细胞周期相关因子CDCA3基因shRNA表达载体的构建及鉴定

Construction and identification of CDCA3 shRNA expression plasmid Vector

目的设计能转录短发夹状RNA(short hairpin RNAs,shRNA)的DNA序列,构建能有效下调CDCA3蛋白表达的shRNA真核表达载体,研究其对CDCA3表达的影响,以便进一步研究CDCA3的功能。方法根据文献获得CDCA3的siRNA靶序列,依据RNA干扰机制和shRNA靶序列的设计原则,以CDCA3基因为靶基因,合成一对编码shRNA的两条DNA序列,经退火后合成DNA双链,再克隆至shRNA表达载体pSIREN-DNRDsRed质粒中,连接产物转化E.coilJM109感受态细胞,然后挑取克隆提质粒,进行酶切鉴定和DNA序列测定。将构建成功的载体通过脂质体介导转染导入人胚肾HEK293细胞,采用Western blot法和Realtime RT-PCR,检测特异性shRNA对CDCA3蛋白在mRNA及蛋白表达水平的抑制效果。结果成功构建靶向CDCA3蛋白的特异shRNA的真核表达载体;转染HEK293细胞后,可显著下调CDCA3在细胞内的mRNA水平及蛋白表达水平。结论 CDCA3蛋白的特异shRNA的真核表达载体能显著下调HEK293细胞内的CDCA3蛋白表达。成功构建CDCA3 shRNA表达载体,为进一步研究CDCA3基因的功能提供了实验基础。

Objective To design the DNA sequence that can transcribe short hairpin RNAs and construct small hairpin RNA（shRNA） eukaryotic expression plasmids which can effectively knock down CDCA3 protein expression and investigate it interfere with CDCA3 expression,and in order to further study the functions of CACA3.Methods According to the reference documents,the siRNA target sequence of CDCA3 is obtained.According to the mechanism of RNA interference and the criteria of designing small hairpin RNA（shRNA）,CDCA3 shRNA template DNA sequences were designed and synthesized.The annealed shRNA template was inserted into expression vector pSIREN-DNRDsRed plasmid.Then transform to the competence E.coil JM109.Recombinant clones were selected and plasmid was extracted.The positive plasmids were identified by enzyme digestion and DNA sequencing.Then the plasmids were transfected into human embryonic kidney HEK293 cells by liposome mediation and the interference with the expressions of CDCA3 mRNA and its protein were detected by Western blot and Real time RT-PCR.Results One distinctive shRNA eukaryon expression plasmid for CDCA3 was constructed successfully.After the transfection,the transcription and expressions of CDCA3 in HEK293 cells were significantly decreased in mRNA and protein levels.Conclusion The specific shRNA eukaryon expression plasmid for CDCA3 can significantly inhibit CDCA3 protein expression in HEK293 cells.We construct shRNA expression plasmid for CDCA3 successfully.This study set up a foundation for experiments to further investigate CDCA3 functions.