Effect Of Colocalization Of HPV18 E2 Protein And HDaxx On Apoptosis

Objective:To provide experimental basis for further studying effect of human papillomavirus type 18 (HPV18) E2 protein on oncogenic mechanism and E2 prophylatic or treatment vaccine, confocal laser microscopy and image software are used to observe or analyse the localization or colocalization of HPV18 E2 protein and hDaxx in the cells,and effect of two proteins on apoptosis of HeLa cells is investigated.Methods:(1) The plasmid of pEGFP-C1/E2 and pDsRed-Monomer-C1 was digested with EcoRâ… and BamHâ… , respectively. The digested products were gotten and purified. After linked by T4 ligase, the linked product was tranfered into E. coli JM109. Then the positive clone was screened, and the vector of pDsRed-Monomer-C1/E2 expressing DsRed-HPV18 E2 fusion protein was constructed.(2) The plasmid of pDsRed-Monomer-C1/E2 was transfected into HeLa cells. Then the expression and localization of HPV18 E2 protein was observed under the inverted fluorescence microscopy. Meanwhile, Western-blot was used to detect the protein expression.(3) The plasmids of pDsRed-Monomer-C1/E2 and pEGFP-C1/hDaxx were co-transfected into HeLa cells, the laser confocal microscopy was used to observe the distribution of HPV18 E2 protein and hDaxx. Then their colocalization was analyzed.(4) The plasmids pcDNA3.1(-)/hDaxx(0.5μg or 1.0μg) and pcDNA3.1(-)/E2(2.0μg)were co-transfected into HeLa cells, and the control group was pcDNA3.1 (-)group or the empty cells. Forty-eight hours later,every group cells were collected and fixed overnight by 75% ethanol at 4℃, and then stained with Propidium Iodide(PI). The apoptotic rate was analyzed using flow cytometry.(5) The plasmids pcDNA3.1(-)/hDaxx (0.5μg or 1.0μg) and pcDNA3.1(-)/E2(2.0μg) were co-transfected into HeLa cells, and the control group was pcDNA3.1 (-)group or the empty cells. Forty-eight hours later, every group cells were collected and operated according to caspase-8 kit manual. The absorbance(A value) of 405 nm wavelength was analyzed using ELISA Reader to indicate the relative activity of caspase-8.Results:(1) The recombinant constructed was digested with two enzymes, molecular weight of the small fragment was about 1100bp, and its gene sequence consistent with those of HPV18 E2 published on the GenBank (NC₀01562). The pDsRed-Monomer-C1/E2 recombinant was successfully constructed.(2) Western-blot showed that the eukaryotic expression vector of pDsRed-Monomer-C1/E2 could express the DsRed-HPV18 E2 fusion protein with molecular weight about 69kD in HeLa cells. E2 proteins localized in the cytoplasms and nucleus of HeLa cells under fluorescence microscopy.(3) By observed under Confocal microscopy, it showed that hDaxx only localized in nucleus when its plasmid was transfected into HeLa cells, but hDaxx and HPV18 E2 protein localized and colocalized in cytoplasm and nucleus when their plasmids were co-transfected into cells.(4) The apoptotic rate of 2μg pcDNA3.1(-)/E2+1.0μg pcDNA3.1(-)/hDaxx group was obviously higher than that of 2μg pcDNA3.1(-)/E2+ 0.5μg pcDNA3.1(-)/ hDaxx group,there was statistically significant(P<0.001). Meanwhile, these two groups were obviously higher than that of other groups (P < 0.001). The apoptotic rate of pcDNA3.1(-)/E2 group was obviously higher than that of pcDNA3.1(-) group or the empty cells group(P<0.001).(5) The caspase-8's relative activity of 2μg pcDNA3.1(-)/E2+1.0μg pcDNA3.1(-)/hDaxx group was obviously higher than that of 2μg pcDNA3.1(-)/E2+0.5μg pcDNA3.1(-)/ hDaxx group, there was statistically significant(P<0.01). Meanwhile, these two groups were obviously higher than that of other groups (P<0.01). The caspase-8's relative activity of pcDNA3.1(-)/E2 group was higher than that of pcDNA3.1(-) group or the empty cells group(P<0.01).Conclusions:(1) The eukaryotic expression recombinant pDsRed-Monomer-C1/E2 was successfully constructed, and the DsRed-HPV18 E2 fusion protein can express in cells.(2) HPV18 E2 protein and hDaxx colocalize in the cytoplasms and nucleus. hDaxx may translocalize from nucleus to cytoplasms by HPV18 E2 protein.(3) The activity of caspase-8 and HeLa cells apoptosis may induce by over-expression of HPV18 E2 protein, and hDaxx may enhance apoptotic rate in a dose-dependent manner.