Arresten在烟草中的表达及其生物学活性分析

Expression of Biologically Active Recombinant Arresten in Nicotiana tabacum

摘要:

采用5￠端引入His-tag的引物从携带有Arresten基因的质粒pCA中扩增血管生成抑制因子Arresten编码基因,构建其植物表达载体pCAMBIAarr并通过冻融法转化根癌农杆菌LBA4404, 获得携带目的基因的重组农杆菌。采用叶盘法以重组农杆菌转化烟草, 在50 mg/mL潮霉素B为选择压力下获得再生烟草植株, 经过Southern 杂交、RT-PCR和Western blotting检测, 获得稳定整合有Arresten编码基因的烟草转基因植株。牛血管内皮细胞BCE增殖抑制实验表明, 采用镍离子螯合次氨基三乙酸亲和层析法从转基因烟草叶片中分离纯化的重组Arresten蛋白具有明显的抑制牛血管内皮细胞增殖的生物活性。

Abstract:

In this report, the biological activity of the recombinant Arresten expressed in Nicotiana tabacum was studied. The gene coding for the tumor angiogenesis inhibitor Arresten was PCR-amplified from the plasmid pCA and its plant expression vector named pCAMBIAarr was constructed by inserting the Arresten cDNA fragment into the NcoI/BstEII sites of the plant binary expression vector pCAMBIA1301. Then pCAMBIAarr was transferred into Agrobacterium tumefacien LBA4404 by the freeze-thaw method. The adventitious shoots and regenerated plants of Nicotiana tabacum with hygromycinB-resistance were obtained via Agrobacterium-mediated leaf disk transformation method. Southern hybridization, RT-PCR and Western blotting analysis showed that the Arresten cDNA was integrated into the genome of some of the regenerated plants and the recombinant Arresten was expressed with a molecular size of 26 kD. Recombinant Arresten purified from transgenic tobacco leaves had an anti-proliferative effect on bovine endothelial cells. We speculate that biologically active recombinant Arresten can be produced by using plants as bioreactors.