KR101359510B1 - Hemocyte-specific promoter and its core active region of bombyx mori for regulation of tissue-specific stage gene expression - Google Patents
Hemocyte-specific promoter and its core active region of bombyx mori for regulation of tissue-specific stage gene expression Download PDFInfo
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Abstract
본 발명은 누에 혈림프 특이적 발현 유전자의 프로모터 및 이 유전자의 최대 프로모터 활성영역에 관한 것이다.
본 발명의 누에 혈림프 특이적 발현 프로모터는 누에로부터 분리하여 서열번호 1로 표현되는 것이 특징이다.
본 발명에 의해, 누에의 혈림프에 특이적으로 발현되어 형질전환 누에로부터 유용한 유전자 재조합 단백질을 생산하도록 한 누에 혈림프 특이적 발현 유전자의 프로모터 및 이 유전자의 최대 프로모터 활성영역이 제공된다.The present invention relates to a promoter of the silkworm hemolymph specific expression gene and the maximum promoter active region of the gene.
The silkworm hemolymph specific expression promoter of the present invention is characterized by being expressed by SEQ ID NO: 1 from silkworm.
By the present invention, there is provided a promoter of silkworm hemolymph specific expressing genes that are specifically expressed in the hemolymph of silkworms to produce a useful recombinant protein from a transgenic silkworm and the maximum promoter active region of the gene.
Description
본 발명은 누에 혈림프 특이적 발현 유전자의 프로모터 및 이 유전자의 최대 프로모터 활성영역에 관한 것으로, 보다 상세하게는 누에의 혈림프에서 특이적으로 발현되어 형질전환 누에로부터 유용한 유전자 재조합 단백질을 생산하도록 한 누에 혈림프 특이적 발현 유전자의 프로모터 및 이 유전자의 최대 프로모터 활성영역에 관한 것이다.
The present invention relates to a promoter of a silkworm hemolymph specific expression gene and a maximum promoter active region of the gene, and more particularly, to specifically express a gene in hemolymph of silkworm to produce a useful recombinant protein from a transgenic silkworm. A promoter of a silkworm hemolymph specific expression gene and a maximum promoter active region of the gene.
누에(silkworm, Bombyx mori)는 나비목 누에나방과에 속하는 누에나방의 유충으로서, 알에서 부화한 유충이 발육하여 번데기가 되고, 성충(나방)이 되어 알을 낳고 일생을 마친다. Silkworm (silkworm, Bombyx) mori ) is a larva of the silkworm moth belonging to the genus Moth, and the larvae hatched from eggs develop into pupae and become adult (moths) to lay eggs and finish their lives.
누에알(silkworm egg)은 고치 생산을 위해서 이용되는 누에의 알로써, 1년에 한 번 발생하는 1화성(voltinism, 곤충이 1년간에 몇 세대를 되풀이하는 성질로 유전형질에 의하여 결정되어 있는 특성)과 두 번 발생하는 2화성, 그리고 여러번 발생하는 다화성이 있다. Silkworm egg (silkworm egg) is a silkworm egg used for the production of cocoons. It is a trait that is determined by genotyping that occurs once a year in voltinism. ), Two occurrences of Mars, and multiple occurrences of Mars.
다화성 누에는 열대나 아열대 지역에 적응한 계통이고 우리나라에서 사육하고 있는 누에는 모두 2화성 계통이다. 2화성 품종은 다화성에 비해 알, 누에, 번데기, 나방이 모두 크고 튼튼하며, 고치 무게, 고치층 무게 등 실용형질이 우수하여 실용품종으로 일컫는다. The polymorphic silkworms are adapted to tropical or subtropical areas, and all of the silkworms raised in Korea are bisexual. 2 Martian varieties are large, strong eggs, silkworms, pupa, and moths compared to polymorphism.
최초의 누에 형질전환 연구로 백란계통의 누에알에 흑란계통의 누에알 전체 게놈 DNA를 주입하여 흑란 유도를 시도하였는데, 당 세대에서는 흑란이 유도되었으나, 다음 세대로 형질이 전이되지 않아 실패하였다. In the first silkworm transformation study, we tried to induce black eggs by injecting the whole genome DNA of black silkworm eggs into the silkworm eggs of the white eggs. In this generation, the black eggs were induced, but failed to transfer to the next generation.
