KR20190088683A - Transgenic silkworms producing recombinant antibacterial adhesive peptide and the method producing the recombinant antibacterial adhesive peptide using the same - Google Patents

Transgenic silkworms producing recombinant antibacterial adhesive peptide and the method producing the recombinant antibacterial adhesive peptide using the same Download PDF

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KR20190088683A
KR20190088683A KR1020180007079A KR20180007079A KR20190088683A KR 20190088683 A KR20190088683 A KR 20190088683A KR 1020180007079 A KR1020180007079 A KR 1020180007079A KR 20180007079 A KR20180007079 A KR 20180007079A KR 20190088683 A KR20190088683 A KR 20190088683A
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박성길
이선혜
피경배
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주식회사 어드밴스드바이오텍
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Abstract

The present invention relates to a transgenic silkworm producing a recombinant antibacterial adhesive protein and a method for producing the recombinant antibacterial adhesive protein using the same. According to the present invention, it is possible to produce an antibacterial adhesive protein which is biocompatible, and has excellent adhesive strength and excellent antibacterial properties.

Description

재조합 항균 접착단백질을 생산하는 형질전환된 누에 및 이를 이용하여 재조합 항균 접착단백질을 생산하는 방법 {Transgenic silkworms producing recombinant antibacterial adhesive peptide and the method producing the recombinant antibacterial adhesive peptide using the same}[0001] The present invention relates to a recombinant antibacterial adhesive peptide, and more particularly, to a recombinant antibacterial adhesive peptide producing the recombinant antibacterial adhesive peptide and a recombinant antibacterial adhesive peptide using the recombinant antibacterial adhesive peptide,

본 발명은 재조합 항균 접착단백질을 생산하는 형질전환된 누에 및 이를 이용하여 재조합 항균 접착단백질을 생산하는 방법에 관한 것으로, 보다 구체적으로는 항균성을 가지는 항균 펩타이드(antibacterial peptide)가 융합된 홍합 접착단백질(Mussel Adhesive Protein)을 생산할 수 있는 형질전환된 누에 및 이를 이용하여 재조합 항균 접착단백질을 생산하는 방법에 관한 것이다. The present invention relates to a transformed silkworm producing recombinant antibacterial adhesive protein and a method for producing recombinant antibacterial adhesive protein using the same. More particularly, the present invention relates to a method for producing recombinant antibacterial adhesive protein using mussel adhesive protein (antibacterial peptide) Mussel Adhesive Protein) and a method for producing the recombinant antibacterial adhesive protein using the same.

누에(Bombyx mori)는 분류학상 곤충각(Insectada), 인시목(lepidoptera), 가잠아과 (Bombyxidae), 가잠아속(Bombyx), 가잠종(mori)에 속한다. 누에는 완전변태 곤충으로 알에서 부화한 유충이 발육하여 번데기가 되고, 성충(나방)이 되어 알을 낳고 일생을 마친다. 누에의 일생은 평균 60일 정도로 비교적 짧아 실험동물로서 이점을 가지고 있다. 이러한 누에는 연구용뿐만 아니라 그 산업적 가치로 인해 형질전환된 누에를 생산하기 위한 다양한 연구가 진행되어 왔다. 누에 형질전환 기술 개발은 일본의 Tamura 등에 의해 나비목곤충인 Trichopusia ni에서 유래한 piggyBac 유전자를 이용하여 형질전환용 전이벡터를 구축하고 이를 다화성 누에 품종의 알에 미세주입(microinjection)하여 최초로 형질전환 누에 제작에 성공하였다.The silkworm (Bombyx mori) belongs to the taxonomic Insectada, lepidoptera, Bombyxidae, Bombyx, and moth species (mori). The silkworm is a complete transformational insect, which develops larvae hatching from eggs, becomes a pupa, becomes an adult (moth), gives birth to an egg, and finishes its life. The life of the silkworm is relatively short, on average 60 days, and it has an advantage as an experimental animal. These silkworms have been subjected to various studies for producing silkworms transformed not only for research but also for their industrial value. In the development of silkworm transgenic technology, Tamura et al. Constructed a transgenic vector for transformation using the piggyBac gene derived from Trichopus ni, a lepidopteran insect, and microinjected it into the eggs of the polyhedrin silkworm varieties to produce transgenic silkworms .

형질전환 동물이란 외래의 유전자가 숙주의 게놈상에 삽입되어 그 형질의 일부가 변한 동물을 말하며 그때의 외래유전자를 트랜스젠(transgene)이라 한다. 1970년 중반부터 체세포나 생식세포에 유전자재조합 바이러스를 사용하여 외래유전자를 도입하기 시작하였고, 1980년도에는 미세주사방법(microinjection)으로 고든에 의해 슈퍼마우스를 생산하게 되었다.A transgenic animal is an animal in which an exogenous gene has been inserted into the genome of a host and a part of its trait has changed, and the foreign gene at that time is called a transgene. In the mid 1970's, we started to import foreign genes by using recombinant viruses in somatic cells or germ cells. In 1980, Gordon produced super-mice by microinjection.

외래유전자를 도입하는 기술로는 인산칼슘법, 전기천공법, DEAE-덱스트란법, 리포좀법, 미세주사법, bombardment법 등이 있다. 상기 방법들 중 DEAE-덱스트란법과 전기 천공법은 세포를 DNA가 열린 구멍을 통해 직접 세포질로 들어가게 하는 방법인데, 이 두 방법에서는 DNA가 손상을 입을 수도 있다. 리포좀을 이용하는 방법은 DNA를 인공지질 소포체인 리포좀을 넣어 세포막과 융합시켜 직접 세포 내로 운반시키는 방법으로 광범위하게 사용되고 있다. 미세주사법은 1세포기 수정란에 미세조작기를 사용하여 난에 손상을 주지 않을 정도의 미세주사침으로 DNA를 직접 주입하는 방법이다. 실용화 단계에 있는 외래유전자 도입기술은 도입되는 외래 유전자들이 성장률 조절, 극한 환경에서의 내성, 유전자 치료에 관련된 것이라면 인류에게 무한한 혜택을 줄 수 있을 것이다.Techniques for introducing a foreign gene include calcium phosphate, electroporation, DEAE-dextran, liposome, microinjection, and bombardment. Among these methods, the DEAE-dextran method and the electroporation method allow the cells to enter the cytoplasm directly through the open hole of the DNA, which may damage the DNA. The method using liposomes is widely used as a method of directly transferring DNA into a cell by fusing the DNA with a liposome, an artificial lipid vesicle, into the cell membrane. The microinjection method is a method of directly injecting DNA with a micro needle to such an extent that it does not damage the eggs by using a micro manipulator in the first embryo transfer embryo. Outpatient gene transfer technology at the practical stage will have infinite benefits to human beings if introduced foreign genes are related to growth rate control, tolerance in extreme environments, gene therapy.

이러한 누에 형질전환과 관련된 선행기술로는 대한민국 등록특허 제10-0267742호(등록일: 2000년 07월 07일, 명칭: 녹색 형광단백질 유전자가 삽입된 재조합 베큘로바이러스를 이용한 형광누에 및 제조방법), 대한민국 등록특허 제10-0323550호(등록일: 2002년 01월 24일, 명칭: 누에의 형질전환방법과 형질전환된 누에), 대한민국 등록특허 10-1480153를 (등록일: 2014년 12월31일, 명칭: 멜리틴 항생펩타이드를 함유한 누에고치를 생산하는 형질전환 누에) 등이 있다.Prior art related to such silkworm transformation is Korean Patent No. 10-0267742 (filed Jul. 7, 2000, entitled "Fluorescent silkworms using recombinant baculovirus inserted with green fluorescent protein gene and preparation method thereof"), Korean Patent No. 10-0323550 (filed on Jan. 24, 2002, entitled: Transformation method of silkworm and transgenic silkworm), Korea Patent No. 10-1480153 (registered on Dec. 31, 2014 : Transgenic silkworms producing silkworm cocoons containing melittin antibiotic peptides).

한편, 해양 생명체인 홍합(mussel)은 접착단백질(adhesive proteins)을 생산 및 분비함으로써 홍합 자신을 바다 속의 바위와 같은 젖은 고체표면에 단단히 부착할 수 있어, 파도의 충격이나 바닷물의 부력 효과에 영향을 받지 않는다. 홍합 접착단백질은 강력한 자연 접착제로 알려져 있으며, 화학 합성 접착제와 비교하였을 때 대부분 에폭시 수지보다 약 두 배 정도의 높은 인장강도를 나타내면서도 휘어질 수 있는 유연성을 지니고 있다. 또한, 홍합 접착단백질은 플라스틱, 유리, 금속, 테플론 및 생체물질 등의 다양한 표면에 접착할 수 있는 능력을 가지고 있으며, 아직까지 화학접착제 개발에서 미완의 과제로 남아 있는 젖은 표면에서의 접착도 몇 분 안에 가능하다. 특히, 홍합의 족사 (byssal thread)를 구성하고 있는 미틸러스 에둘리스 족사 단백질 1 (Mefp-1) 단백질의 경우, 단백질 내에 존재하는 디하이드록시-페닐알라닌 (DOPA) 잔기가 Fe3+ 이온과 결합하면서 표면에 큐티클 층을 구성하고, 이러한 결합에 의한 단단한 코팅층은 족사가 외부 충격에도 견딜 수 있도록 하는데 중요한 역할을 하는 것으로 알려져 있다. 또한, DOPA 잔기가 DOPA-퀴논으로 산화되면서 주변 DOPA 잔기 및 아미노산과 결합하면서 가교작용을 일으키고 이것이 물 속에서도 강력한 결합을 유지할 수 있도록 도와주는 것으로 알려져 있다. 최근에는 이러한 DOPA와 metal의 결합 및 DOPA의 산화반응을 이용하여 젤을 만들거나 생체접착제로 응용하려는 시도 및 연구가 일어나고 있다. 홍합 접착단백질은 별도의 표면처리 없이 다양한 종류의 표면에 접착이 가능하며, 에폭시 수지의 두 배 이상의 인장강도를 가지는 강력한 접착력을 가진다. 또한, 홍합 접착단백질은 수중 접착 능력이 매우 뛰어나기 때문에 화장용 접착제로서 기존의 접착제가 가지지 못하는 우수한 성능을 보유하고 있다. 또 다른 장점으로는 자연계에서 생분해가 가능한 환경친화적 접착제이며, 인체에 무해하고 기존 접착제에서 문제시 되고 있는 톨루엔, 벤젠, 에틸 벤젠, 헥산, 사이클로헥사, 클로로포름, 포름알데히드 등의 용매를 필요로 하지 않는다. On the other hand, marine life mussel produces and secretes adhesive proteins, which can firmly attach the mussel itself to wet solid surfaces such as rocks in the sea, thereby affecting the impact of waves and the buoyancy effect of seawater. I do not accept. Mussel adhesive protein is known as a strong natural adhesive. It has flexibility that can be flexed while showing high tensile strength about twice that of epoxy resin, compared with chemical synthetic adhesive. In addition, mussel adhesive proteins have the ability to adhere to a variety of surfaces, such as plastics, glass, metals, teflon and biomaterials. Adhesion on wet surfaces, which remain an incomplete challenge in chemical adhesive development, It is possible within. Particularly, in the case of the Meitilus edulis herb protein 1 (Mefp-1) protein constituting the byssal thread of the mussel, the dihydroxy-phenylalanine (DOPA) And it is known that the hard coating layer formed by such bonding plays an important role in allowing the footbath to withstand external impacts. In addition, it is known that DOPA residues are oxidized to DOPA-quinone, which binds to surrounding DOPA residues and amino acids to cause crosslinking, which helps maintain strong binding even in water. In recent years, attempts have been made to apply gels or bioadhesives using DOPA-metal bonding and DOPA oxidation reaction. The mussel adhesive protein can be attached to various kinds of surfaces without any surface treatment, and has a strong adhesive force with a tensile strength more than twice that of the epoxy resin. In addition, the mussel adhesive protein has excellent ability to adhere underwater, and therefore has excellent performance that conventional adhesives do not have as a cosmetic adhesive. Another advantage is that it is an environmentally friendly adhesive that can be biodegraded in nature and does not require a solvent such as toluene, benzene, ethylbenzene, hexane, cyclohexa, chloroform, formaldehyde, etc., .

