抗HPV16E6全蛋白单克隆抗体的制备及鉴定

Preparation and Identification of Monoclonal Antibody of Anti-HPV16E6

为了制备特异性识别HPV16E6蛋白的单克隆抗体,为建立HPV早期诊断试剂盒提供有效试剂,通过细胞融合、间接ELISA方法筛选阳性克隆、多次亚克隆得到稳定分泌抗体的单克隆杂交瘤细胞株,体内诱生法大量制备腹水,过UNOsphere SuPrA^(TM)柱和ENrich^(TM)SEC650分子筛柱纯化,通过蛋白电泳鉴定其纯度并测定抗体浓度,并采用Western Blot法和间接免疫荧光法验证纯化后HPV16E6抗体与HPV16型阳性Caski细胞和HPV18型阳性Hela细胞特异性结合。建立了亚型为IgG2a的单克隆HPV16E6-4D4杂交瘤细胞株,两步纯化后得到纯度较高的抗体,可特异性识别HPV16型阳性的Caski细胞中的E6蛋白,不与Hela细胞反应,为在蛋白水平上检测HPV16型病毒提供了新手段。

To prepare monoelonal antibodies specific to HPV16E6 protein, and to provide an effective reagent for the early diagnosis of HPV, HPV16E6 recombinant protein which was antigen inoculated Balb/e mouse, the hybridoma cells were obtained from cell fusion, and Indirect ELISA（iELISA） was adopted to select the positive clones as well as subcloning. After large-scale preparation of ascites, UNOsphere SuPrATM column and ENrichTM SEC650 size exclusion column were adopted to purify antibody. Coomassie blue staining was adopted to identify the purity and the standard curve to calculate the concentration of antibody. Western blot and indirect immunofluorescence were adopted to verify the specific bind of purified antibody E6 to Caski （HPV16 positive） and Hela（ HPV18 positive） cells. The obtained hybridoma ceils named HPV16E6-4D4 which was IgG2a has better antibody purity after two-steps puritification, and could specifically recognize HPV16E6 protein in Caski cells, and not react with Hela cells. The monoclonal antibody against HPV16E6 whole protein has been successfully selected and it provided a new way for the detection of HPV E6 protein at the protein level for its specificity.