초록
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▶ 연구목적 :
○ 우리 고유의 독자적인 누에 형질전환 원천기술 개발 및 이를 이용한 인간 조혈촉진단백질 생산 형질전환누에 개발
○ 누에 조직 • 시기 특이 발현 유전자의 프로모터 개발
○ 인간 조혈촉진단백질의 리모델...
▶ 연구목적 :
○ 우리 고유의 독자적인 누에 형질전환 원천기술 개발 및 이를 이용한 인간 조혈촉진단백질 생산 형질전환누에 개발
○ 누에 조직 • 시기 특이 발현 유전자의 프로모터 개발
○ 인간 조혈촉진단백질의 리모델링 기술개발에 의한 재조합단백질 발현량 및 생물학적 활성 향상
▶ 주요연구내용 :
○ 1세부과제명 : 누에 고유 전이인자 개발 및 인간 조혈촉진단백질 생산 누에 제작
- 누에 고유 전이인자 분리 및 외래유전자에 대한 전이활성 분석
· 누에 자체 전이인자의 분리 및 구조 분석 : 전체 346개의 아미노산으로 구성되어 있으며, 5‘-과 3’- 말단에 ITR과 전이인자 인식부위인 반복 TATA 서열 보유하고 있었음
· 누에 자체 전이인자(BmMarT)의 기능 분석 : EMSA 분석 결과, 재조합 BmMarT는 5‘-ITR과 3’-ITR 둘 다에 강하게 결합된 것을 확인
· BmMarT의 외래유전자에 대한 전이활성 분석 : 누에 배양세포주에서 4.1×10-5의 빈도로 외래유전자에 대한 전이활성을 나타냄
- 우리 고유의 독자적인 누에 형질전환 원천기술 개발
· 농가보급용 누에 실용품종 이용 세계 최초로 형질전환 성공
· 알과 유충에서 형질전환체 선발기술 개발 : 선발 비용과 노동력 최소화
· 최적 미세주사 위치구명에 의한 일본 대비 4배 이상 형질전환 효율 향상
- 원천기술 이용 누에 온몸에서 인간조혈촉진단백질이 생산되는 형질전환누에 개발 : 8세대 형질고정
- 누에 견사선 특이발현 인간 조혈촉진단백질 생산 형질전환누에 제작 : 2세대
○ 2세부과제명 : 누에 특이발현 유전자 프로모터 개발
- 누에 배아시기 특이 또는 열충격에 의한 유전자 발현조절 프로모터 개발
· 형질전환누에의 초기 선발이 가능하여 선발시간 및 비용절감 효과
- 타겟 유전자 발현 조절을 위한 열충격 프로모터 개발
· 열충격에 의해 타겟 유전자의 임의적 고발현 형질전환누에 개발 가능
- 누에 조직 특이적 유전자 발현조절 프로모터 개발
· 조직별 특이적 발현 유전자 선발 : 견사선(3), 혈구(1)와 중장(2) 특이적 발현 유전자 6종 선발
· 기존 조직 특이적 프로모터(BmA3) 보다 고 발현이 가능한 혈림프 특이적 프로모터 개발
○ 1협동과제명 : 인간 조혈촉진단백질의 리모델링 기술 개발
- 인간 조혈촉진단백질 발현 벡터 구축 : SCF, IL-3, G-CSF
- 누에 특이 소포체 전달 신호펩타이드 융합을 통한 G-CSF 리모델링 확립
- G-CSF N-terminal 부분 결손을 통한 G-CSF 리모델링 확립
- 리모델링된 G-CSF의 생산 세포주로부터 생산된 G-CSF를 정제하여 기능성을 검정 및 잠정적인 G-CSF 시제품 확보
- 상업적 항-G-CSF 항체를 대체할 수 있는 항체 확보
- G-CSF 발현을 통제할 수 있는 누에 특이 Hsp70 프로모터 벡터 개발
· 상업적 누에 벡터에 사용되는 프로모터 대체하여 단백질 발현을 통제
Abstract
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In 2,000, a method for stable germline transformation in B. mori was developed using a piggyBac transposon-derived vector (Tamura ...
