KR20220076890A - Recombinant promoter comprising the recombinant promoter region of the mutated ovotransferrin gene and the promoter region of the ovalbumin gene - Google Patents
Recombinant promoter comprising the recombinant promoter region of the mutated ovotransferrin gene and the promoter region of the ovalbumin gene Download PDFInfo
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Abstract
본 발명은 변형 오브트렌스페린 유전자의 재조합 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 위한 재조합 프로모터, 및 상기 재조합 프로모터를 포함하는 벡터에 관한 것으로서, 본 발명의 변형 오브트렌스페린 유전자의 재조합 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터, 및 상기 재조합 프로모터를 포함하는 벡터를 이용하는 경우 형질전환 암탉에서 유용 단백질을 높은 효율로 과발현시킬 수 있다.The present invention relates to a recombinant promoter comprising a recombinant promoter region of a modified obtransferrin gene and a promoter region of an ovalbumin gene, and a vector comprising the recombinant promoter, the recombinant promoter region of the modified obtransferrin gene of the present invention. And when a recombinant promoter including a promoter region of the ovalbumin gene, and a vector including the recombinant promoter are used, useful proteins can be overexpressed in a transgenic hen with high efficiency.
Description
본 발명은 염기서열이 변형된(이하 변형) 오브트렌스페린 유전자의 재조합 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 recombinant promoter region of an obtransferrin gene and a promoter region of an ovalbumin gene whose base sequence is modified (hereinafter modified), and a vector including 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.The development of modern science and technology, especially the development of the medical sector, has brought life extension of people, and the demand for physiologically active proteins useful to people is increasing. Currently, the most common method for producing physiologically active proteins is using E. coli, but the production method using E. coli has a problem in that it cannot produce proteins with a complex structure to which sugars bind, so proteins with complex structures are produced using animal cell lines. . Recently, studies to produce useful proteins using livestock are being conducted, and tangible results are reported in some cases. Most of the animals used for the production of physiologically active protein so far are medium/large livestock, and the method using glands, that is, mammary glands (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 the conventional method for producing useful protein using livestock, there is a problem in that it takes a lot of cost to produce protein using livestock, and the production efficiency is low.
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 각광받고 있으며, 이와 관련된 해당 기술분야의 종래기술로는 닭의 오브알부민 유전자의 프로모터 영역을 이용하여 제조한 프로모터에 관한 미국 등록 특허 제8507749 B2가 있다.On the other hand, chickens have a large number of livestock compared to mammalian livestock, and due to the short generation interval, large-scale reproduction is possible in a single individual in a short period of time. A method of producing useful proteins using chicken eggs has recently been spotlighted, and as a prior art in the related art, U.S. Patent No. 8507749 relates to a promoter prepared using the promoter region of the chicken ovalbumin gene. There is B2.
한편, 본 발명자들은 소형 가축인 닭을 이용하여 유용 단백질을 효과적으로 발현시키기 위해서, 변형 오브트렌스페린 유전자의 재조합 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는, 종래 닭의 오브알부민 유전자의 프로모터를 개량한 재조합 프로모터를 개발함으로써 본 발명을 완성하였다.On the other hand, the present inventors improved the promoter of the conventional chicken ovalbumin gene, including the recombinant promoter region of the modified obtransferrin gene and the promoter region of the ovalbumin gene, in order to effectively express useful proteins using chickens, which are small livestock. The present invention was completed by developing a recombinant promoter.
본 발명은 형질전환 암탉 생산 시 유용 단백질의 효율적인 생산을 위해, 변형 오브트렌스페린 유전자의 재조합 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터를 제공하는 것을 목적으로 한다.An object of the present invention is to provide a recombinant promoter comprising a recombinant promoter region of a modified obtransferrin gene and a promoter region of an ovalbumin gene for efficient production of useful proteins in the production of transgenic hens.
또한, 본 발명은 상기 재조합 프로모터를 포함하는 벡터를 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a vector comprising the recombinant promoter.
또한, 본 발명은 상기 벡터에 의해 형질전환된 닭을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a chicken transformed with the vector.
아울러, 본 발명은 목적 유전자를 포함하는 상기 재조합 벡터를 닭에 형질전환하여, 닭에서 목적 단백질을 발현시키는 방법을 제공하는 것을 목적으로 한다.In addition, an object of the present invention is to provide a method for expressing a target protein in chickens by transforming the recombinant vector containing the target gene into chickens.
상기 목적을 달성하기 위하여, 본 발명은 변형 오브트렌스페린 유전자의 재조합 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터를 제공한다.In order to achieve the above object, the present invention provides a recombinant promoter comprising a recombinant promoter region of a modified obtransferrin gene and a promoter region of an ovalbumin gene.
또한, 본 발명은 상기 재조합 프로모터를 포함하는 벡터를 제공한다.In addition, the present invention provides a vector comprising the recombinant promoter.
또한, 본 발명은 상기 벡터에 의해 형질전환된 닭을 제공한다.In addition, the present invention provides a chicken transformed with the vector.
아울러, 본 발명은 목적 유전자를 포함하는 상기 재조합 벡터를 닭에 형질전환하여, 닭에서 목적 단백질을 발현시키는 방법을 제공한다.In addition, the present invention provides a method for expressing a target protein in chickens by transforming the recombinant vector containing the target gene into a chicken.
본 발명의 변형 오브트렌스페린 유전자의 재조합 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터, 및 상기 재조합 프로모터를 포함하는 벡터를 이용하는 경우 형질전환 암탉에서 유용 단백질을 높은 효율로 과발현시킬 수 있다.When a recombinant promoter comprising a recombinant promoter region of a modified obtransferrin gene and a promoter region of an ovalbumin gene of the present invention, and a vector including the recombinant promoter are used, useful proteins can be overexpressed in transgenic hens with high efficiency. .
도 1은 닭에서 유용 단백질 발현을 위한 재조합 프로모터 제작에 이용하기 위해, 변형 오브트렌스페린 유전자의 프로모터 영역을 선발한 것을 나타낸 도이다.
도 2는 본 발명에서 제조한 재조합 프로모터의 개략도를 나타낸 것이다.
도 3은 HeLa 세포에서 닭의 변형 오브트렌스페린 유전자의 재조합 프로모터 영역과 닭의 오브알부민 유전자의 프로모터 영역이 재조합된 리포터 벡터(Mut-OVOT-pOV)의 프로모터의 활성을 오브알부민 유전자의 프로모터 영역을 포함하는 대조군 리포터 벡터(Mut-4.4kb-pOV)와 비교한 결과를 나타낸 도이다.
도 4는 MES-SA 세포에서 Mut-OVOT-pOV 프로모터의 활성을 대조군인 Mut-4.4kb-pOV와 비교한 결과를 나타낸 도이다.
도 5는 LMH/2A 세포에서 Mut-OVOT-pOV 프로모터의 활성을 대조군인 Mut-4.4kb-pOV와 비교한 결과를 나타낸 도이다.
