do.

본 발명은 패혈증 비브리오균의 플라젤린 단백질과 병원체의 단백질 항원을 융합하여 제조한 재조합 융합 단백질에 관한 것이다. The present invention relates to a recombinant fusion protein prepared by fusing a flagellin protein of sepsis vibrio and a protein antigen of a pathogen.

본 발명에서 사용하는 플라젤린 단백질은 TLR-5 작동제이고 서열번호 1의 아미노산 서열을 갖는 패혈증 비브리오균의 플라젤린 FlaB가 바람직하나 이에만 한정되지 않는다.The flagellin protein used in the present invention is a TLR-5 agonist and is preferably flagellin FlaB of sepsis vibrio having the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

본 발명에서 사용하는 병원체의 단백질 항원으로는 폐렴구균(Streptococcus pneumoniae)의 표면 단백 A(PspA)의 알파 나선 도메인(α-helix domain) 및 폐렴구 균 표면 부착인자 A(PsaA: pneumococcal surface protein A); 인플루엔자 바이러스의 서브유닛 HA(hemagglutinin) 및 NA(neuraminidase); 및 중증급성 호흡기 증후군 바이러스(SARS 바이러스)의 스파이크(S) 단백질 등을 들 수 있으나, 이에만 한정되지 않는다.Protein antigens of the pathogens used in the present invention include alpha-helix domains of surface protein A (PspA) and pneumococcal surface protein A (PsaA) of surface protein A (PspA) of pneumococcal ( Streptococcus pneumoniae ). ; Subunits of influenza virus HA (hemagglutinin) and NA (neuraminidase); And Spike (S) protein of Severe Respiratory Syndrome Virus (SARS virus), but are not limited thereto.

본 발명에서 사용하는 패혈증 비브리오균 플라젤린과 폐렴구균 항원 PspA를 융합한 재조합 단백질은 그 자체로 백신제제로 사용할 수도 있고, 현재 널리 사용되고 있는 폐렴구균의 다당질-단백 결합 백신의 효능을 높일 수 있는 새로운 컨쥬게이션 파트너로 사용될 수도 있다.Recombinant protein fusion of sepsis Vibrio flagellin and pneumococcal antigen PspA used in the present invention can be used as a vaccine formulation by itself, and a novel polysaccharide-protein binding vaccine of pneumococcus, which is widely used now, can enhance the efficacy. It can also be used as a conjugation partner.

본 발명에 의한 재조합 융합 단백질은 패혈증 비브리오균의 플라젤린과 병원체 항원 단백질을 유전자 수준에서 융합시키고 발현 및 분리 정제하여 제조할 수 있다.Recombinant fusion proteins according to the present invention can be prepared by fusing, expressing and separating and purifying flagellin and pathogen antigen proteins of sepsis Vibrio.

본 발명의 실시예에서 상기 재조합 융합 단백질은 서열번호 4의 아미노산 서열을 갖는 FlaB-PspA이거나 또는 서열번호 6의 아미노산 서열을 갖는 PspA-FlaB 일 수 있다.In an embodiment of the present invention, the recombinant fusion protein may be FlaB-PspA having an amino acid sequence of SEQ ID NO: 4 or PspA-FlaB having an amino acid sequence of SEQ ID NO: 6.

본 명세서에서 언급하는 플라젤린은 세균의 운동성을 결정하는 섬모인 플라젤라를 구성하는 단위 분자이며, PspA는 폐렴구균의 표면에 존재하는 단백질 A로서 높은 항원성을 갖고 있다. Flagellin referred to in the present specification is a unit molecule constituting flagella, a cilia that determine the motility of bacteria, and PspA is a protein A present on the surface of pneumococcal and has high antigenicity.

본 발명에서 제공하는 상기 FlaB-PspA 또는 PspA-FlaB 재조합 융합 단백질은 PspA 단독으로 면역한 군에 비해 월등한 항체 생성 및 방어 면역능을 나타낸다.The FlaB-PspA or PspA-FlaB recombinant fusion protein provided by the present invention exhibits superior antibody production and protective immunity compared to the group immunized with PspA alone.

따라서, 본 발명에 의해 제조된 FlaB-PspA 또는 PspA-FlaB 재조합 융합 단백 질은 PspA 항원만을 단독으로 사용했을 때 보다 면역능이 월등히 향상된 폐렴구균 백신을 제공하는데 사용될 수 있다. Thus, the FlaB-PspA or PspA-FlaB recombinant fusion protein prepared by the present invention can be used to provide a pneumococcal vaccine with significantly improved immunity than when using only PspA antigen alone.

또한 본 발명은 상기 재조합 융합 단백질을 유효성분으로 포함하는 점막 투여용 백신에 관한 것이다. 특히 본 발명에서는 비점막(Nasal mucosa) 투여용 백신을 제공할 수 있으며, 폐구균, 인플루엔자 바이러스 또는 중증급성 호흡기 증후군 바이러스(SARS 바이러스)에 대한 백신으로 제형화될 수 있다.The present invention also relates to a vaccine for mucosal administration comprising the recombinant fusion protein as an active ingredient. In particular, the present invention may provide a vaccine for nasal mucosa administration, and may be formulated as a vaccine against pneumococcal, influenza virus or acute respiratory syndrome virus (SARS virus).

본 발명에서 제공하는 점막 투여용 백신은 당업계에 잘 알려진 통상적인 방법으로 제조될 수 있고, 당업계에서 점막 투여용 백신 제조 시 사용할 수 있는 여러 첨가물들을 임의로 더 포함할 수도 있다. The vaccine for mucosal administration provided by the present invention may be prepared by conventional methods well known in the art, and may further optionally include various additives that may be used in the manufacture of vaccine for mucosal administration.

또한 본 발명의 기술구성을 토대로 하여 상기 백신보조제인 플라젤린과 감염질환, 항암 및 여러 가지 질병에 대한 항원을 융합하여 다양한 재조합 융합 단백질을 제조하고 이를 유효성분으로 포함하는 백신을 제공할 수 있을 것이다.In addition, based on the technical configuration of the present invention will be able to provide a vaccine comprising a variety of recombinant fusion protein by fusing the vaccine adjuvant flagellin and antigens for infectious diseases, anti-cancer and various diseases and as an active ingredient .