이후에도 지속적으로 누에 형질전환을 시도하였지만 실패를 계속 하다가, 2000년도에 나비목 곤충 Trichoplusia ni에서 유래한 피기백 전이인자(piggyBac transposon)을 이용하여 다음 세대의 누에 유충에서 표지유전자를 발현하는 누에를 선발함으로써 누에 형질전환에 성공한 바 있다.He continued to try to transform silkworms, but he continued to fail. In 2000, Lepidoptera insect Trichoplusia PiggyBac transposon derived from ni was used to transform silkworms by selecting silkworms that express marker genes in the next generation of silkworm larvae.
현재까지 누에 형질전환체 선발을 위해서 사용되고 있는 프로모터는 누에의 액틴3 프로모터(BmA3)와 초파리의 홑눈과 신경시스템에서 특이적으로 발현하는 3xP3 프로모터가 사용되고 있다.To date, the promoters used for silkworm transformant selection include the
현재 누에 혈림프 세포 특이적으로 발현되는 유전자는 몇 가지가 개발이 되어 있으나, 이 유전자의 발현을 조절하는 프로모터 영역에 대한 연구 및 개발은 미미한 상태로서 특이적으로 본 발명과 같이 전체 조직에서 유전자 발현을 조절하는 BmA3 프로모터에 비해 활성이 우수한 혈림프 특이적 발현 프로모터에 대한 연구는 시행이 된바 없다.
Currently, several genes that are specifically expressed in silkworm hematopoietic cell have been developed, but research and development on the promoter region that regulates the expression of these genes is insignificant, and specifically, gene expression in whole tissues as in the present invention. No studies have been conducted on hemolymph specific expression promoters that are more active than the BmA3 promoter that regulates the regulation.
본 발명의 목적은 누에의 혈림프에 특이적으로 발현되어 형질전환 누에로부터 유용한 유전자 재조합 단백질을 생산하도록 한 누에 혈림프 특이적 발현 유전자의 프로모터 및 이 유전자의 최대 프로모터 활성영역을 제공하기 위한 것이다. It is an object of the present invention to provide a promoter of silkworm hemolymph specific expressing genes which are specifically expressed in the hemolymph of silkworms to produce a useful recombinant protein from a transgenic silkworm and the maximum promoter active region of the gene.
본 발명이 이루고자 하는 기술적 과제들은 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급되지 않은 다른 기술적 과제들은 아래의 기재로부터 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.
상기 목적을 달성하기 위한 본 발명의 누에 혈림프 특이적 발현 프로모터는 누에로부터 분리하여 서열번호 1로 표현되며, 579bp로 이루어진 것이 특징이다.Silkworm hemolymph specific expression promoter of the present invention for achieving the above object is expressed by SEQ ID NO: 1 separated from silkworms, characterized in that consisting of 579bp.
본 발명의 누에 혈림프 특이적 발현 유전자의 최대 프로모터 활성영역은 서열번호 1로 표현되는 누에 혈림프 특이적 발현 프로모터를 포함하여 이루어지는 것이 특징이다.The maximum promoter active region of the silkworm hemolymph specific expression gene of the present invention is characterized by comprising a silkworm hemolymph specific expression promoter represented by SEQ ID NO: 1.
본 발명의 누에 형질전환방법은 상기의 누에 혈림프 특이적 발현 프로모터를 이용하는 것이 특징이다.Silkworm transformation method of the present invention is characterized by using the silkworm hemolymph specific expression promoter.
본 발명의 누에 형질전환체는 상기의 누에 혈림프 특이적 발현 프로모터를 사용하여 유용한 유전자 재조합 단백질을 생산하는 것이 특징이다.
The silkworm transformant of the present invention is characterized by producing a useful recombinant protein using the silkworm hemolymph specific expression promoter.
본 발명에 의해, 누에 혈림프에 특이적으로 발현이 가능한 누에 혈림프 특이적 발현 유전자의 프로모터가 제공되고 이 유전자의 최대 프로모터 활성영역이 확인됨으로써, 혈림프 세포에서 특이적으로 발현하여 원하는 유용한 유전자 재조합 단백질 수득이 가능하고, 그 수득방법 또한 간편하여 유용한 유전자 재조합 단백질을 생산하는 누에 형질전환체 개발에 소요되는 비용과 시간을 절감시킬 수 있다.
