KR20150001391A - Single domain antibody for M13 bacteriophage and immunosensor fusion protein comprising the same - Google Patents
Single domain antibody for M13 bacteriophage and immunosensor fusion protein comprising the same Download PDFInfo
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- KR20150001391A KR20150001391A KR20130074548A KR20130074548A KR20150001391A KR 20150001391 A KR20150001391 A KR 20150001391A KR 20130074548 A KR20130074548 A KR 20130074548A KR 20130074548 A KR20130074548 A KR 20130074548A KR 20150001391 A KR20150001391 A KR 20150001391A
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- South Korea
- Prior art keywords
- protein
- single domain
- bacteriophage
- domain antibody
- gfp
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
Abstract
Description
본 발명은 M13 박테리오파지에 대한 단일 도메인 항체 및 이를 포함하는 면역센서 융합 단백질에 관한 것이다.The present invention relates to a single domain antibody against M13 bacteriophage and an immunosensor fusion protein comprising the same.
M13 박테리오파지를 이용한 파지 전시(phage display)는 특이한 폴리펩타이드(polypeptide)를 조작하여 특정 단백질을 도입하여 이들의 기능을 규명하는데 유용한 실험기술이다(Sidhu, S. S. 2001. Biomolecular Engineering 18:57-63, Sidhu, S. S., Lowman, H. B., Cunningham, B. C., Wells, J. A. 2000. Meth. Enzymol. 328:333-363, Sidhu, S. S. 2000. Curr. Opin. Biotechnol. 11:610-616, Smith, G. P., Petrenko, V. A. 1997. Chem. Rev. 97:391-410). 상기 방법은 유전자를 암호화하는 폴리펩타이드가 파지(phage) 외피 단백질 유전자와 혼합되어 결국 파지 표면에 단백질로 발현되어 드러나게 되는 것을 말한다(Smith, G. P. 1985. Science. 228:1315-1317).Phage display using M13 bacteriophage is an experimental technique useful for identifying specific proteins by manipulating specific polypeptides (Sidhu, SS 2001. Biomolecular Engineering 18: 57-63, Sidhu , SS, Lowman, HB, Cunningham, BC, Wells, JA 2000. Meth. Enzymol. 328: 333-363, Sidhu, SS 2000. Curr. Opin. Biotechnol. 11: 610-616, Smith, GP, Petrenko, VA 1997. Chem. Rev. 97: 391-410). This method refers to the fact that a polypeptide encoding a gene is mixed with a phage coat protein gene and eventually expressed as a protein on the phage surface (Smith, G. P. 1985. Science. 228: 1315-1317).
M13 박테리오파지는 사상형(filamentous) 박테리오파지로 원형의(circular) 외가닥(single strand) DNA를 가진 긴 원통형(cylinder)의 단백질 외피로 구성되어 있고 길이는 1㎛ 내외, 직경은 10㎛보다 작은 구조로 되어 있다(Carlos, F. B., Dennis, R. B., Jamie, K. S., Gregg, J. S. 2001. Phage display, a laboratory manual). 이들의 단백질 외피는 다섯 가지 다른 종류의 단백질로 구성되어 있지만 (P3, P6, P7, P8, P9) 대부분은 gene 8에서 발현되는 P8로 파지의 외피단백질을 구성되어 있다(Marvin D. A., 1998. Curr. Opin. Struct. Biol. 8:150-158). P8의 구조는 α-나선형 입자로 되어 있고 반복적인 정렬로 파지의 긴 외피를 구성하고 있으며(Marvin D. A., 1998. Curr. Opin. Struct. Biol. 8:150-158) 파지의 생존과도 밀접한 관계가 있다(Williams, K. A., Glibowicka, M., Li, Z., Li, H., Khan, A. R., Chen, Y. M. Y. 1995. J. Mol. Biol. 252:6-14., Deberr, C. M., Khan, A. R., Li, Z., Joensson, C., Glibowicka, M. W. 1993. J. Proc. Natl. Acad. Sciii. 90:11648-11652). 파지를 구성하는 다섯 가지 종류의 외피 단백질들은 파지 입자의 구조적 안정성을 위해 존재하고 특히, P3은 기주 세포의 인식과 이들의 감염에 필수적인 역할을 하며 세부분의 도메인(domain)으로 구성되어 있다(Armstrong, J., Perham, R. N., Walker, J. E. 1981. FEBS Lett. 135:167-172). 이 파지들은 F 플라스미드를 가진 특정 대장균의 섬모(pilus)를 통해 감염이 되는데 이 대장균의 섬모와 결합하는 역할을 하는 것이 파지 외피 단백질 P3의 세부분의 도메인 중 두 번째 도메인이다(Jakes, K. S., Davis, N. G., Zinder, N. D. 1988. J. Bacteriol. 170:4231-4238., Stengele, I., Bross, P., Garces, X., Giray, J., Rasched, I. 1990. J. Mol. Biol. 212:143-149). M13 박테리오파지의 복제는 P3이 대장균 세포의 표면에 있는 섬모와 결합할 때 시작되는데 이때 파지의 외피 단백질들은 기주세포의 페리플라즘(periplasm)과 결합을 하여 대장균의 안쪽 세포막(inner membrane)과 바깥쪽 세포막(outer membrane)을 가로지르는 통로 역할을 하게 된다. 이렇게 만들어진 통로를 통해 파지의 외가닥(single strand) DNA가 기주세포 내로 이동을 하게 되며 기주세포 내에서의 여러 가지 효소들에 의해 이중가닥(double strand) DNA가 되어 복제가 되고 많은 수의 파지 DNA가 기주세포내에 축적이 되고 결국은 만들어진 통로를 통해 기주세포를 죽이지 않고 기주 세포외로 나오게 되며 이와 동시에 파지 외피 단백질들(P3, P5, P7, P8, P9)로 둘러싸이게 되어 성숙한 파지 입자가 생성되게 된다(Sachdew, S. S. 2001. Biomolecular Engineering 18:57-63).M13 bacteriophage is a filamentous bacteriophage composed of a long, cylindrical shell of a protein with a single strand of DNA, with a length of less than 1 μm and a diameter of less than 10 μm (Carlos, FB, Dennis, RB, Jamie, KS, Gregg, JS 2001. Phage display, a laboratory manual). Although their protein envelopes are composed of five different proteins (P3, P6, P7, P8, P9), most of them are composed of the envelope proteins of the phage with P8 expressed in gene 8 (Marvin DA, 1998. Curr Opin. Struct Biol., 8: 150-158). The structure of P8 is composed of α-helical particles and constitutes a long shell of the phage by repeated alignment (Marvin DA, 1998. Curr. Opin. Struct. Biol. 8: 150-158) Li, H., Khan, AR, Chen, YMY 1995. J. Mol. Biol. 252: 6-14., Deberr, CM, Khan, AR, Li, Z., Joensson, C., Glibowicka, MW 1993. J. Proc. Natl. Acad Sciii. 90: 11648-11652). Five types of envelope proteins that constitute the phage are present for the structural stability of the phage particles, and in particular, P3 plays an essential role in recognition of the host cells and infection thereof, and consists of three domains (Armstrong , J., Perham, RN, Walker, JE 1981. FEBS Lett. 135: 167-172). These phages are infected through certain E. coli pilus with F plasmids, which is the second domain of the three-part domain of the phage coat protein P3 (Jakes, KS, Davis , NG, Zinder, ND 1988. J. Bacteriol. 170: 4231-4238., Stengele, I., Bross, P., Garces, X., Giray, J., Rasched, I. 1990. J. Mol. Biol 212: 143-149). Replication of the M13 bacteriophage is initiated when P3 binds to the ciliated cells on the surface of E. coli cells, and the envelope proteins of the phage bind to the periplasm of the host cell to form the inner membrane of the E. coli and the outer membrane And serves as a passage through the outer membrane. Through this pathway, the single strand DNA of the phage migrates into the host cell, becomes double stranded DNA by the various enzymes in the host cell, replicates, and a large number of phage DNA (P3, P5, P7, P8, P9), and mature phage particles are generated at the same time as the host cells do not kill (Sachdew, SS 2001. Biomolecular Engineering 18: 57-63).
한편, 단일 도메인 항체에 대한 연구는 1993년에 Hamers-Casterman에 의해 최초 보고(Hamers-Casterman, C. et al. 1993. Nature 363:446-448)되면서 단일 도메인 항체에 대한 연구가 시작되었다. 기존의 항체들은 150kDa이고 재조합 항체들은 25~50kDa이나 낙타, 라마, 상어에서 유래한 단일 도메인 항체는 12~13kDa으로 가장 작은 크기의 항체여서 세포 내로 쉽게 이동이 가능하며(Cortez-Retamozo, V. et al. 2004. Cancer Res. 64:2853-2857,) 유전적 조작이 용이하여 세균과 효모에서 쉽게 발현이 되기도 하며(Arbabi-Ghahroudii, M. et al. 1997. FEBS Lett. 414:521-526) 높은 수용성이며 극도의 pH조건, 90℃까지의 온도조건에서도 안정적인 특징을 가지고 있다(Dumoulin, M. et al. 2002. Protein Sci. 11:500-515, Dumoulin, M. et al. 2003. Nature 424:783-788). On the other hand, studies on single domain antibodies were first reported by Hamers-Casterman in 1993 (Hamers-Casterman, C. et al . 1993. Nature 363: 446-448) and studies on single domain antibodies have begun. Conventional antibodies are 150 kDa and recombinant antibodies are 25 to 50 kDa, whereas single domain antibodies derived from camel, llama and shark are 12 to 13 kDa, which is the smallest antibody and can be easily transferred into cells (Cortez-Retamozo, V. et al . 2004. Cancer Res. 64: 2853-2857). It is easy to genetically manipulate and is easily expressed in bacteria and yeast (Arbabi-Ghahroudii, M. et al . 1997. FEBS Lett. 414: 521-526) is highly water-soluble and has stable characteristics even at extreme pH conditions and temperatures up to 90 ° C (Dumoulin, M. et al . 2002. Protein Sci. 11: 500-515, Dumoulin, M. et al . 2003. Nature 424: 783-788).