본 발명의 일 실시예에서 FlaB-PspA 또는 PspA-FlaB 재조합 융합 단백질의 향상된 면역능을 증명하는 과정은 하기 단계들을 포함한다:In one embodiment of the present invention, the process of demonstrating the enhanced immunity of the FlaB-PspA or PspA-FlaB recombinant fusion protein comprises the following steps:

1) 서열번호 4의 FlaB-PspA 또는 서열번호 6의 PspA-FlaB 재조합 융합 단백질을 제조, 분리하는 단계;1) preparing and isolating FlaB-PspA of SEQ ID NO: 4 or PspA-FlaB recombinant fusion protein of SEQ ID NO: 6;

2) 상기 재조합 융합 단백질 중의 플라젤린의 TLR-5 활성화를 확인하는 단계; 2) confirming TLR-5 activation of flagellin in said recombinant fusion protein;

3) 상기 재조합 융합 단백질의 비강 내 면역 후 항원 특이적 면역반응을 확인하는 단계; 및3) identifying an antigen specific immune response after intranasal immunization of the recombinant fusion protein; And

4) 상기 재조합 융합 단백질의 비강 내 면역 후 마우스에서 야생형 폐렴구균에 대한 숙주 방어능력을 측정하는 단계.4) measuring host defense ability against wild-type pneumococcal in mice following intranasal immunization of the recombinant fusion protein.

이하, 본 발명의 내용을 실시예 및 시험예를 통하여 보다 구체적으로 설명한다. 이들 실시예는 본 발명의 내용을 이해하기 위해 제시되는 것일 뿐 본 발명의 권리범위가 이들 실시예로 한정되는 것은 아니고, 당업계에서 통상적으로 주지된 변형, 치환 및 삽입 등을 수행할 수 있으며, 이에 대한 것도 본 발명의 범위에 포함된다.Hereinafter, the content of the present invention will be described in more detail through examples and test examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

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 또는 Strain or
플라스미드Plasmid 특징Characteristic 출처source 대장균Escherichia coli DH5αDH5α F-recA1; restriction negativeF- recA 1; restriction negative 실험실 기존 보유Existing lab ER2566ER2566 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
Biolab, Inc.New england
Biolab, Inc. 플라스미드Plasmid pCR2.1 TOPOpCR2.1 TOPO PCR TA 클로닝 벡터, 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
Biolab, Inc.New england
Biolab, Inc.

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

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

[실시예 1] 재조합 플라젤린, PspA 및 융합 단백질의 제조 및 분리Example 1 Preparation and Isolation of Recombinant Flagellin, PspA and Fusion Proteins

항원으로서 S. pneumoniae의 PspA 유전자의 알파 나선 도메인(α-helix domain) 부분의 N-말단 또는 C-말단 융합용 DNA 조각을 얻기 위하여, 하기 표 2에 기재된 서열번호 7 및 8의 PspA-N과 서열번호 9 및 10의 PspA-C PCR 프라이머 쌍을 사용하여 N-말단 융합용 또는 C-말단 융합용 PspA 유전자를 포함하고 있는 0.7 kbp의 DNA 조각을 증폭하였다. 즉 각각의 프라이머 쌍을 사용한 PCR 반응은 95℃에서 5분간 초기변성한 후, 95℃에서 30초간 변성, 65℃에서 30초간 어닐링 및 72℃에서 1분간 확장하는 사이클을 30회 반복한 다음 마지막에 72℃에서 10분간 반응시키는 조건으로 실시하였다. 이렇게 증폭된 PspA DNA 조각은 PCR 2.1-TOPO 클로닝 벡터(Invitrogen Co.)에 클로닝하여 pCMM8201 또는 pCMM8202로 명명하였다. In order to obtain a DNA fragment for N-terminal or C-terminal fusion of the alpha-helix domain portion of the PspA gene of S. pneumoniae as an antigen, PspA-N of SEQ ID NOs: 7 and 8 shown in Table 2 below The PspA - C PCR primer pairs of SEQ ID NOs: 9 and 10 were used to amplify a 0.7 kbp DNA fragment containing the PspA gene for N-terminal fusion or C-terminal fusion. In other words, the PCR reaction using each primer pair was initially denatured at 95 ° C. for 5 minutes, followed by 30 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 65 ° C. for 30 seconds and extension at 72 ° C. for 30 minutes. It carried out on the conditions made to react at 72 degreeC for 10 minutes. The amplified PspA DNA fragment was cloned into PCR 2.1-TOPO cloning vector (Invitrogen Co.) and named pCMM8201 or pCMM8202.

백신 보조제로서 패혈증 비브리오균 플라젤린 유전자 flaB의 N-말단 또는 C-말단 융합용 DNA 조각을 얻기 위하여 서열번호 11 및 12의 FlaB-N과 서열번호 13 및 14의 FlaB-C PCR 프라이머 쌍을 사용하여 N-말단 융합용 또는 C-말단 융합용 flaB 유전자를 포함하고 있는 1.1 kbp의 DNA 조각을 증폭하였다. 즉 각각의 프라이머 쌍을 사용한 PCR 반응은 95℃에서 5분간 초기변성한 후, 95℃에서 30초간 변성, 65℃에서 30초간 어닐링 및 72℃에서 1분간 확장하는 사이클을 30회 반복한 다음 마지막에 72℃에서 10분간 반응시키는 조건으로 실시하였다. 이렇게 증폭된 flaB DNA 조각은 PCR 2.1-TOPO 클로닝 벡터(Invitrogen Co.)에 클로닝하여 pCMM8203 또는 pCMM8204로 명명하였다.As a vaccine adjuvant, FlaB - N of SEQ ID NOs: 11 and 12 and FlaB - C PCR primer pairs of SEQ ID NOs: 13 and 14 were used to obtain DNA fragments for N-terminal or C-terminal fusion of sepsis vibrio flagellin gene flaB . A 1.1 kbp DNA fragment containing the flaB gene for N-terminal fusion or C-terminal fusion was amplified. In other words, the PCR reaction using each primer pair was initially denatured at 95 ° C. for 5 minutes, followed by 30 cycles of denaturation at 95 ° C. for 30 seconds, annealing at 65 ° C. for 30 seconds and extension at 72 ° C. for 30 minutes. It carried out on the conditions made to react at 72 degreeC for 10 minutes. The flaB DNA fragment thus amplified was cloned into PCR 2.1-TOPO cloning vector (Invitrogen Co.) and named pCMM8203 or pCMM8204.

