KR102114194B1 - Transgenic silkworms producing silk expressed KillerRed protein - Google Patents
Transgenic silkworms producing silk expressed KillerRed protein Download PDFInfo
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- KR102114194B1 KR102114194B1 KR1020180159942A KR20180159942A KR102114194B1 KR 102114194 B1 KR102114194 B1 KR 102114194B1 KR 1020180159942 A KR1020180159942 A KR 1020180159942A KR 20180159942 A KR20180159942 A KR 20180159942A KR 102114194 B1 KR102114194 B1 KR 102114194B1
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Abstract
Description
본 발명은 킬러레드(KillerRed) 단백질을 발현하는 실크를 생산하는 형질전환 누에, 및 상기 형질전환 누에를 이용하여 킬러레드 단백질을 발현하는 실크를 생산하는 방법에 관한 것이다.The present invention relates to a transformed silkworm producing silk expressing a KillerRed protein, and a method for producing silk expressing a Killerred protein using the transformed silkworm.
누에는 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. 산란된 알에는 방치하면 착색하여 다음해 봄까지 부화하지 않는 것과 착색하지 않고 부화하는 것이 있다. 1년에 몇 회 부화하는가는 각각의 유전자 작용에 의하여 식도 하 신경절 내에서 생산되는 휴면 호르몬의 분비량에 의해 결정된다. 누에알에서 수정 핵은 여러 번 분열하여 분열 핵을 형성하고 핵 주위에 원형질이 둘러싸게 되고 이들은 알의 가장자리를 향하여 이동하게 된다. 예를 들면 개미누에는 산란 후 12시간이 지나면 세포융합(Syncytial blastoderm)이 형성되고 20시간 후면 인공부화법의 하나인 침산 처리가 가능해진다. 산란 후 30시간이면, 완전한 난황세포가 되고 약 10일 후 개미누에로 부화된다.The silkworm belongs to the taxonomy of Insectada, lepidoptera, Bombyxidae, Gambyx and Mori. The silkworm is a fully metamorphosed insect, and the larvae hatched from the eggs develop and become pupae. They become adults (moths), laying eggs and ending their lives. The lifespan of the silkworm is relatively short, on average about 60 days, and has advantages as an experimental animal. The spawned eggs are colored when left untreated, and hatched without coloring until the following spring. How many times a year hatch is determined by the amount of dormant hormone produced in the oesophageal ganglion by each gene action. In the silkworm, the fertile nucleus divides several times to form a fission nucleus, and the protoplasm surrounds the nucleus, which moves toward the edge of the egg. For example, after 12 hours of spawning in the ants, syncytial blastoderm is formed, and after 20 hours, one of the artificial incubation methods, pickling, becomes possible. After 30 hours of spawning, they become fully yolk cells and hatch into ant silkworms after about 10 days.
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. 최근에는 다양한 바이오의약품을 생산하는 형질전환누에가 보고되고 있다. 한편, 누에알에 전이벡터를 주입하는 것은 2000년 Tamura 등이 사용한 미세주입법으로서 미세주입의 위치는 배아의 주공과 후부 사이의 가운데 배면 부분에 주사함으로써 형질전환 효율을 향상시킬 수 있음이 보고된 바 있다. 또한, 형질전환에 사용된 누에 품종인 금옥잠은 잠125와 잠140의 교잡종으로서 일반 원종에 비해 누에알이 크고, 인공사료에 의한 연중 사육이 가능하며, 월년 종의 특징인 염산처리에 의해 부화된다. 그러므로 선발된 누에형질전환체를 장기간 보관할 수 있는 장점을 가지고 있다. Various studies have been conducted to produce silkworms transformed due to their industrial value as well as for research. The development of silkworm transformation technology is the first production of transformed silkworms by constructing a transformation vector for transformation using the piggyBac gene derived from Trichopusia ni , a lepidopteran insect, by Tamura of Japan, etc. To succeed. Recently, transgenic silkworms that produce various biopharmaceuticals have been reported. On the other hand, injecting a transfer vector into silkworm eggs is a micro-injection method used by Tamura in 2000. have. In addition, the silkworm varieties used for transformation, Geumokjam, is a hybrid of Jam 125 and Jam 140, which has a larger silkworm egg than normal species, and can be kept year-round by artificial feed, and hatched by hydrochloric acid treatment, which is a characteristic of monthly species. do. Therefore, it has the advantage of storing the selected silkworm transformant for a long time.
누에는 헤비사슬 피브로인(heavy-chain fibroin), 라이트사슬 피브로인(light-chain fibroin), 및 P25 단백질로 이루어진 견 단백질을 생산한다. 견 단백질 발현은, 특히 피브로인은 5령기 유충의 후부견사선에서 아주 강력한 발현을 하여 전체 체중의 약 20% 이상이나 되는 피프로인 단백질을 생산한다. 이러한 피브로인의 강력하고 조직 특이적인 발현 조절 기작을 해명하는 것은 유전자 발현조절 연구에 중요한 실마리를 제공할 수 있을 것으로 기대되며, 이 조절 기작은 진핵생물에서 유용물질의 대량생산에 이용될 수 있다.The silkworm produces a canine protein consisting of heavy-chain fibroin, light-chain fibroin, and P25 protein. Expression of canine protein, especially fibroin, produces very strong expression in the posterior line of the 5th-stage larva, producing over 20% of the total weight of fiproin protein. Exploring the powerful and tissue-specific expression control mechanism of fibroin is expected to provide an important clue to the study of gene expression control, and this control mechanism can be used for mass production of useful substances in eukaryotic organisms.
피브로인 유전자의 연구는 분자량이 큰 아미노산 배열의 특수성을 이용하여 5령 말기의 후부 견사선으로부터 Suzuki와 Brown에 의하여 처음으로 1972년에 mRNA가 분리되었으며, 이어서 1976년에는 누에의 게놈(genomic) DNA로부터 피브로인 유전자를 클로닝하였다. 그리고 이후의 연구로 가잠에서 H 사슬과 L 사슬이, 야잠에서 H 사슬이 클로닝되어 그 구조와 조절의 연구가 활발하게 진행 중에 있다.In the study of the fibroin gene, mRNA was isolated in 1972 for the first time by Suzuki and Brown from the posterior glial line at the end of the 5th year using the specificity of the amino acid sequence having a large molecular weight, followed by fibroin from the silkworm genomic DNA in 1976. The gene was cloned. In addition, H chain and L chain are cloned in Gazam and H chain is cloned in Jajam, and studies of its structure and regulation are actively underway.
가잠의 H사슬은 16 내지 17 kb의 길이로 2개의 엑손(exon)으로 이루어져 있으며, L 사슬의 경우 13472 bp 길이로 7개의 엑손(exon)으로 이루어져 있다. 피브로인의 H 사슬 유전자의 전사는 배 발생의 25 stage에서 활성화된 후 유충 발생시기에 후부견사선에서 스위치 온과 오프가 반복적으로 일어난다. Nuclear run-on assay를 통한 전사 조절 연구에 의하여 5령기 유충의 후부견사선의 전반부에서 국한되어 전사가 일어남을 밝혀졌다. 그리고 이때 L 사슬이 H 사슬과 같이 전사됨이 밝혀졌다. 피브로인 유전자는 세포 분화 과정에 유전자의 증폭이나 메틸레이션(methylation)과 같은 구조적 변화가 일어나지 않는 것이 확인되었는데, 이것은 피브로인 유전자의 발현이 대부분 전사에 의해 조절된다는 것을 의미한다.Gajam's H chain is 16 to 17 kb long and consists of 2 exons, and for the L chain, it is composed of 7 exons at 13472 bp in length. The transcription of the H chain gene of fibroin is activated at 25 stages of embryo development, and the switch on and off occurs repeatedly in the posterior ray at the time of larval development. A study of transcription regulation through the Nuclear run-on assay revealed that transcription was limited to the first half of the 5th-year-old larva's posterior glial. And at this time, it was found that the L chain is transcribed like the H chain. It was confirmed that the fibroin gene does not undergo structural changes such as amplification or methylation of the gene during cell differentiation, which means that the expression of the fibroin gene is mostly regulated by transcription.
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 a foreign gene has been inserted into the host's genome and a part of its trait has changed. The foreign gene at that time is called a transgene. In the mid 1970s, genetic recombination viruses were introduced into somatic and germ cells to introduce foreign genes, and in 1980, superinjection was produced by Gordon as a microinjection method.
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 foreign genes include calcium phosphate method, electroporation method, DEAE-dextran method, liposome method, micro injection method, and bombardment method. Among the above methods, the DEAE-dextran method and the electroporation method allow a cell to enter the cytoplasm directly through an open hole of the DNA. In these two methods, DNA may be damaged. The method of using liposomes is widely used as a method of inserting DNA into liposomes, which are artificial lipid vesicles, and fusion with a cell membrane to transport them directly into cells. The micro-injection method is a method of directly injecting DNA with a micro-injection needle that does not damage an egg using a micro-manipulator in a 1-cell fertilized egg. The technology for introducing foreign genes in the commercialization stage could provide unlimited benefits to humans if the introduced foreign genes are related to growth rate regulation, resistance in extreme environments, and gene therapy.
