KR101631312B1 - Immunostimulatory Compositions Comprising Recombinant Giardia lamblia binding immunoglobulin protein - Google Patents
Immunostimulatory Compositions Comprising Recombinant Giardia lamblia binding immunoglobulin protein Download PDFInfo
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- KR101631312B1 KR101631312B1 KR1020140072731A KR20140072731A KR101631312B1 KR 101631312 B1 KR101631312 B1 KR 101631312B1 KR 1020140072731 A KR1020140072731 A KR 1020140072731A KR 20140072731 A KR20140072731 A KR 20140072731A KR 101631312 B1 KR101631312 B1 KR 101631312B1
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Abstract
본 발명은 재조합 람블편모충 결합 면역글로블린 단백질을 포함하는 면역증강 조성물에 관한 것이다. 본 발명은 동정된 재조합 람블편모충 결합 면역글로블린 단백질(recombinant G. lamblia binding immunoglobulin protein, rGlBiP)이 마우스 수지상 세포에서 사이토카인 생산 및 수지상 세포의 성숙과 같은 면역반응을 통해 면역 활성화를 크게 향상시킴으로써 면역증진용 제제로서 유용하게 이용될 수 있으며, 본 발명은 재조합 람블편모충 결합 면역글로블린 단백질을 이용하여 면역원성을 갖는 에피토프의 스크리닝 방법 및 이의 항체 스크리닝 방법에 유용하게 이용될 수 있다. The present invention is directed to an immunomodulating composition comprising a recombinant gibb-binding immunoglobulin protein. The present invention relates to a recombinant G. lamblia binding immunoglobulin protein (rGlBiP), which is a recombinant G. lamblia binding immunoglobulin protein (rGlBiP), which enhances immune activation through immune responses such as cytokine production and dendritic cell maturation in mouse dendritic cells, The present invention can be usefully used for screening an epitope having immunogenicity and a method for screening an antibody using the recombinant gabbial binding immunoglobulin protein.
Description
본 발명은 재조합 람블편모충 결합 면역글로블린 단백질을 포함하는 면역증강 조성물에 관한 것이다.
The present invention is directed to an immunomodulating composition comprising a recombinant gibb-binding immunoglobulin protein.
람블편모충(Giardia lamblia)은 인간을 포함하는 포유동물에서 장내 흡수불량 및 설사를 야기하는 원생동물 기생충이다(1). 람블편모충증(Giardiasis)은 무증상부터 심각한 설사까지 다양한 임상적 발현 방식을 갖는다. 람블편모충 영양형은 조직으로 침투하지 못한다 하더라도, 장내 표피세포의 점막 표면은 람블편모충의 항원 발현에 의해 자극 받을 수 있다. 따라서, 숙주 면역체계에 의해 인지된 람블편모충의 항원은 람블편모충의 감염 동안 면역 반응의 조절을 이해하기 위해 그 메커니즘이 해명될 필요가 있다(3). 람블편모충 클론 GS/M-83-H7 및 GlVSP 유래의 변이체 표면 단백질은 람블편모충에 감염된 인간(6, 7) 뿐만 아니라 마우스(4, 5)에서 1차 면역반응을 유도하는 항원으로 고려된다. GlVSP는 GIVSP가 in vivo에서 연속적으로 변화하고, 그렇게 함으로써 람블편모충이 숙주 면역 반응을 회피하게 만들기 때문에 람블편모충 항원의 변이에 원인이 된다. 또한, α -1 지아르딘(giardin)은 원형질막-결합 능력 및 글라이코사미노글라이칸-결합 능력을 갖는 면역우성 단백질이라는 것이 보고되었다(9). 람블편모충에 감염된 환자로부터 수득한 혈청은 또한 88 kDa의 표면 단백질(10) 및 람블편모충으로부터 수득된 지방산 결합 단백질 분획물(11)에 강한 면역 반응을 보여주었다. Giardia lamblia is a protozoan parasite that causes intestinal malabsorption and diarrhea in mammals, including humans (1). Giardiasis has various clinical modes of expression, ranging from asymptomatic to severe diarrhea. Even if the gingival nematode does not penetrate into the tissue, the mucosal surface of the intestinal epidermal cells may be stimulated by the antigen expression of the gingival bract. Therefore, antigens of gabbial glands recognized by the host immune system need to be elucidated to understand the regulation of the immune response during the infection of gabbial glands (3). Mutant surface proteins derived from the gibbsite clone GS / M-83-H7 and GlVSP are considered as antigens inducing primary immune responses in mice (4,5) as well as humans (6,7) infected with gambia biloba. GlVSP causes GIVSP to change continuously in vivo , thereby causing mutation of gambia biloba antigens, since gabbial germs avoid host immune responses. Furthermore, it has been reported that α-1 giardin is an immunoprivile protein having plasma membrane-binding ability and glycosaminoglycan-binding ability (9). The serum obtained from patients infected with gambia bollworm also showed a strong immune response to the surface protein (10) of 88 kDa and the fatty acid binding protein fraction (11) obtained from gambia.
람블편모충의 감염은 대개 자기 제한적이고, 이것은 효과적인 숙주 방어의 존재를 나타낸다(12). 랑포드(Langford) 등(13, 14)은 B 세포 매개 면역 반응이 람블편모충 감염의 조절에 있어서 중요한 역할을 한다는 것을 보고하였다. 감염된 숙주에서 T 세포의 고갈은 또한 람블편모충이 감염된 마우스로부터 제거되는 것을 방해하고, 이것은 만성 람블편모충증을 유도한다(15). 인터루킨-6(이하 IL-6라 함)는 람블편모충에 감염된 마우스에서 향상된 수준으로 발견되었다(16). 람블편모충 감염의 조절은 야생형 마우스 보다 IL-6가 결핍된 마우스에서 더 느렸고(17), 야생형 마우스에서 비만세포(mast cell)는 중요한 역할을 하였다(18). 또한 람블편모충의 방출에 노출된 상피세포가 람블편모충 케모카인(C-C 모티프) 리간드 20을 방출한다는 것이 보고되었다(19). 넉아웃(knock out) 마우스를 이용하여 in vivo 감염과 함께 마우스에서 람블편모충 감염에 대한 마이크로어레이 분석은 iNOS(inducible nitric oxide synthase) 및 기질금속단백질 분해효소 7(matrix metalloprotease, MMP7)이 람블편모충 감염의 조절에 있어서 중요한 역할을 한다는 것을 입증하였다(20).
Infection with gibbsite larvae is usually self-limited, indicating the presence of effective host defense (12). Langford et al. (13, 14) reported that B cell mediated immune responses play an important role in the control of glioblastoma infection. Depletion of T cells in infected hosts also prevents the gibbsite from being removed from infected mice, leading to chronic gibberellosis (15). Interleukin-6 (hereinafter referred to as IL-6) was found at an enhanced level in mice infected with gambia biloba (16). Regulation of glioblastoma infection was slower in mice deficient in IL-6 than in wild-type mice (17), and mast cells in wild-type mice played an important role (18). It has also been reported that epithelial cells exposed to the release of gabbial bollworm release gambrel bilemenia chemokine (CC motif) ligand 20 (19). Microarray analysis of glioblastoma infection in mice with in vivo infection using knock out mice showed that inducible nitric oxide synthase (iNOS) and matrix metalloprotease 7 (MMP7) (20). It has also been shown that it plays an important role in the regulation of inflammation.
한편, 람블편모충에 대한 다중클로날 뮤린(murin) 항체를 이용한 항원 물질의 연구를 위해서, 본 발명자들은 람블편모충 결합 면역글로블린 단백질(G. lamblia binding immunoglobulin protein, GlBiP)을 면역 반응성 단백질로 동정하였다. Meanwhile, for the study of antigenic material using a multiclonal murine antibody against gabbial germs, the present inventors identified G. lamblia binding immunoglobulin protein (GlBiP) as an immunoreactive protein.
포유동물 시스템에서, BiP(binding immunoglobulin protein, 이하 Bip라고 함)는 초기에 포도당 조절 단백질 78(glucose regulated protein 78, Grp78)로 명명되었고(21), 이후 Bip가 면역글로블린 중쇄(heavy chain)에 결합되어 있다는 것이 발견되었을 때, 재명명되었다(22). BiP는 열충격 단백질 70(heat shock protein 70, Hsp70, 이하 Hsp70이라 함) 패밀리의 잘 알려진 멤버이다. 즉, 상기 패밀리 단백질은 소포체에서 분자적 사프론(molecular chaperone)으로 작용한다(23). In the mammalian system, BiP (binding immunoglobulin protein, hereinafter referred to as Bip) was first named glucose regulated protein 78 (Grp78) (21), and then Bip was bound to the immunoglobulin heavy chain When it was discovered that it had been (22). BiP is a well-known member of the heat shock protein 70 (Hsp70, hereinafter referred to as Hsp70) family. That is, the family protein acts as a molecular chaperone in the endoplasmic reticulum (23).
Hsp70 패밀리에 속하는 단백질은 면역반응에서 중요한 항상성 또는 생리학적 신호전달 네트워크를 제공하는 다양한 세포 표면 수용기에 결합함으로써 항상성(homeostatic) 또는 물리적 면역 효과를 발휘하는 염증전 작용 또는 항염증 작용과 함께 강한 세포간 신호전달 분자로 보고되어 왔다(24). Proteins belonging to the Hsp70 family bind to various cell surface receptors that provide important homeostasis or physiological signaling networks in the immune response, resulting in an inflammatory or anti-inflammatory effect that exerts homeostatic or physical immunological effects, Have been reported as signaling molecules (24).
Hsp70는 다양한 원생동물 기생충, 예컨대 리슈마니아(Leishmania), 말라리아원충(Plasmodium), 톡소플라스마 곤디이(Toxoplasma gondii) 및 트리파노소마(Trypanosome spp.)에서 강한 항원이 되는 것으로 알려져 있다(25). Hsp70 is known to be a strong antigen in a variety of protozoan parasites such as Leishmania, Plasmodium, Toxoplasma gondii and Trypanosoma spp. (25).
본 발명자들은 뮤린 수지상 세포의 활성화에 대한 효과를 측정하기 위하여 람블편모충 감염의 적응면역 반응에서 BiP 상동체의 역할에 대하여 람블편모충의 BiP 상동체(homolog)를 평가하였다.
The present inventors evaluated the BiP homologue of the gibbons on the role of the BiP homolog in the adaptive immune response of the gibberellin infection in order to measure the effect on the activation of murine dendritic cells.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 동정된 재조합 람블편모충 결합 면역글로블린 단백질(recombinant G. lamblia binding immunoglobulin protein, rGlBiP)이 마우스 수지상 세포에서 사이토카인 생산 및 수지상 세포의 성숙과 같은 면역반응을 통해 면역 활성화를 크게 향상시킴으로써 면역증진용 제제로서 유용하게 이용될 수 있고, 상기 재조합 람블편모충 결합 면역글로블린 단백질을 이용하여 면역원성을 갖는 에피토프의 스크리닝 방법 및 이의 항체 스크리닝 방법에 유용하게 이용될 수 있다는 것을 규명함으로써, 본 발명을 완성하게 되었다. The present inventors have found that recombinant G. lamblia binding immunoglobulin protein (rGlBiP), which is an identified recombinant G. lamblia binding immunoglobulin protein, significantly enhances immune activation through immune responses such as cytokine production and dendritic cell maturation in mouse dendritic cells, And that the recombinant gabbial binding immunoglobulin protein can be usefully used as an agent for screening an epitope having immunogenic properties and the antibody screening method thereof, .
따라서, 본 발명의 목적은 람블편모충 결합 면역글로블린 단백질(Giardia lamblia binding immunoglobulin protein) 또는 그의 단편의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 면역증진용 약제학적 조성물을 제공하는데 있다. Accordingly, an object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically effective amount of a Giardia lamblia binding immunoglobulin protein or a fragment thereof; And (b) a pharmaceutically acceptable carrier.
본 발명의 다른 목적은 람블편모충 결합 면역글로블린 단백질 또는 그의 단편을 포함하는 면역원성을 가지는 에피토프의 스크리닝 방법을 제공하는데 있다. It is another object of the present invention to provide a method for screening an epitope having an immunogenic property comprising a gambia copepod binding immunoglobulin protein or a fragment thereof.
본 발명의 또 다른 목적은 람블편모충 결합 면역글로블린 단백질 또는 그의 단편을 포함하는 항원에 대한 항체의 스크리닝 방법을 제공하는데 있다.
It is another object of the present invention to provide a method for screening an antibody against an antigen comprising a gibbsite immunoglobulin protein or a fragment thereof.
본 발명의 목적 및 이점은 하기의 발명의 상세한 설명 및 청구범위에 의해 보다 명확하게 된다.
The objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.
본 발명의 일 양태에 따르면, 본 발명은 (a) 람블편모충 결합 면역글로블린 단백질(Giardia lamblia binding immunoglobulin protein) 또는 그의 단편의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 면역증진용 약제학적 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of a Giardia lamblia binding immunoglobulin protein or a fragment thereof; And (b) a pharmaceutically acceptable carrier.
본 발명자들은 동정된 재조합 람블편모충 결합 면역글로블린 단백질(recombinant G. lamblia binding immunoglobulin protein, rGlBiP)이 마우스 수지상 세포에서 사이토카인 생산 및 수지상 세포의 성숙과 같은 면역반응을 통해 면역 활성화를 크게 향상시킴으로써 면역증진용 제제로서 유용하게 이용될 수 있다는 사실을 규명하였다. The present inventors have found that recombinant G. lamblia binding immunoglobulin protein (rGlBiP), which is an identified recombinant G. lamblia binding immunoglobulin protein, significantly enhances immune activation through immune responses such as cytokine production and dendritic cell maturation in mouse dendritic cells, And can be usefully used as a pharmaceutical preparation.
본 명세서에서 용어“면역자극(immunostimulatory)”은 초기 면역 반응을 유도하거나 항원에 대한 기존의 면역 반응을 측정 가능할 정도로 증가시키는 것을 말한다.The term " immunostimulatory " as used herein refers to inducing an initial immune response or increasing the existing immune response to an antigen to a measurable extent.
본 명세서에서 용어“재조합 람블편모충 결합 면역글로블린 단백질(recombinant G. lamblia binding immunoglobulin protein, rGlBiP)”은 람블편모충의 결합 면역글로블린 단밸질(BiP) 상동체(GL50581_3283)(sequence coverage, 36%: MASCOT score, 1227)를 의미하고, 보다 구체적으로 1281bp의 DNA 염기서열을 암호화하는 N-말단 부위(서열목록 제 1서열의 1 내지 427번째 아미노산)와 753bp의 DNA 염기서열을 암호화하는 C-말단 부위(서열목록 제 1서열의 428 내지 677번째 아미노산)를 포함하는 단백질(서열목록 제 1서열)을 의미하며, 재조합 GlBiP 또는 rGlBiP와 동일한 용어로 혼용하여 사용한다. As used herein, the term " recombinant G. lamblia binding immunoglobulin protein (rGlBiP) " refers to a combination immunoglobulin homologous (BiP) homologue (GL50581_3283) (sequence coverage, 36%: MASCOT score, 1227), and more specifically, an N-terminal region (1st to 427th amino acids of Sequence Listing 1) encoding a DNA sequence of 1281 bp and a C-terminal region encoding a DNA base sequence of 753 bp (Amino acid 428 to 677 of the first sequence) (SEQ ID No. 1) and is used in combination with the same terms as recombinant GlBiP or rGlBiP.
본 명세서에서 용어“재조합 람블편모충 결합 면역글로블린 단백질의 단편”은 상기 재조합 람블편모충 결합 면역글로블린 단백질의 일부 아미노산 서열을 포함하는 단백질 단편을 의미하고, 보다 구체적으로 N-말단 또는 C-말단을 포함하는 단백질을 의미한다. As used herein, the term " fragment of recombinant gibbsite binding immunoglobulin protein " means a protein fragment comprising a part of the amino acid sequence of the recombinant gibberellin-binding immunoglobulin protein, and more specifically, Protein.
본 발명의 일구현예에 따르면, 상기 람블편모충 결합 면역글로블린 단백질의 단편은 재조합 람블편모충 결합 면역글로블린 단백질의 C-말단, 즉 서열목록 제1서열의 428 내지 677번째 아미노산을 포함한다. According to one embodiment of the present invention, the fragment of the gibbsite immunoglobulin protein comprises the C-terminus of the recombinant gibberellin-binding immunoglobulin protein, i.e., the 428th to 677th amino acids of the first sequence of the sequence listing.
본 발명의 조성물에는 기타 약물 또는 다른 면역 보조제가 포함되어 추가적인 면역 자극 효과를 제공할 수 있다. 추가되는 면역 보조제는 예를 들어 리포폴리사카라이드(LPS), 프레운트 완전 보조제(Freund's complete adjuvant), 유지, 알루미늄염, 크레스틴(Krestin), 렌티난(Lentinan) 및 AHCC(Active Hexose Correlated Compound)를 포함하나 이에 제한되는 것은 아니다. The composition of the present invention may include other drugs or other adjuvants to provide additional immunostimulatory effects. Additional adjuvants include, for example, lipopolysaccharide (LPS), Freund ' s complete adjuvant, fat, aluminum salts, Krestin, Lentinan and AHCC (Active Hexose Correlated Compound) But are not limited thereto.