도 6은 cOEC 세포에서 Mut-OVOT-pOV 프로모터의 활성을 대조군인 Mut-4.4kb-pOV와 비교한 결과를 나타낸 도이다.
도 7은 LMH/2A 세포에서 에스트로겐 농도(0, 1, 10, 100, 1000 및 3000nM)에 따른 Mut-OVOT-pOV 프로모터의 활성을 나타낸 도이다.1 is a view showing the selection of the promoter region of the modified obtransferrin gene for use in the production of a recombinant promoter for useful protein expression in chickens.
Figure 2 shows a schematic diagram of the recombinant promoter prepared in the present invention.
3 shows the promoter activity of the reporter vector (Mut-OVOT-pOV) in which the recombinant promoter region of the chicken ovalbumin gene and the promoter region of the chicken ovalbumin gene are recombined in HeLa cells, the promoter region of the ovalbumin gene It is a diagram showing the result of comparison with the control reporter vector (Mut-4.4kb-pOV) containing.
4 is a diagram showing the results of comparing the activity of the Mut-OVOT-pOV promoter in MES-SA cells with that of the control, Mut-4.4kb-pOV.
5 is a diagram showing the results of comparing the activity of the Mut-OVOT-pOV promoter in LMH/2A cells with that of the control, Mut-4.4kb-pOV.
6 is a diagram showing the results of comparing the activity of the Mut-OVOT-pOV promoter in cOEC cells with the control Mut-4.4kb-pOV.
7 is a diagram showing the activity of the Mut-OVOT-pOV promoter according to the estrogen concentration (0, 1, 10, 100, 1000 and 3000 nM) in LMH/2A cells.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 변형 오브트렌스페린 유전자의 재조합 프로모터 영역(Mut-OVOT) 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 recombinant promoter region of a modified obtransferrin gene (Mut-OVOT) and a promoter region of an ovalbumin gene, and the recombinant promoter can be used to express a target protein in chickens.
상기 변형 오브트렌스페린 유전자의 재조합 프로모터 영역은 서열번호 2의 염기서열로 표시되는 것을 특징으로 하고, 상기 오브알부민 유전자의 프로모터 영역은 서열번호 3의 염기서열로 표시되는 것을 특징으로 할 수 있다.The recombinant promoter region of the modified obtransferrin gene may be represented by the nucleotide sequence of SEQ ID NO: 2, and the promoter region of the ovalbumin gene may be represented by the nucleotide sequence of SEQ ID NO: 3.
상기 변형 오브트렌스페린 유전자의 재조합 프로모터 영역은 서열번호 1의 염기서열로 표시되는 오브트렌스페린 유전자의 프로모터 영역(OVOT)에 4개의 ERα(Estrogen receptor alpha) 결합 부위를 추가로 도입하는 것일 수 있으나, 이에 제한되지 않는다.The recombinant promoter region of the modified obtransferrin gene may be to additionally introduce four estrogen receptor alpha (ERα) binding sites into the promoter region (OVOT) of the obtransferrin gene represented by the nucleotide sequence of SEQ ID NO: 1, It is not limited thereto.
상기 프로모터 영역 서열의 변이체가 본 발명의 범위 내에 포함된다. 변이체는 염기서열은 변화되지만, 서열번호 1, 2 또는 3의 염기 서열과 유사한 기능적 특성을 갖는 염기 서열이다. 구체적으로, 상기 변형 오브트렌스페린 유전자의 프로모터 영역 염기서열은 서열번호 1의 염기 서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있고, 상기 변형 오브트렌스페린 유전자의 재조합 프로모터 영역 염기서열은 서열번호 2의 염기 서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있고, 상기 오브알부민 유전자의 프로모터 영역 염기서열은 서열번호 3의 염기서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다.Variants of the above promoter region sequences are included within the scope of the present invention. A variant is a nucleotide sequence having a functional characteristic similar to that of the nucleotide sequence of SEQ ID NO: 1, 2 or 3, although the nucleotide sequence is changed. Specifically, the promoter region nucleotide sequence of the modified obtransferrin gene is 70% or more, more preferably 80% or more, still more preferably 90% or more, most preferably 95% or more of the nucleotide sequence of SEQ ID NO: 1 It may include a nucleotide sequence having homology, and the recombinant promoter region nucleotide sequence of the modified obtransferrin gene is 70% or more, more preferably 80% or more, still more preferably 90% to the nucleotide sequence of SEQ ID NO: 2 More preferably, it may include a nucleotide sequence having 95% or more sequence homology, and the promoter region nucleotide sequence of the ovalbumin gene is 70% or more with the nucleotide sequence of SEQ ID NO: 3, more preferably 80% or more , more preferably 90% or more, and most preferably 95% or more of a nucleotide sequence having 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, a 'promoter' is a gene region located upstream of a coding region including a point for initiating transcription in general. It consists of an enhancer that promotes the expression of almost all genes regardless of the region and the location and direction that affect gene expression in response.
본 발명은 형질전환 암탉의 계란에서 유용 단백질 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 smaller size and stronger promoter activity than the ovalbumin promoter that has been used for the production of useful proteins in eggs of transgenic hens. Ovalbumin, the protein most expressed in egg white and each promoter region of the next most frequently expressed obtransferrin (ovotransferrin) was linked.
본 발명의 재조합 프로모터에 포함되어 있는 오브트렌스페린 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 obtransferrin gene included in the recombinant promoter of the present invention, an estrogen response element (ERE) or an estrogen receptor alpha (ERα) binding site may exist.
상기 ERα(Estrogen receptor alpha) 결합 부위는 재조합 프로모터에 포함되어 있는 오브트렌스페린 유전자의 프로모터 영역에 1개 내지 5개가 포함되어 있을 수 있고, 바람직하게는 2개 내지 3개가 포함되어 있을 수 있다.1 to 5 of the estrogen receptor alpha (ERα) binding site may be included in the promoter region of the obtransferrin gene included in the recombinant promoter, and preferably 2 to 3 may be included.
