HER-2基因特异性核酶设计与体外转录载体构建

Construction of Design of HER-2 Specific Ribozyme Genes and Its Transcription Vector in Vitro

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.