KR20100011581A - Recombinant gram-negative bacteria producing outer membrane vesicles and method for preparing outer membrane vesicles tagged with foreign epitopes using the same - Google Patents
Recombinant gram-negative bacteria producing outer membrane vesicles and method for preparing outer membrane vesicles tagged with foreign epitopes using the same Download PDFInfo
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- KR20100011581A KR20100011581A KR20080072858A KR20080072858A KR20100011581A KR 20100011581 A KR20100011581 A KR 20100011581A KR 20080072858 A KR20080072858 A KR 20080072858A KR 20080072858 A KR20080072858 A KR 20080072858A KR 20100011581 A KR20100011581 A KR 20100011581A
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- outer membrane
- recombinant
- bacteria
- lipid
- omv
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Abstract
Description
본 발명은 외막소체를 생산하는 재조합 G(-) 박테리아 및 이를 이용한 다가항원이 실린 외막소체의 제조방법에 관한 것으로서, 더욱 상세하게는 내독성이 제거되어 안전하면서도 다가항원(multi-valent epitope) 전달 및 면역효과 증강을 동시에 유도할 수 있는 외막소체를 생산하는 재조합 G(-) 박테리아 및 이를 이용한 다가항원이 실린 외막소체의 제조방법에 관한 것이다.The present invention relates to a method for producing a recombinant G (-) bacteria producing the outer membrane body and the outer membrane body containing the multivalent antigen using the same, more specifically, the endotoxin is removed safe and multi-valent epitope delivery (multi-valent epitope) And it relates to a recombinant G (-) bacteria to produce an outer membrane body that can induce enhanced immune effect at the same time and to a method for producing an outer membrane body containing a multivalent antigen using the same.
현재까지의 고전적인 감염병 예방백신은 감염 원인균 균체를 불활성화 시킨 사균백신 (killed vaccine)과 균체를 사멸시키지 않고 약독화 (virulence attenuation) 시킨 생균백신 (live-attenuated vaccine)으로 크게 구분되는데, 이러한 구분은 병원체의 숙주내 감염 및 증식패턴에 따라 이를 방어하기 위한 면역반 응의 차별적 유도에 따른 것이다. 그러므로 세포내 병원체 (intracellular pathogen)에 의한 감염병의 예방백신은 효과적인 세포성면역 (cell-mediated immunity)의 유도를 촉진하는 생균백신이나 DNA 백신으로 개발하는 것이고, 숙주세포 외부에서 증식하는 병원체를 방어하기 위하여서는 체액에 분비되는 항체를 통한 체액성면역(humoral immunity) 유도가 필수적이서, 사멸시킨 균체를 포함하는 사균백신 또는 균체에서 주된 세포표면 항원성분만을 추출하여 제조한 서브유닛 (subunit) 백신으로 개발하는 것이 보편화 되어있다. The classic vaccines for infectious diseases to date are largely divided into killed vaccines that inactivate the infecting organisms and live-attenuated vaccines that have been attenuated without killing the cells. Is due to the differential induction of immune responses to defend against pathogen infection and proliferation patterns in the host. Therefore, the prophylactic vaccine of infectious diseases caused by intracellular pathogens is to develop into live vaccines or DNA vaccines that promote the induction of effective cell-mediated immunity, and to protect against pathogens proliferating outside the host cell. In order to induce humoral immunity through antibodies secreted into body fluids, it is essential to develop a subunit vaccine prepared by extracting only the major cell surface antigen components from dead germ vaccines or cells containing killed cells. It is universal.
그러나 이러한 백신 개발에 있어서, 효능과 안전성에 대한 문제점들이 항상 걸림돌로 작용되고 있다. 대표적인 문제점으로는 생균백신 개발에 있어서 항상 대두되는 안전성 문제이다. 생균백신 후보물질은 비록 약독화 되었지만, 여전히 살아있는 병원체이기 때문에 생체내 접종시 백신주로 사용된 생균이 증식 및 활성화되어 자연감염과 유사한 증상을 나타내는 위험성이 있기 때문에, 비록 세포성면역을 자극하는 생균백신의 효능이 크다 할지라도 안전성 문제 때문에 개발 및 시판허가를 받기가 어려운 실정에 있다. 따라서 생균 백신주에 의한 부작용을 배제하면서도 생균백신과 비슷한 세포성면역 유도효과를 나타내는 생체내 비증식성 (non-replicating, acellular) 백신물질 개발의 필요성이 크게 대두되고 있다. 또한, 사균백신이나 서브유닛 백신도 생체내 비증식성 백신물질이지만, 이들은 체액성 면역유도에 효과적이어서 세포성 면역유도가 필요한 질병에서는 고려되지 않는다. 사균백신의 문제점은 또한 사균체 전부를 백신물질로 사용하므로 감염방어에 효과적인 세포표면 항원성분 외에도 많은 종류의 다양한 세포내 단백질들이 포함되기 때문에 자가면역 반응 유도와 같은 부작용을 초래할 수 있다. 서브유닛 백신은 보통 한 종류의 항원 성분만을 내포하며 면역원성이 약하고 세포성 면역 반응의 유도가 어려워 이를 극복하기 위해 고가의 adjuvant를 함께 혼합하여야 함으로 비용측면에서도 불리하다.However, in the development of such a vaccine, the problems of efficacy and safety have always been an obstacle. A representative problem is the safety problem that always comes up in the development of live vaccines. Although live vaccine candidates are attenuated, they are still living pathogens, and because live bacteria used as vaccine strains in vivo inoculate, there is a risk of proliferating and activating symptoms similar to those of natural infection. Although its efficacy is large, it is difficult to obtain development and marketing authorization due to safety issues. Therefore, there is a great need to develop a non-replicating (acellular) vaccine material in vivo, which excludes side effects caused by viable vaccine strains and exhibits cellular immune induction effects similar to viable vaccines. In addition, although the bacterium vaccine or subunit vaccine is a non-proliferative vaccine substance in vivo, they are effective for humoral induction and are not considered in diseases requiring cellular induction. The problem of the vaccinated vaccine also uses all of the bacterium as a vaccine material, and thus, various kinds of intracellular proteins are included in addition to the cell surface antigen components effective for defense against infection. Subunit vaccines usually contain only one type of antigenic component, have low immunogenicity, and are difficult to induce cellular immune responses, and thus, cost-effective adjuvants must be mixed together to overcome them.
이러한 생체내 비증식성 백신들의 단점들을 보완하는 차세대 비세포성 백신 후보물질중의 하나가 외막소체 (OMV)이다. 비록 외막소체 (OMV)가 차세대 백신물질로서 주목받을 수 있는 바이오 나노 입자이지만, 세계적으로 외막소체 (OMV)를 이용한 백신은 아직까지 상용화 되지 못하고, 특정 세균에서 생산된 것만이 임상단계 시험 (효능 및 안전성 검사)을 거치고 있다. 그 이유는 외막소체 (OMV)를 생산하여 백신으로 개발하는 데에 있어 극복해야 할 몇 가지 문제점들이 아직 남아 있기 때문이며, 해결해 나가야 할 문제점 (bottleneck)은 크게 네 가지로 설명될 수 있다. 그 중 하나는 외막소체 (OMV)에 내포된 리포폴리사카라이드 (LPS) 성분 때문에 생기는 내독성 (endotoxicity)을 제거하는 것이고, 그 다음은 세균의 외막으로부터 소량으로 분비되는 OMV의 분비량을 충분히 증가시키도록 하는 것이다. 또한 다가항원성 (multi-valent antigenicity)을 나타내기 위한 선택적인 외부항원을 도입또는 자체 발현하는 재조합 외막소체 (OMV)를 만들 수 있다면 하나의 외막소체(OMV)에 여러 이종 항원들을 내포하게 되므로 혼합백신 형태의 효능을 갖게 된다. 또한 내독성이 제거된 외막소체를 분리하여 이종항원들이나 adjuvants를 외막소체 내강에 삽입하거나 표면에 결합시켜 새로이 재구성한다면, 하나의 외막소체 (OMV)에 다양한 외부항원을 실어 면역체계에 전달할 수 있을 뿐만 아니라 예방하고자 하는 병원 체의 증식패턴 (intracellular 혹은 extracellular)에 따라 효과적인 (세포성 또는 체액성) 면역유도를 할 수 있게 된다. 예를들면 세포성면역 유도를 위한 Th1 adjuvant를 외막소체 (OMV)에 포함시키는 기술을 실현하여 외막소체를 재구성 할 수 있으므로, 이렇게 재구성된 외막소체 (OMV) 백신물질은 차세대 백신으로서의 필요충분조건들을 모두 갖춘 이상적인 백신물질이 될 것이다. One of the next generation of non-cellular vaccine candidates that complements the shortcomings of these in vivo non-proliferative vaccines is the outer membrane body (OMV). Although the outer membrane body (OMV) is a bio-nanoparticle that can be spotlighted as a next-generation vaccine material, vaccines using the outer membrane body (OMV) have not been commercialized yet, and only those produced by specific bacteria have been used in clinical trials. Safety check). The reason for this is that there are still some problems to overcome in producing OMV and developing them as vaccines, and the bottleneck to be solved can be explained in four ways. One of them is to eliminate endotoxicity due to the lipopolysaccharide (LPS) component contained in the outer membrane body (OMV), followed by a sufficient increase in the secretion of small amounts of OMV secreted from the bacterial outer membrane. It is to be. In addition, if one can create a recombinant outer membrane OMV that introduces or self-expresses an optional external antigen to show multi-valent antigenicity, it will contain several heterologous antigens in one outer membrane body (OMV). Efficacy in the form of a vaccine. In addition, by removing the endotoxin from which the endotoxin has been removed, heterologous antigens or adjuvants can be inserted into the lumen of the outer membrane or combined with the surface and newly reconstituted. In addition, effective (cellular or humoral) immunity can be induced depending on the growth pattern (intracellular or extracellular) of the pathogen to be prevented. For example, by implementing the technology of incorporating Th1 adjuvant into the outer membrane body (OMV) for cellular immunity, the outer membrane body can be reconstructed. It would be the ideal vaccine for all.
지금까지의 외막소체 (OMV)의 생산과 이용에 대한 세계적 동향을 간략히 분석하면, 자연생성 OMV 그 자체 또는 여러 종류의 자연생성 OMV를 분리하여 이들을 합체하거나, 또는 인공합성으로 제작된 리포좀 (liposome)과의 합체로 hybrid (liposome + MV)를 생산하는 방법과 이용에 대한 국제특허가 등록되어 있다. 또한 뇌수막염균 (Neisseria meningitidis)으로부터 자연 생성된 OMV를 획득하여, 이를 백신으로 개발하여 시판하기 위한 임상단계 효능시험이 진행되어지고 있으나, 아직까지 뇌수막염균 이외의 그람음성 균에서 상기한 네 가지의 문제점들을 극복하는 발전된 기술을 조합하여 제조한 안전하고 효과적인 OMV 백신은 개발되지 않고 있다. A brief analysis of the global trends in the production and use of outer membrane bodies (OMV) to date is based on the separation of the naturally occurring OMV itself or several kinds of naturally occurring OMVs and coalescing them, or artificially produced liposomes. International patents on the method and use of the hybrid (liposome + MV) production in combination with In addition, the clinical trial efficacy test to obtain a naturally produced OMV from the meningitis ( Nisseria meningitidis ), develop it as a vaccine and market it, but there are still four problems described above in Gram-negative bacteria other than meningitis Safe and effective OMV vaccines made by combining advanced techniques to overcome them have not been developed.
한편, 한국공개특허 제2002-7001441호 및 미국공개특허 제20060216307호에서는, 뇌수막염 원인균 (N. meningitidis)의 msbB (lpxM ) 또는 htrB (lpxL) 등의 유전자를 결실시킨 변이주를 개발하여, lipid A의 구조 변화를 통해 내독성을 낮춘 LPS를 보유하는 외막소체 (OMV) 백신을 개시한 바 있다. 그러나, 이러한 OMV 백신개발은 자연발생적으로 상당한 량의 OMV를 생성하여 분비하는 뇌수막염 균에 한정되어지고 있다. On the other hand, in Korean Patent Publication No. 2002-7001441 and US Patent Publication No. 20060216307, msbB of meningitis causative bacteria ( N. meningitidis ) Mutant strains that have deleted genes such as ( lpxM ) or htrB ( lpxL ) have been developed to disclose an outer membrane vesicle (OMV) vaccine having LPS that has reduced endotoxin through structural changes in lipid A. However, the development of such OMV vaccine is limited to meningitis bacteria that naturally produce and secrete a significant amount of OMV.
그리고, 대장균이나 살모넬라균과 같이 보통의 장관 감염성 그람음성 세균들은 뇌수막염 균과는 달리 자연생성 OMV의 분비량이 매우 적어 백신으로 사용하기 위해서는 OMV의 생성을 충분히 증가시키도록 할 필요가 있다. 또한, 대장균이나 살모넬라균에서는 상기 선행문헌에 개시된 LPS의 lipid A 구조를 변화시키는 msbB 또는 lpxL 유전자 변이체 만으로는 내독성이 충분히 낮은 penta-acyl lipid A 종류를 가진 OMV를 생산 할 수 없는 문제점이 있다. In addition, unlike intestinal infectious Gram-negative bacteria such as Escherichia coli and Salmonella, the secretion of naturally-occurring OMV is very small, unlike meningitis, so it is necessary to sufficiently increase the production of OMV for use as a vaccine. In addition, in Escherichia coli and Salmonella, there is a problem in that msbB or lpxL gene variants that change the lipid A structure of LPS disclosed in the above-described literature cannot produce OMV having a sufficiently low penta-acyl lipid A type.
이에, 본 발명자들은 상기 문제점들을 해결하기 위하여 예의 노력한 결과, 대장균 등의 G(-) 박테리아에서 msbB 유전자와 함께 pagP 유전자를 결실시키면, 외막소체(OMV)에 내포된 리포폴리사카라이드의 내독성(LPS endotoxicity)이 제거된 penta-acylated lipid A 만을 생성할 수 있고, 추가로 degP 유전자를 결실시키면 외막소체(OMV) 생산능을 높일수 있음을 확인하였고, 또한, G(-) 박테리아의 외막단백질에 특정 외부항원을 융합 및 발현시키거나, G(-) 박테리아에서 분리된 외막소체(OMV) 내부공간에 외부항원으로 작용하는 합성 펩티드(synthetic peptide) 또는 상기 합성 펩티드를 코딩하는 DNA 단편을 삽입하면 다가항원을 지닌 외막소체(OMV)를 제조할 수 있음을 확인하고 본 발명을 완성하게 되었다. Accordingly, the present inventors have made diligent efforts to solve the above problems, pagP together with the msbB gene in G (-) bacteria such as E. coli. Deletion of the gene can produce only penta-acylated lipid A from which the lipopolysaccharide contained in the outer membrane vesicle (OMV) has been removed, and in addition, the degP gene can be deleted. It has been confirmed that it can increase the production capacity, and also fusion and expression of a specific external antigen in the outer membrane protein of G (-) bacteria, or acts as an external antigen in the inner space of the outer membrane body (OMV) isolated from the G (-) bacteria When inserting the synthetic peptide (synthetic peptide) or a DNA fragment encoding the synthetic peptide it was confirmed that the outer membrane body (OMV) having a polyvalent antigen can be prepared and completed the present invention.
본 발명의 목적은 안전하면서도 다가항원(multi-valent epitope) 전달 및 면 역효과 증강을 동시에 유도할 수 있는 외막소체를 분비하는 재조합 G(-) 박테리아 및 그 제조방법을 제공하는데 있다.It is an object of the present invention to provide a recombinant G (-) bacteria and a method for preparing the same, which secrete outer membrane bodies capable of inducing safe and multi-valent epitope delivery and enhancing immune effects.
본 발명의 다른 목적은 안전하면서도 다가항원 (multi-valent epitope) 전달 및 면역효과 증강을 동시에 유도할 수 있는 외막소체 제조방법을 제공하는데 있다.Another object of the present invention is to provide a method for preparing outer membrane body that can induce safe and multi-valent epitope delivery and enhanced immune effect at the same time.
본 발명의 또 다른 목적은 상기한 기술의 조합으로 만들어진 백신후보 물질로서의 OMV의 표면에 형광물질 (fluorophore)을 표지 (labeling)하여, 이러한 OMV가 항원운반체로서 생체내에서 흡수, 이동, 및 분해되는 과정을 in vivo imaging 장비로 모니터링 할 수 있도록 하는 외막소체 재구성 방법을 제공하는데 있다.Another object of the present invention is to label fluorophore on the surface of OMV as a vaccine candidate made by the combination of the above techniques, so that such OMV is absorbed, transported and degraded in vivo as an antigen carrier. To provide a method for reconstructing the outer membrane body to monitor the process by in vivo imaging equipment.
상기 목적을 달성하기 위하여, 본 발명은 (a) 외막소체(OMV) 생성능을 가지는 G(-) 박테리아에서 상기 외막소체가 내포하는 리포폴리사카라이드(LPS)의 리피드(lipid) A 생합성에 관여하는 유전자를 결실시킨 G(-) 박테리아 변이주를 제작하는 단계; (b) 상기 G(-) 박테리아의 외막단백질과 외부항원이 융합·발현하도록 구성된 유전자 재조합체를 제작하는 단계; 및 (c) 상기 (b) 단계에서 제작된 유전자 재조합체를 상기 (a) 단계에서 제작된 G(-) 박테리아 변이주에 도입하는 단계를 포함하는 외막단백질과 외부항원을 융합·발현할 수 있는 재조합 G(-) 박테리아의 제조방법을 제공한다. In order to achieve the above object, the present invention (a) is involved in lipid A biosynthesis of lipopolysaccharide (LPS) contained in the envelope membrane in G (-) bacteria having the ability to produce envelope membrane (OMV) Preparing a G (-) bacterial mutant strain which has deleted the gene; (b) preparing a genetic recombinant configured to fuse and express the outer membrane protein of the G (-) bacteria and the external antigen; And (c) introducing the genetic recombinant produced in step (b) into the G (-) bacterial mutant prepared in step (a). Provided are methods for preparing G (-) bacteria.