According to the present invention, a promoter of a silkworm hemolymph specific expression gene capable of specifically expressing silkworm hemolymph is provided and the maximum promoter active region of the gene is identified, thereby making it useful for expressing specifically in hemolymph cells. It is possible to obtain a recombinant protein, and the method of obtaining the recombinant protein is also simple, thereby reducing the cost and time required for developing a silkworm transformant to produce a useful recombinant protein.
도 1은 각 조직 특이적 발현 유전자의 타겟 발굴을 위해 이용되는 BmMDB(Bombyx mori Microarray Database) 웹을 나타낸 도면.
도 2는 누에 조직 특이적 발현 유전자 검증을 위해 도 1에서 발굴된 타겟에 대한 primer를 이용하여 RT-PCR결과를 나타낸 도면.
silk gland: 견사선
Midgut: 중장
Fat body: 지방체
도 3은 정량 PCR(real-time RT-PCR) 방법을 통하여 각 유전자별 혈림프(Hemocyte)에 대해 특이적 발현율을 나타낸 그래프.
도 4는 혈림프 세포 RNA를 이용한 RT-PCR결과를 나타낸 도면.
도 5는 혈림프 세포에 특이적으로 발현하는 유전자(sw17255) 프로모터의 핵심 염기서열을 나타낸 도면.
도 6은 혈림프 세포에 특이적으로 발현하는 유전자(sw17255)의 예상 프로모터 영역의 유전자 분절 크기를 나타낸 도면.
도 7은 유전자 분절을 이용한 프로모터 활성상태를 나타낸 그래프.1 is a diagram showing a Bombyx mori Microarray Database (BmMDB) web used for target discovery of each tissue specific expression gene.
Figure 2 is a diagram showing the results of RT-PCR using primers for the target unearthed in Figure 1 for verification of silkworm tissue-specific expression gene.
silk gland: silk gland
Midgut: Lieutenant General
Fat body: fat body
Figure 3 is a graph showing the specific expression rate for hemolymph (Hemocyte) for each gene through the quantitative PCR (real-time RT-PCR) method.
Figure 4 is a diagram showing the RT-PCR results using hemolymph cell RNA.
Figure 5 shows the core sequence of the gene (sw17255) promoter specifically expressing hemolymph cells.
Figure 6 shows the gene segment size of the expected promoter region of the gene (sw17255) specifically expressing hemolymph cells.
7 is a graph showing promoter activity using gene segments.
본 발명은 혈림프 세포에서 특이적으로 발현하여 원하는 유용한 유전자 재조합 단백질 수득이 가능하고, 그 수득방법 또한 간편하여 유용한 유전자 재조합 단백질을 생산하는 누에 형질전환체 개발에 소요되는 비용과 시간을 절감시킬 수 있도록 하기 위한 목적으로, 하기와 같은 과정을 거쳐 누에 혈림프에 특이적으로 발현이 가능한 누에 혈림프 특이적 발현 유전자의 프로모터를 개발하고, 이 유전자의 최대 프로모터 활성영역을 제공하게 된다.The present invention is specifically expressed in hemolymph cells to obtain a desired useful recombinant protein, the method of obtaining is also easy to reduce the cost and time required to develop a silkworm transformant to produce a useful recombinant protein. For the purpose of this, a promoter of a silkworm hemolymph specific expression gene capable of specifically expressing silkworm hemolymph is developed through the following process, and provides a maximum promoter active region of the gene.
먼저, 1) 누에 혈림프 세포 특이적 발현유전자 발굴한다.First, 1) silkworm hemolymph cell-specific expression genes are identified.
누에 조직(견사선, 중장, 지방체, 혈림프)별로 분리하여 누에 조직 특이적 유전자 발현 양상 분석을 통한 혈림프 특이적 발현 유전자를 분석하게 된다.Hemolymph-specific expression genes are analyzed through silkworm tissue-specific gene expression pattern analysis.
이때, 정량적 유전자 발현측정법(Real-Time RT-PCR)을 이용하여 발현율을 검증한다.At this time, the expression rate is verified using quantitative gene expression measurement (Real-Time RT-PCR).
