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目的:克隆人三元复合因子Net全长基因,构建其重组腺病毒载体。方法 :抽提正常人胰腺组织中总RNA,经RT-PCR扩增出全长Net基因,利用同源重组的方法将腺病毒骨架质粒pBHG35和穿梭质粒pDC316-Net共转染293细胞,获得腺病毒重组质粒Ad5/F35-Net,在293细胞中包装和扩增后,获得高滴度的腺病毒重组质粒Ad5/F35-Net。采用PCR和蛋白印迹法检测Net基因的表达。结果 :用RT-PCR方法 ,从人胰腺癌组织中扩增出1224 bp的cDNA片段,经测序证实为人Net基因。最终构建及包装出高滴度的重组腺病毒载体Ad5/F35-Net。结论:成功克隆了人Net全长基因,并构建其重组腺病毒表达载体,为进一步研究人Net基因在相关肿瘤疾病中的生物学作用奠定了基础。
Objective: To clone full-length human triplet complex factor gene and construct its recombinant adenovirus vector. Methods: Total RNA was extracted from normal pancreas and amplified by RT-PCR. The recombinant adenovirus plasmid pBHG35 and shuttle plasmid pDC316-Net were co-transfected into 293 cells by homologous recombination. The virus recombinant plasmid Ad5 / F35-Net was packaged and expanded in 293 cells to obtain high titer adenovirus recombinant plasmid Ad5 / F35-Net. Net gene expression was detected by PCR and Western blotting. Results: The 1224 bp cDNA fragment was amplified from human pancreatic cancer tissue by RT-PCR and confirmed to be human Net gene by sequencing. The recombinant adenoviral vector Ad5 / F35-Net with high titer was constructed and packaged. CONCLUSION: The full-length human full-length gene was cloned and its recombinant adenovirus expression vector was constructed, which laid the foundation for further study on the biological role of human Net gene in related oncological diseases.