摘要
目的表达人Survivin重组蛋白、制备单克隆抗体及检测Survivin在多种肿瘤细胞中的表达。方法诱导含有pMS-Survivin重组质粒的E.coli.表达人MS2-Survivin重组蛋白,并作为抗原免疫BALB/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合,经ELISA双筛,获得稳定分泌抗人Survivin mAb的杂交瘤细胞株。ELISA检测Survivin mAb的特异性。免疫细胞化学检测多种肿瘤细胞中天然Survivin的表达情况。结果诱导菌在相对分子质量为30KD左右表达出一新的蛋白条带。筛选出2株稳定分泌特异性抗人Survivin mAb的杂交瘤细胞株,亚类鉴定均为IgG1类。两株单克隆抗体的效价均达到6.4×105。ELISA结果显示出抗人Survivin mAb的特异性。在胃癌细胞、肺癌细胞、宫颈癌细胞中均强表达,且主要在细胞浆中。结论成功制备出特异性抗人Survivin mAb,为以后应用于临床检测奠定了基础,也为其进一步开发成诊断性试剂盒奠定了基础。
Objective To express human Survivin protein in E. coll. and to prepare monoclonal antibody against it as well as checking survivin expression in several tumor cell lines. Methods The expression of recombinant plasmid pMS- Survivin was induced in E. coll. and the expressed product was used as antigen to immunize BALB/c mice. Monoclonal antibody against Survivin was prepared by normal hybridoma technology. Specificity of Survivin mAbs was analysed by ELISA. Expression of survivin in tumor cell lines was investigated by immunocytochemical staining. Results SDS- PAGE showed the expression of recombinant protein of human survivin. Two hybridmas producing antibodies against survivin were obtained. IgG isotypes of two mAbs were IgG1. ELISA showed that the mAbs against Survivin were spe- cific for Survivin protein. Survivin expression was proved in cell plasma of several tumor cell lines by immunocytochemi- cal staining, such as gastric cancer, lung cancer, cervical cancer. Conclusion The Survivin mAb prepared by using re- combinant Survivin as antigen may be used for detecting clinical tumor samples. It also makes a foundation to explore a diagnostics kit in the future.
出处
《中国实验诊断学》
2013年第11期1933-1937,共5页
Chinese Journal of Laboratory Diagnosis
基金
国家高技术研究发展计划(编号:2012AA02A504)
首都卫生发展科研专项(2011-1009-03)
首都临床特色应用研究(Z121107005112004)
