Journal of Sericultural and Entomological Science (한국잠사곤충학회지)

Korean Society of Sericultural Science (한국잠사학회)
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Production of the melittin antimicrobial peptide in transgenic silkworm

멜리틴 항균펩타이드를 생산하는 형질전환누에

  • Received : 2015.04.27
  • Accepted : 2015.04.30
  • Published : 2015.04.30
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Abstract

Melittin is the main component of Bee Venom and has antibacterial activity against several bacteria. To produce the melittin antimicrobial peptide, we constructed transgenic silkworm that expressed melittin gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 300 eggs of bivoltin silkworms, Baegokjam. In total, 131 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 36 broods, and we selected 4 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 12 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a melittin as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that melittin possesses high antibacterial activities against gramnegative bacteria.

본 연구의 목적은 누에형질전환 기술을 이용하여 누에체액에서 melittin 항균펩타이드를 생산하는 것으로서, 본 실험에서는 누에유래의 액틴3 프로모터를 이용하여 melittin 항균펩타이드를 발현시켰다. 누에형질전환체 선발을 위해서는 3xP3 promoter와 EGFP 유전자를 이용하여 선발하였고, 300개의 누에알에 microinjection 하여 F1 세대에서 11 bloods의 누에형질전환체를 선발하였다. 선발된 누에형질전환체는 초기배 단계의 눈과 신경조직, 유충과 번데기 그리고 성충의 눈에서 EGFP 형광단백질이 발현되는 것을 확인할 수 있었다. 또한 G2 세대 누에형질전환체를 5령 5일 유충까지 사육 후, 체액을 채취한 후 전처리 하였다. 이 시료를 항균활성검정을 하였고, 총 10마리의 누에를 선발할 수 있었다. 이렇게 선발 된 누에는 서로 교배를 통해서 계대사육을 하였다. 이러한 과정으로 선발된 G3세대 누에형질전환체를 이용하여 앞의 과정과 동일한 방법으로 항균할성을 검정하였다. 그 결과 대조군으로 사용된 시그마사의 melittin(0.016 mg/ml)과 거의 동일한 항균활성을 나타내었다. 이상의 결과에서 melittin 항균펩타이드를 생산하는 누에형질전환체가 성공적으로 제작되었음을 확인할 수 있었다.

Keywords

References

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