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目的克隆人肿瘤转移抑制基因p27kip1,利用基因重组技术,构建其腺相关病毒载体,为进一步研究骨肉瘤的基因治疗建立一个平台。方法从人正常胎盘组织中提取总RNA,RT-PCR获取p27kip1基因(ORF)cDNA序列,并将其克隆至腺相关病毒载体pAAV-MCS,构建人p27kip1基因腺相关病毒载体。结果人胎盘组织抽提总RNA,并通过RT-PCR获得目的基因ORF,经TA克隆后,将目的基因连入pAAV-MCS中,送测序结果无误。结论成功构建的重组质粒pAAV-MCS-p27kip1,将为进一步研究p27kip1蛋白的生物学效应提供基础,为p27kip1基因应用于骨肉瘤的治疗提供实验依据。
Objective To clone human tumor metastasis suppressor gene p27kip1 and construct its adeno-associated virus vector by gene recombination technology, so as to establish a platform for further study on the gene therapy of osteosarcoma. Methods The total RNA was extracted from normal human placenta and the cDNA sequence of p27kip1 gene (ORF) was obtained by RT-PCR and cloned into the adeno-associated virus vector pAAV-MCS to construct human adeno-associated virus vector p27kip1. Results Total RNA was extracted from human placenta and the ORF of the target gene was obtained by RT-PCR. After TA cloning, the target gene was ligated into pAAV-MCS and the sequencing result was correct. Conclusion The constructed recombinant plasmid pAAV-MCS-p27kip1 will provide the foundation for further study of the biological effect of p27kip1 protein and provide experimental evidence for the application of p27kip1 gene in the treatment of osteosarcoma.