바이러스 질병 예방을 위한 식물 경구 백신 연구 동향

한범수,박종석,김형국,하선화,조강진,김용환,김종범,Hahn, Bum-Soo,Park, Jong-Sug,Kim, Hyeong-Kuk,Ha, Sun-Hwa,Cho, Kang-Jin,Kim, Yong-Hwan,Kim, Jong-Bum 한국식물생명공학회 2004 식물생명공학회지 Vol.31 No.2

Transgenic plants have been studied as delivery system for edible vaccine against various diseases. Edible plant vaccines have several potential advantages as follows: an inexpensive source of antigen, easy administration, reduced need for medical personnel, economical to mass produce and easy transport, heat-stable vaccine without refrigerator, generation of systemic and mucosal immunity and safe antigen without fetal animal-virus contaminants. The amount of recombinant antigens in transgenic plants ranged from 0.002 to 0.8% in total soluble protein, depending on promoters for the expression of interested genes and plants to be used for transformation. Throughout the last decade, edible plant vaccine made notable progresses that protect from challenges against virus or bacteria. However edible plant vaccines have still problems that could be solved. First, the strong promoter or inducible promoter or strategy of protein targeting could be solved to improve the low expression of antigens in transgenic plants. Second, the transformation technique of target plant should be developed to be able to eat uncooked. Third, marker-free vector could be constructed to be more safety. In this review we describe advances of edible plant vaccines, focusing on the yields depending on plants/promoters employed and the results of animal/clinical trials, and consider further research for the development of a new plant-derived vaccine.

세균성 질병 예방을 위한 식물 경구 백신 연구 동향

한범수,정영재,노경희,박종석,조강진,김용환,김종범,Hahn Bum-Soo,Jeong Young-Jae,Roh Kyung-Hee,Park Jong-Sug,Cho Kang-Jin,Kim Yong-Hwan,Kim Jong-Bum 한국식물생명공학회 2005 식물생명공학회지 Vol.32 No.4

Plants have considerable advantages for the production of antigenic proteins because they provide an inexpensive source of protein and an easy administration of vaccine. Since a publication describing edible plant vaccine of HBsAg in 1992, a number of laboratories around the world have studied the use of plants as the bioreactor to produce antigenic proteins of human or animal pathogens. Over the last ten years, these works have been mainly focused on three major strategies for the production of antigenic proteins in plants: stable genetic transformation of either the nuclear or plastid genome, or transient expression in plants using viral vectors. As many antigenic proteins have been expressed in tobacco, also several laboratories have succeeded to express genes encoding antigenic proteins in other crop plants: potato, tomato, maize, carrot, soybean and spinach. At present many works for the production of edible plant vaccine against bacteria-mediated diseases have mostly performed the studies of enterotoxins and adhesion proteins. Also the development of new-type antigens (pili, flagella, surface protein, other enterotoxin and exotoxin etc.) is required for various targets and more efficacy to immunize against microorganism pathogens. Many works mostly studied in experimental animals had good results, and phase I clinical trial of LTB clearly indicated its immunogenic ability. On the other hand, edible plant vaccines have still problems remained to be solved. In addition to the accumulation of sufficient antigen in plants, human health, environment and agriculture regulation should be proven. Also oral tolerance, the physiological response to food antigens and commensal flora is the induction of a state of specific immunological unresponsiveness, needs to be addressed before plant-derived vaccine becomes a therapeutic option.

곤충 생약으로부터 항응고 및 항혈전 물질의 탐색

한범수,우숭지,김성환,김영식,Hahn, Bum-Soo,Wu, Song-Ji,Kim, Sung-Whan,Kim, Yeong-Shik 한국생약학회 1999 생약학회지 Vol.30 No.4

The in vitro anticoagulant and fibrinolytic activities of crude extract from insects which have been used as traditional medicines. The extracts of Formica, Huechys and Eupolyph-aga/Steleophaga prolonged activated partial thromboplastin time and thrombin time compared to the value of the control. The fibrinolytic activity of insect extracts was also tested by fibrin plate method. We found that the extracts of Cicadae Periostracum, Eupolyphaga/Steleophdga, Mantidis $O{\ddot{o}}otheca$ and Huechys directly could hydrolyze the fibrin clot without the activation of plasminogen by plasminogen activators.