본 발명의 구체적인 실시예에서, 본 발명자들은 오브트렌스페린 유전자 프로모터 영역 중 에스트로겐 수용체 알파(Esptoren receptor alpha, ERα)가 결합할 수 있는 서열들이 포함된 영역을 탐색하여 5kb 염기서열 중 3곳의 ERα 결합 부위를 확인하였고, 확인된 3곳의 ERα 결합 부위 중 2곳을 포함하는, 서열번호 1의 염기서열로 표시되는 -4,575bp 내지 -3,618bp의 프로모터 영역(OVOT, 958bp)를 일차적으로 선발하였고, 상기 OVOT 영역에 추가로 4개의 ERα 결합 부위를 도입하였으며, 추가로 도입한 ERα 결합 부위는 각 염기서열 간에 거리를 두어 최종적으로 서열번호 2의 염기서열로 표시되는 재조합 영역(Mut-OVOT, 1,002bp)을 확정하였다(도 1 참조). 그 후, 상기 재조합 영역(1,002bp)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 searched for a region containing sequences capable of binding to estrogen receptor alpha (ERα) in the obtransferrin gene promoter region, and ERα binding of 3 of the 5 kb nucleotide sequences The site was confirmed, and the promoter region (OVOT, 958bp) of -4,575bp to -3,618bp represented by the nucleotide sequence of SEQ ID NO: 1, including two of the three confirmed ERα binding sites, was selected, Four additional ERα-binding sites were introduced into the OVOT region, and the additionally introduced ERα-binding sites were separated from each other by a distance between each nucleotide sequence, and finally a recombination region represented by the nucleotide sequence of SEQ ID NO: 2 (Mut-OVOT, 1002 bp). ) was confirmed (see FIG. 1). Thereafter, a recombinant promoter was prepared in which the recombination region (1,002 bp) and a part of the promoter region (2.8 kb) of the chicken ovalbumin gene were sequentially linked (see Example 1).
또한, 본 발명은 변형 오브트렌스페린 유전자의 재조합 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.Further, the present invention provides a vector comprising a recombinant promoter comprising a recombinant promoter region of a modified obtransferrin gene and a promoter region of an 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.A vector including the recombinant promoter may have a smaller size than a vector including a promoter of the prior art.
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)" refers to 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. A vector may be a plasmid, a phage particle, or simply a potential genomic introduction. Upon transformation into an appropriate host, the vector may replicate and function independently of the host genome, or in some cases may 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 vector is a plasmid vector, cosmid vector, HIV (Human immunodeficiency virus) vector, MLV (Murineleukemia virus) vector, ASLV (Avian sarcoma / leukosis) vector, SNV (Spleen necrosis virus) vector, RSV (Rous sarcoma virus) vector, Any one vector selected from the group consisting of MMTV (Mouse mammary tumor virus) vector, adenovirus vector and herpes simplex virus vector, lentivirus vector and episomal 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 an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a specific 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 contain one or more selectable markers. The marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include antibiotic resistance genes such as ampicillin, kanamycin, neomycin, G418, bleomycin, hygromycin, chloramphenicol, but are limited thereto 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 context of the present invention, "operably linked" means that the appropriate molecule is linked in such a way as to enable gene expression when bound to an expression control sequence. Also, "expression control sequence (expression control sequence)" refers to a DNA sequence that controls the expression of an operably linked polynucleotide sequence in a specific host cell. Such regulatory sequences include promoters to effect transcription, optional operator sequences to control transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the 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, the "target protein" refers to any external target protein that is desired to be highly expressed by the promoter of the present invention, and may preferably 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 production of transgenic chickens.
본 발명의 구체적인 실험예에서, 변형 오브트렌스페린 유전자의 재조합 프로모터 영역 및 오브알부민 유전자의 프로모터 영역을 포함하는 재조합 프로모터의 활성을 리포터 분석을 통해 인간세포(HeLa 세포 및 MES-SA 세포)에서 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 프로모터와 비교하여 프로모터의 활성이 유사하였다(도 3, 4 및 실험예 1 참조).In a specific experimental example of the present invention, the activity of a recombinant promoter comprising the recombinant promoter region of the modified obtransferrin gene and the promoter region of the ovalbumin gene was measured in human cells (HeLa cells and MES-SA cells) through reporter analysis. As a result, the activity of the promoter was similar to that of the Kanuma promoter, which includes the entire promoter region of the ovalbumin gene (the 4.4 kb ovalbumin promoter (US Patent No. 8507749B2) containing an estrogen responsive element (ERE)) (Fig. 3, 4 and Experimental Example 1).
또한, 상기 재조합 프로모터를 사용하여 구축한 벡터를 닭의 세포(LMH/2A 세포 및 cOEC 세포)에 도입하여 활성을 비교한 결과, LMH/2A 세포에서 Mut-OVOT-pOV 프로모터는 대조군인 Mut-4.4kb-pOV 프로모터와 대비하여 약 2.31배 높은 유전자 활성을 나타내는 것을 확인하였고(도 5 참조), cOEC 세포에서 Mut-OVOT-pOV 프로모터는 Mut-4.4kb-pOV 프로모터와 비교하여 약 4.03배 높은 유전자 발현 활성을 나타내는 것을 확인하였다(도 6 및 실험예 2 참조).In addition, the vector constructed using the recombinant promoter was introduced into chicken cells (LMH/2A cells and cOEC cells) to compare the activity. As a result, in LMH/2A cells, the Mut-OVOT-pOV promoter was Mut-4.4 as a control. It was confirmed that the gene activity was about 2.31 times higher than that of the kb-pOV promoter (see Fig. 5), and the Mut-OVOT-pOV promoter in cOEC cells was about 4.03 times higher in gene expression compared to the Mut-4.4kb-pOV promoter. It was confirmed that the activity was shown (see FIG. 6 and Experimental Example 2).
추가적으로, 상기 Mut-OVOT-pOV 프로모터가 클로닝된 리포터 벡터를 LMH/2A 세포에 도입하고 약 24시간 후 에스트로겐을 각각의 농도(0, 1, 10, 100, 1,000 및 3,000nM)로 처리한 결과, 100nM 수준의 에스트로겐 농도에서부터 유전자의 발현량이 증가하는 결과를 확인하였다(도 7 참조).Additionally, after introducing the reporter vector cloned into the Mut-OVOT-pOV promoter into LMH/2A cells, approximately 24 hours later, estrogen was treated at respective concentrations (0, 1, 10, 100, 1,000 and 3,000 nM). It was confirmed that the expression level of the gene increased from the estrogen concentration of 100 nM level (see FIG. 7 ).
따라서, 본 발명의 변형 오브트렌스페린 유전자의 재조합 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.Accordingly, the vector containing the recombinant promoter including the recombinant promoter region of the modified obtransferrin gene and the promoter region of the ovalbumin gene of the present invention is smaller in size than the vector containing the promoter of the prior art, and the promoter of the prior art It is possible to overexpress the target protein with high efficiency compared to the vector containing the .
또한, 본 발명은 상기 재조합 프로모터를 포함하는 벡터에 의해 형질전환된 닭을 제공한다.In addition, the present invention provides a chicken transformed by the 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 may be introduced into a blastocyst stage fertilized egg of a chicken through microinjection. The first generation candidate transgenic chicks (mosaic, G0) are produced through the culturing and transplantation of the fertilized eggs, and the first generation candidate transgenic chicks (mosaic, G0) are selected from among the produced male candidates and the trait is transmitted to the gonads through artificial insemination. Generation of transgenic chickens (G1) can be produced and the second generation (G2) and subsequent generations of transgenic chickens having a recombinant promoter and target gene can be continuously produced through a breeding scheme including G1 individuals.