명칭designation 서열
번호order
number 서열order 비고Remarks PspA-NPspA - N 정방향Forward 77 5'-CAT ATG TCT CCC GTA GCC AGT CAG TCT-3'5'- CAT ATG TCT CCC GTA GCC AGT CAG TCT-3 ' 클로닝을 위해 삽입한 제한 효소 NdeI 인식부위를 밑줄로 그어 표시하였음Restriction enzyme Nde I recognition site inserted for cloning is underlined 역방향Reverse 88 5'-GTC GAC CTC ATC AAT CTT ATC ACT TAA CTC TTC AAG-3'5'- GTC GAC CTC ATC AAT CTT ATC ACT TAA CTC TTC AAG-3 ' 클로닝을 위해 삽입한 제한 효소 SalI 인식부위를 밑줄로 그어 표시하였음The restriction enzyme Sal I recognition site inserted for cloning is underlined. PspA-CPspA - C 정방향Forward 99 5'-GTC GAC TCT CCC GTA GCC AGT CAG T-3'5'- GTC GAC TCT CCC GTA GCC AGT CAG T-3 ' 클로닝을 위해 삽입한 제한 효소 SalI 인식부위를 밑줄로 그어 표시하였음The restriction enzyme Sal I recognition site inserted for cloning is underlined. 역방향Reverse 1010 5'-CCC GGG TTA CTC ATC AAT CTT ATC ACT TAA CTC TTC-3'5'- CCC GGG TTA CTC ATC AAT CTT ATC ACT TAA CTC TTC-3 ' 클로닝을 위해 삽입한 제한 효소 SmaI 인식부위를 밑줄로 그어 표시하였음Restriction enzyme Sma I recognition site inserted for cloning is underlined FlaB-N
FlaB - N
정방향Forward 1111 5'-CAT ATG ATG GCA GTG AAT GTA AAT ACA AAC GTA GCA-3'5'- CAT ATG ATG GCA GTG AAT GTA AAT ACA AAC GTA GCA-3 ' 클로닝을 위해 삽입한 제한 효소 NdeI 인식부위를 밑줄로 그어 표시하였음Restriction enzyme Nde I recognition site inserted for cloning is underlined 역방향Reverse 1212 5'-GTC GAC GCC TAG TAG ACT TAG CGC T-3'5'- GTC GAC GCC TAG TAG ACT TAG CGC T-3 ' 클로닝을 위해 삽입한 제한 효소 SalI 인식부위를 밑줄로 그어 표시하였음The restriction enzyme Sal I recognition site inserted for cloning is underlined. FlaB-CFlaB - C 정방향Forward 1313 5'-GTC GAC ATG GCA GTG AAT GTA AAT ACA AAC GTA G-3'5'- GTC GAC ATG GCA GTG AAT GTA AAT ACA AAC GTA G-3 ' 클로닝을 위해 삽입한 제한 효소 SalI 인식부위를 밑줄로 그어 표시하였음The restriction enzyme Sal I recognition site inserted for cloning is underlined. 역방향Reverse 1414 5'-CCC GGG TTA GCC TAG TAG ACT TAG CG-3'5'- CCC GGG TTA GCC TAG TAG ACT TAG CG-3 ' 클로닝을 위해 삽입한 제한 효소 SmaI 인식부위를 밑줄로 그어 표시하였음Restriction enzyme Sma I recognition site inserted for cloning is underlined

FlaB-PspA 융합 단백질 제작을 위하여 pCMM8203의 flaB DNA 조각을 제한 효소 NdeI과 SalI으로 잘라낸 후 동일한 제한 효소로 자른 pTYB12 플라스미드(New England Biolabs)에 클로닝하여 pCMM8205로 명명하였다. 이 플라스미드에 제한 효소 SalI과 SmaI으로 자른 pCMM8202의 PspA DNA 조각을 클로닝하여 pCMM8208이라 명명하였다.To prepare the FlaB-PspA fusion protein, the flaB DNA fragment of pCMM8203 was cut with restriction enzymes Nde I and Sal I and cloned into pTYB12 plasmid (New England Biolabs) cut with the same restriction enzyme and named pCMM8205. The PspA DNA fragment of pCMM8202 cut with restriction enzymes Sal I and Sma I was cloned into this plasmid and named pCMM8208.

PspA-FlaB 융합 단백질 제작을 위하여 pCMM8201의 PspA DNA 조각을 제한 효소 NdeI과 SalI으로 잘라낸 후 동일한 제한 효소로 자른 pTYB12 플라스미드(New England Biolabs)에 클로닝하여 pCMM8206으로 명명하였다. 이 플라스미드에 제한 효소 SalI과 SmaI으로 자른 pCMM8204의 flaB DNA 조각을 클로닝하여 pCMM8207이라 명명하였다. To prepare the PspA-FlaB fusion protein, the PspA DNA fragment of pCMM8201 was cut with restriction enzymes Nde I and Sal I, cloned into pTYB12 plasmid (New England Biolabs) cut with the same restriction enzyme, and named pCMM8206. The flaB DNA fragment of pCMM8204 cut with restriction enzymes Sal I and Sma I was cloned into this plasmid and named pCMM8207.

이들 플라스미드를 ER2566 대장균에 일렉트로포레이션 방법으로 넣어 주고 5-브로모-인돌-3-클로로-이소프로필-β-D-갈락토피라노시드(IPTG) 0.5 mM을 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 서열번호 1의 FlaB, 서열번호 2의 PspA, 서열번호 4의 FlaB-PspA, 및 서열번호 6의 PspA-FlaB 단백질들을 얻었으며, 상기 과정을 도 1에 상세히 도시하였다. 분리된 단백질 내에 함유되어 있는 내독소(endotoxin)는 AffinityPakTM Detoxi GelTM Endotoxin Removing gel(Pirece 사)을 이용하여 제거하여 사용하였다.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. Flasp of SEQ ID NO: 1, PspA of SEQ ID NO: 2 from an intein fusion protein using a chitin bead column and 1,4-dithiothreitol (1,4-DTT) according to the manufacturer's (New England Biolabs Inc.) instructions , FlaB-PspA of SEQ ID NO: 4, and PspA-FlaB proteins of SEQ ID NO: 6 were obtained, and the procedure is shown in detail in FIG. Endotoxins contained in the isolated protein were removed using AffinityPak ™ Detoxi Gel ™ Endotoxin Removing gel (Pirece).

[시험예 1] 플라젤린-PspA 재조합 융합 단백질의 TLR-5 자극 활성Test Example 1 TLR-5 Stimulating Activity of Flagellin-PspA Recombinant Fusion Protein

