KR20170011221A - Vaccine for preventing Klebsiella related disease - Google Patents
Vaccine for preventing Klebsiella related disease Download PDFInfo
- Publication number
- KR20170011221A KR20170011221A KR1020150103471A KR20150103471A KR20170011221A KR 20170011221 A KR20170011221 A KR 20170011221A KR 1020150103471 A KR1020150103471 A KR 1020150103471A KR 20150103471 A KR20150103471 A KR 20150103471A KR 20170011221 A KR20170011221 A KR 20170011221A
- Authority
- KR
- South Korea
- Prior art keywords
- klebsiella
- vaccine
- bns
- protoplast
- ompa
- Prior art date
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Abstract
Description
본 발명은, 클렙시엘라균 감염질환의 예방 또는 치료용 백신을 제조하는 방법, 및 이에 의해 제조된 백신에 관한 것으로, 보다 구체적으로는 크렙시엘라 뉴모니에 외막단백질 A(OmpA)를 함유하는 원형질체 유래 나노소포를 포함하는 백신 조성물, 이의 제조방법에 관한 것이다.The present invention relates to a method for producing a vaccine for the prevention or treatment of Klebsiella spp infectious disease and a vaccine prepared thereby, and more particularly to a vaccine comprising Kpc Derived nano-vesicles, and a method for producing the vaccine composition.
크렙시엘라 뉴모니에(Klebsiella pneumoniae)는 그람음성의 피막을 가진 혐기성 세균으로 입, 피부, 장 등의 정상세균총에서 발견된다. 병원 밖 크렙시엘라 세균에 의해 유발되는 가장 흔한 감염질환은 폐렴이며, 전형적으로 기관지폐렴 및 기관지염의 형태로 나타난다. 크렙시엘라 뉴모니에는 감염 및 출혈을 통해 인간 폐를 파괴할 수 있고, 때로 점액성 객담을 생성한다. 상기 세균은 전형적으로 구강인두에 대량 서식하는 미생물 흡입에 의해 기도로 들어가게 된다. Klebsiella pneumoniae is an anaerobic bacterium with a gram-negative coating and is found in normal flora, including the mouth, skin, and intestines. The most common infectious disease caused by Klebsiella bacteria outside the hospital is pneumonia, typically in the form of bronchopneumonia and bronchitis. Klebsiella pneumoniae can destroy human lungs through infection and bleeding, and sometimes produce mucus sputum. The bacterium typically enters the airways by microbial inhalation, which inhabits the mouth pharynx in large quantities.
크렙시엘라 감염은 대부분 면역력이 약한 사람에게서 나타나며, 항균치료를 수행하더라도 약 50%의 높은 사망률을 나타낸다. 게다가 크렙시엘라 뉴모니에는 비뇨기관, 쓸개관, 및 수술에 의한 상처부위에서도 감염을 유발할 수 있다. 최근에는 크렙시엘라 뉴모니에가 병원내 감염에서 매우 중요한 병원균으로 인식되고 있다. 항생제의 과다한 사용은 다양한 항생제에 내성을 갖는 크렙시엘라 세균의 병원내 감염의 위험을 증가시키는 요인이 될 수 있다. 다양한 약물에 내성을 갖는 크렙시엘라 뉴모니에 감염에 의한 치사율 때문에, 크렙시엘라 감염률을 낮추기 위한 예방용 제제의 개발이 절실하다.Klebsiella infection is most commonly seen in people with weakened immune systems and shows a high mortality rate of about 50% even with antimicrobial treatment. In addition, Klebsiella pneumoniae can also cause infections in the urinary tract, in overview, and in surgical wound areas. Recently, Klebsiella pneumoniae has been recognized as a very important pathogen in hospital infections. Excessive use of antibiotics may increase the risk of intra-hospital infection of Klebsiella bacteria resistant to various antibiotics. Due to the high mortality due to infection with various drug resistant Klebsiella pneumoniae, it is urgent to develop a preventive agent for lowering the Klebsiella infection rate.
백신은 질병에 대한 방어 면역을 생성하기 위해 항원 물질을 투여하는 것으로, 감염성 질병을 예방하기 위해 비용적으로 가장 효율이 높은 방법으로 여겨지고 있다. 상기 방법은, 적응면역의 특징인 특이적인 장기 방어 기억을 유도하는 능력을 가진 능동면역 전략을 사용함으로써, 방어면역이 유도되도록 하는 것이다. 병원균 감염을 근절하기 위한 열쇠는 체액성(또는 항체 매개) 및 세포성(T-세포 매개) 면역과 같은 병원균에 특이적인 면역반응의 활성화이다. Vaccines are considered to be the most cost effective way to prevent infectious diseases by administering antigenic substances to produce defense immunity against disease. The method is to induce protective immunity by using an active immune strategy with the ability to induce specific long-term defense memory, which is characteristic of adaptive immunity. The key to eradicating pathogenic infections is the activation of pathogen-specific immune responses such as humoral (or antibody-mediated) and cellular (T-cell mediated) immune responses.
그람음성균 유래 외막소포(outer membrane vesicles ; OMVs)라고도 알려진 세포밖 소포(extracellular vesicles ;EVs)는 구형의 인지질 이중막으로 20-200 nm의 직경을 가지며, 병원체와 관련된 많은 단백질을 포함한다. 생화학 및 프로테옴 연구를 통해 세균 유래 세포밖 소포는 외막 단백질, 지질다당류(lipopolysaccharide; LPS), 외막 지질, 주변세포질 단백질, DNA, RNA, 및 다른 독성관련 인자로 구성되어 있음이 보고되었다. 최근 수막염균(Neisseria meningitidis), 아시네토박터 바우마니(Acinetobacter baumannii), 포르피로모나스 진지발리스(Porphyromonas gingivalis), 살모넬라 엔테리카(Salmonella enterica), 세로바 티피무륨(serovar Typhimurium), 헬리코박터 파일로리(Helicobacter pylori), 및 비브리오 콜레라(Vibrio cholerae)와 같은 그람음성균 유래 세포밖 소포는 마우스에서 세균 감염에 대한 방어면역을 유도함이 보고된 바 있다. Extracellular vesicles (EVs), also known as outer membrane vesicles (OMVs), derived from Gram-negative bacteria are spherical phospholipid bilayers with a diameter of 20-200 nm and contain many proteins associated with pathogens. Biochemical and proteome studies have shown that the bacterial-derived extracellular vesicles consist of the outer membrane protein, lipopolysaccharide (LPS), outer membrane lipid, surrounding cytoplasmic protein, DNA, RNA, and other toxic related factors. Recent meningitis bacteria (Neisseria meningitidis), Acinetobacter baumannii (Acinetobacter baumannii , Porphyromonas gingivalis , Salmonella enterica , serovar Typhimurium), Helicobacter pylori (Helicobacter pylori), and Vibrio cholera (Vibrio cholerae ) have been reported to induce defense immunity against bacterial infection in mice.
백신으로써 그람음성균 유래 외막소포는 자체의 우수한 효과에도 불구하고 독성문제가 남아있다. 외막소포는 항원으로 작용할 수 있는 다양한 종류의 분자를 포함하지만 어떠한 분자는 심각한 면역반응을 유도하고 염증을 야기시키므로, 패혈증과 같은 국소 또는 전신 염증 반응을 유발할 수 있다. As a vaccine, the outer membrane vesicle derived from gram negative bacteria remains toxic despite its excellent effect. Although outer membrane vesicles contain a wide variety of molecules capable of acting as antigens, some molecules can induce a serious immune response and cause inflammation, thus leading to local or systemic inflammatory responses such as sepsis.
원형질체(Protoplast)는 식물, 세균의 살아있는 세포 물질이며, 이들 세포의 세포벽은 부분적으로 또는 완전히 기계적/효소적인 방법을 사용하여 제거될 수 있다. 따라서, 그람음성균의 원형질체는 세균의 외막과 펩티도글리칸층을 가지지 않으므로, 원형질체가 외막소포의 독성문제를 해결할 수 있는 한편, 외막을 가지고 있지 않으므로 항원이 충분하지 않아 백신으로써 사용하기에는 문제가 있다. 이러한 이유로 원형질체를 백신으로 사용하기 위해서는 항원으로 작용할 수 있는 단백질이 필요하다. Protoplasts are living cell material of plants and bacteria, and the cell walls of these cells can be partially or completely removed using mechanical / enzymatic methods. Therefore, the protoplasts of Gram-negative bacteria do not have a bacterial outer membrane and a peptidoglycan layer. Therefore, the protoplasm can solve the toxicity problem of the outer membrane vesicles, but the outer membrane does not have sufficient antigen. For this reason, proteins that can act as antigens are needed to use protoplasts as vaccines.
이에, 본 발명은 크렙시엘라 뉴모니에 외막단백질 A(OmpA)를 함유하는 원형질체 유래 나노소포를 포함하는 백신 조성물, 및 이의 제조방법을 제공하고자 한다.Accordingly, the present invention provides a vaccine composition comprising a protoplast-derived nano-vasicle containing outer membrane protein A (OmpA) in Klebsiella pneumoniae, and a method for producing the vaccine composition.
또한, 본 발명은 상기 백신을 이용하여 크렙시엘라 속 세균유래 세포밖 소포에 의한 질환 및/또는 크렙시엘라 속 세균에 의한 감염을 효과적으로 예방 또는 치료하는 방법을 제공하고자 한다.The present invention also provides a method for effectively preventing or treating a disease caused by an extracellular vesicle derived from Klebsiella subtilis bacteria and / or an infection caused by a bacterium belonging to the genus Klebsiella using the vaccine.
본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.The technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.
본 발명은, 하기의 단계를 포함하는, 클렙시엘라균 감염질환 예방 또는 치료용 백신의 제조방법을 제공한다:The present invention provides a method for the preparation of a vaccine for the prevention or treatment of Klebsiella infestation disease comprising the steps of:
(a) 클렙시엘라균 유래의 OmpA(Outer membrane protein A) 유전자를 숙주균에 과발현시키는 단계; (b) 상기 과발현된 숙주균에서 외막과 펩티도글리칸층을 제거하여 원형질체를 얻는 단계; 및 (c) 상기 원형질체를 분리하는 단계.(a) Overexpressing OmpA (Outer membrane protein A) gene derived from Klebsiella sp. (b) removing the outer membrane and the peptidoglycan layer from the overexpressed host bacteria to obtain a protoplast; And (c) separating the protoplast.
