Jung Yuchul, Rhee Yong, Auh Chung-Kyoon, Shim Hyekyung, Choi Jung-Jin, Kwon Suk-Tae, Yang Joo-Sung, Kim Donggiun, Kwon Myung-Hee, Kim Yong-Sung, Lee Sukchan
Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Korea.
Plant Cell Rep. 2009 Oct;28(10):1593-602. doi: 10.1007/s00299-009-0758-3. Epub 2009 Aug 18.
We developed an asexual reproductive plant, Kalanchoe pinnata, as a new bioreactor for plant-based molecular farming using a newly developed transformation method. Leaf crenate margins were pin-pricked to infect the plant with the Agrobacterium strain LBA4404 and vacuum infiltration was also applied to introduce the target gene into the plants. Subsequently, the young mother leaf produced new clones at the leaf crenate margins without the need for time- and labor-consuming tissue culture procedures. The average transformation rates were approximately 77 and 84% for pin-prickling and vacuum-infiltration methods, respectively. To functionally characterize an introduced target protein, a nucleic acid hydrolyzing recombinant 3D8 scFv was selected and the plant based 3D8 scFv proteins were purified and analyzed. Based on abzyme analysis, the purified protein expressed with this system had catalytic activity and exhibited all of properties of the protein produced in an E. coli system. This result suggested that vegetatively reproductive K. pinnata can be a novel and potent bioreactor for bio-pharmaceutical proteins.
我们利用一种新开发的转化方法，培育出了一种无性繁殖植物——落地生根，作为基于植物的分子农业的新型生物反应器。用针刺法刺破叶片的圆齿状边缘，以农杆菌菌株LBA4404感染植株，并采用真空渗透法将目标基因导入植物。随后，幼嫩的母叶在叶片圆齿状边缘产生新的克隆体，无需耗时费力的组织培养程序。针刺法和真空渗透法的平均转化率分别约为77%和84%。为了对导入的目标蛋白进行功能表征，选择了一种核酸水解重组3D8单链抗体片段，并对基于植物的3D8单链抗体片段蛋白进行了纯化和分析。基于抗体酶分析，该系统表达的纯化蛋白具有催化活性，表现出了在大肠杆菌系统中产生的蛋白的所有特性。这一结果表明，无性繁殖的落地生根可以成为生产生物制药蛋白的新型高效生物反应器。
