肝癌组织靶向性正义与反义表达载体pEBAF/N-ras的构建  

CONSTRUCTION OF pEBAF EXPRESSION VECTORS OF N RAS SENSE AND ANTISENSE TARGETED TO HEPATOCELLULAR CARCINOMA

作　　者：孔建平[1] 孙汶生[1] 曹英林 宋静 张利宁 马春红 

机构地区：[1]山东大学医学院微生物学与免疫学教研室

出　　处：《山东医科大学学报》2001年第3期193-196,共4页Acta Academiae Medicinae Shandong

基　　金：国家自然科学基金资助课题 (3 9970 3 3 3 )

摘　　要：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 。Objective:To obtain recombinant Epstein Barr virus expression vector pEBAF with N ras cDNA targeted to hepatocelluar carcinoma.Methods:EB virus vector pEBAF which contains human α fetoprotein promoter and enhancer was used as an expression vector.For N ras,the 1.06 kb fragment(including coding region and flanking sequence)from the plasmid pZIP NeoSV(X)1 was excised using BamHI and ligated into the BamHI site of pEBAF treated by phosphorylase in sense and antisense directions.First,poritive clones were selected with restriction map and in situ hybridization.Then DNA sequencing was used for the analysis of its homeogenesis with other related N ras mRNA sequences.Lastly the insert direction of N ras was proved using restriction endonuclease PvuⅡ.Results:The recombinant pEBAF/s N ras and pEBAF/as N ras were both successfully obtained with the molecular biological technique.Conclusion:The two kinds of recombinant pEBAF/N ras expression vectors may lead to targeted expression of N ras on hepatoma and have tissue specificity and exclusiveness for hepatoma.Consequently this will lay foundation for the study of development mechanism and gene therapy of PHC.

关 键 词：基因 RAS 癌 肝细胞 核苷酸类 基因表达 

分 类 号：R73－3[医药卫生—肿瘤]