우리나라 잠사업은 70년대에 연간 5억불을 수출한 최고의 농산물이었으나(세계 5위의 잠업 국가), 국내 잠사업은 급속한 경제성장에 따른 임금상승과 노동력 부족, 농약 사용의 증가에 다른 양잠피해의 속출, 중국산 저가 고치의 수입 증가에 따라 급격히 감소하여 우리나라의 누에고치 생산은 거의 전무한 실정이다. 따라서, 국내 양잠산업의 규모 확대와 양잠농가의 획기적인 소득증대를 위하여 고부가가치의 형질전환누에 개발이 요구되고 있는 실정이다.Korea's latent business was the best agricultural product that exported $ 500 million annually in the 70s (the world's fifth-largest country), but the domestic latent business continued to increase wages due to rapid economic growth, labor shortages, and increased use of pesticides. In the meantime, the production of silkworm cocoon in Korea is almost non-existent due to the sharp decrease in imports of Chinese low-priced cocoon. Therefore, in order to increase the scale of the domestic sericulture industry and to dramatically increase the income of the sericulture farmers, the development of highly value-added transformed silkworms is required.
이에 본 발명자들은 형질전환 누에에 대한 각종 연구를 진행하던 중, 항균성이 있는 단백질인 킬러레드(KillerRed) 단백질을 발현할 수 있는 누에를 이용하여 항균 기능이 있는 기능성 원사의 제조 또는 항균 단백질의 용이한 생산이 가능함을 확인하여 본 발명을 완성하였다. Accordingly, the present inventors are carrying out various studies on transgenic silkworms, using silkworms capable of expressing an antibacterial protein, Killer Red protein, to facilitate the production of functional yarns with antimicrobial functions or the ease of antibacterial proteins. The present invention was completed by confirming that production is possible.
본 발명의 목적은, 고부가가치를 갖는 형질전환누에 개발을 위해, 누에 실크의 주성분인 피브로인(Fibroin) 단백질의 프로모터(pFibH)로 킬러레드 유전자가 발현 조절이 되도록 구성된 재조합 발현벡터를 제작하고 상기 재조합 발현벡터를 이용하여 누에를 형질전환함으로써 킬러레드 단백질을 발현하는 실크를 생산하는 형질전환 누에를 제공하는 데에 있다. An object of the present invention, for the development of a transformed silkworm having a high added value, to produce a recombinant expression vector composed of the killer red gene expression control with the promoter (pFibH) of the fibroin protein, which is the main component of silkworm silk, and to produce the recombinant silkworm It is to provide a transformed silkworm producing silk expressing the killer red protein by transforming the silkworm using an expression vector.
본 발명의 다른 목적은 상기 형질전환 누에를 이용하여 킬러레드 단백질을 발현하는 실크 또는 상기 킬러레드 단백질을 대량으로 생산하는 방법을 제공하는 데에 있다. Another object of the present invention is to provide a method for producing a mass of silk or the killer red protein expressing the killer red protein using the transformed silkworm.
본 발명은 표지 유전자 조절 프로모터, 표지 유전자, 누에 유래의 피브로인(Fibroin) 프로모터 및 킬러레드(KillerRed) 단백질 발현 유전자가 작동가능하게 연결된 유전자 컨스트럭트를 포함하는 재조합 발현벡터를 제공한다.The present invention provides a recombinant expression vector comprising a gene construct operably linked to a marker gene regulatory promoter, a marker gene, a silkworm-derived Fibroin promoter, and a KillerRed protein expression gene.
상기 킬러레드 단백질 발현 유전자는 서열번호 3의 염기서열로 이루어진 것을 특징으로 한다.The killer red protein expression gene is characterized by consisting of the nucleotide sequence of SEQ ID NO: 3.
상기 표지 유전자 조절 프로모터는 3xP3 프로모터인 것을 특징으로 한다.The marker gene regulatory promoter is characterized by being a 3xP3 promoter.
상기 표지 유전자는 EGFP(green fluorescent protein) 유전자인 것을 특징으로 한다.The marker gene is characterized by being a green fluorescent protein (EGFP) gene.
상기 유전자 컨스트럭트는 도 1의 구조로 이루어지는 것을 특징으로 한다.The gene construct is characterized by comprising the structure of FIG. 1.
상기 발현벡터는 piggyBac 벡터인 것을 특징으로 한다.The expression vector is characterized by being a piggyBac vector.
본 발명은 상기 본 발명에 따른 재조합 발현벡터를 누에(Bombyx mori) 알에 형질전환시켜 제조한 것을 특징으로 하는 킬러레드 단백질을 발현하는 실크를 생산하는 형질전환 누에를 제공한다.The present invention provides a transformed silkworm producing silk expressing a killer red protein, characterized in that the recombinant expression vector according to the present invention is transformed into silkworm ( Bombyx mori ) eggs.
또한, 본 발명은, (제1단계) 본 발명에 따른 재조합 발현벡터를 제조하는 단계;In addition, the present invention, (first step) preparing a recombinant expression vector according to the present invention;
(제2단계) 상기 제1단계의 재조합 발현벡터를 누에(Bombyx mori) 알에 형질전환시켜 형질전환된 누에알을 제조하는 단계; 및(Second step) transforming the recombinant expression vector of the first step into silkworm ( Bombyx mori ) eggs to prepare transformed silkworm eggs; And
(제3단계) 상기 제2단계의 형질전환된 누에알을 부화시켜 형질전환 누에를 사육하는 단계;를 포함하는 것을 특징으로 하는 킬러레드 단백질을 발현하는 실크를 생산하는 형질전환 누에의 제조방법을 제공한다.(Third step) hatching the transformed silkworm eggs of the second step to breed the transformed silkworm; A method for producing a transformed silkworm producing silk expressing a killer red protein comprising the steps of: to provide.
상기 제2단계의 형질전환은 미세주입법(microinjection)을 이용하는 것을 특징으로 한다.The second step of transformation is characterized by using a microinjection method.
상기 제3단계에서, a) 발현벡터에 표지 유전자를 도입한 후, 형질전환 누에에서 상기 표지 유전자의 발현을 확인하거나, b) 형질전환 누에에서 킬러레드 단백질 유전자의 발현을 확인하여, 형질전환 누에를 선발하는 단계를 추가적으로 포함하는 것을 특징으로 한다.In the third step, a) after introducing the marker gene into the expression vector, the expression of the marker gene in the transformed silkworm, or b) the expression of the killer red protein gene in the transformed silkworm, transformed silkworm It characterized in that it further comprises the step of selecting.
아울러, 본 발명은 상기 제조방법을 통해 얻은 형질전환된 누에를 사육하여 킬러레드 단백질이 발현된 실크를 획득하는 방법을 제공하며, 이렇게 획득한 실크를단백질 정제하여 항균능이 있는 킬러레드 단백질을 수득하는 방법을 제공한다. In addition, the present invention provides a method of acquiring silk expressing a killer red protein by breeding the transformed silkworm obtained through the above manufacturing method, and purifying the silk thus obtained to obtain a killer red protein having antibacterial activity. Provide a method.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 표지 유전자 조절 프로모터, 표지 유전자, 누에 유래의 피브로인(Fibroin) 프로모터 및 킬러레드(KillerRed) 단백질 발현 유전자가 작동가능하게 연결된 유전자 컨스트럭트를 포함하는 재조합 발현벡터를 제공한다.The present invention provides a recombinant expression vector comprising a gene construct operably linked to a marker gene regulatory promoter, a marker gene, a silkworm-derived Fibroin promoter, and a KillerRed protein expression gene.
상기 재조합 발현벡터에 있어서, 상기 킬러레드(KillerRed) 단백질 발현 유전자는 서열번호 3의 염기서열로 이루어진 것이 바람직하나 이에 한정되지 않으며, 상기 염기서열에서 하나 또는 둘 이상의 염기가 삽입, 결실 또는 치환된 서열로 구성된 것도 포함할 수 있다.In the recombinant expression vector, the killer red (KillerRed) protein expression gene is preferably composed of the nucleotide sequence of SEQ ID NO: 3, but is not limited thereto, one or two or more bases inserted, deleted or substituted in the nucleotide sequence It may also include.
상기 재조합 발현벡터에 있어서, 상기 피브로인 프로모터는 누에 유래 피브로인 H 프로모터(pFibH)를 사용하는 것이 바람직하다.In the recombinant expression vector, the fibroin promoter is preferably a silkworm-derived fibroin H promoter (pFibH).
상기 재조합 발현벡터에 있어서, 상기 표지 유전자 조절 프로모터는 3xP3 프로모터인 것이 바람직하나 이에 한정되지 않으며, 표지 유전자를 발현시킬 수 있는 프로모터는 모두 사용가능하다.In the recombinant expression vector, the labeled gene regulatory promoter is preferably a 3xP3 promoter, but is not limited thereto, and any promoter capable of expressing the labeled gene can be used.
