ABSTRACT
Human manganese superoxide dismutase (hMn-SOD) cDNA was amplified by RT-PCR from total RNA of human liver cell (L02), and cloned into yeast expression vector pPIC9K containing AOX1 promoter and the alpha-factor signal peptide sequence. The resultant pPIC9K-MnSOD was transformed to P. pastoris GS115, screened for Mut+ carrying multiple copies of hMn-SOD. The positive transformants were fermented in flasks and induced by 0.5% methanol. After 4 days of methanol induction, the expressed hMn-SOD was up to 32% of the total proteins in the supernatant by SDS-PAGE with specific activity of 247.7 u/mg.
Subject(s)
Pichia/genetics , Recombinant Proteins/biosynthesis , Superoxide Dismutase/genetics , Cloning, Molecular , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Humans , Pichia/metabolism , Recombinant Proteins/genetics , Superoxide Dismutase/biosynthesisABSTRACT
BACKGROUND & OBJECTIVE: P53 gene is a tumor suppressor gene,and its mutation in human tumor cells is frequently observed. Previous studies revealed that wild type p53 (wt-p53)gene can suppress proliferation ,and induce apoptosis of tumor cells. However,the enhancive effect of wt-p53 on apoptosis of tumor cells is not so obvious when it is used alone. Therefore,it is a new field for tumor research that wt-p53 gene combined with drug is used to enhance apoptosis rate of tumor cells. This study was to investigate the enhancement effect of the combination of wt-p53 and 1,2:5,6-dianhydro-3,4-diacetylgalactitol (DADAG)on apoptosis of human hepatocarcinoma cell line HLE. METHODS: HLE cells were transfected with pUHD10-3-p53 plasmid contained wt-p53 gene,and treated with DADAG. After 96-hour treatment,apoptosis was evaluated by flow cytometry and DNA electrophoresis. RESULTS: The apoptosis rates were: 1.4% in untreated group, 3.5% in pUHD10-3-transfection group, 32.6% in DADAG group, 43.4% in pUHD10-3-p53-transfection group, and 74.6% in pUHD10-3-p53-transfection plus DADAG-treatment group. DNA ladder was observed in pUHD10-3-p53-transfection plus DADAG-treatment group. CONCLUSION: Apoptosis of HLE cells could be induced by both wt-p53 gene and DADAG,and the effect was more obvious when HLE cells were treated by the combination of wt-p53 gene and DADAG.
