表达miR-150shRNA细胞株的初步建立  

Establishment of Cell Line Expressing MiR-150 shRNA

作　　者：代晨[1] 邓文[1] 张彦明[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨陵712100

出　　处：《动物医学进展》2009年第8期1-5,共5页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(30771607);家畜疫病病原生物学国家重点实验室开放基金项目(猪瘟病毒感染相关microRNA的克隆与鉴定)

摘　　要：为了探讨融合microRNA重组干扰载体构建的方法,为今后mi R-150对猪瘟病毒(CSFV)潜在的调控进行研究,利用生物信息学技术筛选出mi R-150,设计并合成了表达mi R-150的两条shRNA序列的DNA单链,将其退火后与干扰载体pGenesil-1连接,构建了含有mi R-150shRNA和绿色荧光蛋白基因的重组干扰质粒pGene-mi R-150shRNA,提取并纯化质粒后,采用脂质体法将重组质粒转染PK-15细胞,用G418抗性筛选。经过酶切鉴定和测序鉴定,表明成功构建了重组干扰质粒pGene-mi R-150shRNA,并在转染24 h后即可检测到绿色荧光。本研究成功得到稳定表达mi R-150的细胞株,所建的方法可以应用于各种microRNA的构建。To explore and establish a method of vector construction, and to research on potential regulation of CSFV by miR-150,miR-150 was selected by bioinformatics, two pairs of single DNA chain encoding short hairpin RNA（shRNA）of miR-150 were designed and synthesized, and were ligated to the interference vector pGenesil-1. The recombinant plasmid pGene-miR-150shRNA was constructed, and transfected into the PK-15 cells by liposome after extraction and purification. The cell line expressing miR-150 was established by screening positive clones in the presence of G418. After enzymatic assay and recovering, the recombinant plasmid pGene-miR-150shRNA was constructed, and the green fluorescence could be detected. All the results above indicated that the method could be used in microRNA vector construction and the cell line expressing miR-150 was established.

关 键 词：猪瘟病毒 miR-150 发夹RNA 转染 

分 类 号：S852.651[农业科学—基础兽医学]