【摘要】 目的:构建人鸟氨酸脱羧酶(ODC)基因第三外显子反义RNA的腺病毒载体。方法:RT-PCR方法克隆出人ODC基因第三外显子的片段,插入pMD18-T载体中,经SalⅠ、BglⅡ酶切,插入穿梭质粒pAd-Track-CMV形成重组质粒pAdTrack-CMV-ODCr。经PmeⅠ酶切线性化后,转入pAdesy-1细菌中与含腺病毒基因骨架的质粒pAdesy-1同源重组。将重组质粒pAdesy-ODCr经PacⅠ酶切后,转染293细胞包装成腺病毒颗粒,经荧光显微镜和PCR方法对重组腺病毒进行鉴定。结果:用RT-PCR方法从人前列腺癌组织中扩增出120bp的cDNA片段,经测序证实为ODC基因第三外显子的片段。重组质粒pAdTrack-CMV-ODCr转入pAdesy-1细菌中获得多个阳性克隆。重组质粒pAdesy-ODCr转染293细胞进行包装,经荧光显微镜观察可见其在293细胞中表达。进一步通过PCR法证实其含有目的基因。结论:成功地构建了ODC基因第三外显子反义RNA重组腺病毒载体,为研究其抗肿瘤的作用奠定了基础。更多还原

【Abstract】 Objective:To construct an antisense RNA recombinant adenovirus vector of the third extron in ODC gene.Methods:Domain of human ODC cDNA was amplified by RT-PCR from prostate cancer tissues and cloned into shuttle vector pAdTrack-CMV by TA clone.Then it was linealized with Pme Ⅰand transformed into pAdeasy-1cells.The identified recombinant adenovirus plasmid pAdeasy-ODCr was digested with PacⅠand transfected to293cells to package recombinant adenovirus particles.The recom-binant adenovirus production was observed by fluorescent microscope,and PCR technique was used to de-tect target gene.Results:A120bp cDNA was amplified by RT-PCR from prostate cancer tissue and the sequencing result showed it was the domain of the third extron in ODC gene.There were some positive recombinant bacterial clones after transformation of pAdeasy-1cell with pAdTrack-CMV-ODCr.Fluorescent microscope observation and PCR conformed that pAdeasy-ODCr could infect293cells and replicate in the cells.Conclusion:The antisense RNA recombinant adenovirus vector of ODC gene has been constructed,and it provides a sound foundation for further study on its antitumor activity.更多还原