이에 본 발명자들은 이러한 홍합 접착단백질과 항균 펩타이드를 접착시킨 항균 접착단백질과 관련하여 대한민국 등록특허 제10-1652263호(등록일: 2016년 8월 24일, 명칭: 항균 펩티드를 포함하는 접착단백질 및 이를 포함하는 항균 코팅 조성물) 및 대한민국 특허공개 제10-2017-0106571호(공개일: 2017년 9월 21일, 명칭: 항균 접착단백질을 유효성분으로 포함하는 여드름 예방 또는 개선을 위한 패치 제형)로서 특허절차를 진행한 바 있다.Accordingly, the present inventors have found that, in connection with the antibacterial adhesive protein to which the mushroom adhesive protein and the antibacterial peptide are adhered, Korean Patent No. 10-1652263 (filed on Aug. 24, 2016, entitled "Antibacterial Peptide- And Patent Application No. 10-2017-0106571 (published on Sep. 21, 2017, entitled " Patch Formulation for Acne Prevention or Improvement Containing Antimicrobial Adhesion Protein as Active Ingredient) " .

여기에 더하여 본 발명자들은 차세대 재조합 단백질 발현 시스템으로 주목받고 있는 누에를 이용하여 상기한 항균 접착단백질을 생산하는 기술을 개발하고자 계속 연구를 진행한 결과 항균 접착단백질를 발현하는 형질전환 누에를 제작함으로써 본 발명을 완성하였다. In addition, the present inventors have continued to develop a technology for producing the above-mentioned antimicrobial adhesive protein using a silkworm, which is attracting attention as a next generation recombinant protein expression system. As a result, they have found that by producing a transgenic silkworm expressing an antibacterial adhesive protein, .

따라서, 본 발명에서 해결하고자 하는 기술적 과제는 재조합 항균 접착단백질을 생산할 수 있는 형질전환 누에를 제공하기 위한 것이다.Accordingly, a technical object of the present invention is to provide a transgenic silkworm capable of producing a recombinant antibacterial adhesive protein.

또한, 본 발명에서 해결하고자 하는 다른 기술적 과제는 상기 형질전환 누에를 이용하여 항균 접착단백질을 생산하는 방법에 관한 것이다.Another object of the present invention is to provide a method for producing an antibacterial adhesive protein using the transgenic silkworm.

상기한 기술적 과제를 해결하기 위하여, 본 발명에서는 서열번호 1 또는 서열번호 2의 염기서열로 이루어진 항균 펩타이드 (antibacterial peptide)와 홍합 접착단백질(Mussel Adhesive Protein)이 융합된 항균 접착단백질의 유전자를 포함하는 재조합 발현벡터를 제공한다.In order to solve the above-mentioned technical problem, the present invention includes a gene of an antibacterial adhesive protein fused with an antibacterial peptide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and a mussel adhesive protein (Mussel Adhesive Protein) To provide a recombinant expression vector.

바람직하게, 상기 재조합 발현벡터는 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm인 것을 특징으로 한다.Preferably, the recombinant expression vector is pXLBacII-DsRED-SER1-MAP13151-AMP-Bm.

또한, 본 발명에서는 상기 항균 접착단백질 유전자를 포함하는 재조합 발현벡터로 형질전환시켜 제조된 항균 접착단백질을 발현하는 형질전환 누에를 제공한다.In addition, the present invention provides a transformed silkworm expressing an antibacterial adhesive protein produced by transforming with a recombinant expression vector containing the antibacterial adhesive protein gene.

또한, 본 발명에서는 (1) 항균 접착단백질의 유전자를 포함하는 재조합 발현벡터를 제조하는 단계; (2) 상기 단계 (1)에서 제조된 발현벡터를 누에알에 형질전환시켜 형질전환된 누에알을 제조하는 단계; 및 (3) 상기 단계 (2)에서 형질전환된 누에알을 부화시켜 형질전환 누에를 제조하는 단계를 포함하는 항균 접착단백질이 발현되는 형질전환 누에의 제조방법을 제공한다.In addition, the present invention provides a method for producing a recombinant expression vector, comprising the steps of (1) preparing a recombinant expression vector comprising a gene of an antibacterial adhesive protein; (2) transforming the expression vector prepared in the step (1) into silkworm eggs to produce transformed silkworm eggs; And (3) hatching the transformed silkworm in the step (2) to produce a transformed silkworm, wherein the transforming silkworm is expressed.

상기한 다른 기술적 과제를 해결하기 위하여, 본 발명에서는 상기 형질전환 누에를 이용하여 항균 접착단백질을 생산하는 방법을 제공한다.According to another aspect of the present invention, there is provided a method for producing an antibacterial adhesive protein using the transgenic silkworm.

본 발명에서는 또한 상기 항균 접착단백질 유전자를 포함하는 재조합 발현벡터로 형질전환시켜 제조된 형질전환 누에로부터 분리된 항균 접착단백질을 유효성분으로 포함하는 항균용 조성물을 제공한다.The present invention also provides an antimicrobial composition comprising, as an active ingredient, an antibacterial adhesive protein isolated from a transformed silkworm produced by transformation with a recombinant expression vector comprising the antibacterial adhesive protein gene.

본 발명에서 "홍합 접착단백질(Mussel Adhesive Protein)"은 그 자체로 사용하거나, 족사 단백질(foot protein, FP) 5(FP-5) 또는 FP-3, FP-6의 C-말단이나 N-말단 혹은 양쪽 모두에 해당하는 제1 펩타이드와 홍합 접착단백질 FP-1, FP-2, FP-4 및 각 단백질의 절편으로 이루어진 군으로부터 선택되는 적어도 하나의 제2 펩타이드가 융합된 융합 단백질로서 사용될 수 있다. In the present invention, "Mussel Adhesive Protein" may be used as such, or may be used as a foot protein (FP) 5 (FP-5) or FP- Or at least one second peptide selected from the group consisting of mussel adhesive proteins FP-1, FP-2, FP-4, and fragments of each protein may be used as the fusion protein .

본 발명의 한 구현예에 따르면, 상기 홍합 접착단백질은 서열번호 4 내지 서열번호 22로 이루어진 군으로부터 선택될 수 있다.According to one embodiment of the present invention, the mussel adhesive protein may be selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 22.

본 발명에서 "항균 펩타이드(antibacterial peptide)"는 유전자 재조합 기술로 홍합 접착단백질의 C-말단이나, N-말단, 또는 양쪽 모두 또는 하이브리드 홍합 접착단백질의 사이에 부가될 수 있다. 예를 들면, 2 개의 FP-1 사이에 1 개의 FP-5이 결합된 구조를 갖는 융합 단백질 FP-151의 경우 FP-1과 FP-5 사이에 항균 펩타이드를 부가할 수 있다. 또한, 양 말단이나 융합 단백질 사이에 서로 다른 항균 펩타이드를 부가할 수 있다. 예를 들면, 본 발명의 항균 펩타이드를 포함하는 접착단백질은 아프리카 개구리 제노퍼스 래비스(Xenopus laevis)의 피부로부터 분리된 α-나선형 23개 아미노산 펩타이드인 마가이닌(Magainin)이나 더마셉틴(Dermaseptin)과 같은 항균 펩타이드를 제한없이 포함할 수 있으며, 또한 인간 디펜신(human defensin), 카세리시딘(cathelicidin) LL-37, 히스타틴(Histatin)과 같은 항균 펩타이드가 포함될 수 있다.In the present invention, an " antibacterial peptide "may be added at the C-terminal, N-terminal, or both, or between hybrid mussel adhesive proteins of a mussel adhesive protein by gene recombination technology. For example, an antimicrobial peptide may be added between FP-1 and FP-5 in the case of a fusion protein FP-151 having a structure in which one FP-5 is bound between two FP-1. In addition, different antimicrobial peptides can be added between the two ends or the fusion protein. For example, the adhesive proteins comprising the antimicrobial peptides of the present invention may be conjugated to the alpha-helical 23 amino acid peptides Magainin or Dermaseptin, isolated from the skin of the African frog genus Xenopus laevis, And may also include antimicrobial peptides such as human defensin, cathelicidin LL-37, histatin, and the like.

본 발명에서 홍합 접착단백질에 융합되는 항균 펩타이드는 자연에서 유래하거나 인공적으로 합성되는 임의의 펩타이드를 제한없이 사용할 수 있다. 본 발명의 한 구현예에 따르면, 상기 항균 펩타이드는 미생물의 세포막을 파괴하거나 세포막을 투과하여 대사 기능을 저해하는 기작을 통해 항균 효과를 발휘한다. In the present invention, the antimicrobial peptide fused to the mussel adhesive protein may be any natural peptide or artificially synthesized peptide without limitation. According to one embodiment of the present invention, the antimicrobial peptide exhibits an antimicrobial effect through a mechanism that disrupts the cell membrane of the microorganism or permeates the cell membrane to inhibit the metabolic function.