M13 박테리오파지는 각종 유전자 라이브러리(library)등을 제작하거나 특정 단백질 기능을 규명하기 위한 유용한 실험 장비로 사용되고 있지만 이들의 반복적인 서열을 가진 외피 단백질의 특성을 이용하여 검출용 면역센서로서의 기능을 증대시킬 수 있는 연구가 미비하다. 따라서 낙타에서 유래한 12kDa 내지 13kDa의 가장 작은 크기의 항체로 세포 내로 이동이 가능하며 유전자 조작이 용이한 단일 도메인 항체와 세포생물학과 분자생물학 분야에서 리포터 및 바이오센서로서 광범위한 역할을 하고 있는 형광 단백질 및 다양한 기능성 단백질을 연결하는 연구를 통해 다양한 시료 등에서 충분한 역할을 담당할 수 있는 검출용 면역센서로서의 기능을 증대시키고자 한다.M13 bacteriophage is used as a useful experimental device for producing various gene libraries or identifying specific protein functions. However, the function of the M13 bacteriophage can be enhanced by using the characteristics of the repetitive sequences of the envelope proteins There are few studies available. Therefore, it is the smallest antibody of 12 kDa to 13 kDa derived from camel. It can be transferred into cells, has a single domain antibody that is easy to manipulate, and has a wide variety of fluorescent proteins and diverse functions as reporters and biosensors in cell biology and molecular biology The study of linking functional proteins is intended to increase the function as an immunosensor for detection which can play a sufficient role in various samples.
이에 본 발명자들은 검출용 면역센서로서의 형광단백질의 증폭을 연구하던 중, 반복적인 서열로 이루어진 M13 박테리오파지의 외피 단백질에 대한 단일 도메인 항체를 개발하여 이를 형광단백질과 연결하면 형광감광이 증가됨을 확인하여 본 발명을 완성하게 되었다. Accordingly, the present inventors have studied the amplification of a fluorescent protein as an immunosensor for detection, and developed a single domain antibody against the envelope protein of the M13 bacteriophage consisting of repeated sequences, and confirmed that fluorescence sensitization was increased by linking with a fluorescent protein Thereby completing the invention.
본 발명의 목적은 서열번호 1 내지 5로 이루어진 아미노산 서열 중 선택된 1종의 아미노산 서열로 이루어진 M13 박테리오파지에 대한 단일 도메인 항체를 제공함에 있다.The object of the present invention is to provide a single domain antibody against the M13 bacteriophage consisting of one selected from the amino acid sequences of SEQ ID NOS: 1 to 5.
본 발명의 다른 목적은 M13 박테리오파지에 대한 단일 도메인 항체; 및Another object of the present invention is to provide a single domain antibody against M13 bacteriophage; And
이에 연결된 단백질-단백질 상호작용이 가능한 단백질, 형광단백질 및 단일 도메인 항체 단백질로 이루어진 군 중에서 선택된 1종의 단백질을 포함하는 융합 단백질을 제공함에 있다.And a protein capable of interacting with the protein, a fluorescent protein, and a single domain antibody protein.
본 발명은 서열번호 1 내지 5로 이루어진 아미노산 서열 중 선택된 1종의 아미노산 서열로 이루어진 M13 박테리오파지에 대한 단일 도메인 항체를 제공한다.The present invention provides a single domain antibody against the M13 bacteriophage consisting of one selected from the amino acid sequences of SEQ ID NOS: 1 to 5.
본 발명은 M13 박테리오파지에 대한 단일 도메인 항체; 및The present invention relates to a single domain antibody against M13 bacteriophage; And
이에 연결된 단백질-단백질 상호작용이 가능한 단백질, 형광단백질 및 단일 도메인 항체 단백질로 이루어진 군 중에서 선택된 1종의 단백질을 포함하는 융합 단백질을 제공한다.A protein capable of protein-protein interaction, a fluorescent protein, and a single domain antibody protein.
본 발명의 M13 박테리오파지에 대한 단일 도메인 항체는 M13 박테리아파지에 대하여 특이적으로 결합하고, 상기 단일 도메인 항체를 형광 단백질과 결합시키는 경우, 형광의 발광이 증폭되어 면역센서를 포함하는 바이오센서로 유용하게 사용할 수 있다. The single domain antibody to the M13 bacteriophage of the present invention binds specifically to the M13 bacterial phage, and when the single domain antibody is bound to the fluorescent protein, the fluorescence emission is amplified and useful as a biosensor including an immune sensor Can be used.
도 1은 단일 도메인 항체가 되는 단일 도메인 가변영역(VHH domain Fv) 부분의 중합효소연쇄반응(PCR) 결과를 나타낸 도이다.
도 2는 M13 박테리오파지에 특이적인 단일 도메인 항체들을 선별하기 위한 ELISA 결과를 나타낸 도이다.
도 3은 M13 박테리오파지에 특이적으로 결합하는 양성 균주들의 중합효소연쇄반응(PCR) 결과를 나타낸 도이다.
도 4는 M13 박테리오파지에 특이적으로 결합하는 단일 도메인 항체 5개의 아미노산 서열을 분석하여 정렬(alignment)한 결과를 나타낸 도이다(다른 아미노산 서열을 가지는 그룹을 실선으로 표시함; 단일 도메인 항체의 주요 부분인 CDR 1, 2, 3는 각 서열 상단에 표시함).
도 5는 linker GFP의 염기 및 아미노산 서열을 나타낸 도이다.
도 6은 linker RFP의 염기 및 아미노산 서열을 나타낸 도이다.
도 7은 LB 평판 배지상에서 M13 박테리오파지에 특이적으로 결합하는 단일 도메인 항체가 GFP와 결합된 융합단백질의 발현 결과를 나타낸 도이다
[1:IPTG(무처리)(a;(2B12+GFP)/pET28a/BL21DE3,b;(2B12+GFP)/pET28a/BL21DE3pLysS,c;(2B12+GFP)/pET28a/Rosseta2DE3,d;(2B12+GFP)/pET28a/Rosseta2DE3pLysS),
2:1mM의 IPTG를 처리하여 20℃에서 36시간 배양함(a;(2B12+GFP)/pET28a/ BL21DE3, b;(2B12+GFP)/pET28a/BL21DE3pLysS, c;(2B12+GFP)/pET28a/Rosseta2DE3, d;(2B12+GFP)/pET28a/Rosseta2DE3pLysS),
3:IPTG(무처리)(a;(2E9+GFP)/pET28a/BL21DE3,b;(2E9+GFP)/pET28a/BL21DE3pLysS, c;(2E9+GFP)/pET28a/Rosseta2DE3, d;(2E9+ GFP)/pET28a/Rosseta2DE3pLysS),
4:1mM IPTG를 처리하여 20℃에서 36시간 배양함 (a;(2E9+GFP)/pET28a/ BL21DE3,b;(2E9+GFP)/pET28a/BL21DE3pLysS,c;(2E9+GFP)/pET28a/Rosseta2DE3,d;(2E9+GFP)/pET28a/Rosseta2DE3pLysS),
5:IPTG(무처리)(a:(53A1+GFP)/pET28a/BL21DE3,b;(53A1+GFP)/pET28a/BL21DE3pLysS,c;(53A1+GFP)/pET28a/Rosseta2DE3, d;(53A1+ GFP)/pET28a/Rosseta2DE3pLysS),
6:1mM IPTG를 처리하여 20℃에서 36시간 배양함(a;(53A1+GFP)/pET28a/ BL21DE3, b;(53A1+GFP)/pET28a/BL21DE3pLysS,c;(53A1+GFP)/pET28a/Rosseta2DE3,d;(53A1+GFP)/pET28a/Rosseta2DE3pLysS),
7:IPTG(무처리)(a;(59B5+GFP)/pET28a/BL21DE3,b;(59B5+GFP)/pET28a/BL21DE3pLysS, c;(59B5+GFP)/pET28a/Rosseta2DE3, d;(59B5+GFP)/pET28a/Rosseta2DE3pLysS),
8:1mM IPTG를 처리하여 20℃에서, 36시간 배양함(a;(59B5+GFP)/pET28a/ BL21DE3,b;(59B5+GFP)/pET28a/BL21DE3pLysS,c;(59B5+GFP)/pET28a/Rosseta2DE3,d;(59B5+GFP)/pET28a/Rosseta2DE3pLysS)].
도 8은 LB 평판 배지상에서 M13 박테리오파지에 특이적으로 결합하는 단일 도메인 항체가 RFP와 결합된 융합단백질의 발현 결과를 나타낸 도이다.
