Journal of Sericultural and Entomological Science (한국잠사곤충학회지)

Korean Society of Sericultural Science (한국잠사학회)
권호 표지

Germ Line Transformation of the Silkworm, Bombyx mori L. with a piggyBac Vector Harboring the Human Lactoferrin Gene

락토페린 유전자도입 piggyBac 벡터에 의한 누에 형질전환

  • Kim, Yong-Soon (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA) ;
  • Sohn, Bong-Hee (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA) ;
  • Kim, Kee-Young (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA) ;
  • Jung, I-Yeon (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA) ;
  • Kim, Mi-Ja (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA) ;
  • Kang, Pil-Don (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, RDA)
  • 김용순 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 손봉희 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 김기영 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 정이연 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 김미자 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 강필돈 (농촌진흥청 농업과학기술원 농업생물부)
  • Published : 2007.12.30
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Abstract

Lactoferrin, an ion-binding 80-kDa glycoprotein, has been suggested to have many biologic activities, such as facilitating ion absorption and having antimicrobial and anti-inflammatory effects. Several of these activities are likely to only be facilitated by human lactoferrin because they depend on the binding of human lactoferrin to specific receptor. To produce recombinant human lactoferrin to animal foods using transgenic silkworm, Bombyx mori L, we have cloned and sequenced the cDNA encoding for a human lactoferrin (HLf) from the mRNA in mammary tumor line (GI-101). As a result, the 2.5-kb fragment of HLf gene was cloned with pGEM-T vector and then this fragment was sequenced. In the nucleotide sequence analysis, single open reading frame of the 2,136-bp encoding for a polypeptide of 712 amino acid residues was detected. On the other hand, we constructed a recombinant plasmid(pPT-HLf), containing human lactoferrin gene for germ line transformation of the silkworm using a piggyBac transposon-derived vector. A nonautonomous helper plasmid encodes the piggyBac transposase. Approximately 6.7% of individuals in the G0 silkworms expressed green fluorescent protein (GFP). PCR analyses of GFP-positive silkworms (G0 and G1) revealed that independent insertions occurred frequently. Furthermore, Western blot analysis showed that the recombinant HLf expressed in hemolymph has the same molecular weight (80 kDa) as a native protein. On the basis of these experiments, expression of HLf in next generation of transgenic silkworm is now in process.

락토페린 cDNA 유전자를 도입시킨 누에 형질전환 실험을 수행한 결과, 다음과 같은 결과를 얻을 수 있었다. 1. 사람 GI-101 세포주의 mRNA로부터 클로닝 된 락토페린 cDNA 유전자의 개시코돈 ATG와 종결코돈 TAA를 포함하는 open reading frame(2,136 bp) 영역을 확인하였다. 2. Sf9 배양세포의 조추출물 시료에 의한 Western blot 분석 결과, 락토페린으로 추정되는 약 80kDa의 단백질 발현을 확인하였다. 3. 누에 형질전환에 높은 전이효율과 활성을 나타내는 트랜스포존을 이용한 전이벡터 pPIGA3GFP를 개조하여 락토페린 cDNA를 삽입시킨 전이벡터 pPT-HLf를 구축하였다. 4. DNA 미량 주사법에 의한 누에 형질전환 개체의 발현 비율은 약 6.7% 정도를 나타냈다. 5. 형질전환 누에(G0) 동일한 세대간 교배 및 처리하지 않은 성충간의 역교배에 의한 차세대(G1) 개체로부터 락토페린 유전자와 동일한 크기의 2.1 kb DNA 단편을 확인 할 수 있었으며, 형질전환 G1 세대의 조추출물 시료에 의한 Western blot 분석 결과, 표준 락토페린 항체와 반응하는 약 80 kDa의 단백질 발현을 확인할 수 있었다.

Keywords

References

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