본 발명의 다른 구현예에 따르면, 미생물의 세포막을 파괴하는 기작을 통해 항균 효과를 발휘하는 임의의 항균 펩타이드는 모두 본 발명에 사용될 수 있다. 바람직하게는, 접착단백질에 융합될 항균 펩타이드는 그램 양성균은 물론 그램 음성균에 효과가 있는 항균 펩타이드 중에서 임의로 선택될 수 있다. 보다 바람직하게는 LWKKWAKKWLKLWKA(서열번호 23), FALALKALKKL(서열번호 24), ILRWPWWPWRRK(서열번호 25), AKRHHGYKRKFH(서열번호 26), KKQRFRHRNRKGYRSQ(서열번호 27), KWLFKKIGAVLKVL(서열번호 28), LVKLVAGIKKFLKWK(서열번호 29), KFKWPW(서열번호 30), GIGAVLKVLTTGLPALISWI(서열번호 31), SWLSKTAKKGAVLKVL(서열번호 32), KKLFKKILKYL(서열번호 33), 및 GLKKLISWIKRAAQQG(서열번호 34)로 이루어진 군으로부터 선택될 수 있다. 본 발명의 바람직한 실시양태에 따르면, 서열번호 27의 항균 펩타이드를 사용하였다. According to another embodiment of the present invention, any antimicrobial peptide exhibiting an antimicrobial effect through a mechanism of destroying the cell membrane of a microorganism can be used in the present invention. Preferably, the antimicrobial peptide to be fused to the adhesive protein can be selected arbitrarily from among antimicrobial peptides effective against gram-positive bacteria as well as gram-negative bacteria. More preferably, LWKKWAKKWLKLWKA (SEQ ID NO: 23), FALALKALKKL (SEQ ID NO: 24), ILRWPWWPWRRK (SEQ ID NO: 25), AKRHHGYKRKFH (SEQ ID NO: 26), KKQRFRHRNRKGYRSQ (SEQ ID NO: 27), KWLFKKIGAVLKVL 29), KFKWPW (SEQ ID NO: 30), GIGAVLKVLTTGLPALISWI (SEQ ID NO: 31), SWLSKTAKKGAVLKVL (SEQ ID NO: 32), KKLFKKILKYL (SEQ ID NO: 33), and GLKKLISWIKRAAQQG (SEQ ID NO: 34). According to a preferred embodiment of the present invention, the antimicrobial peptide of SEQ ID NO: 27 was used.

본 발명에서 "항균 접착단백질"은 항균 펩타이드와 홍합 접착단백질이 융합된 단백질이다. 본 발명의 구현예에 따르면, 상기 항균 접착단백질은 서열번호 1 또는 서열번호 2의 염기시열에 의해 코딩될 수 있으며, 바람직하게는 서열번호 3의 아미노산 서열을 가지는 단백질 또는 이와 기능적 동등물일 수 있다. In the present invention, "antibacterial adhesive protein" is a protein in which an antibacterial peptide and a mussel adhesive protein are fused. According to an embodiment of the present invention, the antimicrobial adhesive protein may be encoded by a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, preferably a protein having the amino acid sequence of SEQ ID NO: 3 or a functional equivalent thereof.

상기 "기능적 동등물"이란, 아미노산의 부가, 치환 또는 결실의 결과, 서열번호 3으로 표시되는 아미노산 서열과 적어도 60%, 바람직하게는 70%, 보다 바람직하게는 80% 이상의 서열 상동성을 갖는 것으로서 본 발명의 항균 접착단백질을 코딩하는 단백질과 실질적으로 동질의 활성을 나타내는 단백질을 의미한다.   As used herein, the term "functional equivalent" means a polypeptide which has at least 60%, preferably 70%, more preferably 80% or more sequence homology with the amino acid sequence of SEQ ID NO: 3 as a result of amino acid addition, substitution or deletion Quot; means a protein that exhibits substantially the same activity as the protein encoding the antibacterial adhesive protein of the present invention.

상기 기능적 동등물에는, 예를 들어, 서열번호 3의 아미노산 서열의 아미노산 중 일부가 치환되거나, 결실 또는 부가된 아미노산 서열 변형체가 포함된다. 아미노산의 치환은 바람직하게는 보존적 치환이다. 천연에 존재하는 아미노산의 보존적 치환의 예는 다음과 같다; 지방족 아미노산(Gly, Ala, Pro), 소수성 아미노산(Ile, Leu, Val), 방향족 아미노산(Phe, Tyr, Trp), 산성 아미노산 (Asp, Glu), 염기성 아미노산(His, Lys, Arg, Gln, Asn) 및 황 함유 아미노산(Cys, Met). 아미노산의 결실은 바람직하게는 본 발명의 항균 접착단백질의 활성에 직접 관여하지 않는 부분에 위치한다. 또한 상기 기능적 동등물의 범위에는 항균 접착단백질의 기본 골격 및 이의 생리활성을 유지하면서 단백질의 일부 화학 구조가 변형된 단백질 유도체도 포함된다. 예를 들어, 본 발명의 단백질의 안정성, 저장성, 휘발성 또는 용해도 등을 변경시키기 위한 구조변경 및 생리활성을 유지하면서 GFP(Green Fluorescent Protein)와 같은 다른 단백질과 융합으로 만들어진 융합단백질 등이 이에 포함된다.Such functional equivalents include, for example, amino acid sequence variants in which a portion of the amino acid sequence of SEQ ID NO: 3 has been substituted, deleted or added. Substitution of amino acids is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). Deletion of the amino acid is preferably located at a site that is not directly involved in the activity of the antimicrobial adhesive protein of the present invention. The functional equivalents also include protein derivatives in which the chemical structure of the protein is modified while maintaining the basic skeleton of the antibacterial adhesive protein and its physiological activity. These include, for example, fusion proteins made by fusion with other proteins such as GFP (Green Fluorescent Protein) while retaining the structural modification and physiological activity to change the stability, storage stability, volatility or solubility of the protein of the present invention .

본 발명의 항균 접착단백질 유전자는 서열번호 1 또는 서열번호 2에 기재된 염기서열을 가지는데, 상기 유전자는 첨부한 서열목록에 기재된 서열번호 1 또는 서열번호 2의 염기서열에 한정되지 않으며, 기능적으로 균등한 코돈 또는 동일한 아미노산을 코딩하는 코돈, 또는 생물학적으로 균등한 아미노산을 코딩하는 코돈을 포함하는 염기서열을 포함한다. 생물학적으로 균등 활성을 갖는 변이를 고려한다면, 본 발명에서 이용되는 염기서열은 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 60%의 상동성, 보다 바람직하게는 70%의 상동성, 보다 더 바람직하게는 80%의 상동성, 가장 바람직하게는 90%의 상동성을 나타내는 서열을 의미한다. The antimicrobial adhesive protein gene of the present invention has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the gene is not limited to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 described in the attached Sequence Listing, A codon coding for one codon or the same amino acid, or a codon coding for a biologically equivalent amino acid. Considering a mutation having biologically equivalent activity, the nucleotide sequence used in the present invention is interpreted to include a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology.

본 발명의 바람직한 실시양태에 따르면, 항균 접착단백질의 유전자로서 서열번호 2의 염기서열을 가지는 MAP13151-AMP-Bm 유전자를 사용하였다.According to a preferred embodiment of the present invention, the MAP13151-AMP-Bm gene having the nucleotide sequence of SEQ ID NO: 2 was used as the gene of the antibacterial adhesive protein.

본 발명의 하나의 구현예에 따르면, 본 발명에서는 서열번호 1 또는 서열번호 2의 염기서열을 가지는 항균 접착단백질의 유전자를 포함하는 누에 발현용 플라스미드에 도입시킨 발현벡터를 제공한다. 상기 플라스미드로는 누에 발현용으로, 그 종류에는 제한이 없다. 본 발명에서는 플라스미드 pXLBacII-DsRED-SER1을 사용하였다.According to one embodiment of the present invention, there is provided an expression vector introduced into a silkworm-expressing plasmid comprising the gene of the antibacterial adhesive protein having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. As the plasmid, silkworm expression is not limited. In the present invention, the plasmid pXLBacII-DsRED-SER1 was used.

본 발명의 바람직한 실시양태에 따르면, 서열번호 2의 염기서열을 가지는 항균 접착단백질의 MAP13151-AMP-Bm 유전자를 포함하는 누에 발현용 발현벡터는 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm이다(도 2).According to a preferred embodiment of the present invention, the expression vector for silkworm expression comprising the MAP13151-AMP-Bm gene of the antimicrobial adhesive protein having the nucleotide sequence of SEQ ID NO: 2 is pXLBacII-DsRED-SER1-MAP13151-AMP-Bm 2).

본 발명에서 "형질전환용 재조합 발현벡터"란 유전자 삽입물이 발현되도록 작동가능하게 연결된 필수적인 조절 요소를 포함하는 유전자 작제물로, 플라스미드 벡터, 코스미드 벡터, 박테리오파지 벡터 및 바이러스 벡터 등을 포함한 통상의 모든 벡터를 포함한다.As used herein, the term "recombinant expression vector for transformation" refers to a gene construct containing an essential regulatory element operatively linked to the expression of a gene insert, and includes all plasmid vectors, cosmid vectors, bacteriophage vectors, Vector.

본 발명의 형질전환용 재조합 벡터는 상기 항균 접착단백질의 유전자가 발현될 수 있도록, 발현조절 서열과 기능적으로 연결되어 있다. 예를 들어, 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널, 인핸서 같은 발현 조절 요소 외에도 막 표적화 또는 분비를 위한 신호 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 또한, 벡터는 선택성 마커를 포함할 수 있으며, 벡터는 자가 복제하거나 숙주 DNA에 통합될 수 있다. 본 발명의 벡터는 당해 기술 분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용한다.The recombinant vector for transformation of the present invention is operably linked to an expression control sequence so that the gene of the antibacterial adhesive protein can be expressed. For example, the vector may comprise a signal sequence or leader sequence for membrane targeting or secretion in addition to an expression regulatory element such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, an enhancer, . In addition, the vector may comprise a selectable marker, and the vector may be self-replicating or integrated into the host DNA. The vector of the present invention can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage are performed using enzymes generally known in the art.

본 발명의 구현예에 따르면, 상기 발현벡터를 이용하여 형질전환된 형질전환 누에를 제공한다.According to an embodiment of the present invention, there is provided a transformed silkworm transformed with said expression vector.

본 발명의 항균 접착단백질이 발현되는 형질전환 누에의 제조방법은 (1) 항균 접착단백질의 유전자를 포함하는 재조합 발현벡터를 제조하는 단계; (2) 상기 단계 (1)에서 제조된 발현벡터를 누에알에 형질전환시켜 형질전환된 누에알을 제조하는 단계; 및 (3) 상기 단계 (2)에서 형질전환된 누에알을 부화시켜 형질전환 누에를 제조하는 단계를 포함하는 것이 특징이다.The method for producing a transformed silkworm in which the antibacterial adhesive protein of the present invention is expressed comprises the steps of: (1) preparing a recombinant expression vector containing a gene of an antibacterial adhesive protein; (2) transforming the expression vector prepared in the step (1) into silkworm eggs to produce transformed silkworm eggs; And (3) hatching the transformed silkworm in the step (2) to produce a transformed silkworm.

본 발명의 바람직한 실시양태에 따르면, 상기 단계 (2)의 형질전환은 미세주입(microinjection)시스템을 이용하는 것이 특징이다.According to a preferred embodiment of the present invention, the transformation of step (2) is characterized by using a microinjection system.

또한, 본 발명의 형질전환에 사용된 누에 품종으로는 어떠한 품종을 사용하여도 무관하나, 바람직하게는 KS08을 사용한다. 상기 누에 품종 KS08은 강건 다수의 특성을 지닌 2화성 일본종과 중국종간 교잡종이다.The silkworm varieties used in the transformation of the present invention may be any variety, but KS08 is preferably used. The silkworm variant KS08 is a two-harmonious Japanese species and an interspecific crossbred species with a robust number of characteristics.