본 발명의 면역 증강용 조성물은 다양한 질환에 적용될 수 있으며, 바람직하게는 (i) 암(예컨대, 위암, 폐암, 유방암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 대장암, 결장암, 자궁경부암, 뇌암, 전립선암, 골암, 피부암, 갑상선암, 부갑상선암 및 요관암); The composition for immuno-enhancement of the present invention can be applied to various diseases, and is preferably used for various diseases such as (i) cancer (such as gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, , Colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, parathyroid cancer and urothelial cancer);
(ii) 바이러스(예컨대 아데노바이러스, 허르페스바이러스(예를 들어 HSV-I, HSV-II, CMV 또는 VZV), 폭스바이러스(예를 들어 천연두(variola), 백시니아 또는 물사마귀바이러스(molluscum contagiosum)와 같은 진성두창바이러스(orthopoxvirus), 피코르나바이러스(예를 들어 리노바이러스 또는 엔테로바이러스)), 오르토믹소바이러스 (예를 들어 인플루엔자바이러스), 파라믹소바이러스(예를 들어 5-파라인플루엔자바이러스, 유핸성 이하선염 바이러스(mumps virus), 홍역 바이러스 및 호흡기합포체 바이러스), 코로나 바이러스(예를 들어 SARS), 파포바바이러스(예를 들어 성기사마귀, 보통사마귀 또는 족저 사마귀를 유발하는 파필로마 바이러스), 헤파드나바이러스(예를 들어 B형간염 바이러스), 플라비바이러스(예를 들어 C형간염 바이러스 또는 뎅기열바이러스(Dengue virus)) 또는 레트로바이러스(예를 들어 HIV와 같은 렌티바이러스))에 의한 감염성 질환;(ii) a virus (e. g., adenovirus, herpes virus (e. g. HSV-I, HSV-II, CMV or VZV), poxvirus (e. g. variola, vaccinia or waterworm virus, molluscum contagiosum ), Orthomixovirus (e. G. Influenza virus), paramyxovirus (e. G., 5-parainfluenza virus, e. G. Mumps virus, measles virus and respiratory syncytial virus), coronavirus (e. G. SARS), papovavirus (e. G. Genital warts, common warts or papillomaviruses causing plantar warts) (E. G., Hepatitis B or hepatitis B virus), flavivirus (e. G. Hepatitis C virus or dengue virus) or Infectious disease caused by Trojan virus (e. G. Lentivirus such as HIV));
(ⅲ) 박테리아(예컨대 에스케리치아, 엔테로박터, 살모넬라, 스타필로코커스, 쉬겔라, 리스테리아, 에어로박터, 헬리코박터, 클레브시엘라, 프로테우스, 수도모나스, 나이세리아, 클로스트리듐, 바실러스, 코리네박테리움, 마이코박테리움, 캠필로박터,비브리오, 세라티아, 프로비덴시아, 크로모박테리움, 브루셀라, 예르시니아, 해모필러스 또는 보르데텔라 속)에 의한 감염성 질환;(Iii) bacteria (such as Escherichia, Enterobacter, Salmonella, Staphylococcus, Schielera, Listeria, Aerobacter, Helicobacter, Clevesiella, Proteus, Capital Monas, Nyseria, Clostridium, Bacillus, Infectious diseases by bacterial, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus or Bordetella);
(ⅳ) 그 밖의 다른 감염성 질병(예를 들어 클라미디아와 칸디다증, 국균증, 히스토마플라스마증 및 크립토콕쿠스 뇌막염을 포함하는 진균성 질병 및 말라리아, 뉴머시스터스성 폐렴, 리슈마니아증, 립토스포리디움증, 톡소포자충증 및 트리파소노마 감염을 포함하는 기생충성 질환); (Iv) fungal diseases including other infectious diseases such as chlamydia and candidiasis, aspergillosis, histoparasplasma and cryptococcus meningitis, and malaria, nemerocystic pneumonia, ricinmaniasis, liposporidium Parasitic diseases, including infections with rhinitis, toxoplasmosis and triphasoma infections);
(ⅴ) 아토피성 피부염 또는 습진, 호산구증가증, 천식, 알레르기, 알레르기비염 및 오멘 증후군과 같은 Th2-매개된 아토피질환;(V) Th2-mediated atopic diseases such as atopic dermatitis or eczema, eosinophilia, asthma, allergies, allergic rhinitis and Omen's syndrome;
(ⅵ) 알로페시아 그레아타(alopecia greata), 강직성 척추염, 항인지질 증후군, 자가면역 아디슨 질환, 부신의 자가면역 질환, 자가면역 용혈성 빈혈, 자가면역 간염, 자가면역 난소염 및 고환염, 자가면역 혈소판감소증, 베체트병, 수포성 유천포창, 심근병증, 복강 스프루우-피부염(celiac sprue-dermatitis), 만성 피로 면역이상 증후군, 만성염증성 탈수초 다발성 신경병증, Churg-Strauss 증후군, 반흔성유천포창, CREST 증후군, 한냉 응집소 질환, 크론씨병, 원판성 낭창, 복태성복합한냉글로불린혈증, 섬유근통-섬유근염, 사구체신염, 그레이브스 질환, 귈레인 바레 증후군, 하시모토 갑상선염, 특발성 폐섬유화증, 특발성 혈소판 감소성 자반증, IgA 신경염, 연소자성 관절염, 편평태선, 홍반성 루푸스, 메니에르병, 혼합성 연결 조직 질환, 다발성 경화증, 타입 I 또는 면역-매개 당뇨병, 중증근무력증, 심상성 천포창, 악성 빈혈, 결정성 다발동맥염, 다발연골염, 자가면역성 다선 증후군, 류마티스 다발성근통, 다발성 근염과 피부근염, 일차성 무감마글로불린혈증, 일차성 담증성 간경변, 건선, 건선성 관절염, 레이노 현상, 라이터 증후군, 류마티스 관절염, 사르코이드증, 공피증, 강직인간 증후군, 전신성 홍반성 루푸스, 홍반성 루푸스, 다가야스 동맥염, 일시적 동맥염, 거대세포 동맥염, 궤양성 대장염, 포도막염, 백반증 및 베게너 육아종증과 같은 자가면역질환; (Vi) alopecia greata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune adison disease, autoimmune disease of adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and testisitis, autoimmune thrombocytopenia Dyspneic neuropathy, Churg-Strauss syndrome, cicatricial pemphigus, CREST syndrome, cervical sprue-dermatitis, chronic fatigue syndrome, chronic inflammatory dehydration neuropathy, Idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA neuritis, thrombocytopenic purpura, idiopathic thrombocytopenia, idiopathic thrombocytopenia, idiopathic thrombocytopenic purpura, glomerulonephritis, Multiple sclerosis, Type I or < RTI ID = 0.0 > immune-mediated < / RTI > Primary amygdalae cirrhosis, psoriasis, chronic myasthenia gravis, chronic myasthenia gravis, acute myasthenia gravis, pemphigus vulgaris, malignant anemia, crystalline polyarteritis, polychondritis, autoimmune multi-line syndrome, rheumatoid polyposis, , Psoriatic arthritis, Raynaud's phenomenon, lighter syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, rigid human syndrome, systemic lupus erythematosus, lupus erythematosus, polyarteritis nodosa, transient arteritis, giant cell arteritis, ulcerative colitis, uveitis , Autoimmune diseases such as vitiligo and Wegener's granulomatosis;
(ⅶ) 천식, 엔세필리티스(encephilitis), 염증성 장염, 만성 폐쇄성 폐질환, 알러지, 폐혈병성 쇼크증, 폐섬유증, 미분화 척추관절증, 미분화 관절병증, 관절염, 염증성 골용해, 및 만성 바이러스 또는 박테리아 감염에 의한 만성 염증을 포함하는 염증성 질환; 및 Inflammatory bowel disease, allergic, pulmonary vascular disease, pulmonary fibrosis, undifferentiated spondyloarthropathies, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic viral or bacterial infections such as asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease, Inflammatory diseases including chronic inflammation due to infection; And
(ⅶ) 켈로이드 및 다른 형태의 흉터생성 억제(예를 들어 만성상처 등의 치유촉진)와 같은 상처 치유와 관련된 질병의 예방 또는 치료에 이용된다.(Iii) for the prevention or treatment of diseases associated with wound healing, such as keloid and other forms of scar formation inhibition (e.g., promoting healing such as chronic wounds).
본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여할 수 있고, 바람직하게는 비경구 투여이고, 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, the pharmaceutical composition may be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, have.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 한편, 본 발명의 약제학적 조성물의 경구 투여량은 바람직하게는 1일 당 0.001-100 mg/kg(체중)이다.The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the oral dosage amount of the pharmaceutical composition of the present invention is preferably 0.001-100 mg / kg (body weight) per day.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
본 발명의 다른 양태에 따르면, 본 발명은 다음의 단계를 포함하는 면역원성을 가지는 에피토프의 스크리닝 방법을 제공한다:According to another aspect of the present invention, the present invention provides a method of screening an immunogenogenic epitope comprising the steps of:
(a) (i) 람블편모충 결합 면역글로블린 단백질(Giardia lamblia binding immunoglobulin protein) 또는 그의 단편과 (ii) 에피토프 후보물질로서의 펩타이드를 이용하여 인간을 제외한 동물을 면역화시키는 단계; 및 (a) immunizing an animal other than a human using (i) a Giardia lamblia binding immunoglobulin protein or a fragment thereof and (ii) a peptide as an epitope candidate substance; And
(b) 상기 면역화된 동물에서의 면역반응을 측정하는 단계.
(b) measuring the immune response in said immunized animal.
본 명세서에서 용어 "에피토프(epitope)"는 항체와 상호작용하는 항원의 부위를 의미한다. 보다 자세하게는, 에피토프는 면역글로블린(immunoglobulin) 또는 T-세포 수용체에 특이적으로 결합할 수 있는 단백질 결정부위 (determinant)를 의미한다. 또한, 본 발명의 에피토프는 면역반응을 증가시킬 수 있는 어떠한 분자 또는 물질 을 포함한다. 예를 들어, 본 발명의 에피토프는 펩타이드, 이들 펩타이드를 인코딩하는 핵산 및 당단백질을 포함하지만, 이에 한정되는 것은 아니다.As used herein, the term "epitope" refers to the site of an antigen that interacts with an antibody. More specifically, an epitope refers to a protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor. In addition, the epitope of the present invention includes any molecule or substance capable of increasing the immune response. For example, epitopes of the invention include, but are not limited to, peptides, nucleic acids encoding these peptides, and glycoproteins.
람블편모충 결합 면역글로블린 단백질(Giardia lamblia binding immunoglobulin protein) 또는 그의 단편과 에피토프 후보물질로서의 단백질에 의해 면역화시키는 방법은 당업계에 알려진 다양한 면역 투여방법을 포함하며, 바람직하게는 비경구 투여이다. 비경구 투여인 경우에는 정맥내 주입, 피하주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다. 본 발명에서 이용되는 "인간을 제외한 동물(non-human animal)"은 당업계에서 일반적으로 이용되는 다양한 동물들을 포함하며, 바람직하게는 포유동물이고, 가장 바람직하게는 마우스, 토끼 또는 래트이다. 본 발명에서 면역화된 동물에서의 면역반응 측정은 예를 들어, 상기 선별된 리포좀-포집된 펩타이드를 투여한 대상 동물로부터 혈청을 채취하여 항-펩타이드 항체(총 IgG, IgG1 및 IgG2a)의 역가를 측정하는 방법을 통해 이루어질 수 있다. 바람직하게는, 항체의 역가를 측정하는 방법은 ELISA(Enzyme-linked immunosorbent assay), 측면이동분석법(Lateral flow test), MIA(Magnetic immunoassay) 및 면역침강법(Immunoprecipitation)을 포함하지만, 이에 한정되는 것은 아니다. 보다 바람직하게는, ELISA 분석법이 이용될 수 있다. 특정 펩타이드 서열이 항-펩타이드 항체의 역가를 증가시키는 경우, 상기 특정 펩타이드는 에피토프 또는 펩타이드 백신으로 간주된다.
Methods for immunizing a Giardia lamblia binding immunoglobulin protein or a fragment thereof with a protein as an epitope candidate substance include various immunization methods known in the art, and preferably parenteral administration. In the case of parenteral administration, it can be administered by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, and the like. As used herein, a "non-human animal" includes various animals commonly used in the art, preferably a mammal, most preferably a mouse, rabbit or rat. In the present invention, the measurement of the immune response in the immunized animal can be performed, for example, by measuring the activity of the anti-peptide antibodies (total IgG, IgG1 and IgG2a) by collecting serum from the animal to which the selected liposome- And the like. Preferably, the method for measuring antibody titer includes, but is not limited to, enzyme-linked immunosorbent assay (ELISA), lateral flow assay, magnetic immunoassay (MIA), and immunoprecipitation no. More preferably, ELISA assays can be used. When a particular peptide sequence increases the titer of an anti-peptide antibody, the particular peptide is considered an epitope or peptide vaccine.
본 발명의 다른 양태에 따르면, 본 발명은 다음의 단계를 포함하는 단백질 항원에 대한 항체의 스크리닝 방법을 제공한다:According to another aspect of the present invention, the present invention provides a method for screening an antibody against a protein antigen comprising the steps of:
(a) (i) 람블편모충 결합 면역글로블린 단백질(Giardia lamblia binding immunoglobulin protein) 또는 그의 단편과 (ii) 에피토프 후보물질로서의 펩타이드를 이용하여 인간을 제외한 동물을 면역화시키는 단계; 및 (a) immunizing an animal other than a human using (i) a Giardia lamblia binding immunoglobulin protein or a fragment thereof and (ii) a peptide as an epitope candidate substance; And
(b) 상기 면역화된 동물에서의 면역반응을 측정하여 면역원성을 나타내는 펩타이드 에피토프를 선별하는 단계;(b) measuring the immune response in said immunized animal to select a peptide epitope exhibiting immunogenicity;
(c) 상기 선별된 펩타이드 에피토프와 분석 대상의 항체를 접촉시키는 단계;(c) contacting the selected peptide epitope with an analyte;
(d) 상기 단계 (e)의 결과물과 상기 단백질 항원을 접촉시키는 단계; 및(d) contacting the result of step (e) with the protein antigen; And
(e) 상기 단백질 항원과 상기 분석 대상의 항체의 결합을 분석하는 단계.
(e) analyzing the binding between the protein antigen and the antibody to be analyzed.
본 발명의 단백질 항원 또는 후보 항체는 검출가능한 표지(detectable label)로 표지화될 수 있다. 예를 들어, 상기 검출가능한 표지는 화학적 표지(예컨대, 바이오틴), 효소 표지(예컨대, 호스래디쉬 퍼옥시다제, 알칼린 포스파타제, 퍼옥시다제, 루시퍼라제, β-갈락토시다제 및 β-글루코시다제), 방사능 표지(예컨대, C14, I125, P32 및 S35), 형광 표지(예컨대, 쿠마린, 플루오레세인, FITC(fluoresein Isothiocyanate), 로다민 6G, 로다민 B, TAMRA(6-carboxy-tetramethyl-rhodamine), Cy-3, Cy-5, Texas Red, Alexa Fluor, DAPI(4,6-diamidino-2-phenylindole), HEX, TET, Dabsyl 및 FAM), 발광 표지, 화학발광(chemiluminescent) 표지, FRET(fluorescence resonance energy transfer) 표지 또는 금속 표지(예컨대, 금 및 은)이다. The protein antigen or candidate antibody of the present invention may be labeled with a detectable label. For example, the detectable label may be a chemical label (e.g., biotin), an enzyme label (such as horseradish peroxidase, alkaline phosphatase, peroxidase, luciferase, beta -galactosidase, Let agent), a radiolabel (e. g., C 14, I 125, P 32 and S 35), fluorescent labels (e.g., coumarin, fluorescein, FITC (fluoresein Isothiocyanate), rhodamine 6G, rhodamine B, TAMRA (6 (4,6-diamidino-2-phenylindole), HEX, TET, Dabsyl and FAM), luminescent markers, chemiluminescence chemiluminescent label, a fluorescence resonance energy transfer (FRET) label or a metal label (e.g., gold and silver).
검출 가능하도록 표지화된 단백질 항원 또는 후보 항체를 이용하는 경우, 단백질 항원과 항체 사이의 결합은 표지로부터 나오는 시그널을 검출하여 분석할 수 있다. 예를 들어, 표지로서 알칼린 포스파타아제가 이용되는 경우에는, BCIP(bromochloroindolylphosphate), 니트로 블루 테트라졸리움(NBT), 나프톨-AS-B1-포스페이트(naphthol-AS-B1-phosphate) 및 ECF(enhanced chemifluorescence)와 같은 발색반응 기질을 이용하여 시그널을 검출한다. 표지로서 호스 래디쉬 퍼옥시다아제가 이용되는 경우에는 클로로나프톨, 아미노에틸카바졸, 디아미노벤지딘, D-루시페린, 루시게닌(비스-N-메틸아크리디늄 니트레이트), 레소루핀 벤질 에테르, 루미놀, 암플렉스레드 시약(10-아세틸-3,7-디하이드록시페녹사진, Pierce), HYR(p-phenylenediamine-HCl and pyrocatechol), TMB(tetramethylbenzidine), ABTS(2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), OPD(ophenylenediamine) 및 나프톨/파이로닌와 같은 기질을 이용하여 시그널을 검출한다. 택일적으로, 분석대상 항체의 단백질 항원과의 결합 여부는 상호작용물(interactants)의 레이블링 없이 분석할 수도 있다. 예를 들어, 마이크로피지오미터(microphysiometer)를 이용하여 분석대상의 항체가 단백질 항원에 결합하는 지 여부를 분석할 수 있다. 마이크로피지오미터는 LAPS(light-addressable potentiometric sensor)를 이용하여 세포가 그의 환경을 산성화하는 속도(acidifying rate)를 측정할 수 있는 분석 도구이다. 산성화 속도의 변화는, 후보 항체와 단백질 항원 사이의 결합에 대한 지시자(indicator)로 이용될 수 있다.When a detectably labeled protein antigen or candidate antibody is used, the binding between the protein antigen and the antibody can be detected by analyzing the signal from the label. For example, when alkaline phosphatase is used as a label, it is preferable to use bromochloroindolylphosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF chemifluorescence) is used to detect the signal. When horseradish peroxidase is used as a label, it is preferable to use chlorinaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorpine benzyl ether, luminol, (P-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di [3 -ethylbenzthiazoline sulfonate], OPD (ophenylenediamine), and naphthol / pyronin. Alternatively, the binding of the analyte to a protein antigen may be analyzed without labeling the interactants. For example, a microphysiometer can be used to analyze whether an analyte binds to a protein antigen. Microphysiometry is an analytical tool that uses a light-addressable potentiometric sensor (LAPS) to measure the acidifying rate of a cell's environment. Changes in the rate of acidification can be used as an indicator of the binding between the candidate antibody and the protein antigen.
단백질 항원에 대한 후보 항체의 결합 능력은 실-시간(real-time) 이분자 상호작용 분석(BIA)를 이용하여 분석할 수 있다. BIA는 상호작용물(interactants; 예컨대, BIAcoreTM)의 레이블링 없이 실-시간으로 특이적 상호작용을 분석하는 기술이다. 표면 플라즈몬 공명(SPR)에서의 변화는 분자들 사이의 실-시간 반응에 대한 지시자(indicator)로 이용될 수 있다.
The binding ability of candidate antibodies to protein antigens can be analyzed using real-time two-dimensional interaction analysis (BIA). BIA is a technique for analyzing specific interactions in real-time without labeling of interactants (eg, BIAcore ™ ). Changes in surface plasmon resonance (SPR) can be used as an indicator of the real-time response between molecules.
본 발명의 특징 및 이점을 요약하면 다음과 같습니다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 람블편모충 결합 면역글로블린 단백질(Giardia lamblia binding immunoglobulin protein) 또는 그의 단편의 약제학적 유효량; 및 약제학적으로 허용되는 담체를 포함하는 면역증진용 약제학적 조성물을 제공한다. (a) a pharmaceutical effective amount of a Giardia lamblia binding immunoglobulin protein or a fragment thereof; And a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for enhancing immunity, comprising a pharmaceutically acceptable carrier.
(b) 본 발명은 동정된 재조합 람블편모충 결합 면역글로블린 단백질(recombinant G. lamblia binding immunoglobulin protein, rGlBiP)이 마우스 수지상 세포에서 사이토카인 생산 및 수지상 세포의 성숙과 같은 면역반응을 통해 면역 활성화를 크게 향상시킴으로써 면역증진용 제제로서 유용하게 이용될 수 있다. (b) The present invention relates to a recombinant G. lamblia binding immunoglobulin protein (rGlBiP), which is a recombinant G. lamblia binding immunoglobulin protein (rGlBiP), significantly enhances immune activation through immune responses such as cytokine production and dendritic cell maturation in mouse dendritic cells And thus can be usefully used as an agent for enhancing immunity.
(c) 본 발명은 재조합 람블편모충 결합 면역글로블린 단백질을 이용하여 면역원성을 갖는 에피토프의 스크리닝 방법 및 이의 항체 스크리닝 방법에 유용하게 이용될 수 있다.
(c) The present invention can be usefully used for screening an epitope having an immunogenic property using the recombinant gabbial binding immunoglobulin protein and an antibody screening method thereof.
도 1은 람블편모충 재조합 BiP 및 항-GlBip 항체의 형성을 나타낸 도이다. 도1a는 재조합 GlBiP는 히스티딘 태그을 이용하여 대장균에서 발현되었고, 탈론(Talon) 친화성 크로마토그래피를 이용하여 정제되었고 이후 Coomassie로 염색된 12% SDS-PAGE를 수행한 것을 나타내었다: (1) Coomassie로 염색된 SDS-PAGE 젤, (2) 항-히스티딘 항체를 이용한 웨스턴 블롯(1:5000 희석), (3) 항-람블편모충을 이용한 웨스턴 블롯(1:1000 희석).
도 1b에서는 병원성이 없는 랫트(CrjBgi: CD[S.D.]IGS, 6주령, 암컷) 복강내주사를 이용하여 정제된 재조합 GlBiP를 다중클로날 항체를 제조하기 위해 항원으로서 사용되었고, 2주에 3번 주사 후, 혈청을 면역화된 랫트로부터 수득하였고, 수득된 항체의 역가를 검사하였다. 항-GlBiP(1;2000 희석)을 이용한 람블편모충 추출물의 웨스턴 블롯을 수행하였다. SM은 단백질 사이즈 마커를 나타내고, 면역반응성 재조합 GlBiP 및 천연의 GlBiP는 화살표 및 화살표머리(arrowhead)로 각각 나타내었다.
도 2는 재조합 GlBiP가 공동자극의 발현 및 MHCII 분자의 발현을 촉발시킨다는 것을 나타내는 도이다. BALB/c 마우스의 경골 및 대퇴골 유래의 마우스 골수 세포는 수지상 세포로 분화되었다. 7일째에, 미성숙 골수유래 수지상 세포(BMDC)(1X106cells/mL)를 재조합 GlBiP(0.1 mg/mL) 또는 LPS (0.5 mg/mL)를 이용하여 자극되었다. 세포를 수득하였고, 이후 PerCP-Cy 5.5-결합된 항-마우스 I-A/I-E (MHC 클래스 II), APC-결합 항-CD80 및 PE-결합 항-CD86로 염색하였고 함께 APC-eFlour780-결합 항-CD11c로 20분 동안 얼음 위에 두었다. 형광을 유세포 분석기로 측정하였고 데이터를 FlowJo 데이터 분석 소프트웨어를 이용하여 분석하였다.
도 2a는 GlBiP 처리에 대한 반응에서 수지상 세포 활성화를 보여주는 대표적인 히스토그램을 나타낸 도이다. 자극 없는 미성숙한 수지상 세포는 낮은 수준의 CD80, CD86 및 MHC class II(하얀 바탕)를 발현하였다. 재조합 GlBiP를 이용한 세포의 처리(회색 바탕)은 양성 대조군인 대장균 및 LPS(검은바탕) 뿐만 아니라 CD80, CD86, and MHC class II의 높은 발현양을 야기하였다.
