【摘要】 目的构建p27^kip1过表达慢病毒载体,观察其感染人晶状体上皮细胞系后p27^kip1的表达情况,为探讨p27^kip1基因转染治疗后发性白内障（PCO）提供实验基础。方法设计p27^kip1基因引物,通过聚合酶链反应（PCR）扩增制备p27^kip1基因片段。利用限制性内切酶消化慢病毒载体得到线性化的慢病毒载体。将目的基因扩增产物与线性化的慢病毒载体通过交换的方法进行重组反应,从而构建含目的基因的重组质粒,命名为GV308-p27^kip1。将GV308-p27^kip1加入感受态细菌进行转化,用PCR方法鉴定平板上的单克隆菌落,对阳性菌落抽提质粒并送公司测序。将阳性菌液扩大培养,抽提以得到高纯度的GV308-p27^kip1,其与两种包装质粒一起共转染293T细胞,从而包装慢病毒。将重组慢病毒以优化感染滴度感染293T细胞和人晶状体上皮细胞,通过Western Blot检测p27^kip1的表达水平。结果对阳性转化子进行PCR鉴定及重组质粒DNA测序,证明GV308-p27^kip1构建成功。通过Western Blot检测,p27^kip1过表达慢病毒载体能明显提高293T细胞中p27^kip1的表达水平。p27^kip1过表达慢病毒载体可高效感染人晶状体上皮细胞并明显上调p27^kip1的表达。结论本方法成功构建了p27^kip1过表达慢病毒载体,并观察到p27^kip1在293T细胞及人晶状体上皮细胞的过表达,为进一步研究p27^kip1过表达对人晶状体上皮细胞生物学功能的影响及基因治疗PCO奠定了基础。更多还原

【Abstract】 Objective To construct and identify a lentiviral vector over-expression p27^kip1 and to observe the expression of p27^kip1 in human lens epithelial cell line,in order to provide experimental basis for the gene therapy of posterior capsular opacifation. Methods p27^kip1 gene primers were designed and the p27^kip1 gene fragment was prepared by polymerase chain reaction（ PCR） amplification. The lentiviral vector was digested with restriction endonucleases to obtain a linearized vector. The recombinant plasmid was named GV308-p27^kip1. The recombinant plasmids were transformed into the competent bacteria,and the monoclonal colonies were identified by PCR. The positive clones were sequenced. 293 T cells and human lens epithelial cells were infected with recombinant lentivirus,and the expression level of p27^kip1 was detected by western blot. Results PCR identification and DNA sequence showed that GV308-p27^kip1 recombinant plasmid was successfully constructed. The expression of p27^kip1 in 293T cells which were infected recombinant lentivirus was significantly increased. p27^kip1 overexpressing lentiviral vector can efficiently infect human lens epithelial cells and up-regulate p27^kip1 expression. Conclusion The p27^kip1 overexpression lentiviral vector is successfully constructed. The over-expression of p27^kip1 in 293T cells and human lens epithelial cells are observed. This made the experimental basis for p27^kip1over-expression on the biological function of human lens epithelial cells and gene therapy for posterior capsular cataract.更多还原