본 발명의 하나의 구현예에 따르면, 상기 형질전환 누에를 이용하여 항균 접착단백질을 생산하는 방법을 제공한다. 상기 형질전환에는 목적하는 재조합 항균 접착단백질을 고수율로 효과적으로 발현시킬 수 있기만 하면 유전공학 분야에서 통상적인 어떠한 방법이라도 사용될 수 있다. According to one embodiment of the present invention, there is provided a method for producing an antibacterial adhesive protein using the transgenic silkworm. Any method that is conventional in the field of genetic engineering can be used for the transformation, as long as it can efficiently express the desired recombinant antimicrobial adhesive protein at a high yield.

본 발명의 바람직한 실시양태에 따르면, 상기 형질전환 누에를 이용하여 생체친화적이면서 접착력이 우수하고 항균성이 향상된 항균 접착단백질을 생산할 수 있다. According to a preferred embodiment of the present invention, it is possible to produce an antibacterial adhesive protein which is biocompatible, excellent in adhesive force and improved in antibacterial property by using the transgenic silkworm.

본 발명의 구현예에 따르면, 상기 항균 접착단백질 유전자를 포함하는 재조합 발현벡터로 형질전환시켜 제조된 형질전환 누에로부터 분리된 항균 접착단백질을 유효성분으로 포함하는 항균용 조성물을 제공한다.According to an embodiment of the present invention, there is provided an antimicrobial composition comprising, as an active ingredient, an antibacterial adhesive protein separated from a transformed silkworm prepared by transformation with a recombinant expression vector containing the antibacterial adhesive protein gene.

이와 같이, 본 발명의 형질전환 누에는 항균 접착단백질을 함유하고 있는 형질전환 누에를 생산함으로써, 생체친화적이면서 접착력이 우수하고 항균성이 우수한 항균 접착단백질을 생산할 수 있다. 또한, 양잠농가는 본 발명을 통해 일반누에와는 차별화된 고부가가치의 항균 접착단백질을 생산하는 형질전환 누에를 사육함으로써 소득향상에 크게 기여할 수 있다.Thus, the transgenic silkworm of the present invention can produce an antibacterial adhesive protein that is biocompatible, excellent in adhesion, and excellent in antibacterial activity by producing a transgenic silkworm containing an antibacterial adhesive protein. In addition, the silkworm farmers can greatly contribute to income improvement by breeding transgenic silkworms which produce high value-added antibacterial adhesive proteins differentiated from ordinary silkworms through the present invention.

도 1은 항균 접착단백질 MAP13151-AMP 유전자의 염기서열과 누에의 코돈사용빈도에 최적화된 MAP13151-AMP-Bm 유전자 염기서열의 상동성을 나타낸 것이다.
도 2는 재조합 항균 접합단백질의 누에 발현용 발현벡터 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm의 모식도이다.
도 3은 형질전화 누에의 알에서 DsRED 마커가 형광 발현되는 것을 보여주는 현미경 사진이다.
도 4는 재조합 발현벡터 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm이 형질전환된 누에 고치에서 항균 기능성 재조합 홍합 접착단백질의 존재를 확인한 결과이다.
도 5는 형질전환된 누에 고치의 세리신 단백질 추출물에서 항균 기능성 재조합 홍합 접착단백질 MAP13151-AMP-Bm을 분리 정제하고 SDS-PAGE 후 쿠마시 블라이언트 블루(Coomassie brilliant blue) 염색법으로 확인한 결과이다. Fig. 1 shows the homology of the MAP13151-AMP-Bm gene sequence optimized for the base sequence of the antibacterial adhesive protein MAP13151-AMP gene and the codon usage frequency of the silkworm.
2 is a schematic diagram of the expression vector pXLBacII-DsRED-SER1-MAP13151-AMP-Bm for silkworm expression of a recombinant antimicrobial conjugated protein.
3 is a photomicrograph showing that the DsRED marker is fluorescently expressed in the egg of the transgenic silkworm.
FIG. 4 shows the results of confirming the presence of an antibacterial functional recombinant mussel adhesive protein in a transformed silkworm cocoa plant transformed with the recombinant expression vector pXLBacII-DsRED-SER1-MAP13151-AMP-Bm.
FIG. 5 shows the results of SDS-PAGE followed by Coomassie brilliant blue staining for isolating and purifying the antibacterial functional recombinant mussel adhesive protein MAP13151-AMP-Bm from the sericin protein extract of transformed silkworm cocoons.

이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다. Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

<실시예 1> 항균 접착단백질 MAP13151-AMP 유전자 확보<Example 1> Generation of antimicrobial adhesive protein MAP13151-AMP gene

항균 펩타이드 (Anti-microbial Peptide; KKQRFRHRNRKGYRSQ)가 융합된 홍합 접착단백질 MAP13151-AMP 유전자 (서열번호 1)를 형질전환된 누에에서 생산하기 위한 유전자를 확보하기 위해 누에의 코돈사용빈도 (codon usage)에 최적화된 염기서열을 지니는 항균 접착단백질 MAP13151-AMP-Bm 유전자 (서열번호 2)를 합성하였다.Optimization of codon usage of silkworms to obtain genes for producing mussel adhesive protein MAP13151-AMP gene (SEQ ID NO: 1) fused with an anti-microbial peptide (KKQRFRHRNRKGYRSQ) in transformed silkworms (SEQ ID NO: 2) of the antimicrobial adhesive protein MAP13151-AMP-Bm having the nucleotide sequence of SEQ ID NO: 2.

도 1은 항균 접착단백질 MAP13151-AMP 유전자의 염기서열과 누에의 코돈사용빈도 (codon usage)에 최적화된 항균 접착단백질 MAP13151-AMP-Bm 유전자 염기서열의 상동성을 나타낸 것이다. 여기에서 보듯이, 항균 접착단백질 MAP13151-AMP 유전자와 염기서열이 누에의 코돈사용빈도에 최적화된 항균 접착단백질 MAP13151-AMP-Bm 유전자의 1,014개 염기서열 중 787개의 염기서열은 동일하며 (유사성 77.6%) 227개의 염기서열이 상이한 것으로 나타났다 (상이성 22.4%).  FIG. 1 shows the homology of the MAP13151-AMP-Bm gene sequence, which is an antibacterial adhesive protein optimized for the nucleotide sequence of the antibacterial adhesive protein MAP13151-AMP gene and the codon usage of the silkworm silkworm. As shown here, 787 nucleotide sequences of the MAP13151-AMP gene and the nucleotide sequence of the MAP13151-AMP-Bm gene, which are optimized for the frequency of codon usage of the silkworm, are identical (77.6% ) 227 nucleotide sequences (22.4%).

<실시예 2> 누에 형질전환용 재조합 발현벡터 구축Example 2 Construction of Recombinant Expression Vector for Silkworm Transformation

항균 접합단백질 MAP13151-AMP-Bm 유전자의 발현을 위해 플라스미드 pXLBacII-DsRED-SER1을 사용하였다. 플라스미드 pXLBacII-DsRED-SER1는 누에 형질전환 도메인인 Bombyx mori 3' 터미널 리피트(terminal repeat), 5' 터미널 리피트를 지니며 Bombyx mori 세리신 1 프로모터(sericin 1 promoter), SV40 poly(A) 시그널 및 형질전환 누에 선별을 위한 DsRED 단백질 발현 카세트를 가지고 있다. The plasmid pXLBacII-DsRED-SER1 was used for expression of the antimicrobial conjugated protein MAP13151-AMP-Bm gene. The plasmid pXLBacII-DsRED-SER1 contains the silkworm transcription domain Bombyx mori 3 'terminal repeats, the 5' terminal repeats, the Bombyx mori sericin 1 promoter, the SV40 poly (A) And a DsRED protein expression cassette for silkworm selection.

상기 실시예 1에서 확보한 항균 접착단백질 MAP13151-AMP-Bm 유전자를 pXLBacII-DsRED-SER1 플라스미드의 NheI, XhoI 제한효소 위치에 클로닝하여 재조합 홍합 접착단백질 MAP13151-AMP-Bm의 누에 발현용 재조합 발현벡터 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm을 수득하였다. The antimicrobial adhesive protein MAP13151-AMP-Bm gene obtained in Example 1 was cloned into the NheI and XhoI restriction sites of the pXLBacII-DsRED-SER1 plasmid, and the recombinant mussel adhesive protein MAP13151-AMP-Bm was transformed into the recombinant expression vector pXLBacII -DsRED-SER1-MAP13151-AMP-Bm.

도 2는 재조합 항균 접합단백질의 누에 발현용 발현벡터 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm의 모식도이다.2 is a schematic diagram of the expression vector pXLBacII-DsRED-SER1-MAP13151-AMP-Bm for silkworm expression of a recombinant antimicrobial conjugated protein.

<실시예 3> 누에 형질전환 및 선별 Example 3 Silkworm Transformation and Screening

형질전환에 사용된 누에 품종은 KS08이며 강건 다수의 특성을 지닌 2화성 일본종과 중국종간 교잡종이었다. 부화 후 1령에서 5령 유충 전 기간은 온도 25℃, 습도 70%로 설정되어진 사육용 인큐베이터에서 사육하였다. 상족 후 화아까지는 온도 23℃, 습도 60%의 상족실에서 개체 보호 후 암ㅇ수나방 교미과정을 거쳐 주사용 누에알을 획득하였다. The silkworm varieties used for the transformation were KS08 and the two species of Japanese Marsh and Chinese interspecific hybrids with robustness. From the 1st to 5th instar larvae after hatching, they were kept in incubation incubator set at 25 ℃ and 70% humidity. The control group was maintained in a temperature of 23 ℃ and a humidity of 60% in the spermatozoa.

상기 실시예 2에서 수득한 재조합 발현벡터 pXLBacII-ECFP-SER1-MAP13151-AMP-Bm을 최종 농도 0.3 μg/μL로 주입 완충액 (0.1 mM sodium phosphate, 5 mM KC1, pH 6.8)에 용해시켰다. 누에 암나방에서 수집한 누에알을 현미경 슬라이드 상에 배치하고 재조합 발현벡터 pXLBacII-eCFP-SER1-MAP13151-AMP-Bm 주입액 2 μL를 전 배수엽 누에 배아 (preblastoderm silkworm embryo)에 World Precision Instruments PV820 pressure regulator (USA), Suruga Seiki M331 micromanipulator (Japan), Narishige HD-21 double pipette holder (Japan)로 구성된 미세주사시스템 (injection system)을 이용하여 주입하였다. The recombinant expression vector pXLBacII-ECFP-SER1-MAP13151-AMP-Bm obtained in Example 2 was dissolved in injection buffer (0.1 mM sodium phosphate, 5 mM KCl, pH 6.8) to a final concentration of 0.3 μg / The silkworm eggs collected from the silkworms were placed on a microscope slide and 2 μL of the recombinant expression vector pXLBacII-eCFP-SER1-MAP13151-AMP-Bm was added to the preblastoderm silkworm embryo with World Precision Instruments PV820 pressure regulator (USA), Suruga Seiki M331 micromanipulator (Japan) and Narishige HD-21 double pipette holder (Japan).

