돼지설사병 방제용 CTB::fasG 형질전환 벼 및 캘러스의 도입유전자 발현
Expression of CTB::fasG Gene against Piglet Bacterial Diarrhea Disease in Transgenic Rice and Its Callus
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돼지설사병 방제용 식물경구백신을 개발하고자 점막면역보조제인 Cholera toxin subunit B (CTB) 유전자와 설사병을유발시키는 대장균 987P-fasG 유전자를 연결시킨 CTB::fasG DNA 카세트로 벼를 형질전환시켰다. 벼에 삽입된 도입유전자의 copy수를 T₁및 T₂세대의 종자를 대상으로 TaqMan probe법으로 분석한 결과 1 개부터 8 개까지의 유전자가 복수로 도입된것으로 확인하였다. 형질전환체 T₁및 T₂세대에서의 mRNA의발현량은 Quantitative real time-PCR로 분석한 결과 기준치에비해 최대 35배까지 나타났다. 또한 형질전환체를 조직배양하여생성된 callus를 장시간 계대배양시키며 저장기간 별로 도입유전자를 확인한 결과 계대배양 9개월까지 도입유전자가 유지됨을확인할 수 있었다. Callus에서의 mRNA의 발현량 역시 기준치의2.6배 정도로 유지됨을 확인하였다. 이와 같이 도입유전자를형질전환체 뿐만 아니라 callus에서도 확인할 수 있었으므로본 연구 결과는 식물유래 경구백신 개발의 기초자료로 활용될수 있을 것으로 판단된다.
Abstract
Antigen production in plant is a safe and effective strategy for vaccine development. In this study, rice transformants were developed for oral vaccine against pigs diarrhea disease. DNA cassette composed with the cholera toxin subunit B (CTB) connected to the 987P-fasG, for stimulating a strong oral immune response, was introduced to rice through Agrobacterium mediated genetic transformation. Copy number analysis by TaqMan real-time PCR for transgenes revealed that transgene of 1 to 8 copies have been introduced into T₁ and T₂ rice seeds. The expression level of mRNA in the transformants T₁ and T₂ generations were up to 35 times higher than the reference value in the result of analysis by Quantitative real time-PCR. In addition, the callus cultured from rice transformants was confirmed that the introduced gene has been maintained till 9-month subculture duration. The amount of mRNA expression value was also confirmed in callus, which was maintained above 2.6 times compared with that of the standard control for a long time. These results provide that the introduced antigen for plant-based vaccine against bacterial diarrhea disease can be maintained in the callus as well as in the transgenic plant and suggest that the callus culture of plant transformant will be an effective way to obtain a plant-derived edible vaccine.
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