[1:ITPG(무처리)(a;(2B12+RFP)/pET28a/BL21DE3,b;(2B12+RFP)/pET28a/BL21DE3pLysS,c;(2B12+RFP)/pET28a/Rosseta2DE3,d;(2B12+ RFP)/pET28a/Rosseta2DE3pLysS),
2:1mM IPTG를 처리하여 20℃에서 36시간 배양함(a;(2B12+RFP)/pET28a/ BL21DE3,b;(2B12+RFP)/pET28a/BL21DE3pLysS,c;(2B12+RFP)/pET28a/Rosseta2DE3,d;(2B12+RFP)/pET28a/Rosseta2DE3pLysS),
3:ITPG(무처리)(a;(2E9+RFP)/pET28a/BL21DE3,b;(2E9+RFP)/pET28a/BL21DE3pLysS, c;(2E9+RFP)/pET28a/Rosseta2DE3, d;(2E9+RFP)/pET28a/Rosseta2DE3pLysS),
4:1mM IPTG를 처리하여 20℃에서 36시간 배양함(a;(2E9+RFP)/pET28a/ BL21DE3,b;(2E9+RFP)/pET28a/BL21DE3pLysS,c;(2E9+RFP)/pET28a/Rosseta2DE3,d;(2E9+RFP)/pET28a/Rosseta2DE3pLysS,
5:ITPG(무처리)(a;(49C2+RFP)/pET28a/BL21DE3,b;(49C2+RFP)/pET28a/BL21DE3pLysS, c;(49C2+RFP)/pET28a/Rosseta2DE3 d;(49C2+RFP)/pET28a/Rosseta2DE3pLysS),
6:1mM ITPG를 처리하여 20℃에서 36시간 배양함(a;(49C2+RFP)/pET28a/ BL21DE3,b;(49C2+RFP)/pET28a/BL21DE3pLysS,c;(49C2+RFP)/pET28a/Rosseta2DE3,d;(49C2+RFP/pET28a/Rosseta2DE3pLysS),
7:IPTG(무처리)(a;(53A1+RFP)/pET28a/BL21DE3,b;(53A1+RFP)/pET28a/BL21DE3pLysS,c;(53A1+RFP)/pET28a/Rosseta2DE3,d;(53A1+ RFP)/pET28a/Rosseta2DE3pLysS),
8:1mM IPTG를 처리하여 20℃에서 36시간 배양함(a;(53A1+RFP)/pET28a/ BL21DE3,b;(53A1+RFP)/pET28a/BL21DE3pLysS,c;(53A1+RFP)/pET28a/Rosseta2DE3,d;(53A1+RFP)/pET28a/Rosseta2DE3pLysS),
9:IPTG(무처리)(a;(59B5+RFP)/pET28a/BL21DE3,b;(59B5+RFP)/pET28a/BL21DE3pLysS, c;(59B5+RFP)/pET28a/Rosseta2DE3, d;(59B5+RFP)/pET28a/Rosseta2DE3pLysS),
10:1mM IPTG를 처리하여 20℃에서 36시간 배양함(a;(59B5+RFP)/pET28a/ BL21DE3,b;(59B5+RFP)/pET28a/BL21DE3pLysS,c;(59B5+RFP)/pET28a/Rosseta2DE3,d;(59B5+RFP)/pET28a/Rosseta2DE3pLysS)].
도 9는 재조합 스트렙타비딘(streptavidin)의 염기 및 아미노산 서열 결과를 나타낸 도이다.
도 10은 M13 박테리오파지에 특이적으로 결합하는 단일 도메인 항체가 GFP와 결합된 융합단백질의 효능에 대한 결과를 나타낸 도이다
[1;PBS 완충액, 2;바이오틴과 결합된 GFP , 3;(2B12+GFP), 4;(2E9+GFP), 5; (53A1+GFP), 6;(2B12+GFP)+(2E9+GFP), 7;(2B12+GFP)+(53A1+GFP), 8;(2E9+GFP)+ (53A1+GFP), 9;(2B12+GFP)+(2E9+GFP)+(53A1+GFP).
도 11은 M13 박테리오파지에 특이적으로 결합하는 단일 도메인 항체가 RFP와 결합된 융합단백질의 효능에 대한 결과이다
[1;PBS 완충액,2;바이오틴과 결합된 RFP, 3;(2B12+RFP), 4;(2E9+RFP), 5;(53A1+RFP), 6;(2B12+RFP)+(2E9+RFP), 7;(2B12+RFP)+(53A1+RFP), 8;(2E9+RFP)+(53A1+RFP), 9;(2B12+RFP)+(2E9+RFP)+(53A1+RFP)]FIG. 1 is a view showing the result of PCR of a single domain variable region (VHH domain Fv) portion as a single domain antibody.
FIG. 2 shows ELISA results for selecting single domain antibodies specific to M13 bacteriophage. FIG.
FIG. 3 is a graph showing the results of PCR for the positive strains that specifically bind to the M13 bacteriophage.
Figure 4 shows the result of alignment of 5 amino acid sequences of single domain antibodies specifically binding to M13 bacteriophage (the groups having different amino acid sequences are indicated by solid lines, the major part of the single
Figure 5 shows the nucleotide and amino acid sequences of linker GFP.
Figure 6 shows the base and amino acid sequence of the linker RFP.
7 is a graph showing the results of expression of a fusion protein in which a single domain antibody specifically binding to M13 bacteriophage is bound to GFP on an LB plate medium
(2B12 + GFP) / pET28a / BL21DE3pLysS, c (2B12 + GFP) / pET28a / Rosseta2DE3, d (2B12 + GFP) / pET28a / BL21DE3, b GFP) / pET28a / Rosseta2DE3pLysS),
(2B12 + GFP) / pET28a / BL21DE3, b (2B12 + GFP) / pET28a / BL21DE3pLysS, c; (2B12 + GFP) / pET28a / Rosseta2DE3, d; (2B12 + GFP) / pET28a / Rosseta2DE3pLysS),
(2E9 + GFP) / pET28a / Rosseta2DE3, d (2E9 + GFP) / pET28a / BL21DE3pLysS, c; (2E9 + GFP) / pET28a / Rosseta2DE3pLysS),
(2E9 + GFP) / pET28a / BL21DE3, b (2E9 + GFP) / pET28a / BL21DE3pLysS, c (2E9 + GFP) / pET28a / Rosseta2DE3 , d; (2E9 + GFP) / pET28a / Rosseta2DE3pLysS),
(53A1 + GFP) / pET28a / Rosseta2DE3, d (53A1 + GFP) / pET28a / BL21DE3pLysS, c; (53A1 + GFP) / pET28a / Rosseta2DE3pLysS),
(53A1 + GFP) / pET28a / BL21DE3, b (53A1 + GFP) / pET28a / BL21DE3pLysS, c (53A1 + GFP) / pET28a / Rosseta2DE3 , d; (53A1 + GFP) / pET28a / Rosseta2DE3pLysS),
(59B5 + GFP) / pET28a / Rosseta2DE3, d (59B5 + GFP) / pET28a / BL21DE3pLysS, c; (59B5 + GFP) ) / pET28a / Rosseta2DE3pLysS),
(59B5 + GFP) / pET28a / BL21DE3, b (59B5 + GFP) / pET28a / BL21DE3pLysS, c; (59B5 + GFP) / pET28a / Rosseta2DE3, d; (59B5 + GFP) / pET28a / Rosseta2DE3pLysS)].
FIG. 8 shows the results of expression of a fusion protein in which a single domain antibody specifically binding to M13 bacteriophage binds to RFP on an LB plate culture medium.
(2B12 + RFP) / pET28a / Rosseta2DE3, d (2B12 + RFP) / pET28a / BL21DE3pLysS, c ) / pET28a / Rosseta2DE3pLysS),
(2B12 + RFP) / pET28a / BL21DE3, b (2B12 + RFP) / pET28a / BL21DE3pLysS, c (2B12 + RFP) / pET28a / Rosseta2DE3 , d; (2B12 + RFP) / pET28a / Rosseta2DE3pLysS),
(2E9 + RFP) / pET28a / Rosseta2DE3, d (2E9 + RFP) / pET28a / BL21DE3pLysS, c: (2E9 + RFP) ) / pET28a / Rosseta2DE3pLysS),
(2E9 + RFP) / pET28a / BL21DE3, b (2E9 + RFP) / pET28a / BL21DE3pLysS, c; (2E9 + RFP) / pET28a / Rosseta2DE3 , d; (2E9 + RFP) / pET28a / Rosseta2DE3pLysS,
(49C2 + RFP) / pET28a / Rosseta2DE3 (49C2 + RFP) / pET28a / BL21DE3pLysS, c (49C2 + RFP) / pET28a / Rosseta2DE3pLysS),
(49C2 + RFP) / pET28a / BL21DE3, b (49C2 + RFP) / pET28a / BL21DE3pLysS, c (49C2 + RFP) / pET28a / Rosseta2DE3 , d (49C2 + RFP / pET28a / Rosseta2DE3pLysS),
(53A1 + RFP) / pET28a / Rosseta2DE3, d (53A1 + RFP) / pET28a / BL21DE3pLysS, c; (53A1 + RFP) / pET28a / Rosseta2DE3pLysS),
(53A1 + RFP) / pET28a / BL21DE3, b (53A1 + RFP) / pET28a / BL21DE3pLysS, c; (53A1 + RFP) / pET28a / Rosseta2DE3 , d; (53A1 + RFP) / pET28a / Rosseta2DE3pLysS),
(59B5 + RFP) / pET28a / Rosseta2DE3, d (59B5 + RFP) / pET28a / BL21DE3pLysS, c ) / pET28a / Rosseta2DE3pLysS),
(59B5 + RFP) / pET28a / BL21DE3, b (59B5 + RFP) / pET28a / BL21DE3pLysS, c (59B5 + RFP) / pET28a / Rosseta2DE3 , d; (59B5 + RFP) / pET28a / Rosseta2DE3pLysS)].
Figure 9 is a diagram showing the base and amino acid sequence results of recombinant streptavidin.
FIG. 10 shows the results of the effect of a fusion protein in which a single domain antibody specifically binding to M13 bacteriophage is bound to GFP
[1; PBS buffer, 2; GFP coupled with biotin, 3; (2B12 + GFP), 4; (2E9 + GFP), 5; (53A1 + GFP), 6; (2B12 + GFP) + (2E9 + GFP), 7; (2B12 + GFP) + (53A1 + GFP), 8; (2E9 + GFP) + (2B12 + GFP) + (2E9 + GFP) + (53A1 + GFP).
