普通质粒载体与Semliki森林病毒复制子载体表达GHRH的比较

新一代Semliki森林病毒RNA复制子源自甲病毒属Semliki森林病毒的基因组，它克服了一些现有的DNA和RNA载体表达效率低的缺点。复制子保留了编码病毒复制酶的基因，结构基因被外源基因所取代。复制酶能够使RNA在胞质中高水平的复制以及外源基因高水平的表达。为了评价SFV复制子载体提高外源基因表达的效力，本研究首先对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和两个普通表达载体pLNCX-LacZ、pIRES-neo-LacZ转染293细胞；将表达GHRH的复制子载体pCMV-Rep-GHRH和普通载体pIRES-GHRH、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 、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。本研究为提高外源基因在真核细胞的表达提供了有益的探索。

Comparison of Conventional Plasmid Vector and Semliki forest virus-derived Vectors in Expressing Growth Hormone Releasing Hormone (GHRH)

The idea and the elements for semliki frost virus(SFV) RNA replicon, a kind of new generation vector, come from the Alphavirus genus. It was designed to overcome the poor efficacy of some current DNA-based and RNA-based 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, creating pIRES-neo-LacZ in the present study. RNA replicon vector pCMV-rep-LacZ and two conventional CMV promoter-based vector pLNCX-LacZ, pIRES-neo-LacZ were transfected by Lipofectin to prepared 293 cells respectively. RNA replicon vector pCMV-Rep-GHRH and two conventional CMV promoter-based vector pCDNA3.1(+)-GHRH, pIRES-neo-GHRH were transfected by Lipofectin to prepared 293.