도 2b에서 막대그래프는 3마리의 마우스가 골수세포 유래 수지상 세포를 이용하기 위해 3번의 독립적인 실험에서의 평균 결과로서 나태난 바와 같이, 상기 표면 마커를 발현하는 세포의 퍼센트 비율을 나타낸다.
도 3은 재조합 GlBiP 자극 마우스 수지상 세포에 의한 사이토카인 생산을 나타낸 도이다. 미성숙한 수지상 세포를 0.1, 1 또는 10 /mL의 재조합 GlBiP를 이용하여 16시간 동안 시험하였다. 음성 대조군으로, 세포를 또한 대장균 LPS(Sigma)로 처리하였다. 세포가 없는 상등액을 수집하였고, 다양한 사이토카인에 대해 ELISA 키트를 이용하여 분석하였다. 음성 대조군은 자극받지 않았던 마우스 미성숙 수지상 세포의 상등액이었다.
(A)는 IL-12, (B)는 TNF-α, (C)는 IL-6 그리고 (d)는 IL-4를 각각 나타낸 도이다. 어세이를 3번 반복하였다. 각각의 실험을 단일 마우스를 이용하여 수행하였고, ELISA를 3번 반복 수행하였다. 3번의 실험이 유사한 패턴의 데이터를 생성한다는 것을 확인한 대표적인 실험을 나타내었다. 실험결과를 대표적인 3번의 실험의 평균±표준편차로 표현하였다.
도 4는 람블편모충 추출물로 자극받은 마우스 수지상 세포에 의한 표면 마커 발현 및 사이토카인 생산을 나타낸다. 미성숙한 수지상 세포를 50 /mL의 람블편모충 추출물로 16시간 동안 조사하였다.
도 4a는 왼쪽 패널은 자극 없이 미성숙한 수지상 세포를 나타내는 반면에 람블편모충으로 처리된 세포 추출물은 오른쪽 패널에 나타내었다. 중간 패널은 양성 대조군, 대장균 LPS로 처리된 세포를 나타낸다.
도 4b는 람블편모충 추출물을 이용하여 처리된 골수세포 유래 수지상 세포의 사이토카인 생산을 나타낸 도이다. 세포가 없는 상등액을 수집하였고 IL-12 및 TNF-α를 위해 ELISA 키트를 이용하여 분석하였다. 상기 분석은 동일한 실험으로 3번 반복되었다. 3번의 실험이 유사한 패턴의 데이터를 생성한다는 것을 확인한 대표적인 실험을 나타내었다. 실험결과를 대표적인 3번의 실험의 평균±표준편차로 표현하였다.
도 5는 재조합 GlBiPrk 골수세포 유래 수지상 세포에서 사이토카인 생산을 유도한다는 것을 인지한다는 것을 나타낸 도이다.
도 5a는 미성숙한 골수세포 유래 수지상 세포를 항-TLR4 항체 또는 항-TLR2 항체을 이용하여 1시간 동안 전-배양하였다. 상대적인 동종 IgG가 대조군으로 사용되었다. 상기 세포를 이후 재조합 GlBiP을 이용하여 16시간 동안 공배양하였고(0.1/mL), 배양 상등액을 TNF-α를 위해 분석하였다.
도 5b는 pFLAG-TLR4를 이용하여 형질감염된 HEK293 세포(5X105)가 추가적인 20시간 동안 재조합 GlBiP를 이용하여 자극 받았았고, 이후 루시퍼레이즈활성을 측정하기 위하여 용해되었다. 대조군으로, 플라스미드 pFLAG-CMV 또는 pFLAG-TLR2가 형질감염 실험에서 포함되었다.
도 5c는 야생형, 동질유전자 TLR4 넉아웃 및 동질유전자 TLR2 KO 마우스 유래의 골수세포 유래 수지상 세포 및 TLR2 넉아웃 골수세포 유래 수지상 세포 유래의 골수세포 유래 수지상 세포를 분리시켰고 이후 재조합 GlBiP로 처리하였다(0.1 /mL). 세포 배양 상등액을 사이토카인, 예컨대 (1) IL-12, (2) TNF-α 및 (3) IL-6의 생산을 분석하기 위하여 수득하였다. 양성 대조군으로, 골수세포 유래 수지상 세포를 LPS로 배양하였다. 분석을 3번 반복하였고, 3번의 실험이 유사한 패턴의 데이터를 생성한다는 것을 확인한 대표적인 실험을 나타내었다. 실험결과를 대표적인 3번의 실험의 평균±표준편차로 표현하였다.
도 6은 재조합 GlBiP에 의해 유도된 사이토카인 생산이 MyD88-의존적이라는 것을 나타내는 도이다.
도 6a는 야생형 BALB/c 마우스 및 동질유전자 MyD88 넉아웃 마우스의 대퇴골 및 정강뼈 유래의 골수세포 유래 수지상 세포는 수지상 세포로 분화되었다. 미성숙한 수지상 세포는 16시간동안 0.1-10mg/mL에서 rGlBiP에 대해 지시된 농도에서 및 LPS에 대해 0.5mg/mL에서 다음 자극을 이용하여 자극받았다. 배양 상등액을 마우스 IL-12 (a), TNF-α (b) IL-6 및 (c) ELISA 키트를 이용하여 사이토카인 생산을 측정하기 위하여 수득하였다.
도 6b는 재조합 GlBiP에 의해 자극받은 THP-1 세포에서 항-TLR4 항체를 이용한 MyD88의 공동-면역침강법을 나타낸 도이다. THP-1 세포의 용해물을 2시간 동안 재조합 GlBiP를 이용하여 배양하였고 항-TLR4 또는 항-MyD88 항체와 반응시켰고 이후 단백질 G 세파로오스(Protein G Sepharose) 비드로 침강시켰다(immunoprecipitation: IP). 면역침전물에서 단백질을 항-MyD88 또는 항-TLR4를 이용하여 웨스턴블롯(WB)에 의해 분석하였다.
도 7은 수지상 세포에서 재조합 GlBiP에 의해 유도된 사이토카인 생산의 역할을 나타낸 도이다.
도 7a 및 도 7b에서 (1) IL-12, (2) TNF-α, (3) ERK1-특이적 억제자, PD98059(도 7a) 및 p38-특이적인 억제자, SB202190(도 7b)에 의해 재조합 GlBiP로 처리된 수지상 세포에서의 생산을 각각 나타내었다.
도 7a 및 도 7b에서 억제자로 처리된 세포의 사이토카인 수준을 비처리된 세포와 비교하였다. 모든 실험에서, 화합물을 용해된 형태로 DMSO에서 첨가하였다. DMSO의 농도는 수지상 세포의 어떠한 세포성 과정에 영향을 주지 않는 농도인 배양 부피 및 농도의 1%를 초과하지 않았다. 도 7a의 (3) 및 도 7b의 (3)는 재조합 GlBiP에 대해 노출할 때, 골수세포 유래 수지상 세포에서 MAPK의 활성화를 나타내었다. 미성숙한 수지상 세포(4X105)를 0.1 /ml의 재조합 GlBiP를 이용하여 0-60분 동안 자극하였고, ERK1/2 활성화(도 7a의 (3)) 또는 p38 활성화(도 7b의 (3))에 대해 웨스턴 블롯에 의해 인산화된 ERK1/2 및 p38에 대한 항체를 이용하여 모니터링하였다(도7B의 (3)). 스티리핑 버퍼(stripping buffer)에서 배양 후, 동일한 세포막을 ERK1/2 및 p38의 전체 형태에 대한 항체를 이용하여 조사하였다.
도 7c는 JNK 억제자(0.2-2μM)로 처리된 재조합 GlBiP를 30분 동안 재조합 GlBiP(0.1 μg/mL)를 이용하여 16시간 동안 자극하였다. 배양 상등액을 IL-12(도 7의 (1)) 및 TNF-α(도 7의 (2)) 생산을 측정하기 위해 수집하였다.
도 8은 재조합 GlBiP로 자극된 재조합 GlBiP에서 NF-κB 및 AP-1의 증가된 결합 활성화를 나타낸 도이다. 핵추출을 미성숙한 수지상 세포(1X105)로부터 제조하였다. 핵 추출물 중 3개의 미생물은 바이오틴-11UTP-표지된 NF-κB 올리고뉴클레오타이드(도 8a) 또는 AP-1 올리고뉴클레오타이드(도 8b)를 이용한 결합 어세이를 위해 이용되었다. 상대적인 양의 비표지된 올리고뉴클레오타이드를 결합의 특이성을 측정하기 위해서 결합 반응에 첨가하였다. 세포를 또한 양성 대조군인 LPS를 이용하여 처리하였다.
도 9는 GlBiP에서 수지상 세포 활성화를 위한 기능적 도메인의 맵핑(mapping)을 나타낸다.
도 9a는 3개의 재조합 GlBiP 단백질의 계략도(패널 A), 즉 전장 재조합 GlBiP(패널 B의 F), 절단된 재조합 GlBiP-N(패널 B의 N) 및 절단된 재조합 GlBiP-C(패널 B의 C)를 각각 나타내었다.
도 9b는 항-람블편모충 항체를 이용한 상기 재조합 GlBiP 단백질의 웨스턴 블롯 분석(1:1,000 dilution)을 나타낸 도이다.
도 9c는 재조합 GlBiP 단백질을 이용하여 자극 받은 마우스 수지상 세포에 의한 사이토카인 생산을 나타낸 도이다. 미성숙한 수지상 세포는 0.1 /mL의 재조합 GlBiP, 재조합 GlBiP-N 또는 재조합 GlBiP-C를 이용하여 16시간 동안 검사하였다. 양성 대조군으로서, 세포를 또한 0.5 /mL의 대장균 LPS를 이용하여 처리하였다. 세포가 없는 상등액을수집하였고, ELISA 키트를 이용하여 TNF-a 및 IL-12에 대해 분석하였다. 음성 대조군은 자극 받지 않은 마우스 미성숙 수지상 세포의 상등액이었다. 분석을 3번 반복하였고, 3번의 실험이 유사한 패턴의 데이터를 생성한다는 것을 확인한 대표적인 실험을 나타내었다. 실험결과를 대표적인 3번의 실험의 평균±표준편차로 표현하였다.
도 10은 재조합 GlBiP에 의해 유도된 수지상 세포 활성화를 통한 T 세포에 의한 사이토카인 생산을 나타낸 도이다. 순진한 T 세포를 BALB/c 마우스 지라(spleen)으로부터 제조하였다. CD4+ T 세포를 CD4(L3T4) MicroBead와 함게 autoMACS를 이용하여 지라세포로부터 분리하였다. 24시간 동안 1 μg/mL의 재조합 GlBiP 단백질로 처리된 수지상 세포(2 X 105cells/well)를 CD4+ T-세포(2X106)를 이용하여 공동배양하였다. 공동배양 중 3일째에, 상등액을 사이토카인 생산을 위해 ELISA로 분석하였다.
도 11은 람블편모충으로 감염된 마우스 유래의 혈청을 이용한 람블편모충 추출물 및 재조합 GlBiP(전장 재조합 GlBiP = N, 재조합 GlBiP-N = C, 재조합 GlBiP-C = C, 각각 1 mg) 단백질의 웨스턴 블롯 분석을 나타낸 도이다(E, 10 mg) . 상기 재조합 GlBiP은 2주 또는 4주 동안의 감염후(post-infection) 람블편모충으로 감염된 마우스로부터 전면역 혈청과 반응하였다.
도 12는 항-람블편모충 항체로 반응시킨 항원성 분자의 동정을 나타낸 도이다.
도 12a는 항-람블편모충 항체를 이용한 람블편모충 추출물의 웨스턴 블롯을 나타낸 도이다. 항-람블편모충 항체를 이용한 람블편모충 추출물의 웨스턴 블롯은 74kDa 및 30KDa에서 2 개의 주요한 면역반응성 밴드 및 63kDa 및 25KDa에서 부수적인 밴드를 보여주었다.
도 12b는 비바-스핀(Viva-spin) 컬럼을 이용한 람블편모충 단백질의 분획을 나타낸 도이다. 상기 비바-스핀 컬럼은 분자량에 따라 단백질을 분리하는 초미세여과(ultrafiltration) 장치이다. 웨스턴 블롯 분석은 항-람블편모충 또는 전면역 혈청을 이용한 상기 분획된 단백질을 이용하여 형성되었다. 별표는 다음 라운드의 크로마토그래피를 위해 선택된 분획물을 나타낸다.
도 12c는 DEAE 세파로오스 양이온 교환 크로마토그래피를 이용한 람블편모충 단백질의 분획을 나타낸다(1,000 kDa-100 kDa). 0.1 M NaCl-0.5 M NaCl로 용출된 단백질은 항-람블편모충 항체 도는 전면역 혈청과 반응하엿다. 별표는 다음 라운드의 크로마토그래피를 위해 선택된 분획물을 나타낸다.
도 12d는 젤 여과 크로마토그래피를 이용하여 람블편모충 단백질의 분획물(0.2 M NaCl로 용출됨)을 나타낸 도이다. 0.1 M NaCl - 0.5 M NaCl로 용출된 단백질을 항-람블편모충 항체 또는 전면역 혈청을 이용하여 반응시켰다. 74 kDa의 면역반응성 단백질 밴드(화살표 머리)를 MALDI-TOF에 의해 분석하였다.
도 13은 재조합 GlBiP로 자극된 마우스 골수세포 유래 수지상 세포에서 사이토카인 생산에 대한 사이토카인 생산에 대한 폴리믹신(polymyxin) B의 효과를 나타낸다. 마우스 미성숙 수지상 세포를 폴리믹신 B를 이용하여 전처리하였고 이후 재조합 GlBiP(0.1 /mL) 또는 LPS(0.5 /mL)를 이용하여 자극하였다. 배양 상등액을 ELISA 키트를 이용한 TNF-α 수준을 분석하기 위해서 수득하였다. 1, Lt; RTI ID = 0.0 > BiP < / RTI > and anti-Glbip antibodies.1A,Recombinant GlBiP was expressed in Escherichia coli using a histidine tag and purified using Talon affinity chromatography followed by 12% SDS-PAGE stained with Coomassie: (1) SDS stained with Coomassie -PAGE gel, (2) western blot (1: 5000 dilution) using anti-histidine antibody, and (3) Western blot (1: 1000 dilution) using anti-gambling compound.
1B, Recombinant GlBiP purified using intraperitoneal injection of a virulent rat (CrjBgi: CD [SD] IGS, 6 weeks old, female) was used as an antigen to prepare a multiclonal antibody, , Sera were obtained from immunized rats and the titer of the antibodies obtained was examined. Western blotting of the gibbsite extract with anti-GlBiP (1; 2000 dilution) was performed. SM represents a protein size marker, and immunoreactive recombinant GlBiP and native GlBiP are represented by arrows and arrowheads, respectively.
2Lt; RTI ID = 0.0 > GlBiP < / RTI > induces expression of co-stimulation and expression of MHCII molecules. The bone marrow cells derived from tibia and femur of BALB / c mice were differentiated into dendritic cells. On day 7, immature bone marrow derived dendritic cells (BMDC) (1 * 10 <6cells / mL) were stimulated with recombinant GlBiP (0.1 mg / mL) or LPS (0.5 mg / mL). Cells were stained with PerCP-Cy5.5-conjugated anti-mouse IA / IE (MHC class II), APC-conjugated anti-CD80 and PE-conjugated anti-CD86, followed by APC-eFlour780-conjugated anti-CD11c For 20 minutes on ice. Fluorescence was measured with a flow cytometer and data were analyzed using FlowJo data analysis software.
2A,Is a representative histogram showing dendritic cell activation in response to GlBiP treatment. Immature dendritic cells without stimulation expressed low levels of CD80, CD86 and MHC class II (white background). Treatment of cells with recombinant GlBiP (gray background) resulted in high expression levels of both CD80, CD86, and MHC class II as well as positive controls, E. coli and LPS (black background).
2B, The bar graph represents the percentage of cells expressing the surface marker, as indicated by the average outcome in three independent experiments for the three mice to utilize dendritic cells derived from bone marrow cells.
3Shows the production of cytokines by recombinant GlBiP stimulated mouse dendritic cells. Immature dendritic cells were tested for 16 hours with 0.1, 1 or 10 / mL of recombinant GlBiP. As a negative control, cells were also treated with E. coli LPS (Sigma). Cell-free supernatants were collected and analyzed for various cytokines using ELISA kits. The negative control was the supernatant of mouse immature dendritic cells that had not been stimulated.
(A)IL-12,(B)RTI ID = 0.0 > TNF-a,(C)IL-6 and(d)Lt; RTI ID = 0.0 > IL-4 < / RTI > The assay was repeated 3 times. Each experiment was performed using a single mouse, and ELISA was repeated three times. A representative experiment confirmed that three experiments generated similar patterns of data. Experimental results were expressed as mean ± standard deviation of three representative experiments.
4Shows surface marker expression and cytokine production by mouse dendritic cells stimulated with glioblastiforme extract. Immature dendritic cells were irradiated for 16 hours with 50 / mL gambling extract.
4AShows the immature dendritic cells on the left panel without stimulation, whereas the cell extract treated with gambia bollworms is shown on the right panel. The middle panel represents the cells treated with the positive control, E. coli LPS.
4BShows the production of cytokines in dendritic cells derived from bone marrow cells treated with a gambia extract. Cell-free supernatants were collected and analyzed for IL-12 and TNF-a using an ELISA kit. The assay was repeated three times in the same experiment. A representative experiment confirmed that three experiments generated similar patterns of data. Experimental results were expressed as mean ± standard deviation of three representative experiments.
5Lt; RTI ID = 0.0 > GlBiPrk < / RTI > bone marrow cell-derived dendritic cells.
5AImmature bone marrow cell-derived dendritic cells were pre-incubated with anti-TLR4 antibody or anti-TLR2 antibody for 1 hour. Relative homologous IgG was used as a control. The cells were then co-cultured (0.1 / mL) for 16 hours with recombinant GlBiP and the culture supernatant was analyzed for TNF- [alpha].
5BWere transfected with HEK293 cells transfected with pFLAG-TLR4 (5X10 < RTI ID = 0.0 >5) Was stimulated with recombinant GlBiP for an additional 20 hours and then dissolved to determine the luciferase activity. As a control, the plasmid pFLAG-CMV or pFLAG-TLR2 was included in the transfection experiment.
5cDerived dendritic cells derived from wild-type, homologous gene TLR4 knockout and homologous gene TLR2 KO mice and bone marrow derived dendritic cells derived from dendritic cells derived from TLR2 knockout bone marrow cells were treated with recombinant GlBiP (0.1 / mL ). Cell culture supernatants were obtained for the analysis of cytokines such as (1) IL-12, (2) TNF-α and (3) IL-6 production. As a positive control, bone marrow cell-derived dendritic cells were cultured with LPS. The analysis was repeated three times and a representative experiment confirmed that three experiments produced similar patterns of data. Experimental results were expressed as mean ± standard deviation of three representative experiments.
6Is a diagram showing that the production of recombinant GlBiP-induced cytokines is MyD88-dependent.
6ADerived dendritic cells derived from the femur and tibial bone of wild-type BALB / c mice and the homogeneous gene MyD88 knockout mice were differentiated into dendritic cells. Immature dendritic cells were stimulated at the indicated concentrations for rGlBiP at 0.1-10 mg / mL for 16 hours and at 0.5 mg / mL for LPS using the following stimulation. The culture supernatants were obtained for the measurement of cytokine production using mouse IL-12 (a), TNF-alpha (b) IL-6 and (c) ELISA kits.
6BShows the co-immunoprecipitation of MyD88 using anti-TLR4 antibody in THP-1 cells stimulated by recombinant GlBiP. Lysates of THP-1 cells were incubated for 2 hours with recombinant GlBiP, reacted with anti-TLR4 or anti-MyD88 antibodies and then precipitated with Protein G Sepharose beads (immunoprecipitation: IP). The proteins in the immunoprecipitates were analyzed by western blot (WB) using anti-MyD88 or anti-TLR4.
7In dendritic cells Lt; / RTI > shows the role of cytokine production induced by recombinant GlBiP.
7AAnd7BTreated with recombinant GlBiP by (1) IL-12, (2) TNF-α, (3) ERK1-specific inhibitor, PD98059 (FIG. 7A) and p38-specific inhibitor, SB202190 And production in dendritic cells, respectively.