Figure 11 shows the results of the efficacy of a fusion protein in which a single domain antibody specifically binding to M13 bacteriophage is bound to RFP
(2B12 + RFP), 4 (2E9 + RFP), 5 (53A1 + RFP), 6; (2B12 + RFP) + (2E9 + RFP) RFP), 7 (2B12 + RFP) + (53A1 + RFP), 8; (2E9 + RFP) + 53A1 + RFP, 9; (2B12 + RFP)
본 발명은 서열번호 1 내지 5로 이루어진 아미노산 서열 중 선택된 1종의 아미노산 서열로 이루어진 M13 박테리오파지에 대한 단일 도메인 항체를 제공한다.The present invention provides a single domain antibody against the M13 bacteriophage consisting of one selected from the amino acid sequences of SEQ ID NOS: 1 to 5.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 M13 박테리오파지에 대한 단일 도메인 항체는 하기와 같은 방법으로 제조한다. 먼저, M13 박테리오파지를 낙타에 감염시킨 후, 낙타의 백혈구를 추출하여, 백혈구로부터 RNA를 추출한다. 특정 항체의 가변부위를 증폭하는 프라이머를 이용하여 상기 RNA에 대하여 RT-PCR을 수행하여 DNA를 증폭한 후, 상기 증폭된 DNA를 제한효소로 절단하고, 벡터에 결합시킨다. 상기 벡터를 전기천공법등을 이용하여 대장균에 형질전환시킨 후, 헬퍼 파지를 대장균에 감염시키고, M13 박테리오파지가 코팅된 플라스크에서 파지와 결합시키고, M13 박테리오파지 외피 단백질에 특이적으로 결합하는 파지를 대장균에 재감염시켜 이를 배양하였고, 상기 대장균으로부터 서열번호 1 내지 5의 아미노산 서열로 이루어진 M13 박테리오파지에 대한 단일 도메인 항체를 발현시켜 상기 단일 도메인 항체를 제조한다.A single domain antibody for the M13 bacteriophage of the present invention is prepared by the following method. First, after infecting the camel with the M13 bacteriophage, the camel's white blood cells are extracted and RNA is extracted from the white blood cells. RT-PCR is performed on the RNA using a primer that amplifies a variable region of a specific antibody to amplify the DNA, and the amplified DNA is digested with a restriction enzyme and bound to a vector. The vector was transformed into Escherichia coli using an electroporation method, and the helper phage was infected with E. coli, bound to the phage in a flask coated with M13 bacteriophage, and the phage specifically binding to the M13 bacteriophage coat protein was transformed into E. coli Re-infected and cultured, and a single domain antibody against the M13 bacteriophage consisting of the amino acid sequence of SEQ ID NOS: 1 to 5 was expressed from the E. coli to prepare the single domain antibody.
상기 서열번호 1 내지 5의 항체를 각각 2B12, 2E9, 49C2, 53A1 및 59B5로 명명하였다. 상기 서열번호 1의 아미노산 서열은 서열번호 6의 염기서열에 의하여 코딩되는 것을 특징으로 하고, 서열번호 2의 아미노산 서열은 서열번호 7의 염기서열에 의하여 코딩되는 것을 특징으로 하고, 서열번호 3의 아미노산 서열은 서열번호 8이 염기서열에 의하여 코딩되는 것을 특징으로 하고, 상기 서열번호 4의 아미노산 서열은 서열번호 9의 염기서열에 의하여 코딩되는 것을 특징으로 하고, 서열번호 5의 아미노산 서열은 서열번호 10의 염기서열에 의하여 코딩되는 것을 특징으로 한다.The antibodies of SEQ ID NOS: 1 to 5 were named 2B12, 2E9, 49C2, 53A1 and 59B5, respectively. Wherein the amino acid sequence of SEQ ID NO: 1 is encoded by the nucleotide sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 2 is encoded by the nucleotide sequence of SEQ ID NO: 7, 4 is characterized in that the amino acid sequence of SEQ ID NO: 4 is encoded by the nucleotide sequence of SEQ ID NO: 9, and the amino acid sequence of SEQ ID NO: Lt; RTI ID = 0.0 > of: < / RTI >
또한, 상기 단일 도메인 항체는 M13 박테리오파지를 특이적으로 인식할 수 있는 범위 내에서 첨부한 서열번호 1 내지 5에 기재된 아미노산 서열의 변이체를 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 개선시키기 위하여 항체의 아미노산 서열에 변화를 줄 수 있다. 이러한 변형은, 예를 들어 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. In addition, the single domain antibody may include variants of the amino acid sequences set forth in SEQ ID NOS: 1 to 5 attached thereto within a range capable of specifically recognizing M13 bacteriophage. For example, the amino acid sequence of an antibody may be altered to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion and / or substitution of the amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like.
또한, 상기 단일 도메인 항체는 서열번호 1 내지 서열번호 5의 아미노산 서열과 60% 이상의 상동성이 있는 아미노산 서열을 갖는 항체를 포함할 수 있으며, 바람직하게는 90% 이상의 상동성을 갖는 아미노산 서열을 갖는 항체일 수 있다.In addition, the single domain antibody may include an antibody having an amino acid sequence having 60% or more homology with the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 5, preferably having an amino acid sequence having 90% or more homology Lt; / RTI >
또한 상기 단일 도메인 항체는 대장균에서 과발현시켜 적은 비용으로 대량생산이 가능하며 단일 도메인만으로 유전자 재조합하여 생산하므로 수용액 내에서 용해도가 높은 장점을 가진 항체를 말한다.In addition, the single domain antibody can be over-expressed in E. coli and can be mass-produced at a low cost. Since it is produced by gene recombination only in a single domain, it has an advantage of high solubility in aqueous solution.
또한, 본 발명의 염기서열은 실질적인 동일성을 나타내는 염기서열을 포함할 수 있으며, 상기 실질적인 동일성은 염기서열과 임의의 다른 서열을 최대한 대응되도록 배열하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 배열된 서열을 분석한 경우에, 최소 80%의 상동성, 보다 바람직하게는 최소 90%의 상동성, 가장 바람직하게는 최소 95%의 상동성을 나타내는 염기서열을 의미한다.
In addition, the nucleotide sequence of the present invention may include a nucleotide sequence that exhibits substantial identity, and the nucleotide sequence may be arranged so as to correspond to any other nucleotide sequence as much as possible, Refers to a nucleotide sequence that exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology, when analyzing a sequence that has been analyzed.
본 발명은 서열번호 1 내지 5로 이루어진 아미노산 서열 중 선택된 1종의 아미노산 서열로 이루어진 M13 박테리오파지에 대한 단일 도메인 항체; 및The present invention provides a single domain antibody against M13 bacteriophage comprising one selected from the amino acid sequences of SEQ ID NOS: 1 to 5; And
이에 연결된 단백질-단백질 상호작용이 가능한 단백질, 형광단백질 및 단일 도메인 항체 단백질로 이루어진 군 중에서 선택된 1종의 단백질을 포함하는 융합 단백질을 제공한다.A protein capable of protein-protein interaction, a fluorescent protein, and a single domain antibody protein.
상기 형광단백질은 적색 형광 단백질(red fluorescent protein), 녹색 형광 단백질(green fluorescent protein) 및 황색 형광 단백질(yellow fluorescent protein)을 포함하나, 이에 한정되지 않는다.The fluorescent protein includes, but is not limited to, a red fluorescent protein, a green fluorescent protein and a yellow fluorescent protein.
또한, 상기 단백질-단백질 상호작용이 가능한 단백질은 스트렙타비딘(stretavidin), 단백질 G(protein G), 단백질 A(protein A), 헤테로 이합체 단백질 AG(hetero dimer protein AG), 헤테로 이합체 단백질 GA(hetero dimer protein GA) 및 헤테로 삼합체 단백질 GAG(hetero trimer protein GAG)로 이루어진 군에서 선택된 1종 이상의 단백질을 포함하나, 이에 한정되지 않는다.The protein capable of protein-protein interaction may be selected from the group consisting of streavidin, protein G, protein A, heterodimer protein AG, heterodimer protein GA dimer protein GA) and a heterotrimeric protein GAG (GAG). However, the present invention is not limited thereto.
상기 융합 단백질은 형광 감광이 일반적인 GFP 또는 RFP보다 증가하여, 면역 센서로서 유용하게 이용할 수 있다.The fusion protein is useful as an immunosensor because fluorescence sensitization is higher than that of general GFP or RFP.
상기 면역 센서란 항원-항체 복합체를 형성하는 성질을 이용한 바이오센서의 일종을 의미한다. 이는 항원-항체 복합체를 형성함으로써 어떤 변화를 전기신호로 변환, 표시하는 것을 기본으로 하고 있으며, 측정방식의 차이에 따른 비표식 면역센서와 표식면역 센서를 모두 포함할 수 있다. 본 발명의 면역 센서는 바람직하게는 표식 면역 센서를 의미하며, 표식제로부터 항원항체 복합체의 형성을 간접적으로 측정할 수 있는 면역 센서임을 특징으로 할 수 있다.The immunosensor refers to a kind of biosensor that uses the property of forming an antigen-antibody complex. This is based on the conversion of any change into an electrical signal by the formation of an antigen-antibody complex, and may include both a non-tagged immune sensor and a tagged immune sensor depending on the difference in measurement method. The immunosensor of the present invention preferably means a labeled immunosensor and may be an immunosensor capable of indirectly measuring the formation of an antigen-antibody complex from a marker.
상기 융합 단백질을 이용할 수 있는 방법에는 바람직하게는, 방사면역측정법(Radio Immuno Assay; RIA), RET(resonance energy transfer), FRET(Forster resonance energy transfer), 단백질-단백질 상호작용을 이용한 단백질 농도 측정 측정 방법, 더 바람직하게는 효소결합면역흡착측정법(enzyme linked immunosorbent assay; ELISA)에 이용할 수 있으나, 이에 제한되지 않는다.
Preferred methods for using the fusion protein include radioimmunoassay (RIA), resonance energy transfer (RET), forster resonance energy transfer (FRET), protein concentration measurement using protein-protein interaction Method, more preferably enzyme-linked immunosorbent assay (ELISA).
