Effect Of Interaction Of Human Papillomavirus Type 16 E6 Protein And HDaxx On Apoptosis

Objective To provide the experimental basis for studying further effects on oncogenic mechanism of human papillomavirus type 16 (HPV16) E6 protein, the interaction of HPV16 E6 protein and hDaxx was detected by yeast two-hybrid system, and its effect on apoptosis was explored by flow cytometry(FCM).Methodsâ‘´Construction of pGADT7/E6: Polymerase chain reaction (PCR) was used to amplify the HPV16 E6-encoding gene which was built in EcoRI and BamHI restriction enzyme sites. After the restriction enzyme reaction, the fragment of E6 gene was ligated with pGADT7 vector. Then the recombinant was transformed into E.coli DH5αand identified by restriction enzyme analysis and sequencing.⑵Construction of pGBKT7/hDaxx: After the restriction enzyme reaction of pGBDU-C1/hDaxx and pGBKT7, the fragment of hDaxx gene was subcloned into bait vector pGBKT7. Then the recombinant was transformed into E.coli DH5αand identified by restriction enzyme analysis.⑶Yeast two-hybrid assay: Four groups of plasmids were divided for cotransforming into yeast AH109, respectively. Group A was pGADT7 and pGBKT7; Group B was pGADT7/E6 and pGBKT7/hDaxx; Group C was pGADT7/T and pGBKT7/Lam; Group D was pGADT7/T and pGBKT7/p53. The transformants were plated on SD/-Trp-Leu plates and incubated at 30℃for 2 to 4 days. Then the positive colonies were steaked to the SD/-Trp-Leu-His plates and SD/-Trp-Leu-His-Ade plates, and incubated at 30℃for 2 to 4 days. The yeast protein extracts were prepared for Western blot to detect the expression of hDaxx and E6 protein in positive colonies of group B.â‘·Flow cytometry assay: The recombinant of pcDNA3.1(-)/E6 (10μg) was respectively cotransfected into Hela cells with pcDNA3.1(-)/hDaxx which was divided four dose groups (0μg,10μg,20μg,30μg). The groups of Hela cells only or that transfected by pcDNA3.1(-) was arranged as control. All groups of Hela cells were cultured in RMPI-1640 containing 5-FU for 36h. Every group cells were collected, respectively, and fixed in 75% ethanol at 4℃overnight. The cells were stained by propidium iodide (PI).Then apoptosis of every group was detected by FCM assay. Apoptotic rate of every group was analyzed by SPSS13.0.Resultsâ‘´Construction of pGADT7/E6: The size and sequence of E6 gene in pGADT7/E6 was identical with that in GenBank (Pubmed NC₀01526) using restriction enzyme analysis and sequencing.⑵Construction of pGBKT7/hDaxx: An approximate 2.2kb DNA fragment could be seen in agrose electrophoresis after restriction enzyme analysis. It showed that hDaxx fragment was already subcloned into bait vector pGBKT7.⑶Interaction of E6 protein and hDaxx: There were white colonies on SD/-Trp-Leu plates of the four groups. But only the yeast of group B and D could grow on SD/-Trp-Leu-His plates and SD/-Trp-Leu-His-Ade plates. The expression of hDaxx or E6 protein was detected in positive colonies of group B by Western blot.â‘·Flow cytometry assay: The apoptotic rate of Hela cells transfected by pcDNA3.1(-)/E6 was much lower than that of controls(P<0.01). Moreover, the apoptotic rate of Hela cells transfected by pcDNA3.1(-)/hDaxx increased by the dose of pcDNA3.1(-)/hDaxx.Conclusionâ‘´The recombinant plasmids of pGADT7/E6 or pGBKT7/hDaxx was successfully constructed, and E6 protein or hDaxx could express correctly in yeast AH109.⑵HPV16 E6 protein and hDaxx did exist interaction in vivo.⑶HPV16 E6 protein partly inhibited the apoptosis of Hela cells induced by 5-FU. The over-expression of hDaxx could enhance the sensitivity of Hela cells expressing E6 protein to apoptosis induced by 5-FU.