24-웰 평판 배양기에 293-T 세포를 1×105 세포씩 분주하여 하룻저녁 배양한 후 Effectene(QIAGEN)을 이용하여 NF-κB-Luc 플라스미드(한양대학교 의과대학 미생물학 교실의 김정목 교수로부터 획득)와 TLR-5 유전자가 클로닝된 p3xFlag-hTLR-5 플라스미드(미국 Wake Forest University School of Medicine, Department of Microbiology and Immunology의 Steven B. Mizel로부터 획득) 및 β-갈락토시다아제(β-galactosidase) 발현 대조군 플라스미드(Clontech)를 동시에 세포내로 도입시켰다. 24시간 추가배양 후 새로운 배지로 교체하고 상기 실시예 1에서 제조하고 IMPACT 시스템으로 분리한 FlaB, PspA-FlaB 및 FlaB-PspA 단백질을 일정 시간 처리한 후 루시퍼라아제(luciferase) 활성을 발광분석기(Luminometer, Berthold사)를 이용해 측정하여 NF-κB 전사 정도를 확인하였으며, 그 결과를 도 2에 나타내었다. 293-T cells were dispensed in 1 × 10 5 cells in a 24-well plate incubator and cultured overnight, followed by NF-κB-Luc plasmid using Effectene (QIAGEN) (acquired from Professor Kim Jung-mok of the Department of Microbiology, Hanyang University College of Medicine) P3xFlag-hTLR-5 plasmid cloned from TLR-5 gene (obtained from Steven B. Mizel of Wake Forest University School of Medicine, Department of Microbiology and Immunology, USA) and β-galactosidase expression control Plasmids (Clontech) were simultaneously introduced into the cells. After 24 hours of additional culture, the medium was replaced with fresh medium, and the luciferase activity was determined by luminometer (Luminometer) after treatment with FlaB, PspA-FlaB and FlaB-PspA proteins prepared in Example 1 and separated by IMPACT system for a certain time. , Berthold) to determine the degree of NF-κB transcription, the results are shown in FIG.

도 2의 결과에서, PspA-FlaB 및 FlaB-PspA 재조합 융합 단백질 모두 FlaB 단독으로 처리했을 때 보다 각각 1.3배 및 3배 높게 NF-κB 전사를 활성화시키는 것으로 보아 PspA-FlaB 또는 FlaB-PspA 재조합 융합 단백질 중의 FlaB가 TLR-5 매개에 의한 NF-κB 전사 활성능을 보유하고 있음은 물론이고 훨씬 그 능력이 향상되었음을 알 수 있었다. In the results of FIG. 2, both PspA-FlaB and FlaB-PspA recombinant fusion proteins activate NF-κB transcription, 1.3-fold and 3-fold higher than when treated with FlaB alone, respectively. FlaB in the TLR-5-mediated NF-κB transcriptional activity, as well as the ability was much improved.

[시험예 2] 플라젤린-PspA 재조합 융합 단백질의 방어 면역능(protective immunity) 검사[Test Example 2] Protective immunity test of flagellin-PspA recombinant fusion protein

SPF(specific pathogen free) 5주령 Balb/c 암컷 마우스의 비강내로 PBS(phosphate buffered saline), 및 실시예 1에서 제조한 2.5 ㎍ PspA, 2.5 ㎍ PspA와 4 ㎍ FlaB의 혼합물, 26.5 ㎍ PspA-FlaB 및 FlaB-PspA를 2주일 간격으로 3회에 걸쳐 각각 면역시키고 마지막으로 면역한 날로부터 14일 후에 마우스의 혈청, 침(saliva) 및 질세척액(vaginal wash)을 채취한 다음 PspA-특이적 전신 면역 반응(systemic immune response)과 점막 면역 반응(mucosal immune response)을 ELISA(Enzyme linked immuno sorbant assay) 방법으로 측정하였고, 3회에 거쳐 예방접종한 마우스에 반수 치사량(lethal dose 50%, LD50)의 200배에 해당하는 야생형 Streptococcus pneumoniae D39를 비강내로 투여하여 14일 동안 마우스의 생존을 관찰하였으며, 그 결과를 도 3a, 3b 및 도 4에 나타내었다. Specific pathogen free (SPF) phosphate buffered saline (PBS) intranasally in 5 week old Balb / c female mice, and 2.5 μg PspA prepared in Example 1, a mixture of 2.5 μg PspA and 4 μg FlaB, 26.5 μg PspA-FlaB and Immunized FlaB-PspA three times at two-week intervals, and 14 days after the last immunization, serum, saliva and vaginal washes of mice were collected and then PspA-specific systemic immune responses. (systemic immune response) and mucosal immune response (mucosal immune response) were measured by the Enzyme linked immunosorbent assay (ELISA) method, and 200 times of lethal dose (LD 50 ) in mice vaccinated three times. The intranasal administration of wild-type Streptococcus pneumoniae D39, which corresponds to the embryo, observed the survival of the mice for 14 days, and the results are shown in FIGS. 3A, 3B and 4.

도 3a 및 도 3b의 결과에서 알 수 있듯이, PBS만을 투여한 대조군(PBS)이나 PspA 단독(P) 또는 PspA와 FlaB 혼합물(P+F)을 투여한 마우스에서 보다 PspA-FlaB(PF) 및 FlaB-PspA(FP) 재조합 융합 단백질을 투여한 마우스에서 PspA 항원 특이적 전신 면역 및 점액성 면역 반응이 높게 나타났다.As can be seen from the results of Figures 3a and 3b, PspA-FlaB (PF) and FlaB than in the control group (PBS) administered only PBS or PspA alone (P) or mice administered PspA and FlaB mixture (P + F) Mice that received the -PspA (FP) recombinant fusion protein showed high PspA antigen-specific systemic and mucosal immune responses.

또한 도 4의 결과로 알 수 있듯이, PBS 만으로 면역한 대조군 마우스(PBS)는 3일 내에 100%가 사망하였고 PspA만을 투여한 마우스(PspA)에서는 약 20% 마우스만이 생존하였고 PspA와 FlaB의 혼합물을 투여한 경우(P+F) 약 20% 생존율을 보였으나 PspA-FlaB(PF) 및 FlaB-PspA(FP) 재조합 융합 단백질을 비강으로 투여한 경우 40% 및 60% 생존하였다.As can be seen from the results of FIG. 4, 100% of control mice (PBS) immunized with PBS alone died within 3 days, and only 20% of mice survived in PspA-only mice (PspA) and a mixture of PspA and FlaB. When administered (P + F), the survival rate was about 20%, but when the PspA-FlaB (PF) and FlaB-PspA (FP) recombinant fusion proteins were administered nasal, they survived 40% and 60%.

본 발명에서는 TLR-5를 자극하여 강력한 점막백신 보조제 효능이 있는 것으로 밝혀진 플라젤린을 백신보조제로, 폐렴구균의 표면 단백질 A(PspA)를 항원으로 하는 폐렴구균에 대한 백신보조제-항원 융합 재조합 단백질 백신을 제공함으로써 PspA 단독으로 투여한 경우보다 훨씬 면역능이 향상된 백신보조제-항원 융합 재조합 단백질 및 이를 포함하는 백신을 개발하였다. 이는 새로운 패러다임의 '감염질환', '자가 면역 질환' 및 '알러지 질환' 등의 치료와 나아가 '항암면역 치료제'등의 개발에 있어 새로운 접근 방법을 제시할 것이며, 기초 연구와 임상연구를 연결해 주는 중요한 고리를 제공할 것이다. 따라서, 본 발명에 의한 결과를 각종 백신 제제에 적용한다면 큰 부가가치를 창출할 수 있을 것이다.In the present invention, a vaccine adjuvant-antigen fusion recombinant protein vaccine against pneumococcal, which is a vaccine adjuvant using flagellin, which has been shown to have a potent mucosal vaccine adjuvant effect by stimulating TLR-5, as an antigen of surface protein A (PspA) of pneumococcus Vaccine adjuvant-antigen fusion recombinant protein and improved vaccines have been developed that provide much improved immunity than PspA alone. This will suggest a new approach in the development of new paradigms such as 'infectious diseases', 'autoimmune diseases' and 'allergic diseases', as well as 'anti-cancer immunity drugs'. Will provide an important link. Therefore, if the results of the present invention are applied to various vaccine formulations, a great added value can be created.

