摘要
目的构建pEGFP-N1-CatSper1重组质粒,在真核细胞中表达CatSper1,利用化学合成siRNA抑制小鼠CatSper1基因的表达,并筛选抑制效果最佳的siRNA序列。方法利用重组PCR克隆小鼠CatSper1全长cDNA,构建到真核表达载体中。生物信息分析软件校正结果,获得三条针对雄性小鼠CatSper1的siRNA序列,合成后分别与重组质粒pEGFP-N1-CatSper1共转染到小鼠成神经瘤N2a细胞株中,通过观察EGFP荧光强度、实时定量聚合酶链反应和WesternBlot等方法,分析干扰效果,筛选能显著降低N2a细胞中外源性CatSper1表达量的siRNA序列。结果成功克隆小鼠CatSper1基因的全长cDNA片段(2,061 bp),并构建到pEGFP-N1表达载体中。三个siRNA干扰组的CatSper1 mRNA和蛋白表达与空白对照组相比均下降,其中以靠近3′端的siRNA干扰作用更为明显,阴性对照siRNA组未引起CatSper1 mRNA和CatSper1蛋白表达明显变化。结论在小鼠神经瘤N2a细胞株中成功表达外源性CatSper1蛋白,并筛选出有效的siRNA,为深入研究CatSper1功能及用于避孕提供参考。
Objective： To find effective siRNA targeting recombinant plasmid pEGFP-N1-CatSperl. mouse CatSperl mRNA that was transcribed by a Methods： The full length of mouse CatSperl cDNA was cloned using recombinant PCR and integrated into a pEGFP-N1 plasmid （pEGFP-N1-CatSperl）. Three siRNAs were predicted with the online bioinformatics analysis and synthesized. The synthesized siRNAs were co-transfected into the N2a cells with the pEGFP-N1-CatSperl plasmid. The interferential effect of siRNAs was determined by EGFP fluorescence intensity, quantitative PCR and Western blot. Results： The full length of mouse CatSperl cDNA was successfully cloned into the pEGFP-N1 plasmid. CatSperl mRNA and protein expressions were substantially inhibited by the siRNA located on the 3＇ of CatSperl mRNA, while all the three siRNAs showed interferential effects. Negative control siRNA did not cause changes in CatSperl mRNA and protein expressions. Conclusion. The three effective siRNAs targeting CatSperl were identified and could be used for the study of the function and contraceptive potential of CatSperl.
出处
《生殖医学杂志》
CAS
2011年第6期501-506,共6页
Journal of Reproductive Medicine
基金
国家自然科学基金项目(30770814)
湖北省计划生育委员会科研基金