2) 상기 발굴한 혈림프 특이적 발현유전자(sw17255)의 예상 프로모터 영역을 확인하여 유전자원을 확보한다.2) Confirm the expected promoter region of the hemolymph specific expression gene (sw17255) found above to secure a gene source.
즉, 생물정보학 방법을 이용하여 개시코돈 상위 약 579bp의 염기서열(서열목록에 첨부)을 분석하고, 누에 게놈을 대상으로 유전자 증폭 방법을 이용하여 유전자원 클로닝 및 염기서열을 확인하고 유전자 분절(Deletion Mutant)을 구성한다.In other words, using the bioinformatics method, the base sequence (attached to the sequence list) of the top 579 bp of the start codon was analyzed, and the gene source cloning and the nucleotide sequence were identified using the gene amplification method for the silkworm genome. Mutant).
3) 상기 구성된 유전자 분절(Deletion mutant)를 통해 최대 활성영역을 분석한다.3) The maximum active region is analyzed through the constructed mutant.
즉, 생물정보학 방법을 이용한 프로모터 영역의 특징을 조사하고, 각 유전자 분절을 주사방식을 이용하여 누에 혈림프 세포에 직접도입하여 프로모터 활성을 검증한 후, 기존에 알려진 프로모터인 대조군(BmA3 프로모터)과 비교하여 본 개발 영역 프로모터 활성을 비교하여 상기 혈림프 특이적 발현 유전자(sw17255)의 최대 프로모터 활성영역을 제공한다.In other words, by investigating the characteristics of the promoter region using bioinformatics method, each gene segment was directly introduced into the silkworm blood lymphocytes using the injection method to verify the promoter activity, and then the control group (BmA3 promoter), which is a known promoter By comparison, the present developmental region promoter activity is compared to provide the maximum promoter active region of the hemolymph specific expression gene (sw17255).
이에, 혈림프 세포에서 특이적으로 발현하여 원하는 유용한 유전자 재조합 단백질 수득이 가능하고, 그 수득방법 또한 간편하여 유용한 유전자 재조합 단백질을 생산하는 형질전환 누에를 이용한 유용 유전자 재조합 단백질의 개발 및 적용에 소요되는 비용과 시간을 절감시킬 수 있게 된다.
Therefore, it is possible to obtain a desired useful recombinant protein by expressing specifically in hematopoietic cells, and the method of obtaining is also simple and useful for the development and application of a useful recombinant protein using a transformed silkworm to produce a useful recombinant protein. This will save you money and time.
이하, 본 발명에 대하여 실시 예를 통하여 상세히 설명하나, 이들이 본 발명의 범위를 제한하는 것은 아니다. Hereinafter, the present invention will be described in detail with reference to Examples, but these are not intended to limit the scope of the present invention.
<실시예 1> 누에 조직별 특이적 발현 유전자 예상 프로모터 영역 유전자원 클로닝(cloning) 및 분석(assay)Example 1 Cloning and Analysis of Predicted Promoter Region Gene Source Specific Expression Gene of Silkworm Tissue
1. 실험방법1. Experimental Method
백옥잠 품종의 각 조직을 미리 분리하고, 도 1에 나타나 있는 BmMDB(Bombyx mori Microarray Database)를 이용하여 각 조직 특이적 발현 유전자의 타겟을 발굴하였다.Each tissue of the white jade species was previously isolated, and the target of each tissue specific expression gene was discovered using the Bombyx mori Microarray Database (BmMDB) shown in FIG. 1.
1) 누에 조직 특이적 발현 유전자 스크리닝1) Silkworm tissue specific expression gene screening
백옥잠 누에 (잠123×잠124 교잡종)의 견사선, 중장, 지방체, 그리고 혈림프 등을 각 조직별로 분리하고 RNA를 추출하였다.The silk gland, middle limb, fat body, and hemolymph of the white jade silkworm (Zam 123 × Zam 124 hybrid) were separated from each tissue and RNA was extracted.
추출한 RNA를 대상으로 하여 cDNA를 합성하여 BmMDB로부터 발굴하였다.CDNA was synthesized from the extracted RNA and extracted from BmMDB.
