Interaction And Its Domain Between Human Papillomavirus Type16E2Protein And Daxx

Objective:To study the interaction between human papillomavirus type16(HPV16)E2proteinand Daxx, and to provide experimental basis for further exploring the effects andmechanisms of their interactions on the progress of carcinogenesis induced by HPV16.Methods：(1) HPV16E2and its gene fragments (amino acids1-201,202-365and249-365) specificprimers including EcoRI and BamHI enzyme sites were designed by software PRIMER5.0.PCR amplification products, which were digested with EcoRI and BamHI, wererespectively inserted into pGADT7. After screening the positive colonies, the yeastexpression plasmid of pGADT7-HPV16E2, pGADT7-HPV16E2TAD, pGADT7-HPV16E2H-DBD or pGADT7-HPV16E2DBD was respectively constructed and identified byenzyme digestion and DNA sequencing.(2)The yeast AH109was transformed with pGBKT7-Daxx, pGADT7, pGADT7-HPV16E2,pGADT7-HPV16E2TAD, pGADT7-HPV16E2H-DBD or pGADT7-HPV16E2DBD usingLiAc method, respectively. The SD/-Trp, SD/-Leu, SD/-His or SD/-Ade dishes plated withthe transformant incubated for2~4days at30℃. The self-activation of every recombinantwas analyzed.(3)The Plasmids were divided into7groups, they were respectively group A(pGADT7+pGBKT7), group B(pGADT7-T+pGBKT7-Lam), group C(pGADT7-T+pGBKT7-p53),group D(pGBKT7-Daxx+pGADT7-HPV16E2), group E(pGBKT7-Daxx+pGADT7-HPV16E2TAD), group F(pGBKT7-Daxx+pGADT7-HPV16E2H-DBD) and group G(pGBKT7-Daxx+pGADT7-HPV16E2DBD). After every group plasmids werecotransformed into yeast AH109, the transformants were plated on SD/-Trp-Leu plates andincubated for2~4days at30℃. The single colony was streaked on SD/-Trp-Leu-His platesand SD/-Trp-Leu-His-Ade plates，respectively. Then the plates incubated for2~4days at 30℃. The domain of HPV16E2protein interacting with Daxx was assessed by the yeastgrowth.(4) About2.2kb Daxx cDNA fragment from pcDNA3.1/Daxx digested with BamHⅠwasinserted into the same site of pet28a. Then the positive colony was screened with BamHⅠor HindⅢ. And the prokaryotic expression vector pet28a/Daxx was constructed. IPTG wasused to induce the expressions of6His-Daxx or GST-HPV16E2fusion proteins for E.coliBL21containing pet28a/Daxx or pGEX6p-1/HPV16E2. Fusion proteins were purified withNi-NTA kit or Resin GST kit. SDS-PAGE and Western blot were used to detect theexpressions of fusion proteins and their purified productions.(5) The complex of0.5μg6His-Daxx and GST-HPV16E2fusion protein and anti-GST oranti-His antibody were used to detect the binding reaction using coimmunoprecipitation(COIP) and Western blot. Anti-Daxx or anti-HPV16E2antibody was as Western blotantibody. The supernatants of lytic Caski cells were collected to analyze the bindingreaction using COIP and Western blot. Anti-Daxx or anti-HPV16E2antibody was as IPantibody, anti-HPV16E2or anti-Daxx antibody was as Western blot antibody.(6) Indirect immunofluorescence assay and image software were applied to observe andanalyze the distribution, location or co-localization of HPV16E2protein and Daxx in Caskicells.Results:(1) DNA sequencing result showed that HPV16E2or its gene fragment was correctlyinserted pGADT7, respectively. The yeast expression vector of pGADT7-HPV16E2,pGADT7-HPV16E2TAD, pGADT7-HPV16E2H-DBD or pGADT7-HPV16E2DBD wasconstructed.(2) The yeast AH109transformed with the bait plasmids, such as pGBKT7, orpGBKT7-Daxx, 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 pGADT7orpGADT7-HPV16E2could grow on the SD/-Leu plates, but not grow on the SD/-Leu,SD/-His, SD/-Ade plates. Every recombinant could not self-activate.(3) The transformants of Group A to Group G could grow on the SD/-Trp-Leu plates. The transformants of Group C, D and E could grow on the SD/-Trp-Leu-His plates andSD/-Trp-Leu-His-Ade plates. There was not any colony on the SD/-Trp-Leu-His plates andSD/-Trp-Leu-His-Ade plates for Group A, B, F and G.(4) The enzyme digestion results showed that Daxx was correctly linked into pet28a, andprokaryotic expression vector pet28a/Daxx was constructed. In E.coli BL21,6His-Daxxfusion protein was induced express by IPTG. COIP and Western blot results showed thatthe bands were about90kDa and70kDa, so there was binding reaction between HPV16E2and Daxx in vitro.(5) Using the supernatant of lytic Caski cells, COIP and Western blot results showed thatthe bands were about80kDa and60kDa, so there was binding reaction between HPV16E2and Daxx in vio.(6) In Caski cells, the red signals of HPV16E2and the green signals of Daxx mainlylocated in the cytoplasms, and a little distribution were in the nuclei. The yellow signalswere observed when the red signals of HPV16E2merged with the green signals of Daxx.Conclusions:(1) pGADT7-HPV16E2, pGADT7-HPV16E2TAD, pGADT7-HPV16E2H+DBD orpGADT7-HPV16E2DBD can respectively express their associated proteins in the yeast.pet28a/Daxx can express the fusion proteins of6His-Daxx in E.coli BL21.(3) HPV16E2protein can bind to Daxx in vivo or in vitro; Daxx interacts with HPV16E2by its N terminal domain (TAD).(4) Both HPV16E2protein and Daxx mainly distribute in the cytoplasm of Caski cells.The colocalizations of Daxx and HPV16E2protein are in the nuclei and cytoplasm ofCaski cells.