植物选择标记基因hpt在大肠杆菌中的融合表达、纯化及活性测定

为对hpt基因编码蛋白进行的安全性评价，建立一种简便、高效的体外微生物表达体系，以获得大量的HPT蛋白，将用于植物转化的hpt基因的全编码序列克隆到原核表达载体pGEX4T-3上，在E．coli BL21中进行诱导表达，所得融合蛋白主要以包涵体的形式存在，且与Glutathione Sephrose 4B纯化介质的结合率不高。后经优化诱导表达的各种条件，成功地获得了可溶性的融合蛋白，该蛋白具有明显的生物活性。经Glutathione Sephrose 4B亲和层析，所得蛋白纯度＞95％。动物实验显示，融合蛋白还具有良好的免疫原性，免疫家兔后可诱导产生高滴度的抗体(＞1：10000)。ELISA及Western blotting检测进一步证实了所获纯化蛋白及其抗体的特性。这为深入进行HPT蛋白的安全性评价打下了良好的基础。

Fusion Expression, Purification and Bioactivity Assay of Plant Selectable Marker Gene hpt in E.coli

To facilitate the safety assessment of HPT protein, we need to develop a simple and efficient purification method to produce gram quantities of this protein in microbes. The same coding sequence used for plant transformation was cloned into prokaryotic expression vector pGEX4T-3 and transfered into E. coli BL21. The fusion proteins were expressed in form of inclusion body and large amount of them couldn't bind to the Glutathione Sephrose 4B resin. Optimizing the purification procedure, the fusion proteins initially produced as inclusion body were converted successfully to soluble protein exhibiting the desired Hygromycin B resistance activity. By Glutathione SepHrose 4B affinity chromatograpHy, the fusion GST-HPT was obtained with the purity of 95 %. Immunization of rabbits with purified fusion protein generated high titer antibodies ( > 1:10,000). The fusion HPT protein and its antibody were charactered by EL ISA and Western blotting. All the work made a good ground for the further research of HPT bio-safety.