발굴된 각 조직별 유전자 타겟 목록은 하기 표 1과 같이 나타났으며, 표 1의 조직 특이적 발현을 검증하기 위한 RT-PCR을 실시하여 검증하여 도 2와 같이 나타났다.The list of gene targets for each tissue was found as shown in Table 1 below, and verified by performing RT-PCR for verifying tissue-specific expression of Table 1 as shown in FIG. 2.
2. 실험결과2. Experimental results
상기 실험결과 도 3에 나타나 있듯이, 누에의 혈림프 특이적 발현을 나타내는 sw17255 유전자는 BmA3 유전자의 발현율에 비해 현저하게 높은 발현율을 나타내었다.As shown in FIG. 3, the sw17255 gene expressing hemolymph specific expression of silkworms showed a significantly higher expression rate than that of the BmA3 gene.
상기 결과를 바탕으로 본 발명은 혈림프 세포 특이적인 유전자 발현 조절이 가능한 프로모터(promoter)를 발굴하고자 하였다.Based on the above results, the present invention was to discover a promoter capable of regulating hemolymph cell specific gene expression.
즉, 도 4에 나타나 있듯이, 혈림프로부터 분리한 조직 특이적 발현 RNA를 이용한 RT-PCR결과, 내부 대조구(internal control)로 사용된 BmA3유전자의 발현과 비교하여 타겟(target) 유전자인 sw17255 유전자의 경우 견사선 특이적 발현을 보임으로 인해 sw17255는 혈림프 세포 특이적 발현유전자임을 확인하였다.That is, as shown in Figure 4, RT-PCR results using tissue-specific expression RNA isolated from hemolymph, compared with the expression of the BmA3 gene used as internal control (internal control) of the target gene sw17255 gene In the case of silk gland specific expression, sw17255 was identified as a hemolymph cell-specific expression gene.
따라서 sw17255 유전자 프로모터의 경우 형질 전환 누에 제작 시 혈림프 세포 특이적 발현 조절이 가능하여 누에 발생 및 생육에 지장 없이 목적 단백질의 발현이 가능하도록 하는 장점이 있을 것으로 사료된다.
Therefore, the sw17255 gene promoter is capable of regulating hemolymph cell-specific expression during the production of transgenic silkworms, and thus, may have the advantage of enabling the expression of the target protein without disrupting silkworm development and growth.
<실시예 2> 누에 혈림프 세포 특이적 발현 유전자 예상 프로모터 영역의 확보Example 2 Securing silkworm hemolymph cell specific expression gene predicted promoter region
우선 미국 국립생물정보센터(NCBI)의 염기서열 정보를 분석하고 개시 코든(codon) 상위의 2,122 bp 영역을 최대 promoter 예상 영역으로 결정하고 게놈 DNA를 이용하여 PCR을 실시하였다. First, the sequence information of the National Institute of Biological Information (NCBI) was analyzed, and the 2,122 bp region above the starting codon was determined as the maximum promoter region, and PCR was performed using genomic DNA.
예상 promoter 영역의 활성을 분석하여 정확한 promoter 염기서열을 확인하기 위하여 유전자 분절(deletion mutant construct)를 구성하였다. Genetic segment mutant constructs were constructed to identify the exact promoter sequence by analyzing the activity of the predicted promoter region.
유전자 분절 실험을 위한 각 프라이머(primer)의 경우 특이적 염기서열에 따라 일정한 크기가 될 수 있도록 지정하였으며, 크기별 PCR 산물은 pGL3-Basic plasmid DNA (Promega, Madison, WI)에 삽입하기 위하여 순방향 프라이머(forward primer)는 Mlu I 그리고 역방향 프라이머(reverse primer)는 Hind III 제한효소 인식 염기서열을 첨가하여 주었다.Each primer for gene segmentation experiment was designated to have a certain size according to specific sequencing, and PCR products of each size were designated as forward primers to insert into pGL3-Basic plasmid DNA (Promega, Madison, WI). Forward primer) was added to Mlu I and reverse primer to Hind III restriction enzyme recognition sequence.
