한국산 고등군류의 성분 연구(19) : Lampteromyces japonicus(Kawam.) Singer의 성분

양문식(Moon-Sik Yang),김병각(Byong-Kak Kim) 한국생약학회 1980 생약학회지 Vol.11 No.1

To investigate constituents of Lampteromyces japonicus (Kawam.) Singer, quantitative analyses of free and total amino acids were carried out with GLC and an amino acid autoanalyzer. Fats of the mushroom were extracted and saponified. Their fatty acids were methylated and were analyzed by column chromatography and GLC.

분자농업의 현황 및 전망

김태금,양문식,Kim, Tae-Geum,Yang, Moon-Sik 한국식물생명공학회 2010 식물생명공학회지 Vol.37 No.3

Molecular farming is production of pharmaceutically and industrially important proteins in plants. Plants and plant cell culture systems have been used as bio-factory to produce recombinant proteins such as monoclonal antibodies, enzymes, vaccines, hormones, interleukins, commercial enzymes and etc. The terms molecular farming, biofarming, molecular pharming, phytomanufacturing, recombinant or plant-made industrials, planta-pharma, plant bioreactors, plant biofactory, and pharmaceutical gardening are used interchangeably. Molecular farming can provide safe and inexpensive pharmaceutical proteins as well as commercial ones. In spite of several advantages of molecular farming such as safety and inexpensive cost, there are also a couple of drawbacks in the existing technology. One of them is low expression level of target gene in plants, which has been improved by optimizing gene-based codon usage, screening of strong promoters, expression of transcription factors, subcellular targeting of target proteins, chloroplast transformation, and transient expression using viral expression system (magnifection). Some plant-based commercial proteins have already been in markets and more than twenty plant-based pharmaceuticals have been in clinical trials, from that we can expect that several plant-based pharmaceutical proteins will be seen in the markets in the near future.

기관 특이적인 Antisense 를 이용한 콩과식물의 질소고정 유전자의 연구

이제성,양문식 ( Je Sung Lee,Moon Sik Yang ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.5

Nodulins are organ-specific plant proteins induced during symbiotic nitrogen fixation in legume plants. Nodulins play both metabolic and structural roles within infected and uninfected nodule cells. Leghemoglobin, the most abundant nodulin, gene and chloramphenicol acetyl transferase (CAT) gene as a reporter were used to demonstrate that antisense regulation system worked in organ-specific manner in transformed and regenerated Lotus. CAT activity was knocked out in only nodules when Lotus corniculatus transformed with CAT gene was retransformed with antisense-CAT gene under the control of leghemoglobin promoter. This system can be used to study the functions of nodulin genes and to develop plants with new traits.

아그로박테리움을 이용한 Actinobacillus pleuropneumoniae ApxIIA (ApxII toxin) 유전자 발현 옥수수 형질전환체 개발

김현아,유한상,양문식,권석윤,김진석,최필선,Kim, Hyun-A,Yoo, Han-Sang,Yang, Moon-Sik,Kwon, Suk-Yoon,Kim, Jin-Seog,Choi, Pil-Son 한국식물생명공학회 2010 식물생명공학회지 Vol.37 No.3

