KR20200074834A - Recombinant promoter comprising the promoter region of the ovotransferrin gene and the promoter region of the ovalbumin gene - Google Patents
Recombinant promoter comprising the promoter region of the ovotransferrin gene and the promoter region of the ovalbumin gene Download PDFInfo
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Abstract
Description
본 발명은 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터, 및 상기 재조합 프로모터를 포함하는 벡터에 관한 것이다.The present invention relates to a recombinant promoter comprising a promoter region of the obtransferin gene and a promoter region of the ovalbumin gene, and a vector comprising the recombinant promoter.
현대 과학기술의 발전은 특히 의료부분의 발전은 사람의 수명연장을 가져왔으며, 더불어 사람에게 유용한 생리 활성 단백질들의 수요가 증가되고 있다. 현재 가장 일반적인 생리 활성 단백질의 생산 방법은 대장균을 이용하는 방법이지만, 대장균을 이용한 생산 방법은 당이 결합하는 복잡한 구조의 단백질은 생산하지 못하는 문제점이 있어 구조가 복잡한 단백질은 동물 세포주를 이용하여 생산하고 있다. 최근에는 가축을 이용하여 유용 단백질을 생산하고자 하는 연구가 진행되고 있으며, 일부에서는 가시적인 성과가 보고되고 있다. 현재까지 생리활성물질 단백질 생산에 활용되고 있는 동물들은 대부분 중/대 가축들로서 분비선 즉 유선(우유)을 이용한 방법이 주로 활용되고 있다.Advances in modern science and technology, especially in the medical field, have resulted in an extended human lifespan, and the demand for bioactive proteins useful to humans is increasing. Currently, the most common method for producing a bioactive protein is a method using E. coli, but the method using E. coli has a problem in that it cannot produce a protein having a complex structure to which sugar is bound, and a protein having a complex structure is produced using animal cell lines. . Recently, studies have been conducted to produce useful proteins using livestock, and in some cases, visible results have been reported. Until now, most of the animals that are used for the production of physiologically active substances are medium/large livestock, and the method using the gland (milk) is mainly used.
종래의 중/대 가축들을 이용하여 유용 단백질을 생산하는 방법을 사용하는 경우 가축을 이용하여 단백질을 생산하는데 비용이 많이 소요되고, 생산 효율이 낮다는 문제가 있었다.In the case of using a method of producing useful protein using conventional medium/large livestock, there is a problem in that it takes a lot of cost to produce protein using livestock and has low production efficiency.
반면, 닭은 포유류 가축들에 비해 산자수가 많고, 세대간격이 짧은 특징으로 인해 단기간에 하나의 개체에서 대량번식이 가능하고, 특히 산란계의 경우 일년 동안 300-330개의 계란을 생산한다는 장점이 있어서, 닭의 계란을 이용하여 유용 단백질을 생산하는 방법이 최근에 각광 받고 있다.On the other hand, chickens have a larger number of livestock than mammalian livestock, and because of their short generation intervals, they can reproduce in large quantities in one individual in a short period of time, and in particular, in the case of laying eggs, they produce 300-330 eggs per year. Methods for producing useful proteins using chicken eggs have recently been spotlighted.
본 발명의 종래기술로는 닭의 오브알부민 유전자의 프로모터 영역을 이용하여 제조한 프로모터에 관한 미국 등록 특허 제8507749 B2가 있다.As a prior art of the present invention, there is US Patent No. 8507749 B2 for a promoter prepared using a promoter region of a chicken ovalbumin gene.
본 발명자들은 소형 가축인 닭을 이용하여 유용 단백질을 효과적으로 발현시키기 위해서, 종래 닭의 오브알부민 유전자의 프로모터를 개량하여 본 발명을 완성하였다.The present inventors completed the present invention by improving the promoter of the ovalbumin gene of a conventional chicken in order to effectively express useful proteins using a small livestock chicken.
본 발명의 목적은 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터를 제공하는 것이다.It is an object of the present invention to provide a recombinant promoter comprising a promoter region of the obtransferin gene and a promoter region of the ovalbumin gene.
본 발명의 다른 목적은 상기 재조합 프로모터를 포함하는 벡터를 제공하는 것이다.Another object of the present invention is to provide a vector containing the recombinant promoter.
본 발명의 다른 목적은 상기 벡터에 의해 형질전환된 닭을 제공하는 것이다.Another object of the present invention is to provide a chicken transformed with the vector.
본 발명의 다른 목적은 목적 유전자를 포함하는 상기 재조합 벡터를 닭에 형질전환하여, 닭에서 목적 단백질을 발현시키는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for expressing a target protein in chicken by transforming the recombinant vector containing the target gene in chicken.
상기 목적을 달성하기 위하여, 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터를 제공한다.In order to achieve the above object, a recombinant promoter including a promoter region of the obtransferin gene and a promoter region of the ovalbumin gene is provided.
또한, 본 발명은 상기 재조합 프로모터를 벡터를 제공한다.In addition, the present invention provides a vector for the recombinant promoter.
또한, 본 발명은 상기 벡터에 의해 형질전환된 닭을 제공한다.In addition, the present invention provides a chicken transformed with the vector.
또한, 본 발명은 목적 유전자를 포함하는 상기 재조합 벡터를 닭에 형질전환하여, 닭에서 목적 단백질을 발현시키는 방법을 제공한다.In addition, the present invention provides a method of expressing a target protein in chicken by transforming the recombinant vector containing the target gene in chicken.
본 발명의 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터, 및 상기 재조합 프로모터를 포함하는 벡터를 이용하는 경우 닭에서 단백질을 높은 효율로 과발현시킬 수 있다.When using the recombinant promoter comprising the promoter region of the obtransferin gene of the present invention and the promoter region of the ovalbumin gene, and the vector containing the recombinant promoter, it is possible to overexpress the protein with high efficiency in chickens.
도 1은 재조합 프로모터에 포함된 오브트렌스페린 유전자의 프로모터 영역에 존재하는 ERα(Estrogen receptor alpha) 결합 부위를 나타낸 것이다.
도 2는 본 발명에서 제조한 재조합 프로모터의 개략도를 나타낸 것이다.
도 3은 닭의 오브트렌스페린 유전자의 프로모터 영역과 닭의 오브알부민 유전자의 프로모터 영역이 재조합된 리포터 벡터(OVOT-pOV)와 오브알부민 유전자의 프로모터 영역을 포함하는 대조군 리포터 벡터(Kanuma)를 비교하여 나타낸 것이다.
도 4는 HeLa 세포에서 OVOT-pOV 프로모터의 활성을 대조군과 비교한 것이다.
도 5는 MES-SA 세포에서 OVOT-pOV 프로모터의 활성을 대조군과 비교한 것이다.
도 6은 cOEC에서 재조합 프로모터(OVOT-pOV)의 활성을 대조군과 비교한 결과를 나타낸 도이다. Figure 1 shows the ERα (Estrogen receptor alpha) binding site present in the promoter region of the obtransferin gene included in the recombinant promoter.
2 shows a schematic diagram of a recombinant promoter prepared in the present invention.
FIG. 3 compares a reporter vector (OVOT-pOV) in which the promoter region of the chicken's obtransferin gene and the chicken ovalbumin gene are recombined, and a control reporter vector (Kanuma) comprising the promoter region of the ovalbumin gene. It is shown.
Figure 4 compares the activity of the OVOT-pOV promoter in HeLa cells with the control.
Figure 5 compares the activity of the OVOT-pOV promoter in MES-SA cells with the control.
6 is a view showing the results of comparing the activity of the recombinant promoter (OVOT-pOV) in cOEC with the control.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터에 관한 것으로서, 상기 재조합 프로모터는 목적 단백질을 닭에서 발현시키는데 사용될 수 있다.The present invention relates to a recombinant promoter comprising a promoter region of the obtransferin gene and a promoter region of the ovalbumin gene, which can be used to express a target protein in chickens.