상기 재조합 발현벡터에 있어서, 상기 표지 유전자는 형광단백질을 발현하는 유전자는 모두 사용가능하며, EGFP(green fluorescent protein) 유전자를 사용하는 것이 더욱 바람직하나 이에 한정되지 않는다.In the recombinant expression vector, all of the genes expressing the fluorescent protein can be used as the marker gene, and it is more preferable to use the green fluorescent protein (EGFP) gene, but is not limited thereto.
본 발명의 한가지 실시예에서는 형질전환체 선발을 위해서, 형광단백질(EGFP)을 표지유전자로 사용하였고, 표지유전자의 발현조절은 눈과 신경시스템에서 특이적으로 발현하는 3xP3 프로모터를 사용하였다. In one embodiment of the present invention, for the selection of transformants, a fluorescent protein (EGFP) was used as a marker gene, and expression control of the marker gene was performed using a 3xP3 promoter specifically expressed in the eye and nervous system.
상기 재조합 발현벡터에 있어서, 상기 유전자 컨스트럭트는 도 1의 구조로 구성된 컨스트럭트를 갖는 것이 바람직하나 이에 한정되지 않는다.In the recombinant expression vector, the gene construct preferably has a construct composed of the structure of FIG. 1, but is not limited thereto.
상기 재조합 발현벡터에 있어서, 상기 유전자 컨스트럭트가 도입되는 발현벡터는 piggyBac 벡터인 것이 바람직하나 이에 한정되지 않는다.In the recombinant expression vector, the expression vector into which the gene construct is introduced is preferably a piggyBac vector, but is not limited thereto.
또한, 본 발명은 본 발명에 따른 상기 재조합 발현벡터를 누에(Bombyx mori) 알에 형질전환시켜 제조한 킬러레드 단백질을 발현하는 실크를 생산하는 형질전환 누에를 제공한다.In addition, the present invention provides a transformed silkworm producing silk expressing a killer red protein prepared by transforming the recombinant expression vector according to the present invention into silkworm ( Bombyx mori ) eggs.
피브로인 H-chain의 긴 반복서열은 소수성 상호작용에 의해 결정구조를 형성하는데, 이러한 구조를 형성하기 위해서는 2가지 요소가 꼭 필요하다. 첫 번째는 H-chain의 N 말단과 C 말단에 실크 속으로 피브로인 분자를 분비시키기 위한 시그날 서열이 있어야 하고, 두 번째는 H-chain C 말단의 20번째 시스테인 잔기와 L-chain N 말단의 172번 시스테인 잔기가 이황화결합을 형성하고 있어야 한다. 따라서 본 발명에서는 이러한 시스템을 기반으로 피브로인 H-chain에서 재조합 단백질인 킬러레드(KillerRed) 단백질 유전자를 발현시키는 누에 형질전환체를 제작할 수 있다. The long repeating sequence of the fibroin H-chain forms a crystal structure by hydrophobic interaction, and two elements are necessary to form such a structure. The first must have a signal sequence for secreting fibroin molecules into the silk at the N- and C-terminals of the H-chain, and the second is the 20th cysteine residue of the H-chain C-terminal and 172 of the L-chain N-terminal The cysteine residue must form a disulfide bond. Therefore, in the present invention, a silkworm transformant expressing the recombinant protein KillerRed protein gene in the fibroin H-chain can be produced based on this system.
또한, 본 발명은In addition, the present invention
(제1단계) 상기 본 발명의 재조합 발현벡터를 제조하는 단계;(First step) preparing a recombinant expression vector of the present invention;
(제2단계) 상기 제1단계의 재조합 발현벡터를 누에(Bombyx mori) 알에 형질전환시켜 형질전환된 누에알을 제조하는 단계; 및(Second step) transforming the recombinant expression vector of the first step into silkworm ( Bombyx mori ) eggs to prepare transformed silkworm eggs; And
(제3단계) 상기 제2단계의 형질전환된 누에알을 부화시켜 형질전환 누에를 사육하는 단계;를 포함하는 것을 특징으로 하는 킬러레드 단백질을 발현하는 실크를 생산하는 형질전환 누에의 제조방법을 제공한다.(Third step) hatching the transformed silkworm eggs of the second step to breed the transformed silkworm; A method for producing a transformed silkworm producing silk expressing a killer red protein comprising the steps of: to provide.
상기 방법에 있어서, 상기 제2단계의 형질전환은 미세주입법(microinjection)을 이용하는 것이 바람직하나 이에 한정되지 않으며, 공지된 형질전환법은 모두 사용가능하다.In the above method, the second step of transformation is preferably a microinjection method (microinjection), but is not limited thereto, and any known transformation method can be used.
상기 방법에 있어서, 상기 제3단계에 있어서, 하기 중 하나의 방법으로 형질전환 누에를 선발하는 단계를 추가적으로 포함할 수 있는데, 이 단계는 a) 발현벡터에 표지 유전자를 도입한 후, 형질전환 누에에서 상기 표지 유전자의 발현을 확인하거나, b) 형질전환 누에에서 킬러레드 단백질 유전자의 발현을 확인하여, 형질전환 누에를 선발하는 단계를 추가적으로 포함할 수 있다. In the above method, in the third step, the method may further include the step of selecting a transformed silkworm by one of the following methods, which step a) after introducing the marker gene into the expression vector, the transformed silkworm. It may further include the step of selecting the transformed silkworm by confirming the expression of the marker gene, or b) confirming the expression of the killer red protein gene in the transformed silkworm.
본 발명의 한가지 실시예에서는 공지된 미세주입법을 이용하여 상기 재조합 발현벡터로 누에알을 형질전환시킨 후, 그 중 일부를 유충으로 부화시켰으며, 그 중 일부 성충이 된 나방들을 서로 교배시켜 F1 세대의 누에알을 획득하였다. 그런 다음, 이들 F1 세대의 누에알의 산란 후 초기배, 유충, 번데기 또는 성충에서 각각 눈 또는 신경조직에서의 표지 유전자의 발현을 관찰함으로써 형질전환체를 선발하였다. 그런 다음, 최종적으로 이들만을 교배시켜 F2세대의 형질전환체를 획득하였다.In one embodiment of the present invention, after transforming silkworm eggs with the recombinant expression vector using a known microinjection method, some of them were hatched as larvae, and among them, some of the adult moths were crossed with each other to generate F1 generation. Obtained silkworm eggs. Then, after spawning of these F1 generation silkworm eggs, transformants were selected by observing the expression of a marker gene in the eye or nerve tissue, respectively, in the early embryo, larva, pupa or adult. Then, finally, only these were crossed to obtain F2 generation transformants.
본 발명의 한가지 실시예에서는 상기 획득한 F2 세대의 형질전환체를 해부하여 후부 견사선을 형광현미경으로 관찰함으로써 킬러레드 단백질을 발현하는 실크의 생산을 확인한 결과, 피브로인을 생성하는 기관인 후부 견사선에서 킬러레드 단백질 고유의 적색 형광의 발현을 관찰할 수 있었다.In one embodiment of the present invention, as a result of dissecting the obtained F2 generation transformant and observing the posterior silk gland with a fluorescence microscope, the production of silk expressing the killer red protein was confirmed, and killer red at the posterior gland that is an organ that produces fibroin The expression of protein-specific red fluorescence could be observed.
아울러, 본 발명은 형질전환된 누에를 사육하여 킬러레드 단백질이 발현된 실크를 획득하는 방법과, 상기 방법으로 획득한 실크를 단백질 정제하여 항균능이 있는 킬러레드 단백질을 수득하는 방법을 제공할 수 있다. In addition, the present invention can provide a method of obtaining a silk expressing a killer red protein by breeding a transformed silkworm, and a method of obtaining a killer red protein having antibacterial activity by protein purification of the silk obtained by the above method. .
상기 단백질 정제 방법은 본 발명이 속한 분야에서 통상의 방법으로 실시될 수 있으며, 대표적으로는 친화성 크로마토그래피 같은 컬럼 정제법을 사용할 수 있다. The protein purification method may be carried out by a conventional method in the field to which the present invention belongs, and representatively, a column purification method such as affinity chromatography may be used.