본 발명은 또한, 상기 방법에 의해 제조되고, 리포폴리사카라이드(LPS)의 리피드(lipid) A 생합성에 관여하는 유전자가 결실되어있고, G(-) 박테리아의 외막단 백질과 외부항원이 융합·발현하도록 구성된 유전자 재조합체가 도입된 것을 특징으로 하는 재조합 G(-) 박테리아를 제공한다.The present invention is also prepared by the above method, the gene involved in lipid A biosynthesis of lipopolysaccharide (LPS) is deleted, and the outer membrane protein and external antigen of G (-) bacteria are fused and Provided are recombinant G (-) bacteria, characterized by the introduction of a genetic recombinant configured to express.
본 발명은 또한, 상기 재조합 G(-) 박테리아를 배양하여 외부항원을 외막단백질과 융합된 형태로 발현시키는 것을 특징으로 하는 외부항원이 결합된 재조합 외막소체(OMV)의 제조방법을 제공한다.The present invention also provides a method for producing an outer antigen-coupled recombinant envelope membrane (OMV), characterized in that the recombinant G (-) bacteria are cultured to express the external antigen in a fused form with the outer membrane protein.
본 발명은 또한, (a) 외막소체(OMV) 생성능을 가지는 G(-) 박테리아에서 상기 외막소체가 내포하는 리포폴리사카라이드(LPS)의 리피드(lipid) A 생합성에 관여하는 유전자를 결실시킨 G(-) 박테리아 변이주를 제작하는 단계; (b) 상기 G(-) 박테리아 변이주로부터 분비되는 외막소체(OMV)를 회수하는 단계; 및 (c) 상기 (b) 단계에서 회수한 외막소체(OMV) 내부공간에 외부항원으로 작용하는 합성 펩티드(synthetic peptide) 또는 상기 합성 펩티드를 코딩하는 DNA 단편을 삽입하는 단계를 포함하는 것을 특징으로 하는 재조합 외막소체(OMV)의 제조방법을 제공한다.The present invention also relates to (a) G in which a gene involved in lipid A biosynthesis of lipopolysaccharide (LPS) contained in the envelope membrane in a G (-) bacterium having an ability to produce envelope membrane (OMV) is present. (-) Preparing a bacterial variant strain; (b) recovering the outer membrane body (OMV) secreted from the G (-) bacterial mutant strain; And (c) inserting a synthetic peptide acting as an external antigen or a DNA fragment encoding the synthetic peptide into an inner space of the outer membrane body (OMV) recovered in step (b). It provides a method for producing a recombinant outer membrane body (OMV).
본 발명은 또한, 상기 재조합 외막소체(OMV)의 제조방법에 의해 제조된 재조합 외막소체의 내부공간(lumen)에 면역증진 효과를 나타내는 애쥬반트(ajuvants)를 삽입하는 것을 특징으로 하는 재조합 외막소체(OMV)의 재구성 방법을 제공한다. The present invention also provides a recombinant outer membrane body, characterized in that the insertion of an adjuvant (ajuvants) exhibiting an immunopromoting effect into the lumen of the recombinant outer membrane body prepared by the method for producing the recombinant outer membrane body (OMV) ( OMV) reconstruction method.
본 발명은 또한, 상기 재조합 외막소체(OMV)의 제조방법에 의해 제조된 재조합 외막소체에 내포된 리포폴리사카라이드(LPS)의 KDO(3-deoxy-D-manno-oct-2-ulosonic acid) 성분에 (a) 단백질 또는 탄수화물로 구성된 항원성 물질 또는 (b) 형광물질(fluorescent dye)을 화학적 커플링시키는 것을 특징으로 하는 재조합 외막소체(OMV)의 재구성 방법을 제공한다.The present invention also relates to KDO (3-deoxy-D- manno- oct-2-ulosonic acid) of lipopolysaccharide (LPS) contained in a recombinant envelope membrane prepared by the method for producing the recombinant envelope membrane (OMV). Provided is a method for reconstructing a recombinant outer membrane body (OMV) characterized by chemically coupling (a) an antigenic substance consisting of a protein or carbohydrate or (b) a fluorescent dye.
본 발명에 따른 재조합 G(-) 박테리아에서 생성된 외막소체(OMV)는 LPS (lipopolysaccharide)의 내독성(endotoxicity)이 제거되어 안전하면서도, 다가항원(multi-valent epitope) 전달능 및 Toll-like receptors를 자극하는 자체적인 면역효과 증강물질을 보유하고 있어, 특정 adjuvant를 혼합하지 않고도 자체적인 면역증강 효과를 가질 수 있어 첨단 바이오 나노소재 백신물질로 유용하다.The outer membrane body (OMV) produced by the recombinant G (-) bacteria according to the present invention is safe by eliminating endotoxicity of the lipopolysaccharide (LPS), and is capable of delivering multi-valent epitope and toll-like receptors. It possesses its own immune effect enhancer that stimulates, and can be used as an advanced bio-nano vaccine material because it can have its own immune-enhancing effect without mixing specific adjuvant.
본 발명은 일 관점에서, (a) 외막소체(OMV) 생성능을 가지는 G(-) 박테리아에서 상기 외막소체가 내포하는 리포폴리사카라이드(LPS)의 리피드(lipid) A 생합성에 관여하는 유전자를 결실시킨 G(-) 박테리아 변이주를 제작하는 단계; (b) 상기 G(-) 박테리아의 외막단백질과 외부항원이 융합·발현하도록 구성된 유전자 재조합체를 제작하는 단계; 및 (c) 상기 (b) 단계에서 제작된 유전자 재조합체를 상기 (a) 단계에서 제작된 G(-) 박테리아 변이주에 도입하는 단계를 포함하는 외막단백질과 외부항원을 융합·발현할 수 있는 재조합 G(-) 박테리아의 제조방법에 관한 것이다.The present invention, in one aspect, (a) deletion of the gene involved in lipid A biosynthesis of lipopolysaccharide (LPS) contained in the envelope membrane in G (-) bacteria having the ability to produce envelope membrane (OMV) Preparing a G (-) bacterial mutant strain; (b) preparing a genetic recombinant configured to fuse and express the outer membrane protein of the G (-) bacteria and the external antigen; And (c) introducing the genetic recombinant produced in step (b) into the G (-) bacterial mutant prepared in step (a). It relates to a method for producing G (-) bacteria.
본 발명에 있어서, 외막소체 (OMV)는 G(-) 박테리아의 외막으로부터 자연생성되어, 소량 분비되는 나노 크기의 막소체 (membrane vesicle)이므로, 외막(outer membrane)에 존재하는 다양한 항원성분들이 자연적으로 내포되어 있다. 이러한 항 원 성분들 중에서 리포폴리사카라이드 (LPS)는 내독성(endotoxicity)을 가지므로, 내독성 부작용이 없는 안전한 백신을 개발하려면 LPS의 구조를 변화시켜서, 내독성을 충분히 낮추도록 해야 한다. 따라서, 본 발명에서는 리포폴리사카라이드 (LPS)의 리피드 (lipid) A 생합성에 관여하는 msbB 유전자를 우선 결실시키고, 이 msbB 변이체에서 PagP 외막효소의 활성화로 인하여 발생하는 palmitated hexa-acyl lipid A 종류의 형성을 차단하는 pagP 유전자를 불활화 (inactivation)하는 변이체를 제작하여 온전히 penta-acyl lipid A만을 생성하게 함으로써 LPS의 내독성을 충분히 낮추는 것이 바람직하다. 즉, 본 발명에 있어서, 상기 리포폴리사카라이드(LPS)의 리피드(lipid) A 생합성에 관여하는 유전자는 lpxL(htrB), lpxM(msbB) 및 pagP로 구성된 군에서 선택되는 하나 이상인 것을 특징으로 한다. In the present invention, the outer membrane body (OMV) is a nano-scale membrane vesicle that is naturally produced from the outer membrane of G (-) bacteria and secreted in a small amount, so that various antigen components present in the outer membrane are naturally It is implied. Among these antigenic components, lipopolysaccharide (LPS) has endotoxicity. Therefore, in order to develop a safe vaccine without endotoxin side effects, the structure of the LPS should be changed to sufficiently lower the endotoxin. Accordingly, in the present invention, the msbB gene involved in lipid A biosynthesis of lipopolysaccharide (LPS) is first deleted, and a palmitated hexa-acyl lipid A type of palmitated hexa-acyl lipid A generated by activation of the PagP envelope membrane enzyme in this msbB variant. It is preferable to sufficiently reduce the endogenous toxicity of LPS by preparing a variant that inactivates the pagP gene that blocks formation, thereby generating only penta-acyl lipid A. That is, in the present invention, the gene involved in lipid A biosynthesis of lipopolysaccharide (LPS) is characterized in that at least one selected from the group consisting of lpxL (htrB) , lpxM (msbB) and pagP . .
상기 PagP는 뇌수막염 균에는 존재하지 않으나, 대장균, 이질균 (Shigella spp.), 살모넬라 등의 세포외막(outer membrane)에 위치하는 효소 단백질로서, lipid A 분자에 팔미테이트(palmitate)를 첨가하는 lipid A palmitoylation 반응을 촉매하는 효소이다(Bishop RE., Mol. Microbiol., 57:900-912, 2005). 상기 PagP는 hexa-acyl lipid A를 가지는 정상균주에서는 효소활성이 매우 낮으나, 외막의 구조적 부실을 초래하는 (penta-acyl lipid A를 가진) msbB(lpxM) 결실 변이체가 만들어지면 그에 따른 외막의 취약성을 보상하기 위해 이 PagP가 특이적으로 활성화되어 penta-acyl lipid A (기질)에 myristate (MsbB에 의한 촉매) 대신 palmitate를 부가시켜 주는 활성이 증가된다. PagP is not present in meningitis bacteria, but is an enzyme protein located in the outer membrane of Escherichia coli, Shigella spp., Salmonella, etc., and lipid A palmitoylation which adds palmitate to lipid A molecules. Enzyme that catalyzes the reaction (Bishop RE., Mol. Microbiol ., 57: 900-912, 2005). The PagP has very low enzymatic activity in normal strains with hexa-acyl lipid A, but when the msbB ( lpxM ) deletion variant (with penta-acyl lipid A) is made, which results in structural loss of the outer membrane, the outer membrane is vulnerable. To compensate, the PagP is specifically activated to increase the activity of adding palmitate to penta-acyl lipid A (substrate) instead of myristate (catalyzed by MsbB).
만약 상기의 lipid A 합성에 관여하는 효소 유전자들을 불활화 하여도 LPS의 내독성이 충분히 제거되지 않는다면 lipid A에 대한 부가적인 구조를 변화시키는 (lipid A structural modification)데 관여하는 lpxXL, lpxF 및 lpxE로 구성된 군에서 선택되는 하나 이상의 유전자를 발현벡터에 클로닝하여, 이를 상기 재조합 G(-) 박테리아 변이주에 추가로 융합(도입)·발현시킬 수 있다.If inactivation of the enzyme genes involved in lipid A synthesis is not sufficiently eliminated the endotoxin of LPS, lpxXL , lpxF and lpxE are involved in the lipid A structural modification. One or more genes selected from the group consisting of these can be cloned into expression vectors, which can be further fused (introduced) and expressed in the recombinant G (-) bacterial strain.
즉, Brucella abortus 균이나 Helicobacter pylori 균으로부터 lpxE 유전자 (lipid A-1 phosphatase)를 발현벡터에 클로닝하여, 본 발명의 일 실시예에서 제작한 변이체(msbB -/pagP -)에 융합·발현시키면, penta-acyl lipid A로의 변환 뿐 만 아니라, lipid A 분자의 글루코사민 1번 탄소에 연결된 phosphate를 부가적으로 제거한 mono-phosphoryl lipid A (penta-acyl)를 창출 할 수 있으므로 내독성을 완전히 제거할 수 있다.That is, Brucella abortus and the spores and Helicobacter pylori gene (lipid A-1 phosphatase) lpxE from the bacteria cloned in the expression vector, the mutant produced in one embodiment of the present invention (msbB - / pagP -) when the fusion-expression to, penta In addition to the conversion to -acyl lipid A, mono-phosphoryl lipid A (penta-acyl) can be completely eliminated by eliminating the phosphate linked to the
상기 유전자중 msbB는 세균의 세포질에서 lipid A를 합성하는 마지막 단계에서 미리스테이트 (myristate) 지방산을 전구물질 (lipid IVA)에 첨가하는데 관여하는 효소이다. 따라서, 이 유전자를 불활성화 (inactivation) 시킨 변이주 (ΔmsbB mutant)에서 생성된 LPS는 wild type 균주의 LPS와 달리 내독성이 거의 소실된다고 보고 된 바 있다 (Raetz CRH & Whitfield C., Annu. Rev. Biochem., 71:635-700, 2002). Among these genes, msbB is an enzyme involved in adding myristate fatty acid to a precursor (lipid IV A ) in the final step of synthesizing lipid A in bacterial cytoplasm. Therefore, LPS generated from the mutant (Δ msbB mutant) that inactivated the gene has been reported to be almost tolerant to the endotoxin unlike the wild type strain LPS (Raetz CRH & Whitfield C., Annu. Rev Biochem , 71: 635-700, 2002).
LPS의 내독성은 lipid A라고 불리는 LPS의 한 부분의 특이적 분자구조에서 기인되며, 이 정상 lipid A의 특이적인 분자구조 중에서 인산화 (bis-phosphorylated) 및 순차적 아실레이션 (acylation) 반응으로 첨가되는 라우레이트 (laurate)와 미리스테이트 (myristate)로 구성된 여섯 개의 지방산 (hexa-acyl chain) 잔기의 존재가 내독성 (endotoxicity)을 결정하는 필수요소라는 사실이 이미 밝혀진 바 있다 (Alexander C. & Rietschel ET., J. Endotoxin. Res., 7:167-202, 2001). 그러므로 msbB 변이주에서 생성되는 lipid A 분자는 myristate 지방산이 부가되지 않아, 다섯 개의 지방산 (penta-acyl chain) 잔기를 가지는 lipid A 구조가 만들어 짐으로, 이 변형된 구조의 lipid A는 사람이나 동물의 선천면역 리셉터(innate immunity receptor)인 toll-like receptor 4 (TLR4)를 자극하는 어고니스트 (agonist)로 작용하지 못한다. 그러나, 병원성 대장균 O157:H7, 이질균 (Shigella spp.) 및 살모넬라 균 등에서는 MsbB 효소 불활성화 만으로는 뇌수막염균 변이체와 같이 penta-acyl lipid A 종류만 생성하지 않으므로, 이들 균종들은 PagP 효소를 추가적으로 불활성화 하는 변이체를 제작하여야 온전히 penta-acyl lipid A를 가진 LPS가 만들어 진다. 이러한 penta-acyl lipid A를 가진 LPS는 정상적인 hexa-acyl lipid A와 TLR4가 결합하여 일어나는 신호전달의 결과 (과도한 염증매개인자들 (pro-inflammatory cytokines)의 발현 자극) 보다 현저히 낮게 TLR4 리셉터를 자극하기 때문에, 염증매개인자들의 과도한 분비로 인한 내독성의 부작용을 제거할 수 있다. 흥미로운 점은 msbB 변이주에서 만들어진 penta-acyl lipid A는 pro-inflammatory cytokine의 과도한 발현을 자극하는 위험신호 (danger signal)로서의 신호전달 작용은 억제되지만, adjuvancity에 관여하는 dendritic cells의 B7 분자들의 발현을 촉진하는 co-stimulatory signal로서의 신호작용은 그대로 유지한다는 것이다. 그러므로 본 발명에서 개시한 병원성 대장균 O157:H7 변 이체에서 생성된 penta-acyl lipid A는 과도한 염증매개성인자들의 분비자극으로 인한 내독성 부작용을 크게 줄이면서도 면역증강효과는 그대로 유지하는 OMV의 주요 구성성분이 된다. Toxicity of LPS is due to the specific molecular structure of a part of LPS called lipid A, which is added to bis- phosphorylated and sequential acylation reactions among the specific molecular structures of normal lipid A. It has already been shown that the presence of six hexa-acyl chain residues consisting of laurate and myristate is an essential factor in determining endotoxicity (Alexander C. & Rietschel ET. , J. Endotoxin.Res. , 7: 167-202, 2001). Therefore, lipid A molecules produced in the msbB mutant are not added to myristate fatty acids, resulting in a lipid A structure having five fatty acid (penta-acyl chain) residues. It does not act as an agonist that stimulates toll-like receptor 4 (TLR4), an innate immunity receptor. However, pathogenic Escherichia coli O157: H7, Shigella spp., And Salmonella do not produce penta-acyl lipid A alone, such as meningitis, by inactivating MsbB enzyme alone. Variant should be prepared to make LPS with penta-acyl lipid A. LPS with such penta-acyl lipid A stimulates the TLR4 receptor significantly lower than the result of signaling associated with normal hexa-acyl lipid A and TLR4 (stimulating the expression of pro-inflammatory cytokines). Therefore, the side effects of the endotoxin caused by excessive secretion of inflammatory mediators can be eliminated. Interestingly, penta-acyl lipid A produced in the msbB mutant inhibits signaling as a danger signal that stimulates overexpression of pro-inflammatory cytokine, but promotes the expression of B7 molecules in dendritic cells involved in adjuvancity. Signaling as a co-stimulatory signal is maintained as it is. Therefore, the penta-acyl lipid A produced in the Escherichia coli O157: H7 variant disclosed in the present invention is a major component of OMV, which greatly reduces the side effects of inflammatory mediators due to excessive inflammatory mediators while maintaining the immune enhancing effect. It becomes an ingredient.
