HER-2/neu基因探针的制备及特异性分析

目的 筛选、制备乳腺癌标志基因--HER-2/neu的标记探针,利用间接荧光原位杂交(FISH)技术,探索其临床应用价值.方法 通过文库构建、多级筛选、PCR鉴定、序列分析等技术获得HER-2/neu基因组片段,缺口转移法制备生物素标记探针,与生物素-亲和素放大系统和多种荧光显色系统组合,针对病理标本、细胞涂片进行间接FISH检测.结果 制备的HER-2/neu基因探针可以特异性地检测乳腺癌阳性、胃癌阳性病理片,也可以特异性检测阳性、阴性细胞标本,并与不同的荧光显色系统结合使杂交信号可以选择性地呈现绿色或红色荧光.结论 高特异性的HER-2/neu基因探针和灵活的显色系统为FISH的临床应用奠定了基础.

Preparation of HER-2/neu Gene Probe and Analysis of Its Specificity

Objective This study was aimed to prepare Biotin-labeled HER-2/neu probes and relative signal amplification by indirect fluorescence in situ hybridization (FISH) and establish detection system for clinical application.Methods HER-2/neu genomic library was first constructed.The positive clones containing HER-2/neu genomic fragments were screened with PCR technique and sequencing, then labeled with Biotin using nick-translation methods.The obtained Biotin-labeled probes and relative biotin-avidin signal amplification and fluorescence detection system were used to establish FISH in tissue samples and cell lines.Results By use of indirect FISH system, the obtained HER-2/neu probes were capable of detecting breast cancer and gastric carcinoma sections and distinguishing positive and negative cell samples specifically.The hybrid signals were selectable in green or red fluorescence.Conclusion HER-2/neu probes with high specificity, flexible signal amplification and detection system may facilitate FISH technique in clinical application.