人Importin8基因真核表达载体的构建及表达

Construction of a recombinant eukaryotic vector of Importin 8 and its expression in HeLa cells

目的克隆人Importin8（IP08）基因，观察IP08与绿色荧光蛋白（GFP）真核表达载体融合蛋白在宫颈癌细胞HeLa内的表达和定位。方法以HeLa细胞总RNA为模板进行逆转录一聚合酶链反应（RT—PCR），扩增全长IP08编码序列，经HindIll和XbaI双酶切后，插入GFP质粒pEGFP—C1．，构建pEGFP-IP08重组表达载体。瞬时转染重组质粒入HeLa细胞，荧光显微镜观察pEGFP．IP08在细胞内的表达与定位。结果测序结果显示经PCR扩增的IP08编码序列完全正确，酶切显示重组质粒pEGFP—IP08构建成功，荧光显微镜显示融合蛋白GFP—IP08主要在细胞核内分布。结论成功克隆IP08基因并构建真核表达载体，证实GFP—IP08蛋白主要定位于HeLa细胞核。

Objective To clone the coding sequence of lmportin 8 （ IPO8 ） cDNA trom HeLa cells and construct a eukaryotic expression vector for its green fluorescent protein （GFP）-IP08 fusion protein ex- pression in HeLa cells. Methods The total RNA was extracted from HeLa cells and the full length cDNA of IPO8 was amplified using reverse transcription-polymerase chain reaction （RT-PCR） , and cloned into green fluorescence protein vector phosphoryIated enhanced green fluorescent protein （pEGFP）-C1 to con- struct recombinant expression plasmid pEGFP-IPO8. After recombinant plasmids were transfected into HeLa cells, the expression of IPO8 was observed by fluorescent microscope. Results DNA sequence anal- ysis showed that the amplified rat IPO8 gene was in concordance with that published on Gene Bank. The results of enzyme digestion and sequence analysis demonstrated that the recombinant plasmid pEGFP-IPO8 was constructed successfully. Fluorescence microscopy revealed the fusion protein GFP-IPO8 was mainly located in the nucleus of HeLa cells. Conclusion The IPO8 gene was cloned successfully and the recombi- nant plasmid were constructed successfully. The fusion protein GFP-IPO8 was demonstrated to be mainly located in the nucleus of HeLa cells, which provides a basis for biological functional study of IPO8 gene.