Preparation Of ATRX-C Antibody And Its Preliminary Applications In Cervical Carcinoma Tissues With HPV16 Positive

[Objective] To explore the effects and mechanisms of ATRX on the progress of cervical carcinoma induced by human papillomavirus type 16(HPV16), ATRX-C antibody was prepared, and the distribution and expression of ATRX in cervical carcinoma of HPV16 positive were studied. [Methods]The specific primers of ATRX gene fragment(amino acids 2193-2492) PCR amplification products were inserted into p ET30 a. The prokaryotic expression vector of p ET30a/ATRX-C2193-2492 was constructed and identified by the PCR, enzyme digestion and DNA sequencing. The p ET30a/ATRX-C2193-2492 was transformed into E.coli BL21. The recombinant ATRX-C2193-2492 protein was induced by IPTG and purified by Ni-NTA affinity column and identified by Western-blot. The female BALB/c mice were immunized with the purified His- ATRX-C2193-2492 protein by multiple sites of hypodermic and peritoneal injection. The mice were immunized again three weeks later. Then the mice were immunized once every week for two weeks. The sera of mice were collected in the day before every immunization or the 7th day after the last injection. ELISA was used to detect the titer of ATRX-C2193-2492 antibody. The antiserum was purified by saturated ammonium sulfate salting-out sedimentation and indentified by Western blot. The cellular location or colocalization of ATRX and Daxx in THP-1 cell were analyzed using the indirect immunofluorescence assay. Immunochemistry assay was used to analyze the distributions and localations of ATRX in the normal cervical epithelial cells, cervical intraepithelial neoplasia grade(CIN), carcinoma in situ and invasive cervical cancers with HPV16 positive. The expression of ATRX protein in normal cervical epithelial cells, CIN, carcinoma in situ and cervical cancers were analyzed by western blot. [Results]The prokaryotic expression vector p ET30a/ATRX-C2193-2492 was constructed and identified by PCR, enzyme digestion and sequencing. About 34 k Da fusion protein was induced by IPTG in recombinant E.coli BL21 which harboring the p ET30a/ATRX-C2193-2492 plasmid. The fusion protein 6His-ATRX-C2193-2492 was purified successfully and was identified by the western blot.After the mice were immunized with purified 6His-ATRX-C2193-2492 production, ELISA results showed that the titer of the specific antibody for 6His-ATRX-C2193-2492 was 1: 12800.The green signals of ATRX mainly distributed in the nucleus of THP-1 cells and the signal was strong. The red fluorescence of Daxx mainly distributed in the nucleus, and there were a small amount of the distribution in the cytoplasm, It showed the yellow signal when the red signal and yellow signal were merged. ATRX mainly located in the nucleus and had high expression in normal cervical tissues and cells; In HPV16 positive cervical intraepithelial neoplasia(CIN) is similar to the expression of tissue cells and normal cells, expression of medium is in in situ carcinoma tissue cells, the main localization in the nucleus and cytoplasm, the express quantity reduce CIN, expression is less in infiltrating carcinoma tissue cells, mainly locating in the cytoplasm, nucleus expression quantity decreased significantly. [Conclusions] ATRX-C antibody was prepared which could be used to detect ATRX.ATRX located in the nucleus of THP-1 cells or normal cervical epithelial cells. In the progress of the cervical cancer, the expression of ATRX gradually decrease.