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유전자 인공합성을 이용한 구제역 유전자 VP1의 제작과 Agrobacterium Vector System을 이용한 담배 형질전환
이은정,임희영,김성훈,강경선,박영두,윤충효,윤병수,Lee, Eun-Jung,Lim, Hee-Young,Kim, Sung-Hoon,Kang, Kyung-Sun,Park, Young-Doo,Yun, Choong-Hyo,Yoon, Byoung-Su 한국식물생명공학회 2004 식물생명공학회지 Vol.31 No.4
FMDV is a viral pathogen that caused foot-and-mouth disease in animals. VP1 is a major capsid protein of FMDV. It is known as one of best materials for the FMDV diagnosis and for the development of protein vaccine. In this study, 633 bp of VP1 gene was modified for the expression of VP1 in plant, based on the VP1 DNA sequence from FMDV taiwan O type and from FMDV isolated vietnam. The. deduced DNA fragment was artificially synthesized using the multiple fragment extension with long-nucleotides. A new plant transgenic vector system, pCAMBIA139011 was constructed on the basis of pBI12l and pCAMBIA1390. Using this vector system and GFP gene or modified VP1 gene, each target gene was introduced into Nicotiana tabacum. The insertion of whole target gene was successfully confirmed in each transgenic plant named GFP-A7 and VP1-4, respectively. The expression level of each gene was estimated by RT-PCR and Real-Time PCR using VP1, GFP specific primers. FMDV는 동물에서 구제역을 일으키는 병원체이며, VP1은 이 바이러스의 주요 capsid단백질이므로 구제역의 진단과 단백질 백신의 개발에 가장 많이 사용되는 재료 중 하나이다. 본 연구는 FMDV taiwan O형과 베트남에서 분리된 FMDV의 VP1 sequence를 기반으로 식물에서 VP1 유전자의 발현을 위하여 633 bp의 VP1유전자로 재편집하였으며, 이를 long-nucleotide를 사용한 multiple fragment extension 방법을 사용하여 인공적인 DNA 단편을 합성하였다. 또한 새로운 식물 형질전환 벡터로 pBI121 과 pCAMBIA1390의 장점을 수용하여, hygromycin 저항성과 CaMV 35S promoter를 포함하는 pCAMBIA II를 제작하였다. 제작된 벡터와 VP1 유전자 및 GFP유전자를 사용하여 담배를 형질 전환시켰고, 각각의 형질전환식물체내에서 전체길이의 target gene(VPl)의 성공적인 삽입을 확인하였다. 각 유전자의 발현은 RT-PCR과 Real-Time PCR의 결과로 측정하였으며, VP1 유전자의 전사가 담배 내에서 이루어졌음과 고효율의 전사체를 만드는 형질전환체 VP1-4를 선별하였다.
방사선 조사에 따른 U-937 세포의 Ceruloplasmin 유전자에서 mRNA 발현 변화
오연경(Youn Kyoung Oh),임희영(Hee Young Lim),김종수(Jong Soo Kim),윤충효(Choong Hyo Yun),김인규(In Gyu Kim),윤병수(Byoung Su Yoon) 한국독성학회 2004 Toxicological Research Vol.20 No.1
Against environmental stress, ceruloplasmin which is a plasma protein, are believed to play central roles in antioxidant- or peroxidase-activity in blood stream to remove free radicals, which<br/> may be caused by exposing of g-irradiation. In human U-937 cells exposed to g-irradiation, the levels of mRNA in ceruloplasmin gene were measured on 0, 4, 12, 24 hr after exposing by using comparative RT-PCR (Reverse transcriptase-polymerase chain reaction) which was achieved to compare with house keeping genes such as b-actin and hprt. After g-irradiation of 100 rads or 200 rads, the total quantities of RNA were increased as dose and time dependent manner. On the contrary, the variation of mRNA expression in ceruloplasmin was not found until 4 hr after irradiation. After 12 hr and 24 hr of irradiation, the levels of mRNA in ceruloplasmin were significantly increased as dose and time dependent manner than un-exposed cells.