【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。更多还原

【Abstract】 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.更多还原