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명의 내용이 하기 실시예에 한정되는 것을 아니다. Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
[[ 실시예Example 1] One] 낙타에서From the camel 분리된 백혈구를 이용한 Separation of leukocytes M13M13 박테리오Bacterium 파지의 항체 제조 Antibody production of phage
1. One. 낙타에서의Camel 면역반응 유도 및 분리된 백혈구를 이용한 Induction of immune response and separation of leukocytes cDNAcDNA 합성 및 단일 도메인 항체 라이브러리 제조 Synthesis and preparation of single domain antibody libraries
M13 박테리이오 파지는 New England Biolabs Inc.에서 구입한 M13K07 파지를 사용하였고, 이를 Escherichia . coli TG1에 감염시켜 증폭시킨 후(4×1014 pfu/ml), PBS 완충액으로 투석하여 항원으로 사용하였고, 사용을 위하여 냉동 보관하면서 사용하였다. 상기 M13K07 파지 1ml(4×1014 pfu/ml)를 동등한 부피의 프로인트 완전 애주번트(Freund`s complete adjuvant)와 혼합하여 안정된 에멀젼(emulsion)을 제조한 후, 몽골에서 사육중인 낙타(Camelus bacterianus)에 2 내지 4군데 분산하여 피하투여하였다. 이 후, 2주 간격으로 3차례 같은 양의 항원을 같은 부피의 프로인트 불완전 애주번트(Freund`s incomplete adjuvant)와 혼합하여 같은 방법으로 투여하였다. 최종 투여 1주일 후, 항응고제(anti-coagulated blood)가 들어있는 수혈팩에 혈액을 채취하였고, 피콜(Ficoll-paque PLUS)을 이용하여 백혈구를 분리하였다.M13 bacteria was used for the M13K07 phage purchased from New England Biolabs Inc., Escherichia . then it amplifies the infection coli TG1 by dialysis (4 × 10 14 pfu / ml ), PBS buffer was used as an antigen, was used as cryopreserved for use. A stable emulsion was prepared by mixing 1 ml of the M13K07 phage (4 x 10 14 pfu / ml) with an equivalent volume of Freund's complete adjuvant, and then transferred to a Camelus bacterianus ) at 2 to 4 sites. Thereafter, the same amount of antigen was mixed with the same volume of Freund's incomplete adjuvant three times at intervals of two weeks and administered in the same manner. One week after the last administration, blood was collected in a transfusion pack containing anti-coagulated blood and white blood cells were separated using Ficoll-paque PLUS.
상기 백혈구로부터 트리졸(trizol)을 이용하여 RNA를 추출한 후, 특이적 역방향 프라이머를 이용하여 cDNA를 합성 한 후, 골격-1(framework-1) 및 골결-4(framwork-4)에 결합하는 프라이머를 이용하여 단일 도메인 항체의 가변영역 (variable region; 이하 Fv)을 94℃에서 1분, 58℃에서 30초, 72℃에서 1분을 한 사이클로 25회를 반복하여 중합효소연쇄반응을 통해 증폭시켰다. 상기 증폭된 부분을 전기영동을 수행하여 이를 확인한 결과를 도 1에 나타내었다.RNA was extracted from the leukocytes using trizol and cDNA was synthesized using a specific reverse primer and primers binding to framework-1 and framework-4 were synthesized , A variable region (hereinafter referred to as Fv) of a single domain antibody was amplified through a polymerase chain reaction by repeating 25 cycles at 94 ° C for 1 minute, 58 ° C for 30 seconds, and 72 ° C for 1 minute . The amplified fragments were subjected to electrophoresis to confirm the results.
도 1에 나타난 바와 같이, 약 350 내지 400bp에서 증폭된 단일 도메인(VHH domain)DNA 밴드를 확인하였다.
As shown in Figure 1, a single domain (VHH domain) DNA band amplified at about 350-400 bp was identified.
2. 2. 패닝(panning)에In panning 의한 특이적인 항체들의 선별 Screening for specific antibodies by
상기 증폭된 DNA 밴드를 NCOΙ 및 NotΙ제한효소를 이용하여 절단하였고, 상기 제한효소로 절단된 DNA를 pCANTAB5E 벡터에 결합시켰다. 상기 재조합된 벡터를 E.coli TG1에 전기천공법(electroporation)을 이용하여 형질전환시켰고, 상기 전기천공법은 25uF, 2.5kV, 200 옴(ohms) 조건에서 수행하였다.The amplified DNA band NCO I and Not I restriction enzyme, and the restriction enzyme-digested DNA was ligated to the pCANTAB5E vector. The recombinant vector was transformed into E. coli TG1 using electroporation, and the electroporation was performed at 25 uF, 2.5 kV, 200 ohms.
상기 형질 전환된 E. coli TG1을 M13KO7 헬퍼 파지(4×1010pfu)에 감염시키고 하루 배양하고 나온 상등액을 폴리에틸렌글리콜(polyethylene glycol)/NaCl 침전법을 이용하여 파지를 침전시켰다. 이 후, BEA(2-Bromoethylamine hydrobromide)로 불활화시킨 M13K07 파지가 코팅되어 있는 T25 플라스크를 이용하여 파지를 결합시키고 PBS-T(0.5% Tween20) 완충액을 이용하여 10회 세척하였다. 이 후 대수기에 있는 E. coli TG1을 넣어 T25 플라스크에 남아있는 파지를 재감염시키고 배양하였다. 이와 같은 과정을 반복함으로써 M13 박테리오파지 외피 단백질(항원)에 특이적으로 결합하는 파지를 증가시켰다.
The transformed E. coli TG1 was infected with M13KO7 helper phage (4 x 10 10 pfu) and cultured for one day. The supernatant was precipitated with polyethylene glycol / NaCl precipitation method. The phages were then combined and washed 10 times with PBS-T (0.5% Tween20) buffer using a T25 flask coated with M13K07 phage-inactivated with BEA (2-Bromoethylamine hydrobromide). After that, E. coli TG1 in the major stage was added and the remaining phage in the T25 flask was reinfected and cultured. This procedure was repeated to increase the phage specifically binding to the M13 bacteriophage coat protein (antigen).
3. 3. ELISAELISA 를 통한 양성 클론(Positive clone ( cloneclone )의 선별) Selection
상기 실시예 1-2에 의한 각 단계별 패닝(panning) 후 파지에 감염된 대장균 TG1을 암피실린(ampicillin, 100μg/ml)이 함유된 SOBAG 평판배지(Bacto tryptone 20g, Bacto yeast extract 5g, NaCl 0.5g, 1M MgCl2 10ml, 2M Glucose 55.6ml, Bacto agar 15g/L)에 도말하였다. 배양된 단일 콜로니(colony)를 무작위적으로 선별하여 새로운 파지에 감염시킨 후 배양하여 나온 상등액으로 ELISA를 수행하였다. 96 웰 면역플레이트는 웰당 100㎕의 항원용 M13K07 파지(BEA 처리하여 불활화 된 것) 현탁액을 코팅하고 차단 완충액(blocking buffer, 5% 탈지우유 포함한 PBS)으로 차단하였다. 그 후 개별 콜로니에서 배양된 파지를 1차적으로 면역플레이트에 붙이고 2차적으로 항-M13-HRP를 사용하여 발색하는 것을 관찰함으로써 파지가 항원으로 사용된 각 균주들과의 결합 유무를 판단하였다. 결과를 도 2에 나타내었다.After panning each step according to Example 1-2, E. coli TG1 infected with phage was inoculated into SOBAG plate medium (ampicillin, 100 μg / ml) containing 20 g of Bacto tryptone, 5 g of Bacto yeast extract, 0.5 g of NaCl,
도 2에 나타낸 바와 같이, ELISA 결과 총 98개의 양성클론을 확보하였다.
As shown in Fig. 2, a total of 98 positive clones were obtained by ELISA.
4. 양성 클론 반응 확인4. Confirm positive clone response
ELISA 결과 양성반응으로 나온 총 98개의 클론들 중, 32개의 클론들에 대하여 벡터 프라이머를 사용하여 중합효소연쇄반응(94℃(1분), 58℃(30초), 72℃(1분), 30 회)을 진행하였으며, 증폭된 산물을 아가로오스 겔상에서 전기영동으로 확인하였다. 이의 결과를 도 3에 나타내었다.Of the total 98 clones from the positive reaction, 32 clones were subjected to polymerase chain reaction (94 ° C. (1 minute), 58 ° C. (30 seconds), 72 ° C. (1 minute) 30 times), and amplified products were confirmed by electrophoresis on agarose gel. The results are shown in Fig.
도 3에 나타낸 바와 같이, 32개의 클론들은 100%의 양성클론 반응을 나타내었다.
As shown in Fig. 3, 32 clones showed 100% positive clone response.
5.5. M13M13 박테리오파지에 대한 단일 도메인 항체 확인 Single Domain Antibody Identification for Bacteriophage
M13 박테리오파지에 대한 단일 도메인 항체를 확인하기 위해서 상기 1 내지 4의 방법으로 단일 도메인 항체들을 가지는 플라스미드를 확보한 후 이들의 염기서열을 분석하고 NCBI(National Center for Biotechnology Information)에서 블라스트 검색을 하여 낙타에서 유래한 단일 도메인 항체들임을 확인하였다. 이들 항체들간의 비교를 위해서 ClustalW2 프로그램으로 서열들을 정렬(alignment)하였다. 결과를 도 4에 나타내었다.In order to confirm a single domain antibody against M13 bacteriophage, plasmids having single domain antibodies were obtained by the
도 4에 나타낸 바와 같이, 확보된 M13 박테리오파지에 대한 단일 도메인 항체 19개의 서열을 분석한 결과 다른 아미노산 서열을 가진 단일 도메인은 하기 표 1의 항체 5가지를 확보하였다.As shown in FIG. 4, 19 single domain antibodies against the obtained M13 bacteriophage were analyzed. As a result, five single antibodies having different amino acid sequences were obtained as shown in Table 1 below.
[[ 실시예Example 2] 2] M13M13 박테리오파지에 대한 재조합 단일 도메인 항체 및 형광단백질의 Recombinant single domain antibodies and fluorescent proteins for bacteriophage 융합단백질의Of the fusion protein 제조. Produce.
1.단일 도메인 항체를 코딩하는 염기서열 1. A nucleotide sequence encoding a single domain antibody 클로닝Cloning
상기 실시예 1에 따라 확보된 M13 박테리오파지에 대한 단일 도메인 항체인 2B12, 2E9, 49C2, 53A1, 59B5의 DNA를 형광 단백질인 GFP, RFP와 연결하기 위하여 2nd-F-NotI 프라이머, 2nd-R-BspEI XhoI 프라이머를 사용하여, 중합효소연쇄반응을 수행하였다(95℃ 1분, 58℃ 30초, 72℃ 1분, 30회). 그 결과 약 450bp의 PCR 산물을 얻었으며 획득한 PCR 산물을 TA 클로닝 한 후, 서열분석을 통해 해당 단일도메인 항체의 서열과 동일함을 확인하였다.