본 발명에서의 특징은 기본적으로 리피드 (lipid) A의 acylation 상태를 hexa-acylation에서 penta-acylation으로 변화시키기 위하여 병원성 대장균 O157:H7 균주를 예시하여 msbB 유전자와 pagP 유전자를 함께 결실시킨 변이체를 제작하였고, 이러한 변이체에서 생성된 penta-acyl lipid A를 가진 LPS의 내독성은 안전하게 백신으로 사용될 만큼 충분히 낮추어 지는 것을 특징으로 한다. A characteristic feature of the present invention was to exemplify the Escherichia coli O157: H7 strain in order to change the acylation state of lipid A from hexa-acylation to penta-acylation, thereby preparing a variant in which the msbB gene and pagP gene were deleted together. Toxicity of LPS with penta-acyl lipid A generated from these variants is characterized by a low enough safety to be used as a vaccine.
또한 이 변이체 (msbB -/pagP::FLAG)에서 생성된 외막소체에는 adjuvancity를 나타내는 성분들이 내포되어 있어 adjuvant를 별도로 혼합하지 않고도 백신효능을 증진시키는 효과를 가진다. 뿐 만 아니라, 외막소체에 포함되는 lipid A의 구조적 다양성을 증가시키기 위해 다양한 그람음성 세균들에서 특이적인 lipid A 구조형성에 관여하는 효소 (예: lpxF, lpxXL 또는 lpxE)를 발현벡터에 클로닝하여, 이들 중에 선택되는 하나 이상의 유전자를 본 발명에서 예시한 변이체 (msbB -/pagP::FLAG)에 도입·발현시킬 수 있다. 예를 들면, Brucella abortus 균에서 lpxE를 클로닝하여 발현시키면, 두개의 인산기가 부착된 lipid A에서 하나가 탈락되어 단일 인산기를 가진 penta-acyl lipid A의 종류를 생성시킬 수 있다. 이러한 lipid A에 대한 분자 구조적 리모델링을 통해 보다 안전하면서도 면역증진 효과를 나타내게 하는 특정 lipid A 구조가 탄생하는 것이므로 이는 선행문건에서 개시한 뇌수막염 균의 msbB 변이체에서 생성되는 lipid A와 그 구조가 다른 새로운 형태의 lipid A 종류를 가지는 외막소체를 생산 할 수 있게 된다. This variant also - the outer membrane vesicles produced in (msbB / pagP :: FLAG) is has the effect of enhancing the vaccine efficacy without mixing the adjuvant it is implied that the components shown adjuvancity separately. In addition, in order to increase the structural diversity of lipid A contained in the outer membrane body, an enzyme (eg , lpxF , lpxXL or lpxE ) that is involved in the formation of specific lipid A structures in various Gram-negative bacteria is cloned into the expression vector. a variant illustrated in the present invention one or more genes selected among these (msbB - / pagP :: FLAG) can be introduced, it expressed in. For example, if Brucella abortus cloned by expressing lpxE from bacteria, are eliminated from one of the two phosphate groups attached to lipid A it is possible to produce a kind of penta-acyl lipid A with a single phosphate group. Molecular structural remodeling of lipid A results in the creation of a specific lipid A structure that provides safer and more immune-promoting effects. This is a new form that differs from the lipid A produced in the msbB variant of meningitis bacteria disclosed in the preceding document. It is possible to produce the outer membrane body having a lipid A type.
본 발명에 있어서, 상기 "결실"은 염색체의 일부가 없어진 것을 의미한다. 세균세포에서 특정 유전자의 기능을 없애는 방법들은 다양하게 연구되어 왔으나, 최근 비교적 간단하고 효율적인 방법으로 주목받고 있는 Datsenko & Wanner가 고안한 one-step PCR inactivation system (PNAS, 97:6640~6645, 2000)을 이용하면 원하는 유전자를 homologous recombination을 이용한 타겟팅으로 용이하게 결실시킬 수 있다 (도 3 참고).In the present invention, the "deletion" means that a part of the chromosome is missing. Various methods of eliminating the function of specific genes in bacterial cells have been studied, but one-step PCR inactivation system devised by Datsenko & Wanner has recently been attracting attention as a relatively simple and efficient method (PNAS, 97: 6640 ~ 6645, 2000). Using can easily delete the desired gene by targeting using homologous recombination (see Figure 3).
상기 재조합 G(-) 박테리아의 제조방법은 외막소체의 크기와 생성량을 증가시키기 위하여 (a) 단계에서 제작된 G(-) 박테리아 변이주의 degP 유전자를 결실시키는 단계를 추가로 포함하는 것을 특징으로 할 수 있다. The method for preparing a recombinant G (-) bacterium may further include the step of deleting the degP gene of the G (-) bacterial strain produced in step (a) to increase the size and production of the outer membrane body. Can be.
DegP는 periplasmic chaperone으로서의 활동과 더불어 단백질 분해효소로서의 역할을 수행하는데, 만약 세균 세포외막 단백질들의 비정상적인 구조형성으로 외막에 포함되지 못하고 periplasm에 머무르게 될 때 이러한 단백질들을 제거하는 단백질 분해효소로서의 활성을 나타낸다. 그러므로 degP 유전자가 결실된 변이체에서는 periplasm에 축적된 비정상적인 외막단백질들이 제대로 제거되지 못하므로 세균의 생장에 위해가 초래되어 이러한 단백질들을 외막소체에 실어 세포 외부로 운반하여 버리려는 특성을 갖는다. 따라서 외막소체의 크기나 생성량이 증가되는 방 향으로 변이체의 적응이 이루어진다고 유추된다.DegP plays a role as a protease along with its activity as a periplasmic chaperone, which acts as a protease that removes these proteins when they remain in the periplasm due to abnormal structure formation of bacterial extracellular membrane proteins. Therefore, in the degP- deleted variant, abnormal outer membrane proteins accumulated in the periplasm could not be removed properly, causing the growth of bacteria and causing these proteins to be carried in the outer membrane cell and transported to the outside of the cell. Therefore, it is inferred that the adaptation of the mutant takes place in the direction of increasing the size and production of the outer membrane body.
degP 유전자의 결실은 앞서 언급한 homologous recombination을 이용한 타겟팅 방법으로 수행할 수 있다.Deletion of the degP gene can be performed by the aforementioned targeting method using homologous recombination.
본 발명에 있어서, 상기 G(-) 박테리아의 세포외막에 공통으로 존재하면서 비교적 발현이 풍부하고, 또한 이미 그 3차원적인 단백질의 원자구조가 밝혀진 대표적인 외막단백질은 OmpA를 예시할 수 있으며, 상기 OmpA 외막단백질은 PagP 외막단백질처럼 8개의 β-strand가 역방향으로 배열되어 접힌 β-배럴 (barrel) 형태의 3차원적 구조를 가지고 있다. 또한, OmpA를 대신하여 유사한 구조적 특징을 가지는 다른 종류의 외막단백질들 (OmpC, OmpF, OmpX, OmpG, OmpT) 중에서 선택되는 하나 이상의 것을 외부항원의 융합·발현을 위한 타겟으로 사용 할 수 있다.In the present invention, a representative outer membrane protein which is commonly present in the extracellular membrane of the G (-) bacteria and is relatively rich in expression, and whose atomic structure of the three-dimensional protein has already been found, may exemplify OmpA. The envelope protein has a three-dimensional structure of folded β-barrels in which eight β-strands are arranged in a reverse direction like PagP envelope proteins. In addition, one or more selected from other types of outer membrane proteins (OmpC, OmpF, OmpX, OmpG, OmpT) having similar structural characteristics in place of OmpA can be used as a target for fusion and expression of the external antigen.
또한, 상기 외부항원은 (a) 인간 또는 동물에서 다양한 감염성 질병을 일으키는 바이러스 또는 병원성 미생물의 주요 단백질 항원성분, (b) 면역세포(예: dendritic cell) 또는 암세포에 발현하는 특이적인 세포표면 마커 및 (c) 면역세포 또는 암세포의 표면에 존재하는 특정 리셉터에 결합하는 리간드 (ligand)로 구성된 군에서 선택되는 것을 사용할 수 있다. 본 발명의 일 실시예에서는 발현 유무를 쉽게 검출할 수 있는 FLAG®를 외부항원으로 사용하였으며, OMV 백신개발을 실제로 할 때는 human papillomavirus의 E7 epitope 등과 같은 예방하고자 하는 병원체의 항원을 외막단백질에 융합시켜 외막소체에 포함시킬수 있다. In addition, the external antigen may include (a) a major protein antigen component of a virus or pathogenic microorganism that causes various infectious diseases in humans or animals, (b) specific cell surface markers that are expressed on immune cells (eg dendritic cells) or cancer cells; (c) Any one selected from the group consisting of ligands that bind to specific receptors present on the surface of immune cells or cancer cells can be used. In one embodiment of the present invention, FLAG ® , which can easily detect the presence or absence of expression, was used as an external antigen, and when an OMV vaccine was actually developed, an antigen of a pathogen to be prevented, such as E7 epitope of human papillomavirus, was fused to an outer membrane protein. It can be included in the outer membrane body.
즉, 본 발명의 일 실시예에서는 외부항원을 외막소체에 도입하기 위하여 OmpA 단백질 유전자에 외부항원인 FLAG 시퀀스가 융합되도록 DNA 재조합체를 구성하고, 이 DNA 단편을 상기한 변이체(msbB -/pagP - ) 내부로 넣어 원래의 ompA 유전자와 homologous recombination이 일어나도록 유도하여 ompA::FLAG를 가진 변이체를 제작하였다. 이러한 ompA::FLAG 변이체의 외막에는 자연적으로 도입하고자 하는 외부항원(FLAG)이 OmpA와 융합되어 풍부하게 발현되므로 결국 상기 재조합 G(-) 박테리아 변이체가 생성하는 외막소체는 예시한 FLAG 외부항원이 그대로 내포되는 것을 특징으로 한다. That is, in one embodiment of the present invention configured to DNA recombinants that the foreign epitope is FLAG sequence in the OmpA gene fusion to introduce a foreign epitope to the outer membrane vesicles, and above this a DNA fragment mutant (msbB - / pagP - ), The homologous recombination with the original ompA gene was induced to produce a variant with ompA :: FLAG. In the outer membrane of the ompA :: FLAG variant, the external antigen (FLAG) to be naturally introduced is fused with OmpA to express abundantly, so the outer membrane body produced by the recombinant G (-) bacterial variant is the same as the FLAG outer antigen. It is characterized by being nested.
본 발명에 있어서, 상기 유전자 재조합체는 외부항원이 외막단백질의 3차원 구조(beta(β)-barrel 형태)상 외부(extracellular loop) 또는 내부인 페리플라즘(periplasm)에 결합되어 발현되는 것을 특징으로 한다. 따라서, 상기와 같이 외막단백질에 외부항원을 융합발현 시키면, 이 변이체의 세포외막에서 분출된 외막소체는 자연적으로 외부항원을 포함하게 됨으로써, 다가항원 (multi-valent epitope) 백신개발을 위한 기반 (platform) 기술로 이용될 수 있다. In the present invention, the genetic recombinant is characterized in that the external antigen is expressed by binding to the periplasm (extracellular loop) or the inner (periplasm) on the three-dimensional structure (beta (β) -barrel form) of the outer membrane protein. It is done. Therefore, when the external antigen is fused and expressed in the outer membrane protein as described above, the outer membrane body ejected from the extracellular membrane of the variant naturally includes the external antigen, thereby providing a platform for the development of a multi-valent epitope vaccine. ) Can be used as a technique.
본 발명에 있어서, 상기 G(-) 박테리아는 pagP 유전자를 보유하는 그람음성 세균으로, 에스케리치아 종 (Escherichia spp .), 살모넬라 종 (Salmonella spp .), 크렙시엘라 뉴모니애 (Klebsiella pneumoniae), 여시니아 종 (Yersinia spp.), 레지오넬라 뉴모필라 (Legionella pneumophila ), 보데텔라 종 (Bordetella spp .), 쉬 겔라 종 (Shigella spp .) 등을 예시할 수 있으며, 다른 균종에 비하여 비교적 LPS의 구조와 기능이 많이 연구된 에스케리치아 종 (Escherichia spp .)을 사용하는 것이 바람직하다. In the present invention, the G (-) bacteria are Gram-negative bacteria carrying the pagP gene, Escherichia spp . ), Salmonella species (Salmonella spp . ), Klebsiella pneumoniae , Yersinia spp. , Legionella pneumoniae pneumophila ) , Bodetella spp. spp . ), Shigella species spp . Escherichia species ( Esherichia spp., Which have been studied for the structure and function of LPS relatively compared to other species). spp . Is preferably used.
상기 에스케리치아 종 (Escherichia spp .)은 병원성 (pathogenicity)을 보유하고, 두개의 msbB 유전자가 게놈(genome)에 존재하는 O157:H7 혈청형 균주를 예시할 수 있다. 상기 E. coli O157:H7균은 세균성 이질을 유발하는 쉬겔라 종 (Shigella spp.)과 같이 특이하게도 두 개의 msbB 효소 유전자를 가지고 있으며, 염색체(chromosome)에 존재하는 msbB1과 plasmid (pO157)에 존재하는 msbB2가 있다 (Kim et al., Infect Immun., 72:1174-1180, 2004). Escherichia spp. spp . ) May illustrate an O157: H7 serotype strain that has pathogenicity and two msbB genes are present in the genome. The E. coli O157: H7 bacteria, like Shigella spp. , Which cause bacterial dysentery, specifically have two msbB enzyme genes, and are present in msbB1 and plasmid (pO157) on the chromosome. a msbB2 to (Kim et al, Infect Immun, 72:.. 1174-1180, 2004).
본 발명은 다른관점에서, 상기 재조합 G(-) 박테리아 제조방법에 의해 제조되고, 리포폴리사카라이드(LPS)의 리피드(lipid) A 생합성에 관여하는 유전자가 결실되어있고, G(-) 박테리아의 외막단백질과 외부항원이 융합·발현하도록 구성된 유전자 재조합체가 도입된 것을 특징으로 하는 재조합 G(-) 박테리아에 관한 것이다.In another aspect, the present invention is prepared by the recombinant G (-) bacteria production method, the gene involved in lipid A biosynthesis of lipopolysaccharide (LPS) is deleted, and The present invention relates to a recombinant G (-) bacterium, characterized in that a genetic recombinant configured to fuse and express an outer membrane protein and an external antigen is introduced.
상기 리포폴리사카라이드(LPS)의 리피드(lipid) A 생합성에 관여하는 유전자, 외막단백질, 외부항원, G(-) 박테리아 등은 앞서 언급한 바와 동일하다. 또한, 상기 재조합 G(-) 박테리아는 리피드(lipid) A의 구조에 관여하는 lpxXL , lpxF 및 lpxE로 구성된 군에서 선택되는 하나 이상의 유전자가 추가로 융합·발현된 것을 특징으로 하고, 상기 재조합 G(-) 박테리아는 외막소체의 크기와 생성량을 증가시 키기 위하여 degP 유전자가 추가로 결실된 것을 특징으로 한다.Genes involved in lipid A biosynthesis of lipopolysaccharide (LPS), outer membrane proteins, external antigens, G (-) bacteria and the like are the same as mentioned above. In addition, the recombinant G (-) bacteria is characterized in that the one or more genes selected from the group consisting of lpxXL , lpxF and lpxE involved in the structure of lipid (lipid A) is further fused and expressed, the recombinant G ( Bacteria are characterized by an additional deletion of the degP gene in order to increase the size and production of the outer membrane body.
본 발명은 또 다른관점에서, 상기 재조합 G(-) 박테리아를 배양하여 외부항원을 외막단백질과 융합된 형태로 발현시키는 것을 특징으로 하는 외부항원이 결합된 재조합 외막소체(OMV)의 제조방법에 관한 것이다.In another aspect, the present invention relates to a method for producing a recombinant outer membrane body (OMV) coupled to an external antigen, characterized in that by culturing the recombinant G (-) bacteria to express the outer antigen in a fused form with the outer membrane protein. will be.
본 발명에 따른 재조합 G(-) 박테리아 변이체로부터 생성된 외막소체(OMV)는 내독성이 낮아 안전하고, 다양한 외부항원을 전달 할 수 있으며, 자체적인 면역증진효과 (adjuvancity)를 지닌 항원전달체이다. 또한, 본 발명에서 생성시킨 외막소체(OMV)는 보통의 바이러스 particle과 유사한 nano-scale 크기 (평균 약 60~70nm)의 막소체 (membrane vesicle)로서, 인공적으로 형성시킨 liposome과 유사하므로 바이오 나노공학 분야에서 주목할만한 운반체 (vehicle)로 사용될 수 있다.Outer membrane bodies (OMV) generated from recombinant G (-) bacterial variants according to the present invention have low endotoxins, are safe, can transmit a variety of external antigens, and are antigen transporters with their own immunostimulating effect (adjuvancity). In addition, the outer membrane body (OMV) produced in the present invention is a nano-scale size (mean about 60 to 70 nm) membrane vesicles similar to those of ordinary virus particles, and is similar to artificially formed liposomes. It can be used as a vehicle notable in the field.