본 발명의 일 구현예에 있어서, 상기 (b) 단계에서 원형질체는 숙주균에 라이소자임을 처리한 후 압출하여 얻는 것을 특징으로 한다.In one embodiment of the present invention, the protoplast in the step (b) is characterized in that the host microorganism is treated with lysozyme and then extruded.
본 발명의 다른 구현예에 있어서, 상기 클렙시엘라균은 클렙시엘라 뉴모니에(Klebsiella pneumoniae)인 것을 특징으로 한다.In another embodiment of the present invention, the Klebsiella sp. Is Klebsiella pneumoniae.
본 발명의 또 다른 구현예에 있어서, 상기 클렙시엘라균 감염질환은 패혈증, 폐렴 및 폐기종으로 이루어진 군에서 선택되는 것을 특징으로 한다.In another embodiment of the present invention, the Klebsiella infection disease is selected from the group consisting of sepsis, pneumonia and emphysema.
본 발명의 또 다른 구현예에 있어서, 상기 숙주균은 대장균(E. coli)인 것을 특징으로 한다.In another embodiment of the present invention, the host microorganism is E. coli .
또한, 본 발명은 상기 방법으로 제조된, 클렙시엘라균 감염질환 예방 또는 치료용 백신을 제공한다.In addition, the present invention provides a vaccine for preventing or treating Klebsiella infectious disease produced by the above method.
본 발명의 일 구현예에 있어서, 상기 백신은 원형질체의 내강(lumen)에 클렙시엘라균 유래의 OmpA(Outer membrane protein A) 단백질이 과발현되어 존재하는 것을 특징으로 한다.In one embodiment of the present invention, the vaccine is characterized in that OmpA (Outer membrane protein A) derived from Klebsiella is overexpressed in the lumen of the protoplast.
본 발명의 다른 구현예에 있어서, 상기 클렙시엘라균은 클렙시엘라 뉴모니에(Klebsiella pneumoniae)인 것을 특징으로 한다.In another embodiment of the present invention, the Klebsiella sp. Is Klebsiella pneumoniae.
본 발명의 또 다른 구현예에 있어서, 상기 클렙시엘라균 감염질환은 패혈증, 폐렴 및 폐기종으로 이루어진 군에서 선택되는 것을 특징으로 한다.In another embodiment of the present invention, the Klebsiella infection disease is selected from the group consisting of sepsis, pneumonia and emphysema.
본 발명의 또 다른 구현예에 있어서, 상기 원형질체는 평균 직경이 200-300nm인 것을 특징으로 한다.In another embodiment of the present invention, the protoplast is characterized by an average diameter of 200-300 nm.
본 발명은 크렙시엘라 뉴모니에 외막단백질 A(OmpA)를 함유하는 원형질체 유래 나노소포를 백신으로 이용함으로써, 면역반응을 조절하여 크렙시엘라 속 세균에 의한 감염 혹은 크렙시엘라 속 세균유래 세포밖 소포에 의한 질병을 효율적으로 예방 또는 치료할 수 있다.The present invention uses a protoplast-derived nano-vesicle containing outer membrane protein A (OmpA) as a vaccine to control the immune response to kill infections caused by Klebsiella subspecies or Klebsiella subfamily cells Diseases caused by vesicles can be efficiently prevented or treated.
도 1은, 크렙시엘라 뉴모니에 유래 세포밖 소포의 독성을, 마우스의 체중(도 1A), 말초 혈액의 백혈구수(도 1B), 생존률(도 1C)로 평가한 결과이다.
도 2는, 크렙시엘라 뉴모니에 OmpA, ABC transporter(ABCT), FepA 유전자의 크기 및 염기서열을 나타낸 것이다.
도 3은, 크렙시엘라 뉴모니에 OmpA, ABCT, FepA이 각각 로딩된 P-BNS(protoplast-bionanosome)에 대하여 TEM(도 3A) 및 DLS(도 3B) 분석을 실시한 결과이다.
도 4는, 야생형 BL21균, 단백질-형질전환된 BL21균, 단백질(OmpA, ABCT, FepA)-형질전환된 P-BNS에 대하여 SDS-PAGE(도 4A) 및 웨스턴 블랏팅(도 4B)을 실시한 결과이다.
도 5는, 크렙시엘라 뉴모니에 OmpA(도 5A), ABCT(도 5B), FepA(도 5C)이 각각 로딩된 P-BNS가 선천성 면역반응을 유도하는지 확인하기 위하여, 대식세포에 P-BNS를 처리후 IL-6와 TNF-α의 발현을 확인한 결과이다.
도 6은, 크렙시엘라 뉴모니에 OmpA(도 6A), ABCT(도 6B), FepA(도 6C)이 각각 로딩된 P-BNS에 대하여 각각 특이적인 IgG 항체가 생성되었는지 확인한 결과이다.
도 7은, 크렙시엘라 뉴모니에 OmpA, ABCT, FepA이 각각 로딩된 P-BNS의 독성을 평가하기 위하여 마우스에 투여후 생존율을 확인한 결과이다.
도 8은, 크렙시엘라 뉴모니에 OmpA이 로딩된 P-BNS에 대하여 T 세포 반응을 평가하기 위하여 IFN-γ, IL-17, IL-4, IL-10의 양을 측정한 결과이다.
도 9는, 크렙시엘라 뉴모니에 ABCT이 로딩된 P-BNS에 대하여 T 세포 반응을 평가하기 위하여 IFN-γ, IL-17, IL-4, IL-10의 양을 측정한 결과이다.
도 10은, 크렙시엘라 뉴모니에 FepA이 로딩된 P-BNS에 대하여 T 세포 반응을 평가하기 위하여 IFN-γ, IL-17, IL-4, IL-10의 양을 측정한 결과이다.
도 11은, 패혈증에 대한 크렙시엘라 뉴모니에 단백질(OmpA(도 11A), ABCT(도 11B), FepA(도 11C)) 함유 P-BNS 백신의 보호 효과를 확인한 결과이다.Fig. 1 shows the results of evaluating the toxicity of extracellular vesicles derived from Klebsiella annuumisii with mouse body weight (Fig. 1A), peripheral blood leukocyte count (Fig. 1B) and survival rate (Fig. 1C).
Fig. 2 shows the size and nucleotide sequence of OmpA, ABC transporter (ABCT) and FepA gene in Kepsiella annumi.
FIG. 3 shows the results of TEM (FIG. 3A) and DLS (FIG. 3B) analysis of P-BNS (protoplast-bionanosome) loaded with OmpA, ABCT and FepA on Kepsiella annumii.
Figure 4 shows the results of SDS-PAGE (Fig. 4A) and Western blotting (Fig. 4B) of wild-type BL21, protein-transformed BL21, protein (OmpA, ABCT, FepA) Results.
Figure 5 shows that P-BNS loaded with OmpA (Fig. 5A), ABCT (Fig. 5B) and FepA (Fig. 5C) in Kepsiella annumii induces congenital immune responses, The expression of IL-6 and TNF-α was examined after treatment with BNS.
FIG. 6 shows the results of confirming whether IgG antibodies specific for P-BNS loaded with OmpA (FIG. 6A), ABCT (FIG. 6B) and FepA (FIG.
FIG. 7 shows the results of confirming the survival rate after administration to mice in order to evaluate the toxicity of P-BNS loaded with OmpA, ABCT and FepA on Krebsiella nymonis.
FIG. 8 shows the results of measuring the amounts of IFN-γ, IL-17, IL-4 and IL-10 in order to evaluate the T cell response to OmpA-loaded P-BNS in Kepsiella annumii.
FIG. 9 shows the results of measuring the amounts of IFN-y, IL-17, IL-4, and IL-10 in order to evaluate T cell response to ABCT-loaded P-BNS in Krebshiella nymonis.
FIG. 10 shows the results of measuring the amounts of IFN-γ, IL-17, IL-4 and IL-10 in order to evaluate the T cell response to FepA-loaded P-BNS in Krebshiella nymonis.
Fig. 11 shows the results of confirming the protective effect of P-BNS vaccine containing Krepsiella nymoniie protein (OmpA (Fig. 11A), ABCT (Fig. 11B) and FepA (Fig. 11C) against sepsis.
그람음성균에서 유래된 EV는 숙주에 다양한 면역반응을 유도할 수 있기 때문에, 최근의 백신 연구는, 그람음성균에서 분비되는 OMV(outer membrane vesicle) 즉 EV(extracellular vesicle)를 vehicle로 이용하는데 집중되어 있다. 그러나, OMV는 LPS(lipopolysaccharide)와 같은 세균독소를 함유하고 있기 때문에 독성문제가 남아있고, 제조상의 어려움도 있다.Since EV derived from Gram-negative bacteria can induce various immune responses in the host, recent vaccine studies have focused on using OMV (outer membrane vesicle), or EV (extracellular vesicle) secreted from Gram-negative bacteria, as a vehicle . However, since OMV contains a bacterial toxin such as LPS (lipopolysaccharide), toxicity problems remain and there are difficulties in manufacturing.
이에, 본 발명에서는, 종래 OMV계 백신의 독성문제를 피하기 위하여, 그람음성균의 원형질막(plasma membrane)만 남겨두고 외막(outer membrane)과 펩티도글리칸층(peptidoglycan)을 포함하는 세포벽(cell wall)을 제거하여 만들어진 원형질체(protoplast)를 이용하였다. 또한, 자연적으로 배출되는 EV는 생산성이 낮기 때문에, 세균 원형질체로부터 인공적으로 EV를 제조하면 생산성도 증대시킬 수 있는 바, 이와 같은 본 발며의 신규 항원 운반 시스템을 P-BNS(protoplast-derived bionanosome)이라 칭한다.Therefore, in the present invention, in order to avoid the toxicity problem of the conventional OMV vaccine, a cell wall containing an outer membrane and a peptidoglycan layer, leaving only a plasma membrane of Gram-negative bacteria, And the protoplast was used. In addition, since the naturally released EV is low in productivity, the productivity of the EV can be increased by artificially producing the EV from the bacterial protoplast. Thus, the novel antigen delivery system of this invention is called protoplast-derived bionanosome (P-BNS) It is called.
