克隆大鼠内皮素B受体基因重组腺病毒穿梭质粒的构建及鉴定

Construction and identification of recombinant adenovirus vector containing EDNRB gGene

目的克隆大鼠内皮素B受体(EDNRB)基因,构建含有EDNRB基因的重组腺病毒载体,为转染神经干细胞和基因治疗先天性巨结肠(HD)奠定基础。方法从大鼠心脏组织中提取总RNA,用RT-PCR方法扩增出约1580bp的片段,并将该片段克隆到载体pAdtrack-CMV中,进行酶切鉴定和序列分析。结果证实成功构建了含有EDNRB基因的重组腺病毒质粒。结论克隆到正确的大鼠EDNRB基因,并构建出重组质粒pAd-EDNRB,为下一步重组腺病毒颗粒和基因治疗打下基础。

[Objective] To clone rat EDNRB gene and recombination of shuttle adenovirus plasmid Pad-EDNRB, then being transfected into neural stem ceils. [Methods] 100 mg brain tissue of a wiatar rat was used to extract to total RNA. A 1 580 bp positive cDNA was obtained by RT-PCR. This fragment was cloned into the shuttle plasmid Pad Tract-CMV. The cloned fragment was sequenced and identified by restriction enzymens and sequerces analysis. [Results] The right rat EDNRB gene was obtained after compared with the sequenced eposited in Genebank. [Conclusion] A recombinant adenovirus vector containing EDNRB was constructed success fully. It lays the foundation for further study on gene therapy.