摘要
目的 分离及克隆胰腺癌基因组中 K- ras基因片段 ,构建重组正反义 K- ras基因逆转录病毒载体并探讨其临床意义。方法 设计两对 PCR引物 ,分别在上下游引物中引进 Bam H1和 Eco R1位点 ,以胰腺癌细胞基因组 DNA为模板扩增 K- ras基因外显子 4 B及侧翼序列 ,并采用重组 DNA技术将目的基因分别定向插入逆转录病毒载体 L ZRSp BMN- Z中。重组克隆通过菌液 PCR和重组质粒限制性内切酶酶切鉴定。结果 正反义 K- ras基因外显子 4 B及侧翼序列成功地克隆入 L ZRSp BMN- Z中。结论 L ZRSp BMN- Z是胰腺癌反义基因治疗中新型候选载体之一。应用 PCR方法获取反义 K- ras基因方便可行 ,可用于重组反义 K- ras基因逆转录病毒载体的构建。
Objective To isolate and clone sense & antisense K-ras gene fragment. Methods Polymerase chain reaction was employed to amplify exon4B and flanking sequence from BxPC-3 pancreatic carcinoma cell line genomic DNA,which was inserted into LZRSpBMN-Z vector using recombinant DNA technique. The recombinant plasmid was analyzed and identified by polymerase chain reaction of E.coli and the recombiant plasmid′s restriction enzyme digestion. Results The sense & antisense exon4B and flanking sequence of K-ras gene were successfully cloned into LZRSpBMN-Z. Conclusions LZRSpBMN-Z vector may be a novel ideal candidate vector for pancreatic carcinoma gene therapy. Antisense K-ras gene fragments cloned by polymerase chain reaction may prove useful in the construction of recombinant cancer gene retroviral vehicle.
出处
《胰腺病学》
2003年第4期203-206,共4页
Chinese JOurnal of Pancreatology