In order to link the DNAs of 2B12, 2E9, 49C2, 53A1 and 59B5, which are single domain antibodies against M13 bacteriophage obtained according to Example 1, with the fluorescent proteins GFP and RFP, 2nd-F-NotI primer, 2nd-R-BspEI Polymerase chain reaction was performed using the XhoI primer (95 ° C for 1 minute, 58 ° C for 30 seconds, 72 ° C for 1 minute, 30 times). As a result, a PCR product of about 450 bp was obtained. The obtained PCR product was TA cloned and confirmed by sequencing to be identical to the sequence of the corresponding single domain antibody.
2.재조합 형광단백질을 코딩하는 염기서열 2. A nucleotide sequence encoding a recombinant fluorescent protein 클로닝Cloning
단일 도메인 항체와 GFP 및 RFP를 연결하기 위해 링커를 사용하여 재조합 형광단백질을 제작하였는데 링커 GFP 제작은 Royal Veterinary and Agricultural University(Denmark)의 Birgit Koch에게서 분양받은 mini-Tn7gfp2/DH5 균주에서 DNA를 추출하여 이를 F-Linker GFP 프라이머와 R-GFP BspEI XhoI 프라이머로 중합효소연쇄반응(PCR)을 통해 증폭하였다. The recombinant fluorescent protein was constructed using a linker to link the single domain antibody to GFP and RFP. The linker GFP was prepared from mini-Tn7 gfp2 / DH5 strain, which was purchased from Birgit Koch of Royal Veterinary and Agricultural University, Denmark And then amplified by PCR using F-Linker GFP primer and R-GFP BspEI XhoI primer.
Linker RFP 제작은 pCMV-tdTomato DNA를 이용하여 F-Linker Tomato 프라이머와 R-Tomato BspEI XhoI 프라이머를 사용하여 중합효소연쇄반응 (95℃ 1분, 58℃ 30초, 72℃ 1분, 30회)하여 준비하였다.Linker RFP was prepared by polymerase chain reaction (95 ° C for 1 minute, 58 ° C for 30 seconds, 72 ° C for 1 minute, 30 times) using pCMV-tdTomato DNA using F-Linker Tomato primer and R-Tomato BspEI XhoI primer Prepared.
각각 증폭된 산물을 아가로오스 겔상에서 전기영동하여 확인한 결과 linker GFP는 약 750bp, linker RFP는 약 1450bp 크기임을 확인하였다(도면 미제시). 확인한 각 산물을 TA 클로닝 벡터에 삽입하여 염기서열을 분석하였다. 이의 결과를 도 5(GFP) 및 도 6(RFP)에 나타내었다.The amplified products were confirmed by electrophoresis on agarose gel. As a result, it was confirmed that linker GFP was about 750 bp and linker RFP was about 1450 bp (not shown). Each of the identified products was inserted into TA cloning vector to analyze the base sequence. The results are shown in Fig. 5 (GFP) and Fig. 6 (RFP).
도 5 및 도 6에 나타난 바와 같이, 재조합 GFP 및 RFP를 제조되었음을 확인하였다. As shown in FIGS. 5 and 6, it was confirmed that recombinant GFP and RFP were produced.
3.단일 도메인 항체 및 재조합 형광단백질의 3. Single Domain Antibodies and Recombinant Fluorescent Protein 융합단백질을The fusion protein 발현하는 세포의 제조 및 상기 단백질의 발현 확인 Preparation of expressing cells and confirmation of expression of the protein
상기 서열이 확인된 linker GFP와 linker RFP는 AgeI/XhoI을 처리하여 BspEI/XhoI 이 처리된 상기 서열이 확인된 재조합 M13 박테리오파지 2B12, 2E9, 49C2, 53A1, 59B5의 DNA와 결합한 후 염기서열을 다시 확인하였다. 이 후, NotI/XhoI 이 처리된 pET-28a 벡터와 결합시키고, 상기 재조합 단일 도메인 항체 및 형광단백질의 융합단백질 발현을 위해 단백질 발현용 E. coli 균주인 BL21DE3, BL21DE3pLysS, Rosseta2DE3, Rosseta2DE3pLysS에 각각 형질전환시켰다. SDS-PAGE를 통해서 단백질 발현 여부를 확인하였으며, 형광 발현 유무를 관찰하기 위해서 1mM IPTG와 카나마이신이 첨가된 LB 평판배지에 접종하여 20℃ 및 37℃에서 각각 형광 유무를 관찰하였다. Linker GFP and linker RFP having the above sequence were ligated with the DNA of recombinant M13 bacteriophage 2B12, 2E9, 49C2, 53A1, and 59B5, which had been subjected to BspEI / XhoI treatment and treated with AgeI / XhoI, Respectively. Thereafter, NotI / XhoI-treated pET-28a vector was ligated and transformed into BL21DE3, BL21DE3pLysS, Rosseta2DE3 and Rosseta2DE3pLysS, which are E. coli strains for protein expression, for expression of the fusion protein of the recombinant single domain antibody and fluorescent protein, respectively . Protein expression was confirmed by SDS-PAGE. Fluorescence was observed at 20 ° C and 37 ° C, respectively, inoculated on LB plate medium supplemented with 1mM IPTG and kanamycin to observe the presence or absence of fluorescence.
결과를 도 7 내지 도 8에 나타내었다.The results are shown in Figs.
도 7에 나타낸 바와 같이, LB 평판 배지상에서 M13 박테리오파지에 대한 단일 도메인 항체와 GFP 형광 단백질이 결합된 융합단백질의 발현을 확인하였다. As shown in Fig. 7, the expression of a fusion protein in which a single domain antibody and a GFP fluorescent protein were bound to M13 bacteriophage on an LB plate medium was confirmed.
도 8에 나타난 바와 같이, 단일 도메인 항체와 RFP 형광 단백질이 결합된 융합단백질의 발현을 확인할 수 있었다.
As shown in FIG. 8, the expression of a fusion protein in which a single domain antibody and an RFP fluorescent protein were combined was confirmed.
[[ 실시예Example 3] 3] GFPGFP 또는 or RFPRFP 가 연결된 Connected M13M13 박테리오파지에 대한 단일 도메인 항체들을 이용한 형광증폭 검정 Fluorescence amplification using single domain antibodies against bacteriophage
1-1.1-1. 스트렙타비딘(streptavidin)의Streptavidin 제조 및 기능 확인 Manufacturing and functional verification
스트렙타비딘(Streptavidin)은 Streptomyces avidinii에서 유래된 것으로 이를 증폭하기 위해서 한국생명공학연구원 생물자원센터에서 S. avidinii(KCTC9757)를 구입하여 사용하였다. 우선 S. avidinii를 YM broth(Yeast extract 3g, Malt extract 3g, Peptone 5g, Dextrose 10g, DW 1)에서 배양한 후 (30℃, 48시간) 균체를 수거하였다. 수거한 균체는 액체 질소를 이용해서 고르게 파쇄한 후 genomic DNA extraction kit(INTRON Biotechnology, Inc.)를 이용해서 genomic DNA를 확보하였다. 확보된 DNA는 F-NcoI STR 프라이머, R-NotI STR 프라이머를 사용하여 PCR[94℃(1분), 58℃(30초), 72℃(1분), 30 회]한 후 약 500 bp의 PCR 산물을 획득하였고, 획득한 PCR 산물을 TA 클로닝 한 후, 서열분석을 통해 스트렙타비딘의 염기서열을 확인하였다. 결과를 도 9에 나타내었다.Streptavidin is known as Streptomyces avidinii and amplified by using S. avidinii (KCTC9757) from Korea Research Institute of Bioscience and Biotechnology. First, S. avidinii was cultured in YM broth (Yeast extract 3 g, Malt extract 3 g, Peptone 5 g, Dextrose 10 g, DW 1) (30 ° C., 48 hours) The collected cells were evenly disrupted using liquid nitrogen and genomic DNA was obtained using a genomic DNA extraction kit (INTRON Biotechnology, Inc.). The obtained DNA was subjected to PCR [94 ° C. (1 minute), 58 ° C. (30 seconds), 72 ° C. (1 minute), and 30 times] using F-NcoI STR primer and R-NotI STR primer, PCR products were obtained, and the obtained PCR products were subjected to TA cloning, followed by sequence analysis to confirm the base sequence of streptavidin. The results are shown in Fig.
도 9에 나타난 바와 같이, 스트렙타비딘을 성공적으로 증폭하였음을 확인하였다.As shown in Fig. 9, it was confirmed that streptavidin was successfully amplified.
1-2. 스트렙타비딘의 클로닝 및 기능확인 1- 2. Streptavidin Cloning and functional verification
상기 스트렙타비딘은 단일 도메인 항체 제작시 사용된 벡터인 pCANTAB5E와 연결하여 클로닝하였다. 재조합된 플라스미드는 E. coli TG1에 형질전환 하였고 형질 전환된 E. coli TG1은 M13K07 헬퍼 파지(41010pfu)에 감염시키고 하루 배양 후 나온 상등액을 실험에 사용하였는데 이 상등액은 폴리에틸렌글리콜(polyethylene glycol)/NaCl 침전법에 의거 파지를 침전시켰으며 PBS로 투석한 후 이를 냉동 보관 하면서 M13 박테리오파지에 의해 분비되는 스트렙타비딘의 실험에 사용하였으며 바이오틴을 이용하여 대장균에서 발현하여 정제한 스트렙타비딘과 동일한 기능을 하는 것을 확인하였다.
