重组穿梭质粒pAdTrack-cmv-rHSG载体的构建及鉴定

Construction and Identification of Recombinant Plasmid pAdTrack-cmv-rHSG Encoding the Rat rHSG Gene

目的构建大鼠增殖抑制基因(rat hyperplasia suppressor gene,rHSG)腺病毒穿梭质粒,为进一步研究体内诱导rHSG在恶性胶质瘤的特异性表达奠定基础。方法提取SD大鼠心肌总RNA,利用RT-PCR方法扩增大鼠增殖抑制基因(rHSG)的全开放读码框区片段,将其亚克隆入T载体,双酶切后与经同样处理的腺病毒穿梭质粒pAdTrack-cmv相连,用酶切和测序进行鉴定。结果扩增rHSG克隆出rHSG基因并成功重组于入腺病毒基因并成功的穿梭质粒。结论构建成功重组大鼠增殖抑制基因腺病毒穿梭质粒载体pAdTrack-cmv-rHSG。

Objective To construct a adenovirus shuttle plasmid encoding rat hyperplasia suppressor gene（rHSG）,and to provide the basis for further researching on rHSG specific expression in the spongioblastoma tissue.Methods The complementary RNA of SD rats Cardiac muscle was extracted.The whole Open reading frame fragment of the rHSG gene was amplified by RT-PCR and subcloned in the pGM-T plasmid by double enzymat cutting.It was identified by enzymatic cutting and PCR products sequencing.Results RHSG gene was amplified by RT-PCR and cloned in the adenovirus shuttle plasmid.The favorite gene sequence was completely consistent with that reported In GENEBANK.Conclusion cDNA of rHSG can be successfully cloned and inserted into pAdTrack-cmv vector.