도 1은 PspA, FlaB, PspA-FlaB 및 FlaB-PspA 재조합 단백질을 제작 및 분리하는 과정을 보여주는 모식도이다.1 is a schematic diagram showing a process for preparing and isolating PspA, FlaB, PspA-FlaB and FlaB-PspA recombinant proteins.

도 2는 본 발명에 따른 PspA-FlaB 및 FlaB-PspA 재조합 융합 단백질의 TLR-5 자극 활성을 측정한 결과를 보여주는 그래프이다. Figure 2 is a graph showing the results of measuring the TLR-5 stimulating activity of the PspA-FlaB and FlaB-PspA recombinant fusion protein according to the present invention.

도 3a 및 3b는 본 발명에 따른 PspA, PspA와 FlaB 혼합물, PspA-FlaB 및 FlaB-PspA 재조합 단백질을 마우스의 비강에 면역한 후 마우스의 혈액 및 여러 가지 점막 샘플을 채취하여 항원 특이적 면역 반응을 ELISA 방법으로 측정한 결과이다. Figure 3a and 3b shows the antigen-specific immune response by taking the blood and various mucosal samples of the mouse after immunizing the nasal cavity of the mouse with the PspA, PspA and FlaB mixture, PspA-FlaB and FlaB-PspA recombinant protein according to the present invention It is the result measured by ELISA method.