또한, 본 발명은 목적 유전자를 포함하는 상기 재조합 벡터를 닭에 형질전환하여, 닭의 상피세포에서 목적 단백질을 발현시키는 방법을 제공한다.In addition, the present invention provides a method of expressing a target protein in chicken epithelial cells by transforming the recombinant vector containing the target gene into chickens.
본 발명에서 상기 변형 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 modified obtransferrin 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 is transformed into chickens, the target protein can be expressed in the epithelial cells of the chicken.
이하, 본 발명을 실시예 및 실험예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것이며, 본 발명의 내용이 실시예 및 실험예에 의해 한정되는 것은 아니다.However, the following Examples and Experimental Examples illustrate the present invention, and the content of the present invention is not limited by the Examples and Experimental Examples.
<실시예 1> 닭에서 유용 단백질 발현을 위한 재조합 프로모터 제작<Example 1> Construction of a recombinant promoter for expression of useful proteins in chickens
<1-1> 변형 오브트렌스페린 유전자의 재조합 영역 확정<1-1> Confirmation of recombination region of modified obtransferrin gene
닭에서 유용 단백질 발현을 위한 재조합 프로모터의 제작에 이용하기 위해, 오브트렌스페린 유전자 프로모터 영역 중 에스트로겐 수용체 알파(Esptoren receptor alpha, ERα)가 결합할 수 있는 서열들이 포함된 영역을 탐색하여 번역 개시 부위를 기준으로 5kb 염기서열 중 3곳의 ERα 결합 부위를 확인하였고, 확인된 3곳의 ERα 결합 부위 중 2곳을 포함하는, 서열번호 1의 염기서열로 표시되는 -4,575bp 내지 -3,618bp의 프로모터 영역(OVOT, 958bp)를 일차적으로 선발하였다.To use in the production of a recombinant promoter for useful protein expression in chickens, the translation initiation site was identified by searching for a region containing sequences capable of binding to estrogen receptor alpha (ERα) among the oftransferrin gene promoter regions. As a reference, three ERα binding sites were identified among the 5 kb base sequence, and a promoter region of -4,575 bp to -3,618 bp represented by the nucleotide sequence of SEQ ID NO: 1, including two of the three confirmed ERα binding sites. (OVOT, 958bp) was primarily selected.
일차적으로 선발된 상기 OVOT 영역에 추가로 4개의 ERα 결합 부위를 도입하였으며, 추가로 도입한 ERα 결합 부위는 각 염기서열 간에 거리를 두어 최종적으로 서열번호 2의 염기서열로 표시되는 재조합 영역(Mut-OVOT, 1,002bp)을 확정하였다(도 1).Four additional ERα binding sites were introduced into the firstly selected OVOT region, and the additionally introduced ERα binding sites were separated by a distance between each nucleotide sequence and finally a recombination region represented by the nucleotide sequence of SEQ ID NO: 2 (Mut- OVOT, 1,002 bp) was confirmed (FIG. 1).
<1-2> 재조합 프로모터 제작 <1-2> Recombinant promoter production
닭의 계란에서 유용 단백질 발현을 위해 상기 실시예 1-1에서 확정된 변형 오브트렌스페린 유전자의 프로모터 영역과 오브알부민 유전자의 프로모터 영역이 결합된 재조합 프로모터를 제작하였다.For the expression of useful proteins in chicken eggs, a recombinant promoter in which the promoter region of the modified obtransferrin gene confirmed in Example 1-1 and the promoter region of the ovalbumin gene were combined was prepared.
구체적으로, ThermoFisher사에 의뢰하여 5'에서 3' 방향으로 Nhe I 제한효소 인식 서열, 서열번호 2의 염기서열로 표시되는 닭의 변형 오브트렌스페린 유전자의 프로모터 영역(1,002bp), 닭의 오브알부민 유전자의 프로모터 영역(2784bp)(서열번호 3), 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(Mut-OVOT-pOV)(도 2). 5'에서 3' 방향으로 닭의 변형 오브트렌스페린 유전자의 프로모터 영역(1,002bp)(서열번호 2), 및 닭의 오브알부민 유전자의 프로모터 영역(2784bp)(서열번호 3)이 결합된 재조합 프로모터의 염기서열은 서열번호 4에 개시되어 있다.Specifically, requested by ThermoFisher, the Nhe I restriction enzyme recognition sequence in the 5' to 3' direction, the promoter region of the chicken modified obtransferrin gene represented by the nucleotide sequence of SEQ ID NO: 2 (1,002 bp), the chicken ovalbumin A polynucleotide in which the promoter region (2784bp) of the gene (SEQ ID NO: 3) and the Xho I restriction enzyme recognition sequence were sequentially linked was synthesized. The prepared polynucleotide was treated with Nhe I and Xho I restriction enzymes and introduced into a reporter analysis vector (pGL4.11, Promega) treated with the same enzyme (Mut-OVOT-pOV) (FIG. 2). In the 5' to 3' direction, the promoter region of the chicken's modified obtransferrin gene (1,002 bp) (SEQ ID NO: 2), and the promoter region of the chicken ovalbumin gene (2784 bp) (SEQ ID NO: 3) of a recombinant promoter bound The base sequence is disclosed in SEQ ID NO: 4.
<1-3> 대조군 벡터 제작<1-3> Control vector construction
공지된 대조군 벡터(Mut-4.4kb-pOV, Kanuma)를 제조하기 위해서 ERE(estrogen responsive element)를 포함하는 1.6kb 크기의 닭 오브알부민 유전자의 프로모터 영역과 2.8kb 크기의 닭 오브알부민 유전자의 프로모터 영역이 연결된 4.4kb 오브알부민 프로모터(미국등록특허 8507749B2)를 상기와 동일한 방식으로 합성하고 리포터 분석용 벡터(pGL4.11)에 클로닝하여 대조군 벡터를 제조하였다. To prepare a known control vector (Mut-4.4kb-pOV, Kanuma), the promoter region of the chicken ovalbumin gene of 1.6kb size and the promoter region of the chicken ovalbumin gene of the size of 2.8kb including ERE (estrogen responsive element) This linked 4.4kb ovalbumin promoter (US Patent No. 8507749B2) was synthesized in the same manner as above and cloned into a vector for reporter analysis (pGL4.11) to prepare a control vector.
상기 구축한 재조합 프로모터 벡터들은 염기서열 분석을 통하여 클로닝하고자 한 상기 프로모터들이 정상적으로 삽입되었음을 확인하였다.In the constructed recombinant promoter vectors, it was confirmed that the promoters to be cloned were normally inserted through nucleotide sequence analysis.
