拟南芥della蛋白编码基因rga和gai的原核表达和多克隆 - 兰州大学.pdf

兰州大学学报: 自然科学版, 2016, 52(3) / 6 月 Journal of Lanzhou University ：Natural Sciences，2016 ，52(3) / June 拟南芥DELLA 蛋白编码基因RGA 和GAI 的 原核表达和多克隆抗体制备 王 玮, 管利萍, 张 静, 侯岁稳 兰州大学生命科学学院, 细胞活动与逆境适应教育部重点实验室, 兰州 730000 摘 要：通过聚合酶链反应方法扩增了RGA 和GAI 两个拟南芥DELLA 蛋白基因, 并将其克隆到 pGEX-4T-3 原核表达载体上, 转化大肠杆菌Rosetta 菌株, 利用异丙基硫代半乳糖苷诱导表达了分子量 为90 kD 的GST-RGA 和分子量为85 kD 的GST-GAI 融合蛋白, 这两种融合蛋白均以包涵体的形式存 在. 将包涵体直接作为抗原免疫新西兰白兔, 制备出多克隆抗体血清, 血清效价超过1:400 000. WB 分 析结果表明这两种抗体可以特异性识别拟南芥RGA 和GAI 蛋白, 为进一步研究DELLA 蛋白的调控 机制奠定了基础. 关键词：拟南芥; DELLA蛋白; 原核表达; 包涵体; 多克隆抗体 中图分类号：Q943.2 文献标识码：A 文章编号: 0455-2059(2016)03-0422-07 DOI: 10.13885/j.issn.0455-2059.2016.03.023 Prokaryotic expression and polyclonal antibodies preparation of the DELLA Protein-coding gene, RGA and GAI, in Arabidopsis Wang Wei, Guan Li-ping, Zhang Jing, Hou Sui-wen Key Laboratory of Cell Activities and Stress Adaptations with the Ministry of Education, School of Life Sciences, Lanzhou University, Lanzhou 730000, China Abstract: Two genes of DELLA proteins, RGA and GAI, were amplified from Arabidopsisc DNA by poly- merase chain reaction method and cloned into the prokaryotic expression vector pGEX-4T-3. The pGEX-4T-3- RGA and pGEX-4T-3-GAI vectors were induced into E.coli Rosetta. The fusion proteins of GST-RGA (protein MW 90 kD) and GST-GAI (protein MW 85 kD) were expressed by induction with IPTG. Both proteins mainly existed in the form of inclusion bodies. Two inclusion bodies were directly used as antigens to immunize rab- bits, after which the anti-RGA andanti-GAI polyclonal antibody serums were generated, whose titers were high- er than 1:400 000. A western blotting analysis indicated that both polyclonal antibodies specifically recognized RGA and GAI, respectively. The prepared polyclonal antibodies lay t