The streptavidin was cloned by linking with pCANTAB5E, a vector used for preparing a single domain antibody. The recombinant plasmid was transformed into E. coli TG1. The transformed E. coli TG1 was infected with M13K07 helper phage (410 10 pfu) and the supernatant from the day after culture was used for the experiment. The supernatant was dissolved in polyethylene glycol / NaCl precipitation method. After dialyzing with PBS, it was used for the experiment of streptavidin secreted by M13 bacteriophage while being kept frozen and the same function as streptavidin purified and expressed in E. coli using biotin .
2. 2. GFPGFP 또는 or RFPRFP 와 Wow 바이오틴과의With biotin 결합 Combination
GFP는 상기의 실시예 2의 linker GFP의 DNA에서 F-NheI GFP 프라이머와 R-GFP BspEI XhoI 프라이머를 이용하여, 중합효소연쇄반응을 통해 증폭하였다. RFP는 실시예 2에서 제작된 linker RFP의 DNA와 F-NheI Tomato 프라이머와 R-Tomato BspEI XhoI 프라이머를 이용하여, 중합효소연쇄반응을 통해 증폭하였다. GFP was amplified by polymerase chain reaction using F-NheI GFP primer and R-GFP BspEI XhoI primer from the linker GFP DNA of Example 2 above. RFP was amplified by polymerase chain reaction using the linker RFP DNA prepared in Example 2, F-NheI Tomato primer and R-Tomato BspEI XhoI primer.
각각 증폭된 산물은 아가로오스 겔(agarose gel)상에서 전기영동으로 확인하고 각 산물을 TA 클로닝 벡터에 삽입하여 염기서열을 확인하였다. 염기서열이 확인된 GFP와 RFP는 NheI/XhoII이 처리된 pET-28a 벡터와 결합하여 단백질 발현용 E. coli 균주인 BL21DE3, BL21DE3pLysS, Rosseta2DE3, Rosseta2DE3pLysS에 각각 형질전환하여 SDS-PAGE를 통해서 발현의 여부를 확인하였다. GFP와 RFP의 정제는 실시예 2와 동일하게 실시하여 순도가 높은 형광 단백질 정제물을 확보하였다.Each amplified product was confirmed by electrophoresis on an agarose gel, and each product was inserted into a TA cloning vector to confirm the base sequence. GFP and RFP were sequenced and transfected into pET-28a vector treated with Nhe I / Xho II and transformed into E. coli strains BL21DE3, BL21DE3pLysS, Rosseta2DE3 and Rosseta2DE3pLysS for protein expression, respectively, and expressed by SDS-PAGE . Purification of GFP and RFP was carried out in the same manner as in Example 2 to obtain fluorescent protein purified product having high purity.
확보된 형광 단백질 GFP 및 RFP 1 mg/ml을 50mM 탄산 완충액(carbonate buffer; pH 9.0)에 투석한 후, 20㎕의 NHS-바이오틴(10mg/ml in DMSO)을 처리하여 암흑상태에서 16시간 동안 4℃에서 반응시키고 PBS 완충액으로 투석하여 형광증폭 체계의 효율성 검증에 대조군으로 사용하였다.
The obtained fluorescent protein GFP and 1 mg / ml of RFP were dialyzed into 50 mM carbonate buffer (pH 9.0), treated with 20 μl of NHS-biotin (10 mg / ml in DMSO) ℃ and dialyzed with PBS buffer, and used as a control for the efficiency verification of the fluorescence amplification system.
3. 3. GFPGFP 또는 or RFPRFP 가 연결된 Connected M13M13 박테리오파지에 대한 단일 도메인 항체들을 이용한 형광증폭 검정 Fluorescence amplification using single domain antibodies against bacteriophage
3-1.3-1. M13M13 박테리오Bacterium 파지에 대한 단일 도메인 항체가 A single domain antibody against phage GFPGFP 또는 or RFPRFP 와 Wow 결합된Combined 융합단백질의 정제 Purification of fusion protein
재조합 균주를 카나마이신이 첨가된 200ml LB 배지에 접종하여 37℃에서 배양하고 흡광도가 약 0.4~0.6이 되었을 때 IPTG를 최종농도 1mM이 되게 첨가하였다. IPTG 첨가 후 20℃에서 36시간 배양하고 난 후 배양액을 초고속원심분리기 (centrifuge)에서 8000rpm으로 20분간 회전시켜 균주를 수거하였다. 수거된 균주에 PBS 완충액(8g NaCl, 0.21g KCl, 1.42 g Na2HPO4, 0.27 g KH2PO4 / 1L, pH 7.4)을 20ml 넣고 현탁한 후에 프렌치프레스(FRENCH PRESS)를 이용하여 균주를 파쇄하였다. 파쇄한 균 현탁액을 초고속원심분리기에서 10000rpm으로 20분간 회전시켜 파쇄된 균 현탁액의 상층액을 회수하였다. 회수된 상층액은 용해 완충액 (50mM NaH2PO4 , 300mM NaCl, 10mM Imidazole, pH 8.0)으로 평형을 유지시킨 6×히스티딘 친화성 크로마토그래피인 Ni-NTA 세파로오스 컬럼(Shepharose column)에 흡착시키고, 세척 완충액(50mM NaH2PO4 , 300mM NaCl, 50mM Imidazole, pH 8.0)을 사용하여 세척하였다. 용출 완충액 (50mM NaH2PO4 , 300mM NaCl, 1M Imidazole, pH 8.0)을 사용하여 재조합 단백질을 회수하였고 PBS 완충액으로 투석하였다. 각 정제 과정의 단백질들은 SDS-PAGE에 의해서 확인하였으며, 순도가 높은 정제물을 선별하여 형광증폭 체계의 효율성 검증에 사용하였다.The recombinant strains were inoculated in 200 ml LB medium supplemented with kanamycin, cultured at 37 ° C., and when the absorbance reached about 0.4 to 0.6, IPTG was added to a final concentration of 1 mM. After adding IPTG, the culture was incubated at 20 ° C for 36 hours. The culture was then centrifuged at 8000 rpm for 20 minutes in a high-speed centrifuge to collect the strain. PBS buffer in the collection strains (8g NaCl, 0.21g KCl, 1.42 g Na2HPO4, 0.27
3-2.형광증폭 검정3-2. Fluorescence amplification test
M13 박테리오파지에 대한 단일 도메인 항체들과 GFP 또는 RFP를 연결해서 발현되어 나온 형광 단일 도메인 항체들의 효능을 검정하기 위해 ELISA를 실시하였다. ELISA was performed to test the efficacy of fluorescent single domain antibodies expressed by linking single domain antibodies against M13 bacteriophage with GFP or RFP.
우선 검은색의 96 웰 면역플레이트에 상기 3-1에서 제조한 M13 박테리오파지에서 전시되는 스트렙타비딘(streptavidin)을 웰당 100㎕씩 코팅하고 차단 완충액(blocking buffer, 5% 탈지우유 포함한 PBS)으로 차단하였다. 그 후 트윈 20(tween 20)이 0.5% 함유된 PBST 완충액으로 세척한 후 정제된 각각의 단백질(M13 박테리오파지에 대한 단일 도메인 항체인 2B12, 2E9 및 53A1와 GFP 또는 RFP를 결합한 융합단백질)을 10μg/ml, 50μg/ml, 100μg/ml 농도로 1시간동안 처리하였다. 그리고 PBST 완충액으로 세척한 후 형광검출기(Flurometer, SpectraMax M2E 384, Molecular Devices)를 이용하여 형광수치를 M13 단일 도메인 항체와 결합된 GFP 단백질은 여기(excitation) 파장(395nm), 방출(emission) 파장(509nm)에서, M13 단일 도메인 항체와 결합된 RFP 단백질은 여기(excitation) 파장(554nm), 방출(emission) 파장(581nm)에서 측정하여 그래프로 나타내었다. 양성 대조군으로 바이오틴과 결합된 GFP 또는 RFP를 이용하였고, 음성 대조군으로 PBS 완충액을 사용하였다. 결과를 도 10 내지 도 11에 나타내었다.First, streptavidin was coated on a 96-well plate on a black 96-well plate prepared in the above-described M13 bacteriophage, and blocked with blocking buffer (PBS containing 5% defatted milk) . After washing with PBST buffer containing 0.5% tween 20, each purified protein (a fusion protein of 2B12, 2E9, and 53A1 with GFP or RFP in combination with single domain antibodies against M13 bacteriophage) was incubated with 10 μg / ml, 50 μg / ml, and 100 μg / ml for 1 hour. After washing with PBST buffer, the fluorescence value was measured using a fluorescence detector (Fluorometer, SpectraMax M2E 384, Molecular Devices). The GFP protein bound to the M13 single domain antibody had an excitation wavelength (395 nm) and an emission wavelength 509 nm), the RFP protein bound to the M13 single domain antibody was graphically measured at excitation wavelength (554 nm) and emission wavelength (581 nm). GFP or RFP conjugated with biotin was used as a positive control, and PBS buffer was used as a negative control. The results are shown in FIG. 10 to FIG.
도 10 내지 도 11에 나타낸 바와 같이, 10μg/ml이상의 처리구부터 확연하게 양성 대조군인 바이오틴과 결합된 GFP 또는 RFP의 형광수치보다 차이가 났다. 특히 각각 단독의 M13 박테리오파지에 대한 단일 도메인 항체와 결합된 GFP 또는 RFP를 처리한 것은 바이오틴과 결합된 GFP, RFP 보다 약 2배, 두 가지 M13 박테리오파지에 대한 단일 도메인 항체와 결합된 GFP 또는 RFP 단백질을 처리한 것에서는 바이오틴과 결합된 GFP 또는 RFP 처리구보다 약 4배, 세가지 M13 박테리오파지에 대한 단일 도메인 항체와 결합된 GFP 또는 RFP 단백질을 처리한 것에서는 바이오틴과 결합된 GFP 또는 RFP 처리구보다 약 6배 이상 형광 수치가 높았다. 이 결과는 박테리오파지에 의한 신호증폭(signal amplication)이 된 것으로 스트렙타비딘(streptavidin)에 대해서 바이오틴과 결합된 GFP 또는 RFP 보다 M13 박테리오파지에 대한 단일 도메인 항체와 결합된 GFP 또는 RFP가 효율적 이용이 가능한 것을 확인하였다.