To develop edible vaccines for swine, the embryogenic calli (type II) derived from HiII genotype were inoculated with A. tumefaciens strain C58C1 containing the binary vector pMYV611, 613, 616, and V621, 622 and 623 respectively. Six of those vectors carry nptII gene which confers resistance to paromomycin and apxIIA gene producing ApxII toxin which is generated in various serum types of A. pleuropneumoniae as a target gene. The 4,120 callus clones for pMYV611, 5,959 callus clones for pMYV613, 7,581 callus clones for pMYV616, 52,329 callus clones for V621, 48,948 callus clones for V622, and 56,188 callus clones for V623 were inoculated. The frequency of positive response clone was confirmed into range of 2.3% - 4.4% for each vectors by NPTII ELISA kit assay, and the selected callus clones of them were finally 3 callus clones from pMYV611 (0.07%), 4 callus clones from pMYV613 (0.07%), 2 callus clones from pMYV616 (0.03%), 51 callus clones from V621 (0.1%), 72 callus clones from V622 (0.15%), and 102 callus clones from V623 (0.18%) respectively. From the selected callus clones of each binary vector, the integration of the apxIIA gene into maize genome was detected from 2 plants of pMYV613 and 2 plants of V623 by Southern blot analysis. 돼지 흉막폐렴백신을 개발하기 위해 옥수수 HiII genotype 으로부터 유도한 type II형의 배발생캘러스를 식물발현벡터 pMYV611, pMYV613, pMYV616, V621, V622 및 V623로 형질전환시킨 Agrobacterium (C58C1)과 공동배양 하였다. 이들 식물발현벡터는 paromomycin 항생제 저항 유전자인 NPTII 선발마커와 표적 유전자로서 흉막폐렴균의 여러 가지 혈청을 생산하는 apxIIA유전자로 재조합하여 구축하였다. 식물발현벡터pMYV611, pMYV613, pMYV616, V621, V622 및V623의 경우 각각 4,120개, 5,959개, 7,581개, 52,329개, 48,948개 및 56,188개의 캘러스 클론을 Agrobacterium과 공동한 후 NPTII assay kit에 의해 nptII유전자의 발현빈도를 조사한 결과 각 벡터별로 2.3-4.4%의 캘러스 클론에서 항체결합 양성반응을 보였고, 이들 중 최종적으로 선발된 형질전환 캘러스 클론은 pMYV611에서 3개 (0.07%), pMYV613에서 4개 (0.07%), pMYV616에서 2개 (0.02%), V621에서 51개 (0.1%), V622에서 72개 (0.15%) 및 V623에서 102개 (0.18%)를 각각 얻었다. 형질전환된 캘러스 클론으로부터 재분화된 식물체에서 유전자 도입여부를 Southern 분석으로 통해 확인한 결과 pMYV613에서 2개 식물체 및 V623에서 얻은 2개 식물체에서 각각 확인되었다.

쥐 섬유육종에서 베타카로틴과 방사선조사 병용의 항종양 효과

권형철(Hyoung-Cheol Kwon),양문식(Moon-Sik Yang) 대한방사선종양학회 2000 Radiation Oncology Journal Vol.18 No.2

