铁蛋白重链亚铁氧化酶活性突变体的构建及鉴定

Construction eukaryotic expression vector of ferritin heavy chain mutant with inactivated ferroxidase activity

[目的]构建小鼠铁蛋白重链(ferritin heavy chain,FTH)亚铁氧化酶活性突变基因的真核表达载体。[方法]针对小鼠FTH基因的亚铁氧化酶活性关键位点(K86、E62和H65),设计并合成6条PCR引物。利用重叠延伸PCR技术,获得小鼠FTH基因的突变体m FTH(K86Q、E62K和H65G),简写为m FTH(3M)。然后将m FTH(3M)插入到实验室已有的C端带Flag标签的真核表达载体pc DNA3中,插入位置为多克隆位点区域中限制性内切酶Bam HⅠ和HindⅢ之间,从而得到亚铁氧化酶活性缺失的FTH真核表达载体pc DNA3-m FTH(3M)-Flag。重组质粒经酶切和测序鉴定正确后,用脂质体lipofectamineTM2000将其转染到RAW264.7细胞中,检测细胞中带有Flag标签的m FTH(3M)蛋白的表达。[结果]重组质粒酶切得到预期片段,测序显示所构建的m FTH(3M)真核表达载体序列正确,细胞内检测到带Flag标签的m FTH(3M)蛋白信号的强烈表达。[结论]实验成功构建了小鼠FTH亚铁氧化酶活性位点基因突变的真核表达载体pc DNA3-m FTH(3M)-Flag。

[ Objective ] To construct the eukaryotic expression vector of mouse ferritin heavy chain （mFTH） mutant with inactivated ferrox- idase activity. [ Methods] The specific primers were designed based on the mouse FTH gene -specific ferroxidase activity sites （K86, E62 and H65 ）. FTH mutant gene mFTH （K86Q, E62K and H65G）, which was abbreviated as mFTH （3M）, with inactivated ferroxidase activi- ty was amplified by Overlap Polymerase Chain Reaction （ Overlap PCR）. The obtained gene was inserted between the restriction endonu- clease BamH I and Hindlll recognition sites on the eukaryotic expression vector pcDNA3 to obtain the eukaryotic expression vector pcD- NA3 -Mfth （3 M） -Flag. Then, the recombinant plasmid was identified by digestion and sequencing, pcDNA3 -mFTH （3M） -Flag and its empty control vector （pcDNA3） were transfected into RAW264.7 cells by lipofectamineTM 2000. mFTH （3M） -Flag expression was detected in RAW264. 7 cells. [ Results ] The recombinant vector showed the expected length of the target fragment and the sequencing re- sult was correct. The high level expression of mFTH （3M） -Flag was detected. [ Conclusion ] The present study showed that a eukaryotic expression vector pcDNA3 -mFTH（3M） -Flag, which containing FTH mutant gene with inactivated ferroxidase activity was constructed successfully.