참외 모상근에서 Biochemically Active Human Tissue-Plasminogen Activator의 발현과 대량생산 탄소원 종류 및 최적농도 규명

한범수(Bum Soo Hahn),전형식(Hyung Sik Jeon),김봉찬(Bong Chan Kim),이상순(Sang Soon Lee),고성준(Sung Joon Ko),김성용(Sung Ryong Kim) 한국원예학회 2010 한국원예학회 학술발표요지 Vol.2010 No.5

연구보문 : 코돈최적화를 통한 합성 조직형 플라스미노겐 활성화인자의 형질전환 담배 내 발현

심준수 ( Joon Soo Sim ),차소영 ( So Young Cha ),한정순 ( Jeong Soon Han ),강솔 ( Sol Kang ),헤마바티 ( He Ma Va Thi ),김용환 ( Yong Hwan Kim ),한범수 ( Bum Soo Hahn ) 한국국제농업개발학회 2010 韓國國際農業開發學會誌 Vol.22 No.4

본 연구에서는 현재 전 세계적으로 혈전용해제로서 널리 사용되어지고 있는 t-PA를 담배에서 생산하기 위해 t-PA 유전자를 코돈최적화를 통해 합성하여 담배 rbcS 프로모터하에서 발현을 유도하였다. 합성된 t-PA 유전자는 발현 벡터를 이용하여 담배 형질전환을 실시하였고, 동물세포 유래 t-PA와의 인공 혈 전용해 활성 및 분자량의 동등성 등의 특성을 규명하였다. 1. 담배의 codon usage에 맞게 t-PA 유전자를 합성하였고, 담배 형질전환체 잎에서 피브린 분해 활성이 있는 효소학적으로 안정된 재조합 t-PA의 발현을 확인하였다. 2. 합성 t-PA 유전자의 담배 genomic DNA내의 삽입과 전사는 PCR과 RT-PCR법으로 측정하였고, t-PA 유전자의 PCR 산물(1.6 kb)을 관찰하여 담배 염색체내에 삽입되어 있음을 확인하였다. 또한 t-PA의 전사체 크기에 해당하는 1.6 kb의 PCR 산물을 관찰하여 담배 잎 내에서 정상적인 전사가 이뤄지고 있음을 확인하였다. 3. 담배 형질전환체 잎의 전체 수용성 단백질 중 합성 t-PA 의 평균 발현양은 0.03 μg/TSP mg으로 측정되었고, 가장 높은 발현양은 0.0903 μg/TSP mg로 관찰하였다. 4. 담배 잎 추출물 내에 존재하는 합성 t-PA 단백질의 피브린 분해 활성은 상업적으로 판매되는 재조합의 t-PA 단백질과 동등한 활성을 나타냈고, 담배 형질전환체들의 피브린 용해 활성의 평균값은 0.2589 cm2 lysis zone을 나타냈고, 피브린 용해 활성의 최대치는 0.9 cm2 lysis zone이었다. 5. 형질전환 담배 잎에서 발현된 재조합 합성 t-PA의 분자량을 분석한 결과 동물세포 유래 t-PA와 유사한 크기의 65 kDa으로 확인하였다. 6. T-PA 단백질을 발현하는 담배 형질전환체들은 식물체의 키, 색깔, 모양, 잎의 개수, 성장속도, 종자의 생산량 등과 같은 표현형에 있어 야생종 담배와 별 다른 차이를 보이지 않았으며 후대에도 안정적인 t-PA 활성을 나타내었다. Human tissue-type plasminogen activator(t-PA) which is found in blood and tissues is one of the proteolytic enzyme. T-PA causes fibrin-specific plasminogen activation that can dissolve blood clots in the vasculature. In order to produce recombinant t-PA(st-PA) proteins in transgenic tobacco plants, the t-PA gene was codon-optimized and cloned into a plant binary vector(p221a-rbcS-st-PA) harboring a tobacco rbcS promoter, a tobacco etch virus leader sequence, an N-terminal signal peptide of the alfala glucose-regulated endoplasmic recticular protein and a 35S terminator. The plasmid was transformed into Agrobacterium tumefaciens EHA105 and then the Agrobacterium-mediated transformation of tobacco leaf discs was performed. The insertion of the st-PA gene in genomic DNA of transgenic tobacco plants and the presence of its transcript were verified by PCR and RT-PCR, respectively. We investigated the fibrinolytic activity of recombinant st-PA proteins and quantified their amount to determine the expression level of st-PA proteins in transgenic tobacco leaves. Enzyme-linked immunosorbent assay determined that the highest level of recombinant st-PA expression was 0.09 μg/total soluble protein(mg) in transgenic tobacco plants. The amount of recombinant st-PA proteins in transgenic plants was estimated to range from 0.001 to 0.009% of total soluble proteins. Immuno-blot analysis of transgenic tobacco leaves revealed a single band of approximately 68 kDa recombinant st-PA proteins. These results demonstrated the expression and in vitro activity of recombinant st-PA in transgenic tobacco plants. Thus it provides the information for the additional production of recombinant pharmaceutical proteins using plant system.