도 4는 본 발명에 따른 PspA, PspA와 FlaB 혼합물, PspA-FlaB 및 FlaB-PspA 재조합 단백질을 마우스의 비강에 면역한 후 Streptococcus pneumoniae D39를 투여한 결과 치사량의 S. pneumoniae로부터 숙주 보호능을 측정한 결과이다. Figure 4 shows the protection of the host against the lethal dose of S. pneumoniae after administration of Streptococcus pneumoniae D39 after immunizing the nasal cavity of mice with PspA, PspA and FlaB mixture, PspA-FlaB and FlaB-PspA recombinant proteins according to the present invention. The result is.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Recombinant fusion protein produced by fusing Vibrio vulnificus flagellin and pathogenic antigens and the mucosal vaccine containing the same as an active ingredient <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 377 <212> PRT <213> Vibrio vulnificus flagellin FlaB <400> 1 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 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> 2 <211> 233 <212> PRT <213> Streptococcus pneumoniae PspA <400> 2 Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala 1 5 10 15 Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys 20 25 30 Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln 35 40 45 Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu 50 55 60 Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln 65 70 75 80 Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp 85 90 95 Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val 100 105 110 Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys 115 120 125 Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu 130 135 140 Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu 145 150 155 160 Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile 165 170 175 Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu 180 185 190 Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala 195 200 205 Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu 210 215 220 Glu Glu Leu Ser Asp Lys Ile Asp Glu 225 230 <210> 3 <211> 1836 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein FlaB-PspA <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 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 cgtcgactct 1140 cccgtagcca gtcagtctaa agctgagaaa gactatgatg cagcgaagaa agatgctaag 1200 aatgcgaaaa aagcagtaga agatgctcaa aaggctttag atgatgcaaa agctgctcag 1260 aaaaaatatg acgaggatca gaagaaaact gaggagaaag ccgcgctaga aaaagcagcg 1320 tctgaagaga tggataaggc agtggcagca gttcaacaag cgtatctagc ctatcaacaa 1380 gctacagaca aagccgcaaa agacgcagca gataagatga tagatgaagc taagaaacgc 1440 gaagaagagg caaaaactaa atttaatact gttcgagcaa tggtagttcc tgagccagag 1500 cagttggctg agactaagaa aaaatcagaa gaagctaaac aaaaagcacc agaacttact 1560 aaaaaactag aagaagctaa agcaaaatta gaagaggctg agaaaaaagc tactgaagcc 1620 aaacaaaaag tggatgctga agaagtcgct cctcaagcta aaatcgctga attggaaaat 1680 caagttcata gactagaaca agagctcaaa gagattgatg agtctgaatc agaagattat 1740 gctaaagaag gtttccgtgc tcctcttcaa tctaaattgg atgccaaaaa agctaaacta 1800 tcaaaacttg aagagttaag tgataagatt gatgag 1836 <210> 4 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein FlaB-PspA <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 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 Val Asp Ser Pro Val Ala Ser 370 375 380 Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala Lys Lys Asp Ala Lys 385 390 395 400 Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys Ala Leu Asp Asp Ala 405 410 415 Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys Thr Glu Glu 420 425 430 Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu Met Asp Lys Ala Val 435 440 445 Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln Gln Ala Thr Asp Lys 450 455 460 Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp Glu Ala Lys Lys Arg 465 470 475 480 Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val Arg Ala Met Val Val 485 490 495 Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys Lys Ser Glu Glu Ala 500 505 510 Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu Glu Glu Ala Lys Ala 515 520 525 Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu Ala Lys Gln Lys Val 530 535 540 Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile Ala Glu Leu Glu Asn 545 550 555 560 Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu Ile Asp Glu Ser Glu 565 570 575 Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala Pro Leu Gln Ser Lys 580 585 590 Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu Glu Glu Leu Ser Asp 595 600 605 Lys Ile Asp Glu 610 <210> 5 <211> 1836 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein PspA-FlaB <400> 5 tctcccgtag ccagtcagtc taaagctgag aaagactatg atgcagcgaa gaaagatgct 60 aagaatgcga aaaaagcagt agaagatgct caaaaggctt tagatgatgc aaaagctgct 120 cagaaaaaat atgacgagga tcagaagaaa actgaggaga aagccgcgct agaaaaagca 180 gcgtctgaag agatggataa ggcagtggca gcagttcaac aagcgtatct agcctatcaa 240 caagctacag acaaagccgc aaaagacgca gcagataaga tgatagatga agctaagaaa 300 cgcgaagaag aggcaaaaac taaatttaat actgttcgag caatggtagt tcctgagcca 360 gagcagttgg ctgagactaa gaaaaaatca gaagaagcta aacaaaaagc accagaactt 420 actaaaaaac tagaagaagc taaagcaaaa ttagaagagg ctgagaaaaa agctactgaa 480 gccaaacaaa aagtggatgc tgaagaagtc gctcctcaag ctaaaatcgc tgaattggaa 540 aatcaagttc atagactaga acaagagctc aaagagattg atgagtctga atcagaagat 600 tatgctaaag aaggtttccg tgctcctctt caatctaaat tggatgccaa aaaagctaaa 660 ctatcaaaac ttgaagagtt aagtgataag attgatgagg tcgacatggc agtgaatgta 720 aatacaaacg tagcagcaat gacagcacag cgttacctga ataacgcaaa cagcgcacaa 780 caaacttcga tggagcgtct gtcttcaggt ttcaaaatca acagtgcaaa agatgacgca 840 gccggtctgc aaatctctaa ccgcttgaac gtacaaagtc gcggtctaga cgttgcggta 900 cgtaacgcca acgacggtat ctcaatcgca caaaccgcag aaggtgcgat gaacgagacc 960 accaacatcc tacaacgtat gcgtgaccta tctctacaat ccgcgaacgg ctcaaactca 1020 aaatcagagc gcgtggcgat tcaagaagaa gtgacagcat tgaatgacga gctaaaccgt 1080 attgcagaaa ccacgtcttt tggtggtaac aagctgctaa acggtactta cggcacgaaa 1140 gcaatgcaaa ttggtgcgga taacggtgaa gcggtcatgc tttcactgaa agacatgcgc 1200 tctgacaacg tgatgatggg cggcgtgagc taccaagctg aagaaggcaa agacaagaac 1260 tggaatgtgg ccgcaggcga caacgacttg acgattgcac tgacagacag ctttggtaac 1320 gagcaagaga tcgaaatcaa cgcgaaagcg ggtgatgaca tcgaagagct agcgacgtac 1380 atcaacggtc aaactgacct tgtaaaagcg tcagtgggtg aaggcggcaa gctacagatc 1440 tttgctggta acaacaaagt tcaaggtgaa attgctttct caggtagcct agctggtgaa 1500 cttggcctag gcgaaggcaa aaacgtcacg gtagacacga ttgacgtgac aaccgtacaa 1560 ggtgcgcaag agtcggtagc gattgtggat gcggcactga aatacgtaga cagccaccgt 1620 gcagagctgg gtgcattcca gaaccgtttc aaccatgcaa tcagcaactt ggacaacatc 1680 aacgaaaacg tgaacgcgtc gaagagccga atcaaagata ccgacttcgc gaaagaaacg 1740 actcagttga ccaagacaca aattctatcg caagcatcaa gttccattct tgcgcaagcg 1800 aaacaagcgc caaactcagc gctaagtcta ctaggc 1836 <210> 6 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein PspA-FlaB <400> 6 Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala 1 5 10 15 Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys 20 25 30 Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln 35 40 45 Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu 50 55 60 Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln 65 70 75 80 Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp 85 90 95 Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val 100 105 110 Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys 115 120 125 Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu 130 135 140 Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu 145 150 155 160 Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile 165 170 175 Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu 180 185 190 Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala 195 200 205 Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu 210 215 220 Glu Glu Leu Ser Asp Lys Ile Asp Glu Val Asp Met Ala Val Asn Val 225 230 235 240 Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg Tyr Leu Asn Asn Ala 245 250 255 Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu Ser Ser Gly Phe Lys 260 265 270 Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu Gln Ile Ser Asn Arg 275 280 285 Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala Val Arg Asn Ala Asn 290 295 300 Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly Ala Met Asn Glu Thr 305 310 315 320 Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser Leu Gln Ser Ala Asn 325 330 335 Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile Gln Glu Glu Val Thr 340 345 350 Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu Thr Thr Ser Phe Gly 355 360 365 Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr Lys Ala Met Gln Ile 370 375 380 Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser Leu Lys Asp Met Arg 385 390 395 400 Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr Gln Ala Glu Glu Gly 405 410 415 Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp Asn Asp Leu Thr Ile 420 425 430 Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu Ile Glu Ile Asn Ala 435 440 445 Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr Tyr Ile Asn Gly Gln 450 455 460 Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly Gly Lys Leu Gln Ile 465 470 475 480 Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile Ala Phe Ser Gly Ser 485 490 495 Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys Asn Val Thr Val Asp 500 505 510 Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln Glu Ser Val Ala Ile 515 520 525 Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His Arg Ala Glu Leu Gly 530 535 540 Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser Asn Leu Asp Asn Ile 545 550 555 560 Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile Lys Asp Thr Asp Phe 565 570 575 Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln Ile Leu Ser Gln Ala 580 585 590 Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala Pro Asn Ser Ala Leu 595 600 605 Ser Leu Leu Gly 610 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PspA N-terminal <400> 7 catatgtctc ccgtagccag tcagtct 27 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PspA N-terminal <400> 8 gtcgacctca tcaatcttat cacttaactc ttcaag 36 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PspA C-terminal <400> 9 gtcgactctc ccgtagccag tcagt 25 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PspA C-terminal <400> 10 cccgggttac tcatcaatct tatcacttaa ctcttc 36 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FlaB N-terminal <400> 11 catatgatgg cagtgaatgt aaatacaaac gtagca 36 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FlaB N-terminal <400> 12 gtcgacgcct agtagactta gcgct 25 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FlaB C-terminal <400> 13 gtcgacatgg cagtgaatgt aaatacaaac gtag 34 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FlaB C-terminal <400> 14 cccgggttag