초록 ▼

$\cdot$ Lactoferrin을 이용한 클로렐라 배양법 특허 등록
$\cdot$ 국내에서 분리한 클로렐라 바이러스로부터 미세조류 형질전환에 이용할 수 있는 CaMV 35S promoter와 유사하거나 그 이상의 활성을 가지는 신종 프로모터 11종 분리
$\cdot$ 신종 프로모터에 관련한 특허 2건 출원
$\cdot$ Lactoferrin을 이용한 배양법과 신종 프로모터에 대한 기술이전 2건 실시
$\cdot$

$\cdot$ Lactoferrin을 이용한 클로렐라 배양법 특허 등록
$\cdot$ 국내에서 분리한 클로렐라 바이러스로부터 미세조류 형질전환에 이용할 수 있는 CaMV 35S promoter와 유사하거나 그 이상의 활성을 가지는 신종 프로모터 11종 분리
$\cdot$ 신종 프로모터에 관련한 특허 2건 출원
$\cdot$ Lactoferrin을 이용한 배양법과 신종 프로모터에 대한 기술이전 2건 실시
$\cdot$ Chlorella vulgaris로부터 유래한 signal peptide 2종 확보
$\cdot$ 확보된 프로모터, signal peptide를 이용하여 EPO, TPA, IGF를 포함한 벡터 제작
$\cdot$ 해양 조류 바이러스인 Feldmannia simplex virus(FsV)의 전체 약 153kb의 genome 분석
$\cdot$ FsV의 142개의 ORF로부터 exonuclease, adenine DNA methyltransferase, integrase, kinase, ligase, methylase 등의 유전자 확보
$\cdot$ Adenine DNA methyltransferase에 관련한 특허 출원

Abstract ▼

The purpose of molecular farming is to provide a safe and inexpensive means for mass production of recombinant pharmaceutical proteins. Heterologous expression systems being used in these days, Escherichia coli, yeast, cultured cells, plant and animals, have their own limits such as lack of biologic

The purpose of molecular farming is to provide a safe and inexpensive means for mass production of recombinant pharmaceutical proteins. Heterologous expression systems being used in these days, Escherichia coli, yeast, cultured cells, plant and animals, have their own limits such as lack of biological activity of the expressed protein, contamination by pathogens and toxins, low level expression, high cost for culture and purification.
Chlorella is a unicellular eukaryotic algae that can be cultured in large scale at low cost. It has been used as healthy food for long time, and there will be less resistant against using this organism as a bioreactor for foreign protein production. In addition, purification of the expressed protein is much easier because of simple cell structure of this organism. Considering all these features, Chlorella is the best system for molecular farming that can be used in future.
The purpose of this research is to construct efficient DNA vectors for microalgae, Chlorella ellipsoidea, transformation, which can used as a bioreactor for the expression of recombinant pharmaceutical proteins.
For this purpose, we have isolated strong promoters from chlorella virus SS-2 and HS-1 that had been isolated from fresh water in Korea. We have isolated 11 new promoters from these two viruses and confirmed that they are equal or stronger than the Cauliflower mosaic virus (CaMV) 35S promoter which is widely used in plant transformation. From this these results, we have applied 2 domestic patients.
In order to make it easy to purify the recombinant protein expressed in transformed chlorella, we have screened secretion signals from chlorella proteins. Also, we have designed two vectors containing signal sequence from eukaryotic proteins and have constructed vectors containing the erythropoietin gene, tissue plasminogen activator gene and insulin like growth factor gene of human.
We also have analyzed the genomic DNA of Feldmannia species virus (FsV), which is composed of 153,259 base pairs and encodes about 128 major open reading frames on both strand. Among the genes encoded by FsV genome, we have characterized the DNA adenine methyltransferase and confirmed its activity in E. coli, and applied a domestic patient.
During the culture of chlorella cells for transformation, we found that lactoferrin can promote the growth of chlorella cell and applied a domestic patient for the results.
The promoters isolated during this research can be used in the construction of vectors for chlorella transformation from which recombinant proteins can be expressed from useful genes including the genes identified from the algal viruses. Also, the proteins such as DNA ligase, DNA polymerase, exonuclease can be expressed in other systems such as baculovirus system and can be used in molecular work. The chlorella transformation system also can be used for the production of proteins including many hormones and vaccine, which are under development.

목차 Contents

• 표지...1
• 제출문...2
• 보고서 초록...3
• 요약문...4
• SUMMARY...7
• CONTENTS...9
• 목차...11
• 제 1 장 연구개발과제의 개요...13
• 제 1 절 서론...13
• 제 2 절 연구개발의 기술적, 경제.산업적, 사회.문화적 중요성...14
• 1. 기술적 측면...14
• 2. 경제$/cdot$산업적 측면...14
• 3. 사회$/cdot$문화적 측면...15
• 제 3 절 연구개발의 목적 및 범위...16
• 제 2 장 국내외 기술개발 현황...17
• 제 1 절 국외의 기술개발 현황...17
• 제 2 절 국내의 기술개발 현황...18
• 제 3 절 당해 기관의 연구...18
• 제 4 절 현재 기술 상태의 취약성...20
• 제 5 절 앞으로의 전망...20
• 제 3 장 연구개발수행 내용 및 결과...22
• 제 1 절 논문...23
• 1. Isolation and characterization of Chlorella virus from fresh water in Korea and application in chlorella transformation system...24
• 2. Activity of early gene promoters from a Korean Chlorella virus isolate in transformed Chlorella algae...39
• 3. Characterization of tRNA gene cluster promoters from a Korean Chlorella virus isolate in transformed Chlorella algae...60
• 4. Identification of the virus integration sites in the host algal chromosome...81
• 5. Complete nucleotide sequence of the small size genome of Feldmannia species Virus(FsV)...96
• 6. Tsolation and characterization of a novel DNA adenine methyltransferase gene from Feldmannia species Virus (FsV)...120
• 제 2 절 특허...135
• 1. 출원...135
• 가. 클로렐라 바이러스 유래 신규 프로모터 및 그 응용...135
• 나. 클로렐라 바이러스 HS-1 유래 초기 발현유전자 프로모터 및 그 응용...151
• 다. 팰드매니아종 바이러스로부터 분리한 신규한 DNA 아데닌 메틸전달효소 유전자...169
• 2. 등록...179
• 가. 락토페린을 함유하는 배양배지 및 이를 이용한 클로렐라의 배양 방법...179
• 제 3 절 기타...186
• 1. Signal sequence의 분리 및 목적단백질의 분비...186
• 제 4 장 목표달성도 및 관련분야에의 기여도...189
• 제 5 장 연구개발결과의 활용계획...191
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보...203
• 제 7 장 참고문헌...206