연구보문 : 식물에서 인체와 유사한 N-glycan 구조를 가진 당단백질 생산에 대한 최근 연구동향

심준수 ( Joon Soo Sim ),신동진 ( Dong Jin Shin ),한범수 ( Bum Soo Hahn ) 한국국제농업개발학회 2011 韓國國際農業開發學會誌 Vol.23 No.3

Several host systems including mammalian cells, yeasts, plants, bacteria, insects and transgenic animals have been employed to produce recombinant therapeutic glycoproteins. Among them, plant system has many advantages such as the low cost, animal pathogens-free, eco-friendly production, and ease of scale up. However, plants are unable to perfectly synthesize human-type N-glycans of therapeutic glycoproteins. It could represent a severe limitation on the use of therapeutic glycoproteins produced from transgenic plants. In this article, we focus on the importance of N-glycosylation, the advantage of molecular farming and its progress strategies, the difference of N-glycan structure between animals and plants, the risk of potential immunogenicity of plant glycoproteins, the strategies to reduce the potential immunogenicity by N-glycosylation engineering including endoplasmic reticulum (ER) targeting of therapeutic glycoproteins, the inhibition of plant-specific glycosyltransferases, and the expression of human specific glycosyltransferases to synthesis galactosylated and sialylated N-glycans in plants.

t-PA 유전자 함유 모상근 생물반응기 이용 대량생산 최적조건 구명

김성용(Sung Ryong Kim),한범수(Bum Soo Hahn),현금선(Keum Sun Hyun),현은아(Eun A Hyeon),김봉찬(Bong Chan Kim),고성준(Sung Jun Ko) 한국원예학회 2009 한국원예학회 학술발표요지 Vol.2009 No.5

연구보문 : 담배 rbcS 프로모터 이용 담배 형질전환체 내에 조직형 플라스미노겐 활성화인자 발현

오창식 ( Chang Sik Oh ),심준수 ( Joon Soo Sim ),민성현 ( Sung Hyun Min ),한정순 ( Jeong Soon Han ),강솔 ( Sol Kang ),차소영 ( So Young Cha ),김용환 ( Yong Hwan Kim ),한범수 ( Bum Soo Hahn ) 한국국제농업개발학회 2010 韓國國際農業開發學會誌 Vol.22 No.2

연구보문 : 참외모상근 내에 인체 조직형 플라스미노겐 활성화인자 변이체들의 발현

헤마바티 ( Hemavathi Ajjappala ),심준수 ( Joon Soo Sim ),김용환 ( Yong Hwan Kim ),한범수 ( Bum Soo Hahn ) 한국국제농업개발학회 2011 韓國國際農業開發學會誌 Vol.23 No.2