<실험예 1> 인간 세포에서 재조합 프로모터의 활성 검증<Experimental Example 1> Verification of the activity of a recombinant promoter in human cells
상기 실시예 1에서 제조한 두 가지 프로모터(Mut-4.4kb-pOV, Mut-OVOT-pOV) 및 음성대조군인 공벡터(Mock, pGL4.11)를 지질 매개성 유전자 도입 방식으로 사람 자궁경관 상피세포주(Human cervix epithelial cells, HeLa) 세포, 또는 사람 자궁 상피세포주(Human uterus epithelial cells, MES-SA) 세포에 도입하고, 24시간 후에 luciferase assay kit(Promega)로 제조사의 프로토콜에 따라 각 프로모터의 활성을 분석하였다.The two promoters (Mut-4.4kb-pOV, Mut-OVOT-pOV) prepared in Example 1 and an empty vector (Mock, pGL4.11) as a negative control were introduced into a human cervical epithelial cell line by a lipid-mediated gene introduction method. After introduction into (Human cervix epithelial cells, HeLa) cells or human uterus epithelial cells (MES-SA) cells, after 24 hours, the activity of each promoter was measured with a luciferase assay kit (Promega) according to the manufacturer's protocol. analyzed.
그 결과, HeLa 세포에서 Mut-OVOT-pOV 프로모터의 활성은 대조군인 Mut-4.4kb-pOV 프로모터의 활성과 거의 유사하였고(도 3), MES-SA 세포에서도 OVOT-pOV 프로모터의 활성은 대조군인 Mut-4.4kb-pOV 프로모터의 활성과 유의적인 차이는 존재하지 않았다(도 4).As a result, the activity of the Mut-OVOT-pOV promoter in HeLa cells was almost similar to that of the control, Mut-4.4kb-pOV promoter (FIG. 3), and the OVOT-pOV promoter activity in MES-SA cells was also similar to that of the control, Mut. There was no significant difference with the activity of the -4.4kb-pOV promoter (FIG. 4).
<실험예 2> 닭의 세포에서 재조합 프로모터의 활성 검증<Experimental Example 2> Verification of recombinant promoter activity in chicken cells
상기 실시예 1에서 제조한 두 가지 프로모터(Mut-4.4kb-pOV, Mut-OVOT-pOV) 및 음성대조군인 공벡터(Mock, pGL4.11)를 지질 매개성 유전자 도입 방식으로 닭 간세포주(Chicken hepatocyte, LMH/2A) 및 닭의 수란관 상피세포주(Chicken oviduct epithelial cells, cOEC)에 도입하고, 24시간 후에 luciferase assay kit(Promega)로 제조사의 프로토콜에 따라 각 프로모터의 활성을 분석하였다.The two promoters (Mut-4.4kb-pOV, Mut-OVOT-pOV) and the negative control empty vector (Mock, pGL4.11) prepared in Example 1 were introduced into a chicken liver cell line (Chicken) by a lipid-mediated gene introduction method. hepatocyte, LMH/2A) and chicken oviduct epithelial cells (cOEC) were introduced, and after 24 hours, the activity of each promoter was analyzed with a luciferase assay kit (Promega) according to the manufacturer's protocol.
그 결과, LMH/2A 세포에서 Mut-OVOT-pOV 프로모터는 대조군인 Mut-4.4kb-pOV 프로모터와 대비하여 약 2.31배 높은 유전자 활성을 나타내는 것을 확인하였다(도 5). 또한, cOEC 세포에서 Mut-OVOT-pOV 프로모터는 Mut-4.4kb-pOV 프로모터와 비교하여 약 4.03배 높은 유전자 발현 활성을 나타내는 것을 확인하였다(도 6).As a result, it was confirmed that the Mut-OVOT-pOV promoter in LMH/2A cells exhibited about 2.31 times higher gene activity compared to the control Mut-4.4kb-pOV promoter ( FIG. 5 ). In addition, it was confirmed that the Mut-OVOT-pOV promoter in cOEC cells exhibited about 4.03 times higher gene expression activity compared to the Mut-4.4kb-pOV promoter ( FIG. 6 ).
추가적으로, 상기 Mut-OVOT-pOV 프로모터가 클로닝된 리포터 벡터를 LMH/2A 세포에 도입하고 약 24시간 후 에스트로겐을 각각의 농도(0, 1, 10, 100, 1,000 및 3,000nM)로 처리한 결과, 100nM 수준의 에스트로겐 농도에서부터 유전자의 발현량이 증가하는 결과를 확인하였다(도 7).Additionally, after introducing the reporter vector cloned into the Mut-OVOT-pOV promoter into LMH/2A cells, approximately 24 hours later, estrogen was treated at respective concentrations (0, 1, 10, 100, 1,000 and 3,000 nM). It was confirmed that the expression level of the gene increased from the estrogen concentration of 100 nM level (FIG. 7).
따라서, 본 발명의 변형 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 including the promoter region of the modified obtransferrin gene and the promoter region of the ovalbumin gene of the present invention is smaller in size than the vector containing the promoter of the prior art, and the promoter of the prior art It is possible to overexpress the target protein with high efficiency compared to the included vector.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Recombinant promoter comprising the recombinant promoter region of the mutated ovotransferrin gene and the promoter region of the ovalbumin gene <130> 2020P-06-001 <160> 4 <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> 1002 <212> DNA <213> Artificial Sequence <220> <223> mutated ovotransferrin promoter DNA sequence <400> 2 gtttggtatt aatgattacc atgagttagc taactggaaa ggggctgaac agcaaatggt 60 ggagttcacg ttgacctatt ggtgacctga cccctttccc gctcgcttga tgcccaaacc 120 tcatttctgg ctgcagaacc atcttgcagc tgaacacaat gcctggtggg ggctgtgcca 180 gcccagctgg gctgcccatg ggctgtgctt gacctgataa gggcaatgag aggctcagcc 240 cctgctcctt gggcctgttc cagcagcgtt gcgcactgcc agccccagga tgtccaaaca 300 gcagcggaag gatctcccaa aacggaggac atagctgaat gcacccttgc ataggtgcag 360 tggatgtggt tcagatctgg gtgcaccttg agtctggctg tgatgtcctt gacctgatat 420 atgatccgct ctgccgccat ctcgttccta tccccttcct gcacagacac tgccagtggt 480 ctgggctctg tcaccgcggg gggacgtctg tgctcctgct ccttctgccc agtacctcca 540 tgtccttgtg agccccagct ttgacctgat atccctctgc actggaaaca acgtggtgtt 600 gggccccctg agccaggtga ccctgctgga gcagggattg ggccagaagg acccgaccac 660 agccattttg ttcctccgtg aaattagcca ccttctctta tgttctgttc ctccaggctg 720 gcccagcatc tccccacagg catatgttgg tagtacttac tgatattgat agtctttctt 780 