본 발명은 킬러레드 단백질을 발현하는 형질전환 누에에 관한 기술로서, 본 발명의 형질전환 누에가 생산하는 실크는 특정 파장의 빛을 쏘이면 어둠 속에서 영롱한 적색 형광을 나타내어 일반 실크에 비해 훨씬 고부가가치를 갖는 고급 패션의류나 벽지 등의 소재에 적용이 가능하다. 특히, 양잠농가는 현재 사육하고 있는 일반 누에와는 차별화된 고품질의 패션의류 등의 소재로 활용할 수 있는 실크를 생산하는 형질전환누에를 사육함으로써 소득향상에 크게 기여할 수 있다. 또한 기존의 형광 색상 발현 실크를 생산하는 누에와 달리 본 발명에서 킬러레드를 발현하는 누에는, 킬러레드 단백질이 항균성이 있어 실크 자체에 항균성을 부가할 수 있어 기능성 원사로서 이용가능하며, 이러한 킬러레드 단백질의 추출을 통해 항균물질의 대량생산이 가능하다는 잇점이 있다. The present invention is a technology related to a transformed silkworm expressing a killer red protein, and the silk produced by the transformed silkworm of the present invention exhibits a bright red fluorescence in the dark when a specific wavelength of light is emitted, thereby providing much higher value than ordinary silk. It can be applied to materials such as high-end fashion clothes and wallpaper. In particular, sericulture farmers can contribute significantly to income growth by breeding transgenic silkworms that produce silk that can be used as a material for high-quality fashion clothing, which is different from ordinary silkworms. In addition, unlike the silkworm producing the existing fluorescent color expressing silk, the silkworm expressing the killer red in the present invention has a killer red protein, so it can add antibacterial properties to the silk itself, and thus can be used as a functional yarn. It has the advantage that mass production of antibacterial substances is possible through the extraction of proteins.
도 1은 본 발명에서 형질전환 누에 제조를 위해 삽입하는 전이벡터(pT121-KillerRed)의 구조를 나타내는 그림이다.
도 2는 F2 세대의 형질전환 누에(F2)가 EGFP 형광 단백질을 발현하는 것을 나타내는 사진으로서, 각각 A는 알, B는 유충, C는 번데기, D는 성충의 사진이며, 알에서는 눈 및 신경계, 유충/번데기/성충에서는 성충에서 각각 형광(화살표)이 나타나는 것이 확인된다.
도 3은 야생형 누에(Wild type)와 킬러레드 유전자가 삽입된 형질전환 누에(T123K)에서의 킬러레드 유전자 발현을 확인한 PCR 결과로서 A의 결과를 B로 수치화하여 나타낸 것이다.
도 4는 F3 세대의 5령 5일 누에를 해부하여 적출한 견사선 사진으로서, A는 명시야(bright field) 조건에서 관찰되는 견사선의 사진이며, B는 적색 형광 시스템 조건에서 관찰되는 견사선 사진이며, 각 사진에서 왼쪽은 야생형 타입 누에, 오른쪽은 형질전환 타입의 누에로부터 적출한 견사선이다.
도 5는 형질전환 누에의 누에고치가 적생형광을 발현하는 것을 나타내는 사진으로서, A는 명시야(bright field) 조건에서 관찰되는 누에고치 사진이며, B는 적색 형광 시스템 조건에서 관찰되는 누에고치 사진이다. 1 is a diagram showing the structure of a transfer vector (pT121-KillerRed) inserted for the production of transformed silkworm in the present invention.
2 is a photograph showing that the transformed silkworm (F2) of the F2 generation expresses the EGFP fluorescent protein, wherein A is an egg, B is a larva, C is a pupa, D is an adult, and the egg is an eye and nervous system, In the larvae / pupae / adult, it is confirmed that fluorescence (arrow) appears in the adult.
3 is a PCR result confirming the expression of a killer red gene in a wild-type silkworm (Wild type) and a transformed silkworm (T123K) into which the killer red gene has been inserted, and the result of A is quantified by B.
FIG. 4 is a photograph of a silkworm extracted by dissecting silkworms on the 5th and 5th days of the F3 generation, A is a photograph of a silkworm observed under bright field conditions, and B is a photograph of silkworm observed under red fluorescent system conditions, In each photo, the left side is the silkworm extracted from the wild type silkworm and the right side is extracted from the transgenic silkworm.
5 is a photograph showing that the cocoon of the transformed silkworm expresses red fluorescence, A is a cocoon photograph observed under bright field conditions, and B is a cocoon photograph observed under red fluorescent system conditions. .
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해지도록, 당업자에게 본 발명의 사상을 충분히 전달하기 위해 제공하는 것이다. Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.
<실시예 1. 실험곤충의 준비> <Example 1. Preparation of experimental insects>
형질전환에 이용된 누에(Bombyx mori)는 원종인 잠123을 사용하였고, 농촌진흥청 표준사육기준(온도, 24~27℃; 상대습도, 70~90%)에 준하여 사육하였다. The silkworm ( Bombyx mori ) used for transformation was used as a native species, Jam123, and was bred in accordance with the Rural Development Administration standard breeding standard (temperature, 24 ~ 27 ℃; relative humidity, 70 ~ 90%).
<실시예 2. 형질전환용 전이벡터의 제작><Example 2. Preparation of transformation vector for transformation>
형질전환에 사용된 전이벡터는 piggyBac 벡터와 helper plasmid 벡터인 pH3PIG를 사용하였다. The transformation vector used for transformation was a piggyBac vector and a helper plasmid vector, pH3PIG.
KillerRed 적색 형광단백질이 발현되는 형진전환누에를 만들기 위하여 피브로인 프로모터 및 유전자 일부와 KillerRed 유전자를 piggyBac 벡터에 클로닝하였다.In order to make a transformed silkworm in which the KillerRed red fluorescent protein is expressed, a part of the fibroin promoter and gene and the KillerRed gene were cloned into a piggyBac vector.
실시예 2-1. pGEMT-pFibH-NTD 제작Example 2-1. pGEMT-pFibH-NTD production
피브로인 프로모터 유전자 서열을 얻기 위하여, N-말단에 피브로인 H 유전자 (nt 61,312 to 63,870 of AF226688)의 인트론 (972 bp)이 포함된 단편은 누에에서 분리된 genomic DNA와 프라이머들 (pFibHN-F: 5′-GGC GCG CCG TGC GTG ATC AGG AAA AAT-3′(27mer))과 (pFibHN-R: 5′-TGC ACC GAC TGC AGC ACT AGT GCT GAA-3′(27mer))을 사용하여 PCR로 서열번호 1의 서열을 증폭하여 분리 후 상기 서열을 pGEM-T Easy Vector)에 클로닝하여 pGEMT-pFibH-NTD를 제작하였다.To obtain the fibroin promoter gene sequence, the fragment containing the intron (972 bp) of the fibroin H gene (nt 61,312 to 63,870 of AF226688) at the N-terminus was genomic DNA and primers separated from silkworms (pFibHN-F: 5 ' SEQ ID NO: 1 by PCR using -GGC GCG CCG TGC GTG ATC AGG AAA AAT-3 '(27mer)) and (pFibHN-R: 5'-TGC ACC GAC TGC AGC ACT AGT GCT GAA-3' (27mer)) After amplifying and separating the sequence of, the sequence was cloned into pGEM-T Easy Vector) to produce pGEMT-pFibH-NTD.
서열번호 1 : SEQ ID NO: 1
실시예 2-2. pGEMT-CTD 제작Example 2-2. pGEMT-CTD production
피브로인 H 유전자의 일부 서열을 얻기 위하여, ORF의 exon 1의 180 bp 3' 말단과 exon 2의 300 bp 3'영역이 포함된 단편을 누에의 genomic DNA와 프라이머들 (pFibHC-F: 5′-AGC GTC AGT TAC GGA GCT GGC AGG GGA-3′(27mer) 과 pFibHC-F: 5′-TAT AGT ATT CTT AGT TGA GAA GGC ATA-3′ (27mer))를 이용하여 서열번호 2의 서열을 증폭하고 pGEM-T Easy Vector에 클로닝하여 pGEMT-CTD를 제작하였다.In order to obtain some sequences of the fibroin H gene, the fragment containing the 180 bp 3 'end of
서열번호 2 : SEQ ID NO: 2:
실시예 2-2. pFibHNC-null 제작Example 2-2. Made by pFibHNC-null
상기 제작한 pGEMT-pFibH-NTD는 Asc I과 BamH I으로, pGEMT-CTD는 Sal I과 Fse I으로 각각 제한효소 처하여 단편을 준비하여 제한효소가 처리된 pBluescriptII SK(-) vector)에 함께 클로닝하여 pFibHNC-null을 제작하였다.The prepared pGEMT-pFibH-NTD is Asc I and BamH I, pGEMT-CTD is Sal I and Fse I, respectively, and a fragment is prepared by restriction enzymes, cloned together into the restriction enzyme-treated pBluescriptII SK (-) vector) pFibHNC-null was produced.
