胰腺癌反义K-ras癌基因片段的分离和克隆

目的：分离及克隆胰腺癌基因组中K—ras基因片段，构建重组反义K—ras癌基因逆转录病毒载体，并探讨其意义。方法：设计2对PCR引物，分别在上下游引物中引进BamHI和EoRI位点，以胰腺癌细胞株BxPC-3基因组DNA为模板扩增K—ras基因外显子1及侧翼序列，并采用重组DNA技术将目的基因插入逆转录病毒载体LZRSpBMN—Z中，并经菌液PCR和限制性内切酶酶切鉴定。结果：K—ras基因外显子1及侧翼序列已成功地克隆入LZRSpBMN—Z中。结论：LZRSpBMN—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目的基因外显子1方便可行，可用于重组反义K—ras癌基因逆转录病毒载体的构建。

The Isolation and Cloning of Antisense K-ras Gene Fragment

Objective: To isolate and clone antisense K-ras gene fragment in pancreatic carcinoma. Methods: Polymerase chain re-action(PCR) was used to amplify exon 1 and flanking sequence of BxPC-3 pancreatic carcinoma cell line genomic DNA and the PCR product was inserted into retrovial vector LZRSpBMN-Z using recombinant DNA technique. The recombinant plasmid was isolated, purified and identified by PCR and restriction enzyme digestion. Results:The exon 1 and flanking sequence of K-ras gene were successfully cloned into the vector LZRSpBMN-Z. Conclusion: The vector LZRSpBMN-Z may be a novel ideal candidate vector for gene therapy of pancreatic carcinoma. Antisense K-ras gene fragment cloned by PCR could be applied to construct of retroviral vector of recombinant.