tcctgattat caatatttgc tggtttgttc ccaccaatct ttaggaaata cccaggaagc 840 aggcatgaac agagctccag cagctccatg attccagcag gagccattga cctgatatca 900 gcccaccgtg ccttcctgtg ttcccttccc aaagcccacc aggaacgtcg cagttcccag 960 ggctgcacag agctgctggt aaccctgtta acctttgggt ta 1002 <210> 3 <211> 2784 <212> DNA <213> Artificial Sequence <220> <223> ovalbumin promoter DNA sequence <400> 3 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> 4 <211> 3786 <212> DNA <213> Artificial Sequence <220> <223> Recombinant promoter DNA sequence <400> 4 gtttggtatt aatgattacc atgagttagc taactggaaa ggggctgaac agcaaatggt 60 ggagttcacg ttgacctatt ggtgacctga cccctttccc gctcgcttga tgcccaaacc 120 tcatttctgg ctgcagaacc atcttgcagc tgaacacaat gcctggtggg ggctgtgcca 180 gcccagctgg gctgcccatg ggctgtgctt gacctgataa gggcaatgag aggctcagcc 240 cctgctcctt gggcctgttc cagcagcgtt gcgcactgcc agccccagga tgtccaaaca 300 gcagcggaag gatctcccaa aacggaggac atagctgaat gcacccttgc ataggtgcag 360 tggatgtggt tcagatctgg gtgcaccttg agtctggctg tgatgtcctt gacctgatat 420 atgatccgct ctgccgccat ctcgttccta tccccttcct gcacagacac tgccagtggt 480 ctgggctctg tcaccgcggg gggacgtctg tgctcctgct ccttctgccc agtacctcca 540 tgtccttgtg agccccagct ttgacctgat atccctctgc actggaaaca acgtggtgtt 600 gggccccctg agccaggtga ccctgctgga gcagggattg ggccagaagg acccgaccac 660 agccattttg ttcctccgtg aaattagcca ccttctctta tgttctgttc ctccaggctg 720 gcccagcatc tccccacagg catatgttgg tagtacttac tgatattgat agtctttctt 780 tcctgattat caatatttgc tggtttgttc ccaccaatct ttaggaaata cccaggaagc 840 aggcatgaac agagctccag cagctccatg attccagcag gagccattga cctgatatca 900 gcccaccgtg ccttcctgtg ttcccttccc aaagcccacc aggaacgtcg cagttcccag 960 ggctgcacag agctgctggt aaccctgtta acctttgggt tattaagtcc tcagacttgg 1020 caaggagaat gtagatttct acagtatata tgttttcaca aaaggaagga gagaaacaaa 1080 agaaaatggc actgactaaa cttcagctag tggtatagga aagtaattct gcttaacaga 1140 gattgcagtg atctctatgt atgtcctgaa gaattatgtt gtactttttt ccctcatttt 1200 taaatcaaac agtgctttac agaggtcaga atggtttctt tactgtttgt caattctatt 1260 atttcaatac agaacaatag cttctataac tgaaatatat ttgctattgt atattatgat 1320 tgtccctcga accatgaaca ctcctccagc tgaatttcac aattcctctg tcatctgcca 1380 ggccattaag ttattcatgg aagatctttg aggaacactg caagttcata tcataaacac 1440 atttgaaatt gagtattgtt ttgcattgta tggagctatg ttttgctgta tcctcagaaa 1500 aaaagtttgt tataaagcat tcacacccat aaaaagatag atttaaatat tccagctata 1560 ggaaagaaag tgcgtctgct cttcactcta gtctcagttg gctccttcac atgcacgctt 1620 ctttatttct cctattttgt caagaaaata ataggtcacg tcttgttctc acttatgtcc 1680 tgcctagcat ggctcagatg cacgttgtac atacaagaag gatcaaatga aacagacttc 1740 tggtctgtta ctacaaccat agtaataagc acactaacta ataattgcta attatgtttt 1800 ccatctccaa ggttcccaca tttttctgtt ttcttaaaga tcccattatc tggttgtaac 1860 tgaagctcaa tggaacatga gcaatatttc ccagtcttct ctcccatcca acagtcctga 1920 tggattagca gaacaggcag aaaacacatt gttacccaga attaaaaact aatatttgct 1980 ctccattcaa tccaaaatgg acctattgaa actaaaatct aacccaatcc cattaaatga 2040 tttctatggc gtcaaaggtc aaacttctga agggaacctg tgggtgggtc acaattcagg 2100 ctatatattc cccagggctc agccagtgtc tgtacataca gctagaaagc tgtattgcct 2160 ttagcactca agctcaaaag gtaagcaact ctctggaatt accttctctc tatattagct 2220 cttacttgca cctaaacttt aaaaaattaa caattattgt gttatgtgtt gtatctttaa 2280 gggtgaagta cctgcgtgat accccctata aaaacttctc acctgtgtat gcattctgca 2340 ctattttatt atgtgtaaaa gctttgtgtt tgttttcagg aggcttattc tttgtgctta 2400 aaatatgttt ttaatttcag aacatcttat cctgtcgttc actatctgat atgctttgca 2460 gtttgcctga ttaacttcta gccctacaga gtgcacagag agcaaaatca tggtgttcag 2520 tgaattctgg ggagttattt taatgtgaaa attctctaga agtttaattc ctgcaaagtg 2580 cagctgctga tcactacaca agataaaaat gtggggggtg cataaacgta tattcttaca 2640 ataatagata catgtgaact tgtatacaga aaagaaaatg agaaaaatgt gtgtgcgtat 2700 actcacacac gtggtcagta aaaacttttg aggggtttaa tacagaaaat ccaatcctga 2760 ggccccagca ctcagtacgc atataaaggg ctgggctctg aaggacttct gactttcaca 2820 gattatataa atctcaggaa agcaactaga ttcatgctgg ctccaaaagc tgtgctttat 2880 ataagcacac tggctataca atagttgtac agttcagctc tttataatag aaacagacag 2940 aacaagtata aatcttctat tggtctatgt catgaacaag aattcattca gtggctctgt 3000 tttatagtaa acattgctat tttatcatgt ctgcatttct cttctgtctg aatgtcacca 3060 ctaaaattta actccacaga aagtttatac tacagtacac atgcatatct ttgagcaaag 3120 caaaccatac ctgaaagtgc aatagagcag aatatgaatt acatgcgtgt ctttctccta 3180 gactacatga ccccatataa attacattcc ttatctattc tgccatcacc aaaacaaagg 3240 taaaaatact tttgaagatc tactcatagc aagtagtgtg caacaaacag atatttctct 3300 acatttattt ttagggaata aaaataagaa ataaaatagt cagcaagcct ctgctttctc 3360 atatatctgt ccaaacctaa agtttactga aatttgctct ttgaatttcc agttttgcaa 3420 gcctatcaga ttgtgtttta atcagaggta ctgaaaagta tcaatgaatt ctagctttca 3480 ctgaacaaaa atatgtagag gcaactggct tctgggacag tttgctaccc aaaagacaac 3540 tgaatgcaaa tacataaata gatttatgaa tatggttttg aacatgcaca tgagaggtgg 3600 atatagcaac agacacatta ccacagaatt actttaaaac tacttgttaa catttaattg 3660 cctaaaaact gctcgtaatt tactgttgta gcctaccata gagtaccctg catggtacta 3720 tgtacagcat tccatcctta cattttcact gttctgctgt ttgctctaga caactcagag 3780 ttcacc 3786 <110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Recombinant promoter