人CD147基因siRNA真核表达质粒的构建及其对HeLa细胞中CD147基因表达的影响

Construction of siRNA Eukaryotic Expression Plasmids Targeting Human CD147 Gene and Their Effects on CD147 Gene Expression in HeLa Cells

目的构建针对人CD147基因的siRNA真核表达质粒,并观察其对HeLa细胞中CD147基因表达的影响。方法根据GenBank中登录的人CD147基因序列,设计并合成针对CD147基因的特异性小干扰片段,并将其定向克隆至带有卡那霉素抗性和增强型绿色荧光蛋白的真核表达载体pGenesil-1中,对重组质粒进行酶切分析和DNA序列测定。用脂质体将重组质粒转染至HeLa细胞中,观察其对HeLa细胞CD147基因mRNA及蛋白表达水平的影响。结果酶切鉴定和测序结果表明,3个表达短发夹RNA的质粒及其阴性对照质粒构建正确。3个siRNA真核表达质粒对HeLa细胞CD147基因mRNA转录水平的抑制率分别为32.5%、66.8%和59.1%;对CD147蛋白表达水平的抑制率分别为28.3%、63.5%和56.2%。结论已成功构建针对人CD147基因的siRNA真核表达质粒,其对HeLa细胞CD147基因的表达具有明显的抑制作用,为进一步研究CD147基因的功能奠定了基础。

Objective To construct the siRNA eukaryotic expression plasmids targeting human CD147 gene and observe their effects on CD147 gene expression in HeLa cells. Methods The specific oligonucleotides of siRNA targeting CD147 gene were designed and synthesized according to the CD147 gene sequence reported in GenBank and directionally cloned into plasmid pGenesil-1 with enhanced green fluorescence protein （EGFP） and Kan genes. The constructed recombinant plasmids were identified by restriction analysis and sequencing then transfected to HeLa cells in mediation of liposome, and their effects on mRNA transcription and protein expression of CD147 were observed. Results Restriction analysis and sequencing proved that three siRNA eukaryotic expression plasmids and their negative control plasmids were constructed correctly. The inhibitory rates of three siRNA eukaryotic expression plasmids on CD147 mRNA transcription in HeLa cells were 32. 5%, 66. 8% and 59. 1%, and those to CD147 protein expression were 28. 3%, 63. 5% and 56. 2%, respectively. Conclusion The siRNA eukaryotic expression plasmids targeting human CD147 gene were successfully constructed and showed significantly inhibitory effect on CD147 gene expression in HeLa cells, which laid a foundation of further study on function of CD147 gene.