Preparation Of Daxx-C Antibody And Its Preliminary Applications In Cervical Carcinoma Cells With HPV16Positive

Objective To provide the basis for further exploring the effect and its mechanism ofDaxx on the progress of cervical carcinoma induced by human papillomavirus type16(HPV16), Daxx-C antibody was developed and the distribution of Daxx in cervicalcarcinoma of HPV16positive was observed, and the location and colocalization of Daxxand ATRX in Caski cells were analyzed.Methods(1) The specific primers of Daxx gene fragment(amino acids626-740) including BamHIand SalI enzyme sites were designed by software PRIMER5.0. PCR amplification productswere inserted into pMD18-T, and the vector of pMD18-T/DM626-740was constructed. Then,DM626-740gene fragments from pMD18-T/DM626-740digested with BamHI and SalI wereinserted into pET28a. The prokaryotic expression vector of pET28a/DM626-740wasconstructed by identification of enzyme digestion and DNA sequence.(2)The plasmid of pET28a/DM626-740was transformed into E. coli BL21. The expressionproductions were induced with IPTG and purified by Ni-NTA kit. Western bolt was used toanalyze expression and purification production of6His-DM626-740fusion protein.(3)A total of4female BALB/c mice were inoculated with the purification of6His-DM626-740by multiple sites of hypodermic injection and peritoneal injection. Threeweeks later, the mice were inoculated again. Then the mice were inoculated once everyweek for two weeks. The sera of mice were collected in the day before every immunizationor the7thday after the last injection. ELISA was used to detect the titer of DM626-740antibody. Saturated ammonium sulfate salting-out sedimentation was used to purify theantiserum. The antiserum purified production was detected using Western blot.(4)The samples of normal cervical epithelial cells, cervical intraepithelial neoplasia grade Ⅰ(CINⅠ), CINⅡ，CINⅢ and cervical cancers were collected. Immunochemistry assaywas used to analyze the distributions and localations of Daxx in the cervical tissue.(5) Indirect immunofluorescence assay was used to analyze the cellular location orcolocalization of ATRX and Daxx in Caski cells under fluorescence microscopy and usingimage software.Results:(1) The enzyme digestion and DNA sequencing results showed that Daxx genefragment(626-740amino acid residues) was correctly linked into pMD18-T or pET28a.The vector of pMD18-T/DM626-740or prokaryotic expression vector of pET28a/DM626-740was constructed respectively.(2)The aim protein of molecular weight of about19kDa expression in E. coli transformedby pET28a/DM626-740and induced expression with IPTG could be detected using anti-Daxxantibody.(3)After the mice were inoculated with6His-DM626-740purified production, ELISA resultshowed that the titer of the specific antibody was1:6400.(4)Under the light microscopy, the brown signals of Daxx distributed in the nuclei ofnormal cervical epithelial cells; Daxx mainly distributed in nuclear membrane and therewere a small amount of Daxx in the nuclei in CINⅠ. Daxx intensively distributed in thecytoplasm and cell membrane in CINⅡ，CINⅢ and cervical cancer.(5) The green signals of Daxx mainly distributed in the cytoplasm of Caski cells. Therewere a small amount of Daxx in the nucleus and nuclear membrane. It showed that therewere strong fluorescence signals in the vicinity of nuclear membrane and regionalaggregation. The red signals of ATRX mainly distributed in the nucleus, and there were asmall amount of the distribution in the cytoplasm. There were the yellow signals when thegreen signals of Daxx and the red signals of ATRX were overlapped.Conclusions(1) The prokaryotic expression vector of pET28a/DM626-740constructed can express the aim protein of6His-DM626-740.(2) The Daxx-C antibody prepared can be used to detect Daxx in the tissue. In the progressof the cervical cancer, Daxx gradually translocates from nucleus into nuclear membrane,cytoplasm and cell membrane. Daxx locates in the cytoplasm and cell membrane in CINⅡ,CINⅢ and cervical cancer.(3) There are colocalizations of Daxx and ATRX in the nuclear membrane of Caski cells.