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

Abstract

Objective To clone the coding sequence of Importin 8 (IPO8) cDNA from HeLa cells and construct a eukaryotic expression vector for its green fluorescent protein (GFP)-IPO8 fusion protein expression 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 phosphorylated enhanced green fluorescent protein (pEGFP)-C1 to construct 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 analysis 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 recombinant 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. Key words: Importin 8 gene; Green fluorescent protein; Fusion protein; Eukaryotic vector