목 적 : 베타카로틴과 방사선조사의 병용효과에 관한 평가를 목적으로, 베타카로틴을 병용한 경우 방사선조사 단독의 경우 보다 세포독성의 차이는 어떠하며, 또한 쥐 섬유육종에서 두 군간의 종양성장의 지연 정도에 어떠한 차이가 있는가를 관찰하고자 본 연구를 시행하였다. 대상 및 방법 : 2% 베타카로틴 유제를 2 mg/ml 으로 만든 다음 단계적으로 희석하여 사용하였으며, 섬유육종세포와 태생 5∼6주의 C3H/N의 실험쥐를 이용하였다. 방사선조사는 6 MV 선형가속기를 이용하였고, 세포내 독성은 쥐 섬유육종세포의 생존을 감소시키는 능력으로 평가하였으며, 베타카로틴 2 mg/ml을 방사선조사 1시간 전 섬유육종세포주에 접촉시켰다. 종양성장 지연 실험을 위하여 베타카로틴과 방사선조사 병용군(n=6)과 방사선조사 단독군(n=5)으로 분류하였으며, 베타카로틴 20 mg/kg을 방사선조사 30분전 섬유육종이 접종된 쥐의 복강내 일회 주사하였고, 방사선조사량은 20 Gy를 주었다. 종양용적은 장경×장경×장경/2 (mm3) 공식을 사용하였으며, 2∼3일 마다 측정하였다. 결 과 : 섬유육종세포에 베타카로틴 0.002, 0.02, 0.2, 2 mg/ml 농도액을 1시간 동안 접촉 후 얻은 각각 생존분율은 0.69±0.07, 0.59±0.08, 0.08±0.008 및 0.02±0.006이었다. 그리고 방사선조사 1시간 전 섬유육종세포에 베타카로틴 2 mg/ml을 접촉한 후 조사량 2, 4, 6 및 8 Gy에서 얻은 각각의 생존분율은 0.13±0.05, 0.03±0.005, 0.01±0.002 및 0.009±0.0008이었으며 방사선조사 단독군의 경우 동일 조사량에서 얻은 생존분율은 각각 0.66±0.05, 0.40±0.04, 0.11±0.01 및 0.03±0.006으로 나타났다(p<0.05). 종양성장의 지연정도를 나타내는 실험에서 섬유육종을 쥐에 접종한 후 종양의 용적이 1,000 mm3 에 달하는 기간은 베타카로틴 병용군과 방사선조사 단독군에서 각각 18일과 19일로 나타났다(p>0.05). 결 론 : 쥐 섬유육종세포에 베타카로틴을 접촉한 경우 세포독성이 나타났으며, 베타카로틴 농도 증가에 따라 세포독성도 증가하였다. 그리고 쥐 섬유육종세포의 세포독성은 베타카로틴 병용군에서 방사선조사 단독군의 경우 보다 부가적으로 증가하였으며, 두 군간에 통계학적으로 현저한 차이를 보였다. 그러나 쥐 섬유육종 성장 지연정도에 있어서 베타카로틴 병용군과 방사선조사 단독군간의 통계학적으로 뚜렷한 차이는 없었다. Purpose :To investigate whether combined beta- carotene with X- irradiation has more enhanced radition response than X- irradiation or not, we performed a experiment about in vitro cytotoxicity of beta- carotene and/or X- irradiation in the fibrosarcoma cells, tumor growth delay of combined beta- caroten with/or X- irradiation in the mouse fibrosarcoma. Materials and Methods :2% emulsion of beta- carotene was serially diluted and used. X- irradiation was given by 6 MeV linear accelerator. The cytotoxicity of beta- carotene in vitro was evaluated from clonogenic assay. To compare the cytotoxicity between combined beta- carotene with X- irradiation and X- irradiation group, 2 mg/ml of beta- carotene was contacted to fibrosarcoma (FSaII) cells for 1 hour before X- irradiation. For the tumor growth delay, single 20 Gy was given to FSaII tumor bearing C3H/N mice whic was classified as beta- crotene with X- irradiation group (n=6) and X- irradiation alone group (n=5). 0.2 ml of 20 mg/kg of beta- carotene were i.p. injected to mice 30 minute before X- irradiation in the beta- crotene with X- irradiation group. The tumor growth delay defined as the time which reach to 1,000 mm3 of tumor volume. Result : (1) Cytotoxicity in vitro; 1) survival fraction at beta- carotene concentration of 0.002, 0.02, 0.2 and 2mg/ml were 0.69±0.07, 0.59±0.08, 0.08±0.008 and 0.02±0.006, respectively. 2) each survival fraction at 2, 4, 6 and 8 Gy in the 2 mg/ml of beta- carotene +X- irradiation group were 0.13±0.05, 0.03±0.005, 0.01±0.002 and 0.009±0.0008, respectively. But each survival fraction at same irradiation dose in the Xirradiation group were 0.66±0.05, 0.40±0.04, 0.11±0.01 and 0.03±0.006, respectively(p<0.05). (2) The time which reach to 1,000 mm3 of tumor volume of beta- carotene + X- irradiation group and X- irradiation alone group were 18, 19 days, respectively(p>0.05). Conclusion :The contact of beta- caroten to FSaII cells showed mild cytotoxicity which was increased according to concentration. The cytotoxicity of combined beta- carotene with X- irradiation more increased than that of X- irradiation, additionaly. And there was significant difference of cytotoxicity between two groups. But there were no significant difference of the growth delay of fibrosarcoma between two groups.

