【摘要】 新一代Semliki森林病毒RNA复制子源自甲病毒属(Alphavirus)Semliki森林病毒的基因组,它克服了一些现有的质粒载体表达效率低的缺点。研究首先对pIRES-neo表达载体用BamHⅠ酶切和去磷酸化,连接LacZ基因,构建了普通真核表达载体pIRES-neo-LacZ;然后采用脂质体介导分别将含报道基因LacZ复制子载体pCMV-rep-LacZ以及两个普通表达载体pLNCX-LacZ和pIRES-neo-LacZ转染293细胞;将表达生长激素释放激素(GHRH)的复制子载体pCMV-Rep-GHRH和普通载体pIRES-GHRH和pCDNA3-GHRH转染293细胞。分别对不同载体转染细胞表达的β-半乳糖苷酶以及利用RIA和RT-PCR对表达的GHRH进行了定量分析,结果表明:复制子载体表达外源基因的效率比非复制子载体高2～3倍。更多还原

【Abstract】 The elements for Semliki forest virus (SFV) RNA replicon, a kind of new generation vector, comes from the Alphavirus genus. It was designed to overcome the poor efficacy of some current plasmid vector. Genes coding for viral replicases are reserved while genes coding for structure proteins are replaced by foreign gene in RNA replicon. High level replication of RNA and expression of foreign gene in cytoplasm are regulated by the replicases. To evaluate the effects of the SFV RNA replicon on the efficiency of gene expression, LacZ gene was inserted into pIRES-neo which digested by BamHⅠand dephosphorylated by shrimp alkaline phosphatase, and pIRES-neo-LacZ vector was constructed. RNA replicon vector pCMV-rep-LacZ and two conventional CMV promoter-based vector (pLNCX-LacZ and pIRES-neo-LacZ) were transfected by Lipofectin to prepared 293 cells, respectively. RNA replicon vector pCMV-Rep-GHRH(Growth hormone releasing hormone) and two conventional CMV promoter-based vector(pCDNA3.1(+)-GHRH and pIRES-neo-GHRH) were transfected by Lipofectin to prepared 293 cells, respectively, too. Results of quantitating for β-galactosidase expressed from transfected cells and quantitating for GHRH expressed from transfected cells by RIA and RT-PCR showed that the express level of RNA replicon vector was superior to ordinary vector and 2~3 times higher than normal plasmid vector. This will provide a beneficial exploring to improve the efficiency of gene expression in eukaryotic cell.更多还原