구멍 난 알은 Helping Hand Super Glue gel (The Faucet Queens, Inc., USA)로 밀봉하였고 표준사육법에 준하여 배자 발육시켰다. 부화 후 인공 사료 (Japan Nosan Co., Japan)에서 유충을 사육하고 같은 계통 내의 자가 교배에 의해 후손 세대를 얻었다. 형질전환된 누에는 550 내지 700 nm 사이의 필터를 가진 Olympus SXZ12 현미경 (Tokyo, Japan)을 사용하여 DsRED 마커의 존재에 의해 형광발현을 확인하였다.Hatch eggs were sealed with Helping Hand Super Glue gel (The Faucet Queens, Inc., USA) and embryonic development was performed according to standard breeding methods. After hatching, larvae were raised in artificial diets (Japan Nosan Co., Japan), and descendants were obtained by self-crossing in the same line. The transformed silkworms were confirmed for fluorescence expression by the presence of DsRED markers using an Olympus SXZ12 microscope (Tokyo, Japan) with filters between 550 and 700 nm.

도 3은 형질전화 누에의 알에서 DsRED 마커가 형광 발현되는 것을 보여주는 현미경 사진이다.3 is a photomicrograph showing that the DsRED marker is fluorescently expressed in the egg of the transgenic silkworm.

<실시예 4> 항균 기능성 재조합 홍합 접착단백질 MAP13151-AMP-Bm 발현 확인Example 4 Expression of Antibacterial Function Recombinant Mussel Adhesion Protein MAP13151-AMP-Bm

재조합 발현벡터 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm이 형질전환된 누에에서 재조합 홍합 접착단백질 MAP13151-AMP-Bm의 발현을 확인하기 위해 형질전환된 누에의 고치의 세리신 층에서 단백질 추출물을 확보하고 재조합 홍합 접착단백질 MAP13151-AMP-Bm의 발현을 확인하였다. 형질전환 누에의 고치 100 mg에 1 mL의 단백질 추출용 완충용액 [50 mM 트리스 (pH 8.0), 8 M 우레아]을 첨가하고 80℃에서 10분간 반응시켰고 원심분리하여 단백질 추출물을 확보하였으며 SDS-PAGE 후 쿠마시에 블라이언트 블루(Coomassie brilliant blue) 염색법으로 염색하여 재조합 홍합 접착단백질 MAP13151-AMP-Bm의 존재를 확인하였다. To confirm the expression of the recombinant mussel adhesive protein MAP13151-AMP-Bm in the recombinant expression vector pXLBacII-DsRED-SER1-MAP13151-AMP-Bm transgenic silkworms, protein extracts were obtained from the sericin layer of transformed silkworm larvae The expression of the recombinant mussel adhesive protein MAP13151-AMP-Bm was confirmed. To 100 mg of transformed silkworm coculture, 1 mL of buffer solution for protein extraction (50 mM Tris (pH 8.0), 8 M urea] was added and reacted at 80 ° C for 10 minutes. The protein extract was obtained by SDS-PAGE And the presence of the recombinant mussel adhesive protein MAP13151-AMP-Bm was confirmed by staining with Coomassie brilliant blue in Fukuma City.

도 4는 재조합 발현벡터 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm이 형질전환된 누에 고치에서 항균 기능성 재조합 홍합 접착단백질의 존재를 확인한 결과이다. 여기에서 보듯이, 재조합 홍합 접착단백질 MAP13151-AMP-Bm은 약 28 kDa의 크기로 발현되었다. 이는 재조합 발현벡터 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm이 형질전환된 누에에 안정적으로 도입되었고 재조합 홍합 접착단백질 MAP13151-AMP-Bm이 형질전환된 누에에서 발현되고 고치의 세리신 층에 존재한다는 것을 의미한다. FIG. 4 shows the results of confirming the presence of an antibacterial functional recombinant mussel adhesive protein in a transformed silkworm cocoa plant transformed with the recombinant expression vector pXLBacII-DsRED-SER1-MAP13151-AMP-Bm. As shown here, the recombinant mussel adhesive protein MAP13151-AMP-Bm was expressed at a size of about 28 kDa. This indicates that the recombinant expression vector pXLBacII-DsRED-SER1-MAP13151-AMP-Bm was stably introduced into the transformed silkworm and that the recombinant mussel adhesion protein MAP13151-AMP-Bm was expressed in the transformed silkworm and was present in the sericin layer of the cocoon it means.

<실시예 5> 재조합 홍합 접착단백질 MAP13151-AMP-Bm 분리 정제 Example 5 Recombinant mussel adhesive protein MAP13151-AMP-Bm isolated and purified

형질전환 누에의 고치 10 g에 100 mL의 단백질 추출용 완충용액 [50 mM Tris (pH 8.0), 8 M urea]을 첨가하고 80℃에서 10분간 반응시킨 후, 원심분리하여 상층액을 수득하였다. 확보된 상층액에 아세트산을 최종농도 25% (v/v)가 되게 첨가하고, 다시 원심분리하여 단백질 추출물을 확보하였다. 분리된 재조합 홍합 접착단백질 추출물 MAP13151-AMP-Bm을 SDS-PAGE 후 쿠마시 블라이언트 블루 염색법으로 염색하여 확인하였다. 도 5는 형질전환된 누에 고치의 세리신 단백질 추출물에서 항균 기능성 재조합 홍합 접착단백질 MAP13151-AMP-Bm을 분리 정제하고 SDS-PAGE 후 쿠마시 블라이언트 블루(Coomassie brilliant blue) 염색법으로 확인한 결과이다. 100 ml of a buffer solution for protein extraction (50 mM Tris (pH 8.0), 8 M urea] was added to 10 g of the transformed silkworm cocoon, followed by reaction at 80 ° C for 10 minutes, followed by centrifugation to obtain a supernatant. Acetic acid was added to the supernatant to a final concentration of 25% (v / v), and centrifuged again to obtain a protein extract. The isolated recombinant mussel adhesive protein extract, MAP13151-AMP-Bm, was identified by SDS-PAGE and stained with Kumasi Blue Blotting. FIG. 5 shows the results of SDS-PAGE followed by Coomassie brilliant blue staining for isolating and purifying the antibacterial functional recombinant mussel adhesive protein MAP13151-AMP-Bm from the sericin protein extract of transformed silkworm cocoons.

또한, 확보된 추출을 통해 재조합 홍합 접착단백질 MAP13151-AMP-Bm을 분리하였고, Prep-LC c18 컬럼을 통해 정제하였다. 정제된 재조합 홍합 접착단백질 MAP13151-AMP-Bm은 HPLC c-18 컬럼을 통해 확인하였다. 도 6은 Prep-LC를 통해 정제된 항균 접착단백질 MAP13151-AMP-Bm의 분석 결과이다.In addition, the recombinant mussel adhesive protein MAP13151-AMP-Bm was isolated from the obtained extract and purified through Prep-LC c18 column. Purified recombinant mussel adhesive protein MAP13151-AMP-Bm was identified on HPLC c-18 column. 6 shows the results of analysis of the antibacterial adhesive protein MAP13151-AMP-Bm purified through Prep-LC.

<실시예 6> 재조합 홍합 접착단백질 MAP13151-AMP-Bm의 항균 기능 실험 Example 6 Antibacterial Activity Test of Recombinant Mussel Adhesion Protein MAP13151-AMP-Bm

항균 기능 시험을 위해 액상시료 기준의 고정농도 시험법을 실시하였다. 먼저 시험액 0.1 mg/mL, 0.01 mg/mL와 대조시험액 (멸균생리식염수)을 멸균한 용기에 넣고, 전배양한 시험균액을 각각 접종하였다 (시험액 : 균액 = 10 : 1). 접종 즉시 대조시험액에서 일부를 채취하여 초기 균수를 확인하였다. 접촉 (4시간) 동안 노출시킨 후 시험액과 대조시험액에서 일부를 채취하여 균수를 확인하였다. For the antimicrobial function test, the fixed concentration test based on the liquid sample was carried out. First, test solutions (0.1 mg / mL, 0.01 mg / mL) and control solution (sterile physiological saline) were placed in a sterilized container, and the pre-cultured test bacteria were inoculated (test solution: bacterium = 10: 1). Immediately after inoculation, a portion of the test solution was collected to confirm the initial number of bacteria. After exposure for 4 hours, part of test solution and control solution was collected to confirm the number of bacteria.

살균력은 하기 수학식 1에 따라 산술하여 백분율로 나타내었다.The sterilization power was expressed as a percentage by the following formula (1).

Figure pat00001
Figure pat00001

상기 식에서,In this formula,

A는 일정시간 접촉 후 대조시험액으로부터의 균수이고,A is the number of bacteria from the control test solution after a certain period of time contact,

B는 일정시간 접촉 후 시험액으로부터의 균수이다.B is the number of bacteria from the test solution after a certain period of contact.

하기 표 1 및 표 2는 각각 시험액 0.1 mg/mL 및 0.01 mg/mL을 사용한 경우의 항균 기능성 재조합 홍합 접착단백질 MAP13151-AMP-Bm의 살균력을 나타낸 것이다. The following Tables 1 and 2 show the sterilizing power of the antibacterial functional recombinant mussel adhesive protein MAP13151-AMP-Bm when 0.1 mg / mL and 0.01 mg / mL of test solutions were used, respectively.

시험균주Test strain 접촉시간Contact time 균수 (CFU/ml)Number of bacteria (CFU / ml) 살균력 (%)Sterility (%) 대조시험액Control Test Solution 시험액Test solution Escherichia coli
(대장균, ATCC 8739)Escherichia coli
(E. coli, ATCC 8739) 초기Early 1.8 x 106 1.8 x 10 6 99.9%99.9% 4시간4 hours 2.0 x 106 2.0 x 10 6 2.5 x 103 2.5 x 10 3 Staphylococcus aureus
(황색포도상구균, ATCC 6538)Staphylococcus aureus
(Staphylococcus aureus, ATCC 6538) 초기Early 2.4 x 106 2.4 x 10 6 98.9%98.9% 4시간4 hours 2.8 x 106 2.8 x 10 6 3.2 x 104 3.2 x 10 4 Pseudomonas aeruginosa
(녹농균, ATCC 9027)Pseudomonas aeruginosa
(Pseudomonas aeruginosa, ATCC 9027) 초기Early 4.1 x 106 4.1 x 10 6 99.9%99.9% 4시간4 hours 4.1 x 106 4.1 x 10 6 3.5 x 103 3.5 x 10 3

시험균주Test strain 접촉시간Contact time 균수 (CFU/ml)Number of bacteria (CFU / ml) 살균력 (%)Sterility (%) 대조시험액Control Test Solution 시험액Test solution Escherichia coli
(대장균, ATCC 8739)Escherichia coli
(E. coli, ATCC 8739) 초기Early 1.8 x 106 1.8 x 10 6 99.9%99.9% 4시간4 hours 2.0 x 106 2.0 x 10 6 1.1 x 103 1.1 x 10 3 Staphylococcus aureus
(황색포도상구균, ATCC 6538)Staphylococcus aureus
(Staphylococcus aureus, ATCC 6538) 초기Early 2.4 x 106 2.4 x 10 6 99.2%99.2% 4시간4 hours 2.8 x 106 2.8 x 10 6 2.2 x 104 2.2 x 10 4 Pseudomonas aeruginosa
(녹농균, ATCC 9027)Pseudomonas aeruginosa
(Pseudomonas aeruginosa, ATCC 9027) 초기Early 4.1 x 106 4.1 x 10 6 99.9%99.9% 4시간4 hours 4.1 x 106 4.1 x 10 6 3.8 x 103 3.8 x 10 3

상기 표 1 및 2에서 보듯이, 형질전환된 누에로부터 분리된 재조합 홍합 접착단백질 MAP13151-AMP-Bm은 살균력이 매우 우수한 것을 확인할 수 있었다.As shown in Tables 1 and 2, it was confirmed that the recombinant mussel adhesive protein MAP13151-AMP-Bm isolated from the transformed silkworm had excellent sterilizing power.