본 발명에서는, 특히 항-크렙시엘라균 백신의 개발을 위한 후보 항원으로써 3가지 단백질을 선별하였다. 즉, OmpA(K. pneumoniae outer membrane protein A), ABCT(K. pneumoniae glutamin ATP-binding cassette transporter perplasmic-binding protein), FepA(K. pneumoniae ferric enterobactin protein A)으로서, 이들 단백질은 모두 외막에 존재하기 때문에 세균 인식을 위한 에피토프(epitopes)로 작용할 수 있다. In the present invention, three proteins were selected as candidate antigens for the development of anti-Klebsiella vaccine. That is, OmpA ( K. pneumoniae outer membrane protein A), ABCT ( K. pneumoniae glutamin ATP-binding cassette transporter perplasmic-binding protein, and FepA ( K. pneumoniae ferric enterobactin protein A). These proteins are all present in the outer membrane and can act as epitopes for bacterial recognition.
크렙시엘라 뉴모니에 OmpA는 엔테로박테리아 과(Enterobacteriaceae family)에서 보존된 단백질로서, 특히 초기 염증반응을 억제하기 위해 선천성 면역반응을 방해함으로써 면역 회피에 중요한 역할을 하는 단백질이다. 크렙시엘라 뉴모니에 ABCT는 ATP-binding cassette family 중 하나로, 대부분의 세균에서 보존된 단백질이며 독성을 가지고 있다. 크렙시엘라 뉴모니에 FepA는 transplastomic 세균 단백질로서 철 수송에 관여하고, 면역원성을 가지므로 백신의 항원으로도 사용할 수 있다.OmpA is a protein conserved in the Enterobacteriaceae family. It is a protein that plays an important role in immune avoidance by interfering with the innate immune response to suppress the initial inflammatory response. ABCT is one of the ATP-binding cassette family, which is a conserved protein in most bacteria and has toxicity. FepA is a transplastomic bacterial protein involved in iron transport and has immunogenicity, so it can also be used as a vaccine antigen.
본 발명에서, 크렙시엘라균 감염에 대한 P-BNS 백신은, 타겟항원(OmpA)을 대장균(E.coli)에 과발현시킨후 원형질체를 제조하여 여기에 OmpA가 로딩되도록 제조할 수 있다. 항원이 로딩된 P-BNS의 in vitro/in vivo 내재 및 적응 면역원성(innate and adaptive immunogenicity)은, 전염증 매개체 유도능이나 항원특이적 적응면역 유도능으로 평가하였다.In the present invention, the P-BNS vaccine against Kefsiella infection can be produced such that a target antigen (OmpA) is overexpressed in E. coli , and then a protoplast is prepared and OmpA is loaded therein. In vitro / in vivo of antigen-loaded P-BNS The innate and adaptive immunogenicity was evaluated by the ability to induce proinflammatory mediators or induce antigen - specific adaptive immunity.
또한, 마우스에서 P-BNS 백신 효율을 검증하기 위하여, K. pneumoniae로 챌린지된 마우스에서 P-BNS 백신 후보로 면역화하였다. 그 결과, 항원-특이 항체 및 T 세포 반응이 유도되었으나, 세균 자체에 의해 유도될 수 있는 치사율은, K. pneumonia에 특이적인 OmpA 항원을 로딩한 P-BNS로는 유도되지 않았다. 또한, OmpA 이외의 다른 항원이 로딩된 경우 역시 치사율이 높았기 때문에 OmpA를 로딩하는 것이 가장 유효한 백신임을 알 수 있었다.In addition, in order to verify P-BNS vaccine efficiency in mice, mice challenged with K. pneumoniae were immunized with P-BNS vaccine candidates. As a result, antigen-specific antibodies and T cell responses were induced, but the mortality that could be induced by the bacteria itself was not induced by P-BNS loaded with OmpA antigen specific to K. pneumonia . In addition, when the other antigens other than OmpA were loaded, it was also confirmed that loading of OmpA was the most effective vaccine because the mortality was high.
본 발명은, (a) 클렙시엘라균 유래의 OmpA(Outer membrane protein A) 유전자를 숙주균에 과발현시키는 단계; (b) 상기 과발현된 숙주균에서 외막과 펩티도글리칸층을 제거하여 원형질체를 얻는 단계; 및 (c) 상기 원형질체를 분리하는 단계를 포함하는, 클렙시엘라균 감염질환 예방 또는 치료용 백신의 제조방법에 관한 것이다.(A) Overexpressing OmpA (Outer membrane protein A) gene derived from Klebsiella sp. (b) removing the outer membrane and the peptidoglycan layer from the overexpressed host bacteria to obtain a protoplast; And (c) separating the protoplast. The present invention also relates to a method for producing a vaccine for preventing or treating Klebsiella spp.
본 발명에서, 원형질체를 제조하는 방법에 특별히 제한은 없으나, 세균에 라이소자임을 처리한 후 필터로 압출하여 얻을 수 있으며, 원심분리, 초원심분리 등의 공정을 더 포함할 수 있다.In the present invention, the method for producing the protoplast is not particularly limited. However, it can be obtained by treating the bacterium with lysozyme and then extruding it with a filter, and it may further include a process such as centrifugation and ultracentrifugation.
본 발명에서, 유전자를 과발현시킬 수 있는 숙주균에 특별히 제한은 없으나, 대장균(E.coli)인 것이 바람직하다.In the present invention, the host bacteria capable of overexpressing the gene are not particularly limited, but E. coli is preferable.
본 발명에서, '클렙시엘라균'이란, 클렙시엘라 속(Genus Klebsiella) 세균이면 특별히 제한은 없으나, 클렙시엘라 뉴모니에(Klebsiella pneumoniae)인 것이 바람직하다.In the present invention, 'Klebsiella sp.' Is not particularly limited as long as it is a genus Klebsiella bacterium, but Klebsiella pneumoniae is preferable.
본 발명에서, '클렙시엘라균 감염질환'이란, 클렙시엘라균 감염에 의해 발생될 수 있는 질환이면 특별히 제한은 없으나, 패혈증, 폐렴 및 폐기종으로 이루어진 군에서 선택되는 것이 바람직하고, 패혈증이 가장 바람직하다.In the present invention, 'Klebsiella infection disease' is not particularly limited as long as it is a disease that can be caused by Klebsiella infection, but it is preferably selected from the group consisting of sepsis, pneumonia and emphysema, desirable.
본 발명에서 '감염질환의 치료 또는 예방'이란, 감염질환의 경감, 완화 및 증상의 개선을 포함하며, 또한 감염질환이 걸릴 가능성을 낮추는 것을 포함하는 의미이다.In the present invention, the term " treatment or prevention of an infectious disease " is meant to include alleviation of an infectious disease, alleviation of symptoms and improvement of symptoms, and lowering the possibility of infectious diseases.
본 발명에서 '클렙시엘라균에서 유래하는 세포밖 소포'란 클렙시엘라균의 배양액에서 분리되거나, 또는 클렙시엘라균로 발효시킨 식품에서 분리될 수 있다. 상기 세균 배양액 또는 상기 세균 첨가 발효식품에서 상기 세포밖 소포를 분리하는 방법은 세포밖 소포를 포함한다면 특별히 제한되지 않으며, 예컨대 배양액이나 발효식품에서, 원심분리, 초고속 원심분리, 필터에 의한 여과, 겔 여과 크로마토그래피, 프리-플로우 전기영동, 모세관 전기영동 등의 방법 및 이들의 조합을 이용하여 세포밖 소포를 분리할 수 있다. 또한, 불순물의 제거를 위한 세척, 수득된 세포밖 소포의 농축 등의 과정을 추가로 포함할 수 있다. 상기 세포밖 소포는 자연적으로 분비된 것이거나, 혹은 인공적으로 분비된 세포밖 소포체를 포함한다.In the present invention, 'extracellular vesicles derived from Klebsiella can be separated from the culture medium of Klebsiella, or can be isolated from foods fermented with Klebsiella. The method of separating the extracellular vesicles from the bacterial culture broth or the fermented food containing the bacteria is not particularly limited as far as it includes extracellular vesicles. For example, in the culture broth or the fermented food, centrifugation, ultracentrifugation, filtration with a filter, Extracellular vesicles can be isolated using methods such as filtration chromatography, pre-flow electrophoresis, capillary electrophoresis, and the like, and combinations thereof. Further, it may further include processes such as washing for removal of impurities and concentration of the obtained extracellular vesicles. The extracellular vesicles include naturally secreted or artificially secreted extracellular endoplasmic reticulum.
본 발명에서, 상기 방법에 의해 제조된 백신의 원형질체는 내강(lumen)에 클렙시엘라균 유래의 단백질이 과발현되어 있으며, 이때 과발현 단백질로는 OmpA, ABC transporter(ABCT), FepA 등이 바람직하며, OmpA이 가장 바람직하다.In the present invention, the protoplast of the vaccine prepared by the above method overexpresses a protein derived from Klebsiella in a lumen, wherein OmpA, ABC transporter (ABCT) and FepA are preferable as the over expressed protein, OmpA is most preferred.
본 발명에서, 상기 방법에 의해 제조된 백신의 원형질체는 평균 직경이 200-300nm일 수 있으나, 바람직하게는 250-300nm이다.In the present invention, the protoplast of the vaccine prepared by the above method may have an average diameter of 200-300 nm, but preferably 250-300 nm.
본 발명에서, 상기 감염질환의 치료 또는 예방용 백신은 약학적 조성물로 제조될 수 있다. 치료 및 예방에 사용하기 위해 본 발명의 원형질체 유래 나노소포(P-BNS) 자체를 투여하는 것이 가능하나, 약학적 조성물의 활성 성분으로서 상기 P-BNS가 포함되는 것이 바람직하다.In the present invention, the vaccine for treating or preventing the infectious disease may be prepared from a pharmaceutical composition. Although it is possible to administer the protoplast-derived nanoparticles (P-BNS) itself for use in therapy and prevention, it is preferred that the P-BNS is included as the active ingredient of the pharmaceutical composition.