comprising the recombinant promoter region of the mutated ovotransferrin gene and the promoter region of the ovalbumin gene <130> 2020P-06-001 <160> 4 <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 tgaacaaat 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> 1002 <212> DNA <213> Artificial Sequence <220> <223> mutated ovotransferrin promoter DNA sequence <400> 2 gtttggtatt aatgattacc atgagttagc taactggaaa ggggctgaac agcaaatggt 60 ggagttcacg ttgacctatt ggtgacctga cccctttccc gctcgcttga tgcccaaacc 120 tcatttctgg ctgcagaacc atcttgcagc tgaacaaat gcctggtggg ggctgtgcca 180 gcccagctgg gctgcccatg ggctgtgctt gacctgataa gggcaatgag aggctcagcc 240 cctgctcctt gggcctgttc cagcagcgtt gcgcactgcc agccccagga tgtccaaaca 300 gcagcggaag gatctcccaa aacggaggac atagctgaat gcacccttgc ataggtgcag 360 tggatgtggt tcagatctgg gtgcaccttg agtctggctg tgatgtcctt gacctgatat 420 atgatccgct ctgccgccat ctcgttccta tccccttcct gcacagacac tgccagtggt 480 ctgggctctg tcaccgcggg gggacgtctg tgctcctgct ccttctgccc agtacctcca 540 tgtccttgtg agccccagct ttgacctgat atccctctgc actggaaaca acgtggtgtt 600 gggccccctg agccaggtga ccctgctgga gcagggattg ggccagaagg acccgaccac 660 agccattttg ttcctccgtg aaattagcca ccttctctta tgttctgttc ctccaggctg 720 gcccagcatc tccccacagg catatgttgg tagtacttac tgatattgat agtctttctt 780 tcctgattat caatatttgc tggtttgttc ccaccaatct ttaggaaata cccaggaagc 840 aggcatgaac agagctccag cagctccatg attccagcag gagccattga cctgatatca 900 gcccaccgtg ccttcctgtg ttcccttccc aaagcccacc aggaacgtcg cagttcccag 960 ggctgcacag agctgctggt aaccctgtta acctttgggt ta 1002 <210> 3 <211> 2784 <212> DNA <213> Artificial Sequence <220> <223> ovalbumin promoter DNA sequence <400> 3 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> 4 <211> 3786 <212> DNA <213> Artificial Sequence <220> <223> Recombinant promoter DNA sequence <400> 4 gtttggtatt aatgattacc atgagttagc taactggaaa ggggctgaac agcaaatggt 60 ggagttcacg ttgacctatt ggtgacctga cccctttccc gctcgcttga tgcccaaacc 120 tcatttctgg ctgcagaacc atcttgcagc tgaacaaat gcctggtggg ggctgtgcca 180 gcccagctgg gctgcccatg ggctgtgctt gacctgataa gggcaatgag aggctcagcc 240 cctgctcctt gggcctgttc cagcagcgtt gcgcactgcc agccccagga tgtccaaaca 300 gcagcggaag gatctcccaa aacggaggac atagctgaat gcacccttgc ataggtgcag 360 tggatgtggt tcagatctgg gtgcaccttg agtctggctg tgatgtcctt gacctgatat 420 atgatccgct ctgccgccat ctcgttccta tccccttcct gcacagacac tgccagtggt 480 ctgggctctg tcaccgcggg gggacgtctg tgctcctgct ccttctgccc agtacctcca 540 tgtccttgtg agccccagct ttgacctgat atccctctgc actggaaaca acgtggtgtt 600 gggccccctg agccaggtga ccctgctgga gcagggattg ggccagaagg acccgaccac 660 agccattttg ttcctccgtg aaattagcca ccttctctta tgttctgttc ctccaggctg 720 gcccagcatc tccccacagg catatgttgg tagtacttac tgatattgat agtctttctt 780 tcctgattat caatatttgc tggtttgttc ccaccaatct ttaggaaata cccaggaagc 840 aggcatgaac agagctccag cagctccatg attccagcag gagccattga cctgatatca 900 gcccaccgtg ccttcctgtg ttcccttccc aaagcccacc aggaacgtcg cagttcccag 960 ggctgcacag agctgctggt aaccctgtta acctttgggt tattaagtcc tcagacttgg 1020 caaggagaat gtagatttct acagtatata tgttttcaca aaaggaagga gagaaacaaa 1080 agaaaatggc actgactaaa cttcagctag tggtatagga aagtaattct gcttaacaga 1140 gattgcagtg atctctatgt atgtcctgaa gaattatgtt gtactttttt ccctcatttt 1200 taaatcaaac agtgctttac agaggtcaga atggtttctt tactgtttgt caattctatt 1260 atttcaatac agaacaatag cttctataac tgaaatatat ttgctattgt atattatgat 1320 tgtccctcga accatgaaca ctcctccagc tgaatttcac aattcctctg tcatctgcca 1380 ggccattaag ttattcatgg aagatctttg aggaacactg caagttcata tcataaacac 1440 atttgaaatt gagtattgtt ttgcattgta tggagctatg ttttgctgta tcctcagaaa 1500 aaaagtttgt tataaagcat tcacacccat aaaaagatag atttaaatat tccagctata 1560 ggaaagaaag tgcgtctgct cttcactcta gtctcagttg gctccttcac atgcacgctt 1620 ctttatttct cctattttgt caagaaaata ataggtcacg tcttgttctc acttatgtcc 1680 tgcctagcat ggctcagatg cacgttgtac atacaagaag gatcaaatga aacagacttc 1740 tggtctgtta ctacaaccat agtaataagc acactaacta ataattgcta attatgtttt 1800 ccatctccaa ggttcccaca tttttctgtt ttcttaaaga tcccattatc tggttgtaac 1860 tgaagctcaa tggaacatga gcaatatttc ccagtcttct ctcccatcca acagtcctga 1920 tggattagca gaacaggcag aaaacacatt gttacccaga attaaaaact aatatttgct 1980 ctccattcaa tccaaaatgg acctattgaa actaaaatct aacccaatcc cattaaatga 2040 tttctatggc gtcaaaggtc aaacttctga agggaacctg tgggtgggtc acaattcagg 2100 ctatatattc cccagggctc agccagtgtc tgtacataca gctagaaagc tgtattgcct 2160 ttagcactca agctcaaaag gtaagcaact ctctggaatt accttctctc tatattagct 2220 cttacttgca cctaaacttt aaaaaattaa caattattgt gttatgtgtt gtatctttaa 2280 gggtgaagta cctgcgtgat accccctata aaaacttctc acctgtgtat gcattctgca 2340 ctattttatt atgtgtaaaa gctttgtgtt tgttttcagg aggcttattc tttgtgctta 2400 aaatatgttt ttaatttcag aacatcttat cctgtcgttc actatctgat atgctttgca 2460 gtttgcctga ttaacttcta gccctacaga gtgcacagag agcaaaatca tggtgttcag 2520 tgaattctgg ggagttattt taatgtgaaa attctctaga agtttaattc ctgcaaagtg 2580 cagctgctga tcactacaca agataaaaat gtggggggtg cataaacgta tattcttaca 2640 ataatagata catgtgaact tgtatacaga aaagaaaatg agaaaaatgt gtgtgcgtat 2700 actcacacac gtggtcagta aaaacttttg aggggtttaa tacagaaaat ccaatcctga 2760 ggccccagca