【摘要】 目的:构建靶向人C lC-2基因小分子干扰RNA(siRNA)真核细胞表达载体。方法:根据DEQOR提供的siRNA设计原则和程序,设计两个靶向C lC-2基因的发夹状siRNA;通过化学合成的方法合成两对分别互补的寡核苷酸链,退火后得到编码该siRNA的C lC-2基因片段。将该片段连接至经BglⅡ和HindⅢ双酶切的真核细胞表达载体pSUPER.puro的多克隆位点,转化扩增提取质粒;对重组质粒进行EcoRⅠ和HindⅢ双酶切电泳鉴定和DNA序列分析鉴定。脂质体L ipofectam ineTM2000介导瞬时转染,通过RT-PCR检测人胶质瘤细胞系BT-325细胞中C lC-2mRNA表达。结果:经酶切电泳鉴定和DNA序列分析鉴定,目的片段与真核细胞表达载体pSUPER.puro连接正确。转染两重组质粒细胞的C lC-2 mRNA表达明显降低。结论:成功构建人C lC-2基因干扰真核表达载体2个,分别命名为pSUPER.puro-siC lC-21和pSUPER.puro-siC lC-22,可用于进一步研究C lC-2基因对人胶质瘤侵袭和迁移活动的影响。更多还原

【Abstract】 Objective :To construct the small interfering RNA（siRNA） eukaryotic expression vectors of human ClC-2 gene. Methods: According to the program and principles of DEQOR about designing siRNA,two pairs ClC-2 mRNA-targeted hairpin siRNA were devised,and the two pairs complemantary oligonucleotide strands of DNA fragments that encoded the above siRNA were synthesized by chemosynthesis.After annealing of the complementary strands,the DNA fragments were connected to the polyclone sites of plasmid eukaryotic expression vector pSUPER.puro that was cut by restriction endonuclease BglⅡ and HindⅢ,followed by transformation,amplification and plasmid extraction in E.coli,and finally,the two recombinant plasmids were identified by agarose gel electrophoresis by means of cutting with EcoRⅠand HindⅢ and by DNA sequence analysis.The plasmids were transfected transiently into human glioma cell line BT-325 cells by Lipofectamine^TM2000.The mRNA expression of ClC-2 gene was detected by（RT-PCR.） Results: The connections between the DNA fragments encoding ClC-2-targeted siRNA and the pSUPER.puro plasmid were correct,as confirmed by agarose gel electrophoresis and DNA sequencing（analysis.） ClC-2 mRNA expression of the BT-325 cells transfected two recombinant vectors was（significantly） decreased.Conclusion: The two RNAi recombinant vectors of human ClC-2 gene were successfully constructed.They were named pSUPER.puro-siClC-21 and pSUPER.puro-siClC-22,respectively.This laid the groundwork for future research about ClC-2 gene affecting invasion and migration of human glioma cells.更多还原