상기 오브트렌스페린 유전자의 프로모터 영역은 서열번호 1의 염기서열을 포함하는 것을 특징으로 하고, 상기 오브알부민 유전자의 프로모터 영역은 서열번호 2의 염기서열을 포함하는 것을 특징으로 한다.The promoter region of the obtransferin gene is characterized by comprising the nucleotide sequence of SEQ ID NO: 1, and the promoter region of the ovalbumin gene is characterized by comprising the nucleotide sequence of SEQ ID NO: 2.
상기 프로모터 영역 서열의 변이체가 본 발명의 범위 내에 포함된다. 변이체는 염기서열은 변화되지만, 서열번호 1 또는 2의 염기 서열과 유사한 기능적 특성을 갖는 염기 서열이다. 구체적으로, 상기 오브트렌스페린 유전자의 프로모터 영역 염기서열은 서열번호 1의 염기 서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있고, 상기 오브알부민 유전자의 프로모터 영역 염기서열은 서열번호 2의 염기서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다.Variants of the promoter region sequence are included within the scope of the present invention. A variant is a nucleotide sequence having functional characteristics similar to that of SEQ ID NO: 1 or 2, although the nucleotide sequence is changed. Specifically, the nucleotide sequence of the promoter region of the obtransferin gene is 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more of the nucleotide sequence of SEQ ID NO: 1 It may include a nucleotide sequence having homology, the promoter region nucleotide sequence of the ovalbumin gene is 70% or more, more preferably 80% or more, even more preferably 90% or more, with the nucleotide sequence of SEQ ID NO: 2, Most preferably, it may include a base sequence having at least 95% sequence homology.
본 발명에 있어, '프로모터(promoter)'란, 일반적으로 전사를 개시하는 지점을 포함하는 암호화 부위의 상류 쪽에 위치하는 유전자 부위로, 흔히 유전자 발현을 조절하는 TATA 박스, CAAT 박스 부위, 외부 자극에 반응하여 유전자 발현에 영향을 주는 부위 및 위치와 방향에 상관없이 거의 모든 유전자의 발현을 촉진하는 인핸서 등으로 이루어져 있다. In the present invention,'promoter (promoter)' is a gene region located generally upstream of a coding region including a point for initiating transcription, and is commonly used for TATA box, CAAT box region, and external stimulation to regulate gene expression. It consists of enhancers that promote the expression of almost all genes regardless of the site, location, and direction that affects gene expression in response.
본 발명은 형질전환 암탉의 계란에서 유용단백질 생산을 위해 기존에 활용되고 있는 오브알부민 프로모터에 비하여 크기가 작고 프로모터의 활성이 강력한 재조합 프로모터로서, 계란 난백에서 가장 많이 발현되는 단백질인 오브알부민(ovalbumin)과 그 다음으로 많이 발현되는 오브트렌스페린(ovotransferrin) 각각의 프로모터 영역을 연결하였다.The present invention is a recombinant promoter having a small size and strong promoter activity compared to the ovalbumin promoter that has been used for the production of useful protein in eggs of transgenic hens, ovalbumin, the most frequently expressed protein in egg eggs. And the promoter region of each of the next most expressed ovotransferrin.
본 발명의 재조합 프로모터에 포함되어 있는 오브알부민 유전자의 프로모터 영역에는 에스트로겐 호르몬에 반응하는 영역(Estrogen response element, ERE) 또는 ERα(Estrogen receptor alpha) 결합 부위가 존재할 수 있다.In the promoter region of the ovalbumin gene included in the recombinant promoter of the present invention, an estrogen response element (ERE) or an ERα (Estrogen receptor alpha) binding site may be present.
상기 ERα(Estrogen receptor alpha) 결합 부위는 재조합 프로모터에 포함되어 있는 오브알부민 유전자의 프로모터 영역에 1개 내지 5개가 포함되어 있을 수 있고, 바람직하게는 2개 내지 3개가 포함되어 있을 수 있다(도 1 참조).The ERα (Estrogen receptor alpha) binding site may contain 1 to 5 in the promoter region of the ovalbumin gene contained in the recombinant promoter, preferably 2 to 3 (FIG. 1). Reference).
본 발명의 구체적인 실시예에서, 본 발명자들은 닭의 오브트렌스페린 유전자의 프로모터 영역의 일부(958 bp)와 닭의 오브알부민 유전자의 프로모터 영역의 일부(2.8 kb)가 순차적으로 연결된 재조합 프로모터를 제조하였다(실시예 1 참조).In a specific embodiment of the present invention, the present inventors prepared a recombinant promoter in which a portion of the promoter region of the chicken's obtransferin gene (958 bp) and a portion of the chicken ovalbumin gene's promoter region (2.8 kb) were sequentially linked. (See Example 1).
본 발명은 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터를 포함하는 벡터를 제공한다. 바람직하게 상기 벡터는 프로모터의 하류(downstream)에 작동 가능하게 연결된 목적 단백질을 암호화하는 유전자를 더 포함할 수 있다.The present invention provides a vector comprising a recombinant promoter comprising a promoter region of the obtransferin gene and a promoter region of the ovalbumin gene. Preferably, the vector may further include a gene encoding a target protein operably linked downstream of the promoter.
상기 재조합 프로모터를 포함하는 벡터는 종래기술의 프로모터를 포함하는 벡터에 비해서 그 크기가 작을 수 있다.The vector containing the recombinant promoter may be smaller in size than the vector containing the prior art promoter.
본 발명에 있어서 "벡터(vector)"는 적합한 숙주 내에서 DNA를 발현시킬 수 있도록 적합한 조절 서열에 작동 가능하게 연결된 DNA 서열을 보유하는 DNA 제조물을 의미한다. 벡터는 플라스미드, 파지 입자, 또는 간단하게 잠재적 게놈 도입물일 수 있다. 적당한 숙주로 형질 전환되면 벡터는 숙주 게놈과 무관하게 복제하고 기능할 수 있거나, 또는 일부 경우에 게놈 그 자체에 통합될 수 있다.In the present invention, "vector (vector)" means a DNA preparation having a DNA sequence operably linked to a suitable regulatory sequence so that the DNA can be expressed in a suitable host. The vector can be a plasmid, phage particle, or simply a potential genome introduction. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself.
상기 벡터는 플라스미드 벡터, 코즈미드 벡터, HIV(Human immunodeficiency virus) 벡터, MLV(Murineleukemia virus) 벡터, ASLV(Avian sarcoma/leukosis) 벡터, SNV(Spleen necrosis virus) 벡터, RSV(Rous sarcoma virus) 벡터, MMTV(Mouse mammary tumor virus) 벡터, 아데노바이러스(Adenovirus) 벡터 및 헤르페스 심플렉스 바이러스(Herpes simplex virus) 벡터, 렌티바이러스(lentivirus) 벡터 및 에피조말(episomal) 벡터로 이루어진 군에서 선택된 어느 하나의 벡터일 수 있다.The vectors include plasmid vectors, cosmid vectors, human immunodeficiency virus (HIV) vectors, Murineleukemia virus (MLV) vectors, Avian sarcoma/leukosis (ASLV) vectors, Spleen necrosis virus (SNV) vectors, Rous sarcoma virus (RSV) vectors, Any vector selected from the group consisting of a mouse mammary tumor virus (MMTV) vector, an adenovirus vector and a herpes simplex virus vector, a lentivirus vector, and an epiisomal vector Can.
본 발명에서 사용된 용어 "발현벡터" 또는 "발현을 위한 벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동 가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다.As used herein, the term "expression vector" or "vector for expression" refers to a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence essential for expressing a coding sequence operably linked in a particular host organism. .
