| 半滑舌鳎ＨＩＦ １α基因的原核表达与多克隆抗体的制备 |
| Prokaryotic Expression of Cynoglossus semilaevis HIF－1 α and Preparation of Rabbit anti-HIF－1 α Polyclonal Antibody |
| 投稿时间：2012-09-28 修订日期：2012-11-06 |
| DOI： |
| 中文关键词:半滑舌鳎 HIF-1α 原核表达 蛋白纯化 Western blot |
| 英文关键词:Cynoglossus semilaevis HIF-1α Prokaryotic expression protein purification western blot |
| 基金项目:国家自然科学基金(31101887);江苏省自然科学基金(BK2011418);江苏省省属高校自然科学基金项目(10KJB240001) |
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| 中文摘要: |
| 根据半滑舌鳎HIF-1αcDNA序列设计特异性引物,从半滑舌鳎肝脏中扩增HIF-1α用于表达片段,连接至载体pMD18-T,分别以KpnI和XholI对含有目的基因的质粒和pET-30a进行酶切,连接并转化大肠杆菌BL21中,构建了半滑舌鳎HIF-1α原核蛋白表达载体。以IPTG进行诱导表达。表达产物通过Ni-NTA亲和层析进行纯化,免疫接种日本大耳白兔,制备了兔抗HIF-1α多克隆抗体。Western blot检测显示该多克隆抗体具有较好的特异性,能够与HIF-1α表达宿主菌蛋白特异性结合。 |
| 英文摘要: |
| The partial sequence of HIF－1 α for expression analysis was cloned from liver cDNA of tongue sole (Cynoglossus semilaevis) using the specific primers and cloned into pMD18-T plasmid. Then，the plasmid and pET-30a vector were digested with Kpn I and Xhol I and ligated ，and subcloned into BL21 .The cells were induced by IPTG and the expression products were purified with Ni一NTA affinity chromatography method. After that，the rabbit was immunized with HIF-1 a protein to prepare the polyclonal antibody. Western blot analysis showed that the anti-HIF一1a polyclonal antibody possessed specificity which could bind specific with the cells contained pET30a-HIF-1a plasmid. These results provided the basis for further study the biological function of tongue sole HIF-1α. |
| 王资生,齐志涛,张启焕,仇 明,赵卫红.2012.半滑舌鳎ＨＩＦ １α基因的原核表达与多克隆抗体的制备[J].水生态学杂志,33(6):103-108. |
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