7AAnd7BThe cytokine levels of the cells treated with the inhibitor were compared to untreated cells. In all experiments, compounds were added in dissolved form in DMSO. The concentration of DMSO did not exceed 1% of the culture volume and concentration, which does not affect any cellular process of dendritic cells. Figures 7 (a) and 7 (b) show activation of MAPK in dendritic cells derived from bone marrow cells when exposed to recombinant GlBiP. Immature dendritic cells (4X105) Was stimulated with 0.1 / ml of recombinant GlBiP for 0-60 min and phosphorylated by Western blot for ERK1 / 2 activation ((3) in FIG. 7a) or p38 activation (3b in FIG. 7b) ERK1 / 2 and p38 (Fig. 7B (3)). After incubation in a stripping buffer, the same cell membranes were examined using antibodies against whole forms of ERK1 / 2 and p38.
7CWere stimulated with recombinant GlBiP (0.1 [mu] g / mL) for 16 hours for 30 minutes with recombinant GlBiP treated with JNK inhibitor (0.2-2 [mu] M). The culture supernatants were collected for measurement of IL-12 (FIG. 7 (1)) and TNF-α (FIG. 7 (2)) production.
8silver Lt; / RTI > B and AP-1 in recombinant GlBiP stimulated recombinant GlBiP. Nuclear extraction was performed on immature dendritic cells (1 * 10 <5). Three of the nuclear extracts were biotin-11Was used for binding assays using UTP-labeled NF-kB oligonucleotides (Figure 8a) or AP-1 oligonucleotides (Figure 8b). Relative amounts of unlabeled oligonucleotides were added to the binding reaction to determine the specificity of the binding. Cells were also treated with a positive control, LPS.
9≪ / RTI > represents the mapping of functional domains for dendritic cell activation in GlBiP.
9A(Panel B), truncated recombinant GlBiP-N (Panel B of N) and truncated recombinant GlBiP-C (Panel C of Panel B) of three recombinant GlBiP proteins, Respectively.
9BWestern blot analysis (1: 1,000 dilution) of the recombinant GlBiP protein using an anti-gambling antibody.
Figure 9c FIG. 5 shows cytokine production by stimulated mouse dendritic cells using recombinant GlBiP protein. FIG. Immature dendritic cells were examined for 16 hours using 0.1 / mL recombinant GlBiP, recombinant GlBiP-N or recombinant GlBiP-C. As a positive control, cells were also treated with 0.5 / mL of E. coli LPS. Cell-free supernatants were collected and analyzed for TNF-a and IL-12 using an ELISA kit. Negative controls were supernatants of unstimulated mouse immature dendritic cells. The analysis was repeated three times and a representative experiment confirmed that three experiments produced similar patterns of data. Experimental results were expressed as mean ± standard deviation of three representative experiments.
10Shows cytokine production by T cells through dendritic cell activation induced by recombinant GlBiP. Naive T cells were prepared from BALB / c mouse spleen. CD4+T cells were isolated from spleen cells using autoMACS with CD4 (L3T4) MicroBead. Dendritic cells treated with 1 [mu] g / ml of recombinant GlBiP protein for 24 hours (2 x 10 <5cells / well) with CD4+T-cells (2 * 10 <6). ≪ / RTI > On
11 Western blot analysis of the gibbsite extract and recombinant GlBiP (full length recombinant GlBiP = N, recombinant GlBiP-N = C, recombinant GlBiP-C = C, 1 mg each) protein using sera derived from mouse infected with gambia (E, 10 mg). The recombinant GlBiP reacted with the full-length sera from mice infected with post-infection glioblastoma for 2 or 4 weeks.
12Is an illustration of the identification of an antigenic molecule that has been reacted with an anti-gambia antibacterial antibody.
12AIs a diagram showing western blotting of a gingival extract of lambs using an anti-gambling antibacterial antibody. Western blot analysis of gabbro extracts using anti-gambling anthelmintic antibodies showed two major immunoreactive bands at 74 kDa and 30 kDa and ancillary bands at 63 kDa and 25 kDa.
12BIs a diagram showing fractions of gambia benthic protein using a Viva-spin column. The Viba-spin column is an ultrafiltration device for separating proteins according to their molecular weights. Western blot analysis was performed using the fractionated proteins using anti-gambled albumin or full-length serum. The asterisk denotes fractions selected for the next round of chromatography.
12C(1,000 kDa-100 kDa) of DEAE sepharose cation exchange chromatography. Proteins eluted with 0.1 M NaCl-0.5 M NaCl were reacted with anti-gambling antiserum or with an immunized sera. The asterisk denotes fractions selected for the next round of chromatography.
12D(Eluted with 0.2 M NaCl) of the gibberellin protein using gel filtration chromatography. Proteins eluted with 0.1 M NaCl - 0.5 M NaCl were reacted with anti-gambling monoclonal antibody or whole blood serum. The 74 kDa immunoreactive protein band (arrowhead) was analyzed by MALDI-TOF.
13Shows the effect of polymyxin B on cytokine production on cytokine production in dendritic cells derived from recombinant GlBiP-stimulated mouse bone marrow cells. Mouse immature dendritic cells were pretreated with PolyMixin B and then stimulated with recombinant GlBiP (0.1 / mL) or LPS (0.5 / mL). Culture supernatants were obtained for analysis of TNF-a levels using an ELISA kit.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
실시예Example
실험 재료 및 실험 방법Materials and Experiments
1. 람블편모충 영향형(1. Adversary affected by lamplight ( G. lamblia G. lamblia trophozoite)의 배양trophozoite
람블편모충 GS 유래의 영양형(ATCC50581, American Type Culture Collection, Mannassas, VA) 균주 유래의 영양체를 TYI-S-33 배지에서 72시간 동안 배양하였다(2% casein digest, 1% yeast extract, 1% glucose, 0.2% NaCl, 0.2% L-cysteine, 0.02% ascorbic acid, K2HPO4, 0.06% KH2PO4, 10% calf serum, and 0.05 mg/mL bovine bile, pH 7.1)(26).
(2% casein digest, 1% yeast extract, 1% glucose) were cultured in TYI-S-33 medium for 72 hours , 0.2% NaCl, 0.2% L-cysteine, 0.02% ascorbic acid, K2HPO4, 0.06% KH2PO4, 10% calf serum and 0.05 mg / mL bovine bile.
2. 항-람블편모충 영양체 추출물의 제조 및 항-람블편모충 항체의 형성 2. Preparation of anti-gambling anthelmintic juice extract and formation of anti-gambling anthelmintic antibody
람블편모충 GS 영양체를 용해 버퍼(PBS: 137 mMNaCl, 1.7 mMKCl, 10 mM Na2HPO4, and 2 mM KH2PO4, pH 7.3)에서 리서스펜션(resusupension)을 수행하였고, 음파처리(sonication)로 용해하였다. 단백질 추출물을 800g에서 5분 동안 원심분리를 하여 제조하였다. 10μg의 추출물을 0.1 ml의 완전한 프로인트 보조제(Freund adjuvant)를 이용하여 혼합하였고(Sigma, St. Louis, MO), 복강내로 무병원체성 마우스 내로 주입하였다(BALB/c 마우스, 6주령 암컷). 1차 면역 후 2주 및 4주에서 두 개의 추가적인 예방 주사를 불완전한 프로인트 보조제와 혼합된 동일한 양의 단백질을 이용하여 수행하였다. 3차 면역한지 1주 후, 혈청을 면역화된 마우스로부터 수득하였고 웨스턴 블롯 분석을 이용하였다.
Resubmission was carried out in a lysis buffer (PBS: 137 mM NaCl, 1.7 mM KCl, 10 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 , pH 7.3) ≪ / RTI > Protein extracts were prepared by centrifugation at 800 g for 5 minutes. 10 μg of the extract was mixed with 0.1 ml of Freund's adjuvant (Sigma, St. Louis, Mo.) and injected intraperitoneally into a non-hospitalized mouse (BALB / c mice, 6 weeks old female). Two additional immunizations at 2 and 4 weeks after primary immunization were performed with the same amount of protein mixed with incomplete Freund's adjuvant. One week after the third immunization, sera were obtained from immunized mice and Western blot analysis was used.
3. 크로마토그래피를 이용한 람블편모충 항원 단백질의 분획3. Chromatographic fractionation of the gambia biloba antigen protein
차등적 컷오프값(1,000 kDa 초과, 1,000 - 100 kDa, 100 - 10 kDa 및 10 kDa 미만)을 갖는 람블편모충 영양체로부터 제조된 단백질 추출물을 4개의 비바 스핀(Viva-spin, Sartorius-StedimBiotech, Goettingen, Germany)을 이용하여 분획하였다. Protein extracts prepared from gambular copra nutrients with differential cut-off values (> 1,000 kDa, 1,000-100 kDa, 100-10 kDa and <10 kDa) were transferred to four Viva-spin, Sartorius-StedimBiotech, Goettingen, Germany).
1,000-100 kDa의 컷오프값을 갖는 비바 스핀 멤브레인(membrane)을 통과했던 단백질을 DEAE 세파로오스 빠른 유속(GE Healthcare Bio-Sciences AB, Uppsala,Sweden) 이온 교환 칼럼(volume 15x1 cm)을 이용하여 결합 버퍼(20mMTris-HCl,pH8.5)로 평형을 맞춘 양이온 교환 크로마토그래피에 의해 연속적으로 분획화하였다. 컬럼에 결합한 단백질을 0.1M에서 0.5 M까지 NaCl의 농도를 증가시키면서 용출하였다. 각각의 분획물을 상술한 항-람블편모충 항체를 이용한 웨스턴 블롯에 의해 분석하였다. Proteins that had passed through a Viva spin membrane with a cutoff value of 1,000-100 kDa were combined using a DEAE Sepharose fast flow rate (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) ion exchange column (volume 15 x 1 cm) And subsequently fractionated by cation exchange chromatography equilibrated with buffer (20 mM Tris-HCl, pH 8.5). Proteins bound to the column were eluted with increasing concentrations of NaCl from 0.1 M to 0.5 M. Each fraction was analyzed by western blotting using the anti-gambling anthelmintic antibody described above.
각각의 분획물을 항-람블편모충을 이용한 웨스턴 블롯에 의해 분석하였다. 0.2M NaCl을 이용하여 용출된 단백질 분획물을 20m MTris-HCl, pH 8.5을 이용하여 평형을 맞춘 40 ml Sephacryl S-200 고해상도 컬럼(GE Healthcare Bio-Sciences AB)(volume 50 x 1 cm)에 적용하였다.Each fraction was analyzed by western blotting with anti-gambling compound. The protein fractions eluted with 0.2 M NaCl were applied to a 40 ml Sephacryl S-200 high resolution column (GE Healthcare Bio-Sciences AB) (volume 50 x 1 cm) equilibrated with 20m MTris-HCl, pH 8.5 .
각각의 분획물의 단백질 농도를 브래드포드 어세이에 의해 측정하였다(Biorad, Hercules,CA). 각각의 정제단계로부터 수득된 분획물을 SDSPAGE에 의해 분리하였고 폴리비닐리덴 플로라이드(polyvinylidene fluoride, PVDF) 멤브레인(Millipore, Billerica, MA)에 옮겼다. 멤브레인을 블로킹(blocking) 용액(1:1,000 dilution, PBS with 5% skim milk, and 0.05% Tween 20)에서 폴리클로날 마우스 항-람블편모충 항체를 이용하여 배양하였고, 알칼라인 포스파테이즈(alkaline phosphatase, AP)-결합 랫트 항-마우스 IgG(Sigma)(1:1000 희석). 면역반응성 단백질을 나이트로-블루 테트라졸리움(nitro-blue tetrazolium, NBT)/5-브로모-4-클로로-3-인돌일포스페이트(BCIP) 시스템(Promega, Madison, WI)을 이용하여 가시화하였다.
Protein concentrations of each fraction were determined by Bradford assay (Biorad, Hercules, Calif.). The fractions obtained from each purification step were separated by SDSPAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, Mass.). Membranes were incubated with polyclonal mouse anti-gambling monoclonal antibody in blocking solution (1: 1,000 dilution, PBS with 5% skim milk, and 0.05% Tween 20), and alkaline phosphatase AP) -binding rat anti-mouse IgG (Sigma) (1: 1000 dilution). Immunoreactive proteins were visualized using nitro-blue tetrazolium (NBT) / 5-bromo-4-chloro-3-indolylphosphate (BCIP) system (Promega, Madison, Wis.).
4. 람블편모충의 면역반응성 단백질의 MALDI-TOF 분석4. MALDI-TOF analysis of immunoreactive proteins of gabbial moths
용출된 젤 여과 크로마토그래피 분획물에서 74 kDa의 면역반응성 단백질을 절개하고 트립신을 이용하여 젤 내에서 분해(in-gel digestion) 처리하였다(Sigma). 소화된 생성물을 MALDI-TOF MS 뿐만 아니라, 4중극 TOF(quadrupole TOF, Q-TOP)에 의해 분석하였다. 이온 스펙트럼 생성물을 정보-의존적 인식(information-dependent acquisition, IDA) 모드에서 수집하였고, Agilent 6530 Accurate-Mass Q-TOP MS을 이용하여 분석하였다. Q-TOP 액체 크로마토그래피-이중 질량분석기(LC-MS/MS) 데이터 세트를 위해, 이중 질량분석 스펙트럼을 본 발명의 마스코트 인하우스 데이터베이스 연구 엔진(GiardiaDB)에 제출하였다. 100 ppm 및 0.2 Da의 질량스펙트럼의 분자량 오차범위는 각각 전구체 이온 및 토막 이온을 위해 이용되었다. 37 초과의 마스코트 이온 스코어를 단백질 동정을 위한 기준으로 이용하였다.
A 74 kDa immunoreactive protein was eluted from the eluted gel filtration chromatography fraction and in-gel digestion was performed using trypsin (Sigma). The digested product was analyzed by MALDI-TOF MS as well as quadrupole TOF (Q-TOP). Ion spectral products were collected in an information-dependent acquisition (IDA) mode and analyzed using an Agilent 6530 Accurate-Mass Q-TOP MS. For the Q-TOP liquid chromatography-dual mass spectrometer (LC-MS / MS) data set, a dual mass spectrometry spectrum was submitted to the mascot in-house database research engine (GiardiaDB) of the present invention. Molecular weight error ranges of mass spectra of 100 ppm and 0.2 Da were used for precursor ions and fragment ions, respectively. A mascot ion score of greater than 37 was used as a reference for protein identification.
5. 재조합 GlBiP 단백질의 형성5. Formation of recombinant GlBiP protein
bipORFGL50581_3283를 포함하는 A2,034-bp의 DNA 단편을 2개의 프라이머, 즉, 프라이머 rBiP_F (5’-CCG GAATTC GATGACGTCTAGTCGCGTTAA-3’: 밑줄친 염기는 EcoRI 위치를 나타냄) 및 프라이머 rBiP_R(5’-CCG CTCGAG GAGCTCATCTTTCTCTGCAT-3’: 밑줄친 염기는 XhoI 위치를 나타냄)을 이용하여 증폭하였고, 이후 pET21b(+) 발현 벡터(Novagen, Darmstadt, Germany) 내로 클로닝을 하였다. The DNA fragment of A2,034-bp containing bipORFGL50581_3283 was amplified by PCR using two primers: primer rBiP_F (5'-CCG GAATTC GATGACGTCTAGTCGCGTTAA-3 ': underlined base represents EcoRI site) and primer rBiP_R (5'-CCG CTCGAG GAGCTCATCTTTCTCTGCAT-3 ': the underlined base represents the XhoI site) and then cloned into the pET21b (+) expression vector (Novagen, Darmstadt, Germany).
또한, bip ORF를 포함하는 DNA 단편을 2 부분으로 분해하였다. bip의 5’-부위(1,280 bp)를 상기 프라이머, 즉, 재조합 BiP-F(5’-CCG GAATTC GGGTGCGAAGGATCATGATGT-3’: 밑줄친 염기는 EcoRI 부위를 나타냄) 및 재조합 BiP-NR (5’-CCG CTCGAG GCTAAGGATGGAGGCCTGCA-3’: 밑줄친 염기는 XhoI 부위를 나타냄)를 이용한 PCR에 의해 람블편모충의 게놈 DNA로부터 증폭하였다. bip의 3’ 지역(754 bp)을 또 다른 세트의 프라이머, 즉, rBiP-CF (5’-CCGGAATTCGGGTGCGAAGGATCATGATGT-3’: EcoRI 부위를 나타냄) 및 재조합 rBiP_R을 이용하여 증폭하였다. In addition, the DNA fragment containing the bip ORF was digested into two fragments. (5'-CCG GAATTC GGGTGCGAAGGATCATGATGT-3 ': underlined base represents the EcoRI site) and recombinant BiP-NR (5'-CCG CTCGAG GCTAAGGATGGAGGCCTGCA-3 ': the underlined base represents the Xho I site) from the genomic DNA of the germ line. The 3 'region (754 bp) of the bip was amplified using another set of primers, rBiP-CF (5'-CCGGAATTCGGGTGCGAAGGATCATGATGT-3': EcoRI site) and recombinant rBiP_R.
수득한 bip DNA 단편을 절단된 재조합 GlBiP 폴리펩타이드를 위한 과발현 플라스미드, 즉, pETBiP-N 및 pETBiP-C를 생성하기 위하여 pET21b(Novagen) 내로 클로닝을 하였다. 상기 재조합 GlBiP를 1 mM의 IPTG(isopropyl thio-β-D-galactoside)를 첨가함으로써 E.colil BL21(DE3)에서 과발현하였고, 매뉴얼에 따라 Talon 친화성 크로마토그래피에 의해 정제하였다(Clonetech, Mountain View,CA). The resulting bip DNA fragment was cloned into pET21b (Novagen) to generate over-expression plasmids for the cleaved recombinant GlBiP polypeptide, i. E. PETBiP-N and pETBiP-C. The recombinant GlBiP was overexpressed in E. coli BL21 (DE3) by adding 1 mM IPTG (isopropyl thio-β-D-galactoside) and purified by Talon affinity chromatography according to the manual (Clonetech, Mountain View, CA).
6. 재조합 GlBiP 활성을 측정하기 이전의 폴리믹신(polymyxin) B를 이용한 엔도톡신의 제거6. Removal of endotoxin using polymyxin B prior to measurement of recombinant GlBiP activity
재조합 GlBiP에 의해 유도된 수지상 세포의 성숙은 제조된 단백질에서 오염을 일트키는 엔도톡신에 기인하는 것이 아니라는 것을 확인하기 위하여 폴리믹신 B(Sigma)(PmB)로 수지상 세포의 전처리를 수행하였다. 수지상 세포를 500 ng/mL LPS 또는 100 ng/mL 재조합 GlBiP를 이용한 반응 전에 1 μg/mL PmB로 상온에서 1시간 동안 전배양하였다. 16 시간 후, 골수세포 유래 수지상 세포의 상등액에서 TNF-α, , IL-12 및 IL-6 수준을 ELISA 키트를 이용하여 측정하였다. 이후 실험에서, 재조합 GlBiP의 박테리아 LPS 오염을 배제하기 위해 세포를 PmB(1 μg/mL)를 이용하여 1시간 동안 전배양하였다. 또한, Limulus Amebocyte Lysate LPS Detection Kit(Lonza, Basel, Switzerland)에 의해 측정하였을 때, LPS 오염이 1.5 EU/mL(1 EU = 100 pg) 미만이었다는 것을 확인하기 위하여 재조합 GlBiP 단백질을 정제하였다. LPS로 오염된 단백질 샘플을 Detoxi-GelTM 엔도톡신 제거 젤(Pierce, Rockford, IL)을 이용하여 추가로 정제하였다.
Pretreatment of dendritic cells with polyMixin B (Sigma) (PmB) was performed to confirm that the maturation of the dendritic cells induced by recombinant GlBiP was not due to endotoxin, which causes contamination in the prepared protein. The dendritic cells were preincubated with 1 μg / mL PmB for 1 hour at room temperature prior to the reaction using 500 ng / mL LPS or 100 ng / mL recombinant GlBiP. After 16 hours, levels of TNF-a, IL-12 and IL-6 in the supernatant of dendritic cells derived from bone marrow cells were measured using an ELISA kit. In subsequent experiments, cells were preincubated with PmB (1 μg / mL) for 1 hour to eliminate bacterial LPS contamination of recombinant GlBiP. Recombinant GlBiP protein was also purified to confirm that LPS contamination was less than 1.5 EU / mL (1 EU = 100 pg) when measured by the Limulus Amebocyte Lysate LPS Detection Kit (Lonza, Basel, Switzerland). Protein samples contaminated with LPS were further purified using a Detoxi-Gel ™ endotoxin removal gel (Pierce, Rockford, Ill.).