발현벡터는 바람직하게는 하나 이상의 선택성 마커를 포함할 수 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질 전환된 세포를 비 형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 그 예로는 암피실린(ampicilin), 카나마이신(kanamycin), 네오마이신(neomycin), G418, 블레오마이신(Bleomycin), 하이그로마이신(hygromycin), 클로람페니콜(chloramphenicol)과 같은 항생제 내성 유전자가 있으나, 이에 한정되는 것은 아니며, 당업자에 의해 적절히 선택 가능하다.The expression vector may preferably include one or more selectable markers. The marker is a nucleic acid sequence having a property that can be selected by a chemical method, and all genes capable of distinguishing transformed cells from non-transformed cells are included. Examples include, but are not limited to, antibiotic resistance genes such as ampicilin, kanamycin, neomycin, G418, bleomycin, hygromycin, chloramphenicol. No, it can be appropriately selected by those skilled in the art.
본 발명에 있어, "작동 가능하게 연결(operably linked)" 된다는 것은 적절한 분자가 발현 조절 서열에 결합할 때 유전자 발현을 가능하게 하는 방식으로 연결되는 것을 의미한다. 또한, "발현 조절 서열(expression control sequence)"이란, 특정한 숙주 세포에서 작동 가능하게 연결된 폴리뉴클레오티드 서열의 발현을 조절하는 DNA 서열을 의미한다. 그러한 조절 서열은 전사를 실시하기 위한 프로모터, 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합 부위를 코딩하는 서열 및 전사 및 해독의 종결을 조절하는 서열을 포함한다.In the present invention, "operably linked (operably linked)" means that the appropriate molecule is linked in a manner that enables gene expression when binding to the expression control sequence. In addition, "expression control sequence (expression control sequence)" means a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a specific host cell. Such regulatory sequences include promoters for conducting transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that regulate termination of transcription and translation.
본 발명에 있어서, "목적 단백질"이란 본 발명의 프로모터에 의해 고 발현되길 원하는 외부 유래의 어떤 표적 단백질을 말하며, 바람직하게는 외인성 단백질이거나 또는 내인성 단백질 및 리포터 단백질일 수 있다. In the present invention, "target protein" refers to any target protein derived from the outside desired to be highly expressed by the promoter of the present invention, and preferably may be an exogenous protein or an endogenous protein and a reporter protein.
본 발명의 상기 벡터는 형질전환 닭 생산용 바이러스 제조를 위한 목적으로 사용될 수 있다.The vector of the present invention can be used for the purpose of producing a virus for producing transgenic chicken.
본 발명의 구체적인 실시예에서, 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터의 활성을 리포터 분석을 통해 인간세포에서 측정한 결과, 오브알부민 유전자의 프로모터 영역 전체를 포함(에스트로겐 반응성 요소(estrogen responsive element, ERE)하는 4.4 kb 오브알부민 프로모터(미국등록특허 8507749B2))하는 카누마 프로모터에 비해서 프로모터의 활성이 높았다(실험예 1 참조).In a specific embodiment of the present invention, as a result of measuring in a human cell through reporter analysis, the activity of a recombinant promoter comprising a promoter region of the obtransferin gene and a promoter region of the ovalbumin gene, the entire promoter region of the ovalbumin gene (Estrogen responsive element (estrogen responsive element, ERE) 4.4 kb ovalbumin promoter (US Pat. No. 8507749B2)) was higher in the activity of the promoter than the canuma promoter (see Experimental Example 1).
또한, 상기 재조합 프로모터를 사용하여 구축한 벡터를 cOEC(Chicken oviduct epithelial cells)에 도입하여 활성을 비교한 결과, cOEC에서 2.31배 높은 유전자 발현 활성을 나타내는 것을 확인하였다(실험예 2 참조).In addition, as a result of introducing the vector constructed using the recombinant promoter into cOEC (Chicken oviduct epithelial cells) and comparing the activity, it was confirmed that it shows a gene expression activity 2.31 times higher in cOEC (see Experimental Example 2).
따라서, 본 발명의 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터가 포함된 벡터는 종래기술의 프로모터가 포함된 벡터에 비해서 그 크기가 작고, 종래기술의 프로모터가 포함된 벡터에 비해서 목적 단백질을 높은 효율로 과발현시킬 수 있다.Therefore, the vector containing the recombinant promoter comprising the promoter region of the obtransferin gene and the promoter region of the ovalbumin gene of the present invention is smaller in size than the vector containing the prior art promoter, and the prior art promoter is included. The target protein can be overexpressed with higher efficiency than the vector.
또한, 본 발명은 상기 재조합 프로모터를 포함하는 벡터에 의해 형질전환된 닭을 제공한다.In addition, the present invention provides a chicken transformed with a vector containing the recombinant promoter.
본 발명에서 형질전환된 닭을 제조하기 위해서, 상기 재조합 프로모터를 포함하는 형질전환 닭 생산용 바이러스 제조를 위한 벡터를 닭의 배반엽단계 수정란에 미세 주입을 통하여 도입할 수 있다. 상기 수정란의 배양 및 이식을 통해 제1세대 후보 형질전환 병아리(mosaic, G0)를 생산하고, 생산된 수컷 후보 중 성선에 그 형질이 전달(Germline transmission)된 개체를 선발하여 인공수정을 통해 제1세대 형질전환 닭(G1)을 생산하고 G1 개체를 포함하는 교배 계획을 통해 재조합 프로모터 및 목적 유전자를 가지는 제2세대(G2) 및 그 이후 세대의 형질전환 닭을 계속 생산할 수 있다.In order to prepare a transgenic chicken in the present invention, a vector for producing a virus for producing a transgenic chicken containing the recombinant promoter can be introduced through micro-injection into the embryonic stage of the embryonic stage of the chicken. Producing first-generation candidate transgenic chicks (mosaic, G0) through cultivation and transplantation of the fertilized eggs, and selecting the individual whose trait is transmitted to the sex glands from the male candidates produced through artificial insemination The generation of transgenic chickens (G1) and the generation of transgenic chickens of the second generation (G2) and subsequent generations with recombinant promoters and target genes can be continued through a breeding scheme involving G1 individuals.
본 발명은 목적 유전자를 포함하는 상기 재조합 벡터를 닭에 형질전환하여, 닭의 상피세포에서 목적 단백질을 발현시키는 방법을 제공한다.The present invention provides a method of expressing a target protein in epithelial cells of a chicken by transforming the recombinant vector containing the target gene in chicken.
본 발명에서 상기 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터는 하류(downstream)에 작동 가능하게 연결된 목적 단백질을 암호화하는 유전자를 더 포함할 수 있고, 상기 재조합 프로모터를 닭에 형질전환시키는 경우, 목적 단백질을 닭의 상피세포에서 발현시킬 수 있다.In the present invention, the recombinant promoter comprising the promoter region of the obtransferin gene and the promoter region of the ovalbumin gene may further include a gene encoding a target protein operably linked downstream, and the recombinant promoter When transformed into chicken, the target protein can be expressed in chicken epithelial cells.
이하, 본 발명을 실시예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples.
단, 하기 실시예는 본 발명을 예시하는 것이며, 본 발명의 내용이 실시예에 의해 한정되는 것은 아니다.However, the following examples illustrate the present invention, and the contents of the present invention are not limited by the examples.
실시예 1: 닭에서 유용단백질 발현을 위한 재조합 프로모터 제작Example 1: Construction of recombinant promoter for expression of useful protein in chickens
닭의 계란에서 유용단백질 발현을 위해 도 1과 같이 오브트렌스페린 유전자의 프로모터 영역과 오브알부민 유전자의 프로모터 영역이 결합된 재조합 프로모터를 제작하였다.For expression of useful proteins in chicken eggs, a recombinant promoter was prepared in which the promoter region of the obtransferin gene and the promoter region of the ovalbumin gene were combined as shown in FIG. 1.