실시예 2-3. pFibHNC-KillerRed 제작Example 2-3. Made by pFibHNC-KillerRed
KillerRed(GeneBank No. ADM52297) 유전자는 Evrogen으로부터 구입한 pKillerRed-B 벡터로부터, PCR 프라이머 KillerRed-F-Not I : 5'-GCG GCC GCA CCA TGG GTT CAG AGG GCG GCC-3'(30 mer) 과 KillerRed-R-Bbvc I : 5'-GCT GAG GAT CCT CGT CGC TAC CGA TGG CGC-3'(30 mer)를 이용하여 서열번호 3의 서열을 증폭하고 Not I과 Bbvc I 제한효소를 처리 후 분리하여 동일한 제한효소가 처리된 pFibHNC-null에 클로닝하여 pFibHNC-KillerRed를 제작하였다. KillerRed (GeneBank No.ADM52297) gene is from pKillerRed-B vector purchased from Evrogen, PCR primer KillerRed-F-Not I: 5'-GCG GCC GCA CCA TGG GTT CAG AGG GCG GCC-3 '(30 mer) and KillerRed -R-Bbvc I: 5'-GCT GAG GAT CCT CGT CGC TAC CGA TGG CGC-3 '(30 mer) was used to amplify the sequence of SEQ ID NO: 3, process the Not I and Bbvc I restriction enzymes and separate them The pFibHNC-KillerRed was produced by cloning the restriction enzyme-treated pFibHNC-null.
서열번호 3 : SEQ ID NO: 3:
실시예 2-3. pT121-KillerRed 제작Example 2-3. Made by pT121-KillerRed
제작된 pFibHNC-KillerRed에 Asc I과 Fse I 제한효소 처리 후 KillerRed 유전자가 포함된 절편을 분리하여 연구실에서 보유중인 pG-3xP3-EGFP에 클로닝하여 pT121-KillerRed로 명명한 도 1의 구조로 이루어진 형질전환용 전이벡터를 완성하였다.After the Asc I and Fse I restriction enzyme treatment was performed on the produced pFibHNC-KillerRed, the fragment containing the KillerRed gene was isolated, cloned into pG-3xP3-EGFP in the laboratory, and transformed with the structure of FIG. 1 named pT121-KillerRed. The dragon transfer vector was completed.
<실시예 3. 형질전환 누에알의 제조 - 전이벡터 미세주사><Example 3. Preparation of transformed silkworm eggs-transfer vector micro injection>
누에 형질전환체의 제작은 2000년 Tamura 등이 사용한 미세주입(microinjection)법을 참고로 실험을 진행하였다(Tamura T et al., 2000). The production of silkworm transformants was conducted in 2000 with reference to the microinjection method used by Tamura et al. (Tamura T et al., 2000).
산란 후 4시간 이내의 누에알을 이용하여, 제작된 pT121-KillerRed 벡터와 Helper 벡터(pHA3PIG)를 1:1로 혼합하여 각각 200 ng/㎕의 농도로 완충용액 (5 mM KCl, 0.5 mM Phosphate buffer, pH 7.0)에 희석 후, 배아의 주공과 후부 사이의 가운데 배 부분에 10~15 nl 가량의 DNA 용액을 주사하였다. 보다 자세하게, 이를 위해 텅스텐 침으로 누에알의 난간에 작은 구멍을 뚫고, 이 구멍에 DNA 용액이 들어있는 미세관(microcapillary)의 끝을 삽입 후, 미세주입기(microinjector)의 공기압을 이용하여 DNA 용액을 알 속으로 주입하였다. The prepared pT121-KillerRed vector and the Helper vector (pHA3PIG) were mixed 1: 1 using the silkworm eggs within 4 hours after spawning, and buffer solutions (5 mM KCl, 0.5 mM Phosphate buffer) at a concentration of 200 ng / µl, respectively. , pH 7.0), a DNA solution of about 10 to 15 nl was injected into the middle fold between the main hole and the rear of the embryo. For more detail, for this purpose, a small hole is made in the railing of the silkworm egg with a tungsten needle, and after inserting the end of a microcapillary containing a DNA solution into the hole, the DNA solution is used using the air pressure of the microinjector. Injected into the eggs.
난간에 생긴 구멍은 Cyanocrylate 접착제를 사용하여 막은 후 누에알은 보습한 패트리디쉬에서 25℃를 유지하여 부화할 때까지 보호하였다.The holes in the railing were closed with a Cyanocrylate adhesive, and the silkworm eggs were protected at 25 ° C. in a moisturized petri dish until incubation.
<실시예 4. 형질전환체 선발><Example 4. Selection of transformants>
실시예 4-1. 계대사육Example 4-1. Passage
T12l-KillerRed 전이벡터 미세주사 후 부화한 240 마리의 유충을 지속적으로 사육하여 성충이 된 나방을 서로 교배하여 F1 세대 40 아구(broods)를 얻었다. 산란 후 7일째 형광현미경을 이용하여 검경한 결과 23개 아구에서 형광이 관찰되었으며, 부화 후 형광이 관찰된 개체들만을 선발하여 사육 후 교배하여 F2 세대 형질전환 누에를 제작하였다. F2 세대 역시 F1세대와 동일하게 현광현미경을 이용한 검경을 통해 형질전환 누에를 선발하고 세대를 전진하였다(표 1 참조). After micro-injection of the T12l-KillerRed transfer vector, 240 hatched larvae were continuously bred to cross each adult moth to obtain F1 generation 40 broods. As a result of spectroscopic examination using a fluorescence microscope on the 7th day after spawning, fluorescence was observed in 23 subgroups. After incubation, only individuals with fluorescence were selected, reared after breeding, and F2 generation transformed silkworms were produced. In the same way as in the F1 generation, the transformed silkworm was selected and advanced through the speculum using a microscope under the same conditions as the F1 generation (see Table 1).
이 때, 형광 발현이 되는 누에의 선발은 LEICA MZ16FA 현미경(Leica사, USA) 및 Microscope MZ FLIII Flourescence Filter EGFP 형광필터(Leica사, USA)를 사용하여, 누에의 세대별 및 시기별로 관찰하여 선발하였다. 기존에 보고된 문헌에 의하면 3xP3 프로모터는 누에 초기 배 단계의 눈과 신경조직, 유충과 번데기 및 성충의 눈에서 작용한다고 알려져 있으므로, 이러한 특징을 근거로 형질전환체를 선발하였다. At this time, the selection of silkworms that exhibited fluorescence was selected by observing each generation and period of silkworms using a LEICA MZ16FA microscope (Leica, USA) and a Microscope MZ FLIII Flourescence Filter EGFP fluorescent filter (Leica, USA). . According to the previously reported literature, the 3xP3 promoter is known to act on the eyes of the early embryonic stage and the nerve tissue, larvae, pupae, and adult eyes, and thus, a transformant was selected based on these characteristics.
<실시예 5. 형질전환 누에의 확인><Example 5. Confirmation of transformed silkworm>
실시예 5-1. 현광현미경을 이용한 EGFP 발현 확인Example 5-1. Confirmation of EGFP expression using a microscope
3xP3 프로모터를 이용하여 발현이 조절되는 EGFP는 유충의 신경계에서 발현되며 특히 눈에서의 발현이 용이하다. 전이벡터가 게놈에 삽입된 형질전환체는 눈에서 EGFP의 발현이 관찰되어 F1 세대부터 현광현미경을 이용하여 관찰 및 선발가능하며 세대를 전진할수록 형질전환체의 출현빈도가 증가한다. EGFP whose expression is regulated using the 3xP3 promoter is expressed in the larva's nervous system and is particularly easy to express in the eye. EGFP expression is observed in the transformant in which the transfection vector is inserted into the genome, and can be observed and selected using a microscope under the F1 generation, and the frequency of transformants increases as the generation progresses.
이에 대해, 도 2에 F2 세대의 형질전환 누에(F2)가 EGFP 형광 단백질을 발현하는 것을 나타내었는데, 각각 A는 알, B는 유충, C는 번데기, D는 성충의 사진이며, 알에서는 눈 및 신경계, 유충/번데기/성충에서는 성충에서 각각 형광(화살표)이 나타나는 것이 확인된다. In contrast, FIG. 2 shows that the transformed silkworm (F2) of the F2 generation expresses the EGFP fluorescent protein, wherein A is an egg, B is a larva, C is a pupa, D is a picture of an adult, and the egg is an eye and In the nervous system, larvae, pupae, and adult, it is confirmed that fluorescence (arrow) appears in the adult.