cctagtagac ttagcg 26 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Recombinant fusion protein produced by fusing Vibrio vulnificus          flagellin and pathogenic antigens and the mucosal vaccine          containing the same as an active ingredient <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 377 <212> PRT <213> Vibrio vulnificus flagellin FlaB <400> 1 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 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> 2 <211> 233 <212> PRT <213> Streptococcus pneumoniae PspA <400> 2 Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala   1 5 10 15 Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys              20 25 30 Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln          35 40 45 Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu      50 55 60 Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln  65 70 75 80 Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp                  85 90 95 Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val             100 105 110 Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys         115 120 125 Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu     130 135 140 Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu 145 150 155 160 Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile                 165 170 175 Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu             180 185 190 Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala         195 200 205 Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu     210 215 220 Glu Glu Leu Ser Asp Lys Ile Asp Glu 225 230 <210> 3 <211> 1836 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein FlaB-PspA <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 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 cgtcgactct 1140 cccgtagcca gtcagtctaa agctgagaaa gactatgatg cagcgaagaa agatgctaag 1200 aatgcgaaaa aagcagtaga agatgctcaa aaggctttag atgatgcaaa agctgctcag 1260 aaaaaatatg acgaggatca gaagaaaact gaggagaaag ccgcgctaga aaaagcagcg 1320 tctgaagaga tggataaggc agtggcagca gttcaacaag cgtatctagc ctatcaacaa 1380 gctacagaca aagccgcaaa agacgcagca gataagatga tagatgaagc taagaaacgc 1440 gaagaagagg caaaaactaa atttaatact gttcgagcaa tggtagttcc tgagccagag 1500 cagttggctg agactaagaa aaaatcagaa gaagctaaac aaaaagcacc agaacttact 1560 aaaaaactag aagaagctaa agcaaaatta gaagaggctg agaaaaaagc tactgaagcc 1620 aaacaaaaag tggatgctga agaagtcgct cctcaagcta aaatcgctga attggaaaat 1680 caagttcata gactagaaca agagctcaaa gagattgatg agtctgaatc agaagattat 1740 gctaaagaag gtttccgtgc tcctcttcaa tctaaattgg atgccaaaaa agctaaacta 1800 tcaaaacttg aagagttaag tgataagatt gatgag 1836 <210> 4 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein FlaB-PspA <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 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 Val Asp Ser Pro Val Ala Ser     370 375 380 Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala Lys Lys Asp Ala Lys 385 390 395 400 Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys Ala Leu Asp Asp Ala                 405 410 415 Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln Lys Lys Thr Glu Glu             420 425 430 Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu Met Asp Lys Ala Val         435 440 445 Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln Gln Ala Thr Asp Lys     450 455 460 Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp Glu Ala Lys Lys Arg 465 470 475 480 Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val Arg Ala Met Val Val                 485 490 495 Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys Lys Ser Glu Glu Ala             500 505 510 Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu Glu Glu Ala Lys Ala         515 520 525 Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu Ala Lys Gln Lys Val     530 535 540 Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile Ala Glu Leu Glu Asn 545 550 555 560 Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu Ile Asp Glu Ser Glu                 565 570 575 Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala Pro Leu Gln Ser Lys             580 585 590 Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu Glu Glu Leu Ser Asp         595 600 605 Lys Ile Asp Glu     610 <210> 5 <211> 1836 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein PspA-FlaB <400> 5 tctcccgtag ccagtcagtc taaagctgag aaagactatg atgcagcgaa gaaagatgct 60 aagaatgcga aaaaagcagt agaagatgct caaaaggctt tagatgatgc aaaagctgct 120 cagaaaaaat atgacgagga tcagaagaaa actgaggaga aagccgcgct agaaaaagca 180 gcgtctgaag agatggataa ggcagtggca gcagttcaac aagcgtatct agcctatcaa 240 caagctacag acaaagccgc aaaagacgca gcagataaga tgatagatga agctaagaaa 300 cgcgaagaag aggcaaaaac taaatttaat actgttcgag caatggtagt tcctgagcca 360 gagcagttgg ctgagactaa gaaaaaatca gaagaagcta aacaaaaagc accagaactt 420 actaaaaaac tagaagaagc taaagcaaaa ttagaagagg ctgagaaaaa agctactgaa 480 gccaaacaaa aagtggatgc tgaagaagtc gctcctcaag ctaaaatcgc tgaattggaa 540 aatcaagttc atagactaga acaagagctc aaagagattg atgagtctga atcagaagat 600 tatgctaaag aaggtttccg tgctcctctt caatctaaat tggatgccaa aaaagctaaa 660 ctatcaaaac ttgaagagtt aagtgataag attgatgagg tcgacatggc agtgaatgta 720 aatacaaacg tagcagcaat gacagcacag cgttacctga ataacgcaaa cagcgcacaa 780 caaacttcga tggagcgtct gtcttcaggt ttcaaaatca acagtgcaaa agatgacgca 840 gccggtctgc aaatctctaa ccgcttgaac gtacaaagtc gcggtctaga cgttgcggta 900 cgtaacgcca acgacggtat ctcaatcgca caaaccgcag aaggtgcgat gaacgagacc 960 accaacatcc tacaacgtat gcgtgaccta tctctacaat ccgcgaacgg ctcaaactca 1020 aaatcagagc gcgtggcgat tcaagaagaa gtgacagcat tgaatgacga gctaaaccgt 1080 attgcagaaa ccacgtcttt tggtggtaac aagctgctaa acggtactta cggcacgaaa 1140 gcaatgcaaa ttggtgcgga taacggtgaa gcggtcatgc tttcactgaa agacatgcgc 1200 tctgacaacg tgatgatggg cggcgtgagc taccaagctg aagaaggcaa agacaagaac 1260 tggaatgtgg ccgcaggcga caacgacttg acgattgcac tgacagacag ctttggtaac 1320 gagcaagaga tcgaaatcaa cgcgaaagcg ggtgatgaca tcgaagagct agcgacgtac 1380 atcaacggtc aaactgacct tgtaaaagcg tcagtgggtg aaggcggcaa gctacagatc 1440 tttgctggta acaacaaagt tcaaggtgaa attgctttct caggtagcct agctggtgaa 1500 cttggcctag gcgaaggcaa aaacgtcacg gtagacacga ttgacgtgac aaccgtacaa 1560 ggtgcgcaag agtcggtagc gattgtggat gcggcactga aatacgtaga cagccaccgt 1620 gcagagctgg gtgcattcca gaaccgtttc aaccatgcaa tcagcaactt ggacaacatc 1680 aacgaaaacg tgaacgcgtc gaagagccga atcaaagata ccgacttcgc gaaagaaacg 1740 actcagttga ccaagacaca aattctatcg caagcatcaa gttccattct tgcgcaagcg 1800 aaacaagcgc caaactcagc gctaagtcta ctaggc 1836 <210> 6 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein PspA-FlaB <400> 6 Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala Ala   1 5 10 15 Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala Gln Lys              20 25 30 Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp Gln          35 40 45 Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser Glu Glu      50 55 60 Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala Tyr Gln  65 70 75 80 Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met Ile Asp                  85 90 95 Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn Thr Val             100 105 110 Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr Lys Lys         115 120 125 Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys Lys Leu     130 135 140 Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala Thr Glu 145 150 155 160 Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala Lys Ile                 165 170 175 Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu Lys Glu             180 185 190 Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe Arg Ala         195 200 205 Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser Lys Leu     210 215 220 Glu Glu Leu Ser Asp Lys Ile Asp Glu Val Asp Met Ala Val Asn Val 225 230 235 240 Asn Thr Asn Val Ala Ala Met Thr Ala Gln Arg Tyr Leu Asn Asn Ala                 245 250 255 Asn Ser Ala Gln Gln Thr Ser Met Glu Arg Leu Ser Ser Gly Phe Lys             260 265 270 Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu Gln Ile Ser Asn Arg         275 280 285 Leu Asn Val Gln Ser Arg Gly Leu Asp Val Ala Val Arg Asn Ala Asn     290 295 300 Asp Gly Ile Ser Ile Ala Gln Thr Ala Glu Gly Ala Met Asn Glu Thr 305 310 315 320 Thr Asn Ile Leu Gln Arg Met Arg Asp Leu Ser Leu Gln Ser Ala Asn                 325 330 335 Gly Ser Asn Ser Lys Ser Glu Arg Val Ala Ile Gln Glu Glu Val Thr             340 345 350 Ala Leu Asn Asp Glu Leu Asn Arg Ile Ala Glu Thr Thr Ser Phe Gly         355 360 365 Gly Asn Lys Leu Leu Asn Gly Thr Tyr Gly Thr Lys Ala Met Gln Ile     370 375 380 Gly Ala Asp Asn Gly Glu Ala Val Met Leu Ser Leu Lys Asp Met Arg 385 390 395 400 Ser Asp Asn Val Met Met Gly Gly Val Ser Tyr Gln Ala Glu Glu Gly                 405 410 415 Lys Asp Lys Asn Trp Asn Val Ala Ala Gly Asp Asn Asp Leu Thr Ile             420 425 430 Ala Leu Thr Asp Ser Phe Gly Asn Glu Gln Glu Ile Glu Ile Asn Ala         435 440 445 Lys Ala Gly Asp Asp Ile Glu Glu Leu Ala Thr Tyr Ile Asn Gly Gln     450 455 460 Thr Asp Leu Val Lys Ala Ser Val Gly Glu Gly Gly Lys Leu Gln Ile 465 470 475 480 Phe Ala Gly Asn Asn Lys Val Gln Gly Glu Ile Ala Phe Ser Gly Ser                 485 490 495 Leu Ala Gly Glu Leu Gly Leu Gly Glu Gly Lys Asn Val Thr Val Asp             500 505 510 Thr Ile Asp Val Thr Thr Val Gln Gly Ala Gln Glu Ser Val Ala Ile         515 520 525 Val Asp Ala Ala Leu Lys Tyr Val Asp Ser His Arg Ala Glu Leu Gly     530 535 540 Ala Phe Gln Asn Arg Phe Asn His Ala Ile Ser Asn Leu Asp Asn Ile 545 550 555 560 Asn Glu Asn Val Asn Ala Ser Lys Ser Arg Ile Lys Asp Thr Asp Phe                 565 570 575 Ala Lys Glu Thr Thr Gln Leu Thr Lys Thr Gln Ile Leu Ser Gln Ala             580 585 590 Ser Ser Ser Ile Leu Ala Gln Ala Lys Gln Ala Pro Asn Ser Ala Leu         595 600 605 Ser Leu Leu Gly     610 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PspA N-terminal <400> 7 catatgtctc ccgtagccag tcagtct 27 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PspA N-terminal <400> 8 gtcgacctca tcaatcttat cacttaactc ttcaag 36 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PspA C-terminal <400> 9 gtcgactctc ccgtagccag tcagt 25 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PspA C-terminal <400> 10 cccgggttac tcatcaatct tatcacttaa ctcttc 36 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FlaB N-terminal <400> 11 catatgatgg cagtgaatgt aaatacaaac gtagca 36 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FlaB N-terminal <400> 12 gtcgacgcct agtagactta gcgct 25 <210> 13 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FlaB C-terminal <400> 13 gtcgacatgg cagtgaatgt aaatacaaac gtag 34 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FlaB C-terminal <400> 14 cccgggttag cctagtagac ttagcg 26  

