大鼠Atoh1基因真核表达载体的构建及转染293T细胞的实验研究  

Construction of a recombinant eukaryotic vector of Atoh1 and its expression in 293-T cells

作　　者：郑国玺[1] 祝康[1] 朱珠[1] 侯瑾[1] 韦俊荣[1] 许珉[1] 

机构地区：[1]西安交通大学医学院第二附属医院耳鼻咽喉头颈外科病院,陕西西安710004

出　　处：《南方医科大学学报》2011年第7期1131-1137,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(30471877);陕西省科技攻关项目(2010K15-08)~~

摘　　要：目的利用基因工程方法克隆SD大鼠Atoh1基因CDS,构建大鼠核转录因子Atoh1的真核表达载体并在真核细胞中表达。方法从SD大鼠结肠粘膜提取总RNA,采用逆转录PCR法扩增Atoh1基因CDS区序列并TA克隆至PMD-19T载体中。测序鉴定后将Atoh1基因连接于含有EGFP和内部核糖体转入位点(internal ribosome entrysite,IRES)的真核细胞表达载体pIRES2-EGFP中,对重组质粒进行酶切鉴定和测序鉴定后,以脂质体介导法转染至293T细胞,荧光显微镜、PT-PCR和Western blot检测其在293T细胞中的表达。结果扩增得到大鼠Atoh1CDS区长1056bp,编码351个氨基酸,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(SNP),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,转染293T细胞48h后,荧光显微镜下观察到荧光蛋白的表达,PT-PCR和Western blot证实Atoh1的mRNA和蛋白能在293T细胞中正确表达。结论成功构建了真核表达载体pAtoh1-IRES2-EGFP,并在293T细胞中成功表达,为进一步研究Atoh1的功能、作用机制及感音神经性耳聋的基因治疗奠定了基础。Objective To clone the coding sequence of Atoh1 cDNA from SD rat and construct a eukaryotic expression vector for its expression in eukaryotic cells.Methods The total RNA was extracted from colonic mucosa of SD rats and the double-stranded cDNA of Atoh1 was amplified using RT-PCR.The cDNA coding sequence was then cloned into PMD-19T vector followed by sequence analysis.The digested fragment,after purification,was linked into the eukaryotic expression vector pIRES2-EGFP containing the EGFP gene and the internal ribosomes site（IRES）.After identification by enzyme digestion and sequence analysis,the recombinant plasmid was transfected into 293T cells via Lipofectamine,and the expression of the target protein was detected under fluorescence microscope,using PT-PCR and Western blotting.Results DNA sequence analysis showed that the amplified rat Atoh1 gene,with a length of 1056 bp of the coding sequence which encoded 351 amino acids,had two base mutations compared to the reference sequences in GenBank,possibly as a result of single nucleotide polymorphisms that induced nonsense mutation without affecting the amino acid sequences or the protein expression.The results of enzyme digestion and sequence analysis demonstrated that the Atoh1 gene was correctly inserted in the eukaryotic expression vector plRES2-EGFP.Fluorescence microscopy and Western blotting both confirmed successful expression of Atohl at the mRNA and protein levels in 293T cells 48 h after transfection with the recombinant plasmid.Conclusion The recombinant plasmid harboring the coding sequence of SD rat Atoh1 gene has been constructed successfully and can be expressed in the 293T cells,which provides a basis for functional study of Atoh1 gene and future researches in gene therapy for sensorineural hearing loss.

关 键 词：感音神经性耳聋 Atoh1 基因克隆 真核表达载体 

分 类 号：R764.43[医药卫生—耳鼻咽喉科]