구체적으로, ThermoFisher사에 의뢰하여 5'에서 3' 방향으로 Nhe I 제한효소 인식 서열, 닭의 오브트렌스페린 유전자의 프로모터 영역(958 bp)(서열번호 1), 닭의 오브알부민 유전자의 프로모터 영역(2784 bp)(서열번호 2), Xho I 제한효소 인식 서열이 순차적으로 연결된 폴리뉴클레오티드를 합성하였다. 상기 제조한 폴리뉴클레오티드에 Nhe I 및 Xho I 제한효소를 처리하고 동일한 효소를 처리한 리포터 분석용 벡터(pGL4.11, Promega)에 도입하였다(OVOT-pOV). 5'에서 3' 방향으로 닭의 오브트렌스페린 유전자의 프로모터 영역(958 bp)(서열번호 1), 및 닭의 오브알부민 유전자의 프로모터 영역(2784 bp)(서열번호 2)이 결합된 재조합 프로모터의 염기서열은 서열번호 3에 개시되어 있다.Specifically, Nhe I restriction enzyme recognition sequence in the 5'to 3'direction, commissioned by ThermoFisher, the promoter region of the chicken's obtransferin gene (958 bp) (SEQ ID NO: 1), the promoter region of the chicken's ovalbumin gene ( 2784 bp) (SEQ ID NO: 2), Xho I restriction enzyme recognition sequence was sequentially linked polynucleotides were synthesized. Nhe I and Xho I restriction enzymes were treated with the prepared polynucleotide and introduced into a reporter analysis vector (pGL4.11, Promega) treated with the same enzyme (OVOT-pOV). In the 5'to 3'direction, the promoter region (958 bp) of the chicken's obtransferin gene (SEQ ID NO: 1) and the promoter region (2784 bp) of the chicken ovalbumin gene (SEQ ID NO: 2) were combined The base sequence is shown in SEQ ID NO: 3.
또한, 공지된 대조군 벡터(카누마, Kanuma)를 제조하기 위해서 ERE(estrogen responsive element)를 포함하는 1.6 kb 크기의 닭 오브알부민 유전자의 프로모터 영역과 2.8 kb 크기의 닭 오브알부민 유전자의 프로모터 영역이 연결된 4.4 kb 오브알부민 프로모터(미국등록특허 8507749B2)를 상기와 동일한 방식으로 합성하고 리포터 분석용 벡터(pGL4.11)에 클로닝하여 대조군 벡터를 제조하였다. 닭의 오브트렌스페린 유전자의 프로모터 영역과 닭의 오브알부민 유전자의 프로모터 영역이 재조합된 벡터(OVOT-pOV)와 오브알부민 유전자의 프로모터 영역을 포함하는 대조군 벡터(kanuma)를 비교하여 도 3에 나타냈다.In addition, to prepare a known control vector (Kanuma, Kanuma), the promoter region of the chicken ovalbumin gene of 1.6 kb size and the promoter region of the chicken ovalbumin gene size of 2.8 kb, which contains an etrogen responsive element (ERE), are linked. A control vector was prepared by synthesizing a 4.4 kb ovalbumin promoter (U.S. Patent No. 8507749B2) in the same manner as above and cloning it into a reporter analysis vector (pGL4.11). The promoter region of the chicken's obtransferin gene and the chicken ovalbumin gene promoter region (OVOT-pOV) and a control vector (kanuma) containing the promoter region of the ovalbumin gene are compared and shown in FIG. 3.
구축한 재조합 프로모터 벡터들은 염기서열 분석을 통하여 클로닝하고자 한 상기 프로모터들이 정상적으로 삽입되었음을 확인하였다.The constructed recombinant promoter vectors were confirmed that the promoters to be cloned were successfully inserted through sequencing.
실험예 1: 인간 세포에서 재조합 프로모터의 활성 검증Experimental Example 1: Validation of recombinant promoter activity in human cells
실시예 1에서 제조한 두 가지 프로모터(Kanuma, OVOT-pOV) 및 음성대조군인 공벡터(Mock, pGL4.11)를 지질 매개성 유전자 도입 방식으로 HeLa(Human cervix epithelial cells) 세포, 또는 MES-SA(Human uterus epithelial cells) 세포에 도입하고, 24시간 후에 luciferase assay kit(Promega)로 제조사의 프로토콜에 따라 각 프로모터의 활성을 분석하였다.Two promoters (Kanuma, OVOT-pOV) prepared in Example 1 and a negative control vector (Mock, pGL4.11) were introduced into lipid-mediated gene in HeLa (Human cervix epithelial cells) cells, or MES-SA (Human uterus epithelial cells) was introduced into cells, and after 24 hours, the activity of each promoter was analyzed according to the manufacturer's protocol with a luciferase assay kit (Promega).
그 결과 HeLa 세포에서 OVOT-pOV 프로모터의 활성은 대조군인 카누마 프로모터의 활성보다 높았고(도 4), MES-SA 세포에서 OVOT-pOV 프로모터의 활성은 대조군인 카누마 프로모터의 활성과 거의 유사했다(도 5).As a result, the activity of the OVOT-pOV promoter in HeLa cells was higher than that of the control Kanuma promoter (FIG. 4), and the activity of the OVOT-pOV promoter in MES-SA cells was almost similar to that of the control Kanuma promoter ( Fig. 5).
실험예 2: 닭의 세포에서 재조합 프로모터의 활성 검증Experimental Example 2: Validation of the activity of the recombinant promoter in chicken cells
실시예 1에서 제조한 두 가지 프로모터(Kanuma, OVOT-pOV) 및 음성대조군인 공벡터(Mock, pGL4.11)를 지질 매개성 유전자 도입 방식으로 cOEC(Chicken oviduct epithelial cells)에 도입하고, 24시간 후에 luciferase assay kit(Promega)로 제조사의 프로토콜에 따라 각 프로모터의 활성을 분석하였다.Two promoters prepared in Example 1 (Kanuma, OVOT-pOV) and a negative control vector (Mock, pGL4.11) were introduced into cOEC (Chicken oviduct epithelial cells) by lipid-mediated gene introduction, and 24 hours. Afterwards, the activity of each promoter was analyzed according to the manufacturer's protocol with a luciferase assay kit (Promega).
그 결과, cOEC에서 2.31배 높은 유전자 발현 활성을 나타내는 것을 확인하였다(도 6).As a result, it was confirmed that cOEC exhibited a 2.31-fold higher gene expression activity (FIG. 6).
따라서, 본 발명의 오브트렌스페린 유전자의 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터가 포함된 벡터는 종래기술의 프로모터가 포함된 벡터에 비해서 그 크기가 작고, 종래기술의 프로모터가 포함된 벡터에 비해서 목적 단백질을 높은 효율로 과발현시킬 수 있다.Therefore, the vector containing the recombinant promoter comprising the promoter region of the obtransferin gene and the promoter region of the ovalbumin gene of the present invention is smaller in size than the vector containing the prior art promoter, and the prior art promoter is included. The target protein can be overexpressed with higher efficiency than the vector.
