关键词: 凋亡诱导因子, 细胞凋亡, 腺病毒载体, K562细胞

Abstract: Objective To construct the recombinant adenovirus Ad-AIF, Ad-DMLS-AIF and Ad-DHBD-AIF and detect their apoptotic effect on leukemia K562 cells. Methods AIF gene and its mutants（DMLS-AIF gene and DHBD-AIF gene）were amplified and cloned into the shuttle plasmid pAd -Track-CMV-HA in turn, after identification by PCR、double-enzyme digestion and sequencing, recombined homology with pAdEasy1 to obtain recombinant adenovirus plasmid and then which were packaged and amplified, adenovirus without exogenous sequence as empty vector. Western blot was used to analyze expression of the recombinant adenovirus in K562 cells; Co-immunoprecipitation assay (CO-IP) were applied to detect the relationship between HSP70 and AIF or DMLS-AIF or DHBD-AIF; Hoechst33258 dye and FCM were applied to detect the apoptotic effect of the recombinant adenovirus on K562 cells. Results The recombinant adenovirus were constructed successfully and expressed in K562 cells; AIF and DMLS-AIF were connected to HSP70 except DHBD-AIF; the chromatin were agglutinated in K562 cells infected with Ad-DHBD-AIF, that were uniformity in K562 cells infected with Ad-AIF or Ad-DMLS-AIF; the apoptotic effect of Ad-DHBD-AIF on K562 cells was higher than that of Ad-AIF and Ad-DMLS-AIF. Conclusion Ad-AIF、Ad-DMLS-AIF and Ad-DHBD-AIF were constructed successfully, the apoptotic effect of Ad-DHBD-AIF on K562 cells were higher significantly comparing with that of Ad-AIF and Ad-DMLS-AIF, which could be as the basis of the further experience of K562 cells anti-apoptosis.

Key words: apoptosis-inducing factor, cell apoptosis, recombinant adenovirus, K562 cells