식물 칼모듈린 체계에 미치는 환경적 요인의 영향

오석흥(Suk Heung Oh),양문식(Moon Sik Yang) 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.1

Transgenic tobacco plants expressing calmodulin derivative(lys→ile 115 calmodulin) and hygromycin resistance genes or hygromycin resistance gene alone(control) were generated by Agrobacterium-mediated DNA transfer. Seeds obtained from the transgenic plants(F_o) were tested for resistance to hygromycin and the expected 3 : 1 ratio was observed. The expression of calmodulin derivative in the tobacco plants was identified by a combined method of Western blot and Chemiluminescence. The effects of surface sterilizers on the germiation of seeds from transgenic tobacco plants were tested in Murashige and Skoog agar medium. Seeds obtained from transgenic tobacoo plants expressing the calmodulin derivative showed no fungi contamination with normal germination by treating with sterilized water alone or sodium hypochlorite(2% effective chlorine). In contrast, seeds from the control transgenic tobacco plants showed severe contamination with fungi by treating with sterilized water alone and showed no contamination with normal germination by treating with sodium hypochlorite(2% chlorine). The effects of calcium concentration on the germination of seeds from transgenic tobacco plants were tested in Murashige and Skoog agar medium. Seeds obtained from transgenic tobacco plants expressing the calmodulin derivative showed better germination frequency than that of the control transgenic tobacco seeds in the medium containing 30 mM CaCl₂. The data raise the possibility that the expression of calmodulin derivative gene in tobacco plants could increase adaptability of the seeds to environmental stresses.

초고압처리에 의한 저염 멸치젓의 품질 변화

임상빈(Sangbin Lim),양문식(Moon-Sik Yang),김수현(Soo-Hyun Kim),목철균(Chulkyoon Mok),우건조(Gun-Jo Woo) 한국식품과학회 2000 한국식품과학회지 Vol.32 No.1

Cryparin 유전자의 promoter 분석을 위한 Cryparin 유전자 치환체의 순수 제조

김명주(Myoung Ju Kim),양문식(Moon Sik Yang),김대혁(Dae Hyuk Kim) 한국균학회 1998 韓國菌學會誌 Vol.26 No.4

N/A

Agrobacterium을 이용한 형질전환 상추의 세포 현탁배양으로부터 hGM-CSF의 생산

김영숙,김미영,권태호,양문식,Kim, Young-Sook,Kim, Mi-Young,Kwon, Tae-Ho,Yang, Moon-Sik 한국식물생명공학회 2003 식물생명공학회지 Vol.30 No.1

hGM-CSF가 식물세포 현탁 배양을 통하여 생산이 가능한지를 조사하기 위하여 hGM-CSF를 포함하고 있는 A. tumerfaciens LBA4404를 가지고 상추에 형질전환시켰다. 형질전환된 상추로부터 캘러스를 유도하여 캘러스를 이용한 세포배양체계를 확립하였다. PCR과 Southern blot analysis 결과 상추에 hGM-CSF 유전자가 도입된 것을 확인하였으며, Northern blot analysis 결과 상추식물체에 hGM-CSF 유전자가 발현됨을 확인하였다. 현탁 배양 세포로부터 분비된 hGM-CSF를 ELISA를 이용하여 측정한 결과 149.0 $\mu\textrm{g}$/L가 생산됨 을 확인하였다 이러한 결과는 상추의 현탁 배양 세포가 hGM-CSF와 같은 치료용 단백질의 생산 숙주로 이용될 수 있음을 보여주었다. Lettuce (Lactuca sativa) was transformed with Agrobacterium tumefacience LBA4404 containing human granulocyte macrophage colony stimulating factor (hGM-CSF) gene to produce in cell suspension cultures. Cell suspension culture was established using callus from transgenic lettuce plant. Integration of hGM-CSF gene into plant chromosome was confirmed through genomic PCR and Southern blot analysis. In addition, Northern blot analysis indicated the expression of the introduced hGM-CSF gene in transformed lettuce. The recombinant hGM-CSF was expressed in transgenic cell cultures derived from transgenic plants as a yield of about 149.0 $\mu\textrm{g}$/L in culture filtrate, which was determined by ELISA. These results demonstrated that transformed lettuce cell suspension cultures could be used as a production system of therapeutic proteins such as hGM-CSF.

합성유전자를 이용한 식물단백질의 향상

김태금(Tae Geum Kim),양문식(Moon Sik Yang) 한국생물공학회 1992 KSBB Journal Vol.7 No.3