Selection of Mutant Silkworm with Oxidation-deficient Haemolymph for Insect Cell Culture

곤충세포 배지 개발을 위한 체액산화지연 돌연변이 누에계통 선발

  • Choi, Ji-Young (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, R.D.A.) ;
  • Kim, Jong-Gill (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, R.D.A.) ;
  • Choi, Young-Cheol (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, R.D.A.) ;
  • Yoon, Hyung-Joo (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, R.D.A.) ;
  • Ahn, Mi-Young (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, R.D.A.) ;
  • Kim, Sam-Eun (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, R.D.A.) ;
  • Hwang, Seok-Jo (Department of Agricultural Biology, National Institute of Agricultural Science and Technology, R.D.A.)
  • 최지영 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 김종길 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 최영철 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 윤형주 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 안미영 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 김삼은 (농촌진흥청 농업과학기술원 농업생물부) ;
  • 황석조 (농촌진흥청 농업과학기술원 농업생물부)
  • Published : 2007.12.30

Abstract

Insect cell culture system has been demonstrated the effective means of producing medical and agricultural products. Furthermore, Fetal bovine serum (FBS) is in wide use in insect cell culture. Silkworm hemolymph was tested to develop as a substitute for FBS and was effective in insect cell growth. Hemolymph is oxidized and darkens visibly during the collection from silkworms due to the activity of tyrosinase in it. Toxic quinones are produced by the oxidation and consequently inhibit the cell growth. Heat treatment can be used to prevent the oxidation; however, the oxidation may occur during the collection of hemolymph before it is heat-treated. Hemolymphs collected from 257 different strains of silkworms were examined to select the slowly oxidized hemolymphs. Hemolymphs collected from mutant strains such as $Y_4$, TBO and $wE^b$ showed relatively slow color changes. Oxidation rates of the hemolymphs were measured by the absorbance change using a spectrophotometer. The absorbance of mutant hemolymph reached the saturation value at $20^{\circ}C$ in each 330 min ($Y_4$), 360 min (TBO) and 450 min ($wE^b$) min, whereas the total oxidation time of the wild-type (Baekokjam) hemolymph at the same temperature was 120 min. The cell growth in the medium supplemented with mutant species hemolmph was more effective that in the medium supplemented with Baekokjam species hemolymph.

누에 체액은 공기 중의 산소와 접하게 되면 도파퀴논(dopaquinone)이라는 세포 성장을 저해하는 물질을 생성하게 된다. 체액의 산화를 방지하기 위해서는 일반적으로 열처리에 의해서 효소를 불활성화 시키는 방법이 이용되고 있으나 대량 채혈 시에는 열처리 이전에 이미 산화가 진행되는 문제점이 있다. 이러한 문제점을 해결하기 위하여 누에 중 체액 산화가 더디게 진행되는 누에 계통을 선발하게 되었다. 누에체액을 채취하여 경과시간별로 산화정도(변색)를 육안으로 확인하여 TBO, $Y_4$, 백안$E^b$과잉지 3품종을 선발하고 spectrophotometer를 이용하여 산화반응 속도를 측정하였다. 백옥잠 체액과 각각의 산화지연 누에 체액의 산화속도를 400 nm에서 측정한 결과 백옥잠은 120분인데 비하여 체액산화 지연계통인 $Y_4$는 330분, TBO는 360분 그리고 백안$E^b$과잉지는 450분에 산화가 완료되었다. 산화지연 누에계통의 체액의 곤충세포 증식에 미치는 영향을 조사한 결과 대규모 채혈한 체액산화지연 계통의 체액은 야성형 계통(백옥잠)에서 소규모 채취한 체액에는 미치지 못하였으나 대규모 채취한 체액보다는 우수하였다.

Keywords

References

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