<110> RURAL DEVELOPMENT ADMINISTRATION <120> Recombinant promoter comprising the promoter region of the ovotransferrin gene and the promoter region of the ovalbumin gene <130> 2019p-06-023 <150> KR 10-2018-0162197 <151> 2018-12-14 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 958 <212> DNA <213> Artificial Sequence <220> <223> ovotransferrin promoter DNA sequence <400> 1 gtttggtatt aatgattacc atgagttagc taactggaaa ggggctgaac agcaaatggt 60 ggagttcacg ttgacctatt ggtgacctga cccctttccc gctcgcttga tgcccaaacc 120 tcatttctgg ctgcagaacc atcttgcagc tgaacacaat gcctggtggg ggctgtgcca 180 gcccagctgg gctgcccatg ggctgtgcag ggcaatgaga ggctcagccc ctgctccttg 240 ggcctgttcc agcagcgttg cgcactgcca gccccaggat gtccaaacag cagcggaagg 300 atctcccaaa acggaggaca tagctgaatg cacccttgca taggtgcagt ggatgtggtt 360 cagatctggg tgcaccttga gtctggctgt gatgtcctat gatccgctct gccgccatct 420 cgttcctatc cccttcctgc acagacactg ccagtggtct gggctctgtc accgcggggg 480 gacgtctgtg ctcctgctcc ttctgcccag tacctccatg tccttgtgag ccccagcttc 540 cctctgcact ggaaacaacg tggtgttggg ccccctgagc caggtgaccc tgctggagca 600 gggattgggc cagaaggacc cgaccacagc cattttgttc ctccgtgaaa ttagccacct 660 tctcttatgt tctgttcctc caggctggcc cagcatctcc ccacaggcat atgttggtag 720 tacttactga tattgatagt ctttctttcc tgattatcaa tatttgctgg tttgttccca 780 ccaatcttta ggaaataccc aggaagcagg catgaacaga gctccagcag ctccatgatt 840 ccagcaggag ccatcagccc accgtgcctt cctgtgttcc cttcccaaag cccaccagga 900 acgtcgcagt tcccagggct gcacagagct gctggtaacc ctgttaacct ttgggtta 958 <210> 2 <211> 2784 <212> DNA <213> Artificial Sequence <220> <223> ovalbumin promoter DNA sequence <400> 2 ttaagtcctc agacttggca aggagaatgt agatttctac agtatatatg ttttcacaaa 60 aggaaggaga gaaacaaaag aaaatggcac tgactaaact tcagctagtg gtataggaaa 120 gtaattctgc ttaacagaga ttgcagtgat ctctatgtat gtcctgaaga attatgttgt 180 acttttttcc ctcattttta aatcaaacag tgctttacag aggtcagaat ggtttcttta 240 ctgtttgtca attctattat ttcaatacag aacaatagct tctataactg aaatatattt 300 gctattgtat attatgattg tccctcgaac catgaacact cctccagctg aatttcacaa 360 ttcctctgtc atctgccagg ccattaagtt attcatggaa gatctttgag gaacactgca 420 agttcatatc ataaacacat ttgaaattga gtattgtttt gcattgtatg gagctatgtt 480 ttgctgtatc ctcagaaaaa aagtttgtta taaagcattc acacccataa aaagatagat 540 ttaaatattc cagctatagg aaagaaagtg cgtctgctct tcactctagt ctcagttggc 600 tccttcacat gcacgcttct ttatttctcc tattttgtca agaaaataat aggtcacgtc 660 ttgttctcac ttatgtcctg cctagcatgg ctcagatgca cgttgtacat acaagaagga 720 tcaaatgaaa cagacttctg gtctgttact acaaccatag taataagcac actaactaat 780 aattgctaat tatgttttcc atctccaagg ttcccacatt tttctgtttt cttaaagatc 840 ccattatctg gttgtaactg aagctcaatg gaacatgagc aatatttccc agtcttctct 900 cccatccaac agtcctgatg gattagcaga acaggcagaa aacacattgt tacccagaat 960 taaaaactaa tatttgctct ccattcaatc caaaatggac ctattgaaac taaaatctaa 1020 cccaatccca ttaaatgatt tctatggcgt caaaggtcaa acttctgaag ggaacctgtg 1080 ggtgggtcac aattcaggct atatattccc cagggctcag ccagtgtctg tacatacagc 1140 tagaaagctg tattgccttt agcactcaag ctcaaaaggt aagcaactct ctggaattac 1200 cttctctcta tattagctct tacttgcacc taaactttaa aaaattaaca attattgtgt 1260 tatgtgttgt atctttaagg gtgaagtacc tgcgtgatac cccctataaa aacttctcac 1320 ctgtgtatgc attctgcact attttattat gtgtaaaagc tttgtgtttg ttttcaggag 1380 gcttattctt tgtgcttaaa atatgttttt aatttcagaa catcttatcc tgtcgttcac 1440 tatctgatat gctttgcagt ttgcctgatt aacttctagc cctacagagt gcacagagag 1500 caaaatcatg gtgttcagtg aattctgggg agttatttta atgtgaaaat tctctagaag 1560 tttaattcct gcaaagtgca gctgctgatc actacacaag ataaaaatgt ggggggtgca 1620 taaacgtata ttcttacaat aatagataca tgtgaacttg tatacagaaa agaaaatgag 1680 aaaaatgtgt gtgcgtatac tcacacacgt ggtcagtaaa aacttttgag gggtttaata 1740 cagaaaatcc aatcctgagg ccccagcact cagtacgcat ataaagggct gggctctgaa 1800 ggacttctga ctttcacaga ttatataaat ctcaggaaag caactagatt catgctggct 1860 ccaaaagctg tgctttatat aagcacactg gctatacaat agttgtacag ttcagctctt 1920 tataatagaa acagacagaa caagtataaa tcttctattg gtctatgtca tgaacaagaa 1980 ttcattcagt ggctctgttt tatagtaaac attgctattt tatcatgtct gcatttctct 2040 tctgtctgaa tgtcaccact aaaatttaac tccacagaaa gtttatacta cagtacacat 2100 gcatatcttt gagcaaagca aaccatacct gaaagtgcaa tagagcagaa tatgaattac 2160 atgcgtgtct ttctcctaga ctacatgacc ccatataaat tacattcctt atctattctg 2220 ccatcaccaa aacaaaggta aaaatacttt tgaagatcta ctcatagcaa gtagtgtgca 2280 acaaacagat atttctctac atttattttt agggaataaa aataagaaat aaaatagtca 2340 gcaagcctct gctttctcat atatctgtcc aaacctaaag tttactgaaa tttgctcttt 2400 gaatttccag ttttgcaagc ctatcagatt gtgttttaat cagaggtact gaaaagtatc 2460 aatgaattct agctttcact gaacaaaaat atgtagaggc aactggcttc tgggacagtt 2520 tgctacccaa aagacaactg aatgcaaata cataaataga tttatgaata tggttttgaa 2580 catgcacatg agaggtggat atagcaacag acacattacc acagaattac tttaaaacta 2640 cttgttaaca tttaattgcc taaaaactgc tcgtaattta ctgttgtagc ctaccataga 2700 gtaccctgca tggtactatg tacagcattc catccttaca ttttcactgt tctgctgttt 2760 gctctagaca actcagagtt cacc 2784 <210> 3 <211> 3742 <212> DNA <213> Artificial Sequence <220> <223> Recombinant promoter DNA sequence <400> 3 gtttggtatt aatgattacc atgagttagc taactggaaa ggggctgaac agcaaatggt 60 ggagttcacg ttgacctatt ggtgacctga cccctttccc gctcgcttga tgcccaaacc 120 tcatttctgg ctgcagaacc atcttgcagc tgaacacaat gcctggtggg ggctgtgcca 180 gcccagctgg gctgcccatg ggctgtgcag ggcaatgaga ggctcagccc ctgctccttg 240 ggcctgttcc agcagcgttg cgcactgcca gccccaggat gtccaaacag cagcggaagg 300 atctcccaaa acggaggaca tagctgaatg