본 발명은 또 다른관점에서, (a) 외막소체(OMV) 생성능을 가지는 G(-) 박테리아에서 상기 외막소체가 내포하는 리포폴리사카라이드(LPS)의 리피드(lipid) A 생합성에 관여하는 유전자를 결실시킨 G(-) 박테리아 변이주를 제작하는 단계; (b) 상기 G(-) 박테리아 변이주로부터 분비되는 외막소체(OMV)를 회수하는 단계; 및 (c) 상기 (b) 단계에서 회수한 외막소체(OMV) 내부공간에 외부항원으로 작용하는 합성 펩티드(synthetic peptide) 또는 상기 합성 펩티드를 코딩하는 DNA 단편을 삽입하는 단계를 포함하는 것을 특징으로 하는 재조합 외막소체(OMV)의 제조방법에 관한 것이다.In still another aspect, the present invention provides a gene involved in lipid A biosynthesis of lipopolysaccharide (LPS) contained in the envelope membrane in G (-) bacteria having the ability to produce envelope membrane (OMV). Preparing a deleted G (-) bacterial mutant; (b) recovering the outer membrane body (OMV) secreted from the G (-) bacterial mutant strain; And (c) inserting a synthetic peptide acting as an external antigen or a DNA fragment encoding the synthetic peptide into an inner space of the outer membrane body (OMV) recovered in step (b). It relates to a method for producing a recombinant envelope membrane (OMV).
본 발명에 따른 재조합 외막소체(OMV)의 제조방법에 있어서, 상기 리포폴리사카라이드(LPS)의 리피드(lipid) A 생합성에 관여하는 유전자, 외막단백질, 외부항원, G(-) 박테리아 등은 앞서 언급한 바와 동일하다. 또한, 상기 재조합 G(-) 박테리아는 리피드(lipid) A의 구조에 관여하는 lpxXL, lpxF 및 lpxE로 구성된 군에서 선택되는 하나 이상의 유전자가 추가로 융합·발현된 것을 특징으로 하고, 상기 재조합 G(-) 박테리아는 외막소체의 크기와 생성량을 증가시키기 위하여 degP 유전자가 추가로 결실된 것을 특징으로 한다.In the method for producing a recombinant outer membrane body (OMV) according to the present invention, genes involved in lipid A biosynthesis of the lipopolysaccharide (LPS), outer membrane protein, external antigen, G (-) bacteria, etc. Same as mentioned. In addition, the recombinant G (-) bacteria is characterized in that the one or more genes selected from the group consisting of lpxXL, lpxF and lpxE involved in the structure of lipid (lipid A) is further fused and expressed, the recombinant G ( -) Bacteria are characterized by an additional deletion of the degP gene in order to increase the size and production of the outer membrane body.
본 발명에 있어서, 상기 재조합 외막소체(OMV)의 제조방법으로 제조된 외막소체(OMV)는 주요 구성성분인 LPS와 lipoprotein 등의 PAMP (pathogen-associated molecular pattern)들로 인하여 TLR4 와 TLR2에 의한 innate immunity를 자극할 수 있다. 따라서, 본 발명에서 개시한 방법으로 외부항원을 OMV에 내포시켜 백신으로 사용하면 OMV의 구성성분인 PAMP와 물리적으로 연결되어 별도의 adjuvant를 혼합하지 않고도 외막소체가 보유한 자체적인 면역증강효과 (adjuvancity)로 인하여 도입한 외부항원에 대한 면역반응을 증대시킬 수 있다. 이러한 이유는 TLR agonists로 작용하는 외막소체의 주요 성분들이 도입한 외부항원과 함께 연결되어 항원제시세포 (APC, antigen-presenting cell)에 탐식되기 때문이다. 이렇게 외막소체를 탐식하여 활성화된 항원제시세포 (예: 수지상세포)들은 항원정보를 전달할 대상인 helper T (Th cell) 세포와 긴밀하게 접촉하여 탐식한 항원에 대한 정보를 강하게 전달 할 수 있는데, 그러한 이유는 TLR을 통한 자극으로 인하여 항원제시세포 표면 에 co-stimulatory 분자들을 발현하여 강한 signal을 전달할 수 있기 때문이다. In the present invention, the outer membrane body (OMV) prepared by the method for producing the recombinant outer membrane body (OMV) is innate by TLR4 and TLR2 due to PAMP (pathogen-associated molecular patterns) such as LPS and lipoprotein as main constituents. Can stimulate immunity. Therefore, when the antigen is contained in the OMV and used as a vaccine by the method disclosed in the present invention, it is physically connected with PAMP, which is a component of the OMV, and has its own immune augmentation effect without mixing adjuvant (adjuvancity). This can increase the immune response to the foreign antigen introduced. This is because the major components of the outer membrane bodies, which act as TLR agonists, are linked with the external antigens introduced into them to feed on antigen-presenting cells (APCs). The antigen-presenting cells (eg, dendritic cells) activated by phagocytosis of the outer membrane body can be in close contact with helper T (Th cell) cells, which are the targets for transferring antigen information, to strongly transmit information on the phagocytosis. This is because co-stimulatory molecules can be expressed on the surface of antigen-presenting cells due to stimulation through TLRs, thereby delivering a strong signal.
본 발명은 다른 관점에서, 상기 재조합 외막소체(OMV)의 제조방법에 의해 제조된 재조합 외막소체의 내부공간(lumen)에 면역증진 효과를 나타내는 애쥬반트(ajuvants)를 삽입하는 것을 특징으로 하는 재조합 외막소체(OMV)의 재구성 방법에 관한 것이다. 예를들면, 세포성면역 유도를 위한 Th1-adjuvant로 작용하는 메틸기가 붙지 않은 CpG DNA (nucleotide sequence) 단편을 OMV 내부에 삽입하여, 바이러스 감염예방 백신과 같이 반드시 세포성 면역유도를 해야 하는 백신으로 이용할 수 있다.In another aspect, the present invention provides a recombinant outer membrane comprising inserting an adjuvant exhibiting an immunopromoting effect into the lumen of the recombinant outer membrane body prepared by the method for producing the recombinant outer membrane body (OMV). It relates to a method for reconstruction of the body (OMV). For example, a non-methylated CpG DNA (nucleotide sequence) fragment that acts as a Th1-adjuvant for induction of cellular immunity is inserted into the OMV to be a vaccine that must undergo cellular immunity, such as a viral infection vaccine. It is available.
본 발명은 또 다른 관점에서, 상기 재조합 외막소체(OMV)의 제조방법에 의해 제조된 재조합 외막소체에 내포된 리포폴리사카라이드(LPS)의 KDO(3-deoxy-D-manno-oct-2-ulosonic acid) 성분에 (a) 단백질 또는 탄수화물로 구성된 항원성 물질 또는 (b) 형광물질(fluorescent dye)을 화학적 커플링시키는 것을 특징으로 하는 재조합 외막소체(OMV)의 재구성 방법에 관한 것이다.In another aspect, the present invention provides a KDO (3-deoxy-D- manno- oct-2-) of lipopolysaccharide (LPS) contained in a recombinant outer membrane body prepared by the method for producing the recombinant outer membrane body (OMV). A method for reconstructing a recombinant outer membrane body (OMV) characterized by chemically coupling (a) an antigenic substance consisting of a protein or carbohydrate or (b) a fluorescent dye to a ulosonic acid) component.
상기 단백질 또는 탄수화물로 구성된 항원성 물질은 말라리아 메로조이트(malaria merozoite) 표면단백질 (MSP), 인간 파필로마바이러스 발암 단백질(human papillomavirus oncogenic protein(E6 또는 E7), 종양-관련 뮤신 글리코펩타이드(tumor-associated mucin glycopeptide, MUC1), 혈액형 관련(blood group- related) 탄수화물 항원 Lewisy (Ley) 등을 예시할 수 있고, 상기 형광물질은 플루오레세인(Fluorescein), 로다민(Rhodamine), 텍사스 레드(Texas Red), Cy3, Cy5.5 등을 예시할 수 있다. The antigenic substance consisting of the protein or carbohydrate is malaria merozoite surface protein (MSP), human papillomavirus oncogenic protein (E6 or E7), tumor-associated mucin glycopeptide (tumor- associated mucin glycopeptide (MUC1), blood group-related carbohydrate antigen Lewis y (Le y ), and the like, and the fluorescent material may be Fluorescein, Rhodamine, Texas Red ( Texas Red), Cy3, Cy5.5, and the like.
표면에 형광물질이 화학적으로 커플링된 재조합 외막소체(OMV)를 이용하면 외막소체가 생체내에서 수지상세포 (dendritic cell)에 탐식되어 항원을 전달하는 과정을 OMV에 표지된 형광물질로부터 live in vivo image를 얻어 관찰 할 수 있다. Using recombinant outer membrane bodies (OMVs) with chemically coupled fluorescent substances on the surface, the outer membrane bodies can detect the dendritic cells and deliver antigens in vivo from fluorescent substances labeled with OMV. You can get an image and observe it.
본 발명에 따른 상기 외막소체(OMV)의 재구성 방법에서, 상기 화학적 커플링은 LPS분자의 한 부분인 lipid A와 core 올리고당 부분을 연결하므로 거의 모든 LPS 분자에 공통으로 존재하는 특이한 sugar인 KDO (3-deoxy-D-manno-oct-2-ulosonic acid)를 이용하는 것을 특징으로 한다. KDO sugar는 반응성이 높은 carboxyl acid residue (COOH group)와 aldehyde group을 가지므로 이 반응성 잔기들을 이용한 화학적 커플링 방법으로 단백질 혹은 탄수화물로 구성된 외부항원이나 혹은 적당한 cross-linker와 결합된 형광물질을 KDO sugar에 커플링 할 수 있다. In the method for reconstructing the outer membrane body (OMV) according to the present invention, the chemical coupling connects lipid A, which is a part of the LPS molecule, with the core oligosaccharide part, so that KDO (3) is a unique sugar commonly present in almost all LPS molecules. -deoxy-D-manno-oct-2-ulosonic acid). Since KDO sugar has highly reactive carboxyl acid residue (COOH group) and aldehyde group, chemical coupling method using these reactive residues is used for the KDO sugar to bind external antigens composed of proteins or carbohydrates or fluorescent materials combined with appropriate cross-linkers. Can be coupled to
일반적으로 이러한 잔기들을 이용한 화학적 커플링 방법들은 여러 선행 학술논문들에서 보고 된 바 있다 (Verheul et al., Infect. Immun. 1991, 59: 843-851). 이를 간략히 소개하면, OMV의 주요 구성성분인 LPS에는 공통적으로 KDO 당이 존재하므로, OMV에 cystamine (NH2-(CH2)2-S-S-(CH2)2-NH2)을 반응시켜 KDO에 존재하는 카르복실기와 cystamine에 존재하는 아미노기 (NH2 group)가 peptide bond를 형성할 때와 같은 축약반응 (condensation)을 일으키게 하여 cystamine에 포함된 thiol (SH) group을 OMV의 LPS에 도입할 수 있다. 또한 OMV에 커플링하고자 하는 외부항원(단백질)이 자발적으로 화학적 coupling이 일으나도록 N-succinimidyl bromoacetate (Br-CO-CH2-O-NHS)과 반응시켜 Protein(외부항원)-[NH-CO-CH2-Br]형태의 결합물이 생성되도록 한다. 이러한 bromoacetyl group이 포함된 외부항원을 치올기(thiol group)가 부가된 OMV와 결합시키면 LPS의 KDO에 부가된 thiol기가 bromoacetyl기와 화학적 coupling반응이 생기므로, 결국 안정한 thioether 결합으로 연결된 OMV와 외부항원의 결합물 (conjugate)이 탄생된다. In general, chemical coupling methods using these residues have been reported in several previous articles (Verheul et al ., Infect. Immun. 1991, 59: 843-851). In brief, KDO sugar is commonly present in LPS, which is a major component of OMV. Therefore, cystamine (NH 2- (CH 2 ) 2 -SS- (CH 2 ) 2 -NH 2 ) is reacted to KDO by OMV. The carboxyl group present and the amino group present in cystamine (NH 2 group) can cause condensation, such as when forming peptide bonds, and the thiol (SH) group contained in cystamine can be introduced into LPS of OMV. In addition, the external antigen (protein) to be coupled to the OMV spontaneously reacts with N-succinimidyl bromoacetate (Br-CO-CH 2 -O-NHS) so that the chemical coupling is spontaneous, and the protein (external antigen)-[NH-CO- To allow the formation of a CH 2 -Br] form of bond. When the external antigen containing the bromoacetyl group is combined with the OMV to which the thiol group is added, the thiol group added to the KDO of LPS undergoes a chemical coupling reaction with the bromoacetyl group. Conjugates are born.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당 업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
[실시 예 1] 병원성 대장균 O157:H7 Sakai 균주 (이하 Sakai)에서 Example 1 In Escherichia Coli O157: H7 Sakai Strain (Hereinafter, Sakai) msbB msbB (( lpxMlpxM ) mutant의 제작 및 표현형 분석) Mutant Fabrication and Phenotypic Analysis
Sakai 균 (KCTC 11344BP)에는 두개의 msbB 유전자가 존재함으로, MsbB 효소의 완전한 불활성화 변이체를 만들려면 염색체(msbB1) 및 플라스미드(msbB2)에 존재하는 각기 msbB 유전자를 불활성화 하여야 한다. 그러므로 이를 위하여, 도 2에 나타낸 전략과 같이 msbB1 및 msbB2를 inactivation시킨 변이주를 제조하였다. Since there are two msbB genes in Sakai (KCTC 11344BP), it is necessary to inactivate the respective msbB genes present in the chromosome ( msbB1 ) and plasmid ( msbB2 ) in order to make a complete inactivating variant of the MsbB enzyme. Therefore, for this purpose, a mutant strain in which msbB1 and msbB2 were inactivated was prepared as in the strategy shown in FIG. 2.
가. end. msbB1msbB1 유전자의 결실 변이주 (Δ Deletion Mutant of Gene (Δ msbB1msbB1 mutant) 제작 mutant production
먼저, 염색체상의 msbB1 유전자를 결실시키기 위하여 M1-Fw 프라이머(서열번호 1: TGTCGCTCTGCTTTCCAGAACGTAGTGAAGCTGAACGCGAGTGTAGGCTGGAGCTGCTTCG)와 M1-Rev 프라이머(서열번호 2: TCTCGACTTCTTCATTCATCCGCCGCGCAATCGTATGATCCATATGAATATCCTCCTTAG)를 사용하여 pKD3(서열번호 3, GenBank accession no. AY048742, 2804 bp sequence)을 주형으로 PCR을 수행하여 pKD3 플라스미드에 존재하는 chloramphenicol (Cm)에 내성을 갖도록 하는 유전자 단위 (Cm-cassette)를 포함하는 PCR 산물을 제조하였다. PCR 조건은 template DNA로서 pKD3를 사용하고, M1-Fw와 M1-Rev 프라이머를 각각 30pico mole 농도로 첨가한 다음, AccuPrime Taq DNA polymerase®(Invitrogen)와 kit에 첨가된 reaction buffer를 혼합하여, 94℃에서 1분간 denaturing, 57℃에서 30초간 annealing, 70℃에서 1분간 extension하는 반복 주기를 한 사이클 삼아 32 사이클 까지 반응시켰다.First, M1-Fw primer in order to fruition the msbB1 gene on the chromosome (SEQ ID NO: 1: TGTCGCTCTGCTTTCCAGAACGTAGTGAAGCTGAACGCGA GTGTAGGCTGGAGCTGCTTCG) and M1-Rev primer: using (SEQ ID NO: 2 TCTCGACTTCTTCATTCATCCGCCGCGCAATCGTATGATC CATATGAATATCCTCCTTAG) pKD3 (SEQ ID NO: 3, GenBank accession no AY048742, PCR was performed using a 2804 bp sequence as a template to prepare a PCR product including a gene unit (Cm-cassette) that is resistant to chloramphenicol (Cm) present in the pKD3 plasmid. For PCR conditions, pKD3 was used as template DNA, M1-Fw and M1-Rev primers were added at 30 pico mole concentration, and AccuPrime Taq DNA polymerase ® (Invitrogen) and reaction buffer added to the kit were mixed at 94 ° C. 1 minute denaturing, annealing at 57 ° C for 30 seconds, and 1 cycle at 70 ° C were repeated for up to 32 cycles.