<110> Advanced BioTech Co.,Ltd. <120> Transgenic silkworms producing recombinant antibacterial adhesive peptide and the method producing the recombinant antibacterial adhesive peptide using the same <130> PA-17-0269 <160> 34 <170> KopatentIn 2.0 <210> 1 <211> 1014 <212> DNA <213> Artificial Sequence <220> <223> MAP13151-AMP <400> 1 atggctagcg ctaaaccgtc ttacccgccg acctacaaag caaaaccctc gtacccaccg 60 acttataagg ctaaacctag ctatccacct acgtacaaag ctaaaccgtc ttacccgccg 120 acttacaaag caaaaccgtc ctaccctccg acctataagg ctaaaccgag ttaccccccg 180 acttacaaag gctgcagggc ggattattat ggtccgaaat atggtccgcc gcgtcgttac 240 ggtggtggca actacaaccg ttatggcgga tcccgtcgtt atggcggtta taaaggctgg 300 aacaacggtt ggaaacgtgg tcgttggggt cgtaaatatt atgaattcgc tagcgctaaa 360 ccgtcttacc cgccgaccta caaagcaaaa ccctcgtacc caccgactta taaggctaaa 420 cctagctatc cacctacgta caaagctaaa ccgtcttacc cgccgactta caaagcaaaa 480 ccgtcctacc ctccgaccta taaggctaaa ccgagttacc ccccgactta caaaggctgc 540 agttctgaag aatacaaggg tggttattac ccaggcaatt cgaaccacta tcattcaggt 600 ggtagttatc acggatccgg ctaccatgga ggatataagg gaaagtatta cggaaaggca 660 aagaaatact attataaata taaaaacagc ggaaaataca agtatctaaa gaaagctaga 720 aaataccata gaaagggtta caagaagtat tatggaggta gcagtgaatt cgaatttgct 780 aaaccgtctt acccgccgac ctacaaagca aaaccctcgt acccaccgac ttataaggct 840 aaacctagct atccacctac gtacaaagct aaaccgtctt acccgccgac ttacaaagca 900 aaaccgtcct accctccgac ctataaggct aaaccgagtt accccccgac ttacaaaaag 960 cttaaaaagc agcgcttccg tcaccgtaac cgcaaaggtt atcgtagcca gtga 1014 <210> 2 <211> 1014 <212> DNA <213> Artificial Sequence <220> <223> MAP13151-AMP-Bm <400> 2 atggctagtg ctaaaccgag ctacccgcct acctacaagg ctaaacctag ctacccgccg 60 acttataaag ccaaaccgtc ctaccccccg acatataaag ctaaaccaag ctacccacct 120 acttacaaag cgaagccgtc ttaccctcca acttacaagg ctaaaccctc ttacccgccc 180 acatacaaag gttgcagggc tgactactac ggcccgaaat acggaccgcc tagaagatac 240 ggtggtggca actacaacag gtacggaggt tcccgcagat acggtggcta caagggatgg 300 aacaacggtt ggaaaagagg caggtggggt cgtaaatact acgaattcgc cagcgccaaa 360 cctagctacc ctcctacata caaagctaag ccgtcctacc ctccgactta caaggccaaa 420 ccttcatacc cgccaacata caaagcaaaa ccgtcatacc ctccgacgta caaagctaag 480 ccatcgtacc ctccgacata caaagccaag ccgtcctacc ctccgaccta caaaggatgc 540 agctctgagg aatacaaagg tggctactac cccggcaact caaaccacta ccactccgga 600 ggttcctacc acggttctgg ttaccacggt ggttacaaag gaaagtacta cggcaaagct 660 aaaaaatact actacaaata caaaaactca ggcaagtaca aatacctgaa aaaagctcgc 720 aaataccatc gcaaaggcta caaaaaatac tacggtggtt cgagcgaatt cgaattcgcc 780 aagccgagct acccacctac atacaaagcg aaaccgtcat acccgccgac atacaaggct 840 aagccaagct accctcctac ttacaaagcc aaaccttcat acccgcctac gtacaaagct 900 aaaccttcct acccgccaac ttataaagca aaaccttctt accccccaac ctacaagaaa 960 ttgaagaaac agcgttttcg acaccgcaac cgaaaaggtt ataggtcaca ataa 1014 <210> 3 <211> 337 <212> PRT <213> Artificial Sequence <220> <223> MAP13151-AMP-Bm <400> 3 Met Ala Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 1 5 10 15 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 20 25 30 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 35 40 45 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly 50 55 60 Cys Arg Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr 65 70 75 80 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Gly Ser Arg Arg Tyr Gly Gly 85 90 95 Tyr Lys Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys 100 105 110 Tyr Tyr Glu Phe Ala Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 115 120 125 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 130 135 140 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 145 150 155 160 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 165 170 175 Tyr Lys Gly Cys Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly 180 185 190 Asn Ser Asn His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr 195 200 205 His Gly Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr 210 215 220 Tyr Lys Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg 225 230 235 240 Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Glu 245 250 255 Phe Glu Phe Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 260 265 270 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 275 280 285 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 290 295 300 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Lys 305 310 315 320 Leu Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg Ser 325 330 335 Gln <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> model peptide of the tandem repeat decapeptide derived from foot protein 1 (FP-1, Mytilus edulis) <400> 4 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 1 5 10 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 2 times repeated sequence derived from foot protein 1 (FP-1, Mytilus edulis) <400> 5 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys 20 <210> 6 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> 6 times repeated sequence derived from foot protein 1 (FP-1, Mytilus edulis) <400> 6 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 50 55 60 <210> 7 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> partial sequence of foot protein type 2 (FP-2, Mytilus californianus) <400> 7 Glu Val His Ala Cys Lys Pro Asn Pro Cys Lys Asn Asn Gly Arg Cys 1 5 10 15 Tyr Pro Asp Gly Lys Thr Gly Tyr Lys Cys Lys Cys Val Gly Gly Tyr 20 25 30 Ser Gly Pro Thr Cys Ala Cys 35 <210> 8 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> Foot protein type 3 (FP-3, Mytilus edulis) <400> 8 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly 1 5 10 15 Gly Asn Tyr Asn Arg Tyr Phe Phe Ser Arg Arg Tyr Gly Gly Tyr Lys 20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 Glu Phe Glu Phe 50 <210> 9 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Foot protein type 3 (FP-3, Mytilus galloprovincialis : mgfp-3A) <400> 9 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly 1 5 10 15 Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp 20 25 30 Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 45 <210> 10 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> Foot protein type 3 (FP-3, Mytilus edulis: mefp-3F) <400> 10 Ala Asp Tyr Tyr Gly Pro Asn Tyr Gly Pro Pro Arg Arg Tyr Gly Gly 1 5 10 15 Gly Asn Tyr Asn Arg Tyr Asn Gly Tyr Gly Gly Gly Arg Arg Tyr Gly 20 25 30 Gly Tyr Lys Gly Trp Asn Asn Gly Trp Asn Arg Gly Arg Arg Gly Lys 35 40 45 Tyr Trp 50 <210> 11 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> Foot protein type 3 (FP-3, Mytilus californianus) <400> 11 Gly Ala Tyr Lys Gly Pro Asn Tyr Asn Tyr Pro Tyr Arg Tyr Gly Gly 1 5 10 15 Lys Tyr Asn Gly Tyr Lys Gly Tyr Pro Arg Gly Tyr Gly Trp Asn Lys 20 25 30 Gly Trp Asn Lys Gly Arg Trp Gly Arg Lys Tyr Tyr 35 40 <210> 12 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> partial sequence from foot protein type 4 (Mytilus californianus) <400> 12 Gly His Val His Arg His Arg Val Leu His Lys His Val His Asn His 1 5 10 15 Arg Val Leu His Lys His Leu His Lys His Gln Val Leu His Gly His 20 25 30 Val His Arg His Gln Val Leu His Lys His Val His Asn His Arg Val 35 40 45 Leu His Lys His Leu His Lys His Gln Val Leu His 50 55 60 <210> 13 <211> 75 <212> PRT <213> Artificial Sequence <220> <223> Foot protein type5 (FP-5, Mytilus edulis) <400> 13 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Tyr Asn Ala Tyr His 1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr 20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys 35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Ala 50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser 65 70 75 <210> 14 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Foot protein 5 (FP-5, Mytilus edulis) <400> 14 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His 1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr 20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys 35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg 50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser 65 70 75 <210> 15 <211> 71 <212> PRT <213> Artificial Sequence <220> <223> Foot protein 5 (FP-5, Mytilus coruscus) <400> 15 Tyr Asp Asp Tyr Ser Asp Gly Tyr Tyr Pro Gly Ser Ala Tyr Asp Tyr 1 5 10 15 Pro Ser Gly Ser His Trp His Gly His Gly Tyr Lys Gly Lys Tyr Tyr 20 25 30 Gly Lys Gly Lys Lys Tyr Tyr Tyr Lys Phe Lys Arg Thr Gly Lys Tyr 35 40 45 Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys 50 55 60 His Tyr Gly Gly Ser Ser Ser 65 70 <210> 16 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> mussel adhesive protein foot protein type5 from (Mytilus galloprovincialis) <400> 16 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His 1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr 20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys 35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Ala 50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser 65 70 75 <210> 17 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> mussel adhesive protein foot protein type 6 <400> 17 Gly Gly Gly Asn Tyr Arg Gly Tyr Cys Ser Asn Lys Gly Cys Arg Ser 1 5 10 15 Gly Tyr Ile Phe Tyr Asp Asn Arg Gly Phe Cys Lys Tyr Gly Ser Ser 20 25 30 Ser Tyr Lys Tyr Asp Cys Gly Asn Tyr Ala Gly Cys Cys Leu Pro Arg 35 40 45 Asn Pro Tyr Gly Arg Val Lys Tyr Tyr Cys Thr Lys Lys Tyr Ser Cys 50 55 60 Pro Asp Asp Phe Tyr Tyr Tyr Asn Asn Lys Gly Tyr Tyr Tyr Tyr Asn 65 70 75 80 Asp Lys Asp Trp Phe Asn Cys Gly Ser Tyr Asn Gly Cys Cys Leu Ala 85 90 95 Ser Gly Tyr <210> 18 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-151, MEFP-5 based) <400> 18 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu 50 55 60 Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ala Tyr His Tyr His Ser Gly 65 70 75 80 Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr 85 90 95 Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys 100 105 110 Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys 115 120 125 Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 130 135 140 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 145 150 155 160 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 165 170 175 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 180 185 190 Tyr Lys <210> 19 <211> 164 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-151, MGFP-5 based) <400> 19 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu 50 55 60 Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly 65 70 75 80 Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr 85 90 95 Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys 100 105 110 Lys Tyr Tyr Gly Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr 115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 130 135 140 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 145 150 155 160 Pro Thr Tyr Lys <210> 20 <211> 192 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-151, MCFP-5 based) <400> 20 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Tyr Asp Gly Tyr 50 55 60 Ser Asp Gly Tyr Tyr Pro Gly Ser Ala Tyr Asn Tyr Pro Ser Gly Ser 65 70 75 80 His Gly Tyr His Gly His Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Gly 85 90 95 Lys Lys Tyr Tyr Tyr Lys Tyr Lys Ala Thr Gly Lys Tyr Lys Tyr Leu 100 105 110 Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly 115 120 125 Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 130 135 140 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 145 150 155 160 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 165 170 175 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 180 185 190 <210> 21 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-131) <400> 21 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly Cys Arg Ala 50 55 60 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly 65 70 75 80 Asn Tyr Asn Arg Tyr Gly Gly Ser Arg Arg Tyr Gly Gly Tyr Lys Gly 85 90 95 Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly 100 105 110 Phe Glu Phe Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 115 120 125 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 130 135 140 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 145 150 155 160 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Lys 165 170 175 Leu <210> 22 <211> 179 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-251) <400> 22 Met Glu Val His Ala Cys Lys Pro Asn Pro Cys Lys Asn Asn Gly Arg 1 5 10 15 Cys Tyr Pro Asp Gly Lys Thr Gly Tyr Lys Cys Lys Cys Val Gly Gly 20 25 30 Tyr Ser Gly Pro Thr Cys Ala Cys Ser Ser Glu Glu Tyr Lys Gly Gly 35 40 45 Tyr Tyr Pro Gly Asn Ser Asn His Tyr His Ser Gly Gly Ser Tyr His 50 55 60 Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala 65 70 75 80 Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu 85 90 95 Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Tyr Tyr Gly Gly 100 105 110 Ser Ser Glu Phe Glu Phe Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 115 120 125 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 130 135 140 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 145 150 155 160 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 165 170 175 Tyr Lys Lys <210> 23 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 1 <400> 23 Leu Trp Lys Lys Trp Ala Lys Lys Trp Leu Lys Leu Trp Lys Ala 1 5 10 15 <210> 24 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 2 <400> 24 Phe Ala Leu Ala Leu Lys Ala Leu Lys Lys Leu 1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 3 <400> 25 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys 1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 4 <400> 26 Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His 1 5 10 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 5 <400> 27 Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg Ser Gln 1 5 10 15 <210> 28 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 6 <400> 28 Lys Trp Leu Phe Lys Lys Ile Gly Ala Val Leu Lys Val Leu 1 5 10 <210> 29 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 7 <400> 29 Leu Val Lys Leu Val Ala Gly Ile Lys Lys Phe Leu Lys Trp Lys 1 5 10 15 <210> 30 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 8 <400> 30 Lys Phe Lys Trp Pro Trp 1 5 <210> 31 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 9 <400> 31 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile 20 <210> 32 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 10 <400> 32 Ser Trp Leu Ser Lys Thr Ala Lys Lys Gly Ala Val Leu Lys Val Leu 1 5 10 15 <210> 33 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 11 <400> 33 Lys Lys Leu Phe Lys Lys Ile Leu Lys Tyr Leu 1 5 10 <210> 34 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 12 <400> 34 Gly Leu Lys Lys Leu Ile Ser Trp Ile Lys Arg Ala Ala Gln Gln Gly 1 5 10 15 &Lt; 110 > Advanced BioTech Co., Ltd. <120> Transgenic silkworms producing recombinant antibacterial adhesive          peptide and the method of producing the recombinant antibacterial          adhesive peptide using the same <130> PA-17-0269 <160> 34 <170> Kopatentin 2.0 <210> 1 <211> 1014 <212> DNA <213> Artificial Sequence <220> <223> MAP13151-AMP <400> 1 atggctagcg ctaaaccgtc ttacccgccg acctacaaag caaaaccctc gtacccaccg 60 acttataagg ctaaacctag ctatccacct acgtacaaag ctaaaccgtc ttacccgccg 120 acttacaaag caaaaccgtc ctaccctccg acctataagg ctaaaccgag ttaccccccg 180 acttacaaag gctgcagggc ggattattat ggtccgaaat atggtccgcc gcgtcgttac 240 ggtggtggca actacaaccg ttatggcgga tcccgtcgtt atggcggtta taaaggctgg 300 aacaacggtt ggaaacgtgg tcgttggggt cgtaaatatt atgaattcgc tagcgctaaa 360 ccgtcttacc cgccgaccta caaagcaaaa ccctcgtacc caccgactta taaggctaaa 420 cctagctatc cacctacgta caaagctaaa ccgtcttacc cgccgactta caaagcaaaa 480 ccgtcctacc