cacccttgca taggtgcagt ggatgtggtt 360 cagatctggg tgcaccttga gtctggctgt gatgtcctat gatccgctct gccgccatct 420 cgttcctatc cccttcctgc acagacactg ccagtggtct gggctctgtc accgcggggg 480 gacgtctgtg ctcctgctcc ttctgcccag tacctccatg tccttgtgag ccccagcttc 540 cctctgcact ggaaacaacg tggtgttggg ccccctgagc caggtgaccc tgctggagca 600 gggattgggc cagaaggacc cgaccacagc cattttgttc ctccgtgaaa ttagccacct 660 tctcttatgt tctgttcctc caggctggcc cagcatctcc ccacaggcat atgttggtag 720 tacttactga tattgatagt ctttctttcc tgattatcaa tatttgctgg tttgttccca 780 ccaatcttta ggaaataccc aggaagcagg catgaacaga gctccagcag ctccatgatt 840 ccagcaggag ccatcagccc accgtgcctt cctgtgttcc cttcccaaag cccaccagga 900 acgtcgcagt tcccagggct gcacagagct gctggtaacc ctgttaacct ttgggttatt 960 aagtcctcag acttggcaag gagaatgtag atttctacag tatatatgtt ttcacaaaag 1020 gaaggagaga aacaaaagaa aatggcactg actaaacttc agctagtggt ataggaaagt 1080 aattctgctt aacagagatt gcagtgatct ctatgtatgt cctgaagaat tatgttgtac 1140 ttttttccct catttttaaa tcaaacagtg ctttacagag gtcagaatgg tttctttact 1200 gtttgtcaat tctattattt caatacagaa caatagcttc tataactgaa atatatttgc 1260 tattgtatat tatgattgtc cctcgaacca tgaacactcc tccagctgaa tttcacaatt 1320 cctctgtcat ctgccaggcc attaagttat tcatggaaga tctttgagga acactgcaag 1380 ttcatatcat aaacacattt gaaattgagt attgttttgc attgtatgga gctatgtttt 1440 gctgtatcct cagaaaaaaa gtttgttata aagcattcac acccataaaa agatagattt 1500 aaatattcca gctataggaa agaaagtgcg tctgctcttc actctagtct cagttggctc 1560 cttcacatgc acgcttcttt atttctccta ttttgtcaag aaaataatag gtcacgtctt 1620 gttctcactt atgtcctgcc tagcatggct cagatgcacg ttgtacatac aagaaggatc 1680 aaatgaaaca gacttctggt ctgttactac aaccatagta ataagcacac taactaataa 1740 ttgctaatta tgttttccat ctccaaggtt cccacatttt tctgttttct taaagatccc 1800 attatctggt tgtaactgaa gctcaatgga acatgagcaa tatttcccag tcttctctcc 1860 catccaacag tcctgatgga ttagcagaac aggcagaaaa cacattgtta cccagaatta 1920 aaaactaata tttgctctcc attcaatcca aaatggacct attgaaacta aaatctaacc 1980 caatcccatt aaatgatttc tatggcgtca aaggtcaaac ttctgaaggg aacctgtggg 2040 tgggtcacaa ttcaggctat atattcccca gggctcagcc agtgtctgta catacagcta 2100 gaaagctgta ttgcctttag cactcaagct caaaaggtaa gcaactctct ggaattacct 2160 tctctctata ttagctctta cttgcaccta aactttaaaa aattaacaat tattgtgtta 2220 tgtgttgtat ctttaagggt gaagtacctg cgtgataccc cctataaaaa cttctcacct 2280 gtgtatgcat tctgcactat tttattatgt gtaaaagctt tgtgtttgtt ttcaggaggc 2340 ttattctttg tgcttaaaat atgtttttaa tttcagaaca tcttatcctg tcgttcacta 2400 tctgatatgc tttgcagttt gcctgattaa cttctagccc tacagagtgc acagagagca 2460 aaatcatggt gttcagtgaa ttctggggag ttattttaat gtgaaaattc tctagaagtt 2520 taattcctgc aaagtgcagc tgctgatcac tacacaagat aaaaatgtgg ggggtgcata 2580 aacgtatatt cttacaataa tagatacatg tgaacttgta tacagaaaag aaaatgagaa 2640 aaatgtgtgt gcgtatactc acacacgtgg tcagtaaaaa cttttgaggg gtttaataca 2700 gaaaatccaa tcctgaggcc ccagcactca gtacgcatat aaagggctgg gctctgaagg 2760 acttctgact ttcacagatt atataaatct caggaaagca actagattca tgctggctcc 2820 aaaagctgtg ctttatataa gcacactggc tatacaatag ttgtacagtt cagctcttta 2880 taatagaaac agacagaaca agtataaatc ttctattggt ctatgtcatg aacaagaatt 2940 cattcagtgg ctctgtttta tagtaaacat tgctatttta tcatgtctgc atttctcttc 3000 tgtctgaatg tcaccactaa aatttaactc cacagaaagt ttatactaca gtacacatgc 3060 atatctttga gcaaagcaaa ccatacctga aagtgcaata gagcagaata tgaattacat 3120 gcgtgtcttt ctcctagact acatgacccc atataaatta cattccttat ctattctgcc 3180 atcaccaaaa caaaggtaaa aatacttttg aagatctact catagcaagt agtgtgcaac 3240 aaacagatat ttctctacat ttatttttag ggaataaaaa taagaaataa aatagtcagc 3300 aagcctctgc tttctcatat atctgtccaa acctaaagtt tactgaaatt tgctctttga 3360 atttccagtt ttgcaagcct atcagattgt gttttaatca gaggtactga aaagtatcaa 3420 tgaattctag ctttcactga acaaaaatat gtagaggcaa ctggcttctg ggacagtttg 3480 ctacccaaaa gacaactgaa tgcaaataca taaatagatt tatgaatatg gttttgaaca 3540 tgcacatgag aggtggatat agcaacagac acattaccac agaattactt taaaactact 3600 tgttaacatt taattgccta aaaactgctc gtaatttact gttgtagcct accatagagt 3660 accctgcatg gtactatgta cagcattcca tccttacatt ttcactgttc tgctgtttgc 3720 tctagacaac tcagagttca cc 3742 <110> RURAL DEVELOPMENT ADMINISTRATION <120> Recombinant promoter comprising the promoter region of the ovotransferrin gene and the promoter region of the ovalbumin gene <130> 2019p-06-023 <150> KR 10-2018-0162197 <151> 2018-12-14 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 958 <212> DNA <213> Artificial Sequence <220> <223> ovotransferrin promoter DNA sequence <400> 1 gtttggtatt aatgattacc atgagttagc taactggaaa ggggctgaac agcaaatggt 60 ggagttcacg ttgacctatt ggtgacctga cccctttccc gctcgcttga tgcccaaacc 120 tcatttctgg ctgcagaacc atcttgcagc tgaacacaat gcctggtggg ggctgtgcca 180 gcccagctgg gctgcccatg ggctgtgcag ggcaatgaga ggctcagccc ctgctccttg 240 ggcctgttcc agcagcgttg cgcactgcca gccccaggat gtccaaacag cagcggaagg 300 atctcccaaa acggaggaca tagctgaatg cacccttgca taggtgcagt ggatgtggtt 360 cagatctggg tgcaccttga gtctggctgt gatgtcctat gatccgctct gccgccatct 420 cgttcctatc cccttcctgc acagacactg ccagtggtct gggctctgtc accgcggggg 480 gacgtctgtg ctcctgctcc ttctgcccag tacctccatg tccttgtgag ccccagcttc 540 cctctgcact ggaaacaacg tggtgttggg ccccctgagc caggtgaccc tgctggagca 600 gggattgggc cagaaggacc cgaccacagc cattttgttc ctccgtgaaa ttagccacct 660 tctcttatgt tctgttcctc caggctggcc cagcatctcc ccacaggcat atgttggtag 720 tacttactga tattgatagt ctttctttcc tgattatcaa tatttgctgg tttgttccca 780 ccaatcttta ggaaataccc aggaagcagg catgaacaga gctccagcag