大鼠Atoh1真核表达载体pAtoh1-IRES2-EGFP的构建及转染293T细胞的研究

The cloning and sequencing of SD rat Atoh1 gene CDS region

目的:利用基因工程方法克隆SD大鼠Atoh1基因编码区序列,构建Atoh1的真核表达载体pA-toh1-IRES2-EGFP,并验证其在293T细胞中的表达。方法:通过RT-PCR从SD大鼠结肠组织内扩增出Atoh1基因全长CDS序列,并TA克隆于PMD-19T载体中。纯化回收的目的片段测序后连接于真核细胞表达载体pIRES2-EGFP中,构建pAtoh1-IRES2-EGFP真核表达载体。再次测序后脂质体介导重组质粒转染293T细胞,荧光显微镜下观察绿色荧光蛋白的表达。结果:测序后将扩增得到的大鼠Atoh1CDS序列与GeneBank公布的参考序列对比,有两处碱基发生突变,但克隆序列编码的氨基酸序列与参考序列完全一致,两处碱基应为单核苷酸多态性,突变为无义突变,不影响蛋白表达。双酶切和测序结果证明Atoh1已正确地克隆到真核表达载体pIRES2-EGFP中,重组质粒转染293T细胞24h后荧光显微镜下观察到绿色荧光蛋白表达。结论:获得正确Atoh1编码序列,真核表达载体pAtoh1-IRES2-EGFP构建成功并可以在293T细胞中表达,为进一步对感音神经性聋的基因治疗奠定了基础。

Objective：To clone Atoh1 gene coding sequence of SD rat and construct the Eukaryotic expression plasmid pAtoh1-IRES2-EGFP,and to study its expression in 293T cells.Method：Total RNA was extracted from colon of SD rat.Atoh1 cDNA was obtained by RT-PCR amplification and subcloned into PMD-19T vector.The purified digested fragment was connected into Eukaryotic expression vector pIRES2-EGFP to construct the recombinant plasmid.The recombinant expression plasmid was identified by enzyme digestion and sequence analysis and then transfected into 293T cells with Lipofectamine.The expression of green fluorescent protein was detected through fluorescence microscope.Result： Compared cloned DNA sequence of Atoh1 gene CDS area with the reference sequences published in GeneBank,there were two base nonsense mutation in the sequence,deduced amino acid of cloning sequences as the same as reference sequences.Two bases should be single nucleotide polymorphism.Results of enzyme digestion and sequencing confirmed the successful construction of the recombinant plasmid.The expression of the green fluorescent protein was observed in the transfected 293T cells 24 h after transfection by fluorescence microscope.Conclusion：plRES2-EGFP-Atoh1 can be constructed and expressed successfully in the 293T cells,which will guide further research on gene therapy for sensorineural hearing loss.