Korean Journal of Plant Tissue Culture (식물조직배양학회지)

The Korean Society of Plant Biotechnology (한국식물생명공학회)
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Expression of Recombinant Erythropoietin Gene in Transgenic Tobacco Plant

형질전환 담배 식물체에서 재조합 erythropoietin 유전자의 발현

  • CHOI, Jang Won (Dept. of Agricultural Chemistry, College of Natural Resources, Taegu University) ;
  • PARK, Hee Sung (Dept. of Plant Breeding and Genetics, Catholic University of Taegu-Hyosung)
  • Published : 1997.01.01
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Abstract

Erythropoietin (EPO) is a glycoprotein that mediates the growth and differentiation of erythroid progenitors. In order to produce recombinant human erythropoietin in tobacco plant, the EPO genomic DNA (5.4 kb) was cloned into plant expression vectors, pBI$\Delta$GUS121, pBD$\Delta$GUS121 and pPEV-1, and introduced in Nicotiana tabacum (var. Xanthi) via Agrobacterium tumefaciens-mediated transformation. After selection on MS media containing kanamycin (Km), 10 Km-resistant plants were obtained per each construct. The correct integration of EPO genomic DNA in the genome of transgenic plant was confirmed by polymerase chain reaction (PCR). Northern blot showed that transcripts of 1.8 kb length were produced in leaves of the plants, but there was no difference of mRNA amount according to promoter number and 5'-untranslated sequence (UTS). The proteins obtained from leaves of transgenic plants were immunologically detected by Western blot using rabbit anti-human EPO polyclonal antibody. The expressed protein appeared as smaller band of apparent mass of 30 kDa as compared to the EPO protein from human urine (37 kDa), suggesting that the modification (glycosylation) system in tobacco plant might be different from that of mammalian cells.

Erythropoietin(EPO)은 적혈구 모세포의 분화와 성장을 중재하는 당단백질이며 담배 식물체에서 재조합 사람 EPO를 생산하기 위해 CaMV 35S promoter를 갖는 발현 vector인 pBI$\Delta$GUS121, pBD$\Delta$ GUS121, pPEV-1을 이용하여 5.4kb의 EPO genomic DNA를 cloning 하였고 Agrobacterium tumefaciens에 의한 형질전환에 의해 Nicotiana tabacum (var. Xanthi)으로 도입되었다. Kanamycin을 포함하는 MS 배지에서 각각의 construct에 대하여 10 km 저항성 식물체들이 얻어졌다. 형질전환된 식물체의 게놈에 EPO genomic DNA의 정확한 결합은 polymerase chain reaction에 의해 332bP의 DNA 조각에 의해 확인되었으며 Northern blot 결과 1.8 kb의 전사체들이 식물체 잎에서 발현 축적되는 것이 확인되었다. Promoter의 수나 5'-UTS 서열에 의한 mRNA 양에는 변화가 없었지만 식물체 게놈에 결합된 위치 및 copy number에 의해 mRNA 수준에 영향을 주는 것으로 밝혀졌다. EPO 항체를 이용한 Western blot 결과 식물체에서 발현된 EPO 단백질의 크기는 동물세포에서 발현된 37kDa 보다 작은 30 kDa 이었다. 이는 식물체에서 modification(glycosylation) system은 동물세포에서와는 다르다는 것을 보여준다.

Keywords

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