七鳃鳗PHB2的真核表达与亚细胞定位

Eukaryotic expression and cellular localization of lamprey PHB2

以东北七鳃鳗心肌总RNA反转录得到的cDNA为模板,PCR扩增PHB2基因全长CDS区,并将其定向克隆至真核表达载体pEGFP-N1上,构建真核重组载体pEGFP-N1-Lm-PHB2.提取重组质粒,去除内毒素,利用脂质体介导的方法转染CHL、HeLa和MCF-7细胞,通过Western Blot检测转染后Lm-PHB2是否表达,Confocal观察EGFP融合蛋白在细胞中的表达及亚细胞定位.结果表明:PCR扩增片段与预期结果相符,双酶切和测序证明真核表达载体构建成功;Western Blot检测到Lm-PHB2蛋白条带,Confocal观察到绿色荧光蛋白GFP,证明真核重组载体成功表达;Lm-PHB2蛋白主要定位在CHL细胞的细胞核,少量存在于基质和线粒体中,而在HeLa和MCF-7细胞中,Lm-PHB2蛋白集中定位在细胞核中.本研究为七鳃鳗Lm-PHB2的功能研究奠定了重要基础.

The cDNA, reverse-transcribed by lamprey myocardial total RNA, was chosen as a template to amplify a full-length CDS region of Lm-PHB2 gene. The eukaryotic expression plasmid （pEGFP-N1-Lm-PHB2） was constructed, extracted without endotoxin and transfected into CHL, HeLa and MCF-7 cells by liposomes. Cellular localization and expression of Lm-PHB2 was observed by Confocal and Western Blot. The results showed that pEGFP-N1-Lm-PHB2 was successfully constructed by PCR, restriction enzyme digestion and sequencing verification. Lm-PHB2 could express in eukaryotic cells, main localization in the nucleus and only a small amount in mitochondria of CHL, however, focused distribution in the nucleus of HeLa and MCF-7. The study lays the vital founda- tion to the functional research of Lm-PHB2 in cells.