유전자 인공합성을 이용한 구제역 유전자 VP1의 제작과 Agrobacterium Vector System을 이용한 담배 형질전환
Construction of FMDV VP1 Gene Using Artificial DNA Aynthesis and Transformation of Nicotiana tabacum Using Agrobacterium Vector System
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초록
FMDV는 동물에서 구제역을 일으키는 병원체이며, VP1은 이 바이러스의 주요 capsid 단백질이므로 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 VP1 유전자로 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 promotor를 포함하는 pCAMBIA II를 제작하였다. 제작된 벡터와 VP1 유전자 및 GFP 유전자를 사용하여 담배를 형질전환시켰고, 각각의 형질전환 식물체내에서 전체길이의 target gene (VP1)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.
Abstract
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, pCAMBIA1390II was constructed on the basis of pBI121 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.
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