| 摘要: |
| 目的:表达具有生物学活性的重组人成纤维细胞生长因子-20(rhFGF-20),并制备rhFGF-20抗血清.方法:应用RT-PCR从人前列腺组织总RNA中扩增FGF-20的cDNA,将其克隆到pET-24a中,在大肠杆菌中诱导表达,经NTA-Ni2+-琼脂糖亲和层析纯化,应用SDS-聚丙烯酰胺凝胶电泳、细胞增殖试验进行分析鉴定,并免疫新西兰雄性白兔制备多克隆抗体,免疫双扩散法测定抗体效价.结果:构建的pET-24a-FGF-20能在大肠杆菌中高表达;诱导表达的rhFGF-20蛋白主要存在于包含体中,经包含体溶解、Ni2+-NTA His-Bind Resins亲和层析后,纯化产物呈单一条带;细胞增殖试验显示其能促进成纤维细胞生长,并具有剂量依赖性(浓度为50～5 000 ng/ml);制备的抗血清效价为1∶32,可与hFGF-20产生免疫反应.结论:大肠杆菌表达的rhFGF-20蛋白具有刺激成纤维细胞增殖的活性,制备的抗血清具有特异性. |
| 关键词: 成纤维细胞生长因子-20、原核表达、免疫血清 |
| DOI：10.3724/SP.J.1008.2006.00950 |
| 投稿时间：2006-01-16修订日期：2006-07-07 |
| 基金项目:上海市自然科学基金(05ZR14149). |
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| Prokaryotic expression, purification and biological activity determination of recombinant human fibroblast growth factor-20 and preparation of its polyclonal antisera |
| JIANG Zhi-guo,YIN Zheng-feng,QIAN Hai-hua,KANG Xiao-yan,LUO Xiang-ji,LI Jin |
| (第二军医大学东方肝胆外科医院分子肿瘤实验室,上海,200438) |
| Abstract: |
| Objective： To express and purify bioactive recombinant human fibroblast growth factor-20 （rhFGF-20）protein and to prepare its polyclonal antisera. Methods： FGF 20 cDNA was amplified from human prostate tissue by RT-PCR and was subcloned into expression vector pET-24a, which was then transformed into the E. coli DE3. Expression of rhFGF-20 protein was induced in E. coli DE3 and the protein was purified by Ni2＋-NTA His-Bind Resins. The purity and biological activity of rh FGF-20 protein were determined by SDS-PAGE and cell-proliferation assay, respectively. Two New Zealand white rabbits were immunized with rhFGF-20 protein to prepare polyclonal antisera and the titer of antibody was determined by double diffusion test. Results： rhFGF-20 protein was efficiently expressed in E. coli DE3 in the form of inclusion body and homogeneous protein was obtained after purification with Ni2-NTA His Bind Resins. Cell proliferation assay indicated that rhFGF-20 dose-dependently （50-5 000 ng/ml） promoted fibroblast cells proliferation. The prepared polyclonal antisera, with a titer of I ： 32, had immunoreatlon with hFGF-20. Conclusion： The expressed rhFGF-20 protein can stimulate the proliferation of fibroblast cells and the prepared antisera are antigen specific |
| Key words: fibroblast growth factor-20 prokaryotic expression immune sera |
