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目的 :用分子克隆技术构建肝癌组织特异性N rascDNA正义与反义表达载体。方法 :用BamHI酶切携带目的基因的质粒 pZIP NeoSV (X) 1,获得 1 0 6kbN rascDNA ;同时以BamHI将载体 pE BAF线性化 ,并行去磷酸化修饰 ,用T4DNA连接酶将两者连接 ,转化感受态细菌DH5α。先以酶切和随机引物法标记的N rascDNA探针进行菌落原位杂交筛选阳性重组克隆 ,再以PvuⅡ酶切鉴定插入方向 ,同时进行DNA测序和同源性分析。结果 :成功构建肝癌组织特异性正义与反义表达载体 pEBAF/s N ras和 pE BAF/as N ras。结论 :成功构建的N rascDNA正 /反义表达载体 ,为原发性肝癌的发生机理和基因治疗研究奠定良好的基础
Objective : To construct a sense and antisense expression vector specific for N rascDNA of hepatocellular carcinoma by molecular cloning technique. Methods: The plasmid pZIP NeoSV (X) 1, which carries the target gene, was digested with BamHI to obtain 106kbN rascDNA. At the same time, the vector pE BAF was linearized with BamHI, dephosphorylated in parallel, and the two were ligated with T4 DNA ligase and transformed. Competent bacteria DH5α. The positive recombinant clones were screened by in situ hybridization with N ras cDNA probes labeled with restriction enzyme and random primers. The insertion direction was identified by PvuII digestion, DNA sequencing and homology analysis were performed. Results : The tissue-specific sense and antisense expression vectors pEBAF/s N ras and pE BAF/as N ras were successfully constructed. Conclusion : The successfully constructed N ras cDNA gene/antisense expression vector lays a good foundation for the study of the mechanism and gene therapy of primary liver cancer.