7. 폴리클로날 항-GlBiP의 제조7. Preparation of polyclonal anti-GlBiP
무병원균 랫트내로 복강내 주사를 통해 폴리클로날 항체를 생산하기 위한 항체로 정제된 재조합 GlBiP를 이용하였다(CrjBgi: CD[S.D.]IGS, 6-weeks-old, females). 2주 간격으로 3번의 주사 후, 혈청을 면역화된 랫트로부터 수득하였고 수득된 항체의 티터(titer)를 시험하였다. 백 나노그램의 재조합 GlBiP를 12% SDS-PAGE에 의해 분리하였고 나이트로셀룰로오스 멤브레인에 옮겼다. 멤브레인을 항-람블편모충 항체(1:1,000 희석) 또는 항-GlBiP 항체(1:2,000 희석), 이어서 AP-결합된 랫트 항-마우스 IgG를 이용하여 배양하였다. 면역반응성 단백질을 상술한 바와 같이 검출하였다.
(CrjBgi: CD [SD] IGS, 6-weeks-old, females) was used as an antibody to produce polyclonal antibodies by intraperitoneal injection into non-pathogenic rats. After three injections every two weeks, sera were obtained from immunized rats and the titer of the antibodies obtained was tested. One hundred nanograms of recombinant GlBiP was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were incubated with anti-gambling monoclonal antibody (1: 1,000 dilution) or anti-GlBiP antibody (1: 2000 dilution) followed by AP-conjugated rat anti-mouse IgG. Immunoreactive proteins were detected as described above.
8. 실험용 마우스8. Experimental mouse
BALB/c 마우스를 OrientBio(Seoul, Korea) 및 TLR2 넉아웃(KO)(TLR2/), TLR4 KO(TLR4/)로부터 수득하였고, 골수성 분화 인자 88(MyD88) 넉아웃 (MyD88/) 마우스를 아키라 박사(S. Akira)에 의해 제공된 BALB/c 마우스로부터 수득하였다(Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Japan). 동물들은 연구기관의 지침 및 한국의 법정준비소요액에 따라 인도적인 보살핌을 받았다.
BALB / c mice were obtained from OrientBio (Seoul, Korea) and TLR2 knockout (KO) (TLR2 /), TLR4 KO (TLR4 /) and myeloid differentiation factor 88 (MyD88) Was obtained from BALB / c mice provided by S. Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Japan). The animals received humanitarian care according to the guidelines of the research institutes and the amount of preparation for the court in Korea.
9. 수지상 세포의 분리 및 배양9. Isolation and culture of dendritic cells
골수세포 유래 수지상 세포를 BALB/c 마우스의 대퇴골 및 경골로부터 제조하였고 적혈구 용출 버퍼에서 리서스펜션을 수행하였다(150 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA, pH 7.4). 원심분리 후, 세포를 100 U/mL의 페니실린/스트렙토마이신(GIBCO, Karsruhe, Germany)으로 보충된 골수세포 유래 수지상 세포(BMDC) 내로 분화시켰고, 10% FBS, 50 μM의 메르캅토에탄올(mercaptoethanol), 0.1 mM의 비필수 아미노산(Sigma), 1 mM HEPES 버퍼 및 20 ng/mL의 과립대식세포집락자극인자(granulocyte-macrophages colony-stimulating factor(GM-CSF: JW CreaGene, Seongnam, Korea))를 2일 마다 RPMI1640 배지에서 보충하였고 37°C, 5% CO2 존재 하에서 배양하였다. 6 또는 7일간의 배양일 동안, 미성숙한 골수세포 유래 수지상 세포를 추가 실험을 위해 수득하였다.
Bone marrow cell-derived dendritic cells were prepared from the femur and tibia of BALB / c mice and resuspended in erythrocyte elution buffer (150 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA, pH 7.4). After centrifugation, the cells were differentiated into bone marrow cell-derived dendritic cells (BMDC) supplemented with 100 U / mL penicillin / streptomycin (GIBCO, Karsruhe, Germany) and resuspended in 10% FBS, 50 μM mercaptoethanol (Sigma), 1 mM HEPES buffer and 20 ng / mL granulocyte-macrophages colony-stimulating factor (GM-CSF: JW CreaGene, Seongnam, Korea) Day in RPMI 1640 medium and cultured in the presence of 5% CO 2 at 37 ° C. For 6 or 7 days of culture days, immature bone marrow cell-derived dendritic cells were obtained for further experiments.
10. 세포 표면 표현형의 유세포 분석(Flow cytometric analysis)10. Flow cytometric analysis of cell surface phenotype
미성숙한 골수세포 유래 수지상 세포를 16시간 동안 재조합 GlBiP(0.1 to 5 μg/mL) 또는 람블편모충 추출물(50 μg/mL)로 처리하였다. 이후, 세포를 수득하였고, PBS로 세척하고 라이브/데드 고정가능 사멸세포 염색 키트live/dead fixable dead cell stain kit (Invitrogen, Carlsbad, CA)를 이용하여 죽은(염색된) 세포의 퍼센트 비율을 측정하기 위해서 얼음 위에서 20분 동안 염색하였다. 이후, 세포를 PBS로 세 번 세척하였고 페리디닌 클로로필(PerCP)-Cy 5.5-결합 항-마우스I-A/I-E(MHC class II) 알로피코시아닌(allophycocyanin, APC)-결합 항-CD80 및 피코에리트린(phycoerythrin, PE)-결합 항-CD86 및 APC-eFlour780-결합 항-CD11c를 이용하여 얼음 상에서 20분 동안 염색하였다. 세포를 PBS로 세 번 세척하였고 500 μL의 FACS(fluorescence-activated cell sorter)에서 리서스펜션을 수행하였다(PBS, 1% BSA, and 0.1% sodium azide). 형광을 유세포 분석기로 측정하였고, 실험데이터를 FlowJo 데이터 분석 소프트웨어를 이용하여 분석하였다.
Immature bone marrow cell-derived dendritic cells were treated with recombinant GlBiP (0.1 to 5 μg / mL) or gabbro extract (50 μg / mL) for 16 h. Cells were then harvested, washed with PBS and assayed for percentage of dead (stained) cells using a live / dead fixable dead cell stain kit (Invitrogen, Carlsbad, Calif.) For 20 minutes on ice. Cells were then washed three times with PBS and incubated with peridinin chlorophyll (PerCP) -Cy5.5-conjugated anti-mouse IA / IE (MHC class II) allophycocyanin (APC) -conjugated anti-CD80 and picoeritrine (phycoerythrin, PE) -conjugated anti-CD86 and APC-eFlour780-conjugated anti-CD11c for 20 minutes on ice. Cells were washed three times with PBS and resuspended in 500 μL of fluorescence-activated cell sorter (PBS, 1% BSA, and 0.1% sodium azide). Fluorescence was measured by flow cytometry and the experimental data were analyzed using FlowJo data analysis software.
11. 사이토카인 농도의 측정11. Measurement of cytokine concentration
미성숙한 마우스 수지상 세포를 48웰 배양 플레이트에서 분주하였고 람블편모충 추출물(50μg/mL), 재조합 GlBiP (0.1-10 μg/mL) 또는 LPS(0.5 μg/mL)를 이용하여 16시간 동안 배양하였다. 상등액을 TNF-α, IL-12, IL-6 및 IL-4를 검출하기 위해 샌드위치 ELISA에 의해 매뉴얼(BD Biosciences, Franklin Lakes, NJ)에 따라 분석하였다.
Immature mouse dendritic cells were seeded on 48-well culture plates and cultured for 16 hours with gibberellic acid extract (50 μg / mL), recombinant GlBiP (0.1-10 μg / mL) or LPS (0.5 μg / mL) The supernatants were analyzed according to the manual (BD Biosciences, Franklin Lakes, NJ) by sandwich ELISA to detect TNF-a, IL-12, IL-6 and IL-4.
12. 항체 블로킹 실험12. Antibody blocking experiment
쥐의 골수세포 유래 수지상 세포를 재조합 GlBiP로 처리된 5 μg/mL의 마우스 TLR4 단일클론 항체(BioLegend, San Diego, CA), 마우스 TLR2 단일클론 항체(BioLegend) 또는 동종 대조군 IgG 항체(BioLegend)를 이용하여 RPMI 1640 무혈청 배지에서 1시간 동안 전배양하였다. 자극을 이용하여 16시간 후, 상등액을 ELISA을 위해 수득하였다.
Dendritic cells derived from mouse bone marrow cells were treated with recombinant GlBiP-treated 5 μg / ml mouse TLR4 monoclonal antibody (BioLegend, San Diego, CA), mouse TLR2 monoclonal antibody (BioLegend) or homologous control IgG antibody (BioLegend) And then pre-cultured in RPMI 1640 serum-free medium for 1 hour. After 16 hours with stimulation, supernatants were obtained for ELISA.
13. NF-κB 리포터 어세이13. NF-κB Reporter Assay
형질감염 하루 전에, HEK293 세포를 12웰 배양 플레이트 상에서 1×105 세포로 플레이팅을 수행하였다. 분주된 세포를 0.5μg의 NF-κB 루시퍼레이즈 플라스미드[pGLIL8p-luc+](27), 5ng의 pCH110(Promega) 및, 0.1μg의 pFLAG-TLR2(28) 또는 pFLAG-TLR4(28)를 이용하여 일시적으로 Lipofectamine 2000 (Invitrogen)에 의해 형질감염시켰다. 대조군으로, pFLAG-CMV1(Invitrogen)을 pFLAG-TLR2 또는 pFLAG-TLR4 대신에 형질감염시켰다. 형질감염한지 24시간 후, 세포를 PBS 없이 재조합 GlBiP(1μg/mL) 또는 LPS (0.5μg/mL)를 이용하여 6시간 동안 자극시켰다. 루시퍼레이즈 활성을 Dual-Luciferase Reporter Assay System(Promega)를 이용하여 매뉴얼에 따라 측정하였다.
One day before transfection, HEK293 cells were plated at 1x10 < 5 > cells on a 12 well culture plate. The dispensed cells were transiently transfected with 0.5 μg of NF-κB Luciferase plasmid [pGLIL8p-luc +] (27), 5 ng of pCH110 (Promega), and 0.1 μg of pFLAG-TLR2 (28) or pFLAG- RTI ID = 0.0 > 2000 < / RTI > (Invitrogen). As a control, pFLAG-CMV1 (Invitrogen) was transfected in place of pFLAG-TLR2 or pFLAG-TLR4. Twenty-four hours after transfection, cells were stimulated with recombinant GlBiP (1 μg / mL) or LPS (0.5 μg / mL) for 6 hours without PBS. Luciferase activity was measured according to the manual using the Dual-Luciferase Reporter Assay System (Promega).
14. 세포주의 배양14. Culture of cell line
인간 배아 신장 세포, HEK 293(CRL-1573; American Type Culture Collection)를 4.5 g/L 포도당, 10% 열로 불활성화된 소태아 혈청(fetal bovine serum, FBS), 100 U/mL 페니실린, 100 μg/mL 스트렙토마이신 및 10 μg/mL 블라스티시딘(blasticidin S, InvivoGen, San Diego, CA)로 보충된 DMEM에서 배양하였다. 인간 단핵세포주, THP-1(TIB-202; American Type Culture Collection)를 37℃, 5% CO2 건조 대기, RPMI-1640(Sigma) 조건에서 배양하였다.
Human embryonic kidney cells, HEK 293 (CRL-1573; American Type Culture Collection), were cultured in DMEM supplemented with 4.5 g / L glucose, 10% heat-inactivated fetal bovine serum (FBS), 100 U / mL penicillin, mL streptomycin and 10 μg / mL blasticidin S (InvivoGen, San Diego, Calif.). Human mononuclear cell line, THP-1 (TIB-202; American Type Culture Collection) was cultured at 37 ° C, 5% CO 2 dry atmosphere, and RPMI-1640 (Sigma) conditions.
15. 공동면역침강법(Coimmunoprecipitation)15. Coimmunoprecipitation (Coimmunoprecipitation)
재조합 GlBiP로 배양된 THP-1 세포를 용해 완충액(10 mMTrisHCl, pH 7.4, 5 mM EDTA, 130 mMNaCl, 1% Triton X-100, 1% 4-amidino-phenyl-methylsulfonyl fluoride, and 1% protease inhibitor)에서 파괴하였고, 4°C에서 하룻밤 동안 항-TLR4 단일항체 Absor 항-MyD88 단일클로날 항체(Cell Signaling, Beverly, MA)를 이용하여 배양하였고, 이후 단백질 G 아가로오스 비드를 이용하여 4°C에서 4시간 동안 침전시켰다. 침전된 단백질을 이후 항-TLR4 또는 항-MyD88 항체를 이용하여 웨스턴 블롯에 의해 분석하였다(Cell Signaling).
THP-1 cells cultured with recombinant GlBiP were lysed in lysis buffer (10 mM Tris HCl, pH 7.4, 5 mM EDTA, 130 mM NaCl, 1% Triton X-100, 1% 4-amidino- phenylmethylsulfonyl fluoride, (Cell Signaling, Beverly, Mass.) And incubated overnight at 4 ° C with protein G agarose beads at 4 ° C ≪ / RTI > for 4 hours. The precipitated proteins were then analyzed by western blotting using anti-TLR4 or anti-MyD88 antibodies (Cell Signaling).
16. 재조합 GlBiP로 처리된 수지상 세포에서 MAPK 경로의 억제16. Inhibition of MAPK pathway in dendritic cells treated with recombinant GlBiP
MAPK의 관련성을 연구하가 위해서, 미성숙한 수지상 세포를 무혈청 RPMI 1640 배지에서 억제자를 이용하여 16시간 동안 전처리하였다. 약제학적 성분을 Calbiochem(Darmstadt, Germany)으로부터 구입하였다. 모든 억제자들을 DMSO에서 용해시켰다. 억제자들을 다음 농도에서 이용하였다. 0.1-1의 SB 202190(선택적 p38 억제자) 또는 PD98059(선택적 ERK1/2 억제자) 및 0.2-2 JNK 억제자 II(선택적 JNK 억제자). 억제자를 이용한 모든 실험에서, 시험된 농도는 배양 부피의 1%를 초과하지 않았고 수지상 세포의 생존능력에 영향을 주지 않았다.
To investigate the relevance of MAPK, immature dendritic cells were pretreated with inhibitor in serum-free RPMI 1640 medium for 16 h. Pharmaceutical ingredients were purchased from Calbiochem (Darmstadt, Germany). All inhibitors were dissolved in DMSO. The inhibitors were used at the following concentrations. 0.1-1, SB 202190 (selective p38 inhibitor) or PD98059 (selective ERK1 / 2 inhibitor) and 0.2-2 JNK inhibitor II (selective JNK inhibitor). In all experiments with inhibitors, the concentrations tested did not exceed 1% of the culture volume and did not affect the viability of dendritic cells.
17. 핵 추출물의 제조 및 젤 이동성 분석(electrophoretic mobility gel shift assay, EMSA) 17. Preparation of nuclear extract and electrophoretic mobility gel shift assay (EMSA)
24웰 배양 플레이트로 분주된 마우스 골수세포 유래 수지상 세포를 다양한 시간(3-60분)으로 재조합 GlBiP를 이용하여 자극하였고, 이후 핵 추출물을 위해 처리하였다(29). 요약해서, 세포를 100 용해 완충액 [10 mM HEPES, 10 mMKCl, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mMdithiothreitol (DTT)]에서 15분 동안 얼음 상에서 용해하였고 12.5의 10% Nonidet P-40을 세포에 첨가하였다. 4°C, 20,000g에서 8분 동안 원심분리 후, 핵 펠렛을 25의 얼음처럼 찬 핵 추출물 버퍼에서 리서스펜션하였고 간헐적인 교반으로 15분 동안 얼음에서 유지하였다. 이후 샘플을 4℃ 얼음에서 5분 동안 원심분리하였고, 결합 어세이까지 상등액을 -70°C에 저장하였다. 바이오틴-표지된 프로브(100 mMTris, 500 mMKCl 및 10 mM DTT, pH 7.5)를 이용하여 결합 어세이를 핵 추출물 결합 버퍼 (100 mMTris, 500 mMKCl 및 10 mM DTT, pH 7.5)에서 수행하였다. 센스 가닥 올리고뉴클레이타이드의 서열는 다음과 같다: NF-κB, 5’-AGTTGAGGGGACTTTCCCAGGC-3’(Promega) 및 AP-1, 5’-CGCTTGATGAGTCAGCCGGAA-3’(Promega). 단백질-DNA 복합체를 0.5×Tris/Boric acid/EDTA (TBE) 버퍼에서 4°C, 6% 폴리아크릴아마이드 젤에서 전기영동하였고, 나일론 멤브레인에 옮겼다. UV-가교결합된 멤브레인을 스트렙토아비딘-서양고추냉이 퍼옥시데이즈 결합(Pierce)를 이용하여 처리하였고, 이후 DNA 밴드를 검출하기 위하여 Light-Shift Chemiluminescent EMSA 키트(Pierce)를 이용하여 X-레이 필름에 노출시켰다. 또한, 핵 추출물을 다양한 시간 동안 0.5 /mL LPS로 자극된 골수세포 유래 수지상 세포로부터 제조하였고, 결합 어세이 동안 양성 대조군으로 이용하였다.
Dendritic cells derived from mouse bone marrow cells, seeded with 24-well culture plates, were stimulated with recombinant GlBiP for various times (3-60 minutes) and then treated for nuclear extracts (29). Briefly, cells were lysed on ice for 15 minutes in 100 lysis buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mM dithiothreitol (DTT)) and 12.5% 10% Nonidet P- . After centrifugation at 4 ° C, 20,000 g for 8 minutes, the nuclear pellet was resuspended in 25 ice-cold nuclear extract buffer and kept on ice for 15 minutes with intermittent agitation. The samples were then centrifuged for 5 minutes at 4 ° C on ice and the supernatant was stored at -70 ° C until binding assays. The binding assays were performed in nuclear extract binding buffer (100 mM Tris, 500 mM KCl and 10 mM DTT, pH 7.5) using biotin-labeled probes (100 mM Tris, 500 mM KCl and 10 mM DTT, pH 7.5). The sequence of the sense strand oligonucleotide is as follows: NF-κB, 5'-AGTTGAGGGGACTTTCCCAGGC-3 '(Promega) and AP-1, 5'-CGCTTGATGAGTCAGCCGGAA-3' (Promega). Protein-DNA complexes were electrophoresed in 0.5 × Tris / Boric acid / EDTA (TBE) buffer at 4 ° C, 6% polyacrylamide gel and transferred to nylon membranes. The UV-crosslinked membranes were treated with streptavidin-horseradish peroxidase conjugate (Pierce), and then analyzed by X-ray film using a Light-Shift Chemiluminescent EMSA kit (Pierce) Exposed. In addition, nuclear extracts were prepared from bone marrow cell-derived dendritic cells stimulated with 0.5 / mL LPS for various times and used as positive controls during binding assays.
18. 혼합된 림프구 반응 18. Mixed lymphocyte reaction
미접촉(naive) T-세포를 BALB/c 마우스 비장으로부터 제조하였다. 비장세포를 적혈구 완충액에서 용해하였다. CD4+ T 세포를 autoMACS(magnetic-activated cell sorting) 분리기에서 CD4(L3T4) MicroBeads(Miltenyi Biotec, Auburn,CA)를 이용하여 분리하였다. 1 μg/mL의 재조합 GlBiP로 처리된 수지상 세포를 24시간 동안 CD4+ T 세포를 이용하여 1:10의 수지상 세포: T 세포 비율에서 공동 배양하였다. 공동 배양 중 3일째에, 상등액을 IL-2 생산을 위하여 ELISA에 의하여 수집하고 분석하였다(30). 또한, 상등액을 Th1 분극(polarization)를 검사하기 위하여 IFN-γ로 분석하였다(31).
Non-contact T-cells were prepared from BALB / c mouse spleen. Splenocytes were lysed in erythrocyte buffer. CD4 + T cells were isolated using CD4 (L3T4) MicroBeads (Miltenyi Biotec, Auburn, Calif.) In an autoMACS (magnetic-activated cell sorting) separator. Dendritic cells treated with 1 μg / mL of recombinant GlBiP were co-cultured with CD4 + T cells for 24 hours at a dendritic cell: T cell ratio of 1:10. On
19. 마우스 감염 어세이 19. Mouse Infection Assay
람블편모충(균주 GS ATCC50581)를 상술한 바와 같이(18) 감염을 위하여 이용하고 배양하였다. BALB/c 마우스를 PBS(phosphate-buffered saline)의 람블편모충 영양체에서 5 X 105G로 위관영양법에 의해 감염시켰다. 2주 및 4주 간격으로, 혈청을 감염된 마우스로부터 수득하였고 람블편모충 또는 재조합 GlBiP 단백질의 용해물을 웨스턴 블롯 분석을 위해 이용하였다.