상기 약학적 조성물은 상기 분리된 P-BNS를 유효성분으로 함유하며, 약학적으로 허용 가능한 담체를 포함할 수 있다. 상기 약학적으로 허용 가능한 담체는 제제시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 사이클로덱스트린, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 등을 포함하지만 이에 한정되지 않으며, 필요에 따라 항산화제, 완충액 등 다른 통상의 첨가제를 더 포함할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제화에 관해서는 레밍턴의 문헌 (Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학적 조성물은 제형에 특별한 제한은 없으나 주사제, 흡입제, 피부 외용제 등으로 제제화할 수 있다. The pharmaceutical composition contains the separated P-BNS as an active ingredient and may include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed. It may also be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions and the like, with the addition of diluents, dispersants, surfactants, binders and lubricants. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into injections, inhalants, external skin preparations, and the like.
본 발명의 약학적 조성물의 투여방법은 특별히 제한되는 것은 아니나, 목적하는 방법에 따라 정맥 내, 피하, 복강 내, 흡입, 피부도포 또는 국소적용과 같이 비경구 투여하거나 경구 투여할 수 있다. The method of administering the pharmaceutical composition of the present invention is not particularly limited, but it may be parenterally or orally administered intravenously, subcutaneously, intraperitoneally, by inhalation, skin application or topical application according to the intended method.
투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 일일 투여량은 치료를 필요로 하는 개체에 투여됨으로써 경감된 질병 상태에 대한 치료에 충분한 본 발명의 치료용 물질의 양을 의미한다. 치료용 물질의 효과적인 양은 특정 화합물, 질병 상태 및 그의 심각도, 치료를 필요로 하는 개체에 따라 달라지며, 이는 당업자에 의해 통상적으로 결정될 수 있다. 비제한적 예로서, 본 발명에 의한 조성물의 인체에 대한 투여량은 환자의 나이, 몸무게, 성별, 투여 형태, 건강 상태 및 질환 정도에 따라 달라질 수 있으며, 몸무게가 70 ㎏인 성인 환자를 기준으로 할 때, 일반적으로는 0.01∼1000 ㎎/일, 바람직하게는 1∼500 ㎎/일이며, 일정시간 간격으로 1일 1회 내지 수회에 분할 투여할 수도 있다.The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The daily dose refers to the amount of the therapeutic substance of the present invention which is sufficient to treat a relieved disease state by being administered to an individual in need of treatment. The effective amount of the therapeutic substance will depend on the particular compound, the disease state and its severity, the individual in need of treatment, which can be routinely determined by one of ordinary skill in the art. As a non-limiting example, the dosage for the human body of the composition according to the present invention may vary depending on the age, weight, sex, dosage form, health condition and disease severity of the patient and is based on adult patients weighing 70 kg , It is generally 0.01 to 1000 mg / day, preferably 1 to 500 mg / day, and may be dividedly administered once to several times a day at predetermined time intervals.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
[[ 실시예Example ]]
실시예Example 1: One: 크렙시엘라Krebscheiler 뉴모니에Nimonie (( K. K. pneumoniaepneumoniae ) 유래 ) Origin 세포밖Extracellular 소포의 독성 Toxicity of vesicles
세포밖 소포의 독성을 확인하기 위하여, 크렙시엘라 뉴모니에 유래 세포밖 소포를 6-7주령의 암컷 야생형 C57BL/6 마우스에 투여 후, 마우스의 체중(Body weight), 말초 혈액의 백혈구수(WBC), 생존률(Survival)을 확인하였다. 이때, 대조군으로는 PBS를 이용하였다.To confirm the toxicity of the extracellular vesicles, the extracellular vesicles derived from Klebsiella nemona were administered to female wild-type C57BL / 6 mice at 6-7 weeks of age, and then the body weight of the mouse, the number of leukocytes WBC) and survival rate (Survival). At this time, PBS was used as a control group.
그 결과, 도 1A에 나타낸 바와 같이, 1 ㎍ 및 5 ㎍의 세포밖 소포 투여 그룹 모두에서 체중 감소를 유도하였으며, 특히 5 ㎍의 세포밖 소포를 투여한 그룹에서는 투여 48시간 후 3 g 이상 몸무게가 감소하였다. 또한, 도 1B에 나타낸 바와 같이, 세포밖 소포를 투여한지 24시간 후 백혈구가 감소되었다. As a result, as shown in Fig. 1A, weight loss was induced in both the 1 占 퐂 and 5 占 퐂 extracellular vesicle administration groups, in particular, in the group administered with 5 占 퐂 extracellular vesicles, Respectively. In addition, as shown in FIG. 1B, leukocytes were reduced 24 hours after administration of the extracellular vesicles.
또한, 세포밖 소포의 치사율을 측정하기 위하여, 더 많은 양(1, 25, 50 ㎍)의 세포밖 소포를 복강 내로 투여한 결과, 도 1C에 나타낸 바와 같이, 50 ㎍, 25 ㎍의 세포밖 소포를 투여한 마우스에서 각각 80%, 20%가 죽었다. In addition, in order to measure the mortality of extracellular vesicles, intraperitoneal administration of a larger amount (1, 25, 50)) of extracellular vesicles resulted in 50 ㎍ and 25 의 of extracellular vesicles Were killed by 80% and 20%, respectively.
이러한 결과는, 세포밖 소포가 패혈증(sepsis)과 같은 면역학적 문제를 일으킬 수 있음을 의미하는 것이며, 따라서 세포밖 소포를 백신으로 사용시 안전성 문제를 고려해야 할 것이다.These results imply that extracellular vesicles may cause immunological problems such as sepsis, and therefore safety issues should be considered when using extracellular vesicles as a vaccine.
실시예Example 2: 2: 크렙시엘라Krebscheiler 뉴모니에Nimonie (( K. K. pneumoniaepneumoniae ) 후보 유전자 ) Candidate genes 클로닝Cloning
크렙시엘라 뉴모니에(ATCC 4208)의 전체 genomic DNA를 얻기 위해서, 박테리아를 OD값이 1.5가 될 때까지 200rpm, 37℃에서 영양배지(Merck)에 배양한 후 배양균을 4℃에서 5,000 × g, 10분 동안 원심분리하였다.To obtain the whole genomic DNA of Klebsiella pneumoniae (ATCC 4208), the bacteria were cultured in nutrient medium (Merck) at 200 rpm and 37 ° C until the OD value reached 1.5, and then cultured at 5,000 × g, and centrifuged for 10 minutes.
박테리아 펠렛(pellet)을 증류수로 재현탁하였고, 100℃에서 10분 동안 가열한 후, 박테리아 외막과 펩티도글리칸층을 포함하는 세포벽을 제거하기 위하여 가열한 박테리아를 10,000 x g에서 10분 동안 원심분리하였다. 상층액의 게놈은 후보 유전자들을 증폭시키기 위해 PCR 주형으로써 사용하였고, K. pneumonia OmpA, FepA 및 ABC transporter 유전자를 후보 유전자로 하여 클로닝하였으며, 각 유전자의 크기와 염기서열을 도 2 및 서열목록(서열번호 1 내지 3)에 나타내었다.The bacterial pellet was resuspended in distilled water and heated at 100 ° C. for 10 minutes and then the heated bacteria were centrifuged at 10,000 × g for 10 minutes to remove the cell walls containing the bacterial outer membrane and the peptidoglycan layer . The genome of the supernatant was used as a PCR template to amplify candidate genes, and K. pneumonia OmpA, FepA and ABC transporter genes were cloned as candidate genes. The size and nucleotide sequence of each gene were shown in Fig. 2 and the sequence listing Nos. 1 to 3).
구체적으로는, 이들 유전자를 제조사의 프로토콜에 따라 KOD FX(TOYOBO) DNA 폴리머라아제, dNTP, 프라이머쌍을 이용하여 PCR 증폭하였으며, 각각의 프라이머쌍을 하기 [표 1]에 나타내었다Specifically, these genes were PCR-amplified using KOD FX (TOYOBO) DNA polymerase, dNTP, and primer pairs according to the manufacturer's protocol, and the respective primer pairs are shown in Table 1 below
Primer Sequence
transporterABC
transporter
PCR 산물은 T-blunt PCR 벡터(T-blunt PCR cloning kit, Solgent)에 삽입한 후, 각각의 플라스미드를 EcoRI(OmpA, ABC transporter) 및 BglII(FepA)로 절단하였다. 절편을 전기영동으로 분리한 후 pET-30a 플라스미드에 삽입하고, 대장균 DH5a에 열충격법으로 형질전환(transformation)하여, 최종적으로 상기 3종류의 후보 유전자를 클로닝하였다.The PCR product was inserted into a T-blunt PCR cloning kit (Solgent), and each plasmid was digested with EcoRI (OmpA, ABC transporter) and BglII (FepA). The sections were separated by electrophoresis, inserted into a pET-30a plasmid, transformed into Escherichia coli DH5a by thermal shock method, and finally the three kinds of candidate genes were cloned.
실시예Example 3: 3: 크렙시엘라Krebscheiler 뉴모니에Nimonie (K. (K. pneumoniaepneumoniae ) 단백질 함유 P-) Protein-containing P- BNS의BNS 특성분석 Character analysis
3-1. 항원이 3-1. Antigens 로딩된Loaded P- P- BNSBNS (protoplast-(protoplast- bionanosomebionanosome ) 제조) Produce
상기 실시예 2에서 얻어진 3종류의 클론을 37℃, 200 rpm, 12시간 동안 Luria Bertani broth (Merch)에서 배양한 후, Exprep plasmid mini prep. Kit (GeneAll)를 이용하여 각각의 플라스미드를 분리하여 대장균 BL21(Real Biotech)에 트랜스펙션시켰다. The three clones obtained in Example 2 were cultured in Luria Bertani broth (Merch) at 37 DEG C and 200 rpm for 12 hours, and then Exprep plasmid mini prep. Kit (GeneAll) was used to isolate each plasmid and transfected into E. coli BL21 (Real Biotech).