실시예 5-2. KillerRed 유전자 발현분석Example 5-2. KillerRed gene expression analysis
선발된 F3 세대 형질전환누에(T123K)를 사육하고 5령 3일째 유충을 액체질소를 이용하여 동결 후 분쇄하여 total RNA를 추출하고, oligo dT promer를 이용하여 total RNA로부터 cDNA를 합성하였다. 이후 700 ng의 cDNA를 이용하여 RT-PCR 및 Real-time PCR을 통해 정상 잠123과 형질전환누에 T123K 사이에서의 KillerRed 유전자 발현을 비교 분석하였고, 이에 대한 결과를 도 3에 나타내었다. The selected F3 generation transformed silkworm (T123K) was bred and the larvae at 5th and 3rd days were frozen and crushed using liquid nitrogen to extract total RNA, and cDNA was synthesized from total RNA using oligo dT promer. Thereafter, the expression of KillerRed gene between
도 3을 참고하면, 야생 형질의 잠123에서는 KillerRed 유전자의 발현이 검출되지 않는 반면 형질전환 누에인 T123K에서는 KillerRed 유전자의 발현이 확인된다. Referring to FIG. 3, expression of the KillerRed gene is not detected in the
실시예 5-3. 견사선 내 KillerRed 단백질 발현분석Example 5-3. Analysis of KillerRed protein expression in the silk gland
일반적으로 실크가 생성되는 과정은 누에의 후부 견사선에서 피브로인이 생성된 후 중부 견사선에서 세리신이 코팅되고 전부 견사선에서 토사공을 통해 밖으로 용출된다. 형질전환누에 T123K의 KillerRed 단백질 발현여부를 확인하기 위하여 F3 세대의 5령 5일 누에를 해부하여 견사선을 적출하고 형광현미경을 이용하여 확인하였다. In general, the process in which silk is produced is fibroin from the silkworm's posterior silkworm, and then sericin is coated on the central silkworm and all elutes out through the earthwork from the silkworm. In order to confirm whether the transformed silkworm T123K expressed the KillerRed protein, the silkworm was dissected by dissecting the 5th and 5th silkworms of the F3 generation and confirmed using a fluorescence microscope.
현미경 확인 사진은 도 4에 나타내었는데, 도 4를 참고하면, 백색광 조건에서 연녹색을 띄는 잠123의 견사선에 비하여 형질전환누에 T123K의 견사선은 연한 적색을 나타내었으며, 형광조건에서 T123K의 견사선은 강한 적색 형광을 발산하여 잠123의 견사선과 극명한 대비를 이루는 것을 확인할 수 있다. The microscopic confirmation picture is shown in FIG. 4, and referring to FIG. 4, the silkworm of the transformed silkworm T123K showed a light red color compared to the silkworm of
실시예 5-4. KillerRed 단백질을 포함하는 누에고치 분석Example 5-4. Analysis of cocoon containing KillerRed protein
F3 세대 형질전환누에 T123K을 고치를 생성할 때 까지 지속적으로 사육하여 얻어진 형질전환누에 T123K의 고치와 잠123의 고치를 형광현미경을 이용하여 확인하였다. 이에 대한 결과는 도 5에 나타내었는데, 도 5를 참고하면, 백색광(자연광) 조건에서 잠123의 고치는 밝은 흰색을 나타내었고 형질전환누에 T123K의 고치는 엷은 적색을 나타내었으며, 형광조건에서는 형질전환누에 T123K의 고치가 진한 적색을 나타내는 것이 확인되었다. The F3 generation transformed silkworm T123K was continuously raised until the cocoon was produced, and the transformed silkworm T123K and sleep 123 cocoon were identified using a fluorescence microscope. The results for this are shown in FIG. 5. Referring to FIG. 5, in the white light (natural light) condition, the cocoon of
이러한 누에고치에 발현되는 KillerRed 단백질은 활성산소(ROS)을 생성하기 때문에 KillerRed 단백질이 포함된 T123K의 고치 또한 활성산소를 생산할 것으로 기대된다. 활성산소는 항균력이 있기 때문에 이와 같은 본 발명의 기술을 통해 T123K의 고치를 이용하여 항균 섬유와 같은 특수 섬유생산이 가능하며, 킬러레드 단백질을 누에고치로부터 분리추출할 경우 천연물 유래의 항균 조성물로도 이용가능함을 알 수 있다.Since the KillerRed protein expressed in the cocoon generates free radicals (ROS), the cocoon of T123K containing the KillerRed protein is also expected to produce free radicals. Since free radicals have antimicrobial activity, it is possible to produce special fibers such as antibacterial fibers using the cocoon of T123K through the technology of the present invention. Also, when killer red protein is separated and extracted from the cocoon, it is also used as an antibacterial composition derived from natural products. You can see that it is available.
실시예 6. 형질전환 누에고치로부터 분리된 KillerRed 단백질의 항균활성Example 6. Antibacterial activity of KillerRed protein isolated from transformed cocoon
F3 세대 형질전환누에 T123K와 잠123가 생산한 누에고치로부터 생사를 정련하여 세리신을 제거하고 14.3 mol/L LiBr 수용액에서 30℃에서 72시간 동안 용해시킨 후 유수 중에서 셀룰로오스 막 투석을 통해 단백질 용액을 준비 후 RDA (radial diffusion assay)법을 이용하여 항균활성을 분석하였다.F3 generation After transforming silkworms from the cocoon produced by the transformed silkworm T123K and Jam123, the sericin was removed, dissolved in 14.3 mol / L LiBr aqueous solution at 30 ° C for 72 hours, and then the protein solution was prepared through dialysis of cellulose membrane in running water, followed by RDA. The antibacterial activity was analyzed using the (radial diffusion assay) method.
RDA법은 Citrate phosphate buffer(9 mM sodium phosphate, 1 mM sodium citrate, pH7.4)와 1%(w/v) type Ⅰ(low electroendosmosis) agarose, 0.03% TSB로 구성된 멸균된 underlay gel에 배양된 대장균(Escherichia coli BL21)을 4 × 106 colony forming units로 넣고 혼합해 준 뒤 사각플레이트에 붓고, underlay gel이 굳으면 지름 3mm의 구멍을 내어 각 누에고치로부터 추출된 단백질 용액 10 ug을 도입시킨 후 상온의 암실에서 530 nm의 녹색광을 6시간 동안 조사하여 KillerRed 단백질의 활성화 및 활성산소의 생성을 유도하였다. 이후, overlay agar(6% TSB, 1% agarose) 10 ㎖을 붓고 37℃에서 14시간 동안 다시 배양하여 clear zone의 직경을 비교하였다.E. coli cultured in sterile underlay gel composed of Citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH7.4), 1% (w / v) type I (low electroendosmosis) agarose, 0.03% TSB ( Escherichia coli BL21) is added to 4 × 10 6 colony forming units, mixed, poured into a square plate, and when the underlay gel hardens, a hole with a diameter of 3 mm is introduced to introduce 10 ug of protein solution extracted from each cocoon, and then at room temperature. Green light of 530 nm was irradiated for 6 hours in the dark room to induce activation of the KillerRed protein and generation of free radicals. Then, 10 ml of overlay agar (6% TSB, 1% agarose) was poured and incubated again at 37 ° C. for 14 hours to compare the diameter of the clear zone.