The gibbsite bacterium (strain GS ATCC 50581) was used and cultured for infection (18) as described above. BALB / c mice were infected by gavage with 5 x 105 g of gabbroic saliva from PBS (phosphate-buffered saline). At two and four week intervals, sera were obtained from infected mice and lysates of gibbbit or recombinant GlBiP protein were used for western blot analysis.
20. 통계분석20. Statistical Analysis
실험결과를 대표적인 3번의 실험의 평균±표준편차로 표현하였다. 실험데이터를 Student’s t-test(SYSTAT program, SIGMAPLOT version 9; Systat Software Inc., Chicago,IL, USA)를 이용하여 쌍으로 비교에 의하여 분석하였다. P-값이 0.05 미만이면, 편차를 유의하게 고려하였다. 0.01 미만의 P값을 갖는 실험데이터는 2개의 별표로 나타내었고 0.01 및 0.05 사이의 P-값을 갖는 실험데이터를 하나의 별표로 나타내었다.
Experimental results were expressed as mean ± standard deviation of three representative experiments. Experimental data were analyzed by pairwise comparison using Student's t-test (SYSTAT program, SIGMAPLOT version 9; Systat Software Inc., Chicago, IL, USA). If the P-value is less than 0.05, the deviation is considered significant. Experimental data with a P value of less than 0.01 are represented by two asterisks and experimental data with P-values between 0.01 and 0.05 are represented by a single asterisk.
[실험 결과][Experiment result]
1. 람블편모충의 항원 단백질로서 결합 면역글로블린 단백질(binding immunoglobulin protein, BiP)의 동정 1. Identification of the binding immunoglobulin protein (BiP) as the antigen protein of the gibblet
본 발명자들은 람블편모충의 항원 분자를 동정하기 위해 일련의 실험을 수행하였다. 람블편모충 추출물을 항원으로서 이용하여 다중클로날 항체(항-람블편모충 항체)를 제조하였다. 제조된 항체와 람블편모충 추출물의 면역블로팅은 74 및 30 kDa의 두 개의 주요한 면역반응성 밴드와, 62 및 25 kDa의 두 개의 부수적인 면역 반응성 단백질을 각각 보여주었다(Supplementary 도 12a). 이후, 람블편모충 추출물을 3가지의 절차를 이용하여 분획하였고, 본 발명자들은 74kDa의 단백질이 항-람블편모충의 강한 면역을 나타낸다는 것을 동정하였다. The present inventors conducted a series of experiments to identify antigen molecules of gabbial biloba. A multiclonal antibody (anti-gambia anthelmintic antibody) was prepared using a gambia bark extract as an antigen. Immunoblotting of the prepared antibody and gibbsite extract showed two major immunoreactive bands of 74 and 30 kDa and two secondary immunoreactive proteins of 62 and 25 kDa, respectively (Supplementary Figure 12a). The gibbsite extract was then fractionated using three procedures, and the present inventors have identified that the 74 kDa protein represents a strong immunity of the anti-giblet copepods.
1000 kDa 초과 및 1000-100 kDa의 컷오프(cut-off)값을 갖는 세포막을 이용하여 비바 스핀(Viva spin) 원심분리를 통해 수득된 웨스턴 블롯(Western blot)의 분석은 74kDa의 면역반응성 단백질이라는 것을 밝혀내었다(도 12b).Analysis of the Western blot obtained via Viva spin centrifugation using a cell membrane with a cut-off value of more than 1000 kDa and 1000-100 kDa is an immunoreactive protein of 74 kDa (Fig. 12B).
대조적으로, 상기 면역반응성 밴드는 면역반응전 혈청 분획물의 웨스턴 블롯 분석에서 존재하지 않았다. 100-10 kDa의 컷오프값을 갖는 세포막을 이용하여 비바 스핀 원심분리를 통해서 수득된 분획물은 15 kDa의 면역반응성 단백질의 면역 반응성 밴드를 나타냈다. 74 kDa를 포함하는 상기 두 밴드 중에서, 본 발명자들은 이후의 분획과정을 위해 1,000-100 kDa의 컷오프값을 갖는 세포막을 통해서 비바 스핀 원심분리로부터 수득된 분획물을 선별하였다. 상기 분획물의 DEAE 세파로오스(sepharose) 양이온 교환 크로마토그래피는 74 kDa의 면역반응성 단백질을 포함하는 두 가지 분획물을 야기하였고, 상기 단백질들을 0.2 or 0.3 M NaCl를 이용하여 용출하였다(도 12c). 0.2 M NaCl에 의해 용출된 단밸질은 20mM Tris-HCl, pH 8.5을 이용하여 평형을 이룬 젤 여과 컬럼 상에 충진되었고 이후 0.2 M NaCl을 이용하여 용출되었을 때, 용출된 단백질(분획물 번호 #22 내지 #30)은 람블편모충 항체에 대한 면역반응을 입증하였다(도 12d). In contrast, the immunoreactive band was not present in Western blot analysis of serum fractions prior to the immune response. The fractions obtained by Vivaspin centrifugation using a cell membrane having a cutoff value of 100-10 kDa exhibited an immunoreactive band of the immunoreactive protein of 15 kDa. Of the two bands containing 74 kDa, we selected fractions obtained from Vivas spin centrifugation through a cell membrane with a cutoff value of 1,000-100 kDa for subsequent fractionation. DEAE sepharose cation exchange chromatography of the fractions resulted in two fractions containing an immunoreactive protein of 74 kDa and the proteins were eluted using 0.2 or 0.3 M NaCl (Figure 12c). The protein eluted with 0.2 M NaCl was loaded onto an equilibrated gel filtration column using 20 mM Tris-HCl, pH 8.5 and then eluted with 0.2 M NaCl to give the eluted protein (fractions # 22 to # 30) demonstrated an immune response against gabbroic antibody (Fig. 12d).
74 kDa의 단백질 밴드를 잘라내어, LC-MS/MS로 분석하였다. 단백질의 LC-MS/MS 분석을 이용한 데이터베이스 분석은 상기 단백질이 람블편모충의 결합 면역글로블린 단밸질(BiP) 상동체(GL50581_3283)(sequence coverage, 36%: MASCOT score, 1227)였다는 것을 나타내었다. The 74 kDa protein band was cut out and analyzed by LC-MS / MS. A database analysis of the protein using LC-MS / MS analysis indicated that the protein was the binding immunoglobulin protein (BiP) homologue (GL50581_3283) (sequence coverage, 36%: MASCOT score, 1227) of gabbial biceps.
따라서, 본 발명자들은 Bip가 이후 실험에서 람블편모충에서 검출한 74 kDa의 면역반응성 단백질에 해당하는 지를 조사하였다.
Therefore, the present inventors investigated whether Bip corresponds to the 74 kDa immunoreactive protein detected in the goby gland in later experiments.
2. 람블편모충의 74kDa인 면역성 단백질로서 GlBiP의 확인2. Identification of GlBiP as an immunogenic protein of 74 kDa of gambia
GlBiP가 항-람블편모충 항체와 반응한 74 kDA의 단백질인지를 결정하기 위하여, 람블편모충의 Bip를 대장균(E. coli)에서 재조합 단백질로서 발현시켰고, 수득된 람블편모충의 BiP를 정제하였다. 정제된 재조합 GlBiP를 본 발명자들이 항-람블편모충은 분획 실험을 통해서 동정하기 위해 사용했던 항-람블편모충이 항-람블편모충 항체와 반응한다는 사실이 발견되었다(도 1a). To determine whether GlBiP was a 74 kDa protein that reacted with the anti-gambia anthelmintic antibody, the Bip of the gibbini bark was expressed as a recombinant protein in E. coli , and the resulting BiP of the gibbini bark was purified. It has been found that the anti-gambia albicans which the present inventors used to identify the purified recombinant GlBiP through fractionation experiments of the anti-gambia bacterium react with the anti-gambia antibacterial antibody (Fig. 1A).
이후 상기 정제된 재조합 GlBiP를 래트에서 특이적인 다중클로날 항체를 제조하기 위한 항체로서 사용하였고, 수득된 항-GlBiP 항체의 특이성을 람블편모충 영향형 추출물의 웨스턴 블롯 분석에 의해 분석하였다. 즉, 람블편모충의 타고난 BiP가 명백히 74kDa의 면역반응성 밴드로 검출되었다(도 1b).
The purified recombinant GlBiP was then used as an antibody to produce a specific multiclonal antibody in rats, and the specificity of the obtained anti-GlBiP antibody was analyzed by western blot analysis of gabbro extract. Namely, the innate BiP of the gibbsite was clearly detected as an immunoreactive band of 74 kDa (Fig. 1B).
3. 재조합 GlBiP에 의한 수지상 세포 성숙 및 염증전 사이토카인 분비의 유도3. Induction of dendritic cell maturation and proinflammatory cytokine secretion by recombinant GlBiP
수지상 세포는 병원균을 인지하고 결과적으로 적절한 T 세포를 활성화시키는 국소 림프절로 이동하는 전문적인 항원제공 세포(antigen-presenting cells, APC)이다. 본 발명자들은 GlBiP가 마우스 유래의 수지상 세포의 성숙을 유도할 수 있는지를 검사하였다. GlBiP의 면역촉진성 활성(immunostiumulatory activity)을 측정하기 위한 어세이를 수행하기 전에, 본 발명자들은 LPS에 대해 대립하는 화합물인 PmB를 이용하여 마우스 골수유래 수지상 세포(bone marrow-derived dendritic cell, BMDC)를 전처리하였고, 0.1 μg/mL 재조합 GlBiP의 존재하에서 24 시간 동안 세포를 배양하였다(도 13). 재조합 rGlBiP의 결함을 갖는 수지상 세포에서 TNF-α의 수준은 PmB 치료에 의한 영향을 받지 않은 반면, TNF-α의 생산은 LPS로 처리된 수지상 세포에서 PmB에 의해 급격하게 영향을 받았다. 따라서, 수지상 세포는 이후 실험에서 LPS의 면역학적 활성을 억제하기 위하여 LPS로 처리된 수지상 세포에서 1 μg/mL에서 PmB로 전처리가 되었다. Dendritic cells are specialized antigen-presenting cells (APCs) that migrate to the local lymph nodes that recognize pathogens and eventually activate the appropriate T cells. The present inventors have examined whether GlBiP can induce the maturation of dendritic cells derived from mice. Prior to conducting an assay to measure the immunostimulatory activity of GlBiP, the present inventors used BMDC (bone marrow-derived dendritic cell, BMDC) using PmB, an opposing compound to LPS, And cells were cultured for 24 hours in the presence of 0.1 μg / mL recombinant GlBiP (FIG. 13). TNF-α levels in dendritic cells with defective recombinant rGlBiP were not affected by PmB treatment, whereas production of TNF-α was dramatically affected by PmB in dendritic cells treated with LPS. Thus, dendritic cells were pretreated with PmB at 1 μg / mL in dendritic cells treated with LPS to inhibit the immunological activity of LPS in subsequent experiments.
이후 실험에서, 본 발명자들은 수지상 세포 활성에 대한 3개의 마커, 즉 CD80, CD86 및 MHC 클래스 II의 발현을 조사하였다(도 2). 골수세포 유래 수지상 세포를 0.1 내지 5μg/mL 범위의 다양한 재조합 GlBiP의 존재하에서 24시간 동안 배양하였고, 이후 상기 표면 마커의 발현에 대해 분석하였다(데이터는 나타내지 않음). 표면 마커 MHCII를 발현하는 세포의 퍼센트 비율은 소량(0.1 μg/mL) 및 다량(5 μg/mL)의 재조합 GlBiP로서 급격하게 증가하지 않았다(51% 내지 68%). In a subsequent experiment, we examined the expression of three markers for dendritic cell activity, CD80, CD86 and MHC class II (FIG. 2). Bone marrow cell-derived dendritic cells were cultured for 24 hours in the presence of various recombinant GlBiP ranging from 0.1 to 5 μg / mL, and then analyzed for expression of the surface markers (data not shown). The percentages of cells expressing surface marker MHCII did not increase sharply (51% to 68%) with small amounts (0.1 μg / mL) and large amounts (5 μg / mL) of recombinant GlBiP.
따라서, 골수세포 유래 수지상 세포를 하기 실험에서 0.1 μg/mL의 재조합 GlBiP를 이용하여 처리하였다. MHC 클래스II의 발현을 보여주는 세포의 퍼센트 비율은 재조합 GlBiP로 처리한 반응에서 28% 내지 65%까지 증가하였다. 재조합 GlBiP을 이용한 자극에서, 공동자극(costimulatory) 분자, 즉 CD86 및 CD80를 발현하는 세포는 또한 각각 21% 내지 59% 및 25% 내지 61%로 증가하였다. 수지상 세포 활성화에 대한 양성 조절로서, LPS(0.5 μg/mL)가 골수세포 유래 수지상 세포를 자극시키기 위해 이용되었고, 골수세포 유래 수지상 세포에서 MHC 클래스 II(53%)와 두 개의 공동자극 마커, CD86(59%) 및 CD80 (49%)를 향상시킨다는 것을 발견하였다. 상기 결과는 람블편모충 BiP가 LPS 만큼 효율적으로 수지상 세포의 활성화를 유도하였다. Therefore, dendritic cells derived from bone marrow cells were treated with 0.1 μg / mL of recombinant GlBiP in the following experiment. Percentage of cells showing MHC class II expression increased from 28% to 65% in the reaction treated with recombinant GlBiP. In stimulation with recombinant GlBiP, cells expressing costimulatory molecules, CD86 and CD80, also increased from 21% to 59% and 25% to 61%, respectively. As a positive control for dendritic cell activation, LPS (0.5 μg / mL) was used to stimulate dendritic cells derived from bone marrow cells, and MHC class II (53%) and two co-stimulatory markers, CD86 (59%) and CD80 (49%). The above results led to the activation of dendritic cells as efficiently as the LPS by the gibbsite biloba BiP.
활성화된 수지상 세포는 사이토카인을 분비하고, 상기 사이토카인은 면역반응에서 중요한 역할을 한다(33). 마우스 골수세포 유래 수지상 세포는 다양한 농도의 재조합 GlBiP을 이용하여 자극받았고(0.1-10 μg/mL), 이후 IL-12, TNF-α, IL-6 및 IL-4 분비에 대해 분석되었다(도 3). 재조합 GlBiP는 대조군 세포 보다 높은 수준의 TNF-α, IL-12 및 IL-6 유의하게 골수세포 유래 수지상 세포를 분비하였다. 특히, 재조합 GlBiP에 결함이 있는 수지상 세포는 용량 의존적 방법으로 매우 다량의 IL-12(70,900 pg/mL)를 분비하였다. 상당한 양의 TNF-α 및 IL-6가 또한 GlBiP가 처리된 수지상 세포에 의해 분비되었다(70,900 pg/mL). 상당한 양의 TNF-α 및 IL-6가 또한 재조합 GlBiP이 처리된 수지상세포에 의해 각각 분비되었다(1,098 및 2,322 pg/mL). 그러나, TNF-α 및 IL-6의 수준은 고농도의 재조합 GlBiP에서 감소하였다. Il-4(79 pg/mL)는 재조합 GlBiP가 처리된 수지상 세포에서 거의 검출되지 않았다. 예상대로, 유의한 양의 IL-12, TNF-α, 및 IL-6가 LPS가 처리된 수지상 세포에 의해 분비되었다. 람블편모충에 대한 반응에서 생산된 사이토카인 프로파일은 람블편모충에 의해 유도된 수지상 세포 활성화가 Th-1 유형 면역 반응을 유도한다는 가능성을 향상시켰다.
Activated dendritic cells secrete cytokines, which play an important role in the immune response (33). Dendritic cells derived from mouse bone marrow cells were stimulated (0.1-10 μg / mL) with various concentrations of recombinant GlBiP and subsequently analyzed for IL-12, TNF-α, IL-6 and IL-4 secretion ). Recombinant GlBiP secreted significantly higher levels of TNF-α, IL-12 and IL-6 than dendritic cells from control cells. In particular, dendritic cells defective in recombinant GlBiP secreted very large amounts of IL-12 (70,900 pg / mL) in a dose-dependent manner. Significant amounts of TNF-a and IL-6 were also secreted by the dendritic cells treated with GlBiP (70,900 pg / mL). Significant amounts of TNF-a and IL-6 were also secreted by the dendritic cells treated with recombinant GlBiP, respectively (1,098 and 2,322 pg / mL). However, the levels of TNF-a and IL-6 decreased in high concentrations of recombinant GlBiP. Il-4 (79 pg / mL) was hardly detected in dendritic cells treated with recombinant GlBiP. As expected, significant amounts of IL-12, TNF-a, and IL-6 were secreted by LPS treated dendritic cells. The cytokine profile produced in response to gabbial larvae improved the likelihood that dendritic cell activation induced by the gambia biloba induces a Th-1 type immune response.
4. 람블편모충 추출물에 의한 마우스 골수유래 수지상세포(bone marrow-derived dendritic cells , BDDC)의 성숙4. Maturation of bone marrow-derived dendritic cells (BDDC) by glioblastiforme extract
본 발명자들은 람블편모충 추출물이 골수세포 유래 수지상 세포의 성숙을 유도할 수 있다는 것을 조사하였다(도 4a). 마우스 골수세포 유래 수지상 세포를 50 μg/mL에서 람블편모충와 경쟁시켰을 때, MHC II 표면 마커 발현하는 세포의 퍼센트 비율은 40% 내지 58%까지 증가하였다. 람블편모충 추출물을 이용한 자극의 경우, 공동자극성 분자, 즉 CD86 및 CD80를 발현하는 세포는 각각 40% 내지 92% 및 70% 내지 92%까지 증가하였다. 또한, 람블편모충 추출물로 자극 받은 세포는 람블편모충이 또한 수지상 세포 활성화를 유도한다는 것을 나타내는 유의한 수준의 TNF-α(129 pg/mL) 및 IL-12 (7,980 pg/mL)를 분비하였다.
The inventors of the present invention investigated that the gingival extract can induce maturation of dendritic cells derived from bone marrow cells (Fig. 4A). When the dendritic cells derived from mouse bone marrow cells were competing with the gambia at 50 μg / mL, the percentage of cells expressing MHC II surface markers increased from 40% to 58%. In the case of stimulation with gabbine extract, co-stimulatory molecules, namely CD86 and CD80 expressing cells, increased by 40% to 92% and 70% to 92%, respectively. Cells stimulated with gabbine extract also secreted significant levels of TNF-α (129 pg / mL) and IL-12 (7,980 pg / mL), indicating that gabbial extract also induces dendritic cell activation.
5. 수지상 세포에 의해 GlBiP-유도된 사이토카인 생산에서 TLR4의 역할5. The role of TLR4 in the production of GlBiP-induced cytokines by dendritic cells
본 발명자들은 람블편모충의 BiP 단백질이 인지하는 방법 및 상기 인지방법이 수지상 세포의 활성화 및 사이토카인 생산을 야기하는 방법를 연구하였다. 특히, 본 발명자들은 수지상 세포에 의해 재조합 GlBiP로 유도된 사이토카인 생산에서 두 가지 Toll 유사 수용기(Toll-like receptors, TLR)의 역할을 조사하였다(도 5a). 골수세포 유래 수지상 세포는 재조합 GlBiP를 이용한 자극 전에 TLR2 또는 TLR4에 특이적인 항체로 처리되었다. 대조군으로서, 골수세포 유래 수지상 세포의 또 다른 세트는 TLR 항체 대신에 아이소타입(isotype) 대조군 IgG로 처리되었고, 이후 동일한 방법으로 재조합 GlBiP를 이용하여 자극 받았다. 상기 재조합 GlBiP가 손상된 수지상 세포는 TNF-α의 생산을 위해 분석되었다. TLR2 항체로 처리된 수지상 세포에서, 재조합 GlBiP로 활성화된 수지상 세포는 아이소타입 IgG(2,554 pg/mL; p= 0.852, Student t-test)을 이용하여 전처리된 활성화된 수지상 세포와 유사한 수준의 TNF-α를 분비하였다. 대조적으로, TLR4 항체로 전처리된 세포는 아이소타입 IgG로 처리된 단백백혈구와 비교해서 TNF-α 생산에서 유의한 감소를 나타내었다(482 pg/mL).The present inventors have studied a method of recognizing BiP proteins of glioblastomae, and a method of inducing dendritic cell activation and cytokine production. In particular, we investigated the role of two Toll-like receptors (TLRs) in the production of recombinant GlBiP-induced cytokines by dendritic cells (Figure 5a). Bone marrow cell-derived dendritic cells were treated with antibodies specific for TLR2 or TLR4 prior to stimulation with recombinant GlBiP. As a control, another set of bone marrow cell-derived dendritic cells was treated with isotype control IgG instead of TLR antibody and then stimulated with recombinant GlBiP in the same manner. The dendritic cells in which the recombinant GlBiP was impaired were analyzed for the production of TNF-a. In dendritic cells treated with the TLR2 antibody, dendritic cells activated with recombinant GlBiP were treated with a level of TNF-α that was similar to pretreated activated dendritic cells using isotype IgG (2,554 pg / mL; p = 0.852, Student t-test) alpha. In contrast, cells pretreated with the TLR4 antibody showed a significant decrease in TNF-α production (482 pg / mL) compared to isotype IgG-treated protein leukocytes.