다시 말해, PCR을 실시하기 위한 주형 게놈(genomic) DNA는 잠124로부터 추출하였고, PCR 수행은 최초 94℃에서 2분 동안 예비 변성을 시킨 다음, 변성반응은 94℃에서 30초, 중합반응은 56℃에서 30초, 그리고 합성반응은 72℃에서 2분동안 반응을 조건으로 하여 20회 반복 수행을 한 후 최종적으로 72℃에서 10분간 반응하여 종결하였다. In other words, the template genomic DNA for PCR was extracted from Zam124, and PCR was preliminarily denatured for 2 minutes at 94 ° C, followed by 30 seconds at 94 ° C, and polymerization at 56 ° C. 30 seconds at ℃, and the synthesis reaction was repeated 20 times with the reaction conditions for 2 minutes at 72 ℃ and finally the reaction was terminated at 72 ℃ for 10 minutes.
PCR 증폭된 유전자는 pGEM-T Easy 벡터(Promega, Madison, WI)에 삽입한 다음 염기서열 분석방법을 이용하여 2,122 bp의 염기서열을 확인하였다. PCR amplified gene was inserted into pGEM-T Easy vector (Promega, Madison, Wis.) And then nucleotide sequence of 2,122 bp was confirmed using sequencing method.
NCBI의 염기서열 정보에 따르면 총 2,122 bp 크기의 염기서열을 나타내었으나, 본 발명에 사용된 잠124를 target으로 한 PCR 증폭결과 동일한 염기서열을 확보할 수 있었다(도 5)According to the base sequence information of the NCBI, the base sequence of a total size of 2,122 bp was shown, but the same base sequence was obtained by PCR amplification targeting the latent 124 used in the present invention (FIG. 5).
확인된 약 2.1 kb의 최대 promoter 예상 영역 유전자는 Mlu I (NEB, Ipswich, MA, USA)과 Hind III (NEB) 제한효소를 사용하여 절단하고 분리하여 pGL3-Basic plasmid DNA의 동일한 제한효소 부위에 삽입하여 완성하였고 이를 pGL3-sw17255(B)로 명명하였다. A maximum promoter predicted region gene of about 2.1 kb identified was cleaved and isolated using Mlu I (NEB, Ipswich, MA, USA) and Hind III (NEB) restriction enzymes and inserted into the same restriction site of pGL3-Basic plasmid DNA. This was completed by the name pGL3-sw17255 (B).
이후 핵심 promoter 영역을 확인하기 위하여 유전자 분절을 구성하였다(도 6). Afterwards, gene segments were constructed to identify key promoter regions (FIG. 6).
서로 다른 크기의 유전자 분절을 위하여 각각 새로운 primer를 제작하였으며, 이 primer들 또한 pGL3-Basic plasmid DNA에 삽입하기 위하여 forward primer는 Mlu I 그리고 reverse primer는 Hind III 제한효소 인식 염기서열을 첨가하여 주었다. PCR 증폭을 위한 주형 DNA는 pGL3-sw17255(B)를 사용하였다. New primers were prepared for different size gene fragments, and the primers also added the Mlu I for the forward primer and the Hind III restriction enzyme recognition sequence for the reverse primer to be inserted into pGL3-Basic plasmid DNA. As template DNA for PCR amplification, pGL3-sw17255 (B) was used.
PCR 증폭된 유전자는 pGL3-Basic plasmid DNA의 동일한 제한효소 부위에 삽입하였다.
PCR amplified gene was inserted into the same restriction enzyme site of pGL3-Basic plasmid DNA.
<실시예 3> 듀얼 루시퍼레이즈 분석방법(DUAL-Luciferase Assay)을 이용한 프로모터(promoter) 활성영역 분석<Example 3> Analysis of promoter active region using the dual luciferase assay method (DUAL-Luciferase Assay)
도 5에 나타낸 각 유전자 분절을 대상으로 루시퍼레이즈(Luciferase)의 활성을 분석함으로써 정확한 promoter의 영역을 확립하고자 하였다. By analyzing the activity of luciferase in each gene segment shown in FIG. 5, an accurate promoter region was established.
설명하면, 5령 2일의 백옥잠 누에는 프로모터의 활성을 분석하기 위해 각 plasmid 유전자를 도입하여 Luciferase Assay 실험에 사용하였다. In order to analyze the activity of Baekjam silkworms at 5 days and 2 days, each plasmid gene was introduced and used in the Luciferase Assay experiment.
