Cloning of Human Gfil cDNA and Construction of Recombinant Lentiviral Expressing Vector Gfil / 华中科技大学学报(医学版)

ABSTRACT

Objective To clone the full-length of human Gill cDNA and construct the recombinant lentiviral expressing vector pLOX-Gfil for eukaryotic expression,providing a basis for further study on the biological functions of Gfil.Methods Total RNA was isolated from K562 cells,and the full-length Gfil cDNA was amplified by RT-PCR and then ligated with pGEM-T vector after retrieve and purification.The ligation product was transformed into competent cells DH5a.The positive recombinant clones were selected and identified by a complementation,restriction endonuclease digestion.The cloning vector and the lentiviral vector pLOX first digested with BarnH I were ligated and transformed.The enzyme and PCR analyses were performed to confirm the recombinant vector,and then DNA sequence analysis.Results A fragment of 1.2 kb was obtained by RT-PCR.The enzyme and PCR analyses revealed that the correct Gfil cDNA was cloned.The sequence of cloned cDNA was identical to the sequence deposited in GenBank (NM005263).Conclusion Gfil was cloned correctly and the recombinant lentiviral vector pLOX-Gfil for eukaryotic expression was constructed successfully.