Construction of recombinant eukaryotic expression plasmid containing human neutral amino acid transporter B~0AT1 gene and establishment of stably transfected Hela cell lines

Objective: To construct an eukaryotic expression vector containing human neutral amino acid transporter B0AT1 gene and obtain a stably transfected Hela cell line expressing human B0AT1.Methods: The human B0AT1 gene was amplified by RT-PCR from human intestinal epithelial cell and digested by EcoRⅠ / XbaⅠ.This digested fragment was inserted into eukaryotic expression vector pcDNA3.1 and then transformed E.coli DH5α.The positive recombinant plasmid was transfected into Hela cell line by LipolectamineTM 2000.Cells stably expressing human B0AT1 were selected by G418 and confirmed by RT-PCR.The biological activities of B0AT1 expressed on Hela cells were confirmed by amino acid transport experiment.Results: Human B0AT1 gene was amplified by RT-PCR and cloned into eukaryotic expression plasmid pcDNA3.1 successfully.And the Hela cell line stably expressing human neutral amino acid transporter B0AT1 was obtained.Conclusion: The pcDNA3.1-B0AT1 transfected Hela cell line is established successfully,laying a foundation for further study of the function and regulation of B0AT1 transporter.