In 2,000, a method for stable germline transformation in B. mori was developed using a piggyBac transposon-derived vector (Tamura et al., 2000). B. mori is therefore a good candidate host for the production of recombinant proteins at an industrial scale. It is easy to handle large numbers of transgenic silkworms because the rearing system was improved by using mulberry leaves and an artificial diet, the adult moths are unable to fly, and the silkworms are unable to live in nature. Recently, papers have shown that the generation of transgenic silkworms that spin recombinant human type III procollagen as a component of the cocoon, an achievement with considerable implications for the mass production of recombinant proteins (Tomita et al., 2003). Also, such as basic fibroblast growth factor (bFGF) (Hino et al. 2006), human serum albumin (HSA) (Ogawa et al. 2007), feline interferon (FeIFN) (Kurihara et al. 2007) and insulin like growth factor-I (hIGF-I) (Zhao et al. 2009) have been successfully expressed in the silk glands of transgenic silkworms. In conclusion, systems based on B. mori could become a major technology for the production of low-cost and high-yield protein, especially in the area of cosmetic, diagnostic, animal-therapeutic and human therapeutic uses. Therefore, to produced the recombinant human granulocyte macrophage colony-stimulating factor 2 (hGM-CSF), two types of transgenic silkworm were designed to be secreted into the whole body and the luman of posterior silk glands. These transgenic silkworms successfully produced recombinant hGM-CSF2 in whole body and cocoon fiber, respectively.
The mulberry silkworm, Bombyx mori, is a powerful model for the analysis of the genetic regulatory mechanism, not to mention one of the most important commercial insects to the silk industry since its first introduction to Korea in 10th century B.C. (Park et al., 2010). Compared to the traditional value of B. mori as a source of silk, its major role in the near future will be as a biomedical insect contributing to the production of biomedical proteins and biomaterials (Park et al., 2011). Therefore, the silkworm might be suited as a host for the mass production of recombinant proteins compared to mammalian and non-mammalian cultured cells. The expressions of target genes in the transgenic insects have to regulate by a time- or tissue-specific manner for effective production of recombinant proteins (Park et al., 2010). This purpose has driven the successful search for several tissue-specific or inducible promoters capable of driving expression in transgenic insects relevant to recombinant production, including the midgut, hemolymph, fat body and salivary glands (Park et al., 2010; Park et al., 2011). In particular, we think that hemolymph-transcribed promoters are ideal candidates to produce of the exogenous genes while it completes its life cycle within the silkworm. Though the data is consistent on most occasions, expressions in specific tissues should be confirmed not only because these data are very useful to researchers working with silkworms, but also because they can be used for the creating transgenic silkworms as a biomedical insect for producing biomaterials, not to mention as well-characterized models for understanding the mechanism for the genetic regulation of tissue-specific development (Park et al., 2010; Park et al., 2011)
Hematopoietic cytokines regulate production of blood cells by stimulating proliferation and differentiation of bone marrow cells. These hematopoietic cytokines have been leading protein drug market over the world. We attempted to develop insect-based expression system of human hematopoietic cytokines such as IL-3, stem cell factor (SCF), and glranulocyte-colony stimulating Factor (G-CSF). For the purpose of maximum expression of human-origin protein in insect cells, we engineered human G-CSF with Bombyx specific endoplasmic reticulum (ER) signal sequence. Bombyx mori-origin cell line, Bm5, was used for this experiment and four different cell lines with wild type G-CSF and three different chimera G-CSF were established. Three different chimera G-CSF were constructed with three Bomyx-origin ER signal sequences of prophenoloxidase activating enzyme (PPAE), protein disulfide isomerase (PDI), and bombyxin (Bx). Secretion of human G-CSF protein from Bm5 cells was greatly improved when Bombyx ER signal sequence was fused to human G-CSF matured protein region. Among the three Bombyx ER signal sequences PPAE's signal sequence showed the greatest production of G-CSF. Also, remodeling in the N-terminal region of matured G-CSF protein indicated important role of secretion and/or production. Ni-sepharose affinity chromatography purified efficiently his-tagged G-CSF and anti-G-CSF antibody was produced for G-CSF protein anssay and purification in the future. Bombyx-origin Hsp70 promoter was linked to bG-CSF that human G-CSF DNA sequence was changed by optimizing codon-usage in Bomyx mori.
