摘要
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 the tumor metastasis suppressor gene p27^kip1 and construct vectors based on adeno-associated virus type 2 （rAAV2） carrying p27^kip1 by gene recombination technology and to investigate gene therapy of osteosarcoma further. Methods Totle RNA was extracted from the human placenta. The p27^kip1 cDNA （ORF） was isolated by RT- PCR and connected into the vector pAAV - MCS. The result was corrected by sequencing. Results Gene p27^kip1 was extracted from the human placenta and connected into the v ector pAAV- MCS through T vector. The result was corrected by digestion of internal restriction enzyme and ressequencing. Conclusion The successful construction of the recombinant plasmid pAAV- MCS- p27^kip1 will benefit further study on the p27^kip1 protein expression and gene therapy of osteosarcoma onwards.
出处
《中国骨与关节损伤杂志》
2009年第2期126-128,共3页
Chinese Journal of Bone and Joint Injury
基金
福建卫生教育联合基金资助(WKJ2005-2-014)
