【摘要】 目的对人Survivin基因进行克隆,制备和鉴定针对人Survivin的单克隆抗体,检测Survivin在肝癌中的表达。方法利用RTPCR方法从人乳腺癌细胞MCF7扩增克隆Survivin基因,构建可表达Survivin蛋白的原核表达载体,在大肠杆菌中表达MSSurvivin融合蛋白。以纯化的融合蛋白免疫BALB/C小鼠,取其脾细胞,用传统杂交瘤技术与小鼠骨髓瘤细胞融合及次黄嘌呤、氨基蝶呤与胸苷选择性培养。单克隆杂交瘤上清经ELISA双筛后,进行亚类和Westernblot分析。免疫化学方法检测Survivin在肝癌中的表达情况。结果克隆了429bp的Survivin全长基因。经ELISA双筛,获得了4株能稳定分泌针对Survivin的单克隆抗体的杂交瘤细胞株,其分泌抗体亚类均为IgG1。Westernblot结果显示,抗Survivin单抗可与MSSurvivin融合蛋白呈特异性结合。免疫细胞化学显示,该单抗与人肝癌HepG2细胞株呈阳性反应,并且可与肝癌组织结合,呈强阳性反应。结论以分子生物学技术克隆了凋亡抑制因子人Survivin基因,通过原核表达产物制备并纯化了针对Survivin的单克隆抗体,Survivin可在肝癌细胞和肝癌组织中表达,为深入研究Survivin的功能和临床检测肿瘤组织及体液中的Survivin奠定了可靠基础。更多还原

【Abstract】 Objective To clone survivin gene, prepare its monoclonal antibody(McAb) and check its expression in liver carcinoma cells.Methods Survivin gene cDNA was amplified by RT-PCR from human breast carcinoma cell line MCF-7 and constructed into prokaryocytic expression vector.Fusion-protein of human Survivin was expressed and used for immunizing BALB/C mice.The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium.ELISA and Western blot were used to screen and identify the McAbs.Immunocytochemical staining was applied for survivin expression in liver carcinoma cells.Results The full survivin gene was cloned. The hybridoma cell that secret specific monoclonal antibody against human surviving was identified.The immunoglobulin subclasses of the McAbs were IgG1.Western blot showed that the McAbs against survivin can specifically react with MS-Survivin fusion protein. The positive reaction was found in hepatocarcinoma cell line HepG2 and hepatocarcinoma tissue by immunocytochemical staining.Conclusion The McAbs against the human Survivin were successfully prepared by a MS2-Survivin fusion protein expressed by E.coli. and the McAb had positive reaction with HepG2 and hepatocarcinoma tissue. It may be a useful tool to study the functions of survivin and check clinical cancer samples.更多还原