ctccatgatt 840 ccagcaggag ccatcagccc accgtgcctt cctgtgttcc cttcccaaag cccaccagga 900 acgtcgcagt tcccagggct gcacagagct gctggtaacc ctgttaacct ttgggtta 958 <210> 2 <211> 2784 <212> DNA <213> Artificial Sequence <220> <223> ovalbumin promoter DNA sequence <400> 2 ttaagtcctc agacttggca aggagaatgt agatttctac agtatatatg ttttcacaaa 60 aggaaggaga gaaacaaaag aaaatggcac tgactaaact tcagctagtg gtataggaaa 120 gtaattctgc ttaacagaga ttgcagtgat ctctatgtat gtcctgaaga attatgttgt 180 acttttttcc ctcattttta aatcaaacag tgctttacag aggtcagaat ggtttcttta 240 ctgtttgtca attctattat ttcaatacag aacaatagct tctataactg aaatatattt 300 gctattgtat attatgattg tccctcgaac catgaacact cctccagctg aatttcacaa 360 ttcctctgtc atctgccagg ccattaagtt attcatggaa gatctttgag gaacactgca 420 agttcatatc ataaacacat ttgaaattga gtattgtttt gcattgtatg gagctatgtt 480 ttgctgtatc ctcagaaaaa aagtttgtta taaagcattc acacccataa aaagatagat 540 ttaaatattc cagctatagg aaagaaagtg cgtctgctct tcactctagt ctcagttggc 600 tccttcacat gcacgcttct ttatttctcc tattttgtca agaaaataat aggtcacgtc 660 ttgttctcac ttatgtcctg cctagcatgg ctcagatgca cgttgtacat acaagaagga 720 tcaaatgaaa cagacttctg gtctgttact acaaccatag taataagcac actaactaat 780 aattgctaat tatgttttcc atctccaagg ttcccacatt tttctgtttt cttaaagatc 840 ccattatctg gttgtaactg aagctcaatg gaacatgagc aatatttccc agtcttctct 900 cccatccaac agtcctgatg gattagcaga acaggcagaa aacacattgt tacccagaat 960 taaaaactaa tatttgctct ccattcaatc caaaatggac ctattgaaac taaaatctaa 1020 cccaatccca ttaaatgatt tctatggcgt caaaggtcaa acttctgaag ggaacctgtg 1080 ggtgggtcac aattcaggct atatattccc cagggctcag ccagtgtctg tacatacagc 1140 tagaaagctg tattgccttt agcactcaag ctcaaaaggt aagcaactct ctggaattac 1200 cttctctcta tattagctct tacttgcacc taaactttaa aaaattaaca attattgtgt 1260 tatgtgttgt atctttaagg gtgaagtacc tgcgtgatac cccctataaa aacttctcac 1320 ctgtgtatgc attctgcact attttattat gtgtaaaagc tttgtgtttg ttttcaggag 1380 gcttattctt tgtgcttaaa atatgttttt aatttcagaa catcttatcc tgtcgttcac 1440 tatctgatat gctttgcagt ttgcctgatt aacttctagc cctacagagt gcacagagag 1500 caaaatcatg gtgttcagtg aattctgggg agttatttta atgtgaaaat tctctagaag 1560 tttaattcct gcaaagtgca gctgctgatc actacacaag ataaaaatgt ggggggtgca 1620 taaacgtata ttcttacaat aatagataca tgtgaacttg tatacagaaa agaaaatgag 1680 aaaaatgtgt gtgcgtatac tcacacacgt ggtcagtaaa aacttttgag gggtttaata 1740 cagaaaatcc aatcctgagg ccccagcact cagtacgcat ataaagggct gggctctgaa 1800 ggacttctga ctttcacaga ttatataaat ctcaggaaag caactagatt catgctggct 1860 ccaaaagctg tgctttatat aagcacactg gctatacaat agttgtacag ttcagctctt 1920 tataatagaa acagacagaa caagtataaa tcttctattg gtctatgtca tgaacaagaa 1980 ttcattcagt ggctctgttt tatagtaaac attgctattt tatcatgtct gcatttctct 2040 tctgtctgaa tgtcaccact aaaatttaac tccacagaaa gtttatacta cagtacacat 2100 gcatatcttt gagcaaagca aaccatacct gaaagtgcaa tagagcagaa tatgaattac 2160 atgcgtgtct ttctcctaga ctacatgacc ccatataaat tacattcctt atctattctg 2220 ccatcaccaa aacaaaggta aaaatacttt tgaagatcta ctcatagcaa gtagtgtgca 2280 acaaacagat atttctctac atttattttt agggaataaa aataagaaat aaaatagtca 2340 gcaagcctct gctttctcat atatctgtcc aaacctaaag tttactgaaa tttgctcttt 2400 gaatttccag ttttgcaagc ctatcagatt gtgttttaat cagaggtact gaaaagtatc 2460 aatgaattct agctttcact gaacaaaaat atgtagaggc aactggcttc tgggacagtt 2520 tgctacccaa aagacaactg aatgcaaata cataaataga tttatgaata tggttttgaa 2580 catgcacatg agaggtggat atagcaacag acacattacc acagaattac tttaaaacta 2640 cttgttaaca tttaattgcc taaaaactgc tcgtaattta ctgttgtagc ctaccataga 2700 gtaccctgca tggtactatg tacagcattc catccttaca ttttcactgt tctgctgttt 2760 gctctagaca actcagagtt cacc 2784 <210> 3 <211> 3742 <212> DNA <213> Artificial Sequence <220> <223> Recombinant promoter DNA sequence <400> 3 gtttggtatt aatgattacc atgagttagc taactggaaa ggggctgaac agcaaatggt 60 ggagttcacg ttgacctatt ggtgacctga cccctttccc gctcgcttga tgcccaaacc 120 tcatttctgg ctgcagaacc atcttgcagc tgaacacaat gcctggtggg ggctgtgcca 180 gcccagctgg gctgcccatg ggctgtgcag ggcaatgaga ggctcagccc ctgctccttg 240 ggcctgttcc agcagcgttg cgcactgcca gccccaggat gtccaaacag cagcggaagg 300 atctcccaaa acggaggaca tagctgaatg cacccttgca taggtgcagt ggatgtggtt 360 cagatctggg tgcaccttga gtctggctgt gatgtcctat gatccgctct gccgccatct 420 cgttcctatc cccttcctgc acagacactg ccagtggtct gggctctgtc accgcggggg 480 gacgtctgtg ctcctgctcc ttctgcccag tacctccatg tccttgtgag ccccagcttc 540 cctctgcact ggaaacaacg tggtgttggg ccccctgagc caggtgaccc tgctggagca 600 gggattgggc cagaaggacc cgaccacagc cattttgttc ctccgtgaaa ttagccacct 660 tctcttatgt tctgttcctc caggctggcc cagcatctcc ccacaggcat atgttggtag 720 tacttactga tattgatagt ctttctttcc tgattatcaa tatttgctgg tttgttccca 780 ccaatcttta ggaaataccc aggaagcagg catgaacaga gctccagcag ctccatgatt 840 ccagcaggag ccatcagccc accgtgcctt cctgtgttcc cttcccaaag cccaccagga 900 acgtcgcagt tcccagggct gcacagagct gctggtaacc ctgttaacct ttgggttatt 960 aagtcctcag acttggcaag gagaatgtag atttctacag tatatatgtt ttcacaaaag 1020 gaaggagaga aacaaaagaa aatggcactg actaaacttc agctagtggt ataggaaagt 1080 aattctgctt aacagagatt gcagtgatct ctatgtatgt cctgaagaat tatgttgtac 1140 ttttttccct catttttaaa tcaaacagtg ctttacagag gtcagaatgg tttctttact 1200 gtttgtcaat tctattattt caatacagaa caatagcttc tataactgaa atatatttgc 1260 tattgtatat tatgattgtc cctcgaacca tgaacactcc tccagctgaa tttcacaatt 1320 cctctgtcat ctgccaggcc attaagttat tcatggaaga tctttgagga acactgcaag 1380 ttcatatcat aaacacattt gaaattgagt attgttttgc attgtatgga gctatgtttt 1440 gctgtatcct cagaaaaaaa gtttgttata aagcattcac