BL21를 OD값이 0.3이 될 때까지 200 rpm, 37℃, 100 ml LB broth(1 mM IPTG)에서 3시간 배양한 후, 3,000 × g, 4℃에서 10분 동안 원심분리하여 얻어진 박테리아 펠렛을 Tris buffer(0.01M Tris HCl, 0.5M sucrose)로 세척하였고, 4℃, 3,000 × g에서 10분 동안 원심분리한 다음 Tris buffer로 재현탁하였다. 재현탁한 박테리아를 37℃, 100 rpm에서 30분 동안 0.01 M EDTA로 처리하고, 4℃, 3,000 × g에서 10분 동안 펠릿화한 후, Tris buffer로 세척하여 펠릿화하였다. The BL21 was cultured in 100 ml LB broth (1 mM IPTG) for 3 hours at 200 rpm and 37 ° C until the OD value reached 0.3, and the bacterial pellet obtained by centrifugation at 3,000 × g for 10 minutes at 4 ° C was treated with Tris (0.01 M Tris HCl, 0.5 M sucrose), centrifuged at 3,000 × g for 10 min at 4 ° C and resuspended in Tris buffer. Resuspended bacteria were treated with 0.01 M EDTA at 100 rpm for 30 min at 37 ° C, pelleted at 4 ° C, 3,000 x g for 10 min, and then washed with Tris buffer for pelleting.
다음으로, 박테리아 펠릿을 Tris buffer로 재현탁하고, 원형질체를 얻기 위하여 37℃, 100 rpm, 2시간 동안 1 mg/ml 라이소자임(lysozyme)(Sigma)을 처리한 후, 10 μm, 5 μm, 및 1 μm 기공 크기의 막을 통해 순서대로 LiposoFast extruder (Avestin)로 압출하였다. 최종적으로, 10% 및 50% opti-prep 밀도구배 배지(OptiPrep)로 초원심분리여 P-BNS를 얻었다.Next, the bacterial pellet was resuspended in Tris buffer and treated with 1 mg / ml lysozyme (Sigma) at 37 ° C, 100 rpm for 2 hours to obtain protoplasts, followed by 10 μM, 5 μM, and 1 μM and then extruded through a membrane with a pore size of μm through a LiposoFast extruder (Avestin). Finally, P-BNS was obtained by ultracentrifugation with 10% and 50% opti-prep density gradient media (OptiPrep).
3-2. 투과 전자 현미경(Transmission electron microscopy, 3-2. Transmission electron microscopy (TEM) TEMTEM ))
실시예 3-1에서 얻은 P-BNS를 PBS로 희석(50μg/ml)시키고, 300-mesh copper grids(EMS)에 10 μl 로딩하였다. 음성염색(negative stain)을 위하여 우라닐아세테이트(2%)를 그리드에 떨어뜨리고, JEM1011 전자 현미경(JEOL)으로 관찰하였다.P-BNS obtained in Example 3-1 was diluted with PBS (50 μg / ml) and loaded with 10 μl into 300-mesh copper grids (EMS). Uranyl acetate (2%) was dropped onto the grid for negative staining and observed with a JEM1011 electron microscope (JEOL).
그 결과, 도 3A에 나타낸 바와 같이, P-BNS는 구형으로서 프로토플라즘((protoplasm)으로 둘러싸여 있음을 확인하였다.As a result, as shown in FIG. 3A, it was confirmed that P-BNS was spherical and surrounded by a protoplasm.
3-3. 동적 3-3. dynamic 광산란Light scattering (Dynamic light scattering, DLS)(Dynamic light scattering, DLS)
실시예 3-1에서 얻은 P-BNS를 PBS로 희석(500 ng/ml)시키고, Zetasizer Nano ZS (Malvern Instruments)을 이용하여 동적 광산란법으로 지름 크기 분포를 측정하고, Dynamic V6 software를 이용하여 결과를 분석하였다.The P-BNS obtained in Example 3-1 was diluted with PBS (500 ng / ml) and the diameter size distribution was measured by dynamic light scattering method using a Zetasizer Nano ZS (Malvern Instruments). Using Dynamic V6 software, Respectively.
그 결과, 도 3B에 나타낸 바와 같이, P-BNS는 평균지름이 263nm임을 알 수 있었다.As a result, as shown in FIG. 3B, it was found that the average diameter of P-BNS was 263 nm.
3-4. SDS-PAGE3-4. SDS-PAGE
실시예 3-1에서 얻은 P-BNS와 대조군으로서의 BL21균을 방사선-면역 침전 어세이버퍼(Thermo Scientific)로 용해시킨 후, 각각 SDS로 가열하고 10% 폴리아크릴아마이드 겔에 로딩하여, 300 mA에서 전기영동하였다. The P-BNS obtained in Example 3-1 and the BL21 as a control were dissolved in a radiation-immunoprecipitation assay buffer (Thermo Scientific), each was heated with SDS, loaded on a 10% polyacrylamide gel, And electrophoresed.
그 결과, 도 4A에 나타낸 바와 같이, 야생형 BL21균, 단백질-형질전환된 BL21균, 단백질-형질전환된 P-BNS 각각은 단백질 프로파일이 서로 상이함을 알 수 있었다As a result, as shown in Fig. 4A, the wild-type BL21 strain, the protein-transformed BL21 strain, and the protein-transformed P-BNS were found to have different protein profiles
3-5. 3-5. 웨스턴Western 블랏팅Blasting
실시예 3-1에서 얻은 P-BNS와 대조군으로서의 BL21균에 대하여, 항-OmpA, 항-ABCT, 항-FepA 항체로 웨스턴 블로팅을 실시하였다.P-BNS obtained in Example 3-1 and BL21 as a control were subjected to Western blotting with anti-OmpA, anti-ABCT and anti-FepA antibodies.
그 결과, 도 4B에 나타낸 바와 같이, 클로닝으로 로딩된 단백질들이 과발현되었음을 알 수 있었다.As a result, as shown in Fig. 4B, it was found that the proteins loaded by cloning were overexpressed.
실시예Example 4: 4: 크렙시엘라Krebscheiler 뉴모니에Nimonie (K. (K. pneumoniaepneumoniae ) 단백질 함유 P-) Protein-containing P- BNS에To BNS 의해 유도된 선천성 면역반응 Congenital immune response induced by
P-BNS를 백신으로서 이용하기 위해서는 면역반응을 유도할 수 있어야 하기 때문에, P-BNS에 의한 선천성 면역반응 유도 수준을 평가하기 위해 Raw 264.7 마우스 대식세포를 사용하여 in vitro 실험을 수행하였다. In order to assess the level of induction of innate immune responses by P-BNS, Raw 264.7 mouse macrophages were used to perform in vitro experiments to be able to induce an immune response in order to use P-BNS as a vaccine.
우선, Raw 264.7 마우스 대식세포(5 x 105cells/well)를 10% FBS와 항생제(100 unit/ml 페니실린 및 100 μg/ml 스트렙토마이신)를 함유한 DMEM으로 37℃에서 24시간 동안 24 well 조직배양플레이트(TPP)에서 배양하였다. 배지 제거 후, P-BNS를 0.1 ng/ml∼10 mg/ml의 다양한 농도로 첨가한 무혈청 DMEM 배지로 갈아주었다. 15시간 후 상기 배양 배지로 ELISA assay를 수행하여 전염증 사이토카인인 IL-6 및 TNF-α의 발현수준을 측정하였다. 이때, 음성대조군으로는 LPS를 이용하였다.First, Raw 264.7 mouse macrophages (5 x 10 5 cells / well) were cultured in DMEM containing 10% FBS and antibiotics (100 units / ml penicillin and 100 μg / ml streptomycin) And cultured in a culture plate (TPP). After removal of the medium, P-BNS was changed to serum-free DMEM medium supplemented with various concentrations of 0.1 ng / ml to 10 mg / ml. After 15 hours, the ELISA assay was performed on the culture medium to measure the levels of the proinflammatory cytokines IL-6 and TNF-a. At this time, LPS was used as a negative control group.
그 결과, 도 5에 나타낸 바와 같이, P-BNS가 처리된 대식세포는 IL-6와 TNF-α의 발현을 효과적으로 유도하였다. 즉, 3종류의 단백질을 과발현하는 각각의 P-BNS 모두 선천성 면역반응을 유도할 수 있었으며, 특히 K. pneumoniae ABCT(도 5B) 또는 FebA(도 5C)에 비하여, K. pneumoniae OmpA가 로딩된 P-BNS의 경우 가장 효과적으로 선천성 면역반응을 유도하였다(도 5A).As a result, as shown in Fig. 5, macrophages treated with P-BNS effectively induced the expression of IL-6 and TNF-a. That is, the comparison with respective to overexpression of the three types of protein-BNS P both were able to induce innate immune responses, especially K. pneumoniae ABCT (FIG. 5B) or FebA (Fig. 5C), K. pneumoniae OmpA loads P -BNS induced the innate immune response most effectively (Figure 5A).
실시예Example 5: 5: 크렙시엘라Krebscheiler 뉴모니에Nimonie (K. (K. pneumoniaepneumoniae ) 단백질 함유 P-) Protein-containing P- BNSBNS 백신에 의한 By vaccine in vivoin vivo 항체 생성 및 P-BNS의 치사율 분석 Antibody production and mortality analysis of P-BNS
5-1. 5-1. 생체내In vivo 항체 생성 확인 Identification of antibody production
생체내(in vivo)에서 P-BNS에 의한 적응 면역반응 유도능을 확인하고, P-BNS의 유효량을 결정하기 위하여, P-BNS를 마우스에 투여하여 면역성을 갖도록 하였다. 또한, 면역화 후 대조군(PBS) 및 P-BNS를 투여한 마우스에서 혈액을 채취하여 P-BNS 특이적 IgG 항체의 농도를 측정하였다.In order to confirm the ability of P-BNS to induce adaptive immune responses in vivo and to determine the effective amount of P-BNS, P-BNS was administered to mice to have immunity. Blood was also collected from the mice to which the control (PBS) and P-BNS were administered after the immunization, and the concentration of the P-BNS specific IgG antibody was measured.
