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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 the K-ras gene fragment of pancreatic cancer genome and construct the recombinant retroviral vector of sense and antisense K-ras gene and to explore its clinical significance. Methods Two pairs of PCR primers were designed. BamHI and EcoRI sites were introduced into the upstream and downstream primers respectively. Exon 4 B and flanking sequences of K-ras gene were amplified by using genomic DNA of pancreatic cancer cells. Recombinant DNA technology The target genes were respectively inserted into the retrovirus vector LZRSp BMN-Z. Recombinant clones were identified by bacterial PCR and restriction endonuclease digestion. Results The positive and negative K-ras gene exon 4 B and flanking sequences were successfully cloned into L ZRSp BMN-Z. Conclusion L ZRSp BMN-Z is one of the new candidate vectors in the treatment of pancreatic cancer antisense gene. It is convenient and feasible to obtain antisense K-ras gene by PCR and can be used to construct retroviral vector of recombinant antisense K-ras.