ctccgaccta taaggctaaa ccgagttacc ccccgactta caaaggctgc 540 agttctgaag aatacaaggg tggttattac ccaggcaatt cgaaccacta tcattcaggt 600 ggtagttatc acggatccgg ctaccatgga ggatataagg gaaagtatta cggaaaggca 660 aagaaatact attataaata taaaaacagc ggaaaataca agtatctaaa gaaagctaga 720 aaataccata gaaagggtta caagaagtat tatggaggta gcagtgaatt cgaatttgct 780 aaaccgtctt acccgccgac ctacaaagca aaaccctcgt acccaccgac ttataaggct 840 aaacctagct atccacctac gtacaaagct aaaccgtctt acccgccgac ttacaaagca 900 aaaccgtcct accctccgac ctataaggct aaaccgagtt accccccgac ttacaaaaag 960 cttaaaaagc agcgcttccg tcaccgtaac cgcaaaggtt atcgtagcca gtga 1014 <210> 2 <211> 1014 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > MAP13151-AMP-Bm <400> 2 atggctagtg ctaaaccgag ctacccgcct acctacaagg ctaaacctag ctacccgccg 60 acttataaag ccaaaccgtc ctaccccccg acatataaag ctaaaccaag ctacccacct 120 acttacaaag cgaagccgtc ttaccctcca acttacaagg ctaaaccctc ttacccgccc 180 acatacaaag gttgcagggc tgactactac ggcccgaaat acggaccgcc tagaagatac 240 ggtggtggca actacaacag gtacggaggt tcccgcagat acggtggcta caagggatgg 300 aacaacggtt ggaaaagagg caggtggggt cgtaaatact acgaattcgc cagcgccaaa 360 cctagctacc ctcctacata caaagctaag ccgtcctacc ctccgactta caaggccaaa 420 ccttcatacc cgccaacata caaagcaaaa ccgtcatacc ctccgacgta caaagctaag 480 ccatcgtacc ctccgacata caaagccaag ccgtcctacc ctccgaccta caaaggatgc 540 agctctgagg aatacaaagg tggctactac cccggcaact caaaccacta ccactccgga 600 ggttcctacc acggttctgg ttaccacggt ggttacaaag gaaagtacta cggcaaagct 660 aaaaaatact actacaaata caaaaactca ggcaagtaca aatacctgaa aaaagctcgc 720 aaataccatc gcaaaggcta caaaaaatac tacggtggtt cgagcgaatt cgaattcgcc 780 aagccgagct accacacctac atacaaagcg aaaccgtcat acccgccgac atacaaggct 840 aagccaagct accctcctac ttacaaagcc aaaccttcat acccgcctac gtacaaagct 900 aaaccttcct acccgccaac ttataaagca aaaccttctt accccccaac ctacaagaaa 960 ttgaagaaac agcgttttcg acaccgcaac cgaaaaggtt ataggtcaca ataa 1014 <210> 3 <211> 337 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > MAP13151-AMP-Bm <400> 3 Met Ala Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro   1 5 10 15 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr              20 25 30 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr          35 40 45 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly      50 55 60 Cys Arg Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr  65 70 75 80 Gly Gly Gly Asn Tyr Asn Arg Tyr Gly Gly Ser Arg Arg Tyr Gly Gly                  85 90 95 Tyr Lys Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys             100 105 110 Tyr Tyr Glu Phe Ala Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys         115 120 125 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro     130 135 140 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 145 150 155 160 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr                 165 170 175 Tyr Lys Gly Cys Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly             180 185 190 Asn Ser Asn His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr         195 200 205 His Gly Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr     210 215 220 Tyr Lys Tyr Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg 225 230 235 240 Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Glu                 245 250 255 Phe Glu Phe Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro             260 265 270 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr         275 280 285 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr     290 295 300 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Lys 305 310 315 320 Leu Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg Ser                 325 330 335 Gln     <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide peptide of the tandem repeat decapeptide derived from foot          protein 1 (FP-1, Mytilus edulis) <400> 4 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys   1 5 10 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 2 times repeated sequence derived from foot protein 1 (FP-1,          Mytilus edulis) <400> 5 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys              20 <210> 6 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> 6 times repeated sequence derived from foot protein 1 (FP-1,          Mytilus edulis) <400> 6 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys              20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr          35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys      50 55 60 <210> 7 <211> 39 <212> PRT <213> Artificial Sequence <220> Partial sequence of foot protein type 2 (FP-2, Mytilus          californianus) <400> 7 Glu Val His Ala Cys Lys Pro Asn Pro Cys Lys Asn Asn Gly Arg Cys   1 5 10 15 Tyr Pro Asp Gly Lys Thr Gly Tyr Lys Cys Lys Cys Val Gly Gly Tyr              20 25 30 Ser Gly Pro Thr Cys Ala Cys          35 <210> 8 <211> 52 <212> PRT <213> Artificial Sequence <220> Foot protein type 3 (FP-3, Mytilus edulis) <400> 8 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly   1 5 10 15 Gly Asn Tyr Asn Arg Tyr Phe Phe Ser Arg Arg Tyr Gly Gly Tyr Lys              20 25 30 Gly Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr          35 40 45 Glu Phe Glu Phe      50 <210> 9 <211> 46 <212> PRT <213> Artificial Sequence <220> Foot protein type 3 (FP-3, Mytilus galloprovincialis: mgfp-3A) <400> 9 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly   1 5 10 15 Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp              20 25 30 Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr          35 40 45 <210> 10 <211> 50 <212> PRT <213> Artificial Sequence <220> Foot protein type 3 (FP-3, Mytilus edulis: mefp-3F) <400> 10 Ala Asp Tyr Tyr Gly Pro Asn Tyr Gly Pro Pro Arg Arg Tyr Gly Gly   1 5 10 15 Gly Asn Tyr Asn Arg Tyr Asn Gly Tyr Gly Gly Gly Arg Arg Tyr Gly              20 25 30 Gly Tyr Lys Gly Trp Asn Asn Gly Trp Asn Arg Gly Arg Arg Gly Lys          35 40 45 Tyr Trp      50 <210> 11 <211> 44 <212> PRT <213> Artificial Sequence <220> Foot protein type 3 (FP-3, Mytilus californianus) <400> 11 Gly Ala Tyr Lys Gly Pro Asn Tyr Asn Tyr Pro Tyr Arg Tyr Gly Gly   1 5 10 15 Lys Tyr Asn Gly Tyr Lys Gly Tyr Pro Arg Gly Tyr Gly Trp Asn Lys              20 25 30 Gly Trp Asn Lys Gly Arg Trp Gly Arg Lys Tyr Tyr          35 40 <210> 12 <211> 60 <212> PRT <213> Artificial Sequence <220> Partial sequence from foot protein type 4 (Mytilus californianus) <400> 12 Gly His Val His Arg His Arg Val Leu His Lys His Val His Asn His   1 5 10 15 Arg Val Leu His Lys His Leu His Lys His Gln Val Leu His Gly His              20 25 30 Val His Arg His Gln Val Leu His Lys His Val His Asn His Arg Val          35 40 45 Leu His Lys His Leu His Lys His Gln Val Leu His      50 55 60 <210> 13 <211> 75 <212> PRT <213> Artificial Sequence <220> Foot protein type 5 (FP-5, Mytilus edulis) <400> 13 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Tyr Asn Ala Tyr His   1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr              20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys          35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Ala      50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser  65 70 75 <210> 14 <211> 76 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Foot protein 5 (FP-5, Mytilus edulis) <400> 14 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His   1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr              20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys          35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg      50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser  65 70 75 <210> 15 <211> 71 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > Foot protein 5 (FP-5, Mytilus coruscus) <400> 15 Tyr Asp Asp Tyr Ser Asp Gly Tyr Tyr Pro Gly Ser Ala Tyr Asp Tyr   1 5 10 15 Pro Ser Gly Ser His Trp His Gly His Gly Tyr Lys Gly Lys Tyr Tyr              20 25 30 Gly Lys Gly Lys Lys Tyr Tyr Tyr Lys Phe Lys Arg Thr Gly Lys Tyr          35 40 45 Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys      50 55 60 His Tyr Gly Gly Ser Ser Ser  65 70 <210> 16 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> mussel adhesive protein protein type 5 from (Mytilus          galloprovincialis) <400> 16 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His   1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr              20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys          35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Ala      50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser  65 70 75 <210> 17 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> mussel adhesive protein foot protein type 6 <400> 17 Gly Gly Asn Tyr Arg Gly Tyr Cys Ser Asn Lys Gly Cys Arg Ser   1 5 10 15 Gly Tyr Ile Phe Tyr Asp Asn Arg Gly Phe Cys Lys Tyr Gly Ser Ser              20 25 30 Ser Tyr Lys Tyr Asp Cys Gly Asn Tyr Ala Gly Cys Cys Leu Pro Arg          35 40 45 Asn Pro Tyr Gly Arg Val Lys Tyr Tyr Cys Thr Lys Lys Tyr Ser Cys      50 55 60 Pro Asp Phe Tyr Tyr Tyr Asn Asn Lys Gly Tyr Tyr Tyr Tyr Asn  65 70 75 80 Asp Lys Asp Trp Phe Asn Cys Gly Ser Tyr Asn Gly Cys Cys Leu Ala                  85 90 95 Ser Gly Tyr             <210> 18 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-151, MEFP-5 based) <400> 18 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys              20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr          35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu      50 55 60 Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ala Tyr His Tyr His Ser Gly  65 70 75 80 Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr                  85 90 95 Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys             100 105 110 Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys         115 120 125 Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys     130 135 140 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 145 150 155 160 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys                 165 170 175 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr             180 185 190 Tyr Lys         <210> 19 <211> 164 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-151, MGFP-5 based) <400> 19 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys              20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr          35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu Glu      50 55 60 Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser Gly  65 70 75 80 Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr                  85 90 95 Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly Lys             100 105 110 Lys Tyr Tyr Gly Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr         115 120 125 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser     130 135 140 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 145 150 155 160 Pro Thr Tyr Lys                 <210> 20 <211> 192 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-151, MCFP-5 based) <400> 20 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys              20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr          35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Tyr Asp Gly Tyr      50 55 60 Ser Asp Gly Tyr Tyr Pro Gly Ser Ser Ala Tyr Asn Tyr Ser Ser Ser Ser Ser  65 70 75 80 His Gly Tyr His Gly His Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Gly                  85 90 95 Lys Lys Tyr Tyr Tyr Lys Tyr Lys Ala Thr Gly Lys Tyr Lys Tyr Leu             100 105 110 Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly         115 120 125 Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys     130 135 140 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 145 150 155 160 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser                 165 170 175 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys             180 185 190 <210> 21 <211> 177 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-131) <400> 21 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys              20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr          35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly Cys Arg Ala      50 55 60 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly  65 70 75 80 Asn Tyr Asn Arg Tyr Gly Gly Ser Arg Arg Tyr Gly Gly Tyr Lys Gly                  85 90 95 Trp Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly             100 105 110 Phe Glu Phe Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro         115 120 125 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr     130 135 140 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 145 150 155 160 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Lys                 165 170 175 Leu     <210> 22 <211> 179 <212> PRT <213> Artificial Sequence <220> <223> hybrid mussel adhesive protein (FP-251) <400> 22 Met Glu Val His Ala Cys Lys Pro Asn Pro Cys Lys Asn Asn Gly Arg   1 5 10 15 Cys Tyr Pro Asp Gly Lys Thr Gly Tyr Lys Cys Lys Cys Val Gly Gly              20 25 30 Tyr Ser Gly Pro Thr Cys Ala Cys Ser Ser Glu Glu Tyr Lys Gly Gly          35 40 45 Tyr Tyr Pro Gly Asn Ser Asn His Tyr His Ser Gly Gly Ser Tyr His      50 55 60 Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala  65 70 75 80 Lys Lys Tyr Tyr Tyr Lys Tyr Lys Tyr Leu                  85 90 95 Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr Lys Tyr Tyr Gly Gly             100 105 110 Ser Ser Glu Phe Glu Phe Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys         115 120 125 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro     130 135 140 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 145 150 155 160 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr                 165 170 175 Tyr Lys Lys             <210> 23 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 1 <400> 23 Leu Trp Lys Lys Trp Ala Lys Lys Trp Leu Lys Leu Trp Lys Ala   1 5 10 15 <210> 24 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 2 <400> 24 Phe Ala Leu Ala Leu Lys Ala Leu Lys Lys Leu   1 5 10 <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 3 <400> 25 Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys   1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 4 <400> 26 Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His   1 5 10 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 5 <400> 27 Lys Lys Gln Arg Phe Arg His Arg Asn Arg Lys Gly Tyr Arg Ser Gln   1 5 10 15 <210> 28 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 6 <400> 28 Lys Trp Leu Phe Lys Lys Ile Gly Ala Val Leu Lys Val Leu   1 5 10 <210> 29 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 7 <400> 29 Leu Val Lys Leu Val Ala Gly Ile Lys Lys Phe Leu Lys Trp Lys   1 5 10 15 <210> 30 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 8 <400> 30 Lys Phe Lys Trp Pro Trp   1 5 <210> 31 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 9 <400> 31 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu   1 5 10 15 Ile Ser Trp Ile              20 <210> 32 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 10 <400> 32 Ser Trp Leu Ser Lys Thr Ala Lys Lys Gly Ala Val Leu Lys Val Leu   1 5 10 15 <210> 33 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 11 <400> 33 Lys Lys Leu Phe Lys Lys Ile Leu Lys Tyr Leu   1 5 10 <210> 34 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial peptides 12 <400> 34 Gly Leu Lys Lys Leu Ile Ser Trp Ile Lys Arg Ala Gln Gln Gly   1 5 10 15

