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-1α and Preparation of Rabbit anti-HIF-1α Polyclonal Antibody

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。

The partial sequence of HIF-1α 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-NTA affinity chromatography method. After that, the rabbit was immunized with HIF-1 ct protein to prepare the polyclonal antibody. Western blot analysis showed that the anti-HIF-lcx polyclonal antibody possessed specificity which could bind specific with the cells contained pET30a- HIF-1 α plasmid. These results provided the basis for further study the biological function of tongue sole HIF-1α.