구체적으로, 100 ng의 P-BNS가 포함된 100 μl의 PBS를 4℃에서 96-well black plate (Greiner Bio-one)에 24시간 동안 코팅하였다. P-BNS로 코팅된 플레이트를 PBS로 세척하고 PBS로 희석한 1% BSA용액을 1시간 동안 처리하여 블로킹 과정을 수행하고 PBS로 다시 세척하였다. Specifically, 100 μl of PBS containing 100 ng of P-BNS was coated on a 96-well black plate (Greiner Bio-one) at 4 ° C for 24 hours. Plates coated with P-BNS were washed with PBS, blocked with 1% BSA solution diluted with PBS for 1 hour, and then washed again with PBS.
대조군(PBS)과 P-BNS이 5 ㎍, 10 ㎍, 40 ㎍ 투여된 마우스로부터 혈액을 분리하고 원심분리하여 혈청과 세포층을 분리한 후, 혈청은 상기 1% BSA 용액으로 1:500 비율로 희석하였으며, P-BNS로 코팅된 플레이트에서 2시간 동안 반응시켰다. 배양 후 플레이트를 세척하고 horseradish peroxidase-conjugated goat anti-mouse IgG(Santa Cruz Biotechnology) 200 ng/ml를 첨가하여 2시간 동안 반응시켰다. 다시 플레이트를 세척한 후 ECL substrate(Thermo Scientific)를 첨가하고 Victor Wallac 1420 apparatus (PerkinElmer)를 이용하여 IgG 항체 역가를 분석하였다. Blood was separated from the mice to which 5 μg, 10 μg, and 40 μg of the control (PBS) and P-BNS were administered, and the serum and the cell layer were separated by centrifugation. The serum was diluted with the 1% BSA solution And reacted on a plate coated with P-BNS for 2 hours. After incubation, the plate was washed and reacted with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) 200 ng / ml for 2 hours. After washing the plate again, ECL substrate (Thermo Scientific) was added and the IgG antibody titer was analyzed using a Victor Wallac 1420 apparatus (PerkinElmer).
그 결과, 도 6에 나타낸 바와 같이, 크렙시엘라 뉴모니에 단백질을 함유한 P-BNS 백신은, 상기 P-BNS에 특이적인 IgG 항체 생성을 유도함을 알 수 있었다.As a result, as shown in FIG. 6, it was found that the P-BNS vaccine containing the protein in Klebsiella nymonii induced the generation of the IgG antibody specific to the P-BNS.
5-2. 치사율 분석(독성평가)5-2. Lethality analysis (toxicity assessment)
P-BNS에 의한 독성을 평가하기 위하여, P-BNS를 50, 100 및 500 ㎍의 고농도로 마우스에 복강투여 한 후 3일 동안 관찰하였다.In order to evaluate the toxicity by P-BNS, P-BNS was intraperitoneally administered to mice at high concentrations of 50, 100 and 500 후 and observed for 3 days.
그 결과, 도 7에 나타낸 바와 같이, 3종류의 단백질이 로딩된 P-BNS가 투여된 마우스 모두 생존함을 확인하였다.As a result, as shown in Fig. 7, it was confirmed that all mice to which P-BNS loaded with three kinds of proteins were alive survived.
실시예Example 6: 6: 크렙시엘라Krebscheiler 뉴모니에Nimonie (K. (K. pneumoniaepneumoniae ) 단백질 함유 P-) Protein-containing P- BNSBNS 백신에 의한 By vaccine in vivoin vivo T 세포 반응 분석 T cell response analysis
생체내(in vivo)에서 P-BNS에 의한 적응 면역반응을 유도한 후, 대조군(PBS) 및 P-BNS를 투여한 마우스에서 사이토카인의 양을 측정하여 T 세포 반응을 평가하였다.After induction of adaptive immune response by P-BNS in vivo , the amount of cytokine was measured in the control (PBS) and P-BNS-administered mice to evaluate the T cell response.
즉, Raw 264.7 마우스 대식세포주 또는 마우스 유래 비장세포로부터 분비된 사이토카인 양을 측정하기 위하여, ELISA duoset(R&D Systems)을 이용해 제조사의 프로토콜에 따라 P-BNS로 자극된 Raw 264.7 세포로부터 IL-6 및 TNF-α의 양을, 또한 항-CD3/항-CD28로 재자극된 비장세포의 배양 배지로부터 IFN-γ, IL-17, IL-4 및 IL-10의 양을 측정하였다.That is, IL-6 and / or IL-6 were isolated from Raw 264.7 cells stimulated with P-BNS according to the manufacturer's protocol using an ELISA duoset (R & D Systems) to measure the amount of cytokine secreted from Raw 264.7 mouse macrophages or mouse- The amount of TNF- [alpha] was measured as well as the amount of IFN- [gamma], IL-17, IL-4 and IL-10 from the culture medium of spleen cells re-stimulated with anti-CD3 / anti-CD28.
이때, 비장세포 재자극 어세이(splenocyte re-stimulation assay) 방법은 다음과 같다. 우선, 마우스로부터 분리한 비장을 5ml 시린지 washing buffer (2.5% FBS, 0.01M HEPES 함유 DMEM)로 100 μm cell strainer (BD Biosciences)에 통과시켜 분해시켰다. 분리된 비장세포에 암모늄 클로라이드 용액(STEM CELL)을 4℃에서 10분간 처리하여 적혈구 세포가 용해되도록 하였다. 얻어진 비장세포를 washing buffer로 세척하고 40 μm cell strainer(BD)로 필터링한 후 10% FBS, 50 μM 2-ME, 0.01 M HEPES 및 항생제(100 unit/ml 페니실린, 100 μg/ml 스트렙토마이신)가 포함된 RPMI 1640 배지를 이용해 24-well 플레이트에서 12시간 동안 배양하였다. 이때 24-well 플레이트는 비장 T 세포를 재자극시키기 위하여 1 μg/ml의 항-CD3(eBioscience)과 1 μg/ml의 항-CD28(eBioscience) 항체로 코팅된 것을 이용하였다. At this time, the splenocyte re-stimulation assay method is as follows. First, the spleen from the mouse was resolved by passing it through a 100 μm cell strainer (BD Biosciences) in a 5 ml syringe washing buffer (DMEM containing 2.5% FBS and 0.01 M HEPES). The separated splenocytes were treated with ammonium chloride solution (STEM CELL) at 4 ° C for 10 minutes to allow the red blood cells to dissolve. The obtained splenocytes were washed with washing buffer and filtered with a 40 μm cell strainer (BD). Then, 10% FBS, 50 μM 2-ME, 0.01 M HEPES and antibiotics (100 unit / ml penicillin, 100 μg / ml streptomycin) And cultured in a 24-well plate for 12 hours using RPMI 1640 medium. At this time, 24-well plates were coated with 1 μg / ml anti-CD3 (eBioscience) and 1 μg / ml anti-CD28 (eBioscience) antibody to re-stimulate spleen T cells.
그 결과, 도 8 내지 10에 나타낸 바와 같이, P-BNS에 특이적으로 생성된 Th1 세포에 의해 생성되는 주요 사이토카인인 IFN-γ의 양은, 3종류의 단백질이 로딩된 P-BNS에서 모두 대조군과 비교하여 투여 용량에 비례하게 증가하였다. As a result, as shown in Figs. 8 to 10, the amount of IFN-y, which is a major cytokine produced by Th1 cells specifically generated in P-BNS, was significantly higher in all three protein-loaded P- Compared with the control group.
그러나, Th17에서 생성되는 주요 사이토카인인 IL-17의 경우, OmpA(도 8) 단백질을 함유한 P-BNS가 투여된 마우스에서만 증가하였을 뿐, ABC transporter(도 9) 및 FepA(도 10) 단백질을 함유한 P-BNS가 투여된 마우스에서는 증가하지 않았다. However, in the case of IL-17, a major cytokine produced in Th17, only ABC transporter (FIG. 9) and FepA (FIG. 10) protein Lt; RTI ID = 0.0 > P-BNS < / RTI >
또한, Th2 세포에서 생성되는 주요 사이토카인인 IL-4와 IP-10의 경우, 3종류의 P-BNS 백신이 투여된 경우 모두 증가하지 않았다. In addition, IL-4 and IP-10, the major cytokines produced by Th2 cells, did not increase in all three P-BNS vaccines.
실시예Example 7: 패혈증에 대한 7: for sepsis 크렙시엘라Krebscheiler 뉴모니에Nimonie (K. (K. pneumoniaepneumoniae ) 단백질 함유 P-BNS 백신의 보호 효과) Protective Effect of Protein-Containing P-BNS Vaccine
크렙시엘라 뉴모니에에 의해 유발되는 패혈증에 대한 P-BNS 백신의 효과를 검증하기 위하여, 다음과 같은 실험을 수행하였다.In order to verify the effect of P-BNS vaccine on sepsis caused by Klebsiella lupumii, the following experiment was conducted.
우선, 마우스에 각각 5, 10 또는 40 ㎍의 P-BNS가 포함된 100 μl의 PBS를 일주일 간격으로 3주 동안 복강투여하고, 마지막 투여 일주일 후 크렙시엘라 뉴모니에를 치사량(1 × 108CFU)으로 접종하고 3일 동안 12시에 생존여부를 체크하였다. First, each of the
그 결과, 도 11에 나타낸 바와 같이, 백신을 투여하지 않은 대조군 마우스(PBS)들은 크렙시엘라 뉴모니에 주입 후 36시간 내에 모두 죽은 반면, OmpA 단백질 함유 P-BNS를 10과 40 ㎍의 농도로 투여한 마우스의 경우에는 각각 80%, 100% 생존하였다(도 11A).As a result, as shown in Fig. 11, the control mice (PBS) without the vaccine were all killed within 36 hours after injection into Krebshiella pneumonia, while the P-BNS containing OmpA protein was administered at a concentration of 10 and 40 로 And 80% and 100%, respectively, of the mice administered (Fig. 11A).
