摘要
目的 :设计并合成人 8-羟基鸟嘌呤 -DNA糖苷酶 (HOGG1)特异性的锤头状核酶 (hammerheadribozyme)基因并构建其真核表达载体。初步探讨其在A5 4 9细胞中的瞬时表达效应。方法 :运用计算机软件人工设计并合成核酶基因 (RZ) ,将核酶基因克隆到真核表达载体pcDNA3 1(+)中 ,阳性重组子由BamHI和EcoRI酶切 ,琼脂糖凝胶电泳鉴定及DNA测序证实。大量抽提阳性重组子并在非脂质体转染试剂FuGENE 6的介导下引入人肺腺癌细胞株A5 4 9,RT -PCR(逆转录多聚酶链反应 )法半定量检测转染细胞中HOGG1mRNA表达的改变。结果 :经酶切电泳及DNA测序证实合成的核酶基因序列正确并已被准确克隆入pcDNA3 .1(+)的BamHI和EcoRI酶切位点之间 ,命名为pcDNA3 .1(+) -RZ。经RT -PCR法检测到转染核酶的靶细胞中HOGG1mRNA表达下降 36 % ,与空白细胞组差异有显著性。结论 :成功合成HOGG1特异性的锤头状核酶基因并构建了该基因的真核表达载体。核酶在A5 4 9细胞内对靶基因HOGG1具有抑制其表达的作用。
Objective:To construct and identify the eukaryotic ex pression vecto r with genes of hammerhead ribozyme targeting human 8-oxoguanine DNA glycosylase 1 (HOGG1) mRNA and to investigate its transient effect on lung cancer A549 cell s.Methods:According to design by computer, two specific restrict ion sites BamH I and EcoR I were added to the both ends of the ribozyme gene, then the modified rib ozyme gene was synthesized and cloned into the eukaryotic expression vector pcDN A3.1(+). The positive recombinants were screened by tolerance of ampicillin, a nd plasmids were extracted from the positive recombinants and digested by BamH I a nd EcoR I, and then were analyzed by agarose gel electrophoresis and DNA sequenc ing. The recombinants were transiently transfected into A549 cells. The change s of HOGG1 mRNA in A549 cells were detected by RT-PCR.Results:Th e recombinants containin g the ribozyme gene were successfully selected by restriction endonuclease digestion and agaro se gel electrophoresis, and were proved by DNA automatic sequencing. The expres s ion of HOGG1 mRNA in A549 transfected ribozyme was 36% less than that in contr ol ce lls.Conclusion:The eukaryotic expression vectors with genes of h ammerhead ribo zyme targeting HOGG1 mRNA were constructed successfully, and it effectively inh ibited the expression of HOGG1 gene in lung cancer A549 cells。
出处
《现代预防医学》
CAS
北大核心
2005年第3期207-209,共3页
Modern Preventive Medicine