마우스 수지상 세포에서, 재조합 GlBiP로 유도된 사이토카인 생산에서 TLR4의 기능을 연구하기 위하여, 본 발명자들은 또한 HEK 293세포를 이용한 in vitro 시스템를 재구성하였고, 상기 시스템에서 TLR2 또는 TLR 4가 공통된 어댑터 단백질(adaptor protein), 즉 MyD88과 함께 발현되었다(34). 대조군으로서, HEK 293 세포의 또 다른 세트는 pFLAG-TLR2 또는 pFLAG-TLR4를 위한 공벡터, pFLAG-CMV1을 이용하여 형질감염되었다. 리포터 유전자를 이용한 사이토카인 생산을 모니터하기 위하여, 상기 시스템은 형질 감염 효율을 이용하여 노말화된 NF-κB에 대한 루시퍼레이즈 리포터를 포함한다. 사이토카인 생산을 유도하기 위한 재조합 람블편모충의 능력은 TLR4를 발현하는 HEK 293 세포, TLR2을 발현하는 HEK 293 세포, 및 대조군 HEK 293 세포의 루시퍼레이즈 활성을 측정함으로서 모니터링 되었다(도 5b). LPS 또는 재조합 GlBiP을 이용한 배양의 경우, pFLAG-CMV를 운반하는 HEK 293는 각각 57 또는 26으로, 상대적으로 감소된 루시퍼레이즈 활성을 나타내었다. TLR2를 발현하는 HEK293 세포는 재조합 GlBiP가 아니라, 오직 LPS와 반응하여 루시퍼레이즈 활성에서 적절한 증가를 보여주었다(루시퍼레이즈 활성에 상대적으로 80 내지 196)). 대조적으로, 재조합 GlBiP 또는 LPS로 자극되는 경우, TLR4를 발현하는 HEK 293 세포는 루시퍼레이즈 활성에서 유의한 증가를 보여주었다(루시퍼레이즈 활성에 상대적으로 각각 771 및 636). In order to study the function of TLR4 in the production of recombinant GlBiP-induced cytokines in mouse dendritic cells, we also reconstructed the in vitro system using HEK 293 cells and found that TLR2 or TLR4 in this system is a common adapter protein protein, that is, MyD88 (34). As a control, another set of HEK 293 cells was transfected with pFLAG-CMV1, a pivotal vector for pFLAG-TLR2 or pFLAG-TLR4. To monitor cytokine production using the reporter gene, the system comprises a Luciferase reporter for NF- [kappa] B, which is normalized using transfection efficiency. The ability of recombinant gambia to induce cytokine production was monitored by measuring the luciferase activity of HEK 293 cells expressing TLR4, HEK 293 cells expressing TLR2, and control HEK 293 cells (Fig. 5B). In the case of culturing using LPS or recombinant GlBiP, HEK 293 carrying pFLAG-CMV exhibited a relatively reduced luciferase activity of 57 or 26, respectively. HEK293 cells expressing TLR2 exhibited an appropriate increase in luciferase activity (80 to 196 relative to luciferase activity), not only with recombinant GlBiP, but with LPS only. In contrast, when stimulated with recombinant GlBiP or LPS, HEK 293 cells expressing TLR4 showed a significant increase in luciferase activity (771 and 636 relative to luciferase activity, respectively).
다음 세트의 실험에서, 본 발명자들은 TLR4 넉아웃 마우스 모델을 이용한 수지상 세포에 의하여 재조합 GlBiP에 의해 유도된 사이토카인 생산에서 TLR4의 역할을 확인하였다(도 5c). TLR4-/- 마우스의 골수세포 유래 수지상 세포는 재조합 GlBiP에 의해 유도된 IL-12 분비에서 급격한 감소(12 pg/mL)를 나타낸 반면에. TLR2-/- 마우스의 골수세포 유래 수지상 세포는 재조합 GlBiP에 대한 반응에서 야생형 골수세포 유래 수지상 세포(24,350 pg/mL)에 대한 IL-12와 유사한 수준(23,671 pg/mL)을 분비하였다. 재조합 GlBiP를 이용한 골수세포 유래 수지상 세포의 자극의 경우, 야생형 세포 및 TLR2 결핍 마우스 유래의 세포는 향상된 TNF-α 생산을 입증한(각각 719 및 682pg/mL) 반면에, TLR-4 결핍 세포는 TNF-α를 생산하지 못하였다(6 pg/mL). 동일한 방법으로, 수지상 세포에 의한 재조합 GlBiP-처리 유도된 IL-6 생산은 야생형 및 TLR2-/- 마우스에서 수득되었다(각각 6,792 및 7,139 pg/mL). 대조적으로, TLR4-/- 마우스 유래의 세포는 예상대로 재조합 GlBiP에 의해 유도된 IL-6 분부의 일부 감소를 4,082 pg/mL로서 나타낸 반면에, LPS 및 TNF-α를 이용한 야생형 골수세포 유래 수지상 세포의 시험은 TNF-α의 감소된 생산을 야기하였다(각각 5,300 및 90 pg/mL). TLR4-/- 마우스 유래의 골수세포 유래 수지상 세포는 LPS에 대한 반응에서 IL-6를 분비할 능력을 상실하였다(87 pg/mL). 상기 데이터는 명확히 TLR4가 람블편모충 Bip에 대한 반응을 위해 요구된다는 것을 나타낸다.
In the next set of experiments, we confirmed the role of TLR4 in the production of recombinant GlBiP-induced cytokines by dendritic cells using the TLR4 knockout mouse model (Fig. 5C). Dendritic cells derived from bone marrow cells of TLR4 - / - mice exhibited a rapid decrease (12 pg / mL) in IL-12 secretion induced by recombinant GlBiP. Bone marrow-derived dendritic cells of TLR2 - / - mice secreted a similar level of IL-12 (23,671 pg / mL) to wild-type bone marrow cell-derived dendritic cells (24,350 pg / mL) in response to recombinant GlBiP. In the case of stimulation of bone marrow cell-derived dendritic cells using recombinant GlBiP, cells from wild-type and TLR2 deficient mice demonstrated improved TNF-a production (719 and 682 pg / mL, respectively), whereas TLR- -α (6 pg / mL). In the same way, recombinant GlBiP-treatment induced IL-6 production by dendritic cells was obtained in wild-type and TLR2 - / - mice (6,792 and 7,139 pg / mL, respectively). In contrast, cells derived from TLR4 - / - mice exhibited a partial reduction of IL-6 fraction induced by recombinant GlBiP as expected at 4,082 pg / mL, whereas wild-type bone marrow cell-derived dendritic cells using LPS and TNF- Resulted in reduced production of TNF-a (5,300 and 90 pg / mL, respectively). Bone marrow cell-derived dendritic cells derived from TLR4 - / - mice lost ability to secrete IL-6 in response to LPS (87 pg / mL). The data clearly indicate that TLR4 is required for response to gibbons Bip.
6. 마우스 수지상 세포 유래의 재조합 GlBiP에 의해 유도된 사이토카인 생산에서 MyD88의 역할6. Role of MyD88 in the production of recombinant GlBiP-induced cytokines from mouse dendritic cells
MyD88은 표면 TLR과 세포내 신호전달 구성성분 사이의 신호전달을 매개하는 일종의 어댑터(adaptor)이다(35). 그러나, 일부 TLR 리간드는 세포내 신호전달 구성요소, 예컨대 TRIF를 MyD88의 관여 없이 활성화시킨다. 본 발명자들은 MyD88가 재조합 GlBiP BiP에 의해 유도된 사이토카인과 관련이 있는 지를 MyD88을 이용하여 조사하였다(도 6a). MyD88로부터 제조된 골수세포 유래 수지상 세포는 LPS를 이용하여 시험하였을 때, 종래 보고된 바외 같이(37) IL-12, TNF-α 또는 IL-6를 생산하지 못하였다. 재조합 GlBiP로 처리되었을 경우, MyD88은 TLR4/BiP 상호작용과 관련된 본질적인 신호전달 구성요소라는 것을 시사한다. MyD88 is an adapter that mediates signal transduction between surface TLRs and intracellular signaling components (35). However, some TLR ligands activate the intracellular signaling component, such as TRIF, without involvement of MyD88. We investigated whether MyD88 is associated with a cytokine induced by recombinant GlBiP BiP using MyD88 (Fig. 6A). When myeloid-derived dendritic cells prepared from MyD88 were tested using LPS, they did not produce IL-12, TNF-a or IL-6 as previously reported (37). When treated with recombinant GlBiP, MyD88 suggests that it is an essential signaling component associated with TLR4 / BiP interaction.
또한, MyD88 및 TLR4간의 상호작용은 재조합 GlBiP로 처리된 수지상 세포에서 TLR4와 MyD88의 물리적 결합을 측정함으로써 모니터링 되었다(도 6b). 재조합 GlBiP를 이용하여 처리된 수지상 세포 유래의 용해물(lasaye)을 항-TLR4 또는 항-MyD88 항체를 이용한 면역침강법을 수행하였다. 침강된 용해물의 웨스턴 블롯 분석은 비처리된 세포와 비교하여 2 내지 3배 더 많은 MyD88가 재조합 GlBiP-처리된 세포로부터 수득한 항-TLR4 항체와 공동-침강된다는 것을 나타내었다. 항-MyD88 항체는 비처리된 세포 보다 재조합 GlBiP로 자극 받은 세포의 두 배 만큼 침강되었다. 상기 결과는 TLR4 또는 MyD88간의 결합이 재조합 GlBiP에 노출된 수지상 세포에서 증가되었다는 것을 입증하는 것이다.
In addition, the interaction between MyD88 and TLR4 was monitored by measuring the physical association of TLR4 and MyD88 in dendritic cells treated with recombinant GlBiP (Fig. 6B). Lysates from dendritic cells treated with recombinant GlBiP were subjected to immunoprecipitation using anti-TLR4 or anti-MyD88 antibodies. Western blot analysis of the precipitated lysate showed that two to three-fold more MyD88 co-precipitates with the anti-TLR4 antibody obtained from recombinant GlBiP-treated cells as compared to untreated cells. The anti-MyD88 antibody precipitated twice as much as the cells stimulated with recombinant GlBiP over untreated cells. The results demonstrate that the binding between TLR4 or MyD88 was increased in dendritic cells exposed to recombinant GlBiP.
7. 마우스 수지상 세포 유래의 재조합 GlBiP에 의해 유도된 사이토카인 생산에서 MAPK의 역할7. The role of MAPKs in the production of cytokines induced by recombinant GlBiP derived from mouse dendritic cells
MAPK는 병원성 미생물에 대한 세포 반응을 촉발하는 특성규명이 잘된 신호전달 카이네이즈(kinase)이다. 진핵세포에서 3개의 잘 알려진 MAPK 서브 패밀리, 즉 ERK1/2, p38 및 JNK이다. 상기 카이네이즈는 다양한 세포외적 자극, 예컨대 물리적 스트레스, 염증성 사이토카인, 성장 호르몬 및 박테리아 성분들에 대한 반응에서 독립적으로 또는 동시에 활성화된다(38). 본 발명자들은 마우스 수지상 세포에서 람블편모충 BiP에 의해 유도된 사이토카인 생산에서 MAPK의 역할을 조사하였다. PD98059에 의한 ERK1/2의 불활성화(ERK1/2의 선택적 억제자)는 마우스 수지상 세포에서 재조합 GlBiP와 반응하여 IL-12 및 TNF-α의 생산을 비처리된 세포에서 각각 14% 및 36%의 수준까지 감소시켰다(도 7a). 이후 본 발명자는 재조합 GlBiP 처리에 대한 반응에서 ERK1/2 MAPK의 활성화 상태를 조사하였다. MAPK is a well-characterized signaling kinase that triggers cellular responses to pathogenic microorganisms. Three well known MAPK subfamilies in eukaryotic cells, ERK1 / 2, p38 and JNK. The kinase is activated independently or simultaneously in response to a variety of extracellular stimuli such as physical stress, inflammatory cytokines, growth hormone and bacterial components (38). The present inventors have investigated the role of MAPK in the production of cytokines induced by gibberellin BiP in mouse dendritic cells. Inactivation of ERK1 / 2 by PD98059 (selective inhibitor of ERK1 / 2) reacted with recombinant GlBiP in mouse dendritic cells to produce IL-12 and TNF-α in 14% and 36% of untreated cells, respectively (Fig. 7A). The present inventors then investigated the activation state of ERK1 / 2 MAPK in response to recombinant GlBiP treatment.
재조합 GlBiP의 노출은 자극 10분 후 최대 활성화를 갖는 ERK1/2의 인산화를 야기하였다. ERK1/2의 인산화는 또한 LPS로 처리된 수지상 세포에서 최대 활성화가 자극 8분 후에 관찬되었을 때 발생하였다. p38 MAPK의 억제자인 SB202190을 이용한 수지상 세포의 전처리는 재조합 GlBiP에 대한 반응에서 비처리된 세포에서 측정된 수준의 12% and 32%까지 각각IL-12 및 TNF-α의 생산의 감소를 야기하였다(도 7b). 재조합 GlBiP에 대한 마우스 수지상 세포의 노출은 자극의 8분 및 10분 사이의 최대활성화를 갖는 p38 인산화를 야기하였다. 마우스 수지상 세포의 LPS 처리는 또한 p38 MAPK의 인산화를 유도하였다. Exposure of recombinant GlBiP resulted in phosphorylation of ERK1 / 2 with maximal activation after 10 minutes of stimulation. Phosphorylation of ERK1 / 2 also occurred when maximal activation was observed 8 min after stimulation in dendritic cells treated with LPS. Pretreatment of dendritic cells with p38 MAPK inhibitor SB202190 resulted in a reduction in the production of IL-12 and TNF-a by 12% and 32%, respectively, in the untreated cells in response to recombinant GlBiP 7b). Exposure of mouse dendritic cells to recombinant GlBiP resulted in p38 phosphorylation with maximal activation between 8 and 10 minutes of stimulation. LPS treatment of mouse dendritic cells also induced phosphorylation of p38 MAPK.
p38 및 ERK1/2 MAPK와 대조적으로, JNK의 분명한 활성화는 재조합 GlBiP를 이용한 마우스 수지상 세포의 배양의 경우 검출되지 않았다(데이터는 나타내지 않음). JNK 억제자 및 JNK 억제자 II를 이용한 마우스 수지상 세포의 전처리는 재조합 GlBiP와 반응하여 IL-12 and TNF-α에서 약간의 감소, 즉 24-28%의 감소를 야기하였다(도 7c). 상기 결과는 MAPK가 수지상 세포 성숙에서 어떤 역할을 한다는 것을 나타내었다. 3개의 MAPK 중에서, p38 및 ERK1/2 MAPK는 재조합 GlBiP에 의해 유도된 면역 반응에 대한 JNK 보다 더 많이 기여하였다.
In contrast to p38 and ERKl / 2 MAPK, the clear activation of JNK was not detected in the case of culturing mouse dendritic cells with recombinant GlBiP (data not shown). Pretreatment of mouse dendritic cells with JNK inhibitor and JNK inhibitor II resulted in a slight decrease in IL-12 and TNF-a, i.e., 24-28% reduction, in response to recombinant GlBiP (Figure 7c). These results indicate that MAPK plays a role in dendritic cell maturation. Of the three MAPKs, p38 and ERK1 / 2 MAPK contributed more than JNK to the immune response induced by recombinant GlBiP.
8. GlBiP에 의해 활성화된 수지상 세포에서 NF-κB 및 AP-1의 증가된 결합 활성8. Increased binding activity of NF-κB and AP-1 in dendritic cells activated by GlBiP
본 발명자들은 두 개의 전사인자, AP-1 및 NF-κB가 람블편모충 BiP 자극에 대해 유사한 결합 부위에 결합하는 지를 EMAS를 이용하여 조사하였다. 핵 추출물을 재조합 GlBiP로 처리된 마우스 골수세포 유래 수지상 세포로부터 제조하였고, 이후 NF-κB 결합 부위를 포함하는 올리고뉴클레오타이드와 함께 배양하였다(도 8a). 이것은 표지된 DNA 단편이 더 느리게 이동하게 만들지만, 이 효과는 초과한 비표지된 NF-κB 결합 부위를 결합 반응에 첨가하였을 때 사라졌다. NF-κB 결합 활성은 또한 예상대로 LPS로 자극된 수지상 세포의 핵 추?물에서 관찰되었다. 재조합 GlBiP로 자극 받은 골수세포 유래 수지상 세포의 핵 추출물은 또한 AP-1 결합 부위를 포함하는 표지된 올리고뉴클레오타이드와 함게 배양되었다(도 8b). 마찬가지로, 상기 핵 추출물은 느리게 움직이는 AP-1 DNA 단편을 생산하였고, 이 상호작용은 특이적인 상호작용을 나타내는 AP-1 결할 부위의 첨가에 의해 방해받았다. LPS로 처리된 수지상 세포로부터 제조된 핵 추출물은 또한 증가된 AP-1 결합 활성을 나타내었다.
The present inventors investigated whether two transcription factors, AP-1 and NF-κB, bind to similar binding sites for gibberellin BiP stimulation, using EMAS. Nuclear extracts were prepared from dendritic cells derived from mouse bone marrow cells treated with recombinant GlBiP and then incubated with oligonucleotides containing NF-κB binding sites (FIG. 8A). This caused the labeled DNA fragment to migrate more slowly, but this effect disappeared when the excess unlabeled NF-κB binding site was added to the binding reaction. NF-κB binding activity was also observed in nuclear extracts of dendritic cells stimulated with LPS as expected. Nuclear extracts of dendritic cells derived from bone marrow cells stimulated with recombinant GlBiP were also incubated with labeled oligonucleotides containing the AP-1 binding site (Figure 8b). Likewise, the nuclear extract produced a slow-moving AP-1 DNA fragment, which was interrupted by the addition of AP-1 knock-in regions that exhibited specific interactions. Nuclear extracts prepared from dendritic cells treated with LPS also exhibited increased AP-1 binding activity.
9. GlBiP의 Hsp70 도메인에 의한 수지상 세포 활성화의 유도9. Induction of dendritic cell activation by the Hsp70 domain of GlBiP
GlBiP은 2개의 추정상의 도메인, 즉, 액틴 결합 도메인(N 말단 부위) 및 열충격 단백질(C 말단 부위)을 각각 포함한다. 전장 재조합 GlBiP 뿐만 아니라, 두 개의 절단된 재조합 GlBiP 단백질, 재조합 GlBiP-N 도메인 및 재조합 GlBiP-C 도메인이 대장균에서 발현되었다(도 9a). 상기 재조합 GlBiP 단백질을 재조합 GlBiP의 동정을 위해 이용된 항-람블편모충 항체로 배양하였을 때, rGlBiP-N 도메인 및 rGlBiP-C 도메인이 대장균에서 발현되었다(도 9a). 상기 재조합 GlBiP 단백질을 항-람블편모충 항체를 이용하여 배양하였을 때(도 12 및 도 1), 전장 rGlBiP 및 rGlBiP-콜로니는 면역반응성 단백질을 나타낸 반면에 그것들은 면역전 혈청을 이용한 웨스턴 블롯 분석에서 존재하지 않았다(도 9b). 또 다른 절단된 재조합 GlBiP, 재조합 GlBiP-N 도메인은 전면역 혈청 또는 항-람블편모충 항체와 반응하지 않았다. GlBiP contains two putative domains, namely an actin binding domain (N-terminal region) and a heat shock protein (C-terminal region), respectively. In addition to full-length recombinant GlBiP, two truncated recombinant GlBiP proteins, a recombinant GlBiP-N domain and a recombinant GlBiP-C domain were expressed in E. coli (Fig. 9A). When the recombinant GlBiP protein was cultured with the anti-gambia antibodia antibody used for identification of the recombinant GlBiP, the rGlBiP-N domain and the rGlBiP-C domain were expressed in E. coli (FIG. 9A). When the recombinant GlBiP protein was cultured using an anti-gambling anthelmintic antibody (FIG. 12 and FIG. 1), the full-length rGlBiP and rGlBiP-colonies showed immunoreactive proteins whereas they did not exist in Western blot analysis using immunized sera (Fig. 9B). Another truncated recombinant GlBiP, recombinant GlBiP-N domain, did not react with the full-length serum or anti-lambda monoclonal antibody.