Claims (7)

패혈증 비브리오균(Vibrio vulnificus)의 플라젤린 단백질과 병원체의 단백질 항원을 융합하여 제조한 재조합 융합 단백질로서, 상기 플라젤린 단백질은 FlaB이고, 병원체의 단백질 항원은 페렴구균(Streptococcus pneumoniae)의 표면 단백 A(PspA)이며, 재조합 융합 단백질은 서열번호 4의 아미노산 서열을 갖는 FlaB-PspA 단백질인 재조합 융합 단백질.A recombinant fusion protein prepared by fusing the flagellin protein of sepsis Vibrio vulnificus and the protein antigen of a pathogen, wherein the flagellin protein is FlaB, and the protein antigen of the pathogen is surface protein A of Streptococcus pneumoniae. PspA), and the recombinant fusion protein is a FlaB-PspA protein having the amino acid sequence of SEQ ID NO: 4. 삭제delete 삭제delete 삭제delete 삭제delete 제 1항에 의한 재조합 융합 단백질을 유효성분으로 포함하는 점막 투여용 백신.A vaccine for mucosal administration comprising the recombinant fusion protein according to claim 1 as an active ingredient. 제 6항에 있어서, 상기 점막은 비점막(Nasal mucosa)임을 특징으로 하는 점막 투여용 백신.The vaccine for mucosal administration according to claim 6, wherein the mucosa is nasal mucosa.
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WO2014084456A1 (en) * 2012-11-30 2014-06-05 전남대학교 산학협력단 Composition comprising recombinant fusion protein of pathogenic antigen protein and flagellin of vibrio vulnificus for preventing, alleviating, or treating aging
KR101733063B1 (en) * 2015-05-11 2017-05-08 전남대학교 산학협력단 Novel bacteria expressing hyaluroniase and use thereof
KR20180064813A (en) 2016-12-06 2018-06-15 전남대학교산학협력단 Linker peptide for binding biomolecule
US11254714B2 (en) 2012-11-30 2022-02-22 Medispan Co., Ltd. Method for inhibiting, improving, or preventing aging using recombinant fusion protein of pathogenic antigen protein and flagellin of Vibrio vulnificus

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KR101997319B1 (en) 2016-06-21 2019-07-08 전남대학교산학협력단 Manufacturing and Applications of flagellin-Adjuvanted Vaccine which induces Conformer recognizing Antibodies

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Infect Immun. 2004 May;72(5):2810-6.*

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014084456A1 (en) * 2012-11-30 2014-06-05 전남대학교 산학협력단 Composition comprising recombinant fusion protein of pathogenic antigen protein and flagellin of vibrio vulnificus for preventing, alleviating, or treating aging
US10407471B2 (en) 2012-11-30 2019-09-10 Industry Foundation Of Chonnam National University Composition comprising recombinant fusion protein of pathogenic antigen protein and flagellin of vibrio vulnificus for preventing, alleviating, or treating aging
US11254714B2 (en) 2012-11-30 2022-02-22 Medispan Co., Ltd. Method for inhibiting, improving, or preventing aging using recombinant fusion protein of pathogenic antigen protein and flagellin of Vibrio vulnificus
KR101733063B1 (en) * 2015-05-11 2017-05-08 전남대학교 산학협력단 Novel bacteria expressing hyaluroniase and use thereof
KR20180064813A (en) 2016-12-06 2018-06-15 전남대학교산학협력단 Linker peptide for binding biomolecule

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