摘要
目的构建人表皮生长因子受体-2(HER-2)特异性核酶(ribozyme,RZ)及其底物基因的体外转录载体.方法设计合成RZ基因,将该RZ克隆到真核表达载体pcDNA3.1(+)中,测序鉴定.应用Trizol试剂提取MDA-MB-453细胞总RNA,RT-PCR扩增靶基因HER-2 cDNA片段,同样插入载体pcDNA3.1(+),测序鉴定.结果经DNA测序分别证实合成的RZ基因序列和通过RT-PCR扩增的HER-2 cDNA序列克隆入pcDNA3.1(+)中.结论构建HER-2特异性RZ真核表达载体及含有HER-2 cDNA的载体,有助于进一步研究RZ对靶基因切割作用及雌激素受体阴性乳腺癌细胞MDA-MB-453中HER-2信号转导通路.
Objective To construct the transcription vectors of genes of HER-2 specific ribozyme and its substrate in vitro. Methods According to design of computer,the ribozyme gene was synthesized,then was cloned into the eukaryotic expression vector peDNA3.1 (+). The positive recombinants were analyzed by DNA sequencing. Trizol reagent facilitated isolation of total RNA from MDA-MB-453 cells. The HER-2 eDNA gene sequence amplified by reverse transcription polymerase chain reaction( RT-PCR) was inserted into the same vector peDNA3.1( + ).The HER-2 positive recombinants were analyzed by DNA sequencing. Results The reeombinants containing the ribozyme gene and the HER-2eDNA gene proved by DNA sequencing were cloned into pcDNA3.1( + ). Conclusion A hammerhead ribozyme,the eukaryotic expression vector with the RZ and in vitro transcription vector with HER-2 gene were constructed. It helps to study deeply cleavage of ribozyme targeting HER-2 mRNA and the HER-2 signal transduetion pathway of estrogen receptor negative breast cancer cells MDA-MB-453.
出处
《北华大学学报(自然科学版)》
CAS
2008年第5期417-419,共3页
Journal of Beihua University(Natural Science)
基金
吉林省教育厅科研基金项目(2006-87)