| Objective:To provide the experimental basis for further studying effects of human papillomavirus type16(HPV16) E6protein on the oncogenic mechanisms, and to explore the domain of Daxx interacting with HPV16E6protein and the location of Daxx and PML in the cervical cancer cells, such as Caski cells and HeLa cells.Methods:(1)The specific primers including Nde I or Sal I restriction enzyme site were designed by the PRIMER5.0software. Polymerase chain reaction (PCR) was used to amplify the fragments(1-240,241-500,501-625and626-740amino acid residues) of Daxx gene. After the PCR productions were digested by the restriction enzymes, the fragments were inserted into the same enzyme sites of pGBKT7, respectively. Then the recombinants were transformed into E.coli JM109and the positive clones were screened. The restriction enzymes digestions and DNA sequencing were used to identify these recombinants.(2)The yeast AH109were transformed with pGBKT7, pGBKT7-Daxx, pGBKT7-Daxx-DM1-240, pGBKT7-Daxx-DM241-500, pGBKT7-Daxx-DM501-625, pGBKT7-Daxx-DM626-740, pGADT7and pGADT7-E6using LiAC, respectively. The SD/-Trp, SD/-Leu, SD/-His and SD/-Ade dishes plated with the transformant incubated for2-4days at30℃. The self-activation of every recombinant was analyzed.(3) The plasmids were divided into eight groups, they were Group A(pGADT7and pGBKT7), Group B(pGADT7-T and pGBKT7-Lam), Group C(pGADT7-T and pGBKT7-p53), Group D(pGADT7-E6and pGBKT7-Daxx), Group E(pGADT7-E6and pGBKT7-Daxx-DM1-240), Group F(pGADT7-E6and pGBKT7-Daxx-DM240-50), Group G(pGADT7-E6and pGBKT7-Daxx-DM501-625) and Group H(pGADT7-E6and pGBKT7-Daxx-DM626-740). After every group plasmids were cotransformed into yeast AH109, the transformants were plated on SD/-Trp-Leu plates and incubated for2-4days at30℃.The single colony was streaked on the SD/-Trp-Leu-His plates and SD/-Trp-Leu-His-Ade plates, respectively. Then the plates incubated for2-4days at30℃. The domain of Daxx interacting with HPV16E6protein was assessed by the yeast growth.(4) Co-immunoprecipitation assay was used to detect the binding of Daxx and HPV16E6proteins in Hela cells transfected with pcDNA3.1(-)/HPV16E6.(5) Indirect immunofluorescence assay was used to analyze the subcellular location of PML and Daxx in HeLa and Caski cells under confocal microscopy. It was also used to analyze the colocalization of Daxx and HPV16E6protein in Caski cells.Results:(1) DNA sequencing results showed that every fragment(1-240,241-500,501-625and626-740amino acid residues) of Daxx gene was correctly inserted into pGBKT7, respectively. The yeast expression vector of pGBKT7-Daxx-DM1-240, pGBKT7-Daxx-DM241-500,pGBKT7-Daxx-DM501-625and pGBKT7-Daxx-DM626-740was respectively constructed.(2) The yeast AH109transformed with the bait plasmids, such as pGBKT7, pGBKT7-Daxx,pGBKT7-Daxx-DM1-240,pGBKT7-Daxx-DM241-500,pGBKT7-Daxx-DM501-625and pGBKT7-Daxx-DM626-740, could grow on the SD/-Leu plates, but not grow on the SD/-Leu, SD/-His, SD/-Ade plates. The yeast AH109transformed with prey plasmids of pGADT7and pGADT7-E6could grow on the SD/-Leu plates, but not grow on the SD/-Leu, SD/-His, SD/-Ade plates. Every recombinant had not the self-activation.(3) The transformants of Group A-Group G could grow on the SD/-Trp-Leu plates. The transformants of Group C, D and H could grow on the SD/-Trp-Leu-His plates and SD/-Trp-Leu-His-Ade plates. There was not any colony on the SD/-Trp-Leu-His plates and SD/-Trp-Leu-His-Ade plates for Group A, B, E, F and G.(4) Western blot’s results showed that Daxx(or HPV16E6protein) bound HPV16E6protein(or Daxx) in HeLa cells.(5) The distribution of HPV16E6and Daxx was not uniform in the nucleus of Caski cells, but regional aggregation. The yellow signals were shown when the red signals of HPV16E6merged with the green signals of Daxx. It showed that HPV16E6protein could colocalize with Daxx in the nuclei and cytoplasm of Caski cells.(6) The green signals of Daxx and the red signals of PML were dispersed uniform in the nuclei and cytoplasm of HeLa cells or Caski cells. There was not the yellow signal when the green signals of Daxx and the red signals of PML were merged.Conclusions:(1)The recombinant of pGBKT7-Daxx-DM1-240, pGBKT7-Daxx-DM241-500, pGBKT7-Daxx-DM501-625, and pGBKT7-Daxx-DM626-740are successfully constructed and can express the target proteins in the yeast.(2) The C-terminal domain of Daxx binds with HPV16E6protein. There is interaction between HPV16E6protein and Daxx.(3) The colocalizations of Daxx and HPV16E6protein are in the nuclei and cytoplasm of Caski cells.(4) There is not colocalization of Daxx and PML in PML-NBs of Caski cells or HeLa cells. |
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