이에 대한 결과는 도 6에 나타내었는데, 형질전환누에 T123K의 고치에서 용출된 KillerRed 단백질은 광 활성화 이전에도 약한 항균활성을 나타내었으며, 동일한 조건에서 T123K의 실크 단백질에 530 nm의 녹색광을 이용하여 활성화시킬 경우 항균활성이 크게 증가하는 것으로 확인되었다. 반면, 형질전환되지 않은 일반 잠 123 누에로부터 얻은 실크 단백질의 항균성은 확인되지 않았다. The results for this are shown in FIG. 6, the KillerRed protein eluted from the cocoon of the transformed silkworm T123K showed weak antimicrobial activity even before light activation, and was activated by using green light of 530 nm for the silk protein of T123K under the same conditions. In the case, it was confirmed that the antibacterial activity was greatly increased. On the other hand, the antibacterial properties of silk proteins obtained from non-transformed
킬러레드 단백질은 자연광인 녹색 및 주황색 광원에 의하여 적색 색상 및 항균활성이 활성화되는데 이와 같은 실험을 통해 누에고치에서 생성된 킬러레드 단백질이 항균 활성을 갖는 온전한 상태임을 확인할 수 있다. The killer red protein activates red color and antibacterial activity by natural green and orange light sources. Through these experiments, it can be confirmed that the killer red protein produced by the cocoon is intact with antibacterial activity.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Transgenic silkworms producing silk expressed KillerRed protein <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 2556 <212> DNA <213> Unknown <220> <223> Bombyx mori <400> 1 cctgcgtgat caggaaaaat gtggaaagct taacgatttt gtcacatttt acttatcaca 60 acttgttttt ataataattc gcttaaatga gcagctatta cttaatctcg tagtggtttt 120 tgacaaaatc agcttcttta gaactaaaat atcatttttt tcgtaatttt tttaatgaaa 180 aatgctctag tgttatacct ttccaaaatc accattaatt aggtagtgtt taagcttgtt 240 gtacaaaact gccacacgca tttttttctc cactgtaggt tgtagttacg cgaaaacaaa 300 atcgttctgt gaaaattcaa acaaaaatat tttttcgtaa aaacacttat caatgagtaa 360 agtaacaatt catgaataat ttcatgtaaa aaaaaaatac tagaaaagga atttttcatt 420 acgagatgct taaaaatctg tttcaaggta gagatttttc gatatttcgg aaaattttgt 480 aaaactgtaa atccgtaaaa ttttgctaaa catatattgt gttgttttgg taagtattga 540 cccaagctat cacctcctgc agtatgtcgt gctaattact ggacacattg tataacagtt 600 ccactgtatt gacaataata aaacctcttc attgacttga gaatgtctgg acagatttgg 660 ctttgtattt ttgatttaca aatgtttttt tggtgattta cccatccaag gcattctcca 720 ggatggttgt ggcatcacgc cgattggcaa acaaaaacta aaatgaaact aaaaagaaac 780 agtttccgct gtcccgttcc tctagtggga gaaagcatga agtaagttct ttaaatatta 840 caaaaaaatt gaacgatatt ataaaattct ttaaaatatt aaaagtaaga acaataagat 900 caattaaatc ataattaatc acattgttca tgatcacaat ttaatttact tcatacgttg 960 tattgttatg ttaaataaaa agattaattt ctatgtaatt gtatctgtac aatacaatgt 1020 gtagatgttt attctatcga aagtaaatac gtcaaaactc gaaaattttc agtataaaaa 1080 ggttcaactt tttcaaatca gcatcagttc ggttccaact ctcaagatga gagtcaaaac 1140 ctttgtgatc ttgtgctgcg ctctgcaggt gagttaatta ttttactatt atttcagaag 1200 gtggccagac gatatcacgg gccacctgat aataagtggt cgccaaaacg cacagatatc 1260 gtaaattgtg ccatttgatt tgtcacgccc gggggggcta cggaataaac tacatttatt 1320 tatttaaaaa atgaacctta gattatgtaa cttgtgattt atttgcgtca aaagtaggca 1380 agatgaatct atgtaaatac ctgggcagac ttgcaatatc ctatttcacc ggtaaatcag 1440 cattgcaata tgcaatgcat attcaacaat atgtaaaaca attcgtaaag catcattaga 1500 aaatagacga aagaaattgc ataaaattat aaccgcatta ttaatttatt atgatatcta 1560 ttaacaattg ctattgcctt tttttcgcaa attataatca ttttcataac ctcgaggtag 1620 cattctgtta cattttaata cattggtatg tgattataac acgagctgcc cactgagttt 1680 ctcgccagat cttctcagtg ggtcgcgtta ccgatcacgt gatagattct atgaagcact 1740 gctcttgtta gggctagtgt tagcaaattc tttcaggttg agtctgagag ctcacctacc 1800 catcggagcg tagctggaat aggctaccag ctaataggta gggaaaacaa agctcgaaac 1860 aagctcaagt aataacaaca taatgtgacc ataaaatctc gtggtgtatg agatacaatt 1920 atgtactttc ccacaaatgt ttacataatt agaatgttgt tcaacttgcc taacgcccca 1980 gctagaacat tcaattatta ctattaccac tactaaggca gtatgtccta actcgttcca 2040 gatcagcgct aacttcgatt gaatgtgcga aatttatagc tcaatatttt agcacttatc 2100 gtattgattt aagaaaaaat tgttaacatt ttgtttcagt atgtcgctta tacaaatgca 2160 aacatcaatg attttgatga ggactatttt gggagtgatg tcactgtcca aagtagtaat 2220 acaacagatg aaataattag agatgcatct ggggcagtta tcgaagaaca aattacaact 2280 aaaaaaatgc aacggaaaaa taaaaaccat ggaatacttg gaaaaaatga aaaaatgatc 2340 aagacgttcg ttataaccac ggattccgac ggtaacgagt ccattgtaga ggaagatgtg 2400 ctcatgaaga cactttccga tggtactgtt gctcaaagtt atgttgctgc tgatgcggga 2460 gcatattctc agagcgggcc atacgtatca aacagtggat acagcactca tcaaggatat 2520 acgagcgatt tcagcactag tgctgcagtc ggtgca 2556 <210> 2 <211> 480 <212> DNA <213> Unknown <220> <223> Bombyx mori <400> 2 agcgtcagtt acggagctgg caggggatac ggacaaggtg caggaagtgc agcttcctct 60 gtgtcatctg cttcatctcg cagttacgac tattctcgtc gtaacgtccg caaaaactgt 120 ggaattccta gaagacaact agttgttaaa ttcagagcac tgccttgtgt gaattgctaa 180 tttttaatat aaaataaccc ttgtttctta cttcgtcctg gatacatcta tgtttttttt 240 ttcgttaata aatgagagca tttaagttat tgtttttaat tacttttttt tagaaaacag 300 atttcggatt ttttgtatgc attttatttg aatgtactaa tataatcaat taatcaatga 360 attcatttat ttaagggata acaataatcc atgaattcac atgcacattt aaaacaaaac 420 taaattacaa taggttcata taaaaacaac aagtatgcct tctcaactaa gaatactata 480 480 <210> 3 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> KillerRed gene <400> 3 atgggttcag agggcggccc cgccctgttc cagagcgaca tgaccttcaa aatcttcatc 60 gacggcgagg tgaacggcca gaagttcacc atcgtggccg acggcagcag caagttcccc 120 cacggcgact tcaacgtgca cgccgtgtgc gagaccggca agctgcccat gagctggaag 180 cccatctgcc acctgatcca gtacggcgag cccttcttcg cccgctaccc cgacggcatc 240 agccatttcg cccaggagtg cttccccgag ggcctgagca tcgaccgcac cgtgcgcttc 300 gagaacgacg gcaccatgac cagccaccac acctacgagc tggacgacac ctgcgtggtg 360 agccgcatca ccgtgaactg cgacggcttc cagcccgacg gccccatcat gcgcgaccag 420 ctggtggaca tcctgcccaa cgagacccac atgttccccc acggccccaa cgccgtgcgc 480 cagctggcct tcatcggctt caccaccgcc gacggcggcc tgatgatggg ccacttcgac 540 agcaagatga ccttcaacgg cagccgcgcc atcgagatcc ccggcccaca cttcgtgacc 600 atcatcacca agcagatgag ggacaccagc gacaagcgcg accacgtgtg ccagcgcgag 660 gtggcctacg cccacagcgt gccccgcatc accagcgcca tcggtagcga cgaggattga 720 720 <110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Transgenic silkworms producing silk expressed KillerRed protein <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 2556 <212> DNA <213> Unknown <220> <223> Bombyx mori <400> 1 cctgcgtgat caggaaaaat gtggaaagct taacgatttt gtcacatttt acttatcaca 60 acttgttttt ataataattc gcttaaatga gcagctatta cttaatctcg tagtggtttt 120 tgacaaaatc agcttcttta gaactaaaat atcatttttt tcgtaatttt tttaatgaaa 180 aatgctctag tgttatacct ttccaaaatc accattaatt aggtagtgtt taagcttgtt 240 gtacaaaact gccacacgca tttttttctc cactgtaggt tgtagttacg cgaaaacaaa 300 atcgttctgt gaaaattcaa acaaaaatat tttttcgtaa aaacacttat caatgagtaa 360 agtaacaatt catgaataat ttcatgtaaa aaaaaaatac tagaaaagga atttttcatt 420 acgagatgct taaaaatctg tttcaaggta gagatttttc gatatttcgg aaaattttgt 480 aaaactgtaa atccgtaaaa ttttgctaaa catatattgt gttgttttgg taagtattga 540 cccaagctat cacctcctgc agtatgtcgt gctaattact ggacacattg tataacagtt 600 ccactgtatt gacaataata aaacctcttc attgacttga gaatgtctgg acagatttgg 660 ctttgtattt ttgatttaca aatgtttttt tggtgattta cccatccaag gcattctcca 720 ggatggttgt ggcatcacgc cgattggcaa acaaaaacta aaatgaaact aaaaagaaac 780 agtttccgct gtcccgttcc tctagtggga gaaagcatga agtaagttct ttaaatatta 840 caaaaaaatt gaacgatatt ataaaattct ttaaaatatt aaaagtaaga acaataagat 900 caattaaatc ataattaatc acattgttca tgatcacaat ttaatttact tcatacgttg 960 tattgttatg ttaaataaaa agattaattt ctatgtaatt gtatctgtac aatacaatgt 1020 gtagatgttt attctatcga aagtaaatac gtcaaaactc