Objective       To construct an eukaryotic expression vector encoding human Bcl-2 and adenovirus E1B 19 kDa interacting protein (BNIP3) gene

and to investigate the effect of BNIP3 over-expression on the chemosensitivity of human colon cancer cell line HT-29.       Methods       The full-length cDNA of BNIP3 was amplified by PCR and cloned into pEGFP-C3 vector using genetic engineering technology. The recombinant expression vector pEGFP-C3/BNIP3 was

confirmed by enzyme digestion and  sequencing

and then was transferred into HT-29 cells by liposome. The expression of BNIP3 was detected by Western blotting. The chemosensitivity of transfected HT-29 cells to 5-Fu and cell proliferation were evaluated by MTT assay. Cell apoptosis was measured by flow cytometry.       Results       An eukaryotic expression vector of BNIP3 was constructed successfully

and the BNIP3 protein was highly expressed in HT-29 cells transfected with pEGFP-C3/BNIP3. The IC

50

of HT-29 cells transfected with pEGFP-C3/BNIP3 incubated with 5-Fu was significantly lower than those of HT-29 cells untransfected or transfected with pEGFP-C3  (120.11±5.45

113.40±4.72 vs  19.08±2.62 μg/mL

P

0.05). The apoptotic rate of HT-29 cells transfected with pEGFP-C3/BNIP3 was significantly decreased compared with HT-29 cells untransfected or transfected with pEGFP-C3  [(5.51±0.32)%

(7.19±0.47)% vs (41.72±1.48)%

P

0.05

]

. Compared with the control cells

HT-29 cells transfected with pEGFP-C3/BNIP3 had significantly reduced cell colony formation  (52±6

49±5 vs 11±3

P

0.05) and decreased cell proliferation rate.       Conclusion       The recombinant expression vector pEGFP-C3/BNIP3 is successfully constructed. Over-expression of BNIP3 can enhance the chemosensitivity of HT-29 cells to 5-Fu.