Expression and apoptosis-inducing effect of Apoptin gene in human breast cancer cell strain 435

Objective To discuss the mechanism of vp3 inducing tumor cells into apoptosis by constructing recombinant expression plasmid pcDvp3 and studying its expression and apoptosis inducing effect on human breast cancer cells 435.Methods (1) Apoptin gene fragment was amplified and cloned into the plasmid pcDNA3.1 to form the recombinant plasmid pcDvp3.The nucleotides sequencing was processed.(2) Hela cells were transfected with pcDvp3 and pcDNA3.1 separately.The vp3 expression was detected by immunofluorescent method.(3) The human breast cancer cell line 435 was transfected with pcDvp3 by liposome.Optical microscope,electron microscope,agarose electrophoresis and flow cytometry were used to detect the apoptosis of the tumor cells.[WT5”HZ]Results (1) The sequence analysis demonstrated that the cloned vp3 was the same as that published in literature and inserted into the vector accurately,which suggested recombinant plasmid pcDvp3 had been constructed successfully.(2) Two days fter the COS cells were transfected with vp3,the obvious fluorescence was seen,while in the pcDNA 3.1 group,no fluorescence was found.(3) Distinct cellular morphological transformation and typical apoptosis bodies could be observed in the tumor cells.Agarose electrophoresis of the DNA extracted from the transfected tumor cells showed typical ladder bands.Flow cytometry analysis showed obvious apoptosis peaks and the highest percentage rate of apoptotic cells was present at the time of 48 h after transfection with the apoptotic rate being 14.42%.Conclusion vp3 could be expressed in vivo and in vitro and effectively induced apoptosis of human breast cancer cell line 435.