다음으로, 제조된 PCR 산물을 정제한 후, pKD46 플라스미드를 함유하는 형질전환 Sakai균 속에 PCR 산물을 도입하기 위하여 electroporation을 실시하였다. Electroporation를 위하여 우선 Sakai균을 배양한 다음, 차가운 10% glycerol solution으로 균체를 washing하여 electro-competent cell을 만들고, pKD46 플라스 미드 1㎍을 혼합하여 얼음 속에서 10분간 방치한 다음, electroporation용 cuvette에 옮겨 GenePulser® Xcell (BioRad) 기기를 사용하여 2.5 kV, 200Ω의 조건으로 전기자극을 가하여 Sakai 균체를 형질전환 시켰다.Next, the purified PCR product was subjected to electroporation in order to introduce the PCR product into the transformed Sakai bacteria containing the pKD46 plasmid. First, incubate Sakai bacteria for electroporation, wash the cells with cold 10% glycerol solution, make electro-competent cells,
Sakai(pKD46) 세균에 들어있는 플라스미드 pKD46에는 상동성을 지닌 DNA 단편끼리 재조합시키는 효소인 Red recombinase가 존재하므로, 도입된 targeting DNA 단편(PCR 산물)이 염색체에 존재하는 msbB1 유전자를 표적화 (targeting) 시키고, Cm에 내성을 지닌 변이체를 제조하였다.The plasmid pKD46 contained in the Sakai (pKD46) bacterium contains red recombinase, an enzyme that recombines homologous DNA fragments. Therefore, the targeting DNA fragment (PCR product) introduced targets the msbB1 gene present on the chromosome. , Variants were prepared that were resistant to Cm.
이러한 유전자 표적화(gene targeting)는 상동성 재조합 (homologous recombination)에 의하여 일어나므로, Sakai균 genome상의 상동성 부분 (msbB1)을 제외한 다른 site에는 변이가 생기지 않는다. 따라서, 인위적으로 특정 유전자를 변이시키고자 할 때 그 유전자와 상동성이 있는 DNA 단편을 제작하여 (msbB1 유전자의 경우 그 DNA sequence의 가운데 부분이 결실되고, 대신 pKD3 유래의 Cm-cassette를 포함하는 DNA 단편이 생기도록 도 2와 같이 제작) 균체내부로 전달하면 타겟 유전자가 도입한 DNA 단편과 교체되어 msbB1 유전자 변이체는 LB-Cm plate에 콜로니를 형성하게 된다. 이러한 Cm 저항성 콜로니들을 다시 한번 순수 분리하여 msbB1 유전자의 타겟 부분이 의도대로 변이가 도입되었는지를 PCR로 확인하였다. Since the gene targeting occurs by homologous recombination, no mutation occurs at other sites except for the homologous portion ( msbB1 ) on the Sakai genome. Therefore, in order to artificially mutate a specific gene, a DNA fragment homologous to the gene is produced (in the case of the msbB1 gene, the middle part of the DNA sequence is deleted, and instead, a DNA including a CK -cassette derived from pKD3). 2 is produced as shown in Figure 2) to deliver the cells inside the cell is replaced with the DNA fragment introduced by the target gene and msbB1 gene variants form colonies on the LB-Cm plate. These Cm resistant colonies were once again isolated purely and confirmed by PCR to determine whether the target portion of the msbB1 gene was introduced as intended.
즉, 변이주의 genomic DNA를 분리하여 변이를 도입한 부분의 외부에서 프라이머를 작성하여 PCR로 증폭된 DNA산물의 크기를 확인하였다 (예상크기 만큼의 증가로 확인). 그런 다음, 최종적으로 genome에 삽입한 Cm-cassette를 다시 잘라내 어(excision) Cm 저항성 마커를 제거하였다. 이를 위하여 변이주를 41˚C의 고온에 배양시켜 온도-민감(temperature-sensitive)형인 pKD46 (AmpR) 플라스미드를 소실시킨 다음, pCP20 (AmpR) 플라스미드를 변이체 내로 도입시켰다. 그 결과, pCP20에 포함된 FLP recombinase에 의해 Cm-cassette 내부 말단에 포함된 FRT (FLP recognition target) 시퀀스가 인식되므로, Cm-cassette가 FLP에 의하여 절단(excision)되어 탈락하는 효소반응이 일어났다. 이렇게 Cm-cassette를 소실시킨 Sakai ΔmsbB1 mutant를 PCR로 다시 확인하고 (예상크기 만큼의 감소), 이를 새로운 msbB2 결실 변이를 위한 parental strain으로 준비하였다.In other words, the genomic DNA of the mutant strain was isolated to prepare a primer outside the region where the mutation was introduced to confirm the size of the DNA product amplified by PCR (confirmed by the increase in the expected size). Then, the Cm-cassette finally inserted into the genome was cut again to remove Cm-resistant markers. To this end, the mutant strains were cultured at a high temperature of 41 ° C. to eliminate the temperature-sensitive pKD46 (Amp R ) plasmid, and then the pCP20 (Amp R ) plasmid was introduced into the variant. As a result, an FLP recognition target (FRT) sequence contained in the inner end of the Cm-cassette was recognized by the FLP recombinase included in pCP20. Thus, an enzyme reaction occurred in which the Cm-cassette was excisioned by FLP and eliminated. The Sakai Δ msbB1 mutant that lost Cm-cassette was reconfirmed by PCR (reduced by expected size) and prepared as a parental strain for a new msbB2 deletion mutation.
나. I. msbB1msbB1 및 And msbB2msbB2 유전자의 결실 변이주 (Δ Deletion Mutant of Gene (Δ msbB1msbB1 /Δ/ Δ msbB2msbB2 mutant) 제작 mutant production
Sakai 균주의 genome에는 두개의 msbB 유전자가 각각 chromosome (msbB1) 과 plasmid pO157 (msbB2)에 존재하므로, Sakai균의 MsbB 효소활성을 완전하게 제거하기 위하여 상기 방법대로 제작한 ΔmsbB1 mutant에 또 다른 msbB2 유전자를 불활화하여 MsbB 효소활성을 완전히 제거시킨 변이체를 제작하였다. 플라스미드 pO157에 위치하는 msbB2 유전자를 변이시키는 방법도 상기 msbB1 결실 변이를 유도하는 방법과 동일한 전략을 사용하여 결실 변이를 유도하였다. 표적하는 msbB2 유전자 말단 부분에 짧은 상동성 (short homology)을 갖도록 약 40 bp의 msbB2 염기서열을 포함시켜 제작한 M2-Fw 프라이머 (서열번호 4: GACGTGGACAGGTATCGGTATTATCTGTGTGTTTGCAATGGTGTAGGCTGGAGCTGCTTCG) 및 M2-Rev 프라 이머 (서열번호 5: TCGCGCAATATGAGCATCACTCTTCTGTCGTATAGCAAGTCATATGAATATCCTCCTTAG)를 사용하여, 상기한 pKD3 template을 주형으로 하여 PCR을 수행하였다. 따라서, 그 결과로 얻어진 PCR 산물은 msbB2 유전자 내부에 Cm-cassette를 포함하는 msbB2 gene targeting용 DNA 단편이 되는 것이다. In the genome of the Sakai strain, two msbB genes are present in the chromosome ( msbB1 ) and plasmid pO157 ( msbB2 ), respectively , and another msbB2 gene in the Δ msbB1 mutant prepared according to the above method to completely remove MsbB enzyme activity of the Sakai strain Inactivated to prepare a variant that completely eliminates the MsbB enzyme activity. Mutation of the msbB2 gene located at plasmid pO157 also induced deletion mutations using the same strategy as inducing msbB1 deletion mutations. M2-Fw primer (SEQ ID NO: 4: GACGTGGACAGGTATCGGTATTATCTGTGTGTTTGCAATG GTGTAGGCTGGAGCTGCTTCG ) and M2-Rev primer (SEQ ID NO: 5) prepared by including about 40 bp of msbB2 sequence to have a short homology at the terminal end of the target msbB2 gene : PCR was performed using the pKD3 template as a template using TCGCGCAATATGAGCATCACTCTTCTGTCGTATAGCAAGT CATATGAATATCCTCCTTAG ). Thus, PCR product obtained as a result would be a DNA fragment for targeting msbB2 gene, which contains a Cm-cassette in the msbB2 gene inside.
다음으로, 제조된 msbB2 gene targeting용 DNA 단편을 ΔmsbB1(pKD46) 균체 내부로 상기한 electroporation 방법을 통하여 삽입하였고, msbB2 유전자의 상동성에 의한 homologous recombination이 유도됨으로써, msbB2 유전자를 표적하여 변이를 도입시킨 Cm-저항성 변이체를 제조하였다. 최종적으로 msbB2 유전자 내부에 삽입한 Cm-cassette를 다시 절단(excision)하여 제거하기 위하여, 상기한 방법으로 고온 배양하여 pKD46을 소실시키고, pCP20 플라스미드를 변이체 내로 도입시켰다. 그 결과, pCP20에 포함된 FLP recombinase에 의해 Cm-cassette 내부 말단에 포함된 FRT (FLP recognition target) 시퀀스가 인식되어, Cm-cassette가 FLP에 의하여 절단(excision)되어 예상된 크기만큼의 유전자 길이가 감소된 msbB2 DNA를 PCR로 증폭하여 확인하였다. Next, were inserted through the above-described electroporation method, the DNA fragment for the manufacture msbB2 gene targeting into the Δ msbB1 (pKD46) bacteria, homologous recombination is induced by homology of the msbB2 gene. Thus, by targeting the msbB2 gene was introduced into the mutant Cm-resistant variants were prepared. Finally, in order to remove the Cm-cassette inserted into the msbB2 gene again by excision, high-temperature culture was carried out by the above-described method to remove pKD46, and the pCP20 plasmid was introduced into the variant. As a result, a FLP recognition target (FRT) sequence included in the inner end of the Cm-cassette is recognized by the FLP recombinase included in pCP20, and the Cm-cassette is excisioned by the FLP to generate a gene length of the expected size. Reduced msbB2 DNA was confirmed by amplification by PCR.
Sakai ΔmsbB1/ΔmsbB2 mutant를 확인하기 위한 PCR 조건은 template DNA로서 mutant에서 분리한 genomic DNA를 사용하고, ΔmsbB1를 확인하기 위한 프라이머 set (MsbB1-Fw primer 서열번호 6: TCGCGAATTCCTCGAGCAGGCCAA와 MsbB1-Rev primer 서열번호 7: CTGAAGCAAGCTTGAACTTATCA)과 ΔmsbB2를 확인하기 위한 프라이머 set (MsbB2-Fw primer 서열번호 8: CTCACTGATCTCGAGACTCTTCC 와 MsbB2-Rev primer 서열 번호 9: GCTGGGTAAGCTTATTATCCTGA)을 각각 30 pico mole 농도로 첨가한 다음, AccuPrime Taq DNA polymeraseㄾ (Invitrogen)와 kit에 첨가된 reaction buffer를 혼합하여, 94℃에서 1분간 denaturing, 57℃에서 30초간 annealing, 70℃에서 1분간 extension하는 반복 주기를 한 사이클 삼아 32 사이클 까지 반응시켰다.PCR conditions for identifying the Sakai Δ msbB1 / Δ msbB2 mutants were genomic DNA isolated from mutants as template DNA, and primer sets for identifying Δ msbB1 (MsbB1-Fw primer SEQ ID NO: 6: TCGCGAATTCCTCGAGCAGGCCAA and MsbB1-Rev) primer SEQ ID No. 7: CTGAAGCAAGCTTGAACTTATCA) and primer set (MsbB2-Fw primer SEQ ID No. 8: CTCACTGATCTCGAGACTCTTCC and MsbB2-Rev primer SEQ ID No. 9: GCTGGGTAAGCTTATTATCCTGA) for the identification of Δ msbB2 , respectively, were added at 30 pico mole concentration, followed by AccuPrime Ta. DNA polymerase ㄾ (Invitrogen) and the reaction buffer added to the kit were mixed and reacted for up to 32 cycles with one cycle of denaturing at 94 ° C for 1 minute, annealing at 57 ° C for 30 seconds, and one minute extension at 70 ° C.
도 3의 결과에서 나타난 바와 같이, Sakai ΔmsbB1/ΔmsbB2 double mutant (이하 Sakai-DM으로 명명; KCTC 11346BP)를 PCR로 검증한 결과, 변이체에서는 각각의 ΔmsbB1 및 ΔmsbB2의 변이가 예상대로 정확히 이루어졌음 알 수 있었다.As shown in the results of FIG. 3, PCR of the Sakai Δ msbB1 / Δ msbB2 double mutant (hereinafter referred to as Sakai-DM; KCTC 11346BP) showed that the mutations of the respective Δ msbB1 and Δ msbB2 were exactly as expected. It could be done.
[실험예 1] 변이주에서 생산된 LPS 표현형 변화 비교 Experimental Example 1 Comparison of LPS Phenotype Changes Produced in Mutant
상기 실시예에서 제작된 Sakai ΔmsbB1 mutant (Sakai-M1)와 ΔmsbB1/ΔmsbB2 double mutant (Sakai-DM)의 LPS 표현형 (phenotype) 변화를 비교하기 위하여, wild type, Sakai-M1, 그리고 Sakai-DM으로부터 각각 LPS를 분리하고, 16% SDS-PAGE gel에 전개시켜 silver stain으로 염색하였다. To compare the LPS phenotype changes of the Sakai Δ msbB1 mutant (Sakai-M1) and Δ msbB1 / Δ msbB2 double mutant (Sakai-DM) prepared in the above examples, wild type, Sakai-M1, and Sakai- Each LPS was isolated from DM and developed on a 16% SDS-PAGE gel and stained with silver stain.
그 결과 도 4에 나타난 바와 같이, 전체적인 LPS pattern은 세 균주 모두 유사하였으며, wild type Sakai 균주와 Sakai-M1은 동일한 smooth LPS를 생성하지만, Sakai-DM은 미리스테이트(myristate) 잔기가 부착되지 않은 만큼의 분자량이 감소되어 SDS-PAGE 상에서 LPS 밴드의 이동이 조금 빠르게 나타났음을 확인하였다.As a result, as shown in Figure 4, the overall LPS pattern was similar to all three strains, wild type Sakai strain and Sakai-M1 produced the same smooth LPS, but Sakai-DM is not attached to myristate residues It was confirmed that the molecular weight of the LPS band shifted slightly faster on the SDS-PAGE.
[실험예 2] LPS의 lipid A 아실화 상태 분석Experimental Example 2 Lipid A Acylation Status of LPS
LPS의 lipid A 부분에 대한 acylation 변화를 관찰하기 위하여 lipid A 분자 에 포함된 인(phosphate) 성분을 방사선 동위원소인 P-32로 표지하여 lipid A 부분만을 LPS로부터 mild-acid hydrolysis 방법으로 해리시켜 순수분리 한 다음, thin-layer chromatography (TLC)로 전개시켜, 소수성 (hydrophobicity) 변화에 따른 TLC 상의 이동 패턴을 통해 lipid A의 acylation 변화를 관찰 하였다. In order to observe the acylation change of lipid A part of LPS, the phosphorus component included in lipid A molecule was labeled with P-32, which is a radioisotope, and only lipid A part was dissociated from LPS by mild-acid hydrolysis. After separation, it was developed by thin-layer chromatography (TLC), and the change in lipid A acylation was observed through the shift pattern on TLC according to the change in hydrophobicity.
그 결과, 도 5에 나타난 바와 같이, MsbB 효소활성의 유무에 따른 미리스테이트(myristate)의 부착여부로 인하여 hydrophobicity에 변화가 초래되어 TLC 전개상의 이동패턴이 변화된 lipid A spot을 관찰 할 수 있었다. Sakai wild type (lane 1)에서는 주로 전형적인 hexa-acyl lipid A 분자들이 생성되었지만, MsbB 효소활성이 완전히 소실된 Sakai-DM (lane 3)에서는 주로 penta-acyl lipid A 분자들과 함께 PagP에 의한 palmitoylated hexa-acyl lipid A가 부분적으로 생성되었으므로, msbB 불활성화 변이체의 예상 표현형을 확인 할 수 있었다.As a result, as shown in Figure 5, the presence of the myristate (myristate) according to the presence or absence of MsbB enzyme activity caused a change in the hydrophobicity was observed lipid A spot was changed the movement pattern on the TLC development. In Sakai wild type (lane 1), mainly hexa-acyl lipid A molecules were produced, but in Sakai-DM (lane 3) where MsbB enzyme activity was completely lost, palmitoylated hexa was induced by PagP together with penta-acyl lipid A molecules. Since partial production of -acyl lipid A resulted in the expected phenotype of the msbB inactivated variant.
특히, 흥미롭게도 Sakai-M1 (lane 2)에서의 lipid A acylation pattern은 ΔmsbB1 결실변이 도입에 따라 상당한 양의 penta-acyl lipid A 종류가 생성되고 있음을 알 수 있었고, 이는 plasmid에 존재하는 msbB2 유전자만으로는 ΔmsbB1 변이체에서 penta-acyl lipid A 전구물질에 myristate를 부가하여 hexa-acyl lipid A 종류로 전환하게 하는 MsbB 효소활성이 상대적으로 낮다는 것을 알 수 있었다. 이러한 Sakai균 변이체의 lipid A 분석 결과를 통하여 pO157에 남겨진 msbB2 유전자의 단독 발현만으로는 lipid A의 완전한 hexa-acylation이 일어나지 않았으며, 생성된 lipid A spot의 강도를 비교해 볼 때 MsbB2의 활성은 MsbB1의 activity에 비하여 약 70% 수준으로 myristate를 부가하는 활성 (penta-acyl form에서 hexa-acyl form으로 전환)이 일어났음을 유추할 수 있었다. 뿐만 아니라, 도 5의 Sakai-DM (lane 3)에서는 약 30% 정도의 lipid A가 PagP에 의하여 세균세포 외막에서 palmitate가 부가된 형태의 hexa-acyl form이 생성됨을 알 수 있었다. In particular, the lipid A acylation pattern in Sakai-M1 (lane 2) showed that a significant amount of penta-acyl lipid A was produced by the introduction of the Δ msbB1 deletion mutation, which indicates that the msbB2 gene present in the plasmid. In Δ msbB1 mutant, MsbB enzymatic activity of converting myristate to hexa-acyl lipid A type by adding myristate to penta-acyl lipid A precursor was relatively low. Liposomal A analysis of these Sakai mutants did not result in complete hexa-acylation of lipid A by expression of the msbB2 gene left in pO157 alone, and MsbB2 activity was determined by comparing the intensity of lipid A spot. It can be inferred that the activity of adding myristate (conversion from penta-acyl form to hexa-acyl form) occurred at about 70%. In addition, in Sakai-DM (lane 3) of FIG. 5, about 30% of lipid A produced hexa-acyl form in which palmitate was added to the bacterial cell outer membrane by PagP.