그러나, ABC transporter 및 FepA 단백질 함유 P-BNS를 투여한 마우스 그룹에서는, 대조군 마우스와 백신이 투여된 마우스 간의 생존률에 있어서 현저한 차이가 나타나지 않았다(도 11B 및 11C).However, no significant difference was observed in the survival rates between the control mice and the mice to which the vaccine was administered in the group of mice administered ABC transporter and P-BNS containing FepA protein (FIGS. 11B and 11C).
따라서, OmpA 단백질 함유 P-BNS이 크렙시엘라 감염질환에 대하여 백신으로서 가장 유효함을 알 수 있었다.Therefore, it was found that P-BNS containing OmpA protein is most effective as a vaccine against Klebsiella infection disease.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해되어야 한다.It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.
<110> Ewha University - Industry Collaboration Foundation <120> Vaccine for preventing Klebsiella related disease <130> MP15-084 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 1071 <212> DNA <213> Homo Sapiens <220> <221> gene <222> (1)..(1071) <223> OmpA <400> 1 atgaaaaaga cagctatcgc gattgcagtg gcactggctg gcttcgctac cgtagcgcag 60 gccgctccga aagataacac ctggtatgca ggtggtaaac tgggttggtc ccagtatcac 120 gacaccggtt tctacggtaa cggtttccag aacaacaacg gtccgacccg taacgatcag 180 cttggtgctg gtgcgttcgg tggttaccag gttaacccgt acctcggttt cgaaatgggt 240 tatgactggc tgggccgtat ggcatataaa ggcagcgttg acaacggtgc tttcaaagct 300 cagggcgttc agctgaccgc taaactgggt tacccgatca ctgacgatct ggacatctac 360 acccgtctgg gcggcatggt ttggcgcgct gactccaaag gcaactacgc ttctaccggc 420 gtttcccgta gcgaacacga cactggcgtt tccccagtat ttgctggcgg cgtagagtgg 480 gctgttactc gtgacatcgc tacccgtctg gaataccagt gggttaacaa catcggcgac 540 gcgggcactg tgggtacccg tcctgataac ggcatgctga gcctgggcgt ttcctaccgc 600 ttcggtcagg aagatgctgc accggttgtt gctccggctc cggctccggc tccggaagtg 660 gctaccaagc acttcaccct gaagtctgac gttctgttca acttcaacaa agctaccctg 720 aaaccggaag gtcagcaggc tctggatcag ctgtacactc agctgagcaa catggatccg 780 aaagacggtt ccgctgttgt tctgggctac accgaccgca tcggttccga agcttacaac 840 cagcagctgt ctgagaaacg tgctcagtcc gttgttgact acctggttgc taaaggcatc 900 ccggctggca aaatctccgc tcgcggcatg ggtgaatcca acccggttac tggcaacacc 960 tgcgacaacg tgaaagctcg cgctgccctg atcgattgcc tggctccgga tcgtcgtgta 1020 gagatcgaag ttaaaggcta caaagaagtt gtaactcagc cggcggctta a 1071 <210> 2 <211> 2229 <212> DNA <213> Homo Sapiens <220> <221> gene <222> (1)..(2229) <223> FepA <400> 2 atgaataaca ggatcaaatc cctggccttg ctggtcaatc tgggaattta cggggttgct 60 tttccgttaa gcgcagcgga aaccgccacc gacgataaaa acagcgccgc tgaagagacc 120 atggtggtca ccgccgccga gcagaacctg caggcgccgg gcgtctccac catcaccgcc 180 gatgagatcc gcaaacgccc cccggcgcgc gacgtctcgg agatcattcg caccatgccg 240 ggggtcaacc tgaccggcaa ctccaccagc ggccagcgcg gcaacaaccg ccagattgat 300 atccgcggca tgggcccgga aaataccctg atcctgatcg acggcaagcc ggtcaccagc 360 cgcaactccg tgcgccttgg ctggcgcggc gagcgcgaca cccgcggcga taccagctgg 420 gtgccgccgg agatgatcga acgtatcgaa gtgattcgcg gcccggccgc cgcccgctac 480 ggcaacggcg ccgccggcgg cgtggtgaat atcatcacca aaaaaaccgg cgatgagtgg 540 cacggctcat ggaacaccta tatgaacgcc ccggagcaca aggatgaagg ctccaccaaa 600 cgcactaact tcagcctcag cggcccgctg ggcggcgatt ttagcttccg cctgttcggt 660 aacctcgaca aaacgcaggc tgacgcctgg gatatcaacc agggccatca gtccgagcgt 720 accgggatct atgccgatac tctgccggcc gggcgcgaag gggtgaaaaa caaaaacatc 780 gatggtctgg tgcgctggga attcgctccg atgcagtcgc tggagtttga ggccggctac 840 agccgccagg gcaacctcta cgccggcgat acccagaaca ccaactccaa cgacctggta 900 aaagagaact acggcaaaga gaccaaccgt ctgtatcgca acacctactc ggttacctgg 960 aacggcgcct gggacaacgg ggtgaccacc agcaactggg cgcagtacga acgcacccgc 1020 aactcgcgca aaggcgaagg cctggccggc ggcaccgagg ggatctttaa cagcaaccag 1080 ttcacggata tcgatctggc ggatgtgatg ctgcacagcg aagtcagcat tcccttcgac 1140 tatctggtta atcagaacct gacgctgggc agcgagtgga accagcagcg gatgaaggat 1200 aacgcctcca atacccaggc gctgtcgggc ggcagcatcc cgggctacga cagcaccggc 1260 cgcagcccgt actcgcaggc ggaaatcttc tcgctgttcg ctgagaacaa catggagctg 1320 accgacacca ccatgctgac cccggcgctg cgtttcgatc atcacagcat cgtgggcaat 1380 aactggagcc cgtccctcaa cctgtcgcag ggcctgtggg atgacttcac gctgaagatg 1440 ggcatcgccc gcgcctataa agcgccgagc ctgtatcaga ccaacccgaa ctacattctc 1500 tacagtaaag gccagggctg ctatgccagt aaagacggct gctatctgca gggtaacgac 1560 gacttaaaag ccgagaccag catcaacaaa gagattggcc tcgagtttaa acgcgacggc 1620 tggctggctg gcgtcacctg gttccgcaac gactaccgca acaagattga agcgggctat 1680 gccccggtct atcaaaacaa taaaggtacc gatctctacc agtgggaaaa cgtgccgaaa 1740 gcggtggtgg aaggtctgga ggggacgttg aacgttccgg tgagcgagac cgtcaactgg 1800 accaacaaca tcacctatat gctgcagagt aagaacaaaa agaccggcga tcgtctgtcg 1860 attatcccgg aatacacgct gaactccacc ctgagctggc aggttcgcga tgacgtttcg 1920 ctgcagtcga ccttcacctg gtacggcaag caggagccga agaagtacaa ctacaagggt 1980 caaccggtca ccggcagcga gaagaacgag gttagcccct acagcatcct cggcctgagc 2040 gcgacctggg acgtcaccaa atacgtcagt ctgaccggcg gcgtggataa cgtcttcgat 2100 aagcgccact ggcgcgcggg caacgcccag accaccgggg gcgccaccgg cacgatgtac 2160 ggcgccggcg ccgagaccta caatgaatcg ggccgcacct ggtacctgag cgtcaacacc 2220 cacttctga 2229 <210> 3 <211> 831 <212> DNA <213> Homo Sapiens <220> <221> gene <222> (1)..(831) <223> ABC transporter <400> 3 atgtgtctga gcgctgtatt aacggtcgct ttttcttatc acgccgtcgc ggccgatctt 60 ccggaaatag aaaaatccgg caccttgaaa gtggcgacgg aagatgatta tgcgccgttt 120 aactttatga ataatggcca ggctgatggc tttaacaaag atatgcttga ggaattacgt 180 aagtacgcaa aattccatgt cgaccagagc atattaccgt ggaccggatt attagcggct 240 gtctccaccg ggcaatacga tatggcctta accggggcgg ttattaccga tgagcggctg 300 aaggtctttg atttcacccc accgtgggcc tccgcgcagc actatttcgt caaacgcgct 360 ggcgatacct cgctaaatac cattgccgat ctcagcggca aaaaagtggg ggtacaggcc 420 gggagcgcgc tgctggcgcg tttgccggag ctgaaggcga tgctggagaa gaccggcggc 480 aagctggggc cggtggtgga gtatccctcc tatccggaag cctacgccga cctggcgaac 540 aagcgactgg attacgtgat taacgtggtg atctcggtaa acgacctggc gaaagccaaa 600 ccgaaggtct tcgccaaagg gctggccgtc tccggaccgg ggtatatggc gtggccgatc 660 ccgaaaaact cgccgcagct gctggcctat atgaccaggt ttatgaacca catgaaggag 720 accggcaagc tcgccgaact gcagaaaaaa tggttcggcg aaacctatga caacctgccg 780 accgaggcga tcaccagccc ggaacagttt cacaaactcg ccggactgta a 831 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for OmpA <400> 4 atgattgcag tggcactggc t 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for OmpA <400> 5 aaagccgccg gctgagttac 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FepA <400> 6 atgacggtcg ctttttctta tcac 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FepA <400> 7 aacagtccgg cgagtttgtg aaa 23 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for ABC transporter <400> 8 agatctttcc ctggcctt 18 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for ABC transporter <400> 9 agatctttca gaagtgggtg ttga 24 <110> Ewha University - Industry Collaboration Foundation <120> Vaccine for preventing Klebsiella related disease <130> MP15-084 <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 1071 <212> DNA <213> Homo Sapiens <220> <221> gene <222> (1). (1071) <223> OmpA <400> 1 cgtagcgcag gccgctccga aagataacac ctggtatgca ggtggtaaac tgggttggtc ccagtatcac 120 gacaccggtt tctacggtaa cggtttccag aacaacaacg gtccgacccg taacgatcag 180 cttggtgctg gtgcgttcgg tggttaccag gttaacccgt acctcggttt cgaaatgggt 240 tatgactggc tgggccgtat ggcatataaa ggcagcgttg acaacggtgc tttcaaagct 300 cagggcgttc agctgaccgc taaactgggt tacccgatca ctgacgatct ggacatctac 360 acccgtctgg gcggcatggt ttggcgcgct gactccaaag gcaactacgc ttctaccggc 420 gtttcccgta gcgaacacga cactggcgtt tccccagtat ttgctggcgg cgtagagtgg 