Claims (6)

서열번호 1 또는 서열번호 2의 염기서열로 이루어진 항균 펩타이드와 홍합 접착단백질이 융합된 항균 접착단백질의 유전자를 포함하는 재조합 발현벡터.A recombinant expression vector comprising a gene of an antibacterial adhesive protein fused with an antimicrobial peptide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and a mussel adhesive protein. 제 1 항에 있어서,
재조합 발현벡터 pXLBacII-DsRED-SER1-MAP13151-AMP-Bm.The method according to claim 1,
The recombinant expression vector pXLBacII-DsRED-SER1-MAP13151-AMP-Bm. 제 1 항 또는 제 2 항에 따른 항균 접착단백질 유전자를 포함하는 재조합 발현벡터로 형질전환시켜 제조된 항균 접착단백질을 발현하는 형질전환 누에. A transformed silkworm expressing an antibacterial adhesive protein produced by transforming a recombinant expression vector comprising the antibacterial adhesive protein gene according to claim 1 or 2. (1) 항균 접착단백질의 유전자를 포함하는 재조합 발현벡터를 제조하는 단계; (2) 상기 단계 (1)에서 제조된 발현벡터를 누에알에 형질전환시켜 형질전환된 누에알을 제조하는 단계; 및 (3) 상기 단계 (2)에서 형질전환된 누에알을 부화시켜 형질전환 누에를 제조하는 단계를 포함하는 항균 접착단백질이 발현되는 형질전환 누에의 제조방법.(1) preparing a recombinant expression vector containing a gene of an antibacterial adhesive protein; (2) transforming the expression vector prepared in the step (1) into silkworm eggs to produce transformed silkworm eggs; And (3) hatching the transformed silkworm in the step (2) to produce a transformed silkworm. 제 3 항에 따른 항균 접착단백질 유전자를 포함하는 재조합 발현벡터로 형질전환시켜 제조된 형질전환 누에를 이용하여 항균 접착단백질을 생산하는 방법.A method for producing an antibacterial adhesive protein using a transgenic silkworm produced by transforming a recombinant expression vector comprising the antibacterial adhesive protein gene according to claim 3. 제 3 항에 따른 항균 접착단백질 유전자를 포함하는 재조합 발현벡터로 형질전환시켜 제조된 형질전환 누에로부터 분리된 항균 접착단백질을 유효성분으로 포함하는 항균용 조성물.An antimicrobial composition comprising an antibacterial adhesive protein isolated from a transformed silkworm prepared by transforming a recombinant expression vector comprising the antibacterial adhesive protein gene according to claim 3 as an active ingredient.
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