[실시 예 2] Sakai O157:H7 균주에서 Example 2 In Sakai O157: H7 Strain degPdegP mutant의 제작 및 표현형 분석 Production and Phenotypic Analysis of Mutants
Sakai O157:H7 균주에서 외막소체(OMV) 생산량을 증가시키고 외막소체의 크기를 증대시키기 위하여 degP 유전자를 결실시키고, 이 변이체에서 생성된 OMV의 크기와 생성량을 조사하였다. DegP gene was deleted in order to increase the production of outer membrane body (OMV) and increase the size of the outer membrane body in the Sakai O157: H7 strain, and the size and production of the OMV generated in this variant were investigated.
degP 변이체를 제작하는 방법은 상기 msbB 변이체 제조와 같은 기본전략으로 targeting DNA 단편을 제작하여 Sakai(pKD46) 및 Sakai-DM(pKD46) 균체내에 각각 전달하여 상동성 재조합 균주 (CmR)를 분리하고, PCR 방법으로 degP 유전자에 정확한 변이가 도입되었는지를 확인하였다. 이를 위하여 먼저 degP 유전자를 변이시킨 DNA 제조합체를 제조하였다. degP 유전자 양쪽 말단에 해당되는 400-bp 이상의 비교적 긴 상동성 (homology)을 갖도록 하는 DNA 단편을 pUC18에 클로닝한 다음, 상동성 단편 사이에 pKD3 유래 Cm-cassette를 삽입하여 degP 결실변이를 위한 재조합 DNA를 제작하였다. 상기 제작된 degP gene targeting용 DNA 단편을 상기한 electroporation 방법을 통하여 균체 내부로 도입하여 homologous recombination이 유도됨으로써, degP::Cm 변이유전자로 치환된 Cm-저항성 변이체를 제조하였다. 상기의 방법으로 Sakai 균 및 Sakai-DM으로부터 ΔdegP::Cm의 변이체를 제작하여 Δ degP::Cm 유전자의 변이에 해당하는 PCR 산물의 정확한 DNA 크기를 확인하였다. ΔdegP::Cm의 변이체를 확인하기 위한 PCR 조건은 주형으로서 각 변이체 유래 genomic DNA를 사용하고, 프라이머들 (For-degP primer 서열번호 10: TGGGTTCCGGCGTCATCATTGAT 와 Rev-degP primer 서열번호 11: CGATCTGCGCAGCCGGAGTGCC)을 각각 30 pico mole 농도로 반응액에 첨가한 다음, AccuPrime Taq DNA polymeraseㄾ (Invitrogen)와 kit에 포함된 중합효소를 혼합하여, 94℃에서 1분간 denaturing, 57℃에서 30초간 annealing, 70℃에서 1분간 extension하는 주기를 한 사이클 삼아 32 사이클 까지 반복 반응시켰다. The method for preparing degP variants is prepared by targeting DNA fragments using the same basic strategy as the preparation of the msbB variant, and delivered to the Sakai (pKD46) and Sakai-DM (pKD46) cells to separate homologous recombinant strains (Cm R ), PCR confirmed that the correct mutation was introduced into the degP gene. To this end, first, a DNA preparation mutated with the degP gene was prepared. DNA fragments having a relatively long homology of 400-bp or more corresponding to both ends of the degP gene were cloned into pUC18, and then a pKD3-derived Cm-cassette was inserted between the homologous fragments to recombinant DNA for degP deletion. Was produced. The prepared DNA fragment for degP gene targeting was introduced into the cell through the electroporation method to induce homologous recombination, thereby preparing Cm-resistant variants substituted with degP :: Cm mutants. By the above method, a Δ degP :: Cm variant was prepared from Sakai and Sakai-DM, and the exact DNA size of the PCR product corresponding to the Δ degP :: Cm gene was confirmed. PCR conditions for identifying variants of Δ degP :: Cm using genomic DNA derived from each variant as a template, primers (For-degP primer SEQ ID NO: 10: TGGGTTCCGGCGTCATCATTGAT and Rev-degP primer SEQ ID NO: 11: CGATCTGCGCAGCCGGAGTGCC) was added to the reaction solution at 30 pico mole concentration, respectively, and then AccuPrime Taq DNA polymerase ㄾ (Invitrogen) and the polymerase included in the kit were mixed, denaturing at 94 ° C for 1 minute, and at 57 ° C. The reaction was repeated up to 32 cycles using one cycle of annealing for 30 seconds and 1 minute extension at 70 ° C.
이러한 degP 변이체에서 생성된 외막소체를 수집하여 전자현미경으로 확인하기 위하여 우선 변이체를 500ml LB broth에 배양한 다음 원심분리 (11,000×G, 30분) 하여 균체와 배양상청액을 분리하였다. 분리한 배양상청액을 0.2㎛ pore size를 가진 membrane filter에 걸러내어 남아있는 균체와 큰 덩어리의 세포 파편들을 제거한 다음 배양상청액에 포함된 외막소체를 회수하기 위해 ammonium sulfate를 가하여 외막소체를 4℃에서 하룻밤 동안 침전시켰다. 침전된 외막소체는 원심분리 (11,000×G, 30분) 로 수집한 다음 20ml의 phosphate-buffered saline (PBS)에 부유시키고 다시 저속 원심분리 (16,000×G, 15분)하여 상청액과 저속침전물을 분리한 후 상청액에 남아있는 외막소체를 초고속 원심분리 (100,000×G, 2시간) 하여 외막소체를 침전시킨 다음 2ml의 증류수에 부유시켜 -20℃에 보관하였다. 상기 방법으로 분리한 외막소체를 전자현미경으로 확인하기 위하여 샘플을 formvar-coated grid 상에서 1% uranyl acetate로 negative stain하여 관찰하였다. In order to collect the outer membrane bodies generated from these degP variants and confirm them by electron microscopy, the strains were first cultured in 500ml LB broth, and then centrifuged (11,000 × G, 30 minutes) to separate the cells and the culture supernatant. The isolated culture supernatant was filtered through a membrane filter having a 0.2 μm pore size to remove remaining cells and large cell debris, and then the ammonium sulfate was added to recover the outer membrane body contained in the culture supernatant. Was precipitated. The precipitated outer membrane body was collected by centrifugation (11,000 × G, 30 minutes), then suspended in 20 ml of phosphate-buffered saline (PBS), and again subjected to low speed centrifugation (16,000 × G, 15 minutes) to separate the supernatant and the slow precipitate. The outer membrane body remaining in the supernatant was then subjected to ultrafast centrifugation (100,000 × G, 2 hours) to precipitate the outer membrane body, and then suspended in 2 ml of distilled water and stored at -20 ° C. In order to confirm the outer membrane body separated by the above method by electron microscopy, the sample was observed by negative staining with 1% uranyl acetate on a formvar-coated grid.
도 6에 나타난 바와 같이 degP가 결실된 변이체(Sakai-degP)에서는 degP가 결실되지 않은 Sakai균 (data not shown) 이나 Sakai-DM에 비하여 외막소체의 생산량 및 생산된 외막소체의 크기의 증가를 확인 할 수 있었다. 하지만 Sakai-DM/ΔdegP::Cm 변이체에서는 오히려 외막소체 생성량이 매우 적어지고 또한 그 크기도 작아지는 것으로 확인 되었으며 (data not shown), 이러한 이유는 모균주인 Sakai-DM의 외막의 취약성이 ΔdegP::Cm 변이유발로 더욱 증가되었기 때문으로 해석된다. 그러므로 Sakai-DM에서 생성되는 외막소체들이 상대적으로 균일한 크기(평균 60 ~ 70 nm)를 가진 것들이 생성됨을 확인하였다(도 6 A). As shown in FIG. 6, in the degP -deleted mutant (Sakai-degP), an increase in the amount of the outer mesobody produced and the size of the produced outer mesobody compared to Sakai (data not shown) or Sakai-DM in which the degP was deleted was confirmed. Could. However, in Sakai-DM / Δ degP :: Cm variant, it was found that the formation of outer membrane body was very small and its size was also reduced (data not shown). This is because the vulnerabilities of the outer membrane of the parent strain Sakai-DM are Δ. It is interpreted that the increase was further caused by the degP :: Cm mutation. Therefore, it was confirmed that the outer membrane bodies produced in Sakai-DM were produced with relatively uniform size (average 60 to 70 nm) (FIG. 6A).
또한, 균체에서 분출되어 나오는 외막소체를 관찰하기 위하여 각 변이체들의 콜로니를 한천배지에 형성시켜 2.5% paraformaldehyde-glutaraldehyde를 함유하는 고정액으로 고정시킨 다음 1% osmium tetroxide로 추가 고정하여 ethanol 탈수과정을 거친 다음 Epon-812 수지에 포매하였다. 포매된 샘플을 ultramicrotome으로 초미세 절단하여 uranyl acetate로 염색한 후 투과전자현미경 (TEM)으로 변이체에서 분출되어 나오는 외막소체를 관찰하였다. 상기한 배양상청액에서 회수한 외막소체들의 전자현미경 관찰결과와 같은 외막소체가 균체 세포외막에서 분출되어 나오는 것을 비교 확인하였다(도 6 B). In addition, in order to observe the outer membrane bodies ejected from the cells, colonies of the variants were formed in agar medium, fixed with a fixed solution containing 2.5% paraformaldehyde-glutaraldehyde, and further fixed with 1% osmium tetroxide, followed by ethanol dehydration. Embedded in Epon-812 resin. The embedded samples were ultrafinely cut with ultramicrotome, stained with uranyl acetate, and the outer membrane bodies were ejected from the mutant with a transmission electron microscope (TEM). It was confirmed that the outer membrane bodies, such as electron microscopic observation results of the outer membrane bodies recovered from the culture supernatant, were ejected from the cell extracellular membrane (FIG. 6B).
[실시예 3] Sakai ΔExample 3 Sakai Δ msbB1msbB1 /Δ/ Δ msbB2msbB2 mutant (Sakai-DM)에서 from mutant (Sakai-DM) pagPpagP ::FLAG tagged mutant의 제작 및 표현형 분석 :: Production and Phenotypic Analysis of FLAG tagged Mutants
상기 실시예 1에서 제작된 Sakai ΔmsbB1/ΔmsbB2 mutant (Sakai-DM)의 lipid A profile에서 PagP 효소의 활성(lipid A palmitoylation)에 의한 부분적인 hexa-acyl lipid A 형태가 출현하였으므로 이러한 hexa-acyl lipid A를 없애고 완전히 penta-acyl lipid A가 형성되도록 Sakai-DM에 새로운 변이(pagP::FLAG)를 도입하여 PagP의 효소 활성을 제거하고, 외부항원으로 FLAG 시퀀스를 도입한 변이체(Sakai-DM/pagP::FLAG)를 제작하였다. In the lipid A profile of the Sakai Δ msbB1 / Δ msbB2 mutant (Sakai-DM) prepared in Example 1, a partial hexa-acyl lipid A form due to the activation of PagP enzyme (lipid A palmitoylation) appeared. A new mutation ( pagP :: FLAG) was introduced into Sakai-DM to eliminate lipid A and form penta-acyl lipid A completely, eliminating the enzyme activity of PagP, and introducing the FLAG sequence into an external antigen (Sakai-DM / pagP :: FLAG) was produced.
가. end. pagPpagP ::FLAG 재조합 유전자 구축:: FLAG Recombinant Gene Construction
Sakai-DM에 새로운 pagP::FLAG 변이를 도입하여 PagP의 효소 활성을 제거하면서 또한 외막단백질에 외부항원을 융합시켜 그 발현정도를 검출할 목적으로 FLAG epitope를 선택하였다. 먼저, 전체 크기의 pagP 유전자를 pUC18 벡터에 클로닝하고, pKD3 유래 Cm-cassette를 동일 플라스미드의 pagP 유전자 다음에 삽입시킨 다음, 이 재조합 플라스미드를 PCR을 이용한 site-directed mutagenesis의 template DNA로 사용하였다. A new pagP :: FLAG mutation was introduced in Sakai-DM to remove the enzyme activity of PagP, and to select the FLAG epitope for the purpose of detecting the expression level by fusing external antigens into the outer membrane protein. First, the full-size pagP gene was cloned into the pUC18 vector, and the pKD3-derived Cm-cassette was inserted after the pagP gene of the same plasmid, and the recombinant plasmid was used as template DNA of site-directed mutagenesis using PCR.
도 7에 나타낸 바와 같이, 15개의 FLAG epitope 아미노산 서열(서열번호 12: DYKDHDGDYKDHDDD)에 대한 DNA 염기서열을 포함하는 78bp 길이를 가진 kinated mutagenic reverse primer (서열번호 13: CGGTCGCTCGTTATAATCGTCATCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCAGCGAAACGTGCATGCCA)를 제작하여, 첫 번째 PCR을 수행하였다. 그 결과로 얻은 PCR 산물 (2차 PCR을 위한 megaprimer)은 본래 PagP 단백질의 loop-1을 구성하는 8개의 아미노산 (YDKEKTDR)이 제외되고, 대신 15개의 FLAG 아미노산이 대체되어 삽입된 형태로 연 결된 pagP::FLAG fusion이 이루어졌음을 확인하였다. 즉, PagP의 loop-1 부분의 original sequence는 WHARFA-(YDKEKTDR)-YNERP로 연결된 아미노산 배열을 가졌으나, 본 실험을 통한 site-directed mutagenesis로 인하여 괄호 안의 8개 아미노산 (YDKEKTDR)이 배제되고, 대신 외부항원인 FLAG sequence가 삽입된 형태 WHARFA-(DYKDHDGDYKDHDDD)-YNERP의 변이를 도입할 수 있었다.As shown in FIG. 7, a kinated mutagenic reverse primer (SEQ ID NO: 13: CGGTCGCTCGTTATA ATCGTCATCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTC AGC, AGAG) was prepared by using a 78 bp long DNA sequence containing the DNA sequences for 15 FLAG epitope amino acid sequences (SEQ ID NO: 12: DYKDHDGDYKDHDDD). First PCR was performed. The resulting PCR product (megaprimer for secondary PCR) excludes the 8 amino acids (YDKEKTDR) that make up loop-1 of the original PagP protein, and instead replaces 15 FLAG amino acids and connects them into the inserted pagP. :: FLAG fusion was confirmed. In other words, the original sequence of the loop-1 portion of PagP had an amino acid sequence linked by WHARFA- (YDKEKTDR) -YNERP, but 8 amino acids (YDKEKTDR) in parentheses were excluded because of site-directed mutagenesis through this experiment. Mutation of the type WHARFA- (DYKDHDGDYKDHDDD) -YNERP with the FLAG sequence as an external antigen was introduced.
나. Sakai-DM (ΔI. Sakai-DM (Δ msbB1msbB1 /Δ/ Δ msbB2msbB2 )에서 )in pagPpagP ::FLAG 변이체의 제작:: Fabrication of FLAG Variants
상기 '가' 단계에서 구축한 재조합 플라스미드(template plasmid)를 주형으로 하여 pagP::FLAG-Cm cassette DNA 부분을 포함하면서, 타겟이 되는 pagP 유전자의 하류 (downstream)에 상동성을 지니도록 P2H2 reverse 프라이머 (서열번호 14: ACACAAATGCTGTGTCGGTTACCAGTACACCAATTGTGGTAC CATATGAATATCCTCCTTAG )를 작성하여 PCR로 증폭시켰다. 그런 다음, 이 PCR 증폭산물을 정제하여 Sakai-DM(pKD46) 균에 electroporation을 통하여 삽입함으로써, 균 내부에서 상동성 재조합 (homologous recombination)이 일어나도록 하여 외부에서 도입한 pagP::FLAG-Cm cassette DNA부분이 genome에 있는 pagP 유전자에 targeting될 수 있도록 하였다. 이러한 결과로 생겨난 Cm-저항성 콜로니들을 PCR 반응을 통해 변이도입 부분을 증폭하여 그 PCR 산물의 크기가 예상대로 증가되었는지를 확인하고, 또한 DNA 염기서열 분석을 통하여 pagP::FLAG 융합이 translation frame에 부합하게 이루어 졌음을 확인하였다. 이렇게 얻어진 Sakai-DM/pagP::FLAG 변이체의 표현형 변화를 확인하기 위하여 P-32 동위원소로 표지한 변이체에서 lipid A 부분을 추출하여 TLC로 전개하여 확인해 본 결과, 예상대로 PagP 효소의 불활성화로 palmitoylation에 의한 lipid A의 부분적인 hexa-acylation이 Sakai-DM/pagP::FLAG 변이체에서는 일어나지 않았음을 확인하였다 (도 8). P2H2 reverse primer containing pagP :: FLAG-Cm cassette DNA moiety using a recombinant plasmid constructed in the step 'A' as a template and having homology to the downstream of the target pagP gene (SEQ ID NO: 14: ACACAAATGCTGTGTCGGTTACCAGTACACCAATTGTGGTAC CATATGAATATCCTCCTTAG ) was prepared and amplified by PCR. Then, the PCR amplification product was purified and inserted into the Sakai-DM (pKD46) bacteria through electroporation, thereby allowing homologous recombination to occur inside the bacteria, thereby introducing pagP :: FLAG-Cm cassette DNA. The part can be targeted to the pagP gene in the genome. The resulting Cm-resistant colonies were amplified by the PCR reaction to amplify the mutagenesis region, and the size of the PCR product was increased as expected. Also, DNA sequencing confirmed that the pagP :: FLAG fusion was matched to the translation frame. It was confirmed that it was done. In order to confirm the phenotypic change of the Sakai-DM / pagP :: FLAG variant thus obtained, lipid A was extracted from T-labeled variants and developed by TLC. It was confirmed that partial hexa-acylation of lipid A by palmitoylation did not occur in Sakai-DM / pagP :: FLAG variant (FIG. 8).
