PARP酶抑制剂未引起整合的外源LacZ基因的丢失  

The inhibitor of PARP enzyme does delete the integrated LacZ pene

作　　者：杨其伟[1] 陈雅文[1] 张学[1] 陈德风[1] 张自立[2] 

机构地区：[1]南开大学分子生物学研究所,天津300071 [2]南开大学生物化学及分子生物学系,天津300071

出　　处：《生物化学杂志》1997年第2期145-149,共5页

基　　金：国家自然科学基金!39480016

摘　　要：使用聚ADP核糖聚合酶（PARP）NAD位点抑制剂苯甲酞胺（BA）研究了降低PARP酶活性对外源LacZ基因整合稳定性的影响．利用DNA体外重组技术将LacZ基因全序列插入到真核表达载体pSV2neo的HindⅢ位点，构建了一个具有真核细胞neo基因筛选标记和LacZ基因的真核表达重组体pSV2neo-beta-gal．将该重组体导入HeLa细胞，经G418筛选获得了能稳定表达β-半乳糖苷酶的HeLa转化细胞系HeLa-beta-gal．使用PARP酶抑制剂苯甲酸胺处理细胞5周，随后进行细胞基因组Southern杂交分析与细胞内容物β-半乳苷酶的活性检测．结果表明，经BA处理的HeLa-beta-gal细胞β-半乳糖苷酶的活性及杂交带密度与未经BA处理的HeLa-beta-gal细胞相比未有明显区别．结合以前的结果可以认为，PARP酶抑制剂BA并不能导致所有外源基因的丢失，且使用PARP酶抑制剂BA引起外源基因的丢失具有选择性．Effect of lowering PARP enzyme activity on stability of integrated exogenous hacZ gene was studied. An eukaryotic expression recombinant (pSV2neo - beta - gal)capable of generating both beta - galactosidase moleculars and neomycin phosphotransferase was constructed by inserting full-length bacterial LacZ gene into Hind Ⅲ site of pSV2neo-beta-gal vector. A stable transformed cell line(HeLa-beta-gal) that constitutively expresses the product of exogenous LacZ gene, beta-galactosidase, was obtained after transfection of HeLa cells with pSV2neo-beta-gal construct, and selection with G418. The obtained HeLa-beta -gal cells were cultured continuously in the presence of 5 mmol/L benzamide, an NAD site inhibitor of PARP enzyme. After treatment for 5 weeks, bet a-galactosidase activity assay and Southern blot analysis were performed. The results showed that there was no difference between HeLa-beta-gal cells and benzamide-treated ones for beta-galactosidase activity as well as exogenous hybridization band intensity. These data in combination with results from previous work suggested that the inhibitor of PARP can not lead to the loss of all kinds of exogenous genes from cell chromosomes and lowering of PARP activity with the use of its inhibitor may have a selective effect on deletion of exogenous genes.

关 键 词：LACZ基因 整合 PARP抑制剂 

分 类 号：Q75[生物学—分子生物学]