상기 절단된 재조합 GlBiP 단백질은 마우스 골수세포 유래 수지상 세포를 자극하기 위하여 이용되었다(도 9c). Hsp70 도메인을 갖는 조합 GlBiP는 자극 받은 수지상 세포로부터 TNF-α 및 IL-12를 유도하는데 효율적이었다. 반면에, 액틴 결합 도메인을 갖는 재조합 GlBiP는 마우스 골수세포 유래 수지상 세포로부터 사이토카인 생산을 자극하지 않았다.
The truncated recombinant GlBiP protein was used to stimulate dendritic cells derived from mouse bone marrow cells (Fig. 9C). Combination GlBiP with the Hsp70 domain was effective in inducing TNF-a and IL-12 from stimulated dendritic cells. On the other hand, recombinant GlBiP having an actin-binding domain did not stimulate cytokine production from dendritic cells derived from mouse bone marrow cells.
10. 재조합 GlBiP 단백질에 의한 수지상 세포 활성화를 통하여 T 세포에 의한 사이토카인 생산의 유도10. Induction of cytokine production by T cells through dendritic cell activation by recombinant GlBiP protein
마우스 지라세포(splenocyte)로부터 수득된 CD4+ T 세포가 재조합 GlBiP 또는 재조합 GlBiP-C 도메인을 이용하여 처리된 마우스 수지상 세포를 이용하여 공동 배양되었을 때, 이들은 IL-2(각각 174 pg/mL or 2,364pg/mL)을 분비하였다(Fig. 10A). 반면에, 재조합 GlBiP-N 도메인으로 처리된 마우스 수지상 세포와 공배양된 CD4+ T 세포는 IL-2를 방출하지 않았다(2 pg/mL). IL-2는 재조합 GlBiP가 손송된 수지상 세포는 IL-2를 생산하지 않았기 때문에 CD4+ T 세포로부터 수득되었지만, 마우스 수지상 세포로부터 수득되지 않았다. 또한, 오직 전장 재조합 GlBiP 또는 재조합 GlBiP-C 도메인과 함께 배양된 T-세포로부터 수득된 배양액은 상당한 양의 IFN-γ 분비를 입증하였다(2,921 pg/mLor 52,469 pg/mL)(Fig. 10B). 동일한 방법으로, 대부분의 IFN-γ는 재조합 GlBiP로 자극된 수지상 세포만이 T 세포 없이 낮은 수준의 IFN-γ를 생산하였기 때문에, T 세포로부터 나온다(68 pg/mL). 상기 결과는 GlBiP가 DC 활성화를 통해서 T 세포에 의한 사이토카인 생산을 유도할 수 있고 C-말단의 Hsp70 도메인은 이 과정에서 중요하다는 것을 나타낸다.
Obtained from mouse splenocytes When CD4 + T cells were co-cultured using recombinant GlBiP or recombinant GlBiP-C domain-treated mouse dendritic cells, they secreted IL-2 (174 pg / mL or 2,364 pg / mL, respectively) (Fig. 10A). On the other hand, CD4 + T cells co-cultured with mouse dendritic cells treated with the recombinant GlBiP-N domain did not release IL-2 (2 pg / mL). IL-2 was obtained from CD4 + T cells, but not from mouse dendritic cells, because dendritic cells transfected with recombinant GlBiP did not produce IL-2. In addition, the culture obtained from T-cells incubated with only the full-length recombinant GlBiP or recombinant GlBiP-C domain demonstrated a significant amount of IFN-γ secretion (2,921 pg / mLor 52,469 pg / mL) (Fig. In the same way, most IFN-γ is derived from T cells (68 pg / mL) because only dendritic cells stimulated with recombinant GlBiP produced low levels of IFN-γ without T cells. The results indicate that GlBiP can induce cytokine production by T cells through DC activation and that the Csp-terminal Hsp70 domain is important in this process.
11. 마우스의 람블편모충 감염에 의한 GlBiP의 Hsp70 도메인에 대한 항체 형성의 촉진11. Promotion of antibody formation against the Hsp70 domain of GlBiP by lambda bract infection in mice
람블편모충 영양형의 마우스내로의 구강 감염 후, 이들의 혈청을 람블편모충 추출물 또는 재조합 GlBiP 단백질을 이용하여 웨스턴 블록에 의해 분석하였다(도 11). 4개의 면역반응성 단백질이 항-람블편모충 항체를 이용한 웨스턴 블롯에서 분자량 74, 58, 55 및 30 kDa에서 관찰되었다(도 12a). 74 kDa의 면역반응성 단백질은 GlBiP로 추정되었다. 상기 혈청을 또한 3개의 재조합 GlBiP와 반응시켰다. 전면역 혈청을 이용한 웨스턴 블롯에서 명백한 면역반응성 단백질이 전혀 존재하지 않는 반면에, 감염된 마우스로부터 수득된 혈청을 이용한 반응은 Hsp70 도메인을 수반하는 절단된 재조합 GlBiP-C 도메인 뿐만 아니라 전장 재조합 GlBiP 단백질에 대한 면역반응성을 입증하였다. 대조적으로, 재조합 GlBiP-N 도메인은 상기 혈청에 대한 어떠한 면역반응성을 나타내지 않았다.
After oral infections into the mice of the glioblastoma enteritidis, their sera were analyzed by Western bloc using gabbial extract or recombinant GlBiP protein (Fig. 11). Four immunoreactive proteins were observed at molecular weights of 74, 58, 55, and 30 kDa in Western blot using anti-gambia anthelmintic antibody (Fig. 12A). The 74 kDa immunoreactive protein was estimated to be GlBiP. The serum was also reacted with three recombinant GlBiPs. While there is no apparent immunoreactive protein in the Western blot using preimmune sera, the reaction with the sera obtained from the infected mouse results in the cleaved recombinant GlBiP-C domain carrying the Hsp70 domain as well as the full-length recombinant GlBiP protein Immunoreactivity was demonstrated. In contrast, the recombinant GlBiP-N domain did not show any immunoreactivity to the serum.
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<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, Yonsei University <120> Immunostimulatory Compositions Comprising Recombinant Giardia lamblia binding immunoglobulin protein <130> PN140275 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 677 <212> PRT <213> Girardia <220> <221> DOMAIN <222> (1)..(677) <223> recombinant Giardia lamblia binding immunoglobulin protein <400> 1 Met Thr Ser Ser Arg Val Asn Gln Lys Glu Tyr Thr Lys Ile Lys Met 1 5 10 15 Ile Ala Leu Val Phe Ala Ala Leu Ala Leu Ala Glu Thr Ile Ile Gly 20 25 30 Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Ala Val Ser Arg Ala Gly 35 40 45 Gln Val Glu Ile Ile Pro Asn Glu Leu Gly Ala Arg Val Thr Pro Ser 50 55 60 Tyr Val Ala Phe Thr Ala Asp Gly Glu Arg Leu Val Gly Asp Ala Ala 65 70 75 80 Lys Asn Tyr Ala Pro Ile Ser Pro Glu Asn Thr Ile Phe Asp Val Lys 85 90 95 Arg Leu Ile Gly Arg Lys Phe Asp Asp Pro Glu Val Gln Lys Asp Met 100 105 110 Lys Leu Leu Pro Tyr Lys Val Ile Asn Lys Asp Gly Arg Pro Phe Val 115 120 125 Gln Leu Ser Gly Thr Asn Leu Pro Lys Glu Leu Gln Xaa Lys Ile Met 130 135 140 Ser Pro Glu Glu Ile Ser Ala Met Val Leu Thr Lys Met Lys Thr Ile 145 150 155 160 Ala Glu Asp Tyr Leu Gly Glu Lys Ile Thr Lys Ala Val Val Thr Val 165 170 175 Pro Ala Tyr Phe Ser Asp Ser Gln Arg Ser Ala Thr Lys Asp Ala Gly 180 185 190 Arg Ile Ala Gly Leu Asp Val Val Arg Ile Ile Asn Glu Pro Thr Ser 195 200 205 Ser Ser Ile Ala Tyr Gly Leu Asp Lys Lys Thr Gln Glu Thr Ser Gly 210 215 220 Lys Ala Lys Asn Ile Leu Val Phe Asp Cys Gly Gly Gly Thr His Asp 225 230 235 240 Val Ser Ile Leu Ser Val Asp Ser Gly Val Phe Glu Val Leu Ala Thr 245 250 255 Ala Gly Asn Thr His Leu Gly Gly Glu Asp Phe Asp Arg Arg Leu Leu 260 265 270 Asp His Phe Ile Ala Ile Phe Lys Lys Lys Asn Asn Ile Asp Leu Ser 275 280 285 Ile Thr Asn Thr Gly Asp Lys Ala Lys Asp Met Ala Val Lys Lys Ala 290 295 300 Ile Ser Arg Leu Arg Arg Glu Ile Glu Ala Gly Lys Arg Gln Leu Ser 305 310 315 320 Thr Ala Ser Ser Val Gln Ile Val Val Asp Ser Leu Ile Asp Gly Val 325 330 335 Asp Phe Ser Glu Ser Leu Thr Arg Ala Lys Phe Glu Glu Leu Asn Ile 340 345 350 Asp Leu Phe Lys Lys Ser Ile Lys Pro Val Glu Gln Val Leu Arg Asp 355 360 365 Ala Lys Leu Lys Thr Thr Asp Ile Asp Glu Val Val Leu Val Gly Gly 370 375 380 Ser Thr Arg Ile Pro Lys Ile Arg Gln Leu Leu Gln Asp Tyr Phe Asn 385 390 395 400 Gly Lys Ala Leu Asn Lys Asp Ile Asn Ala Asp Glu Ala Val Ala Trp 405 410 415 Gly Ala Ala Val Gln Ala Ser Ile Leu Ser Gly Ala Lys Asp His Asp 420 425 430 Val Leu Leu Ile Asp Val Thr Pro Leu Thr Leu Gly Ile Glu Thr Gln 435 440 445 Gly Gly Ile Met Thr Pro Leu Ile Glu Arg Asn Ser Tyr Ile Pro Val 450 455 460 Lys Lys Ser Lys Ile Phe Ser Thr Val Gln Asp Gln Gln Thr Met Val 465 470 475 480 Lys Ile Gln Val Tyr Glu Gly Glu Arg Ser Met Val Lys Asp Asn Asn 485 490 495 Leu Leu Gly Asn Phe Asp Leu Asn Asp Ile Pro Pro Ala Pro Arg Gly 500 505 510 Thr Pro Gln Ile Glu Val Thr Phe Glu Ile Asp Ser Asn Gly Ile Leu 515 520 525 Thr Val Ser Ala Val Glu Lys Ser Ser Gly Lys Glu Glu Ser Ile Thr 530 535 540 Ile Lys Asn Asp Arg Gly Arg Leu Ser Glu Asp Glu Ile Asn Arg Leu 545 550 555 560 Val Arg Glu Ala Glu Glu Phe Ala Glu Glu Asp Lys Ile Asn Arg Glu 565 570 575 Arg Ala Glu Ala Arg Asn Ala Phe Glu Thr Ile Val Ser Ile Thr Thr 580 585 590 Thr Gln Thr Thr Ala Asp Lys Glu Gly Asn Ile Val Asp Lys Ile Ser 595 600 605 Ser Asp Asp Leu Glu Lys Val Lys Glu Ala Ile Lys Glu Ala Gln Asp 610 615 620 Trp Leu Arg Asp Asn Thr Asp Ala Ser Lys Glu Glu Ile Glu Glu Glu 625 630 635 640 Lys Ser Lys Phe Glu Lys Val Val Gln Pro Ile Leu Gly Glu Asn Phe 645 650 655 Gly Arg Ser Ala Ser Ala Gly Gly Ser Gly Pro Glu Tyr Asp Tyr Ala 660 665 670 Glu Lys Asp Glu Leu 675 <110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, Yonsei University <120> Immunostimulatory Compositions Comprising Recombinant Giardia lamblia binding immunoglobulin protein <130> PN140275 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 677 <212> PRT <213> Girardia <220> <221> DOMAIN <222> (1). (677) <223> recombinant Giardia lamblia binding immunoglobulin protein <400> 1 Met Thr Ser Ser Arg Val Asn Gln Lys Glu Tyr Thr Lys Ile Lys Met 1 5 10 15 Ile Ala Leu Ala Leu Ala Leu Ala Glu Thr Ile Ile Gly 20 25 30 Ile Asp Leu Gly Thr Thr Tyr Ser Cys Val Ala Val Ser Arg Ala Gly 35 40 45 Gln Val Glu Ile Ile Pro Asn Glu Leu Gly Ala Arg Val Thr Pro Ser 50 55 60 Tyr Val Ala Phe Thr Ala Asp Gly Glu Arg Leu Val Gly Asp Ala Ala 65 70 75 80 Lys Asn Tyr Ala Pro Ile Ser Pro Glu Asn Thr Ile Phe Asp Val Lys 85 90 95 Arg Leu Ile Gly Arg Lys Phe Asp Asp Pro Glu Val Gln Lys Asp Met 100 105 110 Lys Leu Leu Pro Tyr Lys Val Ile Asn Lys Asp Gly Arg Pro Phe Val 115 120 125 Gln Leu Ser Gly Thr Asn Leu Pro Lys Glu Leu Gln Xaa Lys Ile Met 130 135 140 Ser Pro Glu Glu Ile Ser Ala Met Val Leu Thr Lys Met Lys Thr Ile 145 150 155 160 Ala Glu Asp Tyr Leu Gly Glu Lys Ile Thr Lys Ala Val Val Thr Val 165 170 175 Pro Ala Tyr Phe Ser Asp Ser Gln Arg Ser Ala Thr Lys Asp Ala Gly 180 185 190 Arg Ile Ala Gly Leu Asp Val Val Arg Ile Asn Glu Pro Thr Ser 195 200 205 Ser Ser Ile Ala Tyr Gly Leu Asp Lys Lys Thr Gln Glu Thr Ser Gly 210 215 220 Lys Ala Lys Asn Ile Leu Val Phe Asp Cys Gly Gly Gly Thr His Asp 225 230 235 240 Val Ser Ile Leu Ser Val Asp Ser Gly Val Ghe Val Leu Ala Thr 245 250 255 Ala Gly Asn Thr His Leu Gly Gly Glu Asp Phe Asp Arg Arg Leu Leu 260 265 270 Asp His Phe Ile Ala Ile Phe Lys Lys Lys Asn Asn Ile Asp Leu Ser 275 280 285 Ile Thr Asn Thr Gly Asp Lys Ala Lys Asp Met Ala Val Lys Lys Ala 290 295 300 Ile Ser Arg Leu Arg Arg Glu Ile Glu Ala Gly Lys Arg Gln Leu Ser 305 310 315 320 Thr Ala Ser Ser Val Gln Ile Val Val Asp Ser Leu Ile Asp Gly Val 325 330 335 Asp Phe Ser Glu Ser Leu Thr Arg Ala Lys Phe Glu Glu Leu Asn Ile 340 345 350 Asp Leu Phe Lys Lys Ser Ile Lys Pro Val Glu Gln Val Leu Arg Asp 355 360 365 Ala Lys Leu Lys Thr Thr Asp Ile Asp Glu Val Val Leu Val Gly Gly 370 375 380 Ser Thr Arg Ile Pro Lys Ile Arg Gln Leu Leu Gln Asp Tyr Phe Asn 385 390 395 400 Gly Lys Ala Leu Asn Lys Asp Ile Asn Ala Asp Glu Ala Val Ala Trp 405 410 415 Gly Ala Ala Val Gln Ala Ser Ile Leu Ser Gly Ala Lys Asp His Asp 420 425 430 Val Leu Leu Ile Asp Val Thr Pro Leu Thr Leu Gly Ile Glu Thr Gln 435 440 445 Gly Gly Ile Met Thr Pro Leu Ile Glu Arg Asn Ser Tyr Ile Pro Val 450 455 460 Lys Lys Ser Lys Ile Phe Ser Thr Val Gln Asp Gln Gln Thr Met Val 465 470 475 480 Lys Ile Gln Val Tyr Glu Gly Glu Arg Ser Ser Val Lys Asp Asn Asn 485 490 495 Leu Leu Gly Asn Phe Asp Leu Asn Asp Ile Pro Pro Ala Pro Arg Gly 500 505 510 Thr Pro Gln Ile Glu Val Thr Phe Glu Ile Asp Ser Asn Gly Ile Leu 515 520 525 Thr Val Ser Ala Val Glu Lys Ser Ser Gly Lys Glu Glu Ser Ile Thr 530 535 540 Ile Lys Asn Asp Arg Gly Arg Leu Ser Glu Asp Glu Ile Asn Arg Leu 545 550 555 560 Val Arg Glu Ala Glu Glu Phe Ala Glu Glu Asp Lys Ile Asn Arg Glu 565 570 575 Arg Ala Glu Ala Arg Asn Ala Phe Glu Thr Ile Val Ser Ile Thr Thr 580 585 590 Thr Gln Thr Thr Ala Asp Lys Glu Gly Asn Ile Val Asp Lys Ile Ser 595 600 605 Ser Asp Leu Glu Lys Val Lys Glu Ala Ile Lys Glu Ala Gln Asp 610 615 620 Trp Leu Arg Asp Asn Thr Asp Ala Ser Lys Glu Glu Ile Glu Glu Glu 625 630 635 640 Lys Ser Lys Phe Glu Lys Val Val Gln Pro Ile Leu Gly Glu Asn Phe 645 650 655 Gly Arg Ser Ala Ser Ala Gly Gly Ser Gly Pro Glu Tyr Asp Tyr Ala 660 665 670 Glu Lys Asp Glu Leu 675
Claims (7)
(a) a pharmaceutically effective amount of a Giardia lamblia binding immunoglobulin protein fragment consisting of amino acids 428 to 677 of SEQ ID NO: 1; And (b) a pharmaceutically acceptable carrier.
(a) (i) 서열목록 제1서열의 428 내지 677번째 아미노산으로 구성되는 람블편모충 결합 면역글로블린 단백질(Giardia lamblia binding immunoglobulin protein) 단편과 (ii) 에피토프 후보물질로서의 펩타이드를 이용하여 인간을 제외한 동물을 면역화시키는 단계; 및
(b) 상기 면역화된 동물에서의 면역반응을 측정하는 단계.
A method for screening an epitope having immunogenicity against a gibblet comprising the following steps:
(a) (i) a Giardia lamblia binding immunoglobulin protein fragment consisting of amino acids 428 to 677 of SEQ ID NO: 1 and (ii) a peptide as an epitope candidate substance, ; ≪ / RTI > And
(b) measuring the immune response in said immunized animal.
(a) (i) 서열목록 제1서열의 428 내지 677번째 아미노산으로 구성되는 람블편모충 결합 면역글로블린 단백질(Giardia lamblia binding immunoglobulin protein) 단편과 (ii) 에피토프 후보물질로서의 펩타이드를 이용하여 인간을 제외한 동물을 면역화시키는 단계; 및
(b) 상기 면역화된 동물에서의 면역반응을 측정하여 면역원성을 나타내는 펩타이드 에피토프를 선별하는 단계;
(c) 상기 선별된 펩타이드 에피토프와 분석 대상의 항체를 접촉시키는 단계;
(d) 상기 단계 (c)의 결과물과 상기 단백질 항원을 접촉시키는 단계; 및
(e) 상기 단백질 항원과 상기 분석 대상의 항체의 결합을 분석하는 단계.
A method for screening an antibody against a gibb complex protein antigen comprising the steps of:
(a) (i) a Giardia lamblia binding immunoglobulin protein fragment consisting of amino acids 428 to 677 of SEQ ID NO: 1 and (ii) a peptide as an epitope candidate substance, ; ≪ / RTI > And
(b) measuring the immune response in said immunized animal to select a peptide epitope exhibiting immunogenicity;
(c) contacting the selected peptide epitope with an analyte;
(d) contacting the resultant of step (c) with the protein antigen; And
(e) analyzing the binding between the protein antigen and the antibody to be analyzed.
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