도 9는 상기 TLC 상에서 나타난 lipid A spot에 해당하는 화학적 구조를 도해한 그림이다. 9 is a diagram illustrating the chemical structure corresponding to the lipid A spot shown on the TLC.
도 10은 본 발명의 일 실시예에 따라 제작된 Sakai-DM/pagP::FLAG 변이주의 외막에 발현하는 PagP::FLAG 융합단백질의 2차 구조적 topology를 예측한 그림이다. 외부항원인 FLAG 시퀀스가 PagP의 2차 구조상 loop-1 부분과 reading-frame에 맞게 융합되어, 도입할 외부항원이 외막소체에서 외부환경으로 돌출되어 존재하도록 tagging 하는데 이용될 수 있음을 알 수 있다. 10 is a diagram predicting the secondary structural topology of the PagP :: FLAG fusion protein expressed in the outer membrane of the Sakai-DM / pagP :: FLAG mutant strain prepared according to an embodiment of the present invention. The FLAG sequence, the external antigen, is fused to the loop-1 part of the PagP structure and the reading frame, and it can be used to tagging the external antigen to be protruded from the outer membrane body to the external environment.
다. Sakai-DM/All. Sakai-DM / pagPpagP ::FLAG 변이체에서 PagP::FLAG 융합단백질의 발현Expression of PagP :: FLAG Fusion Proteins in FLAG Variants
외막단백질인 PagP에 FLAG epitope을 외부항원으로 도입한 Sakai-DM 유래 변이주에서 융합단백질인 PagP::FLAG가 발현되어 검출되는지를 알아보기 위하여 FLAG epitope에 대한 monoclonal antibody (Sigma)를 구입하여 Western blot방법으로 검출을 시도하였다. 먼저, 비교하고자 하는 Sakai-DM균과 이 균의 pagP::FLAG 변이주에서 각각 외막 분획 (outer membrane fraction)을 획득하여 발현을 확인한 다음, 외막소체에서도 발현 정도를 확인하였다. 균주를 각기 배양시키고 균체를 수집하여 PBS로 한번 수세시키고, French press로 압착시켜 균체를 완전히 파쇄시켰다. 파쇄된 균액 (lysate)을 원심분리하여 완전히 파쇄되지 않은 cell debris를 제거시 킨 다음, 상층액을 초고속 원심분리(ultracentrifugation)하여, soluble한 세포질 단백질이 포함된 상층액과 파쇄된 멤브레인 (membrane) 조각들로 구성된 insoluble한 침전물(total membrane protein)을 각각 분리하였다. 세균 세포 내막과 외막이 혼합된 분획에 1% N-laurylsarcosine 용액을 1:1 비율로 혼합하여 실온에서 30분간 방치하여 N-laurylsarcosine (detergent)에 soluble한 세포내막 단백질들을 insoluble한 세포외막 단백질들과 원심 분리하였다. 세포외막 단백질들이 풍부한 분획에서 BCA kit (PIERCE)를 이용한 방법으로 단백질 함량을 정량하여 14% SDS-PAGE gel에 전기영동 한 다음, anti-FLAG 단클론성 항체를 이용하여 Western blot을 수행하였다. 그 결과, 변이체의 외막 단백질 분획에서 PagP::FLAG 융합단백질의 존재를 확인할 수 없었다. 이러한 이유는 변이체의 외막 분획에는 Western blot으로 검출 가능한 한계 보다 낮은 량의 PagP::FLAG 융합 단백질이 포함되었기 때문이라고 사료된다. 이러한 추정은 경험적으로 PagP의 발현이 대장균에서는 매우 낮아 PagP 단백질을 과발현 하지 않으면 검출이 되지 않았고, 또한 하기 실시예 4를 통하여 실시 방법상 아무런 문제가 없음을 확인하였기 때문이다. To examine whether PagP :: FLAG, a fusion protein, was expressed and detected in the Sakai-DM-derived mutant strain in which FLAG epitope was introduced into PagP as an outer antigen, a Western blot method was obtained by purchasing a monoclonal antibody (Sigma) against FLAG epitope. Detection was attempted with. First, the outer membrane fractions were obtained from the Sakai-DM and pagP :: FLAG mutant strains to be compared, respectively, and the expression levels were confirmed in the outer membrane bodies. The strains were incubated separately, and the cells were collected, washed once with PBS, and pressed by the French press to completely disrupt the cells. The lysate was centrifuged to remove cell debris that was not completely crushed, and then the supernatant was ultracentrifugated to give supernatant containing soluble cytoplasmic protein and fragmented membrane fragments. Insoluble precipitate (total membrane protein) consisting of each was isolated. 1% N-laurylsarcosine solution was mixed at a ratio of 1% N-laurylsarcosine solution in a mixture of bacterial cell inner and outer membranes, and left at room temperature for 30 minutes to obtain extracellular membrane proteins insoluble in N-laurylsarcosine (detergent). Centrifuged. Protein content was quantified by BCA kit (PIERCE) in the fraction enriched with extracellular membrane proteins, followed by electrophoresis on 14% SDS-PAGE gel, followed by Western blot using anti-FLAG monoclonal antibody. As a result, the presence of PagP :: FLAG fusion protein could not be confirmed in the outer membrane protein fraction of the variant. This may be due to the fact that the outer membrane fraction of the variant contained a lower amount of PagP :: FLAG fusion protein than the limit detected by Western blot. This estimation is because the expression of PagP is very low in Escherichia coli and was not detected without overexpressing the PagP protein, and it was confirmed that there was no problem in the implementation method through Example 4 below.
[실시예 4] Sakai-DM으로부터 Example 4 From Sakai-DM ompAompA ::FLAG tagged mutant의 제작 및 표현형 분석:: Production and Phenotypic Analysis of FLAG tagged Mutants
상기 실시예 3에서 기술한 바와 같은 전략으로 Sakai-DM의 외막에 외부항원인 FLAG epitope을 융합 발현되도록 하기 위하여, 외막에서 풍부하게 발현되는 OmpA 단백질을 FLAG 시퀀스의 융합 파트너로 삼아 새로운 ompA::FLAG 변이주를 제작하였다. In order to ensure that the FLAG epitope fusion strategy expression of foreign antigens in the outer membrane of the Sakai-DM to as described in Example 3, Sanya the OmpA protein abundantly expressed in the outer membrane as a fusion partner in the new FLAG sequence ompA :: FLAG Mutant strains were produced.
가. end. ompAompA ::FLAG 재조합 유전자 구축:: FLAG Recombinant Gene Construction
Sakai-DM에 새로운 변이 (ompA::FLAG)를 도입하여 외막에서 풍부하게 발현하는 외막단백질인 OmpA에 외부항원인 FLAG epitope를 융합시켜 발현하는 변이체를 제조하고자 ompA::FLAG-Cm cassette로 연결되어 구성된 재조합 DNA를 구축하였다. 먼저, ompA 유전자를 pBlueScript SK-II 벡터에 클로닝하고, pKD3 유래 Cm-cassette를 동일 플라스미드의 ompA 유전자 다음에 연결하여 삽입시킨 다음, 상동성 재조합이 용이하게 일어나도록 ompA 유전자 하류 (downstream)의 염기서열에 상동성을 가지는 약 350-bp 크기의 DNA 단편을 Cm-cassette 부분에 연결하여 전체적인 재조합 플라스미드를 제작하였다. 이 재조합 플라스미드 DNA를 site-directed mutagenesis를 위한 PCR의 template로 사용하였다. 상기 실시예 3에서 설명한 바과 같이, 융합하고자 하는 OmpA의 아미노산 부분에 FLAG epitope 아미노산 (DYKDHDGDYKDHDDD)을 연결한 다음, 번역 (translation)이 종료되도록 종결코돈을 포함하는 DNA 염기서열을 가진 78-bp 길이의 kinated mutagenic reverse primer (서열번호 15: GAGCCGGAGTCACTAATCGTCATCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCTTCGCCCTGACCGAAACG)를 제작하여, 1차 PCR을 수행하였다. 그 결과로 얻은 PCR 산물은 2차 PCR을 위한 megaprimer로 사용되어 짐으로써 특정 DNA 시퀀스에 변이가 도입된 DNA 단편을 제조하였다. Introduced a new variant ( ompA :: FLAG) in Sakai-DM and fused with ompA :: FLAG-Cm cassette to prepare a mutant expressing fusion of the outer antigen FLAG epitope to OmpA, an abundantly expressed outer membrane protein. Constructed recombinant DNA was constructed. First, the ompA gene is cloned into the pBlueScript SK-II vector, and the pKD3-derived Cm-cassette is inserted after the ompA gene of the same plasmid, and then inserted into the nucleotide sequence downstream of the ompA gene to facilitate homologous recombination. An about 350-bp sized DNA fragment having homology to the Cm-cassette moiety was used to construct an overall recombinant plasmid. This recombinant plasmid DNA was used as a template for PCR for site-directed mutagenesis. As described in Example 3, a 78-bp length having a DNA base sequence containing a stop codon to terminate the translation after linking the FLAG epitope amino acid (DYKDHDGDYKDHDDD) to the amino acid portion of OmpA to be fused A kinated mutagenic reverse primer (SEQ ID NO: 15: GAGCCGGAGTCACTA ATCGTCATCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTC TTCGCCCTGACCGAAACG) was prepared and primary PCR was performed. The resulting PCR product was used as a megaprimer for secondary PCR to prepare DNA fragments with mutations in specific DNA sequences.
이를 더욱 상세히 설명하면, OmpA의 3차원 접힘 구조상에서 외막을 관통하는 ㅯ-배럴(barrel) 부분 (mature OmpA 아미노산 1번 ~ 170번) 다음에 이어지는 카르복실기 말단(C-terminal) 쪽의 periplasmic 도메인 부분 (아미노산 171번 이하)에 FLAG 시퀀스를 fusion시킴으로써, OmpA의 C-terminal 부분이 truncation 되도록 OmpA::FLAG tagging을 시도하였다. 즉, 외막에 존재하는 OmpA를 구성하는 아미노산 169번부터 174번까지의 6개 아미노산 시퀀스 (RFGQGE: CGTTTCGGTCAGGGCGAA)에 연속해서 FLAG 시퀀스 (DYKDHDGDYKDHDDD: GACTACAAAGACCATGACGGTGATTATAAAGATCATGATGACGAT)를 연결하고, 이어서 OmpA의 아미노산 175번째부터 truncation이 일어나도록 종결코돈(stop codon: TAG-TGA)을 포함한 15-bp의 nucleotide(TAGTGACTCCGGCTC)를 연결시켜 작성한 78-bp 길이를 가진 mutagenic reverse primer (서열번호 15: GAGCCGGAGTCACTAATCGTCATCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCTTCGCCCTGACCGAAACG)를 제작하여, site-directed mutagenesis를 위한 PCR을 수행하였다. 즉, 상기의 방법으로 OmpA::FLAG 융합단백질이 발현되면 OmpA 아미노산 서열 중에서 베타-배럴을 도메인을 포함하는 1번~174번까지의 부분과 연결된 15개의 FLAG 시퀀스를 가진 OmpA::FLAG 외막 단백질이 합성되며, 외부항원으로 도입된 FLAG epitope은 앞선 OmpA의 베타-배럴 구조에 연결되어periplasmic space로 위치하게 됨으로 이러한 외막단백질이 발현된 변이체에서 외막소체를 분리하면 외부항원은 자연적으로 외막소체의 내부공간 (lumen)에 위치하게 된다 (도 12 참조).In more detail, on the three-dimensional folding structure of OmpA, the periplasmic domain portion on the carboxyl terminal (C-terminal) side that follows the membrane-barrel portion (mature
나. Sakai-DM으로부터 I. From Sakai-DM ompAompA ::FLAG 변이체의 제작:: Fabrication of FLAG Variants
상기 '가' 단계에서 구축한 ompA::FLAG 재조합 플라스미드를 주형으로 하여 ompA::FLAG-Cm cassette로 구성된 DNA 부분을 PCR로 증폭하여 이를 정제한 다음, Sakai-DM(pKD46) 균에 electroporation을 통하여 삽입함으로써, 균 내부에서 상동성 재조합 (homologous recombination)이 일어나도록 하여 외부에서 도입한 ompA::FLAG-Cm cassette 부분이 genome에 있는 ompA 유전자에 표적화 될 수 있도록 하였다. 이러한 결과로 생겨난 Cm-저항성 콜로니들을 PCR 반응을 통해 변이도입 부분을 증폭하여 그 크기가 예상대로 증가되었는지를 확인하고, 또한 DNA 염기서열 분석을 통하여 ompA::FLAG 융합이 reading frame에 맞게 이루어 졌음을 확인하였다. Using the ompA :: FLAG recombinant plasmid constructed in the above step 'A' as a template, DNA amplification of the ompA :: FLAG-Cm cassette was amplified and purified by PCR, and then electroporation to the Sakai-DM (pKD46) bacteria. By inserting, homologous recombination takes place inside the fungus so that the ompA :: FLAG-Cm cassette portion introduced from outside can be targeted to the ompA gene in the genome. PCR amplification of the resulting Cm-resistant colonies resulted in amplification of the mutagenesis region to confirm that the size increased as expected, and DNA sequencing confirmed that the ompA :: FLAG fusion was achieved in the reading frame. Confirmed.
다. Sakai-DM/All. Sakai-DM / ompAompA ::FLAG 변이체의 표현형 분석Phenotypic Analysis of FLAG Variants
상기한 바와 같이 제작한 Sakai-DM/ompA::FLAG 변이체의 외막 분획과 이 변이체에서 회수한 외막소체에서 도입한 FLAG 외부항원이 제대로 발현되어 존재하는 지를 알아보기 위하여, 실시예 3과 동일한 방법으로 외막 분획과 외막소체를 분리하였다. OmpA 단백질은 외막에서 풍부하게 발현하는 대표적 단백질이므로 OmpA::FLAG 융합단백질도 이 변이체의 외막에 풍부하게 존재할 것으로 예측하였다. 변이체를 배양하여 외막 분획과 외막소체를 분리한 다음 SDS-PAGE로 단백질을 전개하고 anti-FLAG 단클론성항체를 사용하여 웨스턴블롯을 실시한 결과 외막 분획과 외막소체에서 OmpA::FLAG 융합단백질에 해당하는 약 21 kDa의 분자량을 가진 특이적 밴드를 검출할 수 있었다 (도 11). In order to determine whether the outer membrane fraction of the Sakai-DM / ompA :: FLAG variant prepared as described above and the FLAG external antigen introduced from the outer membrane body recovered from the variant were properly expressed and present in the same manner as in Example 3 The outer membrane fraction and outer membrane body were separated. Since OmpA protein is a representative protein expressed abundantly in the outer membrane, it was predicted that OmpA :: FLAG fusion protein would also be abundantly present in the outer membrane of this variant. After culturing the mutants, the outer membrane fraction and the outer membrane body were separated, the protein was developed by SDS-PAGE, and Western blot was performed using anti-FLAG monoclonal antibody. The outer membrane fraction and the outer membrane body corresponded to the OmpA :: FLAG fusion protein. Specific bands with a molecular weight of about 21 kDa could be detected (FIG. 11).
이로부터, 실시예 3에서와 같이 외막에 발현하는 정도가 매우 낮은 PagP::FLAG 융합단백질은 검출 할 수 없었지만, 외막에서 발현이 풍부한 OmpA::FLAG 융합단백질은 쉽게 검출 할 수 있음을 알 수 있었다.From this, it was found that PagP :: FLAG fusion protein, which is very low in expression in the outer membrane, as in Example 3, was not detected, but that OmpA :: FLAG fusion protein, which is rich in expression in the outer membrane, was easily detected. .
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다. The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
도 1은 본 발명에서 설명하는 외막소체를 생성하는 G(-) 박테리아에서 외막소체가 형성되어 외막으로부터 분출되어 나오는 그림으로, 본 발명에서 개시한 실시예에 따라 변이체들을 제작하여, LPS의 내독성을 제거하고 다가항원성을 지닌 외막소체를 생산하는 기본적인 생산 플랫폼을 간단히 나타낸 모식도이다.1 is an illustration of the outer membrane formed in the G (-) bacteria to produce the outer shell body described in the present invention is ejected from the outer membrane, by producing variants according to the embodiment disclosed in the present invention, LPS It is a schematic diagram showing a basic production platform that removes and produces an outer membrane body with polyvalent antigenicity.