gaaaattttc agtataaaaa 1080 ggttcaactt tttcaaatca gcatcagttc ggttccaact ctcaagatga gagtcaaaac 1140 ctttgtgatc ttgtgctgcg ctctgcaggt gagttaatta ttttactatt atttcagaag 1200 gtggccagac gatatcacgg gccacctgat aataagtggt cgccaaaacg cacagatatc 1260 gtaaattgtg ccatttgatt tgtcacgccc gggggggcta cggaataaac tacatttatt 1320 tatttaaaaa atgaacctta gattatgtaa cttgtgattt atttgcgtca aaagtaggca 1380 agatgaatct atgtaaatac ctgggcagac ttgcaatatc ctatttcacc ggtaaatcag 1440 cattgcaata tgcaatgcat attcaacaat atgtaaaaca attcgtaaag catcattaga 1500 aaatagacga aagaaattgc ataaaattat aaccgcatta ttaatttatt atgatatcta 1560 ttaacaattg ctattgcctt tttttcgcaa attataatca ttttcataac ctcgaggtag 1620 cattctgtta cattttaata cattggtatg tgattataac acgagctgcc cactgagttt 1680 ctcgccagat cttctcagtg ggtcgcgtta ccgatcacgt gatagattct atgaagcact 1740 gctcttgtta gggctagtgt tagcaaattc tttcaggttg agtctgagag ctcacctacc 1800 catcggagcg tagctggaat aggctaccag ctaataggta gggaaaacaa agctcgaaac 1860 aagctcaagt aataacaaca taatgtgacc ataaaatctc gtggtgtatg agatacaatt 1920 atgtactttc ccacaaatgt ttacataatt agaatgttgt tcaacttgcc taacgcccca 1980 gctagaacat tcaattatta ctattaccac tactaaggca gtatgtccta actcgttcca 2040 gatcagcgct aacttcgatt gaatgtgcga aatttatagc tcaatatttt agcacttatc 2100 gtattgattt aagaaaaaat tgttaacatt ttgtttcagt atgtcgctta tacaaatgca 2160 aacatcaatg attttgatga ggactatttt gggagtgatg tcactgtcca aagtagtaat 2220 acaacagatg aaataattag agatgcatct ggggcagtta tcgaagaaca aattacaact 2280 aaaaaaatgc aacggaaaaa taaaaaccat ggaatacttg gaaaaaatga aaaaatgatc 2340 aagacgttcg ttataaccac ggattccgac ggtaacgagt ccattgtaga ggaagatgtg 2400 ctcatgaaga cactttccga tggtactgtt gctcaaagtt atgttgctgc tgatgcggga 2460 gcatattctc agagcgggcc atacgtatca aacagtggat acagcactca tcaaggatat 2520 acgagcgatt tcagcactag tgctgcagtc ggtgca 2556 <210> 2 <211> 480 <212> DNA <213> Unknown <220> <223> Bombyx mori <400> 2 agcgtcagtt acggagctgg caggggatac ggacaaggtg caggaagtgc agcttcctct 60 gtgtcatctg cttcatctcg cagttacgac tattctcgtc gtaacgtccg caaaaactgt 120 ggaattccta gaagacaact agttgttaaa ttcagagcac tgccttgtgt gaattgctaa 180 tttttaatat aaaataaccc ttgtttctta cttcgtcctg gatacatcta tgtttttttt 240 ttcgttaata aatgagagca tttaagttat tgtttttaat tacttttttt tagaaaacag 300 atttcggatt ttttgtatgc attttatttg aatgtactaa tataatcaat taatcaatga 360 attcatttat ttaagggata acaataatcc atgaattcac atgcacattt aaaacaaaac 420 taaattacaa taggttcata taaaaacaac aagtatgcct tctcaactaa gaatactata 480 480 <210> 3 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> KillerRed gene <400> 3 atgggttcag agggcggccc cgccctgttc cagagcgaca tgaccttcaa aatcttcatc 60 gacggcgagg tgaacggcca gaagttcacc atcgtggccg acggcagcag caagttcccc 120 cacggcgact tcaacgtgca cgccgtgtgc gagaccggca agctgcccat gagctggaag 180 cccatctgcc acctgatcca gtacggcgag cccttcttcg cccgctaccc cgacggcatc 240 agccatttcg cccaggagtg cttccccgag ggcctgagca tcgaccgcac cgtgcgcttc 300 gagaacgacg gcaccatgac cagccaccac acctacgagc tggacgacac ctgcgtggtg 360 agccgcatca ccgtgaactg cgacggcttc cagcccgacg gccccatcat gcgcgaccag 420 ctggtggaca tcctgcccaa cgagacccac atgttccccc acggccccaa cgccgtgcgc 480 cagctggcct tcatcggctt caccaccgcc gacggcggcc tgatgatggg ccacttcgac 540 agcaagatga ccttcaacgg cagccgcgcc atcgagatcc ccggcccaca cttcgtgacc 600 atcatcacca agcagatgag ggacaccagc gacaagcgcg accacgtgtg ccagcgcgag 660 gtggcctacg cccacagcgt gccccgcatc accagcgcca tcggtagcga cgaggattga 720 720
Claims (12)
상기 표지 유전자 조절 프로모터는 3xP3 프로모터인 것을 특징으로 하는 재조합 발현벡터.According to claim 1,
The marker gene regulatory promoter is a 3xP3 promoter, characterized in that the recombinant expression vector.
상기 표지 유전자는 EGFP(green fluorescent protein) 유전자인 것을 특징으로 하는 재조합 발현벡터.According to claim 1,
The marker gene is a recombinant expression vector, characterized in that the green fluorescent protein (EGFP) gene.
상기 유전자 컨스트럭트는 도 1의 구조로 이루어지는 것을 특징으로 하는 재조합 발현벡터.According to claim 1,
The gene construct is a recombinant expression vector, characterized in that consisting of the structure of Figure 1.
상기 발현벡터는 piggyBac 벡터인 것을 특징으로 하는 재조합 발현벡터.According to claim 1,
The expression vector is a recombinant expression vector, characterized in that the piggyBac vector.
(제2단계) 상기 제1단계의 재조합 발현벡터를 누에(Bombyx mori) 알에 형질전환시켜 형질전환된 누에알을 제조하는 단계; 및
(제3단계) 상기 제2단계의 형질전환된 누에 알을 부화시켜 형질전환 누에를 사육하는 단계를 포함하는 것을 특징으로 하는 킬러레드 단백질을 발현하는 실크를 생산하는 형질전환 누에의 제조방법.(First step) preparing the recombinant expression vector of any one of claims 1 and 3 to 6;
(Second step) transforming the recombinant expression vector of the first step into silkworm (Bombyx mori) eggs to prepare transformed silkworm eggs; And
(Step 3) A method of producing a silkworm that produces silk expressing a killer red protein, comprising incubating the transformed silkworm egg by hatching the transformed silkworm egg of the second step.
상기 제2단계의 형질전환은 미세주입법(microinjection)을 이용하는 것을 특징으로 하는 실크를 생산하는 형질전환 누에의 제조방법.The method of claim 8,
The second step of transformation is a method of producing a silkworm transforming silk, characterized in that using a microinjection method (microinjection).
상기 제3단계에서 a) 발현벡터에 표지 유전자를 도입한 후, 형질전환 누에에서 상기 표지 유전자의 발현을 확인하거나, b) 형질전환 누에에서 킬러레드 단백질 유전자의 발현을 확인하여, 형질전환 누에를 선발하는 단계를 추가적으로 포함하는 것을 특징으로 하는 킬러레드 단백질을 발현하는 실크를 생산하는 형질전환 누에의 제조방법.The method of claim 8,
In the third step, a) after introducing the marker gene into the expression vector, confirm the expression of the marker gene in the transformed silkworm, or b) confirm the expression of the killer red protein gene in the transformed silkworm, Method of producing a transformed silkworm producing silk expressing a killer red protein, characterized in that it further comprises the step of selecting.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140057790A (en) * | 2012-11-05 | 2014-05-14 | 대한민국(농촌진흥청장) | Transgenic silkworms producing cocoons containing melittins antibiotic peptides |
| KR20150000154A (en) * | 2013-06-24 | 2015-01-02 | 대한민국(농촌진흥청장) | Transgenic silkworms producing antimicrobial peptide |
| KR101510437B1 (en) | 2012-11-05 | 2015-04-10 | 대한민국 | Transgenic silkworms producing red fluorescent cocoons |
| KR101570784B1 (en) | 2013-06-24 | 2015-11-20 | 대한민국 | Transgenic silkworms producing yellow fluorescent cocoons |
| KR101634272B1 (en) | 2014-06-24 | 2016-06-30 | 대한민국 | Transgenic silkworms producing blue fluorescent cocoons |
-
2018
- 2018-12-12 KR KR1020180159942A patent/KR102114194B1/en active IP Right Grant
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140057790A (en) * | 2012-11-05 | 2014-05-14 | 대한민국(농촌진흥청장) | Transgenic silkworms producing cocoons containing melittins antibiotic peptides |
| KR101510437B1 (en) | 2012-11-05 | 2015-04-10 | 대한민국 | Transgenic silkworms producing red fluorescent cocoons |
| KR20150000154A (en) * | 2013-06-24 | 2015-01-02 | 대한민국(농촌진흥청장) | Transgenic silkworms producing antimicrobial peptide |
| KR101570784B1 (en) | 2013-06-24 | 2015-11-20 | 대한민국 | Transgenic silkworms producing yellow fluorescent cocoons |
| KR101634272B1 (en) | 2014-06-24 | 2016-06-30 | 대한민국 | Transgenic silkworms producing blue fluorescent cocoons |
Non-Patent Citations (2)
| Title |
|---|
| Bulina, Maria E., et al. "A genetically encoded photosensitizer." NATURE BIOTECHNOLOGY 24.1 (2006): 95.* * |
| Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P, Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol, 2000, 18, 81~84. |
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