acccataaaa agatagattt 1500 aaatattcca gctataggaa agaaagtgcg tctgctcttc actctagtct cagttggctc 1560 cttcacatgc acgcttcttt atttctccta ttttgtcaag aaaataatag gtcacgtctt 1620 gttctcactt atgtcctgcc tagcatggct cagatgcacg ttgtacatac aagaaggatc 1680 aaatgaaaca gacttctggt ctgttactac aaccatagta ataagcacac taactaataa 1740 ttgctaatta tgttttccat ctccaaggtt cccacatttt tctgttttct taaagatccc 1800 attatctggt tgtaactgaa gctcaatgga acatgagcaa tatttcccag tcttctctcc 1860 catccaacag tcctgatgga ttagcagaac aggcagaaaa cacattgtta cccagaatta 1920 aaaactaata tttgctctcc attcaatcca aaatggacct attgaaacta aaatctaacc 1980 caatcccatt aaatgatttc tatggcgtca aaggtcaaac ttctgaaggg aacctgtggg 2040 tgggtcacaa ttcaggctat atattcccca gggctcagcc agtgtctgta catacagcta 2100 gaaagctgta ttgcctttag cactcaagct caaaaggtaa gcaactctct ggaattacct 2160 tctctctata ttagctctta cttgcaccta aactttaaaa aattaacaat tattgtgtta 2220 tgtgttgtat ctttaagggt gaagtacctg cgtgataccc cctataaaaa cttctcacct 2280 gtgtatgcat tctgcactat tttattatgt gtaaaagctt tgtgtttgtt ttcaggaggc 2340 ttattctttg tgcttaaaat atgtttttaa tttcagaaca tcttatcctg tcgttcacta 2400 tctgatatgc tttgcagttt gcctgattaa cttctagccc tacagagtgc acagagagca 2460 aaatcatggt gttcagtgaa ttctggggag ttattttaat gtgaaaattc tctagaagtt 2520 taattcctgc aaagtgcagc tgctgatcac tacacaagat aaaaatgtgg ggggtgcata 2580 aacgtatatt cttacaataa tagatacatg tgaacttgta tacagaaaag aaaatgagaa 2640 aaatgtgtgt gcgtatactc acacacgtgg tcagtaaaaa cttttgaggg gtttaataca 2700 gaaaatccaa tcctgaggcc ccagcactca gtacgcatat aaagggctgg gctctgaagg 2760 acttctgact ttcacagatt atataaatct caggaaagca actagattca tgctggctcc 2820 aaaagctgtg ctttatataa gcacactggc tatacaatag ttgtacagtt cagctcttta 2880 taatagaaac agacagaaca agtataaatc ttctattggt ctatgtcatg aacaagaatt 2940 cattcagtgg ctctgtttta tagtaaacat tgctatttta tcatgtctgc atttctcttc 3000 tgtctgaatg tcaccactaa aatttaactc cacagaaagt ttatactaca gtacacatgc 3060 atatctttga gcaaagcaaa ccatacctga aagtgcaata gagcagaata tgaattacat 3120 gcgtgtcttt ctcctagact acatgacccc atataaatta cattccttat ctattctgcc 3180 atcaccaaaa caaaggtaaa aatacttttg aagatctact catagcaagt agtgtgcaac 3240 aaacagatat ttctctacat ttatttttag ggaataaaaa taagaaataa aatagtcagc 3300 aagcctctgc tttctcatat atctgtccaa acctaaagtt tactgaaatt tgctctttga 3360 atttccagtt ttgcaagcct atcagattgt gttttaatca gaggtactga aaagtatcaa 3420 tgaattctag ctttcactga acaaaaatat gtagaggcaa ctggcttctg ggacagtttg 3480 ctacccaaaa gacaactgaa tgcaaataca taaatagatt tatgaatatg gttttgaaca 3540 tgcacatgag aggtggatat agcaacagac acattaccac agaattactt taaaactact 3600 tgttaacatt taattgccta aaaactgctc gtaatttact gttgtagcct accatagagt 3660 accctgcatg gtactatgta cagcattcca tccttacatt ttcactgttc tgctgtttgc 3720 tctagacaac tcagagttca cc 3742
Claims (9)
Recombinant promoter comprising the promoter region of the obtransferin gene and the promoter region of the ovalbumin gene.
The recombinant promoter of claim 1, wherein the promoter region of the obtransferin gene comprises the nucleotide sequence of SEQ ID NO:1.
The recombinant promoter of claim 1, wherein the promoter region of the ovalbumin gene comprises the nucleotide sequence of SEQ ID NO: 2.
The recombinant promoter of claim 1, wherein the recombinant promoter is for overexpressing a target protein.
A vector comprising the recombinant promoter of claim 1.
The plasmid vector, cosmid vector, human immunodeficiency virus (HIV) vector, Murineleukemia virus (MLV) vector, Avian sarcoma/leukosis (ASLV) vector, Spleen necrosis virus (SNV) vector, RSV In the group consisting of Rous sarcoma virus (Mouse mammary tumor virus) vector, MMTV (Mouse mammary tumor virus) vector, Adenovirus vector and Herpes simplex virus vector, lentivirus vector and epiisomal vector A vector, characterized in that it is any one vector selected.
Chicken transformed with the vector of claim 6.
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| KR20220076890A (en) * | 2020-12-01 | 2022-06-08 | 대한민국(농촌진흥청장) | Recombinant promoter comprising the recombinant promoter region of the mutated ovotransferrin gene and the promoter region of the ovalbumin gene |
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| KR20090102876A (en) * | 2007-01-26 | 2009-09-30 | 시나게바 바이오파르마, 코포레이션 | Transgene expression in avians |
| KR20110057365A (en) * | 2009-11-24 | 2011-06-01 | 대한민국(농촌진흥청장) | Method for the production of the transgenic chicken containing the human lactoferrin gene |
| KR20120014404A (en) * | 2010-08-09 | 2012-02-17 | 서울대학교산학협력단 | Methods for producing proteins using transgenic avians |
| US8372956B2 (en) * | 1997-10-16 | 2013-02-12 | Synageva Biopharma Corp. | Antibodies produced in a transgenic avian egg white |
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| US8372956B2 (en) * | 1997-10-16 | 2013-02-12 | Synageva Biopharma Corp. | Antibodies produced in a transgenic avian egg white |
| KR20090102876A (en) * | 2007-01-26 | 2009-09-30 | 시나게바 바이오파르마, 코포레이션 | Transgene expression in avians |
| KR20110057365A (en) * | 2009-11-24 | 2011-06-01 | 대한민국(농촌진흥청장) | Method for the production of the transgenic chicken containing the human lactoferrin gene |
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| KR20220076890A (en) * | 2020-12-01 | 2022-06-08 | 대한민국(농촌진흥청장) | Recombinant promoter comprising the recombinant promoter region of the mutated ovotransferrin gene and the promoter region of the ovalbumin gene |
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