480 gctgttactc gtgacatcgc tacccgtctg gaataccagt gggttaacaa catcggcgac 540 gcgggcactg tgggtacccg tcctgataac ggcatgctga gcctgggcgt ttcctaccgc 600 ttcggtcagg aagatgctgc accggttgtt gctccggctc cggctccggc tccggaagtg 660 gctaccaagc acttcaccct gaagtctgac gttctgttca acttcaacaa agctaccctg 720 aaaccggaag gtcagcaggc tctggatcag ctgtacactc agctgagcaa catggatccg 780 aaagacggtt ccgctgttgt tctgggctac accgaccgca tcggttccga agcttacaac 840 cagcagctgt ctgagaaacg tgctcagtcc gttgttgact acctggttgc taaaggcatc 900 ccggctggca aaatctccgc tcgcggcatg ggtgaatcca acccggttac tggcaacacc 960 tgcgacaacg tgaaagctcg cgctgccctg atcgattgcc tggctccgga tcgtcgtgta 1020 gagatcgaag ttaaaggcta caaagaagtt gtaactcagc cggcggctta a 1071 <210> 2 <211> 2229 <212> DNA <213> Homo Sapiens <220> <221> gene ≪ 222 > (1) .. (2229) <223> FepA <400> 2 atgaataaca ggatcaaatc cctggccttg ctggtcaatc tgggaattta cggggttgct 60 tttccgttaa gcgcagcgga aaccgccacc gacgataaaa acagcgccgc tgaagagacc 120 atggtggtca ccgccgccga gcagaacctg caggcgccgg gcgtctccac catcaccgcc 180 gatgagatcc gcaaacgccc cccggcgcgc gacgtctcgg agatcattcg caccatgccg 240 ggggtcaacc tgaccggcaa ctccaccagc ggccagcgcg gcaacaaccg ccagattgat 300 atccgcggca tgggcccgga aaataccctg atcctgatcg acggcaagcc ggtcaccagc 360 cgcaactccg tgcgccttgg ctggcgcggc gagcgcgaca cccgcggcga taccagctgg 420 gtgccgccgg agatgatcga acgtatcgaa gtgattcgcg gcccggccgc cgcccgctac 480 ggcaacggcg ccgccggcgg cgtggtgaat atcatcacca aaaaaaccgg cgatgagtgg 540 cgggctcat ggaacaccta tatgaacgcc ccggagcaca aggatgaagg ctccaccaaa 600 cgcactaact tcagcctcag cggcccgctg ggcggcgatt ttagcttccg cctgttcggt 660 aacctcgaca aaacgcaggc tgacgcctgg gatatcaacc agggccatca gtccgagcgt 720 accgggatct atgccgatac tctgccggcc gggcgcgaag gggtgaaaaa caaaaacatc 780 gatggtctgg tgcgctggga attcgctccg atgcagtcgc tggagtttga ggccggctac 840 agccgccagg gcaacctcta cgccggcgat acccagaaca ccaactccaa cgacctggta 900 aaagagaact acggcaaaga gaccaaccgt ctgtatcgca acacctactc ggttacctgg 960 aacggcgcct gggacaacgg ggtgaccacc agcaactggg cgcagtacga acgcacccgc 1020 aactcgcgca aaggcgaagg cctggccggc ggcaccgagg ggatctttaa cagcaaccag 1080 ttcacggata tcgatctggc ggatgtgatg ctgcacagcg aagtcagcat tcccttcgac 1140 tatctggtta atcagaacct gacgctgggc agcgagtgga accagcagcg gatgaaggat 1200 aacgcctcca atacccaggc gctgtcgggc ggcagcatcc cgggctacga cagcaccggc 1260 cgcagcccgt actcgcaggc ggaaatcttc tcgctgttcg ctgagaacaa catggagctg 1320 accgacacca ccatgctgac cccggcgctg cgtttcgatc atcacagcat cgtgggcaat 1380 aactggagcc cgtccctcaa cctgtcgcag ggcctgtggg atgacttcac gctgaagatg 1440 ggcatcgccc gcgcctataa agcgccgagc ctgtatcaga ccaacccgaa ctacattctc 1500 tacagtaaag gccagggctg ctatgccagt aaagacggct gctatctgca gggtaacgac 1560 gacttaaaag ccgagaccag catcaacaaa gagattggcc tcgagtttaa acgcgacggc 1620 tggctggctg gcgtcacctg gttccgcaac gactaccgca acaagattga agcgggctat 1680 gccccggtct atcaaaacaa taaaggtacc gatctctacc agtgggaaaa cgtgccgaaa 1740 gcggtggtgg aaggtctgga ggggacgttg aacgttccgg tgagcgagac cgtcaactgg 1800 accaacaaca tcacctatat gctgcagagt aagaacaaaa agaccggcga tcgtctgtcg 1860 attatcccgg aatacacgct gaactccacc ctgagctggc aggttcgcga tgacgtttcg 1920 ctgcagtcga ccttcacctg gtacggcaag caggagccga agaagtacaa ctacaagggt 1980 caaccggtca ccggcagcga gaagaacgag gttagcccct acagcatcct cggcctgagc 2040 gcgacctggg acgtcaccaa atacgtcagt ctgaccggcg gcgtggataa cgtcttcgat 2100 aagcgccact ggcgcgcggg caacgcccag accaccgggg gcgccaccgg cacgatgtac 2160 ggcgccggcg ccgagaccta caatgaatcg ggccgcacct ggtacctgag cgtcaacacc 2220 cacttctga 2229 <210> 3 <211> 831 <212> DNA <213> Homo Sapiens <220> <221> gene ≪ 222 > (1) .. (831) <223> ABC transporter <400> 3 atgtgtctga gcgctgtatt aacggtcgct ttttcttatc acgccgtcgc ggccgatctt 60 ccggaaatag aaaaatccgg caccttgaaa gtggcgacgg aagatgatta tgcgccgttt 120 aactttatga ataatggcca ggctgatggc tttaacaaag atatgcttga ggaattacgt 180 aagtacgcaa aattccatgt cgaccagagc atattaccgt ggaccggatt attagcggct 240 gtctccaccg ggcaatacga tatggcctta accggggcgg ttattaccga tgagcggctg 300 aaggtctttg atttcacccc accgtgggcc tccgcgcagc actatttcgt caaacgcgct 360 ggcgatacct cgctaaatac cattgccgat ctcagcggca aaaaagtggg ggtacaggcc 420 gggagcgcgc tgctggcgcg tttgccggag ctgaaggcga tgctggagaa gaccggcggc 480 aagctggggc cggtggtgga gtatccctcc tatccggaag cctacgccga cctggcgaac 540 aagcgactgg attacgtgat taacgtggtg atctcggtaa acgacctggc gaaagccaaa 600 ccgaaggtct tcgccaaagg gctggccgtc tccggaccgg ggtatatggc gtggccgatc 660 ccgaaaaact cgccgcagct gctggcctat atgaccaggt ttatgaacca catgaaggag 720 accggcaagc tcgccgaact gcagaaaaaa tggttcggcg aaacctatga caacctgccg 780 accgaggcga tcaccagccc ggaacagttt cacaaactcg ccggactgta a 831 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for OmpA <400> 4 atgattgcag tggcactggc t 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for OmpA <400> 5 aaagccgccg gctgagttac 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for FepA <400> 6 atgacggtcg ctttttctta tcac 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for FepA <400> 7 aacagtccgg cgagtttgtg aaa 23 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for ABC transporter <400> 8 agatctttcc ctggcctt 18 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for ABC transporter <400> 9 agatctttca gaagtgggtg ttga 24
Claims (10)
(a) 클렙시엘라균 유래의 OmpA(Outer membrane protein A) 유전자를 숙주균에 과발현시키는 단계;
(b) 상기 과발현된 숙주균에서 외막과 펩티도글리칸층을 제거하여 원형질체를 얻는 단계; 및
(c) 상기 원형질체를 분리하는 단계.A method for the preparation of a vaccine for the prevention or treatment of Klebsiella spp infectious disease, comprising the steps of:
(a) Overexpressing OmpA (Outer membrane protein A) gene derived from Klebsiella sp.
(b) removing the outer membrane and the peptidoglycan layer from the overexpressed host bacteria to obtain a protoplast; And
(c) separating the protoplast.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150103471A KR20170011221A (en) | 2015-07-22 | 2015-07-22 | Vaccine for preventing Klebsiella related disease |
| PCT/KR2016/007496 WO2017014468A1 (en) | 2015-07-22 | 2016-07-11 | Vaccine for preventing klebsiella infections |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150103471A KR20170011221A (en) | 2015-07-22 | 2015-07-22 | Vaccine for preventing Klebsiella related disease |
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| Publication Number | Publication Date |
|---|---|
| KR20170011221A true KR20170011221A (en) | 2017-02-02 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020150103471A KR20170011221A (en) | 2015-07-22 | 2015-07-22 | Vaccine for preventing Klebsiella related disease |
Country Status (2)
| Country | Link |
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| KR (1) | KR20170011221A (en) |
| WO (1) | WO2017014468A1 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2785542B1 (en) * | 1998-11-06 | 2001-02-09 | Pf Medicament | USE OF AN ENTEROMACTERY OmpA PROTEIN FOR THE SPECIFIC TARGETING OF A BIOLOGICALLY ACTIVE SUBSTANCE THAT IS ASSOCIATED WITH CELLS PRESENTING ANTIGENS SUCH AS HUMAN DENDRITIC CELLS |
| CN103002879B (en) * | 2010-07-01 | 2016-04-20 | 阿昂梅迪克斯公司 | From microvesicle and the application thereof of cell protoplast |
-
2015
- 2015-07-22 KR KR1020150103471A patent/KR20170011221A/en not_active Application Discontinuation
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2016
- 2016-07-11 WO PCT/KR2016/007496 patent/WO2017014468A1/en active Application Filing
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| WO2017014468A1 (en) | 2017-01-26 |
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