携带GFP和FLAG标签的真核表达载体介导细胞珠蛋白在细胞内表达的定位研究

The difference of localization of GFP-tagged/FLAG-tagged eukaryotic expression vector of cytoglobin in the cells

目的分别构建带有绿色荧光GFP标签和带有FLAG标签的细胞珠蛋白(CYGB)真核表达载体,观察其在人肝星状细胞(LX-2)的表达定位情况,探讨细胞珠蛋白经不同载体介导表达的定位差异。方法提取人肝癌细胞(HepG2)总RNA,通过逆转录-聚合酶链反应(RT-PCR)获得CYGB的cDNA,以cDNA为模板采用PCR法扩增得到CYGB编码序列。将其酶切后分别连接至带有GFP标签和FLAG标签的载体上,酶切与测序鉴定阳性克隆。随后将重组质粒分别瞬时转染LX-2细胞,Western blot鉴定表达情况。利用细胞免疫荧光定位,在荧光显微镜下观察两组定位差异。结果双酶切和测序鉴定表明pEGFP-N1-CYGB和pcDNA3.0-CYGB-FLAG真核表达载体构建成功;经Western blot鉴定,转染的两个载体都能够在LX-2细胞中表达融合蛋白;通过荧光显微镜发现,pEGFP-N1-CYGB和pcDNA3.0-CYGB-FLAG介导的表达产物分别定位在整个细胞和细胞质中。结论可能由于GFP分子量大等多种原因,导致带GFP标签的融合蛋白定位与带FLAG标签的定位不同,带FLAG标签的融合蛋白定位结果更加可信。

Objective To construct eukaryotic expression vector of green fluorescent GFP-tagged and FLAG-tagged cytoglobin （CYGB）, and detect the expression and the difference of localization of fusion protein in human hepatic stellate cells （LX-2）. Methods Total RNA from the human hepatoma cell （HepG2） was extracted and the cDNA was amplified by reverse transcription-polymerase chain reaction（RT-PCR）. The coding sequence of CYGB was amplified by PCR with cDNA as the template and cloned into the GFP-tagged or FLAG-tagged eukaryotic expression vector. The positive clones were identified with enzyme digestion and sequencing. The recombinant plasmids were transfected into LX- 2 cells and were identified the expression of CYGB by Western blot. The localization of the the fusion proteins were observed under fluorescence microscope. Results The recombinant plasmids pEGFP-N1-CYGB and pcDNA3.0-CYGB- FLAG were verified by enzyme digestion and sequence analysis.According to Western blot identification,CYGB fusion proteins can be expressed in LX-2 cells.Under fluorescence microscopy observation we found that the CYGB fusion protein was highly expressed in LX-2 ceils,respectively distributing in the whole cell and cytoplasm. Conclusions Because of the large molecular weight of GFP or other possible reasons, GFP fusion protein＇s localization was different from that of FLAG-tagged fusion protein. The FLAG-tagged labeling may be more reliable.