본 연구는 뇌졸중, 심근경색과 폐색전증에 사용되는 t-PA 단백질 변이체를 참외모상근에서 생산하고자 수행되었다. t-PA 변이체 유전자가 함유된 A. rhizogene으로 형질전환된 참외모상근에서 t-PA 변이체 단백질들이 성공적으로 발현되는 것을 확인하였으며, 발현된 t-PA변이체 단백질들은 동물세포에서 발현되어 상업적으로 판매되는 t-PA와 시험관 내의 인공 혈전 용해 활성이 동등함을 확인할 수 있었다. 1. t-PA 변이체 유전자들의 참외모상근 genomic DNA내의 삽입은 PCR법으로 t-PA 유전자의 PCR 산물(1.6 kb)을 관찰하여 참외 모상근 염색체 내에 삽입되어 있음을 확인하였다. 2. t-PA 변이체 유전자들의 전사는 RT-PCR법을 활용하여 t-PA transcript 크기에 해당하는 1.6 kb의 PCR 산물을 관찰하여 참외모상근에 정상적인 전사가 이뤄지고 있음을 확인하였다. 3. 형질전환된 참외모상근 추출물 내에 존재하는 t-PA 변이체 단백질들의 피브린 분해 활성은 상업적으로 판매되는 재조합의 t-PA 단백질과 동등하였다. 4. 형질전환된 참외모상근의 전체 수용성 단백질 중 t-PA 변이체 단백질의 평균 발현량은 전체 수용성 단백질 mg 당 0.47(t-PAer)과 0.57(t-PAhis6)로 측정되었으며, 가장 높은 발현량은 t-PAhis6로 형질전환된 모상근에서 전체 수용성 단백질 mg 당 0.65 μg으로 관찰되었다. 5. 형질전환된 모상근에서 발현된 재조합 t-PA 변이체 단백질들의 분자량은 동물세포에서 발현된 t-PA와 유사한 59 kDa으로 확인하였다. Human tissue-plasminogen activator (t-PA) is a thrombolytic protein that plays an active role in dissolving fibrin clots in the blood vessels by activating plasminogen to plasmin. Human tissueplasminogen activator, its derivatives and synthetic genes were expressed as enzymatically active form in the hairy roots of Cucumis melo L. cv. Geumssaragi-euncheon (Oriental melon) infected by Agrobacterium rhizogene harboring binary vectors. Hairy roots were produced from the wounded surface of the cotyledon explants of Oriental melon on MS selective medium containing 300 μg/ml kanamycin and 500μg/ml carbenicillin. PCR analysis revealed the insertion of the t-PA genes in genomic DNA of transgenic hairy roots. The presence of the t-PA-specific transcripts in the total RNAs of transgenic hairy roots was confirmed by RT-PCR. Western blot analysis of the transgenic hairy roots showed a single distinct band of 59-kDa of recombinant t-PAs. ELISA experiments demonstrated the maximum level of recombinant t-PA expression up to 0.065% of the total soluble protein in hairy roots transformed by plasmid p221a-t- PAhis6. The highest fibrinolysis of recombinant t-PAs was detected from the hairy roots expressing plasmid p221a-t-PAhis6, respectively. These studies demonstrated that hairy roots could be successfully employed for the mass production of an enzymatically active t-PA.

Agro-morphological Characters, Total Phenolic Content, and Fatty Acid Compositions of Safflower Genetic Resources

Awraris Derbie Assefa,Jung Sook Sung(성정숙),Myeong Chul Lee(이명철),Bum Soo Hahn(한범수),Hyung Jun Noh(노형준),On Sook Hur(허온숙),Na Young Ro(노나영),Ae Jin Hwang(황애진),Bich Saem Kim(김빛샘),Jae Eun Lee(이재은) 한국약용작물학회 2021 한국약용작물학회지 Vol.29 No.1

Background: Safflower is an important crop that does not require rich soils. It grows well in dry soils or arid areas with seasonal rain. Exploring the fatty acid profiles and agro-morphological characteristics of diversified collections of safflower provides baseline data for developing improved varieties. In this study, we investigated the variation in agro-morphological characteristics, fatty acid composition and total phenolic content of the seeds, and the relationship between the agro-morphological and biochemical characteristics. Methods and Results: Agro-morphological characteristics were recorded in the field and laboratory. Total phenolic content was estimated using Folin-Ciocalteu’s method and fatty acids were determined using gas chromatography-mass spectrometry. Orange, red, and white petal colors were observed; orange was the dominant pigment. Wide ranges of other agro-morphological characteristics were also recorded. More than 87% of the accessions contained > 50% linoleic acid while approximately 12% of the accessions contained > 50% oleic acid. A strong correlation was observed between palmitic and linoleic acid, and crude fat and oleic acid. A strong negative correlation was observed between crude fat and linoleic acid, palmitic and oleic acid, and oleic and linoleic acid. Conclusions: Safflower accessions were found to be a poor indicator of essential linolenic acid. The wide variation in agro-morphological and biochemical traits of safflower accessions could potentially help to develop an improved, nutrient-dense safflower cultivar.