摘要
目的 人端粒酶蛋白亚基 (hTERT)单克隆抗体制备及肿瘤检测应用。方法 利用多肽固相合成法 (Fmoc法 )合成hTERT抗原多肽 ,免疫BALB/c小鼠 ,以杂交瘤法制备单克隆抗体。应用ELISA ,Westernblot和免疫组织化学ABC法进行鉴定和肿瘤检测。结果 经合成得到hTERT抗原多肽hTERT9,免疫小鼠及细胞融合后得到抗hTERT9杂交瘤细胞 ;经 3次有限稀释法及ELISA筛选获得抗hTERT9单克隆抗体的杂交瘤细胞H4、G8和A11;H4、G8为IgM类 ,而A11为IgG1。半抗原竞争性抑制实验证明为hTERT9特异性单克隆抗体 ;相对亲和力实验发现G8株亲和力较H4株和A11株强。用H4及G8株单克隆抗体检测HeLa、2 93细胞 ,免疫印迹有特异相对分子质量≈ 12 30 0 0hTERT条带 ,免疫组织化学显示为细胞核着色 ,而正常细胞 2BS无阳性反应。免疫组织化学方法检测人癌组织、癌前病变、良性病变hTERT表达发现 :人肿瘤组织细胞阳性率为 80 31% (10 2 / 12 7) ,且大多为中度阳性和强阳性 ;癌前病变中 ,hTERT有 17 5 % (7/ 4 0 )弱阳性 ;良性肿瘤均为阴性。统计学分析表明 :hTERT在癌组织的表达显著高于癌前及良性病变 (P <0 0 1)。结论 通过固相合成hTERT抗原多肽和杂交瘤技术成功制备抗人端粒酶蛋白亚基hTERT单克隆抗体 ,并能运用于免疫印迹。
Objective To develop monoclonal antibodies against the catalytic subunit of human telomerase hTERT for its expression detection of human tumors Methods A dominant epitope in hTERT (peptide hTERT 9)was automatically synthesized based on Fmoc method, and was used to immunize BALB/c mice Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization of which were performed by Western blotting and immunohistochemical staining Results Antigenic peptide hTERT 9 was synthesized and confirmed by MALDI TOF MS and HPLC analysis Three hybridoma cell lines secreting anti hTERT 9 antibodies designated as H4, G8 and A11 were established after primary screening and consequent three rounds of limited dilution Both of H4 and G8 were IgM, while A11 was IgG1 in isotyping The competitive assay showed that the antibodies were hTERT 9 specific, and the affinity of G8 was stronger than that of H4 and A11 assayed by affinity ranking However, in Western blotting, both of H4 and G8 stained an about 123 000 protein band with HeLa and 293 cell extracts but not with normal 2BS cells Besides, positive staining presented in the nucleus of HeLa, while 2BS was non reactive immunohistochemically The sections from paraffin embedded blocks of 127 cases of human cancer, 40 of precancerous and 19 of benign tumors were in situ stained by G8 antibody, the results showed that the human cancer tissues were 80 31%(102/127)positive in specific nuclear reaction, on the contrary, only a minority of precancerous lesions present weak positive(17 5%, 7/40), and negative in benign tumors (0/19) Conclusions The monoclonal antibodies developed against synthetic peptide were hTERT specific and could recognize both the native and the denatured form Thus their use in immunoblotting or immunohistochemistry for detecting the telomerase hTERT expression of cancer cell and tissues was promising
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2002年第1期50-54,共5页
Chinese Journal of Pathology
基金
国家"十五攻关计划"基金资助项目
