Construction of recombinant plasmid of pcDNA3.1 (+)/HSPT0-Endo/EGFP and its expression in HepG2 cells

Objective To construct the recombinant plasmid of pcDNA3.1 (+)/HSP70-Endo/ EGFP containing gene of endostatin and promoter of heat shock protein 70 gene, and to study its expression in HepG2 cells.Methods The HSP70 promoter (350 bp) was cloned by methods of polymerase chain reaction.Fusion gene of Endo/EGFP was cut from plasmid of pSP-Endo/EGFP using restriction endonucleases of EcoR Ⅰ and Sal Ⅰ , and cloned into multiple clone site of pcDNA3.1 (+), obtaining recombinant plasmid of pcDNA3.1 (+)/HSP70-Endo/EGFP.HepG2 cells were transfected with the recombinant plasmid using lipofectine2000.Following the thermal induction at 37,39,41,43,45 ℃ for 30 min,the expression of EGFP was detected by fluorescence microscope and flow cytometry.The expression of endostatin mRNA was tested by RT-PCR.The concentration of endostatin protein in supernatant was tested by ELISA.Results The subsequence of HSP70 promoter was confirmed with that of AL671762 in GenBank.Hypothermia could induce the up-regulation of Endo/EGFP genes in HepG2 cells, especially heated at 43 ℃,its EGFP expression reached 3.3-fold of 37 ℃ group.The expression of endostatin mRNA in HepG2 cells with thermal induction was higher than that in those without thermal induction.The concentration of endostatin protein in supernatant in 43 ℃ group was (177±28) μg/L,higher than that of 37 ℃ group [(43 ± 11)μg/L].Conclusion Recombinant plasmid of pcDNA3.1 (+)/HSP70-Endo/EGFP was constructed.Hypothermia could induce HSP70 promoter to up-regulate the expression of Endo gene in HepG2 cells. Key words: Carcinoma,hepatocellular;  Gene therapy;  Heat shock protein