植烷酸氧化酶真核表达载体的构建及其细胞内定位

目的构建小鼠植烷酸氧化酶（Phyh）真核表达载体,并观察其在NIH3T3细胞中的表达定位情况。方法提取BALB/c小鼠肝脏组织的总RNA,通过RT-PCR扩增得到Phyh编码序列,将其克隆到真核表达载体pcDNA3上;对阳性克隆进行PCR、酶切和测序鉴定,利用脂质体转染方法转染NIH3T3细胞;使用细胞免疫化学技术对重组质粒pcDNA3/HA-Phyh在NIH3T3细胞中的表达及蛋白定位进行分析。结果 PCR、双酶切和测序鉴定表明,pcDNA3/HA-Phyh真核表达质粒构建正确;转染实验发现,该质粒能够在NIH3T3细胞中表达,表达产物主要定位在细胞质中。结论成功构建了带HA标签的小鼠Phyh真核表达载体,该载体能够在哺乳动物细胞NIH3T3中表达,为进一步研究Phyh的细胞内生物学功能提供了一个重要的工具。

Construction of Eukaryotic Expression Vector of Phytanoyl-CoA Hydroxylase

Objective To construct the eukaryotic expression vector that expresses the fusion protein of phytanoyl-CoA hydroxylase （Phyh）,and determine the cellular localization of the expression product in NIH3T3 cells. Methods Total RNA from the liver of BALB/c mice was extracted and the corresponding coding sequences of mouse Phyh （GenBank accession no. NM-010726） were amplified by RT-PCR and cloned into pcDNA3 with hemagglutinin （HA） tags. Before transfection,pcDNA3/HA-Phyh was identified and confirmed by double digestion with restriction endonuclease and PCR,and the sequence was confirmed by DNA sequencing. The transfected NIH3T3 cells were then cultured,fixed and labeled with an antibody against HA and fluorescence-conjugated secondary antibody to detect the cellular localization of the expression product HA-Phyh. Results Results of PCR,digestion with restriction endonuclease and sequencing indicated that the vector pcDNA3 / HA-Phyh was correctly constructed. The HA-Phyh gene carried by the vector pcDNA3 / HA-Phyh could be expressed in the mammalian cell line NIH3T3,and the protein HA-Phyh was mainly localized in the cytoplasm. Conclusion The eukaryotic expression vector of Phyh fusion protein labeled with HA has been successfully constructed and effectively expressed in mammalian cells. It may lay a foundation for the future study of the intracellular biological functions of Phyh.