As shown in Figs. 10 to 11, the fluorescence value of GFP or RFP bound to biotin as a positive control group was significantly different from the treatment group of 10 쨉 g / ml or more. In particular, the treatment of GFP or RFP bound to a single domain antibody against a single M13 bacteriophage, respectively, resulted in a GFP or RFP protein conjugated with a single domain antibody against two M13 bacteriophages, about twice that of biotin-conjugated GFP and RFP Treated GFP or RFP-conjugated GFP or RFP-conjugated GFP or RFP-treated GFP or RFP-treated GFP or RFP-treated GFP or RFP-treated GFP or RFP-treated GFP or RFP- Fluorescence values were high. This result indicates that GFP or RFP combined with a single domain antibody against M13 bacteriophage can be more efficiently used than GFP or RFP bound to biotin for streptavidin due to signal amplification by bacteriophage Respectively.
<110> JOONGKYEOM
<120> Single domain antibody for M13 bacteriophage and immunosensor
fusion protein comprising the same
<160> 10
<170> KopatentIn 2.0
<210> 1
<211> 122
<212> PRT
<213> Artificial Sequence
<220>
<223> it is 2B12(single domain antibody for M13 bacteriophage) amino
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<400> 1
Glu Ser Gly Gly Gly Ser Val Gln Val Gly Glu Ser Leu Arg Leu Ala
1 5 10 15
Cys Thr Ala Ser Glu Phe Thr Phe Asp Asp Thr Asp Val Gly Trp Tyr
20 25 30
Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser Ser Ile Asn Ser
35 40 45
Asp Gly Gly Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Leu Asp Asn Gly Lys Asn Thr Val Tyr Leu Gln Met Asp Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Asp Ala Lys Val
85 90 95
Lys Ala Gln Ala Leu Gly Thr Leu Leu Cys Arg Ile Leu Gly Asn Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 2
<211> 117
<212> PRT
<213> Artificial Sequence
<220>
<223> it is 2E9(single domain antibody for M13 bacteriophage) amino
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<400> 2
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Thr Ala Ser Gly Phe Thr Phe Ala Asp Ser Asp Met Gly Trp Tyr
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val Ala Ala Ile Asp Ser
35 40 45
Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Val Pro Thr
85 90 95
Tyr Asp Ser Ser Trp Cys Ser Arg Asp Tyr Trp Gly Lys Gly Thr Gln
100 105 110
Val Thr Val Ser Ser
115
<210> 3
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> it is 49C2(single domain antibody for M13 bacteriophage) amino
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<400> 3
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Asp Met Ser Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Ile Asp Ser
35 40 45
Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Met Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Gly Arg Gly
85 90 95
Gly Ser Trp Leu Thr Leu Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 4
<211> 122
<212> PRT
<213> Artificial Sequence
<220>
<223> it is 53A1(single domain antibody for M13 bacteriophage) amino
acid sequence.
<400> 4
Glu Ser Gly Gly Gly Ser Val Gln Val Gly Glu Ser Leu Arg Leu Ala
1 5 10 15
Cys Thr Ala Ser Glu Phe Thr Phe Asp Asp Thr Asp Val Gly Trp Tyr
20 25 30
Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser Ser Ile Asn Ser
35 40 45
Asp Gly Gly Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Leu Asp Asn Gly Lys Asn Thr Val Tyr Leu Gln Met Asp Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ala Asp Ala Lys Val
85 90 95
Lys Ala Gln Ala Leu Gly Thr Leu Leu Cys Arg Ile Leu Gly Asn Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 5
<211> 85
<212> PRT
<213> Artificial Sequence
<220>
<223> it is 59B5(single domain antibody for M13 bacteriophage) amino
acid sequence.
<400> 5
Glu Ser Gly Gly Gly Ser Glu Gly Ala Ala Ala Ile Asp Thr Asp Gly
1 5 10 15
Ser Thr Thr Tyr Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Lys
20 25 30
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro
35 40 45
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Glu Ala Glu Thr Ala
50 55 60
Tyr Gly Gly Arg Cys Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr Gln
65 70 75 80
Val Thr Val Ser Ser
85
<210> 6
<211> 366
<212> DNA
<213> Artificial Sequence
<220>
<223> it is 2B12(single domain antibody for M13 bacteriophage) amino
acid sequence.
<400> 6
gagtctggag gaggctcggt gcaggttgga gagagtctga gactcgcctg tacagcctct 60
gaattcactt ttgatgatac cgacgtgggc tggtaccgcc aggctccagg gaacgagtgt 120
gaattggtct caagtattaa tagtgatggt ggcacatact atacagactc cgtgaagggc 180
cgattcacca tctccctaga taacgggaag aacacggtgt atctgcaaat ggacagcctg 240
aagcctgaag acacggccgt gtattactgc gtggcagatg cgaaagtgaa ggcgcaggct 300
ttggggacgc ttttatgccg cattcttggt aactggggcc agggaaccca ggtcaccgtc 360
tcctca 366
<210> 7
<211> 351
<212> DNA
<213> Artificial Sequence
<220>
<223> it is 2E9(single domain antibody for M13 bacteriophage) amino
acid sequence.
<400> 7
gagtctggag gaggctcggt gcaggctgga gggtctctga gactctcctg tacagcctct 60
ggattcactt ttgctgattc tgacatgggc tggtaccgcc aggctccagg gaaggagcgc 120
gagcgggtcg cagctattga cagtgatggt aggacgagct acgccgactc cgtgaagggc 180
cgattcacca tctccaaaga caacgccaag aacactctgt atctgcaaat gaacagcctg 240
aaacctgagg acactgccat gtactactgt gcggcggggg tcccaacata cgatagtagc 300
tggtgctcca gggactactg gggcaaagga acccaggtca ccgtctcctc a 351
<210> 8
<211> 345
<212> DNA
<213> Artificial Sequence
<220>
<223> it is 49C2(single domain antibody for M13 bacteriophage) DNA
sequence.
<400> 8
gagtctgggg gaggctcggt gcaggctgga gggtctctga gactctcctg tgcagcctct 60
ggattcacat tcagtagcta cgacatgagc tgggtccgcc aggctccagg gaaggggctc 120
gagtgggtct caactattga tagtggtggt ggtagcacat actatgcaga ctccatgaag 180
ggccggttca ccatctccag agacaacgcc aagaacacgc tgtatctgca aatgaacagc 240
ctgaaaactg aggacacggc cgtgtattac tgtgcggcag gtcggggtgg cagctggtta 300
acgcttggtt actggggcca ggggacccag gttaccgtct cctca 345
<210> 9
<211> 366
<212> DNA
<213> Artificial Sequence
<220>
<223> it is 53A1(single domain antibody for M13 bacteriophage) DNA
sequence.
<400> 9
gagtctggag gaggctcggt gcaggttgga gagagtctga gactcgcctg tacagcctct 60
gaattcactt ttgatgatac cgacgtgggc tggtaccgcc aggctccagg gaacgagtgt 120
gaattggtct caagtattaa tagtgatggt ggcacatact atacagactc cgtgaagggc 180
cgattcacca tctccctaga taacgggaag aacacggtgt atctgcaaat ggacagcctg 240
aagcctgaag acacggccgt gtattactgc gtggcagatg cgaaagtgaa ggcgcaggct 300
ttggggacgc ttttatgccg cattcttggt aactggggcc agggaaccca ggtcaccgtc 360
tcctca 366
<210> 10
<211> 255
<212> DNA
<213> Artificial Sequence
<220>
<223> it is 59B5(single domain antibody for M13 bacteriophage) DNA
sequence.
<400> 10
gagtctgggg gaggctcgga gggggccgca gctattgata ctgatggtag cacaacctac 60
gcagactccg tgaggggccg attcaccatc tccaaagaca acgccaagaa cactctttat 120
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc ggcaggggaa 180
gccgagacgg cgtacggtgg taggtgctac ggcatggact actggggcaa aggaacccag 240
gtcaccgtct cctca 255
<110> JOONGKYEOM
<120> Single domain antibody for M13 bacteriophage and immunosensor
fusion protein comprising the same
<160> 10
<170> Kopatentin 2.0
<210> 1
<211> 122
<212> PRT
<213> Artificial Sequence
<220>
<223> it is 2B12 (single domain antibody for M13 bacteriophage) amino
acid sequence.
<400> 1
Glu Ser Gly Gly Gly Ser Val Gln Val Gly Glu Ser Leu
Claims (9)
이에 연결된 단백질-단백질 상호작용이 가능한 단백질, 형광단백질 및 단일 도메인 항체 단백질로 이루어진 군 중에서 선택된 1종의 단백질을 포함하는 융합 단백질.A single domain antibody against the M13 bacteriophage of claim 1; And
A fusion protein comprising one protein selected from the group consisting of a protein capable of protein-protein interaction, a fluorescent protein, and a single domain antibody protein.
The method according to claim 7, wherein the protein capable of protein-protein interaction is selected from the group consisting of streavidin, protein G, protein A, heterodimer protein AG, Wherein the fusion protein is at least one protein selected from the group consisting of a protein GA (heterodimer protein GA) and a heterotrimeric protein GAG.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004533264A (en) * | 2001-07-05 | 2004-11-04 | セルテック アール アンド ディ リミテッド | Expression vector encoding bacteriophage signal peptide |
| US6979534B1 (en) * | 1996-10-11 | 2005-12-27 | The Trustees Of The University Of Pennsylvania | Compositions and methods for detection of antibody binding to cells |
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2013
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6979534B1 (en) * | 1996-10-11 | 2005-12-27 | The Trustees Of The University Of Pennsylvania | Compositions and methods for detection of antibody binding to cells |
| JP2004533264A (en) * | 2001-07-05 | 2004-11-04 | セルテック アール アンド ディ リミテッド | Expression vector encoding bacteriophage signal peptide |
Non-Patent Citations (1)
| Title |
|---|
| Wood, S. C. Engineering Single-Domain Antibodies for Bioactive Paper Application. ?프대학교 석사학위 논문 (2011)* * |
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