도 2는 본 발명의 일 실시예에 따른 변이주 (Sakai ΔmsbB1/ΔmsbB2 mutant)를 제작하기 위하여 사용한 방법을 모식화한 그림으로 G(-) 박테리아에서 염색체 상의 목표 유전자를 선택적으로 결실 (deletion) 또는 불활화 (inactivation) 시키는 기본적 시스템을 나타낸다.FIG. 2 is a diagram schematically illustrating a method used to prepare a mutant strain (Sakai Δ msbB1 / Δ msbB2 mutant) according to an embodiment of the present invention. Selective deletion of a target gene on a chromosome from a G (−) bacterium. Or basic system of inactivation.
도 3은 본 발명의 일 실시예에 따라 제작한 Sakai ΔmsbB1/ΔmsbB2 mutant에서 msbB 유전자 부위의 유전자 변이를 확인하기 위하여 실시한 PCR 산물의 전기영동 사진이다(lanes M: size marker, 1: intact msbB1, 2: ΔmsbB1, 3: intact msbB2, 4: ΔmsbB2).Figure 3 is an electrophoretic image of a PCR product performed to identify the genetic variation of the msbB gene region in Sakai Δ msbB1 / Δ msbB2 mutant prepared according to an embodiment of the present invention (lanes M: size marker, 1: intact msbB1 , 2: Δ msbB1 , 3: intact msbB2 , 4: Δ msbB2 ).
도 4는 본 발명의 일 실시예에 따른 변이주들 (Sakai-M1, Sakai-DM)로부터 분리된 LPS를 SDS-PAGE 방법으로 분자량에 따라 이동시킨 다음 silver stain으로 염색하여 나타낸 사진이다(WT: wild type Sakai, M1: Sakai-M1, DM: Sakai-DM).FIG. 4 is a photograph showing LPS isolated from mutant strains (Sakai-M1, Sakai-DM) according to an embodiment of the present invention and stained with silver stain according to molecular weight by SDS-PAGE method (WT: wild type Sakai, M1: Sakai-M1, DM: Sakai-DM).
도 5는 본 발명의 일 실시예에 따른 변이주들 (Sakai-M1, Sakai-DM)로부터 [32P]로 표지한 lipid A를 분리한 후, lipid A의 acylation 상태를 알아보기 위하여 TLC를 수행한 사진이다(Lanes 1: wild type Sakai, 2: Sakai-M1, 3: Sakai-DM).5 is isolated from lipids labeled [ 32 P] from mutants (Sakai-M1, Sakai-DM) according to an embodiment of the present invention, TLC was performed to determine the acylation state of lipid A Photograph (Lanes 1: wild type Sakai, 2: Sakai-M1, 3: Sakai-DM).
도 6은 본 발명의 일 실시예에 따라 제작된 변이체들을 배양하여 이들로부터 생성되는 외막소체(OMV)를 전자현미경 (TEM)으로 관찰한 사진이다(Panel A: 균체를 배양한 다음 배지 상청액에 분리되어 나온 외막소체를 회수하여 전자현미경으로 관찰한 사진, Panel B: 균체의 외막으로부터 분출되어 나오는 외막소체를 고정하여 관찰한 사진).Figure 6 is a photograph observing the outer membrane body (OMV) produced from them by culturing the variants prepared according to an embodiment of the present invention with an electron microscope (TEM) (Panel A: cultured cells and then separated in the medium supernatant Collected and observed by electron microscope, Panel B: fixed and observed outer membrane body ejected from the outer membrane of the cell).
도 7은 본 발명의 일 실시예에서 개시한 외부항원을 G(-) 박테리아의 외막단백질에 융합발현되도록 재조합 DNA를 제작하여 이 재조합 DNA가 도입된 변이체를 만들기 위한 전략적 모식도이다. Figure 7 is a strategic schematic diagram for making a recombinant DNA prepared by fusion expression of the outer antigen disclosed in one embodiment of the present invention to the outer membrane protein of G (-) bacteria to make a variant introduced into the recombinant DNA.
도 8은 본 발명의 일 실시예에 따라 제작된 변이체들의 lipid A의 acylation 상태를 확인한 TLC 크로마토그래피이다(Lanes 1: wild type Sakai, 2: Sakai-M1(msbB1), 3: Sakai-M2 (msbB2), 4: Sakai-DM/pagP::FLAG, 5: Sakai-DM).8 is TLC chromatography confirming the acylation state of lipid A of the variants prepared according to an embodiment of the present invention (Lanes 1: wild type Sakai, 2: Sakai-M1 ( msbB1 ), 3: Sakai-M2 ( msbB2) ), 4: Sakai-DM / pagP :: FLAG, 5: Sakai-DM).
도 9는 도 8의 TLC 상에서 나타난 lipid A spot에 해당하는 화학적 구조를 도해한 그림이다. 9 is a diagram illustrating a chemical structure corresponding to lipid A spot shown on TLC of FIG. 8.
도 10은 본 발명의 일 실시예에 따라 제작된 Sakai-DM/pagP::FLAG 변이주의 외막에 발현하는 PagP::FLAG 융합단백질의 2차 구조적 topology를 예측한 그림이다.10 is a diagram predicting the secondary structural topology of the PagP :: FLAG fusion protein expressed in the outer membrane of the Sakai-DM / pagP :: FLAG mutant strain prepared according to an embodiment of the present invention.
도 11은 본 발명의 일 실시예에 따라 제작된 Sakai-DM/ompA::FLAG 변이체의 OmpA::FLAG 발현을 확인하기 위한 웨스턴블롯 사진이다.FIG. 11 is a Western blot photograph for confirming OmpA :: FLAG expression of a Sakai-DM / ompA :: FLAG variant prepared according to one embodiment of the present invention. FIG.
도 12는 본 발명의 일 실시예에 따른 OmpA::FLAG 단백질의 변이체 외막에서의 2차 구조적인 topology를 예측한 그림이다.12 is a diagram predicting the secondary structural topology of the outer membrane of the variant of OmpA :: FLAG protein according to an embodiment of the present invention.
<110> Korea Research Institute of Bioscience and Biotechnology <120> Recombinant Gram-negative Bacteria Producing Outer Membrane Vesicles and Method for Preparing Outer Membarne Vesicles Tagged with Foreign Epitopes Using the Same <130> P07-B261 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> M1-Fw primer <400> 1 tgtcgctctg ctttccagaa cgtagtgaag ctgaacgcga gtgtaggctg gagctgcttc 60 g 61 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> M1-Rev primer <400> 2 tctcgacttc ttcattcatc cgccgcgcaa tcgtatgatc catatgaata tcctccttag 60 60 <210> 3 <211> 2804 <212> DNA <213> Artificial Sequence <220> <223> Template plasmid pKD3 <400> 3 agattgcagc attacacgtc ttgagcgatt gtgtaggctg gagctgcttc gaagttccta 60 tactttctag agaataggaa cttcggaata ggaacttcat ttaaatggcg cgccttacgc 120 cccgccctgc cactcatcgc agtactgttg tattcattaa gcatctgccg acatggaagc 180 catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg 240 tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta 300 aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa 360 accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt 420 gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt 480 gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt 540 tcattgccat acgtaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg 600 ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct 660 gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac 720 gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt 780 ccttagctcc tgaaaatctc gacaactcaa aaaatacgcc cggtagtgat cttatttcat 840 tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg 900 cccagggctt cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt 960 ccgtcacagg taggcgcgcc gaagttccta tactttctag agaataggaa cttcggaata 1020 ggaactaagg aggatattca tatggaccat ggctaattcc catgtcagcc gttaagtgtt 1080 cctgtgtcac tgaaaattgc tttgagaggc tctaagggct tctcagtgcg ttacatccct 1140 ggcttgttgt ccacaaccgt taaaccttaa aagctttaaa agccttatat attctttttt 1200 ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt 1260 gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacgttag ccatgagagc 1320 ttagtacgtt agccatgagg gtttagttcg ttaaacatga gagcttagta cgttaaacat 1380 gagagcttag tacgtgaaac atgagagctt agtacgtact atcaacaggt tgaactgcgg 1440 atcttgcggc cgcaaaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 1500 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 1560 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 1620 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 1680 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 1740 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 1800 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 1860 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 1920 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 1980 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 2040 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 2100 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 2160 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 2220 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 2280 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 2340 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 2400 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 2460 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgcat cgatggcccc 2520 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa 2580 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct 2640 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga 2700 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc 2760 ctgacggatg gcctttttgc gtggccagtg ccaagcttgc atgc 2804 <210> 4 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> M2-Fw primer <400> 4 gacgtggaca ggtatcggta ttatctgtgt gtttgcaatg gtgtaggctg gagctgcttc 60 g 61 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> M2-Rev primer <400> 5 tcgcgcaata tgagcatcac tcttctgtcg tatagcaagt catatgaata tcctccttag 60 60 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MsbB1-Fw primer <400> 6 tcgcgaattc ctcgagcagg ccaa 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MsbB1-Rev primer <400> 7 ctgaagcaag cttgaactta tca 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MsbB2-Fw primer <400> 8 ctcactgatc tcgagactct tcc 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MsbB2-Rev primer <400> 9 gctgggtaag cttattatcc tga 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> For-degP primer <400> 10 tgggttccgg cgtcatcatt gat 23 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Rev-degP primer <400> 11 cgatctgcgc agccggagtg cc 22 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> FLAG epitope <400> 12 Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Asp Asp 1 5 10 15 <210> 13 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> kinated mutagenic reverse primer <400> 13 cggtcgctcg ttataatcgt catcatgatc tttataatca ccgtcatggt ctttgtagtc 60 agcgaaacgt gcatgcca 78 <210> 14 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> P2H2 reverse primer <400> 14 acacaaatgc tgtgtcggtt accagtacac caattgtggt accatatgaa tatcctcctt 60 ag 62 <210> 15 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> kinated mutagenic reverse primer <400> 15 gagccggagt cactaatcgt catcatgatc tttataatca ccgtcatggt ctttgtagtc 60 ttcgccctga ccgaaacg 78 <110> Korea Research Institute of Bioscience and Biotechnology <120> Recombinant Gram-negative Bacteria Producing Outer Membrane Vesicles and Method for Preparing Outer Membarne Vesicles Tagged with Foreign Epitopes Using the Same <130> P07-B261 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> M1-Fw primer <400> 1 tgtcgctctg ctttccagaa cgtagtgaag ctgaacgcga gtgtaggctg gagctgcttc 60 g 61 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> M1-Rev primer <400> 2 tctcgacttc ttcattcatc cgccgcgcaa tcgtatgatc catatgaata tcctccttag 60 60 <210> 3 <211> 2804 <212> DNA <213> Artificial Sequence <220> <223> Template plasmid pKD3 <400> 3 agattgcagc attacacgtc ttgagcgatt gtgtaggctg gagctgcttc gaagttccta 60 tactttctag agaataggaa cttcggaata ggaacttcat ttaaatggcg cgccttacgc 120 cccgccctgc cactcatcgc agtactgttg tattcattaa gcatctgccg acatggaagc 180 catcacaaac ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg 240 tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta 300 aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa 360 accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt 420 gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt 480 gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt 540 tcattgccat acgtaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg 600 ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct 660 gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac 720 gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt 780 ccttagctcc tgaaaatctc gacaactcaa aaaatacgcc cggtagtgat cttatttcat 840 tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc caaaagttgg 900 cccagggctt cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt 960 ccgtcacagg taggcgcgcc gaagttccta tactttctag agaataggaa cttcggaata 1020 ggaactaagg aggatattca tatggaccat ggctaattcc catgtcagcc gttaagtgtt 1080 cctgtgtcac tgaaaattgc tttgagaggc tctaagggct tctcagtgcg ttacatccct 1140 ggcttgttgt ccacaaccgt taaaccttaa aagctttaaa agccttatat attctttttt 1200 ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt 1260 gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacgttag ccatgagagc 1320 ttagtacgtt agccatgagg gtttagttcg ttaaacatga gagcttagta cgttaaacat 1380 gagagcttag tacgtgaaac atgagagctt agtacgtact atcaacaggt tgaactgcgg 1440 atcttgcggc cgcaaaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 1500 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 1560 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 1620 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 1680 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 1740 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 1800 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt 1860 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 1920 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 1980 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 2040 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 2100 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag 2160 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 2220 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 2280 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 2340 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 2400 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 2460 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgcat cgatggcccc 2520 ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa 2580 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct 2640 ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga 2700 gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc 2760 ctgacggatg gcctttttgc gtggccagtg ccaagcttgc atgc 2804 <210> 4 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> M2-Fw primer <400> 4 gacgtggaca ggtatcggta ttatctgtgt gtttgcaatg gtgtaggctg gagctgcttc 60 g 61 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> M2-Rev primer <400> 5 tcgcgcaata tgagcatcac tcttctgtcg tatagcaagt catatgaata tcctccttag 60 60 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MsbB1-Fw primer <400> 6 tcgcgaattc ctcgagcagg ccaa 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MsbB1-Rev primer <400> 7 ctgaagcaag cttgaactta tca 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MsbB2-Fw primer <400> 8 ctcactgatc tcgagactct tcc 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> MsbB2-Rev primer <400> 9 gctgggtaag cttattatcc tga 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> For-degP primer <400> 10 tgggttccgg cgtcatcatt gat 23 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Rev-degP primer <400> 11 cgatctgcgc agccggagtg cc 22 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> FLAG epitope <400> 12 Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Asp Asp 1 5 10 15 <210> 13 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> kinated mutagenic reverse primer <400> 13 cggtcgctcg ttataatcgt catcatgatc tttataatca ccgtcatggt ctttgtagtc 60 agcgaaacgt gcatgcca 78 <210> 14 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> P2H2 reverse primer <400> 14 acacaaatgc tgtgtcggtt accagtacac caattgtggt accatatgaa tatcctcctt 60 ag 62 <210> 15 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> kinated mutagenic reverse primer <400> 15 gagccggagt cactaatcgt catcatgatc tttataatca ccgtcatggt ctttgtagtc 60 ttcgccctga ccgaaacg 78
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| KR20080072858A KR101042541B1 (en) | 2008-07-25 | 2008-07-25 | Recombinant Gram-negative Bacteria Producing Outer Membrane Vesicles and Method for Preparing Outer Membrane Vesicles Tagged with Foreign Epitopes Using the Same |
| PCT/KR2008/004811 WO2010010983A1 (en) | 2008-07-25 | 2008-08-19 | Recombinant gram-negative bacteria producing outer membrane vesicles and method for preparing outer membrane vesicles tagged with foreign epitopes using the same |
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Cited By (5)
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| WO2012002760A3 (en) * | 2010-07-01 | 2012-05-03 | 포항공과대학교 산학협력단 | Method for treating and diagnosing cancer by using cell-derived microvesicles |
| KR20160028869A (en) * | 2014-09-04 | 2016-03-14 | 한국과학기술연구원 | Composition and kit for dephosphorylating lipid, and method for the production of dephosphorylated lipid using the same |
| WO2017150755A1 (en) * | 2016-02-29 | 2017-09-08 | (주)비손바이오로직스 | Inactivated trivalent salmonella vaccine composition reduced in endotoxin level for prevention of fowl typhoid and salmonellosis |
| CN111454979A (en) * | 2020-04-03 | 2020-07-28 | 江苏省农业科学院 | Method for improving yield of bacterial outer membrane vesicles and application thereof |
| CN113755514A (en) * | 2021-09-08 | 2021-12-07 | 西北农林科技大学 | Construction method of escherichia coli mutant and preparation method of outer membrane vesicle |
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| EP2617833A1 (en) * | 2012-01-18 | 2013-07-24 | Centre National de la Recherche Scientifique (CNRS) | A method for specifically detecting living bacteria |
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| AU2013320313B2 (en) | 2012-09-18 | 2018-07-12 | Glaxosmithkline Biologicals Sa | Outer membrane vesicles |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| GB9918319D0 (en) * | 1999-08-03 | 1999-10-06 | Smithkline Beecham Biolog | Vaccine composition |
| PL399492A1 (en) * | 2002-08-02 | 2012-11-19 | Glaxosmithkline Biologicals S.A. | Vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2012002760A3 (en) * | 2010-07-01 | 2012-05-03 | 포항공과대학교 산학협력단 | Method for treating and diagnosing cancer by using cell-derived microvesicles |
| US9066971B2 (en) | 2010-07-01 | 2015-06-30 | Postech Academy-Industry Foundation | Method for treating and diagnosing cancer by using cell-derived microvesicles |
| KR20160028869A (en) * | 2014-09-04 | 2016-03-14 | 한국과학기술연구원 | Composition and kit for dephosphorylating lipid, and method for the production of dephosphorylated lipid using the same |
| WO2017150755A1 (en) * | 2016-02-29 | 2017-09-08 | (주)비손바이오로직스 | Inactivated trivalent salmonella vaccine composition reduced in endotoxin level for prevention of fowl typhoid and salmonellosis |
| CN111454979A (en) * | 2020-04-03 | 2020-07-28 | 江苏省农业科学院 | Method for improving yield of bacterial outer membrane vesicles and application thereof |
| CN111454979B (en) * | 2020-04-03 | 2023-06-06 | 江苏省农业科学院 | Method for improving bacterial outer membrane vesicle yield and application thereof |
| CN113755514A (en) * | 2021-09-08 | 2021-12-07 | 西北农林科技大学 | Construction method of escherichia coli mutant and preparation method of outer membrane vesicle |
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| WO2010010983A1 (en) | 2010-01-28 |
| KR101042541B1 (en) | 2011-06-17 |
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