각 1㎍의 유전자 분절용 플라스미드 DNA(deletion mutant plasmid DNA)들과 대조군으로 pGL3-BmA3 plasmid DNA는 세포내 핵산도입시약(Fugene Transfection Reagent)을 사용하여 누에의 체강에 직접 주사기로 주입하여 유전자를 도입하였다. Each 1 μg of gene fragment plasmid DNA and pGL3-BmA3 plasmid DNA were used as a control to inject the gene directly into the silkworm's body cavity using an intracellular Nucleus Transfection Reagent. It was.
레닐라(Renilla)를 발현하는 pRLA-A3 plasmid DNA를 각 실험군과 대조군에 함께 넣은 듀얼-루시퍼레이즈 리포터 분석 키트(Dual-Luciferase Reporter Assay Kit)을 사용하여 프로모터의 활성을 분석하였다. The activity of the promoter was analyzed using a Dual-Luciferase Reporter Assay Kit in which pRLA-A3 plasmid DNA expressing Renilla was added to each experimental group and the control group.
즉, Renilla를 발현하는 pRLA-A3 각 plasmid DNA를 실온에서 Fugene Transfection Reagent와 함께 혼합한 다음, 최종적으로 100㎕ 부피가 될 수 있도록 PBS를 첨가하여 주었다. In other words, Renilla expressing each of the plasmid DNA pRLA-A3 with Fugene Transfection Reagent at room temperature, and then added PBS so that the final volume to 100μl.
100㎕ 부피의 유전자 도입을 위한 혼합액은 이 후 상온에서 15분 동안 더 정치한 다음, 각 누에의 개체에 주사하여 주었다. The mixed solution for the introduction of a 100 μl volume gene was then further left at room temperature for 15 minutes and then injected into each silkworm individual.
이후 적당한 온도에서 72시간 동안 사육하였다. 각 프로모터 후보 유전자의 활성을 측정하기 위하여 채취한 혈림프는 1X PBS로 세척한 세포는 100㎕의 수동적 분해버퍼(Passive Lysis Buffer)를 넣고 세포를 파쇄한 다음 측정기(Luminometer)를 사용하여 루시퍼레이즈(Luciferase)와 레닐라(Renilla)의 활성을 확인하였다.Thereafter, the animals were bred at an appropriate temperature for 72 hours. Hemolymphs collected to measure the activity of each promoter candidate gene were washed with 1X PBS, and 100 μl of passive lysis buffer (Passive Lysis Buffer) was added to the cells, and the cells were disrupted and then luciferase (Luminometer) was used. Luciferase) and Renilla (Renilla) activity was confirmed.
그 결과, 도 7에 나타나 있듯이 예상된 프로모터의 5-말단부위 분절을 이용한 연구를 통하여 전체 유전자 모두를 포함하는 경우 기존의 알려진 BmA3 프로모터와 비교하여 활성이 증가된 특성을 보였으며, 이는 조직 특이적 발현조절을 위한 유용한 프로모터임을 확인하였다.As a result, as shown in FIG. 7, the study using the 5-terminal segment of the expected promoter showed that the activity was increased in comparison with the known BmA3 promoter when all the genes were included, which was tissue specific. It was confirmed that it is a useful promoter for expression control.
상기의 본 발명은 바람직한 실시 예를 중심으로 살펴보았으며, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자는 본 발명의 본질적 기술 범위 내에서 상기 본 발명의 상세한 설명과 다른 형태의 실시 예들을 구현할 수 있을 것이다. 여기서 본 발명의 본질적 기술범위는 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.
The present invention has been described with reference to the preferred embodiments, and those skilled in the art to which the present invention pertains to the detailed description of the present invention and other forms of embodiments within the essential technical scope of the present invention. Could be. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.
서열목록 전자파일 첨부Attach an electronic file to a sequence list
Claims (5)
누에 혈림프 특이적 발현 프로모터.Represented by SEQ ID NO: 1 separated from silkworms,
Silkworm hemolymph specific expression promoter.
누에 혈림프 특이적 발현 프로모터.According to claim 1, wherein SEQ ID NO: 1 consists of 579 bp,
Silkworm hemolymph specific expression promoter.
누에 형질전환체.
Using the silkworm hemolymph specific expression promoter of claim 1 or 2 to produce a useful recombinant protein,
Silkworm transformant.
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