ctcagtacgc atataaaggg ctgggctctg aaggacttct gactttcaca 2820 gattatataa atctcaggaa agcaactaga ttcatgctgg ctccaaaagc tgtgctttat 2880 ataagcacac tggctataca atagttgtac agttcagctc tttataatag aaacagacag 2940 aacaagtata aatcttctat tggtctatgt catgaacaag aattcattca gtggctctgt 3000 tttatagtaa acattgctat tttatcatgt ctgcatttct cttctgtctg aatgtcacca 3060 ctaaaattta actccacaga aagtttatac tacagtacac atgcatatct ttgagcaaag 3120 caaaccatac ctgaaagtgc aatagagcag aatatgaatt acatgcgtgt ctttctccta 3180 gactacatga ccccatataa attacattcc ttatctattc tgccatcacc aaaacaaagg 3240 taaaaatact tttgaagatc tactcatagc aagtagtgtg caacaaacag atatttctct 3300 acatttattt ttagggaata aaaataagaa ataaaatagt cagcaagcct ctgctttctc 3360 atatatctgt ccaaacctaa agtttactga aatttgctct ttgaatttcc agttttgcaa 3420 gcctatcaga ttgtgtttta atcagaggta ctgaaaagta tcaatgaatt ctagctttca 3480 ctgaacaaaa atatgtagag gcaactggct tctgggacag tttgctaccc aaaagacaac 3540 tgaatgcaaa tacataaata gattatgaa tatggttttg aacatgcaca tgagaggtgg 3600 atatagcaac agacacatta ccacagaatt actttaaaac tacttgttaa catttaattg 3660 cctaaaaact gctcgtaatt tactgttgta gcctaccata gagtaccctg catggtacta 3720 tgtacagcat tccatcctta cattttcact gttctgctgt ttgctctaga caactcagag 3780 ttcacc 3786
Claims (8)
A recombinant promoter comprising a recombinant promoter region (Mut-OVOT) of the modified obtransferrin gene represented by the nucleotide sequence of SEQ ID NO: 2 and a promoter region of the ovalbumin gene represented by the nucleotide sequence of SEQ ID NO: 3.
상기 변형 오브트렌스페린 유전자의 재조합 프로모터 영역(Mut-OVOT)은 서열번호 1의 염기서열로 표시되는 오브트렌스페린 유전자의 프로모터 영역(OVOT)에 4개의 ERα(Estrogen receptor alpha) 결합 부위를 추가로 도입하는 것을 특징으로 하는, 재조합 프로모터.
According to claim 1,
In the recombinant promoter region (Mut-OVOT) of the modified obtransferrin gene, four estrogen receptor alpha (ERα) binding sites were additionally introduced into the promoter region (OVOT) of the obtransferrin gene represented by the nucleotide sequence of SEQ ID NO: 1 Recombinant promoter, characterized in that.
상기 재조합 프로모터는 목적 단백질을 과발현시키기 위한 것인, 재조합 프로모터.
According to claim 1,
The recombinant promoter is for overexpressing the target protein, the recombinant promoter.
상기 오브트렌스페린 유전자의 프로모터 영역(OVOT)은 2개의 ERα(Estrogen receptor alpha) 결합 부위를 포함하는 것을 특징으로 하는, 재조합 프로모터.
3. The method of claim 2,
The promoter region (OVOT) of the obtransferrin gene is a recombinant promoter, characterized in that it comprises two Estrogen receptor alpha (ERα) binding sites.
A vector comprising the recombinant promoter of any one of claims 1 to 4.
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 벡터인 것을 특징으로 하는, 벡터.
6. The method of claim 5,
The vector is a plasmid vector, cosmid vector, HIV (Human immunodeficiency virus) vector, MLV (Murineleukemia virus) vector, ASLV (Avian sarcoma / leukosis) vector, SNV (Spleen necrosis virus) vector, RSV (Rous sarcoma virus) vector, Any one vector selected from the group consisting of MMTV (Mouse mammary tumor virus) vector, adenovirus vector and herpes simplex virus vector, lentivirus vector and episomal vector Characterized in that, vector.
A chicken transformed with the vector of claim 5 .
A method of expressing the target protein in chickens by transforming the recombinant vector of claim 5 containing the target gene into a chicken.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110097298A (en) * | 2010-02-25 | 2011-08-31 | 고려대학교 산학협력단 | Expression system comprising dual promoters and overexpression of foreign genes using the same |
| KR20120014404A (en) * | 2010-08-09 | 2012-02-17 | 서울대학교산학협력단 | Methods for producing proteins using transgenic avians |
| KR20200074834A (en) * | 2018-12-14 | 2020-06-25 | 대한민국(농촌진흥청장) | Recombinant promoter comprising the promoter region of the ovotransferrin gene and the promoter region of the ovalbumin gene |
| KR102159050B1 (en) * | 2019-07-12 | 2020-09-24 | 대한민국 | Recombinant promoter comprising the promoter region of the ovomucoid gene and the promoter region of the ovalbumin gene |
| KR102159051B1 (en) * | 2019-07-12 | 2020-09-24 | 대한민국 | Recombinant promoter comprising the promoter region of the lysozyme gene and the promoter region of the ovalbumin gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110097298A (en) * | 2010-02-25 | 2011-08-31 | 고려대학교 산학협력단 | Expression system comprising dual promoters and overexpression of foreign genes using the same |
| KR20120014404A (en) * | 2010-08-09 | 2012-02-17 | 서울대학교산학협력단 | Methods for producing proteins using transgenic avians |
| KR20200074834A (en) * | 2018-12-14 | 2020-06-25 | 대한민국(농촌진흥청장) | Recombinant promoter comprising the promoter region of the ovotransferrin gene and the promoter region of the ovalbumin gene |
| KR102159050B1 (en) * | 2019-07-12 | 2020-09-24 | 대한민국 | Recombinant promoter comprising the promoter region of the ovomucoid gene and the promoter region of the ovalbumin gene |
| KR102159051B1 (en) * | 2019-07-12 | 2020-09-24 | 대한민국 | Recombinant promoter comprising the promoter region of the lysozyme gene and the promoter region of the ovalbumin gene |
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